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hematopoietic stem cell transplantation ( hsct ) has become a common procedure of therapy for patients with hematological malignancies and many other life threatening blood disorders . however , viral and fungal infections associated with the severe immunoablative conditioning used prior to hsct still represent a major challenge ( martelli et al . , 2002 ; seggewiss and einsele , 2010 ) . one approach to address this barrier is to use reduced intensity conditioning ( ric ) . hsct following ric relies on non - myeloablative preparatory regimen that spares a substantial level of the host immunity and thus reduce transplant related mortality ( trm ) by both improving post - transplant immune reconstitution and reducing the toxicity associated with the conditioning agents ( ballen and spitzer , 2010 ; gyurkocza et al . , ric was first developed to enable allogeneic hsct in patients with advanced hematological malignancies who can not withstand myeloablative conditioning because of age and/or performance status . ric protocols also enable the use of hsct in patients with non - malignant disorders , associated with longer life expectancy . in these patients the aim of the conditioning prior to the allogeneic hsct is merely to support sustained donor cell s engraftment for correction of the disease ( steward and jarisch , 2005 ; ringden et al . , 2006 ) , or , as demonstrated in limited number of patients , for the induction of donor chimerism , as a platform for tolerance induction as prelude to organ transplantation ( kawai et al . , nevertheless , currently used ric protocols are still relatively aggressive and therefore it is highly desirable to develop safer preparative regimen protocols that selectively achieve a state of donor - specific unresponsiveness without compromising the overall immune response . one way to achieve a state of donor - specific tolerance uses donor cells endowed with veto activity . the term veto , coined in 1980 by miller ( miller , 1980 ) , relates to the ability of cells to specifically delete t cells directed against antigens ( ags ) of the veto cells themselves , but not against third - party ag ( muraoka and miller , 1983 ; claesson and miller , 1984 ) . the suppression of effector t cells directed against the veto cells is both ag - specific and major histocompatibility complex ( mhc ) restricted , resulting from the unique manner by which the veto cell kills its target . thus , veto activity results from unidirectional recognition of the veto cell by the responding t cell , but not vice versa . therefore , the recognizing t cell , the t cell receptor ( tcr ) of which is directed against the mhc of the veto cell , is killed upon binding to its veto target , due to exchange of signals allowed following this interaction . hence , the use of donor veto cells capable of specifically eliminating only the host anti - donor t cell clones that mediate the transplant rejection while sparing other t cells that can persist and fight infectious pathogens , could offer an effective modality for the induction of transplantation tolerance . initially , veto activity was described for cells within the spleen of athymic nude mice ( miller , 1980 ) . based on this initial observation , various cell types have been shown to mediate veto activity including t lymphocytes , natural killer cells , and dendritic cells . a very strong veto activity was documented for cd8 cytotoxic t cell ( ctl ) lines or clones ( fink et al . , 1984 ; claesson and ropke , 1986 ; claesson and miller , 1989 ) and direct comparison of the veto reactivity of various cell types revealed that ctls have the strongest in vitro veto effect ( reich - zeliger et al . , cells in murine bm and in t cell colonies grown from such bm were shown to mediate veto activity in vitro ( muraoka and miller , 1980 ) and un - separated donor bm was shown to specifically reduce the frequency of anti - donor ctls in grafted mice ( wood et al . , 1992 ) . however , this in vivo tolerizing activity of the bm cells could be attributed to t cells which reside within the bm . these t cells , while potentially mediating beneficial veto activity , also cause a severe multi - system graft versus host disease ( gvhd ) . early attempts to avoid gvhd risk and to apply t cell - depleted bmt ( tdbmt ) in leukemia patients indeed revealed that this benefit of gvhd prevention is offset by increased risk for graft rejection , due to absence of donor t cells within the graft ( gale and reisner , 1986 ; kernan et al . , 1987 ) . however , veto activity could be also assigned to non - t cells within the bm . for example , a series of studies by the group of judy thomas described potent veto activity of cells within the cd8cd16dr subset in the bm of rhesus macaque primates ( thomas et al . , 1991 ; cd8 surface expression was shown to play a pivotal role in the tolerogenic effect of these bm cells . thus , these studies demonstrated that cd8 crosslinking following interaction with donor - reactive ctl precursors ( ctlp ) , elicits upregulation of transforming growth factor-1 ( tgf-1 ) and fas ligand ( fasl ) by these donor bm cells , leading to clonal deletion of the donor - reactive ctlp ( asiedu et al . ( 1998 ) that human hematopoietic cd34 progenitors are endowed with marked veto activity ( figure 1 ) . explaining in part how megadose of purified cd34 cells enables to overcome rejection in recipients of three hla - loci mismatched ( haploidentical ) hsct while avoiding the threat of gvhd ( aversa et al . ( 2005 ) demonstrated that this veto activity is mediated through a tnf- based mechanism . ( 2002 ) demonstrated that veto activity is not only mediated directly by the infused cd34 cells but also by their cd33 progeny , which lose this tolerizing activity upon completion of maturation , at the level of cd14 monocytes or cd13 neutrophils . furthermore , preliminary results suggest that bm - derived immature dendritic cells , previously shown to induce immune tolerance , exhibit marked veto activity on cd8 t cells , in addition to the non - specific suppression of cd4 t cells mediated by the no system ( zangi et al . , 2009 ) . finally , nk cells which were shown to exhibit veto activity upon activation with il-2 , develop and appear early during the post - transplant period ( chrobak and gress , 2001 ; reich - zeliger et al . , 2004a ) . the regulatory activity of cd34 cells : evidence for target specificity . the average ctl response ( sd ) in the presence ( black bars ) or absence ( white bars ) of cd34 cells at a veto - to - responder cell ratio of 0.5 . the veto effect was tested by a limiting dilution assay as follows : equal numbers ( 1 10/ml ) of responder cells and irradiated allogeneic stimulator cells from the donor of the cd34 cells and a third - party donor were co - cultured for 5 days . the responder cells were then cultured again for 7 days under limiting dilution , and the ctl activity was determined by cr - release assay . data represent the average sd of 11 independent experiments using different donor and third - party pairs . a significant difference ( p < 0.001 on t - test compared with control cultures without cd34 cells ) between control cultures and those including cd34 cells was found upon stimulation against donor cells ( rachamim et al . , 1998 ) . thus , based on these observations , the following working hypothesis can be suggested to explain how megadose of cd34 cells can overcome rejection in human recipients of haploidentical hsct . upon administration of purified cd34 cells , the graft supporting veto activity is initially mediated directly by the infused cd34 cells , and subsequently by the cd33 progeny of these cells which grow exponentially within the first few days post - transplant . this second phase of differentiating veto cells also includes cd11c+ immature dendritic cells and other graft facilitating cells . clearly , the number of all these tolerizing cell types emerging after transplantation is proportional to the number of cd34cells infused . the increased engraftment of megadose of hsct is therefore greatly dependent not only on the ability of the initial inoculums of the cd34 cells to veto anti - donor t cells , but also on their ability to seed the bm and to generate as rapidly as possible the second or third derivatives which are required to complete the eradication of host anti - donor t cells . as described above , the use of purified megadose of cd34 hsct has enabled haploidentical transplantation in leukemia patients and was the first demonstration of the potent clinical potential of donor veto cells . however , this approach is currently limited to supra - lethal myeloablative and highly immunosuppressive conditioning protocols ( martelli et al . , 2002 ) . indeed , studies in non - human primates revealed that any significant reduction of the conditioning , to levels acceptable for elderly patients or for patients with non - malignant disorders , would require veto inducing cd34 stem cell numbers which can not be realistically collected from human donors ( gan et al . therefore , other populations of veto cells could have a crucial role in supporting and promoting successful engraftment of purified stem cell transplantation under relatively safer ric . as outlined above , cd8 ctls were shown in vitro to exhibit the strongest veto reactivity ( reich - zeliger et al . , 2004a ) . previous insights on the veto mechanism of cd8 veto ctls , combining anti - cd8 blockade and fasl - mutated veto cells , have suggested that co - expression of cd8 and fasl is required for the veto activity of these cells ( reich - zeliger et al . , 2010,2004a ) . such a mechanism involves initial recognition of the veto cell by the tcr of the effector t cells , leading to expression of fas by the effector t cell upon activation , and thereby enabling fas fasl mediated apoptosis to take place , once inhibitory molecules such as flice - inhibitory protein ( flip ) are down regulated in the effector cell ( reisner et al . , 2006 ) . in addition , the interaction between cd8 on veto ctl and the mhc class i alpha3 domain on the effector cell , is associated with phosphorylation of mek / erk in the latter cell , and with a significant reduction of x - linked inhibitor of apoptosis protein ( xiap ) levels which , in turn , enables even more potent triggering of fas fasl mediated apoptosis in the recognizing effector cell scheme 1 reich - zeliger et al . , 2010 ) . more recent results have indicated that veto activity exhibited by ctls can also be mediated by an additional perforin based mechanism ( milstein et al . , 2010 ) . however , despite their potent veto activity , cd8 ctls can not be used for safe tolerance induction in allogeneic hsct because of their marked gvh reactivity . veto - based deletion of host anti - donor cd8 t cell clones by veto ctl . cd8-mhc class i engagement induces perk , which is associated with reduction of xiap ( apoptosis inhibitor ) levels , thereby enabling fas one approach for generating veto ctls with reduced gvhd reactivity has been described by a series of studies by d. h. fowler ( fowler and gress , 2000 ; erdmann et al . , 2004 ; mariotti et al . , 2008 ) . in these studies , it was demonstrated that by using cd3/cd28 particles under il-2- , il-7- , and il-4-containing medium , veto ctls with tc2 phenotype can be generated . we have previously described an alternative approach to generate ctls with highly reduced gvh reactivity by means of stimulation against third - party stimulators in the absence of exogenous cytokines , followed by further ex vivo expansion using third - party stimulators and il-2 ( bachar - lustig et al . , 2003 ) . this approach was based on the observation that only activated ctlp are capable of surviving the cytokine deprivation in the primary culture , and that these anti - third - party clones further expand throughout the culture . these anti - third - party ctls indeed were shown to be depleted of gvhd while supporting bm engraftment in murine models . in accordance with the veto concept thus , a ctl line originating from a strain other than that of the bm donor failed to prevent graft rejection ( bachar - lustig et al . , 2003 ) . importantly , anti - third - party veto ctls , upon adoptive transfer into tdbmt recipient mice , were shown to eliminate not only host anti - donor nave cells , but also host anti - donor memory cells ( reich - zeliger et al . , 2007 ) . memory t cells , derived from prior exposure to alloantigen or generated by heterologous immunity or lymphopenia - induced proliferation , are believed to be an important part of the barrier preventing the translation of tolerance induction protocols from inbred rodent strains to the clinic ( pantenburg et al . therefore , the ability of veto ctls to overcome memory t cells - mediated rejection could be highly important for their action in clinical settings . furthermore , a recent study of nguyen and geiger ( 2010 ) propose that veto ctl can also effectively promote b cell tolerance . thus , in this study the authors demonstrated that murine cd8 ctls , in addition to their renowned veto activity upon recognizing t cells , can also induce ag - specific elimination of recognizing b cells , both in vitro and in vivo and thus veto ctls may selectively overcome both cellular and humoral graft rejection . nevertheless , despite all these important attributes of the veto cd8 ctls , their in vivo activity was shown to be markedly inferior compared to that exhibited in vitro , requiring the administration of large number of ctls in conjunction with the immunosuppressive drug rapamycin in order to efficiently overcome tdbmt rejection in murine models ( bachar - lustig et al . , 2003 ) . the discrepancy between the ctls veto activity in vitro and in vivo could be explained when considering two limitations of the veto cells : first , the veto activity is mediated through cell to cell contact and , second , host t cells are prone to veto mainly in a window of opportunity of up to 48 h after their activation , thus , once these host t cells develop into mature effector t cells , veto cells can no longer exert their effect ( anderson and zimring , 2006 ) . therefore , while in vitro the veto ctls are directly plated with their cognate targets , upon infusion in vivo the veto ctls need to co - localize with the host t cells within the first 48 h of the rejection process , or else their effect will be hampered . indeed , we recently demonstrated that ctls attained upon long ex vivo culture in the presence of il-2 exhibit a migration pattern different from the one displayed by naive host t cells thereby precluding their co - localization . thus , while nave host t cells home efficiently to the lymph nodes ( lns ) of bmt recipient mice , the veto ctls are excluded from the lns and tend to localize in peripheral sites ( ophir et al . , in order to improve the lns homing potential of anti - third - party cd8 veto cells we developed a new protocol , using il-15 , that favor the induction of central memory phenotype in anti - third - pary cd8 t cells ( ophir et al . , 2010 ) . central memory t cells ( tcm ) express the ln homing receptors cd62l and ccr7 ( sallusto et al . , 1999 ) and similarly to naive t cells , localize to the t cell area of all secondary lymphoid organs ( weninger et al . , 2001 ) . indeed , we demonstrated that these ex vivo induced anti - third - party tcm , in contrast to anti - third - party ctls , home to lns of bmt recipients , where they co - localize with the recipient s endogenous host t cells . moreover , the tcm displayed strong proliferation at the early post - transplant period and subsequently persisted in vivo for more than 1 year post - bmt , in line with their memory phenotype ( ophir et al . most importantly , we demonstrated that tcm derived from ( host donor)f1 mice can specifically and efficiently delete in vivo host tcr - transgenic t cells carrying a tcr transgene with anti - donor specificity . in accordance with the veto concept , this efficient in vivo deletion of anti - donor host t cells , found to be mediated by apoptosis , did not occur when non - specific tcm , not expressing the donor h-2 haplotype , were used ( figure 2 ) . thus , murine tcm perform in vivo veto activity , efficiently deleting host t cells only when the host t cells recognize antigens on the tcm , and not by a non - specific general immunosuppressive effect . taken together , all these attributes of the tcm were shown to translate into improved efficacy in overcoming t cell mediated rejection of murine tdbmt , thereby enabling high survival rate and long - term donor chimerism , without causing gvhd . thus , adoptive transfer of anti - third - party tcm , in the absence of rapamycin treatment significantly abolished host t cells - mediated rejection of fully mismatched nude - bm . this is in sharp contrast to anti - third - party ctls , which fail to enable engraftment unless administered in higher numbers and in conjunction with rapamycin treatment ( bachar - lustig et al we concluded therefore that by generating anti - third - party cd8 cells with a central memory phenotype we were able to dramatically enhance their tolerizing veto activity in vivo . ( a , b ) lethally irradiated c57bl/6 ( h-2 ) mice received 1 10 cd8 purified tcr - transgenic 2c cells ( carrying tcr with anti h-2 specificity ) and 5 10 irradiated balb / c ( h-2 ) splenocytes . the following day , the mice were transplanted with 1 10 c57bl/6-nude bm cells or received , in addition , 5 10 specific , derived from cb6 ( h-2 , black bars ) , or non - specific , derived from c57bl/6 ( h-2 , gray bars ) anti - third - party tcm . recipients were sacrificed 8 days post - transplant , their spleens were harvested , and the deletion of anti - donor 2c t cells was monitored by facs . ( a ) representative result demonstrating the level of surviving ( 7aad ) 2c cells in the absence ( left panel , 2c alone ) or presence of specific tcm ( right panel 2c + specific tcm ) . ( b ) quantification of results demonstrating efficient inhibition of the 2c cells only by data represent average sd of percent inhibition from at least 10 animals in each group , pooled from two independent experiments . ( c , d ) in vivo model was established as in ( a , b ) , but 5 10 purified cd8 2c cells and 2.5 10 irradiated balb / c splenocytes were administrated . recipients were sacrificed 8 days post - transplant , their spleens were harvested and facs analysis of annexin v levels on living ( 7aad ) cd81b2 + 2c cells was conducted . ( c ) representative result demonstrating apoptosis induction upon anti - donor 2c cells by the tcm as evident by annexin v levels on 2c cells in the absence ( left panel , 2c alone ) or presence of specific tcm ( right panel , 2c + specific tcm ) . ( d ) quantification of results measuring annexin v levels on the 2c cells following interactions with specific and non - specific tcm . data present average sd of percent annexin v levels in at least four animals from each group , in one representative experiment , out of three performed . * * p < 0.01 , * * * a second objective which could be attained by administration of donor - derived activated cd8 t cells in the context of hsct is related to the ability of these cells to control residual hematological malignancies . considering that gvl is generally associated with gvhd it was surprising that autologous or allogeneic human and murine anti - third - party veto cd8 t cells , markedly depleted of gvh reactivity , exhibit vigorous responses against different b cell malignancies ( arditti et al . , 2005 ; lask et al . , 2011 ) . the killing of b cell tumors by anti - third - party ctls was shown to involve a unique tcr - independent two steps mechanism . first , long - lasting conjugates are formed between the ctl and the tumor cell . these conjugates are rapidly formed through binding of icam1 on tumor cells by lfa-1 expressed on the veto ctl . second , a slower process of mhc class i - dependent apoptosis is mediated by binding of the mhc class i 2/3 constant region on the tumor cells to the cd8 molecule on the ctl membrane ( arditti et al . , recent results from our laboratory indicate that murine anti - third - party veto tcm can also efficiently eliminate murine b cell lymphoma in vivo , through a similar tcr - independent mechanism ( lask et al . , 2010 ) . hence , such single agent veto cell therapy has the potential to have a double benefit in the context of hsct , namely , promoting and supporting hsct engraftment through specific tolerizing veto activity while mediating effective anti - cancer response , in the absence of gvhd . translation of this therapy , optimizing protocols for the generation of human anti - third - party tcm is now in progress and clinical evaluation will commence in the near future in patients with b cell malignancies . donor derived veto cells represent an attractive and effective modality for the induction of specific tolerance toward donor ags . this approach has been already demonstrated in patients by using megadose of purified veto cd34 stem cells , that can overcome the host s residual immunity surviving the myeloablative conditioning and enable engraftment of hsct across major genetic barriers without the severe threat of gvhd . however , the number of veto cd34 cells that can be harvested is insufficient for overcoming the large numbers of host t cells remaining after ric . therefore , combining megadose of cd34 hsct with other gvhd - depleted veto cells , potentially anti - third - party cd8 tcm , could enable facilitation of engraftment of hsct under ric without the adverse complication of gvhd - producing t cells and without the need for deleterious post - transplant gvhd prophylaxis . it is hoped that this approach could extend the use of hsct to elderly patients with b cell malignancies who can not tolerate intensive protocols and to a variety of patients with non - malignant disorders , associated with longer life expectancy , in whom the current trm associated with hsct may not be ethically justified . use of experimental animals statement : all experiments on live vertebrates were performed in accordance with the weizmann institutional animal care and use committee .

the induction of immune tolerance by specific agents , as opposed to general immune suppression , is a most desirable goal in transplantation biology . one approach to attain this goal is afforded by the use of donor - derived cells endowed with veto activity , which is the ability of a cell to specifically suppress only t cells directed against its antigens . a megadose of purified veto cd34 + hematopoietic stem cells is already used in patients to allow hematopoietic stem cells transplantation ( hsct ) across major genetic barriers , while avoiding severe graft versus host disease ( gvhd ) . however , allowing engraftment of such t cell - depleted hsct under safer reduced intensity conditioning ( ric ) protocols still remains a challenge . therefore , combining megadose of cd34 + hsct with other gvhd - depleted veto cells could enable facilitation of engraftment of hsct under ric without the adverse complication of gvhd . this approach might provide a safer modality for enabling engraftment of hsct , enabling its application in elderly patients who can not tolerate intensive protocols and to a variety of patients with non - malignant disorders , associated with longer life expectancy , in whom the use of a high risk conditioning can not be considered .

INTRODUCTION THE VETO CONCEPT VETO ACTIVITY OF BONE-MARROW CELLS THE USE OF VETO CD8 CD8 TCR-INDEPENDENT KILLING OF MALIGNANT B CELLS BY CD8 CONCLUDING REMARKS Conflict of Interest Statement

hematopoietic stem cell transplantation ( hsct ) has become a common procedure of therapy for patients with hematological malignancies and many other life threatening blood disorders . however , viral and fungal infections associated with the severe immunoablative conditioning used prior to hsct still represent a major challenge ( martelli et al . one approach to address this barrier is to use reduced intensity conditioning ( ric ) . hsct following ric relies on non - myeloablative preparatory regimen that spares a substantial level of the host immunity and thus reduce transplant related mortality ( trm ) by both improving post - transplant immune reconstitution and reducing the toxicity associated with the conditioning agents ( ballen and spitzer , 2010 ; gyurkocza et al . , ric was first developed to enable allogeneic hsct in patients with advanced hematological malignancies who can not withstand myeloablative conditioning because of age and/or performance status . ric protocols also enable the use of hsct in patients with non - malignant disorders , associated with longer life expectancy . in these patients the aim of the conditioning prior to the allogeneic hsct is merely to support sustained donor cell s engraftment for correction of the disease ( steward and jarisch , 2005 ; ringden et al . , 2006 ) , or , as demonstrated in limited number of patients , for the induction of donor chimerism , as a platform for tolerance induction as prelude to organ transplantation ( kawai et al . , nevertheless , currently used ric protocols are still relatively aggressive and therefore it is highly desirable to develop safer preparative regimen protocols that selectively achieve a state of donor - specific unresponsiveness without compromising the overall immune response . one way to achieve a state of donor - specific tolerance uses donor cells endowed with veto activity . the term veto , coined in 1980 by miller ( miller , 1980 ) , relates to the ability of cells to specifically delete t cells directed against antigens ( ags ) of the veto cells themselves , but not against third - party ag ( muraoka and miller , 1983 ; claesson and miller , 1984 ) . the suppression of effector t cells directed against the veto cells is both ag - specific and major histocompatibility complex ( mhc ) restricted , resulting from the unique manner by which the veto cell kills its target . thus , veto activity results from unidirectional recognition of the veto cell by the responding t cell , but not vice versa . therefore , the recognizing t cell , the t cell receptor ( tcr ) of which is directed against the mhc of the veto cell , is killed upon binding to its veto target , due to exchange of signals allowed following this interaction . hence , the use of donor veto cells capable of specifically eliminating only the host anti - donor t cell clones that mediate the transplant rejection while sparing other t cells that can persist and fight infectious pathogens , could offer an effective modality for the induction of transplantation tolerance . initially , veto activity was described for cells within the spleen of athymic nude mice ( miller , 1980 ) . based on this initial observation , various cell types have been shown to mediate veto activity including t lymphocytes , natural killer cells , and dendritic cells . a very strong veto activity was documented for cd8 cytotoxic t cell ( ctl ) lines or clones ( fink et al . , cells in murine bm and in t cell colonies grown from such bm were shown to mediate veto activity in vitro ( muraoka and miller , 1980 ) and un - separated donor bm was shown to specifically reduce the frequency of anti - donor ctls in grafted mice ( wood et al . however , this in vivo tolerizing activity of the bm cells could be attributed to t cells which reside within the bm . these t cells , while potentially mediating beneficial veto activity , also cause a severe multi - system graft versus host disease ( gvhd ) . early attempts to avoid gvhd risk and to apply t cell - depleted bmt ( tdbmt ) in leukemia patients indeed revealed that this benefit of gvhd prevention is offset by increased risk for graft rejection , due to absence of donor t cells within the graft ( gale and reisner , 1986 ; kernan et al . , 1987 ) . however , veto activity could be also assigned to non - t cells within the bm . for example , a series of studies by the group of judy thomas described potent veto activity of cells within the cd8cd16dr subset in the bm of rhesus macaque primates ( thomas et al . thus , these studies demonstrated that cd8 crosslinking following interaction with donor - reactive ctl precursors ( ctlp ) , elicits upregulation of transforming growth factor-1 ( tgf-1 ) and fas ligand ( fasl ) by these donor bm cells , leading to clonal deletion of the donor - reactive ctlp ( asiedu et al . ( 1998 ) that human hematopoietic cd34 progenitors are endowed with marked veto activity ( figure 1 ) . explaining in part how megadose of purified cd34 cells enables to overcome rejection in recipients of three hla - loci mismatched ( haploidentical ) hsct while avoiding the threat of gvhd ( aversa et al . ( 2005 ) demonstrated that this veto activity is mediated through a tnf- based mechanism . ( 2002 ) demonstrated that veto activity is not only mediated directly by the infused cd34 cells but also by their cd33 progeny , which lose this tolerizing activity upon completion of maturation , at the level of cd14 monocytes or cd13 neutrophils . furthermore , preliminary results suggest that bm - derived immature dendritic cells , previously shown to induce immune tolerance , exhibit marked veto activity on cd8 t cells , in addition to the non - specific suppression of cd4 t cells mediated by the no system ( zangi et al . finally , nk cells which were shown to exhibit veto activity upon activation with il-2 , develop and appear early during the post - transplant period ( chrobak and gress , 2001 ; reich - zeliger et al . , 2004a ) . the regulatory activity of cd34 cells : evidence for target specificity . the average ctl response ( sd ) in the presence ( black bars ) or absence ( white bars ) of cd34 cells at a veto - to - responder cell ratio of 0.5 . the veto effect was tested by a limiting dilution assay as follows : equal numbers ( 1 10/ml ) of responder cells and irradiated allogeneic stimulator cells from the donor of the cd34 cells and a third - party donor were co - cultured for 5 days . thus , based on these observations , the following working hypothesis can be suggested to explain how megadose of cd34 cells can overcome rejection in human recipients of haploidentical hsct . upon administration of purified cd34 cells , the graft supporting veto activity is initially mediated directly by the infused cd34 cells , and subsequently by the cd33 progeny of these cells which grow exponentially within the first few days post - transplant . this second phase of differentiating veto cells also includes cd11c+ immature dendritic cells and other graft facilitating cells . the increased engraftment of megadose of hsct is therefore greatly dependent not only on the ability of the initial inoculums of the cd34 cells to veto anti - donor t cells , but also on their ability to seed the bm and to generate as rapidly as possible the second or third derivatives which are required to complete the eradication of host anti - donor t cells . as described above , the use of purified megadose of cd34 hsct has enabled haploidentical transplantation in leukemia patients and was the first demonstration of the potent clinical potential of donor veto cells . however , this approach is currently limited to supra - lethal myeloablative and highly immunosuppressive conditioning protocols ( martelli et al . indeed , studies in non - human primates revealed that any significant reduction of the conditioning , to levels acceptable for elderly patients or for patients with non - malignant disorders , would require veto inducing cd34 stem cell numbers which can not be realistically collected from human donors ( gan et al . therefore , other populations of veto cells could have a crucial role in supporting and promoting successful engraftment of purified stem cell transplantation under relatively safer ric . previous insights on the veto mechanism of cd8 veto ctls , combining anti - cd8 blockade and fasl - mutated veto cells , have suggested that co - expression of cd8 and fasl is required for the veto activity of these cells ( reich - zeliger et al . such a mechanism involves initial recognition of the veto cell by the tcr of the effector t cells , leading to expression of fas by the effector t cell upon activation , and thereby enabling fas fasl mediated apoptosis to take place , once inhibitory molecules such as flice - inhibitory protein ( flip ) are down regulated in the effector cell ( reisner et al . in addition , the interaction between cd8 on veto ctl and the mhc class i alpha3 domain on the effector cell , is associated with phosphorylation of mek / erk in the latter cell , and with a significant reduction of x - linked inhibitor of apoptosis protein ( xiap ) levels which , in turn , enables even more potent triggering of fas fasl mediated apoptosis in the recognizing effector cell scheme 1 reich - zeliger et al . more recent results have indicated that veto activity exhibited by ctls can also be mediated by an additional perforin based mechanism ( milstein et al . however , despite their potent veto activity , cd8 ctls can not be used for safe tolerance induction in allogeneic hsct because of their marked gvh reactivity . veto - based deletion of host anti - donor cd8 t cell clones by veto ctl . cd8-mhc class i engagement induces perk , which is associated with reduction of xiap ( apoptosis inhibitor ) levels , thereby enabling fas one approach for generating veto ctls with reduced gvhd reactivity has been described by a series of studies by d. h. fowler ( fowler and gress , 2000 ; erdmann et al . , 2008 ) . we have previously described an alternative approach to generate ctls with highly reduced gvh reactivity by means of stimulation against third - party stimulators in the absence of exogenous cytokines , followed by further ex vivo expansion using third - party stimulators and il-2 ( bachar - lustig et al . this approach was based on the observation that only activated ctlp are capable of surviving the cytokine deprivation in the primary culture , and that these anti - third - party clones further expand throughout the culture . these anti - third - party ctls indeed were shown to be depleted of gvhd while supporting bm engraftment in murine models . in accordance with the veto concept thus , a ctl line originating from a strain other than that of the bm donor failed to prevent graft rejection ( bachar - lustig et al . memory t cells , derived from prior exposure to alloantigen or generated by heterologous immunity or lymphopenia - induced proliferation , are believed to be an important part of the barrier preventing the translation of tolerance induction protocols from inbred rodent strains to the clinic ( pantenburg et al . therefore , the ability of veto ctls to overcome memory t cells - mediated rejection could be highly important for their action in clinical settings . thus , in this study the authors demonstrated that murine cd8 ctls , in addition to their renowned veto activity upon recognizing t cells , can also induce ag - specific elimination of recognizing b cells , both in vitro and in vivo and thus veto ctls may selectively overcome both cellular and humoral graft rejection . the discrepancy between the ctls veto activity in vitro and in vivo could be explained when considering two limitations of the veto cells : first , the veto activity is mediated through cell to cell contact and , second , host t cells are prone to veto mainly in a window of opportunity of up to 48 h after their activation , thus , once these host t cells develop into mature effector t cells , veto cells can no longer exert their effect ( anderson and zimring , 2006 ) . therefore , while in vitro the veto ctls are directly plated with their cognate targets , upon infusion in vivo the veto ctls need to co - localize with the host t cells within the first 48 h of the rejection process , or else their effect will be hampered . indeed , we recently demonstrated that ctls attained upon long ex vivo culture in the presence of il-2 exhibit a migration pattern different from the one displayed by naive host t cells thereby precluding their co - localization . thus , while nave host t cells home efficiently to the lymph nodes ( lns ) of bmt recipient mice , the veto ctls are excluded from the lns and tend to localize in peripheral sites ( ophir et al . , in order to improve the lns homing potential of anti - third - party cd8 veto cells we developed a new protocol , using il-15 , that favor the induction of central memory phenotype in anti - third - pary cd8 t cells ( ophir et al . , 2010 ) . central memory t cells ( tcm ) express the ln homing receptors cd62l and ccr7 ( sallusto et al . , 1999 ) and similarly to naive t cells , localize to the t cell area of all secondary lymphoid organs ( weninger et al . indeed , we demonstrated that these ex vivo induced anti - third - party tcm , in contrast to anti - third - party ctls , home to lns of bmt recipients , where they co - localize with the recipient s endogenous host t cells . moreover , the tcm displayed strong proliferation at the early post - transplant period and subsequently persisted in vivo for more than 1 year post - bmt , in line with their memory phenotype ( ophir et al . most importantly , we demonstrated that tcm derived from ( host donor)f1 mice can specifically and efficiently delete in vivo host tcr - transgenic t cells carrying a tcr transgene with anti - donor specificity . in accordance with the veto concept , this efficient in vivo deletion of anti - donor host t cells , found to be mediated by apoptosis , did not occur when non - specific tcm , not expressing the donor h-2 haplotype , were used ( figure 2 ) . thus , murine tcm perform in vivo veto activity , efficiently deleting host t cells only when the host t cells recognize antigens on the tcm , and not by a non - specific general immunosuppressive effect . taken together , all these attributes of the tcm were shown to translate into improved efficacy in overcoming t cell mediated rejection of murine tdbmt , thereby enabling high survival rate and long - term donor chimerism , without causing gvhd . thus , adoptive transfer of anti - third - party tcm , in the absence of rapamycin treatment significantly abolished host t cells - mediated rejection of fully mismatched nude - bm . this is in sharp contrast to anti - third - party ctls , which fail to enable engraftment unless administered in higher numbers and in conjunction with rapamycin treatment ( bachar - lustig et al we concluded therefore that by generating anti - third - party cd8 cells with a central memory phenotype we were able to dramatically enhance their tolerizing veto activity in vivo . the following day , the mice were transplanted with 1 10 c57bl/6-nude bm cells or received , in addition , 5 10 specific , derived from cb6 ( h-2 , black bars ) , or non - specific , derived from c57bl/6 ( h-2 , gray bars ) anti - third - party tcm . recipients were sacrificed 8 days post - transplant , their spleens were harvested , and the deletion of anti - donor 2c t cells was monitored by facs . ( a ) representative result demonstrating the level of surviving ( 7aad ) 2c cells in the absence ( left panel , 2c alone ) or presence of specific tcm ( right panel 2c + specific tcm ) . ( c ) representative result demonstrating apoptosis induction upon anti - donor 2c cells by the tcm as evident by annexin v levels on 2c cells in the absence ( left panel , 2c alone ) or presence of specific tcm ( right panel , 2c + specific tcm ) . ( d ) quantification of results measuring annexin v levels on the 2c cells following interactions with specific and non - specific tcm . data present average sd of percent annexin v levels in at least four animals from each group , in one representative experiment , out of three performed . * * p < 0.01 , * * * a second objective which could be attained by administration of donor - derived activated cd8 t cells in the context of hsct is related to the ability of these cells to control residual hematological malignancies . considering that gvl is generally associated with gvhd it was surprising that autologous or allogeneic human and murine anti - third - party veto cd8 t cells , markedly depleted of gvh reactivity , exhibit vigorous responses against different b cell malignancies ( arditti et al . , 2011 ) . hence , such single agent veto cell therapy has the potential to have a double benefit in the context of hsct , namely , promoting and supporting hsct engraftment through specific tolerizing veto activity while mediating effective anti - cancer response , in the absence of gvhd . translation of this therapy , optimizing protocols for the generation of human anti - third - party tcm is now in progress and clinical evaluation will commence in the near future in patients with b cell malignancies . donor derived veto cells represent an attractive and effective modality for the induction of specific tolerance toward donor ags . this approach has been already demonstrated in patients by using megadose of purified veto cd34 stem cells , that can overcome the host s residual immunity surviving the myeloablative conditioning and enable engraftment of hsct across major genetic barriers without the severe threat of gvhd . however , the number of veto cd34 cells that can be harvested is insufficient for overcoming the large numbers of host t cells remaining after ric . therefore , combining megadose of cd34 hsct with other gvhd - depleted veto cells , potentially anti - third - party cd8 tcm , could enable facilitation of engraftment of hsct under ric without the adverse complication of gvhd - producing t cells and without the need for deleterious post - transplant gvhd prophylaxis . it is hoped that this approach could extend the use of hsct to elderly patients with b cell malignancies who can not tolerate intensive protocols and to a variety of patients with non - malignant disorders , associated with longer life expectancy , in whom the current trm associated with hsct may not be ethically justified . use of experimental animals statement : all experiments on live vertebrates were performed in accordance with the weizmann institutional animal care and use committee .

hematopoietic stem cell transplantation ( hsct ) has become a common procedure of therapy for patients with hematological malignancies and many other life threatening blood disorders . however , viral and fungal infections associated with the severe immunoablative conditioning used prior to hsct still represent a major challenge ( martelli et al . hsct following ric relies on non - myeloablative preparatory regimen that spares a substantial level of the host immunity and thus reduce transplant related mortality ( trm ) by both improving post - transplant immune reconstitution and reducing the toxicity associated with the conditioning agents ( ballen and spitzer , 2010 ; gyurkocza et al . , ric was first developed to enable allogeneic hsct in patients with advanced hematological malignancies who can not withstand myeloablative conditioning because of age and/or performance status . ric protocols also enable the use of hsct in patients with non - malignant disorders , associated with longer life expectancy . in these patients the aim of the conditioning prior to the allogeneic hsct is merely to support sustained donor cell s engraftment for correction of the disease ( steward and jarisch , 2005 ; ringden et al . , 2006 ) , or , as demonstrated in limited number of patients , for the induction of donor chimerism , as a platform for tolerance induction as prelude to organ transplantation ( kawai et al . , nevertheless , currently used ric protocols are still relatively aggressive and therefore it is highly desirable to develop safer preparative regimen protocols that selectively achieve a state of donor - specific unresponsiveness without compromising the overall immune response . the term veto , coined in 1980 by miller ( miller , 1980 ) , relates to the ability of cells to specifically delete t cells directed against antigens ( ags ) of the veto cells themselves , but not against third - party ag ( muraoka and miller , 1983 ; claesson and miller , 1984 ) . the suppression of effector t cells directed against the veto cells is both ag - specific and major histocompatibility complex ( mhc ) restricted , resulting from the unique manner by which the veto cell kills its target . thus , veto activity results from unidirectional recognition of the veto cell by the responding t cell , but not vice versa . therefore , the recognizing t cell , the t cell receptor ( tcr ) of which is directed against the mhc of the veto cell , is killed upon binding to its veto target , due to exchange of signals allowed following this interaction . hence , the use of donor veto cells capable of specifically eliminating only the host anti - donor t cell clones that mediate the transplant rejection while sparing other t cells that can persist and fight infectious pathogens , could offer an effective modality for the induction of transplantation tolerance . based on this initial observation , various cell types have been shown to mediate veto activity including t lymphocytes , natural killer cells , and dendritic cells . , 1984 ; claesson and ropke , 1986 ; claesson and miller , 1989 ) and direct comparison of the veto reactivity of various cell types revealed that ctls have the strongest in vitro veto effect ( reich - zeliger et al . , cells in murine bm and in t cell colonies grown from such bm were shown to mediate veto activity in vitro ( muraoka and miller , 1980 ) and un - separated donor bm was shown to specifically reduce the frequency of anti - donor ctls in grafted mice ( wood et al . however , this in vivo tolerizing activity of the bm cells could be attributed to t cells which reside within the bm . these t cells , while potentially mediating beneficial veto activity , also cause a severe multi - system graft versus host disease ( gvhd ) . early attempts to avoid gvhd risk and to apply t cell - depleted bmt ( tdbmt ) in leukemia patients indeed revealed that this benefit of gvhd prevention is offset by increased risk for graft rejection , due to absence of donor t cells within the graft ( gale and reisner , 1986 ; kernan et al . for example , a series of studies by the group of judy thomas described potent veto activity of cells within the cd8cd16dr subset in the bm of rhesus macaque primates ( thomas et al . , 1991 ; cd8 surface expression was shown to play a pivotal role in the tolerogenic effect of these bm cells . thus , these studies demonstrated that cd8 crosslinking following interaction with donor - reactive ctl precursors ( ctlp ) , elicits upregulation of transforming growth factor-1 ( tgf-1 ) and fas ligand ( fasl ) by these donor bm cells , leading to clonal deletion of the donor - reactive ctlp ( asiedu et al . explaining in part how megadose of purified cd34 cells enables to overcome rejection in recipients of three hla - loci mismatched ( haploidentical ) hsct while avoiding the threat of gvhd ( aversa et al . ( 2002 ) demonstrated that veto activity is not only mediated directly by the infused cd34 cells but also by their cd33 progeny , which lose this tolerizing activity upon completion of maturation , at the level of cd14 monocytes or cd13 neutrophils . furthermore , preliminary results suggest that bm - derived immature dendritic cells , previously shown to induce immune tolerance , exhibit marked veto activity on cd8 t cells , in addition to the non - specific suppression of cd4 t cells mediated by the no system ( zangi et al . finally , nk cells which were shown to exhibit veto activity upon activation with il-2 , develop and appear early during the post - transplant period ( chrobak and gress , 2001 ; reich - zeliger et al . the average ctl response ( sd ) in the presence ( black bars ) or absence ( white bars ) of cd34 cells at a veto - to - responder cell ratio of 0.5 . the veto effect was tested by a limiting dilution assay as follows : equal numbers ( 1 10/ml ) of responder cells and irradiated allogeneic stimulator cells from the donor of the cd34 cells and a third - party donor were co - cultured for 5 days . a significant difference ( p < 0.001 on t - test compared with control cultures without cd34 cells ) between control cultures and those including cd34 cells was found upon stimulation against donor cells ( rachamim et al . thus , based on these observations , the following working hypothesis can be suggested to explain how megadose of cd34 cells can overcome rejection in human recipients of haploidentical hsct . upon administration of purified cd34 cells , the graft supporting veto activity is initially mediated directly by the infused cd34 cells , and subsequently by the cd33 progeny of these cells which grow exponentially within the first few days post - transplant . clearly , the number of all these tolerizing cell types emerging after transplantation is proportional to the number of cd34cells infused . the increased engraftment of megadose of hsct is therefore greatly dependent not only on the ability of the initial inoculums of the cd34 cells to veto anti - donor t cells , but also on their ability to seed the bm and to generate as rapidly as possible the second or third derivatives which are required to complete the eradication of host anti - donor t cells . as described above , the use of purified megadose of cd34 hsct has enabled haploidentical transplantation in leukemia patients and was the first demonstration of the potent clinical potential of donor veto cells . indeed , studies in non - human primates revealed that any significant reduction of the conditioning , to levels acceptable for elderly patients or for patients with non - malignant disorders , would require veto inducing cd34 stem cell numbers which can not be realistically collected from human donors ( gan et al . therefore , other populations of veto cells could have a crucial role in supporting and promoting successful engraftment of purified stem cell transplantation under relatively safer ric . previous insights on the veto mechanism of cd8 veto ctls , combining anti - cd8 blockade and fasl - mutated veto cells , have suggested that co - expression of cd8 and fasl is required for the veto activity of these cells ( reich - zeliger et al . such a mechanism involves initial recognition of the veto cell by the tcr of the effector t cells , leading to expression of fas by the effector t cell upon activation , and thereby enabling fas fasl mediated apoptosis to take place , once inhibitory molecules such as flice - inhibitory protein ( flip ) are down regulated in the effector cell ( reisner et al . in addition , the interaction between cd8 on veto ctl and the mhc class i alpha3 domain on the effector cell , is associated with phosphorylation of mek / erk in the latter cell , and with a significant reduction of x - linked inhibitor of apoptosis protein ( xiap ) levels which , in turn , enables even more potent triggering of fas fasl mediated apoptosis in the recognizing effector cell scheme 1 reich - zeliger et al . however , despite their potent veto activity , cd8 ctls can not be used for safe tolerance induction in allogeneic hsct because of their marked gvh reactivity . cd8-mhc class i engagement induces perk , which is associated with reduction of xiap ( apoptosis inhibitor ) levels , thereby enabling fas one approach for generating veto ctls with reduced gvhd reactivity has been described by a series of studies by d. h. fowler ( fowler and gress , 2000 ; erdmann et al . we have previously described an alternative approach to generate ctls with highly reduced gvh reactivity by means of stimulation against third - party stimulators in the absence of exogenous cytokines , followed by further ex vivo expansion using third - party stimulators and il-2 ( bachar - lustig et al . this approach was based on the observation that only activated ctlp are capable of surviving the cytokine deprivation in the primary culture , and that these anti - third - party clones further expand throughout the culture . in accordance with the veto concept thus , a ctl line originating from a strain other than that of the bm donor failed to prevent graft rejection ( bachar - lustig et al . importantly , anti - third - party veto ctls , upon adoptive transfer into tdbmt recipient mice , were shown to eliminate not only host anti - donor nave cells , but also host anti - donor memory cells ( reich - zeliger et al . memory t cells , derived from prior exposure to alloantigen or generated by heterologous immunity or lymphopenia - induced proliferation , are believed to be an important part of the barrier preventing the translation of tolerance induction protocols from inbred rodent strains to the clinic ( pantenburg et al . therefore , the ability of veto ctls to overcome memory t cells - mediated rejection could be highly important for their action in clinical settings . thus , in this study the authors demonstrated that murine cd8 ctls , in addition to their renowned veto activity upon recognizing t cells , can also induce ag - specific elimination of recognizing b cells , both in vitro and in vivo and thus veto ctls may selectively overcome both cellular and humoral graft rejection . nevertheless , despite all these important attributes of the veto cd8 ctls , their in vivo activity was shown to be markedly inferior compared to that exhibited in vitro , requiring the administration of large number of ctls in conjunction with the immunosuppressive drug rapamycin in order to efficiently overcome tdbmt rejection in murine models ( bachar - lustig et al . the discrepancy between the ctls veto activity in vitro and in vivo could be explained when considering two limitations of the veto cells : first , the veto activity is mediated through cell to cell contact and , second , host t cells are prone to veto mainly in a window of opportunity of up to 48 h after their activation , thus , once these host t cells develop into mature effector t cells , veto cells can no longer exert their effect ( anderson and zimring , 2006 ) . therefore , while in vitro the veto ctls are directly plated with their cognate targets , upon infusion in vivo the veto ctls need to co - localize with the host t cells within the first 48 h of the rejection process , or else their effect will be hampered . indeed , we recently demonstrated that ctls attained upon long ex vivo culture in the presence of il-2 exhibit a migration pattern different from the one displayed by naive host t cells thereby precluding their co - localization . thus , while nave host t cells home efficiently to the lymph nodes ( lns ) of bmt recipient mice , the veto ctls are excluded from the lns and tend to localize in peripheral sites ( ophir et al . , in order to improve the lns homing potential of anti - third - party cd8 veto cells we developed a new protocol , using il-15 , that favor the induction of central memory phenotype in anti - third - pary cd8 t cells ( ophir et al . indeed , we demonstrated that these ex vivo induced anti - third - party tcm , in contrast to anti - third - party ctls , home to lns of bmt recipients , where they co - localize with the recipient s endogenous host t cells . moreover , the tcm displayed strong proliferation at the early post - transplant period and subsequently persisted in vivo for more than 1 year post - bmt , in line with their memory phenotype ( ophir et al . most importantly , we demonstrated that tcm derived from ( host donor)f1 mice can specifically and efficiently delete in vivo host tcr - transgenic t cells carrying a tcr transgene with anti - donor specificity . in accordance with the veto concept , this efficient in vivo deletion of anti - donor host t cells , found to be mediated by apoptosis , did not occur when non - specific tcm , not expressing the donor h-2 haplotype , were used ( figure 2 ) . thus , murine tcm perform in vivo veto activity , efficiently deleting host t cells only when the host t cells recognize antigens on the tcm , and not by a non - specific general immunosuppressive effect . taken together , all these attributes of the tcm were shown to translate into improved efficacy in overcoming t cell mediated rejection of murine tdbmt , thereby enabling high survival rate and long - term donor chimerism , without causing gvhd . thus , adoptive transfer of anti - third - party tcm , in the absence of rapamycin treatment significantly abolished host t cells - mediated rejection of fully mismatched nude - bm . this is in sharp contrast to anti - third - party ctls , which fail to enable engraftment unless administered in higher numbers and in conjunction with rapamycin treatment ( bachar - lustig et al we concluded therefore that by generating anti - third - party cd8 cells with a central memory phenotype we were able to dramatically enhance their tolerizing veto activity in vivo . ( a , b ) lethally irradiated c57bl/6 ( h-2 ) mice received 1 10 cd8 purified tcr - transgenic 2c cells ( carrying tcr with anti h-2 specificity ) and 5 10 irradiated balb / c ( h-2 ) splenocytes . the following day , the mice were transplanted with 1 10 c57bl/6-nude bm cells or received , in addition , 5 10 specific , derived from cb6 ( h-2 , black bars ) , or non - specific , derived from c57bl/6 ( h-2 , gray bars ) anti - third - party tcm . ( a ) representative result demonstrating the level of surviving ( 7aad ) 2c cells in the absence ( left panel , 2c alone ) or presence of specific tcm ( right panel 2c + specific tcm ) . ( b ) quantification of results demonstrating efficient inhibition of the 2c cells only by data represent average sd of percent inhibition from at least 10 animals in each group , pooled from two independent experiments . ( c , d ) in vivo model was established as in ( a , b ) , but 5 10 purified cd8 2c cells and 2.5 10 irradiated balb / c splenocytes were administrated . ( c ) representative result demonstrating apoptosis induction upon anti - donor 2c cells by the tcm as evident by annexin v levels on 2c cells in the absence ( left panel , 2c alone ) or presence of specific tcm ( right panel , 2c + specific tcm ) . * * p < 0.01 , * * * a second objective which could be attained by administration of donor - derived activated cd8 t cells in the context of hsct is related to the ability of these cells to control residual hematological malignancies . considering that gvl is generally associated with gvhd it was surprising that autologous or allogeneic human and murine anti - third - party veto cd8 t cells , markedly depleted of gvh reactivity , exhibit vigorous responses against different b cell malignancies ( arditti et al . second , a slower process of mhc class i - dependent apoptosis is mediated by binding of the mhc class i 2/3 constant region on the tumor cells to the cd8 molecule on the ctl membrane ( arditti et al . hence , such single agent veto cell therapy has the potential to have a double benefit in the context of hsct , namely , promoting and supporting hsct engraftment through specific tolerizing veto activity while mediating effective anti - cancer response , in the absence of gvhd . translation of this therapy , optimizing protocols for the generation of human anti - third - party tcm is now in progress and clinical evaluation will commence in the near future in patients with b cell malignancies . this approach has been already demonstrated in patients by using megadose of purified veto cd34 stem cells , that can overcome the host s residual immunity surviving the myeloablative conditioning and enable engraftment of hsct across major genetic barriers without the severe threat of gvhd . however , the number of veto cd34 cells that can be harvested is insufficient for overcoming the large numbers of host t cells remaining after ric . therefore , combining megadose of cd34 hsct with other gvhd - depleted veto cells , potentially anti - third - party cd8 tcm , could enable facilitation of engraftment of hsct under ric without the adverse complication of gvhd - producing t cells and without the need for deleterious post - transplant gvhd prophylaxis . it is hoped that this approach could extend the use of hsct to elderly patients with b cell malignancies who can not tolerate intensive protocols and to a variety of patients with non - malignant disorders , associated with longer life expectancy , in whom the current trm associated with hsct may not be ethically justified .

rolled - up nanotech has become a powerful technology with applications in a broad range of research fields , including optofluidics , micromachinery [ 4 - 6 ] , magnetofluidics , biophysics , nanomechanics , and waveguiding for different spectral ranges and applications [ 10 - 12 ] . large arrays of periodically ordered microtubes can be fabricated by a combination of lithography and deliberate self - rolling of strained layers upon selective underetching [ 1,13 - 15 ] . however , in some cases , it might be important to stimulate tube formation in certain areas and suppress tube formation outside those areas . precise control over the lateral positioning and the number of windings is fundamental for the use of the tubes in optical resonators for sensors [ 16 - 20 ] , hyperlenses , or electrical transport ( curved electron gases ) [ 23 - 27 ] . numerous existing studies offer highly sophisticated methods for precise lateral positioning of rolled - up semiconductor nanostructures by controlling the starting edges [ 13 - 15,28,29 ] , but difficulties arise for thin layers due to the extensive , successive lithography steps involved , and ways to control the stopping point of rolling remain largely lacking without modifying the structure . here , we demonstrate illumination - controlled hydrofluoric acid ( hf ) etching of a buried thin alas sacrificial layer . sufficiently intense light exposure of a certain substrate area leads to a complete suppression of the underetching effect , and as a result , the formation of rolled - up ingaas / gaas tubes can be easily controlled spatially . this method allows for control over the roll - up stopping point and is suitable for very thin sacrificial layers . the aim of this study was to quantify the observed etch - suppression effect ( ese ) and precisely control the release of ingaas / gaas bilayers with an illumination technique . v rolled - up nanotube ( runt ) : when immersed in hf solution , the compressively strained double layer ( a gaas layer on top of a larger - lattice constant inxga1x as layer deposited pseudomorphically on a gaas substrate with an alas sacrificial layer ) releases from the substrate as the sacrificial layer is preferentially removed by the hf at a previously defined starting edge and begins to roll up in order to relax the built - in strain gradient . finally , the bent bilayer forms a tube or scroll after performing a full rotation . the starting edge , which for ingaas / gaas layers should be oriented in the direction for optimal structural integrity of the resulting runts , can be defined either by scratching the surface , thereby laterally exposing the sacrificial layer , or by opening a window through the entire layer structure by photolithography and subsequent wet chemical etching . the tube diameter , i.e. , the size of the cross section of the structure obtained after the etching step has been completed , depends on the thickness and inherent strain of the layers , and can be accurately varied in the nano- to micrometer range [ 1,30 - 32 ] . for precise positioning of runts on the surface , the important parameters in the roll - up process are the sacrificial layer thickness , etching time , and tube diameter . the ratio of inner - to - outer diameter can also be controlled over a wide range , based on the number of rotations performed by the tubes on the surface ( or rolling distance , measured from the initial starting edge see schematic in fig . bilayer rolling due to sacrificial layer removal in hf,3 . rolled - up structure after one full rotation).bschematic of the etching experiment during in situ observation by optical microscopy and result for strained bilayers ( relative ratio of mesa diameter to layer thickness not to size).coptical image of an alas / ingaas / gaas mesa structure etched in a 2.5% hf solution , with 20 microscope objective , for 6 min ; the etch - suppression spot radius is 700 m.doptical images of adjacent mesas from ( b ) ( black box ) , showing an increasing degree of etch suppression toward the center of the illumination here , the experiments were performed using 20 nm in0.33ga0.66as/20 nm gaas structures on alas grown on ( 001 ) gaas substrates by molecular beam epitaxy , where the thickness of the alas was varied from 4 to 20 nm . for experiments pertaining to the measurement of the onset of the etch suppression and rolling distance , the starting edges were defined by mechanical scratching , whereas photolithography and wet chemical etching ( by h3po4:h2o2:h2o solution ) were used for controlling the etch suppression . our specially designed setup involved an hf - resistant trough for etching with hf under light focused by a zeiss optical microscope using different objective lenses of 5 through 50 magnification ( for sample evolution during this process , see fig . other measurements were performed using illumination from a hene laser with maximum output power of 2 mw , as well as a laser with adjustable power ( up to 20 mw ) and wavelength ( 535825 nm ) with focusing optics or an optical fiber to illuminate the surface of the sample . for all measurements , the intensity of illumination was measured beforehand using a calibrated power meter . for samples that were etched , illuminated , and observed under the microscope , the settings on the illumination dial were used and the focused spot sizes for each magnification were assumed to be the same . for the quantitative measurements of tube rolling distance , optical images of the sample outside of solution once the etching was terminated were taken . from these images , the rolling distance could then be measured , which we define as distance from the starting edge to the center of the tube width , assumed to correspond to the stopping point of the underetching . the resolution of the es method was studied by precisely positioning the illumination spot on wet - chemically etched mesa structures of different shapes and sizes ( on the order of 10100 m ) during hf etching . the degree of etching suppression ( given by the coverage area of unetched structures remaining on the sample ) was measured versus distance from the center of illumination . figure 1b shows an optical microscopy image of a structure composed of a 4 nm alas layer and a 40 nm symmetrical strained in0.33ga0.66as / gaas bilayer . the larger circular shape observed on the sample at the end of the hf experiment is formed by original mesa structures that were left unetched , while on the rest of the sample the bilayer mesas are underetched , leading to rolling up as expected . figure 2 , which plots the average rolling distance as a function of intensity for structures with varying alas thickness , illustrates the sudden onset of the ese . the error due to variations in experimental parameters such as quantity of hf solution , scattering of light and effective illumination intensity at the sample , as well as the method for evaluating rolling distances , is estimated to be within 15% . it can be seen that the maximum rolling distance increases as the alas thickness increases , while the ese is only present for sacrificial layers below 10 nm . moreover , the onset of the ese , or the intensity of illumination necessary to suppress the alas etching , is higher for larger thicknesses . onset of illumination effect for 20 nm ingaas/20 nm gaas samples with 4 , 7 , 10 , 12 , 13 , 15 , and 20 nm alas sacrificial layers , etched with 2.5% hf solution using 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . the diamond data point shows a maximum rolling distance previously reported figure 3 shows the maximum rolling distance as a function of the sacrificial layer thickness . in a regime from 5 nm up to a thickness of 15 nm of alas the relationship between the maximum rolling distance and the sacrificial layer thickness is approximately linear , and then saturates for thicker alas layers . although in previous studies ingaas - gaas runts ( rolled - up nanotubes ) have been noticed to exhibit a self - limiting rolling behavior for longer etching times , this limiting was not investigated as function of the sacrificial layer thickness . the fit through the data in the linear increase regime ( r = 0.936 ) yields a unitless slope of 14.2e for the scaling of maximum rolling distance with talas . this indicates that in this first regime the maximum rolling distance is not dominated by intrinsic effects of the rolled - up layer and does not occur due to a fundamental limiting process ( as , e.g. , described by cendula et . therefore , we ascribe the saturation in this regime to dynamic effects either between the rolled - up layer and the substrate or inside the etching solution caused by the process in the gap between the substrate and the rolled - up film . the second regime shows a clear saturation behavior for alas thickness larger than 15 nm . in this regime , the saturation should be dominated by intrinsic effects either from the layer system itself or possibly by some kind of fundamental process yet to be found . the linear scaling of the maximum rolling distance offers the possibility for lateral positioning of the rolled - up tube relative to a defined starting edge . 1a , indicating rolling distancedrollmeasured from the starting edge , alas thicknesstalas , ingaas / gaas bilayer thicknesstbil . , and radius of curvaturerof the rolled - up bilayer furthermore , as shown in fig . 4 , the onset of ese changes for different etching speeds : the higher the hf concentration , the higher the intensity needed for suppression , while the thicker the alas layer , the more abrupt the etching behavior transition from the suppression regime to the illumination independent regime . for eachtalas , up to roughly 15 nm the onset of the ese increases approximately linearly with concentration , and scaling slopes are comparable for different thicknesses ( 12 mw / mm ) . from this , as well as fig . 2 , it can be concluded that the onset of ese occurs in a different regime from that of intrinsic saturation of maximum rolling distance . the onset of the etch - suppression effect ( ese ) versus hf concentration for 20 nm ingaas/20 nm gaas structures with 4 nm and 10 nm alas sacrificial layers , with 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . thelinesthrough the data are guides for the eye we also found that the ese is time dependent , i.e. , the etching time under illumination determines whether further etching will continue once the illumination is removed . in our experiments , we found that for samples where the high intensity light exposure was kept short , the layers were able to start rolling after being removed from the light source . this effect was found to not only depend on the light intensity but also on the thickness of the alas layer , which suggests that the etch suppression is a dynamic process that depends on the access of hf to the alas layer and the exchange of products and reactants in this region . this conclusion is further corroborated by the fact that with increasing alas thickness the rolling distance of the strained bilayer becomes independent of the alas gap size ( fig . the ese was used to position and control the roll up of tubes from strained ingaas / gaas bilayers . the patterning allowed us to create well - defined starting edges suitable for the illumination experiment ( as in fig . , one can obtain both etched regions with rolled - up layers and unetched regions within a small area on the sample . furthermore , this technique is useful for applications since , during the es , the structures remain unchanged other than the blocking at the starting edge . 5 , which is a plot of the degree of etching / suppression versus the distance from the center of the illumination for the 80-m diameter pattern shown in fig . the degree of etch suppression is the percentage of the circular mesa surface , which appears intact in optical images of the etched structures ( see fig . as can be seen from this plot , the length scale on which the ese transitions from no significant suppression to full suppression is 100 m . further refinement is possible either using smaller illumination areas , restricting light access close to the surface of the samples ( for instance , by a shadow mask placed directly above the sample in the solution ) or developing methods to reduce stray light and diffraction in the solution . ( % of area ) versus distance from the center of the illumination for the sample in fig . 1 . thegreyareas indicate a fit based on the average degree of suppression and standard deviation values , for all points below and above a distance of 850 m , differentiating the regimes with ese and no significant ese , respectively figure 1b shows an optical microscopy image of a structure composed of a 4 nm alas layer and a 40 nm symmetrical strained in0.33ga0.66as / gaas bilayer . the larger circular shape observed on the sample at the end of the hf experiment is formed by original mesa structures that were left unetched , while on the rest of the sample the bilayer mesas are underetched , leading to rolling up as expected . figure 2 , which plots the average rolling distance as a function of intensity for structures with varying alas thickness , illustrates the sudden onset of the ese . the error due to variations in experimental parameters such as quantity of hf solution , scattering of light and effective illumination intensity at the sample , as well as the method for evaluating rolling distances , is estimated to be within 15% . it can be seen that the maximum rolling distance increases as the alas thickness increases , while the ese is only present for sacrificial layers below 10 nm . moreover , the onset of the ese , or the intensity of illumination necessary to suppress the alas etching , is higher for larger thicknesses . onset of illumination effect for 20 nm ingaas/20 nm gaas samples with 4 , 7 , 10 , 12 , 13 , 15 , and 20 nm alas sacrificial layers , etched with 2.5% hf solution using 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . the diamond data point shows a maximum rolling distance previously reported figure 3 shows the maximum rolling distance as a function of the sacrificial layer thickness . in a regime from 5 nm up to a thickness of 15 nm of alas the relationship between the maximum rolling distance and the sacrificial layer thickness is approximately linear , and then saturates for thicker alas layers . although in previous studies ingaas - gaas runts ( rolled - up nanotubes ) have been noticed to exhibit a self - limiting rolling behavior for longer etching times , this limiting was not investigated as function of the sacrificial layer thickness . the fit through the data in the linear increase regime ( r = 0.936 ) yields a unitless slope of 14.2e for the scaling of maximum rolling distance with talas . this indicates that in this first regime the maximum rolling distance is not dominated by intrinsic effects of the rolled - up layer and does not occur due to a fundamental limiting process ( as , e.g. , described by cendula et . therefore , we ascribe the saturation in this regime to dynamic effects either between the rolled - up layer and the substrate or inside the etching solution caused by the process in the gap between the substrate and the rolled - up film . the second regime shows a clear saturation behavior for alas thickness larger than 15 nm . in this regime , the saturation should be dominated by intrinsic effects either from the layer system itself or possibly by some kind of fundamental process yet to be found . the linear scaling of the maximum rolling distance offers the possibility for lateral positioning of the rolled - up tube relative to a defined starting edge . 1a , indicating rolling distancedrollmeasured from the starting edge , alas thicknesstalas , ingaas / gaas bilayer thicknesstbil . , and radius of curvaturerof the rolled - up bilayer furthermore , as shown in fig . 4 , the onset of ese changes for different etching speeds : the higher the hf concentration , the higher the intensity needed for suppression , while the thicker the alas layer , the more abrupt the etching behavior transition from the suppression regime to the illumination independent regime . for eachtalas , up to roughly 15 nm the onset of the ese increases approximately linearly with concentration , and scaling slopes are comparable for different thicknesses ( 12 mw / mm ) . from this , as well as fig . 2 , it can be concluded that the onset of ese occurs in a different regime from that of intrinsic saturation of maximum rolling distance . the onset of the etch - suppression effect ( ese ) versus hf concentration for 20 nm ingaas/20 nm gaas structures with 4 nm and 10 nm alas sacrificial layers , with 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . thelinesthrough the data are guides for the eye we also found that the ese is time dependent , i.e. , the etching time under illumination determines whether further etching will continue once the illumination is removed . in our experiments , we found that for samples where the high intensity light exposure was kept short , the layers were able to start rolling after being removed from the light source . this effect was found to not only depend on the light intensity but also on the thickness of the alas layer , which suggests that the etch suppression is a dynamic process that depends on the access of hf to the alas layer and the exchange of products and reactants in this region . this conclusion is further corroborated by the fact that with increasing alas thickness the rolling distance of the strained bilayer becomes independent of the alas gap size ( fig . the ese was used to position and control the roll up of tubes from strained ingaas / gaas bilayers . the patterning allowed us to create well - defined starting edges suitable for the illumination experiment ( as in fig . , one can obtain both etched regions with rolled - up layers and unetched regions within a small area on the sample . furthermore , this technique is useful for applications since , during the es , the structures remain unchanged other than the blocking at the starting edge . the resolution of this method is indicated by fig . 5 , which is a plot of the degree of etching / suppression versus the distance from the center of the illumination for the 80-m diameter pattern shown in fig . the degree of etch suppression is the percentage of the circular mesa surface , which appears intact in optical images of the etched structures ( see fig . as can be seen from this plot , the length scale on which the ese transitions from no significant suppression to full suppression is 100 m . further refinement is possible either using smaller illumination areas , restricting light access close to the surface of the samples ( for instance , by a shadow mask placed directly above the sample in the solution ) or developing methods to reduce stray light and diffraction in the solution . degree of suppression ( % of area ) versus distance from the center of the illumination for the sample in fig . 1 . thegreyareas indicate a fit based on the average degree of suppression and standard deviation values , for all points below and above a distance of 850 m , differentiating the regimes with ese and no significant ese , respectively we investigated the rolling distance as a function of illumination intensity used during the hf etching of alas / ingaas / gaas structures on gaas for different sacrificial layer thicknesses . for alas layers thinner than 10 nm , total suppression of the etching process occurs beyond a threshold intensity that increases with increasing alas thickness . the ese is clearly influenced by the alas - hf reaction rate as well as the physical characteristics of the structure : the larger the sacrificial layer gap and faster the reaction rate , the higher the intensity of illumination needed to suppress the etching . this trend continues up to a point where the reaction is no longer illumination dependent and suppression is no longer possible . a further series of experiments involving etching with filtered light and a focused laser beam of varying wavelengths ( not presented here ) suggests that the ese is preserved for lower energies than the alas band gap and therefore any photochemical effect in hf can not depend on light excitation of the alas material . we have also eliminated the possibility that heating plays a significant role in the ese , since experiments that involve heating samples in hf solution past the boiling point show an enhancement rather than suppression of the etching and hence would counteract this effect . we believe that for the ese found in samples etched with hf for shorter times under illumination levels close to the threshold value from fig . 2 , the strained bilayer remains intact because the sacrificial layer was not underetched at the starting edge , which may happen through the accumulation of solid as and as oxide following from a photochemical interaction at the gaas surface in the presence of hf . the process through which alas sacrificial layers are wet - etched with hydrofluoric acid ( hf ) [ 34 - 43 ] as well as the laser - induced etching of semiconductors by a dilute acid solution [ 44 - 46 ] have been investigated previously in some depth . in hf - alas reactions , any passivating as formed at the surface is usually oxidized and then dissolved , thus allowing new access to the etch front and sustaining etching . but if the rate of as production exceeds that of oxide formation , or if the type of oxide is stable in the hf , then the etching will be inhibited . from laser - induced etching studies , it is clear that porous stable oxide films formed at the semiconductor surface give a time dependence for the process . during the redox reaction , as(iii ) must be complexed by water and dissolved as haso2 and as2o3 , but when the concentration of as(iii ) surpasses the solubility limit , there is precipitation of as at the surface , slowing down the reaction . our results match well with this type of process , and we propose that the ese occurs in a similar fashion : the formation of very small amounts of as or as2o3 at the alas gap , which would nonetheless be enough to block access of hf to films under 10 nm , can effectively suppress the underetching of the alas layer . in accordance with our findings , the thicker the sacrificial layer gap and the faster the hf etching rate , the less likely the ese takes place . in the suppression regime for thinner alas layers , for higher intensities of illumination the photogeneration of holes is more pronounced leading to a faster subsequent passivation ( and reaching of the ese limit with time ) . for thicker alas layers , higher illumination can still lead to changes at the gaas substrate interface but does not hinder the etching of the alas . while the self - limitation of the maximum rolling distance of runts allows for the precise tuning of the number of rotations as a function of the sacrificial layer , the illumination permits the exact positioning of the tube in combination with common lithographic technology . fine - tuning the etch suppression with patterned samples can yield a useful way of precisely controlling the roll up of strained ingaas / gaas bilayers and the entire tube fabrication process , as well as other more general laser - assisted microfabrication applications , with a convenient , customizable method . in this way , it is complementary to pre - pattering of the sample by lithographic means and allows for a full control over the position of the produced runts .

the effect of illumination on the hydrofluoric acid etching of alas sacrificial layers with systematically varied thicknesses in order to release and roll up ingaas / gaas bilayers was studied . for thicknesses of alas below 10 nm , there were two etching regimes for the area under illumination : one at low illumination intensities , in which the etching and releasing proceeds as expected and one at higher intensities in which the etching and any releasing are completely suppressed . the etch suppression area is well defined by the illumination spot , a feature that can be used to create heterogeneously etched regions with a high degree of control , shown here on patterned samples . together with the studied self - limitation effect , the technique offers a way to determine the position of rolled - up micro- and nanotubes independently from the predefined lithographic pattern .

Introduction and Background Experimental Details Results Characteristics of Etching Suppression Effect Using Etch Suppression for Nanotube Positioning Discussion, Uncertainties, and Limitations Conclusion Acknowledgments

rolled - up nanotech has become a powerful technology with applications in a broad range of research fields , including optofluidics , micromachinery [ 4 - 6 ] , magnetofluidics , biophysics , nanomechanics , and waveguiding for different spectral ranges and applications [ 10 - 12 ] . large arrays of periodically ordered microtubes can be fabricated by a combination of lithography and deliberate self - rolling of strained layers upon selective underetching [ 1,13 - 15 ] . precise control over the lateral positioning and the number of windings is fundamental for the use of the tubes in optical resonators for sensors [ 16 - 20 ] , hyperlenses , or electrical transport ( curved electron gases ) [ 23 - 27 ] . numerous existing studies offer highly sophisticated methods for precise lateral positioning of rolled - up semiconductor nanostructures by controlling the starting edges [ 13 - 15,28,29 ] , but difficulties arise for thin layers due to the extensive , successive lithography steps involved , and ways to control the stopping point of rolling remain largely lacking without modifying the structure . here , we demonstrate illumination - controlled hydrofluoric acid ( hf ) etching of a buried thin alas sacrificial layer . sufficiently intense light exposure of a certain substrate area leads to a complete suppression of the underetching effect , and as a result , the formation of rolled - up ingaas / gaas tubes can be easily controlled spatially . this method allows for control over the roll - up stopping point and is suitable for very thin sacrificial layers . the aim of this study was to quantify the observed etch - suppression effect ( ese ) and precisely control the release of ingaas / gaas bilayers with an illumination technique . v rolled - up nanotube ( runt ) : when immersed in hf solution , the compressively strained double layer ( a gaas layer on top of a larger - lattice constant inxga1x as layer deposited pseudomorphically on a gaas substrate with an alas sacrificial layer ) releases from the substrate as the sacrificial layer is preferentially removed by the hf at a previously defined starting edge and begins to roll up in order to relax the built - in strain gradient . finally , the bent bilayer forms a tube or scroll after performing a full rotation . the starting edge , which for ingaas / gaas layers should be oriented in the direction for optimal structural integrity of the resulting runts , can be defined either by scratching the surface , thereby laterally exposing the sacrificial layer , or by opening a window through the entire layer structure by photolithography and subsequent wet chemical etching . , the size of the cross section of the structure obtained after the etching step has been completed , depends on the thickness and inherent strain of the layers , and can be accurately varied in the nano- to micrometer range [ 1,30 - 32 ] . for precise positioning of runts on the surface , the important parameters in the roll - up process are the sacrificial layer thickness , etching time , and tube diameter . the ratio of inner - to - outer diameter can also be controlled over a wide range , based on the number of rotations performed by the tubes on the surface ( or rolling distance , measured from the initial starting edge see schematic in fig . rolled - up structure after one full rotation).bschematic of the etching experiment during in situ observation by optical microscopy and result for strained bilayers ( relative ratio of mesa diameter to layer thickness not to size).coptical image of an alas / ingaas / gaas mesa structure etched in a 2.5% hf solution , with 20 microscope objective , for 6 min ; the etch - suppression spot radius is 700 m.doptical images of adjacent mesas from ( b ) ( black box ) , showing an increasing degree of etch suppression toward the center of the illumination here , the experiments were performed using 20 nm in0.33ga0.66as/20 nm gaas structures on alas grown on ( 001 ) gaas substrates by molecular beam epitaxy , where the thickness of the alas was varied from 4 to 20 nm . for experiments pertaining to the measurement of the onset of the etch suppression and rolling distance , the starting edges were defined by mechanical scratching , whereas photolithography and wet chemical etching ( by h3po4:h2o2:h2o solution ) were used for controlling the etch suppression . for all measurements , the intensity of illumination was measured beforehand using a calibrated power meter . for samples that were etched , illuminated , and observed under the microscope , the settings on the illumination dial were used and the focused spot sizes for each magnification were assumed to be the same . for the quantitative measurements of tube rolling distance , optical images of the sample outside of solution once the etching was terminated were taken . from these images , the rolling distance could then be measured , which we define as distance from the starting edge to the center of the tube width , assumed to correspond to the stopping point of the underetching . the resolution of the es method was studied by precisely positioning the illumination spot on wet - chemically etched mesa structures of different shapes and sizes ( on the order of 10100 m ) during hf etching . the degree of etching suppression ( given by the coverage area of unetched structures remaining on the sample ) was measured versus distance from the center of illumination . figure 1b shows an optical microscopy image of a structure composed of a 4 nm alas layer and a 40 nm symmetrical strained in0.33ga0.66as / gaas bilayer . the larger circular shape observed on the sample at the end of the hf experiment is formed by original mesa structures that were left unetched , while on the rest of the sample the bilayer mesas are underetched , leading to rolling up as expected . it can be seen that the maximum rolling distance increases as the alas thickness increases , while the ese is only present for sacrificial layers below 10 nm . moreover , the onset of the ese , or the intensity of illumination necessary to suppress the alas etching , is higher for larger thicknesses . onset of illumination effect for 20 nm ingaas/20 nm gaas samples with 4 , 7 , 10 , 12 , 13 , 15 , and 20 nm alas sacrificial layers , etched with 2.5% hf solution using 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . in a regime from 5 nm up to a thickness of 15 nm of alas the relationship between the maximum rolling distance and the sacrificial layer thickness is approximately linear , and then saturates for thicker alas layers . although in previous studies ingaas - gaas runts ( rolled - up nanotubes ) have been noticed to exhibit a self - limiting rolling behavior for longer etching times , this limiting was not investigated as function of the sacrificial layer thickness . the fit through the data in the linear increase regime ( r = 0.936 ) yields a unitless slope of 14.2e for the scaling of maximum rolling distance with talas . this indicates that in this first regime the maximum rolling distance is not dominated by intrinsic effects of the rolled - up layer and does not occur due to a fundamental limiting process ( as , e.g. therefore , we ascribe the saturation in this regime to dynamic effects either between the rolled - up layer and the substrate or inside the etching solution caused by the process in the gap between the substrate and the rolled - up film . in this regime , the saturation should be dominated by intrinsic effects either from the layer system itself or possibly by some kind of fundamental process yet to be found . the linear scaling of the maximum rolling distance offers the possibility for lateral positioning of the rolled - up tube relative to a defined starting edge . 1a , indicating rolling distancedrollmeasured from the starting edge , alas thicknesstalas , ingaas / gaas bilayer thicknesstbil . , and radius of curvaturerof the rolled - up bilayer furthermore , as shown in fig . 4 , the onset of ese changes for different etching speeds : the higher the hf concentration , the higher the intensity needed for suppression , while the thicker the alas layer , the more abrupt the etching behavior transition from the suppression regime to the illumination independent regime . 2 , it can be concluded that the onset of ese occurs in a different regime from that of intrinsic saturation of maximum rolling distance . the onset of the etch - suppression effect ( ese ) versus hf concentration for 20 nm ingaas/20 nm gaas structures with 4 nm and 10 nm alas sacrificial layers , with 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . thelinesthrough the data are guides for the eye we also found that the ese is time dependent , i.e. , the etching time under illumination determines whether further etching will continue once the illumination is removed . in our experiments , we found that for samples where the high intensity light exposure was kept short , the layers were able to start rolling after being removed from the light source . this effect was found to not only depend on the light intensity but also on the thickness of the alas layer , which suggests that the etch suppression is a dynamic process that depends on the access of hf to the alas layer and the exchange of products and reactants in this region . this conclusion is further corroborated by the fact that with increasing alas thickness the rolling distance of the strained bilayer becomes independent of the alas gap size ( fig . the ese was used to position and control the roll up of tubes from strained ingaas / gaas bilayers . the patterning allowed us to create well - defined starting edges suitable for the illumination experiment ( as in fig . , one can obtain both etched regions with rolled - up layers and unetched regions within a small area on the sample . furthermore , this technique is useful for applications since , during the es , the structures remain unchanged other than the blocking at the starting edge . 5 , which is a plot of the degree of etching / suppression versus the distance from the center of the illumination for the 80-m diameter pattern shown in fig . the degree of etch suppression is the percentage of the circular mesa surface , which appears intact in optical images of the etched structures ( see fig . as can be seen from this plot , the length scale on which the ese transitions from no significant suppression to full suppression is 100 m . ( % of area ) versus distance from the center of the illumination for the sample in fig . thegreyareas indicate a fit based on the average degree of suppression and standard deviation values , for all points below and above a distance of 850 m , differentiating the regimes with ese and no significant ese , respectively figure 1b shows an optical microscopy image of a structure composed of a 4 nm alas layer and a 40 nm symmetrical strained in0.33ga0.66as / gaas bilayer . the larger circular shape observed on the sample at the end of the hf experiment is formed by original mesa structures that were left unetched , while on the rest of the sample the bilayer mesas are underetched , leading to rolling up as expected . it can be seen that the maximum rolling distance increases as the alas thickness increases , while the ese is only present for sacrificial layers below 10 nm . moreover , the onset of the ese , or the intensity of illumination necessary to suppress the alas etching , is higher for larger thicknesses . onset of illumination effect for 20 nm ingaas/20 nm gaas samples with 4 , 7 , 10 , 12 , 13 , 15 , and 20 nm alas sacrificial layers , etched with 2.5% hf solution using 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . although in previous studies ingaas - gaas runts ( rolled - up nanotubes ) have been noticed to exhibit a self - limiting rolling behavior for longer etching times , this limiting was not investigated as function of the sacrificial layer thickness . the fit through the data in the linear increase regime ( r = 0.936 ) yields a unitless slope of 14.2e for the scaling of maximum rolling distance with talas . this indicates that in this first regime the maximum rolling distance is not dominated by intrinsic effects of the rolled - up layer and does not occur due to a fundamental limiting process ( as , e.g. therefore , we ascribe the saturation in this regime to dynamic effects either between the rolled - up layer and the substrate or inside the etching solution caused by the process in the gap between the substrate and the rolled - up film . in this regime , the saturation should be dominated by intrinsic effects either from the layer system itself or possibly by some kind of fundamental process yet to be found . the linear scaling of the maximum rolling distance offers the possibility for lateral positioning of the rolled - up tube relative to a defined starting edge . 1a , indicating rolling distancedrollmeasured from the starting edge , alas thicknesstalas , ingaas / gaas bilayer thicknesstbil . , and radius of curvaturerof the rolled - up bilayer furthermore , as shown in fig . 4 , the onset of ese changes for different etching speeds : the higher the hf concentration , the higher the intensity needed for suppression , while the thicker the alas layer , the more abrupt the etching behavior transition from the suppression regime to the illumination independent regime . 2 , it can be concluded that the onset of ese occurs in a different regime from that of intrinsic saturation of maximum rolling distance . the onset of the etch - suppression effect ( ese ) versus hf concentration for 20 nm ingaas/20 nm gaas structures with 4 nm and 10 nm alas sacrificial layers , with 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . thelinesthrough the data are guides for the eye we also found that the ese is time dependent , i.e. , the etching time under illumination determines whether further etching will continue once the illumination is removed . in our experiments , we found that for samples where the high intensity light exposure was kept short , the layers were able to start rolling after being removed from the light source . this effect was found to not only depend on the light intensity but also on the thickness of the alas layer , which suggests that the etch suppression is a dynamic process that depends on the access of hf to the alas layer and the exchange of products and reactants in this region . the ese was used to position and control the roll up of tubes from strained ingaas / gaas bilayers . the patterning allowed us to create well - defined starting edges suitable for the illumination experiment ( as in fig . , one can obtain both etched regions with rolled - up layers and unetched regions within a small area on the sample . furthermore , this technique is useful for applications since , during the es , the structures remain unchanged other than the blocking at the starting edge . 5 , which is a plot of the degree of etching / suppression versus the distance from the center of the illumination for the 80-m diameter pattern shown in fig . the degree of etch suppression is the percentage of the circular mesa surface , which appears intact in optical images of the etched structures ( see fig . as can be seen from this plot , the length scale on which the ese transitions from no significant suppression to full suppression is 100 m . degree of suppression ( % of area ) versus distance from the center of the illumination for the sample in fig . thegreyareas indicate a fit based on the average degree of suppression and standard deviation values , for all points below and above a distance of 850 m , differentiating the regimes with ese and no significant ese , respectively we investigated the rolling distance as a function of illumination intensity used during the hf etching of alas / ingaas / gaas structures on gaas for different sacrificial layer thicknesses . for alas layers thinner than 10 nm , total suppression of the etching process occurs beyond a threshold intensity that increases with increasing alas thickness . the ese is clearly influenced by the alas - hf reaction rate as well as the physical characteristics of the structure : the larger the sacrificial layer gap and faster the reaction rate , the higher the intensity of illumination needed to suppress the etching . we have also eliminated the possibility that heating plays a significant role in the ese , since experiments that involve heating samples in hf solution past the boiling point show an enhancement rather than suppression of the etching and hence would counteract this effect . we believe that for the ese found in samples etched with hf for shorter times under illumination levels close to the threshold value from fig . 2 , the strained bilayer remains intact because the sacrificial layer was not underetched at the starting edge , which may happen through the accumulation of solid as and as oxide following from a photochemical interaction at the gaas surface in the presence of hf . the process through which alas sacrificial layers are wet - etched with hydrofluoric acid ( hf ) [ 34 - 43 ] as well as the laser - induced etching of semiconductors by a dilute acid solution [ 44 - 46 ] have been investigated previously in some depth . in hf - alas reactions , any passivating as formed at the surface is usually oxidized and then dissolved , thus allowing new access to the etch front and sustaining etching . but if the rate of as production exceeds that of oxide formation , or if the type of oxide is stable in the hf , then the etching will be inhibited . from laser - induced etching studies , it is clear that porous stable oxide films formed at the semiconductor surface give a time dependence for the process . during the redox reaction , as(iii ) must be complexed by water and dissolved as haso2 and as2o3 , but when the concentration of as(iii ) surpasses the solubility limit , there is precipitation of as at the surface , slowing down the reaction . our results match well with this type of process , and we propose that the ese occurs in a similar fashion : the formation of very small amounts of as or as2o3 at the alas gap , which would nonetheless be enough to block access of hf to films under 10 nm , can effectively suppress the underetching of the alas layer . in accordance with our findings , the thicker the sacrificial layer gap and the faster the hf etching rate , the less likely the ese takes place . in the suppression regime for thinner alas layers , for higher intensities of illumination the photogeneration of holes is more pronounced leading to a faster subsequent passivation ( and reaching of the ese limit with time ) . for thicker alas layers , higher illumination can still lead to changes at the gaas substrate interface but does not hinder the etching of the alas . while the self - limitation of the maximum rolling distance of runts allows for the precise tuning of the number of rotations as a function of the sacrificial layer , the illumination permits the exact positioning of the tube in combination with common lithographic technology . fine - tuning the etch suppression with patterned samples can yield a useful way of precisely controlling the roll up of strained ingaas / gaas bilayers and the entire tube fabrication process , as well as other more general laser - assisted microfabrication applications , with a convenient , customizable method . in this way , it is complementary to pre - pattering of the sample by lithographic means and allows for a full control over the position of the produced runts .

rolled - up nanotech has become a powerful technology with applications in a broad range of research fields , including optofluidics , micromachinery [ 4 - 6 ] , magnetofluidics , biophysics , nanomechanics , and waveguiding for different spectral ranges and applications [ 10 - 12 ] . large arrays of periodically ordered microtubes can be fabricated by a combination of lithography and deliberate self - rolling of strained layers upon selective underetching [ 1,13 - 15 ] . precise control over the lateral positioning and the number of windings is fundamental for the use of the tubes in optical resonators for sensors [ 16 - 20 ] , hyperlenses , or electrical transport ( curved electron gases ) [ 23 - 27 ] . numerous existing studies offer highly sophisticated methods for precise lateral positioning of rolled - up semiconductor nanostructures by controlling the starting edges [ 13 - 15,28,29 ] , but difficulties arise for thin layers due to the extensive , successive lithography steps involved , and ways to control the stopping point of rolling remain largely lacking without modifying the structure . here , we demonstrate illumination - controlled hydrofluoric acid ( hf ) etching of a buried thin alas sacrificial layer . sufficiently intense light exposure of a certain substrate area leads to a complete suppression of the underetching effect , and as a result , the formation of rolled - up ingaas / gaas tubes can be easily controlled spatially . the aim of this study was to quantify the observed etch - suppression effect ( ese ) and precisely control the release of ingaas / gaas bilayers with an illumination technique . v rolled - up nanotube ( runt ) : when immersed in hf solution , the compressively strained double layer ( a gaas layer on top of a larger - lattice constant inxga1x as layer deposited pseudomorphically on a gaas substrate with an alas sacrificial layer ) releases from the substrate as the sacrificial layer is preferentially removed by the hf at a previously defined starting edge and begins to roll up in order to relax the built - in strain gradient . the starting edge , which for ingaas / gaas layers should be oriented in the direction for optimal structural integrity of the resulting runts , can be defined either by scratching the surface , thereby laterally exposing the sacrificial layer , or by opening a window through the entire layer structure by photolithography and subsequent wet chemical etching . , the size of the cross section of the structure obtained after the etching step has been completed , depends on the thickness and inherent strain of the layers , and can be accurately varied in the nano- to micrometer range [ 1,30 - 32 ] . for precise positioning of runts on the surface , the important parameters in the roll - up process are the sacrificial layer thickness , etching time , and tube diameter . the ratio of inner - to - outer diameter can also be controlled over a wide range , based on the number of rotations performed by the tubes on the surface ( or rolling distance , measured from the initial starting edge see schematic in fig . rolled - up structure after one full rotation).bschematic of the etching experiment during in situ observation by optical microscopy and result for strained bilayers ( relative ratio of mesa diameter to layer thickness not to size).coptical image of an alas / ingaas / gaas mesa structure etched in a 2.5% hf solution , with 20 microscope objective , for 6 min ; the etch - suppression spot radius is 700 m.doptical images of adjacent mesas from ( b ) ( black box ) , showing an increasing degree of etch suppression toward the center of the illumination here , the experiments were performed using 20 nm in0.33ga0.66as/20 nm gaas structures on alas grown on ( 001 ) gaas substrates by molecular beam epitaxy , where the thickness of the alas was varied from 4 to 20 nm . for experiments pertaining to the measurement of the onset of the etch suppression and rolling distance , the starting edges were defined by mechanical scratching , whereas photolithography and wet chemical etching ( by h3po4:h2o2:h2o solution ) were used for controlling the etch suppression . our specially designed setup involved an hf - resistant trough for etching with hf under light focused by a zeiss optical microscope using different objective lenses of 5 through 50 magnification ( for sample evolution during this process , see fig . other measurements were performed using illumination from a hene laser with maximum output power of 2 mw , as well as a laser with adjustable power ( up to 20 mw ) and wavelength ( 535825 nm ) with focusing optics or an optical fiber to illuminate the surface of the sample . for samples that were etched , illuminated , and observed under the microscope , the settings on the illumination dial were used and the focused spot sizes for each magnification were assumed to be the same . for the quantitative measurements of tube rolling distance , optical images of the sample outside of solution once the etching was terminated were taken . from these images , the rolling distance could then be measured , which we define as distance from the starting edge to the center of the tube width , assumed to correspond to the stopping point of the underetching . the resolution of the es method was studied by precisely positioning the illumination spot on wet - chemically etched mesa structures of different shapes and sizes ( on the order of 10100 m ) during hf etching . the degree of etching suppression ( given by the coverage area of unetched structures remaining on the sample ) was measured versus distance from the center of illumination . the larger circular shape observed on the sample at the end of the hf experiment is formed by original mesa structures that were left unetched , while on the rest of the sample the bilayer mesas are underetched , leading to rolling up as expected . figure 2 , which plots the average rolling distance as a function of intensity for structures with varying alas thickness , illustrates the sudden onset of the ese . the error due to variations in experimental parameters such as quantity of hf solution , scattering of light and effective illumination intensity at the sample , as well as the method for evaluating rolling distances , is estimated to be within 15% . it can be seen that the maximum rolling distance increases as the alas thickness increases , while the ese is only present for sacrificial layers below 10 nm . moreover , the onset of the ese , or the intensity of illumination necessary to suppress the alas etching , is higher for larger thicknesses . onset of illumination effect for 20 nm ingaas/20 nm gaas samples with 4 , 7 , 10 , 12 , 13 , 15 , and 20 nm alas sacrificial layers , etched with 2.5% hf solution using 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . in a regime from 5 nm up to a thickness of 15 nm of alas the relationship between the maximum rolling distance and the sacrificial layer thickness is approximately linear , and then saturates for thicker alas layers . although in previous studies ingaas - gaas runts ( rolled - up nanotubes ) have been noticed to exhibit a self - limiting rolling behavior for longer etching times , this limiting was not investigated as function of the sacrificial layer thickness . the fit through the data in the linear increase regime ( r = 0.936 ) yields a unitless slope of 14.2e for the scaling of maximum rolling distance with talas . this indicates that in this first regime the maximum rolling distance is not dominated by intrinsic effects of the rolled - up layer and does not occur due to a fundamental limiting process ( as , e.g. therefore , we ascribe the saturation in this regime to dynamic effects either between the rolled - up layer and the substrate or inside the etching solution caused by the process in the gap between the substrate and the rolled - up film . in this regime , the saturation should be dominated by intrinsic effects either from the layer system itself or possibly by some kind of fundamental process yet to be found . the linear scaling of the maximum rolling distance offers the possibility for lateral positioning of the rolled - up tube relative to a defined starting edge . 4 , the onset of ese changes for different etching speeds : the higher the hf concentration , the higher the intensity needed for suppression , while the thicker the alas layer , the more abrupt the etching behavior transition from the suppression regime to the illumination independent regime . for eachtalas , up to roughly 15 nm the onset of the ese increases approximately linearly with concentration , and scaling slopes are comparable for different thicknesses ( 12 mw / mm ) . 2 , it can be concluded that the onset of ese occurs in a different regime from that of intrinsic saturation of maximum rolling distance . the onset of the etch - suppression effect ( ese ) versus hf concentration for 20 nm ingaas/20 nm gaas structures with 4 nm and 10 nm alas sacrificial layers , with 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . thelinesthrough the data are guides for the eye we also found that the ese is time dependent , i.e. in our experiments , we found that for samples where the high intensity light exposure was kept short , the layers were able to start rolling after being removed from the light source . this effect was found to not only depend on the light intensity but also on the thickness of the alas layer , which suggests that the etch suppression is a dynamic process that depends on the access of hf to the alas layer and the exchange of products and reactants in this region . this conclusion is further corroborated by the fact that with increasing alas thickness the rolling distance of the strained bilayer becomes independent of the alas gap size ( fig . the patterning allowed us to create well - defined starting edges suitable for the illumination experiment ( as in fig . furthermore , this technique is useful for applications since , during the es , the structures remain unchanged other than the blocking at the starting edge . 5 , which is a plot of the degree of etching / suppression versus the distance from the center of the illumination for the 80-m diameter pattern shown in fig . the degree of etch suppression is the percentage of the circular mesa surface , which appears intact in optical images of the etched structures ( see fig . as can be seen from this plot , the length scale on which the ese transitions from no significant suppression to full suppression is 100 m . further refinement is possible either using smaller illumination areas , restricting light access close to the surface of the samples ( for instance , by a shadow mask placed directly above the sample in the solution ) or developing methods to reduce stray light and diffraction in the solution . ( % of area ) versus distance from the center of the illumination for the sample in fig . thegreyareas indicate a fit based on the average degree of suppression and standard deviation values , for all points below and above a distance of 850 m , differentiating the regimes with ese and no significant ese , respectively figure 1b shows an optical microscopy image of a structure composed of a 4 nm alas layer and a 40 nm symmetrical strained in0.33ga0.66as / gaas bilayer . the larger circular shape observed on the sample at the end of the hf experiment is formed by original mesa structures that were left unetched , while on the rest of the sample the bilayer mesas are underetched , leading to rolling up as expected . figure 2 , which plots the average rolling distance as a function of intensity for structures with varying alas thickness , illustrates the sudden onset of the ese . the error due to variations in experimental parameters such as quantity of hf solution , scattering of light and effective illumination intensity at the sample , as well as the method for evaluating rolling distances , is estimated to be within 15% . it can be seen that the maximum rolling distance increases as the alas thickness increases , while the ese is only present for sacrificial layers below 10 nm . moreover , the onset of the ese , or the intensity of illumination necessary to suppress the alas etching , is higher for larger thicknesses . onset of illumination effect for 20 nm ingaas/20 nm gaas samples with 4 , 7 , 10 , 12 , 13 , 15 , and 20 nm alas sacrificial layers , etched with 2.5% hf solution using 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . in a regime from 5 nm up to a thickness of 15 nm of alas the relationship between the maximum rolling distance and the sacrificial layer thickness is approximately linear , and then saturates for thicker alas layers . although in previous studies ingaas - gaas runts ( rolled - up nanotubes ) have been noticed to exhibit a self - limiting rolling behavior for longer etching times , this limiting was not investigated as function of the sacrificial layer thickness . the fit through the data in the linear increase regime ( r = 0.936 ) yields a unitless slope of 14.2e for the scaling of maximum rolling distance with talas . this indicates that in this first regime the maximum rolling distance is not dominated by intrinsic effects of the rolled - up layer and does not occur due to a fundamental limiting process ( as , e.g. therefore , we ascribe the saturation in this regime to dynamic effects either between the rolled - up layer and the substrate or inside the etching solution caused by the process in the gap between the substrate and the rolled - up film . in this regime , the saturation should be dominated by intrinsic effects either from the layer system itself or possibly by some kind of fundamental process yet to be found . the linear scaling of the maximum rolling distance offers the possibility for lateral positioning of the rolled - up tube relative to a defined starting edge . 4 , the onset of ese changes for different etching speeds : the higher the hf concentration , the higher the intensity needed for suppression , while the thicker the alas layer , the more abrupt the etching behavior transition from the suppression regime to the illumination independent regime . for eachtalas , up to roughly 15 nm the onset of the ese increases approximately linearly with concentration , and scaling slopes are comparable for different thicknesses ( 12 mw / mm ) . 2 , it can be concluded that the onset of ese occurs in a different regime from that of intrinsic saturation of maximum rolling distance . the onset of the etch - suppression effect ( ese ) versus hf concentration for 20 nm ingaas/20 nm gaas structures with 4 nm and 10 nm alas sacrificial layers , with 20 microscope objective , for 8 min ; the starting edges were produced by mechanical scratching . thelinesthrough the data are guides for the eye we also found that the ese is time dependent , i.e. this effect was found to not only depend on the light intensity but also on the thickness of the alas layer , which suggests that the etch suppression is a dynamic process that depends on the access of hf to the alas layer and the exchange of products and reactants in this region . this conclusion is further corroborated by the fact that with increasing alas thickness the rolling distance of the strained bilayer becomes independent of the alas gap size ( fig . furthermore , this technique is useful for applications since , during the es , the structures remain unchanged other than the blocking at the starting edge . 5 , which is a plot of the degree of etching / suppression versus the distance from the center of the illumination for the 80-m diameter pattern shown in fig . the degree of etch suppression is the percentage of the circular mesa surface , which appears intact in optical images of the etched structures ( see fig . further refinement is possible either using smaller illumination areas , restricting light access close to the surface of the samples ( for instance , by a shadow mask placed directly above the sample in the solution ) or developing methods to reduce stray light and diffraction in the solution . degree of suppression ( % of area ) versus distance from the center of the illumination for the sample in fig . thegreyareas indicate a fit based on the average degree of suppression and standard deviation values , for all points below and above a distance of 850 m , differentiating the regimes with ese and no significant ese , respectively we investigated the rolling distance as a function of illumination intensity used during the hf etching of alas / ingaas / gaas structures on gaas for different sacrificial layer thicknesses . the ese is clearly influenced by the alas - hf reaction rate as well as the physical characteristics of the structure : the larger the sacrificial layer gap and faster the reaction rate , the higher the intensity of illumination needed to suppress the etching . a further series of experiments involving etching with filtered light and a focused laser beam of varying wavelengths ( not presented here ) suggests that the ese is preserved for lower energies than the alas band gap and therefore any photochemical effect in hf can not depend on light excitation of the alas material . we have also eliminated the possibility that heating plays a significant role in the ese , since experiments that involve heating samples in hf solution past the boiling point show an enhancement rather than suppression of the etching and hence would counteract this effect . we believe that for the ese found in samples etched with hf for shorter times under illumination levels close to the threshold value from fig . 2 , the strained bilayer remains intact because the sacrificial layer was not underetched at the starting edge , which may happen through the accumulation of solid as and as oxide following from a photochemical interaction at the gaas surface in the presence of hf . the process through which alas sacrificial layers are wet - etched with hydrofluoric acid ( hf ) [ 34 - 43 ] as well as the laser - induced etching of semiconductors by a dilute acid solution [ 44 - 46 ] have been investigated previously in some depth . but if the rate of as production exceeds that of oxide formation , or if the type of oxide is stable in the hf , then the etching will be inhibited . during the redox reaction , as(iii ) must be complexed by water and dissolved as haso2 and as2o3 , but when the concentration of as(iii ) surpasses the solubility limit , there is precipitation of as at the surface , slowing down the reaction . our results match well with this type of process , and we propose that the ese occurs in a similar fashion : the formation of very small amounts of as or as2o3 at the alas gap , which would nonetheless be enough to block access of hf to films under 10 nm , can effectively suppress the underetching of the alas layer . in accordance with our findings , the thicker the sacrificial layer gap and the faster the hf etching rate , the less likely the ese takes place . in the suppression regime for thinner alas layers , for higher intensities of illumination the photogeneration of holes is more pronounced leading to a faster subsequent passivation ( and reaching of the ese limit with time ) . while the self - limitation of the maximum rolling distance of runts allows for the precise tuning of the number of rotations as a function of the sacrificial layer , the illumination permits the exact positioning of the tube in combination with common lithographic technology . fine - tuning the etch suppression with patterned samples can yield a useful way of precisely controlling the roll up of strained ingaas / gaas bilayers and the entire tube fabrication process , as well as other more general laser - assisted microfabrication applications , with a convenient , customizable method .

the current study utilizes data from the administrative records of a large workers compensation insurance company in the united states that included claims from a variety of different states , industries , organizations , and company sizes . all workers compensation claims with complete data for individuals who had an injury date between january 1 , 2002 , and december 31 , 2008 , were assessed for inclusion . we included all claims with at least 1 day of paid lost work time within 1 year of the injury date . lost work time comprised both days of temporary total disability ( ttd ) and days of temporary partial disability ( tpd ) . two sets of criteria were used to identify lbp claims on the basis of the icd-9 diagnosis codes that were reported in the claimant 's medical service bills for the first 15 days of medical treatment . medical treatment typically began within 2 weeks of the injury date ( 90% of claims ) , although all claimants who received medical treatment within 1 year of the injury date and who had lost work time were included in the database . first , 99,127 claims were classified as a possible lbp claim based on having at least one icd-9 diagnosis code within the first 15 days of medical treatment relating to specific low back injuries or disorders , which are listed in supplemental table 1 . second , from those identified as possible lbp claims , we required that over two - thirds of the claimant 's icd-9 diagnosis codes in the diseases of the nervous system and sense organs ( 320389 ) , diseases of the musculoskeletal system and connective tissue ( 710739 ) , and injury and poisoning ( 800777 ) chapters within the first 15 days of medical treatment be for specific low back injuries or disorders ( supplemental table 1 or nonspecific back injuries or disorders ( supplemental table 2 ) . in total , 76,955 claims met these criteria and approximately 70% of these claims had 100% of their icd-9 diagnosis codes within the first 15 days coming from the codes listed in supplemental tables 1 or 2 . our sample was further restricted to claimants who had only one claim within a single calendar year . for some claimants with multiple claims in a single year , the claims were for different injuries . however , in certain cases , there were multiple episodes of disability for a single claim for individuals resulting from the same injury . this occurred in cases wherein an individual went back to work after an injury but after having returned to work went back out on ttd or tpd . in this study , we only included claims for individuals in whom multiple episodes of disability within the same claim had less than 14 days between episodes , in which case we considered this a single episode . if the duration between episodes was 14 days or longer , these claims were excluded . we also excluded claims for individuals who received a lump sum payment within 1 year of the injury date . claims were excluded in cases wherein the first date of lost work time was more than 1 year after the injury date . finally , only claims for individuals 18 to 80 years of age were included in this study . in total the first lag time is referred to as the reporting lag time and represents the number of days from the date of injury to the date at which the injury was first reported to the workers compensation insurer . the second lag time , which we refer to as the medical services lag time , is the number of days from the date of injury to the date at which a claimant first sought medical care for that injury . the third lag time is referred to as the work disability lag time and represents the number of days from the date of injury to the date at which a claimant first took tpd or ttd . each of the three lag times were categorized into seven categories : 0 days lag ( reference group ) , 1 to 3 days lag , 4 to 6 days lag , 1 week up to 2 weeks lag , 2 weeks up to 30 days lag , 30 days up to 60 days lag , and 60 days up to 1 year lag . the outcome variable was the length of disability , which was calculated from the date that a claimant first took tpd or ttd until the date at which tpd or ttd ended . tpd or ttd was considered to have ended when no disability days were taken for at least 14 days consecutively . for claims in which the length of disability exceeded 1 year the following covariates were used : gender , annual income , industry , litigation status , injury severity , year of injury , age , and tenure . sixteen ordered categories were used to assess annual income : $ 0 to $ 9999 , $ 10000 to $ 19,999 , $ 20,000 to $ 29,999 , $ 30,000 to $ 39,999 , $ 40,000 to $ 49,999 , $ 50,000 to $ 59,999 , $ 60,000 to $ 69,999 , $ 70,000 to $ 79,999 , $ 80,000 to $ 89,999 , $ 90,000 to $ 99,999 , $ 100,000 to $ 109,999 , $ 110,000 to $ 119,999 , $ 120,000 to $ 129,999 , $ 130,000 to $ 139,999 , $ 140,000 to $ 149,999 , and $ 150,000 or more . industry was categorized into 10 groups , including agriculture , forestry and fishing , construction , finance and insurance , manufacturing , mining , retail trade , services , transportation , public administration , and wholesale trade . litigation status was coded 1 if the workers compensation insurer assigned an attorney to the claim and 0 if not . the codes in the more severe and less severe categories can be found in supplemental tables 1 and 2 . injury severity was coded 1 for having at least one more severe diagnosis within the first 15 days of medical treatment and 0 for having only less severe diagnoses within the first 15 days of medical treatment . the more severe codes generally included diagnoses related to herniated disc , lumbar radiculopathy or neuropathy , spinal stenosis , sciatica , or possible instability . the less severe codes generally referred to diagnoses associated with degenerative changes , nonspecific back pain , or miscellaneous diagnoses . in addition , efforts were made to exclude cases of complicated back pain , such as those with diagnoses that were consistent with experiencing multiple work - related injuries or disorders , very severe injuries , or back pain due to cancer , infection , severe trauma , or an autoimmune disorder . since the database included claims from 2002 to 2008 , the analyses were controlled for the year of the injury . tenure was also measured continuously on the basis of a claimant 's organizational tenure at the time of injury . the first lag time is referred to as the reporting lag time and represents the number of days from the date of injury to the date at which the injury was first reported to the workers compensation insurer . the second lag time , which we refer to as the medical services lag time , is the number of days from the date of injury to the date at which a claimant first sought medical care for that injury . the third lag time is referred to as the work disability lag time and represents the number of days from the date of injury to the date at which a claimant first took tpd or ttd . each of the three lag times were categorized into seven categories : 0 days lag ( reference group ) , 1 to 3 days lag , 4 to 6 days lag , 1 week up to 2 weeks lag , 2 weeks up to 30 days lag , 30 days up to 60 days lag , and 60 days up to 1 year lag . the outcome variable was the length of disability , which was calculated from the date that a claimant first took tpd or ttd until the date at which tpd or ttd ended . tpd or ttd was considered to have ended when no disability days were taken for at least 14 days consecutively . for claims in which the length of disability exceeded 1 year , the following covariates were used : gender , annual income , industry , litigation status , injury severity , year of injury , age , and tenure . sixteen ordered categories were used to assess annual income : $ 0 to $ 9999 , $ 10000 to $ 19,999 , $ 20,000 to $ 29,999 , $ 30,000 to $ 39,999 , $ 40,000 to $ 49,999 , $ 50,000 to $ 59,999 , $ 60,000 to $ 69,999 , $ 70,000 to $ 79,999 , $ 80,000 to $ 89,999 , $ 90,000 to $ 99,999 , $ 100,000 to $ 109,999 , $ 110,000 to $ 119,999 , $ 120,000 to $ 129,999 , $ 130,000 to $ 139,999 , $ 140,000 to $ 149,999 , and $ 150,000 or more . industry was categorized into 10 groups , including agriculture , forestry and fishing , construction , finance and insurance , manufacturing , mining , retail trade , services , transportation , public administration , and wholesale trade . litigation status was coded 1 if the workers compensation insurer assigned an attorney to the claim and 0 if not . the codes in the more severe and less severe categories can be found in supplemental tables 1 and 2 . injury severity was coded 1 for having at least one more severe diagnosis within the first 15 days of medical treatment and 0 for having only less severe diagnoses within the first 15 days of medical treatment . the more severe codes generally included diagnoses related to herniated disc , lumbar radiculopathy or neuropathy , spinal stenosis , sciatica , or possible instability . the less severe codes generally referred to diagnoses associated with degenerative changes , nonspecific back pain , or miscellaneous diagnoses . in addition , efforts were made to exclude cases of complicated back pain , such as those with diagnoses that were consistent with experiencing multiple work - related injuries or disorders , very severe injuries , or back pain due to cancer , infection , severe trauma , or an autoimmune disorder . since the database included claims from 2002 to 2008 , the analyses were controlled for the year of the injury . tenure was also measured continuously on the basis of a claimant 's organizational tenure at the time of injury . the relationships between the three lag times and length of disability were estimated using random effects tobit models . we were only interested in following claimants for 1 year from the time of disability onset ; however , for some claimants , disability may not have ended by 365 days and it is necessary for our models to take this into account . tobit models or censored regression models , as they are sometimes referred to , were chosen to deal with the nature of the data used in the current study wherein length of disability was restricted to a range between 3 and 365 days . similar to ordinary least squared regression models ( ols ) , tobit models are generally used when estimating linear relationships between variables ; however , ols generally assumes a continuous distribution without censored values , whereas the tobit model is able to accommodate left- and/or right - censoring of values . ols produces biased estimates when values are censored , while tobit models eliminate that bias . in order to take into account the non - independence of observations across industry groupings , participants drawn from different industry groups may be thought of as representing subsamples within the larger sample . participants disability durations may be clustered within these different industry groups such that the residuals are dependent on one another within groups . random effects models take this clustering into account by allowing the constant to vary across groups , in this case industries , while at the same time , keeping the other estimates fixed . for all lag times , the 0 days lag category was used as the reference group . separate analyses were conducted for each of the three lag times due to concerns about collinearity among the lag times . in addition to the main analyses , differences in the coefficients for the non - reference group lag time categories were assessed using wald tests . analyses were adjusted for gender , annual income , industry , litigation status , injury severity , year of injury , age , and tenure . to reduce issues with multicollinearity , all continuous variables in the models were mean - centered . stata 13 was used to perform the analyses ( stata corporation , college station , tx ) . in the analyses , on the basis of this , we used a more conservative p value of less than 0.001 to represent statistical significance . the age of claimants at the time of injury ranged from 18 to 80 years , with an average age of 40 years ( sd 11.2 years ) . the length of tenure at the time of injury ranged from 0 to 53 years , with an average tenure of 6 years ( sd 7.7 years ) . over half of the claimants had an annual income of $ 20,000 to $ 50,000 and only 1% of claimants had an annual income of over $ 100,000 . the large majority of claims were for less severe low back injuries ( 81% ) and less than 30% ( 28% ) of claims were involved in litigation . the length of disability ranged from 3 to 365 days , with an average length of disability of 96 days ( sd 118.5 days ) . the breakdown of claims in each of the lag time categories is presented in table 1 . for all of the lag times , the greatest percentage of claims had a 1 to 3-day lag time ( 36% reporting lag time ; 36% medical services lag time ; 57% work disability lag time ) . results of the random effects tobit model are presented in table 2 . for the reporting lag time , in comparison to the 0 days lag time category , having a 1 to 3 days lag time was related to a shorter length of disability , whereas having a lag time of 2 weeks up to 30 days , 30 days up to 60 days , or 60 days up to a year was associated with a longer length of disability . for the medical services lag time , having a lag time of 4 to 6 days , 1 week up to 2 weeks , 2 weeks up to 30 days , 30 days up to 60 days , or 60 days up to a year were all related to a longer duration of disability than having 0 days of lag time . finally , for the work disability lag time , in comparison to all other lag time categories , having 0 days of lag time was associated with a shorter length of disability . to further compare the differences in the length of disability across the different lag times , in figure 3 , we plotted the predicted values for the length of disability for each of the lag time categories . we also assessed significant differences across the lag time categories for each of the respective lag times using wald tests . note : this figure is based on the predicted values for the length of disability at each of the respective lag times . although not all of the differences among the lag time groups were statistically significant , across all three of the lag times , the trend was generally positive with the predicted length of disability increasing as the length of the lag increased . there were a few exceptions to this positive trend , specifically , for the 0 days and 1 to 3 days categories in the reporting lag time and the medical services lag time , the predicted length of disability actually decreased slightly as the length of the lag increased ( note : the decrease was only statistically significant for the reporting lag time ) . in addition , the predicted length of disability again decreased slightly going from a lag time of 30 days up to 60 days to a lag time of 60 days up to a year in the medical services and work disability lag times ( note : these decreases were not statistically significant ) . when examining the reporting lag time , there was over a 20-day difference in the predicted length of disability going from a lag time of 1 to 3 days to a lag time of 60 days up to a year . overall , there was roughly a 10-day difference or slightly more for having less than 2 weeks of lag time in reporting the injury compared with having 2 weeks or longer for the lag time . for the medical services lag time , the greatest difference was between having 1 and 3 days of lag time where the predicted length of disability was 43.6 days and having a lag time of 30 days up to 60 days where the predicted length of disability was 66.9 days . in general , having a reporting lag time of 2 weeks or longer was related to a significantly longer predicted length of disability than having a lag time of less than 2 weeks . the largest difference in the predicted length of disability across the lag time categories was for the work disability lag time . the predicted length of disability was close to 40 days less for having a work disability lag time of 0 days compared with having a lag time of 30 days or longer . in addition , the predicted length of disability increased by approximately a week to a week and a half across each of the work disability lag time categories going from the 0 days category up to the 30 days up to 60 days category . in a sample of workers who experienced work - related low back injuries , the current study examined the associations between the length of work disability and three potential delays in the work disability process including the lag times from the work injury to ( 1 ) reporting the injury to an employer , ( 2 ) receiving medical treatment , and ( 3 ) taking time off of work or initiating light duty work . understanding how these lag times are related to the length of disability can help guide health practitioners and employers in work disability case management . for the reporting lag time , our results suggested that the length of disability may be shorter when there is earlier reporting of the injury to the workers compensation insurer , as there was approximately a 10-day difference or more in the predicted length of disability when comparing having a lag time of less than 2 weeks to having a lag time of 2 weeks up to a year . however , within the first 2 weeks of the injury , the predicted length of disability was slightly higher when reporting the injury on the same day it occurred , in comparison to reporting it 1 to 3 days after it occurred , but reporting the injury in the first week was associated with a shorter predicted length of disability than reporting the injury after the first week . one possible explanation for this finding is that workers may attempt to wait to report an injury in hopes that the injury will get better on its own ; however , some workers with more severe injuries may find that the injury does not resolve and ultimately have to report the injury in order to start the workers compensation process . this would result in those workers who delay reporting an injury having more severe injuries that do not get better on their own . similarly , some workers may feel pressure from their supervisors to delay reporting an injury until they are sure that the injury is serious enough to require medical attention and time off of work , which again would result in a greater length of disability for those who delay the report of the injury . these findings for the reporting lag time are somewhat in line with a previous study of workers with low back injury , which reported that earlier injury reporting was related to a greater likelihood of having returned to work at 1 month following the lbp onset . in this previous study , all workers in the sample had reported the injury within 14 days of the pain onset and thus our findings for claimants reporting the injury after 14 days may not be comparable . our results differ from the previous findings as we found an initially higher length of disability , but that was only for a zero - day lag time we measured length of disability and the previous study measured the likelihood of work disability at 1 month after the injury . it is also possible that the differences in findings resulted from differences in the lag time categories used in the current study compared with the study by shaw et al , which measured the lag time continuously . our findings for the lag time between injury and receiving medical treatment were fairly similar to those for the reporting lag time with shorter lag times relating to a shorter length of disability . for the medical services lag time , we found a two and half week increase or more in the predicted length of disability when waiting 2 weeks or longer to receive medical treatment for a low back injury compared with receiving medical treatment within the first week of injury . also of note , the predicted length of disability was about a week shorter when receiving medical treatment in the first 3 days of injury than receiving treatment 1 week to up to 2 weeks after the injury occurred . these findings could reflect that earlier treatment for low back injuries may help to prevent acute lbp from transitioning to chronic lbp . seeking medical treatment for low back injuries in the first 2 weeks can provide workers with assurance about the course of their condition , as well as help to avoid further reinjury . it is possible that those workers who delayed seeking medical treatment for low back injuries did not take proper measures to stop further aggravation to the back injury , which may have ultimately resulted in a greater length of disability . for the medical services lag time , the findings are generally in line with previous studies of medical services lag times , which found that receiving medical treatment within 30 days of injury was associated with improved rtw outcomes . however , another previous study did not find a significant association between rtw and the lag time in receiving medical services when comparing a lag time of zero days with a lag time of 1 to 3 days or 4 days or more . although our findings did not show a difference in the predicted length of disability for a lag time of 1 to 3 days when compared with zero days , we did find differences between a zero days lag time and a lag time of 4 days or more . it is possible that the differences in findings with this previous study could result from the difference in injury types examined . our study focused specifically on low back injury , while the other study included all work - related injuries . in addition , the outcome variable used in the previous study was a dichotomous survey item indicating whether the worker was currently working , whereas we measured the length of disability continuously using retrospective claims data . for the work disability lag time , the predicted length of disability was found to increase by approximately a week to a week and half going across the different lag time categories . for example , the predicted length of disability was approximately a week longer for claimants with a 1 to 3 days lag time than those with a zero days lag time . the predicted length of disability seemed to plateau at 30 days lags time , with a little difference observed between the 30 days up to 60 days lag time group and the 60 days up to 1 year lag time group . it is possible that these findings reflect workers trying to tough it out in hopes of not having to take time off of work due to injury . however , after several days or weeks , workers may then find themselves in a situation in which the injury is too severe to remain at work . as a result , workers in this delayed group may have more severe injuries that lead to a greater length of disability . to our knowledge , no studies exist that examine the association between lag times in initiating work disability and the length of disability . our findings provide a critical first examination of this relationship and indicate that , in general , workers who initiated work disability earlier had a shorter predicted length of disability . the results of the current study suggest that occupational low back injuries should be reported to the workers compensation insurer within the first week the injury occurs . workers who reported the injury within the first 2 weeks after injury had on average a predicted length of disability over 14 days less compared with those whose injuries were reported 30 days or more after injury . with this in mind , interested parties , such as employers or insurers , may consider creating benchmarks for reporting a work - related injury no later than 2 weeks after the injury has occurred . future research may also seek to investigate how to further mitigate barriers to reporting and facilitate trouble - free reporting , as this may help to shorten the lag time in reporting an injury . for the medical services lag time , this study demonstrated that the predicted length of disability was the shortest for individuals who received medical treatment within the first 3 days of injury . approximately 10% of the sample did not receive any medical treatment during the first 2 weeks following the injury . for these individuals , the predicted length of disability was more than 2 weeks longer than those who received medical treatment within the first 3 days after injury . stakeholders might consider systems to ensure that medical care is rendered within the first 3 days after injury , and to investigate the circumstances that lead to delays in seeking treatment . on the contrary , encouraging workers to seek immediate medical care may have unintended consequences such as increasing the quantity of unnecessary tests and procedures , and ultimately delaying returning to work . as our study was correlational , we are unable to assess whether early seeking of medical treatment directly causes shorter disability duration . in addition , the medical services gap in this study focused specifically on seeking any medical treatment . for some claimants , this may have been intensive medical care , which included a large number of tests and procedures , while for others , it may have been more passive where a doctor recommended waiting a few days to see how the injury progressed . our implications must be interpreted cautiously with this in mind . with regard to the work disability lag time , surprisingly , there were approximately 14% of workers who worked for 1 month or more after injury before initiating work disability . for these workers , the predicted length of disability is more than doubled compared with those who initiated work disability the same day as the injury . an explanation for this doubling may be that a more severe reinjury or aggravation of the initial back injury could have occurred as a result of remaining at work after the injury , ultimately leading to an increase in the length of disability . alternatively , some workers who delayed taking time off of work may have tried to self - manage their lbp , but eventually left work as their symptoms failed to resolve . in this case , these workers would represent a group of cases with likely greater severity of lbp . in either case , had workers more promptly taken time off of work or started light duty after the initial injury , then the length of disability may have been shorter . taking into consideration that this study does not account for workers who received medical care but never lost a day of work , for those who are likely to go out on work disability , or who have persistent symptoms that significantly interfere with their function , they should be encouraged to not delay a transition to temporary light duty work , seeking medical care , or taking off work altogether . for disability case managers and employers , more research is needed to understand the reasons for delayed work disability for 30 days or more . it is possible that these individuals may represent unique cases in need of additional resources to fully rtw after this long initial delay . there were a number of strengths to this study , including the use of a wide range of workers compensation claims representing a variety of different industries , companies , and states . however , there are also several important limitations to note . as this study focused on the administrative data from a large , workers compensation insurer , this study does not provide any explanations as to why we observed differences in the lag times . for example , it may be assumed that individuals would seek treatment immediately after a work - related injury , but this study 's findings suggest this is not always the case , although in this study we have little information as to why there was a delay . gaining a better understanding of why there are lag times in the work disability process may help to shorten the length of disability following a work - related low back injury . along these lines , there are several other variables of interest that may confound the relationship between lag times and the length of disability that were not available in the administrative data . some of these factors include the availability of workplace accommodations , the quality of the relationship with the supervisor , the level of physical demands associated with a claimant 's occupation , and a claimant 's motivation to return to work . it is possible that if these variables were available , they may help to explain the relationship between lag times and the length of disability or possibility reduce the strength of the relationship . in addition , our measure of injury severity may be problematic for certain claims , as it could be an indicator of comorbidity . we focused only on the primary diagnosis relating to the main injury with no information about comorbid diagnoses that might impact recovery from the main injury . another limitation associated with using administrative data is that we lacked a true measure of rtw . for analytic purposes , the length of disability was calculated on the basis of indemnity payments , with the assumption that the end of indemnity payments coincided with rtw . although for many the end of indemnity payments is the result of returning to work , it is possible that in some cases , indemnity payments may have ended for a reason other than rtw . a major limitation in this study was that the analyses were limited to workers who at some point had lost work time compensated through the workers compensation system . this was done for practical reasons , as our analyses focused on the length of disability as an outcome , but it is well known that a large majority of workers compensation claims are for medical treatment only . as such , our findings must be interpreted with this in mind as they may only apply to workers who receive wage replacement for time off of work . accordingly , the implications of our findings may be biased , as we only have the length of disability for individuals who did actually experience lost work time and our recommendation of earlier reporting and seeking of medical treatment may be detrimental for individuals who do not ultimately take time off of work , as early medical treatment may result in a greater number of unnecessary services . in addition , it is possible that some workers may have used vacation days to take off work for recovery purposes in order to avoid receiving lower wages in the workers compensation system , and thus , these cases were not included in our sample . along these lines , our sample may suffer from the immortal time bias . in the administrative data , we only have information on individuals who did ultimately have a claim accepted for a given injury . we do not have information on individuals who may have had an injury but never filed a claim . it is possible that some of these individuals that never filed a claim did not do so because they recovered and returned to work relatively quickly after injury and felt that they did not need to file a claim . if this were the case , following our recommendation in all cases of encouraging early reporting of a claim and early commencement of work disability would be problematic , as it would result in more claims and more time away from work cases . it is therefore important to interpret our results with this in mind as our sample is selective to those who did file claims . thus , these recommendations may make sense only for those persons who are likely to file a disability claim . one of the major findings of our study was that the amount of time individuals waited before taking time off of work was related to the length of disability . although this was a major focus of the study , this may also present a potential limitation of our findings , as we are treating the length of disability as independent of when an injury actually occurred in relation to work disability onset . when calculating the length of disability , we utilize the date that lost work time began and the date that lost work time ended , without considering how many days there were between when the injury occurred and when lost work time started . the work disability lag time does include this information directly , but this variable could not be included in the models for the other two lag times because of collinearity concerns . moreover , we have no information about what claimants with lag times higher than zero days were doing in those days before time off work began . some of these individuals may have already been off work but not collecting benefits , others may have been using sick time , while others could have still been at work . our study focused solely on the length of disability on the basis of receiving indemnity payments for lost wages , despite the fact that individuals may have lost wages for which indemnity payments were not received . first , due to the highly skewed distributions , for analytic purposes , the three lag time variables were measured as categorical variables . it would have been more ideal to run models with these variables coded continuously in order to be more informative about how a day or a week of difference in the lag times was related to the length of disability . we attempted to select meaningful categories for our analyses , but it is possible that valuable information was lost in the process . the second issue was that the three lag times were highly collinear and needed to be analyzed in separate models . as a result , in our analyses , we did not consider the relative strength of the different lag time relationships with the length of disability . moreover , there may be combinations among the lag time categories that may further contribute to the relationship with the length of disability . overall , the current study showed that across all three of the lag times , shorter lag times are related to shorter lengths of disability . these findings suggest that in workers with occupational low back injuries that are expected to go out on work disability , the length of disability may be decreased by earlier injury reporting , earlier receipt of medical care , and earlier initiation of light duty work and/or time off work .

objective : the aim of this study is to examine the associations between lag times following occupational low back injury and the length of work disability.methods:in a retrospective cohort study using workers compensation claims , random effects tobit models were used to explore how disability length relates to three lag times : the number of days from the date of injury to reporting the injury , the number of days from the date of injury to medical care , and the number of days from the date of injury to initiating work disability.results:in general , shorter lag times for each of the different lags were related to shorter lengths of disability.conclusions:decreasing the length of the lag times in reporting injuries , receiving medical care , and missing work may help to decrease the length of work disability for workers after low back injury .

METHODS MEASURES Predictor Variables Outcome Variable Covariates ANALYSES RESULTS DISCUSSION IMPLICATIONS STRENGTHS AND LIMITATIONS CONCLUSIONS Supplementary Material

the current study utilizes data from the administrative records of a large workers compensation insurance company in the united states that included claims from a variety of different states , industries , organizations , and company sizes . all workers compensation claims with complete data for individuals who had an injury date between january 1 , 2002 , and december 31 , 2008 , were assessed for inclusion . two sets of criteria were used to identify lbp claims on the basis of the icd-9 diagnosis codes that were reported in the claimant 's medical service bills for the first 15 days of medical treatment . second , from those identified as possible lbp claims , we required that over two - thirds of the claimant 's icd-9 diagnosis codes in the diseases of the nervous system and sense organs ( 320389 ) , diseases of the musculoskeletal system and connective tissue ( 710739 ) , and injury and poisoning ( 800777 ) chapters within the first 15 days of medical treatment be for specific low back injuries or disorders ( supplemental table 1 or nonspecific back injuries or disorders ( supplemental table 2 ) . however , in certain cases , there were multiple episodes of disability for a single claim for individuals resulting from the same injury . in total the first lag time is referred to as the reporting lag time and represents the number of days from the date of injury to the date at which the injury was first reported to the workers compensation insurer . the second lag time , which we refer to as the medical services lag time , is the number of days from the date of injury to the date at which a claimant first sought medical care for that injury . the third lag time is referred to as the work disability lag time and represents the number of days from the date of injury to the date at which a claimant first took tpd or ttd . each of the three lag times were categorized into seven categories : 0 days lag ( reference group ) , 1 to 3 days lag , 4 to 6 days lag , 1 week up to 2 weeks lag , 2 weeks up to 30 days lag , 30 days up to 60 days lag , and 60 days up to 1 year lag . the outcome variable was the length of disability , which was calculated from the date that a claimant first took tpd or ttd until the date at which tpd or ttd ended . for claims in which the length of disability exceeded 1 year the following covariates were used : gender , annual income , industry , litigation status , injury severity , year of injury , age , and tenure . sixteen ordered categories were used to assess annual income : $ 0 to $ 9999 , $ 10000 to $ 19,999 , $ 20,000 to $ 29,999 , $ 30,000 to $ 39,999 , $ 40,000 to $ 49,999 , $ 50,000 to $ 59,999 , $ 60,000 to $ 69,999 , $ 70,000 to $ 79,999 , $ 80,000 to $ 89,999 , $ 90,000 to $ 99,999 , $ 100,000 to $ 109,999 , $ 110,000 to $ 119,999 , $ 120,000 to $ 129,999 , $ 130,000 to $ 139,999 , $ 140,000 to $ 149,999 , and $ 150,000 or more . since the database included claims from 2002 to 2008 , the analyses were controlled for the year of the injury . the first lag time is referred to as the reporting lag time and represents the number of days from the date of injury to the date at which the injury was first reported to the workers compensation insurer . the second lag time , which we refer to as the medical services lag time , is the number of days from the date of injury to the date at which a claimant first sought medical care for that injury . the third lag time is referred to as the work disability lag time and represents the number of days from the date of injury to the date at which a claimant first took tpd or ttd . each of the three lag times were categorized into seven categories : 0 days lag ( reference group ) , 1 to 3 days lag , 4 to 6 days lag , 1 week up to 2 weeks lag , 2 weeks up to 30 days lag , 30 days up to 60 days lag , and 60 days up to 1 year lag . the outcome variable was the length of disability , which was calculated from the date that a claimant first took tpd or ttd until the date at which tpd or ttd ended . for claims in which the length of disability exceeded 1 year , the following covariates were used : gender , annual income , industry , litigation status , injury severity , year of injury , age , and tenure . sixteen ordered categories were used to assess annual income : $ 0 to $ 9999 , $ 10000 to $ 19,999 , $ 20,000 to $ 29,999 , $ 30,000 to $ 39,999 , $ 40,000 to $ 49,999 , $ 50,000 to $ 59,999 , $ 60,000 to $ 69,999 , $ 70,000 to $ 79,999 , $ 80,000 to $ 89,999 , $ 90,000 to $ 99,999 , $ 100,000 to $ 109,999 , $ 110,000 to $ 119,999 , $ 120,000 to $ 129,999 , $ 130,000 to $ 139,999 , $ 140,000 to $ 149,999 , and $ 150,000 or more . since the database included claims from 2002 to 2008 , the analyses were controlled for the year of the injury . the relationships between the three lag times and length of disability were estimated using random effects tobit models . tobit models or censored regression models , as they are sometimes referred to , were chosen to deal with the nature of the data used in the current study wherein length of disability was restricted to a range between 3 and 365 days . separate analyses were conducted for each of the three lag times due to concerns about collinearity among the lag times . analyses were adjusted for gender , annual income , industry , litigation status , injury severity , year of injury , age , and tenure . the length of tenure at the time of injury ranged from 0 to 53 years , with an average tenure of 6 years ( sd 7.7 years ) . the length of disability ranged from 3 to 365 days , with an average length of disability of 96 days ( sd 118.5 days ) . the breakdown of claims in each of the lag time categories is presented in table 1 . for all of the lag times , the greatest percentage of claims had a 1 to 3-day lag time ( 36% reporting lag time ; 36% medical services lag time ; 57% work disability lag time ) . results of the random effects tobit model are presented in table 2 . for the reporting lag time , in comparison to the 0 days lag time category , having a 1 to 3 days lag time was related to a shorter length of disability , whereas having a lag time of 2 weeks up to 30 days , 30 days up to 60 days , or 60 days up to a year was associated with a longer length of disability . finally , for the work disability lag time , in comparison to all other lag time categories , having 0 days of lag time was associated with a shorter length of disability . to further compare the differences in the length of disability across the different lag times , in figure 3 , we plotted the predicted values for the length of disability for each of the lag time categories . we also assessed significant differences across the lag time categories for each of the respective lag times using wald tests . note : this figure is based on the predicted values for the length of disability at each of the respective lag times . although not all of the differences among the lag time groups were statistically significant , across all three of the lag times , the trend was generally positive with the predicted length of disability increasing as the length of the lag increased . there were a few exceptions to this positive trend , specifically , for the 0 days and 1 to 3 days categories in the reporting lag time and the medical services lag time , the predicted length of disability actually decreased slightly as the length of the lag increased ( note : the decrease was only statistically significant for the reporting lag time ) . in addition , the predicted length of disability again decreased slightly going from a lag time of 30 days up to 60 days to a lag time of 60 days up to a year in the medical services and work disability lag times ( note : these decreases were not statistically significant ) . overall , there was roughly a 10-day difference or slightly more for having less than 2 weeks of lag time in reporting the injury compared with having 2 weeks or longer for the lag time . for the medical services lag time , the greatest difference was between having 1 and 3 days of lag time where the predicted length of disability was 43.6 days and having a lag time of 30 days up to 60 days where the predicted length of disability was 66.9 days . in general , having a reporting lag time of 2 weeks or longer was related to a significantly longer predicted length of disability than having a lag time of less than 2 weeks . the largest difference in the predicted length of disability across the lag time categories was for the work disability lag time . the predicted length of disability was close to 40 days less for having a work disability lag time of 0 days compared with having a lag time of 30 days or longer . in addition , the predicted length of disability increased by approximately a week to a week and a half across each of the work disability lag time categories going from the 0 days category up to the 30 days up to 60 days category . in a sample of workers who experienced work - related low back injuries , the current study examined the associations between the length of work disability and three potential delays in the work disability process including the lag times from the work injury to ( 1 ) reporting the injury to an employer , ( 2 ) receiving medical treatment , and ( 3 ) taking time off of work or initiating light duty work . understanding how these lag times are related to the length of disability can help guide health practitioners and employers in work disability case management . for the reporting lag time , our results suggested that the length of disability may be shorter when there is earlier reporting of the injury to the workers compensation insurer , as there was approximately a 10-day difference or more in the predicted length of disability when comparing having a lag time of less than 2 weeks to having a lag time of 2 weeks up to a year . however , within the first 2 weeks of the injury , the predicted length of disability was slightly higher when reporting the injury on the same day it occurred , in comparison to reporting it 1 to 3 days after it occurred , but reporting the injury in the first week was associated with a shorter predicted length of disability than reporting the injury after the first week . one possible explanation for this finding is that workers may attempt to wait to report an injury in hopes that the injury will get better on its own ; however , some workers with more severe injuries may find that the injury does not resolve and ultimately have to report the injury in order to start the workers compensation process . similarly , some workers may feel pressure from their supervisors to delay reporting an injury until they are sure that the injury is serious enough to require medical attention and time off of work , which again would result in a greater length of disability for those who delay the report of the injury . these findings for the reporting lag time are somewhat in line with a previous study of workers with low back injury , which reported that earlier injury reporting was related to a greater likelihood of having returned to work at 1 month following the lbp onset . in this previous study , all workers in the sample had reported the injury within 14 days of the pain onset and thus our findings for claimants reporting the injury after 14 days may not be comparable . our results differ from the previous findings as we found an initially higher length of disability , but that was only for a zero - day lag time we measured length of disability and the previous study measured the likelihood of work disability at 1 month after the injury . our findings for the lag time between injury and receiving medical treatment were fairly similar to those for the reporting lag time with shorter lag times relating to a shorter length of disability . for the medical services lag time , we found a two and half week increase or more in the predicted length of disability when waiting 2 weeks or longer to receive medical treatment for a low back injury compared with receiving medical treatment within the first week of injury . also of note , the predicted length of disability was about a week shorter when receiving medical treatment in the first 3 days of injury than receiving treatment 1 week to up to 2 weeks after the injury occurred . these findings could reflect that earlier treatment for low back injuries may help to prevent acute lbp from transitioning to chronic lbp . it is possible that those workers who delayed seeking medical treatment for low back injuries did not take proper measures to stop further aggravation to the back injury , which may have ultimately resulted in a greater length of disability . for the medical services lag time , the findings are generally in line with previous studies of medical services lag times , which found that receiving medical treatment within 30 days of injury was associated with improved rtw outcomes . however , another previous study did not find a significant association between rtw and the lag time in receiving medical services when comparing a lag time of zero days with a lag time of 1 to 3 days or 4 days or more . although our findings did not show a difference in the predicted length of disability for a lag time of 1 to 3 days when compared with zero days , we did find differences between a zero days lag time and a lag time of 4 days or more . our study focused specifically on low back injury , while the other study included all work - related injuries . in addition , the outcome variable used in the previous study was a dichotomous survey item indicating whether the worker was currently working , whereas we measured the length of disability continuously using retrospective claims data . for the work disability lag time , the predicted length of disability was found to increase by approximately a week to a week and half going across the different lag time categories . for example , the predicted length of disability was approximately a week longer for claimants with a 1 to 3 days lag time than those with a zero days lag time . the predicted length of disability seemed to plateau at 30 days lags time , with a little difference observed between the 30 days up to 60 days lag time group and the 60 days up to 1 year lag time group . to our knowledge , no studies exist that examine the association between lag times in initiating work disability and the length of disability . our findings provide a critical first examination of this relationship and indicate that , in general , workers who initiated work disability earlier had a shorter predicted length of disability . the results of the current study suggest that occupational low back injuries should be reported to the workers compensation insurer within the first week the injury occurs . workers who reported the injury within the first 2 weeks after injury had on average a predicted length of disability over 14 days less compared with those whose injuries were reported 30 days or more after injury . future research may also seek to investigate how to further mitigate barriers to reporting and facilitate trouble - free reporting , as this may help to shorten the lag time in reporting an injury . for the medical services lag time , this study demonstrated that the predicted length of disability was the shortest for individuals who received medical treatment within the first 3 days of injury . for these individuals , the predicted length of disability was more than 2 weeks longer than those who received medical treatment within the first 3 days after injury . stakeholders might consider systems to ensure that medical care is rendered within the first 3 days after injury , and to investigate the circumstances that lead to delays in seeking treatment . for some claimants , this may have been intensive medical care , which included a large number of tests and procedures , while for others , it may have been more passive where a doctor recommended waiting a few days to see how the injury progressed . for these workers , the predicted length of disability is more than doubled compared with those who initiated work disability the same day as the injury . an explanation for this doubling may be that a more severe reinjury or aggravation of the initial back injury could have occurred as a result of remaining at work after the injury , ultimately leading to an increase in the length of disability . in either case , had workers more promptly taken time off of work or started light duty after the initial injury , then the length of disability may have been shorter . taking into consideration that this study does not account for workers who received medical care but never lost a day of work , for those who are likely to go out on work disability , or who have persistent symptoms that significantly interfere with their function , they should be encouraged to not delay a transition to temporary light duty work , seeking medical care , or taking off work altogether . there were a number of strengths to this study , including the use of a wide range of workers compensation claims representing a variety of different industries , companies , and states . as this study focused on the administrative data from a large , workers compensation insurer , this study does not provide any explanations as to why we observed differences in the lag times . gaining a better understanding of why there are lag times in the work disability process may help to shorten the length of disability following a work - related low back injury . along these lines , there are several other variables of interest that may confound the relationship between lag times and the length of disability that were not available in the administrative data . some of these factors include the availability of workplace accommodations , the quality of the relationship with the supervisor , the level of physical demands associated with a claimant 's occupation , and a claimant 's motivation to return to work . it is possible that if these variables were available , they may help to explain the relationship between lag times and the length of disability or possibility reduce the strength of the relationship . for analytic purposes , the length of disability was calculated on the basis of indemnity payments , with the assumption that the end of indemnity payments coincided with rtw . this was done for practical reasons , as our analyses focused on the length of disability as an outcome , but it is well known that a large majority of workers compensation claims are for medical treatment only . accordingly , the implications of our findings may be biased , as we only have the length of disability for individuals who did actually experience lost work time and our recommendation of earlier reporting and seeking of medical treatment may be detrimental for individuals who do not ultimately take time off of work , as early medical treatment may result in a greater number of unnecessary services . one of the major findings of our study was that the amount of time individuals waited before taking time off of work was related to the length of disability . although this was a major focus of the study , this may also present a potential limitation of our findings , as we are treating the length of disability as independent of when an injury actually occurred in relation to work disability onset . when calculating the length of disability , we utilize the date that lost work time began and the date that lost work time ended , without considering how many days there were between when the injury occurred and when lost work time started . our study focused solely on the length of disability on the basis of receiving indemnity payments for lost wages , despite the fact that individuals may have lost wages for which indemnity payments were not received . it would have been more ideal to run models with these variables coded continuously in order to be more informative about how a day or a week of difference in the lag times was related to the length of disability . as a result , in our analyses , we did not consider the relative strength of the different lag time relationships with the length of disability . moreover , there may be combinations among the lag time categories that may further contribute to the relationship with the length of disability . overall , the current study showed that across all three of the lag times , shorter lag times are related to shorter lengths of disability . these findings suggest that in workers with occupational low back injuries that are expected to go out on work disability , the length of disability may be decreased by earlier injury reporting , earlier receipt of medical care , and earlier initiation of light duty work and/or time off work .

lost work time comprised both days of temporary total disability ( ttd ) and days of temporary partial disability ( tpd ) . two sets of criteria were used to identify lbp claims on the basis of the icd-9 diagnosis codes that were reported in the claimant 's medical service bills for the first 15 days of medical treatment . medical treatment typically began within 2 weeks of the injury date ( 90% of claims ) , although all claimants who received medical treatment within 1 year of the injury date and who had lost work time were included in the database . first , 99,127 claims were classified as a possible lbp claim based on having at least one icd-9 diagnosis code within the first 15 days of medical treatment relating to specific low back injuries or disorders , which are listed in supplemental table 1 . second , from those identified as possible lbp claims , we required that over two - thirds of the claimant 's icd-9 diagnosis codes in the diseases of the nervous system and sense organs ( 320389 ) , diseases of the musculoskeletal system and connective tissue ( 710739 ) , and injury and poisoning ( 800777 ) chapters within the first 15 days of medical treatment be for specific low back injuries or disorders ( supplemental table 1 or nonspecific back injuries or disorders ( supplemental table 2 ) . however , in certain cases , there were multiple episodes of disability for a single claim for individuals resulting from the same injury . in this study , we only included claims for individuals in whom multiple episodes of disability within the same claim had less than 14 days between episodes , in which case we considered this a single episode . in total the first lag time is referred to as the reporting lag time and represents the number of days from the date of injury to the date at which the injury was first reported to the workers compensation insurer . the second lag time , which we refer to as the medical services lag time , is the number of days from the date of injury to the date at which a claimant first sought medical care for that injury . the third lag time is referred to as the work disability lag time and represents the number of days from the date of injury to the date at which a claimant first took tpd or ttd . each of the three lag times were categorized into seven categories : 0 days lag ( reference group ) , 1 to 3 days lag , 4 to 6 days lag , 1 week up to 2 weeks lag , 2 weeks up to 30 days lag , 30 days up to 60 days lag , and 60 days up to 1 year lag . the outcome variable was the length of disability , which was calculated from the date that a claimant first took tpd or ttd until the date at which tpd or ttd ended . for claims in which the length of disability exceeded 1 year the following covariates were used : gender , annual income , industry , litigation status , injury severity , year of injury , age , and tenure . sixteen ordered categories were used to assess annual income : $ 0 to $ 9999 , $ 10000 to $ 19,999 , $ 20,000 to $ 29,999 , $ 30,000 to $ 39,999 , $ 40,000 to $ 49,999 , $ 50,000 to $ 59,999 , $ 60,000 to $ 69,999 , $ 70,000 to $ 79,999 , $ 80,000 to $ 89,999 , $ 90,000 to $ 99,999 , $ 100,000 to $ 109,999 , $ 110,000 to $ 119,999 , $ 120,000 to $ 129,999 , $ 130,000 to $ 139,999 , $ 140,000 to $ 149,999 , and $ 150,000 or more . industry was categorized into 10 groups , including agriculture , forestry and fishing , construction , finance and insurance , manufacturing , mining , retail trade , services , transportation , public administration , and wholesale trade . injury severity was coded 1 for having at least one more severe diagnosis within the first 15 days of medical treatment and 0 for having only less severe diagnoses within the first 15 days of medical treatment . in addition , efforts were made to exclude cases of complicated back pain , such as those with diagnoses that were consistent with experiencing multiple work - related injuries or disorders , very severe injuries , or back pain due to cancer , infection , severe trauma , or an autoimmune disorder . the first lag time is referred to as the reporting lag time and represents the number of days from the date of injury to the date at which the injury was first reported to the workers compensation insurer . the second lag time , which we refer to as the medical services lag time , is the number of days from the date of injury to the date at which a claimant first sought medical care for that injury . the third lag time is referred to as the work disability lag time and represents the number of days from the date of injury to the date at which a claimant first took tpd or ttd . each of the three lag times were categorized into seven categories : 0 days lag ( reference group ) , 1 to 3 days lag , 4 to 6 days lag , 1 week up to 2 weeks lag , 2 weeks up to 30 days lag , 30 days up to 60 days lag , and 60 days up to 1 year lag . the outcome variable was the length of disability , which was calculated from the date that a claimant first took tpd or ttd until the date at which tpd or ttd ended . for claims in which the length of disability exceeded 1 year , the following covariates were used : gender , annual income , industry , litigation status , injury severity , year of injury , age , and tenure . sixteen ordered categories were used to assess annual income : $ 0 to $ 9999 , $ 10000 to $ 19,999 , $ 20,000 to $ 29,999 , $ 30,000 to $ 39,999 , $ 40,000 to $ 49,999 , $ 50,000 to $ 59,999 , $ 60,000 to $ 69,999 , $ 70,000 to $ 79,999 , $ 80,000 to $ 89,999 , $ 90,000 to $ 99,999 , $ 100,000 to $ 109,999 , $ 110,000 to $ 119,999 , $ 120,000 to $ 129,999 , $ 130,000 to $ 139,999 , $ 140,000 to $ 149,999 , and $ 150,000 or more . industry was categorized into 10 groups , including agriculture , forestry and fishing , construction , finance and insurance , manufacturing , mining , retail trade , services , transportation , public administration , and wholesale trade . injury severity was coded 1 for having at least one more severe diagnosis within the first 15 days of medical treatment and 0 for having only less severe diagnoses within the first 15 days of medical treatment . in addition , efforts were made to exclude cases of complicated back pain , such as those with diagnoses that were consistent with experiencing multiple work - related injuries or disorders , very severe injuries , or back pain due to cancer , infection , severe trauma , or an autoimmune disorder . we were only interested in following claimants for 1 year from the time of disability onset ; however , for some claimants , disability may not have ended by 365 days and it is necessary for our models to take this into account . tobit models or censored regression models , as they are sometimes referred to , were chosen to deal with the nature of the data used in the current study wherein length of disability was restricted to a range between 3 and 365 days . in order to take into account the non - independence of observations across industry groupings , participants drawn from different industry groups may be thought of as representing subsamples within the larger sample . in addition to the main analyses , differences in the coefficients for the non - reference group lag time categories were assessed using wald tests . in the analyses , on the basis of this , we used a more conservative p value of less than 0.001 to represent statistical significance . the age of claimants at the time of injury ranged from 18 to 80 years , with an average age of 40 years ( sd 11.2 years ) . the length of tenure at the time of injury ranged from 0 to 53 years , with an average tenure of 6 years ( sd 7.7 years ) . for all of the lag times , the greatest percentage of claims had a 1 to 3-day lag time ( 36% reporting lag time ; 36% medical services lag time ; 57% work disability lag time ) . for the reporting lag time , in comparison to the 0 days lag time category , having a 1 to 3 days lag time was related to a shorter length of disability , whereas having a lag time of 2 weeks up to 30 days , 30 days up to 60 days , or 60 days up to a year was associated with a longer length of disability . for the medical services lag time , having a lag time of 4 to 6 days , 1 week up to 2 weeks , 2 weeks up to 30 days , 30 days up to 60 days , or 60 days up to a year were all related to a longer duration of disability than having 0 days of lag time . finally , for the work disability lag time , in comparison to all other lag time categories , having 0 days of lag time was associated with a shorter length of disability . to further compare the differences in the length of disability across the different lag times , in figure 3 , we plotted the predicted values for the length of disability for each of the lag time categories . although not all of the differences among the lag time groups were statistically significant , across all three of the lag times , the trend was generally positive with the predicted length of disability increasing as the length of the lag increased . there were a few exceptions to this positive trend , specifically , for the 0 days and 1 to 3 days categories in the reporting lag time and the medical services lag time , the predicted length of disability actually decreased slightly as the length of the lag increased ( note : the decrease was only statistically significant for the reporting lag time ) . in addition , the predicted length of disability again decreased slightly going from a lag time of 30 days up to 60 days to a lag time of 60 days up to a year in the medical services and work disability lag times ( note : these decreases were not statistically significant ) . when examining the reporting lag time , there was over a 20-day difference in the predicted length of disability going from a lag time of 1 to 3 days to a lag time of 60 days up to a year . for the medical services lag time , the greatest difference was between having 1 and 3 days of lag time where the predicted length of disability was 43.6 days and having a lag time of 30 days up to 60 days where the predicted length of disability was 66.9 days . in general , having a reporting lag time of 2 weeks or longer was related to a significantly longer predicted length of disability than having a lag time of less than 2 weeks . in addition , the predicted length of disability increased by approximately a week to a week and a half across each of the work disability lag time categories going from the 0 days category up to the 30 days up to 60 days category . in a sample of workers who experienced work - related low back injuries , the current study examined the associations between the length of work disability and three potential delays in the work disability process including the lag times from the work injury to ( 1 ) reporting the injury to an employer , ( 2 ) receiving medical treatment , and ( 3 ) taking time off of work or initiating light duty work . for the reporting lag time , our results suggested that the length of disability may be shorter when there is earlier reporting of the injury to the workers compensation insurer , as there was approximately a 10-day difference or more in the predicted length of disability when comparing having a lag time of less than 2 weeks to having a lag time of 2 weeks up to a year . however , within the first 2 weeks of the injury , the predicted length of disability was slightly higher when reporting the injury on the same day it occurred , in comparison to reporting it 1 to 3 days after it occurred , but reporting the injury in the first week was associated with a shorter predicted length of disability than reporting the injury after the first week . one possible explanation for this finding is that workers may attempt to wait to report an injury in hopes that the injury will get better on its own ; however , some workers with more severe injuries may find that the injury does not resolve and ultimately have to report the injury in order to start the workers compensation process . similarly , some workers may feel pressure from their supervisors to delay reporting an injury until they are sure that the injury is serious enough to require medical attention and time off of work , which again would result in a greater length of disability for those who delay the report of the injury . these findings for the reporting lag time are somewhat in line with a previous study of workers with low back injury , which reported that earlier injury reporting was related to a greater likelihood of having returned to work at 1 month following the lbp onset . in this previous study , all workers in the sample had reported the injury within 14 days of the pain onset and thus our findings for claimants reporting the injury after 14 days may not be comparable . our results differ from the previous findings as we found an initially higher length of disability , but that was only for a zero - day lag time we measured length of disability and the previous study measured the likelihood of work disability at 1 month after the injury . it is also possible that the differences in findings resulted from differences in the lag time categories used in the current study compared with the study by shaw et al , which measured the lag time continuously . our findings for the lag time between injury and receiving medical treatment were fairly similar to those for the reporting lag time with shorter lag times relating to a shorter length of disability . for the medical services lag time , we found a two and half week increase or more in the predicted length of disability when waiting 2 weeks or longer to receive medical treatment for a low back injury compared with receiving medical treatment within the first week of injury . also of note , the predicted length of disability was about a week shorter when receiving medical treatment in the first 3 days of injury than receiving treatment 1 week to up to 2 weeks after the injury occurred . seeking medical treatment for low back injuries in the first 2 weeks can provide workers with assurance about the course of their condition , as well as help to avoid further reinjury . for the medical services lag time , the findings are generally in line with previous studies of medical services lag times , which found that receiving medical treatment within 30 days of injury was associated with improved rtw outcomes . however , another previous study did not find a significant association between rtw and the lag time in receiving medical services when comparing a lag time of zero days with a lag time of 1 to 3 days or 4 days or more . although our findings did not show a difference in the predicted length of disability for a lag time of 1 to 3 days when compared with zero days , we did find differences between a zero days lag time and a lag time of 4 days or more . in addition , the outcome variable used in the previous study was a dichotomous survey item indicating whether the worker was currently working , whereas we measured the length of disability continuously using retrospective claims data . for the work disability lag time , the predicted length of disability was found to increase by approximately a week to a week and half going across the different lag time categories . the predicted length of disability seemed to plateau at 30 days lags time , with a little difference observed between the 30 days up to 60 days lag time group and the 60 days up to 1 year lag time group . to our knowledge , no studies exist that examine the association between lag times in initiating work disability and the length of disability . our findings provide a critical first examination of this relationship and indicate that , in general , workers who initiated work disability earlier had a shorter predicted length of disability . the results of the current study suggest that occupational low back injuries should be reported to the workers compensation insurer within the first week the injury occurs . workers who reported the injury within the first 2 weeks after injury had on average a predicted length of disability over 14 days less compared with those whose injuries were reported 30 days or more after injury . for the medical services lag time , this study demonstrated that the predicted length of disability was the shortest for individuals who received medical treatment within the first 3 days of injury . with regard to the work disability lag time , surprisingly , there were approximately 14% of workers who worked for 1 month or more after injury before initiating work disability . an explanation for this doubling may be that a more severe reinjury or aggravation of the initial back injury could have occurred as a result of remaining at work after the injury , ultimately leading to an increase in the length of disability . taking into consideration that this study does not account for workers who received medical care but never lost a day of work , for those who are likely to go out on work disability , or who have persistent symptoms that significantly interfere with their function , they should be encouraged to not delay a transition to temporary light duty work , seeking medical care , or taking off work altogether . there were a number of strengths to this study , including the use of a wide range of workers compensation claims representing a variety of different industries , companies , and states . as this study focused on the administrative data from a large , workers compensation insurer , this study does not provide any explanations as to why we observed differences in the lag times . for example , it may be assumed that individuals would seek treatment immediately after a work - related injury , but this study 's findings suggest this is not always the case , although in this study we have little information as to why there was a delay . gaining a better understanding of why there are lag times in the work disability process may help to shorten the length of disability following a work - related low back injury . some of these factors include the availability of workplace accommodations , the quality of the relationship with the supervisor , the level of physical demands associated with a claimant 's occupation , and a claimant 's motivation to return to work . we focused only on the primary diagnosis relating to the main injury with no information about comorbid diagnoses that might impact recovery from the main injury . for analytic purposes , the length of disability was calculated on the basis of indemnity payments , with the assumption that the end of indemnity payments coincided with rtw . accordingly , the implications of our findings may be biased , as we only have the length of disability for individuals who did actually experience lost work time and our recommendation of earlier reporting and seeking of medical treatment may be detrimental for individuals who do not ultimately take time off of work , as early medical treatment may result in a greater number of unnecessary services . if this were the case , following our recommendation in all cases of encouraging early reporting of a claim and early commencement of work disability would be problematic , as it would result in more claims and more time away from work cases . one of the major findings of our study was that the amount of time individuals waited before taking time off of work was related to the length of disability . although this was a major focus of the study , this may also present a potential limitation of our findings , as we are treating the length of disability as independent of when an injury actually occurred in relation to work disability onset . when calculating the length of disability , we utilize the date that lost work time began and the date that lost work time ended , without considering how many days there were between when the injury occurred and when lost work time started . our study focused solely on the length of disability on the basis of receiving indemnity payments for lost wages , despite the fact that individuals may have lost wages for which indemnity payments were not received . these findings suggest that in workers with occupational low back injuries that are expected to go out on work disability , the length of disability may be decreased by earlier injury reporting , earlier receipt of medical care , and earlier initiation of light duty work and/or time off work .

the lifetime risk of end - stage kidney disease ( eskd ) among african americans is threefold higher than that among whites . differences in access to care and other socioeconomic factors do not entirely account for the markedly higher rates of eskd among african americans which suggests a possible role for genetic factors [ 16 ] . recently , a region of chromosome 22 , which includes apol1 and myh9 genes , was identified using mapping by admixture disequilibrium as a risk locus for non - diabetic forms of kidney disease , including idiopathic and hiv - associated focal segmental glomerular sclerosis ( fsgs ) and kidney disease clinically attributed to hypertension [ 79 ] . genetic variants in the region show very strong associations with non - diabetic kidney disease . specifically , a two - allele haplotype termed g1 consisting of two non - synonymous coding variants rs73885319 ( s342 g ) and rs60910145 ( i384 m ) along with rs71785313a 6 base pair deletion termed g2 and close to g1 in exon 5 of apol1 [ 10 , 11]is likely to account for the majority of risk of non - diabetic kidney disease associated with the variants in this region , but the role of myh9 variants in non - diabetic kidney disease risk remains controversial . regardless , variants in the region are more common in individuals of african descent compared with those of european descent [ 10 , 11 ] . the fact that these variants are absent or less common in other populations has been used to explain the non - replication of these associations in non - african descent populations . the purposes of the current study were , therefore , to examine the frequency of these variants and to determine whether they are associated with ckd among native africans . the source population for both cases and controls was participants in the genetics of hypertension in blacks study , an ongoing project examining genetic variants for blood pressure among adults from the yoruba tribe . participants in the current analyses included 88 and 81 adults with and without non - diabetic kidney disease , respectively . participants were recruited from the university college hospital general medicine and nephrology clinics , university of ibadan , nigeria . the project was reviewed and approved by the institutional review board at the loyola university chicago stritch school of medicine , maywood , il and the joint ethical committee of the university of ibadan / university college hospital , ibadan , nigeria . the consent process was presented in english or yoruba , and written informed consent was obtained from all participants . cases were defined as individuals of the yoruba tribe aged 1670 years with all stages of chronic kidney disease ( ckd ) of at least 3 months duration who met criteria for non - diabetic ckd clinically associated with long - standing hypertension , or ckd in the presence of proteinuria ( proteinuric ckd ) or ckd associated with hiv . proteinuric ckd was defined as presence of spot urine protein / creatinine 2 g / g in the absence of red blood cell ( rbc ) casts or hematuria on urine microscopy or urinalysis and absence of known causes for ckd such as parasitic or other infections or diabetes . clinically diagnosed hypertension - associated ckd was based on history or clinical evidence of long - standing hypertension ( e.g. , electrocardiogram evidence of left ventricular hypertrophy ) and a spot urine protein / creatinine ratio < 2 g / g , in the absence of other known causes for ckd , consistent with the african american study of kidney disease criteria . ckd associated with hiv was defined as a positive hiv test and absence of other known causes for ckd including diabetes and parasitic diseases . women who were pregnant , persons with diabetes mellitus , sickle cell disease , hepatitis b or c , acute kidney injury , or other terminal illnesses such as cancer were excluded . the clinical exam and laboratory studies included three serial blood pressure measurements using an automated device ( omron hem-412c ) previously evaluated in our field settings , anthropometric measures , a complete blood count and metabolic panel , electrocardiogram ( ecg ) , testing for hiv and hepatitis b , urinalysis with microscopy and a bilateral kidney ultrasound when possible . all laboratory analyses in cases were completed at the university college hospital laboratory as part of routine clinical workup . controls consisted of normotensive individuals ( bp < 140/90 in the absence of anti - hypertensive medication use ) of the yoruba tribe without evidence of kidney disease ( serum creatinine < 1.4 and < 1.2 mg / dl in men and women , respectively , and spot urine albumin / creatinine ratio < 30 mg / g ) . all controls had standardized physical exams including three serial blood pressure measurements using the same device as used for the cases ; fasting blood samples and a spot urine specimen were also obtained . serum creatinine was measured in the control specimens at fairview laboratory in minnesota by rate reflectance spectrophotometry using thin film adaptation of the creatinine amidinohydrolase method on the vitros analyzer ( johnson and johnson clinical diagnostics , rochester , ny ) . genotyping was carried out on genomic dna from 88 non - diabetic ckd subjects and 81 non - ckd subjects randomly selected from among the controls described above . the genotyping was performed at the charles r. bronfman institute for personalized medicine ( mount sinai school of medicine , new york , ny ) , using a custom fluidigm 96.96 array platform and abi taqman snp genotyping assays . the assays were originally selected for 96 published disease - associated single nucleotide polymorphisms ( snps ) and included 4 snps on apol1 and 6 snps on myh9 genes which have been reported to be associated with kidney disease . the assays also included variants associated with liver disease , type 2 diabetes or drug response . standard quality control procedures were applied to the genotype data using all snps on the chip . as part of the procedures , 3 samples ( 1 case and 2 controls ) and 2 snps with proportion of missing genotypes greater than 0.1 were filtered out . we also filtered out snps with minor allele frequency less than 0.05 ( n = 23 ) or failing hardy weinberg equilibrium test at 0.01 ( n = 3 ) . the final 68 snps that passed quality control included 9 of the 10 apol1/myh9 variants . since the objective of the current analysis was to examine the association between previously reported kidney disease associated snps and ckd in a sample of nigerians , subsequent screening for associations with ckd was therefore restricted to just the variants on apol1 ( rs9622363 , rs73885319 , rs60910145 and rs71785313 ) and myh9 ( rs11912763 , rs2032487 , rs4821481 , rs5750248 and rs5750250 ) genes . to test for associations between ckd status and each single snp , we fitted logistic regression models in which each snp was presented as a predictor variable whose values were equal to the number of copies of the minor allele ( 0 , 1 , 2 ) ( i.e. , additive mode ) , or presence of at least one copy of the minor allele ( 0 , 1 ) ( i.e. , dominant mode ) or presence of two copies of the minor allele ( 0 , 1 ) ( i.e. , recessive mode ) . the fitted model , which included sex and age as covariates , can be represented as : logit[pr(d = 1 ) ] = + 1 g + 2sex + 3age , where d denotes ckd affection status ; g denotes snp coded as additive , dominant or recessive ; denotes the corresponding coefficient for each variable in the model ( snp , sex or age ) , and its exponential is the corresponding odds ratio . to account for multiple comparisons , the statistical significance of each association test was empirically derived by permuting the data set 10,000 times . to explore the possible associations between ckd status and various joint allelic configurations of multiple snps , we performed haplotype association analysis of the apol1 and myh9 snps . we used the software haploview to compute the estimates of linkage disequilibrium ( ld ) for each pair of snps by the standard d - prime method and to determine the haplotype blocks regions with no evidence of historical recombination events , but significant level of ld . the haplotype blocks were defined by the four - gamete test [ 16 , 17 ] as implemented in haploview . analysis included all haplotypes with frequencies of at least 10 % within the constructed blocks and also the two - allele haplotypes consisting of rs73885319 and rs60910145 that included g1 . all the haplotypes were individually tested for association with ckd by using logistic regression models as described above for tests of genotype association . the source population for both cases and controls was participants in the genetics of hypertension in blacks study , an ongoing project examining genetic variants for blood pressure among adults from the yoruba tribe . participants in the current analyses included 88 and 81 adults with and without non - diabetic kidney disease , respectively . participants were recruited from the university college hospital general medicine and nephrology clinics , university of ibadan , nigeria . the project was reviewed and approved by the institutional review board at the loyola university chicago stritch school of medicine , maywood , il and the joint ethical committee of the university of ibadan / university college hospital , ibadan , nigeria . the consent process was presented in english or yoruba , and written informed consent was obtained from all participants . cases were defined as individuals of the yoruba tribe aged 1670 years with all stages of chronic kidney disease ( ckd ) of at least 3 months duration who met criteria for non - diabetic ckd clinically associated with long - standing hypertension , or ckd in the presence of proteinuria ( proteinuric ckd ) or ckd associated with hiv . proteinuric ckd was defined as presence of spot urine protein / creatinine 2 g / g in the absence of red blood cell ( rbc ) casts or hematuria on urine microscopy or urinalysis and absence of known causes for ckd such as parasitic or other infections or diabetes . clinically diagnosed hypertension - associated ckd was based on history or clinical evidence of long - standing hypertension ( e.g. , electrocardiogram evidence of left ventricular hypertrophy ) and a spot urine protein / creatinine ratio < 2 g / g , in the absence of other known causes for ckd , consistent with the african american study of kidney disease criteria . ckd associated with hiv was defined as a positive hiv test and absence of other known causes for ckd including diabetes and parasitic diseases . women who were pregnant , persons with diabetes mellitus , sickle cell disease , hepatitis b or c , acute kidney injury , or other terminal illnesses such as cancer were excluded . the clinical exam and laboratory studies included three serial blood pressure measurements using an automated device ( omron hem-412c ) previously evaluated in our field settings , anthropometric measures , a complete blood count and metabolic panel , electrocardiogram ( ecg ) , testing for hiv and hepatitis b , urinalysis with microscopy and a bilateral kidney ultrasound when possible . all laboratory analyses in cases were completed at the university college hospital laboratory as part of routine clinical workup . controls consisted of normotensive individuals ( bp < 140/90 in the absence of anti - hypertensive medication use ) of the yoruba tribe without evidence of kidney disease ( serum creatinine < 1.4 and < 1.2 mg / dl in men and women , respectively , and spot urine albumin / creatinine ratio < 30 mg / g ) . all controls had standardized physical exams including three serial blood pressure measurements using the same device as used for the cases ; fasting blood samples and a spot urine specimen were also obtained . serum creatinine was measured in the control specimens at fairview laboratory in minnesota by rate reflectance spectrophotometry using thin film adaptation of the creatinine amidinohydrolase method on the vitros analyzer ( johnson and johnson clinical diagnostics , rochester , ny ) . genotyping was carried out on genomic dna from 88 non - diabetic ckd subjects and 81 non - ckd subjects randomly selected from among the controls described above . the genotyping was performed at the charles r. bronfman institute for personalized medicine ( mount sinai school of medicine , new york , ny ) , using a custom fluidigm 96.96 array platform and abi taqman snp genotyping assays . the assays were originally selected for 96 published disease - associated single nucleotide polymorphisms ( snps ) and included 4 snps on apol1 and 6 snps on myh9 genes which have been reported to be associated with kidney disease . the assays also included variants associated with liver disease , type 2 diabetes or drug response . standard quality control procedures were applied to the genotype data using all snps on the chip . as part of the procedures , 3 samples ( 1 case and 2 controls ) and 2 snps with proportion of missing genotypes greater than 0.1 were filtered out . we also filtered out snps with minor allele frequency less than 0.05 ( n = 23 ) or failing hardy weinberg equilibrium test at 0.01 ( n = 3 ) . the final 68 snps that passed quality control included 9 of the 10 apol1/myh9 variants . since the objective of the current analysis was to examine the association between previously reported kidney disease associated snps and ckd in a sample of nigerians , subsequent screening for associations with ckd was therefore restricted to just the variants on apol1 ( rs9622363 , rs73885319 , rs60910145 and rs71785313 ) and myh9 ( rs11912763 , rs2032487 , rs4821481 , rs5750248 and rs5750250 ) genes . to test for associations between ckd status and each single snp , we fitted logistic regression models in which each snp was presented as a predictor variable whose values were equal to the number of copies of the minor allele ( 0 , 1 , 2 ) ( i.e. , additive mode ) , or presence of at least one copy of the minor allele ( 0 , 1 ) ( i.e. , dominant mode ) or presence of two copies of the minor allele ( 0 , 1 ) ( i.e. , recessive mode ) . the fitted model , which included sex and age as covariates , can be represented as : logit[pr(d = 1 ) ] = + 1 g + 2sex + 3age , where d denotes ckd affection status ; g denotes snp coded as additive , dominant or recessive ; denotes the corresponding coefficient for each variable in the model ( snp , sex or age ) , and its exponential is the corresponding odds ratio . to account for multiple comparisons , the statistical significance of each association test was empirically derived by permuting the data set 10,000 times . to explore the possible associations between ckd status and various joint allelic configurations of multiple snps , we performed haplotype association analysis of the apol1 and myh9 snps . we used the software haploview to compute the estimates of linkage disequilibrium ( ld ) for each pair of snps by the standard d - prime method and to determine the haplotype blocks regions with no evidence of historical recombination events , but significant level of ld . the haplotype blocks were defined by the four - gamete test [ 16 , 17 ] as implemented in haploview . analysis included all haplotypes with frequencies of at least 10 % within the constructed blocks and also the two - allele haplotypes consisting of rs73885319 and rs60910145 that included g1 . all the haplotypes were individually tested for association with ckd by using logistic regression models as described above for tests of genotype association . after excluding participants whose genotype data did not pass quality control , the study sample consisted of a total of 166 subjects ( 87 cases and 79 controls ) . the descriptive characteristics of the cases and controls are presented in table 1 . among both cases and controls , , the ckd subjects tended to be older ( 42.1 vs. 35.2 years ) , heavier ( 23.2 vs. 21.9 kg / m ) and have higher blood pressure ( systolic : 136.6 vs. 111.6 mm hg ) when compared with the non - ckd controls . among the 87 ckd subjects , the physician - reported diagnosis of kidney disease was proteinuric ckd in 35 ( 40.2 % ) , hiv nephropathy in 8 ( 9.2 % ) and clinically attributed hypertensive ckd in 44 ( 50.5 % ) . all the ckd subjects had a negative hepatitis b surface antigen test within 30 days of enrollment in the study . all but 3 cases had stage 5 ckd ( 1 with proteinuric ckd and 2 with ckd clinically attributed to hypertension.table 1descriptive characteristics of study subjectsnon - diabetic ckd cases ( n = 87)controls ( n = 79)all ( n = 166)no . of females ( % ) 41 ( 47 % ) 39 ( 49 % ) 80 ( 48 % ) age ( years ) 42.1 16.935.2 8.238.8 13.9body weight ( kg)61.6 11.159.8 10.860.8 11.0height ( m)1.6 0.11.7 0.11.6 0.1body mass index ( kg / m)23.2 4.621.9 4.222.6 4.4systolic blood pressure ( mm hg ) 136.6 31.0111.6 10.0124.7 26.6diastolic blood pressure ( mm hg ) 88.1 20.766.9 6.178.0 18.8 ckd chronic kidney diseasedata presented as mean standard deviation values are significantly different ( p < 0.05 ) between cases and controls descriptive characteristics of study subjects ckd chronic kidney disease data presented as mean standard deviation values are significantly different ( p < 0.05 ) between cases and controls the frequencies of the risk allele of the 9 apol1/myh9 variants among the cases and controls are listed in table 2 . in the current analyses , the coded alleles are the same as the minor alleles of the variants . for any variant with reported odds ratio ( or ) greater than 1.0 , the coded minor allele is also the risk or disease susceptibility allele . as would be expected , coded risk alleles tended to be more frequent in cases than in controls , for example , 0.442 versus 0.266 and 0.500 versus 0.301 for rs73885319 and rs60910145 , respectively . the variant rs71785313 , which is an apol1 insertion / deletion , had the least frequent minor allele ( 0.105 ) in both cases and controls . results of the covariate - adjusted case / control analyses are also presented in table 2 for additive , dominant and recessive genetic modes of association . we observed significant associations for two apol1 snps rs73885319 and rs60910145 under all three genetic modes of association . both variants have larger effects under the recessive mode ( odds ratios : 3.85 and 3.12 for rs73885319 and rs60910145 , respectively ) when compared with the additive or dominant mode . the two variants are in almost perfect linkage disequilibrium ( d - prime = 1.00 , r = 0.82 ) and as such , when adjusted for either one , the association for the other disappeared.table 2snp associations with non - diabetic chronic kidney disease in 87 cases and 79 controlssnpgeneallelescoded ( minor ) allele ( frequency , % ) hwe p valuecoded allele frequency ( % ) association modeor ( 95 % ci ) p valuecasescontrolsunadjustedcorrected rs9622363 apol1 a / ga ( 27.71)0.78825.8629.75additive0.76 ( 0.451.31)0.3260.875dominant0.88 ( 0.471.66)0.6950.999recessive0.24 ( 0.051.29)0.0970.377rs73885319 apol1 a / ga ( 35.76)1.00044.1926.58additive2.29 ( 1.393.77)0.0010.005dominant2.59 ( 1.345.00)0.0050.025recessive3.85 ( 1.3111.36)0.0150.038rs60910145 apol1 g / tg ( 40.61)0.11450.0030.13additive2.04 ( 1.323.17)0.0010.006dominant2.54 ( 1.314.92)0.0060.034recessive3.12 ( 1.357.20)0.0080.015 g2 : rs71785313 apol1 d / id ( 10.54)1.0008.6212.66additive0.61 ( 0.291.31)0.2070.701dominant0.64 ( 0.291.40)0.2630.816recessiveneneners11912763 myh9 a / ga ( 33.13)1.00038.5127.22additive1.68 ( 1.022.76)0.0400.197dominant2.03 ( 1.063.87)0.0320.183recessive1.70 ( 0.584.94)0.3340.872rs2032487 myh9 t / ct ( 22.12)0.77018.3926.28additive0.68 ( 0.401.16)0.1570.580dominant0.64 ( 0.331.23)0.1770.645recessive0.55 ( 0.142.22)0.4000.934rs4821481 myh9 t / ct ( 22.29)0.77718.3926.58additive0.66 ( 0.391.13)0.1320.532dominant0.61 ( 0.321.18)0.1430.583recessive0.55 ( 0.142.24)0.4070.940rs5750248 myh9 c / tc ( 30.72)1.00025.8636.08additive0.61 ( 0.370.99)0.0470.225dominant0.56 ( 0.291.05)0.0710.354recessive0.46 ( 0.151.41)0.1760.627rs5750250 myh9 a / ga ( 31.82)0.63526.1637.97additive0.56 ( 0.340.94)0.0270.141dominant0.51 ( 0.270.97)0.0400.208recessive0.44 ( 0.141.38)0.1570.576adjusted for age and gender snps single nucleotide polymorphisms , hwe hardy weinberg equilibrium , or odds ratio , ci confidence interval , ne not estimated p values corrected for multiple comparisons snp associations with non - diabetic chronic kidney disease in 87 cases and 79 controls adjusted for age and gender snps single nucleotide polymorphisms , hwe hardy weinberg equilibrium , or odds ratio , ci confidence interval , ne not estimated p values corrected for multiple comparisons the haplotype blocks formed by the apol1/myh9 variants are displayed in fig . 1 . there were two blocks consisting of three apol1 snps and four myh9 snps , respectively . two snps , rs71785313 on apol1 and rs11912763 on myh9 , were not included in any of the blocks . the apol1 block thus included rs9622363 , rs73885319 and rs60910145 , and the myh9 block included rs2032487 , rs4821481 , rs5750248 and rs5750250 . there were a total of five haplotypes each with a frequency of at least 0.10 ( fig . 1 ) . the results of the covariate - adjusted haplotype associations are presented in table 3 . we also present the results for the two - allele haplotypes of rs73885319 and rs60910145 that included the previously reported g1 haplotype . g haplotype of the apol1 snps ( rs9622363rs73885319rs60910145 ) is a significant risk factor for ckd under any mode of association . the ors are 2.26 ( p = 0.005 ) , 2.54 ( p = 0.023 ) and 3.79 ( p = 0.041 ) for the additive , dominant and recessive modes of association , respectively , after correction for multiple comparisons . similarly , the two - allele haplotypes of rs73885319 and rs60910145 otherwise termed g1 , demonstrated strong levels of association with ckd . the ors for the g1:rs73885319rs60910145 ( a g ) haplotype are 2.25 ( p = 0.006 ) , 2.52 ( p = 0.025 ) and 3.80 ( p = 0.041 ) for the additive , dominant and recessive modes of association , respectively . the crude association [ or , 2.67(95 % ci , 0.798.97 ) ] with the compound risk heterozygote state among subjects heterozygous ( 10 cases and 5 controls ) for both the g1:a g and g2:d risk alleles and subjects ( 21 cases and 28 controls ) without any of the risk alleles was not significant ( p = 0.143 ) . t haplotype of g1:rs73885319rs60910145 indicated significant protective association with ckd under the additive ( or = 0.49 , p = 0.005 ) , dominant ( or = 0.40 , p = 0.025 ) and recessive ( or = 0.32 , p = 0.014 ) modes . the implication of this is a significantly high ckd risk for those carrying zero copies of the g1:g t haplotype with an or as high as 3.13 ( p = 0.014 ) . we did not observe significant evidence of association between any of the myh9 haplotypes and ckd after accounting for multiple testing ( table 3).fig . 1plot of linkage disequilibrium between snps in apol1/myh9 region ( top ) and their haplotypes ( bottom)table 3haplotype associations with non - diabetic chronic kidney disease in 87 cases and 79 controlssnp combinationhaplotypehaplotype frequencies ( % ) association modeor ( 95 % ci ) p valuecasescontrolsallunadjustedcorrected apol1 snps rs9622363 | rs73885319 | rs60910145g a g44.2526.9235.81additive2.26 ( 1.373.73)0.0010.005dominant2.54 ( 1.314.92)0.0060.052recessive3.79 ( 1.2811.20)0.0160.024 rs9622363 | rs73885319 | rs60910145a g t25.2930.1327.41additive0.72 ( 0.421.23)0.2310.641dominant0.81 ( 0.431.53)0.5240.392recessive0.24 ( 0.051.27)0.0930.983 rs9622363 | rs73885319 | rs60910145g g t24.7139.7432.16additive0.58 ( 0.370.90)0.0150.063dominant0.49 ( 0.260.93)0.0280.215recessive0.41 ( 0.161.03)0.0570.134 g1 : rs73885319 | rs60910145a g44.1926.9235.98additive2.25 ( 1.363.71)0.0020.005dominant2.52 ( 1.304.88)0.0060.051recessive3.80 ( 1.2911.22)0.0160.026 g1 : rs73885319 | rs60910145g t50.0069.8759.45additive0.49 ( 0.320.76)0.0010.005dominant0.32 ( 0.140.73)0.0070.018recessive0.40 ( 0.210.77)0.0060.031 myh9 snps rs2032487 | rs4821481 | rs5750248 | rs5750250t t c a17.2426.5821.67additive0.62 ( 0.361.07)0.0830.302dominant0.57 ( 0.291.09)0.0910.899recessive0.50 ( 0.112.16)0.3520.373 rs2032487 | rs4821481 | rs5750248 | rs5750250c c t g72.4162.0367.45additive1.66 ( 1.012.74)0.0460.184dominant2.16 ( 0.716.60)0.1760.299recessive1.80 ( 0.953.42)0.0730.609adjusted for age and gender or odds ratio , snps single nucleotide polymorphisms , ci confidence interval p values corrected for multiple comparisons plot of linkage disequilibrium between snps in apol1/myh9 region ( top ) and their haplotypes ( bottom ) haplotype associations with non - diabetic chronic kidney disease in 87 cases and 79 controls adjusted for age and gender or odds ratio , snps single nucleotide polymorphisms , ci confidence interval p values corrected for multiple comparisons in this study , we report the findings from a case / control association analysis of non - diabetic chronic kidney disease and variants in apol1 and myh9 genes in an african sample from southwest nigeria . apol1 and myh9 variants are associated with non - diabetic ckd among african americans [ 79 , 11 , 18 , 19 ] and hispanic americans . the purpose of our study was to investigate the possible evidence of association between these variants and non - diabetic ckd in a sample of africans of the yoruba tribe without history of european admixture . we replicated association with apol1 gene variants previously reported among african americans and hispanic americans [ 10 , 11 ] . the strength of the association between the two - allele haplotype of apol1 variants rs60910145 and rs73885319 ( g1 ) and non - diabetic ckd in the current study is about half of the sevenfold - increased odds of hypertensive kidney disease reported among african americans carrying multiple copies of apol1 risk alleles . stronger associations have been reported with hiv nephropathy [ 11 , 19 ] . since sample size only affects statistical significance of estimates and not the strength of the estimates , the apparent attenuation of the observed association when compared to previous findings among african americans can not be attributed to sample size . to confirm this , we investigated how much power the current study had to detect significant association between a variant with risk allele frequency at least similar to that observed for g1:a g ( i.e. , 0.360 ) , a genetic effect of at least 3.0 under a population risk of 0.00001 . using the software quanto , we estimated that the current study has at least 80 % power to detect association under recessive mode and over 90 % power under dominant or additive mode . we noted that the risk allele in the current sample is different for rs73885319 than previously reported . it should be noted that risk allele frequencies for this variant have been observed to differ substantially across african populations . in an african population sample set consisting of 676 samples from 12 african populations that included cameroon ( 2 ethnic groups ) , congo , ethiopia ( 4 ethnic groups ) , ghana ( 2 ethnic groups ) , malawi , mozambique and sudan , tzur et al . observed that risk allele frequencies differ between groups from same country and also across the populations . in the bulsa and asante populations of ghana , the frequencies are respectively 0.11 and 0.41 , whereas for the ethiopian groups the frequencies were zero . it is possible that non - diabetic kidney disease risk is not mediated by this variant but rather by other alleles in linkage disequilibrium with the variant or the haplotype g1 . as for myh9 , we did not find significant association between non - diabetic ckd and any variant or haplotype after correction for multiple testing . a previous study indicated that g1 and g2 are in strong ld with variants in myh9 , and most of the association previously attributed to myh9 variants or haplotypes with ckd is explained by ld with apol1 variant rs73885319 [ 10 , 11 ] . in the present study , we observed reduction in the strength of ckd association with myh9 variants when we accounted for the apol1 variants by conditioning on either rs73885319 or rs60910145 in the regression models fitted for each of the myh9 variants . we note that the observed low ld ( r 0.12 ) between apol1 and myh9 variants in our study sample may have contributed to the observed non - significant association of myh9 variants with ckd . among african americans , the frequency of the apol1 risk allele is around 0.33 with the high - risk genotype frequency being about 0.11 . previous studies have reported strong associations with apol1 risk alleles g1 ( rs73885319 and rs60910145 ) and g2 ( rs71785313 ) in an autosomal recessive fashion . inheritance of 2 apol1 risk alleles ( g1 and g1 , g1 and g2 , or g2 and g2 ) increases the risk of non - diabetic kidney disease by over sevenfold . for hiv nephropathy , associations may exceed 30-fold . in this study , we noted a fairly strong association between the g1 risk allele in a recessive model but no association was noted between g2 and non - diabetic kidney disease . this is likely due , in part , to the effect of sample size and the observed low minor allele frequency for g2 . it is also possible that differences in the association between apol1 variants and ckd in this study could , in part , be due to misclassification of cases or gene environment interactions but these interpretations remain speculative . in this study , the strength of the associations between 2 snps included in the myh9 e1 risk haplotype and non - diabetic kidney disease , though not significant after adjusting for multiple comparisons , was similar to associations reported in cases with end - stage kidney disease clinically attributed to hypertension . the mhy9 e1 risk haplotype consists of 4 snps ( rs4821480 , rs2032487 , rs4821481 and rs3752462 ) ; robust associations have been documented in several studies between the e1 haplotype and non - diabetic kidney disease including idiopathic and hiv - associated fsgs and ckd clinically attributed to hypertension [ 8 , 9 , 18 , 20 ] . apol1 variants are in strong linkage disequilibrium with myh9 variants , and it remains controversial whether myh9 variants play a direct role in kidney disease risk [ 23 , 24 ] . due to limited resources and poor access to healthcare for non - urban areas of nigeria , many patients who present for diagnostic workup and treatment of kidney disease do not undergo kidney biopsy in ibadan . however , diabetes prevalence is very low in nigeria [ 25 , 26 ] , and patients with evidence of diabetic kidney disease as determined by the treating physician were excluded from participation in study . the small sample size limited the ability to determine associations with specific types of ckd such as hypertensive ckd or focal segmental glomerulosclerosis . however , even with the small sample , fairly strong associations were noted with apol1 variants and the haplotype g1 . the associations between apol1 variants and non - diabetic ckd among nigerians of the yoruba tribe demonstrate that the impact of these genetic factors on ckd risk appear to be independent of the environment . diet , lifestyle and social structure are dramatically different in nigeria compared to the united states and other industrialized countries . hypertension , diabetes and obesity prevalence are markedly lower in nigeria compared to the african american population [ 2529 ] . despite these differences , apol1 variants are associated with non - diabetic ckd in this population . the discovery of the apol1/myh9 chromosomal 22 region as a region harboring genetic variants for non - diabetic ckd risk may be very important . it is possible that further delineation of the role of myh9 and apol1 variants may lead , in the future , to improved screening programs , prevention strategies and clinical interventions for ckd , one of the most common end - organ causes of morbidity and mortality worldwide . the perceived public health importance of these genetic variants is demonstrated by a patent application by the national institutes of health for these variants . this study demonstrates that these variants are also operative for non - diabetic kidney disease risk among nigerians of the yoruba tribe . data suggest that the g1 and g2 variants in the apol1 region emerged in this population several thousand years ago as a result of conferring protection from trypanosoma brucei rhodesiense [ 10 , 31 ] , a story very similar to the rise in frequency of the sickle cell trait as a result of resistance to certain forms of malaria . in conclusion , apol1 risk variants are associated with non - diabetic forms of ckd among nigerians of yoruba ethnicity . further information on apol1/myh9 variants may lead to screening programs which could lead to earlier detection and interventions for non - diabetic kidney disease . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

purposea region of chromosome 22 which includes apol1 and myh9 genes was recently identified as a risk locus for non - diabetic forms of kidney disease , including idiopathic and hiv - associated focal segmental glomerular sclerosis and kidney disease clinically attributed to hypertension among african americans . the purposes of the current study were , therefore , to examine the frequency of these variants and to determine whether they are associated with chronic kidney disease ( ckd ) among native africans.methodsto investigate the possible evidence of association between variants in these genes and non - diabetic ckd among west africans , we performed a case / control analysis in a sample of 166 nigerians without history of european admixture . our study included a total of 9 variants on apol1 ( n = 4 ) and myh9 ( n = 5 ) genes.resultswe observed significantly strong associations with previously reported apol1 variants rs73885319 and rs60910145 , and their two - allele g1 haplotype ( p < 0.005 ) . we did not observe significant evidence of association between non - diabetic ckd and any of the myh9 variants or haplotypes after accounting for multiple testing in our sample.conclusionsin conclusion , apol1 risk variants are associated with non - diabetic forms of ckd among nigerians of yoruba ethnicity . further information on apol1/myh9 variants may lead to screening programs , which could lead to earlier detection and interventions for non - diabetic kidney disease .

Introduction Subjects and methods Study participants and phenotype measurements Genotyping and quality assessment Statistical analyses Results Discussion Conclusions Conflict of interest Open Access

recently , a region of chromosome 22 , which includes apol1 and myh9 genes , was identified using mapping by admixture disequilibrium as a risk locus for non - diabetic forms of kidney disease , including idiopathic and hiv - associated focal segmental glomerular sclerosis ( fsgs ) and kidney disease clinically attributed to hypertension [ 79 ] . genetic variants in the region show very strong associations with non - diabetic kidney disease . specifically , a two - allele haplotype termed g1 consisting of two non - synonymous coding variants rs73885319 ( s342 g ) and rs60910145 ( i384 m ) along with rs71785313a 6 base pair deletion termed g2 and close to g1 in exon 5 of apol1 [ 10 , 11]is likely to account for the majority of risk of non - diabetic kidney disease associated with the variants in this region , but the role of myh9 variants in non - diabetic kidney disease risk remains controversial . the purposes of the current study were , therefore , to examine the frequency of these variants and to determine whether they are associated with ckd among native africans . participants in the current analyses included 88 and 81 adults with and without non - diabetic kidney disease , respectively . cases were defined as individuals of the yoruba tribe aged 1670 years with all stages of chronic kidney disease ( ckd ) of at least 3 months duration who met criteria for non - diabetic ckd clinically associated with long - standing hypertension , or ckd in the presence of proteinuria ( proteinuric ckd ) or ckd associated with hiv . controls consisted of normotensive individuals ( bp < 140/90 in the absence of anti - hypertensive medication use ) of the yoruba tribe without evidence of kidney disease ( serum creatinine < 1.4 and < 1.2 mg / dl in men and women , respectively , and spot urine albumin / creatinine ratio < 30 mg / g ) . the assays were originally selected for 96 published disease - associated single nucleotide polymorphisms ( snps ) and included 4 snps on apol1 and 6 snps on myh9 genes which have been reported to be associated with kidney disease . since the objective of the current analysis was to examine the association between previously reported kidney disease associated snps and ckd in a sample of nigerians , subsequent screening for associations with ckd was therefore restricted to just the variants on apol1 ( rs9622363 , rs73885319 , rs60910145 and rs71785313 ) and myh9 ( rs11912763 , rs2032487 , rs4821481 , rs5750248 and rs5750250 ) genes . to explore the possible associations between ckd status and various joint allelic configurations of multiple snps , we performed haplotype association analysis of the apol1 and myh9 snps . cases were defined as individuals of the yoruba tribe aged 1670 years with all stages of chronic kidney disease ( ckd ) of at least 3 months duration who met criteria for non - diabetic ckd clinically associated with long - standing hypertension , or ckd in the presence of proteinuria ( proteinuric ckd ) or ckd associated with hiv . controls consisted of normotensive individuals ( bp < 140/90 in the absence of anti - hypertensive medication use ) of the yoruba tribe without evidence of kidney disease ( serum creatinine < 1.4 and < 1.2 mg / dl in men and women , respectively , and spot urine albumin / creatinine ratio < 30 mg / g ) . the assays were originally selected for 96 published disease - associated single nucleotide polymorphisms ( snps ) and included 4 snps on apol1 and 6 snps on myh9 genes which have been reported to be associated with kidney disease . since the objective of the current analysis was to examine the association between previously reported kidney disease associated snps and ckd in a sample of nigerians , subsequent screening for associations with ckd was therefore restricted to just the variants on apol1 ( rs9622363 , rs73885319 , rs60910145 and rs71785313 ) and myh9 ( rs11912763 , rs2032487 , rs4821481 , rs5750248 and rs5750250 ) genes . to explore the possible associations between ckd status and various joint allelic configurations of multiple snps , we performed haplotype association analysis of the apol1 and myh9 snps . all but 3 cases had stage 5 ckd ( 1 with proteinuric ckd and 2 with ckd clinically attributed to hypertension.table 1descriptive characteristics of study subjectsnon - diabetic ckd cases ( n = 87)controls ( n = 79)all ( n = 166)no . of females ( % ) 41 ( 47 % ) 39 ( 49 % ) 80 ( 48 % ) age ( years ) 42.1 16.935.2 8.238.8 13.9body weight ( kg)61.6 11.159.8 10.860.8 11.0height ( m)1.6 0.11.7 0.11.6 0.1body mass index ( kg / m)23.2 4.621.9 4.222.6 4.4systolic blood pressure ( mm hg ) 136.6 31.0111.6 10.0124.7 26.6diastolic blood pressure ( mm hg ) 88.1 20.766.9 6.178.0 18.8 ckd chronic kidney diseasedata presented as mean standard deviation values are significantly different ( p < 0.05 ) between cases and controls descriptive characteristics of study subjects ckd chronic kidney disease data presented as mean standard deviation values are significantly different ( p < 0.05 ) between cases and controls the frequencies of the risk allele of the 9 apol1/myh9 variants among the cases and controls are listed in table 2 . the two variants are in almost perfect linkage disequilibrium ( d - prime = 1.00 , r = 0.82 ) and as such , when adjusted for either one , the association for the other disappeared.table 2snp associations with non - diabetic chronic kidney disease in 87 cases and 79 controlssnpgeneallelescoded ( minor ) allele ( frequency , % ) hwe p valuecoded allele frequency ( % ) association modeor ( 95 % ci ) p valuecasescontrolsunadjustedcorrected rs9622363 apol1 a / ga ( 27.71)0.78825.8629.75additive0.76 ( 0.451.31)0.3260.875dominant0.88 ( 0.471.66)0.6950.999recessive0.24 ( 0.051.29)0.0970.377rs73885319 apol1 a / ga ( 35.76)1.00044.1926.58additive2.29 ( 1.393.77)0.0010.005dominant2.59 ( 1.345.00)0.0050.025recessive3.85 ( 1.3111.36)0.0150.038rs60910145 apol1 g / tg ( 40.61)0.11450.0030.13additive2.04 ( 1.323.17)0.0010.006dominant2.54 ( 1.314.92)0.0060.034recessive3.12 ( 1.357.20)0.0080.015 g2 : rs71785313 apol1 d / id ( 10.54)1.0008.6212.66additive0.61 ( 0.291.31)0.2070.701dominant0.64 ( 0.291.40)0.2630.816recessiveneneners11912763 myh9 a / ga ( 33.13)1.00038.5127.22additive1.68 ( 1.022.76)0.0400.197dominant2.03 ( 1.063.87)0.0320.183recessive1.70 ( 0.584.94)0.3340.872rs2032487 myh9 t / ct ( 22.12)0.77018.3926.28additive0.68 ( 0.401.16)0.1570.580dominant0.64 ( 0.331.23)0.1770.645recessive0.55 ( 0.142.22)0.4000.934rs4821481 myh9 t / ct ( 22.29)0.77718.3926.58additive0.66 ( 0.391.13)0.1320.532dominant0.61 ( 0.321.18)0.1430.583recessive0.55 ( 0.142.24)0.4070.940rs5750248 myh9 c / tc ( 30.72)1.00025.8636.08additive0.61 ( 0.370.99)0.0470.225dominant0.56 ( 0.291.05)0.0710.354recessive0.46 ( 0.151.41)0.1760.627rs5750250 myh9 a / ga ( 31.82)0.63526.1637.97additive0.56 ( 0.340.94)0.0270.141dominant0.51 ( 0.270.97)0.0400.208recessive0.44 ( 0.141.38)0.1570.576adjusted for age and gender snps single nucleotide polymorphisms , hwe hardy weinberg equilibrium , or odds ratio , ci confidence interval , ne not estimated p values corrected for multiple comparisons snp associations with non - diabetic chronic kidney disease in 87 cases and 79 controls adjusted for age and gender snps single nucleotide polymorphisms , hwe hardy weinberg equilibrium , or odds ratio , ci confidence interval , ne not estimated p values corrected for multiple comparisons the haplotype blocks formed by the apol1/myh9 variants are displayed in fig . we also present the results for the two - allele haplotypes of rs73885319 and rs60910145 that included the previously reported g1 haplotype . we did not observe significant evidence of association between any of the myh9 haplotypes and ckd after accounting for multiple testing ( table 3).fig . 1plot of linkage disequilibrium between snps in apol1/myh9 region ( top ) and their haplotypes ( bottom)table 3haplotype associations with non - diabetic chronic kidney disease in 87 cases and 79 controlssnp combinationhaplotypehaplotype frequencies ( % ) association modeor ( 95 % ci ) p valuecasescontrolsallunadjustedcorrected apol1 snps rs9622363 | rs73885319 | rs60910145g a g44.2526.9235.81additive2.26 ( 1.373.73)0.0010.005dominant2.54 ( 1.314.92)0.0060.052recessive3.79 ( 1.2811.20)0.0160.024 rs9622363 | rs73885319 | rs60910145a g t25.2930.1327.41additive0.72 ( 0.421.23)0.2310.641dominant0.81 ( 0.431.53)0.5240.392recessive0.24 ( 0.051.27)0.0930.983 rs9622363 | rs73885319 | rs60910145g g t24.7139.7432.16additive0.58 ( 0.370.90)0.0150.063dominant0.49 ( 0.260.93)0.0280.215recessive0.41 ( 0.161.03)0.0570.134 g1 : rs73885319 | rs60910145a g44.1926.9235.98additive2.25 ( 1.363.71)0.0020.005dominant2.52 ( 1.304.88)0.0060.051recessive3.80 ( 1.2911.22)0.0160.026 g1 : rs73885319 | rs60910145g t50.0069.8759.45additive0.49 ( 0.320.76)0.0010.005dominant0.32 ( 0.140.73)0.0070.018recessive0.40 ( 0.210.77)0.0060.031 myh9 snps rs2032487 | rs4821481 | rs5750248 | rs5750250t t c a17.2426.5821.67additive0.62 ( 0.361.07)0.0830.302dominant0.57 ( 0.291.09)0.0910.899recessive0.50 ( 0.112.16)0.3520.373 rs2032487 | rs4821481 | rs5750248 | rs5750250c c t g72.4162.0367.45additive1.66 ( 1.012.74)0.0460.184dominant2.16 ( 0.716.60)0.1760.299recessive1.80 ( 0.953.42)0.0730.609adjusted for age and gender or odds ratio , snps single nucleotide polymorphisms , ci confidence interval p values corrected for multiple comparisons plot of linkage disequilibrium between snps in apol1/myh9 region ( top ) and their haplotypes ( bottom ) haplotype associations with non - diabetic chronic kidney disease in 87 cases and 79 controls adjusted for age and gender or odds ratio , snps single nucleotide polymorphisms , ci confidence interval p values corrected for multiple comparisons in this study , we report the findings from a case / control association analysis of non - diabetic chronic kidney disease and variants in apol1 and myh9 genes in an african sample from southwest nigeria . apol1 and myh9 variants are associated with non - diabetic ckd among african americans [ 79 , 11 , 18 , 19 ] and hispanic americans . the purpose of our study was to investigate the possible evidence of association between these variants and non - diabetic ckd in a sample of africans of the yoruba tribe without history of european admixture . the strength of the association between the two - allele haplotype of apol1 variants rs60910145 and rs73885319 ( g1 ) and non - diabetic ckd in the current study is about half of the sevenfold - increased odds of hypertensive kidney disease reported among african americans carrying multiple copies of apol1 risk alleles . as for myh9 , we did not find significant association between non - diabetic ckd and any variant or haplotype after correction for multiple testing . a previous study indicated that g1 and g2 are in strong ld with variants in myh9 , and most of the association previously attributed to myh9 variants or haplotypes with ckd is explained by ld with apol1 variant rs73885319 [ 10 , 11 ] . we note that the observed low ld ( r 0.12 ) between apol1 and myh9 variants in our study sample may have contributed to the observed non - significant association of myh9 variants with ckd . among african americans , the frequency of the apol1 risk allele is around 0.33 with the high - risk genotype frequency being about 0.11 . previous studies have reported strong associations with apol1 risk alleles g1 ( rs73885319 and rs60910145 ) and g2 ( rs71785313 ) in an autosomal recessive fashion . in this study , we noted a fairly strong association between the g1 risk allele in a recessive model but no association was noted between g2 and non - diabetic kidney disease . in this study , the strength of the associations between 2 snps included in the myh9 e1 risk haplotype and non - diabetic kidney disease , though not significant after adjusting for multiple comparisons , was similar to associations reported in cases with end - stage kidney disease clinically attributed to hypertension . the mhy9 e1 risk haplotype consists of 4 snps ( rs4821480 , rs2032487 , rs4821481 and rs3752462 ) ; robust associations have been documented in several studies between the e1 haplotype and non - diabetic kidney disease including idiopathic and hiv - associated fsgs and ckd clinically attributed to hypertension [ 8 , 9 , 18 , 20 ] . the associations between apol1 variants and non - diabetic ckd among nigerians of the yoruba tribe demonstrate that the impact of these genetic factors on ckd risk appear to be independent of the environment . despite these differences , apol1 variants are associated with non - diabetic ckd in this population . the discovery of the apol1/myh9 chromosomal 22 region as a region harboring genetic variants for non - diabetic ckd risk may be very important . it is possible that further delineation of the role of myh9 and apol1 variants may lead , in the future , to improved screening programs , prevention strategies and clinical interventions for ckd , one of the most common end - organ causes of morbidity and mortality worldwide . this study demonstrates that these variants are also operative for non - diabetic kidney disease risk among nigerians of the yoruba tribe . in conclusion , apol1 risk variants are associated with non - diabetic forms of ckd among nigerians of yoruba ethnicity . further information on apol1/myh9 variants may lead to screening programs which could lead to earlier detection and interventions for non - diabetic kidney disease .

recently , a region of chromosome 22 , which includes apol1 and myh9 genes , was identified using mapping by admixture disequilibrium as a risk locus for non - diabetic forms of kidney disease , including idiopathic and hiv - associated focal segmental glomerular sclerosis ( fsgs ) and kidney disease clinically attributed to hypertension [ 79 ] . specifically , a two - allele haplotype termed g1 consisting of two non - synonymous coding variants rs73885319 ( s342 g ) and rs60910145 ( i384 m ) along with rs71785313a 6 base pair deletion termed g2 and close to g1 in exon 5 of apol1 [ 10 , 11]is likely to account for the majority of risk of non - diabetic kidney disease associated with the variants in this region , but the role of myh9 variants in non - diabetic kidney disease risk remains controversial . cases were defined as individuals of the yoruba tribe aged 1670 years with all stages of chronic kidney disease ( ckd ) of at least 3 months duration who met criteria for non - diabetic ckd clinically associated with long - standing hypertension , or ckd in the presence of proteinuria ( proteinuric ckd ) or ckd associated with hiv . proteinuric ckd was defined as presence of spot urine protein / creatinine 2 g / g in the absence of red blood cell ( rbc ) casts or hematuria on urine microscopy or urinalysis and absence of known causes for ckd such as parasitic or other infections or diabetes . controls consisted of normotensive individuals ( bp < 140/90 in the absence of anti - hypertensive medication use ) of the yoruba tribe without evidence of kidney disease ( serum creatinine < 1.4 and < 1.2 mg / dl in men and women , respectively , and spot urine albumin / creatinine ratio < 30 mg / g ) . since the objective of the current analysis was to examine the association between previously reported kidney disease associated snps and ckd in a sample of nigerians , subsequent screening for associations with ckd was therefore restricted to just the variants on apol1 ( rs9622363 , rs73885319 , rs60910145 and rs71785313 ) and myh9 ( rs11912763 , rs2032487 , rs4821481 , rs5750248 and rs5750250 ) genes . cases were defined as individuals of the yoruba tribe aged 1670 years with all stages of chronic kidney disease ( ckd ) of at least 3 months duration who met criteria for non - diabetic ckd clinically associated with long - standing hypertension , or ckd in the presence of proteinuria ( proteinuric ckd ) or ckd associated with hiv . proteinuric ckd was defined as presence of spot urine protein / creatinine 2 g / g in the absence of red blood cell ( rbc ) casts or hematuria on urine microscopy or urinalysis and absence of known causes for ckd such as parasitic or other infections or diabetes . controls consisted of normotensive individuals ( bp < 140/90 in the absence of anti - hypertensive medication use ) of the yoruba tribe without evidence of kidney disease ( serum creatinine < 1.4 and < 1.2 mg / dl in men and women , respectively , and spot urine albumin / creatinine ratio < 30 mg / g ) . since the objective of the current analysis was to examine the association between previously reported kidney disease associated snps and ckd in a sample of nigerians , subsequent screening for associations with ckd was therefore restricted to just the variants on apol1 ( rs9622363 , rs73885319 , rs60910145 and rs71785313 ) and myh9 ( rs11912763 , rs2032487 , rs4821481 , rs5750248 and rs5750250 ) genes . to test for associations between ckd status and each single snp , we fitted logistic regression models in which each snp was presented as a predictor variable whose values were equal to the number of copies of the minor allele ( 0 , 1 , 2 ) ( i.e. among both cases and controls , , the ckd subjects tended to be older ( 42.1 vs. 35.2 years ) , heavier ( 23.2 vs. 21.9 kg / m ) and have higher blood pressure ( systolic : 136.6 vs. 111.6 mm hg ) when compared with the non - ckd controls . among the 87 ckd subjects , the physician - reported diagnosis of kidney disease was proteinuric ckd in 35 ( 40.2 % ) , hiv nephropathy in 8 ( 9.2 % ) and clinically attributed hypertensive ckd in 44 ( 50.5 % ) . all but 3 cases had stage 5 ckd ( 1 with proteinuric ckd and 2 with ckd clinically attributed to hypertension.table 1descriptive characteristics of study subjectsnon - diabetic ckd cases ( n = 87)controls ( n = 79)all ( n = 166)no . of females ( % ) 41 ( 47 % ) 39 ( 49 % ) 80 ( 48 % ) age ( years ) 42.1 16.935.2 8.238.8 13.9body weight ( kg)61.6 11.159.8 10.860.8 11.0height ( m)1.6 0.11.7 0.11.6 0.1body mass index ( kg / m)23.2 4.621.9 4.222.6 4.4systolic blood pressure ( mm hg ) 136.6 31.0111.6 10.0124.7 26.6diastolic blood pressure ( mm hg ) 88.1 20.766.9 6.178.0 18.8 ckd chronic kidney diseasedata presented as mean standard deviation values are significantly different ( p < 0.05 ) between cases and controls descriptive characteristics of study subjects ckd chronic kidney disease data presented as mean standard deviation values are significantly different ( p < 0.05 ) between cases and controls the frequencies of the risk allele of the 9 apol1/myh9 variants among the cases and controls are listed in table 2 . the two variants are in almost perfect linkage disequilibrium ( d - prime = 1.00 , r = 0.82 ) and as such , when adjusted for either one , the association for the other disappeared.table 2snp associations with non - diabetic chronic kidney disease in 87 cases and 79 controlssnpgeneallelescoded ( minor ) allele ( frequency , % ) hwe p valuecoded allele frequency ( % ) association modeor ( 95 % ci ) p valuecasescontrolsunadjustedcorrected rs9622363 apol1 a / ga ( 27.71)0.78825.8629.75additive0.76 ( 0.451.31)0.3260.875dominant0.88 ( 0.471.66)0.6950.999recessive0.24 ( 0.051.29)0.0970.377rs73885319 apol1 a / ga ( 35.76)1.00044.1926.58additive2.29 ( 1.393.77)0.0010.005dominant2.59 ( 1.345.00)0.0050.025recessive3.85 ( 1.3111.36)0.0150.038rs60910145 apol1 g / tg ( 40.61)0.11450.0030.13additive2.04 ( 1.323.17)0.0010.006dominant2.54 ( 1.314.92)0.0060.034recessive3.12 ( 1.357.20)0.0080.015 g2 : rs71785313 apol1 d / id ( 10.54)1.0008.6212.66additive0.61 ( 0.291.31)0.2070.701dominant0.64 ( 0.291.40)0.2630.816recessiveneneners11912763 myh9 a / ga ( 33.13)1.00038.5127.22additive1.68 ( 1.022.76)0.0400.197dominant2.03 ( 1.063.87)0.0320.183recessive1.70 ( 0.584.94)0.3340.872rs2032487 myh9 t / ct ( 22.12)0.77018.3926.28additive0.68 ( 0.401.16)0.1570.580dominant0.64 ( 0.331.23)0.1770.645recessive0.55 ( 0.142.22)0.4000.934rs4821481 myh9 t / ct ( 22.29)0.77718.3926.58additive0.66 ( 0.391.13)0.1320.532dominant0.61 ( 0.321.18)0.1430.583recessive0.55 ( 0.142.24)0.4070.940rs5750248 myh9 c / tc ( 30.72)1.00025.8636.08additive0.61 ( 0.370.99)0.0470.225dominant0.56 ( 0.291.05)0.0710.354recessive0.46 ( 0.151.41)0.1760.627rs5750250 myh9 a / ga ( 31.82)0.63526.1637.97additive0.56 ( 0.340.94)0.0270.141dominant0.51 ( 0.270.97)0.0400.208recessive0.44 ( 0.141.38)0.1570.576adjusted for age and gender snps single nucleotide polymorphisms , hwe hardy weinberg equilibrium , or odds ratio , ci confidence interval , ne not estimated p values corrected for multiple comparisons snp associations with non - diabetic chronic kidney disease in 87 cases and 79 controls adjusted for age and gender snps single nucleotide polymorphisms , hwe hardy weinberg equilibrium , or odds ratio , ci confidence interval , ne not estimated p values corrected for multiple comparisons the haplotype blocks formed by the apol1/myh9 variants are displayed in fig . the ors for the g1:rs73885319rs60910145 ( a g ) haplotype are 2.25 ( p = 0.006 ) , 2.52 ( p = 0.025 ) and 3.80 ( p = 0.041 ) for the additive , dominant and recessive modes of association , respectively . the crude association [ or , 2.67(95 % ci , 0.798.97 ) ] with the compound risk heterozygote state among subjects heterozygous ( 10 cases and 5 controls ) for both the g1:a g and g2:d risk alleles and subjects ( 21 cases and 28 controls ) without any of the risk alleles was not significant ( p = 0.143 ) . t haplotype of g1:rs73885319rs60910145 indicated significant protective association with ckd under the additive ( or = 0.49 , p = 0.005 ) , dominant ( or = 0.40 , p = 0.025 ) and recessive ( or = 0.32 , p = 0.014 ) modes . 1plot of linkage disequilibrium between snps in apol1/myh9 region ( top ) and their haplotypes ( bottom)table 3haplotype associations with non - diabetic chronic kidney disease in 87 cases and 79 controlssnp combinationhaplotypehaplotype frequencies ( % ) association modeor ( 95 % ci ) p valuecasescontrolsallunadjustedcorrected apol1 snps rs9622363 | rs73885319 | rs60910145g a g44.2526.9235.81additive2.26 ( 1.373.73)0.0010.005dominant2.54 ( 1.314.92)0.0060.052recessive3.79 ( 1.2811.20)0.0160.024 rs9622363 | rs73885319 | rs60910145a g t25.2930.1327.41additive0.72 ( 0.421.23)0.2310.641dominant0.81 ( 0.431.53)0.5240.392recessive0.24 ( 0.051.27)0.0930.983 rs9622363 | rs73885319 | rs60910145g g t24.7139.7432.16additive0.58 ( 0.370.90)0.0150.063dominant0.49 ( 0.260.93)0.0280.215recessive0.41 ( 0.161.03)0.0570.134 g1 : rs73885319 | rs60910145a g44.1926.9235.98additive2.25 ( 1.363.71)0.0020.005dominant2.52 ( 1.304.88)0.0060.051recessive3.80 ( 1.2911.22)0.0160.026 g1 : rs73885319 | rs60910145g t50.0069.8759.45additive0.49 ( 0.320.76)0.0010.005dominant0.32 ( 0.140.73)0.0070.018recessive0.40 ( 0.210.77)0.0060.031 myh9 snps rs2032487 | rs4821481 | rs5750248 | rs5750250t t c a17.2426.5821.67additive0.62 ( 0.361.07)0.0830.302dominant0.57 ( 0.291.09)0.0910.899recessive0.50 ( 0.112.16)0.3520.373 rs2032487 | rs4821481 | rs5750248 | rs5750250c c t g72.4162.0367.45additive1.66 ( 1.012.74)0.0460.184dominant2.16 ( 0.716.60)0.1760.299recessive1.80 ( 0.953.42)0.0730.609adjusted for age and gender or odds ratio , snps single nucleotide polymorphisms , ci confidence interval p values corrected for multiple comparisons plot of linkage disequilibrium between snps in apol1/myh9 region ( top ) and their haplotypes ( bottom ) haplotype associations with non - diabetic chronic kidney disease in 87 cases and 79 controls adjusted for age and gender or odds ratio , snps single nucleotide polymorphisms , ci confidence interval p values corrected for multiple comparisons in this study , we report the findings from a case / control association analysis of non - diabetic chronic kidney disease and variants in apol1 and myh9 genes in an african sample from southwest nigeria . the purpose of our study was to investigate the possible evidence of association between these variants and non - diabetic ckd in a sample of africans of the yoruba tribe without history of european admixture . the strength of the association between the two - allele haplotype of apol1 variants rs60910145 and rs73885319 ( g1 ) and non - diabetic ckd in the current study is about half of the sevenfold - increased odds of hypertensive kidney disease reported among african americans carrying multiple copies of apol1 risk alleles . a previous study indicated that g1 and g2 are in strong ld with variants in myh9 , and most of the association previously attributed to myh9 variants or haplotypes with ckd is explained by ld with apol1 variant rs73885319 [ 10 , 11 ] . in the present study , we observed reduction in the strength of ckd association with myh9 variants when we accounted for the apol1 variants by conditioning on either rs73885319 or rs60910145 in the regression models fitted for each of the myh9 variants . in this study , the strength of the associations between 2 snps included in the myh9 e1 risk haplotype and non - diabetic kidney disease , though not significant after adjusting for multiple comparisons , was similar to associations reported in cases with end - stage kidney disease clinically attributed to hypertension . the mhy9 e1 risk haplotype consists of 4 snps ( rs4821480 , rs2032487 , rs4821481 and rs3752462 ) ; robust associations have been documented in several studies between the e1 haplotype and non - diabetic kidney disease including idiopathic and hiv - associated fsgs and ckd clinically attributed to hypertension [ 8 , 9 , 18 , 20 ] . the associations between apol1 variants and non - diabetic ckd among nigerians of the yoruba tribe demonstrate that the impact of these genetic factors on ckd risk appear to be independent of the environment . it is possible that further delineation of the role of myh9 and apol1 variants may lead , in the future , to improved screening programs , prevention strategies and clinical interventions for ckd , one of the most common end - organ causes of morbidity and mortality worldwide . data suggest that the g1 and g2 variants in the apol1 region emerged in this population several thousand years ago as a result of conferring protection from trypanosoma brucei rhodesiense [ 10 , 31 ] , a story very similar to the rise in frequency of the sickle cell trait as a result of resistance to certain forms of malaria .

the ability of a newborn baby , fresh out of the womb , to attach to the maternal nipple and begin sucking leads many to label the behavior as innate . some extend this concept of innate behavior to include reflexes , denoting fixed action patterns organized as sensory - motor circuits in the brain stem or spinal cord [ 1 , 2 ] , as well as the rhythmic firing of central pattern generators ( cpgs ) , also located in the central nervous system , that produce correlated neuromuscular sucking rhythms . the concept of innate behavior is controversial , to say the least [ 4 , 5 ] . criticisms abound because such behaviors , when carefully observed and measured are not fixed but are highly variable [ 6 , 7 ] . moreover , so - called instinctive behaviors , under the scrutiny of experimental analysis , prove to be based on various forms of prior experience and learning [ 8 , 9 ] . similarly , while the firing of cpgs may correlate with sucking rhythms , it has yet to be shown that such isolated elements actually combine with other discrete components to create the real behavior of the suckling newborn , behavior that adapts to the unpredictable , dynamic geometry of the mother 's body , and behavior in real time and in real contexts . congenital is a more accurate and defensible term with which to denote a capability in behavior that is present at birth . in contrast to innate and instinctive , congenital is more obviously a description of status at birth than it is an explanation of its basis or origins [ 10 , 11 ] . explaining the developmental origins of a congenital capability such as nipple attachment and sucking by a newborn upon confronting for the first time its mother 's external body in highly novel environment is a formidable challenge of clinical significance . the experimental literature , based on studies with non - human animals , contains a wealth of information pertinent to a better understanding of the onset and development of oral feeding skills . this literature includes some impressive findings concerning the onset of nipple attachment and sucking by newborn rat pups . a brief description of the postnatal onset of sucking by rat pups will help frame the forthcoming presentation and analyses : rat pups are born after a gestation period of about three weeks . they are born as litter , averaging 10 pups ( mothers have 12 nipples ) , blind , deaf , and furless , with limited strength and coordination . for about 6 hrs prior to parturition , labor contractions can be seen rippling vertically on the mother 's abdomen or indenting her sides as she stretches her body . more than 100 labor contractions can be observed during a rat 's labor . as each pup emerges from the birth canal , encased in an amniotic sac with the umbilical cord and placenta trailing , the dam assists with licking and gentle tugging . the mother removes the sac by licking and nibbling , consumes the placenta and membranes , licks the pup some more , licks herself , and then repeats the sequence as the next pup emerges . only after all the pups are thus delivered and the placentas are all consumed , does the rat dam turn her attention to the newborns , which she gathers into a clump in the nest and settles over them . we have described and quantified the labor and delivery process in rats , noting the stimulation received by the pups during labor and throughout the birth process . in the nest , with the dam hovering above the pups , the infants are active . they orient to the dam 's ventrum , probe against her body and root along the ventrum until they orally grasp a nipple and suck . it is difficult to observe directly the natural sequence of events that lead to the rat pups ' initial nipple attachments and sucking . when the dam settles on her newly born litter she is typically crouched above the pups within a nest that affords poor visibility . fortunately , the newborn rat 's behavior is robust and orderly ; when placed under suspended artificial surfaces simulating various properties of the dam 's ventrum , newborn rats show an organized repertoire of behaviors . they travel , wriggle , turn on their sides and upside down , ventroflex , probe the surface , and audibly bark , all in a state of heighted behavioral arousal . other studies have been conducted with the dam anesthetized and the pup 's behavior thus isolated for analysis . thus , it is known that olfactory cues present on the rat mother 's nipples and ventrum are necessary and sufficient for newborn pups to locate and orally apprehend a nipple to suck . these odor cues can be removed by washing the nipples and surrounding body surfaces that eliminates suckling [ 14 , 15 ] . nipple attachment and suckling by newborns can be reinstated , however , by painting onto the dam 's ventrum a distillate of the wash taken from the dam 's body or by painting nipples with amniotic fluid or maternal saliva . other substances , both natural and atypical were tried , but no others were effective in reinstating nipple attachment . knowledge that amniotic fluid is a sufficient stimulus to elicit the newborn 's first nipple attachment led to preliminary considerations of two , mutually exclusive possibilities . one was that the key olfactory stimulus is somehow predetermined and that the newborn is correspondingly and inherently prepared ( hard - wired the second possibility was that the perinate responds with nipple attachment and sucking to the amniotic odor stimulus because of its previous experience with amniotic fluid . they reasoned that if amniotic fluid is a behaviorally potent stimulus because the fetus experienced it previously , then if some other odor was similarly experienced , it should have the same behavioral potency as amniotic fluid . they tested this bold hypothesis by adding a novel , lemon - like substance to the amniotic fluid , and then testing whether this chemical would rescue the newborn 's ability to make its first attachment to the washed nipples of a mother rat . the previously validated experimental procedure involved externalizing the uterine horns of a gestational day ( gd ) 20 dam and injecting a small quantity ( 0.2 ml ) of a citral and saline solution through the transparent wall of the uterus into the amniotic fluid . the uteri were replaced in the dam 's peritoneum , the laparotomy incision was closed , and gestation was completed without complications . then , on gd 22 , pups were delivered by caesarean section and placed immediately for one hr in a warmed nest where they were stroked with a soft artist 's brush for 1 hr in the presence of the citral odor . the test procedure involved presenting the caesarean - delivered pups with an anesthetized parturient rat dam ( not the subject pups ' mother ) . if the dam 's nipples were washed , pups did not attach to nipples , but when citral was on the dam , the treated newborns attached to a nipple and sucked ! natural odors of an unwashed dam were not effective for the citral - treated perinates . the new odor had replaced the natural stimulus . they performed an additional experiment in which pups were exposed to citral ( a ) in utero , ( b ) immediately after birth with stroking , or ( c ) both in utero and with postnatal stroking . only pups with the combined experiences attached to the washed , citral - scented nipples and not to the unwashed , normal nipples . pedersen and blass ' study provided important new insights into the initial plasticity of the newborn rats ' sucking , especially the specification of the cues that can activate and direct the behavior . it seems clear that the establishment of the olfactory control of sucking is determined by the experiences of the perinatal rat pup . but , what are the essential experiences for establishing the newborn 's sucking responses to maternal cues ? we adapted elements of our previous investigations of the perinatal rats ' sensory experiences in the uterine environment and of the birth process [ 12 , 1820 ] to demonstrate that specific components of maternal stimulation are sufficient conditions for the odor learning that establishes the newborn 's sucking responses to maternal cues . the present paper is a review of some of this past research as well as a report of additional , previously unpublished data that , together , provide a new view of how the experience of being born creates a context for learning . that is , embedded in flow of events that constitute the birth process are forms and levels of stimulation that , together , create the contingencies for early , rapid learning in the fetus , as it becomes a newborn . we will show that this learning , though general in initial form , is expressed in the natural context of the mother 's body as organized , adaptive , seemingly goal - directed behavior . we will first review an analysis of rat maternal behavior during labor and delivery from which we derived a set of novel tools that enabled us to simulate the major components of vaginal birth . we will also review some of our evidence that fetal and neonatal rats ( perinates ) have sensory capabilities sufficient to experience the birth process , at least the elements that are needed for basic associative learning . then , we will present data showing that the perinates ' responses to simulations of the birth process ( a ) augment nipple attachment and sucking , ( b ) establish odor - guided responses to the mother , and ( c ) induce neural conditions that mediate state transitions between fetal and neonatal behavioral systems which can account for the activation and expression of the newborn 's initial sucking behavior . under laboratory conditions , norway rats typically give birth on the 22nd day of gestation ; by breeding our animals on a known day , we were able to be present with appropriate video arrangements to view and record the dams ' labor and delivery . from these videorecordings , we quantified an average of 144.6 labor contractions during the six hours prior to the birth of the first pup from eight dams . figure 1 illustrates the three types of visible labor contractions in rat dams and shows the average frequency of each during the 6 hrs of labor . behavioral expressions of labor in the rat progress from uterine peristalsis to lordosis contractions followed by vertical contractions that occur in close association with birth of each pup . the brisk , linear decline in intercontraction intervals shown in figure 2 indicates how the contractions quicken as parturition approaches . from our systematic observations , we are able to describe the labor and delivery in the rat . duration of the delivery phase of parturition ( first to last birth ) ranged from 40 to 136 min , with dams delivering and average of 10.1 ( 1.1 ) pups . delivery duration and litter size were positively related ( r = .73 , p < as each pup begins to emerge from the birth canal , the dam typically adopts a head - between - heels posture , which facilitates delivery by enabling the mother to use her teeth to grasp the newborn and extract it from the vagina . mothers lick and handle each pup , removing and consuming the embryonic membranes , activities that produce cutaneous stimulation and augmented evaporative cooling of the newborn 's body . dams provided about 2 min of continuous stimulation to each newborn while participating in its delivery . each pup also received bursts of vestibular stimulation as the dam rotated its body while systematically consuming the products of gestation . during the initial phases of intense tactile and vestibular stimulation , as birth membranes were removed , especially from the head , pups began to emit the robust gasps that are characteristic of the onset of independent respiration , and thereafter they displayed gross movements and audible vocalizations . after the immediate postpartum licking and handling of each newborn , the dam often refocused her attention on previous newborns , providing each one with about 2.5 min of additional licking and handling . overall , licking by the dams was distributed relatively evenly across the pups ' bodies : head ( 39% ) , body ( 24% ) , anogenital area ( 32% ) , ( see for additional details ) . such observations help to specify events to which the newborn rat is exposed during birth . each pup received a protracted bout of repetitive tactile and vestibular stimulation associated with the dam 's handling and licking . during parturition , offspring are exposed to seemingly harsh forms of stimulation related to cooling and with compressions under the weight of the dam 's body . after systematically describing and analyzing the kinds and amounts of stimulation sustained by rat fetuses as they were being delivered vaginally , we endeavored to assess quantitatively some of the most prominent forms of stimulation . among the forms of birth stimuli that we analyzed were uterine compressions , cooling and rewarming , and maternal licking . from these analyses , we created a set of procedures and tools to mimic the biological stimuli that represent the physical bases of the pups ' experience of being born . uterine contractions , for example , were measured by surgically removing a single fetus from one of the paired uterine horns of a g18 rat installing a small balloon in its place in utero . the balloon was connected to a thin polyethylene tube that ran subcutaneously to the dam 's back and was externalized at the nape of the neck . the tubing could be connected to a pressure transducer with which we measured the forces exerted on the fetuses by the mother 's behavior and by uterine contractions . the dams ' contractions ranged from 2 to 30 mm hg . by attaching an inflated balloon to a small , spring - based calibrated scale , we could apply with the balloon surface a reliable force of 15 mm hg to a single fetal rat or to a newborn ( see figure 3 ) . in this way , we established a protocol for simulating a vaginal birth for rat fetuses : 15 , 20 sec - long compressions delivered at a rate of 1 per min , cooling ( 22c ) , stroking with a soft brush ( 2 min ) , and rewarming ( 33c ) . several of our analyses have focused on the how the birth process , beginning with the mother 's labor contractions , helps organize the fetal - to - neonatal transition . breathing and suckling are two vital behavioral adaptations of the newborn . in one set of studies , we applied our tools to study the components of birth that are important in the onset of pulmonary respiration , perhaps the most essential and immediate requirement of the newborn . the respiratory movements present in utero are episodic and unrelated to gas exchange [ 22 , 23 ] . at birth , however , breathing becomes continuous and regulated to meet the newborn 's oxygen requirements . we found that compressions simulating uterine contractions were necessary for initiating breathing in late gestation rat fetuses . the effectiveness of simulated labor contractions could arise from some mechanical ( nonsensory ) effect of the compression , or cutaneous ( sensory ) effects on the offspring . in a study of gentle stroking of cesarean delivered pups ( without simulated labor contractions ) , only 25% nonstroked pups survived for 1 hr postpartum compared to 100% stroked pups supporting a role for sensory stimulation . these observations fit well with reports of adaptive neuroendocrine changes and neurobehavioral advantages in neonates , both term and preterm , exposed to tactile and kinesthetic stimulation [ 25 , 26 ] . paradigm to examine more complex behavioral patterns in newborn rats in a study of how suckling becomes established . fetal rats were either exposed to labor contractions or not then cesarean delivered as described earlier , except that we manipulated postpartum ambient temperature using one of three biologically relevant temperatures . newborns were exposed to the cool room - temperature environment ( 22c ) or to a warmer temperature maintained at nest ( 33c ) or intrauterine ( 36c ) temperature . after 1 hr postpartum exposure to one of the three temperature regimens , pups from all groups were placed at nest temperature then tested for nipple attachment . the 22c condition contained the sequence of thermal exposures experienced by a vaginally born rat pup under typical thermal conditions . the treatment regime , then , was designed to represent the sequence and duration of stimulation that normally occurs prior to and immediately after vaginal birth , leading to the onset of suckling . at 2 hr postpartum , we found that 90% of vaginally delivered pups attached to a nipple ( table 1 ) . the most dramatic effects of prenatal compression were seen between pups that experienced thermal conditions similar to those of normal , vaginally delivered pups ( i.e. , the room temperature condition ) , whereas thermal effects were most evident in pups exposed to atypically warm temperatures ( i.e. , the intrauterine temperature condition ) . these studies link the major postnatal milestones of pulmonary ventilation and suckling to birth experience . we sought to determine the mechanisms underlying the effectiveness of birth stimuli in facilitating the fetus - to - newborn transition . human babies show a surge of plasma catecholamines associated with the stress of being born , a physiological response to labor and squeezing through the birth canal [ 27 , 28 ] . vaginally delivered infants show exhibit both enhanced respiratory performance and increased alertness compared to cesarean - delivered infants whose mothers did not undergo full labor [ 2931 ] . catecholamine concentrations are higher in vaginally delivered human infants as compared to cesarean - delivered infants . we analyzed plasma catecholamines at 0 to 2 hr - old following either : ( a ) vaginal birth , ( b ) cesarean section with simulated labor contractions , or ( c ) cesarean section without labor contractions ( mimicking planned cesarean delivery ) . pups were exposed to the major elements of the rat 's natural birth process , as we have described ( i.e. , umbilical cord occlusion , tactile stimulation and cooling ) . only pups exposed to actual or simulated labor showed an immediate and profound rise in norepinephrine and epinephrine , to levels up to 35% greater than those of noncompressed pups . our results , the first reported in the perinatal rat , closely parallel those reported in human studies and studies using the precocial sheep model [ 27 , 33 ] . labor contractions do more than move the fetus through the birth canal . whether by design ( natural selection ) or by incidental effect , contractions provide a form of stimulation that serves to facilitate two neonatal achievements : pulmonary respiration and suckling . birth stimuli , that is , the range , levels , and patterns of stimulation that comprise the birth process , might have multiple roles in the successful transition from fetal to postnatal life . our simulated birth model incorporates actual forms and levels of sensory and physiological stimuli to which the rat is exposed during natural vaginal birth and allows us to specifically parcel out the effects of labor on postpartum functions . the experience of labor is associated with a number of positive neonatal outcomes , including lung compliance , respiratory integrity [ 3537 ] , blood flow , resistance to oxidative stress , neonatal neurological condition , and complex global eeg patterns . human infants are particularly responsive to odors emanating from their mother 's nipple / areola region and can identify the nipple by smell [ 42 , 43 ] . amniotic fluid and breast odors are regulators of infant sucking behavior , comfort , and distress reactions [ 4446 ] . learning about natural breast odors is enhanced in neonates that experience labor contractions , possibly mediated by ne . together with the results reported herein , these studies support the view that prenatal events associated with labor initiate a cascade of neural , physiological and behavioral changes that assist the neonate 's successful transition to postnatal life events that assist the newborn infant 's adaptation to the extrauterine world . we now describe an original experiment conducted in our laboratory by abel in which individual , externalized , near - term rat fetuses received a combination of the simulated birth stimuli described earlier ( see figure 3 ) while in the presence of the odor citral and then tested for their responses to a rat dam with natural odors , washed of natural odors and with citral added . specifically , while still residing in their amniotic sac and uterine horn that had been gently brought outside the dam 's abdomen , each pup received a series of simulated labor contractions . pups were next removed from the uterus , at which time there occurred a bout of tactile stimulation associated with removal of the birth membranes . following this birth , each pup was stroked with a soft brush , mimicking the normal maternal licking and it also experienced cooling as it would after a natural birth and then rewarming as it would , had it been brought into the nest for maternal brooding . in effect , we created a simulated birth sequence . one set of pups experienced their birth in the presence of citral that was injected into the amniotic fluid prior to the intrauterine compressions and that was in the air around the pup while it was stroked and cooled and rewarmed . alternatively , saline was used instead of an odorant for the littermate control subjects , that otherwise experienced the same birth sequence . the goal was to test the hypothesis that an arbitrary odor , paired with the experience of birth stimuli , would become a conditioned odor capable of evoking nipple attachment behavior from a newborn . our regime of stimulation was a controlled , 135 min analog of pedersen and blass ' 50-hr - long process used to induce a newborn rat 's nipple attachment to novel odor . in contrast to their approach , we were able to specify and control the kinds , quantities , and timing of a specific stimulation sequence , and to observe the perinate at each stage of experimental manipulation . we predicted that a perinatal sequence of experiences in association with an otherwise neutral olfactory cue would lead to rates of nipple attachment to that cue , similar to those of vaginally delivered newborn rat pups to the odor of amniotic fluid . if the outcome of the simulated birth experience was equivalent to a natural delivery , we would consider this a successful empirical demonstration of sufficiency . we will have demonstrated that the experience of a simulated birth , quantitatively comparable to a natural , vaginal birth , is sufficient to establish a conditioned response to an odor that is expressed as nipple attachment and the onset of sucking in an , otherwise , nave newborn . animal experimentation was conducted in accordance with the guidelines of the indiana university institutional animal care and use committee and the nrc guide for the care and use of laboratory animals ( copyright 1996 , national academy of science ) . one hundred twenty - six fetal rats , derived from 24 , time - mated sprague - dawley rat dams ( rattus norvegicus ) were used as subjects . all breeding and maintenance was conducted in the animal behavior laboratory at indiana university . the first day that sperm was detected in a vaginal lavage was recorded as the day of conception ( gestational day [ g]0 ) , with birth expected on g22 . on g21 , pregnant dams were briefly anesthetized with isoflurane ( aerrane , ohmed ppd inc . , an area overlying the lumbar region was shaved and a small ( 3 cm ) dermal incision exposed the vertebral column . to eliminate movement and sensation below the ribcage , 100% ethyl alcohol ( 0.1 ml ) was administered via intrathecal injection between the t12 and l1 vertebrae . after confirming loss of sensation , the female was placed in a plexiglas holding apparatus and her lower body immersed into a heated ( 37.5c 5c ) saline bath . a midline laparotomy was performed and the dams ' paired uterine horns were gently externalized into the bath . figure 4 depicts the prenatal and postnatal manipulations . for each dam , either citral ( sigma chemical co. , st . louis , 50 l in 4 ml / l isotonic saline ) or vehicle alone was injected into the amniotic fluid surrounding target fetuses . beginning with the fetus in the second ovarian position , amniotic sacs of three - to - four adjacent fetuses were injected . a 30 ga hypodermic needle was inserted through the transparent uterine wall and into the amniotic sac near each fetus ' snout . immediately following either citral or saline injections , compressions of 1015 mm hg pressure were administered to fetuses in one uterine horn using a small latex balloon . such pressures are within the range of pressures typically experienced by rat fetuses during labor contractions [ 4951 ] . compressions were delivered at the rate of one , 15 sec compression per min for 15 min . upon completion of the compressions , fetuses were removed individually from the uterus and delivered onto gauze pads moistened with either citral ( 1 ml of 4 ml citral / l isotonic saline ) or isotonic saline - moistened ( 1 ml ) gauze pads ( figure 4 ) . immediately following delivery from the uterus , two cotton - tipped swabs were used to remove the birth membranes from the newborns , umbilical cords tied with surgical silk , and placentas removed . each neonate was stroked with a soft - bristled artist 's brush until respiratory activity was established ( approximately 2 - 3 min per litter ) . next , the newborns experienced temperature fluxes similar to those observed after a natural birth sequence in the laboratory . they were placed onto saline - moistened gauze pads in individual glass dishes ( pyrex 80 40 ) at room temperature ( 22c 0.5c ) for 60 min , then moved to an incubator maintained at nest temperature ( 33c 1c ) for an additional 60 min . at 60 min postpartum , pups that had received pre- and postnatal exposure to citral were placed individually in glass dishes on citral - moistened gauze pads ( 1 ml citral solution / pad ) . these pups remained in the warm citral ambience for 5 min then transferred back to the original dishes containing saline pads in a noncitral incubator for the remainder of the second postnatal hr . pups in both conditions were handled identically throughout the experiment except for the presence of citral . to ensure olfactory isolation , care was taken to maintain separate citral and noncitral incubators during the postnatal exposure and testing periods . physical stimulation in the form of compression or stroking is necessary for the establishment of respiration in newborn rat pups [ 19 , 20 ] . in the present experiments , postnatal stroking was required to elicit respiratory activity in the noncompressed subjects ; equivalent durations of stroking were provided to all groups . to verify that alterations in frequency of suckling onset between compressed and noncompressed newborns were not related to deficits in respiration , respiratory movements were sampled ( 1 min ) at 3 postpartum time points : 10 min postpartum ; 1 hr postpartum ( while at 22c ) ; 2 hr postpartum ( while at 33c ) . approximately 20 min prior to testing nipple attachment , a recently parturient ( 1 - 2 day postpartum ) dam was anesthetized with a ketamine / xylazine mix ( ip ; 100 mg / ml ; 0.9 ml / kg , 20 mg / ml ; 0.5 ml / kg ) and placed within a 33c test incubator in the supine position . at 120 min post - delivery , each pup was gently grasped and held for up to 120 sec with its snout in contact with a nipple of the test dam . successful attachment to a nipple was verified visually and then by testing if the pup maintained oral grasp of the nipple while gently retracted from the dam . following the first attachment trial , the dam was moved to a second heated ( 33c ) incubator and citral - scented gauze pads ( 5 pads ; 1 ml citral solution / pad ) were rubbed across the ventrum , thus infusing the fur with citral odor . the scented pads were then placed alongside the dam , further contributing to the citral odor within the incubator . then , to further verify the effectiveness of perinatal exposure to citral in promoting nipple attachment to a citral - scented dam , one group of compressed but citral - nave newborns was tested first on a citral - scented dam and then on a normal dam . mcnemar chi - square for dependent measures was used to analyze frequency of nipple attachment . posthoc comparisons were made with newman - keuls with a cutoff of p < 0.05 . the nipple attachment test used in the present experiment reveals robust and reliable behavior in newborn rats . the leftmost histogram bar in figure 5 shows that 90% of the vaginally - delivered newborns held before the ventrum of a natural ( unwashed ) anesthetized dam attached to a nipple and suckled . note that this was the first attachment for each pup . thus , this testing method enables rapid and reliable expression of the onset of postnatal ingestion , and the 90% attachment rate following vaginal birth can be used as standard against which we can evaluate the results of the simulated birth experiences . newborn pups that experienced a simulated vaginal birth in the presence of natural amniotic odors , including the regime of in utero compressions caesarean delivery membrane stripping cooling stroking rewarming ( see figure 3 ) attached to a nipple in 89% of the tests , as shown by the first hatched bar in figure 5 . the legend under that bar , amniotic fluid / natural , indicates that these pups experienced unadulterated amniotic fluid odors and were with a natural , unwashed dam . newborns that experienced the a simulated vaginal birth absent compressions in the presence of natural amniotic odors , attached in only about 44% of the trials , which was a significant decrement in relation to littermates treated identically but with the compressions . the contrasting result is seen in the open bar next to the hatched bar in figure 5 . thus , the complete simulated birth sequence ( including compressions ) , produced rates of nipple attachment in newborns that were fully comparable to those in vaginally delivered pups . newborn pups that experienced a simulated vaginal birth in the presence of citral in their amniotic fluid and in the atmosphere during stroking ( figure 4 ) attached to a nipple in 89% of the tests with a citral - scented dam , as shown by the stippled bar in figure 5 . newborns that experienced the simulated vaginal birth absent compressions in the presence of natural amniotic odors , attached in only about 20% of the trials with the citral - scented dam , a significant decrement in relation to littermates treated identically but with compressions . the open bar , adjacent to the stippled bar in figure 5 , shows the contrasting outcome . thus , the simulated birth sequence in the presence of citral , including compressions , produced rates of nipple attachment to a citral - scented dam that were fully comparable to those seen in vaginally - delivered pups and to pups that experienced the full simulated birth in the presence of amniotic odors when tested with a correspondingly natural - scented dam . the rightmost pair of histograms show that newborns that experienced simulated birth stimuli in the presence of citral in their amniotic fluid and in the atmosphere during stroking ( figure 4 ) when tested with a natural scent dam ( no citral during the test ) attached to a nipple in only 20% and 4% of the trials , for the compressed and noncompressed subjects . clearly , newborns that experienced birth in a citral environment were not prepared to attach to nipples on the body of a dam with only the natural scent of the species . but , we know that these newborns are capable of attaching to a nipple , as evidenced by the performance of the simulated birth group depicted by the stippled bar . for the citral - birthed pups , citral had become a necessary stimulus for the initial attachment . respiratory rates at the three , sampled time - points ( 10 , 60 , and 120 min ) were unaffected by either citral or compression ( p > .10 ) . as expected , however , there was a significant increase in respiration within all groups during the final hour at the warmer temperature ( f(2,120 ) = 320.8 , p < .01 ) . the absence of in utero compressions of the fetus was associated with poor performance in the onset of nipple attachment . it might be tempting to conclude that compressions mimicking labor contractions are necessary for efficient initiation of nipple attachment in the newborn , but the present experiment was not designed to allow such a conclusion . although we categorized each operation as a separate form of stimulation , the perinate might be less discriminating in its responsiveness and all forms of stimulation might simply be additive and incrementally increase the level of arousal in the pup . thus , compressions might just add to the experience of general stimulation in the pup and nipple attachment rates might reflect levels of general arousal . even if true , such an effect would not account for the second broad finding , that odors paired with birth stimuli become conditioned stimuli for nipple attachment . the experience of a vaginal birth , real or simulated , appears to give behavioral meaning to the odors experienced in association with the birth stimuli . schaal and colleagues have suggested that amniotic odors provide a bridge from the fetus ' prenatal world to its postnatal environment , and the present results suggest that this bridge is constructed by the experiences embedded in parturition and that they result in a newborn behavior that has been rapidly assembled to follow the bridge to a nipple and the onset of suckling . stimulation associated with labor and delivery plays a key role in assisting the fetus ' transition to postnatal life by inducing and canalizing specific behaviors , and thereby operating as a critical link in the chain of behavioral adjustments required for adaptation to the postnatal habitat . fetal sensory experience appears to set into motion physiological processes that permit the onset of postpartum behavior and the expression of early learning . it makes sense , both logically and scientifically , to discard the idea that suckling is an innate or instinctive or hard - wired behavior in a newborn baby . nevertheless , it is also sensible to revel in the readiness and competence of a newborn mammal to adjust immediately to severance of its umbilical connection to the uterine world and to make an oral connection to the mother 's body and begin suckling . we recognized the ability of a newborn mammal to suckle by designating it as a congenital behavior , that is , present at birth . clearly , suckling is an important congenital behavior worth understanding for it is one of the primary adaptations to newborn life for all infant mammals , serving not only nutritive , immunological , and general physiological functions , but it is also a powerful component of bonding with the mother and creating a social context which supports sensory , motor , and cognitive development . much of what we explored in the present paper are lines of research that have been important in demystifying the kinds of basic processes that can explain the newborn 's congenital abilities to orient to novel features on the mother 's body surface and to initiate the complex , but vital behavior of suckling . from the findings that we reviewed , it can be concluded that the combination of sensory and motor processes that constitute successful suckling are rapidly assembled during the course of perinatal events . we focused on the roles of birth stimuli and specifically on the experience of being born . in the experiments we described , rat pups that did not experience the mechanical and thermal forces associated with vaginal birth failed to make the fetus - to - newborn transition . moreover , by providing individual perinates with a simulated birth experience , it was possible to induce in them the dramatic developmental changes that serve the transition from fetus to newborn . there is now abundant evidence that learning is an important component of the birth transition in rats and in humans . thus , this perinatal learning takes place in the context of the experience of being born . it appears that the set of sensory , endocrine , and neural events that comprise the physiological transition of birth also serve as factors in the perinate 's learning about the odor cues that are present in utero and carry over into the ex utero world . these are the same cues that the newborn then uses to orient to the mother 's body and that stimulate nipple attachment and suckling . we are impressed by the multi - leveled functions of sensory and physiological events of birth , but much more remains to be understood about them and how they operate during parturition . here , we can speculate on some of the implications we see when considering the experimental findings from rats in the context of human births and the onset of suckling . as students of mammalian development , indeed , our past initial analyses of rat parturition ( e.g. , [ 12 , 19 , 20 , 34 , 51 ] ) were shaped by lagercrantz and slotkin 's perspective on the stress of human vaginal birth . the results of our experiments with rats resemble their observations with human birth , and we have been able to take advantage of opportunities to control and manipulate the birth stimuli to gain insights into the embedded and embodied learning processes . we are particularly struck by the contrasting picture presented by prematurely - born infants who enter the postnatal world at a stage of development when their sensorimotor function is not yet prepared for suckling . while it may be beautiful and exciting to witness the eventual onset of sucking in a baby born at less than 30 weeks of age , it is sobering to contemplate the dramatically different factors and unnatural schedule of experiences that direct the prematurely - born baby to suck : a premie 's early postnatal development may be supported by intravenous nutrition and then gastric intubation until the baby presents signs of readiness for oral feeding . nurses , therapists and parents may then use a variety of techniques to gently and gradually facilitate the transition to sucking and ingestion . how different it is for the baby born at term , for whom the process is short , and in many regards , even intense and abrupt . we see great potential in understanding the necessary and sufficient developmental steps as precursors to improved management and guidance of early ingestion . other researchers have compared the development of feeding skills of term babies and prematurely born infants . schaal and colleagues ( cf . , ) , for example , have focused on the absence of pairings of chemosensory cues and nutritive intake when babies are fed by gavage . their perspective extends to many aspects of experience that normally contribute to the integration of breathing , suckling , and swallowing . recognition of such differences may be an important step towards identifying factors that contribute to the problems experienced by some babies and that lead to the higher incidence of feeding disorders in children born prematurely ( cf . , ) .

understanding the developmental origins of congenital capabilities such as sucking is fundamental knowledge that can contribute to improving the clinical management of early feeding and facilitate the onset of oral ingestion . we describe analyses in rats showing that sensory stimulation in utero and during birth establishes the newborn 's sucking responses to maternal cues . we mimicked elements of labor and delivery ( viz . , compressions simulating labor contractions , stroking simulating postnatal maternal licking of the newborn , and postnatal thermal flux ) , and used them to induce postnatal respiration and nipple attachment in caesarian - delivered pups . we report herein new data showing that , by simulating a fetal rat 's experience of being born , specific components of vaginal birth provide sufficient conditions for the odor learning that guides newborn 's sucking responses . in contrast , the absence of in utero compressions was associated with poor sucking onset . knowing how birth stimuli contribute to the first nipple attachment and constitute a context for learning to suckle is an important step toward better management of some early feeding problems . it can serve also as a foundation for understanding the challenges of facilitating sucking by babies born prematurely so that they do not experience the typical contingencies mediating onset of oral ingestion .

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

the ability of a newborn baby , fresh out of the womb , to attach to the maternal nipple and begin sucking leads many to label the behavior as innate . similarly , while the firing of cpgs may correlate with sucking rhythms , it has yet to be shown that such isolated elements actually combine with other discrete components to create the real behavior of the suckling newborn , behavior that adapts to the unpredictable , dynamic geometry of the mother 's body , and behavior in real time and in real contexts . in contrast to innate and instinctive , congenital is more obviously a description of status at birth than it is an explanation of its basis or origins [ 10 , 11 ] . explaining the developmental origins of a congenital capability such as nipple attachment and sucking by a newborn upon confronting for the first time its mother 's external body in highly novel environment is a formidable challenge of clinical significance . the experimental literature , based on studies with non - human animals , contains a wealth of information pertinent to a better understanding of the onset and development of oral feeding skills . this literature includes some impressive findings concerning the onset of nipple attachment and sucking by newborn rat pups . a brief description of the postnatal onset of sucking by rat pups will help frame the forthcoming presentation and analyses : rat pups are born after a gestation period of about three weeks . they are born as litter , averaging 10 pups ( mothers have 12 nipples ) , blind , deaf , and furless , with limited strength and coordination . more than 100 labor contractions can be observed during a rat 's labor . we have described and quantified the labor and delivery process in rats , noting the stimulation received by the pups during labor and throughout the birth process . fortunately , the newborn rat 's behavior is robust and orderly ; when placed under suspended artificial surfaces simulating various properties of the dam 's ventrum , newborn rats show an organized repertoire of behaviors . nipple attachment and suckling by newborns can be reinstated , however , by painting onto the dam 's ventrum a distillate of the wash taken from the dam 's body or by painting nipples with amniotic fluid or maternal saliva . knowledge that amniotic fluid is a sufficient stimulus to elicit the newborn 's first nipple attachment led to preliminary considerations of two , mutually exclusive possibilities . one was that the key olfactory stimulus is somehow predetermined and that the newborn is correspondingly and inherently prepared ( hard - wired the second possibility was that the perinate responds with nipple attachment and sucking to the amniotic odor stimulus because of its previous experience with amniotic fluid . they tested this bold hypothesis by adding a novel , lemon - like substance to the amniotic fluid , and then testing whether this chemical would rescue the newborn 's ability to make its first attachment to the washed nipples of a mother rat . the uteri were replaced in the dam 's peritoneum , the laparotomy incision was closed , and gestation was completed without complications . the test procedure involved presenting the caesarean - delivered pups with an anesthetized parturient rat dam ( not the subject pups ' mother ) . they performed an additional experiment in which pups were exposed to citral ( a ) in utero , ( b ) immediately after birth with stroking , or ( c ) both in utero and with postnatal stroking . pedersen and blass ' study provided important new insights into the initial plasticity of the newborn rats ' sucking , especially the specification of the cues that can activate and direct the behavior . it seems clear that the establishment of the olfactory control of sucking is determined by the experiences of the perinatal rat pup . but , what are the essential experiences for establishing the newborn 's sucking responses to maternal cues ? we adapted elements of our previous investigations of the perinatal rats ' sensory experiences in the uterine environment and of the birth process [ 12 , 1820 ] to demonstrate that specific components of maternal stimulation are sufficient conditions for the odor learning that establishes the newborn 's sucking responses to maternal cues . the present paper is a review of some of this past research as well as a report of additional , previously unpublished data that , together , provide a new view of how the experience of being born creates a context for learning . we will first review an analysis of rat maternal behavior during labor and delivery from which we derived a set of novel tools that enabled us to simulate the major components of vaginal birth . then , we will present data showing that the perinates ' responses to simulations of the birth process ( a ) augment nipple attachment and sucking , ( b ) establish odor - guided responses to the mother , and ( c ) induce neural conditions that mediate state transitions between fetal and neonatal behavioral systems which can account for the activation and expression of the newborn 's initial sucking behavior . under laboratory conditions , norway rats typically give birth on the 22nd day of gestation ; by breeding our animals on a known day , we were able to be present with appropriate video arrangements to view and record the dams ' labor and delivery . from these videorecordings , we quantified an average of 144.6 labor contractions during the six hours prior to the birth of the first pup from eight dams . figure 1 illustrates the three types of visible labor contractions in rat dams and shows the average frequency of each during the 6 hrs of labor . from our systematic observations , we are able to describe the labor and delivery in the rat . delivery duration and litter size were positively related ( r = .73 , p < as each pup begins to emerge from the birth canal , the dam typically adopts a head - between - heels posture , which facilitates delivery by enabling the mother to use her teeth to grasp the newborn and extract it from the vagina . mothers lick and handle each pup , removing and consuming the embryonic membranes , activities that produce cutaneous stimulation and augmented evaporative cooling of the newborn 's body . during the initial phases of intense tactile and vestibular stimulation , as birth membranes were removed , especially from the head , pups began to emit the robust gasps that are characteristic of the onset of independent respiration , and thereafter they displayed gross movements and audible vocalizations . after the immediate postpartum licking and handling of each newborn , the dam often refocused her attention on previous newborns , providing each one with about 2.5 min of additional licking and handling . such observations help to specify events to which the newborn rat is exposed during birth . among the forms of birth stimuli that we analyzed were uterine compressions , cooling and rewarming , and maternal licking . from these analyses , we created a set of procedures and tools to mimic the biological stimuli that represent the physical bases of the pups ' experience of being born . uterine contractions , for example , were measured by surgically removing a single fetus from one of the paired uterine horns of a g18 rat installing a small balloon in its place in utero . the balloon was connected to a thin polyethylene tube that ran subcutaneously to the dam 's back and was externalized at the nape of the neck . in this way , we established a protocol for simulating a vaginal birth for rat fetuses : 15 , 20 sec - long compressions delivered at a rate of 1 per min , cooling ( 22c ) , stroking with a soft brush ( 2 min ) , and rewarming ( 33c ) . several of our analyses have focused on the how the birth process , beginning with the mother 's labor contractions , helps organize the fetal - to - neonatal transition . breathing and suckling are two vital behavioral adaptations of the newborn . in one set of studies , we applied our tools to study the components of birth that are important in the onset of pulmonary respiration , perhaps the most essential and immediate requirement of the newborn . the effectiveness of simulated labor contractions could arise from some mechanical ( nonsensory ) effect of the compression , or cutaneous ( sensory ) effects on the offspring . in a study of gentle stroking of cesarean delivered pups ( without simulated labor contractions ) , only 25% nonstroked pups survived for 1 hr postpartum compared to 100% stroked pups supporting a role for sensory stimulation . after 1 hr postpartum exposure to one of the three temperature regimens , pups from all groups were placed at nest temperature then tested for nipple attachment . the treatment regime , then , was designed to represent the sequence and duration of stimulation that normally occurs prior to and immediately after vaginal birth , leading to the onset of suckling . , the room temperature condition ) , whereas thermal effects were most evident in pups exposed to atypically warm temperatures ( i.e. human babies show a surge of plasma catecholamines associated with the stress of being born , a physiological response to labor and squeezing through the birth canal [ 27 , 28 ] . we analyzed plasma catecholamines at 0 to 2 hr - old following either : ( a ) vaginal birth , ( b ) cesarean section with simulated labor contractions , or ( c ) cesarean section without labor contractions ( mimicking planned cesarean delivery ) . pups were exposed to the major elements of the rat 's natural birth process , as we have described ( i.e. our results , the first reported in the perinatal rat , closely parallel those reported in human studies and studies using the precocial sheep model [ 27 , 33 ] . birth stimuli , that is , the range , levels , and patterns of stimulation that comprise the birth process , might have multiple roles in the successful transition from fetal to postnatal life . the experience of labor is associated with a number of positive neonatal outcomes , including lung compliance , respiratory integrity [ 3537 ] , blood flow , resistance to oxidative stress , neonatal neurological condition , and complex global eeg patterns . learning about natural breast odors is enhanced in neonates that experience labor contractions , possibly mediated by ne . together with the results reported herein , these studies support the view that prenatal events associated with labor initiate a cascade of neural , physiological and behavioral changes that assist the neonate 's successful transition to postnatal life events that assist the newborn infant 's adaptation to the extrauterine world . we now describe an original experiment conducted in our laboratory by abel in which individual , externalized , near - term rat fetuses received a combination of the simulated birth stimuli described earlier ( see figure 3 ) while in the presence of the odor citral and then tested for their responses to a rat dam with natural odors , washed of natural odors and with citral added . pups were next removed from the uterus , at which time there occurred a bout of tactile stimulation associated with removal of the birth membranes . the goal was to test the hypothesis that an arbitrary odor , paired with the experience of birth stimuli , would become a conditioned odor capable of evoking nipple attachment behavior from a newborn . our regime of stimulation was a controlled , 135 min analog of pedersen and blass ' 50-hr - long process used to induce a newborn rat 's nipple attachment to novel odor . in contrast to their approach , we were able to specify and control the kinds , quantities , and timing of a specific stimulation sequence , and to observe the perinate at each stage of experimental manipulation . we predicted that a perinatal sequence of experiences in association with an otherwise neutral olfactory cue would lead to rates of nipple attachment to that cue , similar to those of vaginally delivered newborn rat pups to the odor of amniotic fluid . we will have demonstrated that the experience of a simulated birth , quantitatively comparable to a natural , vaginal birth , is sufficient to establish a conditioned response to an odor that is expressed as nipple attachment and the onset of sucking in an , otherwise , nave newborn . animal experimentation was conducted in accordance with the guidelines of the indiana university institutional animal care and use committee and the nrc guide for the care and use of laboratory animals ( copyright 1996 , national academy of science ) . the first day that sperm was detected in a vaginal lavage was recorded as the day of conception ( gestational day [ g]0 ) , with birth expected on g22 . these pups remained in the warm citral ambience for 5 min then transferred back to the original dishes containing saline pads in a noncitral incubator for the remainder of the second postnatal hr . physical stimulation in the form of compression or stroking is necessary for the establishment of respiration in newborn rat pups [ 19 , 20 ] . following the first attachment trial , the dam was moved to a second heated ( 33c ) incubator and citral - scented gauze pads ( 5 pads ; 1 ml citral solution / pad ) were rubbed across the ventrum , thus infusing the fur with citral odor . the leftmost histogram bar in figure 5 shows that 90% of the vaginally - delivered newborns held before the ventrum of a natural ( unwashed ) anesthetized dam attached to a nipple and suckled . thus , this testing method enables rapid and reliable expression of the onset of postnatal ingestion , and the 90% attachment rate following vaginal birth can be used as standard against which we can evaluate the results of the simulated birth experiences . newborn pups that experienced a simulated vaginal birth in the presence of natural amniotic odors , including the regime of in utero compressions caesarean delivery membrane stripping cooling stroking rewarming ( see figure 3 ) attached to a nipple in 89% of the tests , as shown by the first hatched bar in figure 5 . newborns that experienced the a simulated vaginal birth absent compressions in the presence of natural amniotic odors , attached in only about 44% of the trials , which was a significant decrement in relation to littermates treated identically but with the compressions . thus , the complete simulated birth sequence ( including compressions ) , produced rates of nipple attachment in newborns that were fully comparable to those in vaginally delivered pups . newborn pups that experienced a simulated vaginal birth in the presence of citral in their amniotic fluid and in the atmosphere during stroking ( figure 4 ) attached to a nipple in 89% of the tests with a citral - scented dam , as shown by the stippled bar in figure 5 . newborns that experienced the simulated vaginal birth absent compressions in the presence of natural amniotic odors , attached in only about 20% of the trials with the citral - scented dam , a significant decrement in relation to littermates treated identically but with compressions . thus , the simulated birth sequence in the presence of citral , including compressions , produced rates of nipple attachment to a citral - scented dam that were fully comparable to those seen in vaginally - delivered pups and to pups that experienced the full simulated birth in the presence of amniotic odors when tested with a correspondingly natural - scented dam . the rightmost pair of histograms show that newborns that experienced simulated birth stimuli in the presence of citral in their amniotic fluid and in the atmosphere during stroking ( figure 4 ) when tested with a natural scent dam ( no citral during the test ) attached to a nipple in only 20% and 4% of the trials , for the compressed and noncompressed subjects . the absence of in utero compressions of the fetus was associated with poor performance in the onset of nipple attachment . it might be tempting to conclude that compressions mimicking labor contractions are necessary for efficient initiation of nipple attachment in the newborn , but the present experiment was not designed to allow such a conclusion . although we categorized each operation as a separate form of stimulation , the perinate might be less discriminating in its responsiveness and all forms of stimulation might simply be additive and incrementally increase the level of arousal in the pup . thus , compressions might just add to the experience of general stimulation in the pup and nipple attachment rates might reflect levels of general arousal . even if true , such an effect would not account for the second broad finding , that odors paired with birth stimuli become conditioned stimuli for nipple attachment . the experience of a vaginal birth , real or simulated , appears to give behavioral meaning to the odors experienced in association with the birth stimuli . schaal and colleagues have suggested that amniotic odors provide a bridge from the fetus ' prenatal world to its postnatal environment , and the present results suggest that this bridge is constructed by the experiences embedded in parturition and that they result in a newborn behavior that has been rapidly assembled to follow the bridge to a nipple and the onset of suckling . stimulation associated with labor and delivery plays a key role in assisting the fetus ' transition to postnatal life by inducing and canalizing specific behaviors , and thereby operating as a critical link in the chain of behavioral adjustments required for adaptation to the postnatal habitat . fetal sensory experience appears to set into motion physiological processes that permit the onset of postpartum behavior and the expression of early learning . we recognized the ability of a newborn mammal to suckle by designating it as a congenital behavior , that is , present at birth . clearly , suckling is an important congenital behavior worth understanding for it is one of the primary adaptations to newborn life for all infant mammals , serving not only nutritive , immunological , and general physiological functions , but it is also a powerful component of bonding with the mother and creating a social context which supports sensory , motor , and cognitive development . much of what we explored in the present paper are lines of research that have been important in demystifying the kinds of basic processes that can explain the newborn 's congenital abilities to orient to novel features on the mother 's body surface and to initiate the complex , but vital behavior of suckling . we focused on the roles of birth stimuli and specifically on the experience of being born . in the experiments we described , rat pups that did not experience the mechanical and thermal forces associated with vaginal birth failed to make the fetus - to - newborn transition . moreover , by providing individual perinates with a simulated birth experience , it was possible to induce in them the dramatic developmental changes that serve the transition from fetus to newborn . there is now abundant evidence that learning is an important component of the birth transition in rats and in humans . thus , this perinatal learning takes place in the context of the experience of being born . it appears that the set of sensory , endocrine , and neural events that comprise the physiological transition of birth also serve as factors in the perinate 's learning about the odor cues that are present in utero and carry over into the ex utero world . these are the same cues that the newborn then uses to orient to the mother 's body and that stimulate nipple attachment and suckling . here , we can speculate on some of the implications we see when considering the experimental findings from rats in the context of human births and the onset of suckling . the results of our experiments with rats resemble their observations with human birth , and we have been able to take advantage of opportunities to control and manipulate the birth stimuli to gain insights into the embedded and embodied learning processes . how different it is for the baby born at term , for whom the process is short , and in many regards , even intense and abrupt . we see great potential in understanding the necessary and sufficient developmental steps as precursors to improved management and guidance of early ingestion . , ) , for example , have focused on the absence of pairings of chemosensory cues and nutritive intake when babies are fed by gavage . their perspective extends to many aspects of experience that normally contribute to the integration of breathing , suckling , and swallowing . recognition of such differences may be an important step towards identifying factors that contribute to the problems experienced by some babies and that lead to the higher incidence of feeding disorders in children born prematurely ( cf .

the ability of a newborn baby , fresh out of the womb , to attach to the maternal nipple and begin sucking leads many to label the behavior as innate . some extend this concept of innate behavior to include reflexes , denoting fixed action patterns organized as sensory - motor circuits in the brain stem or spinal cord [ 1 , 2 ] , as well as the rhythmic firing of central pattern generators ( cpgs ) , also located in the central nervous system , that produce correlated neuromuscular sucking rhythms . moreover , so - called instinctive behaviors , under the scrutiny of experimental analysis , prove to be based on various forms of prior experience and learning [ 8 , 9 ] . similarly , while the firing of cpgs may correlate with sucking rhythms , it has yet to be shown that such isolated elements actually combine with other discrete components to create the real behavior of the suckling newborn , behavior that adapts to the unpredictable , dynamic geometry of the mother 's body , and behavior in real time and in real contexts . explaining the developmental origins of a congenital capability such as nipple attachment and sucking by a newborn upon confronting for the first time its mother 's external body in highly novel environment is a formidable challenge of clinical significance . the experimental literature , based on studies with non - human animals , contains a wealth of information pertinent to a better understanding of the onset and development of oral feeding skills . a brief description of the postnatal onset of sucking by rat pups will help frame the forthcoming presentation and analyses : rat pups are born after a gestation period of about three weeks . as each pup emerges from the birth canal , encased in an amniotic sac with the umbilical cord and placenta trailing , the dam assists with licking and gentle tugging . the mother removes the sac by licking and nibbling , consumes the placenta and membranes , licks the pup some more , licks herself , and then repeats the sequence as the next pup emerges . only after all the pups are thus delivered and the placentas are all consumed , does the rat dam turn her attention to the newborns , which she gathers into a clump in the nest and settles over them . we have described and quantified the labor and delivery process in rats , noting the stimulation received by the pups during labor and throughout the birth process . fortunately , the newborn rat 's behavior is robust and orderly ; when placed under suspended artificial surfaces simulating various properties of the dam 's ventrum , newborn rats show an organized repertoire of behaviors . thus , it is known that olfactory cues present on the rat mother 's nipples and ventrum are necessary and sufficient for newborn pups to locate and orally apprehend a nipple to suck . nipple attachment and suckling by newborns can be reinstated , however , by painting onto the dam 's ventrum a distillate of the wash taken from the dam 's body or by painting nipples with amniotic fluid or maternal saliva . one was that the key olfactory stimulus is somehow predetermined and that the newborn is correspondingly and inherently prepared ( hard - wired the second possibility was that the perinate responds with nipple attachment and sucking to the amniotic odor stimulus because of its previous experience with amniotic fluid . they tested this bold hypothesis by adding a novel , lemon - like substance to the amniotic fluid , and then testing whether this chemical would rescue the newborn 's ability to make its first attachment to the washed nipples of a mother rat . the previously validated experimental procedure involved externalizing the uterine horns of a gestational day ( gd ) 20 dam and injecting a small quantity ( 0.2 ml ) of a citral and saline solution through the transparent wall of the uterus into the amniotic fluid . then , on gd 22 , pups were delivered by caesarean section and placed immediately for one hr in a warmed nest where they were stroked with a soft artist 's brush for 1 hr in the presence of the citral odor . they performed an additional experiment in which pups were exposed to citral ( a ) in utero , ( b ) immediately after birth with stroking , or ( c ) both in utero and with postnatal stroking . only pups with the combined experiences attached to the washed , citral - scented nipples and not to the unwashed , normal nipples . pedersen and blass ' study provided important new insights into the initial plasticity of the newborn rats ' sucking , especially the specification of the cues that can activate and direct the behavior . it seems clear that the establishment of the olfactory control of sucking is determined by the experiences of the perinatal rat pup . we adapted elements of our previous investigations of the perinatal rats ' sensory experiences in the uterine environment and of the birth process [ 12 , 1820 ] to demonstrate that specific components of maternal stimulation are sufficient conditions for the odor learning that establishes the newborn 's sucking responses to maternal cues . the present paper is a review of some of this past research as well as a report of additional , previously unpublished data that , together , provide a new view of how the experience of being born creates a context for learning . that is , embedded in flow of events that constitute the birth process are forms and levels of stimulation that , together , create the contingencies for early , rapid learning in the fetus , as it becomes a newborn . we will show that this learning , though general in initial form , is expressed in the natural context of the mother 's body as organized , adaptive , seemingly goal - directed behavior . we will first review an analysis of rat maternal behavior during labor and delivery from which we derived a set of novel tools that enabled us to simulate the major components of vaginal birth . we will also review some of our evidence that fetal and neonatal rats ( perinates ) have sensory capabilities sufficient to experience the birth process , at least the elements that are needed for basic associative learning . then , we will present data showing that the perinates ' responses to simulations of the birth process ( a ) augment nipple attachment and sucking , ( b ) establish odor - guided responses to the mother , and ( c ) induce neural conditions that mediate state transitions between fetal and neonatal behavioral systems which can account for the activation and expression of the newborn 's initial sucking behavior . under laboratory conditions , norway rats typically give birth on the 22nd day of gestation ; by breeding our animals on a known day , we were able to be present with appropriate video arrangements to view and record the dams ' labor and delivery . from these videorecordings , we quantified an average of 144.6 labor contractions during the six hours prior to the birth of the first pup from eight dams . figure 1 illustrates the three types of visible labor contractions in rat dams and shows the average frequency of each during the 6 hrs of labor . behavioral expressions of labor in the rat progress from uterine peristalsis to lordosis contractions followed by vertical contractions that occur in close association with birth of each pup . duration of the delivery phase of parturition ( first to last birth ) ranged from 40 to 136 min , with dams delivering and average of 10.1 ( 1.1 ) pups . delivery duration and litter size were positively related ( r = .73 , p < as each pup begins to emerge from the birth canal , the dam typically adopts a head - between - heels posture , which facilitates delivery by enabling the mother to use her teeth to grasp the newborn and extract it from the vagina . during the initial phases of intense tactile and vestibular stimulation , as birth membranes were removed , especially from the head , pups began to emit the robust gasps that are characteristic of the onset of independent respiration , and thereafter they displayed gross movements and audible vocalizations . during parturition , offspring are exposed to seemingly harsh forms of stimulation related to cooling and with compressions under the weight of the dam 's body . after systematically describing and analyzing the kinds and amounts of stimulation sustained by rat fetuses as they were being delivered vaginally , we endeavored to assess quantitatively some of the most prominent forms of stimulation . among the forms of birth stimuli that we analyzed were uterine compressions , cooling and rewarming , and maternal licking . from these analyses , we created a set of procedures and tools to mimic the biological stimuli that represent the physical bases of the pups ' experience of being born . in this way , we established a protocol for simulating a vaginal birth for rat fetuses : 15 , 20 sec - long compressions delivered at a rate of 1 per min , cooling ( 22c ) , stroking with a soft brush ( 2 min ) , and rewarming ( 33c ) . several of our analyses have focused on the how the birth process , beginning with the mother 's labor contractions , helps organize the fetal - to - neonatal transition . in one set of studies , we applied our tools to study the components of birth that are important in the onset of pulmonary respiration , perhaps the most essential and immediate requirement of the newborn . in a study of gentle stroking of cesarean delivered pups ( without simulated labor contractions ) , only 25% nonstroked pups survived for 1 hr postpartum compared to 100% stroked pups supporting a role for sensory stimulation . these observations fit well with reports of adaptive neuroendocrine changes and neurobehavioral advantages in neonates , both term and preterm , exposed to tactile and kinesthetic stimulation [ 25 , 26 ] . the treatment regime , then , was designed to represent the sequence and duration of stimulation that normally occurs prior to and immediately after vaginal birth , leading to the onset of suckling . we sought to determine the mechanisms underlying the effectiveness of birth stimuli in facilitating the fetus - to - newborn transition . human babies show a surge of plasma catecholamines associated with the stress of being born , a physiological response to labor and squeezing through the birth canal [ 27 , 28 ] . we analyzed plasma catecholamines at 0 to 2 hr - old following either : ( a ) vaginal birth , ( b ) cesarean section with simulated labor contractions , or ( c ) cesarean section without labor contractions ( mimicking planned cesarean delivery ) . pups were exposed to the major elements of the rat 's natural birth process , as we have described ( i.e. our results , the first reported in the perinatal rat , closely parallel those reported in human studies and studies using the precocial sheep model [ 27 , 33 ] . birth stimuli , that is , the range , levels , and patterns of stimulation that comprise the birth process , might have multiple roles in the successful transition from fetal to postnatal life . our simulated birth model incorporates actual forms and levels of sensory and physiological stimuli to which the rat is exposed during natural vaginal birth and allows us to specifically parcel out the effects of labor on postpartum functions . the experience of labor is associated with a number of positive neonatal outcomes , including lung compliance , respiratory integrity [ 3537 ] , blood flow , resistance to oxidative stress , neonatal neurological condition , and complex global eeg patterns . together with the results reported herein , these studies support the view that prenatal events associated with labor initiate a cascade of neural , physiological and behavioral changes that assist the neonate 's successful transition to postnatal life events that assist the newborn infant 's adaptation to the extrauterine world . we now describe an original experiment conducted in our laboratory by abel in which individual , externalized , near - term rat fetuses received a combination of the simulated birth stimuli described earlier ( see figure 3 ) while in the presence of the odor citral and then tested for their responses to a rat dam with natural odors , washed of natural odors and with citral added . following this birth , each pup was stroked with a soft brush , mimicking the normal maternal licking and it also experienced cooling as it would after a natural birth and then rewarming as it would , had it been brought into the nest for maternal brooding . one set of pups experienced their birth in the presence of citral that was injected into the amniotic fluid prior to the intrauterine compressions and that was in the air around the pup while it was stroked and cooled and rewarmed . in contrast to their approach , we were able to specify and control the kinds , quantities , and timing of a specific stimulation sequence , and to observe the perinate at each stage of experimental manipulation . we predicted that a perinatal sequence of experiences in association with an otherwise neutral olfactory cue would lead to rates of nipple attachment to that cue , similar to those of vaginally delivered newborn rat pups to the odor of amniotic fluid . if the outcome of the simulated birth experience was equivalent to a natural delivery , we would consider this a successful empirical demonstration of sufficiency . we will have demonstrated that the experience of a simulated birth , quantitatively comparable to a natural , vaginal birth , is sufficient to establish a conditioned response to an odor that is expressed as nipple attachment and the onset of sucking in an , otherwise , nave newborn . upon completion of the compressions , fetuses were removed individually from the uterus and delivered onto gauze pads moistened with either citral ( 1 ml of 4 ml citral / l isotonic saline ) or isotonic saline - moistened ( 1 ml ) gauze pads ( figure 4 ) . to verify that alterations in frequency of suckling onset between compressed and noncompressed newborns were not related to deficits in respiration , respiratory movements were sampled ( 1 min ) at 3 postpartum time points : 10 min postpartum ; 1 hr postpartum ( while at 22c ) ; 2 hr postpartum ( while at 33c ) . then , to further verify the effectiveness of perinatal exposure to citral in promoting nipple attachment to a citral - scented dam , one group of compressed but citral - nave newborns was tested first on a citral - scented dam and then on a normal dam . the leftmost histogram bar in figure 5 shows that 90% of the vaginally - delivered newborns held before the ventrum of a natural ( unwashed ) anesthetized dam attached to a nipple and suckled . thus , this testing method enables rapid and reliable expression of the onset of postnatal ingestion , and the 90% attachment rate following vaginal birth can be used as standard against which we can evaluate the results of the simulated birth experiences . newborn pups that experienced a simulated vaginal birth in the presence of natural amniotic odors , including the regime of in utero compressions caesarean delivery membrane stripping cooling stroking rewarming ( see figure 3 ) attached to a nipple in 89% of the tests , as shown by the first hatched bar in figure 5 . newborns that experienced the a simulated vaginal birth absent compressions in the presence of natural amniotic odors , attached in only about 44% of the trials , which was a significant decrement in relation to littermates treated identically but with the compressions . thus , the complete simulated birth sequence ( including compressions ) , produced rates of nipple attachment in newborns that were fully comparable to those in vaginally delivered pups . newborn pups that experienced a simulated vaginal birth in the presence of citral in their amniotic fluid and in the atmosphere during stroking ( figure 4 ) attached to a nipple in 89% of the tests with a citral - scented dam , as shown by the stippled bar in figure 5 . newborns that experienced the simulated vaginal birth absent compressions in the presence of natural amniotic odors , attached in only about 20% of the trials with the citral - scented dam , a significant decrement in relation to littermates treated identically but with compressions . thus , the simulated birth sequence in the presence of citral , including compressions , produced rates of nipple attachment to a citral - scented dam that were fully comparable to those seen in vaginally - delivered pups and to pups that experienced the full simulated birth in the presence of amniotic odors when tested with a correspondingly natural - scented dam . the rightmost pair of histograms show that newborns that experienced simulated birth stimuli in the presence of citral in their amniotic fluid and in the atmosphere during stroking ( figure 4 ) when tested with a natural scent dam ( no citral during the test ) attached to a nipple in only 20% and 4% of the trials , for the compressed and noncompressed subjects . clearly , newborns that experienced birth in a citral environment were not prepared to attach to nipples on the body of a dam with only the natural scent of the species . it might be tempting to conclude that compressions mimicking labor contractions are necessary for efficient initiation of nipple attachment in the newborn , but the present experiment was not designed to allow such a conclusion . schaal and colleagues have suggested that amniotic odors provide a bridge from the fetus ' prenatal world to its postnatal environment , and the present results suggest that this bridge is constructed by the experiences embedded in parturition and that they result in a newborn behavior that has been rapidly assembled to follow the bridge to a nipple and the onset of suckling . stimulation associated with labor and delivery plays a key role in assisting the fetus ' transition to postnatal life by inducing and canalizing specific behaviors , and thereby operating as a critical link in the chain of behavioral adjustments required for adaptation to the postnatal habitat . nevertheless , it is also sensible to revel in the readiness and competence of a newborn mammal to adjust immediately to severance of its umbilical connection to the uterine world and to make an oral connection to the mother 's body and begin suckling . clearly , suckling is an important congenital behavior worth understanding for it is one of the primary adaptations to newborn life for all infant mammals , serving not only nutritive , immunological , and general physiological functions , but it is also a powerful component of bonding with the mother and creating a social context which supports sensory , motor , and cognitive development . much of what we explored in the present paper are lines of research that have been important in demystifying the kinds of basic processes that can explain the newborn 's congenital abilities to orient to novel features on the mother 's body surface and to initiate the complex , but vital behavior of suckling . from the findings that we reviewed , it can be concluded that the combination of sensory and motor processes that constitute successful suckling are rapidly assembled during the course of perinatal events . in the experiments we described , rat pups that did not experience the mechanical and thermal forces associated with vaginal birth failed to make the fetus - to - newborn transition . it appears that the set of sensory , endocrine , and neural events that comprise the physiological transition of birth also serve as factors in the perinate 's learning about the odor cues that are present in utero and carry over into the ex utero world . here , we can speculate on some of the implications we see when considering the experimental findings from rats in the context of human births and the onset of suckling . the results of our experiments with rats resemble their observations with human birth , and we have been able to take advantage of opportunities to control and manipulate the birth stimuli to gain insights into the embedded and embodied learning processes . while it may be beautiful and exciting to witness the eventual onset of sucking in a baby born at less than 30 weeks of age , it is sobering to contemplate the dramatically different factors and unnatural schedule of experiences that direct the prematurely - born baby to suck : a premie 's early postnatal development may be supported by intravenous nutrition and then gastric intubation until the baby presents signs of readiness for oral feeding .

kohn sham density - functional theory ( ks dft ) is the method most widely used in electronic structure calculations , due to its modest computational cost combined with an accuracy that is often competitive with much more expensive ab initio methods . the accuracy of the method is limited by the quality of approximations required to describe the quantum mechanical exchange and correlation ( xc ) interactions of electrons . a large number of density - functional approximations ( dfas ) for the xc energy have been developed in recent decades . the simplest dfas are based on the local density approximation ( lda ) , as proposed by ks in their 1965 paper , in which the xc energy is approximated as a functional of the density at a given point in space . the generalized gradient approximations ( ggas ) go beyond the lda by modeling the xc energy as a functional of the local density and its first derivative . the meta - ggas are closely related but their functional forms are also dependent on the ks kinetic energy density and/or , less commonly , the laplacian of the electron density . further developments led to the introduction of the occupied ks orbitals as ingredients for the xc energy ( hybrid functionals and self - interaction corrections , ) , and more recently also the virtual ks orbitals ( double - hybrid functionals and random - phase approximations ) . local hybrid functionals are also an interesting alternative approach to construct hybrid methods that are pertinent to the context of this work . the inclusion of additional dependencies in xc functionals has often resulted in significant improvements in their accuracy for general calculations . however , these improvements can not be described as systematic in the same way that the accuracy of an ab initio calculation may be systematically improved by considering a larger number of excited determinants ; some dfas give excellent results for particular systems but perform very poorly otherwise , and vice versa . there also remain many problems that none of the currently available dfas can accurately model . an important example of this , which is pertinent to this work , are strong correlation effects , commonly found in systems with near - degenerate orbitals such as the d- and f - block elements , but also in systems where chemical bonds are being broken or formed . in the present work , the problem of constructing dfas accurately for systems with and without strong correlation is examined by considering the adiabatic connection ( ac ) at the local level , i.e. , at each point of space . the ac , discussed in subsection 2.1 , provides an exact expression for the exchange and correlation energies by considering the changes that occur as the strength of electron interaction is smoothly increased from zero . this formalism has provided the basis for the development of several dfas , which attempt to interpolate the ac between the non - interacting and physical systems in order to estimate the xc energy . an advantage of the ac formalism crucial to our construction is that it allows the problem of strong correlation to be addressed in a more direct way , by creating interpolation models that are explicitly dependent on the strongly interacting limit , in addition to the non - interacting limit , of the ac . the strongly interacting limit of the ac has recently become the subject of much interest . the properties of the ac integrand in this limit reveal a highly nonlocal density dependence of correlation effects that can not be obtained from the standard ( semi)local or hybrid functionals . study of the strongly interacting limit in dft has focused on the strictly correlated electrons ( sce ) functional , where the electrons have an infinite interaction strength . this limit is of particular interest from a theoretical point of view as it can be studied exactly for one - dimensional systems and may be closely approximated in systems with spherical or cylindrical symmetry . these studies show that in the limit of infinite interaction strength certain integrals of the density appear in the exchange correlation functionals , revealing a mathematical structure very different from the one of the usual semilocal or orbital - dependent approximations . the nonlocal radius ( nlr ) functional proposed in ref ( 43 ) approximates the sce functional with a model that retains some of the sce nonlocality , introducing integrals of the spherically averaged density around a reference electron . the inclusion of the nlr functional into global and local interpolations along the adiabatic connection has been very recently explored by zhou et al . in another recent work , kong and proynov constructed a functional combining the information from the becke13 model and approximating local quantities along the ac . the aim of the present work is to start a systematic study of local interpolation models along the adiabatic connection , using at a first stage exact input ingredients , thus disentangling the errors due to the interpolation models from those due to the approximate ingredients . the local ac for several closed - shell atoms has been recently computed to high accuracy between the non - interacting and physical systems using the legendre fenchel formulation of dft due to lieb , and the lieb maximization method of refs ( 4952 ) . local information pertaining to the strongly interacting limit is calculated using the sce functional , and together these quantities are used to calculate local analogues of some established global ac interpolation functionals . we also discuss how to approximate crucial local ingredients such as the initial slope of the local adiabatic curve . in section 2 , relevant theoretical background is given including an overview of the ac formalism and the construction of dfas from both global and local variants of the ac . techniques for computing quantities along the ac are discussed , including the determination of the local ac as introduced in ref ( 47 ) . in section 2.3 the construction of a local model for the ac is discussed , considering the non - interacting and strong - interaction limits carefully in this context . finally the forms of some local interpolation models , taken from successful existing global models , are introduced . in section 4 the performance of these models is assessed for the helium and beryllium isoelectronic series and for dissociation of the h2 molecule , a system that typifies the failure of present dfas to properly account for strong correlation . the ac was proposed in a series of reports , which suggested that further insight into electronic correlations in dft may be gained by considering a system at constant electron density as the interaction strength is smoothly scaled between zero , i.e. , the ks auxiliary system , and the full physical interaction strength . this scaling of the interaction strength is achieved by the introduction of a simple coupling - constant coefficient , , such that the hamiltonian for any given is written as1where t is the kinetic energy operator , is the physical electron - interaction operator , and v is the operator representing an external potential v that binds the electron density at , such that it is always equal to the density of the physically interacting system ( = 1 , ) . as the value of is smoothly increased from zero to one , the system evolves adiabatically through a family of -dependent wave functions to the physical system described by 1 . given a hamiltonian , one can define the corresponding -dependent universal density functional aswhere eq 2b follows from the application of the hellmann this allows the well - known ac formula to be derived , yielding the following exact expression for the xc energy of an electronic system,3where is the ( global ) ac integrand , given by4[ ] is the ground - state wave function of in eq 1 , and u[ ] is the hartree ( coulomb ) energy . the ac integrand may be characterized by several features that can be exactly defined : the expansion of in the non - interacting limit is given by5while its expansion in the strongly interacting limit can be expressed as6here , the non - interacting terms and are the exchange energy and twice the second - order correlation energy given by grling their analogues at the strongly interacting limit , and , have been studied in refs ( 33 , 34 , and 38 ) and will be discussed further in section 3.2 . in addition to these asymptotic limits , the behavior of under uniform coordinate scaling is also well - defined , as discussed in ref ( 57 ) . to construct practical dfas one could consider modeling the integrand of eq 4 using a function that interpolates between the limits of eq 5 and eq 6 . the sce limit is of particular importance in the present work ; however , one could also consider models that intercept any other known point on the adiabatic connection for > 0 . several attempts to develop dfas based on these ideas have been put forward in the literature ; see , e.g. , refs ( 30 , 31 , 33 , 34 , and 5862 ) . each form makes a choice of a simple model function and the parameters on which to base the model . these parameters often include the known exact expressions for the parameters in eq 5 , and , since these may be computed from the set of kohn sham orbitals ( { p } ) and orbital energies ( { p } ) . the calculation of requires the gl2 correlation energy , which leads to a computational cost similar to the second - order mller plesset ( mp2 ) model used in ab initio quantum chemistry . the parameters in the sce limit entering eq 6 are clearly also of special interest in this context , and they can be computed numerically for atomic systems and molecules with cylindrical symmetry . more frequently dfas are derived for points along the ac with > 0 , often by employing scaling relations to derive forms from existing dfas . a similar strategy can also be used to approximate by a dfa ; see for example ref ( 60 ) . in tandem with choosing a set of exact or approximate values to parametrize a model for the ac one must also choose an appropriate model function for the ac integrand . a number of these have been suggested and many have been benchmarked in practical applications . one of the simplest ( and most often used ) is that of a [ 1/1 ] pad , as suggested by ernzerhof . a range of forms were suggested by cohen et al . and tested using approximate parametrizations , leading to the mcy1 functional in which a [ 1/1 ] pad model is employed . peach et al . attempted to disentangle approximations in the choice of parameters from those in the choice of model ac function by utilizing nearly exact ks orbitals and orbital energies derived from full configuration - interaction data to calculate and and the corresponding interacting wave functions to evaluate via eq 4 . our present study follows a similar philosophy , but applied to local , rather than global , interpolations . seidl and co - workers were the first to make use of the strong - interaction limit ( although approximated at a semilocal level , using the so - called point - charge - plus - continuum , or pc , functional ) in constructing a global ac model , known as the interaction strength interpolation ( isi ) functional . the revisi model and the models of liu and burke were later constructed to take account of the dependence of the second term of eq 6 , which was not correctly described by the isi approach . teale et al . also proposed forms for the ac integrand based on the structure of traditional ab initio methodologies and parametrized these forms to intercept values of the ac at any > 0 . the majority of these models for the global ac suffer from the fact they are not size consistent in practice . this deficiency arises from a nonlinear dependence on the parameters , , and a chosen approximation to . when these global parameters enter in a nonlinear fashion ( often as ratios ) , then size consistency is difficult to achieve . one route forward is to construct local ac models , which can replace these global parameters with local values defined at each point in space and may be more amenable to the construction of models that recover size consistency ( at least in the usual density - functional sense ) . the ac expression for the xc energy of a system as given in eq 3 describes a global quantity , integrated over the coupling constant . however , it may equally be written as the spatial integral of a local quantity analogous to the local value of an xc functional . to this effect , eq 3 may be rewritten as7where w(r ) is the energy density at a given . it is well - known that the energy density can not be uniquely defined ; an arbitrary number of terms may be added to w(r ) , yet an identical recovered if their spatial integral is zero . thus , any such energy densities are only defined within a particular gauge , and only energy densities defined in the same gauge may be meaningfully compared . in the context of the present work , it is both convenient and physically meaningful to define wxc,(r ) in the gauge of the electrostatic potential of the exchange correlation hole,9and p2(r , r ) is the pair density obtained from the wave function []10the definition of energy densities in the gauge of the xc hole is well - established in the literature , and further discussion may be found in refs ( 29 , 72 , and 73 ) . the coupling - constant - averaged ( -averaged ) xc energy density is defined as11since the spatial integral of the product of this quantity and the density yields the xc energy , the same quantity may be considered as a target to be modeled by xc functionals , although ggas and metaggas often aim at energy densities within different definitions . given the invariance of the exchange energy density to electron - interaction strength , eq 11 may be trivially resolved into separate exchange and correlation terms as12the aim of the local interpolation schemes examined in this work is to approximate wxc(r ) and wc(r ) through interpolating the local ac . in principle , this approach is analogous to that of the global ac interpolation schemes previously discussed , but rather than depending on quantities pertaining to the global ac , they are instead constructed from their local equivalents , w(r ) . obviously , a local interpolation will only be meaningful if all of the local terms are defined in the same gauge . it is again both convenient and logical to define all local quantities in the gauge of eq 8 , as in which highly accurate energy densities w(r ) in the range 0 1 have previously been calculated , and additionally can be computed for small systems in the limit . at = 0 , the energy density in the gauge of eq 8 is the exchange energy density w0(r ) = wx(r ) , often denoted x(r ) in the literature ( also equal to 1/2 the nonlocal slater potential ) , which is the crucial ingredient of local hybrid functionals . accurate and efficient computational schemes for this quantity have become available in recent years . in a way , local interpolation models can be viewed as local hybrids that carefully address the gauge problem . the local equivalent of is not as simple to define , yet is an essential component of ac interpolation schemes as it provides a measure of the departure from exchange - only behavior , in other words provides the information from which the correlation energy is approximated . while many global models use gl2 theory for this purpose , its dependence on global quantities makes it unclear how it could be applied to a local interpolation scheme . this is discussed in detail in section 3.1.2 . in order to assess the quality of our local interpolation functionals , it is necessary to have accurate data of energy densities , defined in the gauge of the xc hole . these may be acquired by the method of lieb maximization , described in refs ( 5052 ) . the lieb maximization is an optimization algorithm developed using the convex conjugate functional defined by lieb in ref ( 48 ) as the legendre fenchel transform to the energy , in which the density and potential v are conjugate variables , belonging to the dual vector spaces14and e[v ] is the energy yielded by a given electronic structure calculation at potential v(r ) . this convex conjugate formulation follows from the concavity of variationally determined energy e[v ] in v , from which lieb showed that f[ ] must be convex in . furthermore , the conjugate functional to a nonconcave energy , such as that which may result from a nonvariational calculation , remains well - defined as it is necessarily convex . a subsequent legendre fenchel transform of f[ ] yields the concave envelope ( least concave upper bound ) to e[v ] ; hence unique solutions to f[ ] can always be obtained . in the lieb maximization , the optimized density (r ) is obtained by maximizing f[ ] with respect to variations in the potential v(r ) , rather than by minimizing e[v ] with respect to (r ) as is usually the case . therefore , at convergence , the potential v(r ) in eq 13b is that which yields (r ) . in the present work , lieb maximizations have been carried out at a number of points along the ac in the range 0 1 ; hence the density is constrained such that = =1 , resulting in a -dependent optimizing potential . in order to effectively optimize with respect to the potential , we parametrize it by using the method of wu and yang ( wy ) as15where vext(r ) is the external potential due to nuclei , vref(r ) is a reference potential chosen to ensure that v(r ) has the correct asymptotic behavior , and { gt } are a set of gaussian functions with coefficients { bt}. in all calculations in this work we choose the potential expansion basis set to be identical to the primary orbital basis set . amaldi potential , and f[ ] is optimized with respect to the coefficients of the potential basis { bt}. additionally , convergence is accelerated through the use of the newton method described in refs ( 5052 ) . the relaxed - lagrangian formulation of helgaker and jrgensen is used to obtain relaxed densities for nonvariational wave functions , which serve as input to the lieb functional and are used in the determination of the derivatives required for its optimization . in this work , lieb maximization calculations are performed using the implementation of refs ( 5052 ) in a development version of the dalton quantum chemistry software package , in which e[v ] is computed by using coupled - cluster singles and doubles ( ccsd ) and full configuration - interaction ( fci ) wave functions . at convergence , where the optimizing potential is such that = =1 , the relaxed -interacting one- and two - particle density matrices are computed , with which the -dependent xc energy densities may be obtained as16 as described in section 2.3 , the initial slope of the ac is an important part of many global ac models , in which it may be calculated directly by gl2 perturbation theory ; however there is no analogous expression that yields the local equivalent , and we give such an expression in section 3.1.2 . here , the local initial slope of the xc energy density that is given in eq 8 is defined as17and is related to the global slope , hence the gl2 correlation energy , by18 in this study w0(r ) is numerically approximated by the method of finite difference , with a series of w(r ) for 1 . the resulting local slopes in the h2 molecule with bond lengths of 1.4 and 6.0 au are plotted along the h h bond in figure 1 , along with the densities from which they are calculated , at the fci level of theory and in the uncontracted aug - cc - pcvtz basis set . in both cases , the local slope is greatest in magnitude at the nuclei , as has been seen previously in atoms . it can be seen that the magnitude of the local slope is significantly larger in the stretched h2 molecule , mirroring observations previously made of the global ac in the dissociating hydrogen molecule . plots comparing the values of (r ) and w0(r ) , with respect to the distance from the bond midpoint , z / a.u . , along the principal axis of the h2 molecule with bond lengths of 1.4 au ( upper panel ) and 6.0 au ( lower panel ) . the local slopes in the he and be isoelectronic series are plotted in figures 2 and 3 , respectively . it is clear that , with increasing nuclear charge , the charge densities in both series become increaslingly contracted . the x - axis in both plots has been scaled by nuclear charge , highlighting a contrast in their behavior with respect to the uniform scaling condition,19which holds for nondegenerate ks systems . in figure 2 , it can be seen that the slope of the ac for the he series becomes less negative with increasing z , tending to an asymptotic value as z , consistent with the scaling relation of eq 19 . however , the slope of the ac in the be isoelectronic series becomes more negative with increasing z , indicating that the scaling relation is not satisfied by this series . plots of w0(r ) for the helium isoelectronic series , with nuclear charges 1 z 10 , and with radial distance from the nucleus r / a.u . scaled by nuclear charge . plots of w0(r ) for the beryllium isoelectronic series , with nuclear charges 4 z 10 , and with radial distance from the nucleus r / a.u . scaled by nuclear charge . while it is useful to numerically approximate the local slope for the purposes of evaluating local interpolation schemes , such functionals would only be viable for mainstream use in dft calculations if they can be described by simple functional forms . in global models , the initial slope can be calculated directly from the occupied and virtual ks orbitals according to gl2 theory,20where the indices i , j and a , b pertain to occupied and virtual ks orbitals , respectively , vxks is the local ks potential , and vxhf the nonlocal hartree the first term in eq 20 is analogous to the correlation energy given by mp2 theory , in which p and p are canonical hf orbitals and eigenvalues rather than ks ones . the second term accounts for the difference between the ks and hf exchange potentials and has a form similar to a singles term in many - body perturbation theory . previous studies of gl2 theory have found that the second term , although non - negligible , is small in magnitude relative to the mp2-like term evaluated with the ks orbitals . therefore , it follows that an approximation to the gl2 correlation energy may be obtained by evaluating the mp2 correlation energy with the ks orbitals and eigenvalues , ecgl2 ecmp2 . given that an approximation to the global ac slope may be obtained from an mp2-like calculation , it follows that an approximation to the local ac slope may be obtained by deriving a local form of this expression . while mp2 theory treats perturbations of the wave function , the analysis may be extended to energy densities in the gauge of the xc hole by means of eq 10 , as the substitution of eq 16 into eq 17 yields the following,21where p2(r , r ) is the derivative of the pair density at = 0,22notice that eq 21 ensures that w0(r ) is in the gauge of the electrostatic potential of the xc hole . given a non - interacting ground - state wave function , the perturbed wave function for || | | can be appproximated by the series expansion23if one assumes that is nondegenerate and has the form of a single slater determinant , the first - order correction to the wave function is given by24restricting the space of k(0 ) to doubly - excited determinants reduces this expression to25 in mp2 theory , contributions to the correlation energy from singly - excited determinants are necessarily zero due to brillouin s theorem . however , this is not strictly true in gl2 theory as the singles term in eq 20 makes a small , but nonzero , contribution to the gl2 correlation energy . as such , considering only double excitations in the model for the local slope can only yield approximations to the local slope ; spatial integration of this quantity will not return the exact gl2 correlation energy . condon rules to eq 25 allows it to be rewritten as26where the coefficient to ijab may be identified as an mp2 doubles amplitude tijab,27to obtain p2(r , r ) , it is necessary to take the derivative of the pair density corresponding to the perturbed wave function , + . substituting this into eq 10 and rearranging the resulting expressions yields the following,28where we assume that and are real and p2(r , r ) = n(n 1 ) ij(r ri ) (r rj ) is the pair - density operator . substituting eq 26 into this expression gives29which may then be resolved into the following orbital - explicit expression,30where i a is the kronecker over two spin functions : i*(ms ) a(ms ) dms = ia . substituting eq 30 into eq 21 finally results in an expression for the local slope,31where vabij(r ) is the antisymmetrized orbital potential,32multiplying the right - hand side of eq 31 by the density and integrating over all space , we recover twice the mp2-like expression . this is not an exact expression for the local slope , as the second term of eq 20 is not accounted for . however , the omitted term is generally small relative to the mp2-like term and vanishes entirely for two - electron systems ; hence the expression for the local slope in eq 31 should , in principle , be a fair approximation of the exact local slope . in future work we will implement and test eq 31 against the numerical results in section 3.1.1 . the doubles amplitudes tijab are readily obtainable from standard quantum chemical packages , and the potential vabij(r ) can also be readily calculated ; however , it would likely be computationally expensive to evaluate on a numerical grid . to reduce this cost , a range of techniques , commonly used to accelerate the calculation of integrals in linear - scaling software packages , may be employed . we note that the behavior of the local slopes presented in figures 1 , 2 , and 3 may be rationalized in a manner similar to that commonly discussed for global models in terms of eq 20 . this is because of the key role of the doubles amplitude tijab in eq 31 . the doubles amplitude has a dependence on the orbitals and orbital energies that is similar to that of the gl2 energy in eq 20 . we see in figure 1 that the local slope of the hydrogen molecule displays the minima at the nuclei . equation 31 , which is exact for two - electron systems , can be used to rationalize this observation . for closed shell two - electron systems with only one virtual orbital , eq 31 is simplified as follows:33even if we used a minimal orbital basis for the evaluation of the expression given in eq 33 for the hydrogen molecule , we would see that the local slope is most negative at the two nuclei , for any bond length . while this effect is captured with the minimal basis , the same minimal basis model would incorrectly describe the slope at the bond midpoint . for example , in the top panel of figure 1 we see that w0(r ) is less than 0 at the bond midpoint of h2 at r = 1.4 . within the minimal basis , the local slope would be exactly 0 for any r , as the antibonding 2(r ) orbital which enters eq 33 has a node at the bond midpoint . we also see in figure 2 that the correlation energy density for the he isoelectronic series scales quickly toward an asymptotic constant as z increases . furthermore , the local slope decays smoothly with distance from the nucleus , reflecting the behavior of vabij(r ) . the ks homo lumo gap is known to increase as z increases from 4 to 10 , from which one would expect the correlation energy to become less negative according to the behavior of tijab . in the core region this behavior holds ; however in the valence region the trend is opposite , with the correlation energy density becoming more negative with increasing z. this suggests that the numerator of tijab and the spatial dependence of vabij(r ) due to the form of the ks orbitals are dominant in this region , provided that eq 31 is sufficiently accurate for the be isoelectronic series . in recent years , the exact strong - coupling limit of the ac has been intensively studied . this limit reveals a new structure for the xc functional : instead of the traditional ingredients of dfas ( local density , density gradients , ks kinetic energy density , and occupied and unoccupied ks orbitals ) , it is observed that certain integrals of the density appear in this limit , encoding highly nonlocal information . tests on model physical and chemical systems ( electrons confined in low - dimensional geometries and low - density , ultracold dipolar systems , simple stretched bonds and anions ) have shown that taking into account this exact behavior can pave the way for the solution of the strong correlation problem in dft . however , the exact information encoded in the infinite coupling limit , described by the sce functional , does not come for free : the sce problem is ultra - nonlocal , and , although sparse in principle , its nonlinearity makes its exact evaluation for general three - dimensional geometry a complex task . a possible route to find suitable algorithms relies on the fact that constructing the exact sce functional for a given density is equivalent to solving an optimal transport ( or mass transportation theory ) problem with a cost function given by the coulomb interaction . this equivalence has triggered interest from the applied mathematics community working on optimal transport problems , which has led to the suggestion of several algorithms , together with very interesting exact results . so far , the sce solution is known exactly for one - dimensional systems . for spherically symmetric systems , a conjectured solution that is very close to the exact one ( and it is in many cases , but not always , exact ) has been proposed and used to address interesting physical problems . using algorithms and ideas from optimal transport , the sce problem for the hydrogen molecule along the dissociation curve has just recently been solved and both the global and local sce quantities have been computed . a more practical way to proceed is to build approximations for the sce functional inspired by its exact form , as it was done in the construction of the nlr functional . it corresponds to the wave function that minimizes the hamiltonian of eq 1 when . one can argue that the sce system is a better starting point than the kohn sham system for the description of very strongly correlated systems . the sce functional is defined as34the xc part can be easily extracted from , as . the ks sce approximation , proposed in ref ( 35 ) , uses the sce functional to approximate the hartree and exchange correlation energy , and it is equivalent to setting for all . it has been shown that ks sce yields good energies for systems where correlation plays a dominant role , such as electrons confined in low - density nanodevices or extremely stretched bonds . on the other hand , ks sce treats moderately and weakly correlated systems very poorly , giving energies that are unacceptably too low . a less drastic approximation is to construct a model , in such a way that its limit is given by the exact or approximate value of , as done in the pioneering work of seidl et al . analogously , one can also model w(r ) , imposing that its limit is given by w(r ) . this latter approach is the main object of the following sections . in the sce limit , the electrons are infinitely or perfectly correlated and their positions are given by an infinite superposition of classical configurations . the basic idea is that the electronic positions are all determined by a collective variable r , a feature that is captured by the so - called comotion functionsfi(r ) . if a reference electron is at r , then the position of all of the other electrons in the system will be given by ri = fi(r ) . since the electrons are perfectly correlated , the probability of finding the reference electron at r has to be the same as the probability of finding the ith electron at fi(r ) . therefore , the comotion functions have to satisfy the following differential equation:35for more details on the comotion functions , including their group properties , see refs ( 29 , 91 , 99 ) , and ( 33 ) . in terms of the comotion functions , the sce functional is given by36despite the high nonlocality of the sce functional , evident from eq 35 , we can easily compute its functional derivative from the following expression37equation 36 suggests the following energy density in the sce limit:38where vh(r ) is the hartree potential . this expression is indeed in the gauge of the xc hole potential of eq 8 , as proven in ref ( 29 ) . being derived from a wave function , the w(r ) energy density decays like , similar to the physical ( = 1 ) and the exchange ( = 0 ) energy densities of eq 16 . its functional derivative , eq 37 , has also the correct asymptotic behavior . to solve the sce problem for spherically symmetric systems ( the he and be isoelectronic series considered in this work ) , we have used the approach presented in ref ( 33 ) , which is exact if n = 2 . for atomic densities with n > 2 it could be either a very good approximation for the true minimum of eq 34 or , again , the exact result . for the h2 molecule we have used the results of ref ( 37 ) , where the sce energy density has been computed by obtaining the comotion function from the dual kantorovich formulation of the sce problem . the local interpolation models tested in this work are largely simple translations of the well - established global interpolation models into a local form . this was done for the model of seidl , perdew , and levy ( spl ) , the simplified model of liu and burke , which will be referred to here as the lb model , and the pad[1/1 ] model . each of the energy densities resulting from the three mentioned models is constructed from three local parameters , a , b , and c , which are defined in the gauge of the xc hole . in addition to these , we constructed a local form of the two - legged representation which , given some value of w1(r ) , takes the form whenever we used the two - legged representation to model the local ac in this work , we did it by incorporating the interpolated w1(r ) of the lb model : w1(r ) w1lb(r ) . by doing the local interpolation this way , we use the following three input quantities : w0(r ) , w0(r ) , and w(r ) and circumvent the direct utilization of the full interacting energy density , w1(r ) . in each of these four models , integration of w(r ) with respect to coupling constant gives the -averaged energy density wxc(r ) , which , if spatially integrated according to eq 7 , yields the xc energy exc[ ] . an important observation in the translation of global to local models is that while the following global inequalities are always satisfied,40their local counterparts do not necessarily satisfy these same inequalities . it has previously been observed for the hooke s atom series that , in the tail regions of the density , w(r ) can be less negative than w1(r ) . in this work , the crossing of w(r ) with wxc(r ) , w1(r ) , and w0(r ) has only been observed in the tail regions of the density and is thought to be an artifact of the numerical instability that occurs where the density is very small . although the helium isoelectronic series is a set of only two - electron systems , it is a useful series to consider in evaluating the local interpolation models as most standard dfas incorrectly characterize the hydride ion ( h ) , failing to predict its existence as a bound electronic system . here , local interpolation models are constructed from energy densities acquired by the lieb maximization at the fci level , as described in section 2.4 , in the range 0 1 and at = by evaluating the sce functional on the = 1 density , also at the fci level of theory . in table 2 , the correlation energies given by local forms of the spl , lb , two - legged representation ( the column labeled two - leg ) and pad[1/1 ] models ( the latter parametrized using the accurate values for w1(r ) , in order to compare with models that , instead , use the information ) are given , along with that given by the global spl model and the fci correlation energy for comparison . these data show that the local interpolation correlation energies are in close agreement with the fci reference values ; the mean absolute errors ( mae ) of the local interpolation models are 2.0 , 1.5 , 0.5 , and 0.1 mh , for the two - legged representation , spl , lb , and pad[1/1 ] models , respectively . as would be expected , the local pad[1/1 ] is the most accurate of the models , given that it is derived from the full interacting energy density . these data further suggest that the local lb model is marginally superior to the local spl and the two - legged representation . however , comparing the global and local models shows a slightly lower error for the global model ; the local spl model has an mae of 1.5 mh , compared to 1.3 mh for the global model . figure 4 compares the fci wc(r ) with that of the local lb and spl models , for the helium atom . this reflects the numerical data in table 2 , both being very close to the fci energy density but with slightly lower error in the lb model . plots comparing the fci , local lb , and local spl -averaged correlation energy density in the helium atom . the changes in correlation energy across the beryllium isoelectronic series are somewhat more complicated than those in the helium isoelectronic series , and its explanation involves the interplay of several effects . with increasing nuclear charge , the density becomes increasingly contracted , suggesting that the correlation energy should approach the high - density limit for very large z. however , this is accompanied by a changing ks homo lumo gap , here the energy difference between 2s and 2p orbitals , which increases from z = 4 13 before decreasing with higher z values . table 3 shows the reference and interpolated ec results for the be isoelectronic series , with z in the range 410 . the wave function for values between 0 and 1 has been computed in the same way as that for the he isoelectronic series , however at the ccsd level of theory rather than fci . as for the helium series , the local pad[1/1 ] that uses w1(r ) however , in contrast to the findings for he isoelectronic series , the local interpolation models are much more accurate than the global models . for example , in the case of f the global spl model has an mae of 47.4 mh , whereas the error for the local spl model is 3.5 mh . the local two - legged interpolation underestimates the correlation energies of the elements of the given series . the local spl and lb interpolation models appear to qualitatively capture the shell structure of wc(r ) ; however , in some regions it overestimates the reference value while in other regions the converse is the case . the error cancellation that results from this is the most likely explanation for the superior accuracy of the local models in comparison to the global models . plots comparing the ccsd , local lb , and local spl -averaged correlation energy density in the beryllium atom . despite the development of dft into the most widely applied electronic structure method , and the wealth of xc dfas that have been developed , there are some systems for which no combination of dfas provide an accurate description . standard dfas become increasingly inaccurate with greater h h bond length , reflecting a fundamental flaw of dfas in their inability to properly treat strong correlation . it has been seen previously that ks sce correctly predicts the dissociation of h2 in a spin - restricted formalism ; however at equilibrium geometry the energies it predicts are extremely low and the bond lengths predicted are overly short . the overall accuracy of ks - sce for h2 dissociation can be substantially improved by the addition of nonlocal corrections . figure 6 shows the dissociation curves for h2 given by the local interpolation models , along with those given by hf , fci , and the pbe functional for comparison . the computational details are the same as those of the he isoelectronic series , and the pbe , hf , and fci curves have been obtained from the dalton quantum chemistry package all within the uncontracted aug - cc - pcvtz basis set . burke , two - legged representation combined with the liu burke model , pad[1/1 ] with w1(r ) . it can be seen in figure 6 that all of the interpolation models correctly predict the dissociation of h2 , which follows from their inclusion of w(r ) . in global ac models , at infinite separation the initial slope diverges as a result of the vanishing homo lumo gap and the spl and lb models reduce to , yielding the exact energies . however , the dissociation curves produced by the local models approach the fci curve slowly , resulting in an unphysical bump-like feature . this is a well - known failing of dft , having been observed with other functionals , such as the random - phase approximation and even the global pad[1/1 ] model with . it can be seen in figure 6 that this is not remedied by the local interpolation approach , as the curve obtained by the local pad[1/1 ] also exhibits this unphysical bump , as does that given by the local spl model and , to a lesser extent , the local lb model . to analyze why the intermediate region is less accurately described by the local interpolation methods than the equilibrium and stretched region , we show in figure 7 the correlation component of the local ac at one of the nuclei of the hydrogen molecule at different bond lengths : r = 1.4 au ( at equilibrium ) , r = 5.0 au ( the intermediate region ) , and r = 13.0 au ( stretched bond ) . the structure of the three local ac curves at one of the nuclei is very similar to the structure of the corresponding global ac curves . from the given figure we see that at equilibrium the local ac is almost linear , so we can expect that even a single line segment approximation to the local ac : w(r ) w0(r ) + w0(r ) would properly capture the shape of the given local ac curve . the local ac curve at the nuclei of the stretched h2 exhibits the characteristic l - shape , which was also observed in the case of the corresponding global ac curve . we would expect that the two - legged representation would capture the given local ac very well , but even a single line segment approximation , w(r ) w(r ) , this time coming from the strong coupling limit , would be highly accurate for the stretched h2 . in contrast to the local ac curves of the stretched and h2 at equilibrium , the curvature of the local ac curve at the intermediate bond length is highly pronounced . the shapes of the local ac curves at the nuclei mirror the difference in correlation regimes present in the hydrogen molecule at different bond lengths . while in h2 at equilibrium and at very stretched bond length , correlation is almost purely dynamical and almost purely static , respectively , in the intermediate dissociation region there is a subtle interplay between the dynamical and static correlation . fci local correlation ac curves at one of the nuclei of h2 for different internuclear separations , r. in the intermediate region of the dissociation curve , where the unphysical bump is present , the local two - legged representation model is more accurate than the local pad[1/1 ] which we always use here with w1(r ) . this may be understood by comparing the exact local ac data with the interpolated quantities . the top panel of figure 8 shows the difference between wfci(r ) and that of each of the local interpolation models , along the h h bond at the 5.0 au geometry , as a function of the distance from the bond midpoint z. this difference w(r ) = wfci(r ) wmodel(r ) , is multiplied by the density to represent an energy per volume element . it shows that the local spl energy density is the one that most overestimates w(r ) . the error is smaller for the lb model and even more so for the local pad[1/1 ] model . the error is smallest in the two - legged model , obtained using the w1(r ) of the local lb . furthermore , there is the error cancellation in the two - legged model , as there are regions where the w(r ) of this model underestimates wfci(r ) . plots of the difference between fci and interpolated -averaged energy densities , w(z ) = wfci(z ) wmodel(z ) , with respect to the distance from the bond midpoint , z / a.u . ( upper panel ) , and the local ac curves at one of the nuclei of the fci and local interpolation models ( lower panel ) , both in h2 with a 5.0 au bond length . it can also be seen that the curves shown in the top panel of figure 8 have a maximum at the nucleus ( z = 2.5 a.u . ) . focusing on this region , it appears that the fci curve meets that of the pad[1/1 ] at = 1 and that the two - legged representation curve meets that of the lb model also at = 1 . this follows from the construction of the pad[1/1 ] and two - legged curves from w1fci(r ) and w1lb(r ) , respectively . all curves , except for that of the two - legged model , lie above the fci curve . in the case of the two - legged interpolation model , the first line segment is below the fci curve , as a result of eq 39a and the convexity of the given local ac curve . the second line segment that starts at x 0.1 the resulting error cancellation makes it clear why the two - legged representation appears more accurate than the other models . in this work we have studied local interpolations along the adiabatic connection for the he and be isoelectronic series and the hydrogen molecule , by using accurate input local quantities computed in the gauge of the electrostatic potential of the xc hole and comparing the results with nearly exact energy densities defined in the same way . in order to obtain approximations to the local ac over the physical regime ( 0 1 ) , we constructed interpolation models between the weak and strong coupling limits of dft . the weak coupling energy densities were obtained using the lieb variation principle , while the strong coupling limit energy densities were obtained using the strictly correlated electrons ( sce ) approach . the inclusion of the sce information in density - functional approximations helps to ensure their ability to capture the strong correlation effects . unlike previous attempts in this direction that used global ( integrated over all space ) input quantities to model the ac , the local approach is more amenable to the construction of approximations that do not violate size consistency , at least in the usual dft sense . since the aim here is to work in a restricted formalism , avoiding mimicking strong correlation with symmetry breaking , some care must be taken when discussing size consistency . in fact , strictly speaking , in a restricted framework the energy densities of the second - order perturbation theory and exact exchange are not intensive quantities in the presence of near degeneracy , which is the main challenge of capturing strong correlation within dft . in future work we will test different approximations for the sce energy densities and the local slope . the development of algorithms for solution of the sce problem is a very active research field . in spite of the recent improvements , we still lack an algorithm that will solve the sce problem for general 3d molecular geometries at low computational cost . however , a good candidate to approximate the sce energy density in the gauge of the xc hole potential is the nonlocal radius functional ( nlr ) , which has been already implemented and used in ref ( 44 ) . in addition to numerically exploring the local ac we have also reported the local weak - coupling slope of the adiabatic connection and derived an approximate expression for it in terms of occupied and unoccupied orbitals . this quantity is very important to signal the amount of correlation at each point of space . in our future work

the construction of density - functional approximations is explored by modeling the adiabatic connection locally , using energy densities defined in terms of the electrostatic potential of the exchange correlation hole . these local models are more amenable to the construction of size - consistent approximations than their global counterparts . in this work we use accurate input local ingredients to assess the accuracy of a range of local interpolation models against accurate exchange correlation energy densities . the importance of the strictly correlated electrons ( sce ) functional describing the strong coupling limit is emphasized , enabling the corresponding interpolated functionals to treat strong correlation effects . in addition to exploring the performance of such models numerically for the helium and beryllium isoelectronic series and the dissociation of the hydrogen molecule , an approximate analytic model is presented for the initial slope of the local adiabatic connection . comparisons are made with approaches based on global models , and prospects for future approximations based on the local adiabatic connection are discussed .

Introduction Theoretical Background Modeling the Local AC Results Conclusion and Perspectives

a large number of density - functional approximations ( dfas ) for the xc energy have been developed in recent decades . the simplest dfas are based on the local density approximation ( lda ) , as proposed by ks in their 1965 paper , in which the xc energy is approximated as a functional of the density at a given point in space . the generalized gradient approximations ( ggas ) go beyond the lda by modeling the xc energy as a functional of the local density and its first derivative . further developments led to the introduction of the occupied ks orbitals as ingredients for the xc energy ( hybrid functionals and self - interaction corrections , ) , and more recently also the virtual ks orbitals ( double - hybrid functionals and random - phase approximations ) . in the present work , the problem of constructing dfas accurately for systems with and without strong correlation is examined by considering the adiabatic connection ( ac ) at the local level , i.e. an advantage of the ac formalism crucial to our construction is that it allows the problem of strong correlation to be addressed in a more direct way , by creating interpolation models that are explicitly dependent on the strongly interacting limit , in addition to the non - interacting limit , of the ac . study of the strongly interacting limit in dft has focused on the strictly correlated electrons ( sce ) functional , where the electrons have an infinite interaction strength . the inclusion of the nlr functional into global and local interpolations along the adiabatic connection has been very recently explored by zhou et al . the aim of the present work is to start a systematic study of local interpolation models along the adiabatic connection , using at a first stage exact input ingredients , thus disentangling the errors due to the interpolation models from those due to the approximate ingredients . we also discuss how to approximate crucial local ingredients such as the initial slope of the local adiabatic curve . in section 2 , relevant theoretical background is given including an overview of the ac formalism and the construction of dfas from both global and local variants of the ac . in section 2.3 the construction of a local model for the ac is discussed , considering the non - interacting and strong - interaction limits carefully in this context . finally the forms of some local interpolation models , taken from successful existing global models , are introduced . in section 4 the performance of these models is assessed for the helium and beryllium isoelectronic series and for dissociation of the h2 molecule , a system that typifies the failure of present dfas to properly account for strong correlation . this scaling of the interaction strength is achieved by the introduction of a simple coupling - constant coefficient , , such that the hamiltonian for any given is written as1where t is the kinetic energy operator , is the physical electron - interaction operator , and v is the operator representing an external potential v that binds the electron density at , such that it is always equal to the density of the physically interacting system ( = 1 , ) . given a hamiltonian , one can define the corresponding -dependent universal density functional aswhere eq 2b follows from the application of the hellmann this allows the well - known ac formula to be derived , yielding the following exact expression for the xc energy of an electronic system,3where is the ( global ) ac integrand , given by4[ ] is the ground - state wave function of in eq 1 , and u[ ] is the hartree ( coulomb ) energy . the ac integrand may be characterized by several features that can be exactly defined : the expansion of in the non - interacting limit is given by5while its expansion in the strongly interacting limit can be expressed as6here , the non - interacting terms and are the exchange energy and twice the second - order correlation energy given by grling their analogues at the strongly interacting limit , and , have been studied in refs ( 33 , 34 , and 38 ) and will be discussed further in section 3.2 . the sce limit is of particular importance in the present work ; however , one could also consider models that intercept any other known point on the adiabatic connection for > 0 . one route forward is to construct local ac models , which can replace these global parameters with local values defined at each point in space and may be more amenable to the construction of models that recover size consistency ( at least in the usual density - functional sense ) . thus , any such energy densities are only defined within a particular gauge , and only energy densities defined in the same gauge may be meaningfully compared . in the context of the present work , it is both convenient and physically meaningful to define wxc,(r ) in the gauge of the electrostatic potential of the exchange correlation hole,9and p2(r , r ) is the pair density obtained from the wave function []10the definition of energy densities in the gauge of the xc hole is well - established in the literature , and further discussion may be found in refs ( 29 , 72 , and 73 ) . given the invariance of the exchange energy density to electron - interaction strength , eq 11 may be trivially resolved into separate exchange and correlation terms as12the aim of the local interpolation schemes examined in this work is to approximate wxc(r ) and wc(r ) through interpolating the local ac . obviously , a local interpolation will only be meaningful if all of the local terms are defined in the same gauge . in order to assess the quality of our local interpolation functionals , it is necessary to have accurate data of energy densities , defined in the gauge of the xc hole . in all calculations in this work we choose the potential expansion basis set to be identical to the primary orbital basis set . at convergence , where the optimizing potential is such that = =1 , the relaxed -interacting one- and two - particle density matrices are computed , with which the -dependent xc energy densities may be obtained as16 as described in section 2.3 , the initial slope of the ac is an important part of many global ac models , in which it may be calculated directly by gl2 perturbation theory ; however there is no analogous expression that yields the local equivalent , and we give such an expression in section 3.1.2 . here , the local initial slope of the xc energy density that is given in eq 8 is defined as17and is related to the global slope , hence the gl2 correlation energy , by18 in this study w0(r ) is numerically approximated by the method of finite difference , with a series of w(r ) for 1 . it can be seen that the magnitude of the local slope is significantly larger in the stretched h2 molecule , mirroring observations previously made of the global ac in the dissociating hydrogen molecule . plots of w0(r ) for the helium isoelectronic series , with nuclear charges 1 z 10 , and with radial distance from the nucleus r / a.u . in global models , the initial slope can be calculated directly from the occupied and virtual ks orbitals according to gl2 theory,20where the indices i , j and a , b pertain to occupied and virtual ks orbitals , respectively , vxks is the local ks potential , and vxhf the nonlocal hartree the first term in eq 20 is analogous to the correlation energy given by mp2 theory , in which p and p are canonical hf orbitals and eigenvalues rather than ks ones . while mp2 theory treats perturbations of the wave function , the analysis may be extended to energy densities in the gauge of the xc hole by means of eq 10 , as the substitution of eq 16 into eq 17 yields the following,21where p2(r , r ) is the derivative of the pair density at = 0,22notice that eq 21 ensures that w0(r ) is in the gauge of the electrostatic potential of the xc hole . as such , considering only double excitations in the model for the local slope can only yield approximations to the local slope ; spatial integration of this quantity will not return the exact gl2 correlation energy . however , the omitted term is generally small relative to the mp2-like term and vanishes entirely for two - electron systems ; hence the expression for the local slope in eq 31 should , in principle , be a fair approximation of the exact local slope . we note that the behavior of the local slopes presented in figures 1 , 2 , and 3 may be rationalized in a manner similar to that commonly discussed for global models in terms of eq 20 . we see in figure 1 that the local slope of the hydrogen molecule displays the minima at the nuclei . for closed shell two - electron systems with only one virtual orbital , eq 31 is simplified as follows:33even if we used a minimal orbital basis for the evaluation of the expression given in eq 33 for the hydrogen molecule , we would see that the local slope is most negative at the two nuclei , for any bond length . in the core region this behavior holds ; however in the valence region the trend is opposite , with the correlation energy density becoming more negative with increasing z. this suggests that the numerator of tijab and the spatial dependence of vabij(r ) due to the form of the ks orbitals are dominant in this region , provided that eq 31 is sufficiently accurate for the be isoelectronic series . this limit reveals a new structure for the xc functional : instead of the traditional ingredients of dfas ( local density , density gradients , ks kinetic energy density , and occupied and unoccupied ks orbitals ) , it is observed that certain integrals of the density appear in this limit , encoding highly nonlocal information . tests on model physical and chemical systems ( electrons confined in low - dimensional geometries and low - density , ultracold dipolar systems , simple stretched bonds and anions ) have shown that taking into account this exact behavior can pave the way for the solution of the strong correlation problem in dft . using algorithms and ideas from optimal transport , the sce problem for the hydrogen molecule along the dissociation curve has just recently been solved and both the global and local sce quantities have been computed . a more practical way to proceed is to build approximations for the sce functional inspired by its exact form , as it was done in the construction of the nlr functional . being derived from a wave function , the w(r ) energy density decays like , similar to the physical ( = 1 ) and the exchange ( = 0 ) energy densities of eq 16 . the local interpolation models tested in this work are largely simple translations of the well - established global interpolation models into a local form . in addition to these , we constructed a local form of the two - legged representation which , given some value of w1(r ) , takes the form whenever we used the two - legged representation to model the local ac in this work , we did it by incorporating the interpolated w1(r ) of the lb model : w1(r ) w1lb(r ) . by doing the local interpolation this way , we use the following three input quantities : w0(r ) , w0(r ) , and w(r ) and circumvent the direct utilization of the full interacting energy density , w1(r ) . in this work , the crossing of w(r ) with wxc(r ) , w1(r ) , and w0(r ) has only been observed in the tail regions of the density and is thought to be an artifact of the numerical instability that occurs where the density is very small . although the helium isoelectronic series is a set of only two - electron systems , it is a useful series to consider in evaluating the local interpolation models as most standard dfas incorrectly characterize the hydride ion ( h ) , failing to predict its existence as a bound electronic system . here , local interpolation models are constructed from energy densities acquired by the lieb maximization at the fci level , as described in section 2.4 , in the range 0 1 and at = by evaluating the sce functional on the = 1 density , also at the fci level of theory . these data show that the local interpolation correlation energies are in close agreement with the fci reference values ; the mean absolute errors ( mae ) of the local interpolation models are 2.0 , 1.5 , 0.5 , and 0.1 mh , for the two - legged representation , spl , lb , and pad[1/1 ] models , respectively . figure 4 compares the fci wc(r ) with that of the local lb and spl models , for the helium atom . the changes in correlation energy across the beryllium isoelectronic series are somewhat more complicated than those in the helium isoelectronic series , and its explanation involves the interplay of several effects . as for the helium series , the local pad[1/1 ] that uses w1(r ) however , in contrast to the findings for he isoelectronic series , the local interpolation models are much more accurate than the global models . the error cancellation that results from this is the most likely explanation for the superior accuracy of the local models in comparison to the global models . figure 6 shows the dissociation curves for h2 given by the local interpolation models , along with those given by hf , fci , and the pbe functional for comparison . the computational details are the same as those of the he isoelectronic series , and the pbe , hf , and fci curves have been obtained from the dalton quantum chemistry package all within the uncontracted aug - cc - pcvtz basis set . it can be seen in figure 6 that all of the interpolation models correctly predict the dissociation of h2 , which follows from their inclusion of w(r ) . in global ac models , at infinite separation the initial slope diverges as a result of the vanishing homo lumo gap and the spl and lb models reduce to , yielding the exact energies . to analyze why the intermediate region is less accurately described by the local interpolation methods than the equilibrium and stretched region , we show in figure 7 the correlation component of the local ac at one of the nuclei of the hydrogen molecule at different bond lengths : r = 1.4 au ( at equilibrium ) , r = 5.0 au ( the intermediate region ) , and r = 13.0 au ( stretched bond ) . we would expect that the two - legged representation would capture the given local ac very well , but even a single line segment approximation , w(r ) w(r ) , this time coming from the strong coupling limit , would be highly accurate for the stretched h2 . the shapes of the local ac curves at the nuclei mirror the difference in correlation regimes present in the hydrogen molecule at different bond lengths . fci local correlation ac curves at one of the nuclei of h2 for different internuclear separations , r. in the intermediate region of the dissociation curve , where the unphysical bump is present , the local two - legged representation model is more accurate than the local pad[1/1 ] which we always use here with w1(r ) . the top panel of figure 8 shows the difference between wfci(r ) and that of each of the local interpolation models , along the h h bond at the 5.0 au geometry , as a function of the distance from the bond midpoint z. this difference w(r ) = wfci(r ) wmodel(r ) , is multiplied by the density to represent an energy per volume element . ( upper panel ) , and the local ac curves at one of the nuclei of the fci and local interpolation models ( lower panel ) , both in h2 with a 5.0 au bond length . in this work we have studied local interpolations along the adiabatic connection for the he and be isoelectronic series and the hydrogen molecule , by using accurate input local quantities computed in the gauge of the electrostatic potential of the xc hole and comparing the results with nearly exact energy densities defined in the same way . in order to obtain approximations to the local ac over the physical regime ( 0 1 ) , we constructed interpolation models between the weak and strong coupling limits of dft . the weak coupling energy densities were obtained using the lieb variation principle , while the strong coupling limit energy densities were obtained using the strictly correlated electrons ( sce ) approach . the inclusion of the sce information in density - functional approximations helps to ensure their ability to capture the strong correlation effects . unlike previous attempts in this direction that used global ( integrated over all space ) input quantities to model the ac , the local approach is more amenable to the construction of approximations that do not violate size consistency , at least in the usual dft sense . in future work we will test different approximations for the sce energy densities and the local slope . in addition to numerically exploring the local ac we have also reported the local weak - coupling slope of the adiabatic connection and derived an approximate expression for it in terms of occupied and unoccupied orbitals .

kohn sham density - functional theory ( ks dft ) is the method most widely used in electronic structure calculations , due to its modest computational cost combined with an accuracy that is often competitive with much more expensive ab initio methods . further developments led to the introduction of the occupied ks orbitals as ingredients for the xc energy ( hybrid functionals and self - interaction corrections , ) , and more recently also the virtual ks orbitals ( double - hybrid functionals and random - phase approximations ) . however , these improvements can not be described as systematic in the same way that the accuracy of an ab initio calculation may be systematically improved by considering a larger number of excited determinants ; some dfas give excellent results for particular systems but perform very poorly otherwise , and vice versa . an important example of this , which is pertinent to this work , are strong correlation effects , commonly found in systems with near - degenerate orbitals such as the d- and f - block elements , but also in systems where chemical bonds are being broken or formed . an advantage of the ac formalism crucial to our construction is that it allows the problem of strong correlation to be addressed in a more direct way , by creating interpolation models that are explicitly dependent on the strongly interacting limit , in addition to the non - interacting limit , of the ac . these studies show that in the limit of infinite interaction strength certain integrals of the density appear in the exchange correlation functionals , revealing a mathematical structure very different from the one of the usual semilocal or orbital - dependent approximations . the aim of the present work is to start a systematic study of local interpolation models along the adiabatic connection , using at a first stage exact input ingredients , thus disentangling the errors due to the interpolation models from those due to the approximate ingredients . the local ac for several closed - shell atoms has been recently computed to high accuracy between the non - interacting and physical systems using the legendre fenchel formulation of dft due to lieb , and the lieb maximization method of refs ( 4952 ) . in section 4 the performance of these models is assessed for the helium and beryllium isoelectronic series and for dissociation of the h2 molecule , a system that typifies the failure of present dfas to properly account for strong correlation . this scaling of the interaction strength is achieved by the introduction of a simple coupling - constant coefficient , , such that the hamiltonian for any given is written as1where t is the kinetic energy operator , is the physical electron - interaction operator , and v is the operator representing an external potential v that binds the electron density at , such that it is always equal to the density of the physically interacting system ( = 1 , ) . given a hamiltonian , one can define the corresponding -dependent universal density functional aswhere eq 2b follows from the application of the hellmann this allows the well - known ac formula to be derived , yielding the following exact expression for the xc energy of an electronic system,3where is the ( global ) ac integrand , given by4[ ] is the ground - state wave function of in eq 1 , and u[ ] is the hartree ( coulomb ) energy . the ac integrand may be characterized by several features that can be exactly defined : the expansion of in the non - interacting limit is given by5while its expansion in the strongly interacting limit can be expressed as6here , the non - interacting terms and are the exchange energy and twice the second - order correlation energy given by grling their analogues at the strongly interacting limit , and , have been studied in refs ( 33 , 34 , and 38 ) and will be discussed further in section 3.2 . attempted to disentangle approximations in the choice of parameters from those in the choice of model ac function by utilizing nearly exact ks orbitals and orbital energies derived from full configuration - interaction data to calculate and and the corresponding interacting wave functions to evaluate via eq 4 . seidl and co - workers were the first to make use of the strong - interaction limit ( although approximated at a semilocal level , using the so - called point - charge - plus - continuum , or pc , functional ) in constructing a global ac model , known as the interaction strength interpolation ( isi ) functional . in the context of the present work , it is both convenient and physically meaningful to define wxc,(r ) in the gauge of the electrostatic potential of the exchange correlation hole,9and p2(r , r ) is the pair density obtained from the wave function []10the definition of energy densities in the gauge of the xc hole is well - established in the literature , and further discussion may be found in refs ( 29 , 72 , and 73 ) . the coupling - constant - averaged ( -averaged ) xc energy density is defined as11since the spatial integral of the product of this quantity and the density yields the xc energy , the same quantity may be considered as a target to be modeled by xc functionals , although ggas and metaggas often aim at energy densities within different definitions . given the invariance of the exchange energy density to electron - interaction strength , eq 11 may be trivially resolved into separate exchange and correlation terms as12the aim of the local interpolation schemes examined in this work is to approximate wxc(r ) and wc(r ) through interpolating the local ac . at = 0 , the energy density in the gauge of eq 8 is the exchange energy density w0(r ) = wx(r ) , often denoted x(r ) in the literature ( also equal to 1/2 the nonlocal slater potential ) , which is the crucial ingredient of local hybrid functionals . the lieb maximization is an optimization algorithm developed using the convex conjugate functional defined by lieb in ref ( 48 ) as the legendre fenchel transform to the energy , in which the density and potential v are conjugate variables , belonging to the dual vector spaces14and e[v ] is the energy yielded by a given electronic structure calculation at potential v(r ) . in order to effectively optimize with respect to the potential , we parametrize it by using the method of wu and yang ( wy ) as15where vext(r ) is the external potential due to nuclei , vref(r ) is a reference potential chosen to ensure that v(r ) has the correct asymptotic behavior , and { gt } are a set of gaussian functions with coefficients { bt}. at convergence , where the optimizing potential is such that = =1 , the relaxed -interacting one- and two - particle density matrices are computed , with which the -dependent xc energy densities may be obtained as16 as described in section 2.3 , the initial slope of the ac is an important part of many global ac models , in which it may be calculated directly by gl2 perturbation theory ; however there is no analogous expression that yields the local equivalent , and we give such an expression in section 3.1.2 . here , the local initial slope of the xc energy density that is given in eq 8 is defined as17and is related to the global slope , hence the gl2 correlation energy , by18 in this study w0(r ) is numerically approximated by the method of finite difference , with a series of w(r ) for 1 . the resulting local slopes in the h2 molecule with bond lengths of 1.4 and 6.0 au are plotted along the h h bond in figure 1 , along with the densities from which they are calculated , at the fci level of theory and in the uncontracted aug - cc - pcvtz basis set . in global models , the initial slope can be calculated directly from the occupied and virtual ks orbitals according to gl2 theory,20where the indices i , j and a , b pertain to occupied and virtual ks orbitals , respectively , vxks is the local ks potential , and vxhf the nonlocal hartree the first term in eq 20 is analogous to the correlation energy given by mp2 theory , in which p and p are canonical hf orbitals and eigenvalues rather than ks ones . while mp2 theory treats perturbations of the wave function , the analysis may be extended to energy densities in the gauge of the xc hole by means of eq 10 , as the substitution of eq 16 into eq 17 yields the following,21where p2(r , r ) is the derivative of the pair density at = 0,22notice that eq 21 ensures that w0(r ) is in the gauge of the electrostatic potential of the xc hole . given a non - interacting ground - state wave function , the perturbed wave function for || | | can be appproximated by the series expansion23if one assumes that is nondegenerate and has the form of a single slater determinant , the first - order correction to the wave function is given by24restricting the space of k(0 ) to doubly - excited determinants reduces this expression to25 in mp2 theory , contributions to the correlation energy from singly - excited determinants are necessarily zero due to brillouin s theorem . for closed shell two - electron systems with only one virtual orbital , eq 31 is simplified as follows:33even if we used a minimal orbital basis for the evaluation of the expression given in eq 33 for the hydrogen molecule , we would see that the local slope is most negative at the two nuclei , for any bond length . in the core region this behavior holds ; however in the valence region the trend is opposite , with the correlation energy density becoming more negative with increasing z. this suggests that the numerator of tijab and the spatial dependence of vabij(r ) due to the form of the ks orbitals are dominant in this region , provided that eq 31 is sufficiently accurate for the be isoelectronic series . this limit reveals a new structure for the xc functional : instead of the traditional ingredients of dfas ( local density , density gradients , ks kinetic energy density , and occupied and unoccupied ks orbitals ) , it is observed that certain integrals of the density appear in this limit , encoding highly nonlocal information . tests on model physical and chemical systems ( electrons confined in low - dimensional geometries and low - density , ultracold dipolar systems , simple stretched bonds and anions ) have shown that taking into account this exact behavior can pave the way for the solution of the strong correlation problem in dft . however , the exact information encoded in the infinite coupling limit , described by the sce functional , does not come for free : the sce problem is ultra - nonlocal , and , although sparse in principle , its nonlinearity makes its exact evaluation for general three - dimensional geometry a complex task . in terms of the comotion functions , the sce functional is given by36despite the high nonlocality of the sce functional , evident from eq 35 , we can easily compute its functional derivative from the following expression37equation 36 suggests the following energy density in the sce limit:38where vh(r ) is the hartree potential . this was done for the model of seidl , perdew , and levy ( spl ) , the simplified model of liu and burke , which will be referred to here as the lb model , and the pad[1/1 ] model . in addition to these , we constructed a local form of the two - legged representation which , given some value of w1(r ) , takes the form whenever we used the two - legged representation to model the local ac in this work , we did it by incorporating the interpolated w1(r ) of the lb model : w1(r ) w1lb(r ) . in each of these four models , integration of w(r ) with respect to coupling constant gives the -averaged energy density wxc(r ) , which , if spatially integrated according to eq 7 , yields the xc energy exc[ ] . in this work , the crossing of w(r ) with wxc(r ) , w1(r ) , and w0(r ) has only been observed in the tail regions of the density and is thought to be an artifact of the numerical instability that occurs where the density is very small . although the helium isoelectronic series is a set of only two - electron systems , it is a useful series to consider in evaluating the local interpolation models as most standard dfas incorrectly characterize the hydride ion ( h ) , failing to predict its existence as a bound electronic system . here , local interpolation models are constructed from energy densities acquired by the lieb maximization at the fci level , as described in section 2.4 , in the range 0 1 and at = by evaluating the sce functional on the = 1 density , also at the fci level of theory . in table 2 , the correlation energies given by local forms of the spl , lb , two - legged representation ( the column labeled two - leg ) and pad[1/1 ] models ( the latter parametrized using the accurate values for w1(r ) , in order to compare with models that , instead , use the information ) are given , along with that given by the global spl model and the fci correlation energy for comparison . these data show that the local interpolation correlation energies are in close agreement with the fci reference values ; the mean absolute errors ( mae ) of the local interpolation models are 2.0 , 1.5 , 0.5 , and 0.1 mh , for the two - legged representation , spl , lb , and pad[1/1 ] models , respectively . with increasing nuclear charge , the density becomes increasingly contracted , suggesting that the correlation energy should approach the high - density limit for very large z. however , this is accompanied by a changing ks homo lumo gap , here the energy difference between 2s and 2p orbitals , which increases from z = 4 13 before decreasing with higher z values . the computational details are the same as those of the he isoelectronic series , and the pbe , hf , and fci curves have been obtained from the dalton quantum chemistry package all within the uncontracted aug - cc - pcvtz basis set . it can be seen in figure 6 that this is not remedied by the local interpolation approach , as the curve obtained by the local pad[1/1 ] also exhibits this unphysical bump , as does that given by the local spl model and , to a lesser extent , the local lb model . to analyze why the intermediate region is less accurately described by the local interpolation methods than the equilibrium and stretched region , we show in figure 7 the correlation component of the local ac at one of the nuclei of the hydrogen molecule at different bond lengths : r = 1.4 au ( at equilibrium ) , r = 5.0 au ( the intermediate region ) , and r = 13.0 au ( stretched bond ) . from the given figure we see that at equilibrium the local ac is almost linear , so we can expect that even a single line segment approximation to the local ac : w(r ) w0(r ) + w0(r ) would properly capture the shape of the given local ac curve . fci local correlation ac curves at one of the nuclei of h2 for different internuclear separations , r. in the intermediate region of the dissociation curve , where the unphysical bump is present , the local two - legged representation model is more accurate than the local pad[1/1 ] which we always use here with w1(r ) . the top panel of figure 8 shows the difference between wfci(r ) and that of each of the local interpolation models , along the h h bond at the 5.0 au geometry , as a function of the distance from the bond midpoint z. this difference w(r ) = wfci(r ) wmodel(r ) , is multiplied by the density to represent an energy per volume element . in this work we have studied local interpolations along the adiabatic connection for the he and be isoelectronic series and the hydrogen molecule , by using accurate input local quantities computed in the gauge of the electrostatic potential of the xc hole and comparing the results with nearly exact energy densities defined in the same way . in fact , strictly speaking , in a restricted framework the energy densities of the second - order perturbation theory and exact exchange are not intensive quantities in the presence of near degeneracy , which is the main challenge of capturing strong correlation within dft . however , a good candidate to approximate the sce energy density in the gauge of the xc hole potential is the nonlocal radius functional ( nlr ) , which has been already implemented and used in ref ( 44 ) . in addition to numerically exploring the local ac we have also reported the local weak - coupling slope of the adiabatic connection and derived an approximate expression for it in terms of occupied and unoccupied orbitals .

this randomized controlled trial was conducted in five primary care clinics affiliated with the edmonton south side primary care network in edmonton , canada . these primary care teams were akin to the patient - centered medical home ( 15 ) and consisted of physicians and nurses who had support from dietitians , physiotherapists , and social workers as needed . patients were eligible if they had type 2 diabetes , were regularly seen by the primary care team , and did not qualify for urgent specialist referral and assessment ( according to protocol , a fasting blood glucose 17 mmol / l , blood pressure 220/120 mmhg , or triglycerides 15 mmol / l ) . we excluded patients who were followed in specialty clinics for diabetes , hypertension , or dyslipidemia ; who were cognitively impaired ; who were not responsible for their own medication administration ; or who were unable to communicate in english . blood pressure screening was not conducted during patient recruitment to minimize contamination of controls . because type 2 diabetic patients have higher blood pressure levels than the general population ( 1 ) eligible patients were identified from the clinic roster , and a clinic staff member made initial contact to tell patients about the study . patients were told that the study was designed to help improve medication therapy for heart disease risk in patients with type 2 diabetes . the specific focus of the study , hypertension , was listed among other risk factors for heart disease . the university of alberta health research ethics board approved the study protocol , and all participants gave written informed consent . pharmacists were given access to the patient 's clinical chart after consent to participate in the study was obtained . a central randomization service ( www.epicore.ualberta.ca ) provided computer - generated random sequences stratified by the primary care clinic for treatment allocation . pharmacists , analysts , and investigators were unaware of the block size and allocation sequence to preserve allocation concealment . control patients received usual care by the primary care team without contributions from study pharmacists , except for standardized blood pressure measurements at the end of the follow - up period ( see below ) . two pharmacists providing the intervention program held a bachelor 's degree in pharmacy , were certified diabetes educators , and had practiced in community pharmacies for over 5 years . both pharmacists completed structured online training courses for hypertension and diabetes management ( www.pharmalearn.com ) and reviewed the canadian hypertension education program and canadian diabetes association guideline recommendations prior to starting the study ( 16,17 ) . the intervention program began with an in - person visit with a study pharmacist to identify all prescription , nonprescription , complementary , and alternative medications . pharmacists also measured the patient 's height , weight , heart rate , and blood pressure . blood pressure was measured according to the canadian hypertension education program recommendations using the bptru bpm-100 ( vsm med tech , coquitlam , bc ) automated machine set to report the average of five measurements at 1-min intervals ( 16 ) . pharmacists then formulated guideline - concordant recommendations to optimize medication management of blood pressure and other cardiovascular risk factors . these recommendations were discussed with the primary care physician who was responsible for authorizing medication changes . interim contact with intervention patients was made at the discretion of the pharmacist , physician , or patient and could be conducted via telephone or in person . interim contacts were used to determine whether medication changes were implemented and to address questions or concerns since the previous encounter ( e.g. , side effects , adverse events , or adherence issues ) . pharmacists recorded the date , duration , and nature of each contact with a study patient . after 1 year , all patients were seen in the primary care clinic to review medications , measure blood pressure with the automated machine , and obtain a fasting blood sample to measure blood glucose , a1c , and cholesterol profile . patients also reported the number of encounters with specialists , other health care professionals , regional health care resources , emergency - room visits , and hospitalizations during the previous year . the primary outcome was achievement of a clinically important reduction in blood pressure , defined as a 10% decrease in systolic blood pressure at 1 year ( 18 ) . secondary outcomes included the absolute change in systolic blood pressure from baseline to 1 year , achievement of recommended blood pressure targets ( < 130/80 mmhg ) ( 8) , and antihypertensive medication changes . we also measured the change in predicted 10-year risk of cardiovascular disease using the uk prospective diabetes study ( ukpds ) risk engine ( 19 ) . baseline and follow - up values for a1c , systolic blood pressure , total cholesterol , and hdl cholesterol were used to calculate the change in the ukpds risk engine score . our sample size was based on observations from a previous study ( 20 ) examining the effect of a diabetes intervention program aimed at physicians . in that study , 40% of intervention patients and 25% of control patients achieved a 10% decrease in blood pressure at 6 months . although the current study follow - up was twice as long and the intervention was directed at patients , we estimated that the event rates would be similar . with a two - sided of 0.05 and 80% power , we estimated the total sample size would be 300 patients . we used statistics to test for between - group differences in the primary outcome . the association between treatment group and achievement of the primary outcome was also examined using a logistic regression model to calculate an odds ratio ( or ) and 95% ci . based on the results of related trials in our region ( 13,20,21 ) , we assumed that patient - related outcomes were statistically independent of one another , with intracluster correlation coefficients < 0.01 . all patients were evaluated in the groups to which they were randomly allocated according to the intention - to - treat principle . missing data were replaced by carrying the last observation forward . to test the robustness of our observations , we restricted our analyses to patients who completed the full study protocol and patients with inadequately controlled hypertension at baseline . first , we used indicator variables to directly adjust for all 18 family physicians involved in the study in a multivariate logistic regression model . second , we used generalized estimating equation methodology to account for the potential correlations of outcomes among patients treated by the same physician . none of the sensitivity analyses changed the direction , magnitude , or statistical significance of our findings ; therefore , we report only our prespecified analyses . a p value of < 0.05 was considered statistically significant , and pasw statistics version 18.0 ( spss , chicago , il ) was used for all analyses . a central randomization service ( www.epicore.ualberta.ca ) provided computer - generated random sequences stratified by the primary care clinic for treatment allocation . pharmacists , analysts , and investigators were unaware of the block size and allocation sequence to preserve allocation concealment . control patients received usual care by the primary care team without contributions from study pharmacists , except for standardized blood pressure measurements at the end of the follow - up period ( see below ) . two pharmacists providing the intervention program held a bachelor 's degree in pharmacy , were certified diabetes educators , and had practiced in community pharmacies for over 5 years . both pharmacists completed structured online training courses for hypertension and diabetes management ( www.pharmalearn.com ) and reviewed the canadian hypertension education program and canadian diabetes association guideline recommendations prior to starting the study ( 16,17 ) . the intervention program began with an in - person visit with a study pharmacist to identify all prescription , nonprescription , complementary , and alternative medications . pharmacists also measured the patient 's height , weight , heart rate , and blood pressure . blood pressure was measured according to the canadian hypertension education program recommendations using the bptru bpm-100 ( vsm med tech , coquitlam , bc ) automated machine set to report the average of five measurements at 1-min intervals ( 16 ) . pharmacists then formulated guideline - concordant recommendations to optimize medication management of blood pressure and other cardiovascular risk factors . these recommendations were discussed with the primary care physician who was responsible for authorizing medication changes . baseline characteristics were obtained from the clinic chart . interim contact with intervention patients was made at the discretion of the pharmacist , physician , or patient and could be conducted via telephone or in person . interim contacts were used to determine whether medication changes were implemented and to address questions or concerns since the previous encounter ( e.g. , side effects , adverse events , or adherence issues ) . pharmacists recorded the date , duration , and nature of each contact with a study patient . after 1 year , all patients were seen in the primary care clinic to review medications , measure blood pressure with the automated machine , and obtain a fasting blood sample to measure blood glucose , a1c , and cholesterol profile . patients also reported the number of encounters with specialists , other health care professionals , regional health care resources , emergency - room visits , and hospitalizations during the previous year . the primary outcome was achievement of a clinically important reduction in blood pressure , defined as a 10% decrease in systolic blood pressure at 1 year ( 18 ) . secondary outcomes included the absolute change in systolic blood pressure from baseline to 1 year , achievement of recommended blood pressure targets ( < 130/80 mmhg ) ( 8) , and antihypertensive medication changes . we also measured the change in predicted 10-year risk of cardiovascular disease using the uk prospective diabetes study ( ukpds ) risk engine ( 19 ) . baseline and follow - up values for a1c , systolic blood pressure , total cholesterol , and hdl cholesterol were used to calculate the change in the ukpds risk engine score . our sample size was based on observations from a previous study ( 20 ) examining the effect of a diabetes intervention program aimed at physicians . in that study , 40% of intervention patients and 25% of control patients achieved a 10% decrease in blood pressure at 6 months . although the current study follow - up was twice as long and the intervention was directed at patients , we estimated that the event rates would be similar . with a two - sided of 0.05 and 80% power , we estimated the total sample size would be 300 patients . we used statistics to test for between - group differences in the primary outcome . the association between treatment group and achievement of the primary outcome was also examined using a logistic regression model to calculate an odds ratio ( or ) and 95% ci . based on the results of related trials in our region ( 13,20,21 ) , we assumed that patient - related outcomes were statistically independent of one another , with intracluster correlation coefficients < 0.01 . all patients were evaluated in the groups to which they were randomly allocated according to the intention - to - treat principle . missing data were replaced by carrying the last observation forward . to test the robustness of our observations , we restricted our analyses to patients who completed the full study protocol and patients with inadequately controlled hypertension at baseline . first , we used indicator variables to directly adjust for all 18 family physicians involved in the study in a multivariate logistic regression model . second , we used generalized estimating equation methodology to account for the potential correlations of outcomes among patients treated by the same physician . none of the sensitivity analyses changed the direction , magnitude , or statistical significance of our findings ; therefore , we report only our prespecified analyses . a p value of < 0.05 was considered statistically significant , and pasw statistics version 18.0 ( spss , chicago , il ) was used for all analyses . we enrolled 260 patients between 28 february 2006 and 6 december 2007 and randomly allocated 131 to the intervention and 129 to the control group ( fig . the main reasons for exclusion were that the pharmacist could not contact eligible patients ( 700 of 1,183 [ 59% ] ) and patient refusal ( 211 of 1,183 [ 18% ] ) . there were 21 intervention patients ( 14 withdrew , 6 were lost to follow - up , and 1 died ) and 16 control patients ( 10 withdrew and 6 were lost to follow - up ) who did not complete the study ( p > 0.05 for all comparisons ) . there were no differences in age , sex , diabetes duration , or baseline blood pressure between the patients who did or did not complete the study . icd-9 , international classification of diseases , 9th revision ; bp , blood pressure ; itt , intention to treat . there were 149 ( 57.3% ) women , mean ( sd ) age was 59.1 11.6 years , bmi was 32.5 6.5 kg / m , diabetes duration was 5.5 6.5 years , and a1c was 7.4 1.4% . the mean blood pressure at baseline was 129.4 15.3/74.1 10.4 mmhg , with 82 of 131 ( 63% ) intervention patients and 71 of 129 ( 55% ) control patients having inadequately controlled hypertension ( 130/80 mmhg ; p = 0.22 for difference ) . of 107 patients with a blood pressure < 130/80 mmhg , 76 were taking one or more antihypertensive medications at baseline . baseline characteristics data are means sd for continuous variables and n ( % ) for categorical variables . over 1 year , there was a statistically significant reduction in systolic blood pressure for intervention patients ( mean decrease 7.4 mmhg [ 95% ci 4.610.2 ] ; p < 0.001 ) but not for control patients ( 2.5 mmhg [ 0.1 to 5.2 ] ; p = 0.06 ) ( supplementary fig . the between - group difference in systolic blood pressure change at 1 year was 4.9 mmhg ( 95% ci 1.08.7 ; p = 0.01 ) ( supplementary table 2 ) in favor of the intervention . the primary outcome was achieved by 48 of 131 ( 37% ) intervention patients and 30 of 129 ( 23% ) control patients ( or 1.91 [ 95% ci 1.113.28 ] ; p = 0.02 ) ( fig . the absolute difference of 14% translates to a number needed to treat ( nnt ) of seven patients followed for 1 year by a pharmacist to achieve one additional patient with better blood pressure control compared with usual care . proportion of patients achieving a 10% decrease in systolic blood pressure at 1 year ( primary outcome ) . limiting our analyses to 223 patients who completed the mean decrease in systolic blood pressure was 7.7 mmhg ( 95% ci 4.510.9 ; p < 0.001 ) for intervention patients and 2.8 mmhg ( 0.2 to 5.8 ; p = 0.07 ) for control patients ( p = 0.03 for between - group differences ) . more intervention patients achieved the primary outcome ( 41 of 110 [ 37% ] ) compared with control patients ( 30 of 113 [ 27% ] ) ; however , this difference was not statistically significant ( p = 0.09 ) . we observed a larger treatment effect when blood pressure changes were examined in 153 patients with inadequately controlled hypertension at baseline . mean blood pressure at baseline was 138.7 11.4/78.8 9.4 mmhg for intervention patients and 137.9 14.1/78.1 11.4 mmhg for control patients ( p > 0.05 ) . all other baseline characteristics for these 153 patients were well balanced between the two groups ( p > 0.05 for all comparisons ) . systolic blood pressure decreased a mean of 13.9 mmhg ( 95% ci 10.617.1 ; p < 0.001 ) for intervention patients and 6.7 mmhg ( 3.210.1 ; p < 0.001 ) for control patients ( p = 0.002 for between - group differences ) ( supplementary fig . the primary outcome was achieved by 41 of 82 ( 50% ) intervention patients and 20 of 71 ( 28% ) control patients ( or 2.55 [ 95% ci 1.305.01 ] ; p = 0.007 ) ( fig . moreover , among these 153 patients , 44 of 82 ( 54% ) intervention patients and 21 of 71 ( 30% ) control patients achieved recommended blood pressure targets at 1 year ( 2.76 [ 1.415.39 ] ; p = 0.003 ; nnt = 4 ) . fifty - five ( 42% ) intervention patients had 85 changes and 32 ( 25% ) control patients had 44 changes to their antihypertensive medication regimen ( or 2.19 [ 95% ci 1.303.71 ] ; p = 0.003 ) ( supplementary fig . 2 ) . among those with uncontrolled hypertension at baseline , only 61 of 153 ( 40% ) had changes . the most common antihypertensive medications added to a patient 's regimen were ramipril ( 10 patients ) , hydrochlorothiazide ( 9 patients ) , and irbesartan ( 8 patients ) . although changes in glycemic control and lipid parameters all favored the intervention , other than blood pressure control , none achieved statistical significance ( supplementary table 2 ) . using the ukpds risk engine ( 19 ) , there was a statistically significant reduction in predicted 10-year risk of cardiovascular events for intervention patients ( mean decrease 2.7% [ 95% ci 1.53.9 ] ; p < 0.001 ) but not for control patients ( 1.2% [ 0.1 to 2.4 ] ; p = 0.06 ) . the between - group difference was 1.5% ( 95% ci 0.2 to 3.3 ; p = 0.005 ) in favor of the intervention ( supplementary table 2 ) . the total number of health care related contacts was 1,439 for intervention patients and 420 for control patients ( p < 0.01 ; supplementary table 3 ) . however , 1,442 ( 77.6% ) of these contacts were either protocol driven ( baseline and 1-year follow - up ) visits or interim contacts between intervention patients and study pharmacists . there were no differences in emergency - room visits ( 11 [ 8.4% ] vs. 11 [ 8.5% ] ) , hospitalizations ( 4 [ 3.1% ] vs. five [ 3.9% ] ) , or all - cause mortality ( 1 [ 0.8% ] vs. 0 [ 0% ] ) between groups during the study . there were 149 ( 57.3% ) women , mean ( sd ) age was 59.1 11.6 years , bmi was 32.5 6.5 kg / m , diabetes duration was 5.5 6.5 years , and a1c was 7.4 1.4% . the mean blood pressure at baseline was 129.4 15.3/74.1 10.4 mmhg , with 82 of 131 ( 63% ) intervention patients and 71 of 129 ( 55% ) control patients having inadequately controlled hypertension ( 130/80 mmhg ; p = 0.22 for difference ) . of 107 patients with a blood pressure < 130/80 mmhg , 76 were taking one or more antihypertensive medications at baseline . baseline characteristics data are means sd for continuous variables and n ( % ) for categorical variables . over 1 year , there was a statistically significant reduction in systolic blood pressure for intervention patients ( mean decrease 7.4 mmhg [ 95% ci 4.610.2 ] ; p < 0.001 ) but not for control patients ( 2.5 mmhg [ 0.1 to 5.2 ] ; p = 0.06 ) ( supplementary fig . the between - group difference in systolic blood pressure change at 1 year was 4.9 mmhg ( 95% ci 1.08.7 ; p = 0.01 ) ( supplementary table 2 ) in favor of the intervention . the primary outcome was achieved by 48 of 131 ( 37% ) intervention patients and 30 of 129 ( 23% ) control patients ( or 1.91 [ 95% ci 1.113.28 ] ; p = 0.02 ) ( fig . the absolute difference of 14% translates to a number needed to treat ( nnt ) of seven patients followed for 1 year by a pharmacist to achieve one additional patient with better blood pressure control compared with usual care . proportion of patients achieving a 10% decrease in systolic blood pressure at 1 year ( primary outcome ) . limiting our analyses to 223 patients who completed the mean decrease in systolic blood pressure was 7.7 mmhg ( 95% ci 4.510.9 ; p < 0.001 ) for intervention patients and 2.8 mmhg ( 0.2 to 5.8 ; p = 0.07 ) for control patients ( p = 0.03 for between - group differences ) . more intervention patients achieved the primary outcome ( 41 of 110 [ 37% ] ) compared with control patients ( 30 of 113 [ 27% ] ) ; however , this difference was not statistically significant ( p = 0.09 ) . we observed a larger treatment effect when blood pressure changes were examined in 153 patients with inadequately controlled hypertension at baseline . mean blood pressure at baseline was 138.7 11.4/78.8 9.4 mmhg for intervention patients and 137.9 14.1/78.1 11.4 mmhg for control patients ( p > 0.05 ) . all other baseline characteristics for these 153 patients were well balanced between the two groups ( p > 0.05 for all comparisons ) . systolic blood pressure decreased a mean of 13.9 mmhg ( 95% ci 10.617.1 ; p < 0.001 ) for intervention patients and 6.7 mmhg ( 3.210.1 ; p < 0.001 ) for control patients ( p = 0.002 for between - group differences ) ( supplementary fig . the primary outcome was achieved by 41 of 82 ( 50% ) intervention patients and 20 of 71 ( 28% ) control patients ( or 2.55 [ 95% ci 1.305.01 ] ; p = 0.007 ) ( fig . moreover , among these 153 patients , 44 of 82 ( 54% ) intervention patients and 21 of 71 ( 30% ) control patients achieved recommended blood pressure targets at 1 year ( 2.76 [ 1.415.39 ] ; p = 0.003 ; nnt = 4 ) . fifty - five ( 42% ) intervention patients had 85 changes and 32 ( 25% ) control patients had 44 changes to their antihypertensive medication regimen ( or 2.19 [ 95% ci 1.303.71 ] ; p = 0.003 ) ( supplementary fig . 2 ) . among those with uncontrolled hypertension at baseline , only 61 of 153 ( 40% ) had changes . the most common antihypertensive medications added to a patient 's regimen were ramipril ( 10 patients ) , hydrochlorothiazide ( 9 patients ) , and irbesartan ( 8 patients ) . although changes in glycemic control and lipid parameters all favored the intervention , other than blood pressure control , none achieved statistical significance ( supplementary table 2 ) . using the ukpds risk engine ( 19 ) , there was a statistically significant reduction in predicted 10-year risk of cardiovascular events for intervention patients ( mean decrease 2.7% [ 95% ci 1.53.9 ] ; p < 0.001 ) but not for control patients ( 1.2% [ 0.1 to 2.4 ] ; the between - group difference was 1.5% ( 95% ci 0.2 to 3.3 ; p = 0.005 ) in favor of the intervention ( supplementary table 2 ) . the total number of health care related contacts was 1,439 for intervention patients and 420 for control patients ( p < 0.01 ; supplementary table 3 ) . however , 1,442 ( 77.6% ) of these contacts were either protocol driven ( baseline and 1-year follow - up ) visits or interim contacts between intervention patients and study pharmacists . there were no differences in emergency - room visits ( 11 [ 8.4% ] vs. 11 [ 8.5% ] ) , hospitalizations ( 4 [ 3.1% ] vs. five [ 3.9% ] ) , or all - cause mortality ( 1 [ 0.8% ] vs. 0 [ 0% ] ) between groups during the study . to our knowledge , this is the largest randomized controlled trial reporting the effect of adding pharmacists to primary care teams on blood pressure control in type 2 diabetic patients . on average , most patients were relatively well controlled in terms of a1c , blood pressure , and other cardiovascular risk factors ; a reflection of both the quality of usual care in this primary care network and the fact that study participants tend to be healthier than nonparticipants . nevertheless , adding pharmacists to primary care teams resulted in more intervention patients achieving a clinically important reduction in systolic blood pressure at 1 year compared with control patients . the absolute difference of 14% translates to an nnt of seven , and absolute benefits were even greater among those who had inadequately controlled hypertension ( i.e. , 22% improvement , nnt of five ) . glycemic control , cholesterol management , and predicted 10-year risk of cardiovascular disease all showed a trend toward improvement with the pharmacist intervention . a 10% reduction in systolic blood pressure is considered clinically worthwhile , ( 18 ) and , if sustained for another 4 years , would be associated with an 25% reduction in cardiovascular events ( 22 ) . our observations are broadly consistent with three previous studies ( 1214 ) that examined pharmacist contributions to diabetic hypertension management . all three studies reported a significant difference in systolic blood pressure change between groups and favored pharmacist intervention ( supplementary fig . , we might have seen a greater difference in systolic blood pressure change between groups if we had excluded people with normal blood pressure or well - controlled hypertension ( n = 107 [ 41% ] ) or included those with elevated hypertension ( 220/120 mmhg ) . we believe that the success of this study can be attributed to three critical components of the pharmacist intervention . these evidence - based resources provided a validated , external benchmark to identify treatment options . second , pharmacists discussed their recommendations directly with primary care physicians and other health care professionals , which is considered an essential component of successful management programs ( 23 ) . discussions facilitated a richer exchange of patient - specific ideas compared with more impersonal e - mails or faxes that are commonly used by pharmacists in the community . third , the frequency of follow - up contact was tailored to the patient 's needs . we have found that the effects of an intervention decay over time without direct , continuous involvement and individualized support of clinicians ( 20,21,24 ) . first , we examined relatively short - term changes in surrogate measures rather than harder longer - term clinical end points such as myocardial infarction , stroke , or death . this limitation may be ameliorated somewhat by the significant changes in the ukpds risk score observed with the intervention . second , there was the possibility of contamination or cointervention because both intervention and control patients were drawn from the same primary care team . although we considered a cluster - randomized trial , we estimated that there were not enough primary care teams to carry out such a study . contamination would only tend to bias to the null , and without the pharmacist 's active intervention it is unlikely that the primary care team would pay greater attention than usual to blood pressure control in those with diabetes . third , our 14% drop - out rate was high but consistent with other randomized controlled trials of pharmacist involvement in diabetic hypertension management ( 1114 ) . withdrawal rates were similar between groups , and there were no significantly different characteristics between patients who withdrew or completed the study . fourth , our intervention was conducted in a jurisdiction with universal health care coverage and set within established primary care teams or patient - centered medical homes , so usual care was already much better than reported in the previous literature . nonetheless , there was still room for improvement , and adding pharmacists to this team did improve care . it is likely , therefore , that the intervention would have an even greater effect when implemented in settings with a lower baseline quality of care . last , the multifaceted nature of our intervention program makes it difficult to attribute the observed differences to a specific component . we believe the next stage in this line of research could be an active comparator study examining the effects of pharmacists , perhaps with prescriptive autonomy , relative to other case managers . working in collaboration with the patient , primary care physician , and other health care professionals , pharmacists can have a significant , positive impact on blood pressure management in type 2 diabetes . we believe our results are applicable to a broad range of patients with type 2 diabetes managed in primary care settings and can be extended to nondiabetic patients with inadequately controlled hypertension . first , we examined relatively short - term changes in surrogate measures rather than harder longer - term clinical end points such as myocardial infarction , stroke , or death . this limitation may be ameliorated somewhat by the significant changes in the ukpds risk score observed with the intervention . second , there was the possibility of contamination or cointervention because both intervention and control patients were drawn from the same primary care team . although we considered a cluster - randomized trial , we estimated that there were not enough primary care teams to carry out such a study . contamination would only tend to bias to the null , and without the pharmacist 's active intervention it is unlikely that the primary care team would pay greater attention than usual to blood pressure control in those with diabetes . third , our 14% drop - out rate was high but consistent with other randomized controlled trials of pharmacist involvement in diabetic hypertension management ( 1114 ) . withdrawal rates were similar between groups , and there were no significantly different characteristics between patients who withdrew or completed the study . fourth , our intervention was conducted in a jurisdiction with universal health care coverage and set within established primary care teams or patient - centered medical homes , so usual care was already much better than reported in the previous literature . nonetheless , there was still room for improvement , and adding pharmacists to this team did improve care . it is likely , therefore , that the intervention would have an even greater effect when implemented in settings with a lower baseline quality of care . last , the multifaceted nature of our intervention program makes it difficult to attribute the observed differences to a specific component . we believe the next stage in this line of research could be an active comparator study examining the effects of pharmacists , perhaps with prescriptive autonomy , relative to other case managers . our observations support the addition of pharmacists to primary care teams . working in collaboration with the patient , primary care physician , and other health care professionals , pharmacists can have a significant , positive impact on blood pressure management in type 2 diabetes . we believe our results are applicable to a broad range of patients with type 2 diabetes managed in primary care settings and can be extended to nondiabetic patients with inadequately controlled hypertension .

objectiveto evaluate the effect of adding pharmacists to primary care teams on the management of hypertension and other cardiovascular risk factors in patients with type 2 diabetes.research design and methodswe conducted a randomized controlled trial with blinded ascertainment of outcomes within primary care clinics in edmonton , canada . pharmacists performed medication assessments and limited history and physical examinations and provided guideline - concordant recommendations to optimize medication management . follow - up contact was completed as necessary . control patients received usual care . the primary outcome was a 10% decrease in systolic blood pressure at 1 year.resultsa total of 260 patients were enrolled , 57% were women , the mean age was 59 years , diabetes duration was 6 years , and blood pressure was 129/74 mmhg . forty - eight of 131 ( 37% ) intervention patients and 30 of 129 ( 23% ) control patients achieved the primary outcome ( odds ratio 1.9 [ 95% ci 1.13.3 ] ; p = 0.02 ) . among 153 patients with inadequately controlled hypertension at baseline , intervention patients ( n = 82 ) were significantly more likely than control patients ( n = 71 ) to achieve the primary outcome ( 41 [ 50% ] vs. 20 [ 28% ] ; 2.6 [ 1.35.0 ] ; p = 0.007 ) and recommended blood pressure targets ( 44 [ 54% ] vs. 21 [ 30% ] ; 2.8 [ 1.45.4 ] ; p = 0.003 ) . the 10-year risk of cardiovascular disease , based on changes to the uk prospective diabetes study risk engine , were predicted to decrease by 3% for intervention patients and 1% for control patients ( p = 0.005).conclusionssignificantly more patients with type 2 diabetes achieved better blood pressure control when pharmacists were added to primary care teams , which suggests that pharmacists can make important contributions to the primary care of these patients .

RESEARCH DESIGN AND METHODS Randomization Control patients Intervention Follow-up Outcomes Statistical analysis RESULTS Patient characteristics Blood pressure changes Antihypertensive medication changes Other outcomes CONCLUSIONS Limitations Conclusion

this randomized controlled trial was conducted in five primary care clinics affiliated with the edmonton south side primary care network in edmonton , canada . these primary care teams were akin to the patient - centered medical home ( 15 ) and consisted of physicians and nurses who had support from dietitians , physiotherapists , and social workers as needed . patients were eligible if they had type 2 diabetes , were regularly seen by the primary care team , and did not qualify for urgent specialist referral and assessment ( according to protocol , a fasting blood glucose 17 mmol / l , blood pressure 220/120 mmhg , or triglycerides 15 mmol / l ) . because type 2 diabetic patients have higher blood pressure levels than the general population ( 1 ) eligible patients were identified from the clinic roster , and a clinic staff member made initial contact to tell patients about the study . patients were told that the study was designed to help improve medication therapy for heart disease risk in patients with type 2 diabetes . control patients received usual care by the primary care team without contributions from study pharmacists , except for standardized blood pressure measurements at the end of the follow - up period ( see below ) . pharmacists also measured the patient 's height , weight , heart rate , and blood pressure . pharmacists then formulated guideline - concordant recommendations to optimize medication management of blood pressure and other cardiovascular risk factors . after 1 year , all patients were seen in the primary care clinic to review medications , measure blood pressure with the automated machine , and obtain a fasting blood sample to measure blood glucose , a1c , and cholesterol profile . the primary outcome was achievement of a clinically important reduction in blood pressure , defined as a 10% decrease in systolic blood pressure at 1 year ( 18 ) . secondary outcomes included the absolute change in systolic blood pressure from baseline to 1 year , achievement of recommended blood pressure targets ( < 130/80 mmhg ) ( 8) , and antihypertensive medication changes . we also measured the change in predicted 10-year risk of cardiovascular disease using the uk prospective diabetes study ( ukpds ) risk engine ( 19 ) . baseline and follow - up values for a1c , systolic blood pressure , total cholesterol , and hdl cholesterol were used to calculate the change in the ukpds risk engine score . our sample size was based on observations from a previous study ( 20 ) examining the effect of a diabetes intervention program aimed at physicians . in that study , 40% of intervention patients and 25% of control patients achieved a 10% decrease in blood pressure at 6 months . the association between treatment group and achievement of the primary outcome was also examined using a logistic regression model to calculate an odds ratio ( or ) and 95% ci . to test the robustness of our observations , we restricted our analyses to patients who completed the full study protocol and patients with inadequately controlled hypertension at baseline . control patients received usual care by the primary care team without contributions from study pharmacists , except for standardized blood pressure measurements at the end of the follow - up period ( see below ) . pharmacists also measured the patient 's height , weight , heart rate , and blood pressure . blood pressure was measured according to the canadian hypertension education program recommendations using the bptru bpm-100 ( vsm med tech , coquitlam , bc ) automated machine set to report the average of five measurements at 1-min intervals ( 16 ) . pharmacists then formulated guideline - concordant recommendations to optimize medication management of blood pressure and other cardiovascular risk factors . after 1 year , all patients were seen in the primary care clinic to review medications , measure blood pressure with the automated machine , and obtain a fasting blood sample to measure blood glucose , a1c , and cholesterol profile . the primary outcome was achievement of a clinically important reduction in blood pressure , defined as a 10% decrease in systolic blood pressure at 1 year ( 18 ) . secondary outcomes included the absolute change in systolic blood pressure from baseline to 1 year , achievement of recommended blood pressure targets ( < 130/80 mmhg ) ( 8) , and antihypertensive medication changes . we also measured the change in predicted 10-year risk of cardiovascular disease using the uk prospective diabetes study ( ukpds ) risk engine ( 19 ) . baseline and follow - up values for a1c , systolic blood pressure , total cholesterol , and hdl cholesterol were used to calculate the change in the ukpds risk engine score . our sample size was based on observations from a previous study ( 20 ) examining the effect of a diabetes intervention program aimed at physicians . in that study , 40% of intervention patients and 25% of control patients achieved a 10% decrease in blood pressure at 6 months . the association between treatment group and achievement of the primary outcome was also examined using a logistic regression model to calculate an odds ratio ( or ) and 95% ci . to test the robustness of our observations , we restricted our analyses to patients who completed the full study protocol and patients with inadequately controlled hypertension at baseline . there were 21 intervention patients ( 14 withdrew , 6 were lost to follow - up , and 1 died ) and 16 control patients ( 10 withdrew and 6 were lost to follow - up ) who did not complete the study ( p > 0.05 for all comparisons ) . there were no differences in age , sex , diabetes duration , or baseline blood pressure between the patients who did or did not complete the study . there were 149 ( 57.3% ) women , mean ( sd ) age was 59.1 11.6 years , bmi was 32.5 6.5 kg / m , diabetes duration was 5.5 6.5 years , and a1c was 7.4 1.4% . the mean blood pressure at baseline was 129.4 15.3/74.1 10.4 mmhg , with 82 of 131 ( 63% ) intervention patients and 71 of 129 ( 55% ) control patients having inadequately controlled hypertension ( 130/80 mmhg ; p = 0.22 for difference ) . of 107 patients with a blood pressure < 130/80 mmhg , 76 were taking one or more antihypertensive medications at baseline . over 1 year , there was a statistically significant reduction in systolic blood pressure for intervention patients ( mean decrease 7.4 mmhg [ 95% ci 4.610.2 ] ; p < 0.001 ) but not for control patients ( 2.5 mmhg [ 0.1 to 5.2 ] ; p = 0.06 ) ( supplementary fig . the between - group difference in systolic blood pressure change at 1 year was 4.9 mmhg ( 95% ci 1.08.7 ; p = 0.01 ) ( supplementary table 2 ) in favor of the intervention . the primary outcome was achieved by 48 of 131 ( 37% ) intervention patients and 30 of 129 ( 23% ) control patients ( or 1.91 [ 95% ci 1.113.28 ] ; p = 0.02 ) ( fig . the absolute difference of 14% translates to a number needed to treat ( nnt ) of seven patients followed for 1 year by a pharmacist to achieve one additional patient with better blood pressure control compared with usual care . proportion of patients achieving a 10% decrease in systolic blood pressure at 1 year ( primary outcome ) . limiting our analyses to 223 patients who completed the mean decrease in systolic blood pressure was 7.7 mmhg ( 95% ci 4.510.9 ; p < 0.001 ) for intervention patients and 2.8 mmhg ( 0.2 to 5.8 ; p = 0.07 ) for control patients ( p = 0.03 for between - group differences ) . more intervention patients achieved the primary outcome ( 41 of 110 [ 37% ] ) compared with control patients ( 30 of 113 [ 27% ] ) ; however , this difference was not statistically significant ( p = 0.09 ) . we observed a larger treatment effect when blood pressure changes were examined in 153 patients with inadequately controlled hypertension at baseline . mean blood pressure at baseline was 138.7 11.4/78.8 9.4 mmhg for intervention patients and 137.9 14.1/78.1 11.4 mmhg for control patients ( p > 0.05 ) . all other baseline characteristics for these 153 patients were well balanced between the two groups ( p > 0.05 for all comparisons ) . systolic blood pressure decreased a mean of 13.9 mmhg ( 95% ci 10.617.1 ; p < 0.001 ) for intervention patients and 6.7 mmhg ( 3.210.1 ; p < 0.001 ) for control patients ( p = 0.002 for between - group differences ) ( supplementary fig . the primary outcome was achieved by 41 of 82 ( 50% ) intervention patients and 20 of 71 ( 28% ) control patients ( or 2.55 [ 95% ci 1.305.01 ] ; p = 0.007 ) ( fig . moreover , among these 153 patients , 44 of 82 ( 54% ) intervention patients and 21 of 71 ( 30% ) control patients achieved recommended blood pressure targets at 1 year ( 2.76 [ 1.415.39 ] ; p = 0.003 ; nnt = 4 ) . fifty - five ( 42% ) intervention patients had 85 changes and 32 ( 25% ) control patients had 44 changes to their antihypertensive medication regimen ( or 2.19 [ 95% ci 1.303.71 ] ; p = 0.003 ) ( supplementary fig . among those with uncontrolled hypertension at baseline , only 61 of 153 ( 40% ) had changes . using the ukpds risk engine ( 19 ) , there was a statistically significant reduction in predicted 10-year risk of cardiovascular events for intervention patients ( mean decrease 2.7% [ 95% ci 1.53.9 ] ; p < 0.001 ) but not for control patients ( 1.2% [ 0.1 to 2.4 ] ; p = 0.06 ) . the between - group difference was 1.5% ( 95% ci 0.2 to 3.3 ; p = 0.005 ) in favor of the intervention ( supplementary table 2 ) . the total number of health care related contacts was 1,439 for intervention patients and 420 for control patients ( p < 0.01 ; supplementary table 3 ) . however , 1,442 ( 77.6% ) of these contacts were either protocol driven ( baseline and 1-year follow - up ) visits or interim contacts between intervention patients and study pharmacists . there were 149 ( 57.3% ) women , mean ( sd ) age was 59.1 11.6 years , bmi was 32.5 6.5 kg / m , diabetes duration was 5.5 6.5 years , and a1c was 7.4 1.4% . the mean blood pressure at baseline was 129.4 15.3/74.1 10.4 mmhg , with 82 of 131 ( 63% ) intervention patients and 71 of 129 ( 55% ) control patients having inadequately controlled hypertension ( 130/80 mmhg ; p = 0.22 for difference ) . of 107 patients with a blood pressure < 130/80 mmhg , 76 were taking one or more antihypertensive medications at baseline . over 1 year , there was a statistically significant reduction in systolic blood pressure for intervention patients ( mean decrease 7.4 mmhg [ 95% ci 4.610.2 ] ; p < 0.001 ) but not for control patients ( 2.5 mmhg [ 0.1 to 5.2 ] ; p = 0.06 ) ( supplementary fig . the between - group difference in systolic blood pressure change at 1 year was 4.9 mmhg ( 95% ci 1.08.7 ; p = 0.01 ) ( supplementary table 2 ) in favor of the intervention . the primary outcome was achieved by 48 of 131 ( 37% ) intervention patients and 30 of 129 ( 23% ) control patients ( or 1.91 [ 95% ci 1.113.28 ] ; p = 0.02 ) ( fig . the absolute difference of 14% translates to a number needed to treat ( nnt ) of seven patients followed for 1 year by a pharmacist to achieve one additional patient with better blood pressure control compared with usual care . proportion of patients achieving a 10% decrease in systolic blood pressure at 1 year ( primary outcome ) . limiting our analyses to 223 patients who completed the mean decrease in systolic blood pressure was 7.7 mmhg ( 95% ci 4.510.9 ; p < 0.001 ) for intervention patients and 2.8 mmhg ( 0.2 to 5.8 ; p = 0.07 ) for control patients ( p = 0.03 for between - group differences ) . more intervention patients achieved the primary outcome ( 41 of 110 [ 37% ] ) compared with control patients ( 30 of 113 [ 27% ] ) ; however , this difference was not statistically significant ( p = 0.09 ) . we observed a larger treatment effect when blood pressure changes were examined in 153 patients with inadequately controlled hypertension at baseline . mean blood pressure at baseline was 138.7 11.4/78.8 9.4 mmhg for intervention patients and 137.9 14.1/78.1 11.4 mmhg for control patients ( p > 0.05 ) . all other baseline characteristics for these 153 patients were well balanced between the two groups ( p > 0.05 for all comparisons ) . systolic blood pressure decreased a mean of 13.9 mmhg ( 95% ci 10.617.1 ; p < 0.001 ) for intervention patients and 6.7 mmhg ( 3.210.1 ; p < 0.001 ) for control patients ( p = 0.002 for between - group differences ) ( supplementary fig . the primary outcome was achieved by 41 of 82 ( 50% ) intervention patients and 20 of 71 ( 28% ) control patients ( or 2.55 [ 95% ci 1.305.01 ] ; p = 0.007 ) ( fig . moreover , among these 153 patients , 44 of 82 ( 54% ) intervention patients and 21 of 71 ( 30% ) control patients achieved recommended blood pressure targets at 1 year ( 2.76 [ 1.415.39 ] ; p = 0.003 ; nnt = 4 ) . fifty - five ( 42% ) intervention patients had 85 changes and 32 ( 25% ) control patients had 44 changes to their antihypertensive medication regimen ( or 2.19 [ 95% ci 1.303.71 ] ; p = 0.003 ) ( supplementary fig . among those with uncontrolled hypertension at baseline , only 61 of 153 ( 40% ) had changes . using the ukpds risk engine ( 19 ) , there was a statistically significant reduction in predicted 10-year risk of cardiovascular events for intervention patients ( mean decrease 2.7% [ 95% ci 1.53.9 ] ; p < 0.001 ) but not for control patients ( 1.2% [ 0.1 to 2.4 ] ; the between - group difference was 1.5% ( 95% ci 0.2 to 3.3 ; p = 0.005 ) in favor of the intervention ( supplementary table 2 ) . the total number of health care related contacts was 1,439 for intervention patients and 420 for control patients ( p < 0.01 ; supplementary table 3 ) . however , 1,442 ( 77.6% ) of these contacts were either protocol driven ( baseline and 1-year follow - up ) visits or interim contacts between intervention patients and study pharmacists . to our knowledge , this is the largest randomized controlled trial reporting the effect of adding pharmacists to primary care teams on blood pressure control in type 2 diabetic patients . on average , most patients were relatively well controlled in terms of a1c , blood pressure , and other cardiovascular risk factors ; a reflection of both the quality of usual care in this primary care network and the fact that study participants tend to be healthier than nonparticipants . nevertheless , adding pharmacists to primary care teams resulted in more intervention patients achieving a clinically important reduction in systolic blood pressure at 1 year compared with control patients . the absolute difference of 14% translates to an nnt of seven , and absolute benefits were even greater among those who had inadequately controlled hypertension ( i.e. glycemic control , cholesterol management , and predicted 10-year risk of cardiovascular disease all showed a trend toward improvement with the pharmacist intervention . a 10% reduction in systolic blood pressure is considered clinically worthwhile , ( 18 ) and , if sustained for another 4 years , would be associated with an 25% reduction in cardiovascular events ( 22 ) . all three studies reported a significant difference in systolic blood pressure change between groups and favored pharmacist intervention ( supplementary fig . , we might have seen a greater difference in systolic blood pressure change between groups if we had excluded people with normal blood pressure or well - controlled hypertension ( n = 107 [ 41% ] ) or included those with elevated hypertension ( 220/120 mmhg ) . second , pharmacists discussed their recommendations directly with primary care physicians and other health care professionals , which is considered an essential component of successful management programs ( 23 ) . third , the frequency of follow - up contact was tailored to the patient 's needs . second , there was the possibility of contamination or cointervention because both intervention and control patients were drawn from the same primary care team . contamination would only tend to bias to the null , and without the pharmacist 's active intervention it is unlikely that the primary care team would pay greater attention than usual to blood pressure control in those with diabetes . fourth , our intervention was conducted in a jurisdiction with universal health care coverage and set within established primary care teams or patient - centered medical homes , so usual care was already much better than reported in the previous literature . nonetheless , there was still room for improvement , and adding pharmacists to this team did improve care . working in collaboration with the patient , primary care physician , and other health care professionals , pharmacists can have a significant , positive impact on blood pressure management in type 2 diabetes . we believe our results are applicable to a broad range of patients with type 2 diabetes managed in primary care settings and can be extended to nondiabetic patients with inadequately controlled hypertension . second , there was the possibility of contamination or cointervention because both intervention and control patients were drawn from the same primary care team . contamination would only tend to bias to the null , and without the pharmacist 's active intervention it is unlikely that the primary care team would pay greater attention than usual to blood pressure control in those with diabetes . fourth , our intervention was conducted in a jurisdiction with universal health care coverage and set within established primary care teams or patient - centered medical homes , so usual care was already much better than reported in the previous literature . nonetheless , there was still room for improvement , and adding pharmacists to this team did improve care . our observations support the addition of pharmacists to primary care teams . working in collaboration with the patient , primary care physician , and other health care professionals , pharmacists can have a significant , positive impact on blood pressure management in type 2 diabetes . we believe our results are applicable to a broad range of patients with type 2 diabetes managed in primary care settings and can be extended to nondiabetic patients with inadequately controlled hypertension .

these primary care teams were akin to the patient - centered medical home ( 15 ) and consisted of physicians and nurses who had support from dietitians , physiotherapists , and social workers as needed . patients were eligible if they had type 2 diabetes , were regularly seen by the primary care team , and did not qualify for urgent specialist referral and assessment ( according to protocol , a fasting blood glucose 17 mmol / l , blood pressure 220/120 mmhg , or triglycerides 15 mmol / l ) . because type 2 diabetic patients have higher blood pressure levels than the general population ( 1 ) eligible patients were identified from the clinic roster , and a clinic staff member made initial contact to tell patients about the study . patients were told that the study was designed to help improve medication therapy for heart disease risk in patients with type 2 diabetes . the specific focus of the study , hypertension , was listed among other risk factors for heart disease . control patients received usual care by the primary care team without contributions from study pharmacists , except for standardized blood pressure measurements at the end of the follow - up period ( see below ) . two pharmacists providing the intervention program held a bachelor 's degree in pharmacy , were certified diabetes educators , and had practiced in community pharmacies for over 5 years . both pharmacists completed structured online training courses for hypertension and diabetes management ( www.pharmalearn.com ) and reviewed the canadian hypertension education program and canadian diabetes association guideline recommendations prior to starting the study ( 16,17 ) . the intervention program began with an in - person visit with a study pharmacist to identify all prescription , nonprescription , complementary , and alternative medications . blood pressure was measured according to the canadian hypertension education program recommendations using the bptru bpm-100 ( vsm med tech , coquitlam , bc ) automated machine set to report the average of five measurements at 1-min intervals ( 16 ) . after 1 year , all patients were seen in the primary care clinic to review medications , measure blood pressure with the automated machine , and obtain a fasting blood sample to measure blood glucose , a1c , and cholesterol profile . patients also reported the number of encounters with specialists , other health care professionals , regional health care resources , emergency - room visits , and hospitalizations during the previous year . the primary outcome was achievement of a clinically important reduction in blood pressure , defined as a 10% decrease in systolic blood pressure at 1 year ( 18 ) . secondary outcomes included the absolute change in systolic blood pressure from baseline to 1 year , achievement of recommended blood pressure targets ( < 130/80 mmhg ) ( 8) , and antihypertensive medication changes . we also measured the change in predicted 10-year risk of cardiovascular disease using the uk prospective diabetes study ( ukpds ) risk engine ( 19 ) . baseline and follow - up values for a1c , systolic blood pressure , total cholesterol , and hdl cholesterol were used to calculate the change in the ukpds risk engine score . our sample size was based on observations from a previous study ( 20 ) examining the effect of a diabetes intervention program aimed at physicians . in that study , 40% of intervention patients and 25% of control patients achieved a 10% decrease in blood pressure at 6 months . although the current study follow - up was twice as long and the intervention was directed at patients , we estimated that the event rates would be similar . the association between treatment group and achievement of the primary outcome was also examined using a logistic regression model to calculate an odds ratio ( or ) and 95% ci . based on the results of related trials in our region ( 13,20,21 ) , we assumed that patient - related outcomes were statistically independent of one another , with intracluster correlation coefficients < 0.01 . all patients were evaluated in the groups to which they were randomly allocated according to the intention - to - treat principle . to test the robustness of our observations , we restricted our analyses to patients who completed the full study protocol and patients with inadequately controlled hypertension at baseline . first , we used indicator variables to directly adjust for all 18 family physicians involved in the study in a multivariate logistic regression model . second , we used generalized estimating equation methodology to account for the potential correlations of outcomes among patients treated by the same physician . control patients received usual care by the primary care team without contributions from study pharmacists , except for standardized blood pressure measurements at the end of the follow - up period ( see below ) . two pharmacists providing the intervention program held a bachelor 's degree in pharmacy , were certified diabetes educators , and had practiced in community pharmacies for over 5 years . both pharmacists completed structured online training courses for hypertension and diabetes management ( www.pharmalearn.com ) and reviewed the canadian hypertension education program and canadian diabetes association guideline recommendations prior to starting the study ( 16,17 ) . the intervention program began with an in - person visit with a study pharmacist to identify all prescription , nonprescription , complementary , and alternative medications . blood pressure was measured according to the canadian hypertension education program recommendations using the bptru bpm-100 ( vsm med tech , coquitlam , bc ) automated machine set to report the average of five measurements at 1-min intervals ( 16 ) . after 1 year , all patients were seen in the primary care clinic to review medications , measure blood pressure with the automated machine , and obtain a fasting blood sample to measure blood glucose , a1c , and cholesterol profile . patients also reported the number of encounters with specialists , other health care professionals , regional health care resources , emergency - room visits , and hospitalizations during the previous year . the primary outcome was achievement of a clinically important reduction in blood pressure , defined as a 10% decrease in systolic blood pressure at 1 year ( 18 ) . secondary outcomes included the absolute change in systolic blood pressure from baseline to 1 year , achievement of recommended blood pressure targets ( < 130/80 mmhg ) ( 8) , and antihypertensive medication changes . we also measured the change in predicted 10-year risk of cardiovascular disease using the uk prospective diabetes study ( ukpds ) risk engine ( 19 ) . baseline and follow - up values for a1c , systolic blood pressure , total cholesterol , and hdl cholesterol were used to calculate the change in the ukpds risk engine score . our sample size was based on observations from a previous study ( 20 ) examining the effect of a diabetes intervention program aimed at physicians . in that study , 40% of intervention patients and 25% of control patients achieved a 10% decrease in blood pressure at 6 months . although the current study follow - up was twice as long and the intervention was directed at patients , we estimated that the event rates would be similar . the association between treatment group and achievement of the primary outcome was also examined using a logistic regression model to calculate an odds ratio ( or ) and 95% ci . based on the results of related trials in our region ( 13,20,21 ) , we assumed that patient - related outcomes were statistically independent of one another , with intracluster correlation coefficients < 0.01 . all patients were evaluated in the groups to which they were randomly allocated according to the intention - to - treat principle . to test the robustness of our observations , we restricted our analyses to patients who completed the full study protocol and patients with inadequately controlled hypertension at baseline . first , we used indicator variables to directly adjust for all 18 family physicians involved in the study in a multivariate logistic regression model . second , we used generalized estimating equation methodology to account for the potential correlations of outcomes among patients treated by the same physician . we enrolled 260 patients between 28 february 2006 and 6 december 2007 and randomly allocated 131 to the intervention and 129 to the control group ( fig . there were 21 intervention patients ( 14 withdrew , 6 were lost to follow - up , and 1 died ) and 16 control patients ( 10 withdrew and 6 were lost to follow - up ) who did not complete the study ( p > 0.05 for all comparisons ) . there were no differences in age , sex , diabetes duration , or baseline blood pressure between the patients who did or did not complete the study . there were 149 ( 57.3% ) women , mean ( sd ) age was 59.1 11.6 years , bmi was 32.5 6.5 kg / m , diabetes duration was 5.5 6.5 years , and a1c was 7.4 1.4% . the mean blood pressure at baseline was 129.4 15.3/74.1 10.4 mmhg , with 82 of 131 ( 63% ) intervention patients and 71 of 129 ( 55% ) control patients having inadequately controlled hypertension ( 130/80 mmhg ; p = 0.22 for difference ) . over 1 year , there was a statistically significant reduction in systolic blood pressure for intervention patients ( mean decrease 7.4 mmhg [ 95% ci 4.610.2 ] ; p < 0.001 ) but not for control patients ( 2.5 mmhg [ 0.1 to 5.2 ] ; p = 0.06 ) ( supplementary fig . the between - group difference in systolic blood pressure change at 1 year was 4.9 mmhg ( 95% ci 1.08.7 ; p = 0.01 ) ( supplementary table 2 ) in favor of the intervention . the primary outcome was achieved by 48 of 131 ( 37% ) intervention patients and 30 of 129 ( 23% ) control patients ( or 1.91 [ 95% ci 1.113.28 ] ; p = 0.02 ) ( fig . the absolute difference of 14% translates to a number needed to treat ( nnt ) of seven patients followed for 1 year by a pharmacist to achieve one additional patient with better blood pressure control compared with usual care . limiting our analyses to 223 patients who completed the mean decrease in systolic blood pressure was 7.7 mmhg ( 95% ci 4.510.9 ; p < 0.001 ) for intervention patients and 2.8 mmhg ( 0.2 to 5.8 ; p = 0.07 ) for control patients ( p = 0.03 for between - group differences ) . more intervention patients achieved the primary outcome ( 41 of 110 [ 37% ] ) compared with control patients ( 30 of 113 [ 27% ] ) ; however , this difference was not statistically significant ( p = 0.09 ) . systolic blood pressure decreased a mean of 13.9 mmhg ( 95% ci 10.617.1 ; p < 0.001 ) for intervention patients and 6.7 mmhg ( 3.210.1 ; p < 0.001 ) for control patients ( p = 0.002 for between - group differences ) ( supplementary fig . the primary outcome was achieved by 41 of 82 ( 50% ) intervention patients and 20 of 71 ( 28% ) control patients ( or 2.55 [ 95% ci 1.305.01 ] ; p = 0.007 ) ( fig . moreover , among these 153 patients , 44 of 82 ( 54% ) intervention patients and 21 of 71 ( 30% ) control patients achieved recommended blood pressure targets at 1 year ( 2.76 [ 1.415.39 ] ; p = 0.003 ; nnt = 4 ) . fifty - five ( 42% ) intervention patients had 85 changes and 32 ( 25% ) control patients had 44 changes to their antihypertensive medication regimen ( or 2.19 [ 95% ci 1.303.71 ] ; p = 0.003 ) ( supplementary fig . using the ukpds risk engine ( 19 ) , there was a statistically significant reduction in predicted 10-year risk of cardiovascular events for intervention patients ( mean decrease 2.7% [ 95% ci 1.53.9 ] ; p < 0.001 ) but not for control patients ( 1.2% [ 0.1 to 2.4 ] ; p = 0.06 ) . the between - group difference was 1.5% ( 95% ci 0.2 to 3.3 ; p = 0.005 ) in favor of the intervention ( supplementary table 2 ) . the total number of health care related contacts was 1,439 for intervention patients and 420 for control patients ( p < 0.01 ; supplementary table 3 ) . however , 1,442 ( 77.6% ) of these contacts were either protocol driven ( baseline and 1-year follow - up ) visits or interim contacts between intervention patients and study pharmacists . there were no differences in emergency - room visits ( 11 [ 8.4% ] vs. 11 [ 8.5% ] ) , hospitalizations ( 4 [ 3.1% ] vs. five [ 3.9% ] ) , or all - cause mortality ( 1 [ 0.8% ] vs. 0 [ 0% ] ) between groups during the study . there were 149 ( 57.3% ) women , mean ( sd ) age was 59.1 11.6 years , bmi was 32.5 6.5 kg / m , diabetes duration was 5.5 6.5 years , and a1c was 7.4 1.4% . over 1 year , there was a statistically significant reduction in systolic blood pressure for intervention patients ( mean decrease 7.4 mmhg [ 95% ci 4.610.2 ] ; p < 0.001 ) but not for control patients ( 2.5 mmhg [ 0.1 to 5.2 ] ; p = 0.06 ) ( supplementary fig . the between - group difference in systolic blood pressure change at 1 year was 4.9 mmhg ( 95% ci 1.08.7 ; p = 0.01 ) ( supplementary table 2 ) in favor of the intervention . the primary outcome was achieved by 48 of 131 ( 37% ) intervention patients and 30 of 129 ( 23% ) control patients ( or 1.91 [ 95% ci 1.113.28 ] ; p = 0.02 ) ( fig . the absolute difference of 14% translates to a number needed to treat ( nnt ) of seven patients followed for 1 year by a pharmacist to achieve one additional patient with better blood pressure control compared with usual care . limiting our analyses to 223 patients who completed the mean decrease in systolic blood pressure was 7.7 mmhg ( 95% ci 4.510.9 ; p < 0.001 ) for intervention patients and 2.8 mmhg ( 0.2 to 5.8 ; p = 0.07 ) for control patients ( p = 0.03 for between - group differences ) . more intervention patients achieved the primary outcome ( 41 of 110 [ 37% ] ) compared with control patients ( 30 of 113 [ 27% ] ) ; however , this difference was not statistically significant ( p = 0.09 ) . systolic blood pressure decreased a mean of 13.9 mmhg ( 95% ci 10.617.1 ; p < 0.001 ) for intervention patients and 6.7 mmhg ( 3.210.1 ; p < 0.001 ) for control patients ( p = 0.002 for between - group differences ) ( supplementary fig . the primary outcome was achieved by 41 of 82 ( 50% ) intervention patients and 20 of 71 ( 28% ) control patients ( or 2.55 [ 95% ci 1.305.01 ] ; p = 0.007 ) ( fig . moreover , among these 153 patients , 44 of 82 ( 54% ) intervention patients and 21 of 71 ( 30% ) control patients achieved recommended blood pressure targets at 1 year ( 2.76 [ 1.415.39 ] ; p = 0.003 ; nnt = 4 ) . fifty - five ( 42% ) intervention patients had 85 changes and 32 ( 25% ) control patients had 44 changes to their antihypertensive medication regimen ( or 2.19 [ 95% ci 1.303.71 ] ; p = 0.003 ) ( supplementary fig . using the ukpds risk engine ( 19 ) , there was a statistically significant reduction in predicted 10-year risk of cardiovascular events for intervention patients ( mean decrease 2.7% [ 95% ci 1.53.9 ] ; p < 0.001 ) but not for control patients ( 1.2% [ 0.1 to 2.4 ] ; the between - group difference was 1.5% ( 95% ci 0.2 to 3.3 ; p = 0.005 ) in favor of the intervention ( supplementary table 2 ) . the total number of health care related contacts was 1,439 for intervention patients and 420 for control patients ( p < 0.01 ; supplementary table 3 ) . there were no differences in emergency - room visits ( 11 [ 8.4% ] vs. 11 [ 8.5% ] ) , hospitalizations ( 4 [ 3.1% ] vs. five [ 3.9% ] ) , or all - cause mortality ( 1 [ 0.8% ] vs. 0 [ 0% ] ) between groups during the study . to our knowledge , this is the largest randomized controlled trial reporting the effect of adding pharmacists to primary care teams on blood pressure control in type 2 diabetic patients . on average , most patients were relatively well controlled in terms of a1c , blood pressure , and other cardiovascular risk factors ; a reflection of both the quality of usual care in this primary care network and the fact that study participants tend to be healthier than nonparticipants . nevertheless , adding pharmacists to primary care teams resulted in more intervention patients achieving a clinically important reduction in systolic blood pressure at 1 year compared with control patients . glycemic control , cholesterol management , and predicted 10-year risk of cardiovascular disease all showed a trend toward improvement with the pharmacist intervention . , we might have seen a greater difference in systolic blood pressure change between groups if we had excluded people with normal blood pressure or well - controlled hypertension ( n = 107 [ 41% ] ) or included those with elevated hypertension ( 220/120 mmhg ) . first , we examined relatively short - term changes in surrogate measures rather than harder longer - term clinical end points such as myocardial infarction , stroke , or death . fourth , our intervention was conducted in a jurisdiction with universal health care coverage and set within established primary care teams or patient - centered medical homes , so usual care was already much better than reported in the previous literature . we believe the next stage in this line of research could be an active comparator study examining the effects of pharmacists , perhaps with prescriptive autonomy , relative to other case managers . working in collaboration with the patient , primary care physician , and other health care professionals , pharmacists can have a significant , positive impact on blood pressure management in type 2 diabetes . we believe our results are applicable to a broad range of patients with type 2 diabetes managed in primary care settings and can be extended to nondiabetic patients with inadequately controlled hypertension . fourth , our intervention was conducted in a jurisdiction with universal health care coverage and set within established primary care teams or patient - centered medical homes , so usual care was already much better than reported in the previous literature . we believe the next stage in this line of research could be an active comparator study examining the effects of pharmacists , perhaps with prescriptive autonomy , relative to other case managers . working in collaboration with the patient , primary care physician , and other health care professionals , pharmacists can have a significant , positive impact on blood pressure management in type 2 diabetes . we believe our results are applicable to a broad range of patients with type 2 diabetes managed in primary care settings and can be extended to nondiabetic patients with inadequately controlled hypertension .

healthy aging is defined as aging without disease . with the current attempts to increase the life span , understanding the molecular interactions and mechanisms involved in normal brain aging continues to be a challenge . cerebral aging is a complex and heterogenous process that is associated with a high degree of interindividual variability . the last 20 years have witnessed a great increase in our knowledge of its basic mechanisms . functional analyses have identified signaling pathways acting as master regulators of aging and lifespan that are conserved in many animals , suggesting that the rate of aging is not inevitably fixed , but is plastic and open to modifications . based on experimental evidence , the evolution of aging is probably the result of determinants of neuronal vulnerability , which include altered protein interaction networks , mitochondria , reactive oxidative species and intracellular calcium homeostasis , autophagy , signal transduction pathways , stem cell proliferation , and stress resistance mechanisms . perturbations in the functional state of these processes may lead to a state of decreased homeostatic reserve , where the aged neurons could still maintain adequate function during normal activity , although they become vulnerable . neurons have significant homeostatic control of essential physiological functions like synaptic excitability , gene expression , and metabolic regulation . any deviation in these physiological events can have severe consequences , as observed in aging . a recent study in a large cohort of > 10 000 persons showed that a measurable decline in generalized cortical function is already present by 45 to 49 years of age , with evidence of faster decline in older people . dementia due to alzheimer 's disease ( ad ) is preceded by about 5 to 6 years of accelerated decline of multiple cognitive functions ; by contrast , little decline is evident in persons who do not develop ad . compromised brain energy metabolism / oxygen delivery to neurons and blood flow differences in the regions most vulnerable to neurodegeneration are possible mechanisms of progression from healthy to unhealthy the human brain is uniquely powerful with respect to cognitive abilities , yet many neuronal networks , in particular the hippocampal and neocortical circuits that mediate such complex functions , are highly vulnerable to aging . loss of neurons , now recognized to be more modest than previously suggested , mainly involves these specific neuroanatomical areas . cognition and its decline associated with brain aging also seems to be variable and possibly open to modifications . studies in humans and animal models suggest that age - related cognitive decline is more likely to be associated with alterations in synaptic connectivity than with neuronal loss and white matter changes . according to recent studies , alterations of intracellular -secretase mediated signaling pathways may be involved in synaptic pathogenesis of ad , and apolipoprotein e is suggested to enhance the toxic effects of oligomeric amyloid beta ( a ) , causing synapse loss , a major correlate of cognitive decline in ad . although dementia - associated hallmarks of ad pathology ( neuritic plaques and neurofibrillary tangles ) become less prominent with increasing age , synaptic marker abnormalities in dementia remain constant and may represent an independent substrate of dementia spanning all ages . these and other changes induce functional network disruptions in degenerative dementia , suggesting that disease progress is transmitted by neural pathways . postmortem and in vivo magnetic resonance imaging ( mri ) studies of healthy brains have reported different location , extent , and severity of these changes with aging , some brain regions with greater activation being linked to better cognitive performance . besides hemispheric asymmetry reduction they indicated increased activity in ( pre)frontal regions , suggesting posterior - anterior shift models of functional brain aging . there is a strong relationship between cognitive ability and cortical fine structure in the prefrontal cortex . postmortem studies of human brains revealed more prominent age - related changes in the anterior and posterior white matter , but not in gray matter volumes , histology showing less severe changes than the imaging methods . while in previous studies postmortem mri of white matter lesions ( wmls ) was less sensitive than pathology , more recent ones showed that postmortem mri is a valid tool for the assessment of subcortical pathologies . mri investigations showed widespread age - related changes in prefrontal cortex and white matter , somatosensory cortex , and , to a lesser degree , in motor cortex , the prefrontal white matter being most susceptible to the influence of age . in cognitively normal elderly subjects , wmls were inversely correlated with gray matter volume , with greatest volume loss in the frontal cortex . both advancing age and hypertension predict higher wml load , which is itself associated with gray matter atrophy . low white matter grade and ventricular grade on mri are powerful determinants of long - term survival among older individuals . recent functional neuroimaging studies indicated reduced cortical activation in the default - mode network for mild cognitive impairment patients , compared with age - matched healthy elderly persons , mainly in the retrosplenial region / posterior cingulate cortex , left hippocampus , and bilateral inferior and middle frontal areas , while increased activation for patients was observed in the medial prefrontal and bilateral middle temporal/ angular cortex , probably as a compensatory mechanism . this region is of particular interest given its contribution to memory function , working memory decline being a common complaint in healthy aging and one of the earliest signs of ad . impaired hippocampal synaptic function is an early detectable pathologic alteration , well before amyloid plaque accumulation and cell death . positive relationships emerged consistently between the hippocampal formation , global cognition , and memory , and between frontal measures and executive function . the hippocampal formation and the papez circuit are targeted differentially by diseases of late life . volumetric mri of temporal and parietal brain structures distinguishes ad patients from healthy subjects , volumetry of the left and right hippocampus providing the highest diagnostic accuracy in separating these groups . recent advances in imaging techniques ( diffusion tensor imaging [ dti ] and magnetization transfer imaging [ mti ] ) indicate that age - related small - vessel disease is a diffuse process affecting the whole brain and that wmls are probably only the tip of the iceberg , while decreased gray matter diffusivity might be a potential new biomarker for early ad . age - related neuronal dysfunction involves a host of subtle changes such as reduction in the complexity of dendritic arborization and length , decrease in spine numbers and related synaptic densities , changes involving receptors , neurotransmitters , cytology , electric transmission , vascular or alzheimer - related changes , and myelin dystrophy . together , these multiple alterations in the brain may lead to age - related cognitive dysfunction . however , every lesion in the nervous system triggers an endogenous neuroprotective reaction , combining neuroplasticity and neurogenesis , which are initiated and regulated by neurotrophic factors in a multimodal way . extrusion of misfolded and aggregated ( toxic ) proteins may be a protective strategy of aging neurons . it is increasingly recognized that the correlation between neuropathological lesions and cognition is modest and accounts for about a quarter of the variance in cognition of older adults . concerning factors that modify or mediate the association between neuropathology and cognition , it was hypothesized that the concept of resilient aging can be useful to understand mechanisms that underlie healthy aging amidst disease - related pathology . some individuals maintain normal cognitive function despite significant brain pathology , while others suffer varying degrees of cognitive and neurological deterioration . many aged people do not exhibit cognitive impairment or other symptoms of disease and live normal lives , but nonetheless display pathological changes that are characteristic of ad , parkinson 's disease ( pd ) , cerebrovascular disease ( cvd ) , or other disorders . although the best morphologic correlates of cognitive impairment / dementia are ; ( i ) the number of neocortical neurofibrillary tangles ( nfts ) ; and ( ii ) loss of synapses , between 8% and 45% of nondemented , often cognitively stable older adults were found to have ad - related pathologies . many of them showed only minimal to mild neuritic changes corresponding to braak tau stages 0-iv , while 31% to 88% showed national institute for aging and reagan institute ( nia - ri ) criteria of no likelihood for ad criteria . the frequency of intermediate likelihood of ad criteria ranged from 11.9% to 35.8% , and only 1.5 to 3% were scored as having a high likelihood of ad . the presence of ad lesions in nondemented aged individuals may represent ad at a stage prior to clinical expression ( presymptomatic or unrecognized early forms ) . this is supported by observations that the mechanisms responsible for these changes in nondemented elderly appear similar if not identical to those found in ad , and their distribution corresponds to the hierarchical topographical procession associated with symptomatic ad . the concept of preclinical ad pathology has been further solidified in biomarker studies using csf a-42 and more directly in vivo positron emission tomography ( pet ) amyloid scanning , demonstrating that 20% to 30% of healthy elderly subjects have elevated pib signals indicative of extensive amyloid deposition . these data suggest a high frequency of preclinical ad pathology in normal elderly similar to that seen in clinico - pathologic cohorts . they further suggest that preclinical changes are not static , but progress over time . among 555 nondemented persons with false - positive pathological nia - ri high likelihood for ad , only 1.6% corresponded to braak stage v , 0.5% to stage vi , and 2.6% to stage v - vi , while in other studies between 35% and 88% were nia - ri negative ; 18% to 25% met the consortium to establish a registry for alzheimer 's disease ( cerad ) criteria for ad . review of the data from national disease coordinating center ( ndcc ) database and the nun study emphasized that there may be no documented example of truly end - stage neurofibrillary pathology with intact cognition . although in the adult changes in thought ( act ) and nun studies , nondemented seniors with severe ad pathology ( mean age of 89.156.9 to 90.805.2 years ) amounted to 8% and 12% , respectively , most of them showed neuritic braak stage v , and frontal nft counts were slightly lower than in a comparable dementia group . moreover , review of clinical data from those studies revealed that most of the seniors classified as nondemented were indeed significantly memory - impaired . a recent study of nondemented elderly demonstrated 62% with low and 28% with high nft levels70 ; 87 nondemented elderly ( mean age 87 5.9 years ; mean mmse 28.3 ) showed mean braak stage 3.00.9 , a total nft score of 4.52.5 , and mean neuritic density of 1.31.1 , whereas ad cases showed much higher cortical neuritic and striatal amyloid plaque scores . the 90 + study revealed significantly less severe a , -synuclein , and tpd-43 pathologies , and hippocampal sclerosis in nondemented subjects , while a distribution showed no essential differences ; nondemented individuals had limited hippocampal tau and neocortical a pathology . a recent clinicopathologic study of 296 persons without cognitive impairment of the religious order study ( ros ) and the memory and aging project ( map ) showed a common presence of ad pathology and macroscopic infarctions . amyloid load was related to global cognition ( p<0.05 ) , with only a trend for nfts ( p = 0.08 ) , while nfts and macroscopic infarctions were related to episodic memory ( p = 0.03 and 0.02 , respectively ) ; ad pathology and a load to working memory ( p = 0.02 and 0.03 , respectively ) . comparing the biochemistry of ad and nondemented nonagenerians revealed the lack of clear amyloid - related pathological/ biochemical determination between both groups . a personal retrospective study of 100 nondemented elderly ( mean age 81.235.47 years , mean mini mental state examination ( mmse ) score 29 ) revealed negative khachaturian criteria and cerad stage 0 in 83% and 86% , respectively , only 13% with cerad stage a and 1% stage b. braak neuritic stages ranged from 0 to iv with an average score of 2.30.8 . 12% were scored nia - ri low , and only 2% intermediate likelihood for ad . thus , mounting evidence from clinicopathologic studies support the view that ad is a continuous spectrum between asymptomatic lesions in cognitively normal elderly and dementia , with mild cognitive impairment ( mci ) as a transition phase between them . although correlations between cognitive deficits and the severity and extension of senile plaques ( sp ) and nfts ( see ref 42 ) have been found , at least in those brains without other pathologies , the distinction between physiological ( in nondemented subjects ) and pathological aging ( pa ) is difficult . a postmortem classification for individuals reported to be cognitively normal before death , their brains showing plaque pathology similar in extent to ad with only minimal cortical tau pathology , may also be difficult . recent biochemical studies found extensive overlap with only subtle quantitative differencies between a levels , peptide profiles , solubility , and oligomeric assemblies in pa and ad brains , suggesting that pa represents an initial prodromal stage of ad and that these individuals would eventually develop clinical symptoms , if they lived long enough , or an inherent individual resistance to the toxic effects of a. recent studies suggest that two independent processes ( synapse - mediated and apoe - mediated ) may contribute to region - specific a accumulation in nondemented individuals , and may influence the mechanisms of the regional vulnerability to a accumulation , which is prevented by apoe . a coding mutation ( a673 t ) in the app gene that reduces the p - cleavage of app may protect against ad and also against cognitive decline in the elderly without ad . older persons with overall normal cognitive function and preclinical ad changes by brain autopsy usually have lower scores on cognitive function tests , particularly episodic and working memory . a biomarker studies also confirmed the relations between preclinical ad and cognition , and a clinicopathologic study indicated that elders with ad changes but without overt dementia are more likely to have memory complaints . the definition of nondemented subjects with ad pathology raises important questions regarding the cognitive profile of these people who are relatively protected from the devastating effects of ad - related lesions . normal aging process , such that plaques and tangles are secondary to aging or that the primary aging effect is on synapses and neurons independent of these morphological ad markers . ad is indeed a disease that accompanies human aging , but it is not an inevitable consequence of it . however , the suggestion that plaques and tangles may cause this disorder is oversimplified or even wrong , since accumulating evidence suggests that ad pathology represents effect rather than cause or at least a host response to injury , equaling adaptive or neuroprotective reactions . many studies emphasize multiple additional pathologies in nondemented elders , in particular cerebrovascular lesions ( cvls ) , eg , small or large cerebral infarctions , lacunes , wmls , in 22 up to almost 100% . evaluation of 336 cognitively normal ( cn ) seniors from four studies revealed moderately to frequent neuritic plaque density in 47% ; of these 6% also had braak stages v or vi ; medullary , nigral , and cortical lewy bodies in 15% , 8% , and 4% , respectively ; cerebral microinfarcts in 33% and high - level cerebral microinfarcts in 10% . the burden of brain lesions and comorbidities varied widely within each study but was similar across studies . among 418 nondemented participants of the religious order study ( mean age 88.55.3 years ) , 35% showed macroscopical infarcts , 8% microinfarcts , 14.8% arteriosclerosis , 5.7% both , only 37.5% being free of cvls . up to 75% of cn seniors had various degrees of cerebral amyloid angiopathy ( caa ) , occasional hippocampal sclerosis , lewy body pathologies in up to 18% , argyrophilic grains in up to 23 % , and mixed pathologies in 7% to 14.8% . in a small autopsy series of cn elders , among 100 nondemented seniors , mild , moderate and severe intracranial atherosclerosis was present in 31% , 17% , and 6% , respectively , lacunar state in basal ganglia and/or white matter in 73% , hippocampal sclerosis in 3% , whereas only 9% were free of cvls . lewy bodies were observed in 5% , tau pathology in brain stem in 60% , and mixed cerebral pathologies ( cvls and moderate neuritic braak stages ) in 6% . a recent british nondemented sample ( n = 53 ; mean age 81.57.4 years ; mmse score 27 - 30 ) showed maximum score neuritic plaques in 32% to 49% , nfts in hippocampus and neocortex in 81% and 30.8% , respectively , white matter changes 55% to 83.7% , small vascular disease 45% , infarcts 13.7% , lacunes 6% , and hemorrhages 10% . thus , clinically silent pathology is widespread in normal aging , and the term healthy aging is inappropriate at the cellular level , and is manifested by regional heterogeneity in the scenario of general volume loss in the human brain . aging is associated with progressive loss in function across multiple systems , including sensation , cognition , memory , motor control , and affect . the traditional view has been that functional decline in aging is unavoidable because it is a direct consequence of brain machinery wearing down over time . in recent years , however , an alternative perspective has emerged that , based on extensive experimental work , argues that as people age , brain plasticity processes with negative consequences begin to dominate brain functioning . four core factors reduced schedules of brain activity , noisy processing , weakened modulatory control , and negative learning interact to create a self - reinforcing downward spiral of degraded brain function . these interrelated functions promote plastic changes in the brain that result in substantial improvement in function and/or recovery from functional losses . neuroplasticity can be defined as the ability of the nervous system to respond to intrinsic and extrinsic stimuli by reorganizing its structure , function , and connections . it is both a substrate of learning and memory and a mediator of responses to neuronal attrition and injury ( compensatory plasticity ) . this continuous process in reaction to neuronal activity and injury involves modulation of structural and functional processes of dendrites , axons , and synapses . however , mechanisms of neuroplasticity may vary with age , and occur in many variations and in many contexts , while common areas of plasticity that emerge across diverse cns conditions include experience dependence and circuit training . contrary to assumptions that changes in brain networks are possible only during crucial periods of development , recent research has supported the idea of a permanent plastic brain . novel experience , altered afferent input due to environmental changes , and learning new skills are now recognized as modulators of brain function and underlying neuroanatomic circuitry . results in animal experiments and discovery of increases in gray and white matter in the adult human brain as a result of learning and exercise have reinforced the old concept of cognitive reserve , that is , the ability to reinforce brain volume in certain areas and thus provide a greater threshold for age - dependent deficits , or the capacity of the brain to manage pathology or age - related changes , thereby minimizing clinical manifestation . the concept of cognitive reserve and a broader theory of brain reserve was originally proposed to help explain epidemiological data indicating that individuals who engaged higher levels of mental and physical activity via education , occupation , and recreation were associated with slower cognitive decline in healthy aging and are at lower risk of developing ad and other forms of dementia . the aging process that results in loss of synapses and possible neurons may be far more detrimental for those with little brain reserve as compared with those with a high one . the construct of cognitive reserve is a set of variables including intelligence , education , and mental stimulation which putatively allows the brain to adapt to underlying pathologies by maintaining cognitive function despite underlying neuronal changes . it also indicates a resilience to neuropathological damage , and could be defined as the ability to optimize or maximize performance through effective recruitment of brain networks and/or alternative cognitive strategies . childhood cognition , educational attainment , and adult occupation all contribute to cognitive reserve independently . enriched environment and physical activity influence the rate of neurogenesis in adult animal model hippocampi . in people with high reserve structural and functional brain imaging studies have revealed selective changes in aging brain that reflect neural decline as well as compensatory neural recruitment , representing possible neural substrates of cognitive reserve , but its neural basis is still a topic of ongoing research . while aging is associated with reductions in cortical thickness , white matter integrity , transmitter activity , and functional engagement in the hippocampus and occipital areas , there are compensatory increases in frontal functional engagement that correlate with better behavioral performance in the elderly . those cortical regions most consistently shrinking in aging prefrontal and parietal cortices are the same regions showing increased regional activation in aging , suggesting that losses in regional brain integrity drive functional reorganization through changes in processing strategy . cognitive reserve allows individuals greater neural efficacy , greater neural capacities , and the ability for compensation via the recruitment of additional brain regions . frontal and supramarginal cortical activity has been suggested to compensate for an age - related decrease in inferior - frontal junction recruitment of verbal fluency processing . larger brain and hippocampal values , and neuronal hypertrophy were associated with preserved cognitive function despite a high burden of ad pathology ( asymptomatic ad ) . the structural and functional imaging correlates of cognitive and brain reserve hypothesis have recently been reviewed . a complementary hypothesis of metabolic reserve is characterized by neuronal circuits that respond adaptively to perturbations in cellular energy metabolism and thereby protect against declining function , mediated by neurotrophic factor signaling , and glucose metabolism . increased basal forebrain metabolism in mci is an evidence for brain reserve in incipient dementia . neuroprotective effects of noradrenaline both in vivo and in vitro suggest noradrenaline 's key role in mediating cognitive reserve by disease compensation , modification , or a combination of both , a viable hypothesis . the structural elements that embody plasticity include synaptic efficacy and remodeling , synaptogenesis , neurite extension including axonal sprouting and dendritic remodeling , neurogenesis , and recruitment from neural progenitor cells . phenomenological processes that manifest plasticity are : synapse , neurite , neuronal cell bodies , anterograde and retrograde transport , cell interactions ( neuron - glia ) , neuronal networks , and related activities . they include intraneuronal , interneuronal , and intercellular signaling through glia , and involve extracellular matrix molecules , immunoglobulins , myelin - associated inhibitors , tyrosine kinase receptors , neurotrophic and growth factors , inflammatory cytokines , and neurotransmitters . these processes are regulated by cell - autonomous and intercellular programs that mediate responses of neuronal cells to environmental input . by generating energy and regulating subcellular ca and redox homeostasis , mitochondria may play important roles in controlling fundamental plasticity processes , including neuronal and synaptic differentiation , neurite outgrowth , neurotransmitter release , and dendritic remodeling . receptor protein tyrosine phosphorylase ( rptp ) regulates synapse structure , function , and plasticity . emerging data suggest that mitochondria emit molecular signals , eg , reactive oxygen species , proteins , and lipid mediators that can act locally or travel to distant targets . disorders in mitochondrial functions and signalling may play roles in impaired neuroplasticity and neurodegeneration . both aging and a that as a normal product of neuronal metabolism has an essential regulatory function at the synapse , independently decrease neuronal plasticity . the major growth of a burden occurs during a preclinical stage of ad , prior to the onset of ad - related symptoms . it is associated with lower cognitive performance both in ad patients and normal elderly , but the association is modified by cognitive reserve , suggesting that this may be protective against amyloid - related cognitive impairment . on the other hand , endogenous a is necessary for hippocampal plasticity and memory within the normal cns , due to regulation of transmitter release , activation of nicotinic acetylcholine receptors , and a-42 production . the basis of age - related toxicity partly resides in mitochondrial dysfunction and an oxidative shift in mitochondrial and cytoplasmic redox potential . in turn , signaling through phosphorylated extracellular signal - regulated protein kinases is affected along with an age - independent increase in phosphorylated cyclic adenosine monophosphate ( camp ) response element - binding protein . furthermore , the production of inflammatory mediators ( inflammatory cytokines , interleukins , neurotrophins ) , activation of glia and other immune cells disrupting the delicate balance needed for the physiological action of immune processes produces direct effects on neural plasticity and neurogenesis , facilitating many forms of neuropathology associated with normal aging as well as neurodegenerative diseases . recent evidence shows that key regulations of communication between neuron and microglia disruption in the aged brain may be one of the factors that precedes and initiates the increase in chronic inflammatory states underlying age - related impairments of cognition and hippocampal neurogenesis . effective treatments that dampen inflammatory activity are expected to have beneficial effects on cognitive performance and neural plasticity . functional recovery of synaptic circuitry requires that reactive synaptogenesis not exacerbate dysfunction , since aberrant misconnection by innervating the wrong target may cause misguided synaptogenesis , and inhibition of sprouting may be protective by sequestering dysfunctional neurons . aberrant , excessive , insufficient , or mistimed plasticity may represent the pathogenic cause of neurodevelopmental and neurodegenerative disorders . neuroplasticity is impaired in patients with ad and pd as a result of diminished growth factor expression and failure of delayed nonsynaptic neural plasticity mechanisms . understanding normative changes in brain structure that occur as a result of environmental changes is pivotal to understanding the ability of the brain to adapt . studies in animals and humans revealed dramatic effects of environmental enrichment , increased physical exercise documenting positive effects of mental and physical exercise , mediating brain and cognitive reserve , thus showing no compromise in daily life despite higher a plaque load . other studies in animal models showed preventive or therapeutic action of environmental enrichment counteracting a pathology by different molecular mechanisms and by mitigating alzheimer - like pathology , and increasing synaptic immunore activity due to reduction of cerebral oxidative stress . examination of synaptic physiology revealed that environmental experience significantly enhanced axonal transport in hippocampal and cortical neurons after enrichment , enhanced hippocampus long - term potentiation , without notable alterations in synaptic transmission . these data suggest that environmental modulation can rescue the impaired phenotype of the ad brain and that induction of brain plasticity may represent therapeutic and preventive avenues in ad . recent studies demonstrated that the magnitude of the contribution of education is greater than the negative impact of either neuropathological burden of ad or cvls with standardized regression weights of -0.14 for hyperintensities and -0.20 for hippocampal atrophy . however , a large clinicopathologic study at 27 ad centers found no evidence of larger education - related differences in cognitive function when ad pathology was more advanced , suggesting that the advances of cognitive reserve may ultimately be overwhelmed by ad pathology . neurogenesis or the birth of new neural cells was thought to occur only in the developing nervous system , but recent studies have demonstrated that it does indeed continue into and throughout adult life . however , the age of olfactory bulb neurones , that are assumed to be derived from neuroblasts via the rostral migratory stream ( rms ) , has been assessed recently by measuring the levels of nuclear bomb test - derived 14c in genomic dna . data from this study suggest that there is very limited , if any , postnatal neurogenesis in the human olfactory bulb . certain areas of the brain may retain pluripotent precursors with the capacity to self - renew and differentiate into new neural lineages in adult mammals , nonhuman primates , and humans . physical activity causes a robust increase in neurogenesis in the dentate gyrus of the hippocampus , a process that would implement a form of network plasticity analogous to that at the synaptic level , but occurring at the cellular network level . neurogenesis represents a key factor of adult brain to response to environmental stimuli , and abnormalities in neurogenesis have been detected in neurodegenerative disorders such as ad . it occurs in the subventricular zone and the subgranuiar layer of the hippocampus , and follows a multistep process probably in five stages , including proliferation , differentiation , migrating , targeting , and integration phases , respectively . stimuli that entail an increase in neuronal activity have been shown to stimulate neurogenesis and enhance survival of new neurons in the adult mammalian hippocampus . the incorporation of functional adult - generated neurons into existing neural networks provides higher capacity for plasticity , while they favor the encoding and storage of certain types of memories . although neurogenesis continues throughout life , its rate declines with increasing age , and the proportion of neuronal stem cells that survive to become mature neuronal ceils is reduced . this may be due to intrinsic decline in neuronal stem cell responsiveness to stimulating environmental cues , to a decrease in or disappearance of these environmental cues , or to accumulation of inhibitory factors . intrinsic properties of neural progenitor cells such as gene transcription and telomere activity change with age , which may contribute to decline in neurogenesis . while most studies indicated a correlation between decreased hippocampal neurogenesis and impaired performance in hippocampus - dependent cognitive tasks in age mice , few have demonstrated that young and aged mice are equivalent in their cognitive ability . the lack of neuronal ability to divide may be overcome by replacing damaged neurons or by restoring their function . thus , kittappa et al revisited the molecular mechanisms responsible for neuronal renewal from stem cells , which are present in specific niches within the adult brain . the authors provided the novel notion that even non - terminaliy differentiated neural stem cells play roles in the regeneration of neurons and their synaptic function by mechanisms beyond mere cell replacement . these cells signal specific survival pathways that are worth investigating in search for novel therapeutic strategies against neurodegeneration . according to this notion , noninvasive tools to follow up synaptic function in the living brain are therefore essential for our better understanding of neuronal regeneration . although neuronal turnover is reduced in every neurogenic region of the aged brain , neuronal precursor cells clearly survive , remain responsive to growth factors and other physiological stimuli , and can increase their activity in response to damage . exploration of the regulation of neuronal progenitor cells in the aging brain is critical not only for understanding age - related cognitive deficits , but also for progress toward the goal of using the brain 's regenerative potential to restore functional loss . dysregulated or impaired neurogenesis may compromise plasticity and neuronal function in the hippocampus and other neuronal systems , and exacerbate neuronal vulnerability . interestingly , increasing evidence suggests that molecular players in ad , including presenilin1 , amyloid precursor protein , and its metabolites , play a role in adult neurogenesis , while alterations in tau phosporylation may interfere with the potential role of tau proteins in neuronal maturation and differentiation . this indicates a crosstalk between signaling molecules involved in both neurogenesis and neurodegeneration , and the ways by which ad - linked dysfunction of these signaling molecules affect neurogenesis in the adult brain . in ad , both increased and decreased neurogenesis has been reported and cholinergic activity may be involved in neurogenesis . however , most of these new neurons die , and fibrillar a-42 seems to be involved in generating an inappropriate environment for those neurons to mature . these findings open up prospects for new strategies that can increase neurogenesis in pathologic processes in the aging brain . recent studies confirming the assumption that cholinergic pathology has a detrimental influence on neurogenesis suggest an attenuation of stem cells together with compensatory increased proliferation that , however , does not result in an increased number of migratory neuroblasts and differentiated neurons in ad . there are indications that neurogenesis is impaired in pd , which might be due to a lack of dopamine in the subventricular zone , but recent studies did not find evidence that dopamine has a direct effect on human stem cell proliferation in vitro . thus , it was concluded that the number of adult neural stem cells is probably not diminished , and the proliferative capacity of the subventricular zone is maintained in the parkinsonian brain . neural stem cells have been identified also in areas where neurogenesis does not occur under physiological conditions , such as the midbrain and striatum , suggesting that they may have the potential to be used as a non - invasive cell replacement therapy in pd . recent studies have shown that the deleterious effects of -synuclein on newly generated neurons , in particular on their dendritic outgrowth and spine development , thus having negative impact on adult neurogenesis and neuronal maturation . further elucidation of the mechanisms regulating the synaptic integration of adult - born neurons is not only crucial for our understanding of the age- and disease - related neuroplasticity / brain plasticity , but also provides a framework for the manipulation and monitoring of endogenous adult neurogenesis as well as grafted cells potential therapeutic applications . a major problem in studying aging is how to separate the effects of aging from disease . cerebral aging is a complex and heterogenous process that is associated with a high variety of molecular interactions , morphological , and functional changes , summarized in table i. the interrelations between them need further elucidation . brain aging results in loss of synapses and possible neurons , which is associated with structural changes in cerebral areas and neural neworks that are essential for cognitive and memory function . many cognitively unimpaired eldery subjects are involved by alzheimer - related or other pathologies of various severity and extent . knowing the substrate of the resilience to cognitive decline in the presence of abundant ad and/or mixed pathology might be crucial not only for the understanding of the pathophysiology of nondemented aged people , but also to discover new prophylactic and/or therapeutic targets for aging processes . as expected from the significant clinicopathologic correlations of synaptic and neuronal loss in ad , high - pathology nondemented controls have preserved densities of synaptophysinlabeled presynaptic terminals and dendritic spines as compared with ad dementia patients with a similar burden of plaques and tangles . greater amounts of specific presynaptic proteins and distinct protein - protein intreactions may be components of cognitive reserve that reduce the risk of dementia with aging . they may have no significant neuronal loss , not even in vulnerable regions , such as the entorhinal cortex and hippocampus , and have lower levels of neuroinflammatory markers than pathology - matched ad patients . this resistance to ad pathology has also been related to a nucleolar , nuclear , and cell body hypertrophy of the hippocampal and cortical neurons , suggestive of a compensatory metabolic activation to face the neurotoxic effects of ad lesions . resilience to ad is also attributed to genetic factors , particularly apolipoprotein e2 and combinations of other genetic polymorphisms . premorbid brain volume has been found to provide protection against clinical manifestation of dementia despite evidence of ad pathology , supporting the brain reserve hypothesis of resilience to ad . although multiple factors and possible interventions may influence cognitive reserve and susceptibility to dementia , much work is required on the mechanisms of action in order to determine which , if any , may improve the clinical and epidemiological picture . on the other hand , the unique observation of a cognitively intact woman aged 115 years with only slight tau pathology corresponding to braak stage ii , almost no plaques or vascular changes , and normal neuron count in the locus ceruleus indicates that the limits of human cognitive function extend far beyond the range that is currently enjoyed by most individuals and that brain disease , even in supercentenarians , is not inevitable . the association between vulnerability and protective factors varies with age , since the effects of these factors on the risk for ad may differ in younger ( age < 80 ) versus older ( age > 80 ) individuals . the understanding of the dynamic of these factors at different age periods will be essential for the implementation of primary prevention treatments for ad . the importance of understanding ageing and the complex interplay of multiple influences on successful cognitive ageing is clear . understanding how brain reserve might be influenced to minimize this is a crucial prerequisite to meaningful research in dementia and illustrates how life - long intellectual engagement can mitigate the negative impact of brain pathology even on healthy ageing . the neuronal underpinning of the dynamic compensatory mechanism opens the possibility for strategic interventions based on environmental approaches . future work should measure the contribution of more diverse influences on cognitive reserve that might operate in early and midlife , such as socioeconomic conditions and social relationships , which might be modified through public education in order to have a positive impact on the looming public health disaster that is dementia . recent studies in a nondemented population have shown that intellectual and physical activity lifestyle factors were not assessed with ad biomarkers , while intellectual lifestyle factors explained the variability in the cognitive performance , providing evidence that lifestyle activities may delay the onset of dementia , but do not significantly influence the expression of ad pathophysiology . the neuropathological distinction between nondemented , cognitively intact , and cognitively impaired / demented subjects , elucidation of the relationship of additional pathologies with minor often clinically latent ad lesions observed in many but not all elderly persons without cognitive impairment is important , allowing further insights into the mechanisms of brain plasticity and the basic mechanisms of adult neurogenesis warrants further experimental and prospective , well documented clinico - pathological studies of elderly individuals . in this continuously growing field , new acquisitions , derived from basic research and clinical grounds , on cognitive reserve mechanisms , neuroplasticity , and the potential application of novel therapeutic targets in neurodegeneration and aging disorders are necessary . as a basis for potential prophylactic and therapeutic options for brain aging ,

cerebral aging is a complex and heterogenous process related to a large variety of molecular changes involving multiple neuronal networks , due to alterations of neurons ( synapses , axons , dendrites , etc ) , particularly affecting strategically important regions , such as hippocampus and prefrontal areas . a substantial proportion of nondemented , cognitively unimpaired elderly subjects show at least mild to moderate , and rarely even severe , alzheimer - related lesions , probably representing asymptomatic preclinical alzheimer 's disease , and/or mixed pathologies . while the substrate of resilience to cognitive decline in the presence of abundant pathologies has been unclear , recent research has strengthened the concept of cognitive or brain reserve , based on neuroplasticity or the ability of the brain to manage or counteract age - related changes or pathologies by reorganizing its structure , connections , and functions via complex molecular pathways and mechanisms that are becoming increasingly better understood . part of neuroplasticity is adult neurogenesis in specific areas of the brain , in particular the hippocampal formation important for memory function , the decline of which is common even in healthy aging . to obtain further insights into the mechanisms of brain plasticity and adult neurogenesis , as the basis for prevention and potential therapeutic options , is a major challenge of modern neurosciences .

Introduction Neuropathology findings in cognitively normal aged subjects Brain aging and neuroplasticity The concept of cognitive reserve Structural basis of neuroplasticity Neurogenesis in the aging brain Conclusions and outlook

healthy aging is defined as aging without disease . cerebral aging is a complex and heterogenous process that is associated with a high degree of interindividual variability . based on experimental evidence , the evolution of aging is probably the result of determinants of neuronal vulnerability , which include altered protein interaction networks , mitochondria , reactive oxidative species and intracellular calcium homeostasis , autophagy , signal transduction pathways , stem cell proliferation , and stress resistance mechanisms . perturbations in the functional state of these processes may lead to a state of decreased homeostatic reserve , where the aged neurons could still maintain adequate function during normal activity , although they become vulnerable . dementia due to alzheimer 's disease ( ad ) is preceded by about 5 to 6 years of accelerated decline of multiple cognitive functions ; by contrast , little decline is evident in persons who do not develop ad . compromised brain energy metabolism / oxygen delivery to neurons and blood flow differences in the regions most vulnerable to neurodegeneration are possible mechanisms of progression from healthy to unhealthy the human brain is uniquely powerful with respect to cognitive abilities , yet many neuronal networks , in particular the hippocampal and neocortical circuits that mediate such complex functions , are highly vulnerable to aging . studies in humans and animal models suggest that age - related cognitive decline is more likely to be associated with alterations in synaptic connectivity than with neuronal loss and white matter changes . according to recent studies , alterations of intracellular -secretase mediated signaling pathways may be involved in synaptic pathogenesis of ad , and apolipoprotein e is suggested to enhance the toxic effects of oligomeric amyloid beta ( a ) , causing synapse loss , a major correlate of cognitive decline in ad . postmortem studies of human brains revealed more prominent age - related changes in the anterior and posterior white matter , but not in gray matter volumes , histology showing less severe changes than the imaging methods . mri investigations showed widespread age - related changes in prefrontal cortex and white matter , somatosensory cortex , and , to a lesser degree , in motor cortex , the prefrontal white matter being most susceptible to the influence of age . in cognitively normal elderly subjects , wmls were inversely correlated with gray matter volume , with greatest volume loss in the frontal cortex . recent functional neuroimaging studies indicated reduced cortical activation in the default - mode network for mild cognitive impairment patients , compared with age - matched healthy elderly persons , mainly in the retrosplenial region / posterior cingulate cortex , left hippocampus , and bilateral inferior and middle frontal areas , while increased activation for patients was observed in the medial prefrontal and bilateral middle temporal/ angular cortex , probably as a compensatory mechanism . this region is of particular interest given its contribution to memory function , working memory decline being a common complaint in healthy aging and one of the earliest signs of ad . positive relationships emerged consistently between the hippocampal formation , global cognition , and memory , and between frontal measures and executive function . recent advances in imaging techniques ( diffusion tensor imaging [ dti ] and magnetization transfer imaging [ mti ] ) indicate that age - related small - vessel disease is a diffuse process affecting the whole brain and that wmls are probably only the tip of the iceberg , while decreased gray matter diffusivity might be a potential new biomarker for early ad . age - related neuronal dysfunction involves a host of subtle changes such as reduction in the complexity of dendritic arborization and length , decrease in spine numbers and related synaptic densities , changes involving receptors , neurotransmitters , cytology , electric transmission , vascular or alzheimer - related changes , and myelin dystrophy . together , these multiple alterations in the brain may lead to age - related cognitive dysfunction . however , every lesion in the nervous system triggers an endogenous neuroprotective reaction , combining neuroplasticity and neurogenesis , which are initiated and regulated by neurotrophic factors in a multimodal way . concerning factors that modify or mediate the association between neuropathology and cognition , it was hypothesized that the concept of resilient aging can be useful to understand mechanisms that underlie healthy aging amidst disease - related pathology . many aged people do not exhibit cognitive impairment or other symptoms of disease and live normal lives , but nonetheless display pathological changes that are characteristic of ad , parkinson 's disease ( pd ) , cerebrovascular disease ( cvd ) , or other disorders . although the best morphologic correlates of cognitive impairment / dementia are ; ( i ) the number of neocortical neurofibrillary tangles ( nfts ) ; and ( ii ) loss of synapses , between 8% and 45% of nondemented , often cognitively stable older adults were found to have ad - related pathologies . the presence of ad lesions in nondemented aged individuals may represent ad at a stage prior to clinical expression ( presymptomatic or unrecognized early forms ) . the concept of preclinical ad pathology has been further solidified in biomarker studies using csf a-42 and more directly in vivo positron emission tomography ( pet ) amyloid scanning , demonstrating that 20% to 30% of healthy elderly subjects have elevated pib signals indicative of extensive amyloid deposition . among 555 nondemented persons with false - positive pathological nia - ri high likelihood for ad , only 1.6% corresponded to braak stage v , 0.5% to stage vi , and 2.6% to stage v - vi , while in other studies between 35% and 88% were nia - ri negative ; 18% to 25% met the consortium to establish a registry for alzheimer 's disease ( cerad ) criteria for ad . although correlations between cognitive deficits and the severity and extension of senile plaques ( sp ) and nfts ( see ref 42 ) have been found , at least in those brains without other pathologies , the distinction between physiological ( in nondemented subjects ) and pathological aging ( pa ) is difficult . recent biochemical studies found extensive overlap with only subtle quantitative differencies between a levels , peptide profiles , solubility , and oligomeric assemblies in pa and ad brains , suggesting that pa represents an initial prodromal stage of ad and that these individuals would eventually develop clinical symptoms , if they lived long enough , or an inherent individual resistance to the toxic effects of a. recent studies suggest that two independent processes ( synapse - mediated and apoe - mediated ) may contribute to region - specific a accumulation in nondemented individuals , and may influence the mechanisms of the regional vulnerability to a accumulation , which is prevented by apoe . a coding mutation ( a673 t ) in the app gene that reduces the p - cleavage of app may protect against ad and also against cognitive decline in the elderly without ad . the definition of nondemented subjects with ad pathology raises important questions regarding the cognitive profile of these people who are relatively protected from the devastating effects of ad - related lesions . however , the suggestion that plaques and tangles may cause this disorder is oversimplified or even wrong , since accumulating evidence suggests that ad pathology represents effect rather than cause or at least a host response to injury , equaling adaptive or neuroprotective reactions . many studies emphasize multiple additional pathologies in nondemented elders , in particular cerebrovascular lesions ( cvls ) , eg , small or large cerebral infarctions , lacunes , wmls , in 22 up to almost 100% . among 418 nondemented participants of the religious order study ( mean age 88.55.3 years ) , 35% showed macroscopical infarcts , 8% microinfarcts , 14.8% arteriosclerosis , 5.7% both , only 37.5% being free of cvls . up to 75% of cn seniors had various degrees of cerebral amyloid angiopathy ( caa ) , occasional hippocampal sclerosis , lewy body pathologies in up to 18% , argyrophilic grains in up to 23 % , and mixed pathologies in 7% to 14.8% . thus , clinically silent pathology is widespread in normal aging , and the term healthy aging is inappropriate at the cellular level , and is manifested by regional heterogeneity in the scenario of general volume loss in the human brain . aging is associated with progressive loss in function across multiple systems , including sensation , cognition , memory , motor control , and affect . the traditional view has been that functional decline in aging is unavoidable because it is a direct consequence of brain machinery wearing down over time . in recent years , however , an alternative perspective has emerged that , based on extensive experimental work , argues that as people age , brain plasticity processes with negative consequences begin to dominate brain functioning . neuroplasticity can be defined as the ability of the nervous system to respond to intrinsic and extrinsic stimuli by reorganizing its structure , function , and connections . this continuous process in reaction to neuronal activity and injury involves modulation of structural and functional processes of dendrites , axons , and synapses . however , mechanisms of neuroplasticity may vary with age , and occur in many variations and in many contexts , while common areas of plasticity that emerge across diverse cns conditions include experience dependence and circuit training . contrary to assumptions that changes in brain networks are possible only during crucial periods of development , recent research has supported the idea of a permanent plastic brain . novel experience , altered afferent input due to environmental changes , and learning new skills are now recognized as modulators of brain function and underlying neuroanatomic circuitry . results in animal experiments and discovery of increases in gray and white matter in the adult human brain as a result of learning and exercise have reinforced the old concept of cognitive reserve , that is , the ability to reinforce brain volume in certain areas and thus provide a greater threshold for age - dependent deficits , or the capacity of the brain to manage pathology or age - related changes , thereby minimizing clinical manifestation . the concept of cognitive reserve and a broader theory of brain reserve was originally proposed to help explain epidemiological data indicating that individuals who engaged higher levels of mental and physical activity via education , occupation , and recreation were associated with slower cognitive decline in healthy aging and are at lower risk of developing ad and other forms of dementia . the construct of cognitive reserve is a set of variables including intelligence , education , and mental stimulation which putatively allows the brain to adapt to underlying pathologies by maintaining cognitive function despite underlying neuronal changes . it also indicates a resilience to neuropathological damage , and could be defined as the ability to optimize or maximize performance through effective recruitment of brain networks and/or alternative cognitive strategies . childhood cognition , educational attainment , and adult occupation all contribute to cognitive reserve independently . while aging is associated with reductions in cortical thickness , white matter integrity , transmitter activity , and functional engagement in the hippocampus and occipital areas , there are compensatory increases in frontal functional engagement that correlate with better behavioral performance in the elderly . frontal and supramarginal cortical activity has been suggested to compensate for an age - related decrease in inferior - frontal junction recruitment of verbal fluency processing . the structural elements that embody plasticity include synaptic efficacy and remodeling , synaptogenesis , neurite extension including axonal sprouting and dendritic remodeling , neurogenesis , and recruitment from neural progenitor cells . phenomenological processes that manifest plasticity are : synapse , neurite , neuronal cell bodies , anterograde and retrograde transport , cell interactions ( neuron - glia ) , neuronal networks , and related activities . receptor protein tyrosine phosphorylase ( rptp ) regulates synapse structure , function , and plasticity . on the other hand , endogenous a is necessary for hippocampal plasticity and memory within the normal cns , due to regulation of transmitter release , activation of nicotinic acetylcholine receptors , and a-42 production . the basis of age - related toxicity partly resides in mitochondrial dysfunction and an oxidative shift in mitochondrial and cytoplasmic redox potential . furthermore , the production of inflammatory mediators ( inflammatory cytokines , interleukins , neurotrophins ) , activation of glia and other immune cells disrupting the delicate balance needed for the physiological action of immune processes produces direct effects on neural plasticity and neurogenesis , facilitating many forms of neuropathology associated with normal aging as well as neurodegenerative diseases . recent evidence shows that key regulations of communication between neuron and microglia disruption in the aged brain may be one of the factors that precedes and initiates the increase in chronic inflammatory states underlying age - related impairments of cognition and hippocampal neurogenesis . understanding normative changes in brain structure that occur as a result of environmental changes is pivotal to understanding the ability of the brain to adapt . other studies in animal models showed preventive or therapeutic action of environmental enrichment counteracting a pathology by different molecular mechanisms and by mitigating alzheimer - like pathology , and increasing synaptic immunore activity due to reduction of cerebral oxidative stress . these data suggest that environmental modulation can rescue the impaired phenotype of the ad brain and that induction of brain plasticity may represent therapeutic and preventive avenues in ad . however , a large clinicopathologic study at 27 ad centers found no evidence of larger education - related differences in cognitive function when ad pathology was more advanced , suggesting that the advances of cognitive reserve may ultimately be overwhelmed by ad pathology . however , the age of olfactory bulb neurones , that are assumed to be derived from neuroblasts via the rostral migratory stream ( rms ) , has been assessed recently by measuring the levels of nuclear bomb test - derived 14c in genomic dna . certain areas of the brain may retain pluripotent precursors with the capacity to self - renew and differentiate into new neural lineages in adult mammals , nonhuman primates , and humans . physical activity causes a robust increase in neurogenesis in the dentate gyrus of the hippocampus , a process that would implement a form of network plasticity analogous to that at the synaptic level , but occurring at the cellular network level . neurogenesis represents a key factor of adult brain to response to environmental stimuli , and abnormalities in neurogenesis have been detected in neurodegenerative disorders such as ad . it occurs in the subventricular zone and the subgranuiar layer of the hippocampus , and follows a multistep process probably in five stages , including proliferation , differentiation , migrating , targeting , and integration phases , respectively . this may be due to intrinsic decline in neuronal stem cell responsiveness to stimulating environmental cues , to a decrease in or disappearance of these environmental cues , or to accumulation of inhibitory factors . although neuronal turnover is reduced in every neurogenic region of the aged brain , neuronal precursor cells clearly survive , remain responsive to growth factors and other physiological stimuli , and can increase their activity in response to damage . exploration of the regulation of neuronal progenitor cells in the aging brain is critical not only for understanding age - related cognitive deficits , but also for progress toward the goal of using the brain 's regenerative potential to restore functional loss . dysregulated or impaired neurogenesis may compromise plasticity and neuronal function in the hippocampus and other neuronal systems , and exacerbate neuronal vulnerability . interestingly , increasing evidence suggests that molecular players in ad , including presenilin1 , amyloid precursor protein , and its metabolites , play a role in adult neurogenesis , while alterations in tau phosporylation may interfere with the potential role of tau proteins in neuronal maturation and differentiation . this indicates a crosstalk between signaling molecules involved in both neurogenesis and neurodegeneration , and the ways by which ad - linked dysfunction of these signaling molecules affect neurogenesis in the adult brain . these findings open up prospects for new strategies that can increase neurogenesis in pathologic processes in the aging brain . there are indications that neurogenesis is impaired in pd , which might be due to a lack of dopamine in the subventricular zone , but recent studies did not find evidence that dopamine has a direct effect on human stem cell proliferation in vitro . thus , it was concluded that the number of adult neural stem cells is probably not diminished , and the proliferative capacity of the subventricular zone is maintained in the parkinsonian brain . neural stem cells have been identified also in areas where neurogenesis does not occur under physiological conditions , such as the midbrain and striatum , suggesting that they may have the potential to be used as a non - invasive cell replacement therapy in pd . recent studies have shown that the deleterious effects of -synuclein on newly generated neurons , in particular on their dendritic outgrowth and spine development , thus having negative impact on adult neurogenesis and neuronal maturation . further elucidation of the mechanisms regulating the synaptic integration of adult - born neurons is not only crucial for our understanding of the age- and disease - related neuroplasticity / brain plasticity , but also provides a framework for the manipulation and monitoring of endogenous adult neurogenesis as well as grafted cells potential therapeutic applications . a major problem in studying aging is how to separate the effects of aging from disease . cerebral aging is a complex and heterogenous process that is associated with a high variety of molecular interactions , morphological , and functional changes , summarized in table i. the interrelations between them need further elucidation . brain aging results in loss of synapses and possible neurons , which is associated with structural changes in cerebral areas and neural neworks that are essential for cognitive and memory function . many cognitively unimpaired eldery subjects are involved by alzheimer - related or other pathologies of various severity and extent . knowing the substrate of the resilience to cognitive decline in the presence of abundant ad and/or mixed pathology might be crucial not only for the understanding of the pathophysiology of nondemented aged people , but also to discover new prophylactic and/or therapeutic targets for aging processes . they may have no significant neuronal loss , not even in vulnerable regions , such as the entorhinal cortex and hippocampus , and have lower levels of neuroinflammatory markers than pathology - matched ad patients . this resistance to ad pathology has also been related to a nucleolar , nuclear , and cell body hypertrophy of the hippocampal and cortical neurons , suggestive of a compensatory metabolic activation to face the neurotoxic effects of ad lesions . resilience to ad is also attributed to genetic factors , particularly apolipoprotein e2 and combinations of other genetic polymorphisms . premorbid brain volume has been found to provide protection against clinical manifestation of dementia despite evidence of ad pathology , supporting the brain reserve hypothesis of resilience to ad . on the other hand , the unique observation of a cognitively intact woman aged 115 years with only slight tau pathology corresponding to braak stage ii , almost no plaques or vascular changes , and normal neuron count in the locus ceruleus indicates that the limits of human cognitive function extend far beyond the range that is currently enjoyed by most individuals and that brain disease , even in supercentenarians , is not inevitable . understanding how brain reserve might be influenced to minimize this is a crucial prerequisite to meaningful research in dementia and illustrates how life - long intellectual engagement can mitigate the negative impact of brain pathology even on healthy ageing . the neuronal underpinning of the dynamic compensatory mechanism opens the possibility for strategic interventions based on environmental approaches . the neuropathological distinction between nondemented , cognitively intact , and cognitively impaired / demented subjects , elucidation of the relationship of additional pathologies with minor often clinically latent ad lesions observed in many but not all elderly persons without cognitive impairment is important , allowing further insights into the mechanisms of brain plasticity and the basic mechanisms of adult neurogenesis warrants further experimental and prospective , well documented clinico - pathological studies of elderly individuals .

functional analyses have identified signaling pathways acting as master regulators of aging and lifespan that are conserved in many animals , suggesting that the rate of aging is not inevitably fixed , but is plastic and open to modifications . based on experimental evidence , the evolution of aging is probably the result of determinants of neuronal vulnerability , which include altered protein interaction networks , mitochondria , reactive oxidative species and intracellular calcium homeostasis , autophagy , signal transduction pathways , stem cell proliferation , and stress resistance mechanisms . perturbations in the functional state of these processes may lead to a state of decreased homeostatic reserve , where the aged neurons could still maintain adequate function during normal activity , although they become vulnerable . dementia due to alzheimer 's disease ( ad ) is preceded by about 5 to 6 years of accelerated decline of multiple cognitive functions ; by contrast , little decline is evident in persons who do not develop ad . compromised brain energy metabolism / oxygen delivery to neurons and blood flow differences in the regions most vulnerable to neurodegeneration are possible mechanisms of progression from healthy to unhealthy the human brain is uniquely powerful with respect to cognitive abilities , yet many neuronal networks , in particular the hippocampal and neocortical circuits that mediate such complex functions , are highly vulnerable to aging . studies in humans and animal models suggest that age - related cognitive decline is more likely to be associated with alterations in synaptic connectivity than with neuronal loss and white matter changes . according to recent studies , alterations of intracellular -secretase mediated signaling pathways may be involved in synaptic pathogenesis of ad , and apolipoprotein e is suggested to enhance the toxic effects of oligomeric amyloid beta ( a ) , causing synapse loss , a major correlate of cognitive decline in ad . although dementia - associated hallmarks of ad pathology ( neuritic plaques and neurofibrillary tangles ) become less prominent with increasing age , synaptic marker abnormalities in dementia remain constant and may represent an independent substrate of dementia spanning all ages . postmortem and in vivo magnetic resonance imaging ( mri ) studies of healthy brains have reported different location , extent , and severity of these changes with aging , some brain regions with greater activation being linked to better cognitive performance . postmortem studies of human brains revealed more prominent age - related changes in the anterior and posterior white matter , but not in gray matter volumes , histology showing less severe changes than the imaging methods . mri investigations showed widespread age - related changes in prefrontal cortex and white matter , somatosensory cortex , and , to a lesser degree , in motor cortex , the prefrontal white matter being most susceptible to the influence of age . recent functional neuroimaging studies indicated reduced cortical activation in the default - mode network for mild cognitive impairment patients , compared with age - matched healthy elderly persons , mainly in the retrosplenial region / posterior cingulate cortex , left hippocampus , and bilateral inferior and middle frontal areas , while increased activation for patients was observed in the medial prefrontal and bilateral middle temporal/ angular cortex , probably as a compensatory mechanism . volumetric mri of temporal and parietal brain structures distinguishes ad patients from healthy subjects , volumetry of the left and right hippocampus providing the highest diagnostic accuracy in separating these groups . recent advances in imaging techniques ( diffusion tensor imaging [ dti ] and magnetization transfer imaging [ mti ] ) indicate that age - related small - vessel disease is a diffuse process affecting the whole brain and that wmls are probably only the tip of the iceberg , while decreased gray matter diffusivity might be a potential new biomarker for early ad . age - related neuronal dysfunction involves a host of subtle changes such as reduction in the complexity of dendritic arborization and length , decrease in spine numbers and related synaptic densities , changes involving receptors , neurotransmitters , cytology , electric transmission , vascular or alzheimer - related changes , and myelin dystrophy . concerning factors that modify or mediate the association between neuropathology and cognition , it was hypothesized that the concept of resilient aging can be useful to understand mechanisms that underlie healthy aging amidst disease - related pathology . although the best morphologic correlates of cognitive impairment / dementia are ; ( i ) the number of neocortical neurofibrillary tangles ( nfts ) ; and ( ii ) loss of synapses , between 8% and 45% of nondemented , often cognitively stable older adults were found to have ad - related pathologies . the concept of preclinical ad pathology has been further solidified in biomarker studies using csf a-42 and more directly in vivo positron emission tomography ( pet ) amyloid scanning , demonstrating that 20% to 30% of healthy elderly subjects have elevated pib signals indicative of extensive amyloid deposition . among 555 nondemented persons with false - positive pathological nia - ri high likelihood for ad , only 1.6% corresponded to braak stage v , 0.5% to stage vi , and 2.6% to stage v - vi , while in other studies between 35% and 88% were nia - ri negative ; 18% to 25% met the consortium to establish a registry for alzheimer 's disease ( cerad ) criteria for ad . although in the adult changes in thought ( act ) and nun studies , nondemented seniors with severe ad pathology ( mean age of 89.156.9 to 90.805.2 years ) amounted to 8% and 12% , respectively , most of them showed neuritic braak stage v , and frontal nft counts were slightly lower than in a comparable dementia group . a recent study of nondemented elderly demonstrated 62% with low and 28% with high nft levels70 ; 87 nondemented elderly ( mean age 87 5.9 years ; mean mmse 28.3 ) showed mean braak stage 3.00.9 , a total nft score of 4.52.5 , and mean neuritic density of 1.31.1 , whereas ad cases showed much higher cortical neuritic and striatal amyloid plaque scores . the 90 + study revealed significantly less severe a , -synuclein , and tpd-43 pathologies , and hippocampal sclerosis in nondemented subjects , while a distribution showed no essential differences ; nondemented individuals had limited hippocampal tau and neocortical a pathology . a recent clinicopathologic study of 296 persons without cognitive impairment of the religious order study ( ros ) and the memory and aging project ( map ) showed a common presence of ad pathology and macroscopic infarctions . amyloid load was related to global cognition ( p<0.05 ) , with only a trend for nfts ( p = 0.08 ) , while nfts and macroscopic infarctions were related to episodic memory ( p = 0.03 and 0.02 , respectively ) ; ad pathology and a load to working memory ( p = 0.02 and 0.03 , respectively ) . a personal retrospective study of 100 nondemented elderly ( mean age 81.235.47 years , mean mini mental state examination ( mmse ) score 29 ) revealed negative khachaturian criteria and cerad stage 0 in 83% and 86% , respectively , only 13% with cerad stage a and 1% stage b. braak neuritic stages ranged from 0 to iv with an average score of 2.30.8 . although correlations between cognitive deficits and the severity and extension of senile plaques ( sp ) and nfts ( see ref 42 ) have been found , at least in those brains without other pathologies , the distinction between physiological ( in nondemented subjects ) and pathological aging ( pa ) is difficult . recent biochemical studies found extensive overlap with only subtle quantitative differencies between a levels , peptide profiles , solubility , and oligomeric assemblies in pa and ad brains , suggesting that pa represents an initial prodromal stage of ad and that these individuals would eventually develop clinical symptoms , if they lived long enough , or an inherent individual resistance to the toxic effects of a. recent studies suggest that two independent processes ( synapse - mediated and apoe - mediated ) may contribute to region - specific a accumulation in nondemented individuals , and may influence the mechanisms of the regional vulnerability to a accumulation , which is prevented by apoe . evaluation of 336 cognitively normal ( cn ) seniors from four studies revealed moderately to frequent neuritic plaque density in 47% ; of these 6% also had braak stages v or vi ; medullary , nigral , and cortical lewy bodies in 15% , 8% , and 4% , respectively ; cerebral microinfarcts in 33% and high - level cerebral microinfarcts in 10% . among 418 nondemented participants of the religious order study ( mean age 88.55.3 years ) , 35% showed macroscopical infarcts , 8% microinfarcts , 14.8% arteriosclerosis , 5.7% both , only 37.5% being free of cvls . up to 75% of cn seniors had various degrees of cerebral amyloid angiopathy ( caa ) , occasional hippocampal sclerosis , lewy body pathologies in up to 18% , argyrophilic grains in up to 23 % , and mixed pathologies in 7% to 14.8% . in a small autopsy series of cn elders , among 100 nondemented seniors , mild , moderate and severe intracranial atherosclerosis was present in 31% , 17% , and 6% , respectively , lacunar state in basal ganglia and/or white matter in 73% , hippocampal sclerosis in 3% , whereas only 9% were free of cvls . lewy bodies were observed in 5% , tau pathology in brain stem in 60% , and mixed cerebral pathologies ( cvls and moderate neuritic braak stages ) in 6% . a recent british nondemented sample ( n = 53 ; mean age 81.57.4 years ; mmse score 27 - 30 ) showed maximum score neuritic plaques in 32% to 49% , nfts in hippocampus and neocortex in 81% and 30.8% , respectively , white matter changes 55% to 83.7% , small vascular disease 45% , infarcts 13.7% , lacunes 6% , and hemorrhages 10% . thus , clinically silent pathology is widespread in normal aging , and the term healthy aging is inappropriate at the cellular level , and is manifested by regional heterogeneity in the scenario of general volume loss in the human brain . results in animal experiments and discovery of increases in gray and white matter in the adult human brain as a result of learning and exercise have reinforced the old concept of cognitive reserve , that is , the ability to reinforce brain volume in certain areas and thus provide a greater threshold for age - dependent deficits , or the capacity of the brain to manage pathology or age - related changes , thereby minimizing clinical manifestation . the concept of cognitive reserve and a broader theory of brain reserve was originally proposed to help explain epidemiological data indicating that individuals who engaged higher levels of mental and physical activity via education , occupation , and recreation were associated with slower cognitive decline in healthy aging and are at lower risk of developing ad and other forms of dementia . in people with high reserve structural and functional brain imaging studies have revealed selective changes in aging brain that reflect neural decline as well as compensatory neural recruitment , representing possible neural substrates of cognitive reserve , but its neural basis is still a topic of ongoing research . while aging is associated with reductions in cortical thickness , white matter integrity , transmitter activity , and functional engagement in the hippocampus and occipital areas , there are compensatory increases in frontal functional engagement that correlate with better behavioral performance in the elderly . those cortical regions most consistently shrinking in aging prefrontal and parietal cortices are the same regions showing increased regional activation in aging , suggesting that losses in regional brain integrity drive functional reorganization through changes in processing strategy . neuroprotective effects of noradrenaline both in vivo and in vitro suggest noradrenaline 's key role in mediating cognitive reserve by disease compensation , modification , or a combination of both , a viable hypothesis . it is associated with lower cognitive performance both in ad patients and normal elderly , but the association is modified by cognitive reserve , suggesting that this may be protective against amyloid - related cognitive impairment . on the other hand , endogenous a is necessary for hippocampal plasticity and memory within the normal cns , due to regulation of transmitter release , activation of nicotinic acetylcholine receptors , and a-42 production . furthermore , the production of inflammatory mediators ( inflammatory cytokines , interleukins , neurotrophins ) , activation of glia and other immune cells disrupting the delicate balance needed for the physiological action of immune processes produces direct effects on neural plasticity and neurogenesis , facilitating many forms of neuropathology associated with normal aging as well as neurodegenerative diseases . recent evidence shows that key regulations of communication between neuron and microglia disruption in the aged brain may be one of the factors that precedes and initiates the increase in chronic inflammatory states underlying age - related impairments of cognition and hippocampal neurogenesis . studies in animals and humans revealed dramatic effects of environmental enrichment , increased physical exercise documenting positive effects of mental and physical exercise , mediating brain and cognitive reserve , thus showing no compromise in daily life despite higher a plaque load . recent studies demonstrated that the magnitude of the contribution of education is greater than the negative impact of either neuropathological burden of ad or cvls with standardized regression weights of -0.14 for hyperintensities and -0.20 for hippocampal atrophy . however , a large clinicopathologic study at 27 ad centers found no evidence of larger education - related differences in cognitive function when ad pathology was more advanced , suggesting that the advances of cognitive reserve may ultimately be overwhelmed by ad pathology . however , the age of olfactory bulb neurones , that are assumed to be derived from neuroblasts via the rostral migratory stream ( rms ) , has been assessed recently by measuring the levels of nuclear bomb test - derived 14c in genomic dna . physical activity causes a robust increase in neurogenesis in the dentate gyrus of the hippocampus , a process that would implement a form of network plasticity analogous to that at the synaptic level , but occurring at the cellular network level . exploration of the regulation of neuronal progenitor cells in the aging brain is critical not only for understanding age - related cognitive deficits , but also for progress toward the goal of using the brain 's regenerative potential to restore functional loss . interestingly , increasing evidence suggests that molecular players in ad , including presenilin1 , amyloid precursor protein , and its metabolites , play a role in adult neurogenesis , while alterations in tau phosporylation may interfere with the potential role of tau proteins in neuronal maturation and differentiation . this indicates a crosstalk between signaling molecules involved in both neurogenesis and neurodegeneration , and the ways by which ad - linked dysfunction of these signaling molecules affect neurogenesis in the adult brain . recent studies confirming the assumption that cholinergic pathology has a detrimental influence on neurogenesis suggest an attenuation of stem cells together with compensatory increased proliferation that , however , does not result in an increased number of migratory neuroblasts and differentiated neurons in ad . there are indications that neurogenesis is impaired in pd , which might be due to a lack of dopamine in the subventricular zone , but recent studies did not find evidence that dopamine has a direct effect on human stem cell proliferation in vitro . thus , it was concluded that the number of adult neural stem cells is probably not diminished , and the proliferative capacity of the subventricular zone is maintained in the parkinsonian brain . neural stem cells have been identified also in areas where neurogenesis does not occur under physiological conditions , such as the midbrain and striatum , suggesting that they may have the potential to be used as a non - invasive cell replacement therapy in pd . recent studies have shown that the deleterious effects of -synuclein on newly generated neurons , in particular on their dendritic outgrowth and spine development , thus having negative impact on adult neurogenesis and neuronal maturation . further elucidation of the mechanisms regulating the synaptic integration of adult - born neurons is not only crucial for our understanding of the age- and disease - related neuroplasticity / brain plasticity , but also provides a framework for the manipulation and monitoring of endogenous adult neurogenesis as well as grafted cells potential therapeutic applications . cerebral aging is a complex and heterogenous process that is associated with a high variety of molecular interactions , morphological , and functional changes , summarized in table i. the interrelations between them need further elucidation . brain aging results in loss of synapses and possible neurons , which is associated with structural changes in cerebral areas and neural neworks that are essential for cognitive and memory function . knowing the substrate of the resilience to cognitive decline in the presence of abundant ad and/or mixed pathology might be crucial not only for the understanding of the pathophysiology of nondemented aged people , but also to discover new prophylactic and/or therapeutic targets for aging processes . as expected from the significant clinicopathologic correlations of synaptic and neuronal loss in ad , high - pathology nondemented controls have preserved densities of synaptophysinlabeled presynaptic terminals and dendritic spines as compared with ad dementia patients with a similar burden of plaques and tangles . this resistance to ad pathology has also been related to a nucleolar , nuclear , and cell body hypertrophy of the hippocampal and cortical neurons , suggestive of a compensatory metabolic activation to face the neurotoxic effects of ad lesions . on the other hand , the unique observation of a cognitively intact woman aged 115 years with only slight tau pathology corresponding to braak stage ii , almost no plaques or vascular changes , and normal neuron count in the locus ceruleus indicates that the limits of human cognitive function extend far beyond the range that is currently enjoyed by most individuals and that brain disease , even in supercentenarians , is not inevitable . the association between vulnerability and protective factors varies with age , since the effects of these factors on the risk for ad may differ in younger ( age < 80 ) versus older ( age > 80 ) individuals . future work should measure the contribution of more diverse influences on cognitive reserve that might operate in early and midlife , such as socioeconomic conditions and social relationships , which might be modified through public education in order to have a positive impact on the looming public health disaster that is dementia . recent studies in a nondemented population have shown that intellectual and physical activity lifestyle factors were not assessed with ad biomarkers , while intellectual lifestyle factors explained the variability in the cognitive performance , providing evidence that lifestyle activities may delay the onset of dementia , but do not significantly influence the expression of ad pathophysiology . the neuropathological distinction between nondemented , cognitively intact , and cognitively impaired / demented subjects , elucidation of the relationship of additional pathologies with minor often clinically latent ad lesions observed in many but not all elderly persons without cognitive impairment is important , allowing further insights into the mechanisms of brain plasticity and the basic mechanisms of adult neurogenesis warrants further experimental and prospective , well documented clinico - pathological studies of elderly individuals . in this continuously growing field , new acquisitions , derived from basic research and clinical grounds , on cognitive reserve mechanisms , neuroplasticity , and the potential application of novel therapeutic targets in neurodegeneration and aging disorders are necessary .

the exocrine pancreas functions are the synthesis , storage , and secretion of digestive enzymes into the lumen of the gastrointestinal tract where the enzymes are responsible for converting foodstuffs ingested into aqueous soluble molecules that can be absorbed by the intestinal epithelium . the two key epithelial cell types of the exocrine pancreas involved in digestive enzyme secretion are the acinar cell and the duct cell . the acinar cell is the site of digestive enzyme synthesis , storage , and regulated secretion while the duct cell provides ions and water necessary for transporting the acinar cell secreted products in the pancreatic ductal system to the intestinal lumen . ductal cell ion secretions are rich in nahco3 which is involved in neutralization of gastric acid emptied into the duodenum . the neutralization of gastric acid is necessary because the pancreatic enzymes have optimal activity at neutral ph . the acinar cell of the exocrine pancreas has the greatest rate of protein synthesis of any mammalian organ and thus is endowed by a highly developed endoplasmic reticulum ( er ) system ( palade , 1975 ; case , 1978 ) . in addition to its functions in performing protein synthesis and processing , the er is the major storage site for intracellular calcium which when released into the cytoplasm is the mediator of regulated secretion of stored digestive enzymes into the pancreatic ductal system ( petersen and tepikin , 2008 ) . each protein synthesized in the er must undergo specific secondary modifications and folding before it can be transported to destination organelles such as golgi , zymogen granule ( storage for the digestive enzymes ) , and lysosome ; or membrane sites . further , because of variation in the demand for protein synthesis as a function of diet ; and because protein processing in the er could be adversely affected by environmental factors such as alcohol , smoking , altered metabolism , and xenobiotics , it is likely that there are regulatory systems within the er that allow it to adapt its functions to such stressors . alcohol abuse and smoking are key risk factors in the epidemiology of both diseases ( go et al . , 2005 ; pandol et al . , 2009 ; yadav and whitcomb , 2010 ) . in the case of alcohol abuse the increased risk for pancreatic cancer occurs largely through the effect of alcohol abuse causing chronic forms of pancreatitis ( lowenfels et al . , smoking also contributes to the development of pancreatitis and is a major risk factor for pancreatic cancer independent of pancreatitis ( lowenfels et al . recent epidemiologic studies demonstrate that smoking accelerates the development of pancreatitis in alcoholic patients and may have an additive or multiplicative effect when combined with alcohol to cause pancreatitis ( maisonneuve et al . , 2005 ; yadav and whitcomb , 2010 ) . the mechanisms underlying the effects of alcohol and smoking on the development of pancreatic diseases an important and unexplained observation is that only a small proportion of heavy drinkers / smokers develop pancreatic diseases ( pandol et al . , 2007 ) . although the reason for lack of development of pathology in the majority of those who drink and smoke is unknown , we hypothesize that an adaptive unfolded protein response ( upr ) is sufficiently robust in most individuals to prevent pathology . proteins enter the er as unfolded polypeptides that require further processing for activation and targeting to the appropriate organelle or membrane site . further , the er is faced with several challenges in completing these functions with high fidelity . figure 1 illustrates many of these challenges which are often referred to as er stress that we have applied to our hypothesis of er stress for the exocrine pancreas . at the bottom of the figure a key implication from the figure is that upr activation leads to an adaptive response in the er . in this figure we hypothesize potential er stressors in the pancreas from what is known about er stressors in general ( ron and walter , 2007 ) . for example , it is likely that increases in protein synthesis rates that occur to replenish digestive enzyme stores after a meal will increase the demand for protein folding resulting in increased need for synthesis and functionality of chaperones and foldases in the er . because the rate of unfolded / misfolded protein formation would also increase with increased proteins synthesized , the er quality control system to degrade these unusable proteins called er - associated protein degradation ( erad ) is required to be present and functional to rid the cell from accumulation of permanently misfolded and unfolded proteins that could present toxicity to the cell . for example , an increased folding demand will result when there is a need for increased digestive enzyme synthesis . . altered er calcium levels as occurs during forms of pancreatitis can also cause er stress . on the left hand side of the figure these include genetic mutations in digestive enzymes ; alcohol abuse ; smoking ; metabolic disorders such as diabetes and hyperlipidemia ; xenobiotics such as drugs and intestinal bacterial metabolites ; and reactive oxygen species that are generated in many of these situations as well as during acute and chronic pancreatitis . these pancreatic er stressors lead to further accumulation of unfolded and misfolded proteins which , in turn , lead to further activation of the unfolded proteins response in an attempt to adapt the pancreas to function in the face of the er stressors . there are several genetic , environmental , and disease - related stressors illustrated in figure 1 that occur in the pancreas that are likely significantly increase er stress requiring the acinar cell to activate its adaptive upr or face the possibility of cellular pathologies . mutations in key protease digestive enzymes are known to lead to chronic forms of pancreatitis ( hereditary pancreatitis ) and increased rate of pancreatic cancer ( whitcomb , 2010 ) . in fact , a recent report ( kereszturi et al . , 2009 ) demonstrates that a mutation in human cationic trypsinogen causes er stress in pancreatic cells . these results support a hypothesis that hereditary pancreatitis results from er stress caused by a mutated protease . another example is altered er calcium levels that occur in experimental models of pancreatitis ( sutton et al . , 2008 ) . calcium is stored in the er by its resident chaperones and foldases which act as low affinity , high capacity ca - binding proteins ( gorlach et al . , 2006 ) . importantly for er stress as discussed below , their chaperone and foldase functions are markedly altered with perturbations in er calcium homeostasis . thus , alterations in er calcium stores in cells can lead to pathologic consequences at least in part because of the er stress induced . further , as discussed in more detail below , manipulation of er calcium stores in pancreatic acinar cells has been shown to activate er stress signals . other potential stressors listed in figure 1 include alcohol , smoking , metabolic disorders , and xenobiotics as well as reactive oxygen species ( ros ) . except for information on alcohol abuse recently published ( lugea et al . , 2010 ) and reviewed below , there is little information on whether these factors affecting the pancreas indeed cause er stress and whether pathology results from an insufficiently robust upr . however , alcohol , smoking , diabetes , obesity , hyperlipidemia , and drugs account for large percentage of the burden of pancreatic diseases ( pandol et al . , 2007 ) ; and ros participate in their pathogenesis . thus , the potential for roles of er stress in pancreatic disease pathogenesis is great . the adaptive upr has three major types of outputs ( marciniak et al . , 2006 ; ron and walter , 2007 ; rutkowski and kaufman , 2007 ; kaser et al . , 2008 ; kim et al . , 2008 these include : ( 1 ) upregulation of the expression and function of chaperones and foldases to augment the folding and export capacity of the er ; ( 2 ) activation of the er - associated protein degradation ( erad ) system to rid the er of accumulated unfolded and misfolded proteins ; and ( 3 ) a global reduction in translation of mrna to decrease the processing demand for newly synthesized proteins . the upr also activates cell death programs under conditions of severe and/or prolonged er stress when the adaptive upr responses are exceeded or when a dysfunctional upr is unable to correct the folding disorders presented to it . as illustrated in figure 2 , the mammalian upr is initiated mainly by three er stress sensor transducers located in the membrane of the er ( ron and walter , 2007 ; rutkowski and kaufman , 2007 ) . these three are inositol - requiring protein-1 ( ire1 ) , activating transcription factor-6 ( atf6 ) , and protein kinase rna ( pkr)-like er kinase ( perk ) . in each case the transmembrane sensor transducer determines the folding status of proteins in the er lumen and transmits this information across the er membrane to the cell cytosol . in some cases , the transmembrane sensor transducer is silenced by binding of an er chaperone called immunoglobulin - binding protein ( bip ) to its luminal domain . bip is also known as also known as 78 kda glucose - regulated protein ( grp78 ) . er stress unfolded and misfolded proteins compete for binding bip resulting in removing its silencing effect resulting in activation of the sensor . are inositol - requiring protein-1 ( ire1 ) , activating transcription factor-6 ( atf6 ) , and protein kinase rna ( pkr)-like er kinase ( perk ) . these sensor transducers is followed specific pathways resulting in transcriptional regulation of chaperones , foldases , and components of er - associated protein degradation ( erad ) system and lipid synthesis for expansion of the er mediated by the combined effects of ire1 and atf6 ; or translational attenuation and transcriptional upregulation pathways involved in antioxidant synthesis and cell death through the transcription factor c / ebp homologous protein ( chop ) as in the case of perk . the participants in the pathways involved in the downstream effects of ire1 , atf6 , and perk activation include x - box binding protein1 ( xbp1 ) , site-1 and site-2 proteases ( sp1/sp2 ) , and eukaryotic translation initiation factor-2a ( eif-2a ) , and activating transcription factor-4 ( atf4 ) as discussed in the text . activation of the er transmembrane protein ire1 initiates a response to increase the expression of er chaperones and foldases in order to assist in protein folding and transport ( figure 2 ) . er stress induces ire1 homodimerization and trans - autophosphorylation to activate its rnase activity toward the mrna for unspliced x - box binding protein1 ( xbp1-u ) . activated ire1 removes a 26-nucleotide intron from xbp1-u resulting in a mrna that translates into a potent transcription factor , spliced xbp1 ( xbp1-s ; yoshida et al . , 2001 ; calfon et al . , 2002 ; xbp1-s , in turn , binds to er stress element ( erse ) and the upr element ( upre ) dna binding sites to upregulate many upr target genes such as the chaperones bip / grp78 and grp94 and the gene encoding xbp1-u ( yoshida et al . , 2001 , 2003 ; calfon et al . , 2002 ; lee et al . , this ability to increase transcription of xbp1-u leads to more substrate for expression of the xbp1-s transcription factor thus augmenting this protective response . the ire1/xbp1 pathway also leads to increased expression of foldases such as protein disulfide isomerase ( pdi ) , enzymes for lipid synthesis for expanding the er membrane and er capacity , components of the er - associated degradation ( erad ) , all protective mechanism to lessen er stress ( yoshida et al . , the importance of xbp1 for the function of the pancreatic acinar cell has been demonstrated in xbp1 deficient mice with a transgene expressing xbp1 in hepatocytes to prevent embryonic lethality ( lee et al . , 2005 ) . these mice show abnormalities limited to the secretory organs , the exocrine pancreas , and salivary gland . the er in the pancreatic acinar cells in these animals is poorly developed accompanied by a decreased in the expression of er chaperones and marked apoptosis of the cells . activating transcription factor-6 represents a second er transmembrane protein that is sensitive to er stress ( figure 2 ) . the n - terminal cytoplasmic domain contains a dna transcription - activating domain while the c - terminal luminal domain is sensitive to er stress . release from bip permits atf6 transport to the golgi compartment where it is cleaved by site-1 and site-2 proteases ( sp1/sp2 ) to a 50- to 60-kda fragment that migrates to the nucleus to activate transcription of xbp1-u and other upr target genes ( shen et al . , 2002 ) . thus , the ire1 and atf6 pathways work in concert to upregulate an er protective response utilizing xbp1 . as indicated , xbp1-s is a potent transcription activator for many upr target genes including the molecular chaperone bip . the increased expression allows more bip available to regulate the er sensors . as discussed here , bip has several functions including acting as a chaperone , a luminal sensor of unfolded proteins , a regulator of activation of upr pathways upr , and a regulator of its own expression . recently , we found that genetic inhibition of bip leads to augmented severity of experimental pancreatitis showing the importance of bip in cellular protective responses ( ye et al . , 2010 ) . the perk plays a key role in adjusting the cell to er stress by causing a significant attenuation of general protein synthesis ( figure 2 ) . the activation of perk by autophosphorylation ( thr ) leads to its phosphorylation of the alpha subunit of the eukaryotic translation initiation factor-2a the non - phosphorylated form of eif-2a in its gtp - bound form is essential for translation initiation because it recruits the first trna ( trna ) to the ribosomal subunits to start translation of the attached mrna . phosphorylation of eif2a at ser51 by perk blocks eif2a - mediated initiation resulting in a general inhibition of protein synthesis . cells with genetic deletion of perk or cells containing eif2a with position 51 containing alanine instead of serine to prevent phosphorylation do not attenuate protein synthesis with er stress ( harding et al . , 2000 ; scheuner et al . , 2001 ) . as a consequence , cells are more sensitive to er stress pointing out a potential protective role of the perk signaling pathway in the er stress response . of note , previous studies in acinar cells have demonstrated that concentrations of cholecystokinin peptides which cause pancreatitis also inhibit translational initiation ; and that this block in translation is mediated by phosphorylation of eif2a ( perkins et al . , 1997 ; sans et al . , 2002 ) . of interest , the phosphorylation of eif2a is augmented by depletion of calcium from er stores , showing that this manipulation of er calcium homeostasis can initiate er stress . because cholecystokinin as well as acetylcholine can also deplete er calcium stores , the findings suggest that changes in er calcium provide an er stress signal in the pancreatic acinar cell . persistent phosphorylation of eif2a leads to specific translational upregulation of atf4 that targets genes involved in antioxidant effects including synthesis of glutathione ( harding et al . , atf4 also upregulates the expression of the transcription factor c / ebp homologous protein ( chop ) which induces apoptosis ( oyadomari and mori , 2004 ) . the mechanisms of the erad system to rid the er of accumulated unfolded and misfolded proteins is complex , debated , and only partly understood in any cell ( brodsky and wojcikiewicz , 2009 ; yoshida and tanaka , 2010 ) . the degradation of erad substrates requires the multi - catalytic 26s protease called the proteasome . the proteasomes are not located in the er but in the cytoplasm adjacent to the er . proteasomes act complementarily to the lysosome / vacuole to mediate the disassembly of proteins for recycling of their amino acids . the mechanisms of delivery of proteins from the er to proteasomes are under active investigation and likely require a proteinaceous channel in the er membrane needed to transfer proteins to the cytoplasmic located proteasomes . the modification with ubiquitin both aids in transfer and targets a protein to the proteasome . of importance , we recently reported ( lugea et al . , 2010 ) that alcohol feeding in animals with genetic inhibition of xbp1 expression led to a marked decrease in expression of er degradation - enhancing alpha - mannosidase - like 1 ( edem1 ) , a key participant involved in erad and targeting unfolded / misfolded proteins to the proteasome ( yoshida and tanaka , 2010 ) . these findings suggest a key role for erad in the adaptive response of the exocrine pancreas to environmental stressors . previous studies ( kubisch et al . , 2006 ) demonstrated activation of all three er stress sensor transducers ( i.e. , ire1 , atf6 , and perk ) and their downstream pathways during the development of pancreatitis in experimental models pancreatitis suggesting involvement of er stress responses in this disorder . however , there was no information on er stress signals and the upr in providing adaptation of environmental insults such as alcohol abuse . previous reports ( gukovsky et al . , 2008 ) have demonstrated that long - term ethanol feeding in animal models causes little evidence of damage of the pancreatic parenchyma as determined by histology and blood concentrations of pancreatic digestive enzymes . for these reasons we designed studies to determine if alcohol abuse causes er stress and activates the upr in the exocrine pancreas ; and studies to investigate the role of upr signals in adapting the pancreas to the effects of alcohol abuse ( lugea et al . , 2010 ) . for these studies rats and mice were fed control diets or ethanol containing diets for 46 weeks using the tsukamoto french intra - gastric model ( tsukamoto et al . , 1985 ) which provides continuous feeding of the diets to the animals . as reportedly previously ( gukovsky et al . , 2008 ) , light microscopic examination of the exocrine pancreatic tissue demonstrated no obvious injury . however , careful structural examination by electron microscopy demonstrated extensive distention of the er of acinar cells and altered distribution of zymogen granules . in addition , an investigation of the redox status of the er demonstrated that there was a significant decrease in reduced and an increase in oxidized glutathione indicating that the alcohol feeding has a significant oxidative effect in the er of the acinar cell . taken together , these findings indicated that ethanol has the oxidative effect in the er of acinar cells associated with morphologic signs of er stress . we then evaluated the effect of the alcohol treatment on the sensors and signals of the upr . the major findings were significant effects of alcohol feeding on ire1 and xbp1-s expression with smaller effects on perk induction ( lugea et al . , 2010 ) . also , we found that a key oxido - reductase , pdi , was significantly upregulated in the pancreas of the ethanol - fed animals . further , we found that a much greater proportion of the pdi was in its oxidized state in the ethanol - fed animals compared to control fed . pdi is critical for protein folding by catalyzing disulfide bond formation , a key step for maturation of proteins in the secretory pathway ( ellgaard and ruddock , 2005 ) . that is , pdi requires reduced glutathione to sustain its catalytic redox cycling ( zhang and kaufman , 2008 ) . the combination of these findings suggest that pdi activity and thus appropriate folding of proteins in the secretory pathway of the acinar cell are impaired due to the oxidizing effects of alcohol feeding . the findings that alcohol feeding increased ire1 and xbp1-s expression suggested to us the possibility that these sensors and signals are important for adapting the er of the acinar cell to the er stress caused by the alcohol feeding . in order to test this possibility we obtained and tested animals with heterozygous deficiency in xbp1 . homozygous deletion of xbp1 is lethal and pancreatic tissue specific deletion leads to lack of development of the pancreas ( lee et al . , 2005 ) . we hypothesized that there would be an incomplete adaptive response to alcohol feeding in the heterozygous animals . in contrast to wild - type animals , there were marked morphological and biochemical effects of alcohol feeding in animals with heterozygous deletion of xbp1 . of note , alcohol feeding increased levels of xbp1-s in the pancreas of heterozygous animals to the same level observed in wild - type animals receiving the control diet but not the augmented levels observed in the pancreas with alcohol feeding in the wild - type animals . thus , the heterozygous deletion specifically prevented the ethanol feeding - induced augmentation in xbp1-s levels in the pancreas . the predominant morphologic abnormalities in the xbp1 deficient animals receiving alcohol were a disorganized ultrastructure with decreased number of zymogen granules with the remaining ones inappropriately scattered throughout the cell and not localized to their normal apical position . there was extensive dilation of the er with occasional dense luminal inclusions , a hallmark of er stress , and a significant accumulation of autophagic vacuoles . there was also a marked decrease in levels of the digestive enzyme amylase corresponding to the decrease number of digestive enzyme storing zymogen granules . these areas showed acinar cell necrosis , apoptosis , and inflammation with replacement by stromal cells and regenerating ductular structures indicative of a response to severe injury . an analysis of the upr revealed that xbp1 deficiency prevented alcohol 's effect to increase pdi while it markedly promoted perk and eif2a phosphorylation and expression of atf4 , all features of prolonged and severe er stress ( harding et al . , 2003 ) . also of importance , xbp1 deficiency decreased levels of edem1 , a key participant in the erad pathway for degradation of misfolded proteins . of note , deficiencies in edam1 have been shown to result in increased degradation of misfolded proteins via autophagy ( hetz et al . , 2009 ) which may explain the increased autophagy observed in our studies . the results of the in vivo studies described above showing the role of the upr in adapting the pancreas to alcohol treatments allow us to propose the model illustrated in figure 3 . we suggest that alcohol abuse usually does not cause pancreatic disease because of an adaptive upr predominated by ire1 and xbp1 . the oxidative stress caused by alcohol metabolism in the er activates ire1 leading to splicing of xbp1 mrna resulting in translation of the active transcription factor , xbp1-s . this transcription factor in turn mediates the expression of chaperones , pdi , erad proteins ( for example edem1 ) , and increased lipid synthesis to expand the capacity of the er . these should all result in an attenuation of the er stress . when there is an insufficient upr response as shown in our experiments using animals with deficient xbp1 , there is unresolved and augmented er stress resulting in perk activation causing global protein synthesis inhibition and even atf4-mediated activation of cell death pathways via chop expression . these are only a few events that must be occurring in the cascade of pathobiologic events resulting in the significant tissue injury we observed . this figure illustrates the results of our experiments in alcohol fed animals showing er stress from ethanol and its metabolites , acetaldehyde , and fatty acid ethanol ester ( faee ) , by inducing an oxidative state in the er mediating misfolding and unfolding of proteins and oxidation of lipids . the er stress leads to upregulation of the ire1 pathway leading to splicing of xbp1 mrna resulting in the translation of the transcription factor xbp1-s which is transported to the nucleus where it upregulates the expression of chaperones , oxido - reductases / foldases such as pdi , erad proteins , and lipid synthetic enzymes through dna binding elements , er stress element ( erse ) and the upr element ( upre ) . the activation of this pathway leads to adaptation of the pancreas to the stress of ethanol abuse and prevents pancreatic injury and disease . the findings related to the oxidative state of glutathione and pdi provide important insights for er stress and the upr in the exocrine pancreas . figure 4 illustrates the mechanism of oxidative folding of nascent proteins ( ellgaard and ruddock , 2005 ; hatahet and ruddock , 2009 ; shimizu and hendershot , 2009 ) . the ability of pdi to mediate folding of a nascent protein is a function of its ability to catalyze disulfide pdi recognizes and binds to nascent unfolded proteins , semi - folded proteins and misfolded proteins but not correctly folded proteins through mechanisms that are not entirely known . through electron transfer , oxidized pdi catalyzes disulfide bridge formation of the substrate protein as shown in the center of figure 4 . oxidative folding in the er . this figure illustrates the er oxido - reductases involved in oxidative folding of nascent proteins in the er . the oxido - reductases pdi and ero1 are coupled and mediate the disulfide bridge formation and protein folding of proteins such as digestive enzymes synthesized in the er . another product of these coupled reactions is ros . reduced glutathione is necessary to reduce incorrectly placed disulfide bonds so that the protein can be recycled for correct disulfide bridge formation and folding . the re - oxidation of pdi is catalyzed by another oxido - reductase called er oxidase ( ero1 ) as shown on the left side of figure 4 . ero1 is a flavo - enzyme that is induced by the upr and it is directly oxidized by molecular oxygen in a fad - dependent reaction ( shimizu and hendershot , 2009 ; tavender and bulleid , 2010 ) . fad is synthesized in the cytoplasm and easily enters the er . in the process ero1 delivers the electrons that have been transferred from pdi to molecular oxygen with resultant ros formation . it is known that pdi function and oxidative folding are dependent on ero1 ( chakravarthi and bulleid , 2004 ; tavender and bulleid , 2010 ) . as illustrated on the right side of figure 4 , the reduction of disulfides is important for the reduction of incorrect disulfide bonds that can form during folding ( chakravarthi and bulleid , 2004 ) . illustrated at the top of figure 4 is the suggestion that glutathione may be involved in regeneration of the oxidized state of pdi in some systems . the activities and direction of the reaction catalyzed by oxido - reductases such and pdi and ero1 are dependent on the oxidative state of their catalytic sites as well as the local environments of the catalytic sites . the results from the experiments in the exocrine pancreas do not yet provide information on the detail of the oxidative state of the oxido - reductases or about their kinetics during the er stress produced by alcohol treatments . however , the initial findings of a marked increase in the ratio of the oxidized to the reduced form of glutathione in the er and the increase in the oxidized form of pdi suggest the likelihood of significant disturbances in oxido - reductase enzyme kinetics which would lead to the findings described . in closing , we hypothesize that alcohol abuse and many other causes of pancreatitis including genetic mutations in digestive enzymes , smoking , metabolic disorders such as diabetes and hypertriglyceridemia , and drugs may cause the disease at least in part by causing er stress . the key question related to why some individuals develop disease while others do not is not known . it is certainly possible that alterations in key components of the upr through mutation ( or environmental factors ) are present in the pancreas of those individuals who develop tissue injury in fact , genetic variants of xbp1 have been associated with the risk of development of inflammatory bowel disease ( kaser et al . , 2008 ) supporting this possibility . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the exocrine pancreas has the greatest protein synthetic capacity of any mammalian organ and is challenged with the synthesis , processing and transporting a large load of digestive enzymes . based on recent findings we present a hypothesis proposing that mutations in the digestive enzymes and environmental risks impacting the pancreas ( i.e. , alcohol abuse , smoking , metabolic disorders , and drugs ) cause endoplasmic reticulum ( er ) stress . we review recent findings showing that in normal pancreas the er stress resulting from alcohol abuse leads to an adaptive unfolded protein response ( upr ) allowing for maintenance of protein synthesis , processing , and transport . however , when key pathways necessary for the adaptive upr are altered , the exocrine cell of the pancreas is unable to maintain these processes and cellular pathology results . these findings may explain why some individuals with alcohol abuse disorders develop organ injury and disease while most do not . further , the findings allow us to hypothesize that the upr in the exocrine pancreas adapts the protein synthetic machinery of the er stress resulting from mutational and environmental stressors . when the ability of the upr to adapt to the stressors is exceeded , pathologic pathways and disease develop .

Cellular Functions of the Exocrine Pancreas Alcohol Abuse, Smoking, and the Diseases of the Exocrine Pancreas Overview of ER Stressors The Adaptive Unfolded Protein Response Alcohol Abuse, ER Stress, and the UPR of the Exocrine Pancreas Conclusion Conflict of Interest Statement

the exocrine pancreas functions are the synthesis , storage , and secretion of digestive enzymes into the lumen of the gastrointestinal tract where the enzymes are responsible for converting foodstuffs ingested into aqueous soluble molecules that can be absorbed by the intestinal epithelium . the two key epithelial cell types of the exocrine pancreas involved in digestive enzyme secretion are the acinar cell and the duct cell . the acinar cell is the site of digestive enzyme synthesis , storage , and regulated secretion while the duct cell provides ions and water necessary for transporting the acinar cell secreted products in the pancreatic ductal system to the intestinal lumen . the acinar cell of the exocrine pancreas has the greatest rate of protein synthesis of any mammalian organ and thus is endowed by a highly developed endoplasmic reticulum ( er ) system ( palade , 1975 ; case , 1978 ) . in addition to its functions in performing protein synthesis and processing , the er is the major storage site for intracellular calcium which when released into the cytoplasm is the mediator of regulated secretion of stored digestive enzymes into the pancreatic ductal system ( petersen and tepikin , 2008 ) . each protein synthesized in the er must undergo specific secondary modifications and folding before it can be transported to destination organelles such as golgi , zymogen granule ( storage for the digestive enzymes ) , and lysosome ; or membrane sites . further , because of variation in the demand for protein synthesis as a function of diet ; and because protein processing in the er could be adversely affected by environmental factors such as alcohol , smoking , altered metabolism , and xenobiotics , it is likely that there are regulatory systems within the er that allow it to adapt its functions to such stressors . in the case of alcohol abuse the increased risk for pancreatic cancer occurs largely through the effect of alcohol abuse causing chronic forms of pancreatitis ( lowenfels et al . , smoking also contributes to the development of pancreatitis and is a major risk factor for pancreatic cancer independent of pancreatitis ( lowenfels et al . although the reason for lack of development of pathology in the majority of those who drink and smoke is unknown , we hypothesize that an adaptive unfolded protein response ( upr ) is sufficiently robust in most individuals to prevent pathology . proteins enter the er as unfolded polypeptides that require further processing for activation and targeting to the appropriate organelle or membrane site . further , the er is faced with several challenges in completing these functions with high fidelity . figure 1 illustrates many of these challenges which are often referred to as er stress that we have applied to our hypothesis of er stress for the exocrine pancreas . at the bottom of the figure a key implication from the figure is that upr activation leads to an adaptive response in the er . in this figure we hypothesize potential er stressors in the pancreas from what is known about er stressors in general ( ron and walter , 2007 ) . for example , it is likely that increases in protein synthesis rates that occur to replenish digestive enzyme stores after a meal will increase the demand for protein folding resulting in increased need for synthesis and functionality of chaperones and foldases in the er . because the rate of unfolded / misfolded protein formation would also increase with increased proteins synthesized , the er quality control system to degrade these unusable proteins called er - associated protein degradation ( erad ) is required to be present and functional to rid the cell from accumulation of permanently misfolded and unfolded proteins that could present toxicity to the cell . on the left hand side of the figure these include genetic mutations in digestive enzymes ; alcohol abuse ; smoking ; metabolic disorders such as diabetes and hyperlipidemia ; xenobiotics such as drugs and intestinal bacterial metabolites ; and reactive oxygen species that are generated in many of these situations as well as during acute and chronic pancreatitis . these pancreatic er stressors lead to further accumulation of unfolded and misfolded proteins which , in turn , lead to further activation of the unfolded proteins response in an attempt to adapt the pancreas to function in the face of the er stressors . there are several genetic , environmental , and disease - related stressors illustrated in figure 1 that occur in the pancreas that are likely significantly increase er stress requiring the acinar cell to activate its adaptive upr or face the possibility of cellular pathologies . mutations in key protease digestive enzymes are known to lead to chronic forms of pancreatitis ( hereditary pancreatitis ) and increased rate of pancreatic cancer ( whitcomb , 2010 ) . these results support a hypothesis that hereditary pancreatitis results from er stress caused by a mutated protease . calcium is stored in the er by its resident chaperones and foldases which act as low affinity , high capacity ca - binding proteins ( gorlach et al . thus , alterations in er calcium stores in cells can lead to pathologic consequences at least in part because of the er stress induced . further , as discussed in more detail below , manipulation of er calcium stores in pancreatic acinar cells has been shown to activate er stress signals . other potential stressors listed in figure 1 include alcohol , smoking , metabolic disorders , and xenobiotics as well as reactive oxygen species ( ros ) . , 2010 ) and reviewed below , there is little information on whether these factors affecting the pancreas indeed cause er stress and whether pathology results from an insufficiently robust upr . however , alcohol , smoking , diabetes , obesity , hyperlipidemia , and drugs account for large percentage of the burden of pancreatic diseases ( pandol et al . thus , the potential for roles of er stress in pancreatic disease pathogenesis is great . the adaptive upr has three major types of outputs ( marciniak et al . , 2008 these include : ( 1 ) upregulation of the expression and function of chaperones and foldases to augment the folding and export capacity of the er ; ( 2 ) activation of the er - associated protein degradation ( erad ) system to rid the er of accumulated unfolded and misfolded proteins ; and ( 3 ) a global reduction in translation of mrna to decrease the processing demand for newly synthesized proteins . the upr also activates cell death programs under conditions of severe and/or prolonged er stress when the adaptive upr responses are exceeded or when a dysfunctional upr is unable to correct the folding disorders presented to it . as illustrated in figure 2 , the mammalian upr is initiated mainly by three er stress sensor transducers located in the membrane of the er ( ron and walter , 2007 ; rutkowski and kaufman , 2007 ) . in each case the transmembrane sensor transducer determines the folding status of proteins in the er lumen and transmits this information across the er membrane to the cell cytosol . er stress unfolded and misfolded proteins compete for binding bip resulting in removing its silencing effect resulting in activation of the sensor . these sensor transducers is followed specific pathways resulting in transcriptional regulation of chaperones , foldases , and components of er - associated protein degradation ( erad ) system and lipid synthesis for expansion of the er mediated by the combined effects of ire1 and atf6 ; or translational attenuation and transcriptional upregulation pathways involved in antioxidant synthesis and cell death through the transcription factor c / ebp homologous protein ( chop ) as in the case of perk . the participants in the pathways involved in the downstream effects of ire1 , atf6 , and perk activation include x - box binding protein1 ( xbp1 ) , site-1 and site-2 proteases ( sp1/sp2 ) , and eukaryotic translation initiation factor-2a ( eif-2a ) , and activating transcription factor-4 ( atf4 ) as discussed in the text . activation of the er transmembrane protein ire1 initiates a response to increase the expression of er chaperones and foldases in order to assist in protein folding and transport ( figure 2 ) . , 2002 ; xbp1-s , in turn , binds to er stress element ( erse ) and the upr element ( upre ) dna binding sites to upregulate many upr target genes such as the chaperones bip / grp78 and grp94 and the gene encoding xbp1-u ( yoshida et al . , this ability to increase transcription of xbp1-u leads to more substrate for expression of the xbp1-s transcription factor thus augmenting this protective response . the ire1/xbp1 pathway also leads to increased expression of foldases such as protein disulfide isomerase ( pdi ) , enzymes for lipid synthesis for expanding the er membrane and er capacity , components of the er - associated degradation ( erad ) , all protective mechanism to lessen er stress ( yoshida et al . , the importance of xbp1 for the function of the pancreatic acinar cell has been demonstrated in xbp1 deficient mice with a transgene expressing xbp1 in hepatocytes to prevent embryonic lethality ( lee et al . these mice show abnormalities limited to the secretory organs , the exocrine pancreas , and salivary gland . the er in the pancreatic acinar cells in these animals is poorly developed accompanied by a decreased in the expression of er chaperones and marked apoptosis of the cells . the perk plays a key role in adjusting the cell to er stress by causing a significant attenuation of general protein synthesis ( figure 2 ) . the activation of perk by autophosphorylation ( thr ) leads to its phosphorylation of the alpha subunit of the eukaryotic translation initiation factor-2a the non - phosphorylated form of eif-2a in its gtp - bound form is essential for translation initiation because it recruits the first trna ( trna ) to the ribosomal subunits to start translation of the attached mrna . phosphorylation of eif2a at ser51 by perk blocks eif2a - mediated initiation resulting in a general inhibition of protein synthesis . cells with genetic deletion of perk or cells containing eif2a with position 51 containing alanine instead of serine to prevent phosphorylation do not attenuate protein synthesis with er stress ( harding et al . as a consequence , cells are more sensitive to er stress pointing out a potential protective role of the perk signaling pathway in the er stress response . of interest , the phosphorylation of eif2a is augmented by depletion of calcium from er stores , showing that this manipulation of er calcium homeostasis can initiate er stress . because cholecystokinin as well as acetylcholine can also deplete er calcium stores , the findings suggest that changes in er calcium provide an er stress signal in the pancreatic acinar cell . the mechanisms of the erad system to rid the er of accumulated unfolded and misfolded proteins is complex , debated , and only partly understood in any cell ( brodsky and wojcikiewicz , 2009 ; yoshida and tanaka , 2010 ) . the proteasomes are not located in the er but in the cytoplasm adjacent to the er . the mechanisms of delivery of proteins from the er to proteasomes are under active investigation and likely require a proteinaceous channel in the er membrane needed to transfer proteins to the cytoplasmic located proteasomes . these findings suggest a key role for erad in the adaptive response of the exocrine pancreas to environmental stressors . , 2006 ) demonstrated activation of all three er stress sensor transducers ( i.e. , ire1 , atf6 , and perk ) and their downstream pathways during the development of pancreatitis in experimental models pancreatitis suggesting involvement of er stress responses in this disorder . however , there was no information on er stress signals and the upr in providing adaptation of environmental insults such as alcohol abuse . , 2008 ) have demonstrated that long - term ethanol feeding in animal models causes little evidence of damage of the pancreatic parenchyma as determined by histology and blood concentrations of pancreatic digestive enzymes . for these reasons we designed studies to determine if alcohol abuse causes er stress and activates the upr in the exocrine pancreas ; and studies to investigate the role of upr signals in adapting the pancreas to the effects of alcohol abuse ( lugea et al . , 1985 ) which provides continuous feeding of the diets to the animals . , 2008 ) , light microscopic examination of the exocrine pancreatic tissue demonstrated no obvious injury . however , careful structural examination by electron microscopy demonstrated extensive distention of the er of acinar cells and altered distribution of zymogen granules . in addition , an investigation of the redox status of the er demonstrated that there was a significant decrease in reduced and an increase in oxidized glutathione indicating that the alcohol feeding has a significant oxidative effect in the er of the acinar cell . taken together , these findings indicated that ethanol has the oxidative effect in the er of acinar cells associated with morphologic signs of er stress . we then evaluated the effect of the alcohol treatment on the sensors and signals of the upr . also , we found that a key oxido - reductase , pdi , was significantly upregulated in the pancreas of the ethanol - fed animals . further , we found that a much greater proportion of the pdi was in its oxidized state in the ethanol - fed animals compared to control fed . the combination of these findings suggest that pdi activity and thus appropriate folding of proteins in the secretory pathway of the acinar cell are impaired due to the oxidizing effects of alcohol feeding . the findings that alcohol feeding increased ire1 and xbp1-s expression suggested to us the possibility that these sensors and signals are important for adapting the er of the acinar cell to the er stress caused by the alcohol feeding . homozygous deletion of xbp1 is lethal and pancreatic tissue specific deletion leads to lack of development of the pancreas ( lee et al . of note , alcohol feeding increased levels of xbp1-s in the pancreas of heterozygous animals to the same level observed in wild - type animals receiving the control diet but not the augmented levels observed in the pancreas with alcohol feeding in the wild - type animals . thus , the heterozygous deletion specifically prevented the ethanol feeding - induced augmentation in xbp1-s levels in the pancreas . the predominant morphologic abnormalities in the xbp1 deficient animals receiving alcohol were a disorganized ultrastructure with decreased number of zymogen granules with the remaining ones inappropriately scattered throughout the cell and not localized to their normal apical position . there was extensive dilation of the er with occasional dense luminal inclusions , a hallmark of er stress , and a significant accumulation of autophagic vacuoles . there was also a marked decrease in levels of the digestive enzyme amylase corresponding to the decrease number of digestive enzyme storing zymogen granules . an analysis of the upr revealed that xbp1 deficiency prevented alcohol 's effect to increase pdi while it markedly promoted perk and eif2a phosphorylation and expression of atf4 , all features of prolonged and severe er stress ( harding et al . the results of the in vivo studies described above showing the role of the upr in adapting the pancreas to alcohol treatments allow us to propose the model illustrated in figure 3 . we suggest that alcohol abuse usually does not cause pancreatic disease because of an adaptive upr predominated by ire1 and xbp1 . the oxidative stress caused by alcohol metabolism in the er activates ire1 leading to splicing of xbp1 mrna resulting in translation of the active transcription factor , xbp1-s . this transcription factor in turn mediates the expression of chaperones , pdi , erad proteins ( for example edem1 ) , and increased lipid synthesis to expand the capacity of the er . these should all result in an attenuation of the er stress . when there is an insufficient upr response as shown in our experiments using animals with deficient xbp1 , there is unresolved and augmented er stress resulting in perk activation causing global protein synthesis inhibition and even atf4-mediated activation of cell death pathways via chop expression . this figure illustrates the results of our experiments in alcohol fed animals showing er stress from ethanol and its metabolites , acetaldehyde , and fatty acid ethanol ester ( faee ) , by inducing an oxidative state in the er mediating misfolding and unfolding of proteins and oxidation of lipids . the er stress leads to upregulation of the ire1 pathway leading to splicing of xbp1 mrna resulting in the translation of the transcription factor xbp1-s which is transported to the nucleus where it upregulates the expression of chaperones , oxido - reductases / foldases such as pdi , erad proteins , and lipid synthetic enzymes through dna binding elements , er stress element ( erse ) and the upr element ( upre ) . the activation of this pathway leads to adaptation of the pancreas to the stress of ethanol abuse and prevents pancreatic injury and disease . the findings related to the oxidative state of glutathione and pdi provide important insights for er stress and the upr in the exocrine pancreas . the ability of pdi to mediate folding of a nascent protein is a function of its ability to catalyze disulfide pdi recognizes and binds to nascent unfolded proteins , semi - folded proteins and misfolded proteins but not correctly folded proteins through mechanisms that are not entirely known . through electron transfer , oxidized pdi catalyzes disulfide bridge formation of the substrate protein as shown in the center of figure 4 . oxidative folding in the er . this figure illustrates the er oxido - reductases involved in oxidative folding of nascent proteins in the er . the oxido - reductases pdi and ero1 are coupled and mediate the disulfide bridge formation and protein folding of proteins such as digestive enzymes synthesized in the er . reduced glutathione is necessary to reduce incorrectly placed disulfide bonds so that the protein can be recycled for correct disulfide bridge formation and folding . fad is synthesized in the cytoplasm and easily enters the er . as illustrated on the right side of figure 4 , the reduction of disulfides is important for the reduction of incorrect disulfide bonds that can form during folding ( chakravarthi and bulleid , 2004 ) . the results from the experiments in the exocrine pancreas do not yet provide information on the detail of the oxidative state of the oxido - reductases or about their kinetics during the er stress produced by alcohol treatments . however , the initial findings of a marked increase in the ratio of the oxidized to the reduced form of glutathione in the er and the increase in the oxidized form of pdi suggest the likelihood of significant disturbances in oxido - reductase enzyme kinetics which would lead to the findings described . in closing , we hypothesize that alcohol abuse and many other causes of pancreatitis including genetic mutations in digestive enzymes , smoking , metabolic disorders such as diabetes and hypertriglyceridemia , and drugs may cause the disease at least in part by causing er stress . the key question related to why some individuals develop disease while others do not is not known . it is certainly possible that alterations in key components of the upr through mutation ( or environmental factors ) are present in the pancreas of those individuals who develop tissue injury in fact , genetic variants of xbp1 have been associated with the risk of development of inflammatory bowel disease ( kaser et al . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the exocrine pancreas functions are the synthesis , storage , and secretion of digestive enzymes into the lumen of the gastrointestinal tract where the enzymes are responsible for converting foodstuffs ingested into aqueous soluble molecules that can be absorbed by the intestinal epithelium . the two key epithelial cell types of the exocrine pancreas involved in digestive enzyme secretion are the acinar cell and the duct cell . the acinar cell is the site of digestive enzyme synthesis , storage , and regulated secretion while the duct cell provides ions and water necessary for transporting the acinar cell secreted products in the pancreatic ductal system to the intestinal lumen . the acinar cell of the exocrine pancreas has the greatest rate of protein synthesis of any mammalian organ and thus is endowed by a highly developed endoplasmic reticulum ( er ) system ( palade , 1975 ; case , 1978 ) . in addition to its functions in performing protein synthesis and processing , the er is the major storage site for intracellular calcium which when released into the cytoplasm is the mediator of regulated secretion of stored digestive enzymes into the pancreatic ductal system ( petersen and tepikin , 2008 ) . each protein synthesized in the er must undergo specific secondary modifications and folding before it can be transported to destination organelles such as golgi , zymogen granule ( storage for the digestive enzymes ) , and lysosome ; or membrane sites . further , because of variation in the demand for protein synthesis as a function of diet ; and because protein processing in the er could be adversely affected by environmental factors such as alcohol , smoking , altered metabolism , and xenobiotics , it is likely that there are regulatory systems within the er that allow it to adapt its functions to such stressors . in the case of alcohol abuse the increased risk for pancreatic cancer occurs largely through the effect of alcohol abuse causing chronic forms of pancreatitis ( lowenfels et al . , smoking also contributes to the development of pancreatitis and is a major risk factor for pancreatic cancer independent of pancreatitis ( lowenfels et al . recent epidemiologic studies demonstrate that smoking accelerates the development of pancreatitis in alcoholic patients and may have an additive or multiplicative effect when combined with alcohol to cause pancreatitis ( maisonneuve et al . the mechanisms underlying the effects of alcohol and smoking on the development of pancreatic diseases an important and unexplained observation is that only a small proportion of heavy drinkers / smokers develop pancreatic diseases ( pandol et al . although the reason for lack of development of pathology in the majority of those who drink and smoke is unknown , we hypothesize that an adaptive unfolded protein response ( upr ) is sufficiently robust in most individuals to prevent pathology . figure 1 illustrates many of these challenges which are often referred to as er stress that we have applied to our hypothesis of er stress for the exocrine pancreas . at the bottom of the figure a key implication from the figure is that upr activation leads to an adaptive response in the er . in this figure we hypothesize potential er stressors in the pancreas from what is known about er stressors in general ( ron and walter , 2007 ) . for example , it is likely that increases in protein synthesis rates that occur to replenish digestive enzyme stores after a meal will increase the demand for protein folding resulting in increased need for synthesis and functionality of chaperones and foldases in the er . because the rate of unfolded / misfolded protein formation would also increase with increased proteins synthesized , the er quality control system to degrade these unusable proteins called er - associated protein degradation ( erad ) is required to be present and functional to rid the cell from accumulation of permanently misfolded and unfolded proteins that could present toxicity to the cell . on the left hand side of the figure these include genetic mutations in digestive enzymes ; alcohol abuse ; smoking ; metabolic disorders such as diabetes and hyperlipidemia ; xenobiotics such as drugs and intestinal bacterial metabolites ; and reactive oxygen species that are generated in many of these situations as well as during acute and chronic pancreatitis . these pancreatic er stressors lead to further accumulation of unfolded and misfolded proteins which , in turn , lead to further activation of the unfolded proteins response in an attempt to adapt the pancreas to function in the face of the er stressors . there are several genetic , environmental , and disease - related stressors illustrated in figure 1 that occur in the pancreas that are likely significantly increase er stress requiring the acinar cell to activate its adaptive upr or face the possibility of cellular pathologies . mutations in key protease digestive enzymes are known to lead to chronic forms of pancreatitis ( hereditary pancreatitis ) and increased rate of pancreatic cancer ( whitcomb , 2010 ) . thus , alterations in er calcium stores in cells can lead to pathologic consequences at least in part because of the er stress induced . further , as discussed in more detail below , manipulation of er calcium stores in pancreatic acinar cells has been shown to activate er stress signals . , 2010 ) and reviewed below , there is little information on whether these factors affecting the pancreas indeed cause er stress and whether pathology results from an insufficiently robust upr . however , alcohol , smoking , diabetes , obesity , hyperlipidemia , and drugs account for large percentage of the burden of pancreatic diseases ( pandol et al . thus , the potential for roles of er stress in pancreatic disease pathogenesis is great . , 2008 these include : ( 1 ) upregulation of the expression and function of chaperones and foldases to augment the folding and export capacity of the er ; ( 2 ) activation of the er - associated protein degradation ( erad ) system to rid the er of accumulated unfolded and misfolded proteins ; and ( 3 ) a global reduction in translation of mrna to decrease the processing demand for newly synthesized proteins . as illustrated in figure 2 , the mammalian upr is initiated mainly by three er stress sensor transducers located in the membrane of the er ( ron and walter , 2007 ; rutkowski and kaufman , 2007 ) . these three are inositol - requiring protein-1 ( ire1 ) , activating transcription factor-6 ( atf6 ) , and protein kinase rna ( pkr)-like er kinase ( perk ) . in each case the transmembrane sensor transducer determines the folding status of proteins in the er lumen and transmits this information across the er membrane to the cell cytosol . are inositol - requiring protein-1 ( ire1 ) , activating transcription factor-6 ( atf6 ) , and protein kinase rna ( pkr)-like er kinase ( perk ) . these sensor transducers is followed specific pathways resulting in transcriptional regulation of chaperones , foldases , and components of er - associated protein degradation ( erad ) system and lipid synthesis for expansion of the er mediated by the combined effects of ire1 and atf6 ; or translational attenuation and transcriptional upregulation pathways involved in antioxidant synthesis and cell death through the transcription factor c / ebp homologous protein ( chop ) as in the case of perk . the participants in the pathways involved in the downstream effects of ire1 , atf6 , and perk activation include x - box binding protein1 ( xbp1 ) , site-1 and site-2 proteases ( sp1/sp2 ) , and eukaryotic translation initiation factor-2a ( eif-2a ) , and activating transcription factor-4 ( atf4 ) as discussed in the text . activation of the er transmembrane protein ire1 initiates a response to increase the expression of er chaperones and foldases in order to assist in protein folding and transport ( figure 2 ) . , 2002 ; xbp1-s , in turn , binds to er stress element ( erse ) and the upr element ( upre ) dna binding sites to upregulate many upr target genes such as the chaperones bip / grp78 and grp94 and the gene encoding xbp1-u ( yoshida et al . the ire1/xbp1 pathway also leads to increased expression of foldases such as protein disulfide isomerase ( pdi ) , enzymes for lipid synthesis for expanding the er membrane and er capacity , components of the er - associated degradation ( erad ) , all protective mechanism to lessen er stress ( yoshida et al . , the importance of xbp1 for the function of the pancreatic acinar cell has been demonstrated in xbp1 deficient mice with a transgene expressing xbp1 in hepatocytes to prevent embryonic lethality ( lee et al . the er in the pancreatic acinar cells in these animals is poorly developed accompanied by a decreased in the expression of er chaperones and marked apoptosis of the cells . release from bip permits atf6 transport to the golgi compartment where it is cleaved by site-1 and site-2 proteases ( sp1/sp2 ) to a 50- to 60-kda fragment that migrates to the nucleus to activate transcription of xbp1-u and other upr target genes ( shen et al . as discussed here , bip has several functions including acting as a chaperone , a luminal sensor of unfolded proteins , a regulator of activation of upr pathways upr , and a regulator of its own expression . recently , we found that genetic inhibition of bip leads to augmented severity of experimental pancreatitis showing the importance of bip in cellular protective responses ( ye et al . the perk plays a key role in adjusting the cell to er stress by causing a significant attenuation of general protein synthesis ( figure 2 ) . the activation of perk by autophosphorylation ( thr ) leads to its phosphorylation of the alpha subunit of the eukaryotic translation initiation factor-2a the non - phosphorylated form of eif-2a in its gtp - bound form is essential for translation initiation because it recruits the first trna ( trna ) to the ribosomal subunits to start translation of the attached mrna . as a consequence , cells are more sensitive to er stress pointing out a potential protective role of the perk signaling pathway in the er stress response . of note , previous studies in acinar cells have demonstrated that concentrations of cholecystokinin peptides which cause pancreatitis also inhibit translational initiation ; and that this block in translation is mediated by phosphorylation of eif2a ( perkins et al . of interest , the phosphorylation of eif2a is augmented by depletion of calcium from er stores , showing that this manipulation of er calcium homeostasis can initiate er stress . because cholecystokinin as well as acetylcholine can also deplete er calcium stores , the findings suggest that changes in er calcium provide an er stress signal in the pancreatic acinar cell . persistent phosphorylation of eif2a leads to specific translational upregulation of atf4 that targets genes involved in antioxidant effects including synthesis of glutathione ( harding et al . the mechanisms of the erad system to rid the er of accumulated unfolded and misfolded proteins is complex , debated , and only partly understood in any cell ( brodsky and wojcikiewicz , 2009 ; yoshida and tanaka , 2010 ) . the mechanisms of delivery of proteins from the er to proteasomes are under active investigation and likely require a proteinaceous channel in the er membrane needed to transfer proteins to the cytoplasmic located proteasomes . , 2010 ) that alcohol feeding in animals with genetic inhibition of xbp1 expression led to a marked decrease in expression of er degradation - enhancing alpha - mannosidase - like 1 ( edem1 ) , a key participant involved in erad and targeting unfolded / misfolded proteins to the proteasome ( yoshida and tanaka , 2010 ) . these findings suggest a key role for erad in the adaptive response of the exocrine pancreas to environmental stressors . , ire1 , atf6 , and perk ) and their downstream pathways during the development of pancreatitis in experimental models pancreatitis suggesting involvement of er stress responses in this disorder . , 2008 ) have demonstrated that long - term ethanol feeding in animal models causes little evidence of damage of the pancreatic parenchyma as determined by histology and blood concentrations of pancreatic digestive enzymes . for these reasons we designed studies to determine if alcohol abuse causes er stress and activates the upr in the exocrine pancreas ; and studies to investigate the role of upr signals in adapting the pancreas to the effects of alcohol abuse ( lugea et al . however , careful structural examination by electron microscopy demonstrated extensive distention of the er of acinar cells and altered distribution of zymogen granules . in addition , an investigation of the redox status of the er demonstrated that there was a significant decrease in reduced and an increase in oxidized glutathione indicating that the alcohol feeding has a significant oxidative effect in the er of the acinar cell . taken together , these findings indicated that ethanol has the oxidative effect in the er of acinar cells associated with morphologic signs of er stress . we then evaluated the effect of the alcohol treatment on the sensors and signals of the upr . also , we found that a key oxido - reductase , pdi , was significantly upregulated in the pancreas of the ethanol - fed animals . further , we found that a much greater proportion of the pdi was in its oxidized state in the ethanol - fed animals compared to control fed . pdi is critical for protein folding by catalyzing disulfide bond formation , a key step for maturation of proteins in the secretory pathway ( ellgaard and ruddock , 2005 ) . the combination of these findings suggest that pdi activity and thus appropriate folding of proteins in the secretory pathway of the acinar cell are impaired due to the oxidizing effects of alcohol feeding . the findings that alcohol feeding increased ire1 and xbp1-s expression suggested to us the possibility that these sensors and signals are important for adapting the er of the acinar cell to the er stress caused by the alcohol feeding . of note , alcohol feeding increased levels of xbp1-s in the pancreas of heterozygous animals to the same level observed in wild - type animals receiving the control diet but not the augmented levels observed in the pancreas with alcohol feeding in the wild - type animals . thus , the heterozygous deletion specifically prevented the ethanol feeding - induced augmentation in xbp1-s levels in the pancreas . the predominant morphologic abnormalities in the xbp1 deficient animals receiving alcohol were a disorganized ultrastructure with decreased number of zymogen granules with the remaining ones inappropriately scattered throughout the cell and not localized to their normal apical position . there was extensive dilation of the er with occasional dense luminal inclusions , a hallmark of er stress , and a significant accumulation of autophagic vacuoles . an analysis of the upr revealed that xbp1 deficiency prevented alcohol 's effect to increase pdi while it markedly promoted perk and eif2a phosphorylation and expression of atf4 , all features of prolonged and severe er stress ( harding et al . the results of the in vivo studies described above showing the role of the upr in adapting the pancreas to alcohol treatments allow us to propose the model illustrated in figure 3 . the oxidative stress caused by alcohol metabolism in the er activates ire1 leading to splicing of xbp1 mrna resulting in translation of the active transcription factor , xbp1-s . this transcription factor in turn mediates the expression of chaperones , pdi , erad proteins ( for example edem1 ) , and increased lipid synthesis to expand the capacity of the er . when there is an insufficient upr response as shown in our experiments using animals with deficient xbp1 , there is unresolved and augmented er stress resulting in perk activation causing global protein synthesis inhibition and even atf4-mediated activation of cell death pathways via chop expression . this figure illustrates the results of our experiments in alcohol fed animals showing er stress from ethanol and its metabolites , acetaldehyde , and fatty acid ethanol ester ( faee ) , by inducing an oxidative state in the er mediating misfolding and unfolding of proteins and oxidation of lipids . the er stress leads to upregulation of the ire1 pathway leading to splicing of xbp1 mrna resulting in the translation of the transcription factor xbp1-s which is transported to the nucleus where it upregulates the expression of chaperones , oxido - reductases / foldases such as pdi , erad proteins , and lipid synthetic enzymes through dna binding elements , er stress element ( erse ) and the upr element ( upre ) . the activation of this pathway leads to adaptation of the pancreas to the stress of ethanol abuse and prevents pancreatic injury and disease . the findings related to the oxidative state of glutathione and pdi provide important insights for er stress and the upr in the exocrine pancreas . figure 4 illustrates the mechanism of oxidative folding of nascent proteins ( ellgaard and ruddock , 2005 ; hatahet and ruddock , 2009 ; shimizu and hendershot , 2009 ) . the ability of pdi to mediate folding of a nascent protein is a function of its ability to catalyze disulfide pdi recognizes and binds to nascent unfolded proteins , semi - folded proteins and misfolded proteins but not correctly folded proteins through mechanisms that are not entirely known . the oxido - reductases pdi and ero1 are coupled and mediate the disulfide bridge formation and protein folding of proteins such as digestive enzymes synthesized in the er . ero1 is a flavo - enzyme that is induced by the upr and it is directly oxidized by molecular oxygen in a fad - dependent reaction ( shimizu and hendershot , 2009 ; tavender and bulleid , 2010 ) . as illustrated on the right side of figure 4 , the reduction of disulfides is important for the reduction of incorrect disulfide bonds that can form during folding ( chakravarthi and bulleid , 2004 ) . illustrated at the top of figure 4 is the suggestion that glutathione may be involved in regeneration of the oxidized state of pdi in some systems . the activities and direction of the reaction catalyzed by oxido - reductases such and pdi and ero1 are dependent on the oxidative state of their catalytic sites as well as the local environments of the catalytic sites . the results from the experiments in the exocrine pancreas do not yet provide information on the detail of the oxidative state of the oxido - reductases or about their kinetics during the er stress produced by alcohol treatments . however , the initial findings of a marked increase in the ratio of the oxidized to the reduced form of glutathione in the er and the increase in the oxidized form of pdi suggest the likelihood of significant disturbances in oxido - reductase enzyme kinetics which would lead to the findings described . in closing , we hypothesize that alcohol abuse and many other causes of pancreatitis including genetic mutations in digestive enzymes , smoking , metabolic disorders such as diabetes and hypertriglyceridemia , and drugs may cause the disease at least in part by causing er stress . it is certainly possible that alterations in key components of the upr through mutation ( or environmental factors ) are present in the pancreas of those individuals who develop tissue injury in fact , genetic variants of xbp1 have been associated with the risk of development of inflammatory bowel disease ( kaser et al .

a large fraction of all dna damages are formed at 2-deoxyguanosines ( dgs ) . of the four common nucleosides in dna , oxidation takes place most easily at dg residues , giving rise to a variety of products including 7,8-dihydro-8-oxo-2-deoxyguanosine ( 8-oxo - dg ) ( figure 1 ) . 8-oxo - dg is more susceptible to oxidation than dg , and it generates a number of secondary oxidation products . the pathway leading to 8-oxo - dg is believed to involve a c8-hydroxyl radical , which also forms fapydg ( figure 1 ) . methylating and ethylating agents preferentially react at n7 , but they also alkylate o of dg , and the fraction of alkylation at o increases with harder the n7-methyl - dg ( n7-me - dg ) adduct is unstable , which either depurinates to form an abasic site or undergoes ring opening to generate mefapydg ( figure 1 ) . interestingly , a vast majority of the bulky adducts are formed at either n7 or the exocyclic n position of dg . the unstable dg - n7 adducts formed by the metabolically activated aromatic amines and nitro compounds rearrange to stable dg - c8 adducts , while minor adducts at the n position of dg have also been isolated . in contrast , a majority of the metabolically activated epoxides of polycyclic aromatic hydrocarbons ( pahs ) form the dg - n adducts as the major products . metabolically activated aflatoxin b1 , however , forms the primary dg - n7 adduct , which undergoes ring opening to a stable formamidopyrimidine ( fapy ) derivative . like the pah epoxides , the antitumor agent mitomycin c ( mc ) containing an aziridine ring preferentially forms the dg - n adducts . the genotoxicity and mutagenicity of many of these adducts have been investigated in prokaryotic and eukaryotic cells for the last three decades . replication of these dna lesions do not follow a unifying mechanism , and each lesion exhibits a characteristic mutational spectrum . however , increasingly it became clear that the mutational signature of a dna lesion is directly related to the identity of the dna polymerase(s ) that bypass it and the mechanism of its nucleotide insertion and extension , though additional factors such as dna sequence context play a role as well . a human cell contains at least 17 different dna polymerases ( pols ) to perform different functions of the cell , which include dna replication of undamaged and damaged dna , replication as part of various dna repair pathways , recombination , telomere maintenance , and other tasks . on the basis of sequence homology , pols have been divided into seven families ( a , b , c , d , x , y , and rt ) , of which c family pols were only found in prokaryotes . in eukaryotes , the b - family enzymes are important since pol and pol of this family carry out a large fraction of nuclear dna replication , whereas pol is involved in initiation and priming . these three pols are essential for dna replication in eukaryotes . in the current model of dna replication , pol carries out a majority of leading strand dna replication of the undamaged genome , whereas pol primarily replicates the lagging strand . however , this model has recently been challenged , and data supporting the involvement of pol in both leading and lagging strand replication have been presented . the discovery of translesion synthesis ( tls ) dna pols in the 1990s invigorated the area of replication of dna lesions , and since then , numerous articles have been published on the catalytic and noncatalytic roles of these pols in the context of damaged dna replication . lesion bypass is carried out principally by the y - family pols , although x- and b - family pols are also frequently involved . like the replicative pols , these pols possess right - handed topology with the active site located in the palm domain , except that the active site is much larger in order to accommodate the dna lesions . unlike the replicative pols , in which the finger and thumb domains ensure correct pairing with the incoming nucleotide , they are shorter and make little interaction with the template and the incoming dntp , thereby reducing the pol s ability to discriminate the accuracy of nucleotide insertion . a little finger domain assists to stabilize the y - family pol on dna . an important aspect of the y - family pols and pol of the b - family is that they lack the 35 proofreading function , making them error - prone but letting them carry out tls . from the perspective of tls , dna lesions can be broadly divided between weak and strong replication blocks . small dna lesions such as o - me - dg and 8-oxo - dg stall but do not completely stop dna synthesis , whereas most bulky dna lesions , such as the adducts formed by the pahs and aromatic amines , are much stronger replication blocks and require the assistance of tls pols to bypass . the current paradigm on tls is as follows . when a processive dna pol encounters a blocking lesion , the pol dissociates , and a tls pol binds to the dna and incorporates a dntp opposite the lesion . in many cases , the same pol continues elongation for a few more bases before dissociating , while in other occasions this tls pol is replaced by another tls pol for the elongation steps . tls pols exhibit higher rate of errors on unmodified templates and are also highly error - prone when bypassing most dna lesions . soon after bypassing the lesion , the processive pol returns to continue dna synthesis . however , the actual process of pol switching is still speculative , and many related questions remain unanswered at the present time . during cellular replication , the fork utilizes many proteins , including dna pol , helicase , and single strand binding proteins , to name a few . a prerequisite for tls is the rad6/rad18-mediated monoubiquitination of proliferating cell nuclear antigen ( pcna ) at the highly conserved lysine k164 . y - family pols contain ubiquitin - binding domains that confer affinity to monoubiquitinated pcna . in mammalian cells , in addition , two human rad5-related proteins , snf2 histone - linker phd - finger ring - finger helicase ( shprh ) and helicase - like transcription factor ( hltf ) , transform monoubiquitinated pcna into the polyubiquitinated form . additional dna damage response pathways , including shprh / hltf - mediated template switching , also depend on pcna ubiquitination . so , when replication by pol or pol is blocked by a dna lesion , pcna is monoubiquitinated by the rad6-rad18 protein complex and promotes the switch to a tls pol at the damage site ( figure 2 ) . evidently , the activity of the tls pols must be tightly regulated so that they only gain access to genomic dna when there is dna damage . for example , monoubiquitination of pol inhibits its interaction with pcna , thereby preventing its activity on undamaged dna , but monoubiquitination is downregulated by the dna damaging agents . this mechanism allows optimal availability of nonubiquitinated and active pol following dna damage . post - translational regulation of these proteins is an area where much emphasis has recently been placed . despite the predominant role of these bypass pols in tls , it is also worth noting that there is evidence that replicative pols ( such as pol ) may take part in some tls events . abridged tls scheme using pol as an example of the tls polymerase . oxidative stress generates many different dna lesions , but 8-oxo - dg is the most widely studied dna lesion formed by reactive oxygen species such as hydroxyl radicals ( figure 1 ) . 8-oxo - dg does not strongly block dna synthesis in eukaryotic cells , as reflected by the number of progeny derived from replication of singly adducted vectors . crystallographic studies using a high fidelity pol indicated that 8-oxo - dg adopts syn conformation at the preinsertion stage and pairs preferentially with adenine via hoogsteen base pairing in the pol active site . however , in vitro experiments using yeast pol showed that only about 10% tls takes place in the absence of any accessory proteins . even in the presence of pcna , steady - state reactions of calf - thymus dna pol were decreased by a factor of 12 for datp and dctp incorporation opposite 8-oxo - dg . the major dna pols in mammalian cells , pol , pol , and pol extend an 8-oxo - g : a pair more efficiently than the correct 8-oxo - g : c pair.in vitro experiments showed that pol is inefficient in nucleotide insertions opposite 8-oxo - dg , but it can efficiently extend from the nucleotides inserted opposite it by pol . yet , in human cells tls of 8-oxo - dg is largely error - free ( mutation frequency ( mf ) 1% in duplex dna and 420% in single stranded dna ) . several repair systems , including base excision repair and mismatch repair , excise 8-oxo - dg from duplex dna , justifying low mf , but most repair systems are inefficient in 8-oxo - dg repair in single - stranded dna . one might wonder why the tls of 8-oxo - dg in single - stranded dna is mostly error - free . the answer came from in vitro and cellular experiments , which determined a crucial role of pol , an x - family enzyme , in 8-oxo - dg bypass . the preference for dctp incorporation over either datp or dgtp incorporation opposite 8-oxo - dg is 12-fold by pol . however , it is remarkable that in the presence of the accessory proteins , human pcna and replication protein a ( rpa ) , correct incorporation of dctp over other dntps opposite 8-oxo - dg increased to 1200-fold by pol . in a similar vein , pcna and rpa increased the preference for dctp over datp or dgtp incorporation opposite 8-oxo - dg by pol from 2.5-fold to 68-fold . on the basis of these results and additional data from mouse embryonic fibroblasts and human cell lines , it was suggested that the switch from pol involved pol and not pol or pol since mutations by 8-oxo - dg increased considerably in pol knockout or knockdown cells . in yeast chromosome , however , the switch to pol , which replicates 8-oxo - dg with an accuracy of 94% , was reported . in the absence of pol , dna pol -interacting protein 2 ( poldip2 , also known as pdip38 ) physically interacts with pol and increases the efficiency of elongation past 8-oxo - dg by pol , suggesting an important role of this protein in pol switch and elongation steps during tls . if pol ( in the presence of the accessory proteins ) were the only pol that bypasses 8-oxo - dg , mf would have dropped to less than 1% . the 420% mf , which depends on the dna sequence context and the type of assay , in single - stranded dna indicates , however , that in addition to pol , other pols bypass the lesion . in human embryonic kidney ( hek ) 293 t cells , depending on the dna sequence context , we observed 3850% increase in mutations induced by 8-oxo - dg , upon knockdown of pol . it is interesting that g t mutations were not significantly increased in pol knockdown cells . the increase in mutations was primarily due to an increase in dinucleotide deletions , involving the lesion and one of its neighboring bases . others have also reported targeted one - base or small deletions in the absence of pol . it appears , therefore , that pol prevents these deletions induced by 8-oxo - dg . in addition to the dna pols , an additional factor is the participation of a homologue of muty glycosylase . muty human homologue ( mutyh ) shares 41% and 79% of sequence homology to its e. coli counterpart muty and mouse homologue mmyh , respectively . muty removes adenine from the 8-oxo - g : a mispair , which allows another chance to incorporate c opposite 8-oxo - dg by a pol . in a study in human lymphoblastoid cells , replication of 8-oxo - dg generated 14% mutants , including 6% g t and 2% targeted single - base deletions . overexpression of mutyh reduced the g t mutations , but the deletions remained unaffected , which also suggests the role of an unidentified dna pol in the 8-oxo - dg induced deletions . while the role of these deletions in human diseases is unknown , inherited variants of mutyh in a family affected by colon cancers show a pattern of high g : c t : a mutations implicating a role of unrepaired 8-oxo - dg lesions in human cancer . is generated at comparable levels under many conditions to 8-oxo - dg , but only a limited number of biological studies have been conducted with this lesion . bypass efficiency of purine - ring opened fapydg is slower than 8-oxo - dg . like 8-oxo - dg , however , the mf is highly dependent on the dna sequence context . for example , the mf of fapydg in the tg*t sequence is significantly higher than when it is located in the tg*a sequence in both simian ( cos-7 ) and human embryonic ( 293 t ) kidney cells . in human cells , in some sequence contexts fapydg is more mutagenic than 8-oxo - dg , while in others the opposite is true . the major difference between the two lesions , however , is that knockdown of pol reduced the level of g t mutations induced by fapydg , in contrast to an increase in mf for 8-oxo - dg . this suggests that pol is involved in a significant fraction of fapydg induced g t mutations , whereas it carries out error - free bypass of 8-oxo - dg . it is interesting , however , that the level of small deletions increases upon replication of either 8-oxo - dg or fapydg in human cells in which pol was knocked down . unlike 8-oxo - dg , which adopts syn conformation to pair with adenine , a structural study of the carbocyclic analogue of fapydg by a high fidelity pol ( bst pol i ) showed that the lesion maintains its anti conformation of the glycosidic bond during both error - free and error - prone replication . most biological assays indicate that n7-me - dg is not mutagenic but that its ring - opened derivative mefapydg ( figure 1 ) is mutagenic.in vitro assays showed that the mefapydg is a strong block to the high fidelity replicative dna polymerases at both the insertion and the extension steps . however , hpol and hpol as well as hrev1 and ypol together can carry out facile tls . with hpol and hpol , the predominant replication product is the error - free extension product , whereas hrev1 and ypol together accomplish entirely error - free tls . up to 29% mutagenic tls , including each of the targeted base changes and one - nucleotide deletion products , were identified from replication products generated by hpol and hpol . in cos-7 cells , mefapydg induces g t mutations and single and dinucleotide deletions as do 8-oxo - dg and fapydg . however , cellular experiments in human cells analogous to fapydg have not been performed with mefapydg , and it would certainly be interesting to compare the replicative properties of fapydg with mefapydg using the same approach . o - methyl - dg ( o - me - dg ) is one of the first mutagenic dna lesions identified as a result of dna methylation ( figure 1 ) . it is highly mutagenic but is quickly repaired in a cell by multiple repair systems . a great deal of circumstantial evidence indicates that it plays a role in the etiology of human cancer . using an intrachromosomal probe , 19% g a mutations were detected after replication of a site - specific o - me - dg in chinese hamster ovary cells deficient in the repair enzyme o - alkylguanine - dna alkyltransferase , but in repair proficient cells , mutation frequency dropped to an 1% level . like 8-oxo - dg , it allows partial bypass of several purified dna polymerases , but pol is only slightly inhibited in vitro and inserts dctp and dttp equally well opposite o - me - dg . however , pol is strongly blocked one base before o - me - dg.o - me - dg also is a strong but not absolute block of human pol , and even though hpol inserts dttp more efficiently than dctp opposite the lesion , it preferentially extends the correct o - me - g : c pair . in the absence of accessory proteins , the human tls pol and pol produce mainly one - base incorporation products opposite this lesion , but hpol is much more efficient . steady - state kinetic analysis showed similar efficiencies of insertion of dctp and dttp opposite o - me - dg by hpol and hpol , whereas hpol showed a higher preference for dttp insertion . genetic studies in yeast implicate both pol and pol in the tls of o - me - dg , even though biochemical studies suggest that hpol is more efficient than hpol . similar to 8-oxo - dg , in yeast pol is very inefficient at inserting nucleotides opposite o - me - dg , but it can efficiently extend from the nucleotides inserted opposite it by pol . as a result , the most efficient bypass can be accomplished in vitro when both pol and pol were used for the tls of templates containing o - me - dg . even though this perspective is focused on dg lesions , it may be pertinent to mention investigations that established a specialized role of pol in efficient and error - free bypass of uv light - induced cis - syn cyclobutane pyrimidine dimers ( cpds ) . this is due to pol s unique ability to accommodate both pyrimidine residues of this bulky lesion in its active site and perform accurate and efficient tls . pol and pol , on the other hand , provide an alternate , albeit highly error - prone , pathway of tls of cpds . in the absence of pol and pol , tls of cpds carried out by pol is error - free , and mutations decrease to the background level . mutations in this gene ( polh ) result in xpv , a variant type of the genetic disease , xeroderma pigmentosum , which is characterized by extreme sensitivity to uv light . no other dna pol exhibits such a precise and dedicated role , but the main characteristics of the other bypass pols have been established . one example is the ability of pol in the error - free bypass of dg - n lesions ( discussed later ) . the enlarged active site of pol allows it to accommodate even the cisplatin - derived large intrastrand n7pt crystal structure analysis showed that to allow the lesion to fit in its active site , pol goes through a backbone rearrangement to stabilize the lesion and incorporate dctp opposite the two guanines . however , it also shows that the rigid backbone of the ternary complex with pol does not allow extension , which necessitates another tls pol such as pol to extend it . the potent hepatocarcinogen aflatoxin b1 ( afb1 ) forms two major dna adducts upon metabolic activation of afb1 to afb1 - 8,9-epoxide by the liver cytochrome p450 enzymes ( figure 3 ) . the primary dna adduct , afb1n7-dg , is formed at the n7 position of dg . this adduct is chemically unstable due to the positive charge at n7 , which can undergo either spontaneous depurination to generate abasic sites or ring opening to form afb1fapy - dg ( figure 3 ) . both these adducts are mutagenic in simian kidney ( cos-7 ) cells when the adduct is located in a ttg*aa sequence , but afb1fapy - dg induces 97% mutations compared to 45% mutations by the afb1n7-dg adduct . interestingly , in vitro tls assays showed that pol bypasses afb1n7-dg in an error - free manner , whereas it is responsible for the erroneous bypass of afb1fapy - dg . because of the importance of these adducts in human cancer , additional structural , genetic , and in vitro studies on the two dna adducts in the future would certainly be of significant interest . pahs are ubiquitous in our environment , and many of them , notably those with a bay or fjord region , are highly mutagenic and carcinogenic . the most extensively studied pah is benzo[a]pyrene ( bp ) , an extremely carcinogenic chemical , which upon metabolic activation binds to dna , predominantly at the n position of dg ( figure 4 ) . bp is metabolized by the mammalian monooxygenase enzymes to form the diastereomeric anti- and syn - benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide ( bpde ) . the metabolically activated ( + ) -anti bpde is a potent mutagen and the most tumorigenic metabolite of bp . it is believed to be the ultimate carcinogenic form that leads to trans- and cis - dg - n adducts ( figure 4 ) . the principal mutation in mammalian cells induced by the major dg adducts of bp is g : c t : a transversion . bp adducts are strong blocks of replication by replicative pols , but the tls pols can bypass them at varying efficiencies . in vitro studies using either hpol or hpol showed that the bpde dg adducts allow slow bypass , which results in a high frequency of nucleotide misincorporations . in yeast , however , ( )-anti - bpde mutagenesis requires pol and partially involves pol , but pol mainly contributes to deletions and insertions of 13 nucleotides . in contrast , pol performs accurate and reasonably efficient replication of the bpde dg adducts . the extent of bypass drops , and mutagenesis increases significantly in human and murine cells lacking pol . pol s catalytic site , unlike that of pol , can only accommodate one watson crick base pair . however , it is capable of tls of many dg - n adducts , including the dna adducts formed by bp . specifically , for the ( + ) -trans - anti - dg - n - bpde adduct , genetic , in vitro kinetics , and structural studies show that pol performs efficient and accurate tls . for the mutagenic tls , genetic evidence suggests that a non - y family pol inserts a wrong nucleotide ( datp or dttp ) opposite the adduct but that extension is performed cooperatively by pol and rev1 . crystal structure analyses of the ( + ) -trans - anti - dg - n - bpde adduct showed that the active site of pol is opened up at the minor groove side of the primer the amino acid residues of the protein in the minor groove side of dna stabilizes the hydrophobic bpde ring and maintains watson crick base pairing with an incoming dctp for accurate replication . pol also bypasses many other dg - n adducts accurately and efficiently , which includes n-(1-carboxyethyl)-dg and n - furfuryl - dg as well as much bulkier adducts formed by iq and mitomycin c ( discussed in the next section ) . we have recently studied the minor , albeit persistent , dg - n adduct ( figure 5 ) formed by the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline ( iq ) , a heterocyclic aromatic amine formed during high temperature cooking of meat , and two dg - nadducts ( figure 6 ) formed by the antitumor agent , mitomycin c ( mc ) , and its metabolite , 27-diaminomitosene ( 2,7-dam ) . structures of the dg - n adducts formed by mitomycin c and its metabolite 2,7-diaminomitosene . the dg - n - iq adduct was studied in the three different guanines of the nari restriction site ( 5-g1g2cg3cc-3 ) . as shown in table 1 , in hek293 t cells mf increases upon knockdown of only pol , whereas knockdown of pol , pol , pol , or rev1 results in a reduction in mf . the greatest reduction in mf occurred when pol , pol , and rev1 were concurrently knocked down . this suggests that pol is involved in error - free bypass of the dg - n adduct formed by iq , whereas pol , pol , and rev1 cooperatively carried out mutagenic tls . similar results were obtained with the mitomycin c adducts ( table 1 ) , indicating that they also follow analogous mechanisms . it was also established that with the increasing bulk of the dg - n adducts , the misincorporation frequency of datp relative to dctp increases significantly . taken together , there seems to be a predictable pattern of error - free and error - prone tls of dg - n adducts by the tls pols . exceptions to this rule , however , are the minor groove adducts -hydroxy-1,n - propano - dg and trans-4-hydroxy-2-nonenal - dg , in which case pol is inefficient in nucleotide insertion opposite the lesion , but it efficiently acts as an extender . in both these cases , pol can insert dctp opposite the lesions but is inefficient in extending the g*:c pair . in contrast , pol is unable to insert a nucleotide opposite these lesions , but it can extend the g*:c pair . thus , the sequential act of pol and pol promotes efficient and error - free tls of these lesions . it is noteworthy that these are cyclic adducts with a covalent bond with n1 in addition to n of dg , suggesting that pol one of the most extensively studied dna adduct is dg - c8-aaf ( figure 7 ) , the dg - c8 adduct formed by n - acetyl-2-aminofluorene ( aaf ) , which induces frameshift mutation in bacteria and human cells , but in simian kidney ( cos-7 ) cells , when the adduct is placed in a single stranded plasmid , it causes largely g t mutations . however , in a subsequent study , also in cos-7 cells but in duplex dna , at the third guanine of 5-ggg-3 and 5-ggcgcc-3 ( nari site ) , 1 and 2 frameshift mutations , respectively , were detected . the frameshift mutations at the 5-ggg-3 sequence are dependent on pol but not pol or pol . furthermore , this pol -mediated erroneous pathway requires rad18 and ubiquitination of pcna . on the other hand , tls is only partially dependent on pol and rad18 when the adduct is situated at the nari site this indicates that the same adduct may follow different mechanisms for mutagenesis in different sequence contexts . structures of the dg - c8 adducts formed by n - acetyl-2-aminofluorene , 1-nitropyrene , and 3-nitrobenzanthrone . the mechanism of both 1 and 2 frameshifts was suggested to follow a slipped frameshift intermediate , and while most pols are inefficient in extending such an intermediate , pol can extend them , albeit slowly . in duplex dna , dg - c8-aaf is known to rotate the guanine base to syn conformation , in contrast to an overwhelming anti conformation of an unmodified dg . biophysical and computational studies indicate that syn conformation in a base - displaced intercalated structure of the dg adduct allows formation of stable slipped intermediates . such intermediates , upon elongation , would cause frameshift mutations , the major types of mutations detected in bacteria and occasionally in mammalian cells ( or cell - free extracts ) . the role of pol in bypassing misaligned adducts has been explored , which showed that depending on the base sequence , a cytosine inserted opposite the dg - c8 lesion slips to generate a 1 , 2 , or 3 frameshift intermediate that pol can continue to replicate , in spite of a bulge . in a crystal structure study , however , pol was able to incorporate dctp opposite the dg - c8-aaf adduct , in which tls occured without rotation of the adduct into the anti conformation , and only one hydrogen bond was formed between the lesion and dctp . this structural investigation recognized pol s ability to perform error - free replication of dg - c8-aaf , in addition to its propensity to carry out frameshifts . like the dg - n adducts , the roles of tls dna pols in bypassing the c8-dg adduct ( dg - c8-iq ) ( figure 5 ) formed by iq were explored at the g1- , g2- , or g3-positions of the nari recognition sequence after replication in hek293 t cells . mf was the highest ( 50% ) when the adduct was placed at g3 , compared to 18% and 24% mf when the adduct was located at g1 and g2 , respectively , inducing mainly g t transversions at each site . mf of dg - c8-iq was reduced in varying degrees upon sirna knockdown of pol , pol - , pol - , or rev1-knockdown cells ( table 2 ) , indicating that these pols are involved in error - prone synthesis of this adduct . in contrast , mf was increased by 826% in pol knockdown cells , suggesting that pol bypasses the lesion accurately . largest % change was noted with pol and pol simultaneous knockdown , which gave 70% reduction in mf . upon simultaneous knockdown of pol , pol , and rev 1 , a synergy was observed in that mf was reduced by more than 90% in each case ( table 2 ) . in vitro experiments using yeast pol confirmed that it can extend the g3*:a pair more efficiently than the g3*:c pair , although it is inefficient at nucleotide incorporation opposite dg - c8-iq . it is , therefore , conceivable that pol and pol cooperatively carry out the majority of the error - prone tls of dg - c8-iq , whereas rev1 may play a noncatalytic role in assembling the tls pols . by contrast , pol is involved mostly in its error - free bypass . similar experiments have also been conducted with dg - c83-aba , the major dna adduct formed by the carcinogen 3-nitrobenzanthrone ( 3-nba ) ( figure 7 ) . like dg - c8-iq , dg - c83-aba induces g t as the major type of mutations in human cells . however , the polymerase knockdown results are different . pol and pol were found to be the major contributors of the mutagenic tls of dg - c83-aba since mf dropped by 70% , when these pols were simultaneously knocked down , although mf actually increased upon knockdown of pol alone . in contrast , pol is involved in the error - free bypass of the lesion since mf increased by 60% in pol knockdown cells . a recent in vitro presteady state kinetic investigation showed that hpol and hpol efficiently bypassed a site - specifically placed dg - c83-aba , whereas hpol and hrev1 were severely stalled by the lesion . crystal structure analysis of dg - c83-aba at the insertion stage of hpol showed that the adduct is wedged at the hydrophobic cleft in the active site in anti conformation stabilized by a hydrogen bond between the c8 amino group and the phosphate , while the 2-deoxyribose adopts c3-endo pucker . this structure provides a model for an accurate but slow bypass of the adduct by pol . the structure of an erroneous bypass of dg - c83-aba by a pol is yet to be solved . we postulate that both pol and pol conduct error - free tls of dg - c83-aba . however , pol also extends mispairs generated by incorporation of datp by pol opposite the adduct . it is noteworthy that single - nucleotide incorporation opposite a dg - c83-aba lesion catalyzed by hpol in vitro showed that at short reaction time frames incorporation of dctp is greater than datp but that with longer time incorporation of these two nucleotides becomes comparable . rev1 likewise is important for mutagenesis , as reflected by 60% reduction in mf upon rev1 knockdown , but as with dg - c8-iq , it probably plays a noncatalytic role by physically interacting with the other two y - family pols . the noncatalytic role of rev1 was indicated by its inability to bypass the lesion in vitro . therefore , the c8-dg adducts dg - c8-iq and dg - c83-aba do not behave the same way with different polymerases . as mentioned earlier on the mechanism of frameshift mutations induced by dg - c8-aaf , many bulky adducts formed at the c8 position of dg , such as dg - c8-iq and dg - c83-aba , rotate the base to syn conformation , which is believed to play a structural role in frameshift mutations observed in bacteria . more frequently in mammalian cells , however , these adducts induce base substitutions . since these purine lesions rotate to syn conformation , one can anticipate a role of pol in bypassing them , as this enzyme uses hoogsteen base pairing to select the incoming nucleotide . pol can bypass only small dg - n adducts since n is oriented toward the major groove , and rotation to syn is inhibited for bulky dg - n adducts . in contrast , bulky dg - c8 adducts can be accommodated in the pol active site more efficiently . an example of pol s potential involvement in dg - c8 adduct bypass is its interaction with dg - c8-ap , the major adduct formed by the environmental carcinogen , 1-nitropyrene ( 1-np ) ( figure 7 ) . dg - c8-ap , like the other dg - c8 adduct mentioned earlier , induces predominantly g t mutations in simian and human embryonic kidney cells . the adduct , as other bulky dg - c8 adducts , exists in syn conformation in a base - displaced intercalated solution structure . replication of dg - c8-ap stalls when in vitro bypass is conducted by the tls pols . of the human tls pols , hpol is most proficient in bypassing it in vitro , but hpol and hpol can incorporate a nucleotide opposite the lesion . crystal structure analyses showed that dctp incorporation opposite dg - c8-ap forces the adduct to rotate to the anti conformation to avoid steric hindrance at the minor groove side . however , this structure inhibits further extension , due to a clash with the little finger domain of the enzyme . in contrast , the adduct can maintain syn conformation when datp is inserted , in which the adenine is stacked above the pyrene ring intercalated in the helix . this structure allows further extension . therefore , error - prone replication of dg - c8-ap potentially may occur by two tls pols , with pol being involved in the insertion stage . another dg - c8 adduct , ( 5s)-8,5-cyclo - dg , a cyclic dna adduct containing a covalent bond between c8 of guanine and 5 c of 2-deoxyribose , was investigated in human cells , which showed that pol , pol , and pol but not pol are involved in tls . unlike the dg - n adducts , therefore , a pattern for tls of the dg - c8 adducts could not be determined . for example , pol is involved in extension of the correct pair of the dg - c83-aba , whereas it extends the wrong pair with dg - c8-iq . studies on additional dg - c8 adducts might give us a clue as to why they fail to follow a unifying mechanism of tls . genetic studies in repair and replication competent cells provide data on the outcome of the damage , and a comparison of these in genetically altered cells ( including knockout or knockdown of specific genes ) has been employed to investigate the role of each tls pol . in vitro experiments using purified pols and accessory proteins elucidate how each pol can deal with the dna damage , whereas structural and computational studies give a more intimate snapshot of the lesion bypass . each approach has its limitations , and consequently , combined approaches are essential to comprehend the mechanism of tls of a dna lesion . mechanistic information on replication of the dna lesions is critical to follow the underlying process for the development of cancer , aging , and various other diseases . these fundamental studies are now paving the way to application of the acquired knowledge toward therapeutic application , as inhibiting the activity of some of the tls pols may enhance the effect of an antitumor agent . as yet , more tls work has been done with the pols from prokaryotes and archaea than from eukaryotes . it is certain that this dynamic area of research is still in its early stage and will continue to enrich the field of toxicology with many novel findings .

with the discovery of translesion synthesis dna polymerases , great strides have been made in the last two decades in understanding the mode of replication of various dna lesions in prokaryotes and eukaryotes . a database search indicated that approximately 2000 articles on this topic have been published in this period . this includes research involving genetic and structural studies as well as in vitro experiments using purified dna polymerases and accessory proteins . it is a daunting task to comprehend this exciting and rapidly emerging area of research . even so , as the majority of dna damage occurs at 2-deoxyguanosine residues , this perspective attempts to summarize a subset of this field , focusing on the most relevant eukaryotic dna polymerases responsible for their bypass .

Introduction TLS of Small and Ring-Opened dG Lesions TLS of Bulky dG Lesions Concluding Comments

in contrast , a majority of the metabolically activated epoxides of polycyclic aromatic hydrocarbons ( pahs ) form the dg - n adducts as the major products . the genotoxicity and mutagenicity of many of these adducts have been investigated in prokaryotic and eukaryotic cells for the last three decades . replication of these dna lesions do not follow a unifying mechanism , and each lesion exhibits a characteristic mutational spectrum . however , increasingly it became clear that the mutational signature of a dna lesion is directly related to the identity of the dna polymerase(s ) that bypass it and the mechanism of its nucleotide insertion and extension , though additional factors such as dna sequence context play a role as well . a human cell contains at least 17 different dna polymerases ( pols ) to perform different functions of the cell , which include dna replication of undamaged and damaged dna , replication as part of various dna repair pathways , recombination , telomere maintenance , and other tasks . on the basis of sequence homology , pols have been divided into seven families ( a , b , c , d , x , y , and rt ) , of which c family pols were only found in prokaryotes . in eukaryotes , the b - family enzymes are important since pol and pol of this family carry out a large fraction of nuclear dna replication , whereas pol is involved in initiation and priming . in the current model of dna replication , pol carries out a majority of leading strand dna replication of the undamaged genome , whereas pol primarily replicates the lagging strand . however , this model has recently been challenged , and data supporting the involvement of pol in both leading and lagging strand replication have been presented . the discovery of translesion synthesis ( tls ) dna pols in the 1990s invigorated the area of replication of dna lesions , and since then , numerous articles have been published on the catalytic and noncatalytic roles of these pols in the context of damaged dna replication . like the replicative pols , these pols possess right - handed topology with the active site located in the palm domain , except that the active site is much larger in order to accommodate the dna lesions . unlike the replicative pols , in which the finger and thumb domains ensure correct pairing with the incoming nucleotide , they are shorter and make little interaction with the template and the incoming dntp , thereby reducing the pol s ability to discriminate the accuracy of nucleotide insertion . small dna lesions such as o - me - dg and 8-oxo - dg stall but do not completely stop dna synthesis , whereas most bulky dna lesions , such as the adducts formed by the pahs and aromatic amines , are much stronger replication blocks and require the assistance of tls pols to bypass . tls pols exhibit higher rate of errors on unmodified templates and are also highly error - prone when bypassing most dna lesions . evidently , the activity of the tls pols must be tightly regulated so that they only gain access to genomic dna when there is dna damage . this mechanism allows optimal availability of nonubiquitinated and active pol following dna damage . despite the predominant role of these bypass pols in tls , it is also worth noting that there is evidence that replicative pols ( such as pol ) may take part in some tls events . oxidative stress generates many different dna lesions , but 8-oxo - dg is the most widely studied dna lesion formed by reactive oxygen species such as hydroxyl radicals ( figure 1 ) . 8-oxo - dg does not strongly block dna synthesis in eukaryotic cells , as reflected by the number of progeny derived from replication of singly adducted vectors . crystallographic studies using a high fidelity pol indicated that 8-oxo - dg adopts syn conformation at the preinsertion stage and pairs preferentially with adenine via hoogsteen base pairing in the pol active site . however , in vitro experiments using yeast pol showed that only about 10% tls takes place in the absence of any accessory proteins . the major dna pols in mammalian cells , pol , pol , and pol extend an 8-oxo - g : a pair more efficiently than the correct 8-oxo - g : c pair.in vitro experiments showed that pol is inefficient in nucleotide insertions opposite 8-oxo - dg , but it can efficiently extend from the nucleotides inserted opposite it by pol . the answer came from in vitro and cellular experiments , which determined a crucial role of pol , an x - family enzyme , in 8-oxo - dg bypass . however , it is remarkable that in the presence of the accessory proteins , human pcna and replication protein a ( rpa ) , correct incorporation of dctp over other dntps opposite 8-oxo - dg increased to 1200-fold by pol . on the basis of these results and additional data from mouse embryonic fibroblasts and human cell lines , it was suggested that the switch from pol involved pol and not pol or pol since mutations by 8-oxo - dg increased considerably in pol knockout or knockdown cells . in the absence of pol , dna pol -interacting protein 2 ( poldip2 , also known as pdip38 ) physically interacts with pol and increases the efficiency of elongation past 8-oxo - dg by pol , suggesting an important role of this protein in pol switch and elongation steps during tls . if pol ( in the presence of the accessory proteins ) were the only pol that bypasses 8-oxo - dg , mf would have dropped to less than 1% . the 420% mf , which depends on the dna sequence context and the type of assay , in single - stranded dna indicates , however , that in addition to pol , other pols bypass the lesion . others have also reported targeted one - base or small deletions in the absence of pol . overexpression of mutyh reduced the g t mutations , but the deletions remained unaffected , which also suggests the role of an unidentified dna pol in the 8-oxo - dg induced deletions . is generated at comparable levels under many conditions to 8-oxo - dg , but only a limited number of biological studies have been conducted with this lesion . like 8-oxo - dg , however , the mf is highly dependent on the dna sequence context . for example , the mf of fapydg in the tg*t sequence is significantly higher than when it is located in the tg*a sequence in both simian ( cos-7 ) and human embryonic ( 293 t ) kidney cells . it is interesting , however , that the level of small deletions increases upon replication of either 8-oxo - dg or fapydg in human cells in which pol was knocked down . most biological assays indicate that n7-me - dg is not mutagenic but that its ring - opened derivative mefapydg ( figure 1 ) is mutagenic.in vitro assays showed that the mefapydg is a strong block to the high fidelity replicative dna polymerases at both the insertion and the extension steps . however , hpol and hpol as well as hrev1 and ypol together can carry out facile tls . o - methyl - dg ( o - me - dg ) is one of the first mutagenic dna lesions identified as a result of dna methylation ( figure 1 ) . it is highly mutagenic but is quickly repaired in a cell by multiple repair systems . a great deal of circumstantial evidence indicates that it plays a role in the etiology of human cancer . using an intrachromosomal probe , 19% g a mutations were detected after replication of a site - specific o - me - dg in chinese hamster ovary cells deficient in the repair enzyme o - alkylguanine - dna alkyltransferase , but in repair proficient cells , mutation frequency dropped to an 1% level . like 8-oxo - dg , it allows partial bypass of several purified dna polymerases , but pol is only slightly inhibited in vitro and inserts dctp and dttp equally well opposite o - me - dg . however , pol is strongly blocked one base before o - me - dg.o - me - dg also is a strong but not absolute block of human pol , and even though hpol inserts dttp more efficiently than dctp opposite the lesion , it preferentially extends the correct o - me - g : c pair . in the absence of accessory proteins , the human tls pol and pol produce mainly one - base incorporation products opposite this lesion , but hpol is much more efficient . as a result , the most efficient bypass can be accomplished in vitro when both pol and pol were used for the tls of templates containing o - me - dg . even though this perspective is focused on dg lesions , it may be pertinent to mention investigations that established a specialized role of pol in efficient and error - free bypass of uv light - induced cis - syn cyclobutane pyrimidine dimers ( cpds ) . this is due to pol s unique ability to accommodate both pyrimidine residues of this bulky lesion in its active site and perform accurate and efficient tls . no other dna pol exhibits such a precise and dedicated role , but the main characteristics of the other bypass pols have been established . one example is the ability of pol in the error - free bypass of dg - n lesions ( discussed later ) . interestingly , in vitro tls assays showed that pol bypasses afb1n7-dg in an error - free manner , whereas it is responsible for the erroneous bypass of afb1fapy - dg . because of the importance of these adducts in human cancer , additional structural , genetic , and in vitro studies on the two dna adducts in the future would certainly be of significant interest . the most extensively studied pah is benzo[a]pyrene ( bp ) , an extremely carcinogenic chemical , which upon metabolic activation binds to dna , predominantly at the n position of dg ( figure 4 ) . the metabolically activated ( + ) -anti bpde is a potent mutagen and the most tumorigenic metabolite of bp . it is believed to be the ultimate carcinogenic form that leads to trans- and cis - dg - n adducts ( figure 4 ) . bp adducts are strong blocks of replication by replicative pols , but the tls pols can bypass them at varying efficiencies . however , it is capable of tls of many dg - n adducts , including the dna adducts formed by bp . specifically , for the ( + ) -trans - anti - dg - n - bpde adduct , genetic , in vitro kinetics , and structural studies show that pol performs efficient and accurate tls . crystal structure analyses of the ( + ) -trans - anti - dg - n - bpde adduct showed that the active site of pol is opened up at the minor groove side of the primer the amino acid residues of the protein in the minor groove side of dna stabilizes the hydrophobic bpde ring and maintains watson crick base pairing with an incoming dctp for accurate replication . pol also bypasses many other dg - n adducts accurately and efficiently , which includes n-(1-carboxyethyl)-dg and n - furfuryl - dg as well as much bulkier adducts formed by iq and mitomycin c ( discussed in the next section ) . the dg - n - iq adduct was studied in the three different guanines of the nari restriction site ( 5-g1g2cg3cc-3 ) . similar results were obtained with the mitomycin c adducts ( table 1 ) , indicating that they also follow analogous mechanisms . it is noteworthy that these are cyclic adducts with a covalent bond with n1 in addition to n of dg , suggesting that pol one of the most extensively studied dna adduct is dg - c8-aaf ( figure 7 ) , the dg - c8 adduct formed by n - acetyl-2-aminofluorene ( aaf ) , which induces frameshift mutation in bacteria and human cells , but in simian kidney ( cos-7 ) cells , when the adduct is placed in a single stranded plasmid , it causes largely g t mutations . furthermore , this pol -mediated erroneous pathway requires rad18 and ubiquitination of pcna . on the other hand , tls is only partially dependent on pol and rad18 when the adduct is situated at the nari site this indicates that the same adduct may follow different mechanisms for mutagenesis in different sequence contexts . this structural investigation recognized pol s ability to perform error - free replication of dg - c8-aaf , in addition to its propensity to carry out frameshifts . in vitro experiments using yeast pol confirmed that it can extend the g3*:a pair more efficiently than the g3*:c pair , although it is inefficient at nucleotide incorporation opposite dg - c8-iq . it is , therefore , conceivable that pol and pol cooperatively carry out the majority of the error - prone tls of dg - c8-iq , whereas rev1 may play a noncatalytic role in assembling the tls pols . like dg - c8-iq , dg - c83-aba induces g t as the major type of mutations in human cells . crystal structure analysis of dg - c83-aba at the insertion stage of hpol showed that the adduct is wedged at the hydrophobic cleft in the active site in anti conformation stabilized by a hydrogen bond between the c8 amino group and the phosphate , while the 2-deoxyribose adopts c3-endo pucker . it is noteworthy that single - nucleotide incorporation opposite a dg - c83-aba lesion catalyzed by hpol in vitro showed that at short reaction time frames incorporation of dctp is greater than datp but that with longer time incorporation of these two nucleotides becomes comparable . rev1 likewise is important for mutagenesis , as reflected by 60% reduction in mf upon rev1 knockdown , but as with dg - c8-iq , it probably plays a noncatalytic role by physically interacting with the other two y - family pols . the noncatalytic role of rev1 was indicated by its inability to bypass the lesion in vitro . as mentioned earlier on the mechanism of frameshift mutations induced by dg - c8-aaf , many bulky adducts formed at the c8 position of dg , such as dg - c8-iq and dg - c83-aba , rotate the base to syn conformation , which is believed to play a structural role in frameshift mutations observed in bacteria . in contrast , bulky dg - c8 adducts can be accommodated in the pol active site more efficiently . the adduct , as other bulky dg - c8 adducts , exists in syn conformation in a base - displaced intercalated solution structure . replication of dg - c8-ap stalls when in vitro bypass is conducted by the tls pols . of the human tls pols , hpol is most proficient in bypassing it in vitro , but hpol and hpol can incorporate a nucleotide opposite the lesion . however , this structure inhibits further extension , due to a clash with the little finger domain of the enzyme . in contrast , the adduct can maintain syn conformation when datp is inserted , in which the adenine is stacked above the pyrene ring intercalated in the helix . therefore , error - prone replication of dg - c8-ap potentially may occur by two tls pols , with pol being involved in the insertion stage . genetic studies in repair and replication competent cells provide data on the outcome of the damage , and a comparison of these in genetically altered cells ( including knockout or knockdown of specific genes ) has been employed to investigate the role of each tls pol . in vitro experiments using purified pols and accessory proteins elucidate how each pol can deal with the dna damage , whereas structural and computational studies give a more intimate snapshot of the lesion bypass . mechanistic information on replication of the dna lesions is critical to follow the underlying process for the development of cancer , aging , and various other diseases . as yet , more tls work has been done with the pols from prokaryotes and archaea than from eukaryotes . it is certain that this dynamic area of research is still in its early stage and will continue to enrich the field of toxicology with many novel findings .

of the four common nucleosides in dna , oxidation takes place most easily at dg residues , giving rise to a variety of products including 7,8-dihydro-8-oxo-2-deoxyguanosine ( 8-oxo - dg ) ( figure 1 ) . methylating and ethylating agents preferentially react at n7 , but they also alkylate o of dg , and the fraction of alkylation at o increases with harder the n7-methyl - dg ( n7-me - dg ) adduct is unstable , which either depurinates to form an abasic site or undergoes ring opening to generate mefapydg ( figure 1 ) . the unstable dg - n7 adducts formed by the metabolically activated aromatic amines and nitro compounds rearrange to stable dg - c8 adducts , while minor adducts at the n position of dg have also been isolated . however , increasingly it became clear that the mutational signature of a dna lesion is directly related to the identity of the dna polymerase(s ) that bypass it and the mechanism of its nucleotide insertion and extension , though additional factors such as dna sequence context play a role as well . a human cell contains at least 17 different dna polymerases ( pols ) to perform different functions of the cell , which include dna replication of undamaged and damaged dna , replication as part of various dna repair pathways , recombination , telomere maintenance , and other tasks . on the basis of sequence homology , pols have been divided into seven families ( a , b , c , d , x , y , and rt ) , of which c family pols were only found in prokaryotes . the discovery of translesion synthesis ( tls ) dna pols in the 1990s invigorated the area of replication of dna lesions , and since then , numerous articles have been published on the catalytic and noncatalytic roles of these pols in the context of damaged dna replication . like the replicative pols , these pols possess right - handed topology with the active site located in the palm domain , except that the active site is much larger in order to accommodate the dna lesions . unlike the replicative pols , in which the finger and thumb domains ensure correct pairing with the incoming nucleotide , they are shorter and make little interaction with the template and the incoming dntp , thereby reducing the pol s ability to discriminate the accuracy of nucleotide insertion . an important aspect of the y - family pols and pol of the b - family is that they lack the 35 proofreading function , making them error - prone but letting them carry out tls . small dna lesions such as o - me - dg and 8-oxo - dg stall but do not completely stop dna synthesis , whereas most bulky dna lesions , such as the adducts formed by the pahs and aromatic amines , are much stronger replication blocks and require the assistance of tls pols to bypass . in mammalian cells , in addition , two human rad5-related proteins , snf2 histone - linker phd - finger ring - finger helicase ( shprh ) and helicase - like transcription factor ( hltf ) , transform monoubiquitinated pcna into the polyubiquitinated form . even in the presence of pcna , steady - state reactions of calf - thymus dna pol were decreased by a factor of 12 for datp and dctp incorporation opposite 8-oxo - dg . the major dna pols in mammalian cells , pol , pol , and pol extend an 8-oxo - g : a pair more efficiently than the correct 8-oxo - g : c pair.in vitro experiments showed that pol is inefficient in nucleotide insertions opposite 8-oxo - dg , but it can efficiently extend from the nucleotides inserted opposite it by pol . yet , in human cells tls of 8-oxo - dg is largely error - free ( mutation frequency ( mf ) 1% in duplex dna and 420% in single stranded dna ) . several repair systems , including base excision repair and mismatch repair , excise 8-oxo - dg from duplex dna , justifying low mf , but most repair systems are inefficient in 8-oxo - dg repair in single - stranded dna . however , it is remarkable that in the presence of the accessory proteins , human pcna and replication protein a ( rpa ) , correct incorporation of dctp over other dntps opposite 8-oxo - dg increased to 1200-fold by pol . on the basis of these results and additional data from mouse embryonic fibroblasts and human cell lines , it was suggested that the switch from pol involved pol and not pol or pol since mutations by 8-oxo - dg increased considerably in pol knockout or knockdown cells . in the absence of pol , dna pol -interacting protein 2 ( poldip2 , also known as pdip38 ) physically interacts with pol and increases the efficiency of elongation past 8-oxo - dg by pol , suggesting an important role of this protein in pol switch and elongation steps during tls . the 420% mf , which depends on the dna sequence context and the type of assay , in single - stranded dna indicates , however , that in addition to pol , other pols bypass the lesion . in human embryonic kidney ( hek ) 293 t cells , depending on the dna sequence context , we observed 3850% increase in mutations induced by 8-oxo - dg , upon knockdown of pol . while the role of these deletions in human diseases is unknown , inherited variants of mutyh in a family affected by colon cancers show a pattern of high g : c t : a mutations implicating a role of unrepaired 8-oxo - dg lesions in human cancer . for example , the mf of fapydg in the tg*t sequence is significantly higher than when it is located in the tg*a sequence in both simian ( cos-7 ) and human embryonic ( 293 t ) kidney cells . the major difference between the two lesions , however , is that knockdown of pol reduced the level of g t mutations induced by fapydg , in contrast to an increase in mf for 8-oxo - dg . unlike 8-oxo - dg , which adopts syn conformation to pair with adenine , a structural study of the carbocyclic analogue of fapydg by a high fidelity pol ( bst pol i ) showed that the lesion maintains its anti conformation of the glycosidic bond during both error - free and error - prone replication . most biological assays indicate that n7-me - dg is not mutagenic but that its ring - opened derivative mefapydg ( figure 1 ) is mutagenic.in vitro assays showed that the mefapydg is a strong block to the high fidelity replicative dna polymerases at both the insertion and the extension steps . however , cellular experiments in human cells analogous to fapydg have not been performed with mefapydg , and it would certainly be interesting to compare the replicative properties of fapydg with mefapydg using the same approach . o - methyl - dg ( o - me - dg ) is one of the first mutagenic dna lesions identified as a result of dna methylation ( figure 1 ) . using an intrachromosomal probe , 19% g a mutations were detected after replication of a site - specific o - me - dg in chinese hamster ovary cells deficient in the repair enzyme o - alkylguanine - dna alkyltransferase , but in repair proficient cells , mutation frequency dropped to an 1% level . however , pol is strongly blocked one base before o - me - dg.o - me - dg also is a strong but not absolute block of human pol , and even though hpol inserts dttp more efficiently than dctp opposite the lesion , it preferentially extends the correct o - me - g : c pair . steady - state kinetic analysis showed similar efficiencies of insertion of dctp and dttp opposite o - me - dg by hpol and hpol , whereas hpol showed a higher preference for dttp insertion . even though this perspective is focused on dg lesions , it may be pertinent to mention investigations that established a specialized role of pol in efficient and error - free bypass of uv light - induced cis - syn cyclobutane pyrimidine dimers ( cpds ) . the enlarged active site of pol allows it to accommodate even the cisplatin - derived large intrastrand n7pt crystal structure analysis showed that to allow the lesion to fit in its active site , pol goes through a backbone rearrangement to stabilize the lesion and incorporate dctp opposite the two guanines . the potent hepatocarcinogen aflatoxin b1 ( afb1 ) forms two major dna adducts upon metabolic activation of afb1 to afb1 - 8,9-epoxide by the liver cytochrome p450 enzymes ( figure 3 ) . both these adducts are mutagenic in simian kidney ( cos-7 ) cells when the adduct is located in a ttg*aa sequence , but afb1fapy - dg induces 97% mutations compared to 45% mutations by the afb1n7-dg adduct . because of the importance of these adducts in human cancer , additional structural , genetic , and in vitro studies on the two dna adducts in the future would certainly be of significant interest . specifically , for the ( + ) -trans - anti - dg - n - bpde adduct , genetic , in vitro kinetics , and structural studies show that pol performs efficient and accurate tls . for the mutagenic tls , genetic evidence suggests that a non - y family pol inserts a wrong nucleotide ( datp or dttp ) opposite the adduct but that extension is performed cooperatively by pol and rev1 . crystal structure analyses of the ( + ) -trans - anti - dg - n - bpde adduct showed that the active site of pol is opened up at the minor groove side of the primer the amino acid residues of the protein in the minor groove side of dna stabilizes the hydrophobic bpde ring and maintains watson crick base pairing with an incoming dctp for accurate replication . pol also bypasses many other dg - n adducts accurately and efficiently , which includes n-(1-carboxyethyl)-dg and n - furfuryl - dg as well as much bulkier adducts formed by iq and mitomycin c ( discussed in the next section ) . we have recently studied the minor , albeit persistent , dg - n adduct ( figure 5 ) formed by the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline ( iq ) , a heterocyclic aromatic amine formed during high temperature cooking of meat , and two dg - nadducts ( figure 6 ) formed by the antitumor agent , mitomycin c ( mc ) , and its metabolite , 27-diaminomitosene ( 2,7-dam ) . the dg - n - iq adduct was studied in the three different guanines of the nari restriction site ( 5-g1g2cg3cc-3 ) . this suggests that pol is involved in error - free bypass of the dg - n adduct formed by iq , whereas pol , pol , and rev1 cooperatively carried out mutagenic tls . taken together , there seems to be a predictable pattern of error - free and error - prone tls of dg - n adducts by the tls pols . exceptions to this rule , however , are the minor groove adducts -hydroxy-1,n - propano - dg and trans-4-hydroxy-2-nonenal - dg , in which case pol is inefficient in nucleotide insertion opposite the lesion , but it efficiently acts as an extender . it is noteworthy that these are cyclic adducts with a covalent bond with n1 in addition to n of dg , suggesting that pol one of the most extensively studied dna adduct is dg - c8-aaf ( figure 7 ) , the dg - c8 adduct formed by n - acetyl-2-aminofluorene ( aaf ) , which induces frameshift mutation in bacteria and human cells , but in simian kidney ( cos-7 ) cells , when the adduct is placed in a single stranded plasmid , it causes largely g t mutations . however , in a subsequent study , also in cos-7 cells but in duplex dna , at the third guanine of 5-ggg-3 and 5-ggcgcc-3 ( nari site ) , 1 and 2 frameshift mutations , respectively , were detected . the role of pol in bypassing misaligned adducts has been explored , which showed that depending on the base sequence , a cytosine inserted opposite the dg - c8 lesion slips to generate a 1 , 2 , or 3 frameshift intermediate that pol can continue to replicate , in spite of a bulge . in a crystal structure study , however , pol was able to incorporate dctp opposite the dg - c8-aaf adduct , in which tls occured without rotation of the adduct into the anti conformation , and only one hydrogen bond was formed between the lesion and dctp . like the dg - n adducts , the roles of tls dna pols in bypassing the c8-dg adduct ( dg - c8-iq ) ( figure 5 ) formed by iq were explored at the g1- , g2- , or g3-positions of the nari recognition sequence after replication in hek293 t cells . mf of dg - c8-iq was reduced in varying degrees upon sirna knockdown of pol , pol - , pol - , or rev1-knockdown cells ( table 2 ) , indicating that these pols are involved in error - prone synthesis of this adduct . it is , therefore , conceivable that pol and pol cooperatively carry out the majority of the error - prone tls of dg - c8-iq , whereas rev1 may play a noncatalytic role in assembling the tls pols . pol and pol were found to be the major contributors of the mutagenic tls of dg - c83-aba since mf dropped by 70% , when these pols were simultaneously knocked down , although mf actually increased upon knockdown of pol alone . crystal structure analysis of dg - c83-aba at the insertion stage of hpol showed that the adduct is wedged at the hydrophobic cleft in the active site in anti conformation stabilized by a hydrogen bond between the c8 amino group and the phosphate , while the 2-deoxyribose adopts c3-endo pucker . it is noteworthy that single - nucleotide incorporation opposite a dg - c83-aba lesion catalyzed by hpol in vitro showed that at short reaction time frames incorporation of dctp is greater than datp but that with longer time incorporation of these two nucleotides becomes comparable . as mentioned earlier on the mechanism of frameshift mutations induced by dg - c8-aaf , many bulky adducts formed at the c8 position of dg , such as dg - c8-iq and dg - c83-aba , rotate the base to syn conformation , which is believed to play a structural role in frameshift mutations observed in bacteria . an example of pol s potential involvement in dg - c8 adduct bypass is its interaction with dg - c8-ap , the major adduct formed by the environmental carcinogen , 1-nitropyrene ( 1-np ) ( figure 7 ) . another dg - c8 adduct , ( 5s)-8,5-cyclo - dg , a cyclic dna adduct containing a covalent bond between c8 of guanine and 5 c of 2-deoxyribose , was investigated in human cells , which showed that pol , pol , and pol but not pol are involved in tls . genetic studies in repair and replication competent cells provide data on the outcome of the damage , and a comparison of these in genetically altered cells ( including knockout or knockdown of specific genes ) has been employed to investigate the role of each tls pol . these fundamental studies are now paving the way to application of the acquired knowledge toward therapeutic application , as inhibiting the activity of some of the tls pols may enhance the effect of an antitumor agent .

medical schools in many parts of the world have begun to incorporate disaster - related topics into their curricula . in 2003 , the association of american medical colleges ( aamc ) in the united states recommended that bioterrorism education be included in all medical school programs . in germany , federal laws have been enacted requiring that medical students be familiar with disaster medicine principles . analysis of the peer - reviewed literature indicates that few curricula for medical student disaster medicine education have been published . the aamc report on bioterrorism training is more of an outline of educational objectives and activities than a specific curriculum . its target group is us medical students in their last year of training , and builds on their growing knowledge and skills . covering a few aspects of medical disaster management , parrish and colleagues provide a description of a medical school course for 2nd year us students by briefly listing major subject headings . first taught by military experts , this course includes simulation exercises , such as a refugee scenario , and an infectious disease outbreak tracking exercise . in this format , however , operation execution appears to be the priority with less focus on medical care delivery . markenson et al . suggest core competencies and subject areas for terrorism , disaster , and public health emergency preparedness for health - care students based on professional school outlines . referencing a proficiencies list , curricular matrices are proposed , but no steps or samples for implementation are presented . in a course for senior dental students , glotzer and et al . describe core competencies and dentists roles within disaster response efforts focusing on bioterrorism ; participation in a 4-h disaster life support course of the american medical association is also recommended to provide guidance regarding incidents with weapons of mass destruction . because a fully developed and comprehensive medical school disaster medicine curriculum was not available , the german government commissioned the development of a core medical student disaster medicine curriculum that could serve as a standardized template . pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . the final curriculum resulted from these pilots , which were formally developed according to the six - step approach to curriculum development . this included general and targeted needs assessment , definition of goals and objectives , choice of educational methods , and pilot implementation and evaluation . the pilots contents were mainly based on government - funded research projects in which all curricula from german medical schools and postgraduate public health physician courses were evaluated for inclusion of disaster medicine - related themes . the physicians of all ( n = 477 ) german county disaster management agencies and district health authorities were also surveyed for additional input into needed disaster medicine competencies . with a nearly 100% response rate from the physicians surveyed , more than 92% expressed the necessity for better education . this resulted in the development of educational concepts for improving disaster preparedness at the under- and postgraduate level [ 7 , 8 ] . key components compiled here , disaster medicine topics of contemporary relevance , and expertise gained from student emergency medicine training were then combined to generate an independent lecture series . this was finally adapted into a disaster medicine curriculum for a 4th - year german medical school program . students are thus expected to have had 3 years of clinical training in medical school when participating in the course . the implementation experience with the pilots was straightforward : the university dean and the department chair fully supported the idea of providing an introduction to the practices of disaster medicine at the student level . course realization could thus readily be achieved : a core curricular team of physicians was assembled and scheduled all tasks . internal and external professionals with expertise in subject areas were identified and invited to become teaching faculty . after coordination with medical school program planners , facilities for the in - house curriculum units ( rooms , equipment ) were organized with the lecture support staff . for the external units , collaborations with rescue and disaster response agencies were established to permit common exercises . for exchange and drill of non - physician professionals and students , agencies provided equipment , faculty , and their trainees to serve as mock victims for a mass casualty simulation . finally , feedback on the curriculum from all participants was utilized to make alterations and improvements as needed . when the government requested a medical student disaster education plan , we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses : the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine , but to enhance their understanding of what occurs in a disaster , and where the limits and challenges faced by those delivering medical care under austere conditions lie . second , improving knowledge of public health - relevant information on radiological / nuclear , biological , chemical , explosive , and other disaster - related health risks appeared to be important . third , the opportunity to refresh and extend familiarity with basic and advanced life support , and to provide some limited experience with triage decision making was an additional benefit . finally and as a long - term goal , we wished to enhance student interest in pursuing further disaster medicine expertise . it was determined that scientific evidence wherever available should be incorporated and combined with professional expertise to build the content basis of the new curriculum [ 9 , 10 ] . following analysis of international guidelines on disaster response training , peer - reviewed publications [ 35 , 12 , 13 ] , and programs such as the us department of homeland security , federal emergency management agency ( fema ) , and american medical association , learning objectives were fixed . after cross - referencing objectives with quality assurance criteria for medical education [ 1416 ] , topics were structured into a framework represented by subject headings . to identify the content details , a systematic literature search in medline , embase , and the cochrane library with the key terms student , education , training , curriculum , course , competency , disaster , catastrophe , mass casualty , medicine , medical , preparedness , plans , and outcome was conducted . due to the relatively small number of relevant publications , complementary internet searches were performed , and resources from websites such as from the fema program were included . curricular committee consensus on those entities regarded to be most important was required for item inclusion . a blueprint for the core contents was then constructed in the form of modules with the goals as a matrix . because learning and memory benefit from multifaceted , stimulating environments [ 17 , 18 ] , we also decided to diversify the instructive strategies . this led us to include interactive discussions , teaching of problem - solving strategies , reviews of real - life experience , role playing , and exposure to exercises in real - world activities were assigned to suitable content modules , with a minimum of one - third of the curricular time being reserved for hands - on training . these units were designed to be performed with other professionals , such as fire fighters . to evaluate the effectiveness of the curriculum , the results of a knowledge - based , pre - program written examination were compared with post - program examination results . because the course was created to be part of the obligatory 4th - year german medical school program , the testing at course completion also served to fulfil requirements of the german educational system . course evaluations for faculty and students were drafted to be used for further curricular developments [ 6 , 14 ] . as a way to approach administrative authorities ( also outside of germany ) , fig . 1 shows the input and adoption of the final curricular version after review by interdisciplinary professionals and governmental bodies involved in disaster health care . our final curricular proposal was accepted by the leading board of the german civil protection committee . small modifications were then made according to the wishes of the president and members of the german society of disaster medicine and the civil protection committee . the revision was then sent to governmental authorities , whose feedback produced another iteration in the process . the outcome was fine - tuned into formal alignment with global standards for quality improvement in medical education . these included alignment with medical school mission statements , instructional methods of preparing students for life - long , self - directed learning , or the provision of learning environments that resemble real - world scenarios . following final civil protection committee and governmental approval , a directive was provided to all directors of universities with medical schools instructing them to implement the curriculum ( february 2007 ) . 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . the final curriculum resulted from these pilots , which were formally developed according to the six - step approach to curriculum development . this included general and targeted needs assessment , definition of goals and objectives , choice of educational methods , and pilot implementation and evaluation . the pilots contents were mainly based on government - funded research projects in which all curricula from german medical schools and postgraduate public health physician courses were evaluated for inclusion of disaster medicine - related themes . the physicians of all ( n = 477 ) german county disaster management agencies and district health authorities were also surveyed for additional input into needed disaster medicine competencies . with a nearly 100% response rate from the physicians surveyed , more than 92% expressed the necessity for better education . this resulted in the development of educational concepts for improving disaster preparedness at the under- and postgraduate level [ 7 , 8 ] . key components compiled here , disaster medicine topics of contemporary relevance , and expertise gained from student emergency medicine training were then combined to generate an independent lecture series . this was finally adapted into a disaster medicine curriculum for a 4th - year german medical school program . students are thus expected to have had 3 years of clinical training in medical school when participating in the course . the implementation experience with the pilots was straightforward : the university dean and the department chair fully supported the idea of providing an introduction to the practices of disaster medicine at the student level . course realization could thus readily be achieved : a core curricular team of physicians was assembled and scheduled all tasks . internal and external professionals with expertise in subject areas were identified and invited to become teaching faculty . after coordination with medical school program planners , facilities for the in - house curriculum units ( rooms , equipment ) were organized with the lecture support staff . for the external units , collaborations with rescue and disaster response agencies were established to permit common exercises . for exchange and drill of non - physician professionals and students , agencies provided equipment , faculty , and their trainees to serve as mock victims for a mass casualty simulation . finally , feedback on the curriculum from all participants was utilized to make alterations and improvements as needed . when the government requested a medical student disaster education plan , we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses : the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine , but to enhance their understanding of what occurs in a disaster , and where the limits and challenges faced by those delivering medical care under austere conditions lie . second , improving knowledge of public health - relevant information on radiological / nuclear , biological , chemical , explosive , and other disaster - related health risks appeared to be important . third , the opportunity to refresh and extend familiarity with basic and advanced life support , and to provide some limited experience with triage decision making was an additional benefit . finally and as a long - term goal , we wished to enhance student interest in pursuing further disaster medicine expertise . it was determined that scientific evidence wherever available should be incorporated and combined with professional expertise to build the content basis of the new curriculum [ 9 , 10 ] . following analysis of international guidelines on disaster response training , peer - reviewed publications [ 35 , 12 , 13 ] , and programs such as the us department of homeland security , federal emergency management agency ( fema ) , and american medical association , learning objectives were fixed . after cross - referencing objectives with quality assurance criteria for medical education [ 1416 ] , topics were structured into a framework represented by subject headings . to identify the content details , a systematic literature search in medline , embase , and the cochrane library with the key terms student , education , training , curriculum , course , competency , disaster , catastrophe , mass casualty , medicine , medical , preparedness , plans , and outcome was conducted . due to the relatively small number of relevant publications , complementary internet searches were performed , and resources from websites such as from the fema program were included . curricular committee consensus on those entities regarded to be most important was required for item inclusion . a blueprint for the core contents was then constructed in the form of modules with the goals as a matrix . because learning and memory benefit from multifaceted , stimulating environments [ 17 , 18 ] , we also decided to diversify the instructive strategies . this led us to include interactive discussions , teaching of problem - solving strategies , reviews of real - life experience , role playing , and exposure to exercises in real - world activities were assigned to suitable content modules , with a minimum of one - third of the curricular time being reserved for hands - on training . these units were designed to be performed with other professionals , such as fire fighters . to evaluate the effectiveness of the curriculum , the results of a knowledge - based , pre - program written examination were compared with post - program examination results . because the course was created to be part of the obligatory 4th - year german medical school program , the testing at course completion also served to fulfil requirements of the german educational system . course evaluations for faculty and students were drafted to be used for further curricular developments [ 6 , 14 ] . as a way to approach administrative authorities ( also outside of germany ) , fig . 1 shows the input and adoption of the final curricular version after review by interdisciplinary professionals and governmental bodies involved in disaster health care . our final curricular proposal was accepted by the leading board of the german civil protection committee . small modifications were then made according to the wishes of the president and members of the german society of disaster medicine and the civil protection committee . the revision was then sent to governmental authorities , whose feedback produced another iteration in the process . the outcome was fine - tuned into formal alignment with global standards for quality improvement in medical education . these included alignment with medical school mission statements , instructional methods of preparing students for life - long , self - directed learning , or the provision of learning environments that resemble real - world scenarios . following final civil protection committee and governmental approval , a directive was provided to all directors of universities with medical schools instructing them to implement the curriculum ( february 2007 ) . 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . the final curriculum resulted from these pilots , which were formally developed according to the six - step approach to curriculum development . this included general and targeted needs assessment , definition of goals and objectives , choice of educational methods , and pilot implementation and evaluation . the pilots contents were mainly based on government - funded research projects in which all curricula from german medical schools and postgraduate public health physician courses were evaluated for inclusion of disaster medicine - related themes . the physicians of all ( n = 477 ) german county disaster management agencies and district health authorities were also surveyed for additional input into needed disaster medicine competencies . with a nearly 100% response rate from the physicians surveyed , more than 92% expressed the necessity for better education . this resulted in the development of educational concepts for improving disaster preparedness at the under- and postgraduate level [ 7 , 8 ] . key components compiled here , disaster medicine topics of contemporary relevance , and expertise gained from student emergency medicine training were then combined to generate an independent lecture series . this was finally adapted into a disaster medicine curriculum for a 4th - year german medical school program . students are thus expected to have had 3 years of clinical training in medical school when participating in the course . the implementation experience with the pilots was straightforward : the university dean and the department chair fully supported the idea of providing an introduction to the practices of disaster medicine at the student level . course realization could thus readily be achieved : a core curricular team of physicians was assembled and scheduled all tasks . internal and external professionals with expertise in subject areas were identified and invited to become teaching faculty . after coordination with medical school program planners , facilities for the in - house curriculum units ( rooms , equipment ) were organized with the lecture support staff . for the external units , collaborations with rescue and disaster response agencies were established to permit common exercises . for exchange and drill of non - physician professionals and students , agencies provided equipment , faculty , and their trainees to serve as mock victims for a mass casualty simulation . finally , feedback on the curriculum from all participants was utilized to make alterations and improvements as needed . when the government requested a medical student disaster education plan , we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses : the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine , but to enhance their understanding of what occurs in a disaster , and where the limits and challenges faced by those delivering medical care under austere conditions lie . second , improving knowledge of public health - relevant information on radiological / nuclear , biological , chemical , explosive , and other disaster - related health risks appeared to be important . third , the opportunity to refresh and extend familiarity with basic and advanced life support , and to provide some limited experience with triage decision making was an additional benefit . finally and as a long - term goal , we wished to enhance student interest in pursuing further disaster medicine expertise . it was determined that scientific evidence wherever available should be incorporated and combined with professional expertise to build the content basis of the new curriculum [ 9 , 10 ] . following analysis of international guidelines on disaster response training , peer - reviewed publications [ 35 , 12 , 13 ] , and programs such as the us department of homeland security , federal emergency management agency ( fema ) , and american medical association , after cross - referencing objectives with quality assurance criteria for medical education [ 1416 ] , topics were structured into a framework represented by subject headings . to identify the content details , a systematic literature search in medline , embase , and the cochrane library with the key terms student , education , training , curriculum , course , competency , disaster , catastrophe , mass casualty , medicine , medical , preparedness , plans , and outcome was conducted . due to the relatively small number of relevant publications , complementary internet searches were performed , and resources from websites such as from the fema program were included . curricular committee consensus on those entities regarded to be most important was required for item inclusion . a blueprint for the core contents was then constructed in the form of modules with the goals as a matrix . because learning and memory benefit from multifaceted , stimulating environments [ 17 , 18 ] , we also decided to diversify the instructive strategies . this led us to include interactive discussions , teaching of problem - solving strategies , reviews of real - life experience , role playing , and exposure to exercises in real - world environments . activities were assigned to suitable content modules , with a minimum of one - third of the curricular time being reserved for hands - on training . these units were designed to be performed with other professionals , such as fire fighters . to evaluate the effectiveness of the curriculum , the results of a knowledge - based , pre - program written examination were compared with post - program examination results . because the course was created to be part of the obligatory 4th - year german medical school program , the testing at course completion also served to fulfil requirements of the german educational system . course evaluations for faculty and students were drafted to be used for further curricular developments [ 6 , 14 ] . as a way to approach administrative authorities ( also outside of germany ) , fig . 1 shows the input and adoption of the final curricular version after review by interdisciplinary professionals and governmental bodies involved in disaster health care . our final curricular proposal was accepted by the leading board of the german civil protection committee . small modifications were then made according to the wishes of the president and members of the german society of disaster medicine and the civil protection committee . the revision was then sent to governmental authorities , whose feedback produced another iteration in the process . the outcome was fine - tuned into formal alignment with global standards for quality improvement in medical education . these included alignment with medical school mission statements , instructional methods of preparing students for life - long , self - directed learning , or the provision of learning environments that resemble real - world scenarios . following final civil protection committee and governmental approval , a directive was provided to all directors of universities with medical schools instructing them to implement the curriculum ( february 2007 ) . 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies the creation of the final curriculum resulted in a course of 14 modules with each unit requiring approximately 2 h time : modules 13 ( table 1a ) introduce the essentials of disaster medicine terminology , disaster assistance and law , management , and multidisciplinary coordination and communication systems . as key competencies , the tasks of assistance agencies , functional response roles , scopes of responsibility , lines of authority , logistical needs for mass casualties , and triage methods are taught . students are familiarized with operational issues of disaster response strategies including problem - based learning discussions ( pbld ) . table 1modules 114modules , learning activityeducational goalcore contentsa medical student disaster medicine curriculum , modules 13module 1 , lecture and pbldintroduction to disaster medicine , terminologyto learn definitions of disaster medicine and to develop an understanding for general disaster managementglossary and common disaster medicine definitions , differences between disaster and emergency medicine , different phases of disaster managementdisaster assistanceprinciples of disaster assistancedisaster assistance organization , assistance agencies and structuretypologyto consider the heterogeneity of disastersnatural and technological disasters , terrorism , man - made disasters , civilian disorders , environmental and other threatslaws and regulationsto understand the legal environment and regulations for civil protection and disaster preparednessregulative and administrative issues for civil disaster protection , warfare disaster protection laws , rescue service and hospital laws , governmental resources , and authoritiesmodule 2 , lecture and pblddisaster medical managementto realize the architecture and organizational management necessary for coping with mass casualty incidents and large - scale acutely ill patients in a coordinated waymass casualty disposition , treatment area , transport issuesfunctional operationstasks of rapid - intervention - units ( sanitary / psychosocial care)incident command systemsdisaster contingency plans command and control structures , functional operations centercoordination structurescoordination , integration , and cooperation of multi - agency rescue and assistance responsefunctional rolesfunctional response roles , e.g. , lead emergency physician , organizational leader , and technical command postinformation managementcommunication , coordinationmodule 3 , lecture and pbldspecific disaster medicineto develop skills in principles of tactically managing mass casualties and large numbers of patients suffering , e.g. , from combined conventional trauma and thermal injuriespatient assessment and triage : triage levels , tags , registration ; primary emergency care , multi - tasking and operational management phases ( e.g. , patient collection , treatment area , transport)tactic disaster medical managementlogistical requirements for care of burn - injuries , mine blast , and missile - hit victims ( including high - speed bullet injuries ) , mass trauma managementb medical student disaster medicine curriculum , modules 48module 4 , pbld and interactive reviewhospital preparedness and disaster management planningto follow orders and principles of hospital alarm and evacuation planshospital disaster lawshospital alarm planshospital preparedness plans for- management of external disasters with mass casualties suffering from multiple injuries , intoxication , infections , and/or radioactive contamination- management of in - house disasters with fire incidents and hospital evacuationmodule 5 , pbld and interactive reviewpresentation of past disasters and review of assistance experiencesto evaluate and understand feasibility issues of providing medical support and health care in the field and under disaster conditions based on experience from worldwide disaster assistance operationspresentation of past disasters and disaster assistance experience gained in the field , e.g. , from lead emergency physicians , operations in earthquake assistance , explosions , highly contagious infectious diseases , repatriation flights , and with the german federal armed forces medical corps in world crisis regionsmodule 6 , experiential trainingpreclinical and clinical triage exerciseto perform triage decisions in reality simulationtriage training exercise : real or virtual scenario simulation rescue exercise , e.g. , explosion with mass casualties and blast , mechanical , and thermal injuries of all triage levelsmodule 7 , experiential trainingevacuation exerciseto apply operational principles and steps of action for evacuation procedurescommand post exercise , real or virtual : e.g. , evacuation organization and evacuation of a hospital , school , part of town , etc.module 8 , lecture and pbldlife - saving disaster emergency medical careto learn concepts of life - saving emergency disaster medical careunder disaster conditions : provision of life - saving emergency medical care to adults and children , e.g. , shock therapy , pain treatment , and sedationc medical student disaster medicine curriculum , modules 911module 9 , lecture and pbldspecific disaster emergency medical care for various situations including bioterrorism incidentsto get to know principles of specific disaster medical caredisaster emergency medical first aid , specific measuressurgical and medical treatments of burn and thermal injuries and illnesses from explosive , warfare and biological agentsepidemiology and approaches to terrorist attacks , weapons , and highly contagious infectious diseases , sentinel casesmodule 10 , lecture and pbldradiological and nuclear threats , accidents with radioactive material , radiation illness and syndrome , decontaminationto learn principles and basic medical care for management of incidents with radiologic / nuclear agents and contaminated victimsspecific dangers of radiological / nuclear agents , associated illnesses and radiation syndromeself protection , protection and detection equipment , special intervention unitsfirst aid medical treatment , isolation and radioactive decontamination , decontamination operations in case of mass trauma combined with contamination injuriesexperiential trainingdecontamination after radiation exposureto be exposed to practical aspects and procedures of decontaminationdecontamination exercise or decontamination demonstration , e.g. , in nuclear power plant or with the fire brigadesmodule 11 , lecture and pbldchemical and toxicological threats from hazardous materials and goods , transport risks , acute poisoning and toxic syndromesto get to know principles of medical countermeasures for management of incidents with dangerous chemical substances , hazardous materials and goodsidentification and risk assessment of hazardous materials , chemicals and goods , and associated toxic syndromesmanagement of acute intoxications and poisonings , threats from specific poisonsself protection , precautionspoisoning epidemiology , risk assessment for mass intoxicationsfirst aid medical treatment , e.g. , enhanced elimination of toxins , use of antidotes , adjunctive services , e.g. , poison emergency centers and toxicology laboratoriesexperiential trainingdecontamination after chemical poisoningto experience exposure to decontamination procedurestriage in case of mass casualties with toxic syndromesdecontamination measures and exercised medical student disaster medicine curriculum , modules 1214module 12 , lecture , pbld , and interactive reviewethics and professionalismto develop familiarity with ethical codes and the duty of care relevant to disaster conditionsgeneva convention and amended protocols , ethical codes of conduct , humanitarian imperatives , social , moral , and ethical challenges of disastersquality assuranceto understand quality improvement efforts and risk management programs for disaster medical responsequality control performance indicators , incident monitoring , tools for risk and critical event assessment , structured improvement approachesappropriate documentationmodule 13 , pbld and interactive reviewto comprehend concepts of psychic stress responsecase presentations for identification of critical incident stress reactions and review of therapeutic interventionspsychosocial careto learn techniques to deal with psychic reactions caused by exposure to disaster scenariostreatment approaches to acute and delayed critical incident stress reactions , acute and chronic stress syndromes , post - traumatic stress disordersto recognize need for help and to initiate psychosocial supportstructure and tasks of psychosocial emergency intervention unitsoperational strategies of psychosocial treatmentmodule 14 , course completionclosing examinationto demonstrate gain of knowledge and skillsstudent final examination , oral and/or written testevaluation of educational successto assess educational value of coursecomparison of pre - program versus post - program student test resultslearner and educator assessment of courseto maintain continuous curriculum development and improvement in course qualitystudent and faculty summative and formative course evaluation modules 4 and 5 ( table 1b ) discuss hospital alarm and preparedness plans , with a focus on in - house and external incidents requiring high surge capacity . as core competencies , notification and mobilization orders are taught , and concepts of an institutional emergency plan are incorporated . interactive review of past major community - threatening events and experiences from worldwide disaster and relief operations are utilized to built recognition of disaster health - care and medical assistance feasibility issues . students learn the risks and limitations of relief operations , while focusing on the challenge of implementing care in the field . modules 6 and 7 ( table 1b ) address knowledge and skills for triage and evacuation through participation in exercises . students conduct triage and assume functional roles in real or virtual experiential training units . to provide a hands - on experience and to increase student understanding of what occurs in a mass casualty management situation , we simulate a triage exercise in a small - group format ( four students / two emergency physicians ) as shown in fig . 2 . in brief , our interdisciplinary executed scenario is conducted at connected hallways and the hospital grounds in the setting of poor illumination , smoke , and loud noises . well - trained , life - like moulage volunteers simulate casualties presenting with various disorders , from superficial injuries to moribund conditions , medical illnesses , and emotional trauma . to maximize the accuracy of the scenario , simulation patients have 20% severe , 20% moderate , and 40% light injuries , as is most typically seen in real - world events . in the multi - task exposure , students are first subjected to an instructive exercise , regarding role assignment , scenario description , protection and triage directives , and radio communication orders . they are not provided with any patient information prior to interaction with the mock victims , but are made aware of a constrained timeframe for the tasks . to be authentic , students must put on full protective gear , helmets , and gloves . the hands - on exercise is then run in a step - by - step manner , starting with patient search : after they are located , patients must be checked for vital functions and examined . following differential diagnosis , they must be triaged , and treatment goals and management priorities must be defined . the compressed timeframe exercise ends with a radio announcement . as a debriefing exercise , students receive performance assessment with respect to future roles as physician , collaborator , and communicator in a contextual feed - back . 2algorithm of student immersion in mass casualty rescue simulation exercise algorithm of student immersion in mass casualty rescue simulation exercise modules 8 and 9 ( table 1b and c ) teach life - saving and specific emergency medical care for various disaster situations , containing first aid and specific approaches to injuries , burns , explosions , blasts , and illnesses from warfare and biological agents . emphasising terrorist attacks and highly contagious infectious diseases , core competencies for care of explosive , warfare , and biological incidents are included . minimally acceptable treatment and limitations for patient - tailored therapy in case of mass casualties are discussed . modules 10 and 11 ( table 1c ) teach the dangers and management of radiological / nuclear and chemical hazardous materials . options for protection , first aid , decontamination , enhanced elimination , and antidotes are reviewed . students learn how victims with combined trauma and toxic injury are triaged . at a nuclear power plant or fire department , the design and state of preparedness of such facilities are evaluated , and equipment is demonstrated . to provide a practical experience of a decontamination procedure and to get students personally involved in how it feels to work in protective gear , we offer a combined unit of didactic , interactive , and hands - on training at the fire department ( fig . 3 ) . after incident presentation , a lecture on radiological / nuclear and chemical hazardous substances is given . technical equipment , mobile units , protective gear , and contamination detection devices are demonstrated . use of protective gear is explained and a decontamination suite described . as hands - on exercises , students put on full protective gear and gas masks . being both protected and hampered by this gear , they must attempt to manage the airway , intubate , and ventilate mannequins . this hands - on program serves as a real - world simulation experience , reproduced by ( 1 ) heat load under the multilayer over - garment , ( 2 ) enhanced personal respiratory efforts , ( 3 ) restricted visual fields because of the gas mask , and ( 4 ) interference with manual dexterity by the chemical protective gloves . after debriefing , the module ends with an interactive review and analysis of past incidents . 3flowchart of the module with the fire fighters or at a nuclear power plant focussed on education of incident management with radioactive and hazardous chemical substances flowchart of the module with the fire fighters or at a nuclear power plant focussed on education of incident management with radioactive and hazardous chemical substances modules 12 and 13 ( table 1d ) deal with challenges of ethical , professional , psychosocial , and quality control criteria of medical disaster management themes . students learn critical aspects of acute and delayed stress disorders ( anxiety and grief ) , and the psychosocial resources , such as crisis intervention units , available to provide specialised assistance . to understand quality improvement efforts , students gain familiarity with assessment tools and incident monitoring to evaluate functional components of disaster response systems . module 14 ( table 1d ) is designed to assess the educational success of the course . students demonstrate the acquired knowledge and attitudes in an oral and/or written examination . according to the german regulations , each medical school has the freedom to design the format of the examinations chosen for their student evaluations . to our knowledge , this curriculum is one of the first publications of a comprehensive medical student disaster medicine curriculum that covers major contemporary aspects of basic disaster medicine management . the developed course offers a cross - professional design in a readily adaptable structure , is focussed on actual scenarios , and has been aligned with quality criteria for improved teaching approaches [ 14 , 16 , 19 ] . at a time when medical schools in numerous countries are struggling to find a place for disaster medicine in their programs , this educational topic is worthy of research and discussion . our work may be used as an educational resource , with the opportunity to adapt the basic template to various locations , threats , and systems . an introduction to care principles will be of value to preparedness in any locale , even though disaster response plans and conditions may differ among countries , or states and cities of a single country . the curriculum also provides resources for endeavors of organizations such as the section in disaster medicine of the european society for emergency medicine , which has as a current goal the development of a standard european disaster medicine curriculum for medical school undergraduates . the analysis of the implementation process and curricular success has been pre - defined for a period of 5 years , and was begun 3 years ago . however , all medical schools that have implemented the curriculum have so far reported that they now teach disaster medicine components with a scope and depth much broader than within emergency medicine , pointing out the additional value for disaster preparedness and disaster medical management . seven of them report that they have implemented a fully independent course , and five of the medical schools base their teachings on an educational program that is in complete conformity with the curriculum developed . because derivation and details for medical student competency definitions differ widely [ 3 , 4 , 6 , 11 ] , we used a consensus - building approach for outlining competencies and thus the framework for the educational goals of this curriculum . requests of the education committee working group of the world association for disaster and emergency medicine , disaster medicine - related competencies as indicated in emergency medicine or radiology curricula , courses recommended by professional organizations , the literature and expert opinion were addressed as far as available [ 7 , 2123 ] . deriving subject matter from thus pre - defined goals provided the advantage of readily defining the main targets for core contents . the competency structured design also permits flexibility in addressing a multiplicity of disaster situations , including floods , hurricanes , earthquakes , and surveillance - related health problems , as well as care for vulnerable populations such as children and hospitalised patients . educational goals were weighted in terms of time allocation to assist medical school planners operating with more limited discretionary curricular time . in our opinion and circumstances , a 14-module course composed of 2-h units provides adequate student education and mock disaster exposure in a reasonable period . some themes important for disaster preparedness are also addressed in other medical school programs , such as in medical microbiology , toxicology , and environmental medicine . the topics we targeted for this curriculum , however , do not overlap with other student programs . emergency medicine curricula for medical students , when present in medical school curricula at all , do not typically focus on disaster medicine or disaster response . introduction to emergency medicine focuses on maximum possible , individual patient - tailored care [ 9 , 21 , 23 ] and if included this is intended to address disaster medicine conditions in which extraordinary treatment demands exceed resources , and where incidents of great magnitude requiring external assistance for management may reach epidemiological dimensions . when planning the experiential units , we attempted to ensure that costs were minimized : for cost containment , selection of a teaching cadre with background in emergency or disaster medicine who collaborates with local disaster response agencies is most effective . additional teachers can join in , and after receiving instructor training , faculty development may be supported through ongoing participation in areas of disaster medicine interest . exercise scenarios may not be available in all medical school environments , but can , in most cases , be established through a joint training / learning venture with existing response agencies . because agencies are obliged to carry out simulation exercises and drill their own staff , curriculum units can be performed with mutual benefit . exercise costs can thus be kept low , and mainly derive from transport to external locations . for an estimation of costs , table 2 provides activity - based descriptions for organizing and implementing the curriculum . table 2task assignment for estimation of organizational burden and curricular implementation costscurricular activitymission descriptionprogram managementdevelopment of a local organizational structure responsible for curricular activities : team recruitment , appointment of managers and departmental staff in - charge , task assignmentidentification of internal and external experts and invitation as teaching cadreestablishment of collaboration with local disaster response agencies and planning of common external units , exercises , and simulationsteaching and preceptor assignment for internal and external unitsinstitution of curriculum within medical school program and coordination of units with program plannersorganization of facilities and equipment for in - house units with university s lecture support stafforganization of transport to external unitsmanagement of administrative and reporting issuesif necessary : establishment of financial recording , billing , and reimbursement systemfaculty development / continuous professional growthfuture faculty development through involvement in existing teaching activities , plans , and team meetingsfaculty instructor trainingoptional : professional growth through participation in related internal and external developmental programs for university educators and disaster medicine preparednessquality assurance / continuous curricular developmentdevelopment of data collection systemanalysis of ongoing evaluations from learners and educatorsuse of course assessments and experiences for orientation , end - of - course team meetings , and future progressstudent supportprovision of curricular schedule and learning objectivesprovision of lecture compilation ( syllabus or cd ) as course content workbook and base for self - directed learningdevelopment of data collection system and analysis of student examination datadevelopment of reporting system for examination gradesmaintenance of partnerships / collaborationscontinuous encouragement and support for internal and external partnerships and collaborationsrunning of administrative / overhead expendituresif necessary : management of direct and indirect expenditures , such as telecommunication costsother pursuitsdesigning of plans for course revisions or curricular enhancements , such as integration of e - learning methods task assignment for estimation of organizational burden and curricular implementation costs there are several limitations to this work . . nevertheless , educational efforts for important medical topic areas have to be started at some point in medical school , and this can be evaluated over time . of course , the provision of one - time disaster medicine training will only allow for a passing familiarity with terms and concepts . to retain competencies , it will be necessary to refresh and assess knowledge and skills periodically , and we are planning to implement educational refresher courses in our german national program . moreover at present , we can not comment on how well the curriculum performed , and what the overall success of the course will turn out to be . moreover , exposing students to mock disaster scenarios and simulation exercises is far from real - world conditions . certainly , students will not intubate patients in full protective gear in real disasters and will of course not be included in first rescue response teams in compromised environments . nevertheless , based on our experience as university educators , they benefit from the knowledge and skill - building hands - on exercises that may serve as a basic training for future medical practice in all kinds of emergency situations . our students also receive clear instruction that they are not trained search and rescue personnel . for our course , we thus try to prevent the possibility that simulation - based medical education experiences could cause student over - confidence and misjudgement of competency . in its first design , our curriculum consists of traditional , face - to - face , instructor - student interactions and hands - on experiences , and does not use electronic ( e- ) learning methods . e - technology holds the promise for distance education of disaster response concepts and virtual reality simulations . because blended curricular formats integrating e - methods may enhance inter - professional exchange without compromising pedagogy , they may be included in future curricular versions . a curriculum is provided for medical student disaster medicine education at an advanced level of medical school training , along with information regarding the process involved in creating such a program at a national level . we wish to emphasize that the curriculum has been designed for medical students in a particular system and that there are various concepts for basic disaster medicine education . due to its comprehensive , interdisciplinary nature , instructive design , and flexible structure , however , the curriculum can benefit other health - care professional systems by serving as a model curriculum , and facilitating refinement and testing of other existing , but perhaps as yet unpublished models .

backgrounddisaster medicine education is an enormous challenge , but indispensable for disaster preparedness.aimswe aimed to develop and implement a disaster medicine curriculum for medical student education that can serve as a peer - reviewed , structured educational guide and resource . additionally , the process of designing , approving and implementing such a curriculum is presented.methodsthe six - step approach to curriculum development for medical education was used as a formal process instrument . recognized experts from professional and governmental bodies involved in disaster health care provided input using disaster - related physician training programs , scientific evidence if available , proposals for education by international disaster medicine organizations and their expertise as the basis for content development.resultsthe final course consisted of 14 modules composed of 2-h units . the concepts of disaster medicine , including response , medical assistance , law , command , coordination , communication , and mass casualty management , are introduced . hospital preparedness plans and experiences from worldwide disaster assistance are reviewed . life - saving emergency and limited individual treatment under disaster conditions are discussed . specifics of initial management of explosive , war - related , radiological / nuclear , chemical , and biological incidents emphasizing infectious diseases and terrorist attacks are presented . an evacuation exercise is completed , and a mass casualty triage is simulated in collaboration with local disaster response agencies . decontamination procedures are demonstrated at a nuclear power plant or the local fire department , and personal decontamination practices are exercised . mannequin resuscitation is practiced while personal protective equipment is utilized . an interactive review of professional ethics , stress disorders , psychosocial interventions , and quality improvement efforts complete the training.conclusionsthe curriculum offers medical disaster education in a reasonable time frame , interdisciplinary format , and multi - experiential course . it can serve as a template for basic medical student disaster education . because of its comprehensive but flexible structure , it should also be helpful for other health - care professional student disaster education programs .

Introduction Methods and curriculum development The dynamic process of curriculum development occurred in several stages Initial curriculum development and pilot testing Revision of pilot curriculum and proposal for the government Adoption of final curriculum Results, course content and implementation Discussion Conclusion

analysis of the peer - reviewed literature indicates that few curricula for medical student disaster medicine education have been published . because a fully developed and comprehensive medical school disaster medicine curriculum was not available , the german government commissioned the development of a core medical student disaster medicine curriculum that could serve as a standardized template . the final curriculum resulted from these pilots , which were formally developed according to the six - step approach to curriculum development . this was finally adapted into a disaster medicine curriculum for a 4th - year german medical school program . for exchange and drill of non - physician professionals and students , agencies provided equipment , faculty , and their trainees to serve as mock victims for a mass casualty simulation . when the government requested a medical student disaster education plan , we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses : the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine , but to enhance their understanding of what occurs in a disaster , and where the limits and challenges faced by those delivering medical care under austere conditions lie . second , improving knowledge of public health - relevant information on radiological / nuclear , biological , chemical , explosive , and other disaster - related health risks appeared to be important . following analysis of international guidelines on disaster response training , peer - reviewed publications [ 35 , 12 , 13 ] , and programs such as the us department of homeland security , federal emergency management agency ( fema ) , and american medical association , learning objectives were fixed . to identify the content details , a systematic literature search in medline , embase , and the cochrane library with the key terms student , education , training , curriculum , course , competency , disaster , catastrophe , mass casualty , medicine , medical , preparedness , plans , and outcome was conducted . 1 shows the input and adoption of the final curricular version after review by interdisciplinary professionals and governmental bodies involved in disaster health care . 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . the final curriculum resulted from these pilots , which were formally developed according to the six - step approach to curriculum development . the pilots contents were mainly based on government - funded research projects in which all curricula from german medical schools and postgraduate public health physician courses were evaluated for inclusion of disaster medicine - related themes . this was finally adapted into a disaster medicine curriculum for a 4th - year german medical school program . for exchange and drill of non - physician professionals and students , agencies provided equipment , faculty , and their trainees to serve as mock victims for a mass casualty simulation . when the government requested a medical student disaster education plan , we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses : the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine , but to enhance their understanding of what occurs in a disaster , and where the limits and challenges faced by those delivering medical care under austere conditions lie . second , improving knowledge of public health - relevant information on radiological / nuclear , biological , chemical , explosive , and other disaster - related health risks appeared to be important . following analysis of international guidelines on disaster response training , peer - reviewed publications [ 35 , 12 , 13 ] , and programs such as the us department of homeland security , federal emergency management agency ( fema ) , and american medical association , learning objectives were fixed . to identify the content details , a systematic literature search in medline , embase , and the cochrane library with the key terms student , education , training , curriculum , course , competency , disaster , catastrophe , mass casualty , medicine , medical , preparedness , plans , and outcome was conducted . 1 shows the input and adoption of the final curricular version after review by interdisciplinary professionals and governmental bodies involved in disaster health care . 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . the final curriculum resulted from these pilots , which were formally developed according to the six - step approach to curriculum development . the pilots contents were mainly based on government - funded research projects in which all curricula from german medical schools and postgraduate public health physician courses were evaluated for inclusion of disaster medicine - related themes . this was finally adapted into a disaster medicine curriculum for a 4th - year german medical school program . for exchange and drill of non - physician professionals and students , agencies provided equipment , faculty , and their trainees to serve as mock victims for a mass casualty simulation . when the government requested a medical student disaster education plan , we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses : the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine , but to enhance their understanding of what occurs in a disaster , and where the limits and challenges faced by those delivering medical care under austere conditions lie . second , improving knowledge of public health - relevant information on radiological / nuclear , biological , chemical , explosive , and other disaster - related health risks appeared to be important . following analysis of international guidelines on disaster response training , peer - reviewed publications [ 35 , 12 , 13 ] , and programs such as the us department of homeland security , federal emergency management agency ( fema ) , and american medical association , after cross - referencing objectives with quality assurance criteria for medical education [ 1416 ] , topics were structured into a framework represented by subject headings . to identify the content details , a systematic literature search in medline , embase , and the cochrane library with the key terms student , education , training , curriculum , course , competency , disaster , catastrophe , mass casualty , medicine , medical , preparedness , plans , and outcome was conducted . 1 shows the input and adoption of the final curricular version after review by interdisciplinary professionals and governmental bodies involved in disaster health care . 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies the creation of the final curriculum resulted in a course of 14 modules with each unit requiring approximately 2 h time : modules 13 ( table 1a ) introduce the essentials of disaster medicine terminology , disaster assistance and law , management , and multidisciplinary coordination and communication systems . table 1modules 114modules , learning activityeducational goalcore contentsa medical student disaster medicine curriculum , modules 13module 1 , lecture and pbldintroduction to disaster medicine , terminologyto learn definitions of disaster medicine and to develop an understanding for general disaster managementglossary and common disaster medicine definitions , differences between disaster and emergency medicine , different phases of disaster managementdisaster assistanceprinciples of disaster assistancedisaster assistance organization , assistance agencies and structuretypologyto consider the heterogeneity of disastersnatural and technological disasters , terrorism , man - made disasters , civilian disorders , environmental and other threatslaws and regulationsto understand the legal environment and regulations for civil protection and disaster preparednessregulative and administrative issues for civil disaster protection , warfare disaster protection laws , rescue service and hospital laws , governmental resources , and authoritiesmodule 2 , lecture and pblddisaster medical managementto realize the architecture and organizational management necessary for coping with mass casualty incidents and large - scale acutely ill patients in a coordinated waymass casualty disposition , treatment area , transport issuesfunctional operationstasks of rapid - intervention - units ( sanitary / psychosocial care)incident command systemsdisaster contingency plans command and control structures , functional operations centercoordination structurescoordination , integration , and cooperation of multi - agency rescue and assistance responsefunctional rolesfunctional response roles , e.g. , patient collection , treatment area , transport)tactic disaster medical managementlogistical requirements for care of burn - injuries , mine blast , and missile - hit victims ( including high - speed bullet injuries ) , mass trauma managementb medical student disaster medicine curriculum , modules 48module 4 , pbld and interactive reviewhospital preparedness and disaster management planningto follow orders and principles of hospital alarm and evacuation planshospital disaster lawshospital alarm planshospital preparedness plans for- management of external disasters with mass casualties suffering from multiple injuries , intoxication , infections , and/or radioactive contamination- management of in - house disasters with fire incidents and hospital evacuationmodule 5 , pbld and interactive reviewpresentation of past disasters and review of assistance experiencesto evaluate and understand feasibility issues of providing medical support and health care in the field and under disaster conditions based on experience from worldwide disaster assistance operationspresentation of past disasters and disaster assistance experience gained in the field , e.g. , from lead emergency physicians , operations in earthquake assistance , explosions , highly contagious infectious diseases , repatriation flights , and with the german federal armed forces medical corps in world crisis regionsmodule 6 , experiential trainingpreclinical and clinical triage exerciseto perform triage decisions in reality simulationtriage training exercise : real or virtual scenario simulation rescue exercise , e.g. , evacuation organization and evacuation of a hospital , school , part of town , etc.module 8 , lecture and pbldlife - saving disaster emergency medical careto learn concepts of life - saving emergency disaster medical careunder disaster conditions : provision of life - saving emergency medical care to adults and children , e.g. , shock therapy , pain treatment , and sedationc medical student disaster medicine curriculum , modules 911module 9 , lecture and pbldspecific disaster emergency medical care for various situations including bioterrorism incidentsto get to know principles of specific disaster medical caredisaster emergency medical first aid , specific measuressurgical and medical treatments of burn and thermal injuries and illnesses from explosive , warfare and biological agentsepidemiology and approaches to terrorist attacks , weapons , and highly contagious infectious diseases , sentinel casesmodule 10 , lecture and pbldradiological and nuclear threats , accidents with radioactive material , radiation illness and syndrome , decontaminationto learn principles and basic medical care for management of incidents with radiologic / nuclear agents and contaminated victimsspecific dangers of radiological / nuclear agents , associated illnesses and radiation syndromeself protection , protection and detection equipment , special intervention unitsfirst aid medical treatment , isolation and radioactive decontamination , decontamination operations in case of mass trauma combined with contamination injuriesexperiential trainingdecontamination after radiation exposureto be exposed to practical aspects and procedures of decontaminationdecontamination exercise or decontamination demonstration , e.g. , in nuclear power plant or with the fire brigadesmodule 11 , lecture and pbldchemical and toxicological threats from hazardous materials and goods , transport risks , acute poisoning and toxic syndromesto get to know principles of medical countermeasures for management of incidents with dangerous chemical substances , hazardous materials and goodsidentification and risk assessment of hazardous materials , chemicals and goods , and associated toxic syndromesmanagement of acute intoxications and poisonings , threats from specific poisonsself protection , precautionspoisoning epidemiology , risk assessment for mass intoxicationsfirst aid medical treatment , e.g. , poison emergency centers and toxicology laboratoriesexperiential trainingdecontamination after chemical poisoningto experience exposure to decontamination procedurestriage in case of mass casualties with toxic syndromesdecontamination measures and exercised medical student disaster medicine curriculum , modules 1214module 12 , lecture , pbld , and interactive reviewethics and professionalismto develop familiarity with ethical codes and the duty of care relevant to disaster conditionsgeneva convention and amended protocols , ethical codes of conduct , humanitarian imperatives , social , moral , and ethical challenges of disastersquality assuranceto understand quality improvement efforts and risk management programs for disaster medical responsequality control performance indicators , incident monitoring , tools for risk and critical event assessment , structured improvement approachesappropriate documentationmodule 13 , pbld and interactive reviewto comprehend concepts of psychic stress responsecase presentations for identification of critical incident stress reactions and review of therapeutic interventionspsychosocial careto learn techniques to deal with psychic reactions caused by exposure to disaster scenariostreatment approaches to acute and delayed critical incident stress reactions , acute and chronic stress syndromes , post - traumatic stress disordersto recognize need for help and to initiate psychosocial supportstructure and tasks of psychosocial emergency intervention unitsoperational strategies of psychosocial treatmentmodule 14 , course completionclosing examinationto demonstrate gain of knowledge and skillsstudent final examination , oral and/or written testevaluation of educational successto assess educational value of coursecomparison of pre - program versus post - program student test resultslearner and educator assessment of courseto maintain continuous curriculum development and improvement in course qualitystudent and faculty summative and formative course evaluation modules 4 and 5 ( table 1b ) discuss hospital alarm and preparedness plans , with a focus on in - house and external incidents requiring high surge capacity . interactive review of past major community - threatening events and experiences from worldwide disaster and relief operations are utilized to built recognition of disaster health - care and medical assistance feasibility issues . to provide a hands - on experience and to increase student understanding of what occurs in a mass casualty management situation , we simulate a triage exercise in a small - group format ( four students / two emergency physicians ) as shown in fig . well - trained , life - like moulage volunteers simulate casualties presenting with various disorders , from superficial injuries to moribund conditions , medical illnesses , and emotional trauma . the hands - on exercise is then run in a step - by - step manner , starting with patient search : after they are located , patients must be checked for vital functions and examined . 2algorithm of student immersion in mass casualty rescue simulation exercise algorithm of student immersion in mass casualty rescue simulation exercise modules 8 and 9 ( table 1b and c ) teach life - saving and specific emergency medical care for various disaster situations , containing first aid and specific approaches to injuries , burns , explosions , blasts , and illnesses from warfare and biological agents . emphasising terrorist attacks and highly contagious infectious diseases , core competencies for care of explosive , warfare , and biological incidents are included . modules 10 and 11 ( table 1c ) teach the dangers and management of radiological / nuclear and chemical hazardous materials . at a nuclear power plant or fire department , the design and state of preparedness of such facilities are evaluated , and equipment is demonstrated . this hands - on program serves as a real - world simulation experience , reproduced by ( 1 ) heat load under the multilayer over - garment , ( 2 ) enhanced personal respiratory efforts , ( 3 ) restricted visual fields because of the gas mask , and ( 4 ) interference with manual dexterity by the chemical protective gloves . 3flowchart of the module with the fire fighters or at a nuclear power plant focussed on education of incident management with radioactive and hazardous chemical substances flowchart of the module with the fire fighters or at a nuclear power plant focussed on education of incident management with radioactive and hazardous chemical substances modules 12 and 13 ( table 1d ) deal with challenges of ethical , professional , psychosocial , and quality control criteria of medical disaster management themes . to understand quality improvement efforts , students gain familiarity with assessment tools and incident monitoring to evaluate functional components of disaster response systems . to our knowledge , this curriculum is one of the first publications of a comprehensive medical student disaster medicine curriculum that covers major contemporary aspects of basic disaster medicine management . at a time when medical schools in numerous countries are struggling to find a place for disaster medicine in their programs , this educational topic is worthy of research and discussion . the curriculum also provides resources for endeavors of organizations such as the section in disaster medicine of the european society for emergency medicine , which has as a current goal the development of a standard european disaster medicine curriculum for medical school undergraduates . however , all medical schools that have implemented the curriculum have so far reported that they now teach disaster medicine components with a scope and depth much broader than within emergency medicine , pointing out the additional value for disaster preparedness and disaster medical management . requests of the education committee working group of the world association for disaster and emergency medicine , disaster medicine - related competencies as indicated in emergency medicine or radiology curricula , courses recommended by professional organizations , the literature and expert opinion were addressed as far as available [ 7 , 2123 ] . the competency structured design also permits flexibility in addressing a multiplicity of disaster situations , including floods , hurricanes , earthquakes , and surveillance - related health problems , as well as care for vulnerable populations such as children and hospitalised patients . in our opinion and circumstances , a 14-module course composed of 2-h units provides adequate student education and mock disaster exposure in a reasonable period . some themes important for disaster preparedness are also addressed in other medical school programs , such as in medical microbiology , toxicology , and environmental medicine . emergency medicine curricula for medical students , when present in medical school curricula at all , do not typically focus on disaster medicine or disaster response . when planning the experiential units , we attempted to ensure that costs were minimized : for cost containment , selection of a teaching cadre with background in emergency or disaster medicine who collaborates with local disaster response agencies is most effective . table 2task assignment for estimation of organizational burden and curricular implementation costscurricular activitymission descriptionprogram managementdevelopment of a local organizational structure responsible for curricular activities : team recruitment , appointment of managers and departmental staff in - charge , task assignmentidentification of internal and external experts and invitation as teaching cadreestablishment of collaboration with local disaster response agencies and planning of common external units , exercises , and simulationsteaching and preceptor assignment for internal and external unitsinstitution of curriculum within medical school program and coordination of units with program plannersorganization of facilities and equipment for in - house units with university s lecture support stafforganization of transport to external unitsmanagement of administrative and reporting issuesif necessary : establishment of financial recording , billing , and reimbursement systemfaculty development / continuous professional growthfuture faculty development through involvement in existing teaching activities , plans , and team meetingsfaculty instructor trainingoptional : professional growth through participation in related internal and external developmental programs for university educators and disaster medicine preparednessquality assurance / continuous curricular developmentdevelopment of data collection systemanalysis of ongoing evaluations from learners and educatorsuse of course assessments and experiences for orientation , end - of - course team meetings , and future progressstudent supportprovision of curricular schedule and learning objectivesprovision of lecture compilation ( syllabus or cd ) as course content workbook and base for self - directed learningdevelopment of data collection system and analysis of student examination datadevelopment of reporting system for examination gradesmaintenance of partnerships / collaborationscontinuous encouragement and support for internal and external partnerships and collaborationsrunning of administrative / overhead expendituresif necessary : management of direct and indirect expenditures , such as telecommunication costsother pursuitsdesigning of plans for course revisions or curricular enhancements , such as integration of e - learning methods task assignment for estimation of organizational burden and curricular implementation costs there are several limitations to this work . a curriculum is provided for medical student disaster medicine education at an advanced level of medical school training , along with information regarding the process involved in creating such a program at a national level . we wish to emphasize that the curriculum has been designed for medical students in a particular system and that there are various concepts for basic disaster medicine education . due to its comprehensive , interdisciplinary nature , instructive design , and flexible structure , however , the curriculum can benefit other health - care professional systems by serving as a model curriculum , and facilitating refinement and testing of other existing , but perhaps as yet unpublished models .

suggest core competencies and subject areas for terrorism , disaster , and public health emergency preparedness for health - care students based on professional school outlines . describe core competencies and dentists roles within disaster response efforts focusing on bioterrorism ; participation in a 4-h disaster life support course of the american medical association is also recommended to provide guidance regarding incidents with weapons of mass destruction . because a fully developed and comprehensive medical school disaster medicine curriculum was not available , the german government commissioned the development of a core medical student disaster medicine curriculum that could serve as a standardized template . the pilots contents were mainly based on government - funded research projects in which all curricula from german medical schools and postgraduate public health physician courses were evaluated for inclusion of disaster medicine - related themes . the implementation experience with the pilots was straightforward : the university dean and the department chair fully supported the idea of providing an introduction to the practices of disaster medicine at the student level . when the government requested a medical student disaster education plan , we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses : the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine , but to enhance their understanding of what occurs in a disaster , and where the limits and challenges faced by those delivering medical care under austere conditions lie . second , improving knowledge of public health - relevant information on radiological / nuclear , biological , chemical , explosive , and other disaster - related health risks appeared to be important . following analysis of international guidelines on disaster response training , peer - reviewed publications [ 35 , 12 , 13 ] , and programs such as the us department of homeland security , federal emergency management agency ( fema ) , and american medical association , learning objectives were fixed . to identify the content details , a systematic literature search in medline , embase , and the cochrane library with the key terms student , education , training , curriculum , course , competency , disaster , catastrophe , mass casualty , medicine , medical , preparedness , plans , and outcome was conducted . this led us to include interactive discussions , teaching of problem - solving strategies , reviews of real - life experience , role playing , and exposure to exercises in real - world activities were assigned to suitable content modules , with a minimum of one - third of the curricular time being reserved for hands - on training . to evaluate the effectiveness of the curriculum , the results of a knowledge - based , pre - program written examination were compared with post - program examination results . because the course was created to be part of the obligatory 4th - year german medical school program , the testing at course completion also served to fulfil requirements of the german educational system . these included alignment with medical school mission statements , instructional methods of preparing students for life - long , self - directed learning , or the provision of learning environments that resemble real - world scenarios . 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . the implementation experience with the pilots was straightforward : the university dean and the department chair fully supported the idea of providing an introduction to the practices of disaster medicine at the student level . when the government requested a medical student disaster education plan , we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses : the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine , but to enhance their understanding of what occurs in a disaster , and where the limits and challenges faced by those delivering medical care under austere conditions lie . second , improving knowledge of public health - relevant information on radiological / nuclear , biological , chemical , explosive , and other disaster - related health risks appeared to be important . following analysis of international guidelines on disaster response training , peer - reviewed publications [ 35 , 12 , 13 ] , and programs such as the us department of homeland security , federal emergency management agency ( fema ) , and american medical association , learning objectives were fixed . to identify the content details , a systematic literature search in medline , embase , and the cochrane library with the key terms student , education , training , curriculum , course , competency , disaster , catastrophe , mass casualty , medicine , medical , preparedness , plans , and outcome was conducted . this led us to include interactive discussions , teaching of problem - solving strategies , reviews of real - life experience , role playing , and exposure to exercises in real - world activities were assigned to suitable content modules , with a minimum of one - third of the curricular time being reserved for hands - on training . to evaluate the effectiveness of the curriculum , the results of a knowledge - based , pre - program written examination were compared with post - program examination results . because the course was created to be part of the obligatory 4th - year german medical school program , the testing at course completion also served to fulfil requirements of the german educational system . these included alignment with medical school mission statements , instructional methods of preparing students for life - long , self - directed learning , or the provision of learning environments that resemble real - world scenarios . 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies pilot versions of the curriculum were initially developed and utilized with faculty and student feedback over a 2-year period . when the government requested a medical student disaster education plan , we offered a clear vision for goals and objectives of a comprehensive curriculum of core issues of disaster medicine based on our experience from the pilot courses : the first aim was not only to increase medical students familiarity with the ideas and practices of disaster medicine , but to enhance their understanding of what occurs in a disaster , and where the limits and challenges faced by those delivering medical care under austere conditions lie . second , improving knowledge of public health - relevant information on radiological / nuclear , biological , chemical , explosive , and other disaster - related health risks appeared to be important . following analysis of international guidelines on disaster response training , peer - reviewed publications [ 35 , 12 , 13 ] , and programs such as the us department of homeland security , federal emergency management agency ( fema ) , and american medical association , after cross - referencing objectives with quality assurance criteria for medical education [ 1416 ] , topics were structured into a framework represented by subject headings . to identify the content details , a systematic literature search in medline , embase , and the cochrane library with the key terms student , education , training , curriculum , course , competency , disaster , catastrophe , mass casualty , medicine , medical , preparedness , plans , and outcome was conducted . this led us to include interactive discussions , teaching of problem - solving strategies , reviews of real - life experience , role playing , and exposure to exercises in real - world environments . to evaluate the effectiveness of the curriculum , the results of a knowledge - based , pre - program written examination were compared with post - program examination results . because the course was created to be part of the obligatory 4th - year german medical school program , the testing at course completion also served to fulfil requirements of the german educational system . these included alignment with medical school mission statements , instructional methods of preparing students for life - long , self - directed learning , or the provision of learning environments that resemble real - world scenarios . 1final curriculum development indicating stages of refinements and input from various professional and governmental bodies final curriculum development indicating stages of refinements and input from various professional and governmental bodies the creation of the final curriculum resulted in a course of 14 modules with each unit requiring approximately 2 h time : modules 13 ( table 1a ) introduce the essentials of disaster medicine terminology , disaster assistance and law , management , and multidisciplinary coordination and communication systems . as key competencies , the tasks of assistance agencies , functional response roles , scopes of responsibility , lines of authority , logistical needs for mass casualties , and triage methods are taught . table 1modules 114modules , learning activityeducational goalcore contentsa medical student disaster medicine curriculum , modules 13module 1 , lecture and pbldintroduction to disaster medicine , terminologyto learn definitions of disaster medicine and to develop an understanding for general disaster managementglossary and common disaster medicine definitions , differences between disaster and emergency medicine , different phases of disaster managementdisaster assistanceprinciples of disaster assistancedisaster assistance organization , assistance agencies and structuretypologyto consider the heterogeneity of disastersnatural and technological disasters , terrorism , man - made disasters , civilian disorders , environmental and other threatslaws and regulationsto understand the legal environment and regulations for civil protection and disaster preparednessregulative and administrative issues for civil disaster protection , warfare disaster protection laws , rescue service and hospital laws , governmental resources , and authoritiesmodule 2 , lecture and pblddisaster medical managementto realize the architecture and organizational management necessary for coping with mass casualty incidents and large - scale acutely ill patients in a coordinated waymass casualty disposition , treatment area , transport issuesfunctional operationstasks of rapid - intervention - units ( sanitary / psychosocial care)incident command systemsdisaster contingency plans command and control structures , functional operations centercoordination structurescoordination , integration , and cooperation of multi - agency rescue and assistance responsefunctional rolesfunctional response roles , e.g. , lead emergency physician , organizational leader , and technical command postinformation managementcommunication , coordinationmodule 3 , lecture and pbldspecific disaster medicineto develop skills in principles of tactically managing mass casualties and large numbers of patients suffering , e.g. , patient collection , treatment area , transport)tactic disaster medical managementlogistical requirements for care of burn - injuries , mine blast , and missile - hit victims ( including high - speed bullet injuries ) , mass trauma managementb medical student disaster medicine curriculum , modules 48module 4 , pbld and interactive reviewhospital preparedness and disaster management planningto follow orders and principles of hospital alarm and evacuation planshospital disaster lawshospital alarm planshospital preparedness plans for- management of external disasters with mass casualties suffering from multiple injuries , intoxication , infections , and/or radioactive contamination- management of in - house disasters with fire incidents and hospital evacuationmodule 5 , pbld and interactive reviewpresentation of past disasters and review of assistance experiencesto evaluate and understand feasibility issues of providing medical support and health care in the field and under disaster conditions based on experience from worldwide disaster assistance operationspresentation of past disasters and disaster assistance experience gained in the field , e.g. , from lead emergency physicians , operations in earthquake assistance , explosions , highly contagious infectious diseases , repatriation flights , and with the german federal armed forces medical corps in world crisis regionsmodule 6 , experiential trainingpreclinical and clinical triage exerciseto perform triage decisions in reality simulationtriage training exercise : real or virtual scenario simulation rescue exercise , e.g. , explosion with mass casualties and blast , mechanical , and thermal injuries of all triage levelsmodule 7 , experiential trainingevacuation exerciseto apply operational principles and steps of action for evacuation procedurescommand post exercise , real or virtual : e.g. , evacuation organization and evacuation of a hospital , school , part of town , etc.module 8 , lecture and pbldlife - saving disaster emergency medical careto learn concepts of life - saving emergency disaster medical careunder disaster conditions : provision of life - saving emergency medical care to adults and children , e.g. , shock therapy , pain treatment , and sedationc medical student disaster medicine curriculum , modules 911module 9 , lecture and pbldspecific disaster emergency medical care for various situations including bioterrorism incidentsto get to know principles of specific disaster medical caredisaster emergency medical first aid , specific measuressurgical and medical treatments of burn and thermal injuries and illnesses from explosive , warfare and biological agentsepidemiology and approaches to terrorist attacks , weapons , and highly contagious infectious diseases , sentinel casesmodule 10 , lecture and pbldradiological and nuclear threats , accidents with radioactive material , radiation illness and syndrome , decontaminationto learn principles and basic medical care for management of incidents with radiologic / nuclear agents and contaminated victimsspecific dangers of radiological / nuclear agents , associated illnesses and radiation syndromeself protection , protection and detection equipment , special intervention unitsfirst aid medical treatment , isolation and radioactive decontamination , decontamination operations in case of mass trauma combined with contamination injuriesexperiential trainingdecontamination after radiation exposureto be exposed to practical aspects and procedures of decontaminationdecontamination exercise or decontamination demonstration , e.g. , in nuclear power plant or with the fire brigadesmodule 11 , lecture and pbldchemical and toxicological threats from hazardous materials and goods , transport risks , acute poisoning and toxic syndromesto get to know principles of medical countermeasures for management of incidents with dangerous chemical substances , hazardous materials and goodsidentification and risk assessment of hazardous materials , chemicals and goods , and associated toxic syndromesmanagement of acute intoxications and poisonings , threats from specific poisonsself protection , precautionspoisoning epidemiology , risk assessment for mass intoxicationsfirst aid medical treatment , e.g. , poison emergency centers and toxicology laboratoriesexperiential trainingdecontamination after chemical poisoningto experience exposure to decontamination procedurestriage in case of mass casualties with toxic syndromesdecontamination measures and exercised medical student disaster medicine curriculum , modules 1214module 12 , lecture , pbld , and interactive reviewethics and professionalismto develop familiarity with ethical codes and the duty of care relevant to disaster conditionsgeneva convention and amended protocols , ethical codes of conduct , humanitarian imperatives , social , moral , and ethical challenges of disastersquality assuranceto understand quality improvement efforts and risk management programs for disaster medical responsequality control performance indicators , incident monitoring , tools for risk and critical event assessment , structured improvement approachesappropriate documentationmodule 13 , pbld and interactive reviewto comprehend concepts of psychic stress responsecase presentations for identification of critical incident stress reactions and review of therapeutic interventionspsychosocial careto learn techniques to deal with psychic reactions caused by exposure to disaster scenariostreatment approaches to acute and delayed critical incident stress reactions , acute and chronic stress syndromes , post - traumatic stress disordersto recognize need for help and to initiate psychosocial supportstructure and tasks of psychosocial emergency intervention unitsoperational strategies of psychosocial treatmentmodule 14 , course completionclosing examinationto demonstrate gain of knowledge and skillsstudent final examination , oral and/or written testevaluation of educational successto assess educational value of coursecomparison of pre - program versus post - program student test resultslearner and educator assessment of courseto maintain continuous curriculum development and improvement in course qualitystudent and faculty summative and formative course evaluation modules 4 and 5 ( table 1b ) discuss hospital alarm and preparedness plans , with a focus on in - house and external incidents requiring high surge capacity . to provide a hands - on experience and to increase student understanding of what occurs in a mass casualty management situation , we simulate a triage exercise in a small - group format ( four students / two emergency physicians ) as shown in fig . 2algorithm of student immersion in mass casualty rescue simulation exercise algorithm of student immersion in mass casualty rescue simulation exercise modules 8 and 9 ( table 1b and c ) teach life - saving and specific emergency medical care for various disaster situations , containing first aid and specific approaches to injuries , burns , explosions , blasts , and illnesses from warfare and biological agents . to provide a practical experience of a decontamination procedure and to get students personally involved in how it feels to work in protective gear , we offer a combined unit of didactic , interactive , and hands - on training at the fire department ( fig . this hands - on program serves as a real - world simulation experience , reproduced by ( 1 ) heat load under the multilayer over - garment , ( 2 ) enhanced personal respiratory efforts , ( 3 ) restricted visual fields because of the gas mask , and ( 4 ) interference with manual dexterity by the chemical protective gloves . 3flowchart of the module with the fire fighters or at a nuclear power plant focussed on education of incident management with radioactive and hazardous chemical substances flowchart of the module with the fire fighters or at a nuclear power plant focussed on education of incident management with radioactive and hazardous chemical substances modules 12 and 13 ( table 1d ) deal with challenges of ethical , professional , psychosocial , and quality control criteria of medical disaster management themes . students learn critical aspects of acute and delayed stress disorders ( anxiety and grief ) , and the psychosocial resources , such as crisis intervention units , available to provide specialised assistance . the curriculum also provides resources for endeavors of organizations such as the section in disaster medicine of the european society for emergency medicine , which has as a current goal the development of a standard european disaster medicine curriculum for medical school undergraduates . requests of the education committee working group of the world association for disaster and emergency medicine , disaster medicine - related competencies as indicated in emergency medicine or radiology curricula , courses recommended by professional organizations , the literature and expert opinion were addressed as far as available [ 7 , 2123 ] . introduction to emergency medicine focuses on maximum possible , individual patient - tailored care [ 9 , 21 , 23 ] and if included this is intended to address disaster medicine conditions in which extraordinary treatment demands exceed resources , and where incidents of great magnitude requiring external assistance for management may reach epidemiological dimensions . table 2task assignment for estimation of organizational burden and curricular implementation costscurricular activitymission descriptionprogram managementdevelopment of a local organizational structure responsible for curricular activities : team recruitment , appointment of managers and departmental staff in - charge , task assignmentidentification of internal and external experts and invitation as teaching cadreestablishment of collaboration with local disaster response agencies and planning of common external units , exercises , and simulationsteaching and preceptor assignment for internal and external unitsinstitution of curriculum within medical school program and coordination of units with program plannersorganization of facilities and equipment for in - house units with university s lecture support stafforganization of transport to external unitsmanagement of administrative and reporting issuesif necessary : establishment of financial recording , billing , and reimbursement systemfaculty development / continuous professional growthfuture faculty development through involvement in existing teaching activities , plans , and team meetingsfaculty instructor trainingoptional : professional growth through participation in related internal and external developmental programs for university educators and disaster medicine preparednessquality assurance / continuous curricular developmentdevelopment of data collection systemanalysis of ongoing evaluations from learners and educatorsuse of course assessments and experiences for orientation , end - of - course team meetings , and future progressstudent supportprovision of curricular schedule and learning objectivesprovision of lecture compilation ( syllabus or cd ) as course content workbook and base for self - directed learningdevelopment of data collection system and analysis of student examination datadevelopment of reporting system for examination gradesmaintenance of partnerships / collaborationscontinuous encouragement and support for internal and external partnerships and collaborationsrunning of administrative / overhead expendituresif necessary : management of direct and indirect expenditures , such as telecommunication costsother pursuitsdesigning of plans for course revisions or curricular enhancements , such as integration of e - learning methods task assignment for estimation of organizational burden and curricular implementation costs there are several limitations to this work . in its first design , our curriculum consists of traditional , face - to - face , instructor - student interactions and hands - on experiences , and does not use electronic ( e- ) learning methods . due to its comprehensive , interdisciplinary nature , instructive design , and flexible structure , however , the curriculum can benefit other health - care professional systems by serving as a model curriculum , and facilitating refinement and testing of other existing , but perhaps as yet unpublished models .

depression , a commonly seen psychological health problem across the world , prevents the functionality , creativity , happiness , and satisfaction of individuals , reduces their quality of life , and leads to losses in the work force ( 1 ) . pregnancy , one of the important processes in women , is a natural event as well as a period during which many biological and psychosocial changes are experienced . the risk of the many factors that may cause depression is high because of its stress and anxiety ( 1 - 3 ) . besides , the neuroendocrinological and psychosocial changes that pregnancy causes are too many compared to other periods of life ( 4 , 5 ) . high levels of norepinephrine and cortisol decrease blood flow into the uterus and thus cause severe obstetric and neonatal problems for both the pregnant woman and the fetus ( 6 - 8 ) . these problems may be listed as follows : spontaneous abortion , antenatal bleeding , increased uterine artery resistance , preeclampsia , eclampsia , fetal death , low apgar score , newborns with low birth weight and high levels of cortisol , neonatal growth retardation , and babies that require neonatal intensive care ( 6 , 9 , 10 ) . international studies emphasize that many cases of depression are among women aged 18 - 44 years and that depression comprises fecundity periods such as pregnancy , birth , and puerperality ( 1 , 8) . the incidence of depression and its symptoms ranges between 8% and 38% ( 7 , 11 - 14 ) in the world . this incidence varies between 12% and 36% in turkey ( 1 , 2 , 15 ) . among the factors that increase depression risk during pregnancy are history of depression , younger ages of mothers , low socioeconomic status , being exposed to violence before and during pregnancy , disharmony between couples , living alone , having experienced a miscarriage in the past , undesired pregnancy or ambivalent thoughts about pregnancy , having many children , and a lack or absence of social support ( 1 , 2 , 14 ) . social support is described as financial , emotional , and mental support given to somebody by others ( 3 , 16 ) . social support positively and directly affects one s health whether there is stress or not and protects psychological well - being by decreasing or balancing the damages brought about by the stressors caused by life events ( 3 , 17 ) . the conducted studies indicate a close correlation between increased depression levels and insufficient levels of social support during pregnancy ( 10 , 18 , 19 ) . insufficient social support during pregnancy deteriorates the psychological health of the pregnant woman and affects negatively her quality of life , has a poor effect on eating habits , and leads to an increase in the use of alcohol , smoking , and substance use ( 3 , 10 ) . depression , one of the health problems frequently happens among pregnant women , is a crucial problem , which should be carefully dealt with , diagnosed early and treated soon because it has an adverse effect on the wellness of the pregnant women , paves the way for postpartum depression , may become chronic , and increases the risk of attempted suicide ( 1 , 2 , 20 ) . depression can be prevented and treated if health care professionals can detect the factors that increase the risk of depression during pregnancy at an early period ( 1 , 21 ) . the aim of the current study was to determine depressive symptom levels of the pregnant women and the sociodemographic and obstetric risk factors that might lead to depression , as well as exploring the correlation between social support and pregnancy depression . this study aimed to determine the frequency of depression symptoms , and its risk factors . the population of this study consisted of the pregnant women who presented at the maternity hospital of trabzon from may 21 to june 13 , 2008 . the study was conducted in a large hospital in northeastern turkey and almost all pregnant women in this region , particularly those living in the vicinity of trabzon province , received antenatal care in that hospital . trabzon maternity hospital is a public hospital with approximately 6188 deliveries ( 3170 vaginal delivery and 3018 cesarean ) per year . it was chosen for this study as it is the largest obstetrics hospital in the area with a 300 beds . the present study design was cross - sectional based on time and descriptive according to the purpose of the study ( 22 , 23 ) . the sample of this study was calculated according to the formula in which the number of individuals in the population is unknown ( equation 1 ) . where , p = 0.22 ( according to research conducted earlier , the incidence of depression in pregnancy in turkey is 22% ) , q = 1 - 0.22 = 0.78 , d = 0.052 = 0.025 , and n = 3.84 ( 0.78 0.22 ) / 0.025 = 263 ( 22 , 23 ) . according to the results of the above formula , 3 more women were added to the sample in case of probable data loss and as a result , a total 266 pregnant women comprised the research sample . in the literature , it is stated that a maximum of 10% data more than the determined sample size can be enrolled ( 22 , 23 ) . pregnant women who accepted to participate in the study were selected by simple random method . the inclusion criteria besides being pregnant were as follows : their labor had not started and without any pregnancy complications ( placenta previa , preeclampsia , fetal distress , etc . ) without any known psychiatric or neurological disorders that would interfere with the completion of the measurements . we aimed to recruit all pregnant women who met the inclusion criteria , but those who used psychiatric drugs during pregnancy ( 3 women ) , those whose labor had started ( 28 women ) and those who did not accept participating in the study for any reason ( 10 women ) were excluded from the study . the questionnaire form included a descriptive information form that included sociodemographic information , the beck depression inventory ( bdi ) , and the multidimensional scale of perceived social support ( mspss ) . it included questions related to the sociodemographic characteristics of the study participants and their medical and obstetric history . after reviewing the relevant literature ( 1 , 2 , 6 , 11 , 15 , 24 - 26 ) , this form was developed by the researcher to study the background and sociodemographic characteristics of the pregnant women . after the reviewing the literature , the researchers designed a three - part questionnaire form . the first part was composed of a descriptive information form , which addressed sociodemographic , obstetric characteristics , and some special situations of pregnancy . the second part was bdi to determine pregnancy depression risks and the third part comprised the mspss . ( 1961 ) ( 27 ) and includes 21 self - evaluation statements about the symptoms of depression and each item scores from 0 to 3 . the aim of the inventory is not to diagnose depression but to measure the severity of the depression symptoms objectively . a score between 0 and 9 indicates no depression ; a score between 10 and 16 indicates a mild level of depression ; a score between 17 and 24 indicates a moderate level of depression ; and a score of 25 or higher indicates a severe level of depression ( major ) . the turkish adaptation , reliability , and validity tests of the inventory were performed by hisli and the cut - off point in the study was accepted as 17 . a score of 17 detects depressive symptoms that require medical treatment with an exactness of 90% ( 28 ) . it is rated based on a 7-point likert scale with responses ranging from absolutely no to absolutely yes ( 1 - 7 points ) . there are 3 subscales of mspss ( family support , friend support , and significant other support ) and each subscale is composed of four statements . the lowest score to be obtained from each subscale is 4 whereas the highest score is 28 . the lowest score to be obtained from the scale is 12 whereas the highest score is 84 . a higher score indicates a higher level of perceived social support while a lower score indicates no perceived social support or a lack of perceived social support ( 29 ) . the turkish adaptation , reliability , and validity tests of the scale were confirmed by eker et al . each pregnant woman was contacted by that observer and provided with a detailed explanation of the purpose and procedure of the study . the questionnaire form , bdi , and mspss used for the data collection were filled in by the researchers using face - to - face interviews with the pregnant women in a separate and quiet room . if the pregnant women were unable to complete the questionnaire form on their own , the researchers read out the questionnaire items to the women and recorded the answers . smirnov test was used to determine normal distribution of the data . in the statistical evaluation , some characteristics of the pregnant women ( age , education , etc . ) and mean bdi score were found not to conform to the normal distribution . data were evaluated using the mann - whitney u , kruskal - wallis tests , and the percentage , mean , standard deviation , median , mean rank , minimum and maximum values were also calculated . the relationship between pregnant women bdi total scores and social support and subscale total score was evaluated using the pearson correlation analysis . written informed consent was obtained from pregnant women , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki as reflected in a prior approval by the institution of human research committee . the aim of the research was explained to the pregnant women and they were informed that if they preferred not to continue , they could withdraw from the study any time they wished . the present study design was cross - sectional based on time and descriptive according to the purpose of the study ( 22 , 23 ) . the sample of this study was calculated according to the formula in which the number of individuals in the population is unknown ( equation 1 ) . where , p = 0.22 ( according to research conducted earlier , the incidence of depression in pregnancy in turkey is 22% ) , q = 1 - 0.22 = 0.78 , d = 0.052 = 0.025 , and n = 3.84 ( 0.78 0.22 ) / 0.025 = 263 ( 22 , 23 ) . according to the results of the above formula , the research sample would be 263 pregnant women . in this study , 3 more women were added to the sample in case of probable data loss and as a result , a total 266 pregnant women comprised the research sample . in the literature , it is stated that a maximum of 10% data more than the determined sample size can be enrolled ( 22 , 23 ) . pregnant women who accepted to participate in the study were selected by simple random method . the inclusion criteria besides being pregnant were as follows : their labor had not started and without any pregnancy complications ( placenta previa , preeclampsia , fetal distress , etc . ) without any known psychiatric or neurological disorders that would interfere with the completion of the measurements . we aimed to recruit all pregnant women who met the inclusion criteria , but those who used psychiatric drugs during pregnancy ( 3 women ) , those whose labor had started ( 28 women ) and those who did not accept participating in the study for any reason ( 10 women ) were excluded from the study . the questionnaire form included a descriptive information form that included sociodemographic information , the beck depression inventory ( bdi ) , and the multidimensional scale of perceived social support ( mspss ) . it included questions related to the sociodemographic characteristics of the study participants and their medical and obstetric history . after reviewing the relevant literature ( 1 , 2 , 6 , 11 , 15 , 24 - 26 ) , this form was developed by the researcher to study the background and sociodemographic characteristics of the pregnant women . after the reviewing the literature , the researchers designed a three - part questionnaire form . the first part was composed of a descriptive information form , which addressed sociodemographic , obstetric characteristics , and some special situations of pregnancy . the second part was bdi to determine pregnancy depression risks and the third part comprised the mspss . ( 1961 ) ( 27 ) and includes 21 self - evaluation statements about the symptoms of depression and each item scores from 0 to 3 . the aim of the inventory is not to diagnose depression but to measure the severity of the depression symptoms objectively . a score between 0 and 9 indicates no depression ; a score between 10 and 16 indicates a mild level of depression ; a score between 17 and 24 indicates a moderate level of depression ; and a score of 25 or higher indicates a severe level of depression ( major ) . the turkish adaptation , reliability , and validity tests of the inventory were performed by hisli and the cut - off point in the study was accepted as 17 . a score of 17 detects depressive symptoms that require medical treatment with an exactness of 90% ( 28 ) . it is rated based on a 7-point likert scale with responses ranging from absolutely no to absolutely yes ( 1 - 7 points ) . there are 3 subscales of mspss ( family support , friend support , and significant other support ) and each subscale is composed of four statements . the lowest score to be obtained from each subscale is 4 whereas the highest score is 28 . the lowest score to be obtained from the scale is 12 whereas the highest score is 84 . a higher score indicates a higher level of perceived social support while a lower score indicates no perceived social support or a lack of perceived social support ( 29 ) . the turkish adaptation , reliability , and validity tests of the scale were confirmed by eker et al . each pregnant woman was contacted by that observer and provided with a detailed explanation of the purpose and procedure of the study . the questionnaire form , bdi , and mspss used for the data collection were filled in by the researchers using face - to - face interviews with the pregnant women in a separate and quiet room . if the pregnant women were unable to complete the questionnaire form on their own , the researchers read out the questionnaire items to the women and recorded the answers . smirnov test was used to determine normal distribution of the data . in the statistical evaluation , some characteristics of the pregnant women ( age , education , etc . ) and mean bdi score were found not to conform to the normal distribution . data were evaluated using the mann - whitney u , kruskal - wallis tests , and the percentage , mean , standard deviation , median , mean rank , minimum and maximum values were also calculated . the relationship between pregnant women bdi total scores and social support and subscale total score was evaluated using the pearson correlation analysis . written informed consent was obtained from pregnant women , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki as reflected in a prior approval by the institution of human research committee . the aim of the research was explained to the pregnant women and they were informed that if they preferred not to continue , they could withdraw from the study any time they wished . after these explanations , 266 pregnant women consented to participate in the study voluntarily . of studied pregnant women , 84.9% were housewives , 54.5% had primary school or secondary school certificates , 51.9% were primigravida , and 68.8% were in the third trimester . table 1 presents the distribution of depressive symptom severity and the mean bdi scores of the pregnant women . the mean bdi scores of the pregnant women was found to be 11.12 6.65 . depression symptom severity of 18.2% of the pregnant women was at a level that required treatment and the mean bdi score in this group was 21.62 5.24 ( table 1 ) . ( % ) or mean sd . the effect of sociodemographic characteristics of the pregnant women on the mean bdi score is presented in table 2 . there were significant differences between the mean bdi scores and variables of educational degrees , employment status and husbands educational level , among the groups ( p < 0.05 ) , whereas no difference existed between mean bdi scores and variables of age , husbands occupation , perceived economical income , length of marriage and family type among the groups ( p > 0.05 ) ( table 2 ) the effects of obstetric characteristics of the pregnant women upon mean bdi scores are presented in table 3 . there were statistically significant differences between mean bdi scores and variables of having an undesired pregnancy , having a chronic disease before pregnancy , experiencing pregnancy - related problems , having a child with disability or having relatives whose children were disabled ( p < 0.05 ) . the mean bdi scores of those who had a miscarriage , cesarean delivery , felt unready for motherhood , and those who knew the sex of the baby but were dissatisfied with it were low though not statistically significant ( p > 0.05 ) ( table 3 ) . mann - whitney u test . kruskal - wallis test . table 4 showed the effect of some special situations of the pregnant women upon the mean bdi score . the correlation between smoking during pregnancy and the mean bdi score was statistically significant ( p < 0.05 ) . however , mean bdi scores were high , though not statistically significant , among pregnant women whose type of pregnancy was different , were exposed to physical - psychological violence by their husbands , were betrayed by their husbands , and those that did not like their appearance due to weight gain during pregnancy ( p > 0.05 ) ( table 4 ) . the highest perceived social support of the pregnant women came from significant others / husband ( 24.63 5.29 ) , family ( 24.10 5.59 ) and friends ( 19.22 7.19 ) . a high significant correlation existed between the mean total mspss score and the mean bdi score of the pregnant women ( p < 0.001 ) ( table 5 ) . abbreviations : mspss , multidimensional scale of perceived social support . found to be extremely and negatively significant . using the pearson correlation analysis . depression , one of the frequently seen health problems among women , is experienced by women in fecundity periods and its incidence increases with pregnancy . in the studies that investigated the incidence of depression during pregnancy in different cultures , the rate of depression was found to be 7.5% in china , 8.1% in korea , 17.9% in hungary , 30% in canada , 20% in the usa , and 19.6% in brazil ( 5 , 11 - 14 , 31 ) . as for turkey , the incidence of depression during pregnancy ranges from 12% to 36% ( 1 , 2 , 15 ) . our study detected an 18.2% rate in the type of pregnancy depression that required medical treatment . the depression level detected by the current study and mean depression scores were similar to some studies while different from others ; the reason for this discrepancy may be due to the different culture of the studied societies and or the use of the different measurement methods to detect depression . it is emphasized in some studies that these factors , including age , low socioeconomic status , negative life experiences , lack of a job with satisfactory income , family problems , low educational status of pregnant women and their husbands augment the severity of the depression symptoms ( 20 , 32 - 34 ) . in our study , the educational status of the pregnant women and their husbands as well as the employment status of the pregnant woman were detected as the factors that increased the severity of the depression symptoms . ( 34 ) noted that pregnancy depression was seen more among pregnant women who had low educational level and worked at a job with a unsatisfactory income . current or past history of pregnancy ( miscarriage or abortion ) , unplanned pregnancy , having a chronic disease and emotional and physical problems experienced during pregnancy are obstetric risk factors for pregnancy depression ( 2 , 8 , 20 , 25 ) . similar to the literature , our study indicated that unplanned pregnancy , having a chronic disease and pregnancy - related problems increased the severity of depression among pregnant women ( p < 0.05 ) . many studies highlighted that pregnancy depression is found more among women who have an unplanned and undesired pregnancy , have a chronic disease and face problems in the current pregnancy ( 15 , 18 , 24 , 35 ) . our study pointed to the fact that pregnant women with a disabled child or relatives with child disability had increased depression symptom severity . the literature states that pregnant women who themselves have a child with mental / physical disability or have first degree relatives or close friends having a child with mental / physical disability is an important risk factor that affects pregnancy depression ( 21 ) . in the studies of raina et al . ( 36 ) , karadavut and uneri ( 26 ) , and pistav akmese et al . ( 37 ) in which mothers with children with disability were investigated , it was found that these mothers had a trait anxiety level above the average level because ambiguities related to what kind of problems the disabled children will meet in the future may cause trait anxiety and depression in the family . the type of marriage may be regarded as a risk factor for pregnancy depression . in the literature review , no study was found in which the type of marriage ( disapproved marriage : running away and getting married to somebody that the family members are opposed to ; or approved marriage : getting married to somebody that the family members approve of ) had been examined . in our study , nearly 14% of the participating women were in a disapproved marriage and their mean bdi scores were found to be higher than in the other type of marriage . if the fact that these pregnant women in disapproved marriages were adolescent at the time of the marriage is taken into consideration , the higher mean bdi scores of these women may relate to the possibility that they had poor / insufficient social support from their families . weight gain during pregnancy , experiencing domestic violence , history of physical , emotional and sexual violence , betrayal by the husband , smoking , and alcohol and substance use are some of the social factors that affect pregnancy depression ( 14 , 21 , 38 , 39 ) . the studies conducted show that weight gain during pregnancy causes dissatisfaction among pregnant women and consequently depressive symptoms increase during pregnancy ( 4 , 39 , 40 ) . ( 18 ) , lancaster et al . ( 34 ) , karmaliani et al . ( 38 ) , and leigh et al . ( 41 ) reported that depression was seen more among women who gained excessive weight , had smoked and were subjected to psychological and physical violence during pregnancy . likewise , in our study , it was found that a statistically significant correlation existed between smoking and depression symptom levels ( p < 0.05 ) . depression symptom severity was higher among pregnant women who were subjected to psychological and physical violence , betrayed by their husbands , gained excessive weight during pregnancy and not satisfied with it ; yet , the differences were not statistically significant . social support provided by the husband , family and/or friends during pregnancy comforts pregnant women emotionally and mentally and enables them to use social sources more , helping them to cope with stressors and anxiety more easily and paving the way for their transition into motherhood roles ( 1 , 17 , 19 , 25 , 35 ) . in the similar studies , it is emphasized that there is a correlation between social support during pregnancy and depression and anxiety levels and also lack of social support augments levels of depression and anxiety ( 1 , 3 , 19 , 20 ) . the total mspss score of the pregnant women in our study was 67.89 14.26 . it was found out that the pregnant women got the highest social support first from significant persons in their lives ( their husbands ) ( 24.63 5.29 ) , second from their families ( 24.10 5.59 ) , and finally from their friends ( 19.22 7.19 ) . furthermore , the social support scores obtained from these 3 groups affected mean bdi scores of the pregnant women ( p < 0.001 ) . this finding was in agreement with the literature . to conclude , this means that mean bdi scores decreases as mspss scores increases . in other words as the perceived social support increases , the psychological problems caused by stressful life events decrease . in a study in canada , it was reported that both pregnancy depression risks and postpartum depression risks considerably increased among those with low social support levels during pregnancy ( 17 ) . ( 18 ) carried out in germany measured the social support scores of 896 pregnant women who were in the first trimester of pregnancy . after this follow up , it was found that the pregnant women with low social support had higher levels of depression symptoms and a decreased quality of life and smoked more during the pregnancy compared to those with high social support . in the randomized study of leigh et al . ( 41 ) conducted in australia on 367 pregnant women , it was found that depressive symptom levels were higher among the women who had poor or no social support compared to those with moderate and high levels of social support . in conclusion , it was found that one - fifth of the pregnant women had a depressive symptom level ( 17 and 18.8% ) that required medical treatment and such sociodemographic and obstetric factors as the pregnant women s educational level , employment status , husbands educational level , presence of a chronic disease , having problems during pregnancy , whether the pregnancy was planned or not , and having a child with disability or having relatives who had children with disability affect severity of depressive symptoms . in addition , a significant correlation was found between the social support given during pregnancy and decreased depressive symptom severity . the study was conducted in one city with a selected group of women who resided in the city center and therefore , its generalizability is considerably limited as the sample group was small . also , the other limitation of the study was that detailed psychiatric examinations and diagnosis were not performed but only the women 's depression and anxiety levels were measured with scales and inventories . we are of the opinion , however , that the study will shed light on relevant studies in the future as it provides information about the situation and frequency of depression in the pregnant women of one city in our country .

background : women are seriously subjected to psychiatric diseases during pregnancy and depression is the most prevailing one among these diseases . there is a relation between the social support and depression in pregnancy whose predisposing factors are genetic , psychological , biological , environmental , and hormonal.objectives:this study aimed to determine the frequency of depression symptoms , and its risk factors . also it studied the correlation between social support and pregnancy depression.patients and methods : this research is a descriptive cross - sectional study . it was conducted on 266 pregnant women selected by simple random method from all pregnant women admitted at the maternity hospital of trabzon , turkey from may 21 to june 13 , 2008 . the data were collected with a questionnaire form , the beck depression inventory ( bdi ) , and the multidimensional scale of perceived social support ( mspss).results : the mean bdi score of the pregnant women was 11.12 6.65 . according to the bdi , 46.2% of the pregnant women had no depression symptoms , 34.59% of them had mild , 13.91% had moderate , and 4.89% had severe level of depression symptoms . it was found that such factors as the educational level of the pregnant women and their husbands , having an undesired pregnancy , suffering from a chronic disease before pregnancy , presence of pregnancy - related problems , having a child with disability or having relatives whose children had disability , and smoking during pregnancy were the risk factors affecting the severity of the depression symptoms and these results were statistically significant ( p < 0.05 ) . on the other hand , the mean mspss score was 67.89 14.26 and it was found that the pregnant women got the highest social support from their husbands . it was found that there was a significant correlation between bdi and mspss total score and its subscale scores ( p < 0.05).conclusions : according to this study , one - fifth of pregnant women were found to experience depressive symptoms , which require treatment during pregnancy , and the factors such as having no support from relatives was found to be associated with the severity of depressive symptoms during pregnancy .

1. Background 2. Objectives 3. Patients and Methods 3.1. Sample 3.2. Procedure and Data Collection 3.3. Questionnaire Form 3.4. Procedure 3.5. Statistical Analysis 3.6. Ethical Consideration 4. Results 5. Discussion

pregnancy , one of the important processes in women , is a natural event as well as a period during which many biological and psychosocial changes are experienced . the risk of the many factors that may cause depression is high because of its stress and anxiety ( 1 - 3 ) . international studies emphasize that many cases of depression are among women aged 18 - 44 years and that depression comprises fecundity periods such as pregnancy , birth , and puerperality ( 1 , 8) . among the factors that increase depression risk during pregnancy are history of depression , younger ages of mothers , low socioeconomic status , being exposed to violence before and during pregnancy , disharmony between couples , living alone , having experienced a miscarriage in the past , undesired pregnancy or ambivalent thoughts about pregnancy , having many children , and a lack or absence of social support ( 1 , 2 , 14 ) . the conducted studies indicate a close correlation between increased depression levels and insufficient levels of social support during pregnancy ( 10 , 18 , 19 ) . insufficient social support during pregnancy deteriorates the psychological health of the pregnant woman and affects negatively her quality of life , has a poor effect on eating habits , and leads to an increase in the use of alcohol , smoking , and substance use ( 3 , 10 ) . depression , one of the health problems frequently happens among pregnant women , is a crucial problem , which should be carefully dealt with , diagnosed early and treated soon because it has an adverse effect on the wellness of the pregnant women , paves the way for postpartum depression , may become chronic , and increases the risk of attempted suicide ( 1 , 2 , 20 ) . depression can be prevented and treated if health care professionals can detect the factors that increase the risk of depression during pregnancy at an early period ( 1 , 21 ) . the aim of the current study was to determine depressive symptom levels of the pregnant women and the sociodemographic and obstetric risk factors that might lead to depression , as well as exploring the correlation between social support and pregnancy depression . this study aimed to determine the frequency of depression symptoms , and its risk factors . the population of this study consisted of the pregnant women who presented at the maternity hospital of trabzon from may 21 to june 13 , 2008 . the study was conducted in a large hospital in northeastern turkey and almost all pregnant women in this region , particularly those living in the vicinity of trabzon province , received antenatal care in that hospital . it was chosen for this study as it is the largest obstetrics hospital in the area with a 300 beds . the present study design was cross - sectional based on time and descriptive according to the purpose of the study ( 22 , 23 ) . the sample of this study was calculated according to the formula in which the number of individuals in the population is unknown ( equation 1 ) . where , p = 0.22 ( according to research conducted earlier , the incidence of depression in pregnancy in turkey is 22% ) , q = 1 - 0.22 = 0.78 , d = 0.052 = 0.025 , and n = 3.84 ( 0.78 0.22 ) / 0.025 = 263 ( 22 , 23 ) . according to the results of the above formula , 3 more women were added to the sample in case of probable data loss and as a result , a total 266 pregnant women comprised the research sample . pregnant women who accepted to participate in the study were selected by simple random method . we aimed to recruit all pregnant women who met the inclusion criteria , but those who used psychiatric drugs during pregnancy ( 3 women ) , those whose labor had started ( 28 women ) and those who did not accept participating in the study for any reason ( 10 women ) were excluded from the study . the questionnaire form included a descriptive information form that included sociodemographic information , the beck depression inventory ( bdi ) , and the multidimensional scale of perceived social support ( mspss ) . it included questions related to the sociodemographic characteristics of the study participants and their medical and obstetric history . after reviewing the relevant literature ( 1 , 2 , 6 , 11 , 15 , 24 - 26 ) , this form was developed by the researcher to study the background and sociodemographic characteristics of the pregnant women . the first part was composed of a descriptive information form , which addressed sociodemographic , obstetric characteristics , and some special situations of pregnancy . the second part was bdi to determine pregnancy depression risks and the third part comprised the mspss . the aim of the inventory is not to diagnose depression but to measure the severity of the depression symptoms objectively . a score between 0 and 9 indicates no depression ; a score between 10 and 16 indicates a mild level of depression ; a score between 17 and 24 indicates a moderate level of depression ; and a score of 25 or higher indicates a severe level of depression ( major ) . the turkish adaptation , reliability , and validity tests of the inventory were performed by hisli and the cut - off point in the study was accepted as 17 . a higher score indicates a higher level of perceived social support while a lower score indicates no perceived social support or a lack of perceived social support ( 29 ) . the questionnaire form , bdi , and mspss used for the data collection were filled in by the researchers using face - to - face interviews with the pregnant women in a separate and quiet room . if the pregnant women were unable to complete the questionnaire form on their own , the researchers read out the questionnaire items to the women and recorded the answers . smirnov test was used to determine normal distribution of the data . in the statistical evaluation , some characteristics of the pregnant women ( age , education , etc . ) and mean bdi score were found not to conform to the normal distribution . data were evaluated using the mann - whitney u , kruskal - wallis tests , and the percentage , mean , standard deviation , median , mean rank , minimum and maximum values were also calculated . the relationship between pregnant women bdi total scores and social support and subscale total score was evaluated using the pearson correlation analysis . written informed consent was obtained from pregnant women , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki as reflected in a prior approval by the institution of human research committee . the aim of the research was explained to the pregnant women and they were informed that if they preferred not to continue , they could withdraw from the study any time they wished . the present study design was cross - sectional based on time and descriptive according to the purpose of the study ( 22 , 23 ) . the sample of this study was calculated according to the formula in which the number of individuals in the population is unknown ( equation 1 ) . where , p = 0.22 ( according to research conducted earlier , the incidence of depression in pregnancy in turkey is 22% ) , q = 1 - 0.22 = 0.78 , d = 0.052 = 0.025 , and n = 3.84 ( 0.78 0.22 ) / 0.025 = 263 ( 22 , 23 ) . according to the results of the above formula , the research sample would be 263 pregnant women . in this study , 3 more women were added to the sample in case of probable data loss and as a result , a total 266 pregnant women comprised the research sample . pregnant women who accepted to participate in the study were selected by simple random method . without any known psychiatric or neurological disorders that would interfere with the completion of the measurements . we aimed to recruit all pregnant women who met the inclusion criteria , but those who used psychiatric drugs during pregnancy ( 3 women ) , those whose labor had started ( 28 women ) and those who did not accept participating in the study for any reason ( 10 women ) were excluded from the study . the questionnaire form included a descriptive information form that included sociodemographic information , the beck depression inventory ( bdi ) , and the multidimensional scale of perceived social support ( mspss ) . it included questions related to the sociodemographic characteristics of the study participants and their medical and obstetric history . after reviewing the relevant literature ( 1 , 2 , 6 , 11 , 15 , 24 - 26 ) , this form was developed by the researcher to study the background and sociodemographic characteristics of the pregnant women . the first part was composed of a descriptive information form , which addressed sociodemographic , obstetric characteristics , and some special situations of pregnancy . the second part was bdi to determine pregnancy depression risks and the third part comprised the mspss . the aim of the inventory is not to diagnose depression but to measure the severity of the depression symptoms objectively . a score between 0 and 9 indicates no depression ; a score between 10 and 16 indicates a mild level of depression ; a score between 17 and 24 indicates a moderate level of depression ; and a score of 25 or higher indicates a severe level of depression ( major ) . the turkish adaptation , reliability , and validity tests of the inventory were performed by hisli and the cut - off point in the study was accepted as 17 . a higher score indicates a higher level of perceived social support while a lower score indicates no perceived social support or a lack of perceived social support ( 29 ) . the questionnaire form , bdi , and mspss used for the data collection were filled in by the researchers using face - to - face interviews with the pregnant women in a separate and quiet room . if the pregnant women were unable to complete the questionnaire form on their own , the researchers read out the questionnaire items to the women and recorded the answers . smirnov test was used to determine normal distribution of the data . in the statistical evaluation , some characteristics of the pregnant women ( age , education , etc . ) and mean bdi score were found not to conform to the normal distribution . data were evaluated using the mann - whitney u , kruskal - wallis tests , and the percentage , mean , standard deviation , median , mean rank , minimum and maximum values were also calculated . the relationship between pregnant women bdi total scores and social support and subscale total score was evaluated using the pearson correlation analysis . written informed consent was obtained from pregnant women , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki as reflected in a prior approval by the institution of human research committee . the aim of the research was explained to the pregnant women and they were informed that if they preferred not to continue , they could withdraw from the study any time they wished . table 1 presents the distribution of depressive symptom severity and the mean bdi scores of the pregnant women . the mean bdi scores of the pregnant women was found to be 11.12 6.65 . depression symptom severity of 18.2% of the pregnant women was at a level that required treatment and the mean bdi score in this group was 21.62 5.24 ( table 1 ) . the effect of sociodemographic characteristics of the pregnant women on the mean bdi score is presented in table 2 . there were significant differences between the mean bdi scores and variables of educational degrees , employment status and husbands educational level , among the groups ( p < 0.05 ) , whereas no difference existed between mean bdi scores and variables of age , husbands occupation , perceived economical income , length of marriage and family type among the groups ( p > 0.05 ) ( table 2 ) the effects of obstetric characteristics of the pregnant women upon mean bdi scores are presented in table 3 . there were statistically significant differences between mean bdi scores and variables of having an undesired pregnancy , having a chronic disease before pregnancy , experiencing pregnancy - related problems , having a child with disability or having relatives whose children were disabled ( p < 0.05 ) . the mean bdi scores of those who had a miscarriage , cesarean delivery , felt unready for motherhood , and those who knew the sex of the baby but were dissatisfied with it were low though not statistically significant ( p > 0.05 ) ( table 3 ) . table 4 showed the effect of some special situations of the pregnant women upon the mean bdi score . the correlation between smoking during pregnancy and the mean bdi score was statistically significant ( p < 0.05 ) . however , mean bdi scores were high , though not statistically significant , among pregnant women whose type of pregnancy was different , were exposed to physical - psychological violence by their husbands , were betrayed by their husbands , and those that did not like their appearance due to weight gain during pregnancy ( p > 0.05 ) ( table 4 ) . the highest perceived social support of the pregnant women came from significant others / husband ( 24.63 5.29 ) , family ( 24.10 5.59 ) and friends ( 19.22 7.19 ) . a high significant correlation existed between the mean total mspss score and the mean bdi score of the pregnant women ( p < 0.001 ) ( table 5 ) . abbreviations : mspss , multidimensional scale of perceived social support . depression , one of the frequently seen health problems among women , is experienced by women in fecundity periods and its incidence increases with pregnancy . in the studies that investigated the incidence of depression during pregnancy in different cultures , the rate of depression was found to be 7.5% in china , 8.1% in korea , 17.9% in hungary , 30% in canada , 20% in the usa , and 19.6% in brazil ( 5 , 11 - 14 , 31 ) . as for turkey , the incidence of depression during pregnancy ranges from 12% to 36% ( 1 , 2 , 15 ) . the depression level detected by the current study and mean depression scores were similar to some studies while different from others ; the reason for this discrepancy may be due to the different culture of the studied societies and or the use of the different measurement methods to detect depression . it is emphasized in some studies that these factors , including age , low socioeconomic status , negative life experiences , lack of a job with satisfactory income , family problems , low educational status of pregnant women and their husbands augment the severity of the depression symptoms ( 20 , 32 - 34 ) . in our study , the educational status of the pregnant women and their husbands as well as the employment status of the pregnant woman were detected as the factors that increased the severity of the depression symptoms . ( 34 ) noted that pregnancy depression was seen more among pregnant women who had low educational level and worked at a job with a unsatisfactory income . current or past history of pregnancy ( miscarriage or abortion ) , unplanned pregnancy , having a chronic disease and emotional and physical problems experienced during pregnancy are obstetric risk factors for pregnancy depression ( 2 , 8 , 20 , 25 ) . similar to the literature , our study indicated that unplanned pregnancy , having a chronic disease and pregnancy - related problems increased the severity of depression among pregnant women ( p < 0.05 ) . many studies highlighted that pregnancy depression is found more among women who have an unplanned and undesired pregnancy , have a chronic disease and face problems in the current pregnancy ( 15 , 18 , 24 , 35 ) . our study pointed to the fact that pregnant women with a disabled child or relatives with child disability had increased depression symptom severity . the literature states that pregnant women who themselves have a child with mental / physical disability or have first degree relatives or close friends having a child with mental / physical disability is an important risk factor that affects pregnancy depression ( 21 ) . ( 37 ) in which mothers with children with disability were investigated , it was found that these mothers had a trait anxiety level above the average level because ambiguities related to what kind of problems the disabled children will meet in the future may cause trait anxiety and depression in the family . in our study , nearly 14% of the participating women were in a disapproved marriage and their mean bdi scores were found to be higher than in the other type of marriage . if the fact that these pregnant women in disapproved marriages were adolescent at the time of the marriage is taken into consideration , the higher mean bdi scores of these women may relate to the possibility that they had poor / insufficient social support from their families . weight gain during pregnancy , experiencing domestic violence , history of physical , emotional and sexual violence , betrayal by the husband , smoking , and alcohol and substance use are some of the social factors that affect pregnancy depression ( 14 , 21 , 38 , 39 ) . the studies conducted show that weight gain during pregnancy causes dissatisfaction among pregnant women and consequently depressive symptoms increase during pregnancy ( 4 , 39 , 40 ) . likewise , in our study , it was found that a statistically significant correlation existed between smoking and depression symptom levels ( p < 0.05 ) . depression symptom severity was higher among pregnant women who were subjected to psychological and physical violence , betrayed by their husbands , gained excessive weight during pregnancy and not satisfied with it ; yet , the differences were not statistically significant . social support provided by the husband , family and/or friends during pregnancy comforts pregnant women emotionally and mentally and enables them to use social sources more , helping them to cope with stressors and anxiety more easily and paving the way for their transition into motherhood roles ( 1 , 17 , 19 , 25 , 35 ) . in the similar studies , it is emphasized that there is a correlation between social support during pregnancy and depression and anxiety levels and also lack of social support augments levels of depression and anxiety ( 1 , 3 , 19 , 20 ) . the total mspss score of the pregnant women in our study was 67.89 14.26 . it was found out that the pregnant women got the highest social support first from significant persons in their lives ( their husbands ) ( 24.63 5.29 ) , second from their families ( 24.10 5.59 ) , and finally from their friends ( 19.22 7.19 ) . furthermore , the social support scores obtained from these 3 groups affected mean bdi scores of the pregnant women ( p < 0.001 ) . in other words as the perceived social support increases , the psychological problems caused by stressful life events decrease . in a study in canada , it was reported that both pregnancy depression risks and postpartum depression risks considerably increased among those with low social support levels during pregnancy ( 17 ) . ( 18 ) carried out in germany measured the social support scores of 896 pregnant women who were in the first trimester of pregnancy . after this follow up , it was found that the pregnant women with low social support had higher levels of depression symptoms and a decreased quality of life and smoked more during the pregnancy compared to those with high social support . ( 41 ) conducted in australia on 367 pregnant women , it was found that depressive symptom levels were higher among the women who had poor or no social support compared to those with moderate and high levels of social support . in conclusion , it was found that one - fifth of the pregnant women had a depressive symptom level ( 17 and 18.8% ) that required medical treatment and such sociodemographic and obstetric factors as the pregnant women s educational level , employment status , husbands educational level , presence of a chronic disease , having problems during pregnancy , whether the pregnancy was planned or not , and having a child with disability or having relatives who had children with disability affect severity of depressive symptoms . in addition , a significant correlation was found between the social support given during pregnancy and decreased depressive symptom severity . the study was conducted in one city with a selected group of women who resided in the city center and therefore , its generalizability is considerably limited as the sample group was small . also , the other limitation of the study was that detailed psychiatric examinations and diagnosis were not performed but only the women 's depression and anxiety levels were measured with scales and inventories . we are of the opinion , however , that the study will shed light on relevant studies in the future as it provides information about the situation and frequency of depression in the pregnant women of one city in our country .

depression , a commonly seen psychological health problem across the world , prevents the functionality , creativity , happiness , and satisfaction of individuals , reduces their quality of life , and leads to losses in the work force ( 1 ) . pregnancy , one of the important processes in women , is a natural event as well as a period during which many biological and psychosocial changes are experienced . the risk of the many factors that may cause depression is high because of its stress and anxiety ( 1 - 3 ) . high levels of norepinephrine and cortisol decrease blood flow into the uterus and thus cause severe obstetric and neonatal problems for both the pregnant woman and the fetus ( 6 - 8 ) . these problems may be listed as follows : spontaneous abortion , antenatal bleeding , increased uterine artery resistance , preeclampsia , eclampsia , fetal death , low apgar score , newborns with low birth weight and high levels of cortisol , neonatal growth retardation , and babies that require neonatal intensive care ( 6 , 9 , 10 ) . international studies emphasize that many cases of depression are among women aged 18 - 44 years and that depression comprises fecundity periods such as pregnancy , birth , and puerperality ( 1 , 8) . the incidence of depression and its symptoms ranges between 8% and 38% ( 7 , 11 - 14 ) in the world . among the factors that increase depression risk during pregnancy are history of depression , younger ages of mothers , low socioeconomic status , being exposed to violence before and during pregnancy , disharmony between couples , living alone , having experienced a miscarriage in the past , undesired pregnancy or ambivalent thoughts about pregnancy , having many children , and a lack or absence of social support ( 1 , 2 , 14 ) . social support positively and directly affects one s health whether there is stress or not and protects psychological well - being by decreasing or balancing the damages brought about by the stressors caused by life events ( 3 , 17 ) . the conducted studies indicate a close correlation between increased depression levels and insufficient levels of social support during pregnancy ( 10 , 18 , 19 ) . insufficient social support during pregnancy deteriorates the psychological health of the pregnant woman and affects negatively her quality of life , has a poor effect on eating habits , and leads to an increase in the use of alcohol , smoking , and substance use ( 3 , 10 ) . depression , one of the health problems frequently happens among pregnant women , is a crucial problem , which should be carefully dealt with , diagnosed early and treated soon because it has an adverse effect on the wellness of the pregnant women , paves the way for postpartum depression , may become chronic , and increases the risk of attempted suicide ( 1 , 2 , 20 ) . depression can be prevented and treated if health care professionals can detect the factors that increase the risk of depression during pregnancy at an early period ( 1 , 21 ) . the aim of the current study was to determine depressive symptom levels of the pregnant women and the sociodemographic and obstetric risk factors that might lead to depression , as well as exploring the correlation between social support and pregnancy depression . this study aimed to determine the frequency of depression symptoms , and its risk factors . the population of this study consisted of the pregnant women who presented at the maternity hospital of trabzon from may 21 to june 13 , 2008 . the study was conducted in a large hospital in northeastern turkey and almost all pregnant women in this region , particularly those living in the vicinity of trabzon province , received antenatal care in that hospital . the present study design was cross - sectional based on time and descriptive according to the purpose of the study ( 22 , 23 ) . the sample of this study was calculated according to the formula in which the number of individuals in the population is unknown ( equation 1 ) . where , p = 0.22 ( according to research conducted earlier , the incidence of depression in pregnancy in turkey is 22% ) , q = 1 - 0.22 = 0.78 , d = 0.052 = 0.025 , and n = 3.84 ( 0.78 0.22 ) / 0.025 = 263 ( 22 , 23 ) . according to the results of the above formula , 3 more women were added to the sample in case of probable data loss and as a result , a total 266 pregnant women comprised the research sample . in the literature , it is stated that a maximum of 10% data more than the determined sample size can be enrolled ( 22 , 23 ) . we aimed to recruit all pregnant women who met the inclusion criteria , but those who used psychiatric drugs during pregnancy ( 3 women ) , those whose labor had started ( 28 women ) and those who did not accept participating in the study for any reason ( 10 women ) were excluded from the study . the questionnaire form included a descriptive information form that included sociodemographic information , the beck depression inventory ( bdi ) , and the multidimensional scale of perceived social support ( mspss ) . it included questions related to the sociodemographic characteristics of the study participants and their medical and obstetric history . after reviewing the relevant literature ( 1 , 2 , 6 , 11 , 15 , 24 - 26 ) , this form was developed by the researcher to study the background and sociodemographic characteristics of the pregnant women . after the reviewing the literature , the researchers designed a three - part questionnaire form . the first part was composed of a descriptive information form , which addressed sociodemographic , obstetric characteristics , and some special situations of pregnancy . the second part was bdi to determine pregnancy depression risks and the third part comprised the mspss . ( 1961 ) ( 27 ) and includes 21 self - evaluation statements about the symptoms of depression and each item scores from 0 to 3 . the aim of the inventory is not to diagnose depression but to measure the severity of the depression symptoms objectively . a score between 0 and 9 indicates no depression ; a score between 10 and 16 indicates a mild level of depression ; a score between 17 and 24 indicates a moderate level of depression ; and a score of 25 or higher indicates a severe level of depression ( major ) . the turkish adaptation , reliability , and validity tests of the inventory were performed by hisli and the cut - off point in the study was accepted as 17 . there are 3 subscales of mspss ( family support , friend support , and significant other support ) and each subscale is composed of four statements . a higher score indicates a higher level of perceived social support while a lower score indicates no perceived social support or a lack of perceived social support ( 29 ) . the questionnaire form , bdi , and mspss used for the data collection were filled in by the researchers using face - to - face interviews with the pregnant women in a separate and quiet room . if the pregnant women were unable to complete the questionnaire form on their own , the researchers read out the questionnaire items to the women and recorded the answers . data were evaluated using the mann - whitney u , kruskal - wallis tests , and the percentage , mean , standard deviation , median , mean rank , minimum and maximum values were also calculated . the relationship between pregnant women bdi total scores and social support and subscale total score was evaluated using the pearson correlation analysis . written informed consent was obtained from pregnant women , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki as reflected in a prior approval by the institution of human research committee . the aim of the research was explained to the pregnant women and they were informed that if they preferred not to continue , they could withdraw from the study any time they wished . the present study design was cross - sectional based on time and descriptive according to the purpose of the study ( 22 , 23 ) . the sample of this study was calculated according to the formula in which the number of individuals in the population is unknown ( equation 1 ) . where , p = 0.22 ( according to research conducted earlier , the incidence of depression in pregnancy in turkey is 22% ) , q = 1 - 0.22 = 0.78 , d = 0.052 = 0.025 , and n = 3.84 ( 0.78 0.22 ) / 0.025 = 263 ( 22 , 23 ) . according to the results of the above formula , the research sample would be 263 pregnant women . in this study , 3 more women were added to the sample in case of probable data loss and as a result , a total 266 pregnant women comprised the research sample . in the literature , it is stated that a maximum of 10% data more than the determined sample size can be enrolled ( 22 , 23 ) . we aimed to recruit all pregnant women who met the inclusion criteria , but those who used psychiatric drugs during pregnancy ( 3 women ) , those whose labor had started ( 28 women ) and those who did not accept participating in the study for any reason ( 10 women ) were excluded from the study . the questionnaire form included a descriptive information form that included sociodemographic information , the beck depression inventory ( bdi ) , and the multidimensional scale of perceived social support ( mspss ) . it included questions related to the sociodemographic characteristics of the study participants and their medical and obstetric history . after reviewing the relevant literature ( 1 , 2 , 6 , 11 , 15 , 24 - 26 ) , this form was developed by the researcher to study the background and sociodemographic characteristics of the pregnant women . after the reviewing the literature , the researchers designed a three - part questionnaire form . the second part was bdi to determine pregnancy depression risks and the third part comprised the mspss . ( 1961 ) ( 27 ) and includes 21 self - evaluation statements about the symptoms of depression and each item scores from 0 to 3 . the aim of the inventory is not to diagnose depression but to measure the severity of the depression symptoms objectively . a score between 0 and 9 indicates no depression ; a score between 10 and 16 indicates a mild level of depression ; a score between 17 and 24 indicates a moderate level of depression ; and a score of 25 or higher indicates a severe level of depression ( major ) . the turkish adaptation , reliability , and validity tests of the inventory were performed by hisli and the cut - off point in the study was accepted as 17 . a higher score indicates a higher level of perceived social support while a lower score indicates no perceived social support or a lack of perceived social support ( 29 ) . the questionnaire form , bdi , and mspss used for the data collection were filled in by the researchers using face - to - face interviews with the pregnant women in a separate and quiet room . if the pregnant women were unable to complete the questionnaire form on their own , the researchers read out the questionnaire items to the women and recorded the answers . data were evaluated using the mann - whitney u , kruskal - wallis tests , and the percentage , mean , standard deviation , median , mean rank , minimum and maximum values were also calculated . the relationship between pregnant women bdi total scores and social support and subscale total score was evaluated using the pearson correlation analysis . the aim of the research was explained to the pregnant women and they were informed that if they preferred not to continue , they could withdraw from the study any time they wished . table 1 presents the distribution of depressive symptom severity and the mean bdi scores of the pregnant women . depression symptom severity of 18.2% of the pregnant women was at a level that required treatment and the mean bdi score in this group was 21.62 5.24 ( table 1 ) . the effect of sociodemographic characteristics of the pregnant women on the mean bdi score is presented in table 2 . there were significant differences between the mean bdi scores and variables of educational degrees , employment status and husbands educational level , among the groups ( p < 0.05 ) , whereas no difference existed between mean bdi scores and variables of age , husbands occupation , perceived economical income , length of marriage and family type among the groups ( p > 0.05 ) ( table 2 ) the effects of obstetric characteristics of the pregnant women upon mean bdi scores are presented in table 3 . there were statistically significant differences between mean bdi scores and variables of having an undesired pregnancy , having a chronic disease before pregnancy , experiencing pregnancy - related problems , having a child with disability or having relatives whose children were disabled ( p < 0.05 ) . the mean bdi scores of those who had a miscarriage , cesarean delivery , felt unready for motherhood , and those who knew the sex of the baby but were dissatisfied with it were low though not statistically significant ( p > 0.05 ) ( table 3 ) . table 4 showed the effect of some special situations of the pregnant women upon the mean bdi score . the correlation between smoking during pregnancy and the mean bdi score was statistically significant ( p < 0.05 ) . however , mean bdi scores were high , though not statistically significant , among pregnant women whose type of pregnancy was different , were exposed to physical - psychological violence by their husbands , were betrayed by their husbands , and those that did not like their appearance due to weight gain during pregnancy ( p > 0.05 ) ( table 4 ) . the highest perceived social support of the pregnant women came from significant others / husband ( 24.63 5.29 ) , family ( 24.10 5.59 ) and friends ( 19.22 7.19 ) . a high significant correlation existed between the mean total mspss score and the mean bdi score of the pregnant women ( p < 0.001 ) ( table 5 ) . depression , one of the frequently seen health problems among women , is experienced by women in fecundity periods and its incidence increases with pregnancy . in the studies that investigated the incidence of depression during pregnancy in different cultures , the rate of depression was found to be 7.5% in china , 8.1% in korea , 17.9% in hungary , 30% in canada , 20% in the usa , and 19.6% in brazil ( 5 , 11 - 14 , 31 ) . as for turkey , the incidence of depression during pregnancy ranges from 12% to 36% ( 1 , 2 , 15 ) . our study detected an 18.2% rate in the type of pregnancy depression that required medical treatment . the depression level detected by the current study and mean depression scores were similar to some studies while different from others ; the reason for this discrepancy may be due to the different culture of the studied societies and or the use of the different measurement methods to detect depression . it is emphasized in some studies that these factors , including age , low socioeconomic status , negative life experiences , lack of a job with satisfactory income , family problems , low educational status of pregnant women and their husbands augment the severity of the depression symptoms ( 20 , 32 - 34 ) . in our study , the educational status of the pregnant women and their husbands as well as the employment status of the pregnant woman were detected as the factors that increased the severity of the depression symptoms . current or past history of pregnancy ( miscarriage or abortion ) , unplanned pregnancy , having a chronic disease and emotional and physical problems experienced during pregnancy are obstetric risk factors for pregnancy depression ( 2 , 8 , 20 , 25 ) . similar to the literature , our study indicated that unplanned pregnancy , having a chronic disease and pregnancy - related problems increased the severity of depression among pregnant women ( p < 0.05 ) . many studies highlighted that pregnancy depression is found more among women who have an unplanned and undesired pregnancy , have a chronic disease and face problems in the current pregnancy ( 15 , 18 , 24 , 35 ) . the literature states that pregnant women who themselves have a child with mental / physical disability or have first degree relatives or close friends having a child with mental / physical disability is an important risk factor that affects pregnancy depression ( 21 ) . ( 37 ) in which mothers with children with disability were investigated , it was found that these mothers had a trait anxiety level above the average level because ambiguities related to what kind of problems the disabled children will meet in the future may cause trait anxiety and depression in the family . in the literature review , no study was found in which the type of marriage ( disapproved marriage : running away and getting married to somebody that the family members are opposed to ; or approved marriage : getting married to somebody that the family members approve of ) had been examined . in our study , nearly 14% of the participating women were in a disapproved marriage and their mean bdi scores were found to be higher than in the other type of marriage . if the fact that these pregnant women in disapproved marriages were adolescent at the time of the marriage is taken into consideration , the higher mean bdi scores of these women may relate to the possibility that they had poor / insufficient social support from their families . weight gain during pregnancy , experiencing domestic violence , history of physical , emotional and sexual violence , betrayal by the husband , smoking , and alcohol and substance use are some of the social factors that affect pregnancy depression ( 14 , 21 , 38 , 39 ) . the studies conducted show that weight gain during pregnancy causes dissatisfaction among pregnant women and consequently depressive symptoms increase during pregnancy ( 4 , 39 , 40 ) . likewise , in our study , it was found that a statistically significant correlation existed between smoking and depression symptom levels ( p < 0.05 ) . depression symptom severity was higher among pregnant women who were subjected to psychological and physical violence , betrayed by their husbands , gained excessive weight during pregnancy and not satisfied with it ; yet , the differences were not statistically significant . social support provided by the husband , family and/or friends during pregnancy comforts pregnant women emotionally and mentally and enables them to use social sources more , helping them to cope with stressors and anxiety more easily and paving the way for their transition into motherhood roles ( 1 , 17 , 19 , 25 , 35 ) . in the similar studies , it is emphasized that there is a correlation between social support during pregnancy and depression and anxiety levels and also lack of social support augments levels of depression and anxiety ( 1 , 3 , 19 , 20 ) . it was found out that the pregnant women got the highest social support first from significant persons in their lives ( their husbands ) ( 24.63 5.29 ) , second from their families ( 24.10 5.59 ) , and finally from their friends ( 19.22 7.19 ) . furthermore , the social support scores obtained from these 3 groups affected mean bdi scores of the pregnant women ( p < 0.001 ) . in a study in canada , it was reported that both pregnancy depression risks and postpartum depression risks considerably increased among those with low social support levels during pregnancy ( 17 ) . after this follow up , it was found that the pregnant women with low social support had higher levels of depression symptoms and a decreased quality of life and smoked more during the pregnancy compared to those with high social support . ( 41 ) conducted in australia on 367 pregnant women , it was found that depressive symptom levels were higher among the women who had poor or no social support compared to those with moderate and high levels of social support . in conclusion , it was found that one - fifth of the pregnant women had a depressive symptom level ( 17 and 18.8% ) that required medical treatment and such sociodemographic and obstetric factors as the pregnant women s educational level , employment status , husbands educational level , presence of a chronic disease , having problems during pregnancy , whether the pregnancy was planned or not , and having a child with disability or having relatives who had children with disability affect severity of depressive symptoms . the study was conducted in one city with a selected group of women who resided in the city center and therefore , its generalizability is considerably limited as the sample group was small . we are of the opinion , however , that the study will shed light on relevant studies in the future as it provides information about the situation and frequency of depression in the pregnant women of one city in our country .

when imagining time and their own life events , humans do not only retrieve or predict when events have occurred or will occur , but also automatically project themselves on an imagined mental time line . self - time travelling can thus be regarded as the ability to transpose one 's habitual self - location in time to different temporal locations in the past or the future . emphasizing the role of perspective taking , this ability to change one 's own temporal egocentric perspective has been proposed to share a common mechanism with the ability to change one 's own spatial egocentric perspective . in both domains , people would use existing representations as templates for processing and understanding new information , in order to plan their short- and long - term behaviors . on this account , the same processes that subserve simulation of the self at a different location in space would also subserve simulation of the self at a different point in time . an interesting question , inspired by this parallel , is whether , similarly to taking another person 's spatial perspective , people can also adopt the temporal perspective of another person when travelling through time . studies investigating spatial perspective taking indicate that people can overcome their own position in space to adopt another person 's spatial perspective ( e.g. , ) . when the scene includes another person , for instance , people may spontaneously describe spatial relations from that person 's perspective despite the very real presence of their own [ 5 , 6 ] . these and other findings suggest that spatial perspective taking may induce an alterocentric remapping , that is , remapping of objects and locations to an alterocentric frame of reference . to our best knowledge , no study has so far investigated whether a similar remapping may take place in the temporal domain . in other words , whether similarly to taking another person 's spatial perspective , people can take another person 's temporal perspective when travelling through time . to address this issue , in the present study we directly compared self- and other - perspective time travelling with the goal of testing whether / how the representation of time varies as a function of perspective taking . the psychological ability to traverse temporal distances is dependent upon a cognitive representation of time that has been suggested to be spatial in nature ( e.g. , [ 2 , 812 ] ; for an overview see ) . in this conceptualization , time travel is characterized by three basic components : ( a ) temporal location , that is , the point in ( space- ) time from where the travel originates [ 8 , 14 , 15 ] ; ( b ) motion direction , that is , the direction along which projection takes place ( either backwards or forwards with respect to the temporal location ) [ 8 , 14 ] ; and ( c ) temporal duration , that is , the temporal interval to be travelled ( for a description of similar concepts , see also ) . within this framework , it has been demonstrated that rather than being mapped to space in a uniform manner , spatiotemporal representation presents areas of different granularity . for instance , christian and colleagues found that when asked to temporally locate events on a time line , participants used more space ( -time ) to represent one year in the present than in the past or the future . in a similar vein , arzy and colleagues showed that , irrespective of the temporal location , participants were faster and more accurate when asked to retrieve an event or a face located forwards rather than backwards ( relative future effects in ; see also ) . in addition , the speed of self - projection in time has been shown to depend logarithmically on the temporal distance between the imagined self - location in time and the location of the imagined event / face to retrieve . these patterns relate to the self - referenced topography of space - time mapping . to investigate whether similar patterns also apply to other - projection in time , here , we asked participants to imagine themselves or someone else at a specific point in time ( i.e. , past , present , or future ) and to operate a notional time machine for travelling either backwards or forwards as to reach a target destination ( i.e. , one , three , or five years back / ahead ) . we used the travel duration as a proxy for how space - time representation depended on the perspective taking ( self- versus other - perspective ) . based on previous evidence that the amount of space used to represent time varies as a function of self - relevance , we expected that travelling in the self - perspective would take longer than travelling in the other - perspective . moreover , we hypothesized that this effect of perspective would be greater when the travel originated in the present than in the past or future . in spite of these differences , however , we also expected similarities between self- and other - perspective time travelling . in particular , we predicted that , for both self- and other - perspective , travel duration would increase as a function of the temporal distance to be travelled . finally , we expected that , in both perspectives and regardless of temporal location , travelling would be facilitated for the forwards motion direction ( see relative future effect ) . twenty - five participants ( 15 females , aged between 20 and 26 years , mean sd 23 1.7 years ) from the university of turin took part in the study . all participants were right - handed and had normal or corrected to normal vision and no history of neurological or psychiatric disorders . all participants gave written informed consent before inclusion in the study , which was conducted in accordance with the principles of the revised helsinki declaration and approved by the ethical committee of the university of turin . participants were seated at a desk approximately 50 cm away from a 17 computer screen ( refresh rate = 60 hz ) . at the beginning of the experiment , they were told that their task was to operate a notional time machine to travel through time . next , they were given instructions about the temporal perspective ( self versus other ) and the time locations at which to imagine themselves . on other - perspective trials , a female coexperimenter was seated at a desk placed perpendicularly with respect to participant 's desk ( at a distance of ~1.5 m ) , in front of a computer monitor connected to participant 's monitor . while sitting at their desk , participants could not see what was displayed onto the monitor in front of the coexperimenter . to control for possible effects related to age difference , participants were recruited as to be similar in age to the coexperimenter . at the beginning of the experiment , each trial began with the instruction to imagine oneself ( for self - perspective trials ) or the other person ( for other - perspective trials ) at specific past ( i.e. , the day of your / coexperimenter 's 10th birthday ) , present ( i.e. , today ) , or future location in time ( i.e. , the day of your / coexperimenter 's 50th birthday ) . as participants were in their late adolescence / early adulthood , past and future locations corresponded to two stages of development markedly distinct from their own ( i.e. , a point in middle childhood and a point in middle adulthood , resp . ) . from these locations , participants were instructed to move one , three , or five years , either backwards or forwards . to support the experience of travelling through time , an animated star - field display was projected onto the screen in front of the participant [ 17 , 19 , 20 ] . the display consisted of approximately 1000 randomly positioned white dots on a black background ( see figure 1 ) . the dots ( i.e. , stars ) were animated ( 25 fps ) so as to appear to move , on a linear trajectory , either toward ( i.e. , centripetally ) or away from ( i.e. , centrifugally ) the center of the display , corresponding to the experience of backwards and forwards self - motion ( figure 1 ) . journeys in the past were accompanied by backwards optic flow , while those in future were accompanied by forwards flow ( for a similar paradigm see ) . on other - perspective trials , the same star - field display was projected onto the screen in front of participant and onto the screen in front of the coexperimenter . in both self- and other - perspective trials , the participant was instructed to look at the screen in front of him / her for the entire duration of the experiment . at a self - paced interval , participants were requested to press a button ( i.e. , space bar ) to begin the time travel ( i.e. , initiate the optic flow ) and press it again once they felt they had reached the target ( i.e. , stop the optic flow ) . the time travel duration was calculated as the interval between these two events . finally , to ensure participants ' compliance with the task requests , in 20% of the experimental trials ( a total of 72 trials per each participant , with 18 trials per each block ) , we asked participants to estimate the actual duration of their last time travel ( in ms ) . each of the four blocks comprised 90 trials for a total of 360 trials . for each perspective condition ( self , other ) , ten trials for each temporal location ( past , present , and future ) by motion direction ( backwards , forwards ) by temporal duration ( one , three , and five years ) combination were administered . within each block , temporal location presentation was fully randomized and administered in miniblock of 6 trials ( i.e. , one trial for each temporal duration by motion direction combination ) . to familiarize participants with the procedure , at the beginning of the experiment a practice session was administered ( 6 self - perspective and 6 other - perspective - trials , one trial for each motion direction by temporal duration combination ) . pittsburgh , pa , usa ) running on a pc was used to present trials and record the duration of the time travel ( i.e. , the time elapsed between starting and stopping the time machine ) . the experiment lasted about 60 minutes . to cope with the high variability within the range of travel duration ( 39421304 ms ) , we converted individual temporal intervals data into z - scores , based on means and standard deviations computed over all trials per each participant . since each standardized coefficient scales appropriately to adjust for the disparity in the variable sizes , this procedure makes it possible to bring all of the variables into proportion with one another without losing the possibility to directly compare participants ' performance across conditions . the mean z - scores were then averaged separately for each trial type ( i.e. , temporal location by each motion direction by temporal duration in both perspectives ) . the investigation of standardized data distribution using one - sample kolmogorov - smirnov goodness - of - fit tests did not show any significant difference , suggesting that data distribution within the sample was gaussian ( 0.196 < ps > 0.998 ) . travel duration z - scores were then submitted to a repeated measures anova with temporal location ( past , present , and future ) , motion direction ( backwards , forwards ) , temporal duration ( 1 , 3 , and 5 years ) , and perspective ( self , other ) as within subjects factors . main effects were used to explore the means of interest ( post hoc t - test ) , and bonferroni 's corrections ( level of p < 0.05 ) were applied . in addition , to obtain an indirect measure of participants ' compliance with the task requests over the experimental session , we analyzed the data from the control task to test whether participants ' ability to reproduce time intervals decreased / increased over the four blocks . to this aim , we first computed a temporal accuracy estimation index , defined as the difference between the actual duration of the travel and the corresponding temporal estimation given by the participant at the control task trial ( i.e. , 20% of the total amount of trials : 18 trials per each block for a total of 72 trials by each participant ) . then , we submitted this index to a repeated measures anova with block ( i.e. , first , second , third , and fourth ) as within subjects factor . participants ' travel duration varied as a function of temporal location so that travels originating in the present moment were longer than travels originating in the past or in the future ( main effect of temporal location : f(2,48 ) = 3.621 ; p = 0.034 ; partial eta square = 0.131 ; mean z - scores = 0.078 , 0.004 , and 0.082 , resp . ; ps < 0.05 ) . moreover , temporal duration of journeys was longer for travelling forwards than backwards ( main effect of motion direction : f(1,24 ) = 7.305 ; p = 0.012 ; partial eta square = 0.233 ; mean z - scores = 0.038 versus 0.038 , resp . ; ps < 0.05 ) . regardless of the temporal origin of the journey and its direction , the travel duration increased as a function of the number of years to travel ( main effect of temporal duration : f(2,48 ) = 277.747 ; p < 0.001 ; partial eta square = 0.920 ; mean z scores = 0.86 for 1 year ; 0.01 for 3 years ; 0.86 for 5 years ; see figure 2 , ps < 0.05 ) . as predicted , neither motion direction nor temporal duration main effect were further qualified by significant interactions by perspective . in contrast , the effect of temporal location varied between self- and other - perspective ( perspective by temporal location interaction : f(2,48 ) = 6.014 ; p = 0.005 ; partial eta square = 0.200 ) . for self - perspective trials , indeed , travel duration was longer when the travel started either in the past or in the present rather than in the future ( 0.034 and 0.163 versus 0.101 , resp . ; see figure 2 ; ps < 0.05 ) . for other - perspective trials , in contrast , no similar modulation of travel duration by temporal location was reported ( 0.007 , 0.025 , and 0.062 for present , past , and future location ; figure 2 ; ps > 0.05 ) . moreover , travel duration was on average longer for the self - perspective than for the other - perspective when the travel started in the present , but not when it started in the past or in the future ( self / present versus other / present = 0.163 versus 0.007 , resp . ; p < 0.05 ; self / past versus other / past = 0.034 versus 0.025 and self / future versus other / future = 0.101 versus 0.062 , resp . ; ps > 0.05 ) . finally , the exploration of the significant temporal location by temporal duration interaction ( f(4,96 ) = 3.780 ; p = 0.007 ; partial eta square = 0.136 ) revealed that there was no difference across temporal locations when travels lasted either 1 year or 3 years ( 1 year : past = 0.897 ; present = 0.793 ; future = 0.912 and 3 years : past = 0.016 ; present = 0.057 ; future = 0.075 ; ps > 0.05 ) . in contrast , when participants were requested to cover a 5-year distance , the trip took longer when it started in the past or present rather than in the future ( 5 years : past = 0.926 and present = 0.968 versus future = 0.74 ; ps < 0.5 ) . neither the main effect of perspective ( f(1,24 ) = 1.588 ; p > 0.05 ; partial eta square = 0.062 ) nor the remaining two- , three- , or four - way interactions were found to be significant ( all fs < 1.484 ; 0.213 < ps > 0.942 ; all partial eta squares < 0.058 ) . control task . anova revealed no significant effect of block ( f3,72 = 2.212 ; p > 0.05 ; partial eta square = 0.084 ) on temporal accuracy estimation index , suggesting that participants ' compliance to the task request remained stable throughout the experiment . in this study , we assessed whether and how self- and other - projections in time map onto similar or different spatiotemporal representations . for the properties of this underlying representation to emerge , we used as a proxy the time taken to move through time ( i.e. , travel duration ) and manipulated three basic properties of time journeys : temporal location ( past , present , or future ) , motion direction ( backwards or forwards ) , and temporal duration ( one , three , or five years ) . our results suggest that self- and other - projections hinge on different temporal representations depending on the temporal location , that is , on where in time the mental travel originates . specifically , for self - perspective , participants took longer to cover identical distances when the travel started in the past or in present compared as to when it started in the future . for other - perspective , in contrast , travel duration was not modulated by temporal location . this effect may reflect the tendency to form higher - level construals of information about remote future event . in this respect , trope and liberman suggest that the greater the temporal distance from a future event , the more likely is the event to be represented abstractly in terms of a few general features that convey the perceived essence of events rather than in terms of more concrete and incidental details of the event . in the same vein , d'argembeau and van der linden provided evidence that projecting oneself in a specific positive or negative experience results in a richer representation when the event is expected to be experienced in the near future rather than in a more distant future . on this account , participants would travel more rapidly from a remote future location because they would simulate future events in more abstract and general terms . this is further supported by the consideration that , in contrast with remembering of past events whose features are already integrated , simulating remote future events requires the combination of disparate details gleaned from a variety of episodic sources . forming abstract representations of remote future event may thus serve a specific adaptive role in reducing the costs required to integrate unrelated details into a coherent future representation . over and above this , self- and other - projection also scaled differently for mental travels taking place in the proximity of the present moment . for the present location , regardless of motion direction , one year was indeed longer in the self - time rather than in the other - time . this was not the case for one - year travels originating from a past or future location . this pattern is in agreement with the finding that participants represent self - time as occupying a greater amount of space than an equivalent period related to others . of direct relevance to the present study , christian and colleagues asked three different groups of participants to time - travel from the present moment to their own birthdays or to the birthdays of either a close friend or a hypothetical stranger of a similar age as theirs . it was found that , irrespective of motion direction ( either in the past or in the future ) , time relevant to self was represented as occupying more space than time relevant to others ( i.e. , best friend or unfamiliar other ) . on a closer examination , however , this effect of self - relevance was only evident for temporally close events ( i.e. , 10 years before or after the present moment ) , but not more distant events ( i.e. , the day of 8th or 58th birthday ) . taken together with our results , this suggests that a distinctive relationship bounds the self to the now . as observed by nez and cooperrider , indeed , within an internal perspective , the ego is always and inherently colocated within the now . it is perhaps therefore not surprising that self - projection is more embedded in the representation of the present time compared to other - projection . on a related note , this could explain why recent autobiographical memories tend to be recalled from the first - person perspective , while more remote memories , particularly early childhood memories , are more likely to be recalled from a third - person perspective [ 2427 ] . despite these differences , however , self- and other - projections also shared many similarities in structure . first , for both self- and other - perspectives , travel durations increased as a function of temporal duration ( one year < three years < five years ) . with only one exception ( i.e. , five years from past and present lasts longer than five years from future ) , this effect was not modulated by the temporal location ( i.e. , past , present , and future ) . this is consistent with the idea that computation of temporal quantities rests on a common system for magnitude processing , which is ( at least in part ) independent of self - relevant content specificity and episodic memory processes . the finding that 5-year travel duration lasted less when it originated in remote future might be taken to suggest that , as far as remote future is concerned , the spatial representation of time is compressed towards the anchoring point . experiments using multiple temporal locations in remote past and future might help to address this issue . second , in both self- and other - perspectives , independent of temporal location , travelling forwards took longer than travelling backwards . the neural system subserving mental time travel has been proposed to have evolved to anticipate and pilot our behavior rather than primarily encoding the past [ 8 , 29 , 30 ] . in line with this , processing of events has been shown to be future oriented across present , past , and future self - location [ 8 , 14 ] . for example , when asked to judge whether an event takes place before ( relative past ) or after ( relative future ) an imagined self - location in time , participants are typically faster and more accurate for relative future than for relative past events . at first sight , the finding that travelling forwards took longer than travelling backwards may seem contradictory to response facilitation for future events . however , both phenomena may be parsimoniously interpreted as resulting from an anisometric pattern of internal spatial representation of relative past and future events , such that the representational medium is compressed towards the relative past and dilated towards the relative future . compression towards the relative past would explain why travelling backwards lasts less , but also why judgements are more difficult ( i.e. , slower and less accurate ) to the relative past . along the same lines , dilation towards the relative future may account for both the increase in travel duration for travelling forwards and the relative future response facilitation . an alternative , not mutually exclusive , explanation of the motion direction effect refers to discrepancy between external time direction ( i.e. , roughly , the time of the calendar ) and subjective time direction ( i.e. , the personal time which is measured by the traveler 's wristwatch ; ) potentially experienced when travelling backwards . when participants are requested to time - travel towards relative future , the external time and their subjective time move towards the same direction ( see also , for similar concepts ) . the subjective time may therefore be expected to add to the external time , extending the travel duration . in contrast , when participants travel towards relative past , the external time goes backwards , while their subjective time moves forwards . , the effect of motion direction would reflect the relative ( forward ) motion of the traveler making journeys end later in the future and earlier in the past . at this stage , both these hypotheses remain speculative and require further study for elaboration and validation . a growing body of evidence indicates that self - projection in space and in time might rest on a common neural network and share similar cognitive processes and representations . in the visuospatial domain , it has been documented that people can transpose their own actual point of view and navigate space from the perspective of someone else . the current findings suggest that a similar ability might also exist in temporal domain , supporting the notion of temporal perspective taking . by contrasting time travelling from self- and other - perspective , we found evidence that temporal representation underlying one 's own projection shares many of the same characteristics of the temporal representation underlying another person 's projection . despite the fact that a greater sensitivity to temporal location for representing time in self- rather than in other - perspective emerges , when considering more abstract properties as direction and magnitude , self- and other - time exhibit a similar structure . further research is warranted to clarify whether and to what extent these effects are sensitive to the degree of similarity between the self and the other person . for example , it will be important for future research to determine how travelling in time from the perspective of a younger or older person impacts on travel duration ( depending , e.g. , on whether the past / future of the participant overlaps with the present of the other person ) . a second issue to be addressed in future studies self - projection in time has shown to recruit a network of brain areas in distinct time periods including the occipitotemporal , temporoparietal , and anteromedial temporal cortices [ 4 , 8 ] . for example , it has been reported that during mental time travel the left lateral parietal cortex is differentially activated by nonpresent subjective times compared with present ( past and future > present ) . capitalizing on the finding that left parietal cortex supports first - person perspective simulation , these results have been interpreted to suggest that the parietal cortex is specifically related to transformations in subjective time . moreover it has been demonstrated that temporal self - projection into the personal past recruits greater ventral medial prefrontal cortex ( mpcf ) whereas self - projection into another person 's perspective recruits greater dorsal mpcf . asking participants to simulate mentally past , present , and future time from their own versus another person 's perspective might help to clarify how transformations in subjective and nonsubjective time are represented in left parietal cortex and to elucidate the exact contribution of the ventral and dorsal subregions of mpcf to self- versus other - projection . finally , the hypothesis of a partial overlap between self- and other - mechanisms for projection in time could be tested in neuropsychiatric patients with temporal orientation failures . to the extent that self- and other - projection rely on a common neural mechanism , self - referenced and other - referenced disorientation may be expected to share common fundamental characteristics .

recent years have seen accumulating evidence for the proposition that people process time by mapping it onto a linear spatial representation and automatically project themselves on an imagined mental time line . here , we ask whether people can adopt the temporal perspective of another person when travelling through time . to elucidate similarities and differences between time travelling from one 's own perspective or from the perspective of another person , we asked participants to mentally project themselves or someone else ( i.e. , a coexperimenter ) to different time points . three basic properties of mental time travel were manipulated : temporal location ( i.e. , where in time the travel originates : past , present , and future ) , motion direction ( either backwards or forwards ) , and temporal duration ( i.e. , the distance to travel : one , three , or five years ) . we found that time travels originating in the present lasted longer in the self- than in the other - perspective . moreover , for self - perspective , but not for other - perspective , time was differently scaled depending on where in time the travel originated . in contrast , when considering the direction and the duration of time travelling , no dissimilarities between the self- and the other - perspective emerged . these results suggest that self- and other - projection , despite some differences , share important similarities in structure .

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

when imagining time and their own life events , humans do not only retrieve or predict when events have occurred or will occur , but also automatically project themselves on an imagined mental time line . self - time travelling can thus be regarded as the ability to transpose one 's habitual self - location in time to different temporal locations in the past or the future . emphasizing the role of perspective taking , this ability to change one 's own temporal egocentric perspective has been proposed to share a common mechanism with the ability to change one 's own spatial egocentric perspective . on this account , the same processes that subserve simulation of the self at a different location in space would also subserve simulation of the self at a different point in time . an interesting question , inspired by this parallel , is whether , similarly to taking another person 's spatial perspective , people can also adopt the temporal perspective of another person when travelling through time . studies investigating spatial perspective taking indicate that people can overcome their own position in space to adopt another person 's spatial perspective ( e.g. when the scene includes another person , for instance , people may spontaneously describe spatial relations from that person 's perspective despite the very real presence of their own [ 5 , 6 ] . these and other findings suggest that spatial perspective taking may induce an alterocentric remapping , that is , remapping of objects and locations to an alterocentric frame of reference . to our best knowledge , no study has so far investigated whether a similar remapping may take place in the temporal domain . in other words , whether similarly to taking another person 's spatial perspective , people can take another person 's temporal perspective when travelling through time . to address this issue , in the present study we directly compared self- and other - perspective time travelling with the goal of testing whether / how the representation of time varies as a function of perspective taking . in this conceptualization , time travel is characterized by three basic components : ( a ) temporal location , that is , the point in ( space- ) time from where the travel originates [ 8 , 14 , 15 ] ; ( b ) motion direction , that is , the direction along which projection takes place ( either backwards or forwards with respect to the temporal location ) [ 8 , 14 ] ; and ( c ) temporal duration , that is , the temporal interval to be travelled ( for a description of similar concepts , see also ) . for instance , christian and colleagues found that when asked to temporally locate events on a time line , participants used more space ( -time ) to represent one year in the present than in the past or the future . in a similar vein , arzy and colleagues showed that , irrespective of the temporal location , participants were faster and more accurate when asked to retrieve an event or a face located forwards rather than backwards ( relative future effects in ; see also ) . in addition , the speed of self - projection in time has been shown to depend logarithmically on the temporal distance between the imagined self - location in time and the location of the imagined event / face to retrieve . to investigate whether similar patterns also apply to other - projection in time , here , we asked participants to imagine themselves or someone else at a specific point in time ( i.e. , past , present , or future ) and to operate a notional time machine for travelling either backwards or forwards as to reach a target destination ( i.e. , one , three , or five years back / ahead ) . we used the travel duration as a proxy for how space - time representation depended on the perspective taking ( self- versus other - perspective ) . based on previous evidence that the amount of space used to represent time varies as a function of self - relevance , we expected that travelling in the self - perspective would take longer than travelling in the other - perspective . moreover , we hypothesized that this effect of perspective would be greater when the travel originated in the present than in the past or future . in spite of these differences , however , we also expected similarities between self- and other - perspective time travelling . in particular , we predicted that , for both self- and other - perspective , travel duration would increase as a function of the temporal distance to be travelled . finally , we expected that , in both perspectives and regardless of temporal location , travelling would be facilitated for the forwards motion direction ( see relative future effect ) . twenty - five participants ( 15 females , aged between 20 and 26 years , mean sd 23 1.7 years ) from the university of turin took part in the study . at the beginning of the experiment , they were told that their task was to operate a notional time machine to travel through time . next , they were given instructions about the temporal perspective ( self versus other ) and the time locations at which to imagine themselves . on other - perspective trials , a female coexperimenter was seated at a desk placed perpendicularly with respect to participant 's desk ( at a distance of ~1.5 m ) , in front of a computer monitor connected to participant 's monitor . at the beginning of the experiment , each trial began with the instruction to imagine oneself ( for self - perspective trials ) or the other person ( for other - perspective trials ) at specific past ( i.e. , the day of your / coexperimenter 's 10th birthday ) , present ( i.e. , today ) , or future location in time ( i.e. as participants were in their late adolescence / early adulthood , past and future locations corresponded to two stages of development markedly distinct from their own ( i.e. from these locations , participants were instructed to move one , three , or five years , either backwards or forwards . to support the experience of travelling through time , an animated star - field display was projected onto the screen in front of the participant [ 17 , 19 , 20 ] . , stars ) were animated ( 25 fps ) so as to appear to move , on a linear trajectory , either toward ( i.e. on other - perspective trials , the same star - field display was projected onto the screen in front of participant and onto the screen in front of the coexperimenter . in both self- and other - perspective trials , the participant was instructed to look at the screen in front of him / her for the entire duration of the experiment . at a self - paced interval , participants were requested to press a button ( i.e. , space bar ) to begin the time travel ( i.e. finally , to ensure participants ' compliance with the task requests , in 20% of the experimental trials ( a total of 72 trials per each participant , with 18 trials per each block ) , we asked participants to estimate the actual duration of their last time travel ( in ms ) . for each perspective condition ( self , other ) , ten trials for each temporal location ( past , present , and future ) by motion direction ( backwards , forwards ) by temporal duration ( one , three , and five years ) combination were administered . within each block , temporal location presentation was fully randomized and administered in miniblock of 6 trials ( i.e. , one trial for each temporal duration by motion direction combination ) . to familiarize participants with the procedure , at the beginning of the experiment a practice session was administered ( 6 self - perspective and 6 other - perspective - trials , one trial for each motion direction by temporal duration combination ) . pittsburgh , pa , usa ) running on a pc was used to present trials and record the duration of the time travel ( i.e. to cope with the high variability within the range of travel duration ( 39421304 ms ) , we converted individual temporal intervals data into z - scores , based on means and standard deviations computed over all trials per each participant . , temporal location by each motion direction by temporal duration in both perspectives ) . travel duration z - scores were then submitted to a repeated measures anova with temporal location ( past , present , and future ) , motion direction ( backwards , forwards ) , temporal duration ( 1 , 3 , and 5 years ) , and perspective ( self , other ) as within subjects factors . main effects were used to explore the means of interest ( post hoc t - test ) , and bonferroni 's corrections ( level of p < 0.05 ) were applied . in addition , to obtain an indirect measure of participants ' compliance with the task requests over the experimental session , we analyzed the data from the control task to test whether participants ' ability to reproduce time intervals decreased / increased over the four blocks . to this aim , we first computed a temporal accuracy estimation index , defined as the difference between the actual duration of the travel and the corresponding temporal estimation given by the participant at the control task trial ( i.e. then , we submitted this index to a repeated measures anova with block ( i.e. participants ' travel duration varied as a function of temporal location so that travels originating in the present moment were longer than travels originating in the past or in the future ( main effect of temporal location : f(2,48 ) = 3.621 ; p = 0.034 ; partial eta square = 0.131 ; mean z - scores = 0.078 , 0.004 , and 0.082 , resp . moreover , temporal duration of journeys was longer for travelling forwards than backwards ( main effect of motion direction : f(1,24 ) = 7.305 ; p = 0.012 ; partial eta square = 0.233 ; mean z - scores = 0.038 versus 0.038 , resp . regardless of the temporal origin of the journey and its direction , the travel duration increased as a function of the number of years to travel ( main effect of temporal duration : f(2,48 ) = 277.747 ; p < 0.001 ; partial eta square = 0.920 ; mean z scores = 0.86 for 1 year ; 0.01 for 3 years ; 0.86 for 5 years ; see figure 2 , ps < 0.05 ) . as predicted , neither motion direction nor temporal duration main effect were further qualified by significant interactions by perspective . in contrast , the effect of temporal location varied between self- and other - perspective ( perspective by temporal location interaction : f(2,48 ) = 6.014 ; p = 0.005 ; partial eta square = 0.200 ) . for self - perspective trials , indeed , travel duration was longer when the travel started either in the past or in the present rather than in the future ( 0.034 and 0.163 versus 0.101 , resp . for other - perspective trials , in contrast , no similar modulation of travel duration by temporal location was reported ( 0.007 , 0.025 , and 0.062 for present , past , and future location ; figure 2 ; ps > 0.05 ) . moreover , travel duration was on average longer for the self - perspective than for the other - perspective when the travel started in the present , but not when it started in the past or in the future ( self / present versus other / present = 0.163 versus 0.007 , resp . finally , the exploration of the significant temporal location by temporal duration interaction ( f(4,96 ) = 3.780 ; p = 0.007 ; partial eta square = 0.136 ) revealed that there was no difference across temporal locations when travels lasted either 1 year or 3 years ( 1 year : past = 0.897 ; present = 0.793 ; future = 0.912 and 3 years : past = 0.016 ; present = 0.057 ; future = 0.075 ; ps > 0.05 ) . in contrast , when participants were requested to cover a 5-year distance , the trip took longer when it started in the past or present rather than in the future ( 5 years : past = 0.926 and present = 0.968 versus future = 0.74 ; ps < 0.5 ) . in this study , we assessed whether and how self- and other - projections in time map onto similar or different spatiotemporal representations . for the properties of this underlying representation to emerge , we used as a proxy the time taken to move through time ( i.e. , travel duration ) and manipulated three basic properties of time journeys : temporal location ( past , present , or future ) , motion direction ( backwards or forwards ) , and temporal duration ( one , three , or five years ) . our results suggest that self- and other - projections hinge on different temporal representations depending on the temporal location , that is , on where in time the mental travel originates . specifically , for self - perspective , participants took longer to cover identical distances when the travel started in the past or in present compared as to when it started in the future . for other - perspective , in contrast , travel duration was not modulated by temporal location . in this respect , trope and liberman suggest that the greater the temporal distance from a future event , the more likely is the event to be represented abstractly in terms of a few general features that convey the perceived essence of events rather than in terms of more concrete and incidental details of the event . in the same vein , d'argembeau and van der linden provided evidence that projecting oneself in a specific positive or negative experience results in a richer representation when the event is expected to be experienced in the near future rather than in a more distant future . over and above this , self- and other - projection also scaled differently for mental travels taking place in the proximity of the present moment . for the present location , regardless of motion direction , one year was indeed longer in the self - time rather than in the other - time . of direct relevance to the present study , christian and colleagues asked three different groups of participants to time - travel from the present moment to their own birthdays or to the birthdays of either a close friend or a hypothetical stranger of a similar age as theirs . it was found that , irrespective of motion direction ( either in the past or in the future ) , time relevant to self was represented as occupying more space than time relevant to others ( i.e. on a closer examination , however , this effect of self - relevance was only evident for temporally close events ( i.e. , 10 years before or after the present moment ) , but not more distant events ( i.e. as observed by nez and cooperrider , indeed , within an internal perspective , the ego is always and inherently colocated within the now . it is perhaps therefore not surprising that self - projection is more embedded in the representation of the present time compared to other - projection . on a related note , this could explain why recent autobiographical memories tend to be recalled from the first - person perspective , while more remote memories , particularly early childhood memories , are more likely to be recalled from a third - person perspective [ 2427 ] . despite these differences , however , self- and other - projections also shared many similarities in structure . first , for both self- and other - perspectives , travel durations increased as a function of temporal duration ( one year < three years < five years ) . with only one exception ( i.e. , five years from past and present lasts longer than five years from future ) , this effect was not modulated by the temporal location ( i.e. , past , present , and future ) . the finding that 5-year travel duration lasted less when it originated in remote future might be taken to suggest that , as far as remote future is concerned , the spatial representation of time is compressed towards the anchoring point . second , in both self- and other - perspectives , independent of temporal location , travelling forwards took longer than travelling backwards . in line with this , processing of events has been shown to be future oriented across present , past , and future self - location [ 8 , 14 ] . for example , when asked to judge whether an event takes place before ( relative past ) or after ( relative future ) an imagined self - location in time , participants are typically faster and more accurate for relative future than for relative past events . however , both phenomena may be parsimoniously interpreted as resulting from an anisometric pattern of internal spatial representation of relative past and future events , such that the representational medium is compressed towards the relative past and dilated towards the relative future . compression towards the relative past would explain why travelling backwards lasts less , but also why judgements are more difficult ( i.e. an alternative , not mutually exclusive , explanation of the motion direction effect refers to discrepancy between external time direction ( i.e. , roughly , the time of the calendar ) and subjective time direction ( i.e. , the personal time which is measured by the traveler 's wristwatch ; ) potentially experienced when travelling backwards . when participants are requested to time - travel towards relative future , the external time and their subjective time move towards the same direction ( see also , for similar concepts ) . in contrast , when participants travel towards relative past , the external time goes backwards , while their subjective time moves forwards . , the effect of motion direction would reflect the relative ( forward ) motion of the traveler making journeys end later in the future and earlier in the past . a growing body of evidence indicates that self - projection in space and in time might rest on a common neural network and share similar cognitive processes and representations . in the visuospatial domain , it has been documented that people can transpose their own actual point of view and navigate space from the perspective of someone else . the current findings suggest that a similar ability might also exist in temporal domain , supporting the notion of temporal perspective taking . by contrasting time travelling from self- and other - perspective , we found evidence that temporal representation underlying one 's own projection shares many of the same characteristics of the temporal representation underlying another person 's projection . despite the fact that a greater sensitivity to temporal location for representing time in self- rather than in other - perspective emerges , when considering more abstract properties as direction and magnitude , self- and other - time exhibit a similar structure . further research is warranted to clarify whether and to what extent these effects are sensitive to the degree of similarity between the self and the other person . for example , it will be important for future research to determine how travelling in time from the perspective of a younger or older person impacts on travel duration ( depending , e.g. a second issue to be addressed in future studies self - projection in time has shown to recruit a network of brain areas in distinct time periods including the occipitotemporal , temporoparietal , and anteromedial temporal cortices [ 4 , 8 ] . for example , it has been reported that during mental time travel the left lateral parietal cortex is differentially activated by nonpresent subjective times compared with present ( past and future > present ) . capitalizing on the finding that left parietal cortex supports first - person perspective simulation , these results have been interpreted to suggest that the parietal cortex is specifically related to transformations in subjective time . moreover it has been demonstrated that temporal self - projection into the personal past recruits greater ventral medial prefrontal cortex ( mpcf ) whereas self - projection into another person 's perspective recruits greater dorsal mpcf . asking participants to simulate mentally past , present , and future time from their own versus another person 's perspective might help to clarify how transformations in subjective and nonsubjective time are represented in left parietal cortex and to elucidate the exact contribution of the ventral and dorsal subregions of mpcf to self- versus other - projection . finally , the hypothesis of a partial overlap between self- and other - mechanisms for projection in time could be tested in neuropsychiatric patients with temporal orientation failures . to the extent that self- and other - projection rely on a common neural mechanism , self - referenced and other - referenced disorientation may be expected to share common fundamental characteristics .

self - time travelling can thus be regarded as the ability to transpose one 's habitual self - location in time to different temporal locations in the past or the future . emphasizing the role of perspective taking , this ability to change one 's own temporal egocentric perspective has been proposed to share a common mechanism with the ability to change one 's own spatial egocentric perspective . in both domains , people would use existing representations as templates for processing and understanding new information , in order to plan their short- and long - term behaviors . on this account , the same processes that subserve simulation of the self at a different location in space would also subserve simulation of the self at a different point in time . an interesting question , inspired by this parallel , is whether , similarly to taking another person 's spatial perspective , people can also adopt the temporal perspective of another person when travelling through time . when the scene includes another person , for instance , people may spontaneously describe spatial relations from that person 's perspective despite the very real presence of their own [ 5 , 6 ] . these and other findings suggest that spatial perspective taking may induce an alterocentric remapping , that is , remapping of objects and locations to an alterocentric frame of reference . to our best knowledge , no study has so far investigated whether a similar remapping may take place in the temporal domain . to address this issue , in the present study we directly compared self- and other - perspective time travelling with the goal of testing whether / how the representation of time varies as a function of perspective taking . the psychological ability to traverse temporal distances is dependent upon a cognitive representation of time that has been suggested to be spatial in nature ( e.g. in this conceptualization , time travel is characterized by three basic components : ( a ) temporal location , that is , the point in ( space- ) time from where the travel originates [ 8 , 14 , 15 ] ; ( b ) motion direction , that is , the direction along which projection takes place ( either backwards or forwards with respect to the temporal location ) [ 8 , 14 ] ; and ( c ) temporal duration , that is , the temporal interval to be travelled ( for a description of similar concepts , see also ) . within this framework , it has been demonstrated that rather than being mapped to space in a uniform manner , spatiotemporal representation presents areas of different granularity . for instance , christian and colleagues found that when asked to temporally locate events on a time line , participants used more space ( -time ) to represent one year in the present than in the past or the future . in a similar vein , arzy and colleagues showed that , irrespective of the temporal location , participants were faster and more accurate when asked to retrieve an event or a face located forwards rather than backwards ( relative future effects in ; see also ) . in addition , the speed of self - projection in time has been shown to depend logarithmically on the temporal distance between the imagined self - location in time and the location of the imagined event / face to retrieve . these patterns relate to the self - referenced topography of space - time mapping . to investigate whether similar patterns also apply to other - projection in time , here , we asked participants to imagine themselves or someone else at a specific point in time ( i.e. we used the travel duration as a proxy for how space - time representation depended on the perspective taking ( self- versus other - perspective ) . based on previous evidence that the amount of space used to represent time varies as a function of self - relevance , we expected that travelling in the self - perspective would take longer than travelling in the other - perspective . moreover , we hypothesized that this effect of perspective would be greater when the travel originated in the present than in the past or future . in spite of these differences , however , we also expected similarities between self- and other - perspective time travelling . in particular , we predicted that , for both self- and other - perspective , travel duration would increase as a function of the temporal distance to be travelled . finally , we expected that , in both perspectives and regardless of temporal location , travelling would be facilitated for the forwards motion direction ( see relative future effect ) . twenty - five participants ( 15 females , aged between 20 and 26 years , mean sd 23 1.7 years ) from the university of turin took part in the study . all participants gave written informed consent before inclusion in the study , which was conducted in accordance with the principles of the revised helsinki declaration and approved by the ethical committee of the university of turin . at the beginning of the experiment , they were told that their task was to operate a notional time machine to travel through time . next , they were given instructions about the temporal perspective ( self versus other ) and the time locations at which to imagine themselves . on other - perspective trials , a female coexperimenter was seated at a desk placed perpendicularly with respect to participant 's desk ( at a distance of ~1.5 m ) , in front of a computer monitor connected to participant 's monitor . to control for possible effects related to age difference , participants were recruited as to be similar in age to the coexperimenter . at the beginning of the experiment , each trial began with the instruction to imagine oneself ( for self - perspective trials ) or the other person ( for other - perspective trials ) at specific past ( i.e. , the day of your / coexperimenter 's 10th birthday ) , present ( i.e. as participants were in their late adolescence / early adulthood , past and future locations corresponded to two stages of development markedly distinct from their own ( i.e. to support the experience of travelling through time , an animated star - field display was projected onto the screen in front of the participant [ 17 , 19 , 20 ] . , centrifugally ) the center of the display , corresponding to the experience of backwards and forwards self - motion ( figure 1 ) . journeys in the past were accompanied by backwards optic flow , while those in future were accompanied by forwards flow ( for a similar paradigm see ) . on other - perspective trials , the same star - field display was projected onto the screen in front of participant and onto the screen in front of the coexperimenter . in both self- and other - perspective trials , the participant was instructed to look at the screen in front of him / her for the entire duration of the experiment . finally , to ensure participants ' compliance with the task requests , in 20% of the experimental trials ( a total of 72 trials per each participant , with 18 trials per each block ) , we asked participants to estimate the actual duration of their last time travel ( in ms ) . for each perspective condition ( self , other ) , ten trials for each temporal location ( past , present , and future ) by motion direction ( backwards , forwards ) by temporal duration ( one , three , and five years ) combination were administered . within each block , temporal location presentation was fully randomized and administered in miniblock of 6 trials ( i.e. to familiarize participants with the procedure , at the beginning of the experiment a practice session was administered ( 6 self - perspective and 6 other - perspective - trials , one trial for each motion direction by temporal duration combination ) . pittsburgh , pa , usa ) running on a pc was used to present trials and record the duration of the time travel ( i.e. to cope with the high variability within the range of travel duration ( 39421304 ms ) , we converted individual temporal intervals data into z - scores , based on means and standard deviations computed over all trials per each participant . since each standardized coefficient scales appropriately to adjust for the disparity in the variable sizes , this procedure makes it possible to bring all of the variables into proportion with one another without losing the possibility to directly compare participants ' performance across conditions . travel duration z - scores were then submitted to a repeated measures anova with temporal location ( past , present , and future ) , motion direction ( backwards , forwards ) , temporal duration ( 1 , 3 , and 5 years ) , and perspective ( self , other ) as within subjects factors . main effects were used to explore the means of interest ( post hoc t - test ) , and bonferroni 's corrections ( level of p < 0.05 ) were applied . in addition , to obtain an indirect measure of participants ' compliance with the task requests over the experimental session , we analyzed the data from the control task to test whether participants ' ability to reproduce time intervals decreased / increased over the four blocks . to this aim , we first computed a temporal accuracy estimation index , defined as the difference between the actual duration of the travel and the corresponding temporal estimation given by the participant at the control task trial ( i.e. participants ' travel duration varied as a function of temporal location so that travels originating in the present moment were longer than travels originating in the past or in the future ( main effect of temporal location : f(2,48 ) = 3.621 ; p = 0.034 ; partial eta square = 0.131 ; mean z - scores = 0.078 , 0.004 , and 0.082 , resp . moreover , temporal duration of journeys was longer for travelling forwards than backwards ( main effect of motion direction : f(1,24 ) = 7.305 ; p = 0.012 ; partial eta square = 0.233 ; mean z - scores = 0.038 versus 0.038 , resp . regardless of the temporal origin of the journey and its direction , the travel duration increased as a function of the number of years to travel ( main effect of temporal duration : f(2,48 ) = 277.747 ; p < 0.001 ; partial eta square = 0.920 ; mean z scores = 0.86 for 1 year ; 0.01 for 3 years ; 0.86 for 5 years ; see figure 2 , ps < 0.05 ) . in contrast , the effect of temporal location varied between self- and other - perspective ( perspective by temporal location interaction : f(2,48 ) = 6.014 ; p = 0.005 ; partial eta square = 0.200 ) . for self - perspective trials , indeed , travel duration was longer when the travel started either in the past or in the present rather than in the future ( 0.034 and 0.163 versus 0.101 , resp . for other - perspective trials , in contrast , no similar modulation of travel duration by temporal location was reported ( 0.007 , 0.025 , and 0.062 for present , past , and future location ; figure 2 ; ps > 0.05 ) . moreover , travel duration was on average longer for the self - perspective than for the other - perspective when the travel started in the present , but not when it started in the past or in the future ( self / present versus other / present = 0.163 versus 0.007 , resp . ; p < 0.05 ; self / past versus other / past = 0.034 versus 0.025 and self / future versus other / future = 0.101 versus 0.062 , resp . finally , the exploration of the significant temporal location by temporal duration interaction ( f(4,96 ) = 3.780 ; p = 0.007 ; partial eta square = 0.136 ) revealed that there was no difference across temporal locations when travels lasted either 1 year or 3 years ( 1 year : past = 0.897 ; present = 0.793 ; future = 0.912 and 3 years : past = 0.016 ; present = 0.057 ; future = 0.075 ; ps > 0.05 ) . in contrast , when participants were requested to cover a 5-year distance , the trip took longer when it started in the past or present rather than in the future ( 5 years : past = 0.926 and present = 0.968 versus future = 0.74 ; ps < 0.5 ) . neither the main effect of perspective ( f(1,24 ) = 1.588 ; p > 0.05 ; partial eta square = 0.062 ) nor the remaining two- , three- , or four - way interactions were found to be significant ( all fs < 1.484 ; 0.213 < ps > 0.942 ; all partial eta squares < 0.058 ) . anova revealed no significant effect of block ( f3,72 = 2.212 ; p > 0.05 ; partial eta square = 0.084 ) on temporal accuracy estimation index , suggesting that participants ' compliance to the task request remained stable throughout the experiment . in this study , we assessed whether and how self- and other - projections in time map onto similar or different spatiotemporal representations . for the properties of this underlying representation to emerge , we used as a proxy the time taken to move through time ( i.e. , travel duration ) and manipulated three basic properties of time journeys : temporal location ( past , present , or future ) , motion direction ( backwards or forwards ) , and temporal duration ( one , three , or five years ) . our results suggest that self- and other - projections hinge on different temporal representations depending on the temporal location , that is , on where in time the mental travel originates . specifically , for self - perspective , participants took longer to cover identical distances when the travel started in the past or in present compared as to when it started in the future . in this respect , trope and liberman suggest that the greater the temporal distance from a future event , the more likely is the event to be represented abstractly in terms of a few general features that convey the perceived essence of events rather than in terms of more concrete and incidental details of the event . in the same vein , d'argembeau and van der linden provided evidence that projecting oneself in a specific positive or negative experience results in a richer representation when the event is expected to be experienced in the near future rather than in a more distant future . this is further supported by the consideration that , in contrast with remembering of past events whose features are already integrated , simulating remote future events requires the combination of disparate details gleaned from a variety of episodic sources . over and above this , self- and other - projection also scaled differently for mental travels taking place in the proximity of the present moment . for the present location , regardless of motion direction , one year was indeed longer in the self - time rather than in the other - time . this pattern is in agreement with the finding that participants represent self - time as occupying a greater amount of space than an equivalent period related to others . of direct relevance to the present study , christian and colleagues asked three different groups of participants to time - travel from the present moment to their own birthdays or to the birthdays of either a close friend or a hypothetical stranger of a similar age as theirs . it was found that , irrespective of motion direction ( either in the past or in the future ) , time relevant to self was represented as occupying more space than time relevant to others ( i.e. on a closer examination , however , this effect of self - relevance was only evident for temporally close events ( i.e. taken together with our results , this suggests that a distinctive relationship bounds the self to the now . it is perhaps therefore not surprising that self - projection is more embedded in the representation of the present time compared to other - projection . on a related note , this could explain why recent autobiographical memories tend to be recalled from the first - person perspective , while more remote memories , particularly early childhood memories , are more likely to be recalled from a third - person perspective [ 2427 ] . first , for both self- and other - perspectives , travel durations increased as a function of temporal duration ( one year < three years < five years ) . this is consistent with the idea that computation of temporal quantities rests on a common system for magnitude processing , which is ( at least in part ) independent of self - relevant content specificity and episodic memory processes . the finding that 5-year travel duration lasted less when it originated in remote future might be taken to suggest that , as far as remote future is concerned , the spatial representation of time is compressed towards the anchoring point . second , in both self- and other - perspectives , independent of temporal location , travelling forwards took longer than travelling backwards . in line with this , processing of events has been shown to be future oriented across present , past , and future self - location [ 8 , 14 ] . however , both phenomena may be parsimoniously interpreted as resulting from an anisometric pattern of internal spatial representation of relative past and future events , such that the representational medium is compressed towards the relative past and dilated towards the relative future . along the same lines , dilation towards the relative future may account for both the increase in travel duration for travelling forwards and the relative future response facilitation . , the effect of motion direction would reflect the relative ( forward ) motion of the traveler making journeys end later in the future and earlier in the past . a growing body of evidence indicates that self - projection in space and in time might rest on a common neural network and share similar cognitive processes and representations . in the visuospatial domain , it has been documented that people can transpose their own actual point of view and navigate space from the perspective of someone else . by contrasting time travelling from self- and other - perspective , we found evidence that temporal representation underlying one 's own projection shares many of the same characteristics of the temporal representation underlying another person 's projection . despite the fact that a greater sensitivity to temporal location for representing time in self- rather than in other - perspective emerges , when considering more abstract properties as direction and magnitude , self- and other - time exhibit a similar structure . further research is warranted to clarify whether and to what extent these effects are sensitive to the degree of similarity between the self and the other person . for example , it will be important for future research to determine how travelling in time from the perspective of a younger or older person impacts on travel duration ( depending , e.g. , on whether the past / future of the participant overlaps with the present of the other person ) . a second issue to be addressed in future studies self - projection in time has shown to recruit a network of brain areas in distinct time periods including the occipitotemporal , temporoparietal , and anteromedial temporal cortices [ 4 , 8 ] . for example , it has been reported that during mental time travel the left lateral parietal cortex is differentially activated by nonpresent subjective times compared with present ( past and future > present ) . capitalizing on the finding that left parietal cortex supports first - person perspective simulation , these results have been interpreted to suggest that the parietal cortex is specifically related to transformations in subjective time . moreover it has been demonstrated that temporal self - projection into the personal past recruits greater ventral medial prefrontal cortex ( mpcf ) whereas self - projection into another person 's perspective recruits greater dorsal mpcf . asking participants to simulate mentally past , present , and future time from their own versus another person 's perspective might help to clarify how transformations in subjective and nonsubjective time are represented in left parietal cortex and to elucidate the exact contribution of the ventral and dorsal subregions of mpcf to self- versus other - projection . finally , the hypothesis of a partial overlap between self- and other - mechanisms for projection in time could be tested in neuropsychiatric patients with temporal orientation failures . to the extent that self- and other - projection rely on a common neural mechanism , self - referenced and other - referenced disorientation may be expected to share common fundamental characteristics .

the development of bioinformatics has been initially driven not only by the enormous quantity of data that the biologist community was able to produce during the last decades , but also by the necessity of finding approaches to organize and better analyze these huge datasets . although the protein structures constitute small datasets with respect to many other data encountered in biology , they nevertheless represent a challenge for the data analysis , as the relative positions of atomic coordinates in a protein structure take values in the continuous three - dimensional ( 3d ) space . the large variability of protein features is obvious from the variety of physicochemical properties among a given family of proteins . furthermore , the full understanding of a protein function requires , in addition of the knowledge of its structure , the knowledge of the internal dynamics and thus of the conformational landscape of the protein , which correspond to large datasets . graphs are traditionally used for modeling biological datasets , as for the analysis of protein protein and molecular interaction networks , for description of drug function , for the description of interactions within a protein , for the description of the hierarchy of local minima in the conformational space . in the description of protein conformational space , the determination of such a graph is hampered by the need to ( i ) simplify the protein local geometry without loss of information and ( ii ) find a generic approach for graph determination , while preserving the specificity of each protein . in contrast , the description of protein structure and dynamics through graphs would allow one to ( i ) relate structure description , conformational variability , and protein function ; ( ii ) unify the structural and dynamical representations ; and ( iii ) obtain , for a given protein , a model that could be interfaced with the graphs described at the cellular level , as the interactome network . in order to investigate the points quoted above , we have been using several processing tools to describe the graphs underlying the structural and dynamical features of the d - ala : d - lac ( vana ) ligase:(i)the self - organizing maps , to convert the conformational space in a two - dimensional ( 2d ) map;(ii)the louvain greedy algorithm , to determine kinetic clusters in the conformational space;(iii)the girvan newmann algorithm , to determine contact communities within the protein structure , which was already used in other structural objects ; and(iv)the analysis of hydrogen bonds within the protein structure , using a machine - learning approach ( random forest).the conformational space has been explored using an enhanced sampling approach : the temperature - accelerated molecular dynamics ( tamd ) . the self - organizing maps , to convert the conformational space in a two - dimensional ( 2d ) map ; the louvain greedy algorithm , to determine kinetic clusters in the conformational space ; the girvan newmann algorithm , to determine contact communities within the protein structure , which was already used in other structural objects ; and the analysis of hydrogen bonds within the protein structure , using a machine - learning approach ( random forest ) . the d - ala : d - lac ligase ( vana ) is present in cases of resistance to the glycopeptide antibiotic vancomycin in enterococcus faetium and staphylococcus aureus . vana synthesizes a modified precursor d - ala - d - lac instead of the usual d - ala - d - ala , synthesized by using a d - ala : d - ala ligase . this depsipeptide is then fixed at the end of the n - acetyl - muramyl - l - ala - d - glu - l - lys - d - ala - d - lac monomers involved in the building of the peptidoglycan , giving rise to a fully efficient cell wall while preventing the binding of vancomycin . the x - ray crystallographic structure of vana ( figure 1a ) includes the domains n - terminal ( residues a2g121 shown in blue ) , central ( residues c122s211 shown in red and yellow ) , and c - terminal ( residues g212a342 shown in black and green ) . the -loop ( shown in green in figure 1a , residues l236a256 ) is part of the c - terminal domain and closes the binding site where the ligase enzymatic reaction occurs . the two - layer -sandwich ( residues a149q208 ) is a region opposite to the -loop in the structure and colored yellow in figure 1a . the binding site is located at the interface between n - terminal , central , and c - terminal domains . concerted motions of the opposite domain and of the -loop allow the opening of the binding cavity to release the product of the catalytic reaction and accept new ligands . ( a ) three - dimensional ( 3d ) view of the x - ray crystallographic structure of vana , colored according to its domains : the n - terminal [ a2g121 ] shown in blue , the c - terminal [ g212a342 ] shown in black , which includes the -loop [ l236a256 ] shown in green , and the central domain [ c122s211 ] shown in red , which includes the opposite domain [ a149q208 ] shown in yellow . the disulfide bridge c52c64 , located in the n - terminal domain , is shown with magenta labels ( bottom right ) . ( b ) localization of the collective variables ( cv ) used for the different tamd calculations on a cartoon view of vana extracted at the end of a 10 ns md trajectory . the three structural cv are shown in orange and the five cv obtained from contact communities calculations are shown in cyan . the bioinformatics approaches described above have been applied to md and tamd trajectories recorded on vana . several graph models describing the structural architecture , internal dynamics , and the opening of the -loop , have been established . these models give an extended view of the structural and dynamical features of vana and agree with the experimental knowledge available for the protein function . the starting point of the simulations was the x - ray crystallographic structure of the d - ala : d - lac ligase ( vana ) from enterococcus faecium bm4147 vana ( pdb i d : 1e4e ) . the co - crystallized ligands , adp and phosphinate ( 1(s)-aminoethyl-(2-carboxypropyl)phosphoryl - phosphinic acid ) , located in the active site were removed . the c52c64 disulfide bridge , observed in the crystal was disrupted to be as close as possible to the physiological state of the d - ala : d - ala ligase . the force field charmm22 including the correction map ( cmap ) was used . explicit tip3p solvent water molecules were added to the systems using a cutoff of 10 . the molecular dynamics ( md ) and the temperature - accelerated molecular dynamics ( tamd ) trajectories were recorded using namd 2.7b2 . a cutoff of 12 and a switching distance of 10 were defined for nonbonded interactions . long - range electrostatic interactions were calculated with the particule mesh ewald ( pme ) protocol . before starting the initial md trajectories , it was first minimized using 1000 steps , then thermalized by heating the system from 0 to 300 k over 30 ps , with a time step of 1 fs . the system then is equilibrated in the npt ensemble for 100 ps with a time step of 2 fs before a 40 ns md simulation . the temperature was maintained at 300 k using a langevin thermostat , and the 1 atm pressure was regulated using the langevin piston nose the shake algorithm kept all covalent bonds involving hydrogens rigid , so an integration time step of 2 fs was used for all md simulations . atomic coordinates were saved every picosecond . at the end of the first 10 ns of the md trajectory , five independent 30-ns temperature - accelerated molecular dynamics ( tamd ) the tamd approach is an enhanced sampling approach , based on the parallel evolution of the protein coordinates x in a classical md simulation and of the target values z for the collective variables (x):1where x are the physical variables ( atomic coordinates ) of the system , (x ) are the collective variables , and z the instantaneous target values of the collective variables . m is the mass matrix , v(x ) is the empirical classical potential of the system , (t ) denotes white noise ( i.e. , gaussian processes with mean 0 and covariance of p(t)p(t) = (t t ) , with p = x , z ) , > 0 is the so - called spring force constant , and > 0 are friction coefficients of the langevin thermostats , = kbt , and = kbt , where kb is the boltzmann constant and t and t represent the temperatures . equation 1 describes the motion of x and z under the extended potential2it was shown in ref ( 29 ) that , by adjusting the parameter , so that z(t ) (x(t ) ) , and the friction coefficient so that the value of z moves slower than that of x , one can generate a trajectory z(t ) in z - space that effectively moves at the artificial temperature t on the free - energy hyper - surface f(z ) , which is defined at the physical temperature t. hence , by construction , the limiting equation for z(t ) in eq 1 samples the distribution e. then , using t > t in eq 1 ) accelerates the exploration of the free - energy landscape by the z(t ) trajectory , as energy barriers can be crossed more easily . the value for the artificial friction on the z variables can be determined following the principle that the separation of time scales between x and z must be such that the x have time to equilibrate before the z values move substantially . in practice , we proceeded as suggested in ref ( 57 ) , i.e. , we ran short standard md trajectories with the collective variables restrained at (x ) = z fixed , and monitored the mean force estimators gj(n ) defined for each collective variable j as3where j(x(ti ) ) is the instantaneous value at time ti of the collective variable . the time required for gj(n ) to reach a plateau ( see figure s1 in the supporting information ) allows one to extract the characteristic time of relaxation of the cartesian variables to a fixed value of the variables z , and hence an estimate of to ensure the time - scales separation /. as the estimator ( described in eq 3 ) converges in 5000 simulation time steps ( 0.002 ps ) , a friction of 50 ps , corresponding to a characteristic time of 0.02 ps , is sufficient to allow system relaxation . the tamd approach was implemented in namd using a tcl script . in tamd , the evolution of the usual md equation , at 300 k , was coupled to the evolution of collective variables at a much higher temperature . several sets of collective variables were used , which were all geometric centers located in different protein regions . the friction coefficient , = 0.5 ps , and the physical thermal energy , = 0.6 kcal / mol , are the parameters of the conventional langevin thermostat , which allow one to obtain a simulation temperature of 300 k. the restraint force constant is set to = 100 kcal/(mol ) . tamd trajectories were run using a value of 20 kcal mol for the artificial thermal energy of the langevin thermostat attached to the collective variables . this thermal energy corresponds to an artificial temperature t of 10 060 k. despite the high temperature values used for the langevin thermostat attached to the collective variables , it is not expected that the folded structure of vana would be destabilized , as a large friction ( = 50 ps ) is used for this thermostat , along with the high force constant ( = 100 kcal/(mol ) to restraint the collective variable coordinates to the collective variables . in that way , we reduce the risk of system instability due to large deviation of the collective variables (x ) from their target values z. the following method has been used to determine the contact communities of vana along each recorded trajectory . at each trajectory frame , a contact is set up for all -carbon pairs closer than 12 , and the frequency of contacts is calculated along the trajectory . the protein structure is then considered as a graph , where the residues c constitute the vertices and the edges are weighted by the frequency of contacts between c atoms along the trajectories . newman algorithm , as implemented in the program python , allows one to divide , in an iterative way , the graph into contact communities . first , all possible shortest paths are calculated between the c and the betweenness of each edge , which is defined as the number of shortest paths crossing this edge , is computed . the algorithm then removes the edge exhibiting the most important betweenness and includes the two edge vertices into the same community . several runs of the algorithm are performed to remove the edge of highest betweenness until no edges remain . at the end of the process , the initial dynamic map of frequency of contacts has been split into contact communities of amino acids that are strongly connected . the self - organizing maps ( som ) approach was used to cluster the conformations generated along md and tamd trajectories . the som algorithm allows the mapping of the conformational space on a periodic subspace of reduced dimensions : a 50 50 map . 341 341 pairwise square euclidean distance matrices d were calculated for the 341 c atoms of vana , for each frame of the trajectory . to compress the data , a covariance matrix c was computed from each d. its four eigenvectors , corresponding to the first four significant eigenvalues ni were kept . for each trajectory frame t , the resulting compressed 4 341 matrix d vi=1, ... ,4 , stored as a vector vt , contains the conformational descriptors and is used to cluster the protein conformations . the som was trained in two phases with the following parameters : ( i ) a map size of 50 50 with periodic boundaries , initialized randomly with a constant learning rate of 0.5 and a radius of 6.250 for the first phase ( 180 000 iterations ) , and ( ii ) an exponential decrease of learning rate ( starting at 0.25 ) and radius ( starting at 3.125 ) for the second phase ( 360 000 iterations ) . after the random initialization of the map , vectors of conformational descriptors vt described above , were presented to the map in random order , and the neuron closest to the presented vt was updated , as well as the neighbor neurons to preserve the coherence of the clustering . at the end of the calculation , each neuron of the som contains a average vector vt corresponding to a mixture of clustered protein conformations . the unified distance matrix ( u - matrix ) representation was computed to display the som topology on a bidimensional matrix . in the u - matrix , each node shows the local similarity between the corresponding neighboring som neurons , i.e. , the mean distance between the node and its eight neighbors . a flooding algorithm was then used to aggregate the u - matrix basins , and to reject outside the regions corresponding to nonsimilar neurones , leading to a continuous map representation while preserving the inherent som topology . the som were additionally processed in two ways in order to determine graphs describing ( i ) the kinetics of the conformational space sampled and ( ii ) the opening path between the closed and open conformations of vana . the graph related to the kinetics of the conformational space sampled was determined in the following way . the som neurons define the microstates , and each structure along a given md or tamd trajectory is assigned to a given neuron . the element tij of the transition matrix , depicting the transition between neurons i and j , is defined as the number of i j transitions divided by the number of starts from neuron i. the transition matrix can be represented as a weighted graph , with the weight of the vertex ij being given by tij . the obtained graph is then partitioned using the greedy algorithm of louvain , in order to maximize the graph modularity . the modularity is a value between 1 and + 1 , measuring the density of edges inside the partitions , compared to the density of edges outside the partitions . the greedy algorithm of louvain optimizes the modularity in two phases . in the first phase , each som neuron then , for each som neuron u , the variation of modularity is evaluated when u is removed from its cluster and placed to the cluster of each of its neighbors . if no gain of modularity is possible , u remains in its cluster . in the second phase , a new graph is built by merging the som neurons belonging to the same cluster the weights of the resulting graph are computed by summing the weights of the links between nodes in the corresponding two clusters . the opening path between the vana states displaying open and closed -loops was determined in the following way . edges between som neurons were weighted by the value of the corresponding element of the u - matrix , which measures the local similarity between protein conformations . the starting point was the som node u corresponding to the starting point of all trajectories , with closed -loop . the final point of the path was chosen as the medoid of the som kinetic cluster 15 which will be described in section 3.3 . the medoid is the neuron whose average distance to all the neurons in the cluster is minimal . the shortest path is computed using the dijkstra algorithm , using the similarity between neurons as a distance . finally , the path defined from som neurons was converted to a series of vana conformations by replacing each neuron by the vana conformation exhibiting the smallest euclidean distance between its vector of conformational descriptors vt and the average of the neuron vector vt. the path describing the opening has been analyzed to detect the most critical hydrogen bonds for the conformational change . for that purpose , along the opening path , a representative conformation was extracted from each kinetic cluster obtained above using the louvain greedy algorithm . this representative conformation was chosen as the medoid of the path conformations belonging to this kinetic cluster . on each of these vana conformations , hydrogen bonds have been detected using criteria based on a survey of small - molecule crystal structures . a hydrogen bond is supposed to be established if the donor acceptor and the hydrogen acceptor distances are respectively smaller than 4.0 and 3.0 . a random forest ( rf ) machine learning approach was used to calculate the importance of each hydrogen bond for predicting to which kinetic cluster the representative conformation belongs . the information on established and disrupted hydrogen bonds was encoded as a boolean vector for each conformation populating the path . the predicted value for each vector was the identifier of the kinetic cluster . the rf calculation was performed using the python package scikit - learn ( scikit-learn.org ) . the number of trees in the forest was set to 10 , with a gini criterion to measure the quality of a split . the number of features used when searching for the best split was set to 40 , which is approximately the square root of the length of the boolean vectors ( 40 ) . once the training done , the importance of each hydrogen bond to define a kinetic cluster has been computed . the substrates , atp , d - ala , d - lac , d - alanyl - phosphate ( d - ala(p ) ) , the transition - state analogue phosphinate or phy , the product of the reaction , d - ala - d - lac , and the allosteric binder , were formatted in mol2 with chimera 1.4 and marvinsketch 5.1 ( www.chemaxon.com/products/marvin/marvinsketch ) for docking . ucsf dock 6.5 was used to perform ligand docking vana conformations along the opening path obtained as described at the end of the section 2.5 . chimera was used to add hydrogens , check atom assignment , and assign partial charges consistent with the amber - ff99sb force field . chimera was also used to produce mol2 format files for the ligands and the selected conformations of the receptor . the dms software program generated the molecular surface of the receptor , using a radius probe of 1.4 . spheres then were calculated around the receptor with the dock 6.5 command sphgen with radius probe values varying between 1.4 and 4 . spheres were selected within a radius of 10 around the geometric center defined by the residues e15 , k170 , r289 , n303 , e304 , n306 , which are close to positions observed for the ligands ( adp , phosphinate ) in 1e4e . the grid encoding van der waals and electrostatic interactions was precalculated with the grid tool in a box containing the selected spheres . the dock program builds up to 500 flexible ligand docking orientations , on the precalculated grid interaction map . the ligand poses were then re - scored with the implementation of the hawkins molecular mechanics generalized born surface area ( mm - gbsa ) score , implemented in ucsf dock 6.5 . the use of the enhanced sampling approach tamd requires the definition of collective variables . in the present work , these variables were chosen as geometric centers of -carbons located in various vana regions . these regions were detected ( table 1 ) from an analysis of the x - ray crystallographic structure of vana ( pdb i d : 1e4e ) or from the contact communities determined by the girvan starting from these regions , two sets of geometric centers were determined ( see table s1 in the supporting information ) : structural collective variables ( cvn - xr , cvo - xr , and cv-xr ) and dynamical collective variables ( cv-com , cve0-com , cve1-com , cvm - com , and cvo - com ) . five independent 30-ns temperature - accelerated molecular dynamics ( tamd ) simulations were launched using various combinations of both sets of collective variables ( see table s2 in the supporting information ) . the first five domain definitions are derived from the analysis of the x - ray ( xr ) crystallographic structure1e4e . the structural collective variables cvn - xr , cvo - xr , and cv-xr ( table s1 and figure 1 ) were respectively defined on the n - terminal domain , opposite domain , and -loop , chosen from a direct observation of the pdb structure 1e4e . this choice is supported by several observations on x - ray crystallographic structures and md trajectories . first , the -loop , containing cv-xr , displays diverse orientations in x - ray crystallographic structures of d - ala : d - ala ligases . second , the opposite region ( residues 149208 ) was chosen to define cvo - xr , as this region moves apart from the protein core , as published in a previous work . the dynamical collectives variables were derived from the contact communities calculated using the girvan newman algorithm along a 30-ns md trajectory : these communities are described in more detail below . the corresponding geometric centers are located in the -loop ( cv-com ) , in the n - terminal and c - terminal domains ( cve0-com , cve1-com ) , and in the middle ( cvm - com ) and opposite ( cvo - com ) domains ( see table s1 and figure 1 ) . the contact community analysis based on the girvan newman algorithm allowed one to divide vana in five communities either in md or in tamd simulations , except in tamd_on , where four communities were observed ( see figure 2 ) . these communities are variable from one simulation to another , but involve similar protein regions for all trajectories ( see table s3 in the supporting information ) , even though different sets of collective variables were used during each tamd trajectory . the two ends_0-com and ends_1-com communities are interlaced in the protein sequence , and contain residues from the structural definition of the n- and c - terminal regions . the opposite - com community is located in the opposite domain , while the -com community corresponds to the -loop and part of the c - terminal . the last community , middle - com ( see table s3 ) , located in the middle of the protein and partially superimposed with the central structural domain central - xr ( table 1 ) , is detected in all trajectories except tamd_on . the definition of contact communities are slightly different from the definitions of structural domains , except opposite - com , almost superimposed to the domain opposite - xr ( table 1 ) . the good fit of opposite - com to opposite - xr is expected as the opposite domain was previously detected from an analysis of md trajectories . the same color code was kept for the communities both on the 3d structures and on the graphs : the communities mainly located in the n - terminal region ( numbers 0 and 1 ) are shown in blue and red ; the middle ( number 2 ) community is shown in magenta , if it exists ; the opposite region is shown in yellow ( number 3 ) ; the -loop and the main part of the c - terminal are shown in green ( number 4 ) . projection of the communities calculated on a 30-ns trajectory of vana for ( a ) md , ( c ) tamd_on , ( e ) tamd_n , ( g ) tamd_on , ( i ) tamd_md , and ( k ) tamd_5cv . also shown is a graph of the interconnectivity calculated between the different communities for ( b ) md , ( d ) tamd_on , ( f ) tamd_n , ( h ) tamd_on , ( j ) tamd_md , and ( l ) tamd_5cv . the collective variables ( cv ) used for tamd trajectories are represented by orange balls when they were derived from structural calculations and cyan balls if they were obtained from the communities calculations . the contact communities graph is connected by edges ( figure 2 ) , which depict the frequency of contact between -carbons belonging to two different communities . thus , the edge thickness gives a qualitative indication of the relative influences that the communities have on each other . overall , the same pattern of influences between communities is observed in all trajectories ( figure 2 ) . the community corresponding to the -loop is always strongly linked with the opposite community , as reflected by the high betweenness . the opposite domain is itself connected to the ends communities detected into the n- and c - terminal domains ( shown in red and blue in figure 2 ) . the definitions of structural , dynamical collective variables and of contact communities determined on the trajectory tamd_n are depicted ( figure 3 ) using a color code . the definitions corresponding to the opposite domain ( yellow ) and to the -loop ( green ) are similar for the three sets of definition . also , similar middle or central domains ( magenta ) are detected between dynamical collective variables and contact communities . definition of collective variables ( cv ) and of contact communities displayed on the vana sequence . the first line contains the definition of structural collective variables ( cvn - xr , cvo - xr , cv-xr : see table s1 ) determined from an analysis of the structure 1e4e . the second line contains the definition of dynamical collective variables ( cve0-com , cve1-com , cvm - com , cvo - com , cv-com : table s1 ) determined from a community analysis using the girvan the third line contains the definition of communities ( ends_oc , ends_1c , middle_c , opposite_c , _c : see table s3 ) determined by the girvan the following color code is used . for the structural cv : cvn - xr ( blue ) , cvo - xr ( yellow ) , and cv-xr ( green ) . for the dynamical cv : cve0-com ( blue ) , cve1-com ( red ) , cvm - com ( magenta ) , cvo - com ( yellow ) , and cv-com ( green ) . for the tamd_n communities : ends_oc ( blue ) , ends_1c ( red ) , middle_c ( magenta ) , opposite_c ( yellow ) , and _c ( green ) . the existence of helices and strands has been monitored along the md and tamd trajectories ( see table s4 in the supporting information ) . most of the secondary structure elements are present more than 80% of the time , at the exception of 5 -strands , which are destabilized in the md as well as in the tamd trajectories . thus , the folded structure of vana is not specifically altered by the use of the tamd , as has been already noticed in section section 2.2 . the 180 000 frames of vana generated either along the md or tamd trajectories were subjected to a som clustering . the analysis of som permits one to determine six clusters of conformations ( see figure 4 ) . for each cluster , the average vana conformation has been drawn in tube representation , where the tube width and color depend on the conformational local variability ( root - mean - square fluctuation ( rmsf ) , ) within the cluster . the color varies from blue ( rmsf close to 1 ) to red , corresponding to the maximal fluctuation in a given cluster ( e.g. , cluster 1 , 13 ; cluster 2 , 13.3 ; cluster 3 , 15.7 ; cluster 4 , 7.9 ; cluster 5 , 8.0 ; cluster 6 , 8.4 ) . a permanent feature of the entire conformational landscape of vana is the large internal mobility of the -loop . this agrees with the apo form of vana simulated : the -loop tendency to open is expected to play an important role in the substrate processing . the root mean square deviation ( rmsd ) from the starting conformation of the trajectories is shown in a prune - green heat map ( in ) . the conformation sets associated with the medoid of each cluster are depicted in putty cartoons . on the cartoons , the root - mean - square fluctuation ( rmsf ) of the backbone is represented by the width of the main chain and by a blue green red color scale corresponding to the rmsf values within the corresponding som cluster . the average conformation of this cluster is characterized by three regions displaying large local rmsf : the -loop , the opposite domain , and three loops [ residues i43v48 ] , [ residues p71h76 ] , [ residues n83h84 ] . a first series of clusters , represented by clusters 1 , 2 , and 3 , displays significant opening of the -loop , with the loop being the most open in clusters 1 and 3 . in all of these clusters , the protein internal mobility remains concentrated on the -loop ( with maximal rmsf values of 13 in cluster 1 and 15.7 in cluster 3 ) and the other regions are much less mobile , except the opposite domain ( maximal rmsf value of 8.0 ) , the other maxima remaining 45 . thus , after only 30 ns of simulation , the tamd trajectories have been able to reach conformations displaying a wide opening of the -loop . these conformations are similar to the x - ray crystallographic structures published on the ttddl d - ala : d - ala ligase ( pdb i d : 2yzg ) . the second series of clusters , which is represented by clusters 5 and 6 , displays conformations with semiopen or semiclosed -loop , similar to the x - ray crystallographic structure of the d - ala : d - ala ligase in ref ( 47 ) ( pdb i d : 2zdg ) . the averaged conformations of clusters 5 and 6 display large mobility of the -loop , as well as that of a few regions of the protein : the opposite domain and the three loops previously detected in cluster 4 : [ residues i43v48 ] , [ residues p71h76 ] , [ residues n83h84 ] . the various trajectories explored the u - matrix differently ( see figure 5 ) . the larger cluster , cluster 4 , was sampled by the different trajectories , but each one sampled distinct areas . the md trajectory explored mainly cluster 4 , keeping the coordinate rmsd value as low as 2.5 , with respect to the starting point ( figure 4 ) , and performing few incursions into cluster 6 . this result agrees with the previously recorded md trajectories in the absence of the disulfide bridge c52c64 . the starting points are shown in pink and the ending ones are shown in magenta . the blue green red color scale represents the local root - mean - square deviation ( rmsd ) , from the starting structure for each structure ( values shown are given in ) . although all tamd trajectories started from the same conformation , the different choices for the collective variables , as well as the random evolution of md simulations , induced distinct explorations of the conformational space . in that respect , the trajectories tamd_on and tamd_5cv visited mainly cluster 4 , containing the starting conformation . the trajectories tamd_n and tamd_on explored clusters 1 , 2 , and 3 , corresponding to the opening of the -loop . the trajectory tamd_mn explored regions 5 and 6 . therefore , it seems that the geometric center of the -loop is a required collective variable to obtain the loop opening . frames extracted from tamd_n are plotted in figure s2 in the supporting information , and reveals that , before the full opening , the -loop undergoes a sideways movement . overall , the cluster analysis of md and tamd trajectories provides an exploration of several possible models for -loop mobility . indeed , protein conformations with fully open loop are obtained along with conformations displaying mobile closed -loop , corresponding to several conformational states explored by apo vana . the opening of the vana binding cavity was monitored by following the values of the angles and between the centers of mass of the entire protein vana ( c ) , of the opposite domain ( o ) , of the n - terminal ( n ) , and of -loop ( ) ( figure 6a ) . the values of and angles were projected on the u - matrix ( see figures 6b and 6c ) . an increased value for corresponds to an opening of the -loop , while an increased value for corresponds to a displacement of the opposite domain apart from the vana structure core . ( a ) tube representation of vana with the -loop in green and the opposite domain in yellow . their own centers of mass is marked with a ball of the same color and respectively called and o. the center of mass of the entire protein vana is called c ( shown in red ) and the center of mass of the n - terminal region , called n ( shown in blue ) . ( b , c ) projections of the angles on the som using a prune - green heat map : ( panel ( b ) ) and ( panel ( c ) ) . some of the structural clusters previously determined from the som analysis ( figure 4 ) display homogeneous angle values while other clusters show much more heterogeneous values ( see figures 6b and 6c ) . cluster 3 , which contains some of the most open conformations of vana ( figure 4 ) is very homogeneous . it exhibits the widest opening ( 55 ) for the angle ( figure 6c ) , while ( figure 6b ) is shrunk with a value of 52 , showing the opposite domain moving apart , with respect to the protein core , while the -loop is still closed . unlike cluster 3 , clusters 1 and 2 , containing open -loops , the and are mostly mirrored , with large values ( green regions in figure 6c ) corresponding to small values ( violet regions in figure 6b ) and vice versa . this is the sign of an anticorrelation between the -loop and opposite domain displacements . nevertheless , some regions of figures 6b and 6c in clusters 1 and 2 display the same color , corresponding to simultaneous shrinkage or expansion of the two protein domains . for the conformations displaying the most closed -loop , sampled in clusters 4 , 5 , and 6 , there is mainly little opening of the angles and . to analyze the kinetics of the conformational exchange in vana , the protein conformations were clustered by the louvain greedy algorithm , taking into account the time order of the dynamic simulations , as described in section 2 . in that way , the conformations populating each kinetic cluster were sampled along the same trajectory , which is a sign that the different tamd trajectories explored various aspects of the conformational kinetics . the division of som according to the kinetic clusters ( figure 7 ) display patterns quite similar to the ones observed for the projection of the angle or on the som ( see figures 6b and 6c ) , which proves that the overall system kinetics is mainly determined by these angle variations . however , the kinetics clustering brings additional information , with respect to the conformational clustering performed by som . indeed , three clusters ( 1 , 5 , and 7 ) display nonconnected regions on the som , respectively labeled 1 and 1 , 5 , 5 , and 5 , and 7 , 7 , and 7 on figure 7 , putting in evidence fast conformational equilibrium between distinct conformational regions . the representative conformations extracted from the nonconnected regions of each of three clusters , display conformational variability in precise regions of vana , as the l and loops and the opposite domain ( o ) . different types of movements for these regions are observed within the three clusters , as shown by the superimposed representative conformations ( figure 7 ) . kinetic clustering of the vana conformation using the louvain greedy algorithm on the som neurones . for the three clusters , including nonconnected regions ( 1 , 5 , and 7 ) , the disconnected regions are labeled , respectively , as 1 and 1 , 5 to 5 , and 7 to 7. the representative conformations corresponding to each disconnected region are drawn superimposed in cartoons . starting from the kinetic clustering of som map and using a procedure described in section 2 , a path relating the conformations of vana with closed and open -loop has been traced on the u - matrix ( see figure 8a ) . the opening path starts from the kinetic cluster 5 ( figure 7 ) , passes through clusters 7 , 2 , and 3 , and ends up in cluster 15 . the conformations sampled along this path correspond to a slight translational move of the -loop ( conformational cluster 2 in figure 4 ) and then to a rotation of the loop on the side ( conformational cluster 1 in figure 4 ) . note that the path through conformational clusters 2 and 1 presents the advantage of permitting a large opening , which allows the substrates to easily enter into the active site . the medoids of each clusters , labeled from a to f , are shown in red and their minimum spanning link is shown in red . ( b ) gbsa score ( in kcal / mol ) for the molecules involved in the enzymatic reaction : the substrates d - ala , d - ala-(p ) , d - lac ; the reaction intermediate homologous , phy ; the product of the enzymatic reaction d - ala - d - lac ; and an allosteric inhibitor . the gbsa score is plotted along the conformations labeled from 0 to 60 , extracted from the opening path . since the opening of the vana binding site is directly related to the protein function , we analyzed the path with respect to the interaction of vana with the substrates , inhibitors , and reaction intermediate . the relative importance of hydrogen bonds within vana along the path then was statistically evaluated , and connected to experimental observations . several ligands ( d - ala , d - ala(p ) ) , d - lac , phy , d - ala - d - lac , and an allosteric inhibitor ) were docked into the vana conformations extracted from the path and the poses scored using the gbsa interaction energy ( figure 8b ) , according to the procedure described in section 2 . the score profile displayed by the allosteric inhibitor ( green curve in figure 8b ) is quite negative and constant . similarly , the score profile of d - ala ( red curve in figure 8b ) is also negative and does not display much variation along the path , which is in agreement with the fact that d - ala is not specific of vana , but rather binds to all proteins of the d - ala : d - lac ligase family . in contrast , the other ligands d - ala(p ) , d - lac , phy , and d - ala - d - lac all display profiles , becoming mostly negative in cluster e of the path , after the -loop opening ( see figure 8b ) . before this opening , the reaction product d - ala - d - lac ( orange curve in figure 8b ) displays repulsion for vana , which agrees with the release of the product after reaction . the intermediate of reaction , phy , displays a behavior similar to that of the other compounds . six conformations , labeled a to f , were picked up in each of the kinetic clusters crossed by the path ( figure 8a ) . on these conformations , a random forest approach , described in section 2 , was used to determine the relative importance of hydrogen bonds for the kinetic cluster prediction ( figure 9 ) . the most important hydrogen bonds are mainly located in the n - terminal domain , in the opposite domain , and in the -loop , which reflects the displacements of these domains described above . in addition , some important hydrogen bonds are observed in the c - terminal region . most important hydrogen bonds for the prediction of the kinetic cluster along the opening path . the protein structure is displayed in trace , with the opposite domain ( residues [ 149208 ] ) colored orange and the -loop ( residues [ 236256 ] ) colored green . the hydrogen bonds within the -loop and the opposite domain are colored cyan , and the hydrogen bonds between these protein domains and other protein regions are colored red . the hydrogen bonds connecting residues from different regions have been colored red in figure 9 . from this outline , the breaking of interactions between protein domains can be followed along the opening path in order to give a description of the kinetic events . the two interactions e250k22 ( between -loop and n - terminal region ) and e207-y137 ( between the opposite domain and the n - terminal region ) are broken in the protein conformation labeled c ( figure 9 ) . on the other hand , hydrogen bonds e207y137 , k203d132 , r174d105 , and , to a lesser extent , r174e104 are formed in the two conformations e and f at the end of the path . the change from the first set of hydrogen bonds to the second set gives a description of the opening , involving only few residues , and can be compared to the patterns of experimental mutations observed for vana . the e250a mutation induces a slight decrease in experimental catalytic efficiency , which would agree with the importance of the e250k22 interaction along the opening of the -loop . the only limited decrease experimentally observed could arise from a possible reorganization of the vana structure , which would be due to the presence of residues compensating for the mutation effect . besides , in the x - ray crystallographic structure of vana , it was observed that the residues e15 , s177 , and h244 are involved in a network of hydrogen bonds preventing the entrance of water molecules that could impair the catalytic reaction by hydrolyzing the ligands . the residues k22 and e250 detected in the present analysis , are located , respectively , in the vicinity of e15 and h244 , and could play a similar role . the analysis of the trajectories in the frame of graph theory has permitted the determination of an opening path of vana , allowing the entrance of substrates in the binding site . the path found agrees with the interaction energy profiles observed for various vana ligands . the d - ala : d - lac ligase vana was analyzed by molecular modeling and various algorithmic tools , in order to obtain a phenomenological description of the protein internal dynamics and conformational landscape , based on graph models . the comparison of md and tamd trajectories reveals the efficiency of tamd to perform enhanced sampling of the protein conformational space . as expected , the regions of conformational space explored during tamd trajectories are closely dependent on the collective variables used . in particular , the opening of the -loop seems to be favored if a geometric center of the -loop is included into the collective variables . the exploration of the conformational landscape has permitted us to describe two different modes of -loop opening : in one mode , opens through a translation , whereas in the other , a translation of is followed by a rotation . the partial opening of the -loop has been previously observed spontaneously in md trajectories in the presence of the crystallographic disulfide bridge c52c64 . the moving of the opposite domain , closely related to the opening of the active site , was also observed in these md trajectories . mentioned that this disulfide bridge was unexpected , because vana is a bacterial intracellular enzyme that should behave in a reducing environment incompatible with the formation of the bridge . the enhanced sampling approach taken here made it possible to observe the opening in the absence of disulfide bridge . the dynamics features observed along the opening path , as the mobility of the opposite domain , are similar to the observations previously made in the presence of the disulfide bridge . the protein internal dynamics along the opening of the active site seems to be closely related to the relative mobility of the -loop and of the opposite domain , as shown by the conformational clustering ( figure 4 ) , by the importance of the angles and ( figure 6 ) , to describe the protein kinetics ( figure 7 ) , and by the analysis of hydrogen bonds along the opening path ( figures 8 and 9 ) . md and tamd trajectories of d - ala : d - lac ligase vana have been analyzed using various algorithms . graph models describe the protein architecture and behavior in the conformational landscape , as well as along the conformational change related to the opening of the -loop . the contact communities detected by analysis of the contacts along the trajectories display a pattern of connections relating the -loop to the middle domain , which acts as a hub to establish connection to the opposite and the n- and c - terminal domains . this pattern is conserved in most of the trajectories , whereas contrasted internal dynamics are observed in these protein regions over the conformational space ( figure 4 ) . indeed , the -loop is always quite mobile whereas other protein regions display large ( clusters 5 and 6 ) to small ( clusters 1 , 2 and 3 ) internal mobility ( figure 4 ) . the various graphs obtained on the contact communities , or on the som , display characteristics similar to those observed in other bioinformatics graphs obtained in different contexts , for example , in hub , middle - com , observed in the graph of contact communities ( figure 2 ) . the graph of hydrogen bonds along the opening path reveals that all residues establishing discriminating hydrogen bonds are connected to < 4 other residues ( figure 9 ) , a property of small world also encountered in chemo - informatics networks based on the ligand - set similarities . several approaches have been proposed in the literature to describe the conformational space of proteins as graph of local minima . the analysis performed in ref ( 22 ) is based on principal component analysis ( pca ) of protein motion . however , the pca - based analysis detects only linear correlation , whereas som can capture nonlinear correlations . the method proposed here is related to the conformational space network ( csn ) , which was proposed by yin et al . however , these authors used discrete structural class to cluster conformations . similarly , in ref ( 20 ) , the structures were clustered using an all - atom rmsd cutoff of 2.0 . in the present paper , we defined the so - called microstates as the elements of the som grid . in addition , from an analysis of conformational transitions between som neurons , a method to detect the kinetics cluster is proposed , and put in evidence fast conformational exchange . the graphs proposed here could be used in a systematic way in proteins for which structural information can be obtained , in order to insert these protein structural graphs into larger graphs as the ones observed in protein such model stacking would permit to relate directly phenotypic information to physicochemical interactions at the atomic level . in the case of vana , the graphs provide a model of the open / closed motion of the -loop , allowing one to perform the synthesis between various information . the influence of specific residues and/or conformations in such graphs provides candidates for directed mutagenesis studies . the md and tamd trajectories allows an exploration of the vana conformational space , which induces the observation of the -loop opening . as the closed loop blocks the entrance of the active site , understanding the way the loop is opening gives a qualitative view of the kinetics of the vana enzymatic function . in the enhanced sampling approach , the time scale of opening events observed along tamd trajectories is biased and can not be used to give quantitative information on the opening kinetics . however , on the other hand , the conformations extracted along the opening path of the -loop , can be used for docking purposes . indeed , during the -loop opening , the entire architecture of the vana structure , as well as the active site geometry change . docking ligands on the active site pocket modified by the -loop opening would block this site into an inactive conformation and would orient the docking prediction toward effective inhibitors of the vana function . the protein conformations sampled during the opening path are available from the authors upon request . the d - ala : d - lac ligase vana have been exhaustively investigated by molecular dynamics and enhanced sampling simulations , in order to propose outlines of ( i ) protein architecture and ( ii ) protein conformational landscape . these two types of analyses have been conducted in parallel and give consistent results . the conformational landscape of vana is characterized by a large mobility of the -loop , which displays different translational and rotational motions , with respect to the remaining part of the protein . this conformational view of the landscape is completed by a slightly different kinetic view , which fully agrees with an angular description of the relative mobility of the opposite domain and -loop . the importance of the relative motions of the opposite domain and -loop is further enforced by the contact communities analysis of the protein structure , showing a large influence between these two regions . overall , the numerical and statistical tools used here provide parallel descriptions of the protein structure and of the protein conformational landscape , which are in global agreement .

the d - ala : d - lac ligase , vana , plays a critical role in the resistance of vancomycin . indeed , it is involved in the synthesis of a peptidoglycan precursor , to which vancomycin can not bind . the reaction catalyzed by vana requires the opening of the so - called -loop , so that the substrates can enter the active site . here , the conformational landscape of vana is explored by an enhanced sampling approach : the temperature - accelerated molecular dynamics ( tamd ) . analysis of the molecular dynamics ( md ) and tamd trajectories recorded on vana permits a graphical description of the structural and kinetics aspects of the conformational space of vana , where the internal mobility and various opening modes of the -loop play a major role . the other important feature is the correlation of the -loop motion with the movements of the opposite domain , defined as containing the residues a149q208 . conformational and kinetic clusters have been determined and a path describing the -loop opening was extracted from these clusters . the determination of this opening path , as well as the relative importance of hydrogen bonds along the path , permit one to propose some key residue interactions for the kinetics of the -loop opening .

Introduction Materials and Methods Results Discussion Conclusion

furthermore , the full understanding of a protein function requires , in addition of the knowledge of its structure , the knowledge of the internal dynamics and thus of the conformational landscape of the protein , which correspond to large datasets . graphs are traditionally used for modeling biological datasets , as for the analysis of protein protein and molecular interaction networks , for description of drug function , for the description of interactions within a protein , for the description of the hierarchy of local minima in the conformational space . in the description of protein conformational space , the determination of such a graph is hampered by the need to ( i ) simplify the protein local geometry without loss of information and ( ii ) find a generic approach for graph determination , while preserving the specificity of each protein . in contrast , the description of protein structure and dynamics through graphs would allow one to ( i ) relate structure description , conformational variability , and protein function ; ( ii ) unify the structural and dynamical representations ; and ( iii ) obtain , for a given protein , a model that could be interfaced with the graphs described at the cellular level , as the interactome network . in order to investigate the points quoted above , we have been using several processing tools to describe the graphs underlying the structural and dynamical features of the d - ala : d - lac ( vana ) ligase:(i)the self - organizing maps , to convert the conformational space in a two - dimensional ( 2d ) map;(ii)the louvain greedy algorithm , to determine kinetic clusters in the conformational space;(iii)the girvan newmann algorithm , to determine contact communities within the protein structure , which was already used in other structural objects ; and(iv)the analysis of hydrogen bonds within the protein structure , using a machine - learning approach ( random forest).the conformational space has been explored using an enhanced sampling approach : the temperature - accelerated molecular dynamics ( tamd ) . the self - organizing maps , to convert the conformational space in a two - dimensional ( 2d ) map ; the louvain greedy algorithm , to determine kinetic clusters in the conformational space ; the girvan newmann algorithm , to determine contact communities within the protein structure , which was already used in other structural objects ; and the analysis of hydrogen bonds within the protein structure , using a machine - learning approach ( random forest ) . the d - ala : d - lac ligase ( vana ) is present in cases of resistance to the glycopeptide antibiotic vancomycin in enterococcus faetium and staphylococcus aureus . vana synthesizes a modified precursor d - ala - d - lac instead of the usual d - ala - d - ala , synthesized by using a d - ala : d - ala ligase . this depsipeptide is then fixed at the end of the n - acetyl - muramyl - l - ala - d - glu - l - lys - d - ala - d - lac monomers involved in the building of the peptidoglycan , giving rise to a fully efficient cell wall while preventing the binding of vancomycin . concerted motions of the opposite domain and of the -loop allow the opening of the binding cavity to release the product of the catalytic reaction and accept new ligands . ( a ) three - dimensional ( 3d ) view of the x - ray crystallographic structure of vana , colored according to its domains : the n - terminal [ a2g121 ] shown in blue , the c - terminal [ g212a342 ] shown in black , which includes the -loop [ l236a256 ] shown in green , and the central domain [ c122s211 ] shown in red , which includes the opposite domain [ a149q208 ] shown in yellow . the bioinformatics approaches described above have been applied to md and tamd trajectories recorded on vana . several graph models describing the structural architecture , internal dynamics , and the opening of the -loop , have been established . these models give an extended view of the structural and dynamical features of vana and agree with the experimental knowledge available for the protein function . the starting point of the simulations was the x - ray crystallographic structure of the d - ala : d - lac ligase ( vana ) from enterococcus faecium bm4147 vana ( pdb i d : 1e4e ) . the c52c64 disulfide bridge , observed in the crystal was disrupted to be as close as possible to the physiological state of the d - ala : d - ala ligase . the molecular dynamics ( md ) and the temperature - accelerated molecular dynamics ( tamd ) trajectories were recorded using namd 2.7b2 . at the end of the first 10 ns of the md trajectory , five independent 30-ns temperature - accelerated molecular dynamics ( tamd ) the tamd approach is an enhanced sampling approach , based on the parallel evolution of the protein coordinates x in a classical md simulation and of the target values z for the collective variables (x):1where x are the physical variables ( atomic coordinates ) of the system , (x ) are the collective variables , and z the instantaneous target values of the collective variables . , gaussian processes with mean 0 and covariance of p(t)p(t) = (t t ) , with p = x , z ) , > 0 is the so - called spring force constant , and > 0 are friction coefficients of the langevin thermostats , = kbt , and = kbt , where kb is the boltzmann constant and t and t represent the temperatures . this thermal energy corresponds to an artificial temperature t of 10 060 k. despite the high temperature values used for the langevin thermostat attached to the collective variables , it is not expected that the folded structure of vana would be destabilized , as a large friction ( = 50 ps ) is used for this thermostat , along with the high force constant ( = 100 kcal/(mol ) to restraint the collective variable coordinates to the collective variables . the som were additionally processed in two ways in order to determine graphs describing ( i ) the kinetics of the conformational space sampled and ( ii ) the opening path between the closed and open conformations of vana . the graph related to the kinetics of the conformational space sampled was determined in the following way . finally , the path defined from som neurons was converted to a series of vana conformations by replacing each neuron by the vana conformation exhibiting the smallest euclidean distance between its vector of conformational descriptors vt and the average of the neuron vector vt. the path describing the opening has been analyzed to detect the most critical hydrogen bonds for the conformational change . for that purpose , along the opening path , a representative conformation was extracted from each kinetic cluster obtained above using the louvain greedy algorithm . the substrates , atp , d - ala , d - lac , d - alanyl - phosphate ( d - ala(p ) ) , the transition - state analogue phosphinate or phy , the product of the reaction , d - ala - d - lac , and the allosteric binder , were formatted in mol2 with chimera 1.4 and marvinsketch 5.1 ( www.chemaxon.com/products/marvin/marvinsketch ) for docking . these regions were detected ( table 1 ) from an analysis of the x - ray crystallographic structure of vana ( pdb i d : 1e4e ) or from the contact communities determined by the girvan starting from these regions , two sets of geometric centers were determined ( see table s1 in the supporting information ) : structural collective variables ( cvn - xr , cvo - xr , and cv-xr ) and dynamical collective variables ( cv-com , cve0-com , cve1-com , cvm - com , and cvo - com ) . five independent 30-ns temperature - accelerated molecular dynamics ( tamd ) simulations were launched using various combinations of both sets of collective variables ( see table s2 in the supporting information ) . first , the -loop , containing cv-xr , displays diverse orientations in x - ray crystallographic structures of d - ala : d - ala ligases . the opposite - com community is located in the opposite domain , while the -com community corresponds to the -loop and part of the c - terminal . the same color code was kept for the communities both on the 3d structures and on the graphs : the communities mainly located in the n - terminal region ( numbers 0 and 1 ) are shown in blue and red ; the middle ( number 2 ) community is shown in magenta , if it exists ; the opposite region is shown in yellow ( number 3 ) ; the -loop and the main part of the c - terminal are shown in green ( number 4 ) . a permanent feature of the entire conformational landscape of vana is the large internal mobility of the -loop . this agrees with the apo form of vana simulated : the -loop tendency to open is expected to play an important role in the substrate processing . the average conformation of this cluster is characterized by three regions displaying large local rmsf : the -loop , the opposite domain , and three loops [ residues i43v48 ] , [ residues p71h76 ] , [ residues n83h84 ] . in all of these clusters , the protein internal mobility remains concentrated on the -loop ( with maximal rmsf values of 13 in cluster 1 and 15.7 in cluster 3 ) and the other regions are much less mobile , except the opposite domain ( maximal rmsf value of 8.0 ) , the other maxima remaining 45 . thus , after only 30 ns of simulation , the tamd trajectories have been able to reach conformations displaying a wide opening of the -loop . the second series of clusters , which is represented by clusters 5 and 6 , displays conformations with semiopen or semiclosed -loop , similar to the x - ray crystallographic structure of the d - ala : d - ala ligase in ref ( 47 ) ( pdb i d : 2zdg ) . the averaged conformations of clusters 5 and 6 display large mobility of the -loop , as well as that of a few regions of the protein : the opposite domain and the three loops previously detected in cluster 4 : [ residues i43v48 ] , [ residues p71h76 ] , [ residues n83h84 ] . although all tamd trajectories started from the same conformation , the different choices for the collective variables , as well as the random evolution of md simulations , induced distinct explorations of the conformational space . an increased value for corresponds to an opening of the -loop , while an increased value for corresponds to a displacement of the opposite domain apart from the vana structure core . to analyze the kinetics of the conformational exchange in vana , the protein conformations were clustered by the louvain greedy algorithm , taking into account the time order of the dynamic simulations , as described in section 2 . in that way , the conformations populating each kinetic cluster were sampled along the same trajectory , which is a sign that the different tamd trajectories explored various aspects of the conformational kinetics . the representative conformations extracted from the nonconnected regions of each of three clusters , display conformational variability in precise regions of vana , as the l and loops and the opposite domain ( o ) . ( b ) gbsa score ( in kcal / mol ) for the molecules involved in the enzymatic reaction : the substrates d - ala , d - ala-(p ) , d - lac ; the reaction intermediate homologous , phy ; the product of the enzymatic reaction d - ala - d - lac ; and an allosteric inhibitor . since the opening of the vana binding site is directly related to the protein function , we analyzed the path with respect to the interaction of vana with the substrates , inhibitors , and reaction intermediate . the relative importance of hydrogen bonds within vana along the path then was statistically evaluated , and connected to experimental observations . similarly , the score profile of d - ala ( red curve in figure 8b ) is also negative and does not display much variation along the path , which is in agreement with the fact that d - ala is not specific of vana , but rather binds to all proteins of the d - ala : d - lac ligase family . in contrast , the other ligands d - ala(p ) , d - lac , phy , and d - ala - d - lac all display profiles , becoming mostly negative in cluster e of the path , after the -loop opening ( see figure 8b ) . before this opening , the reaction product d - ala - d - lac ( orange curve in figure 8b ) displays repulsion for vana , which agrees with the release of the product after reaction . on these conformations , a random forest approach , described in section 2 , was used to determine the relative importance of hydrogen bonds for the kinetic cluster prediction ( figure 9 ) . the most important hydrogen bonds are mainly located in the n - terminal domain , in the opposite domain , and in the -loop , which reflects the displacements of these domains described above . from this outline , the breaking of interactions between protein domains can be followed along the opening path in order to give a description of the kinetic events . on the other hand , hydrogen bonds e207y137 , k203d132 , r174d105 , and , to a lesser extent , r174e104 are formed in the two conformations e and f at the end of the path . the e250a mutation induces a slight decrease in experimental catalytic efficiency , which would agree with the importance of the e250k22 interaction along the opening of the -loop . besides , in the x - ray crystallographic structure of vana , it was observed that the residues e15 , s177 , and h244 are involved in a network of hydrogen bonds preventing the entrance of water molecules that could impair the catalytic reaction by hydrolyzing the ligands . the analysis of the trajectories in the frame of graph theory has permitted the determination of an opening path of vana , allowing the entrance of substrates in the binding site . the d - ala : d - lac ligase vana was analyzed by molecular modeling and various algorithmic tools , in order to obtain a phenomenological description of the protein internal dynamics and conformational landscape , based on graph models . the exploration of the conformational landscape has permitted us to describe two different modes of -loop opening : in one mode , opens through a translation , whereas in the other , a translation of is followed by a rotation . the moving of the opposite domain , closely related to the opening of the active site , was also observed in these md trajectories . the dynamics features observed along the opening path , as the mobility of the opposite domain , are similar to the observations previously made in the presence of the disulfide bridge . the protein internal dynamics along the opening of the active site seems to be closely related to the relative mobility of the -loop and of the opposite domain , as shown by the conformational clustering ( figure 4 ) , by the importance of the angles and ( figure 6 ) , to describe the protein kinetics ( figure 7 ) , and by the analysis of hydrogen bonds along the opening path ( figures 8 and 9 ) . md and tamd trajectories of d - ala : d - lac ligase vana have been analyzed using various algorithms . graph models describe the protein architecture and behavior in the conformational landscape , as well as along the conformational change related to the opening of the -loop . the contact communities detected by analysis of the contacts along the trajectories display a pattern of connections relating the -loop to the middle domain , which acts as a hub to establish connection to the opposite and the n- and c - terminal domains . in the present paper , we defined the so - called microstates as the elements of the som grid . in the case of vana , the graphs provide a model of the open / closed motion of the -loop , allowing one to perform the synthesis between various information . the md and tamd trajectories allows an exploration of the vana conformational space , which induces the observation of the -loop opening . as the closed loop blocks the entrance of the active site , understanding the way the loop is opening gives a qualitative view of the kinetics of the vana enzymatic function . in the enhanced sampling approach , the time scale of opening events observed along tamd trajectories is biased and can not be used to give quantitative information on the opening kinetics . however , on the other hand , the conformations extracted along the opening path of the -loop , can be used for docking purposes . indeed , during the -loop opening , the entire architecture of the vana structure , as well as the active site geometry change . the d - ala : d - lac ligase vana have been exhaustively investigated by molecular dynamics and enhanced sampling simulations , in order to propose outlines of ( i ) protein architecture and ( ii ) protein conformational landscape . the conformational landscape of vana is characterized by a large mobility of the -loop , which displays different translational and rotational motions , with respect to the remaining part of the protein . the importance of the relative motions of the opposite domain and -loop is further enforced by the contact communities analysis of the protein structure , showing a large influence between these two regions .

the development of bioinformatics has been initially driven not only by the enormous quantity of data that the biologist community was able to produce during the last decades , but also by the necessity of finding approaches to organize and better analyze these huge datasets . although the protein structures constitute small datasets with respect to many other data encountered in biology , they nevertheless represent a challenge for the data analysis , as the relative positions of atomic coordinates in a protein structure take values in the continuous three - dimensional ( 3d ) space . furthermore , the full understanding of a protein function requires , in addition of the knowledge of its structure , the knowledge of the internal dynamics and thus of the conformational landscape of the protein , which correspond to large datasets . graphs are traditionally used for modeling biological datasets , as for the analysis of protein protein and molecular interaction networks , for description of drug function , for the description of interactions within a protein , for the description of the hierarchy of local minima in the conformational space . in the description of protein conformational space , the determination of such a graph is hampered by the need to ( i ) simplify the protein local geometry without loss of information and ( ii ) find a generic approach for graph determination , while preserving the specificity of each protein . in contrast , the description of protein structure and dynamics through graphs would allow one to ( i ) relate structure description , conformational variability , and protein function ; ( ii ) unify the structural and dynamical representations ; and ( iii ) obtain , for a given protein , a model that could be interfaced with the graphs described at the cellular level , as the interactome network . in order to investigate the points quoted above , we have been using several processing tools to describe the graphs underlying the structural and dynamical features of the d - ala : d - lac ( vana ) ligase:(i)the self - organizing maps , to convert the conformational space in a two - dimensional ( 2d ) map;(ii)the louvain greedy algorithm , to determine kinetic clusters in the conformational space;(iii)the girvan newmann algorithm , to determine contact communities within the protein structure , which was already used in other structural objects ; and(iv)the analysis of hydrogen bonds within the protein structure , using a machine - learning approach ( random forest).the conformational space has been explored using an enhanced sampling approach : the temperature - accelerated molecular dynamics ( tamd ) . the self - organizing maps , to convert the conformational space in a two - dimensional ( 2d ) map ; the louvain greedy algorithm , to determine kinetic clusters in the conformational space ; the girvan newmann algorithm , to determine contact communities within the protein structure , which was already used in other structural objects ; and the analysis of hydrogen bonds within the protein structure , using a machine - learning approach ( random forest ) . the d - ala : d - lac ligase ( vana ) is present in cases of resistance to the glycopeptide antibiotic vancomycin in enterococcus faetium and staphylococcus aureus . this depsipeptide is then fixed at the end of the n - acetyl - muramyl - l - ala - d - glu - l - lys - d - ala - d - lac monomers involved in the building of the peptidoglycan , giving rise to a fully efficient cell wall while preventing the binding of vancomycin . the x - ray crystallographic structure of vana ( figure 1a ) includes the domains n - terminal ( residues a2g121 shown in blue ) , central ( residues c122s211 shown in red and yellow ) , and c - terminal ( residues g212a342 shown in black and green ) . ( a ) three - dimensional ( 3d ) view of the x - ray crystallographic structure of vana , colored according to its domains : the n - terminal [ a2g121 ] shown in blue , the c - terminal [ g212a342 ] shown in black , which includes the -loop [ l236a256 ] shown in green , and the central domain [ c122s211 ] shown in red , which includes the opposite domain [ a149q208 ] shown in yellow . the starting point of the simulations was the x - ray crystallographic structure of the d - ala : d - lac ligase ( vana ) from enterococcus faecium bm4147 vana ( pdb i d : 1e4e ) . at the end of the first 10 ns of the md trajectory , five independent 30-ns temperature - accelerated molecular dynamics ( tamd ) the tamd approach is an enhanced sampling approach , based on the parallel evolution of the protein coordinates x in a classical md simulation and of the target values z for the collective variables (x):1where x are the physical variables ( atomic coordinates ) of the system , (x ) are the collective variables , and z the instantaneous target values of the collective variables . , gaussian processes with mean 0 and covariance of p(t)p(t) = (t t ) , with p = x , z ) , > 0 is the so - called spring force constant , and > 0 are friction coefficients of the langevin thermostats , = kbt , and = kbt , where kb is the boltzmann constant and t and t represent the temperatures . equation 1 describes the motion of x and z under the extended potential2it was shown in ref ( 29 ) that , by adjusting the parameter , so that z(t ) (x(t ) ) , and the friction coefficient so that the value of z moves slower than that of x , one can generate a trajectory z(t ) in z - space that effectively moves at the artificial temperature t on the free - energy hyper - surface f(z ) , which is defined at the physical temperature t. hence , by construction , the limiting equation for z(t ) in eq 1 samples the distribution e. then , using t > t in eq 1 ) accelerates the exploration of the free - energy landscape by the z(t ) trajectory , as energy barriers can be crossed more easily . the friction coefficient , = 0.5 ps , and the physical thermal energy , = 0.6 kcal / mol , are the parameters of the conventional langevin thermostat , which allow one to obtain a simulation temperature of 300 k. the restraint force constant is set to = 100 kcal/(mol ) . this thermal energy corresponds to an artificial temperature t of 10 060 k. despite the high temperature values used for the langevin thermostat attached to the collective variables , it is not expected that the folded structure of vana would be destabilized , as a large friction ( = 50 ps ) is used for this thermostat , along with the high force constant ( = 100 kcal/(mol ) to restraint the collective variable coordinates to the collective variables . the som was trained in two phases with the following parameters : ( i ) a map size of 50 50 with periodic boundaries , initialized randomly with a constant learning rate of 0.5 and a radius of 6.250 for the first phase ( 180 000 iterations ) , and ( ii ) an exponential decrease of learning rate ( starting at 0.25 ) and radius ( starting at 3.125 ) for the second phase ( 360 000 iterations ) . after the random initialization of the map , vectors of conformational descriptors vt described above , were presented to the map in random order , and the neuron closest to the presented vt was updated , as well as the neighbor neurons to preserve the coherence of the clustering . the element tij of the transition matrix , depicting the transition between neurons i and j , is defined as the number of i j transitions divided by the number of starts from neuron i. the transition matrix can be represented as a weighted graph , with the weight of the vertex ij being given by tij . finally , the path defined from som neurons was converted to a series of vana conformations by replacing each neuron by the vana conformation exhibiting the smallest euclidean distance between its vector of conformational descriptors vt and the average of the neuron vector vt. the path describing the opening has been analyzed to detect the most critical hydrogen bonds for the conformational change . the substrates , atp , d - ala , d - lac , d - alanyl - phosphate ( d - ala(p ) ) , the transition - state analogue phosphinate or phy , the product of the reaction , d - ala - d - lac , and the allosteric binder , were formatted in mol2 with chimera 1.4 and marvinsketch 5.1 ( www.chemaxon.com/products/marvin/marvinsketch ) for docking . these regions were detected ( table 1 ) from an analysis of the x - ray crystallographic structure of vana ( pdb i d : 1e4e ) or from the contact communities determined by the girvan starting from these regions , two sets of geometric centers were determined ( see table s1 in the supporting information ) : structural collective variables ( cvn - xr , cvo - xr , and cv-xr ) and dynamical collective variables ( cv-com , cve0-com , cve1-com , cvm - com , and cvo - com ) . the structural collective variables cvn - xr , cvo - xr , and cv-xr ( table s1 and figure 1 ) were respectively defined on the n - terminal domain , opposite domain , and -loop , chosen from a direct observation of the pdb structure 1e4e . the corresponding geometric centers are located in the -loop ( cv-com ) , in the n - terminal and c - terminal domains ( cve0-com , cve1-com ) , and in the middle ( cvm - com ) and opposite ( cvo - com ) domains ( see table s1 and figure 1 ) . the last community , middle - com ( see table s3 ) , located in the middle of the protein and partially superimposed with the central structural domain central - xr ( table 1 ) , is detected in all trajectories except tamd_on . the same color code was kept for the communities both on the 3d structures and on the graphs : the communities mainly located in the n - terminal region ( numbers 0 and 1 ) are shown in blue and red ; the middle ( number 2 ) community is shown in magenta , if it exists ; the opposite region is shown in yellow ( number 3 ) ; the -loop and the main part of the c - terminal are shown in green ( number 4 ) . the second line contains the definition of dynamical collective variables ( cve0-com , cve1-com , cvm - com , cvo - com , cv-com : table s1 ) determined from a community analysis using the girvan the third line contains the definition of communities ( ends_oc , ends_1c , middle_c , opposite_c , _c : see table s3 ) determined by the girvan the following color code is used . on the cartoons , the root - mean - square fluctuation ( rmsf ) of the backbone is represented by the width of the main chain and by a blue green red color scale corresponding to the rmsf values within the corresponding som cluster . the average conformation of this cluster is characterized by three regions displaying large local rmsf : the -loop , the opposite domain , and three loops [ residues i43v48 ] , [ residues p71h76 ] , [ residues n83h84 ] . in all of these clusters , the protein internal mobility remains concentrated on the -loop ( with maximal rmsf values of 13 in cluster 1 and 15.7 in cluster 3 ) and the other regions are much less mobile , except the opposite domain ( maximal rmsf value of 8.0 ) , the other maxima remaining 45 . the second series of clusters , which is represented by clusters 5 and 6 , displays conformations with semiopen or semiclosed -loop , similar to the x - ray crystallographic structure of the d - ala : d - ala ligase in ref ( 47 ) ( pdb i d : 2zdg ) . the averaged conformations of clusters 5 and 6 display large mobility of the -loop , as well as that of a few regions of the protein : the opposite domain and the three loops previously detected in cluster 4 : [ residues i43v48 ] , [ residues p71h76 ] , [ residues n83h84 ] . the opening of the vana binding cavity was monitored by following the values of the angles and between the centers of mass of the entire protein vana ( c ) , of the opposite domain ( o ) , of the n - terminal ( n ) , and of -loop ( ) ( figure 6a ) . their own centers of mass is marked with a ball of the same color and respectively called and o. the center of mass of the entire protein vana is called c ( shown in red ) and the center of mass of the n - terminal region , called n ( shown in blue ) . to analyze the kinetics of the conformational exchange in vana , the protein conformations were clustered by the louvain greedy algorithm , taking into account the time order of the dynamic simulations , as described in section 2 . the division of som according to the kinetic clusters ( figure 7 ) display patterns quite similar to the ones observed for the projection of the angle or on the som ( see figures 6b and 6c ) , which proves that the overall system kinetics is mainly determined by these angle variations . indeed , three clusters ( 1 , 5 , and 7 ) display nonconnected regions on the som , respectively labeled 1 and 1 , 5 , 5 , and 5 , and 7 , 7 , and 7 on figure 7 , putting in evidence fast conformational equilibrium between distinct conformational regions . for the three clusters , including nonconnected regions ( 1 , 5 , and 7 ) , the disconnected regions are labeled , respectively , as 1 and 1 , 5 to 5 , and 7 to 7. the representative conformations corresponding to each disconnected region are drawn superimposed in cartoons . starting from the kinetic clustering of som map and using a procedure described in section 2 , a path relating the conformations of vana with closed and open -loop has been traced on the u - matrix ( see figure 8a ) . ( b ) gbsa score ( in kcal / mol ) for the molecules involved in the enzymatic reaction : the substrates d - ala , d - ala-(p ) , d - lac ; the reaction intermediate homologous , phy ; the product of the enzymatic reaction d - ala - d - lac ; and an allosteric inhibitor . several ligands ( d - ala , d - ala(p ) ) , d - lac , phy , d - ala - d - lac , and an allosteric inhibitor ) were docked into the vana conformations extracted from the path and the poses scored using the gbsa interaction energy ( figure 8b ) , according to the procedure described in section 2 . similarly , the score profile of d - ala ( red curve in figure 8b ) is also negative and does not display much variation along the path , which is in agreement with the fact that d - ala is not specific of vana , but rather binds to all proteins of the d - ala : d - lac ligase family . in contrast , the other ligands d - ala(p ) , d - lac , phy , and d - ala - d - lac all display profiles , becoming mostly negative in cluster e of the path , after the -loop opening ( see figure 8b ) . besides , in the x - ray crystallographic structure of vana , it was observed that the residues e15 , s177 , and h244 are involved in a network of hydrogen bonds preventing the entrance of water molecules that could impair the catalytic reaction by hydrolyzing the ligands . the exploration of the conformational landscape has permitted us to describe two different modes of -loop opening : in one mode , opens through a translation , whereas in the other , a translation of is followed by a rotation . the protein internal dynamics along the opening of the active site seems to be closely related to the relative mobility of the -loop and of the opposite domain , as shown by the conformational clustering ( figure 4 ) , by the importance of the angles and ( figure 6 ) , to describe the protein kinetics ( figure 7 ) , and by the analysis of hydrogen bonds along the opening path ( figures 8 and 9 ) . the contact communities detected by analysis of the contacts along the trajectories display a pattern of connections relating the -loop to the middle domain , which acts as a hub to establish connection to the opposite and the n- and c - terminal domains . the various graphs obtained on the contact communities , or on the som , display characteristics similar to those observed in other bioinformatics graphs obtained in different contexts , for example , in hub , middle - com , observed in the graph of contact communities ( figure 2 ) . the graphs proposed here could be used in a systematic way in proteins for which structural information can be obtained , in order to insert these protein structural graphs into larger graphs as the ones observed in protein such model stacking would permit to relate directly phenotypic information to physicochemical interactions at the atomic level .

about 20 years ago , for reasons now lost in the mists of the 20th century , i wrote a review about the ribosome for nature . ribosomes had been discovered in the mid-1950s and , until the late 1960s , ribosome research was a major part of molecular biology . by the late 1960s it had emerged that ribosomes are the polymerases that catalyze protein synthesis under mrna control . satisfied with that level of understanding , most who had worked on protein synthesis during the ' golden age ' of molecular biology sought greener pastures in the years thereafter , and interest in the ribosome waned . the thesis of my review , which was entitled ' the ribosome returns ' , was that the ribosome field was poised for advances so dramatic that it would regain the prominence it had last enjoyed in the mid-1960s . in 1988 first , the discovery of ribozymes in the late 1970s had stimulated the interest of biochemists and molecular biologists in rna - containing objects generally , and the ribosome is the most important rna - containing object of them all . second , the shortage of structural information that had for so long plagued the ribosome field seemed ready to end . a month or so ago , i agreed to write a successor to ' the ribosome returns ' for journal of biology , but shortly thereafter i started having second thoughts . as yogi berra is alleged to have said , " it is hard to make predictions , especially about the future " . by writing a successor to ' the ribosome returns ' i would be in the embarrassing position of calling attention to an ancient review , the very title of which was a prediction . below i provide a personal account of what happened in the ribosome field between 1988 and 2000 and my assessment of where the field stands today . by 1988 , a lot had been learned about the three - dimensional organization of the ribosome . the shapes of the two ribosomal subunits , and of the complex they form during protein synthesis , were known at low resolution ( figure 1 ) , and it was understood that protein synthesis occurs in the gap between the two subunits . the secondary structures of the ribosomal rnas ( rrnas ) had been worked out , and sites on rrnas where ribosomal proteins bind had been identified . in addition , the structures of several ribosomal proteins and a few rrna fragments were known at atomic resolution in isolation . however , no one was so deluded as to imagine that structural information of this sort would ever explain ribosome function . the ribosome at low resolution the images shown here are photographs of plaster models of the two ribosomal subunits made by james lake . they were derived from his em images of the two ribosomal subunits from e. coli . ( a ) the large subunit ( left ) and the small subunit ( right ) with some of their landmarks indicated . the only two approaches for addressing the need for structural information that seemed promising in the 1980s were x - ray crystallography and electron microscopy . the first ribosome crystals , reported by yonath , wittmann and colleagues in 1980 , diffracted poorly ; but , as the years went by , crystals were obtained of ribosomes and ribosomal subunits from many prokaryotic species ( for example ) , and the resolutions of the diffraction patterns of the best of them improved . the unit cells of ribosome crystals are very large and , consequently , ribosome crystals diffract x - rays so weakly that useful data can be collected from them only at synchrotron light sources . in 1980 the technology for doing macro - molecular crystallography at synchrotrons was primitive , but major advances were made in the 1980s and thereafter ( for example ) , and by 1988 the technology needed for ribosome crystallography was falling into place . electron microscopy seemed promising because methods were being developed for obtaining three - dimensional electron density maps of biological objects from their two - dimensional electron microscopic ( em ) images . although the theory of image reconstruction is simple , its application to objects like the ribosome , for which the images to be reconstructed are those of isolated , randomly oriented particles , was fraught with difficulties . nevertheless , by 1988 it seemed likely that ribosome reconstructions would eventually emerge with resolutions high enough to allow trnas to be visualized bound to the ribosome . once that threshold was crossed , it seemed to me that em would start contributing to our understanding of protein synthesis . my optimism notwithstanding , nothing published between 1988 and 1995 would have led the unbiased observer to conclude that the ribosome was likely to return any time soon . the advances made in em reconstructions did not seem dramatic , and the papers published on ribosome crystallography were records of frustration . ribosome crystallography had run aground on the shoals of the classic problem in macromolecular crystallography , the so - called phase problem , and it was unclear if it would ever get unstuck . crystal structures are three - dimensional models of molecules that are generated by fitting chemical structure into experimentally determined , three - dimensional maps that display the distributions of electrons in those molecules . electron density maps can be computed from crystal diffraction data only if the phases associated with each of the tens of thousands of reflections in such datasets are known . if there is no prior knowledge about the three - dimensional structure of a macromolecule , phases must be measured experimentally . in the end , the experimental technique that contributed the most to solving ribosome crystal structures was the heavy atom multiple isomorphous replacement ( mir ) method that perutz devised in the 1950s to solve the structure of hemoglobin . however , in 1988 , it was unclear that mir , or any other approach to phasing , such as anomalous scattering ( as ) , would ever work for the ribosome . everything else being equal , the larger a macromolecule , the harder it is to phase its diffraction pattern experimentally ; and by crystallographic standards , ribosomes were and are huge . ad hoc ribosome meetings have been held at different venues around the world for decades . in my estimation , none of them was more important than the ribosome conference that took place in victoria , bc , canada , in the summer of 1995 . there , frank and his colleagues presented the reconstructions they had just obtained from em images of ribosomes embedded in vitreous ice . even though the resolutions of these reconstructions were modest by today 's standards , about 25 , their superiority over their predecessors , which had been derived from images of negatively stained particles , was striking . no significant progress was reported on the crystallographic front at victoria , but it was clear that this area too was heating up . a group at yale , of which i was a member , had just begun working with ribosome crystals , and gossip at the meeting revealed that we were not alone . most notably , by the time i left victoria , i was convinced that the ribosome phase problem was soluble . molecular replacement is a computational method for using the structure of one macromolecule to estimate the phases of the reflections in the diffraction pattern produced by a crystal of another macromolecule of related structure . why not use a cryo - em reconstruction of the ribosome to phase ribosome diffraction patterns by molecular replacement ? although this approach would yield phases only up to the resolutions of the em reconstruction used , which was likely to be low by crystallographic standards , once the proverbial foot was in the proverbial door that far , higher resolution electron density maps would surely follow . the first successful phasing of a ribosomal diffraction pattern was reported in 1998 . that paper presents a 9 resolution electron density map of the large ribosomal subunit from haloarcula marismortui , which was obtained using crystals that had been first described 13 years earlier . the phases used to compute that map were measured by miras methods using crystals into which heavy metal cluster compounds had been soaked . the crucial step in all phasing experiments is determination of the locations in the unit cell where heavy metals and/or anomalous scatters reside . this is normally done using patterson methods , but in this instance , in order to prove that these sites had been correctly located , a second , independent approach was used . phases were obtained by molecular replacement using an em electron density map of the h. marismortui large ribosomal subunit . using those phases , a difference electron density map was computed that displayed only the electrons belonging to the heavy atoms in the unit cell . the patterson - derived positions for these heavy atoms corresponded exactly to the positions found by molecular replacement . even though little molecular detail can be made out in any 9 resolution electron density map , the fundamental accuracy of this 9 resolution electron density map was beyond question . because nothing motivates scientists more powerfully than the knowledge that their problem can be solved , ribosome crystallography advanced rapidly thereafter . by the summer of 1999 , the resolution of the electron density maps available for the large ribosomal subunit from h. marismortui had improved to 5.0 , and using heavy atom isomorphous replacement methods , ramakrishnan and colleagues had obtained a 5.5 resolution electron density map for the small ribosomal subunit from thermus thermophilus . a 7.5 resolution electron density map of the 70s ribosome from t. thermophilus appeared that same year . in august , a 2.4 resolution structure was published of the large ribosomal subunit from h. marismortui . ( figure 2 shows what the improvements in the resolutions of the electron density maps for this object between 1998 and 2000 meant in terms of their interpretability . ) at the beginning of september , an imperfect , 3.3 resolution structure of the small ribosomal subunit from t. thermophilus was presented , and 3 weeks thereafter a significantly more accurate , 3.1 resolution version of that same subunit appeared that had been independently determined . the effect of improvements in resolution on ribosome electron density maps . ( a - c ) views of the entire surface of the large ribosomal subunit of h. marismortui that interacts with the small ribosomal subunit . ( a ) an em reconstruction of that subunit that has a resolution of 20 . ( b ) an x - ray crystallographic image of the same particle , also at a resolution of 20 . ( c ) the same as ( b ) except for the resolution , which is 9 . ( d ) a view of the face of the large subunit at a resolution of 5 , with the positions of l1 and l10 indicated in yellow . ( e ) electron density corresponding to a helix of 23s rrna at a resolution of 5 . ( f ) the electron density for a helix at a resolution of 2.4 . since 2000 , many ribosome crystal structures have been deposited in the protein data bank ( pdb ) . i think of seven of them as founder structures : that is , the first atomic resolution structure obtained from a particular ribosome crystal by a particular laboratory . the three structures that appeared in 2000 are founder structures as are , first , a structure of the large ribosomal subunit from deinococcus radiodurans , second , two independently determined structures for the 70s ribosome from t. thermophilus [ 18 - 20 ] , and third , a structure of the 70s ribosome from escherichia coli . the rest of the ribosome structures in the pdb are those of founder particles with substrates , substrate analogs , protein factors and inhibitors bound , and all of them were generated using effectively the same crystals that produced the corresponding founder structures . ( once the structure of some crystal has been solved , experimental phase determination is not required to solve the structures of derivatives of that crystal . ) since 2000 , the goal of ribosome crystallographers has been the construction of a movie of protein synthesis , the individual frames of which are atomic resolution structures of the ribosome in every state it visits as protein synthesis progresses . this movie is far from complete , mostly because the crystals needed to determine most of its frames are still not available ; but it is not for lack of trying . besides not having all the frames needed to make the complete movie of protein synthesis , what else do we not know about ribosomes crystallographically ? in the first place , there are no crystal structures for eukaryotic ribosomes , and in a world controlled by sponsors fixated on homo sapiens this is not a good thing . in addition , surprisingly , there is still no complete crystal structure for a large ribosomal subunit . neither of the two lateral arms of the large ribosomal subunit ( figure 1 ) can be visualized at high resolution in crystal structures of isolated large subunits ; they wiggle too much . the conformation of the l1 arm is stabilized when the small subunit binds to the large and , for that reason , its structure is known in at least some of the conformations it adopts during protein synthesis . much less the l11 protuberance includes the complex that ribosomal protein l10 forms with four to six copies of ribosomal protein l7/l12 ( the number depends on species ) . the l10 complex is very important functionally , but only its l10 portion has ever been visualized crystallographically , even partially , and its distal components are so flexible that they may never be visualized , either crystallographically or by em . however , structures are available for much of the l10 complex in isolation , and when those structures are added to the structure of the rest of the large subunit structure in silico , the effect is startling , to put it mildly ( figure 3 ) . we have no idea what this part of the ribosome does to promote protein synthesis . there are structures available for most of the complex that l10 forms with the several copies of l7/l12 that interact with it in the ribosome as isolated proteins . these structures have been added to the structure of the large ribosomal subunit from h. marismortui in silico . the small ribosomal subunit is much more dynamic conformationally than the large ribosomal subunit , and its flexibility is vital for its function ( see below ) , but we have crystal structures for the entire object in several of its states . it is much easier to study the functional complexes of the ribosome by electron microscopy than it is to investigate them crystallographically . electron microscopists do not need crystals , and the amounts of material they consume are orders of magnitude less than crystallographers require . finally , image sorting techniques now exist that make it possible to obtain reconstructions of ribosomes in specific functional states , starting with images of samples in which only a fraction of the particles present are in that state ; pure samples are no longer required . consequently , as far as the making of the movie of protein synthesis is concerned , the electron microscopists are well ahead of the crystallographers and , in addition , em images of eukaryotic ribosomes exist ( for example ) . three - dimensional electron density maps obtained by em are qualitatively similar to crystallographic electron density maps , and they are commonly used to generate ( quasi ) atomic resolution models of ribosomes , even though none of them has a resolution high enough to allow an atomic resolution model of the ribosome to be constructed from it de novo . em - derived models of the ribosome are generated by fitting crystal structures into em electron density . this procedure leads to difficulties only in those parts of a structure where you are most in need of information , namely where the structure of the em object diverges from the crystal structure(s ) used to model it . the other major problem faced by consumers of em images is variation in resolution , which makes comparison of images difficult . since 1995 , substantial advances have been made in image reconstruction , and newer reconstructions generally have higher resolutions than older reconstructions . one reason is that the labor per image required to generate a reconstruction has fallen , and resolution improves as the number of images merged grows . the reconstruction process assumes that the images under analysis are different views of the same three - dimensional object . no increase in the number of images processed can overcome resolution limitations caused by particle - to - particle variations in structure , unless the variation involves discrete conformational states , in which case image sorting may save the day . like gaul , protein synthesis divides naturally into three parts : initiation , elongation and termination ( figure 4 ) . during initiation , the two subunits of the ribosome are assembled into a complex that has an aminoacylated initiator trna and an mrna bound , ready to make the first peptide bond of a protein . during termination , a completed protein is released from its trna , and the ribosome assembly dismantled so that its components can be recycled . the steps of the elongation phase , which is the part of the process we will discuss here , constitute a cycle that must be repeated for every peptide bond formed . ( a movie showing the three phases of protein synthesis has been created by the ramakrishnan group at the mrc laboratory of molecular biology and can be accessed from their website . ) the purpose of the references provided here , which are entirely to recent articles and reviews , is to give the reader an entre into the relevant literature , rather than an outline of the history of the field . the intiation of translation is complete once an aminoacyl trna charged with formylated methionine has been placed in the p site bound to the initiating aug codon of an mrna . elongation begins when a second aminoacyl trna recognizes its cognate codon and binds in the a site . this is followed by transfer of the amino acid from the trna in the p site to form a peptide bond with the amino acid attached to the trna in the a site , and translocation of the two trnas into the e and the p site , respectively . this sequence is iterated until a stop codon is encountered , the completed protein is released , and the ribosome disassembles , at termination of the translation cycle ( not shown ) . the ribosome catalyzes two chemical reactions : the aminolysis of the ester bonds that link nascent polypeptides to trnas during protein synthesis , and the hydrolysis of those same bonds . the amino group used for the aminolysis reaction is the -amino group of an amino acid that is ester - linked through its -carboxyl group to the 3 ' oh of the 3'-terminal nucleotide of a trna . the products of this reaction are a peptide that is one residue longer than it was before the reaction , ester - linked to the trna that carried the amino acid into the reaction , and a trna that has nothing attached to it . because this reaction transfers a peptide from one trna to another , it is referred to as the peptidyl transferase reaction . in the second reaction , which occurs only during the termination phase of protein synthesis , the nucleophile is water , instead of an -amino group , and peptides are transferred to water ( that is , the last trna is released from the newly synthesized protein ) . both the peptidyl transferase reaction and the hydrolysis reaction occur at the same site on the ribosome , its peptidyl transferase center ( ptc ) , about which we now know quite a lot . ( 1 ) the ptc is located in the center of the subunit interface surface of the large ribosomal subunit . ( 2 ) although rna - protein interactions are essential for stabilizing the conformation of the ptc , it is composed entirely of rna : the ribosome is a ribozyme . ( 3 ) the catalytic properties of the ptc are not modulated by interactions between the two subunits . ( 4 ) the ptc includes a site that accommodates peptidyl trnas , the p site , and a site to which aminoacyl trnas bind , the a site ( see figure 4 ) . ( 5 ) both the a site and the p site of the ptc interact primarily with the 3 ' terminal cca sequence that is common to all trnas . thus , ( 6 ) when the a site of the ptc is empty and its p site is occupied , the ptc adopts a conformation that protects the ester bonds of peptidyl trnas from nucleophilic attack . a conformational change accompanies the binding of aminoacyl trnas to the a site ( as well as the binding of release factors to the ribosome ) that exposes the ester bond in the p site to nucleophilic attack . ( 7 ) beyond positioning substrates properly , the ptc seems to do little to enhance the rate of peptide bond formation . ( 8) the group that makes the largest chemical contribution to the rate of the peptidyl transferase reaction is the 2 ' oh of the 3'-terminal a of the trna in the p site , which facilitates the removal of a proton from the attacking amino group and the addition of a proton to the leaving group , which is the 3 ' oh of the trna bound in the p site . ( 9 ) on the ribosome , the peptidyl transferase reaction proceeds at a rate that is about 10times faster than the rate of similar reactions in solution . ( 10 ) once substrates are bound appropriately to the ptc , the peptidyl transferase reaction occurs at a rate that is at least ten times faster than the overall rate of protein synthesis in living cells , which is about 20 s. ( 11 ) at neutral ph , the ester bond of an aminoacyl trna is a high energy bond , but the ester bond in a peptidyl trna is not . given that the net effect of the peptidyl transferase reaction is the destruction of a high energy ester bond and the creation of a lower energy peptide bond , the forward direction of the peptide formation is strongly favored thermodynamically . this description of the peptidyl transferase reaction raises as many questions as it answers . what keeps nascent peptides from inhibiting their own synthesis by filling up the ptc ? how does the peptidyl trna product of the peptidyl transferase reaction move from the a site , where it forms , to the p site , where it must reside if another amino acid is to be added to the nascent peptide chain ? product clearance is thought to be the simplest of these issues . as nascent peptides form , they insert into a cavity called the peptide exit tunnel , which extends from the back of the ptc all the way through the body of the large ribosomal subunit . it is not until the length of nascent peptides exceeds about 40 amino acids that their amino - terminal sequences emerge on the far side of the ribosome and start engaging with the apparatus that ensures protein folding and/or export . as far as we know , nascent poly - peptides diffuse down the tunnel in response to the nudge they are given as each peptide bond forms , but there are hints that it may be more interesting . two recent crystal structures have provided insights into how ribosomes carrying completed proteins are recognized and the ester bond linking the protein to trnas is hydrolyzed , which is the ultimate step in product clearance . the trna movements that reset the ptc after each round of peptide bond formation are still only partially understood . discussions of this process , which is called translocation , are best begun by reminding the reader that trnas are l - shaped rnas that vary considerably in sequence but are nearly identical in shape . one arm of the l , the acceptor stem , includes the 3 ' terminal cca sequence mentioned earlier . the other arm , the anticodon stem , terminates with a loop that includes an anticodon , which is the 3'-nucleotide sequence that pairs with mrna codons during protein synthesis . ( aminoacyl trna synthetases ensure that the amino acids that get esterified to the acceptor stems of trnas are the ones encoded by mrna triplets complementary to the anticodon sequences of those trnas . ) messenger rnas bind to the small ribosomal subunit in the region where its head joins its body ( figure 1 ) , and the place on the small subunit where trna anticodons interact with mrna codons is called the decoding center . the a site and the p site of the decoding center are the locations where the anticodons of aminoacyl trnas and peptidyl trnas , respectively , are bound to the small ribosomal subunit just before peptide transfer occurs . ( trnas are l - shaped because there is a prominent ridge on the large subunit separating the ptc from the decoding center of the small subunit that only an l - shaped molecule can surmount . ) translocation on the large subunit precedes translocation on the small subunit , and it seems to be a spontaneous , diffusive process . after peptide bond formation , the acceptor stems of both trnas in the ptc move towards the l1 arm of the large ribosomal subunit . the cca sequence of the discharged trna moves from the p site of the ptc to the so - called e ( exit ) site of the large ribosomal subunit , which can bind only deacylated trnas , and the cca - peptide moiety of the peptidyl trna in the a site moves to the p site of the ptc . the a - to - p motion of peptidyl trnas is accompanied by a 180 rotation of cca sequences relative to the bodies of trnas . large - subunit translocation correlates with a rotation of about 10 of the small subunit relative to the large in the direction of the l1 arm , which is called ratcheting . the ratchet motion also seems to result from thermal diffusion , and it is unclear how tightly ratcheting is coupled to large - subunit translocation . nevertheless , the data suggest that both must occur before small subunit translocation can take place . large - subunit translocation leaves the ribosome in a hybrid state , in the sense that the acceptor stem of the peptidyl trna is in a p site of the ptc while its anticodon end occupies the a site of the decoding center ; the acceptor stem of the discharged trna is in the large - subunit e site while its anticodon is in the p site of the decoding center ( figure 4 ) . first , it advances the ribosome by three nucleotides along the mrna to which it is bound in the 3 ' direction , which places a new codon in the a site of the decoding center . second , it resolves the hybrid state by making the anticodon end of peptidyl trna move from the a site to the p site of the decoding center , and the anticodon end of the deacylated trna move from the p site of the decoding center to the e site of the small subunit . the anticodon of the trna that moves from the a site to the p site of the decoding center remains associated with its codon in the mrna so that the register in which the mrna is being translated is maintained . it is unclear whether the anticodons of trnas in the e site actually interact with mrna or not ; there are biochemical data indicating they do , but the structural data are ambiguous . although small - subunit translocation can occur spontaneously , the spontaneous process is painfully slow . like all the major steps in protein synthesis , it is catalyzed in the cell by a g protein . the g protein in this case , which is ef - g in bacteria and ef-2 in eukaryotes , binds to the ribosome with a gtp bound that is hydrolyzed in the process . em images of ef - g / ribosome complexes , which are all we have , show that this tadpole - shaped molecule binds to the ribosome with its head ( which includes its gtpase site ) bound to the large ribosomal subunit at the base of the l11 arm . that part of the ef - g binding site includes the sarcin - ricin loop ( srl ) of 23s/28s rrna ( figure 3 ) that , for reasons still unclear , is critically important for the activity of all of the g - protein factors that interact with the ribosome during protein synthesis . the distal end of the tail of the tadpole inserts into the a site of the decoding center of the small subunit . the binding of all proteins that interact with the ef - g binding site is promoted by the l10 complex and the rest of the l11 arm , but the details remain to be worked out . in solution the gtpase activity of ef - g is very low , but it increases dramatically when the factor binds to the ribosome . thus , shortly after ef - ggtp binds to the ribosome , its gtp hydrolyzes . this causes ef - g to undergo a major conformational change that seems to push the anticodon stems of trnas across the decoding center , dragging the mrna with them . two additional events ensue : the ribosome unratchets ; and ef - ggdp is released into solution . biochemical data suggest , and em structures confirm , that the conformational changes that accompany ef - g - assisted translocation are more complicated than this account of translocation seems to require , but until the relevant atomic resolution structures become available , we are unlikely to understand them properly . once translocation is complete , the ribosome is ready to bind a new aminoacyl trna , and if there is a deacylated trna in the e site of the ribosome , aminoacyl trna binding is accompanied by release of that trna into solution . biochemical data suggest that these two processes interact with each other , and structural data show that trna release correlates with conformational changes in the l1 arm of the large subunit . aminoacyl trnas are delivered to the ribosome by a second g protein , which is called ef - tu in bacteria and ef-1 in eukaryotes . the complex that ef - tu forms with aminoacyl trna ( and gtp ) , the so - called ternary complex , resembles ef - g in its overall shape , with the anticodon stem of the ternary complex corresponding to the tail of the ef - g tadpole and its ef - tu / acceptor stem portion resembling the head . as far as we know , the ternary complex binds to the ribosome the same way that ef - ggtp does . its ef - tu / acceptor stem portion associates with the srl region of the large subunit and its anticodon stem extends into the a site of the decoding center . if the anticodon of the trna in a ternary complex base - pairs properly with the mrna sequence in the a site of the decoding center , in other words if the codon and anticodon are cognate , a conformational change occurs that stimulates gtp cleavage and release of ef - tugdp from the ribosome . the aminoacyl trna left behind is oriented so that its aminoacyl end is far from the a site of the ptc ; the large reorientation required to place its acceptor stem in the ptc is called accommodation . once accommodation has occurred , the system is ready for the formation of the next peptide bond , which ensues quickly thereafter . the binding of cognate aminoacyl trnas to the ribosome is the rate - limiting step in protein synthesis , and from the point of view of information transfer , it is the most important . given that diffusion is the process that brings ternary complexes to the ribosome , for every encounter with a cognate complex that results in accommodation there will be many encounters with non - cognate ternary complexes that must not result in accommodation if mrnas are to be translated correctly . the reason is that once the wrong aminoacyl trna enters the a site of the ptc , the wrong amino acid will be inserted into the nascent protein : the ptc does not discriminate . the only way in which trna selection can lead to accurate translation is if the ribosome binds cognate complexes much more tightly than non - cognate complexes , and it does . ternary complex selection depends on base - pairing between the sequence of the codon in the a site of the decoding center and trna anticodon sequences . however , it has been realized for decades that the difference in free energy between the formation of a perfect watson - crick helix three base - pairs long and the formation of a three - base - pair duplex in which one of the base juxtapositions is non - canonical is not large enough to explain the accuracy of translation . the crystal structures and em structures have both provided important insights into this aspect of protein synthesis . we know from crystal structures of trnas bound to the ribosome in the accommodated state that when a cognate interaction occurs in the a site of the decoding center , the conformation of the small subunit changes so that two of its rna bases form base - triples with the first two base - pairs of the codon - anticodon helix that form in that center . these interactions , which strongly stabilize the two base - pairs in question , can not occur unless the bases juxtaposed in that helix form watson - crick pairs . for that reason , as had long been suspected , the difference in free energy between cognate pairing and non - cognate pairing is much bigger on the ribosome than it is in solution . the orientation of the anticodon stem of a trna in the pre - accommodation state is very different from its orientation after it has accommodated . this question has been answered by em images that show that before accommodation , the anticodon stems of trnas bend in such a way that their anticodon sequences will interact with the decoding site the same way they do after accommodation . thus , the response of the a site of the decoding center to anticodons appears to be the same no matter whether the trna carrying them is in the pre- or post - accommodation conformation . a vitally important consequence of the conformational change just alluded to is that , by some mechanism we do not understand , it activates the gtpase activity of ef - tu . thus , if a cognate interaction has occurred in the decoding center , the ef - tu in a ternary complex will quickly hydrolyze its gtp and leave the ribosome so that the aminoacyl trna can accommodate . if the interaction between codon and anticodon is non - cognate , gtp hydrolysis is much slower , and the probability is correspondingly high that the non - cognate ternary complex will diffuse away from the ribosome intact , before gtp hydrolysis occurs . suppose that against the odds , a non - cognate ternary complex delivers its aminoacyl trna to the ribosome in the same way that a cognate complex does . before that aminoacyl trna can engage in peptide - bond formation , it must accommodate and , as far as we know , the only interactions that keep accommodating aminoacyl trnas from diffusing away from the ribosome are their codon - anticodon interactions . if that interaction is non - cognate , it will not be stabilized by interactions with the small subunit . it follows that non - cognate aminoacyl trnas are more likely to fall off the ribosome during accommodation than cognate aminoacyl trnas . thus , base - pairing is used to discriminate cognate from non - cognate aminoacyl trnas twice every time an aminoacyl trna is delivered to the a site of the ptc . the understanding of decoding that has emerged from the combination of structural and biochemical investigations is a spectacular example of what everyone hoped would happen once the structure of the ribosome was understood at atomic resolution . however , as the above account shows , our understanding of even that aspect of the elongation cycle remains incomplete . among the many other mysteries still unsolved are the mechanisms of action of lepa , which is a protein factor similar to ef - g that promotes reverse translocation , an unexpected phenomenon believed to contribute to fidelity ; and the tmrna system that rescues ribosomes that have become stuck when translating a broken mrna molecule . the devotees of the ribosome will not run out of interesting problems to investigate for a while yet . my understanding of protein synthesis has been shaped by conversations with my colleagues , especially thomas steitz and rachel green .

since the mid-1990s , insights obtained from electron microscopy and x - ray crystallography have transformed our understanding of how the most important ribozyme in the cell , the ribosome , catalyzes protein synthesis . this review provides a brief account of how this structural revolution came to pass , and the impact it has had on our understanding of how the ribosome decodes messenger rnas .

None How the structural drought ended What is known about ribosome structure today? Structure and function during the elongation phase of protein synthesis How the accuracy of decoding is ensured Acknowledgements

about 20 years ago , for reasons now lost in the mists of the 20th century , i wrote a review about the ribosome for nature . by the late 1960s it had emerged that ribosomes are the polymerases that catalyze protein synthesis under mrna control . satisfied with that level of understanding , most who had worked on protein synthesis during the ' golden age ' of molecular biology sought greener pastures in the years thereafter , and interest in the ribosome waned . the thesis of my review , which was entitled ' the ribosome returns ' , was that the ribosome field was poised for advances so dramatic that it would regain the prominence it had last enjoyed in the mid-1960s . in 1988 first , the discovery of ribozymes in the late 1970s had stimulated the interest of biochemists and molecular biologists in rna - containing objects generally , and the ribosome is the most important rna - containing object of them all . second , the shortage of structural information that had for so long plagued the ribosome field seemed ready to end . by writing a successor to ' the ribosome returns ' i would be in the embarrassing position of calling attention to an ancient review , the very title of which was a prediction . below i provide a personal account of what happened in the ribosome field between 1988 and 2000 and my assessment of where the field stands today . the shapes of the two ribosomal subunits , and of the complex they form during protein synthesis , were known at low resolution ( figure 1 ) , and it was understood that protein synthesis occurs in the gap between the two subunits . the secondary structures of the ribosomal rnas ( rrnas ) had been worked out , and sites on rrnas where ribosomal proteins bind had been identified . the ribosome at low resolution the images shown here are photographs of plaster models of the two ribosomal subunits made by james lake . the only two approaches for addressing the need for structural information that seemed promising in the 1980s were x - ray crystallography and electron microscopy . the first ribosome crystals , reported by yonath , wittmann and colleagues in 1980 , diffracted poorly ; but , as the years went by , crystals were obtained of ribosomes and ribosomal subunits from many prokaryotic species ( for example ) , and the resolutions of the diffraction patterns of the best of them improved . in 1980 the technology for doing macro - molecular crystallography at synchrotrons was primitive , but major advances were made in the 1980s and thereafter ( for example ) , and by 1988 the technology needed for ribosome crystallography was falling into place . electron microscopy seemed promising because methods were being developed for obtaining three - dimensional electron density maps of biological objects from their two - dimensional electron microscopic ( em ) images . although the theory of image reconstruction is simple , its application to objects like the ribosome , for which the images to be reconstructed are those of isolated , randomly oriented particles , was fraught with difficulties . once that threshold was crossed , it seemed to me that em would start contributing to our understanding of protein synthesis . the advances made in em reconstructions did not seem dramatic , and the papers published on ribosome crystallography were records of frustration . ribosome crystallography had run aground on the shoals of the classic problem in macromolecular crystallography , the so - called phase problem , and it was unclear if it would ever get unstuck . in the end , the experimental technique that contributed the most to solving ribosome crystal structures was the heavy atom multiple isomorphous replacement ( mir ) method that perutz devised in the 1950s to solve the structure of hemoglobin . everything else being equal , the larger a macromolecule , the harder it is to phase its diffraction pattern experimentally ; and by crystallographic standards , ribosomes were and are huge . in my estimation , none of them was more important than the ribosome conference that took place in victoria , bc , canada , in the summer of 1995 . most notably , by the time i left victoria , i was convinced that the ribosome phase problem was soluble . although this approach would yield phases only up to the resolutions of the em reconstruction used , which was likely to be low by crystallographic standards , once the proverbial foot was in the proverbial door that far , higher resolution electron density maps would surely follow . by the summer of 1999 , the resolution of the electron density maps available for the large ribosomal subunit from h. marismortui had improved to 5.0 , and using heavy atom isomorphous replacement methods , ramakrishnan and colleagues had obtained a 5.5 resolution electron density map for the small ribosomal subunit from thermus thermophilus . ( figure 2 shows what the improvements in the resolutions of the electron density maps for this object between 1998 and 2000 meant in terms of their interpretability . ) at the beginning of september , an imperfect , 3.3 resolution structure of the small ribosomal subunit from t. thermophilus was presented , and 3 weeks thereafter a significantly more accurate , 3.1 resolution version of that same subunit appeared that had been independently determined . ( b ) an x - ray crystallographic image of the same particle , also at a resolution of 20 . i think of seven of them as founder structures : that is , the first atomic resolution structure obtained from a particular ribosome crystal by a particular laboratory . the rest of the ribosome structures in the pdb are those of founder particles with substrates , substrate analogs , protein factors and inhibitors bound , and all of them were generated using effectively the same crystals that produced the corresponding founder structures . since 2000 , the goal of ribosome crystallographers has been the construction of a movie of protein synthesis , the individual frames of which are atomic resolution structures of the ribosome in every state it visits as protein synthesis progresses . in the first place , there are no crystal structures for eukaryotic ribosomes , and in a world controlled by sponsors fixated on homo sapiens this is not a good thing . however , structures are available for much of the l10 complex in isolation , and when those structures are added to the structure of the rest of the large subunit structure in silico , the effect is startling , to put it mildly ( figure 3 ) . we have no idea what this part of the ribosome does to promote protein synthesis . there are structures available for most of the complex that l10 forms with the several copies of l7/l12 that interact with it in the ribosome as isolated proteins . it is much easier to study the functional complexes of the ribosome by electron microscopy than it is to investigate them crystallographically . electron microscopists do not need crystals , and the amounts of material they consume are orders of magnitude less than crystallographers require . consequently , as far as the making of the movie of protein synthesis is concerned , the electron microscopists are well ahead of the crystallographers and , in addition , em images of eukaryotic ribosomes exist ( for example ) . three - dimensional electron density maps obtained by em are qualitatively similar to crystallographic electron density maps , and they are commonly used to generate ( quasi ) atomic resolution models of ribosomes , even though none of them has a resolution high enough to allow an atomic resolution model of the ribosome to be constructed from it de novo . one reason is that the labor per image required to generate a reconstruction has fallen , and resolution improves as the number of images merged grows . during initiation , the two subunits of the ribosome are assembled into a complex that has an aminoacylated initiator trna and an mrna bound , ready to make the first peptide bond of a protein . during termination , a completed protein is released from its trna , and the ribosome assembly dismantled so that its components can be recycled . ( a movie showing the three phases of protein synthesis has been created by the ramakrishnan group at the mrc laboratory of molecular biology and can be accessed from their website . ) elongation begins when a second aminoacyl trna recognizes its cognate codon and binds in the a site . this is followed by transfer of the amino acid from the trna in the p site to form a peptide bond with the amino acid attached to the trna in the a site , and translocation of the two trnas into the e and the p site , respectively . this sequence is iterated until a stop codon is encountered , the completed protein is released , and the ribosome disassembles , at termination of the translation cycle ( not shown ) . the ribosome catalyzes two chemical reactions : the aminolysis of the ester bonds that link nascent polypeptides to trnas during protein synthesis , and the hydrolysis of those same bonds . in the second reaction , which occurs only during the termination phase of protein synthesis , the nucleophile is water , instead of an -amino group , and peptides are transferred to water ( that is , the last trna is released from the newly synthesized protein ) . both the peptidyl transferase reaction and the hydrolysis reaction occur at the same site on the ribosome , its peptidyl transferase center ( ptc ) , about which we now know quite a lot . ( 4 ) the ptc includes a site that accommodates peptidyl trnas , the p site , and a site to which aminoacyl trnas bind , the a site ( see figure 4 ) . a conformational change accompanies the binding of aminoacyl trnas to the a site ( as well as the binding of release factors to the ribosome ) that exposes the ester bond in the p site to nucleophilic attack . ( 8) the group that makes the largest chemical contribution to the rate of the peptidyl transferase reaction is the 2 ' oh of the 3'-terminal a of the trna in the p site , which facilitates the removal of a proton from the attacking amino group and the addition of a proton to the leaving group , which is the 3 ' oh of the trna bound in the p site . ( 9 ) on the ribosome , the peptidyl transferase reaction proceeds at a rate that is about 10times faster than the rate of similar reactions in solution . ( 10 ) once substrates are bound appropriately to the ptc , the peptidyl transferase reaction occurs at a rate that is at least ten times faster than the overall rate of protein synthesis in living cells , which is about 20 s. ( 11 ) at neutral ph , the ester bond of an aminoacyl trna is a high energy bond , but the ester bond in a peptidyl trna is not . given that the net effect of the peptidyl transferase reaction is the destruction of a high energy ester bond and the creation of a lower energy peptide bond , the forward direction of the peptide formation is strongly favored thermodynamically . two recent crystal structures have provided insights into how ribosomes carrying completed proteins are recognized and the ester bond linking the protein to trnas is hydrolyzed , which is the ultimate step in product clearance . one arm of the l , the acceptor stem , includes the 3 ' terminal cca sequence mentioned earlier . the other arm , the anticodon stem , terminates with a loop that includes an anticodon , which is the 3'-nucleotide sequence that pairs with mrna codons during protein synthesis . messenger rnas bind to the small ribosomal subunit in the region where its head joins its body ( figure 1 ) , and the place on the small subunit where trna anticodons interact with mrna codons is called the decoding center . after peptide bond formation , the acceptor stems of both trnas in the ptc move towards the l1 arm of the large ribosomal subunit . the cca sequence of the discharged trna moves from the p site of the ptc to the so - called e ( exit ) site of the large ribosomal subunit , which can bind only deacylated trnas , and the cca - peptide moiety of the peptidyl trna in the a site moves to the p site of the ptc . large - subunit translocation leaves the ribosome in a hybrid state , in the sense that the acceptor stem of the peptidyl trna is in a p site of the ptc while its anticodon end occupies the a site of the decoding center ; the acceptor stem of the discharged trna is in the large - subunit e site while its anticodon is in the p site of the decoding center ( figure 4 ) . first , it advances the ribosome by three nucleotides along the mrna to which it is bound in the 3 ' direction , which places a new codon in the a site of the decoding center . second , it resolves the hybrid state by making the anticodon end of peptidyl trna move from the a site to the p site of the decoding center , and the anticodon end of the deacylated trna move from the p site of the decoding center to the e site of the small subunit . it is unclear whether the anticodons of trnas in the e site actually interact with mrna or not ; there are biochemical data indicating they do , but the structural data are ambiguous . like all the major steps in protein synthesis , it is catalyzed in the cell by a g protein . the g protein in this case , which is ef - g in bacteria and ef-2 in eukaryotes , binds to the ribosome with a gtp bound that is hydrolyzed in the process . that part of the ef - g binding site includes the sarcin - ricin loop ( srl ) of 23s/28s rrna ( figure 3 ) that , for reasons still unclear , is critically important for the activity of all of the g - protein factors that interact with the ribosome during protein synthesis . thus , shortly after ef - ggtp binds to the ribosome , its gtp hydrolyzes . biochemical data suggest , and em structures confirm , that the conformational changes that accompany ef - g - assisted translocation are more complicated than this account of translocation seems to require , but until the relevant atomic resolution structures become available , we are unlikely to understand them properly . once translocation is complete , the ribosome is ready to bind a new aminoacyl trna , and if there is a deacylated trna in the e site of the ribosome , aminoacyl trna binding is accompanied by release of that trna into solution . biochemical data suggest that these two processes interact with each other , and structural data show that trna release correlates with conformational changes in the l1 arm of the large subunit . the complex that ef - tu forms with aminoacyl trna ( and gtp ) , the so - called ternary complex , resembles ef - g in its overall shape , with the anticodon stem of the ternary complex corresponding to the tail of the ef - g tadpole and its ef - tu / acceptor stem portion resembling the head . as far as we know , the ternary complex binds to the ribosome the same way that ef - ggtp does . if the anticodon of the trna in a ternary complex base - pairs properly with the mrna sequence in the a site of the decoding center , in other words if the codon and anticodon are cognate , a conformational change occurs that stimulates gtp cleavage and release of ef - tugdp from the ribosome . the aminoacyl trna left behind is oriented so that its aminoacyl end is far from the a site of the ptc ; the large reorientation required to place its acceptor stem in the ptc is called accommodation . the binding of cognate aminoacyl trnas to the ribosome is the rate - limiting step in protein synthesis , and from the point of view of information transfer , it is the most important . given that diffusion is the process that brings ternary complexes to the ribosome , for every encounter with a cognate complex that results in accommodation there will be many encounters with non - cognate ternary complexes that must not result in accommodation if mrnas are to be translated correctly . the only way in which trna selection can lead to accurate translation is if the ribosome binds cognate complexes much more tightly than non - cognate complexes , and it does . however , it has been realized for decades that the difference in free energy between the formation of a perfect watson - crick helix three base - pairs long and the formation of a three - base - pair duplex in which one of the base juxtapositions is non - canonical is not large enough to explain the accuracy of translation . we know from crystal structures of trnas bound to the ribosome in the accommodated state that when a cognate interaction occurs in the a site of the decoding center , the conformation of the small subunit changes so that two of its rna bases form base - triples with the first two base - pairs of the codon - anticodon helix that form in that center . for that reason , as had long been suspected , the difference in free energy between cognate pairing and non - cognate pairing is much bigger on the ribosome than it is in solution . the orientation of the anticodon stem of a trna in the pre - accommodation state is very different from its orientation after it has accommodated . thus , the response of the a site of the decoding center to anticodons appears to be the same no matter whether the trna carrying them is in the pre- or post - accommodation conformation . thus , if a cognate interaction has occurred in the decoding center , the ef - tu in a ternary complex will quickly hydrolyze its gtp and leave the ribosome so that the aminoacyl trna can accommodate . if the interaction between codon and anticodon is non - cognate , gtp hydrolysis is much slower , and the probability is correspondingly high that the non - cognate ternary complex will diffuse away from the ribosome intact , before gtp hydrolysis occurs . suppose that against the odds , a non - cognate ternary complex delivers its aminoacyl trna to the ribosome in the same way that a cognate complex does . before that aminoacyl trna can engage in peptide - bond formation , it must accommodate and , as far as we know , the only interactions that keep accommodating aminoacyl trnas from diffusing away from the ribosome are their codon - anticodon interactions . the understanding of decoding that has emerged from the combination of structural and biochemical investigations is a spectacular example of what everyone hoped would happen once the structure of the ribosome was understood at atomic resolution . however , as the above account shows , our understanding of even that aspect of the elongation cycle remains incomplete . among the many other mysteries still unsolved are the mechanisms of action of lepa , which is a protein factor similar to ef - g that promotes reverse translocation , an unexpected phenomenon believed to contribute to fidelity ; and the tmrna system that rescues ribosomes that have become stuck when translating a broken mrna molecule . my understanding of protein synthesis has been shaped by conversations with my colleagues , especially thomas steitz and rachel green .

satisfied with that level of understanding , most who had worked on protein synthesis during the ' golden age ' of molecular biology sought greener pastures in the years thereafter , and interest in the ribosome waned . the thesis of my review , which was entitled ' the ribosome returns ' , was that the ribosome field was poised for advances so dramatic that it would regain the prominence it had last enjoyed in the mid-1960s . in 1988 first , the discovery of ribozymes in the late 1970s had stimulated the interest of biochemists and molecular biologists in rna - containing objects generally , and the ribosome is the most important rna - containing object of them all . by writing a successor to ' the ribosome returns ' i would be in the embarrassing position of calling attention to an ancient review , the very title of which was a prediction . the shapes of the two ribosomal subunits , and of the complex they form during protein synthesis , were known at low resolution ( figure 1 ) , and it was understood that protein synthesis occurs in the gap between the two subunits . the secondary structures of the ribosomal rnas ( rrnas ) had been worked out , and sites on rrnas where ribosomal proteins bind had been identified . the only two approaches for addressing the need for structural information that seemed promising in the 1980s were x - ray crystallography and electron microscopy . the first ribosome crystals , reported by yonath , wittmann and colleagues in 1980 , diffracted poorly ; but , as the years went by , crystals were obtained of ribosomes and ribosomal subunits from many prokaryotic species ( for example ) , and the resolutions of the diffraction patterns of the best of them improved . in 1980 the technology for doing macro - molecular crystallography at synchrotrons was primitive , but major advances were made in the 1980s and thereafter ( for example ) , and by 1988 the technology needed for ribosome crystallography was falling into place . although the theory of image reconstruction is simple , its application to objects like the ribosome , for which the images to be reconstructed are those of isolated , randomly oriented particles , was fraught with difficulties . electron density maps can be computed from crystal diffraction data only if the phases associated with each of the tens of thousands of reflections in such datasets are known . in the end , the experimental technique that contributed the most to solving ribosome crystal structures was the heavy atom multiple isomorphous replacement ( mir ) method that perutz devised in the 1950s to solve the structure of hemoglobin . molecular replacement is a computational method for using the structure of one macromolecule to estimate the phases of the reflections in the diffraction pattern produced by a crystal of another macromolecule of related structure . although this approach would yield phases only up to the resolutions of the em reconstruction used , which was likely to be low by crystallographic standards , once the proverbial foot was in the proverbial door that far , higher resolution electron density maps would surely follow . that paper presents a 9 resolution electron density map of the large ribosomal subunit from haloarcula marismortui , which was obtained using crystals that had been first described 13 years earlier . by the summer of 1999 , the resolution of the electron density maps available for the large ribosomal subunit from h. marismortui had improved to 5.0 , and using heavy atom isomorphous replacement methods , ramakrishnan and colleagues had obtained a 5.5 resolution electron density map for the small ribosomal subunit from thermus thermophilus . ( figure 2 shows what the improvements in the resolutions of the electron density maps for this object between 1998 and 2000 meant in terms of their interpretability . ) at the beginning of september , an imperfect , 3.3 resolution structure of the small ribosomal subunit from t. thermophilus was presented , and 3 weeks thereafter a significantly more accurate , 3.1 resolution version of that same subunit appeared that had been independently determined . ( a - c ) views of the entire surface of the large ribosomal subunit of h. marismortui that interacts with the small ribosomal subunit . ( d ) a view of the face of the large subunit at a resolution of 5 , with the positions of l1 and l10 indicated in yellow . the three structures that appeared in 2000 are founder structures as are , first , a structure of the large ribosomal subunit from deinococcus radiodurans , second , two independently determined structures for the 70s ribosome from t. thermophilus [ 18 - 20 ] , and third , a structure of the 70s ribosome from escherichia coli . the rest of the ribosome structures in the pdb are those of founder particles with substrates , substrate analogs , protein factors and inhibitors bound , and all of them were generated using effectively the same crystals that produced the corresponding founder structures . since 2000 , the goal of ribosome crystallographers has been the construction of a movie of protein synthesis , the individual frames of which are atomic resolution structures of the ribosome in every state it visits as protein synthesis progresses . neither of the two lateral arms of the large ribosomal subunit ( figure 1 ) can be visualized at high resolution in crystal structures of isolated large subunits ; they wiggle too much . the conformation of the l1 arm is stabilized when the small subunit binds to the large and , for that reason , its structure is known in at least some of the conformations it adopts during protein synthesis . much less the l11 protuberance includes the complex that ribosomal protein l10 forms with four to six copies of ribosomal protein l7/l12 ( the number depends on species ) . however , structures are available for much of the l10 complex in isolation , and when those structures are added to the structure of the rest of the large subunit structure in silico , the effect is startling , to put it mildly ( figure 3 ) . there are structures available for most of the complex that l10 forms with the several copies of l7/l12 that interact with it in the ribosome as isolated proteins . finally , image sorting techniques now exist that make it possible to obtain reconstructions of ribosomes in specific functional states , starting with images of samples in which only a fraction of the particles present are in that state ; pure samples are no longer required . consequently , as far as the making of the movie of protein synthesis is concerned , the electron microscopists are well ahead of the crystallographers and , in addition , em images of eukaryotic ribosomes exist ( for example ) . three - dimensional electron density maps obtained by em are qualitatively similar to crystallographic electron density maps , and they are commonly used to generate ( quasi ) atomic resolution models of ribosomes , even though none of them has a resolution high enough to allow an atomic resolution model of the ribosome to be constructed from it de novo . this procedure leads to difficulties only in those parts of a structure where you are most in need of information , namely where the structure of the em object diverges from the crystal structure(s ) used to model it . no increase in the number of images processed can overcome resolution limitations caused by particle - to - particle variations in structure , unless the variation involves discrete conformational states , in which case image sorting may save the day . the steps of the elongation phase , which is the part of the process we will discuss here , constitute a cycle that must be repeated for every peptide bond formed . ( a movie showing the three phases of protein synthesis has been created by the ramakrishnan group at the mrc laboratory of molecular biology and can be accessed from their website . ) the purpose of the references provided here , which are entirely to recent articles and reviews , is to give the reader an entre into the relevant literature , rather than an outline of the history of the field . this is followed by transfer of the amino acid from the trna in the p site to form a peptide bond with the amino acid attached to the trna in the a site , and translocation of the two trnas into the e and the p site , respectively . the amino group used for the aminolysis reaction is the -amino group of an amino acid that is ester - linked through its -carboxyl group to the 3 ' oh of the 3'-terminal nucleotide of a trna . the products of this reaction are a peptide that is one residue longer than it was before the reaction , ester - linked to the trna that carried the amino acid into the reaction , and a trna that has nothing attached to it . in the second reaction , which occurs only during the termination phase of protein synthesis , the nucleophile is water , instead of an -amino group , and peptides are transferred to water ( that is , the last trna is released from the newly synthesized protein ) . both the peptidyl transferase reaction and the hydrolysis reaction occur at the same site on the ribosome , its peptidyl transferase center ( ptc ) , about which we now know quite a lot . ( 2 ) although rna - protein interactions are essential for stabilizing the conformation of the ptc , it is composed entirely of rna : the ribosome is a ribozyme . thus , ( 6 ) when the a site of the ptc is empty and its p site is occupied , the ptc adopts a conformation that protects the ester bonds of peptidyl trnas from nucleophilic attack . a conformational change accompanies the binding of aminoacyl trnas to the a site ( as well as the binding of release factors to the ribosome ) that exposes the ester bond in the p site to nucleophilic attack . ( 8) the group that makes the largest chemical contribution to the rate of the peptidyl transferase reaction is the 2 ' oh of the 3'-terminal a of the trna in the p site , which facilitates the removal of a proton from the attacking amino group and the addition of a proton to the leaving group , which is the 3 ' oh of the trna bound in the p site . ( 10 ) once substrates are bound appropriately to the ptc , the peptidyl transferase reaction occurs at a rate that is at least ten times faster than the overall rate of protein synthesis in living cells , which is about 20 s. ( 11 ) at neutral ph , the ester bond of an aminoacyl trna is a high energy bond , but the ester bond in a peptidyl trna is not . given that the net effect of the peptidyl transferase reaction is the destruction of a high energy ester bond and the creation of a lower energy peptide bond , the forward direction of the peptide formation is strongly favored thermodynamically . how does the peptidyl trna product of the peptidyl transferase reaction move from the a site , where it forms , to the p site , where it must reside if another amino acid is to be added to the nascent peptide chain ? it is not until the length of nascent peptides exceeds about 40 amino acids that their amino - terminal sequences emerge on the far side of the ribosome and start engaging with the apparatus that ensures protein folding and/or export . as far as we know , nascent poly - peptides diffuse down the tunnel in response to the nudge they are given as each peptide bond forms , but there are hints that it may be more interesting . messenger rnas bind to the small ribosomal subunit in the region where its head joins its body ( figure 1 ) , and the place on the small subunit where trna anticodons interact with mrna codons is called the decoding center . the a site and the p site of the decoding center are the locations where the anticodons of aminoacyl trnas and peptidyl trnas , respectively , are bound to the small ribosomal subunit just before peptide transfer occurs . ( trnas are l - shaped because there is a prominent ridge on the large subunit separating the ptc from the decoding center of the small subunit that only an l - shaped molecule can surmount . ) the cca sequence of the discharged trna moves from the p site of the ptc to the so - called e ( exit ) site of the large ribosomal subunit , which can bind only deacylated trnas , and the cca - peptide moiety of the peptidyl trna in the a site moves to the p site of the ptc . large - subunit translocation correlates with a rotation of about 10 of the small subunit relative to the large in the direction of the l1 arm , which is called ratcheting . large - subunit translocation leaves the ribosome in a hybrid state , in the sense that the acceptor stem of the peptidyl trna is in a p site of the ptc while its anticodon end occupies the a site of the decoding center ; the acceptor stem of the discharged trna is in the large - subunit e site while its anticodon is in the p site of the decoding center ( figure 4 ) . first , it advances the ribosome by three nucleotides along the mrna to which it is bound in the 3 ' direction , which places a new codon in the a site of the decoding center . second , it resolves the hybrid state by making the anticodon end of peptidyl trna move from the a site to the p site of the decoding center , and the anticodon end of the deacylated trna move from the p site of the decoding center to the e site of the small subunit . the anticodon of the trna that moves from the a site to the p site of the decoding center remains associated with its codon in the mrna so that the register in which the mrna is being translated is maintained . it is unclear whether the anticodons of trnas in the e site actually interact with mrna or not ; there are biochemical data indicating they do , but the structural data are ambiguous . the g protein in this case , which is ef - g in bacteria and ef-2 in eukaryotes , binds to the ribosome with a gtp bound that is hydrolyzed in the process . em images of ef - g / ribosome complexes , which are all we have , show that this tadpole - shaped molecule binds to the ribosome with its head ( which includes its gtpase site ) bound to the large ribosomal subunit at the base of the l11 arm . that part of the ef - g binding site includes the sarcin - ricin loop ( srl ) of 23s/28s rrna ( figure 3 ) that , for reasons still unclear , is critically important for the activity of all of the g - protein factors that interact with the ribosome during protein synthesis . the binding of all proteins that interact with the ef - g binding site is promoted by the l10 complex and the rest of the l11 arm , but the details remain to be worked out . this causes ef - g to undergo a major conformational change that seems to push the anticodon stems of trnas across the decoding center , dragging the mrna with them . biochemical data suggest , and em structures confirm , that the conformational changes that accompany ef - g - assisted translocation are more complicated than this account of translocation seems to require , but until the relevant atomic resolution structures become available , we are unlikely to understand them properly . once translocation is complete , the ribosome is ready to bind a new aminoacyl trna , and if there is a deacylated trna in the e site of the ribosome , aminoacyl trna binding is accompanied by release of that trna into solution . biochemical data suggest that these two processes interact with each other , and structural data show that trna release correlates with conformational changes in the l1 arm of the large subunit . the complex that ef - tu forms with aminoacyl trna ( and gtp ) , the so - called ternary complex , resembles ef - g in its overall shape , with the anticodon stem of the ternary complex corresponding to the tail of the ef - g tadpole and its ef - tu / acceptor stem portion resembling the head . if the anticodon of the trna in a ternary complex base - pairs properly with the mrna sequence in the a site of the decoding center , in other words if the codon and anticodon are cognate , a conformational change occurs that stimulates gtp cleavage and release of ef - tugdp from the ribosome . the binding of cognate aminoacyl trnas to the ribosome is the rate - limiting step in protein synthesis , and from the point of view of information transfer , it is the most important . given that diffusion is the process that brings ternary complexes to the ribosome , for every encounter with a cognate complex that results in accommodation there will be many encounters with non - cognate ternary complexes that must not result in accommodation if mrnas are to be translated correctly . however , it has been realized for decades that the difference in free energy between the formation of a perfect watson - crick helix three base - pairs long and the formation of a three - base - pair duplex in which one of the base juxtapositions is non - canonical is not large enough to explain the accuracy of translation . we know from crystal structures of trnas bound to the ribosome in the accommodated state that when a cognate interaction occurs in the a site of the decoding center , the conformation of the small subunit changes so that two of its rna bases form base - triples with the first two base - pairs of the codon - anticodon helix that form in that center . thus , the response of the a site of the decoding center to anticodons appears to be the same no matter whether the trna carrying them is in the pre- or post - accommodation conformation . a vitally important consequence of the conformational change just alluded to is that , by some mechanism we do not understand , it activates the gtpase activity of ef - tu . thus , if a cognate interaction has occurred in the decoding center , the ef - tu in a ternary complex will quickly hydrolyze its gtp and leave the ribosome so that the aminoacyl trna can accommodate . if the interaction between codon and anticodon is non - cognate , gtp hydrolysis is much slower , and the probability is correspondingly high that the non - cognate ternary complex will diffuse away from the ribosome intact , before gtp hydrolysis occurs . before that aminoacyl trna can engage in peptide - bond formation , it must accommodate and , as far as we know , the only interactions that keep accommodating aminoacyl trnas from diffusing away from the ribosome are their codon - anticodon interactions . the understanding of decoding that has emerged from the combination of structural and biochemical investigations is a spectacular example of what everyone hoped would happen once the structure of the ribosome was understood at atomic resolution . among the many other mysteries still unsolved are the mechanisms of action of lepa , which is a protein factor similar to ef - g that promotes reverse translocation , an unexpected phenomenon believed to contribute to fidelity ; and the tmrna system that rescues ribosomes that have become stuck when translating a broken mrna molecule .

although the conventional surgical treatment is still the most utilized and effective treatment for colorectal malignancy , the procedure may lead to prolonged hospital stay , increased medical costs , and medical resource overuse due to the aggressiveness of the approach and the high risk of developing postoperative nutrition disorders . patients are often dissatisfied with their functional recovery and the incidence of complications is 20 to 30 % [ 1 , 2 ] . although the incidence of conventional complications such as anastomotic leakage has decreased with the improvements of surgical instruments and techniques , postoperative psychiatric complications such as delirium are frequent , particularly in the elderly ( 70 years ) . as reported previously , about 1050 % of advanced - age patients undergoing surgical treatment may develop delirium postoperatively [ 3 - 5 ] . postoperative delirium is a reversible and fluctuating acute brain syndrome characterized by changes in consciousness , orientation , attention , memory , sensory perception , thinking , emotion , and volition . the occurrence of this condition may lead to prolonged hospital stay and unfavorable prognosis , and may develop into chronic brain syndrome ( dementia ) . although the mechanism of delirium remains unclear , it has been clearly demonstrated that multiple factors are involved [ 7 , 8 ] . besides advanced age , a recent study showed that systemic stress and inflammatory response might play an important role in the development of this condition [ 9 , 10 ] . it has been reported that the serum levels of an inflammatory biomarkers , including interleukin-6 ( il-6 ) , were positively correlated to the incidence of delirium . moreover , therefore , reduction of the perioperative stress and inflammatory response may minimize the occurrence of delirium . it has been found in several clinical trials that fast - track surgery ( fts ) not only facilitates the physical rehabilitation of the patients with colorectal malignancies , but also prevents upregulation of proinflammatory cytokines including il-6 , with reduced stress response and inflammation [ 13 , 14 ] . moreover , krenk et al . have shown that delirium was not observed in fast - track hip and knee arthroplasty in elderly patients . however , there is little data on whether fts can prevent or protect elderly patients with colorectal carcinoma from developing delirium after colorectal surgery . in the present randomized trial , we studied whether fts could prevent or reduce the occurrence of postoperative delirium as well as other complications in elderly patients with colorectal carcinoma and evaluated the role of il-6 in postoperative delirium . a total of 240 elderly patients with colorectal carcinoma , with ages ranging from 70 to 88 years ( mean , 75.18 years ; 150 men and 90 women ) , admitted to the fourth hospital of hebei medical university for open curative resection between 2008 and 2011 , were included in the present study . these patients were randomly assigned into the traditional therapy group ( n = 120 ) and the fts group ( n = 120 ) . eligible patients were randomly assigned to each group in a 1:1 ratio ( fig . 1flow diagram of study flow diagram of study patients with a history of dementia , parkinson s disease , alcohol intake of 250 g / day , long - term use of sleeping pills or anxiolytics , and those who received anesthesia within the past 30 days were excluded from the study during the initial screening . those enrolled patients who were subsequently given intraoperative blood transfusion or were admitted to the intensive care unit ( icu ) for further treatment after operation were also excluded from analysis ( fig . 1 ) . of the 240 study participants , 115 were diagnosed with colon cancer and 125 with rectal cancer . patients in the two groups had comparable baseline characteristics including gender , age , site of lesion , tnm classification of malignant tumours ( tnm staging ) , and surgical procedure ( table 1 ) . in the current study , a preoperative routine cranial magnetic resonance imaging ( mri ) scan was performed for all the patients in both groups , and a repeat mri scan performed for those who developed postoperative delirium to exclude cerebrovascular stroke or other central nervous system ( cns ) conditions as the etiology of delirium.table 1clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233)characteristicsfts ( n = 117)traditional ( n = 116 ) p valueage75.66 4.1874.78 4.010.054gender male76700.467 female4146site of lesions colon57580.845 rectum6058tnm staging i13190.633 ii5247 iii3738 iv1512surgical operation colectomy52530.930 dixon3936 miles2627hypertension26330.275diabetes11140.511 clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233 ) this study was approved by the ethics committee of the fourth hospital of hebei medical university . the length of hospital stay ( los ) and time to pass flatus were documented . the incidence of complications including pulmonary infection , urinary tract infection , intestinal obstruction , anastomotic leakage , heart failure , and deep venous thrombosis ( dvt ) were also monitored and documented . all patients were followed up and all parameters were obtained from all patients . the diagnosis of postoperative delirium as the focus of this study is described below . the perioperative managements for patients of both groups including preoperative preparation , anesthesia , pain control , and postoperative managements are compared and summarized in table 2 . the management of the fts group differed from the traditionally managed group in the several ways : ( 1 ) bowel preparation with oral purgatives instead of a mechanical enema ; ( 2 ) thoracic epidural anesthesia and postoperative analgesic maintenance via the epidural catheter ( ropivacaine , 2 mg / ml maintained for 48 h , controlled to 610 ml ( 1220 mg ) per hour and opium - derived agents were excluded ) ; ( 3 ) no nasogastric tube insertion ; ( 4 ) no drainage tube placement with the exception of low rectal anastomosis ; ( 5 ) water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days ( pod ) with regular diet on pod 3 ; ( 6 ) early urine catheter withdrawal ( at pod 12 ) ; and ( 7 ) early out - of - bed mobilization ( i.e. , walking).table 2comparison of fast - track and traditional perioperative care protocolstraditionalfast trackpreoperative preparationliquid diet for 3 daysoral purgativesmechanical enema(1time / day ) for 3 consecutive daysno mechanical enemafasting at 8 hnormal meal until 6 h before surgerydrink deprivation 4 h before surgerynormal carbohydrate drink until 2 h before surgeryroutine nasogastric tube insertionno nasogastric tube insertionoral antibiotics administration for 3 daysno antibioticsanesthesiageneralthoracic epiduralpain controlfentanyl0.25 mg / mlropivacaine2mg / mlmidazolam0 . 5 mg / mlnefopam1.0 mg / mlvia pceavia pciafor 48 hfor 48 hopium - derived agents were excludedroutine drainage tube placementno routine drainage tube placementpostoperative managementdiet : liquid diet intake after recovery of bowel movementdiet : water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days , regular diet on pod 3urinary catheter withdrawal at 3 to 5 daysurinary catheter withdrawal on pod 12out - of - bed mobilization at 3 to 5 daysout - of - bed mobilization on pod 1 comparison of fast - track and traditional perioperative care protocols the mental status and cognitive function was evaluated in accordance with the delirium rating scale - revised-98 ( drs - r-98 ) . the drs - r-98 evaluates the cognitive domain of delirium by recourse to specific evaluations for attention , orientation , short - term memory , long - term memory , and visuospatial ability . drs - r-98 has a high sensitivity ( 91100 % ) and specificity ( 85100 % ) for detection of delirium . the drs - r-98 scoring was performed on the day of admission and then daily from pod 1 for 5 days . the presence or absence of delirium was evaluated by a psychiatrist and a nurse based on the criteria specified in drs - r-98 . fasting peripheral venous blood samples ( 5 ml ) were collected 1 day preoperatively and on pod 1 , 2 , and 3 . the blood samples were centrifuged at 3,000 rpm for 5 min , and the sera obtained were preserved at 20c for later use . the serum il-6 levels were determined using sandwich elisa with the reagents purchased from invitrogen , camarillo , ca , usa . the results are expressed as mean sd . a total of 240 elderly patients with colorectal carcinoma , with ages ranging from 70 to 88 years ( mean , 75.18 years ; 150 men and 90 women ) , admitted to the fourth hospital of hebei medical university for open curative resection between 2008 and 2011 , were included in the present study . these patients were randomly assigned into the traditional therapy group ( n = 120 ) and the fts group ( n = 120 ) . eligible patients were randomly assigned to each group in a 1:1 ratio ( fig . 1flow diagram of study flow diagram of study patients with a history of dementia , parkinson s disease , alcohol intake of 250 g / day , long - term use of sleeping pills or anxiolytics , and those who received anesthesia within the past 30 days were excluded from the study during the initial screening . those enrolled patients who were subsequently given intraoperative blood transfusion or were admitted to the intensive care unit ( icu ) for further treatment after operation were also excluded from analysis ( fig . 1 ) . of the 240 study participants , 115 were diagnosed with colon cancer and 125 with rectal cancer . patients in the two groups had comparable baseline characteristics including gender , age , site of lesion , tnm classification of malignant tumours ( tnm staging ) , and surgical procedure ( table 1 ) . in the current study , a preoperative routine cranial magnetic resonance imaging ( mri ) scan was performed for all the patients in both groups , and a repeat mri scan performed for those who developed postoperative delirium to exclude cerebrovascular stroke or other central nervous system ( cns ) conditions as the etiology of delirium.table 1clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233)characteristicsfts ( n = 117)traditional ( n = 116 ) p valueage75.66 4.1874.78 4.010.054gender male76700.467 female4146site of lesions colon57580.845 rectum6058tnm staging i13190.633 ii5247 iii3738 iv1512surgical operation colectomy52530.930 dixon3936 miles2627hypertension26330.275diabetes11140.511 clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233 ) this study was approved by the ethics committee of the fourth hospital of hebei medical university . the length of hospital stay ( los ) and time to pass flatus were documented . the incidence of complications including pulmonary infection , urinary tract infection , intestinal obstruction , anastomotic leakage , heart failure , and deep venous thrombosis ( dvt ) were also monitored and documented . all patients were followed up and all parameters were obtained from all patients . the diagnosis of postoperative delirium as the focus of this study is described below . the perioperative managements for patients of both groups including preoperative preparation , anesthesia , pain control , and postoperative managements are compared and summarized in table 2 . the management of the fts group differed from the traditionally managed group in the several ways : ( 1 ) bowel preparation with oral purgatives instead of a mechanical enema ; ( 2 ) thoracic epidural anesthesia and postoperative analgesic maintenance via the epidural catheter ( ropivacaine , 2 mg / ml maintained for 48 h , controlled to 610 ml ( 1220 mg ) per hour and opium - derived agents were excluded ) ; ( 3 ) no nasogastric tube insertion ; ( 4 ) no drainage tube placement with the exception of low rectal anastomosis ; ( 5 ) water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days ( pod ) with regular diet on pod 3 ; ( 6 ) early urine catheter withdrawal ( at pod 12 ) ; and ( 7 ) early out - of - bed mobilization ( i.e. , walking).table 2comparison of fast - track and traditional perioperative care protocolstraditionalfast trackpreoperative preparationliquid diet for 3 daysoral purgativesmechanical enema(1time / day ) for 3 consecutive daysno mechanical enemafasting at 8 hnormal meal until 6 h before surgerydrink deprivation 4 h before surgerynormal carbohydrate drink until 2 h before surgeryroutine nasogastric tube insertionno nasogastric tube insertionoral antibiotics administration for 3 daysno antibioticsanesthesiageneralthoracic epiduralpain controlfentanyl0.25 mg / mlropivacaine2mg / mlmidazolam0 . 5 mg / mlnefopam1.0 mg / mlvia pceavia pciafor 48 hfor 48 hopium - derived agents were excludedroutine drainage tube placementno routine drainage tube placementpostoperative managementdiet : liquid diet intake after recovery of bowel movementdiet : water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days , regular diet on pod 3urinary catheter withdrawal at 3 to 5 daysurinary catheter withdrawal on pod 12out - of - bed mobilization at 3 to 5 daysout - of - bed mobilization on pod 1 comparison of fast - track and traditional perioperative care protocols the mental status and cognitive function was evaluated in accordance with the delirium rating scale - revised-98 ( drs - r-98 ) . the drs - r-98 evaluates the cognitive domain of delirium by recourse to specific evaluations for attention , orientation , short - term memory , long - term memory , and visuospatial ability . drs - r-98 has a high sensitivity ( 91100 % ) and specificity ( 85100 % ) for detection of delirium . the drs - r-98 scoring was performed on the day of admission and then daily from pod 1 for 5 days . the presence or absence of delirium was evaluated by a psychiatrist and a nurse based on the criteria specified in drs - r-98 . fasting peripheral venous blood samples ( 5 ml ) were collected 1 day preoperatively and on pod 1 , 2 , and 3 . the blood samples were centrifuged at 3,000 rpm for 5 min , and the sera obtained were preserved at 20c for later use . the serum il-6 levels were determined using sandwich elisa with the reagents purchased from invitrogen , camarillo , ca , usa . the results are expressed as mean sd . the measurement data are represented as mean sd . the intergroup comparison was performed using the wilcoxon rank - sum test . the age , gender , site of lesions , tnm staging , surgical procedures , and co - morbidity including hypertension and diabetes between the two groups were comparable , without statistically significant differences ( table 1 ) . four patients were excluded from the traditional group : two patients received an intraoperative blood transfusion and the other two were admitted to icu because of pulmonary infection . three patients were excluded from the fts group : one received an intraoperative blood transfusion , and the other two were admitted to icu because of pulmonary infection . thus , a total of 233 patients including 116 patients in the traditional group and 117 patients in the fts group were included this study ( fig . 1 ) . the mean los of the fts group and traditional therapy group was 9.01 1.75 and 13.21 1.32 days , respectively ( p < 0.001 ) . the time to pass flatus in the fts group was significantly shorter than in the traditional therapy group ( 48.50 9.59 vs. 77.66 7.18 h ; p < 0.001 ) . on the pod 1 , the level of serum albumin in the fts group was higher than that in the traditional therapy group ( 28.05 2.82 vs. 26.26 4.12 ; p < 0.001 ) . meanwhile , the glucose in the fts group was lower than that in the traditional therapy group ( 8.30 2.49 vs. 10.25 2.43 ; p < 0.001 ) . no significant difference of liver or renal function was observed between the two groups after operation ( table 3).table 3comparison of postoperative recovery and complications between the fts and traditional groupfts ( 117)traditional ( 116 ) p valuelos ( day)9.01 1.7513.21 1.32<0.001functional recovery time to pass flatus ( h)48.50 9.5977.66 7.18<0.001 serum albumin ( g / l)28.05 2.8226.26 4.12<0.001 glucose ( mmol / l)8.30 2.4910.25 2.43<0.001 alt ( iu / l)34.65 12.2534.88 11.820.738 ast ( iu / l)30.43 10.7829.47 10.400.356 cr ( mol / l)77.05 23.8075.11 25.040.675 bun ( mmol / l)5.63 3.605.62 3.080.831complications ( cases ) infection of incision680.570 pulmonary infection6190.006 urinary infection5130.047 anastomotic leakage321.000 intestinal obstruction460.736 heart failure4130.022 dvt470.340 alt alanine transaminase , ast aspartate transaminase comparison of postoperative recovery and complications between the fts and traditional group alt alanine transaminase , ast aspartate transaminase the postoperative complications were followed and documented based on clavien dindo classification system , including infection , intestinal obstruction , anastomotic leakage , heart failure , and dvt . six pulmonary infections were observed in the fts group , significantly fewer than the 19 cases in the traditional therapy group ( p = 0.006 ) . the incidence of urinary infections was lower in fts group than that in the traditional therapy group ( 5 vs. 13 ; p = 0.047 ) . the incidence of heart failure was much higher in the traditional therapy group than that in the fts group ( 13 vs. 4 , p = 0.022 ) . however , no statistical differences were found in the incidences of incision infection , bowel obstruction , anastomotic leakage , and dvt between the two groups . the results are summarized in table 3 . using the drs - r-98 scoring system , a total 19 cases of postoperative delirium were observed , 15 in the traditional therapy group ( 12.9 % ) and 4 in the fts group ( 3.4 % , p = 0.008 ) ( table 4 ) . in the traditional therapy group , nine incidents of delirium were observed at pod 1 , five at pod 2 , and one at pod 3 , while for the fts group , three incidents of delirium were observed at pod 1 and one at pod 2 ( table 4).table 4incidence of postoperative delirium in the fts and traditional grouppodftstraditional p valuepod 1390.073pod 2150.211pod 3010.498total ( % of analyzed cases)4 ( 3.4 % ) 15 ( 12.9 % ) 0.008 incidence of postoperative delirium in the fts and traditional group repeat mri scanning for those with postoperative delirium did not show any cns changes ( data not shown ) . this indicates that postoperative delirium was not due to apparent organic cns involvements , e.g. , cerebrovascular stroke , after the surgery . the treatments for delirium included intensive nursing care , and for , severe cases , haloperidol ( 0.5 mg ) was intramuscularly administered and repeated if necessary at an interval of 30 to 60 min . the delirium - related symptoms resolved in all 19 patients after the treatments . as proinflammatory cytokines particularly il-6 have been reported to be involved in the development of postoperative delirium [ 10 , 19 - 21 ] , we determined the serum il-6 levels in both the fts and the traditional groups . the preoperative baseline il-6 levels in both groups were very similar . for both group , the serum il-6 level peaked at pod 1 , followed by a rapid decline thereafter . this il-6 peak correlated with the highest incidence of delirium on pod 1 ( fig . 2 ) . although the il-6 level in fst group decreased to approximately the baseline level by pod 3 , the il-6 level of the traditional group was still well above the baseline level ( fig . 2 ) the serum il-6 levels in the traditional therapy group at pod 1 , 2 , and 3 were all significantly higher than those in the fts group ( p < 0.001 in all 3 pod days ) . together , these results showed that enhanced il-6 level correlated with the development of postoperative delirium in the elderly patients undergoing colorectal surgery.fig . 2serum il-6 levels in the fts group and traditional group . * * p < pre preoperation , pod postoperative day serum il-6 levels in the fts group and traditional group . * the postoperative complications were followed and documented based on clavien dindo classification system , including infection , intestinal obstruction , anastomotic leakage , heart failure , and dvt . six pulmonary infections were observed in the fts group , significantly fewer than the 19 cases in the traditional therapy group ( p = 0.006 ) . the incidence of urinary infections was lower in fts group than that in the traditional therapy group ( 5 vs. 13 ; p = 0.047 ) . the incidence of heart failure was much higher in the traditional therapy group than that in the fts group ( 13 vs. 4 , p = 0.022 ) . however , no statistical differences were found in the incidences of incision infection , bowel obstruction , anastomotic leakage , and dvt between the two groups . using the drs - r-98 scoring system , a total 19 cases of postoperative delirium were observed , 15 in the traditional therapy group ( 12.9 % ) and 4 in the fts group ( 3.4 % , p = 0.008 ) ( table 4 ) . in the traditional therapy group , nine incidents of delirium were observed at pod 1 , five at pod 2 , and one at pod 3 , while for the fts group , three incidents of delirium were observed at pod 1 and one at pod 2 ( table 4).table 4incidence of postoperative delirium in the fts and traditional grouppodftstraditional p valuepod 1390.073pod 2150.211pod 3010.498total ( % of analyzed cases)4 ( 3.4 % ) 15 ( 12.9 % ) 0.008 incidence of postoperative delirium in the fts and traditional group repeat mri scanning for those with postoperative delirium did not show any cns changes ( data not shown ) . this indicates that postoperative delirium was not due to apparent organic cns involvements , e.g. , cerebrovascular stroke , after the surgery . the treatments for delirium included intensive nursing care , and for , severe cases , haloperidol ( 0.5 mg ) was intramuscularly administered and repeated if necessary at an interval of 30 to 60 min . as proinflammatory cytokines particularly il-6 have been reported to be involved in the development of postoperative delirium [ 10 , 19 - 21 ] , we determined the serum il-6 levels in both the fts and the traditional groups . the preoperative baseline il-6 levels in both groups were very similar . for both group , the serum il-6 level peaked at pod 1 , followed by a rapid decline thereafter . this il-6 peak correlated with the highest incidence of delirium on pod 1 ( fig . 2 ) . although the il-6 level in fst group decreased to approximately the baseline level by pod 3 , the il-6 level of the traditional group was still well above the baseline level ( fig . 2 ) the serum il-6 levels in the traditional therapy group at pod 1 , 2 , and 3 were all significantly higher than those in the fts group ( p < 0.001 in all 3 pod days ) . together , these results showed that enhanced il-6 level correlated with the development of postoperative delirium in the elderly patients undergoing colorectal surgery.fig . 2serum il-6 levels in the fts group and traditional group . * * p < 0.001 traditional vs. fts group . pre preoperation , pod postoperative day serum il-6 levels in the fts group and traditional group . * in the present randomized trial , patients of advanced age ( 70 years ) with colorectal carcinoma were treated with either fts ( 117 patients ) or traditional approaches ( 116 patients ) . the patients managed with fts had significant shorter los and fast recovery of bowel movement ( table 3 ) . importantly , there was a decrease in the incidence of postoperative complications including pulmonary infection , urinary infection , and heart failure ( table 3 ) , which is consistent with previous reports [ 22 , 23 ] . notably , fts treatment was also associated with a decreased frequency of postoperative delirium ( table 4 ) . fts did not cause the same degree of increase in serum level of the proinflammatory cytokine il-6 ( fig . 2 ) . delirium is an acute brain syndrome most commonly seen in the elderly , particularly in those with underlying organic brain pathology and/or functional impairments . the incidence of postoperative delirium varies depending on the surgical and anesthetic approaches used and ranges from 10 to 24 % in gastrointestinal surgery [ 4 , 5 , 25 , 26 ] . predisposing factors include age ; comorbid disease ; and cognitive , visual , and hearing disorders [ 7 , 8 , 27 , 28 ] . in our trial , all the patients in both groups were of advanced age ( 70 years ) and were more prone to developing delirium after surgery . the precipitating factors include the type and extensiveness of the surgical procedure , anesthetic protocol , opioid pain killer usage , inflammatory response , infection , pain , sleep disturbance , nutritional condition , and electrolyte homeostasis [ 29 , 30 ] . since the highest frequency of delirium occurred early on pod 1 , we believe that preoperative preparation , surgical approach , anesthesia , and pain control protocols were among the most important factors , although other subsequent complications ( e.g. , postoperative infection ) might also play a role most likely later during the course . in our study , patients in both groups underwent similar open colorectal surgery ( table 1 ) , the extensiveness of the surgery and trauma incurred in both groups were similar and unlikely to be a contributing factor . pain and sleep disturbance reported in both groups were comparable ( data not shown ) , so they were not a likely explanation for the discrepancy of incidence of delirium between the two groups . it has been reported that early feeding in open colon resection in the elderly resulted in shorter los and reduced postoperative morbidity , so it is possible that early feeding in the fst group might be contributing to the overall lower postoperative complications including delirium . the two groups received different preoperative preparation ( table 2 ) . in the traditional group , mechanical enema and nasogastric tube insertion were applied , while in the fts group , only oral purgatives was given and no nasogastric tube was used . it is conceivable that these less - invasive procedures used in the fts group could potentially decrease the stress response , and be a factor in the lower occurrence of postoperative delirium . a major difference in the management of patients in the two groups was the different anesthesia approach , pain control , and drug selection ( table 2 ) . it is unclear whether general anesthesia per se might play a role in postoperative delirium , but it was possible that it might cause more extensive systematic stress response than epidural anesthesia applied in the fts group [ 32 , 33 ] . in addition to anesthesia methods , intraoperative and postoperative medication may affect the mental status of patients [ 34 , 35 ] . for patients with traditional therapy , opioid drugs including morphine , dolantin , and fentanyl that were used during general anesthesia and postoperative pain management might contribute to the occurrence of delirium . epidural anesthesia and analgesia may block the sympathetic response that has been reported to be involved in delirium . the analgesic agent used in fts group was ropivacaine , which can alleviate moderate to severe postoperative pains effectively and safely . together , these measures can help reduce the various stimuli to the patients during surgical injury , minimize postoperative inflammatory reaction , and facilitate functional recovery . therefore , integrated measures should be applied to prevent elderly colorectal patients from developing postoperative delirium . excessive release of proinflammatory cytokines such as tnf- , il-1 , il-6 , and il-8 during systemic inflammation can affect brain functions and promote the development of delirium . to investigate whether fts reduced the incidence of delirium by minimizing the inflammatory response , serum il-6 levels were also determined . we found that enhanced il-6 level correlated with the development of postoperative delirium and peaked on pod 1 in the elderly patients undergoing colorectal surgery ( fig . 2 and table 4 ) . fts significantly reduced the level of il-6 increase as compared to the traditional therapy group ( fig . 2 ) . this is consistent with the previous reports that elderly patients who develop postoperative delirium may exhibit an elevated serum il-6 level [ 39 , 40 ] . it is well known that il-6 is an endogenous cytokine released by monocytes , t cells , and vascular endothelial cells and is a major inducer of the inflammation . il-6 can promote the differentiation of lymphocytes and amplify the inflammatory response leading to tissue damage . in this study , there was a correlation between elevated levels of il-6 and delirium , both of which were present at reduced levels in the fts treatment group . less invasive preoperative preparation ( no mechanic enema , no nasogastric tube insertion , etc . ) , use of epidural instead of general anesthesia , and avoidance of opioids for anesthesia and pain control could all contribute to the reduced level of il-6 in the fts group . it has been reported that opioids can stimulate il-6 production in both animal and human studies [ 43 - 47 ] , so anesthesia and analgesia without utilizing opioid drugs may be an important factor for reduced il-6 levels and reduced postoperative delirium in patients managed with fts . although we only examined il-6 level in the peripheral blood , which may not directly represent the intracerebral conditions accurately , however , it has been shown that il-6 can readily cross the blood fts shortens the los , facilitates the recovery of bowel movement , and reduces occurrence of postoperative delirium and other complications in elderly patients with colorectal carcinoma . the lower incidence of delirium in the fts group is likely attributable to the reduced systemic stress and inflammatory response mediated by il-6 .

objectivethis study aims to investigate the role of fast - track surgery in preventing the development of postoperative delirium and other complications in elderly patients with colorectal carcinoma.methodsa total of 240 elderly patients with colorectal carcinoma ( aged 70 years ) undergoing open colorectal surgery was randomly assigned into two groups , in which the patients were managed perioperatively either with traditional or fast - track approaches . the length of hospital stay ( los ) and time to pass flatus were compared . the incidence of postoperative delirium and other complications were evaluated . serum interleukin-6 ( il-6 ) levels were determined before and after surgery.resultsthe los was significantly shorter in the fast - track surgery ( fts ) group than that in the traditional group . the recovery of bowel movement ( as indicated by the time to pass flatus ) was faster in the fts group . the postoperative complications including pulmonary infection , urinary infection and heart failure were significantly less frequent in the fts group . notably , the incidence of postoperative delirium was significantly lower in patients with the fast track therapy ( 4/117 , 3.4 % ) than with the traditional therapy ( 15/116 , 12.9 % ; p = 0.008 ) . the serum il-6 levels on postoperative days 1 , 2 , and 3 in patients with the fast - track therapy were significantly lower than those with the traditional therapy ( p < 0.001).conclusionscompared to traditional perioperative management , fast - track surgery decreases the los , facilitates the recovery of bowel movement , and reduces occurrence of postoperative delirium and other complications in elderly patients with colorectal carcinoma . the lower incidence of delirium is at least partly attributable to the reduced systemic inflammatory response mediated by il-6 .

Introduction Patients and methods Patients and study design Patient perioperative monitoring Perioperative management of the traditional and FTS group Diagnostic criteria of delirium Serum IL-6 determination Statistical analysis Results Incidence of complications Incidence of postoperative delirium Serum IL-6 level Discussion Conflicts of interest

although the incidence of conventional complications such as anastomotic leakage has decreased with the improvements of surgical instruments and techniques , postoperative psychiatric complications such as delirium are frequent , particularly in the elderly ( 70 years ) . it has been reported that the serum levels of an inflammatory biomarkers , including interleukin-6 ( il-6 ) , were positively correlated to the incidence of delirium . it has been found in several clinical trials that fast - track surgery ( fts ) not only facilitates the physical rehabilitation of the patients with colorectal malignancies , but also prevents upregulation of proinflammatory cytokines including il-6 , with reduced stress response and inflammation [ 13 , 14 ] . have shown that delirium was not observed in fast - track hip and knee arthroplasty in elderly patients . however , there is little data on whether fts can prevent or protect elderly patients with colorectal carcinoma from developing delirium after colorectal surgery . in the present randomized trial , we studied whether fts could prevent or reduce the occurrence of postoperative delirium as well as other complications in elderly patients with colorectal carcinoma and evaluated the role of il-6 in postoperative delirium . a total of 240 elderly patients with colorectal carcinoma , with ages ranging from 70 to 88 years ( mean , 75.18 years ; 150 men and 90 women ) , admitted to the fourth hospital of hebei medical university for open curative resection between 2008 and 2011 , were included in the present study . these patients were randomly assigned into the traditional therapy group ( n = 120 ) and the fts group ( n = 120 ) . in the current study , a preoperative routine cranial magnetic resonance imaging ( mri ) scan was performed for all the patients in both groups , and a repeat mri scan performed for those who developed postoperative delirium to exclude cerebrovascular stroke or other central nervous system ( cns ) conditions as the etiology of delirium.table 1clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233)characteristicsfts ( n = 117)traditional ( n = 116 ) p valueage75.66 4.1874.78 4.010.054gender male76700.467 female4146site of lesions colon57580.845 rectum6058tnm staging i13190.633 ii5247 iii3738 iv1512surgical operation colectomy52530.930 dixon3936 miles2627hypertension26330.275diabetes11140.511 clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233 ) this study was approved by the ethics committee of the fourth hospital of hebei medical university . the length of hospital stay ( los ) and time to pass flatus were documented . the incidence of complications including pulmonary infection , urinary tract infection , intestinal obstruction , anastomotic leakage , heart failure , and deep venous thrombosis ( dvt ) were also monitored and documented . the management of the fts group differed from the traditionally managed group in the several ways : ( 1 ) bowel preparation with oral purgatives instead of a mechanical enema ; ( 2 ) thoracic epidural anesthesia and postoperative analgesic maintenance via the epidural catheter ( ropivacaine , 2 mg / ml maintained for 48 h , controlled to 610 ml ( 1220 mg ) per hour and opium - derived agents were excluded ) ; ( 3 ) no nasogastric tube insertion ; ( 4 ) no drainage tube placement with the exception of low rectal anastomosis ; ( 5 ) water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days ( pod ) with regular diet on pod 3 ; ( 6 ) early urine catheter withdrawal ( at pod 12 ) ; and ( 7 ) early out - of - bed mobilization ( i.e. 5 mg / mlnefopam1.0 mg / mlvia pceavia pciafor 48 hfor 48 hopium - derived agents were excludedroutine drainage tube placementno routine drainage tube placementpostoperative managementdiet : liquid diet intake after recovery of bowel movementdiet : water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days , regular diet on pod 3urinary catheter withdrawal at 3 to 5 daysurinary catheter withdrawal on pod 12out - of - bed mobilization at 3 to 5 daysout - of - bed mobilization on pod 1 comparison of fast - track and traditional perioperative care protocols the mental status and cognitive function was evaluated in accordance with the delirium rating scale - revised-98 ( drs - r-98 ) . the serum il-6 levels were determined using sandwich elisa with the reagents purchased from invitrogen , camarillo , ca , usa . a total of 240 elderly patients with colorectal carcinoma , with ages ranging from 70 to 88 years ( mean , 75.18 years ; 150 men and 90 women ) , admitted to the fourth hospital of hebei medical university for open curative resection between 2008 and 2011 , were included in the present study . these patients were randomly assigned into the traditional therapy group ( n = 120 ) and the fts group ( n = 120 ) . in the current study , a preoperative routine cranial magnetic resonance imaging ( mri ) scan was performed for all the patients in both groups , and a repeat mri scan performed for those who developed postoperative delirium to exclude cerebrovascular stroke or other central nervous system ( cns ) conditions as the etiology of delirium.table 1clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233)characteristicsfts ( n = 117)traditional ( n = 116 ) p valueage75.66 4.1874.78 4.010.054gender male76700.467 female4146site of lesions colon57580.845 rectum6058tnm staging i13190.633 ii5247 iii3738 iv1512surgical operation colectomy52530.930 dixon3936 miles2627hypertension26330.275diabetes11140.511 clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233 ) this study was approved by the ethics committee of the fourth hospital of hebei medical university . the length of hospital stay ( los ) and time to pass flatus were documented . the incidence of complications including pulmonary infection , urinary tract infection , intestinal obstruction , anastomotic leakage , heart failure , and deep venous thrombosis ( dvt ) were also monitored and documented . the management of the fts group differed from the traditionally managed group in the several ways : ( 1 ) bowel preparation with oral purgatives instead of a mechanical enema ; ( 2 ) thoracic epidural anesthesia and postoperative analgesic maintenance via the epidural catheter ( ropivacaine , 2 mg / ml maintained for 48 h , controlled to 610 ml ( 1220 mg ) per hour and opium - derived agents were excluded ) ; ( 3 ) no nasogastric tube insertion ; ( 4 ) no drainage tube placement with the exception of low rectal anastomosis ; ( 5 ) water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days ( pod ) with regular diet on pod 3 ; ( 6 ) early urine catheter withdrawal ( at pod 12 ) ; and ( 7 ) early out - of - bed mobilization ( i.e. 5 mg / mlnefopam1.0 mg / mlvia pceavia pciafor 48 hfor 48 hopium - derived agents were excludedroutine drainage tube placementno routine drainage tube placementpostoperative managementdiet : liquid diet intake after recovery of bowel movementdiet : water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days , regular diet on pod 3urinary catheter withdrawal at 3 to 5 daysurinary catheter withdrawal on pod 12out - of - bed mobilization at 3 to 5 daysout - of - bed mobilization on pod 1 comparison of fast - track and traditional perioperative care protocols the mental status and cognitive function was evaluated in accordance with the delirium rating scale - revised-98 ( drs - r-98 ) . fasting peripheral venous blood samples ( 5 ml ) were collected 1 day preoperatively and on pod 1 , 2 , and 3 . the serum il-6 levels were determined using sandwich elisa with the reagents purchased from invitrogen , camarillo , ca , usa . three patients were excluded from the fts group : one received an intraoperative blood transfusion , and the other two were admitted to icu because of pulmonary infection . thus , a total of 233 patients including 116 patients in the traditional group and 117 patients in the fts group were included this study ( fig . the mean los of the fts group and traditional therapy group was 9.01 1.75 and 13.21 1.32 days , respectively ( p < 0.001 ) . the time to pass flatus in the fts group was significantly shorter than in the traditional therapy group ( 48.50 9.59 vs. 77.66 7.18 h ; p < 0.001 ) . on the pod 1 , the level of serum albumin in the fts group was higher than that in the traditional therapy group ( 28.05 2.82 vs. 26.26 4.12 ; p < 0.001 ) . meanwhile , the glucose in the fts group was lower than that in the traditional therapy group ( 8.30 2.49 vs. 10.25 2.43 ; p < 0.001 ) . no significant difference of liver or renal function was observed between the two groups after operation ( table 3).table 3comparison of postoperative recovery and complications between the fts and traditional groupfts ( 117)traditional ( 116 ) p valuelos ( day)9.01 1.7513.21 1.32<0.001functional recovery time to pass flatus ( h)48.50 9.5977.66 7.18<0.001 serum albumin ( g / l)28.05 2.8226.26 4.12<0.001 glucose ( mmol / l)8.30 2.4910.25 2.43<0.001 alt ( iu / l)34.65 12.2534.88 11.820.738 ast ( iu / l)30.43 10.7829.47 10.400.356 cr ( mol / l)77.05 23.8075.11 25.040.675 bun ( mmol / l)5.63 3.605.62 3.080.831complications ( cases ) infection of incision680.570 pulmonary infection6190.006 urinary infection5130.047 anastomotic leakage321.000 intestinal obstruction460.736 heart failure4130.022 dvt470.340 alt alanine transaminase , ast aspartate transaminase comparison of postoperative recovery and complications between the fts and traditional group alt alanine transaminase , ast aspartate transaminase the postoperative complications were followed and documented based on clavien dindo classification system , including infection , intestinal obstruction , anastomotic leakage , heart failure , and dvt . six pulmonary infections were observed in the fts group , significantly fewer than the 19 cases in the traditional therapy group ( p = 0.006 ) . the incidence of urinary infections was lower in fts group than that in the traditional therapy group ( 5 vs. 13 ; p = 0.047 ) . the incidence of heart failure was much higher in the traditional therapy group than that in the fts group ( 13 vs. 4 , p = 0.022 ) . using the drs - r-98 scoring system , a total 19 cases of postoperative delirium were observed , 15 in the traditional therapy group ( 12.9 % ) and 4 in the fts group ( 3.4 % , p = 0.008 ) ( table 4 ) . in the traditional therapy group , nine incidents of delirium were observed at pod 1 , five at pod 2 , and one at pod 3 , while for the fts group , three incidents of delirium were observed at pod 1 and one at pod 2 ( table 4).table 4incidence of postoperative delirium in the fts and traditional grouppodftstraditional p valuepod 1390.073pod 2150.211pod 3010.498total ( % of analyzed cases)4 ( 3.4 % ) 15 ( 12.9 % ) 0.008 incidence of postoperative delirium in the fts and traditional group repeat mri scanning for those with postoperative delirium did not show any cns changes ( data not shown ) . as proinflammatory cytokines particularly il-6 have been reported to be involved in the development of postoperative delirium [ 10 , 19 - 21 ] , we determined the serum il-6 levels in both the fts and the traditional groups . 2 ) the serum il-6 levels in the traditional therapy group at pod 1 , 2 , and 3 were all significantly higher than those in the fts group ( p < 0.001 in all 3 pod days ) . together , these results showed that enhanced il-6 level correlated with the development of postoperative delirium in the elderly patients undergoing colorectal surgery.fig . * * p < pre preoperation , pod postoperative day serum il-6 levels in the fts group and traditional group . * the postoperative complications were followed and documented based on clavien dindo classification system , including infection , intestinal obstruction , anastomotic leakage , heart failure , and dvt . six pulmonary infections were observed in the fts group , significantly fewer than the 19 cases in the traditional therapy group ( p = 0.006 ) . the incidence of urinary infections was lower in fts group than that in the traditional therapy group ( 5 vs. 13 ; p = 0.047 ) . the incidence of heart failure was much higher in the traditional therapy group than that in the fts group ( 13 vs. 4 , p = 0.022 ) . using the drs - r-98 scoring system , a total 19 cases of postoperative delirium were observed , 15 in the traditional therapy group ( 12.9 % ) and 4 in the fts group ( 3.4 % , p = 0.008 ) ( table 4 ) . in the traditional therapy group , nine incidents of delirium were observed at pod 1 , five at pod 2 , and one at pod 3 , while for the fts group , three incidents of delirium were observed at pod 1 and one at pod 2 ( table 4).table 4incidence of postoperative delirium in the fts and traditional grouppodftstraditional p valuepod 1390.073pod 2150.211pod 3010.498total ( % of analyzed cases)4 ( 3.4 % ) 15 ( 12.9 % ) 0.008 incidence of postoperative delirium in the fts and traditional group repeat mri scanning for those with postoperative delirium did not show any cns changes ( data not shown ) . as proinflammatory cytokines particularly il-6 have been reported to be involved in the development of postoperative delirium [ 10 , 19 - 21 ] , we determined the serum il-6 levels in both the fts and the traditional groups . 2 ) the serum il-6 levels in the traditional therapy group at pod 1 , 2 , and 3 were all significantly higher than those in the fts group ( p < 0.001 in all 3 pod days ) . together , these results showed that enhanced il-6 level correlated with the development of postoperative delirium in the elderly patients undergoing colorectal surgery.fig . pre preoperation , pod postoperative day serum il-6 levels in the fts group and traditional group . * in the present randomized trial , patients of advanced age ( 70 years ) with colorectal carcinoma were treated with either fts ( 117 patients ) or traditional approaches ( 116 patients ) . importantly , there was a decrease in the incidence of postoperative complications including pulmonary infection , urinary infection , and heart failure ( table 3 ) , which is consistent with previous reports [ 22 , 23 ] . in our trial , all the patients in both groups were of advanced age ( 70 years ) and were more prone to developing delirium after surgery . in the traditional group , mechanical enema and nasogastric tube insertion were applied , while in the fts group , only oral purgatives was given and no nasogastric tube was used . it is conceivable that these less - invasive procedures used in the fts group could potentially decrease the stress response , and be a factor in the lower occurrence of postoperative delirium . for patients with traditional therapy , opioid drugs including morphine , dolantin , and fentanyl that were used during general anesthesia and postoperative pain management might contribute to the occurrence of delirium . to investigate whether fts reduced the incidence of delirium by minimizing the inflammatory response , serum il-6 levels were also determined . we found that enhanced il-6 level correlated with the development of postoperative delirium and peaked on pod 1 in the elderly patients undergoing colorectal surgery ( fig . , use of epidural instead of general anesthesia , and avoidance of opioids for anesthesia and pain control could all contribute to the reduced level of il-6 in the fts group . although we only examined il-6 level in the peripheral blood , which may not directly represent the intracerebral conditions accurately , however , it has been shown that il-6 can readily cross the blood fts shortens the los , facilitates the recovery of bowel movement , and reduces occurrence of postoperative delirium and other complications in elderly patients with colorectal carcinoma . the lower incidence of delirium in the fts group is likely attributable to the reduced systemic stress and inflammatory response mediated by il-6 .

although the conventional surgical treatment is still the most utilized and effective treatment for colorectal malignancy , the procedure may lead to prolonged hospital stay , increased medical costs , and medical resource overuse due to the aggressiveness of the approach and the high risk of developing postoperative nutrition disorders . it has been found in several clinical trials that fast - track surgery ( fts ) not only facilitates the physical rehabilitation of the patients with colorectal malignancies , but also prevents upregulation of proinflammatory cytokines including il-6 , with reduced stress response and inflammation [ 13 , 14 ] . in the present randomized trial , we studied whether fts could prevent or reduce the occurrence of postoperative delirium as well as other complications in elderly patients with colorectal carcinoma and evaluated the role of il-6 in postoperative delirium . a total of 240 elderly patients with colorectal carcinoma , with ages ranging from 70 to 88 years ( mean , 75.18 years ; 150 men and 90 women ) , admitted to the fourth hospital of hebei medical university for open curative resection between 2008 and 2011 , were included in the present study . 1flow diagram of study flow diagram of study patients with a history of dementia , parkinson s disease , alcohol intake of 250 g / day , long - term use of sleeping pills or anxiolytics , and those who received anesthesia within the past 30 days were excluded from the study during the initial screening . patients in the two groups had comparable baseline characteristics including gender , age , site of lesion , tnm classification of malignant tumours ( tnm staging ) , and surgical procedure ( table 1 ) . in the current study , a preoperative routine cranial magnetic resonance imaging ( mri ) scan was performed for all the patients in both groups , and a repeat mri scan performed for those who developed postoperative delirium to exclude cerebrovascular stroke or other central nervous system ( cns ) conditions as the etiology of delirium.table 1clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233)characteristicsfts ( n = 117)traditional ( n = 116 ) p valueage75.66 4.1874.78 4.010.054gender male76700.467 female4146site of lesions colon57580.845 rectum6058tnm staging i13190.633 ii5247 iii3738 iv1512surgical operation colectomy52530.930 dixon3936 miles2627hypertension26330.275diabetes11140.511 clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233 ) this study was approved by the ethics committee of the fourth hospital of hebei medical university . the management of the fts group differed from the traditionally managed group in the several ways : ( 1 ) bowel preparation with oral purgatives instead of a mechanical enema ; ( 2 ) thoracic epidural anesthesia and postoperative analgesic maintenance via the epidural catheter ( ropivacaine , 2 mg / ml maintained for 48 h , controlled to 610 ml ( 1220 mg ) per hour and opium - derived agents were excluded ) ; ( 3 ) no nasogastric tube insertion ; ( 4 ) no drainage tube placement with the exception of low rectal anastomosis ; ( 5 ) water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days ( pod ) with regular diet on pod 3 ; ( 6 ) early urine catheter withdrawal ( at pod 12 ) ; and ( 7 ) early out - of - bed mobilization ( i.e. , walking).table 2comparison of fast - track and traditional perioperative care protocolstraditionalfast trackpreoperative preparationliquid diet for 3 daysoral purgativesmechanical enema(1time / day ) for 3 consecutive daysno mechanical enemafasting at 8 hnormal meal until 6 h before surgerydrink deprivation 4 h before surgerynormal carbohydrate drink until 2 h before surgeryroutine nasogastric tube insertionno nasogastric tube insertionoral antibiotics administration for 3 daysno antibioticsanesthesiageneralthoracic epiduralpain controlfentanyl0.25 mg / mlropivacaine2mg / mlmidazolam0 . 5 mg / mlnefopam1.0 mg / mlvia pceavia pciafor 48 hfor 48 hopium - derived agents were excludedroutine drainage tube placementno routine drainage tube placementpostoperative managementdiet : liquid diet intake after recovery of bowel movementdiet : water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days , regular diet on pod 3urinary catheter withdrawal at 3 to 5 daysurinary catheter withdrawal on pod 12out - of - bed mobilization at 3 to 5 daysout - of - bed mobilization on pod 1 comparison of fast - track and traditional perioperative care protocols the mental status and cognitive function was evaluated in accordance with the delirium rating scale - revised-98 ( drs - r-98 ) . a total of 240 elderly patients with colorectal carcinoma , with ages ranging from 70 to 88 years ( mean , 75.18 years ; 150 men and 90 women ) , admitted to the fourth hospital of hebei medical university for open curative resection between 2008 and 2011 , were included in the present study . 1flow diagram of study flow diagram of study patients with a history of dementia , parkinson s disease , alcohol intake of 250 g / day , long - term use of sleeping pills or anxiolytics , and those who received anesthesia within the past 30 days were excluded from the study during the initial screening . in the current study , a preoperative routine cranial magnetic resonance imaging ( mri ) scan was performed for all the patients in both groups , and a repeat mri scan performed for those who developed postoperative delirium to exclude cerebrovascular stroke or other central nervous system ( cns ) conditions as the etiology of delirium.table 1clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233)characteristicsfts ( n = 117)traditional ( n = 116 ) p valueage75.66 4.1874.78 4.010.054gender male76700.467 female4146site of lesions colon57580.845 rectum6058tnm staging i13190.633 ii5247 iii3738 iv1512surgical operation colectomy52530.930 dixon3936 miles2627hypertension26330.275diabetes11140.511 clinical data of the fast track surgery ( fts ) and traditional therapy groups ( n = 233 ) this study was approved by the ethics committee of the fourth hospital of hebei medical university . the management of the fts group differed from the traditionally managed group in the several ways : ( 1 ) bowel preparation with oral purgatives instead of a mechanical enema ; ( 2 ) thoracic epidural anesthesia and postoperative analgesic maintenance via the epidural catheter ( ropivacaine , 2 mg / ml maintained for 48 h , controlled to 610 ml ( 1220 mg ) per hour and opium - derived agents were excluded ) ; ( 3 ) no nasogastric tube insertion ; ( 4 ) no drainage tube placement with the exception of low rectal anastomosis ; ( 5 ) water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days ( pod ) with regular diet on pod 3 ; ( 6 ) early urine catheter withdrawal ( at pod 12 ) ; and ( 7 ) early out - of - bed mobilization ( i.e. , walking).table 2comparison of fast - track and traditional perioperative care protocolstraditionalfast trackpreoperative preparationliquid diet for 3 daysoral purgativesmechanical enema(1time / day ) for 3 consecutive daysno mechanical enemafasting at 8 hnormal meal until 6 h before surgerydrink deprivation 4 h before surgerynormal carbohydrate drink until 2 h before surgeryroutine nasogastric tube insertionno nasogastric tube insertionoral antibiotics administration for 3 daysno antibioticsanesthesiageneralthoracic epiduralpain controlfentanyl0.25 mg / mlropivacaine2mg / mlmidazolam0 . 5 mg / mlnefopam1.0 mg / mlvia pceavia pciafor 48 hfor 48 hopium - derived agents were excludedroutine drainage tube placementno routine drainage tube placementpostoperative managementdiet : liquid diet intake after recovery of bowel movementdiet : water was allowed from 6 h postoperation , liquid diet in the morning and semiliquid diet at noon and evening of the first and second postoperative days , regular diet on pod 3urinary catheter withdrawal at 3 to 5 daysurinary catheter withdrawal on pod 12out - of - bed mobilization at 3 to 5 daysout - of - bed mobilization on pod 1 comparison of fast - track and traditional perioperative care protocols the mental status and cognitive function was evaluated in accordance with the delirium rating scale - revised-98 ( drs - r-98 ) . the drs - r-98 evaluates the cognitive domain of delirium by recourse to specific evaluations for attention , orientation , short - term memory , long - term memory , and visuospatial ability . the mean los of the fts group and traditional therapy group was 9.01 1.75 and 13.21 1.32 days , respectively ( p < 0.001 ) . the time to pass flatus in the fts group was significantly shorter than in the traditional therapy group ( 48.50 9.59 vs. 77.66 7.18 h ; p < 0.001 ) . on the pod 1 , the level of serum albumin in the fts group was higher than that in the traditional therapy group ( 28.05 2.82 vs. 26.26 4.12 ; p < 0.001 ) . meanwhile , the glucose in the fts group was lower than that in the traditional therapy group ( 8.30 2.49 vs. 10.25 2.43 ; p < 0.001 ) . no significant difference of liver or renal function was observed between the two groups after operation ( table 3).table 3comparison of postoperative recovery and complications between the fts and traditional groupfts ( 117)traditional ( 116 ) p valuelos ( day)9.01 1.7513.21 1.32<0.001functional recovery time to pass flatus ( h)48.50 9.5977.66 7.18<0.001 serum albumin ( g / l)28.05 2.8226.26 4.12<0.001 glucose ( mmol / l)8.30 2.4910.25 2.43<0.001 alt ( iu / l)34.65 12.2534.88 11.820.738 ast ( iu / l)30.43 10.7829.47 10.400.356 cr ( mol / l)77.05 23.8075.11 25.040.675 bun ( mmol / l)5.63 3.605.62 3.080.831complications ( cases ) infection of incision680.570 pulmonary infection6190.006 urinary infection5130.047 anastomotic leakage321.000 intestinal obstruction460.736 heart failure4130.022 dvt470.340 alt alanine transaminase , ast aspartate transaminase comparison of postoperative recovery and complications between the fts and traditional group alt alanine transaminase , ast aspartate transaminase the postoperative complications were followed and documented based on clavien dindo classification system , including infection , intestinal obstruction , anastomotic leakage , heart failure , and dvt . six pulmonary infections were observed in the fts group , significantly fewer than the 19 cases in the traditional therapy group ( p = 0.006 ) . the incidence of urinary infections was lower in fts group than that in the traditional therapy group ( 5 vs. 13 ; p = 0.047 ) . the incidence of heart failure was much higher in the traditional therapy group than that in the fts group ( 13 vs. 4 , p = 0.022 ) . using the drs - r-98 scoring system , a total 19 cases of postoperative delirium were observed , 15 in the traditional therapy group ( 12.9 % ) and 4 in the fts group ( 3.4 % , p = 0.008 ) ( table 4 ) . in the traditional therapy group , nine incidents of delirium were observed at pod 1 , five at pod 2 , and one at pod 3 , while for the fts group , three incidents of delirium were observed at pod 1 and one at pod 2 ( table 4).table 4incidence of postoperative delirium in the fts and traditional grouppodftstraditional p valuepod 1390.073pod 2150.211pod 3010.498total ( % of analyzed cases)4 ( 3.4 % ) 15 ( 12.9 % ) 0.008 incidence of postoperative delirium in the fts and traditional group repeat mri scanning for those with postoperative delirium did not show any cns changes ( data not shown ) . as proinflammatory cytokines particularly il-6 have been reported to be involved in the development of postoperative delirium [ 10 , 19 - 21 ] , we determined the serum il-6 levels in both the fts and the traditional groups . although the il-6 level in fst group decreased to approximately the baseline level by pod 3 , the il-6 level of the traditional group was still well above the baseline level ( fig . 2 ) the serum il-6 levels in the traditional therapy group at pod 1 , 2 , and 3 were all significantly higher than those in the fts group ( p < 0.001 in all 3 pod days ) . together , these results showed that enhanced il-6 level correlated with the development of postoperative delirium in the elderly patients undergoing colorectal surgery.fig . six pulmonary infections were observed in the fts group , significantly fewer than the 19 cases in the traditional therapy group ( p = 0.006 ) . the incidence of urinary infections was lower in fts group than that in the traditional therapy group ( 5 vs. 13 ; p = 0.047 ) . the incidence of heart failure was much higher in the traditional therapy group than that in the fts group ( 13 vs. 4 , p = 0.022 ) . using the drs - r-98 scoring system , a total 19 cases of postoperative delirium were observed , 15 in the traditional therapy group ( 12.9 % ) and 4 in the fts group ( 3.4 % , p = 0.008 ) ( table 4 ) . in the traditional therapy group , nine incidents of delirium were observed at pod 1 , five at pod 2 , and one at pod 3 , while for the fts group , three incidents of delirium were observed at pod 1 and one at pod 2 ( table 4).table 4incidence of postoperative delirium in the fts and traditional grouppodftstraditional p valuepod 1390.073pod 2150.211pod 3010.498total ( % of analyzed cases)4 ( 3.4 % ) 15 ( 12.9 % ) 0.008 incidence of postoperative delirium in the fts and traditional group repeat mri scanning for those with postoperative delirium did not show any cns changes ( data not shown ) . as proinflammatory cytokines particularly il-6 have been reported to be involved in the development of postoperative delirium [ 10 , 19 - 21 ] , we determined the serum il-6 levels in both the fts and the traditional groups . 2 ) the serum il-6 levels in the traditional therapy group at pod 1 , 2 , and 3 were all significantly higher than those in the fts group ( p < 0.001 in all 3 pod days ) . together , these results showed that enhanced il-6 level correlated with the development of postoperative delirium in the elderly patients undergoing colorectal surgery.fig . importantly , there was a decrease in the incidence of postoperative complications including pulmonary infection , urinary infection , and heart failure ( table 3 ) , which is consistent with previous reports [ 22 , 23 ] . the precipitating factors include the type and extensiveness of the surgical procedure , anesthetic protocol , opioid pain killer usage , inflammatory response , infection , pain , sleep disturbance , nutritional condition , and electrolyte homeostasis [ 29 , 30 ] . since the highest frequency of delirium occurred early on pod 1 , we believe that preoperative preparation , surgical approach , anesthesia , and pain control protocols were among the most important factors , although other subsequent complications ( e.g. in our study , patients in both groups underwent similar open colorectal surgery ( table 1 ) , the extensiveness of the surgery and trauma incurred in both groups were similar and unlikely to be a contributing factor . it has been reported that early feeding in open colon resection in the elderly resulted in shorter los and reduced postoperative morbidity , so it is possible that early feeding in the fst group might be contributing to the overall lower postoperative complications including delirium . in the traditional group , mechanical enema and nasogastric tube insertion were applied , while in the fts group , only oral purgatives was given and no nasogastric tube was used . we found that enhanced il-6 level correlated with the development of postoperative delirium and peaked on pod 1 in the elderly patients undergoing colorectal surgery ( fig . although we only examined il-6 level in the peripheral blood , which may not directly represent the intracerebral conditions accurately , however , it has been shown that il-6 can readily cross the blood fts shortens the los , facilitates the recovery of bowel movement , and reduces occurrence of postoperative delirium and other complications in elderly patients with colorectal carcinoma .

imbalances in the levels of excitatory and inhibitory neurotransmitters , such as serotonin , dopamine , and gaba , can lead to severe cns disorders like epilepsy , schizophrenia , anxiety , and depression . tackling cns diseases related to the gabaergic system is most commonly achieved by using drugs of the benzodiazepine family ( e.g. , diazepam ) , which allosterically modulates the pentameric gabaa receptor ( gabaa - r ) . however , an alternative way of enhancing gaba action is inhibition of the corresponding neurotransmitter uptake system . in the case of the gaba transporter ( gat ) family , four gaba reuptake transporter subtypes ( gat13 , bgt1 ) and one vesicular carrier exist in mammalian organisms . the gat family belongs to the neurotransmitter : sodium symporters ( nss ) which is organized as oligomers at the plasma membrane while , in contrast to the gabaa - r , functions as a monomer . usually , nss transporters use a sodium gradient for uphill transport of neurotransmitters out of the synaptic cleft . in certain cases , a reverse transport mode is also known , releasing neurotransmitter in a nonvesicular way . at present , only one drug targeting this receptor , the anticonvulsant tiagabine , is on the market . tiagabine selectively inhibits gat1 , the most abundant gat subtype in the human brain . an x - ray crystallographic structure is not yet available for any member of the gat family , but a number of homology models have been constructed . the molecular basis of tiagabine action , however , remains elusive , as experimental evidence for proposed binding modes is still lacking . furthermore , ligand - based exploration of inhibitor scaffolds is limited by the low tolerance of this transporter for inhibitor modification . on the basis of a set of tiagabine analogs from literature sources , we recently investigated ligand - based structure activity relationships of the compound class . briefly , binary qsar allowed classification of gaba uptake inhibitors into active and inactive bins by using the degree of rigidity and polarity distribution as main descriptors . with the increasing knowledge provided by the x - ray structures of analogous transport proteins , structure - based approaches for elucidating the molecular basis of drug , we describe a binding hypothesis of tiagabine in gat1 and its successful validation by in silico screening . the closest transporter proteins related to hgat1 for which structures are available are the bacterial leucine amino acid transporter protein , leutaa , and the drosophila dopamine transporter , ddat . despite its lower overall sequence identity , closer substrate relationship and significantly higher resolution of 2.00 vs 2.95 several sequence alignments between hgat1 and leutaa have been published , and all alignments are almost identical within the conserved central substrate binding cavity . both template candidates were available in an open - to - out conformation , thus granting access to bulky inhibitor molecules . suitable templates for the intracellular n- and c - terminal domains of hgat1 are not available and thus were not included in the final homology model . because of the differing stoichiometry of eukaryotic nss family members for cl , the leut structure ( pdb code : 3f3a ) was modified by engineering a chloride binding site using structural information from crystal structure of the ddat and topological information from the literature . on the basis of a combination of low b - values and proximity to the binding site or stabilization of adjacent domains , several water molecules were selected and kept in the template file . finally , a known disulfide bridge between c164 and c173 of el-2 was defined . modeller was used to generate 100 models , which were ranked according to their respective discrete optimized protein energy ( dope ) score for estimating the geometric quality . for the 10 highest ranked models , additional quality checks were performed using the model assessment tools of the swiss - model server . models with core residues showing disallowed geometry according to the ramachandran plot were omitted . the remaining models were visually inspected for their ability to reflect residue proximity and accessibility data from literature . in addition , the models were evaluated regarding the orientation of nonconserved polar residues in tm regions . subsequently , the best structure was selected according to aforementioned criteria and subjected to a soft minimization protocol for relaxation of the system . focused sampling of the conformational space in the putative tiagabine interaction site tiagabine can not be accommodated in the occluded state of the transporter , as both the extracellular gate between r69 and d451 as well as the upper lid of the binding side , formed by the bulky f294 side chain , are limiting the available space . in addition , preliminary md simulations of the transporter model in the apo open - to - out state led to rearrangement of the gating residues impeding subsequent placement of compounds larger than substrates like gaba , guvacine , or nipecotic acid . thus , tiagabine was placed into the central cavity using glide prior to 30 ns of molecular dynamics simulations , which was used for validating and equilibrating the model . subsequently , 10 representative snapshots for the last 10 nanoseconds of the run were extracted based on maximum rmsd diversity of binding pocket residues . thus , focused sampling of the conformational space in the binding site could be achieved , using the snapshots as input structures for subsequent docking experiments . the constrained gaba analogs , nipecotic acid and guvacine ( 2 , 3 ) , are potent uptake inhibitors in vitro but are unable to penetrate the blood brain barrier . in contrast , tiagabine and the selective gat-1 inhibitor sk&f 89976-a ( 4 , 5 ) that contain bulky aromatic substituents are pure inhibitors that are not transported ( figure 1 ) . a large number of systematically modified derivatives of the basic tiagabine scaffold have since been synthesized and tested . these derivatives contain a conformationally restricted gaba - mimetic nipecotic acid or guvacine moiety , a 48 atom linker , and a large , mostly diaromatic , hydrophobic moiety . a total of 162 compounds were extracted from the literature , spanning an activity range from low nanomolar to millimolar ic50 values , each of them tested under comparable assay conditions . ligands exhibiting substantial activity differences linked to distinct structural changes in the key regions shown in figure 2 were selected for subsequent experimental data guided docking . chemical structures and literature ic50 values of ligands with key modifications in linker length , polarity , and rigidity of the aromatic moiety . an important observation was the dramatic activity loss caused by introduction of a direct link between the two aromatic moieties ( 5 vs 7 ) . in contrast , enhanced activity had been reported for introduction of a polar region in the linker . this is exemplified by compound pair 8 and 9 , as well as compound 6 , being the closest available derivative to reference compound 5 . in terms of activity , extending the linker length was well tolerated because compounds 5 , 8 , and 10 gave potent inhibitors . finally , exchange of a benzene by a pyridine leads to a dramatic loss of activity ( 10 vs 11 ) . these activity differences should also be reflected by respective differences in the ligand / protein interaction pattern and thus aid in the prioritization of the docking poses . docking into 10 snapshots derived from the hgat-1tiagabine complex was performed in a sequential ensemble - like manner using gold , thereby allowing for minor movements of the backbone and focused sampling of the binding site side chain orientations . the binding site was defined within a 10 radius around the simulated tiagabine coordinates . two water molecules were kept optional , as they had turned out to be stably involved in the hydrogen bonding network during the previous md simulation . however , in the subsequent docking runs , no direct contribution of these two water molecules to the binding of the selected ligands was observed . side chain orientations of possible interaction partners for polar linker compounds 6 , 8 , 10 , and 11 were addressed individually . conformational sampling of the binding site had been performed with tiagabine as ligand , which lacks a corresponding electronegative moiety in the linker . hence , the full range of conformational flexibility of the r69 , y139 , y140 , and s452 side chains was explored using the internal rotamer library of the gold software package . for each of ligands 611 , 100 docking poses per snapshot were generated and ranked by chemscore , which provides parameters for a putative interaction with sodium , in analogy to leut . however , relying on just a single scoring function bears the risk of missing relevant poses , especially when no experimentally derived complexes for redocking studies are available . thus , all poses were reranked using rank - by - rank consensus scoring that included goldscore , chemplp , london dg , gbvi , and xscore scoring functions . analysis of the 10 top ranked poses per ligand among the ensemble docking results clearly indicated a common binding mode for tiagabine analogs ( figure 3 ) . as already expected from literature results , the most prominent interaction was coordination of the na1 sodium cation by the negatively charged acid moiety , which fulfills an octahedral geometry , together with side chain atoms of n66 , s295 , and n327 , as well as the backbone carbonyl oxygens of a61 and s295 . the majority of observed poses showed an interaction between the positively charged nipecotic acid nitrogen and the backbone of f294 , while the carboxylate group interacting with na1 was in an equatorial conformation . in contrast , poses with the r - configured carboxy group sampled in an axial conformation tended to form an intramolecular hydrogen bond with the charged nitrogen atom . while no clear preference for one of the two carboxylate orientations could be deduced from scoring values , x - ray and nmr studies of nipecotic acid indicated a preferred equatorial configuration , which would be more pronounced by adding a bulky moiety like the biaromatic tail . in addition , skovstrup et al . reported less stable behavior of axial - configured tiagabine poses in molecular dynamics simulations . docking poses of tiagabine ( turquoise ) and analogs ( orange ) in 10 md snapshots of the hgat-1 model . the upper boundary of the binding pocket is formed by a bent region of el-4 , extending into the hydrophobic cavity with the backbone of g360 as a ceiling beam , hence separating it into two pockets , each of which is able to accommodate a single hydrophobic aromatic rings . as it can be seen in figure 3 , the ligand transporter interaction in one hydrophobic pocket is stabilized by a interaction with the side chain of y139 , as well as by a cavity able to accommodate a small o - substituent as present in 5 and 6 . this cavity is confined by the side chains of i143 and y140 , the latter being a residue known to be also important for ligand recognition ( see figure 3 ) . the second cavity is mainly shaped by the hydrophobic side chains of w68 , f294 , and a358 ( not shown ) . because of the relative torsion of the two pockets , poses of 7 tended to encounter an initial steric clash that was relieved after energy minimization of the complexes , whereas compounds with a terminal bis-5-methyl - thienyl ( 5 , 6 ) or diphenyl ( 810 ) group were able to bind in a conformation near their global energy minimum ( see figure 4 ) , thus explaining the activity cliff between 5 and 7 . dihedral energy landscape of 5-methyl - thiophen dihedral angles ; configuration of the docking pose of 5 is marked in yellow . the positive effect of a polar atom in the linker moiety on binding seems to be the result of several factors . transient interactions with residues in the entry path might play a significant role but are not reflected by the docking poses . had indicated a transient interaction with the r69 side chain upon entry in the binding site . hence , docking poses biased toward an interaction between this residue and one of the electronegative linker atoms in 6 , 8 , 10 , and 11 were generated , turning out to be possible but rather short - lived in short md runs due to reset forces of the basic side chain ( data not shown ) . to address the relatively low activity of compound 11 , per - atom contributions to g in the binding pose an unfavorable effect of the pyridine nitrogen in the hydrophobic receptor environment was indicated ( see supporting information , figure s3 ) . in addition , repulsive forces between negative partial charges of the aromatic nitrogen atom and the oxime moiety might force the pyridine ring in a sterically unfavorable orientation . taken together , the common orientation of the compound class was in agreement with the structure activity relationships of the ligand set and the topology of the extended substrate binding pocket . to further probe the proposed binding mode against pharmacologically relevant chemical space , the 3d orientation of the main tiagabine binding features was extracted from the docked complex and encoded in a four - feature pharmacophore model using ligandscout . two hydrophobic features were placed in the respective cavities occupied by the thiophene moieties . finally , a positively ionizable feature was placed on the basic nitrogen to mirror the compound s zwitterionic character ( figure 5 , the model is available for download at http://pharminfo.univie.ac.at ) . pharmacophore model of tiagabine : hydrophobic ( yellow ) , positive ( blue ) , and negative ( red ) ionizable features in context with the gat1 substrate binding site . the sensitivity of the pharmacophore model was validated by a decoy set generated using the dud - e platform ( http://dude.docking.org ) , retrieving just the compounds with known gat-1 activity . to test the predictive value of the model , a commercial vendor database consisting of 1.7 million compounds , as well as the drugbank index covering 1491 marketed drugs , a total of 79 and eight compounds , respectively , matched the pharmacophore query and passed the pains filter for frequent hitters . subsequently , the virtual hits were docked into a representative md snapshot of the homology model , from which the tiagabine pharmacophore model had been derived . ligand interaction fingerprints ( plif ) for the calculated poses were retrieved using moe . the interaction fingerprints were used to filter out compounds , which did not show an interaction with na1 . this reduced the hit list to 13 compounds for the enamine database available in sufficient purity , and seven compounds for drugbank , respectively ( see figure 6 ) . for the latter , tiagabine , three thyroid hormones ( liothyronine , levothyroxine , dextrothyroxine ) , two angiotensin conversion enzyme ( ace ) inhibitors ( ramipril , perindopril ) , and an antihistaminergic drug ( bepotastine ) were retrieved . on the basis of pharmacophoric fit and docking performance ( details in supporting information , table s2 ) , bepotastine , ramipril , and liothyronine were selected for biological testing . compounds tested in the [ h]-gaba uptake assay : enamine ( red frame ) and drugbank hits ( blue frame ) , and reference compounds ( black frame ) . inhibitory potency of the selected compounds was evaluated by an uptake inhibition assay of radiolabeled gaba in hek cells stably expressing rgat1 . first , the ic50 value for tiagabine ( 5 ) in the test system was determined to be 0.64 0.07 m . this was about a factor of 10 higher than the value reported for the unspecific rat synaptosome assay used by andersen et al . subsequently , compounds were measured at a concentration of 100 m against 5 as standard . diazepam ( 28 ) and tiagabine ( 5 ) were used as negative and positive control . as illustrated in figure 7 , one of the commercial screening compounds , 18 , weakly reduced uptake to just below 80% of saline . one drugbank substance , 27a ( liothyronine , a thyroid hormone also known as t3 ) , turned out to significantly inhibit radioligand uptake , which prompted the acquisition and testing of other commercially available derivatives 27b d . among those , the other bioactive hormone levothyroxine ( 27b ) showed reduced uptake , albeit weaker than 27a . the representative of the ace inhibitors and the antihistaminergic drug bepotastine were essentially inactive . remaining uptake of [ h]-gaba in the presence of 100 m of the respective compound ( n = 3 ) . the ic50 value for liothyronine derived from a dose response curve was 13 1.7 m ( figure 8) , providing a direct link between reported general effects of thyroid hormones on gaba uptake and the inhibitory action of 27a on the gat-1 subtype . inhibition curves of tiagabine ( ic50 0.64 0.07 m , white squares ) and liothyronine ( ic50 13.0 1.7 m , black squares ) . as illustrated in figure 9 , the pharmacophoric depiction of 27a reveals that one of the required hydrophobic features is not , as one would expect , the aromatic ring of the 3,5-diiodophenyl moiety , but rather a lipophilic iodine substituent , which could barely be deduced from chemical similarity measures . relying on an appropriate position of the second ring relative to the interacting iodine atom , this type of interaction is also possible for 27b but not for 27d . the 3,3,5-substituted variant of the t3 layout is missing the second substituent on the proximal ring which is responsible for inducing the bioactive conformation . activity relationships of thyroid hormone derivatives both at thyroid hormone receptors and gabaergic rat brain synaptosomes . apparently , the steric requirements in both systems are remarkably similar , relying on correct substitution pattern , stereochemistry , and degree of lipophilicity , possibly also limiting the relative efficacy of 27c . the most remarkable difference between the observed crystallographic hormone binding mode of 27a ( pdb code 3uvv ) and the gat1 binding hypothesis can be found in the 4 position . the presence of the phenolic hydroxyl group is crucial for hormone receptor binding but not for interaction with the transport protein . regarding compounds 1224 , the most crucial property among those molecules seems to be the distance between the positively ionizable group and the first occurrence of lipophilic bulk , which is in close analogy to the linker in tiagabine analogs . distal from the nitrogen atom ( as seen from the negatively ionizable group , which also can be a tetrazole moiety in a reasonable distance ) , usually just one heavy atom separates the positive charge from the next aromatic moiety ( 12 , 19 , 20 ) , or branching position ( 13 , 21 , 24 ) . alternatively , it is part of separate ring system not directly carrying the acidic moiety ( 1417 , 22 ) . with a slightly increased distance between the aromatic ring and the carboxylate group , 18 displays some weak activity . this observation also extends to the inactive drugbank compounds , as the nitrogen atoms of 25 and 26 are both connected to the hydrophobic part by space demanding linkers , whereas the first aromatic ring of thyroid hormones is at a distance of two heavy atoms . the presence of a pyridine moiety known to be unfavorable from the 1011 compound pair could further limit the potential of 25 despite its remarkable structural similarity to the reference compounds . just as for the bulk of a cyclopentane moiety attached to the polar part of 26 , this might considerably inhibit optimal positioning in the binding site . to further assess the degree of similarity between the compounds retrieved and the reference compound tiagabine , we calculated the tanimoto similarity values based on chemical fingerprints ( maccs , fp2 , fp4 ) derived from openbabel . the similarity between tiagabine and liothyronine as well as the slightly active 18 , on the basis of maccs keys , was 25.4 and 51.5% , respectively . inhibitory potency of the selected compounds was evaluated by an uptake inhibition assay of radiolabeled gaba in hek cells stably expressing rgat1 . first , the ic50 value for tiagabine ( 5 ) in the test system was determined to be 0.64 0.07 m . this was about a factor of 10 higher than the value reported for the unspecific rat synaptosome assay used by andersen et al . subsequently , compounds were measured at a concentration of 100 m against 5 as standard . diazepam ( 28 ) and tiagabine ( 5 ) were used as negative and positive control . as illustrated in figure 7 , one of the commercial screening compounds , 18 , weakly reduced uptake to just below 80% of saline . one drugbank substance , 27a ( liothyronine , a thyroid hormone also known as t3 ) , turned out to significantly inhibit radioligand uptake , which prompted the acquisition and testing of other commercially available derivatives 27b d . among those , the other bioactive hormone levothyroxine ( 27b ) showed reduced uptake , albeit weaker than 27a . the representative of the ace inhibitors and the antihistaminergic drug bepotastine were essentially inactive . remaining uptake of [ h]-gaba in the presence of 100 m of the respective compound ( n = 3 ) . the ic50 value for liothyronine derived from a dose response curve was 13 1.7 m ( figure 8) , providing a direct link between reported general effects of thyroid hormones on gaba uptake and the inhibitory action of 27a on the gat-1 subtype . inhibition curves of tiagabine ( ic50 0.64 0.07 m , white squares ) and liothyronine ( ic50 13.0 1.7 m , black squares ) . as illustrated in figure 9 , the pharmacophoric depiction of 27a reveals that one of the required hydrophobic features is not , as one would expect , the aromatic ring of the 3,5-diiodophenyl moiety , but rather a lipophilic iodine substituent , which could barely be deduced from chemical similarity measures . relying on an appropriate position of the second ring relative to the interacting iodine atom , this type of interaction is also possible for 27b but not for 27d . the 3,3,5-substituted variant of the t3 layout is missing the second substituent on the proximal ring which is responsible for inducing the bioactive conformation . activity relationships of thyroid hormone derivatives both at thyroid hormone receptors and gabaergic rat brain synaptosomes . apparently , the steric requirements in both systems are remarkably similar , relying on correct substitution pattern , stereochemistry , and degree of lipophilicity , possibly also limiting the relative efficacy of 27c . the most remarkable difference between the observed crystallographic hormone binding mode of 27a ( pdb code 3uvv ) and the gat1 binding hypothesis can be found in the 4 position . the presence of the phenolic hydroxyl group is crucial for hormone receptor binding but not for interaction with the transport protein . , the most crucial property among those molecules seems to be the distance between the positively ionizable group and the first occurrence of lipophilic bulk , which is in close analogy to the linker in tiagabine analogs . distal from the nitrogen atom ( as seen from the negatively ionizable group , which also can be a tetrazole moiety in a reasonable distance ) , usually just one heavy atom separates the positive charge from the next aromatic moiety ( 12 , 19 , 20 ) , or branching position ( 13 , 21 , 24 ) . alternatively , it is part of separate ring system not directly carrying the acidic moiety ( 1417 , 22 ) . with a slightly increased distance between the aromatic ring and the carboxylate group , 18 displays some weak activity . this observation also extends to the inactive drugbank compounds , as the nitrogen atoms of 25 and 26 are both connected to the hydrophobic part by space demanding linkers , whereas the first aromatic ring of thyroid hormones is at a distance of two heavy atoms . the presence of a pyridine moiety known to be unfavorable from the 1011 compound pair could further limit the potential of 25 despite its remarkable structural similarity to the reference compounds . just as for the bulk of a cyclopentane moiety attached to the polar part of 26 , this might considerably inhibit optimal positioning in the binding site . to further assess the degree of similarity between the compounds retrieved and the reference compound tiagabine , we calculated the tanimoto similarity values based on chemical fingerprints ( maccs , fp2 , fp4 ) derived from openbabel . the similarity between tiagabine and liothyronine as well as the slightly active 18 , on the basis of maccs keys , was 25.4 and 51.5% , respectively . with the increasing number of x - ray structures available for transmembrane transporters , structure - based computational models have provided valuable insights into the molecular basis of ligand transporter interaction . within this article , we propose a binding mode of the antiepileptic drug tiagabine in gat1 by including knowledge from ligand - based studies into the prioritization process for docking poses . subsequent pharmacophore - based virtual screening followed by experimental testing further confirmed the validity of the pose by identifying a commonly used drug ( liothyronine ) as an inhibitor of gat1 . strikingly , liothyronine has been described long ago as potential gat inhibitor without major activity on other neurotransmitter reuptake systems ( dopamine , serotonin , choline , aspartate ) , but final experimental confirmation for subtype gat1 since has been lacking . compounds with significantly higher chemical similarity retrieved in a commercial vendor library all prove inactive , further implying that selective transport inhibition of the protein can only be tackled from the side of steric feature arrangement . furthermore , the results indicate that , apart from privileged tricyclic antidepressant scaffolds known to more or less unspecifically inhibit neurotransmitter uptake , not many drugs on the market are likely to interact with gat1 . the gat1 models were constructed using the crystal structure of leut as template which shows highest available resolution in an open - to - out state of the transporter . when assessing the differences between available sequence alignments of leut and hgat1 ( uniprot entries o67854 and p30531 , respectively ) , no differences for residues in the central binding cavity were observed , except for a one - residue gap in the middle of leut - tm10 , either placed over gat1-g457 , s456 , or a455 . as it has been optimized for gat1 and assessment of the sequence identity between hgat-1 and rgat-1 , the first being the effective protein of interest , the second the one used in cell assays , stated 100% sequence identity for the observed core region and 97.9% for the whole modeled sequence ( see supporting information , figure s4 ) . the crystal structure of leut retrieved from the pdb ( www.pdb.org , accession code 3f3a ) was mutated in silico at position 290 using moe with a serine side chain orientation corresponding to the former glutamic acid . a chlorine ion was placed at the coordinates of the previous center of the e290 side chain , then further optimized according to interaction potential calculations , giving cl as probe . tethering the backbone , s290 and its surrounding residues were carefully energy minimized for final optimization of the local coordinates . the models were built using modeller9v8 in the automodel class , including water molecules , the chloride ion , and two cobound sodium ions as nonprotein atoms . a disulfide bridge was defined between c164 and c173 using a modeller patch command . one hundred models were generated using very thorough vtfm ( variable target function method ) optimization , as provided in modeller . output models were ranked according to dope score , and the top 10 were further assessed by the swiss - model server . according to procheck results and ramachandran plots , models with disallowed backbone geometries in transmembrane regions were omitted . hydrogen atom assignment and soft energy minimization of the raw models was performed within moe using ligx and the charmm27 all - atom force field , otherwise with default settings . the selected complex was inserted into a pre - equilibrated and solvated popc membrane by applying the program g_membed , using the gromos 53a6 united - atom force field and periodic boundary conditions . all simulations were performed at 310 k. the system was neutralized by adding sodium and chlorine ions to a final salt concentration of 150 mm . gradually , position restraints on the main complex were reduced from 500 kj mol nm ( 500 ps ) to 250 kj mol nm ( 500 ps ) . after an equilibration phase without restraints , a fully stable system was achieved after 20 ns . between 21 and 30 ns of the production run , the frames were clustered according to rmsd of residues within a radius of 7 around the ligand . one showed a stable h - bond with y60 ( 89% occupancy ; distance 3.5 ; angle 60 ) , another one was directly attached and showed an almost equally stable h - bond interaction . protonation states were sampled according to possible states in a physiological ph range of 7.2 0.2 using ligprep . these states were cross - checked with the major microspecies calculated by the chemaxon web - tool chemicalize.org . primary placement of tiagabine was done using glide in standard precision ( sp ) mode using default settings . the receptor grid was defined around binding site residues 6063 , 6466 , 136 , 140 , 294297 , 300 , 396 , and 400 . docking and goldscore / chemplp rescoring in the 10 md snapshots were performed with gold 5.0.1 , using chemscore as primary scoring function . early termination was disabled , keeping the 100 best solutions per ligand and snapshot . external rescoring was performed using xscore ( xscore ) and moe ( london dg , gbvi ) . consensus scores were calculated by summing up indices assigned according to respective ranks within a scoring function . secondary docking runs for investigating side chain orientations for specific interactions with polar linker moieties were performed by ( a ) constraining residues y139 , y140 , and s456 to the internal library of allowed rotamers in gold and ( b ) for investigating interactions as reported by skovstrup et al . , likewise rotamer rotations of r69 and f294 were allowed but with an additional distance constraint of 1.53.5 ( spring constant 5 ) between the r69 guanidine function and the polar linker moiety . determination of the potential energy landscape for different dihedral angle configurations was performed with gaussian 09 . after initial geometry optimization with hf/3 - 21 g implemented in the software package , configurations with an increment of 15 were calculated using the m06 - 2x hybrid functional and the 6 - 31 g * basis set . pharmacophore models were built using ligandscout 3.0 . for assembling the customized decoy library , 50 decoys per active compound ( 511 ) were compiled in smiles format using the dud - e platform ( dude.docking.org ) . the nonredundant compounds with similar physicochemical properties but dissimilar 2-d topology for each input line were extracted from the zinc database . the retrieved set of decoys and the active compounds as smiles were assembled and processed to a ligandscout screening database using the maximum number of possible conformers . the drugbank database was downloaded from the web site www.drugbank.ca and consisted of 1491 entries ( version of june 2013 ) . enamine advanced and hts screening collections were obtained from the download site at www.enamine.net ( n = 1719682 ; version 032013 ) . ligandscout command line modules idbgen and iscreen were used for conformation generation and for performing the pharmacophore screening . training set , decoy compilation , and drugbank compounds were prepared using omega - best settings ( max 500 conformations ) , enamine advanced and hts databases were compiled with omega - fast settings ( max 25 conformations ) ( www.eyesopen.com , ) . path - based ( fp2 ) and substructure - based ( maccs , fp4 ) similarity fingerprinting was performed using openbabel 2.3.1 . primary checking for pan assay interference ( pains ) compounds was done by uploading retrieved virtual screening hits to the pains remover web service ( available at http://cbligand.org/pains ) , returning no suspicious compounds . protein screening compounds were purchased from enamine ( enamine ltd . , riga , latvia ) , fluka / sigma - aldrich ( sigma - aldrich co. , saint louis , mo ) , and avachem ( avachem scientific , san antonio , tx ) , all with a purity 95% ( see supporting information , table s4 ) . cloned cells ( 4 10 cells / well ) were seeded and grown at 37 on poly(d)-lysine coated standard plasticware 24 h in advance . uptake of [ h]-gaba into was measured in the presence of 100 m of the compounds , while unspecific uptake was defined as uptake in the presence of 100 m tiagabine . for ic50 determination tested concentrations were : 5 , 0.001 , 0.01 , 0.1 , 0.3 , 1 , 10 , 100 m ; 27a , 0.01 , 0.1 , 0.3 , 1 , 3 , 10 , 30 , 100 m . after 3 min preincubation , [ h]-gaba ( 35 ci / mmol , perkinelmer , boston , ma ) in a final concentration of 0.015 m was added . uptake was stopped by adding ice - cold krebs - hepes buffer ( 10 mm hepes adjusted to ph 7.4 with 35.9 mm solid naoh , 120 mm nacl , 3 mm kcl , 2 mm cacl2 , 2 mm mgso4 , and 2 mm d - glucose as supplement ) . cells were lysed with 1% sds ( sodium dodecyl sulfate ) solution , taken up in 2 ml of scintillation cocktail ( rotiszint eco plus , carl roth gmbh , karlsruhe , germany ) and counted in a standard liquid scintillation counter ( packard tricarb 2300tr , packard instruments ) .

elevating gaba levels in the synaptic cleft by inhibiting its reuptake carrier gat1 is an established approach for the treatment of cns disorders like epilepsy . with the increasing availability of crystal structures of transmembrane transporters , structure - based approaches to elucidate the molecular basis of ligand transporter interaction also become feasible . experimental data guided docking of derivatives of the gat1 inhibitor tiagabine into a protein homology model of gat1 allowed derivation of a common binding mode for this class of inhibitors that is able to account for the distinct structure activity relationship pattern of the data set . translating essential binding features into a pharmacophore model followed by in silico screening of the drugbank identified liothyronine as a drug potentially exerting a similar effect on gat1 . experimental testing further confirmed the gat1 inhibiting properties of this thyroid hormone .

Introduction Results and Discussion Experimental Testing Conclusions Experimental Section

imbalances in the levels of excitatory and inhibitory neurotransmitters , such as serotonin , dopamine , and gaba , can lead to severe cns disorders like epilepsy , schizophrenia , anxiety , and depression . however , an alternative way of enhancing gaba action is inhibition of the corresponding neurotransmitter uptake system . in the case of the gaba transporter ( gat ) family , four gaba reuptake transporter subtypes ( gat13 , bgt1 ) and one vesicular carrier exist in mammalian organisms . the gat family belongs to the neurotransmitter : sodium symporters ( nss ) which is organized as oligomers at the plasma membrane while , in contrast to the gabaa - r , functions as a monomer . usually , nss transporters use a sodium gradient for uphill transport of neurotransmitters out of the synaptic cleft . tiagabine selectively inhibits gat1 , the most abundant gat subtype in the human brain . an x - ray crystallographic structure is not yet available for any member of the gat family , but a number of homology models have been constructed . the molecular basis of tiagabine action , however , remains elusive , as experimental evidence for proposed binding modes is still lacking . furthermore , ligand - based exploration of inhibitor scaffolds is limited by the low tolerance of this transporter for inhibitor modification . on the basis of a set of tiagabine analogs from literature sources , we recently investigated ligand - based structure activity relationships of the compound class . with the increasing knowledge provided by the x - ray structures of analogous transport proteins , structure - based approaches for elucidating the molecular basis of drug , we describe a binding hypothesis of tiagabine in gat1 and its successful validation by in silico screening . suitable templates for the intracellular n- and c - terminal domains of hgat1 are not available and thus were not included in the final homology model . because of the differing stoichiometry of eukaryotic nss family members for cl , the leut structure ( pdb code : 3f3a ) was modified by engineering a chloride binding site using structural information from crystal structure of the ddat and topological information from the literature . on the basis of a combination of low b - values and proximity to the binding site or stabilization of adjacent domains , several water molecules were selected and kept in the template file . for the 10 highest ranked models , additional quality checks were performed using the model assessment tools of the swiss - model server . subsequently , the best structure was selected according to aforementioned criteria and subjected to a soft minimization protocol for relaxation of the system . focused sampling of the conformational space in the putative tiagabine interaction site tiagabine can not be accommodated in the occluded state of the transporter , as both the extracellular gate between r69 and d451 as well as the upper lid of the binding side , formed by the bulky f294 side chain , are limiting the available space . in addition , preliminary md simulations of the transporter model in the apo open - to - out state led to rearrangement of the gating residues impeding subsequent placement of compounds larger than substrates like gaba , guvacine , or nipecotic acid . subsequently , 10 representative snapshots for the last 10 nanoseconds of the run were extracted based on maximum rmsd diversity of binding pocket residues . thus , focused sampling of the conformational space in the binding site could be achieved , using the snapshots as input structures for subsequent docking experiments . a large number of systematically modified derivatives of the basic tiagabine scaffold have since been synthesized and tested . ligands exhibiting substantial activity differences linked to distinct structural changes in the key regions shown in figure 2 were selected for subsequent experimental data guided docking . chemical structures and literature ic50 values of ligands with key modifications in linker length , polarity , and rigidity of the aromatic moiety . an important observation was the dramatic activity loss caused by introduction of a direct link between the two aromatic moieties ( 5 vs 7 ) . in contrast , enhanced activity had been reported for introduction of a polar region in the linker . finally , exchange of a benzene by a pyridine leads to a dramatic loss of activity ( 10 vs 11 ) . these activity differences should also be reflected by respective differences in the ligand / protein interaction pattern and thus aid in the prioritization of the docking poses . two water molecules were kept optional , as they had turned out to be stably involved in the hydrogen bonding network during the previous md simulation . however , in the subsequent docking runs , no direct contribution of these two water molecules to the binding of the selected ligands was observed . conformational sampling of the binding site had been performed with tiagabine as ligand , which lacks a corresponding electronegative moiety in the linker . hence , the full range of conformational flexibility of the r69 , y139 , y140 , and s452 side chains was explored using the internal rotamer library of the gold software package . analysis of the 10 top ranked poses per ligand among the ensemble docking results clearly indicated a common binding mode for tiagabine analogs ( figure 3 ) . as already expected from literature results , the most prominent interaction was coordination of the na1 sodium cation by the negatively charged acid moiety , which fulfills an octahedral geometry , together with side chain atoms of n66 , s295 , and n327 , as well as the backbone carbonyl oxygens of a61 and s295 . in contrast , poses with the r - configured carboxy group sampled in an axial conformation tended to form an intramolecular hydrogen bond with the charged nitrogen atom . the upper boundary of the binding pocket is formed by a bent region of el-4 , extending into the hydrophobic cavity with the backbone of g360 as a ceiling beam , hence separating it into two pockets , each of which is able to accommodate a single hydrophobic aromatic rings . as it can be seen in figure 3 , the ligand transporter interaction in one hydrophobic pocket is stabilized by a interaction with the side chain of y139 , as well as by a cavity able to accommodate a small o - substituent as present in 5 and 6 . because of the relative torsion of the two pockets , poses of 7 tended to encounter an initial steric clash that was relieved after energy minimization of the complexes , whereas compounds with a terminal bis-5-methyl - thienyl ( 5 , 6 ) or diphenyl ( 810 ) group were able to bind in a conformation near their global energy minimum ( see figure 4 ) , thus explaining the activity cliff between 5 and 7 . the positive effect of a polar atom in the linker moiety on binding seems to be the result of several factors . transient interactions with residues in the entry path might play a significant role but are not reflected by the docking poses . had indicated a transient interaction with the r69 side chain upon entry in the binding site . hence , docking poses biased toward an interaction between this residue and one of the electronegative linker atoms in 6 , 8 , 10 , and 11 were generated , turning out to be possible but rather short - lived in short md runs due to reset forces of the basic side chain ( data not shown ) . to address the relatively low activity of compound 11 , per - atom contributions to g in the binding pose an unfavorable effect of the pyridine nitrogen in the hydrophobic receptor environment was indicated ( see supporting information , figure s3 ) . in addition , repulsive forces between negative partial charges of the aromatic nitrogen atom and the oxime moiety might force the pyridine ring in a sterically unfavorable orientation . taken together , the common orientation of the compound class was in agreement with the structure activity relationships of the ligand set and the topology of the extended substrate binding pocket . to further probe the proposed binding mode against pharmacologically relevant chemical space , the 3d orientation of the main tiagabine binding features was extracted from the docked complex and encoded in a four - feature pharmacophore model using ligandscout . two hydrophobic features were placed in the respective cavities occupied by the thiophene moieties . pharmacophore model of tiagabine : hydrophobic ( yellow ) , positive ( blue ) , and negative ( red ) ionizable features in context with the gat1 substrate binding site . the sensitivity of the pharmacophore model was validated by a decoy set generated using the dud - e platform ( http://dude.docking.org ) , retrieving just the compounds with known gat-1 activity . to test the predictive value of the model , a commercial vendor database consisting of 1.7 million compounds , as well as the drugbank index covering 1491 marketed drugs , a total of 79 and eight compounds , respectively , matched the pharmacophore query and passed the pains filter for frequent hitters . subsequently , the virtual hits were docked into a representative md snapshot of the homology model , from which the tiagabine pharmacophore model had been derived . ligand interaction fingerprints ( plif ) for the calculated poses were retrieved using moe . this reduced the hit list to 13 compounds for the enamine database available in sufficient purity , and seven compounds for drugbank , respectively ( see figure 6 ) . for the latter , tiagabine , three thyroid hormones ( liothyronine , levothyroxine , dextrothyroxine ) , two angiotensin conversion enzyme ( ace ) inhibitors ( ramipril , perindopril ) , and an antihistaminergic drug ( bepotastine ) were retrieved . on the basis of pharmacophoric fit and docking performance ( details in supporting information , table s2 ) , bepotastine , ramipril , and liothyronine were selected for biological testing . inhibitory potency of the selected compounds was evaluated by an uptake inhibition assay of radiolabeled gaba in hek cells stably expressing rgat1 . this was about a factor of 10 higher than the value reported for the unspecific rat synaptosome assay used by andersen et al . as illustrated in figure 7 , one of the commercial screening compounds , 18 , weakly reduced uptake to just below 80% of saline . one drugbank substance , 27a ( liothyronine , a thyroid hormone also known as t3 ) , turned out to significantly inhibit radioligand uptake , which prompted the acquisition and testing of other commercially available derivatives 27b d . the representative of the ace inhibitors and the antihistaminergic drug bepotastine were essentially inactive . remaining uptake of [ h]-gaba in the presence of 100 m of the respective compound ( n = 3 ) . as illustrated in figure 9 , the pharmacophoric depiction of 27a reveals that one of the required hydrophobic features is not , as one would expect , the aromatic ring of the 3,5-diiodophenyl moiety , but rather a lipophilic iodine substituent , which could barely be deduced from chemical similarity measures . the 3,3,5-substituted variant of the t3 layout is missing the second substituent on the proximal ring which is responsible for inducing the bioactive conformation . activity relationships of thyroid hormone derivatives both at thyroid hormone receptors and gabaergic rat brain synaptosomes . the most remarkable difference between the observed crystallographic hormone binding mode of 27a ( pdb code 3uvv ) and the gat1 binding hypothesis can be found in the 4 position . the presence of the phenolic hydroxyl group is crucial for hormone receptor binding but not for interaction with the transport protein . the presence of a pyridine moiety known to be unfavorable from the 1011 compound pair could further limit the potential of 25 despite its remarkable structural similarity to the reference compounds . just as for the bulk of a cyclopentane moiety attached to the polar part of 26 , this might considerably inhibit optimal positioning in the binding site . the similarity between tiagabine and liothyronine as well as the slightly active 18 , on the basis of maccs keys , was 25.4 and 51.5% , respectively . first , the ic50 value for tiagabine ( 5 ) in the test system was determined to be 0.64 0.07 m . this was about a factor of 10 higher than the value reported for the unspecific rat synaptosome assay used by andersen et al . one drugbank substance , 27a ( liothyronine , a thyroid hormone also known as t3 ) , turned out to significantly inhibit radioligand uptake , which prompted the acquisition and testing of other commercially available derivatives 27b d . the representative of the ace inhibitors and the antihistaminergic drug bepotastine were essentially inactive . remaining uptake of [ h]-gaba in the presence of 100 m of the respective compound ( n = 3 ) . relying on an appropriate position of the second ring relative to the interacting iodine atom , this type of interaction is also possible for 27b but not for 27d . the 3,3,5-substituted variant of the t3 layout is missing the second substituent on the proximal ring which is responsible for inducing the bioactive conformation . activity relationships of thyroid hormone derivatives both at thyroid hormone receptors and gabaergic rat brain synaptosomes . the most remarkable difference between the observed crystallographic hormone binding mode of 27a ( pdb code 3uvv ) and the gat1 binding hypothesis can be found in the 4 position . the presence of the phenolic hydroxyl group is crucial for hormone receptor binding but not for interaction with the transport protein . just as for the bulk of a cyclopentane moiety attached to the polar part of 26 , this might considerably inhibit optimal positioning in the binding site . the similarity between tiagabine and liothyronine as well as the slightly active 18 , on the basis of maccs keys , was 25.4 and 51.5% , respectively . with the increasing number of x - ray structures available for transmembrane transporters , structure - based computational models have provided valuable insights into the molecular basis of ligand transporter interaction . within this article , we propose a binding mode of the antiepileptic drug tiagabine in gat1 by including knowledge from ligand - based studies into the prioritization process for docking poses . subsequent pharmacophore - based virtual screening followed by experimental testing further confirmed the validity of the pose by identifying a commonly used drug ( liothyronine ) as an inhibitor of gat1 . compounds with significantly higher chemical similarity retrieved in a commercial vendor library all prove inactive , further implying that selective transport inhibition of the protein can only be tackled from the side of steric feature arrangement . the gat1 models were constructed using the crystal structure of leut as template which shows highest available resolution in an open - to - out state of the transporter . as it has been optimized for gat1 and assessment of the sequence identity between hgat-1 and rgat-1 , the first being the effective protein of interest , the second the one used in cell assays , stated 100% sequence identity for the observed core region and 97.9% for the whole modeled sequence ( see supporting information , figure s4 ) . the crystal structure of leut retrieved from the pdb ( www.pdb.org , accession code 3f3a ) was mutated in silico at position 290 using moe with a serine side chain orientation corresponding to the former glutamic acid . tethering the backbone , s290 and its surrounding residues were carefully energy minimized for final optimization of the local coordinates . the models were built using modeller9v8 in the automodel class , including water molecules , the chloride ion , and two cobound sodium ions as nonprotein atoms . hydrogen atom assignment and soft energy minimization of the raw models was performed within moe using ligx and the charmm27 all - atom force field , otherwise with default settings . the selected complex was inserted into a pre - equilibrated and solvated popc membrane by applying the program g_membed , using the gromos 53a6 united - atom force field and periodic boundary conditions . between 21 and 30 ns of the production run , the frames were clustered according to rmsd of residues within a radius of 7 around the ligand . these states were cross - checked with the major microspecies calculated by the chemaxon web - tool chemicalize.org . docking and goldscore / chemplp rescoring in the 10 md snapshots were performed with gold 5.0.1 , using chemscore as primary scoring function . determination of the potential energy landscape for different dihedral angle configurations was performed with gaussian 09 . after initial geometry optimization with hf/3 - 21 g implemented in the software package , configurations with an increment of 15 were calculated using the m06 - 2x hybrid functional and the 6 - 31 g * basis set . the drugbank database was downloaded from the web site www.drugbank.ca and consisted of 1491 entries ( version of june 2013 ) . uptake of [ h]-gaba into was measured in the presence of 100 m of the compounds , while unspecific uptake was defined as uptake in the presence of 100 m tiagabine .

imbalances in the levels of excitatory and inhibitory neurotransmitters , such as serotonin , dopamine , and gaba , can lead to severe cns disorders like epilepsy , schizophrenia , anxiety , and depression . in the case of the gaba transporter ( gat ) family , four gaba reuptake transporter subtypes ( gat13 , bgt1 ) and one vesicular carrier exist in mammalian organisms . the gat family belongs to the neurotransmitter : sodium symporters ( nss ) which is organized as oligomers at the plasma membrane while , in contrast to the gabaa - r , functions as a monomer . on the basis of a set of tiagabine analogs from literature sources , we recently investigated ligand - based structure activity relationships of the compound class . with the increasing knowledge provided by the x - ray structures of analogous transport proteins , structure - based approaches for elucidating the molecular basis of drug , we describe a binding hypothesis of tiagabine in gat1 and its successful validation by in silico screening . the closest transporter proteins related to hgat1 for which structures are available are the bacterial leucine amino acid transporter protein , leutaa , and the drosophila dopamine transporter , ddat . despite its lower overall sequence identity , closer substrate relationship and significantly higher resolution of 2.00 vs 2.95 several sequence alignments between hgat1 and leutaa have been published , and all alignments are almost identical within the conserved central substrate binding cavity . suitable templates for the intracellular n- and c - terminal domains of hgat1 are not available and thus were not included in the final homology model . because of the differing stoichiometry of eukaryotic nss family members for cl , the leut structure ( pdb code : 3f3a ) was modified by engineering a chloride binding site using structural information from crystal structure of the ddat and topological information from the literature . on the basis of a combination of low b - values and proximity to the binding site or stabilization of adjacent domains , several water molecules were selected and kept in the template file . focused sampling of the conformational space in the putative tiagabine interaction site tiagabine can not be accommodated in the occluded state of the transporter , as both the extracellular gate between r69 and d451 as well as the upper lid of the binding side , formed by the bulky f294 side chain , are limiting the available space . in addition , preliminary md simulations of the transporter model in the apo open - to - out state led to rearrangement of the gating residues impeding subsequent placement of compounds larger than substrates like gaba , guvacine , or nipecotic acid . thus , tiagabine was placed into the central cavity using glide prior to 30 ns of molecular dynamics simulations , which was used for validating and equilibrating the model . thus , focused sampling of the conformational space in the binding site could be achieved , using the snapshots as input structures for subsequent docking experiments . the constrained gaba analogs , nipecotic acid and guvacine ( 2 , 3 ) , are potent uptake inhibitors in vitro but are unable to penetrate the blood brain barrier . these derivatives contain a conformationally restricted gaba - mimetic nipecotic acid or guvacine moiety , a 48 atom linker , and a large , mostly diaromatic , hydrophobic moiety . a total of 162 compounds were extracted from the literature , spanning an activity range from low nanomolar to millimolar ic50 values , each of them tested under comparable assay conditions . ligands exhibiting substantial activity differences linked to distinct structural changes in the key regions shown in figure 2 were selected for subsequent experimental data guided docking . chemical structures and literature ic50 values of ligands with key modifications in linker length , polarity , and rigidity of the aromatic moiety . these activity differences should also be reflected by respective differences in the ligand / protein interaction pattern and thus aid in the prioritization of the docking poses . docking into 10 snapshots derived from the hgat-1tiagabine complex was performed in a sequential ensemble - like manner using gold , thereby allowing for minor movements of the backbone and focused sampling of the binding site side chain orientations . however , in the subsequent docking runs , no direct contribution of these two water molecules to the binding of the selected ligands was observed . hence , the full range of conformational flexibility of the r69 , y139 , y140 , and s452 side chains was explored using the internal rotamer library of the gold software package . analysis of the 10 top ranked poses per ligand among the ensemble docking results clearly indicated a common binding mode for tiagabine analogs ( figure 3 ) . as already expected from literature results , the most prominent interaction was coordination of the na1 sodium cation by the negatively charged acid moiety , which fulfills an octahedral geometry , together with side chain atoms of n66 , s295 , and n327 , as well as the backbone carbonyl oxygens of a61 and s295 . the majority of observed poses showed an interaction between the positively charged nipecotic acid nitrogen and the backbone of f294 , while the carboxylate group interacting with na1 was in an equatorial conformation . in contrast , poses with the r - configured carboxy group sampled in an axial conformation tended to form an intramolecular hydrogen bond with the charged nitrogen atom . while no clear preference for one of the two carboxylate orientations could be deduced from scoring values , x - ray and nmr studies of nipecotic acid indicated a preferred equatorial configuration , which would be more pronounced by adding a bulky moiety like the biaromatic tail . the upper boundary of the binding pocket is formed by a bent region of el-4 , extending into the hydrophobic cavity with the backbone of g360 as a ceiling beam , hence separating it into two pockets , each of which is able to accommodate a single hydrophobic aromatic rings . as it can be seen in figure 3 , the ligand transporter interaction in one hydrophobic pocket is stabilized by a interaction with the side chain of y139 , as well as by a cavity able to accommodate a small o - substituent as present in 5 and 6 . because of the relative torsion of the two pockets , poses of 7 tended to encounter an initial steric clash that was relieved after energy minimization of the complexes , whereas compounds with a terminal bis-5-methyl - thienyl ( 5 , 6 ) or diphenyl ( 810 ) group were able to bind in a conformation near their global energy minimum ( see figure 4 ) , thus explaining the activity cliff between 5 and 7 . hence , docking poses biased toward an interaction between this residue and one of the electronegative linker atoms in 6 , 8 , 10 , and 11 were generated , turning out to be possible but rather short - lived in short md runs due to reset forces of the basic side chain ( data not shown ) . to address the relatively low activity of compound 11 , per - atom contributions to g in the binding pose an unfavorable effect of the pyridine nitrogen in the hydrophobic receptor environment was indicated ( see supporting information , figure s3 ) . taken together , the common orientation of the compound class was in agreement with the structure activity relationships of the ligand set and the topology of the extended substrate binding pocket . to further probe the proposed binding mode against pharmacologically relevant chemical space , the 3d orientation of the main tiagabine binding features was extracted from the docked complex and encoded in a four - feature pharmacophore model using ligandscout . finally , a positively ionizable feature was placed on the basic nitrogen to mirror the compound s zwitterionic character ( figure 5 , the model is available for download at http://pharminfo.univie.ac.at ) . pharmacophore model of tiagabine : hydrophobic ( yellow ) , positive ( blue ) , and negative ( red ) ionizable features in context with the gat1 substrate binding site . the sensitivity of the pharmacophore model was validated by a decoy set generated using the dud - e platform ( http://dude.docking.org ) , retrieving just the compounds with known gat-1 activity . to test the predictive value of the model , a commercial vendor database consisting of 1.7 million compounds , as well as the drugbank index covering 1491 marketed drugs , a total of 79 and eight compounds , respectively , matched the pharmacophore query and passed the pains filter for frequent hitters . subsequently , the virtual hits were docked into a representative md snapshot of the homology model , from which the tiagabine pharmacophore model had been derived . for the latter , tiagabine , three thyroid hormones ( liothyronine , levothyroxine , dextrothyroxine ) , two angiotensin conversion enzyme ( ace ) inhibitors ( ramipril , perindopril ) , and an antihistaminergic drug ( bepotastine ) were retrieved . on the basis of pharmacophoric fit and docking performance ( details in supporting information , table s2 ) , bepotastine , ramipril , and liothyronine were selected for biological testing . inhibitory potency of the selected compounds was evaluated by an uptake inhibition assay of radiolabeled gaba in hek cells stably expressing rgat1 . one drugbank substance , 27a ( liothyronine , a thyroid hormone also known as t3 ) , turned out to significantly inhibit radioligand uptake , which prompted the acquisition and testing of other commercially available derivatives 27b d . the ic50 value for liothyronine derived from a dose response curve was 13 1.7 m ( figure 8) , providing a direct link between reported general effects of thyroid hormones on gaba uptake and the inhibitory action of 27a on the gat-1 subtype . as illustrated in figure 9 , the pharmacophoric depiction of 27a reveals that one of the required hydrophobic features is not , as one would expect , the aromatic ring of the 3,5-diiodophenyl moiety , but rather a lipophilic iodine substituent , which could barely be deduced from chemical similarity measures . apparently , the steric requirements in both systems are remarkably similar , relying on correct substitution pattern , stereochemistry , and degree of lipophilicity , possibly also limiting the relative efficacy of 27c . the most remarkable difference between the observed crystallographic hormone binding mode of 27a ( pdb code 3uvv ) and the gat1 binding hypothesis can be found in the 4 position . regarding compounds 1224 , the most crucial property among those molecules seems to be the distance between the positively ionizable group and the first occurrence of lipophilic bulk , which is in close analogy to the linker in tiagabine analogs . distal from the nitrogen atom ( as seen from the negatively ionizable group , which also can be a tetrazole moiety in a reasonable distance ) , usually just one heavy atom separates the positive charge from the next aromatic moiety ( 12 , 19 , 20 ) , or branching position ( 13 , 21 , 24 ) . this observation also extends to the inactive drugbank compounds , as the nitrogen atoms of 25 and 26 are both connected to the hydrophobic part by space demanding linkers , whereas the first aromatic ring of thyroid hormones is at a distance of two heavy atoms . to further assess the degree of similarity between the compounds retrieved and the reference compound tiagabine , we calculated the tanimoto similarity values based on chemical fingerprints ( maccs , fp2 , fp4 ) derived from openbabel . the similarity between tiagabine and liothyronine as well as the slightly active 18 , on the basis of maccs keys , was 25.4 and 51.5% , respectively . inhibitory potency of the selected compounds was evaluated by an uptake inhibition assay of radiolabeled gaba in hek cells stably expressing rgat1 . one drugbank substance , 27a ( liothyronine , a thyroid hormone also known as t3 ) , turned out to significantly inhibit radioligand uptake , which prompted the acquisition and testing of other commercially available derivatives 27b d . the ic50 value for liothyronine derived from a dose response curve was 13 1.7 m ( figure 8) , providing a direct link between reported general effects of thyroid hormones on gaba uptake and the inhibitory action of 27a on the gat-1 subtype . as illustrated in figure 9 , the pharmacophoric depiction of 27a reveals that one of the required hydrophobic features is not , as one would expect , the aromatic ring of the 3,5-diiodophenyl moiety , but rather a lipophilic iodine substituent , which could barely be deduced from chemical similarity measures . apparently , the steric requirements in both systems are remarkably similar , relying on correct substitution pattern , stereochemistry , and degree of lipophilicity , possibly also limiting the relative efficacy of 27c . the most remarkable difference between the observed crystallographic hormone binding mode of 27a ( pdb code 3uvv ) and the gat1 binding hypothesis can be found in the 4 position . , the most crucial property among those molecules seems to be the distance between the positively ionizable group and the first occurrence of lipophilic bulk , which is in close analogy to the linker in tiagabine analogs . distal from the nitrogen atom ( as seen from the negatively ionizable group , which also can be a tetrazole moiety in a reasonable distance ) , usually just one heavy atom separates the positive charge from the next aromatic moiety ( 12 , 19 , 20 ) , or branching position ( 13 , 21 , 24 ) . this observation also extends to the inactive drugbank compounds , as the nitrogen atoms of 25 and 26 are both connected to the hydrophobic part by space demanding linkers , whereas the first aromatic ring of thyroid hormones is at a distance of two heavy atoms . to further assess the degree of similarity between the compounds retrieved and the reference compound tiagabine , we calculated the tanimoto similarity values based on chemical fingerprints ( maccs , fp2 , fp4 ) derived from openbabel . with the increasing number of x - ray structures available for transmembrane transporters , structure - based computational models have provided valuable insights into the molecular basis of ligand transporter interaction . within this article , we propose a binding mode of the antiepileptic drug tiagabine in gat1 by including knowledge from ligand - based studies into the prioritization process for docking poses . strikingly , liothyronine has been described long ago as potential gat inhibitor without major activity on other neurotransmitter reuptake systems ( dopamine , serotonin , choline , aspartate ) , but final experimental confirmation for subtype gat1 since has been lacking . compounds with significantly higher chemical similarity retrieved in a commercial vendor library all prove inactive , further implying that selective transport inhibition of the protein can only be tackled from the side of steric feature arrangement . furthermore , the results indicate that , apart from privileged tricyclic antidepressant scaffolds known to more or less unspecifically inhibit neurotransmitter uptake , not many drugs on the market are likely to interact with gat1 . when assessing the differences between available sequence alignments of leut and hgat1 ( uniprot entries o67854 and p30531 , respectively ) , no differences for residues in the central binding cavity were observed , except for a one - residue gap in the middle of leut - tm10 , either placed over gat1-g457 , s456 , or a455 . as it has been optimized for gat1 and assessment of the sequence identity between hgat-1 and rgat-1 , the first being the effective protein of interest , the second the one used in cell assays , stated 100% sequence identity for the observed core region and 97.9% for the whole modeled sequence ( see supporting information , figure s4 ) . secondary docking runs for investigating side chain orientations for specific interactions with polar linker moieties were performed by ( a ) constraining residues y139 , y140 , and s456 to the internal library of allowed rotamers in gold and ( b ) for investigating interactions as reported by skovstrup et al . training set , decoy compilation , and drugbank compounds were prepared using omega - best settings ( max 500 conformations ) , enamine advanced and hts databases were compiled with omega - fast settings ( max 25 conformations ) ( www.eyesopen.com , ) . uptake of [ h]-gaba into was measured in the presence of 100 m of the compounds , while unspecific uptake was defined as uptake in the presence of 100 m tiagabine . for ic50 determination tested concentrations were : 5 , 0.001 , 0.01 , 0.1 , 0.3 , 1 , 10 , 100 m ; 27a , 0.01 , 0.1 , 0.3 , 1 , 3 , 10 , 30 , 100 m .

since the introduction of the concept or sublethally injured or viable but nonculturable ( vbnc ) cells by byrd and colwell in the 1980 's , there is increasing evidence for the existence of such a state in microbes , particularly in the aquatic environment that elicits a myriad of interrelated sub - lethal microbial stresses such as nutrient starvation and osmotic stress [ 2 , 3 ] ( table 1 ) . this is a cause for concern because of evidence that microbial pathogens in such a state may still retain their capacity to cause infections after ingestion by fish , animals , or by humans , despite their inability to grow under conditions employed in laboratory - based procedures for determining their presence in water . albeit currently unknown in terms of its severity or scope , it is now generally appreciated that heavily stressed pathogenic microbial species existing in a vbnc ( or not immediately culturable state ) may potentially pose as yet an undefined risk to public health , which is attested by the fact that there is increasing evidence to support the viewpoint that stressed cells in this quiescent state may actually be more virulent than well - fed laboratory - tamed microorganisms due to augmented virulence factor expression . were the first to bring experimental evidence of the existence of vbnc state in pathogenic bacteria , where they showed that e. coli and v. cholera cells that were suspended in artificial seawater quickly lost their ability to grow on the culture media normally used for their detection . according to oliver , a bacterium in the vbnc state is defined as a cell which is metabolically active , which being incapable of undergoing the cellular division required for growth in or on a medium normally supporting grown of that cell . besnard et al . suggest that the transition to the vbnc state in l. monocytogenes represents a survival strategy that bacteria can adopt under adverse conditions ( starvation , salt stress , etc . ) . vbnc microorganisms are considered to represent a subpopulation of cells that are unable to grow in the usual culture media and can not resuscitate by traditional resuscitation techniques , but yet remain physically active for several functions such as cellular elongation , respiratory chain activity [ 6 , 7 , 17 ] , or incorporation of radio - labelled substrates . for example , cappelier and coworkers recently reported that avirulent vbnc cells of l. monocytogenes incubated in filtered sterilized distilled water need the presence of an embryo to be recovered in egg yolk and regain virulence after recovery . the vbnc state was observed after a 25 to 47 days incubation period ( concentration of culturable cells less than 1 colony forming unit per ml ) . as microorganisms are extremely diverse and dynamic , it is not surprising that the many different types of microbial species present in the water environment exist in a number of physiological states that possess different requirements for survival and to sustain growth . indeed , the number of waterborne bacteria in which the vbnc state has been reported has greatly increased , particularly in recent times that reflect technological advances . for instance , campylobacter jejuni has been reported to exist in two different cellular morphotypes , where the atypical coccus - form ( currently associated with nongrowing vbnc state ) occurs in water under extended nutrient depletion conditions . a number of different research groups have reported that these atypical culture forms are still capable of infection mice and poultry . moreover , rowan and coworkers recently reported on that different culture morphotypes of l. monocytogenes generated in water after exposure to novel pulsed - plasma gas - discharge treatment can survive internalization by human polymorphonuclear leukocytes . while lindbck et al . reported that the ability to enter into an avirulent vbnc form is widespread among l. monocytogenes isolated from salmon , patients and the environment . l. monocytogenes were tested for virulence in a cell plaque assay and by intraperitoneally inoculation in immunodeficient rag1 mice . moreover , moreno et al . described successions in cellular alterations in helicobacter pylori nctc 11637 after inoculation into chlorinated drinking water . they concluded that h. pylori could survive disinfection practices normally used in drinking water treatment in the vbnc form , which would allow them to reach final consumption points and , at the same time , enable them to be undetectable by culture methods . whereas kastberg et al . recently reported that l. monocytogenes cells , whether planktonic or attached , were homogenous with respect to sensitivity to acidic disinfectants at the single - cell level . directvisualization of actively respiring bacteria is gaining in popularity amongst research groups investigating this vbnc state [ 3 , 6 , 7 ] . researchers have exploited use of metabolic staining to reveal an underestimation in the level of microbial survival compared to similar samples cultured on traditional agar plates . recent research in our laboratory has also shown that subpopulations of waterborne pathogens such as e. coli and pseudomonas spp . treated with novel pulsed - power disinfection technologies ( such as use of pulsed - plasma gas - discharge technology that will be expanded on later in this paper ) were capable of reducing the redox dye 5-cyano-2 , 3-ditolyl tetrazolium chloride ( ctc ) that is an indicator of electron acceptor function , yet similarly treated samples were unable to form colonies on a variety of laboratory - based culture media [ 6 , 7 ] . this corroborates more recent research undertaken by sawaya and coworkers who used a combined 4'6-diamidion-2-phenylindole ( dapi ) and ctc stains to highlight the occurrence of physiologically active bacteria in river and wastewater treatment plants that were much higher than those obtained by plate counting . these researchers also reported that microscopic viable bacteria were more chlorine resistant than culturable bacteria . that said , a demonstration of active respiration does not necessarily infer that these stressed bacteria are capable of future growth . as numerous researchers continue to report on the use of redox stains for highlighting differences in agar - plate counts , it is important that we holistically explore and identify specific microbial cues at the cellular level which govern the transition to the vbnc state along with exploiting commensurate advances in media formulations that are tailored for optimal resuscitation of these sublethally stressed cells ( cited in [ 10 , 24 ] ) . the latter authors showed that the addition of a commercially available antioxidant oxyrase and a heat - stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine , resuscitated e. coli , and salmonella enteric serovar typhimurium that were stressed by prolonged incubation in water microcosms . advancing the earlier pioneering work of dodd et al . and aldsworth et al . , who previously postulated that self - destruction or cell suicide may be attributed to sublethally stressed or damage microbes being incapable of coping with oxidative burst when rapidly growing on nutrient rich media , it is important that we also exploit advances in toxicology ( such as methods exploring cellular apoptosis and necrosis , cell membrane lipid peroxidation assays , and nuclear chromatin / comet assays ) that will provide valuable insights into the possible role of intracellular free radicals in combination with the direct physical action of the applied environmental stress on the generation and persistence of vbnc organisms in water . indeed , servais and coworkers recently reported that certain environmental factors such as nutrient scarcity and solar irradiation lead to a high proportion of vbnc e. coli in freshwater . the latter fecal microorganisms are brought into freshwater environments mainly through wastewater release , surface runoff , and soil leaching . interestingly , modified guidelines for bathing water states that enumeration of e. coli will replace total coliforms and fecal ( also called thermotolerant ) coliforms as bacterial indicators of water quality . the latter authors articulated that the number of e. coli in freshwater is systematically underestimated by traditional culture - based methods such as multiple tube fermentation and or membrane filtration techniques ) , which is cause for concern from a public health perspective . on a related theme , numerous researchers have also recently reported on the occurrence of bacterial autophagy ( i.e. , microbial adaptation to autophagic microbicidal host immune cell defences ) , which is an important cell survival process initiated during nitrogen starvation conditions . among the minority of bacteria that have been discovered it is estimated that more than 90% are as yet nonculturable as attested by the fact that international committee on systematic bacteriology has recognised a new category for nonculturable bacteria that it named candidatus for which phylogenetic relatedness has been determined by amplification and sequence analysis of prokaryotic rna genes with universal prokaryotic primers and authenticity has been verified by use of in situ probes . such nonculturable organisms may only be detected by use of such molecular techniques based on probes such as 16s and 23s rrnas or on determination of mrna , either by quantitative real - time pcr and/or by fluorescent techniques such as in situ hybridization ( fish ) , microradiography , epifluorescence microscopy , and flow cytometry [ 2 , 11 , 12 ] . while fiksdal and tryland advocated use of rapid enzyme assays for monitoring of water quality , which may also detect organisms in the injured or vbnc state . garcia - armisen and servais showed that the ratio of direct viable- ( dvc- ) fish count and the culturable count increased with decreasing abundance of culturable e. coli in river water , and therefore the slope of the linear - log - log correlation of dvc - fish versus colony forming unit numbers was less than one . the authors hypothesized that the more stressful conditions , such as nutrient deprivation and increase solar stress at low turbidities met in low contaminated environments , were responsible for the larger fraction of vbnc e.coli . as mentioned previously by many research groups [ 13 , 2933 ] , all field trials plotting log -d - galactosidase ( galase ) activity or log -d - glucuronidase ( gluase ) activity versus log culturable target bacteria in fresh or marine water have shown regression straight lines with a slope less than one , suggesting that enzyme activity calculated per culturable indicator bacteria increases when their numbers decrease ( e.g. , when sewage effluent becomes diluted in receiving waters ) . pure culture studies of e. coli have also shown that after exposure to other types of extrinsic stress such as chlorination , the galase activity is less reduced than the direct viable count . while zimmerman and coworkers demonstrated that e. coli can be present in higher numbers in recreational water samples using fluorescent antibody direct viable counting that what are detected with standard culture methods . the latter advances the early landmark study of bjergbaek and roslev who reported on the occurrence and persistence of vbnc e. coli in nondisinfected drinking water using different cultivation dependent methods , fluorescence in situ hybridization ( fish ) using specific olignucleotide probes , direct viable counts ( dvc ) , and by enumeration of gfp - tagged e. coli ( green fluorescent protein , gfp ) . these studies specifically reemphasises the need for a rapid , accurate , and precise method for detecting health risks to humans from contaminated water . other likely candidate methods for efficient and rapid - detection of vbnc bacteria in the aquatic and soil environments include rna - based genotypic approaches . recently reported on the rapid and accurate quantification of vbnc pathogens in biosolids via monitoring and quantifying stress - related genes in salmonella spp . using cdna microarrays combined with quantitative reverse transcription polymerase chain reaction ( qrt - pcr ) . okabe and shimazu also describe detection of host - specific bacteroides - prevotella 16s rrna genetic markers ( total , human , cow- and pig - specific ) as promising alternative indicators for identifying the sources of fecal pollution in environmental water because of their abundance in the feces of warm - blooded animals . the authors clearly state that detection of aforementioned genetic markers mainly reflected the presence of vbnc bacteroides - prevotella cells in water , suggesting that seasonal and geographical variations in persistence of these host - specific bacteroiides - prevotella 16s rrna genetic markers must be considered if used as alternative fecal indicators in environmental waters . this corroborates emerging studies that suggest that increases in coliform concentration after stp and dewatering processes may be attributed to cells going into vbnc state implying traditional coliform enumeration methods are not sufficient to determine number of viable cells . this also reinforces common viewpoint shared by many scientists that a variety of problematic bacteria can enter vbnc states as a survival response when exposed to deleterious environmental stresses such as nutrient starvation , osmotic stress , and so forth . however , in order to gain a greater appreciation of environmental and public health risks associated with persistence of microbial pathogens in different culturable states , it is imperative that we acquire an understanding of the interrelated molecular responses governing tolerance to these applied sub - lethal stressors . such as detailed investigations reported by brackman et al . who recently found that autoinducer- ( ai- ) 2 quorum - sensing inhibitors affected starvation response and reduce virulence in several vibrio species , most likely by interfering the signal transduction pathway at the level of luxpq . the aforementioned detection methods are not as yet commonly used for routine measurement as they either are not simple enough and/or the equipment is too expensive . therefore , in terms of enabling effective risk - based assessment and environmental management to address the occurrence and persistence of vbnc pathogens in water , critical data must be acquired to convert vbnc - phase potential pathogens from unknown to known and defined hazards . in terms of satisfying these information gaps , accurate enumeration of total microbial load and real time identification to the various types present in water are pivotal to hazard identification and characterization , which will contribute significantly to assuring water safety . regarding the latter , smeets et al . recently described improved methods for modelling drinking water safety using a robust quantitative microbial risk assessment ( qmra ) , where a case study monitored campylobacter data for rapid sand filtration and ozonation processes . this study showed that currently applied methods do not predict monitored data used for validation . therefore , underestimation in the levels of problematic microbes in water , and failure to identify the presence of pathogenic organisms in representative water samples also pose significant threats to public health . greater information is also required on elucidating the existence of commonly shared cellular mechanisms ( and associated gene expression regulators and gene markers ) that govern cellular conversion to this vbnc state . moreover , there is a dearth of knowledge regarding specific underlying molecular and associated cellular mechanisms governing transition and persistence of waterborne microorganisms in this vbnc state , in addition to obviously establishing what specific environmental conditions or triggers cause these changes in culturable state . greater studies are also required to investigate the presence and role of this vbnc state in microbial pathogens that cause disease in the aquatic natural setting such as in fish.taking for example , flavobacterium psychrophilum , the causative agent of rainbow trout fly syndrome and cold water disease in salmonids , where subpopulations were still culturable after starvation for 300 days in sterilised fresh water . the virulence of starved f. psychrophilum was maintained for at least 7 days after the transfer of the bacterial cells to fresh water . the vnbc state was earlier reported for this fish pathogen by madetoja and wiklund who revealed differences between enumeration using advanced immunoflourescence and genetic probes ( i.e. , nested pcr ) compared to that of using traditional agar plate cultivation . while the long - term survival cellular responses to salinity , temperature and starvation have been studies in the eel pathogen vibrio vulnificus for over a decade . to combat such severe microbial infections affecting fish , many researchers have advocated water recirculation and good management as potential methods to avoid disease outbreaks particularly with f. psychrophilum . thus , highlighting the importance of augmenting water quality through improvements in wastewater treatment , which should be augmented in efficiency and capacity to cater for under - appreciated and for emerging microbial threats . to comprehensively address such issues , detailed analysis of the proteome that arise during this morphological transition will enable identification and characterization of key proteins that are responsible for this vbnc state . in addition , tandem use of microarray gene analysis along with real - time quantitative pcr will help unravel specific gene functions and will pin - point over - arching regulatory framework(s ) . the aforementioned studies should also be carried out in parallel with developing improved protocols for resuscitating vbnc organisms , particularly availing of advances made in fermentation technology such as exploiting chemostat - based bioreactor approaches that can simulate and monitor multiple environmental stresses ( either applied simultaneously or sequentially ) over extended time periods . indeed , kooi and coworkers recently exploited use of a chemostat to report on the dynamic behaviour of simple aquatic ecosystems with emphasis on nutrient recycling in order to monitor toxicants . thus , use of the latter provides a useful vehicle for investigating the effects of deleterious microbial stressors on the structure and functioning of ecosystems . other related studies that merit attention include the development of more rapid , user - friendly , field - based technologies that will reliably and repeatably detect and quantify various waterborne pathogens that may persist in different culture states . less than four hours was recommended as rapid by noble and weisberg in a recent review of rapid detection methods for bacteria in recreational waters . due to plethora of different detection methods currently being developed combined with obvious interlaboratory variability in terms of associated operating protocols , it is imperative that we develop and agree upon a standardized battery of reliable tools for detecting and quantifying culturable and nonculturable bacteria so as to establish future unified , quantitative - risk management protocols for monitoring our aquatic environments . the latter will greatly facilitate our endeavours to collectively comply with emerging environmental policies such as eu 's water framework directive . cryptosporidium species have emerged over the past decades as major waterborne pathogens causing gastroenteritis in humans . the occurrence of the environmentally resistant thick - walled oocyst stage of this organism has become a worldwide concern due to its resistance to disinfection with chlorine at concentrations typically applied in drinking water treatment plants ( 2 to 6 thus , the control of cryptosporidium oocysts remains a major challenge for drinking water utilities due to fact that the common chemical disinfectants - free and combined chlorine , when used singly , are practically ineffective for inactivating this protozoan under conditions encountered in most treatment facilities . this protozoan is transmitted via the faecal - oral route , where consumption of contaminated drinking water and use of recreational water are major sources of infection . cryptosporidium oocysts are resistant to conventional disinfectants at concentrations and exposure times commonly used , and their infectious doses in humans have been estimated to be as low as 30 oocysts . indeed , more than 160 waterborne outbreaks of cryptosporidiosis have been reported globally , with the greatest documentation occurring in the us and the uk [ 46 , 48 ] . this situation has become a major concern for water authorities and consequently , significant modifications to drinking water regulations have been proposed for the detection and surveillance of this protozoan in the us and in other developed countries . the new european drinking water directive advocates that all the state members should provide drinking water supplies with absence of pathogenic organisms . however problems associated with the determination of oocysts viability / infectivity make the establishment of maximum acceptable concentrations very difficult , and concentrations of 330 oocysts/100 litres in treated water have been proposed as action levels . recent contamination of drinking water supplies in the west of ireland have led to a significant number of confirmed cases of cryptosporidiosis , intimating that the use of conventional decontamination methods failed to eliminate this enteroparasite in treated water ( cited in garvey et al . ) . this situation has become a major concern for water authorities and consequently , significant modifications to drinking water regulations have been proposed for the detection and surveillance of this protozoan in the us and in other developed countries . development of alternative methods of cryptosporidium disinfection for water applications ( such as ozone and/or uv ) has been hindered by the uncertainty surrounding efficacy of using in vitro surrogate viability assays due to their overestimation of oocysts survivors posttreatments and the lack of critical data on the preferred use of in vitro cell culture and/or in vivo animal - based infectivity assays to determine interrelated factors governing repeatable disinfection of oocysts suspended in water . although recent studies that utilised at least 20 different cell lines have advocated the preferential use of the human ileocecal adenocarcinoma hct-8 cell line as an equivalent in vitro method to that of using the gold standard mouse assay for measuring infectivity of cryptosporidium , there has been limited evidence to date on the combined use of these approaches for assessing critical operational parameters governing pulsed uv light ( puv ) as a means of disinfecting water contaminated with this enteroparasite . this study demonstrated that there is good agreement between use of in vitro culture - qpcr and the scid - mouse - infectivity assays for evaluating the disinfection efficacy of pulsed uv for inactivating c. parvum suspended in saline , thus reducing the requirement to unnecessarily use animals for these particular research studies . however , the authors recommended that pressing investigations are needed to comprehensively demonstrate efficacy of using this novel disinfection uv approach for treating lower concentrations of infectious oocysts under dynamic conditions found in wwtps and in the aquatic environment . development of a reliable and repeatable method of measuring fluence values from uv - delivered pulses for water disinfection applications is also required , and should take on board critical interrelated factors identified previously by bolton and linden for continuous low- ( lp ) and medium - pressure ( mp ) uv units . development of puv has recently received attention as a potentially novel strategy for decontaminating water as it offers many benefits including rapid microbial reductions and efficiency of energy usage due to underpinning high peak - power dissipation during treatments [ 55 , 56 ] . indeed , use of ultraviolet ( uv ) light have become widely accepted as alternative methods to chlorination for wastewater disinfection [ 57 , 58 ] . there are also over 2,000 wastewater treatment plants worldwide using either lp or mp ultra - violet technologies . recent studies investigating continuous - use uv lamp technology has demonstrated the effectiveness of uv in inactivating pathogens in wastewater . research has also shown that , to ensure permanent inactivation and prevent the recovery of microorganisms following exposure to uv , a broad polychromatic spectrum of uv wavelengths is necessary such as doses delivered by mp and puv systems . these wavelengths inflict irreparable damage not only on cellular dna , but on other molecules such as enzymes as well . moreover , numerous studies have also highlighted limitations of decontamination techniques such as conventional low pressure mercury lamps designed to produce energy at 254 nm ( called monochromatic or germicidal light ) that include microbial repair and the necessity for lengthy durations of exposure to obtain suitable levels of decontamination . more recently , medium - pressure mercury uv lamps have been used because of their much higher germicidal uv power per unit length and because of their ability to emit polychromatic light comprising germicidal wavelengths from 200 to 300 nm ) . this approach kills microorganisms by using ultrashort duration pulses of an intense broadband emission spectrum that is rich in uv - c germicidal light ( 200 to 280 nm band ) . puv is produced using techniques that multiply power manifold by storing electricity in a capacitor over relatively long times ( fractions of a second ) and releasing it in a short time ( millionths or thousandths of a second ) using sophisticated pulse compression techniques [ 56 , 60 , 61 ] . the emitted flash has a high peak power and usually consists of wavelengths from 200 to 1100 nm broad spectrum light enriched with shorter germicidal wavelengths [ 62 , 63 ] . this technology has received several names in the scientific literature : pulsed uv light [ 6466 ] , high intensity broad - spectrum pulsed light , pulsed light , intense pulsed light , and pulsed white light . despite the fact that uv light appears effective for inactivating waterborne enteropathogens , including cryptosporidium spp . it is recognised that many organisms have mechanisms for repairing light - induced dna damage . however , rochelle et al . recently reported that cryptosporidium spp . oocysts could neither repair uv light - induced damage nor regain infectivity under standard conditions used for storage and distribution of treated drinking water . this is despite the fact that both c. parvum and c. hominis were shown to harbour genes encoding uv repair proteins for mechanisms such as nucleotide excision repair and photolyase enzymes . indeed , irradiated oocysts were unable to regain preirradiation levels of infectivity , following exposure to broad array of potential repair conditions , such as prolonged incubation , preinfection excystation triggers , and post - uv holding periods . . reported that adaptive microbial survival ( tailing phenomenon ) occurs when samples are treated in high turbidity solutions using continuous uv sources whereas tailing did not occur when similar samples were treated with pulsed xenon lamp . in terms of potential future alleviation strategies for combating c. parvum in water , it is likely the combined use mp and puv technologies would offer considerable advantages as the next - generation decontamination bolt - on approach including rapid processing and efficiency of oocyst destruction , and should accommodate dynamic operational requirements at wwtp level . use of ozone is gaining in popularity as an alternative or complementary approach for disinfection in drinking water facilities worldwide . some landmark studies have recently reported on the possible efficacy of using ozone for destroying cryptosporidium oocysts . however , there are very limited published findings to date that holistically investigate critical factors governing the effective and repeatable destruction of this recalcitrant enteroparasite in drinking water supplies using ozone or other oxidative agents . of those researchers that have reported on this complex oxidation process , it would appear that using ozone singly or combined with ( or without ) free chlorine or monochloramine ( in addition to other synergistic factors such as disinfection concentration , contact time , dissolved organic content concentration , ph , and temperature ) are of critical importance for the inactivation of treated c. parvum [ 72 , 73 ] . despite limited understanding of the dynamic complexities underpinning advanced oxidation processes , ozonation is often preferred to chlorination because former leads to smaller concentrations of potentially harmful halogenated disinfection by - products . yargeau and leclair also recently reported that use of ozone appears to be a promising technique for degradation of antibiotics , even in wastewater . after 4.5 min of ozone treatment , the concentration of sulphamethoxazole was below the hplc detection limit of 0.6 mgl indicating a degradation efficiency higher than 99.24% . intense research has been recently focused on the development of novel pulsed plasma gas discharge technology as a complementary means of treating water containing unwanted microbial pathogens and chemicals . pulsed plasma gas - discharge ( ppgd ) technology involves applying high voltage pulses to gas - injected test liquids resulting in the formation of a plasma that causes free radicals such as dissolved ozone and hydrogen peroxide , free electrons , ultraviolet light ( uv ) , acoustic shock waves , and electric fields at levels between 1050 kv / cm to be generated in the test liquids ( figure 1 ) . rowan and coworkers has been shown that application of ppgd successfully reduces unwanted campylobacter and salmonella spp . in poultry wash water . ppgd ( akin to puv ) is an enabling technology that requires transient generation of high voltage and high current that in turn results in the generation of large peak powers ranging from megawatts to terrawatts . depending upon the application ( ppgd or puv ) , a pulse generator will deliver a large energy level on a single shot basis or alternatively will deliver a modest amount of energy ( 110 j ) at a repetition rate from 10 to 10,000 pulses per second . thus ppgd offers a radical new approach to energy delivery that involves the use of repetitive switching techniques to deliver stored energy in intense ultrashort bursts ( 85100 nanoseconds ) . during each pulse , very high levels of peak power are generated ( 1020 mw ) , and treatment is achieved using the required number of pulses , which is favourable in terms of requirements for new energy efficient technologies for the fast - approaching post peak oil era . other research groups have also reported on the successful use of pulsed high voltage for decontaminating microbial populations and phenol in liquid solutions . interestingly , research in our laboratory has shown that the myriad of biocidal properties generated by use of ppgd technology has also demonstrated promising results for the removal of prions on surgical material intimating potentially other healthcare applications for this decontamination approach . however , a nonoptimized pulsed plasma - gas discharge process may also lead to the formation of undesirable by - products such as brominated organic compounds and halogenates in treated water . brominated organic compounds are also considered potentially harmful and remain the subject of international study in order to elucidate mechanistic and kinetic information regarding their formation during ozonation so as to identify effective strategies for their reduction or elimination . considerable attention should be given to reducing or eliminating the occurrence of harmful by - products of ozone during this project via adjustments in sparged - gas composition , decreasing ph and complementary use of pulsed uv technology . surprisingly , despite increased interest in the development of nonthermal advanced oxidative processes ( such as corona plasma discharges , ozone combined with h2o2 , low / medium pressure uv combined with h2o2 , etc . ) , this author has been unable to source any published reports on possible toxicological issues associated with use of these new technologies for disinfection applications in healthcare , agri - food or the environment . on a related point , our research group has recently reported on the occurrence of vbnc state for food and water - borne microorganisms generated in liquid suspensions after separate exposure to ppgd and pulse electric field technologies [ 6 , 7 ] . although antibiotics have been used for decades , only recently has an increasing number of studies highlighted the lack of understanding and knowledge about persistence of antibiotics in the aquatic environment and the potential environmental risks that this may pose . particularly as antibiotics are often poorly degraded or removed in conventional wastewater treatment plants ( wwtps ) which causes formation of toxic degradation products that may impact negatively on the aquatic environment and public health . antibiotics in the broader sense are chemotherapeutic agents that inhibit or abolish the growth of microorganisms , and have been used extensively in human and veterinary medicine as well as aquaculture for the purpose of preventing or treating microbial infections . wise estimated antibiotic consumption worldwide to lie between 100,000 and 200,000 ton per annum . the reader is also directed to the recently published landmark reviews of kmmerer [ 83 , 84 ] for further detailed information on the possible input , occurrence , fate , and effects of antibiotics in the aquatic environment . the concentrations of antibiotics in municipal sewage and in sewage treatment plants are typically lower by a factor of 100 compared to hospital effluent . while bacterial resistance to antibiotics have also been found in soil . some studies revealed that many different types of antibiotics are biodegradable under aerobic or anaerobic conditions , while other related research has reported that certain aquatic microbes can actually use these free active compounds as a sole carbon source . however , research has also shown that the concentrations and activity spectrum of compounds found in the environment does not correlate with the presence of resistant bacteria isolated from the same environments . concern over the development of secondary resistance to commonly - used antibiotics ( either through vertical and/or horizontal gene transfer among related and unrelated bacteria ) in the environment with potential for adversely affecting aquatic and terrestrial organisms along with the potential for a cyclic unwanted return to humans again via consumption of drinking water has stimulated numerous research groups to investigate these important issues . kmmerer clearly articulated that the issue of acquired resistance was nearly always addressed in publications by describing the presence of antibiotics in the environment , with often mere speculation as to the possible relationship between low concentrations of antibiotics in water and emergence of resistance . in truth , however , the trend of accumulating and accelerating resistance to antibiotics is of concern as this is also juxtaposed by the marked reduction in mankinds ' current arsenal of effective tools for combating this phenomenon . from a review of best published literature it would appear that possibly the most significant undefined risk in terms of antibiotics are microorganisms harbouring multiple antibiotic resistance genes such as vancomycin - resistant enterococci ( vre ) , methicillin - resistant staphylococcus aureus ( mrsa ) , and multiresistant pseudomonads that are living in close proximity to each other ( such as in biofilms sewage sludge flocs ) as opposed to the free active compounds present at low concentrations in the aquatic environment . indeed , davison provided evidence that antibiotic resistance is already present in natural environments and that it can be exchanged between bacteria for at least a decade . therefore , under intense discussion is the possibility that nutrient - poor , oxygen - limited and cold aquatic environments exemplified by sewage sludge may be selecting for and acting as a reservoir for slowing growing resistant organisms . this environment provides for a higher biodiversity of microbial types and numbers affording a greater probability for accelerated exchange of antimicrobial drug resistance and virulence determinants in the presence of other neighbouring microbes deficient in such deleterious properties . indeed , ohlsen et al . revealed that antibiotics even at sub - inhibitory concentrations can have an impact on cell function and can stimulate expression of quiescent virulence factors or potentiate transfer to an antibiotic resistant state . the latter is strongly aligned with findings from unrelated environmental stress studies which previously showed that many food and waterborne microbial species can sense and adapt to related and unrelated sub - lethal environmental stresses ( such as osmotic stress or starvation ) through complex quorum sensing and respond through an orchestrated controlled subsequence of preferential gene expression . thus , not alone may the latter confer microbial tolerance to the applied stress through a process of elevating activities in important genes that regulate key house - keeping functions such as osmotic stress response and protection vital proteins ( chaperone ) , but such environmental tempering may also act as stimuli for up - regulating microbial virulence factor expression . the classic example being l. monocytogenes , where its ' transcriptional activator prfa up - regulates virulence factor expression in the presence of a known stress ( e.g. , body temperature ) , yet down - regulates transcription of pathogenic determinants in the presence of soil - related carbohydrates . interestingly , zupan and raspor recently reported on the development of an invasion - agar assay for determining in vitro invasiveness of saccharomyces cerevisiae , and showed that this yeast augmented virulence when exposed to temperatures typical of human fever ( 37 to 39c ) yet exhibited strong repression effect on invasion in the presence of salts , anoxia and some preservatives . the aforementioned also highlights the extreme versatility of microbial species present in complex communities ( such as in biofilms in wwtps ) to rapidly change and adapt when confronted with a sustained external selective pressure . kmmerer stated that there is a dearth of information on the interrelated factors governing the fate and effects of antibiotics in the environment ( i.e. , microbial ecology ) , highlighting that the majority of published studies to date are limited to single compound approaches . he also recently postulated that the effects of antibiotics in wwtps or in the aquatic environment may be under - estimated , particularly as there is evidence that microbial exposure to antibiotics from the same group or from different groups may result in additive effects . for -lactams it has been shown that their potency is much higher in the presence of 5-fluorouracil , a cytotoxic compound also present in sewage in concentrations at the mg or gl range . use of biocides such as triclosan and quaternary ammonium compounds used in hospitals and homes may also select for antibiotic resistance in microbial pathogens . additionally , there is no published information as yet on the potential impact of low levels of other pharmaceutically active compounds ( such as endocrine disrupting chemicals ) on development of antibiotic resistance as the former also tolerate sewage treatment . there is also limited information on how long bacteria maintain antibiotic resistance in the absence of continued selective pressure for that resistance . that said , recent findings also suggest that bacteria which have already have become resistant through the application of antibiotics will not necessarily have a selective advantage in sewage treatment [ 92 , 93 ] . interestingly kmmerer reported that resistance was found to be high in hospital effluents and in sewage treatment plants , yet hospital effluents contribute to less than 1% of the total amount of municipal waste suggesting that hospitals are not the main source of resistant bacteria in municipal sewage . the latter would also suggest that antibiotic usage in the community accounts for the main input of resistant bacteria into municipal sewage . the question should also be posed as to whether or not multi - drug resistant bacteria that transcend to the quiescent vbnc state after periods of extended exposure to a nutrient depleted stressful environment ( such as in water or in soil ) are still capable of gene transfer and whether or not these important molecular determinants remain unaltered and stable . kmmerer also reported that the concentration of antibiotics may be much higher if the active compounds are persistent and accumulate , for example , by sorption to solid surfaces such sewage sludge , sediments , or soil . while most antibiotics tested to date have not been biodegradable under aerobic conditions(kmmerer [ 83 , 94 , 95]),biodegradability has been poor for most of the compounds investigated in laboratory tests , including some to the -lactams . out of 16 antibiotics tested , only benzyl penicillin ( penicillin g ) was completely mineralized in a combination test . no evidence of biodegradation for tetracycline was observed during a biodegradability test , and sorption was found to be the principal removal mechanisms for tetracycline in activated sludge . substances extensively applied in fish farming had long half - lives in soil and sediment as reported in several investigations . however , some substances were at least partly degradable . maki et al . found that ampicillin , deoxycycline , oxytetracycline , and thiamphenicol were significantly degraded , while josamycin remained at initial levels . despite the aforegoing , it has yet to be established that permanent exposure of antibiotics in sewage treatment systems promotes the development of antibiotic resistance and selective effects on bacterial communities . studies have also revealed that bacteria that are resistant to antibiotics are present in surface water . furthermore , antimicrobial resistance has also been found in marine bacteria and bacteria living in estuaries or coastal waters polluted with sewage water . other researchers have reported that antibiotic resistance genes ( args ) can be found in a region where no selection pressure exists . whereas pruden et al . noted that tetracycline args tet(w ) and tet(o ) were present in treated drinking water and recycled wastewater , suggesting that these are potential pathways for the spread of args to and from humans . high loads of antibiotics in sediments at concentrations potent enough to inhibit the growth of bacteria have been reported for aquaculture . while antibiotic - resistant bacteria have been detected in drinking water supplies as far back as the 1980s . in fish farming sector such as aquaculture and mariculture , the widespread use of antibiotics for treating bacterial diseases has been associated with the development of resistance in a range of bacterial pathogens including aeromonas hydrophilia , aeromonas salmonicida , edwardsiella tarda , edwardsiella icttaluri , vibrio anguillarum , vibrio salmonicida , pasteurella piscida and yersinia ruckeri . there are also considerable gaps in current knowledge concerning the possible transfer of chemical contaminants and microbial pathogens ( including those harbouring args ) into the food chain through land spreading of some treated organic municipal and industrial material on agricultural land used for food production . the reader is directed to the comprehensive report produced recently by the food safety authority of ireland that addresses critical issues underpinning these putative concerns . other pharmaceutically active chemicals . a marked observation from this paper is the pressing need for greater risk - based assessment and for new and/or improved alleviation strategies for the optimal decontamination of wastewater at treatment plant level so as to reduce or eliminate the microbial ( and their associated metabolite ) risks to public health . in the context of improving wastewater treatment and planning for safe drinking supplies , one must also acknowledge growing international concerns about the release of certain chemicals into the aquatic environment that may result in alterations in the reproductive health of humans and wildlife [ 108110 ] . the environmental presence of such man - made and naturally occurring compounds , properly referred to as endocrine disrupting chemicals ( edcs ) , can mimic or interfere with the binding and action of natural hormones , thus disrupting normal physiological processes [ 111 , 112 ] . consumption of water contaminated with edcs may also cause reproductive disorders in humans as such chemicals have the ability to mimic the function of natural estrogens as well as disrupting the synthesis and metabolism of hormones by binding to hormonal receptors ( cited in [ 108 , 110 ] ) . thyroid system - disrupting activity in water from municipal domestic sewage treatment plants was also detected recently .the list of known edcs is extensive and includes natural and synthetic steroid hormones ( such as 17 -estradiol(e2),estrone ( e1 ) , and 17 -ethinylestradiol ( ee2 ) ) , phytoextrogens , pesticides , pharmaceuticals , and surfactants , all of which have been detected worldwide in processed water from domestic treatment plants ( wwtps ) at the ng / l level that may cause abnormalities to aquatic organisms [ 113 , 114 ] . edcs were recently detected in effluents from wwtps and from receiving waters at levels exceeding ng / l in ireland . recent studies conducted by fogarty and mcgee have demonstrated that fish habituating downstream of waste - water treatment plants ( wwtps ) in the midlands region in ireland exhibited delayed spermatogenesis compared with fish upstream and intersex ( feminization ) was discovered in roach [ 115117 ] . the scientific community has particularly focused on estrogenic edcs ( i.e. , compounds interacting with the human estrogen receptor ) , which enter the environment from a variety of sources including effluent discharge pipes , agricultural runoff and landfills . in particular , such edcs are considered to be dominant contributors to estrogenic activity in treated water from wwtps and have been found in treated effluent at the ng / l level [ 113 , 118 ] . whilst intensive conventional - based treatment approaches have investigated many interrelated factors in the design and operating conditions of wwtps for reduction or elimination of edcs in treated wastewater , none have reported on their effective removal . moreover , due to the common practice of residual chlorine in drinking water distribution , halongated by - products such as haloacetic acids and trihalomethanes form that exhibit a range of potential carcinogenic potentials ( cited in ) . the european commission ( com 706 ) acknowledged that there is an urgent need for further scientific research to denature deconjugated edcs in the environment . therefore , it is essential that such compounds be efficiently and effectively removed from processed water discharged from wwtps . on a similar theme , yamamoto and coworkers recently reported the persistence of 8 pharmaceuticals with relatively high ecological risk and high consumption ( namely acetaminophen , atenolol , carbamazepine , ibuprofen , ifenprodil , indomethacin , metenamicacid , and propranolol ) using river water . previous researchers have recently shown that use of similar non - thermal corona discharge processes such as the ppgd technology may be potentially effective at destroying structurally - related organic compounds such as dyes , phenol and aniline in aqueous solutions [ 121125 ] . other nonplasma related studies have shown that the use of uva can also facilitate removal of edcs including e1 , e2 , and ee2 from water by photolysis [ 123 , 126 ] . given that the majority of known edcs are phenolic in chemical structure , the benzene rings underpinning these edcs should be rapidly photodegraded by hydroxyl radical attack during ppgd treatments . it is worth noting that the use of ozone become widely accepted as alternative methods to chlorination for wastewater disinfection [ 57 , 58 ] , and ozone has traditionally been applied in drinking water treatment plants for disinfection . while other researchers have demonstrated the production of short - lived , high - oxidative , plasmochemical elements in pulsed - plasma - treated test liquids such as water and effluent for bacterial decontamination , no study to date has reported on the use of corona discharges singly or combined with pulsed uv light as a novel approach for the destruction of established and emerging microbial threats to public health in water or effluents . these dynamic environmental fate studies must be carried out in tandem with experiments that ascertain the likelihood of by - product toxicant formation and unwanted persistence in water , and address these associated undefined risks by either adjusting existing and/or implementing new complementary alleviation strategies . there is a pressing need to generate critical data in gaps highlighted above via provision of funding to establish consortia of meaningful stakeholders comprising scientists , engineers , and end - users in order to facilitate policy makers and implementers ( suchaswater managers ) strive towards meeting ambitious objectives set for the eu 's water framework directive that requires member states to attain a good ecological status for all water bodies by 2015 . while a global response to emerging environmental problems has been positive with the provision of substantial research funding been made available to the broad and diverse researcher communities ( such as eu fp7 initiatives ) , one obvious real challenge that still remains is how does mankind harness and channel vast amounts of relevant sophisticated data that is produced and disseminated from a multiplicity of research groups worldwide into meaningful streamlined forums that can be shared and understood by all in a timely fashion . exploiting advances in information technology will assist greatly with this challenge and will drive effective risk - based assessment , evaluation , and management of present and emerging problems for the aquatic environment . currently , there is insufficient information available to reach definitive conclusion on the significance and impact of the vbnc state organisms and antibiotic resistant bacteria in the environment which would allow for the assessment of the potential risk related , for instance , to human health and ecosystem functions . indeed impact of antibiotics present in the aquatic environment on the frequency of resistance transfer is questionable , with greater concern placed on the input of resistant bacteria into the environment from different sources . this suggests that greater emphasis should be placed on reducing the likelihood of antibiotic resistance occurrence in the first instance at the point of source by prudent management and careful rotation of antibiotic usage in both the hospital and community settings and by systematic continued monitoring of resistance , which will collectively impact positively on public health and wellbeing . the lack of understanding and knowledge surrounding a broad range of established and emerging risks to the aquatic environment is apparent . for example , mena and gerba recently reviewed best published data for risk assessing the opportunist pathogen p. aeruginosa in water and concluded that process of estimating risk is currently significantly constrained because of the absence of specific ( quantitative ) occurrence data for pseudomonads . in order to gain a greater appreciation and understanding of the critical environmental fate and associated impact of such undefined and variable microbial risks , more controlled laboratory investigations are needed to be undertaken in combination with conducting field studies . there is also pressing need to obtain definitive data on proposed risk to public health and to the aquatic environment combined with developing appropriate short- and long - term mathematical and computer models with capacity for monitoring and predicting ecotoxicological effects of these microbial stressors , particularly under dynamic naturalistic settings akin to those recently described by bontje and coworkers and gevaert et al . it is quite apparent that proper judgement of the impact of microbial pathogens , their metabolites , and pharmaceutically active compounds require a thorough evaluation of their risk and hazard . regarding the latter , where risk is normally expressed as the ratio between the predicted environmental concentration of the active ingredient ( ai ) and its predicted no - effect concentration . hazard is expressed in terms of ai 's persistence , potential for bioaccumulation , and ecotoxicity . the combined use of monitoring data aligned with development and application of dynamic pollutant ( contaminant ) fate models is recommended . pharmaceutical producers should also highlight environmental precaution when designing new more environmental friendly ais , and that the environmental data should be transparent to the general public . in terms of holistic monitoring and prediction , we must also factor in seasonal environmental changes such as atmospheric and oceanic processes that occur in response to increasing greenhouse gases to map disease potentiation dynamics as this will aid development of appropriate strategies for controlling microbial risks across a range of human and natural systems . indeed , sedas recently showed that climate also influences the abundance and ecology of pathogens , and the links between pathogens and changing ocean conditions , including human disease such as cholera . it is recognised that environmental risk - based management is typically uncertain due to different perceptions of the risk problem and our limited knowledge about the inter - play of biological , physical and chemical processes underlying these risks . while a plethora of real - time quantitative microbial detection tools combined with more rapid and efficient decontamination approaches are on the horizon , we must be equally prudent about exhaustively confirming their efficacy such as agreed standards for sensitivity and reliability for detection , and eco - friendliness for decontamination . it is clear and apparent that the emergence of such complex problems has heralded a new dawn in research and innovation ( i.e. , a marked departure from the solitary phd student environment ) , where collection , analysis and timely dissemination of such vast and meaningful information can only be effectively addressed using a well - managed consortia of networked researchers from various complementary disciplines by way of using a plethora of appropriate frontier funding initiatives such those offered by the eu ( http://cordis.europa.eu/fp7/home.html ) . while it is recognised that undertaking such far - reaching cross - boundary initiatives will enable the potential impact , implications and future proofing of established and emerging risks to be managed and catered for properly . is must be equally recognised that effectively managing and harnessing the potential of such diverse consortia comprising academics , industrial partners ( smes to multinationals ) , policy makers and so forth pose significant logistic and complex challenges , for example , attempting to holistically cater for all stakeholders in a united global society on a single theme who have different needs and goals are real challenges . therefore , such important issues must also be managed with a strong over - arching foresight , particularly in the context of embracing and exploiting advances in the communication and information technology landscape so that we can accommodate and provide for the real - time flow of knowledge to all stakeholders in order to identify potential synergies , emerging trends ( problematic , beneficial or otherwise ) , and/or opportunities .

this timely review primarily addresses important but presently undefined microbial risks to public health and to the natural environment . it specifically focuses on current knowledge , future outlooks and offers some potential alleviation strategies that may reduce or eliminate the risk of problematic microbes in their viable but nonculturable ( vbnc ) state and cryptosporidium oocysts in the aquatic environment . as emphasis is placed on water quality , particularly surrounding efficacy of decontamination at the wastewater treatment plant level , this review also touches upon other related emerging issues , namely , the fate and potential ecotoxicological impact of untreated antibiotics and other pharmaceutically active compounds in water . deciphering best published data has elucidated gaps between science and policy that will help stakeholders work towards the european union 's water framework directive ( 2000/60/ec ) , which provides an ambitious legislative framework for water quality improvements within its region and seeks to restore all water bodies to good ecological status by 2015 . future effective risk - based assessment and management , post definition of the plethora of dynamic inter - related factors governing the occurrence , persistence and/or control of these presently undefined hazards in water will also demand exploiting and harnessing tangential advances in allied disciplines such as mathematical and computer modeling that will permit efficient data generation and transparent reporting to be undertaken by well - balanced consortia of stakeholders .

1. Viable But Nonculturable Forms of Waterborne Bacteria 2. The Waterborne Enteroparasite: Cryptosporidium 3. Antibiotics in the Aquatic Environments 4. Conclusion and Outlooks

since the introduction of the concept or sublethally injured or viable but nonculturable ( vbnc ) cells by byrd and colwell in the 1980 's , there is increasing evidence for the existence of such a state in microbes , particularly in the aquatic environment that elicits a myriad of interrelated sub - lethal microbial stresses such as nutrient starvation and osmotic stress [ 2 , 3 ] ( table 1 ) . albeit currently unknown in terms of its severity or scope , it is now generally appreciated that heavily stressed pathogenic microbial species existing in a vbnc ( or not immediately culturable state ) may potentially pose as yet an undefined risk to public health , which is attested by the fact that there is increasing evidence to support the viewpoint that stressed cells in this quiescent state may actually be more virulent than well - fed laboratory - tamed microorganisms due to augmented virulence factor expression . as microorganisms are extremely diverse and dynamic , it is not surprising that the many different types of microbial species present in the water environment exist in a number of physiological states that possess different requirements for survival and to sustain growth . they concluded that h. pylori could survive disinfection practices normally used in drinking water treatment in the vbnc form , which would allow them to reach final consumption points and , at the same time , enable them to be undetectable by culture methods . treated with novel pulsed - power disinfection technologies ( such as use of pulsed - plasma gas - discharge technology that will be expanded on later in this paper ) were capable of reducing the redox dye 5-cyano-2 , 3-ditolyl tetrazolium chloride ( ctc ) that is an indicator of electron acceptor function , yet similarly treated samples were unable to form colonies on a variety of laboratory - based culture media [ 6 , 7 ] . this corroborates more recent research undertaken by sawaya and coworkers who used a combined 4'6-diamidion-2-phenylindole ( dapi ) and ctc stains to highlight the occurrence of physiologically active bacteria in river and wastewater treatment plants that were much higher than those obtained by plate counting . as numerous researchers continue to report on the use of redox stains for highlighting differences in agar - plate counts , it is important that we holistically explore and identify specific microbial cues at the cellular level which govern the transition to the vbnc state along with exploiting commensurate advances in media formulations that are tailored for optimal resuscitation of these sublethally stressed cells ( cited in [ 10 , 24 ] ) . , who previously postulated that self - destruction or cell suicide may be attributed to sublethally stressed or damage microbes being incapable of coping with oxidative burst when rapidly growing on nutrient rich media , it is important that we also exploit advances in toxicology ( such as methods exploring cellular apoptosis and necrosis , cell membrane lipid peroxidation assays , and nuclear chromatin / comet assays ) that will provide valuable insights into the possible role of intracellular free radicals in combination with the direct physical action of the applied environmental stress on the generation and persistence of vbnc organisms in water . the latter authors articulated that the number of e. coli in freshwater is systematically underestimated by traditional culture - based methods such as multiple tube fermentation and or membrane filtration techniques ) , which is cause for concern from a public health perspective . such nonculturable organisms may only be detected by use of such molecular techniques based on probes such as 16s and 23s rrnas or on determination of mrna , either by quantitative real - time pcr and/or by fluorescent techniques such as in situ hybridization ( fish ) , microradiography , epifluorescence microscopy , and flow cytometry [ 2 , 11 , 12 ] . while fiksdal and tryland advocated use of rapid enzyme assays for monitoring of water quality , which may also detect organisms in the injured or vbnc state . pure culture studies of e. coli have also shown that after exposure to other types of extrinsic stress such as chlorination , the galase activity is less reduced than the direct viable count . the latter advances the early landmark study of bjergbaek and roslev who reported on the occurrence and persistence of vbnc e. coli in nondisinfected drinking water using different cultivation dependent methods , fluorescence in situ hybridization ( fish ) using specific olignucleotide probes , direct viable counts ( dvc ) , and by enumeration of gfp - tagged e. coli ( green fluorescent protein , gfp ) . other likely candidate methods for efficient and rapid - detection of vbnc bacteria in the aquatic and soil environments include rna - based genotypic approaches . this also reinforces common viewpoint shared by many scientists that a variety of problematic bacteria can enter vbnc states as a survival response when exposed to deleterious environmental stresses such as nutrient starvation , osmotic stress , and so forth . however , in order to gain a greater appreciation of environmental and public health risks associated with persistence of microbial pathogens in different culturable states , it is imperative that we acquire an understanding of the interrelated molecular responses governing tolerance to these applied sub - lethal stressors . therefore , in terms of enabling effective risk - based assessment and environmental management to address the occurrence and persistence of vbnc pathogens in water , critical data must be acquired to convert vbnc - phase potential pathogens from unknown to known and defined hazards . in terms of satisfying these information gaps , accurate enumeration of total microbial load and real time identification to the various types present in water are pivotal to hazard identification and characterization , which will contribute significantly to assuring water safety . therefore , underestimation in the levels of problematic microbes in water , and failure to identify the presence of pathogenic organisms in representative water samples also pose significant threats to public health . greater studies are also required to investigate the presence and role of this vbnc state in microbial pathogens that cause disease in the aquatic natural setting such as in fish.taking for example , flavobacterium psychrophilum , the causative agent of rainbow trout fly syndrome and cold water disease in salmonids , where subpopulations were still culturable after starvation for 300 days in sterilised fresh water . thus , highlighting the importance of augmenting water quality through improvements in wastewater treatment , which should be augmented in efficiency and capacity to cater for under - appreciated and for emerging microbial threats . the aforementioned studies should also be carried out in parallel with developing improved protocols for resuscitating vbnc organisms , particularly availing of advances made in fermentation technology such as exploiting chemostat - based bioreactor approaches that can simulate and monitor multiple environmental stresses ( either applied simultaneously or sequentially ) over extended time periods . other related studies that merit attention include the development of more rapid , user - friendly , field - based technologies that will reliably and repeatably detect and quantify various waterborne pathogens that may persist in different culture states . the latter will greatly facilitate our endeavours to collectively comply with emerging environmental policies such as eu 's water framework directive . the occurrence of the environmentally resistant thick - walled oocyst stage of this organism has become a worldwide concern due to its resistance to disinfection with chlorine at concentrations typically applied in drinking water treatment plants ( 2 to 6 thus , the control of cryptosporidium oocysts remains a major challenge for drinking water utilities due to fact that the common chemical disinfectants - free and combined chlorine , when used singly , are practically ineffective for inactivating this protozoan under conditions encountered in most treatment facilities . this situation has become a major concern for water authorities and consequently , significant modifications to drinking water regulations have been proposed for the detection and surveillance of this protozoan in the us and in other developed countries . development of alternative methods of cryptosporidium disinfection for water applications ( such as ozone and/or uv ) has been hindered by the uncertainty surrounding efficacy of using in vitro surrogate viability assays due to their overestimation of oocysts survivors posttreatments and the lack of critical data on the preferred use of in vitro cell culture and/or in vivo animal - based infectivity assays to determine interrelated factors governing repeatable disinfection of oocysts suspended in water . however , the authors recommended that pressing investigations are needed to comprehensively demonstrate efficacy of using this novel disinfection uv approach for treating lower concentrations of infectious oocysts under dynamic conditions found in wwtps and in the aquatic environment . moreover , numerous studies have also highlighted limitations of decontamination techniques such as conventional low pressure mercury lamps designed to produce energy at 254 nm ( called monochromatic or germicidal light ) that include microbial repair and the necessity for lengthy durations of exposure to obtain suitable levels of decontamination . some landmark studies have recently reported on the possible efficacy of using ozone for destroying cryptosporidium oocysts . however , there are very limited published findings to date that holistically investigate critical factors governing the effective and repeatable destruction of this recalcitrant enteroparasite in drinking water supplies using ozone or other oxidative agents . pulsed plasma gas - discharge ( ppgd ) technology involves applying high voltage pulses to gas - injected test liquids resulting in the formation of a plasma that causes free radicals such as dissolved ozone and hydrogen peroxide , free electrons , ultraviolet light ( uv ) , acoustic shock waves , and electric fields at levels between 1050 kv / cm to be generated in the test liquids ( figure 1 ) . , this author has been unable to source any published reports on possible toxicological issues associated with use of these new technologies for disinfection applications in healthcare , agri - food or the environment . although antibiotics have been used for decades , only recently has an increasing number of studies highlighted the lack of understanding and knowledge about persistence of antibiotics in the aquatic environment and the potential environmental risks that this may pose . particularly as antibiotics are often poorly degraded or removed in conventional wastewater treatment plants ( wwtps ) which causes formation of toxic degradation products that may impact negatively on the aquatic environment and public health . the reader is also directed to the recently published landmark reviews of kmmerer [ 83 , 84 ] for further detailed information on the possible input , occurrence , fate , and effects of antibiotics in the aquatic environment . some studies revealed that many different types of antibiotics are biodegradable under aerobic or anaerobic conditions , while other related research has reported that certain aquatic microbes can actually use these free active compounds as a sole carbon source . kmmerer clearly articulated that the issue of acquired resistance was nearly always addressed in publications by describing the presence of antibiotics in the environment , with often mere speculation as to the possible relationship between low concentrations of antibiotics in water and emergence of resistance . from a review of best published literature it would appear that possibly the most significant undefined risk in terms of antibiotics are microorganisms harbouring multiple antibiotic resistance genes such as vancomycin - resistant enterococci ( vre ) , methicillin - resistant staphylococcus aureus ( mrsa ) , and multiresistant pseudomonads that are living in close proximity to each other ( such as in biofilms sewage sludge flocs ) as opposed to the free active compounds present at low concentrations in the aquatic environment . thus , not alone may the latter confer microbial tolerance to the applied stress through a process of elevating activities in important genes that regulate key house - keeping functions such as osmotic stress response and protection vital proteins ( chaperone ) , but such environmental tempering may also act as stimuli for up - regulating microbial virulence factor expression . , body temperature ) , yet down - regulates transcription of pathogenic determinants in the presence of soil - related carbohydrates . kmmerer stated that there is a dearth of information on the interrelated factors governing the fate and effects of antibiotics in the environment ( i.e. he also recently postulated that the effects of antibiotics in wwtps or in the aquatic environment may be under - estimated , particularly as there is evidence that microbial exposure to antibiotics from the same group or from different groups may result in additive effects . additionally , there is no published information as yet on the potential impact of low levels of other pharmaceutically active compounds ( such as endocrine disrupting chemicals ) on development of antibiotic resistance as the former also tolerate sewage treatment . the question should also be posed as to whether or not multi - drug resistant bacteria that transcend to the quiescent vbnc state after periods of extended exposure to a nutrient depleted stressful environment ( such as in water or in soil ) are still capable of gene transfer and whether or not these important molecular determinants remain unaltered and stable . while most antibiotics tested to date have not been biodegradable under aerobic conditions(kmmerer [ 83 , 94 , 95]),biodegradability has been poor for most of the compounds investigated in laboratory tests , including some to the -lactams . a marked observation from this paper is the pressing need for greater risk - based assessment and for new and/or improved alleviation strategies for the optimal decontamination of wastewater at treatment plant level so as to reduce or eliminate the microbial ( and their associated metabolite ) risks to public health . in the context of improving wastewater treatment and planning for safe drinking supplies , one must also acknowledge growing international concerns about the release of certain chemicals into the aquatic environment that may result in alterations in the reproductive health of humans and wildlife [ 108110 ] . thyroid system - disrupting activity in water from municipal domestic sewage treatment plants was also detected recently .the list of known edcs is extensive and includes natural and synthetic steroid hormones ( such as 17 -estradiol(e2),estrone ( e1 ) , and 17 -ethinylestradiol ( ee2 ) ) , phytoextrogens , pesticides , pharmaceuticals , and surfactants , all of which have been detected worldwide in processed water from domestic treatment plants ( wwtps ) at the ng / l level that may cause abnormalities to aquatic organisms [ 113 , 114 ] . , compounds interacting with the human estrogen receptor ) , which enter the environment from a variety of sources including effluent discharge pipes , agricultural runoff and landfills . in particular , such edcs are considered to be dominant contributors to estrogenic activity in treated water from wwtps and have been found in treated effluent at the ng / l level [ 113 , 118 ] . whilst intensive conventional - based treatment approaches have investigated many interrelated factors in the design and operating conditions of wwtps for reduction or elimination of edcs in treated wastewater , none have reported on their effective removal . the european commission ( com 706 ) acknowledged that there is an urgent need for further scientific research to denature deconjugated edcs in the environment . previous researchers have recently shown that use of similar non - thermal corona discharge processes such as the ppgd technology may be potentially effective at destroying structurally - related organic compounds such as dyes , phenol and aniline in aqueous solutions [ 121125 ] . while other researchers have demonstrated the production of short - lived , high - oxidative , plasmochemical elements in pulsed - plasma - treated test liquids such as water and effluent for bacterial decontamination , no study to date has reported on the use of corona discharges singly or combined with pulsed uv light as a novel approach for the destruction of established and emerging microbial threats to public health in water or effluents . there is a pressing need to generate critical data in gaps highlighted above via provision of funding to establish consortia of meaningful stakeholders comprising scientists , engineers , and end - users in order to facilitate policy makers and implementers ( suchaswater managers ) strive towards meeting ambitious objectives set for the eu 's water framework directive that requires member states to attain a good ecological status for all water bodies by 2015 . while a global response to emerging environmental problems has been positive with the provision of substantial research funding been made available to the broad and diverse researcher communities ( such as eu fp7 initiatives ) , one obvious real challenge that still remains is how does mankind harness and channel vast amounts of relevant sophisticated data that is produced and disseminated from a multiplicity of research groups worldwide into meaningful streamlined forums that can be shared and understood by all in a timely fashion . exploiting advances in information technology will assist greatly with this challenge and will drive effective risk - based assessment , evaluation , and management of present and emerging problems for the aquatic environment . currently , there is insufficient information available to reach definitive conclusion on the significance and impact of the vbnc state organisms and antibiotic resistant bacteria in the environment which would allow for the assessment of the potential risk related , for instance , to human health and ecosystem functions . indeed impact of antibiotics present in the aquatic environment on the frequency of resistance transfer is questionable , with greater concern placed on the input of resistant bacteria into the environment from different sources . this suggests that greater emphasis should be placed on reducing the likelihood of antibiotic resistance occurrence in the first instance at the point of source by prudent management and careful rotation of antibiotic usage in both the hospital and community settings and by systematic continued monitoring of resistance , which will collectively impact positively on public health and wellbeing . the lack of understanding and knowledge surrounding a broad range of established and emerging risks to the aquatic environment is apparent . for example , mena and gerba recently reviewed best published data for risk assessing the opportunist pathogen p. aeruginosa in water and concluded that process of estimating risk is currently significantly constrained because of the absence of specific ( quantitative ) occurrence data for pseudomonads . in order to gain a greater appreciation and understanding of the critical environmental fate and associated impact of such undefined and variable microbial risks , more controlled laboratory investigations are needed to be undertaken in combination with conducting field studies . there is also pressing need to obtain definitive data on proposed risk to public health and to the aquatic environment combined with developing appropriate short- and long - term mathematical and computer models with capacity for monitoring and predicting ecotoxicological effects of these microbial stressors , particularly under dynamic naturalistic settings akin to those recently described by bontje and coworkers and gevaert et al . it is quite apparent that proper judgement of the impact of microbial pathogens , their metabolites , and pharmaceutically active compounds require a thorough evaluation of their risk and hazard . it is recognised that environmental risk - based management is typically uncertain due to different perceptions of the risk problem and our limited knowledge about the inter - play of biological , physical and chemical processes underlying these risks . , a marked departure from the solitary phd student environment ) , where collection , analysis and timely dissemination of such vast and meaningful information can only be effectively addressed using a well - managed consortia of networked researchers from various complementary disciplines by way of using a plethora of appropriate frontier funding initiatives such those offered by the eu ( http://cordis.europa.eu/fp7/home.html ) . while it is recognised that undertaking such far - reaching cross - boundary initiatives will enable the potential impact , implications and future proofing of established and emerging risks to be managed and catered for properly . is must be equally recognised that effectively managing and harnessing the potential of such diverse consortia comprising academics , industrial partners ( smes to multinationals ) , policy makers and so forth pose significant logistic and complex challenges , for example , attempting to holistically cater for all stakeholders in a united global society on a single theme who have different needs and goals are real challenges . therefore , such important issues must also be managed with a strong over - arching foresight , particularly in the context of embracing and exploiting advances in the communication and information technology landscape so that we can accommodate and provide for the real - time flow of knowledge to all stakeholders in order to identify potential synergies , emerging trends ( problematic , beneficial or otherwise ) , and/or opportunities .

since the introduction of the concept or sublethally injured or viable but nonculturable ( vbnc ) cells by byrd and colwell in the 1980 's , there is increasing evidence for the existence of such a state in microbes , particularly in the aquatic environment that elicits a myriad of interrelated sub - lethal microbial stresses such as nutrient starvation and osmotic stress [ 2 , 3 ] ( table 1 ) . albeit currently unknown in terms of its severity or scope , it is now generally appreciated that heavily stressed pathogenic microbial species existing in a vbnc ( or not immediately culturable state ) may potentially pose as yet an undefined risk to public health , which is attested by the fact that there is increasing evidence to support the viewpoint that stressed cells in this quiescent state may actually be more virulent than well - fed laboratory - tamed microorganisms due to augmented virulence factor expression . were the first to bring experimental evidence of the existence of vbnc state in pathogenic bacteria , where they showed that e. coli and v. cholera cells that were suspended in artificial seawater quickly lost their ability to grow on the culture media normally used for their detection . treated with novel pulsed - power disinfection technologies ( such as use of pulsed - plasma gas - discharge technology that will be expanded on later in this paper ) were capable of reducing the redox dye 5-cyano-2 , 3-ditolyl tetrazolium chloride ( ctc ) that is an indicator of electron acceptor function , yet similarly treated samples were unable to form colonies on a variety of laboratory - based culture media [ 6 , 7 ] . as numerous researchers continue to report on the use of redox stains for highlighting differences in agar - plate counts , it is important that we holistically explore and identify specific microbial cues at the cellular level which govern the transition to the vbnc state along with exploiting commensurate advances in media formulations that are tailored for optimal resuscitation of these sublethally stressed cells ( cited in [ 10 , 24 ] ) . the latter authors showed that the addition of a commercially available antioxidant oxyrase and a heat - stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine , resuscitated e. coli , and salmonella enteric serovar typhimurium that were stressed by prolonged incubation in water microcosms . , who previously postulated that self - destruction or cell suicide may be attributed to sublethally stressed or damage microbes being incapable of coping with oxidative burst when rapidly growing on nutrient rich media , it is important that we also exploit advances in toxicology ( such as methods exploring cellular apoptosis and necrosis , cell membrane lipid peroxidation assays , and nuclear chromatin / comet assays ) that will provide valuable insights into the possible role of intracellular free radicals in combination with the direct physical action of the applied environmental stress on the generation and persistence of vbnc organisms in water . among the minority of bacteria that have been discovered it is estimated that more than 90% are as yet nonculturable as attested by the fact that international committee on systematic bacteriology has recognised a new category for nonculturable bacteria that it named candidatus for which phylogenetic relatedness has been determined by amplification and sequence analysis of prokaryotic rna genes with universal prokaryotic primers and authenticity has been verified by use of in situ probes . such nonculturable organisms may only be detected by use of such molecular techniques based on probes such as 16s and 23s rrnas or on determination of mrna , either by quantitative real - time pcr and/or by fluorescent techniques such as in situ hybridization ( fish ) , microradiography , epifluorescence microscopy , and flow cytometry [ 2 , 11 , 12 ] . garcia - armisen and servais showed that the ratio of direct viable- ( dvc- ) fish count and the culturable count increased with decreasing abundance of culturable e. coli in river water , and therefore the slope of the linear - log - log correlation of dvc - fish versus colony forming unit numbers was less than one . as mentioned previously by many research groups [ 13 , 2933 ] , all field trials plotting log -d - galactosidase ( galase ) activity or log -d - glucuronidase ( gluase ) activity versus log culturable target bacteria in fresh or marine water have shown regression straight lines with a slope less than one , suggesting that enzyme activity calculated per culturable indicator bacteria increases when their numbers decrease ( e.g. the latter advances the early landmark study of bjergbaek and roslev who reported on the occurrence and persistence of vbnc e. coli in nondisinfected drinking water using different cultivation dependent methods , fluorescence in situ hybridization ( fish ) using specific olignucleotide probes , direct viable counts ( dvc ) , and by enumeration of gfp - tagged e. coli ( green fluorescent protein , gfp ) . okabe and shimazu also describe detection of host - specific bacteroides - prevotella 16s rrna genetic markers ( total , human , cow- and pig - specific ) as promising alternative indicators for identifying the sources of fecal pollution in environmental water because of their abundance in the feces of warm - blooded animals . the authors clearly state that detection of aforementioned genetic markers mainly reflected the presence of vbnc bacteroides - prevotella cells in water , suggesting that seasonal and geographical variations in persistence of these host - specific bacteroiides - prevotella 16s rrna genetic markers must be considered if used as alternative fecal indicators in environmental waters . however , in order to gain a greater appreciation of environmental and public health risks associated with persistence of microbial pathogens in different culturable states , it is imperative that we acquire an understanding of the interrelated molecular responses governing tolerance to these applied sub - lethal stressors . therefore , in terms of enabling effective risk - based assessment and environmental management to address the occurrence and persistence of vbnc pathogens in water , critical data must be acquired to convert vbnc - phase potential pathogens from unknown to known and defined hazards . moreover , there is a dearth of knowledge regarding specific underlying molecular and associated cellular mechanisms governing transition and persistence of waterborne microorganisms in this vbnc state , in addition to obviously establishing what specific environmental conditions or triggers cause these changes in culturable state . greater studies are also required to investigate the presence and role of this vbnc state in microbial pathogens that cause disease in the aquatic natural setting such as in fish.taking for example , flavobacterium psychrophilum , the causative agent of rainbow trout fly syndrome and cold water disease in salmonids , where subpopulations were still culturable after starvation for 300 days in sterilised fresh water . the aforementioned studies should also be carried out in parallel with developing improved protocols for resuscitating vbnc organisms , particularly availing of advances made in fermentation technology such as exploiting chemostat - based bioreactor approaches that can simulate and monitor multiple environmental stresses ( either applied simultaneously or sequentially ) over extended time periods . due to plethora of different detection methods currently being developed combined with obvious interlaboratory variability in terms of associated operating protocols , it is imperative that we develop and agree upon a standardized battery of reliable tools for detecting and quantifying culturable and nonculturable bacteria so as to establish future unified , quantitative - risk management protocols for monitoring our aquatic environments . the occurrence of the environmentally resistant thick - walled oocyst stage of this organism has become a worldwide concern due to its resistance to disinfection with chlorine at concentrations typically applied in drinking water treatment plants ( 2 to 6 thus , the control of cryptosporidium oocysts remains a major challenge for drinking water utilities due to fact that the common chemical disinfectants - free and combined chlorine , when used singly , are practically ineffective for inactivating this protozoan under conditions encountered in most treatment facilities . however problems associated with the determination of oocysts viability / infectivity make the establishment of maximum acceptable concentrations very difficult , and concentrations of 330 oocysts/100 litres in treated water have been proposed as action levels . recent contamination of drinking water supplies in the west of ireland have led to a significant number of confirmed cases of cryptosporidiosis , intimating that the use of conventional decontamination methods failed to eliminate this enteroparasite in treated water ( cited in garvey et al . ) development of alternative methods of cryptosporidium disinfection for water applications ( such as ozone and/or uv ) has been hindered by the uncertainty surrounding efficacy of using in vitro surrogate viability assays due to their overestimation of oocysts survivors posttreatments and the lack of critical data on the preferred use of in vitro cell culture and/or in vivo animal - based infectivity assays to determine interrelated factors governing repeatable disinfection of oocysts suspended in water . although recent studies that utilised at least 20 different cell lines have advocated the preferential use of the human ileocecal adenocarcinoma hct-8 cell line as an equivalent in vitro method to that of using the gold standard mouse assay for measuring infectivity of cryptosporidium , there has been limited evidence to date on the combined use of these approaches for assessing critical operational parameters governing pulsed uv light ( puv ) as a means of disinfecting water contaminated with this enteroparasite . this study demonstrated that there is good agreement between use of in vitro culture - qpcr and the scid - mouse - infectivity assays for evaluating the disinfection efficacy of pulsed uv for inactivating c. parvum suspended in saline , thus reducing the requirement to unnecessarily use animals for these particular research studies . in terms of potential future alleviation strategies for combating c. parvum in water , it is likely the combined use mp and puv technologies would offer considerable advantages as the next - generation decontamination bolt - on approach including rapid processing and efficiency of oocyst destruction , and should accommodate dynamic operational requirements at wwtp level . pulsed plasma gas - discharge ( ppgd ) technology involves applying high voltage pulses to gas - injected test liquids resulting in the formation of a plasma that causes free radicals such as dissolved ozone and hydrogen peroxide , free electrons , ultraviolet light ( uv ) , acoustic shock waves , and electric fields at levels between 1050 kv / cm to be generated in the test liquids ( figure 1 ) . concern over the development of secondary resistance to commonly - used antibiotics ( either through vertical and/or horizontal gene transfer among related and unrelated bacteria ) in the environment with potential for adversely affecting aquatic and terrestrial organisms along with the potential for a cyclic unwanted return to humans again via consumption of drinking water has stimulated numerous research groups to investigate these important issues . from a review of best published literature it would appear that possibly the most significant undefined risk in terms of antibiotics are microorganisms harbouring multiple antibiotic resistance genes such as vancomycin - resistant enterococci ( vre ) , methicillin - resistant staphylococcus aureus ( mrsa ) , and multiresistant pseudomonads that are living in close proximity to each other ( such as in biofilms sewage sludge flocs ) as opposed to the free active compounds present at low concentrations in the aquatic environment . the latter is strongly aligned with findings from unrelated environmental stress studies which previously showed that many food and waterborne microbial species can sense and adapt to related and unrelated sub - lethal environmental stresses ( such as osmotic stress or starvation ) through complex quorum sensing and respond through an orchestrated controlled subsequence of preferential gene expression . thus , not alone may the latter confer microbial tolerance to the applied stress through a process of elevating activities in important genes that regulate key house - keeping functions such as osmotic stress response and protection vital proteins ( chaperone ) , but such environmental tempering may also act as stimuli for up - regulating microbial virulence factor expression . interestingly , zupan and raspor recently reported on the development of an invasion - agar assay for determining in vitro invasiveness of saccharomyces cerevisiae , and showed that this yeast augmented virulence when exposed to temperatures typical of human fever ( 37 to 39c ) yet exhibited strong repression effect on invasion in the presence of salts , anoxia and some preservatives . the question should also be posed as to whether or not multi - drug resistant bacteria that transcend to the quiescent vbnc state after periods of extended exposure to a nutrient depleted stressful environment ( such as in water or in soil ) are still capable of gene transfer and whether or not these important molecular determinants remain unaltered and stable . in fish farming sector such as aquaculture and mariculture , the widespread use of antibiotics for treating bacterial diseases has been associated with the development of resistance in a range of bacterial pathogens including aeromonas hydrophilia , aeromonas salmonicida , edwardsiella tarda , edwardsiella icttaluri , vibrio anguillarum , vibrio salmonicida , pasteurella piscida and yersinia ruckeri . a marked observation from this paper is the pressing need for greater risk - based assessment and for new and/or improved alleviation strategies for the optimal decontamination of wastewater at treatment plant level so as to reduce or eliminate the microbial ( and their associated metabolite ) risks to public health . in the context of improving wastewater treatment and planning for safe drinking supplies , one must also acknowledge growing international concerns about the release of certain chemicals into the aquatic environment that may result in alterations in the reproductive health of humans and wildlife [ 108110 ] . thyroid system - disrupting activity in water from municipal domestic sewage treatment plants was also detected recently .the list of known edcs is extensive and includes natural and synthetic steroid hormones ( such as 17 -estradiol(e2),estrone ( e1 ) , and 17 -ethinylestradiol ( ee2 ) ) , phytoextrogens , pesticides , pharmaceuticals , and surfactants , all of which have been detected worldwide in processed water from domestic treatment plants ( wwtps ) at the ng / l level that may cause abnormalities to aquatic organisms [ 113 , 114 ] . recent studies conducted by fogarty and mcgee have demonstrated that fish habituating downstream of waste - water treatment plants ( wwtps ) in the midlands region in ireland exhibited delayed spermatogenesis compared with fish upstream and intersex ( feminization ) was discovered in roach [ 115117 ] . whilst intensive conventional - based treatment approaches have investigated many interrelated factors in the design and operating conditions of wwtps for reduction or elimination of edcs in treated wastewater , none have reported on their effective removal . on a similar theme , yamamoto and coworkers recently reported the persistence of 8 pharmaceuticals with relatively high ecological risk and high consumption ( namely acetaminophen , atenolol , carbamazepine , ibuprofen , ifenprodil , indomethacin , metenamicacid , and propranolol ) using river water . previous researchers have recently shown that use of similar non - thermal corona discharge processes such as the ppgd technology may be potentially effective at destroying structurally - related organic compounds such as dyes , phenol and aniline in aqueous solutions [ 121125 ] . while other researchers have demonstrated the production of short - lived , high - oxidative , plasmochemical elements in pulsed - plasma - treated test liquids such as water and effluent for bacterial decontamination , no study to date has reported on the use of corona discharges singly or combined with pulsed uv light as a novel approach for the destruction of established and emerging microbial threats to public health in water or effluents . there is a pressing need to generate critical data in gaps highlighted above via provision of funding to establish consortia of meaningful stakeholders comprising scientists , engineers , and end - users in order to facilitate policy makers and implementers ( suchaswater managers ) strive towards meeting ambitious objectives set for the eu 's water framework directive that requires member states to attain a good ecological status for all water bodies by 2015 . while a global response to emerging environmental problems has been positive with the provision of substantial research funding been made available to the broad and diverse researcher communities ( such as eu fp7 initiatives ) , one obvious real challenge that still remains is how does mankind harness and channel vast amounts of relevant sophisticated data that is produced and disseminated from a multiplicity of research groups worldwide into meaningful streamlined forums that can be shared and understood by all in a timely fashion . currently , there is insufficient information available to reach definitive conclusion on the significance and impact of the vbnc state organisms and antibiotic resistant bacteria in the environment which would allow for the assessment of the potential risk related , for instance , to human health and ecosystem functions . this suggests that greater emphasis should be placed on reducing the likelihood of antibiotic resistance occurrence in the first instance at the point of source by prudent management and careful rotation of antibiotic usage in both the hospital and community settings and by systematic continued monitoring of resistance , which will collectively impact positively on public health and wellbeing . for example , mena and gerba recently reviewed best published data for risk assessing the opportunist pathogen p. aeruginosa in water and concluded that process of estimating risk is currently significantly constrained because of the absence of specific ( quantitative ) occurrence data for pseudomonads . in order to gain a greater appreciation and understanding of the critical environmental fate and associated impact of such undefined and variable microbial risks , more controlled laboratory investigations are needed to be undertaken in combination with conducting field studies . there is also pressing need to obtain definitive data on proposed risk to public health and to the aquatic environment combined with developing appropriate short- and long - term mathematical and computer models with capacity for monitoring and predicting ecotoxicological effects of these microbial stressors , particularly under dynamic naturalistic settings akin to those recently described by bontje and coworkers and gevaert et al . in terms of holistic monitoring and prediction , we must also factor in seasonal environmental changes such as atmospheric and oceanic processes that occur in response to increasing greenhouse gases to map disease potentiation dynamics as this will aid development of appropriate strategies for controlling microbial risks across a range of human and natural systems . while a plethora of real - time quantitative microbial detection tools combined with more rapid and efficient decontamination approaches are on the horizon , we must be equally prudent about exhaustively confirming their efficacy such as agreed standards for sensitivity and reliability for detection , and eco - friendliness for decontamination . , a marked departure from the solitary phd student environment ) , where collection , analysis and timely dissemination of such vast and meaningful information can only be effectively addressed using a well - managed consortia of networked researchers from various complementary disciplines by way of using a plethora of appropriate frontier funding initiatives such those offered by the eu ( http://cordis.europa.eu/fp7/home.html ) . is must be equally recognised that effectively managing and harnessing the potential of such diverse consortia comprising academics , industrial partners ( smes to multinationals ) , policy makers and so forth pose significant logistic and complex challenges , for example , attempting to holistically cater for all stakeholders in a united global society on a single theme who have different needs and goals are real challenges . therefore , such important issues must also be managed with a strong over - arching foresight , particularly in the context of embracing and exploiting advances in the communication and information technology landscape so that we can accommodate and provide for the real - time flow of knowledge to all stakeholders in order to identify potential synergies , emerging trends ( problematic , beneficial or otherwise ) , and/or opportunities .

to develop methods for mapping a qtl to a phylogenetic tree , we begin with several simplifying assumptions : the taxa are represented by inbred lines , the tree relating the taxa is known without error , the quantitative trait of interest is affected by a single diallelic qtl , and there are no background effects ( i.e. , the effect of the qtl is the same in the different crosses in which it is segregating ) . we consider the case of intercrosses among pairs of taxa , consider only autosomal loci , and assume a common genetic map . the basic idea , illustrated in figure 1 , is that each possible location for the origin of a diallelic qtl on the tree corresponds to a different partition of the taxa into two groups , with the two groups corresponding to the two qtl alleles . for different partitions , the qtl will segregate in different sets of crosses . in the case of very large crosses , with each having high power to detect the qtl , if present , we could simply consider the crosses individually and use the pattern of presence / absence of qtl to identify the correct partition of the taxa . note that one does not need data on all possible crosses . for the case illustrated in figure 1 , with four taxa , it would be sufficient to consider the crosses a b , a c , and b d , as with just these three crosses , the five possible partitions have distinct patterns of presence / absence of the qtl . in the following , we focus on partitions of the taxa into two groups , in place of locations of the qtl on the tree . illustration of the basic concepts behind the mapping of a qtl to a phylogenetic tree . on the left the locations of possible origins of a diallelic qtl are indicated by the numbers 15 . in the table on the right , we indicate the presence or absence of a qtl in each of the six possible crosses among pairs of taxa , according to the location of the qtl on the tree . each possible qtl location on the tree corresponds to a partition of the taxa into two groups . given limited resources and crosses of limited size , there will be incomplete power to detect the qtl in a given cross , and so the naive approach based on the presence or absence of the qtl in the different crosses will likely be misleading . a more formal approach , in which the likelihoods for the different possible partitions are evaluated and compared , will provide a clear assessment of the evidence for the different locations for the qtl on the tree . consider a particular location in the genome as the site of a putative qtl , and consider a particular partition of the taxa into two qtl alleles . we assume a linear model with normally distributed errorsyij=i+aij+dij+ij , where yij is the phenotype for individual j in cross i , i the average phenotype in cross i , and are the additive and dominance effects of the qtl , respectively , and the ij are independent and identically distributed normal ( 0 , ) . the aij and dij denote encodings of the qtl genotypes , with aij = dij = 0 if the qtl is not segregating in cross i. for convenience , we call the two qtl alleles defined by the partition as the high allele ( h ) and the low allele ( l ) , although we wo nt actually constrain the high allele to increase the phenotype . if the qtl is segregating in cross i , then we take aij = 1 , 0 , or + 1 , if individual j has qtl genotype gij = ll , hl , or hh , respectively , and dij = 1 if individual j has qtl genotype hl and dij = 0 otherwise . for most putative qtl locations , the qtl genotypes are not be observed , but we may calculate ( e.g. , by a hidden markov model ) the conditional probabilities of the qtl genotypes given the available multipoint marker genotype data , pijk = pr(gij = k|mij ) . it is critical that we have a common map for the set of crosses , so that a putative qtl location is clearly defined in all crosses . it is not necessary , however , that the same markers be used in all crosses or that they be informative in all crosses . we may then use standard interval mapping ( lander and botstein 1989 ) or an approximation such as haley knott regression ( haley and knott 1992 ) to fit the model , estimate the parameters i , , , and , and calculate a lod score , lod( ) , where denotes the partition of the taxa and denotes the location of the putative qtl . the lod score is the log10 likelihood comparing the hypothesis of a single qtl at that location to the null hypothesis of no qtl but with the multiple crosses allowed to have separate phenotypic means , that is , yij normal ( i , ) . ( 2005 ) , in that one recodes the genotypes in the crosses in which the qtl is segregating , stacks them on top of one another , as if they were a single intercross , and performs interval mapping with cross indicators as additive covariates . the only difference is that we are considering all possible partitions of the taxa , while li et al . there is one technicality : the crosses in which the qtl does not segregate also need to be included in the likelihood , and they contribute to the estimate of the residual variance . we thus consider each possible partition , , one at a time , and scan the genome to obtain a set of lod curves , lod( ) . we summarize these at the chromosome level , calculating the maximum lod score for partition on chromosome i , mi = maxi lod( ) . the maximum on chromosome i , maxmi , indicates the evidence for a qtl on chromosome i. to evaluate the relative support of the different partitions , we use an approximate bayes procedure . assuming the presence of a single diallelic qtl on chromosome i , we assign equal prior probabilities to the different possible partitions , , treat the profile log likelihoods mi ( in which we have maximized over all nuisance parameters , including the location of the qtl on the chromosome ) as if they were true log likelihoods , and obtain posterior probabilities by taking 10mi and rescaling so that they sum to 1 . that is , we further use these approximate posterior probabilities to form a 95% bayesian credible set of partitions . one could assign unequal prior probabilities to the partitions , for example , based on the branch lengths in the assumed phylogenetic tree , giving more weight to longer branches . one might also use a prior on partitions that assigns greater weight to partitions induced by the tree and lesser ( but nonzero ) weight to the other ( possibly more numerous ) partitions . the 95% credible set of partitions is relevant only if there is sufficient evidence for a qtl on that chromosome . to evaluate the evidence for a qtl , we consider the maximum of the mi on chromosome i and derive a significance threshold , adjusting for the genome scan , by a stratified permutation test ( churchill and doerge 1994 ) . the permutation test is stratified in that we permute the phenotype data , relative to the genotype data , separately in each cross . for each permutation replicate , we calculate the lod curve for each possible partition and then take the maximum lod score across the genome and across partitions . the 95th percentile of these permutation results may be used as a significance threshold , or we may calculate a p - value that accounts for the search across partitions and across the genome . one may restrict the analyses to the set of partitions induced by the assumed phylogenetic tree , or one may consider all possible partitions of the taxa into two groups . for example , for the four - taxon tree in figure 1 , one may consider only the five partitions that correspond to qtl locations on the tree , as in the accompanying table , or one may also consider the two additional partitions , ac|bd and ad|bc . the consideration of all possible partitions will be accompanied by some loss of power , particularly if there is a large number of taxa . however , the correct phylogenetic tree will seldom be known with certainty and will likely vary along the genome , particularly if the taxa are closely related . moreover , if there is strong support for one of the partitions that is not associated with a qtl location on the assumed phylogenetic tree , one would certainly want to know this . thus , we are inclined to always consider all possible partitions and not focus on those induced by an assumed phylogenetic tree . in this section , we address a theoretical question of considerable interest : which subsets of crosses are sufficient to identify the location of a qtl on the phylogenetic tree ? with very large crosses , we can exactly determine which crosses are segregating a qtl and which are not . as discussed in the introduction for example , for the case in figure 1 , if one performs only the crosses a b , a c , and a d , the ideal results perfectly discriminate among the possible locations of the qtl on the tree . however , if one performs only the crosses a b , a c , and b c , several of the possible partitions of strains exhibit the same pattern of presence / absence of qtl and so are confounded . it is useful , in considering this problem , to represent a set of crosses by a graph , with nodes corresponding to taxa and edges indicating a cross between two taxa . three possible choices of a subset of five crosses among the six taxa are displayed in figure 2 , b d . a phylogenetic tree with six taxa ( a ) and three possible choices of five crosses among the six taxa , with nodes denoting taxa and edges denoting crosses ( b d ) . a sufficient condition for identifying the true partition of the strains is the use of a set of crosses that connect all of the taxa , as in figure 2b . choose an arbitrary taxon ( e.g. , a ) and assign it an arbitrary qtl allele . with sufficient numbers of individuals in each cross , we may determine whether the qtl is segregating in a cross , which indicates that the two taxa have different qtl alleles , or is not segregating , which indicates that the two strains have the same qtl allele . thus , one may move between taxa connected by a cross and assign qtl alleles , and so if the set of crosses connect all of the taxa , one can assign qtl alleles to all taxa and so identify the correct partition of taxa . if the set of crosses are not connected ( as in figure 2 , c and d ) , then some partitions of taxa will be confounded . for example , for the crosses in figure 2c , the partition abc|def will give the same set of qtl results as under the null hypothesis of no qtl . other pairs of partitions are confounded in this example , such as ab|cdef and abdef|c . if one is considering all possible partitions of the taxa ( and not just those induced by the tree ) , then graph connectivity is also a necessary condition for identifying the true partition : if the crosses do not connect all taxa there will always be some partitions that are confounded . however , if one focuses solely on those partitions induced by the tree ( that is , partitions that result from a split on an edge in the tree ) , then it is not necessary that the crosses connect all taxa . for the pairs of partitions that are confounded with this choice of crosses , no more than one of each pair corresponds to a split on the tree in figure 2a ; each possible partition induced by the tree gives a distinct set of qtl results for these crosses . moreover , in this case one may omit any one of the three crosses , b c , b e , c e : only four crosses are necessary to distinguish among the nine partitions induced by the tree in figure 2a . that the crosses connect all taxa is a necessary and sufficient criterion to distinguish among all possible partitions , but it is not a necessary condition to distinguish among the partitions induced by the tree . note that a cross between two taxa corresponds to a path along the tree from one leaf to another . further , the qtl will be segregating in crosses whose paths go through the edge with the qtl , but it will not be segregating in crosses whose paths do not go through that edge . a necessary and sufficient criterion for a set of crosses to distinguish the partitions induced by the tree ( i.e. , to distinguish the possible locations of the qtl on the tree ) is that each edge is covered by at least one cross and that no two edges appear only together . if an edge was not covered by a cross , then a qtl on that edge could not be distinguished from the null model , of no qtl . if two edges only appear together in crosses , then those two qtl locations can not be distinguished . note that a cross in which the qtl is segregating will limit the possible qtl locations to the edges on the corresponding path through the tree . as every pair of edges along such a path will appear separately in different crosses , we see that the specific edge containing the qtl may be identified . for n taxa ( with n3 ) , the minimal number of crosses to distinguish among all possible partitions is n1 . to distinguish among the partitions induced by the tree , the minimal number of crosses is 2n/3 ( the smallest integer that is greater than 2n/3 ; a proof appears in the appendix ) . for n5 , these are the same ; for n6 , fewer crosses are needed to distinguish among the tree partitions . as discussed in the previous section , we recommend that one not restrict oneself to the partitions induced by the tree but rather always consider all possible partitions , possibly with different prior weights . as a result , we recommend that one use , at a minimum , a set of crosses that connect all taxa . however , this is based on the assumption of a small number of taxa . if the number of taxa , n , is large , the total number of non - null partitions ( 2 1 ) will vastly exceed the number of partitions induced by the tree ( 2n 3 ) , and so there is great potential advantage in focusing on the tree partitions . of course , in practice crosses are of finite size and so one can not identify the true partition of the taxa without some degree of uncertainty . in the next section we explore , via computer simulation , the relative performance of the proposed method with different possible choices of crosses . we begin by comparing our proposed method to the naive approach of considering the crosses individually and comparing the pattern of presence / absence of a qtl in the crosses to what is expected for different possible partitions . we then compare the performance of our approach with all possible crosses to different choices of a minimal set of crosses . we consider the case of four taxa and use of all six possible intercrosses among pairs of taxa , with 75 individuals per cross ( a total sample size of 450 ) . we consider a single autosome of length 127 cm , with markers at an approximately 10-cm spacing , and with a single diallelic qtl placed in the center of an interval between two markers , near the middle of the chromosome . the qtl alleles were assumed to act additively ( that is , no dominance ) , and the percentage phenotypic variance explained by the qtl , in the crosses in which it was segregating , was 10% . we assumed either the partition a|bcd or ab|cd ; other possible partitions are equivalent to one of these . to reduce computation time , we used haley knott regression ( haley and knott 1992 ) for all simulation studies , with lod score calculations performed on a 1-cm grid . for the naive approach , we applied a given significance threshold and inferred the presence or absence of a qtl in a cross if the maximum lod score on the chromosome was above or below the threshold , respectively . if the presence / absence pattern matched that for a possible partition , that partition was inferred . for the proposed approach , we applied a given significance threshold on maxm and then formed a 95% bayesian credible set of partitions , using equal prior probabilities on all seven possible partitions . if maxm was greater than the threshold but the 95% credible set did not contain the truth , the result was considered a false positive . the results , based on 10,000 simulations , are displayed in figure 3 as receiver operating characteristic ( roc ) curves : the power ( the rate of true positives ) vs. the false positive rate , for varying significance thresholds . we display two sets of curves for the proposed method : for the dashed curves , the power indicates that maxm exceeded the threshold and the true partition was contained within the 95% credible set ; the dotted curves are more stringent and require that the credible set contained only the true partition . points are plotted at the results with a nominal 5% significance threshold , adjusting for an autosomal genome scan , with the genome modeled after the mouse and the thresholds estimated by 10,000 simulations under the null hypothesis of no qtl . ( the estimated thresholds are displayed in supporting information , table s1 and table s2 . ) estimated receiver operating characteristic ( roc ) curves for the naive method ( solid curves ) , the proposed method , with power indicating that the true partition is contained within the 95% credible set ( dashed curves ) , and the proposed method , with power indicating that the 95% credible set contains only the true partition ( dotted curves ) , in the case of four taxa , with each of the six possible intercrosses having a sample size of 75 , and a qtl responsible for 10% of the phenotypic variance in the crosses in which it is segregating . the red and blue curves correspond to the case that the true partition is a|bcd and ab|cd , respectively . the roc curves for the naive method form interesting shapes , with the lower part of each corresponding to low thresholds and the upper part corresponding to high thresholds , and indicate terrible performance : the false positive rate is well controlled , but power is low . the problem is that , with only moderate power to detect the qtl in a given cross , one has low power to detect the qtl in all of the crosses in which it is segregating , which is necessary to identify the correct partition of the taxa . lowering the significance threshold below the 5% level helps somewhat , but the power to detect the true partition is no higher than 21% . the naive approach might actually perform better if one considered a smaller set of crosses , but we have not explored this further . the proposed method performs reasonably well , and the false positive rate is well controlled at the nominal 5% significance threshold ( the points in figure 3 ) . lowering the threshold could give some improvement in power while maintaining the false - positive rate below the target level , at least in the simulated situations . in the previous section , we noted that it is not necessary to use all possible crosses among taxa . to distinguish among all possible partitions , one need sets of crosses that connect all taxa and are of minimal size ( i.e. , n1 crosses for n taxa ) are called minimal sets . we now turn to the question of whether it is better to use all crosses , with a smaller number of individuals per cross , or a minimal set of crosses , with a larger number of individuals per cross . we use the same general settings as for the simulations comparing the proposed method to the naive approach , with four taxa and the true partition being either a|bcd or ab|cd , but here we vary the total sample size among 300 , 450 , and 600 individuals , and we vary the percentage phenotype variance explained by the qtl from 2.5 to 15% . we consider either all six crosses or a minimal set of three crosses , and we consider all 16 choices of three crosses that include all four taxa . we also compared the consideration of all seven possible partitions , or just the five partitions induced by the tree in figure 1 . we estimated 5% genome - wide significance thresholds by simulations under the null hypothesis of no qtl ( see table s2 ) . figure 4 displays the simulation results , as a function of the effect of the qtl , for the case that the total sample size was 450 ( i.e. , 75 individuals per cross when considering all crosses and 150 individuals per cross when considering a minimal set of three crosses ) and when all possible partitions were considered . the results with other sample sizes and with analysis restricted to the five partitions induced by the tree in figure 1 are shown in figure s1 , figure s2 , figure s3 , figure s4 , figure s5 , and figure s6 . the top of each figure indicates the power ( the chance that maxm exceeded its threshold and the true partition was contained in the 95% credible set ) ; the middle indicates the exact power ( the chance that maxm exceeded its threshold and that the credible set contained only the true partition ) ; the bottom indicates the false positive rate . the left and right correspond to the true partition being a|bcd or ab|cd , respectively . the black dashed curves correspond to the use of all six possible crosses ; the solid curves correspond to the different choices of a minimal set of three crosses , with blue , red , and green corresponding to cases in which 3 , 2 , or 1 of the crosses are segregating a qtl . estimated power ( top ) , exact power ( middle ) , and false - positive rates ( bottom ) in the case of four taxa with a total sample size of 450 , as a function of the percentage phenotypic variance explained by the qtl . the other curves are for the various choices of a minimal set of three crosses , with the curves in blue , red , and green corresponding to cases in which three , two , and one of the crosses are segregating the qtl , respectively . the results are based on 10,000 simulation replicates , with analyses considering all possible partitions of the taxa . in choosing among the possible minimal sets of crosses , power is highest when a larger number of crosses are segregating the qtl . for a fixed total sample size , the use of all possible crosses ( with fewer individuals per cross ) has better performance than the worst of the possible minimal sets of crosses , but is not as good as the best of the possible minimal sets of crosses . the use of all possible crosses has greater power when the true partition is ab|cd ( in which case four of the six crosses are segregating the qtl ) than when the true partition is a|bcd ( in which case three of the six crosses are segregating the qtl ) . the false - positive rate ( figure 4 , bottom ) is well controlled throughout . the use of a total sample size of 300 or 600 gives qualitatively similar results ( see figure s1 , figure s2 , figure s3 , figure s4 , figure s5 , and figure s6 ; figure s7 and figure s8 contain the false negative rates ) , although we note that while a larger sample size results in a great improvement in power , it gives only a slight improvement in the chance that the credible set includes only the true partition . restricting the analysis to the five partitions induced by the tree has little effect on power ( compare figure s1 and figure s2 ) , but improves the chance that the credible set includes only the true partition ( compare figure s3 and figure s4 ) , and results in a somewhat lower false - positive rate ( compare figure s5 and figure s6 ) . the performance of the proposed method with different possible choices of minimal crosses is largely predicted by the number of crosses that are segregating a qtl : the solid curves of a given color ( which indicates the number of crosses segregating a qtl ) are largely coincident , but there are some differences ( red curves in figure 4 , middle right ) . to explore this further , the results for the individual choices of crosses , when the percentage phenotypic variance explained by the qtl is 10% and the total sample size is 450 , are displayed in figure 5 . ( for other sample sizes and for the analyses restricted to the partitions induced by the tree in figure 1 , see figure s9 , figure s10 , figure s11 , figure s12 , figure s13 , figure s14 , figure s15 , and figure s16 . ) detailed results on the estimated power ( top ) , exact power ( middle ) , and false positive rates ( bottom ) , for individual choices of crosses , in the case of four taxa with a total sample size of 450 , and with the qtl being responsible for 10% of the phenotypic variance in crosses in which it is segregating . blue , red , and green correspond to cases in which three , two , and one of the crosses are segregating the qtl , respectively . the results are based on 10,000 simulation replicates , with analyses considering all possible partitions of the taxa . the black vertical line segments indicate 95% confidence intervals . in the case that the true partition is ab|cd , there are some differences among the choices of three crosses when two of the three are segregating the qtl , in terms of the chance that the 95% credible set contains only the true partition ( figure 5 , middle ) . for example , the use of the crosses a b , a c , and b d gives exact power of 50% , while the use of a b , a c , and a d gives , we need to consider the sign of the qtl effect in different crosses for the true partition and the best alternative partition ; these are shown in table 1 . if the true partition is ab|cd , with c and d having an allele that results in an increase in the phenotype , the a b cross does not segregate a qtl , while each of a c , b d , and a d have a segregating qtl with the latter taxon in each cross increasing the phenotype . with the crosses a b , a c , and a d , the best alternative partition after ab|cd would be a|bcd , in which a c and a d are also segregating the qtl , but a b should also be segregating the qtl , and note that for both partitions ab|cd and a|bcd , the qtl has effect in the same direction in the a c and a d crosses . on the other hand , with the crosses a b , a c , and b d ( which was seen to have better performance ) , in the only alternative partition with two crosses segregating a qtl , ad|bc , the two crosses should have qtl effects in opposite directions ( the a and d alleles both result in a decrease in phenotype ) , and so this should be easy to distinguish from the ab|cd partition . for this choice of three crosses , all other partitions have a qtl segregating in just one of a c or b d but not both . as a result , the chance that the credible set contains only the true partition is slightly higher . for each partition , the taxa to the right of the vertical bar have the high allele . for each cross , the sign of the effect is for the right vs. the left taxon . while no such differences among the choices of minimal crosses are seen when the true partition is a|bcd and all possible partitions are considered in the analysis , these sorts of differences do arise when the analysis is restricted to the five partitions induced by the tree in figure 1 . we consider the case of four taxa and use of all six possible intercrosses among pairs of taxa , with 75 individuals per cross ( a total sample size of 450 ) . we consider a single autosome of length 127 cm , with markers at an approximately 10-cm spacing , and with a single diallelic qtl placed in the center of an interval between two markers , near the middle of the chromosome . the qtl alleles were assumed to act additively ( that is , no dominance ) , and the percentage phenotypic variance explained by the qtl , in the crosses in which it was segregating , was 10% . we assumed either the partition a|bcd or ab|cd ; other possible partitions are equivalent to one of these . to reduce computation time , we used haley knott regression ( haley and knott 1992 ) for all simulation studies , with lod score calculations performed on a 1-cm grid . recombination was simulated assuming no crossover interference . for the naive approach , we applied a given significance threshold and inferred the presence or absence of a qtl in a cross if the maximum lod score on the chromosome was above or below the threshold , respectively . if the presence / absence pattern matched that for a possible partition , that partition was inferred . for the proposed approach , we applied a given significance threshold on maxm and then formed a 95% bayesian credible set of partitions , using equal prior probabilities on all seven possible partitions . if maxm was greater than the threshold but the 95% credible set did not contain the truth , the result was considered a false positive . the results , based on 10,000 simulations , are displayed in figure 3 as receiver operating characteristic ( roc ) curves : the power ( the rate of true positives ) vs. the false positive rate , for varying significance thresholds . we display two sets of curves for the proposed method : for the dashed curves , the power indicates that maxm exceeded the threshold and the true partition was contained within the 95% credible set ; the dotted curves are more stringent and require that the credible set contained only the true partition . points are plotted at the results with a nominal 5% significance threshold , adjusting for an autosomal genome scan , with the genome modeled after the mouse and the thresholds estimated by 10,000 simulations under the null hypothesis of no qtl . ( the estimated thresholds are displayed in supporting information , table s1 and table s2 . ) estimated receiver operating characteristic ( roc ) curves for the naive method ( solid curves ) , the proposed method , with power indicating that the true partition is contained within the 95% credible set ( dashed curves ) , and the proposed method , with power indicating that the 95% credible set contains only the true partition ( dotted curves ) , in the case of four taxa , with each of the six possible intercrosses having a sample size of 75 , and a qtl responsible for 10% of the phenotypic variance in the crosses in which it is segregating . the red and blue curves correspond to the case that the true partition is a|bcd and ab|cd , respectively . the roc curves for the naive method form interesting shapes , with the lower part of each corresponding to low thresholds and the upper part corresponding to high thresholds , and indicate terrible performance : the false positive rate is well controlled , but power is low . the problem is that , with only moderate power to detect the qtl in a given cross , one has low power to detect the qtl in all of the crosses in which it is segregating , which is necessary to identify the correct partition of the taxa . lowering the significance threshold below the 5% level helps somewhat , but the power to detect the true partition is no higher than 21% . the naive approach might actually perform better if one considered a smaller set of crosses , but we have not explored this further . the proposed method performs reasonably well , and the false positive rate is well controlled at the nominal 5% significance threshold ( the points in figure 3 ) . lowering the threshold could give some improvement in power while maintaining the false - positive rate below the target level , at least in the simulated situations . in the previous section , we noted that it is not necessary to use all possible crosses among taxa . to distinguish among all possible partitions , one need sets of crosses that connect all taxa and are of minimal size ( i.e. , n1 crosses for n taxa ) are called minimal sets . we now turn to the question of whether it is better to use all crosses , with a smaller number of individuals per cross , or a minimal set of crosses , with a larger number of individuals per cross . we use the same general settings as for the simulations comparing the proposed method to the naive approach , with four taxa and the true partition being either a|bcd or ab|cd , but here we vary the total sample size among 300 , 450 , and 600 individuals , and we vary the percentage phenotype variance explained by the qtl from 2.5 to 15% . we consider either all six crosses or a minimal set of three crosses , and we consider all 16 choices of three crosses that include all four taxa . we also compared the consideration of all seven possible partitions , or just the five partitions induced by the tree in figure 1 . we estimated 5% genome - wide significance thresholds by simulations under the null hypothesis of no qtl ( see table s2 ) . figure 4 displays the simulation results , as a function of the effect of the qtl , for the case that the total sample size was 450 ( i.e. , 75 individuals per cross when considering all crosses and 150 individuals per cross when considering a minimal set of three crosses ) and when all possible partitions were considered . the results with other sample sizes and with analysis restricted to the five partitions induced by the tree in figure 1 are shown in figure s1 , figure s2 , figure s3 , figure s4 , figure s5 , and figure s6 . the top of each figure indicates the power ( the chance that maxm exceeded its threshold and the true partition was contained in the 95% credible set ) ; the middle indicates the exact power ( the chance that maxm exceeded its threshold and that the credible set contained only the true partition ) ; the bottom indicates the false positive rate . the left and right correspond to the true partition being a|bcd or ab|cd , respectively . the black dashed curves correspond to the use of all six possible crosses ; the solid curves correspond to the different choices of a minimal set of three crosses , with blue , red , and green corresponding to cases in which 3 , 2 , or 1 of the crosses are segregating a qtl . estimated power ( top ) , exact power ( middle ) , and false - positive rates ( bottom ) in the case of four taxa with a total sample size of 450 , as a function of the percentage phenotypic variance explained by the qtl . the other curves are for the various choices of a minimal set of three crosses , with the curves in blue , red , and green corresponding to cases in which three , two , and one of the crosses are segregating the qtl , respectively . the results are based on 10,000 simulation replicates , with analyses considering all possible partitions of the taxa . in choosing among the possible minimal sets of crosses , power is highest when a larger number of crosses are segregating the qtl . for a fixed total sample size , the use of all possible crosses ( with fewer individuals per cross ) has better performance than the worst of the possible minimal sets of crosses , but is not as good as the best of the possible minimal sets of crosses . the use of all possible crosses has greater power when the true partition is ab|cd ( in which case four of the six crosses are segregating the qtl ) than when the true partition is a|bcd ( in which case three of the six crosses are segregating the qtl ) . the false - positive rate ( figure 4 , bottom ) is well controlled throughout . the use of a total sample size of 300 or 600 gives qualitatively similar results ( see figure s1 , figure s2 , figure s3 , figure s4 , figure s5 , and figure s6 ; figure s7 and figure s8 contain the false negative rates ) , although we note that while a larger sample size results in a great improvement in power , it gives only a slight improvement in the chance that the credible set includes only the true partition . restricting the analysis to the five partitions induced by the tree has little effect on power ( compare figure s1 and figure s2 ) , but improves the chance that the credible set includes only the true partition ( compare figure s3 and figure s4 ) , and results in a somewhat lower false - positive rate ( compare figure s5 and figure s6 ) . the performance of the proposed method with different possible choices of minimal crosses is largely predicted by the number of crosses that are segregating a qtl : the solid curves of a given color ( which indicates the number of crosses segregating a qtl ) are largely coincident , but there are some differences ( red curves in figure 4 , middle right ) . to explore this further , the results for the individual choices of crosses , when the percentage phenotypic variance explained by the qtl is 10% and the total sample size is 450 , are displayed in figure 5 . ( for other sample sizes and for the analyses restricted to the partitions induced by the tree in figure 1 , see figure s9 , figure s10 , figure s11 , figure s12 , figure s13 , figure s14 , figure s15 , and figure s16 . ) detailed results on the estimated power ( top ) , exact power ( middle ) , and false positive rates ( bottom ) , for individual choices of crosses , in the case of four taxa with a total sample size of 450 , and with the qtl being responsible for 10% of the phenotypic variance in crosses in which it is segregating . blue , red , and green correspond to cases in which three , two , and one of the crosses are segregating the qtl , respectively . the results are based on 10,000 simulation replicates , with analyses considering all possible partitions of the taxa . the black vertical line segments indicate 95% confidence intervals . in the case that the true partition is ab|cd , there are some differences among the choices of three crosses when two of the three are segregating the qtl , in terms of the chance that the 95% credible set contains only the true partition ( figure 5 , middle ) . for example , the use of the crosses a b , a c , and b d gives exact power of 50% , while the use of a b , a c , and a d gives , we need to consider the sign of the qtl effect in different crosses for the true partition and the best alternative partition ; these are shown in table 1 . if the true partition is ab|cd , with c and d having an allele that results in an increase in the phenotype , the a b cross does not segregate a qtl , while each of a c , b d , and a d have a segregating qtl with the latter taxon in each cross increasing the phenotype . with the crosses a b , a c , and a d , the best alternative partition after ab|cd would be a|bcd , in which a c and a d are also segregating the qtl , but a b should also be segregating the qtl , and note that for both partitions ab|cd and a|bcd , the qtl has effect in the same direction in the a c and a d crosses . on the other hand , with the crosses a b , a c , and b d ( which was seen to have better performance ) , in the only alternative partition with two crosses segregating a qtl , ad|bc , the two crosses should have qtl effects in opposite directions ( the a and d alleles both result in a decrease in phenotype ) , and so this should be easy to distinguish from the ab|cd partition . for this choice of three crosses , all other partitions have a qtl segregating in just one of a c or b d but not both . as a result , the chance that the credible set contains only the true partition is slightly higher . for each partition , the taxa to the right of the vertical bar have the high allele . for each cross , the sign of the effect is for the right vs. the left taxon . while no such differences among the choices of minimal crosses are seen when the true partition is a|bcd and all possible partitions are considered in the analysis , these sorts of differences do arise when the analysis is restricted to the five partitions induced by the tree in figure 1 . these data concern four intercrosses among five inbred mouse strains , cast / ei ( c ) , dba/2 ( d ) , i / lnj ( i ) , pera / ei ( p ) , and 129s1/svimj ( s ) . the four intercrosses performed were c d , c s , d p , and i p. the c d and c s crosses were all males and had 277 and 275 mice , respectively . the d p and i p crosses had approximately equal numbers of males and females and had a total of 282 and 322 mice , respectively . as in li et al . ( 2005 ) , we focus on a single phenotype , the square root of plasma hdl cholesterol . note that the four intercrosses form a daisy chain , s c d p i , and so satisfy the connectedness condition necessary for inference of the correct partition of the strains at a diallelic qtl . ( 2009 ) , with marker locations obtained using the mouse map converter at the jackson laboratory ( http://cgd.jax.org/mousemapconverter ) . we used standard interval mapping ( lander and botstein 1989 ) and considered all 15 possible partitions of the five strains , without attempting to infer a phylogenetic tree relating the strains . to handle the two sexes , we included sex as an additive covariate ( that is , we allowed for a shift in the average phenotype between the sexes and assumed no qtl sex interaction ) . we used permutation tests with 10,000 replicates to obtain 5% significance thresholds for the individual crosses and for maxmi . the estimated significance thresholds for the individual crosses were approximately 3.44 for all four crosses ; the estimated threshold on maxmi was 5.39 . ( 2005 ) , we focused on chromosomes 1 , 2 , 4 , 5 , 6 , and 11 . the lod curves for the top five partitions on each chromosome are in figure 6 , middle . the posterior probabilities of the different partitions , assuming the presence of a single diallelic qtl , are on the right . in all cases , ( 2005 ) : lod curves for individual crosses ( left ) , lod curves for the top five partitions ( middle ) , and approximate posterior probabilities for each partition ( right ) . the partitions corresponding to the five lod curves in the middle are indicated on the right . the labeled points on the right indicate the partitions included in the 95% bayesian credible sets . on the left and in the middle , dashed horizontal lines are plotted at the 5% significance thresholds . for chromosome 1 , significant evidence for a qtl is seen in the crosses c s and d p but not in c d or i p. by the naive approach , we would infer the partition cd|ips , and this is the partition that li et al . our proposed method does give this partition the highest posterior probability ( 57% ) , but also gives reasonable weight to the alternative ps|cdi ( posterior probability 39% ) , in which case the qtl would also be segregating in the i p cross . for chromosome 2 , we see a qtl just in cross c d. by the naive approach ( given the set of crosses performed ) , we would infer the partition cs|dip , which is the partition that li et al . however , by the proposed method , cs|dip has a posterior probability of only 20% , while the partition c|sdip ( in which the qtl would also be present in the c s cross ) has a posterior probability of 80% . for chromosome 4 , we have evidence for a qtl in all four crosses ( although in the cross i p , the maximum lod score was 3.42 , just missing the threshold of 3.44 ) . if we assume that there is no qtl segregating in i p , we would infer the partition ds|cip , while if we take the evidence for a qtl in i p as sufficient , we would infer the partition cp|dis , and this is the partition that li et al . the latter is the partition with the highest posterior probability ( 78% ) , while the former has posterior probability 7% , and a third partition , c|dips , in which case the qtl is segregating in neither i p nor d p , has posterior probability 16% . for chromosome 5 , we see a qtl only in cross i p , and so by the naive approach we would infer the partition i|cdps ; this partition does have the highest posterior probability ( 83% ) and was the partition that li et al . but the maximum lod score for this partition was 3.98 , which does nt meet the 5% significance threshold . ( the genome - scan - adjusted p - value was 0.37 . ) thus , by our proposed approach , we would not infer the presence of a qtl . but if we do allow that there is a qtl , two other partitions are contained within the 95% credible set : dp|cis , with posterior probability 9% , in which case the qtl is also segregating in the cross c d , and is|cdp , with posterior probability 6% , in which case the qtl is also segregating in the cross c s. for chromosome 6 , we have significant evidence for a qtl only in cross c d ( the other three crosses have maximum lod scores of 1.51.9 on chromosome 6 ) , and so the naive method would give the partition cs|dip , which has posterior probability < 0.01% and is not contained in the 95% credible set . the partitions with highest posterior are c|dips ( 47% ) , with the qtl also segregating in c s , and ci|dps ( 45% ) , with the qtl also segregating in c s and i p. the 95% credible set also contains a third partition , ds|cip , with posterior probability 7% . ( 2005 ) had assumed the partition c|dips , which is the partition with highest posterior probability . for chromosome 11 , there was significant evidence for a qtl only in the cross i p , although the cross d p has a maximum lod score of 3.16 ( corresponding to a genome - scan - adjusted p - value of 0.093 ) . the naive approach would give the partition i|cdps , which has posterior probability 0.9% and is not contained in the 95% credible set . if we consider the evidence for a qtl in d p to be sufficient , we would infer the partition p|cdis , which has posterior 16% and was the one that li et al . the partition with highest posterior probability is di|cps ( posterior probability 60% ) , in which case the qtl is also segregating in c d. the 95% credible set also contains the partition ps|cdi ( posterior probability 21% ) , in which case the qtl is segregating in c s but not c d. as with chromosome 5 , the maximum lod score across partitions ( 4.70 ) does not meet our 5% significance threshold , and so by our proposed method we would not infer the presence of a qtl . we have described a formal approach for the joint analysis of multiple crosses to map the origin of qtl alleles to a position on a phylogenetic tree . our approach unites qtl mapping with phylogenetic comparative methods to provide a view of the genetic mechanism underlying phenotypic evolution . further , our approach partitions taxa according to their qtl allele , facilitating haplotype analyses for the fine mapping of qtl . in addition , as part of this work , we have begun to evaluate a variety of experimental design issues for such research , which provides some guidance to researchers seeking to take advantage of this approach . the goal of the work in li et al . the key difficulty in applying this idea is that one must define a unique partition of the strains into the two qtl alleles , a priori . in the presence of multiple qtl , the phenotypes of the strains can not be trusted for inferring the qtl alleles , and in the current application , the six qtl partition the five strains in diverse ways . ( 2005 ) used the pattern of qtl in the different crosses to infer the appropriate partition , which we have ( perhaps overly harshly ) characterized as the naive approach . we have proposed a formal method for comparing the different possible partitions . for two of the six loci , we find that the partition with strongest support is different from that assumed by li et al . ( 2005 ) , and for all six loci there are multiple partitions with reasonable support . as seen in figure 6 , middle , the different partitions can have quite different lod curves and so provide different information on the likely location of the qtl . thus , our formal approach to identifying the well - supported partitions can improve localization of a qtl . moreover , one could combine the information from the multiple partitions to better define the location of the qtl , taking account of the uncertainty in the partition . furthermore , while the application of these ideas to evolutionary studies remains our primary interest , the more straightforward application is in biomedical or agricultural research , as in li et al . ( 2005 ) , for the combined use of multiple crosses to more precisely map a qtl and , subsequently , with an inferred partition ( or partitions ) of strains in hand , to inform the analysis of the haplotypes of the strains ( see , for example , burgess - herbert et al . the results are also valuable for the design of future experiments , if additional crosses are to be performed . our approach has some similarities to the use of local phylogenetic trees to define possible partitions of multiple alleles ( pan et al . 2009 ; zhang et al . 2012 ) and to coalescent - based approaches ( zllner and pritchard 2005 ) for genome - wide association studies . the key distinction of our method is that we seek not just to establish association but also to identify the appropriate partition and so define the origin of the mutant qtl allele on the local phylogenetic tree . in our approach , the qtl location on the tree is not a nuisance parameter but rather is the target of inference . in our simulation studies , we compared the use , for a fixed total sample size , of all possible crosses to different choices of a minimal set of crosses . depending on the underlying true partition of taxa at a qtl , one can choose a minimal set of crosses with considerably higher power . however , given the prior uncertainty in the true partition , and the possibility of multiple qtl that each partition the taxa differently , it is prudent to consider all or at least a larger number of possible crosses . an even more important experimental design question , which we have not considered here , is how to choose which taxa , out of a large number of related taxa , to consider , in the effort to characterize the genetic architecture of a quantitative trait . the approach could be adapted for the analysis of a set of backcrosses , although these would likely need to be of a special form , with the f1 hybrids all crossed to a common parent . there are a number of additional ways in which our analytical framework could be extended . most quantitative traits are affected by multiple qtl , rather than single qtl as assumed here . the restriction that a qtl has a common effect in all crosses in which it segregates might be relaxed , particularly for traits that are heavily shaped by epistasis , such as hybrid sterility and hybrid inviability ( coyne and orr 2004 ) . prior distributions of qtl partitions could incorporate phylogenetic branch lengths ( taxa separated by shorter evolutionary distances are more likely to share qtl alleles ) as well as topologies . finally , future developments might account for variation in the tree . this variation includes both statistical uncertainty associated with phylogenetic inference and real phylogenetic discordance across the genome , which results from incomplete lineage sorting and introgression in recently diverged taxa ( pamilo and nei 1988 ; maddison 1997 ; pollard et al . 2006 ; white et al . 2009 ) . the power of reconstructing qtl evolution as well as the increasing capacity for genetic mapping of complex traits and phylogenetic reconstruction should provide motivation for these extensions in the evolutionary , biomedical , and agricultural communities . software incorporating the proposed methods are available as part of r / qtl ( broman et al . 2003 , http://www.rqtl.org ) , an add - on package to the general statistical software r ( r development core team 2010 ) .

despite advances in genetic mapping of quantitative traits and in phylogenetic comparative approaches , these two perspectives are rarely combined . the joint consideration of multiple crosses among related taxa ( whether species or strains ) not only allows more precise mapping of the genetic loci ( called quantitative trait loci , qtl ) that contribute to important quantitative traits , but also offers the opportunity to identify the origin of a qtl allele on the phylogenetic tree that relates the taxa . we describe a formal method for combining multiple crosses to infer the location of a qtl on a tree . we further discuss experimental design issues for such endeavors , such as how many crosses are required and which sets of crosses are best . finally , we explore the method s performance in computer simulations , and we illustrate its use through application to a set of four mouse intercrosses among five inbred strains , with data on hdl cholesterol .

Methods Theory Simulations Comparison to naive approach All crosses Application Discussion Supplementary Material

to develop methods for mapping a qtl to a phylogenetic tree , we begin with several simplifying assumptions : the taxa are represented by inbred lines , the tree relating the taxa is known without error , the quantitative trait of interest is affected by a single diallelic qtl , and there are no background effects ( i.e. the basic idea , illustrated in figure 1 , is that each possible location for the origin of a diallelic qtl on the tree corresponds to a different partition of the taxa into two groups , with the two groups corresponding to the two qtl alleles . in the case of very large crosses , with each having high power to detect the qtl , if present , we could simply consider the crosses individually and use the pattern of presence / absence of qtl to identify the correct partition of the taxa . in the following , we focus on partitions of the taxa into two groups , in place of locations of the qtl on the tree . illustration of the basic concepts behind the mapping of a qtl to a phylogenetic tree . in the table on the right , we indicate the presence or absence of a qtl in each of the six possible crosses among pairs of taxa , according to the location of the qtl on the tree . each possible qtl location on the tree corresponds to a partition of the taxa into two groups . consider a particular location in the genome as the site of a putative qtl , and consider a particular partition of the taxa into two qtl alleles . we may then use standard interval mapping ( lander and botstein 1989 ) or an approximation such as haley knott regression ( haley and knott 1992 ) to fit the model , estimate the parameters i , , , and , and calculate a lod score , lod( ) , where denotes the partition of the taxa and denotes the location of the putative qtl . we thus consider each possible partition , , one at a time , and scan the genome to obtain a set of lod curves , lod( ) . the maximum on chromosome i , maxmi , indicates the evidence for a qtl on chromosome i. to evaluate the relative support of the different partitions , we use an approximate bayes procedure . assuming the presence of a single diallelic qtl on chromosome i , we assign equal prior probabilities to the different possible partitions , , treat the profile log likelihoods mi ( in which we have maximized over all nuisance parameters , including the location of the qtl on the chromosome ) as if they were true log likelihoods , and obtain posterior probabilities by taking 10mi and rescaling so that they sum to 1 . one may restrict the analyses to the set of partitions induced by the assumed phylogenetic tree , or one may consider all possible partitions of the taxa into two groups . moreover , if there is strong support for one of the partitions that is not associated with a qtl location on the assumed phylogenetic tree , one would certainly want to know this . in this section , we address a theoretical question of considerable interest : which subsets of crosses are sufficient to identify the location of a qtl on the phylogenetic tree ? with very large crosses , we can exactly determine which crosses are segregating a qtl and which are not . as discussed in the introduction for example , for the case in figure 1 , if one performs only the crosses a b , a c , and a d , the ideal results perfectly discriminate among the possible locations of the qtl on the tree . it is useful , in considering this problem , to represent a set of crosses by a graph , with nodes corresponding to taxa and edges indicating a cross between two taxa . a phylogenetic tree with six taxa ( a ) and three possible choices of five crosses among the six taxa , with nodes denoting taxa and edges denoting crosses ( b d ) . a sufficient condition for identifying the true partition of the strains is the use of a set of crosses that connect all of the taxa , as in figure 2b . thus , one may move between taxa connected by a cross and assign qtl alleles , and so if the set of crosses connect all of the taxa , one can assign qtl alleles to all taxa and so identify the correct partition of taxa . for the pairs of partitions that are confounded with this choice of crosses , no more than one of each pair corresponds to a split on the tree in figure 2a ; each possible partition induced by the tree gives a distinct set of qtl results for these crosses . a necessary and sufficient criterion for a set of crosses to distinguish the partitions induced by the tree ( i.e. for n taxa ( with n3 ) , the minimal number of crosses to distinguish among all possible partitions is n1 . as a result , we recommend that one use , at a minimum , a set of crosses that connect all taxa . of course , in practice crosses are of finite size and so one can not identify the true partition of the taxa without some degree of uncertainty . in the next section we explore , via computer simulation , the relative performance of the proposed method with different possible choices of crosses . we begin by comparing our proposed method to the naive approach of considering the crosses individually and comparing the pattern of presence / absence of a qtl in the crosses to what is expected for different possible partitions . we then compare the performance of our approach with all possible crosses to different choices of a minimal set of crosses . we consider the case of four taxa and use of all six possible intercrosses among pairs of taxa , with 75 individuals per cross ( a total sample size of 450 ) . for the naive approach , we applied a given significance threshold and inferred the presence or absence of a qtl in a cross if the maximum lod score on the chromosome was above or below the threshold , respectively . estimated receiver operating characteristic ( roc ) curves for the naive method ( solid curves ) , the proposed method , with power indicating that the true partition is contained within the 95% credible set ( dashed curves ) , and the proposed method , with power indicating that the 95% credible set contains only the true partition ( dotted curves ) , in the case of four taxa , with each of the six possible intercrosses having a sample size of 75 , and a qtl responsible for 10% of the phenotypic variance in the crosses in which it is segregating . the problem is that , with only moderate power to detect the qtl in a given cross , one has low power to detect the qtl in all of the crosses in which it is segregating , which is necessary to identify the correct partition of the taxa . the naive approach might actually perform better if one considered a smaller set of crosses , but we have not explored this further . we use the same general settings as for the simulations comparing the proposed method to the naive approach , with four taxa and the true partition being either a|bcd or ab|cd , but here we vary the total sample size among 300 , 450 , and 600 individuals , and we vary the percentage phenotype variance explained by the qtl from 2.5 to 15% . the black dashed curves correspond to the use of all six possible crosses ; the solid curves correspond to the different choices of a minimal set of three crosses , with blue , red , and green corresponding to cases in which 3 , 2 , or 1 of the crosses are segregating a qtl . the other curves are for the various choices of a minimal set of three crosses , with the curves in blue , red , and green corresponding to cases in which three , two , and one of the crosses are segregating the qtl , respectively . for a fixed total sample size , the use of all possible crosses ( with fewer individuals per cross ) has better performance than the worst of the possible minimal sets of crosses , but is not as good as the best of the possible minimal sets of crosses . the performance of the proposed method with different possible choices of minimal crosses is largely predicted by the number of crosses that are segregating a qtl : the solid curves of a given color ( which indicates the number of crosses segregating a qtl ) are largely coincident , but there are some differences ( red curves in figure 4 , middle right ) . detailed results on the estimated power ( top ) , exact power ( middle ) , and false positive rates ( bottom ) , for individual choices of crosses , in the case of four taxa with a total sample size of 450 , and with the qtl being responsible for 10% of the phenotypic variance in crosses in which it is segregating . blue , red , and green correspond to cases in which three , two , and one of the crosses are segregating the qtl , respectively . for example , the use of the crosses a b , a c , and b d gives exact power of 50% , while the use of a b , a c , and a d gives , we need to consider the sign of the qtl effect in different crosses for the true partition and the best alternative partition ; these are shown in table 1 . if the true partition is ab|cd , with c and d having an allele that results in an increase in the phenotype , the a b cross does not segregate a qtl , while each of a c , b d , and a d have a segregating qtl with the latter taxon in each cross increasing the phenotype . on the other hand , with the crosses a b , a c , and b d ( which was seen to have better performance ) , in the only alternative partition with two crosses segregating a qtl , ad|bc , the two crosses should have qtl effects in opposite directions ( the a and d alleles both result in a decrease in phenotype ) , and so this should be easy to distinguish from the ab|cd partition . for the naive approach , we applied a given significance threshold and inferred the presence or absence of a qtl in a cross if the maximum lod score on the chromosome was above or below the threshold , respectively . estimated receiver operating characteristic ( roc ) curves for the naive method ( solid curves ) , the proposed method , with power indicating that the true partition is contained within the 95% credible set ( dashed curves ) , and the proposed method , with power indicating that the 95% credible set contains only the true partition ( dotted curves ) , in the case of four taxa , with each of the six possible intercrosses having a sample size of 75 , and a qtl responsible for 10% of the phenotypic variance in the crosses in which it is segregating . the roc curves for the naive method form interesting shapes , with the lower part of each corresponding to low thresholds and the upper part corresponding to high thresholds , and indicate terrible performance : the false positive rate is well controlled , but power is low . the problem is that , with only moderate power to detect the qtl in a given cross , one has low power to detect the qtl in all of the crosses in which it is segregating , which is necessary to identify the correct partition of the taxa . we use the same general settings as for the simulations comparing the proposed method to the naive approach , with four taxa and the true partition being either a|bcd or ab|cd , but here we vary the total sample size among 300 , 450 , and 600 individuals , and we vary the percentage phenotype variance explained by the qtl from 2.5 to 15% . we consider either all six crosses or a minimal set of three crosses , and we consider all 16 choices of three crosses that include all four taxa . the black dashed curves correspond to the use of all six possible crosses ; the solid curves correspond to the different choices of a minimal set of three crosses , with blue , red , and green corresponding to cases in which 3 , 2 , or 1 of the crosses are segregating a qtl . the other curves are for the various choices of a minimal set of three crosses , with the curves in blue , red , and green corresponding to cases in which three , two , and one of the crosses are segregating the qtl , respectively . for a fixed total sample size , the use of all possible crosses ( with fewer individuals per cross ) has better performance than the worst of the possible minimal sets of crosses , but is not as good as the best of the possible minimal sets of crosses . the use of all possible crosses has greater power when the true partition is ab|cd ( in which case four of the six crosses are segregating the qtl ) than when the true partition is a|bcd ( in which case three of the six crosses are segregating the qtl ) . the performance of the proposed method with different possible choices of minimal crosses is largely predicted by the number of crosses that are segregating a qtl : the solid curves of a given color ( which indicates the number of crosses segregating a qtl ) are largely coincident , but there are some differences ( red curves in figure 4 , middle right ) . detailed results on the estimated power ( top ) , exact power ( middle ) , and false positive rates ( bottom ) , for individual choices of crosses , in the case of four taxa with a total sample size of 450 , and with the qtl being responsible for 10% of the phenotypic variance in crosses in which it is segregating . for example , the use of the crosses a b , a c , and b d gives exact power of 50% , while the use of a b , a c , and a d gives , we need to consider the sign of the qtl effect in different crosses for the true partition and the best alternative partition ; these are shown in table 1 . if the true partition is ab|cd , with c and d having an allele that results in an increase in the phenotype , the a b cross does not segregate a qtl , while each of a c , b d , and a d have a segregating qtl with the latter taxon in each cross increasing the phenotype . on the other hand , with the crosses a b , a c , and b d ( which was seen to have better performance ) , in the only alternative partition with two crosses segregating a qtl , ad|bc , the two crosses should have qtl effects in opposite directions ( the a and d alleles both result in a decrease in phenotype ) , and so this should be easy to distinguish from the ab|cd partition . these data concern four intercrosses among five inbred mouse strains , cast / ei ( c ) , dba/2 ( d ) , i / lnj ( i ) , pera / ei ( p ) , and 129s1/svimj ( s ) . we used standard interval mapping ( lander and botstein 1989 ) and considered all 15 possible partitions of the five strains , without attempting to infer a phylogenetic tree relating the strains . for chromosome 1 , significant evidence for a qtl is seen in the crosses c s and d p but not in c d or i p. by the naive approach , we would infer the partition cd|ips , and this is the partition that li et al . for chromosome 2 , we see a qtl just in cross c d. by the naive approach ( given the set of crosses performed ) , we would infer the partition cs|dip , which is the partition that li et al . if we assume that there is no qtl segregating in i p , we would infer the partition ds|cip , while if we take the evidence for a qtl in i p as sufficient , we would infer the partition cp|dis , and this is the partition that li et al . for chromosome 5 , we see a qtl only in cross i p , and so by the naive approach we would infer the partition i|cdps ; this partition does have the highest posterior probability ( 83% ) and was the partition that li et al . thus , by our proposed approach , we would not infer the presence of a qtl . but if we do allow that there is a qtl , two other partitions are contained within the 95% credible set : dp|cis , with posterior probability 9% , in which case the qtl is also segregating in the cross c d , and is|cdp , with posterior probability 6% , in which case the qtl is also segregating in the cross c s. for chromosome 6 , we have significant evidence for a qtl only in cross c d ( the other three crosses have maximum lod scores of 1.51.9 on chromosome 6 ) , and so the naive method would give the partition cs|dip , which has posterior probability < 0.01% and is not contained in the 95% credible set . the partition with highest posterior probability is di|cps ( posterior probability 60% ) , in which case the qtl is also segregating in c d. the 95% credible set also contains the partition ps|cdi ( posterior probability 21% ) , in which case the qtl is segregating in c s but not c d. as with chromosome 5 , the maximum lod score across partitions ( 4.70 ) does not meet our 5% significance threshold , and so by our proposed method we would not infer the presence of a qtl . we have described a formal approach for the joint analysis of multiple crosses to map the origin of qtl alleles to a position on a phylogenetic tree . in addition , as part of this work , we have begun to evaluate a variety of experimental design issues for such research , which provides some guidance to researchers seeking to take advantage of this approach . in the presence of multiple qtl , the phenotypes of the strains can not be trusted for inferring the qtl alleles , and in the current application , the six qtl partition the five strains in diverse ways . ( 2005 ) used the pattern of qtl in the different crosses to infer the appropriate partition , which we have ( perhaps overly harshly ) characterized as the naive approach . moreover , one could combine the information from the multiple partitions to better define the location of the qtl , taking account of the uncertainty in the partition . ( 2005 ) , for the combined use of multiple crosses to more precisely map a qtl and , subsequently , with an inferred partition ( or partitions ) of strains in hand , to inform the analysis of the haplotypes of the strains ( see , for example , burgess - herbert et al . the key distinction of our method is that we seek not just to establish association but also to identify the appropriate partition and so define the origin of the mutant qtl allele on the local phylogenetic tree . in our simulation studies , we compared the use , for a fixed total sample size , of all possible crosses to different choices of a minimal set of crosses . depending on the underlying true partition of taxa at a qtl , one can choose a minimal set of crosses with considerably higher power . however , given the prior uncertainty in the true partition , and the possibility of multiple qtl that each partition the taxa differently , it is prudent to consider all or at least a larger number of possible crosses . an even more important experimental design question , which we have not considered here , is how to choose which taxa , out of a large number of related taxa , to consider , in the effort to characterize the genetic architecture of a quantitative trait . the approach could be adapted for the analysis of a set of backcrosses , although these would likely need to be of a special form , with the f1 hybrids all crossed to a common parent . the power of reconstructing qtl evolution as well as the increasing capacity for genetic mapping of complex traits and phylogenetic reconstruction should provide motivation for these extensions in the evolutionary , biomedical , and agricultural communities .

to develop methods for mapping a qtl to a phylogenetic tree , we begin with several simplifying assumptions : the taxa are represented by inbred lines , the tree relating the taxa is known without error , the quantitative trait of interest is affected by a single diallelic qtl , and there are no background effects ( i.e. the basic idea , illustrated in figure 1 , is that each possible location for the origin of a diallelic qtl on the tree corresponds to a different partition of the taxa into two groups , with the two groups corresponding to the two qtl alleles . in the case of very large crosses , with each having high power to detect the qtl , if present , we could simply consider the crosses individually and use the pattern of presence / absence of qtl to identify the correct partition of the taxa . for the case illustrated in figure 1 , with four taxa , it would be sufficient to consider the crosses a b , a c , and b d , as with just these three crosses , the five possible partitions have distinct patterns of presence / absence of the qtl . in the table on the right , we indicate the presence or absence of a qtl in each of the six possible crosses among pairs of taxa , according to the location of the qtl on the tree . given limited resources and crosses of limited size , there will be incomplete power to detect the qtl in a given cross , and so the naive approach based on the presence or absence of the qtl in the different crosses will likely be misleading . a more formal approach , in which the likelihoods for the different possible partitions are evaluated and compared , will provide a clear assessment of the evidence for the different locations for the qtl on the tree . we assume a linear model with normally distributed errorsyij=i+aij+dij+ij , where yij is the phenotype for individual j in cross i , i the average phenotype in cross i , and are the additive and dominance effects of the qtl , respectively , and the ij are independent and identically distributed normal ( 0 , ) . the aij and dij denote encodings of the qtl genotypes , with aij = dij = 0 if the qtl is not segregating in cross i. for convenience , we call the two qtl alleles defined by the partition as the high allele ( h ) and the low allele ( l ) , although we wo nt actually constrain the high allele to increase the phenotype . we may then use standard interval mapping ( lander and botstein 1989 ) or an approximation such as haley knott regression ( haley and knott 1992 ) to fit the model , estimate the parameters i , , , and , and calculate a lod score , lod( ) , where denotes the partition of the taxa and denotes the location of the putative qtl . the lod score is the log10 likelihood comparing the hypothesis of a single qtl at that location to the null hypothesis of no qtl but with the multiple crosses allowed to have separate phenotypic means , that is , yij normal ( i , ) . ( 2005 ) , in that one recodes the genotypes in the crosses in which the qtl is segregating , stacks them on top of one another , as if they were a single intercross , and performs interval mapping with cross indicators as additive covariates . assuming the presence of a single diallelic qtl on chromosome i , we assign equal prior probabilities to the different possible partitions , , treat the profile log likelihoods mi ( in which we have maximized over all nuisance parameters , including the location of the qtl on the chromosome ) as if they were true log likelihoods , and obtain posterior probabilities by taking 10mi and rescaling so that they sum to 1 . to evaluate the evidence for a qtl , we consider the maximum of the mi on chromosome i and derive a significance threshold , adjusting for the genome scan , by a stratified permutation test ( churchill and doerge 1994 ) . for example , for the four - taxon tree in figure 1 , one may consider only the five partitions that correspond to qtl locations on the tree , as in the accompanying table , or one may also consider the two additional partitions , ac|bd and ad|bc . as discussed in the introduction for example , for the case in figure 1 , if one performs only the crosses a b , a c , and a d , the ideal results perfectly discriminate among the possible locations of the qtl on the tree . with sufficient numbers of individuals in each cross , we may determine whether the qtl is segregating in a cross , which indicates that the two taxa have different qtl alleles , or is not segregating , which indicates that the two strains have the same qtl allele . if the number of taxa , n , is large , the total number of non - null partitions ( 2 1 ) will vastly exceed the number of partitions induced by the tree ( 2n 3 ) , and so there is great potential advantage in focusing on the tree partitions . we begin by comparing our proposed method to the naive approach of considering the crosses individually and comparing the pattern of presence / absence of a qtl in the crosses to what is expected for different possible partitions . we display two sets of curves for the proposed method : for the dashed curves , the power indicates that maxm exceeded the threshold and the true partition was contained within the 95% credible set ; the dotted curves are more stringent and require that the credible set contained only the true partition . estimated receiver operating characteristic ( roc ) curves for the naive method ( solid curves ) , the proposed method , with power indicating that the true partition is contained within the 95% credible set ( dashed curves ) , and the proposed method , with power indicating that the 95% credible set contains only the true partition ( dotted curves ) , in the case of four taxa , with each of the six possible intercrosses having a sample size of 75 , and a qtl responsible for 10% of the phenotypic variance in the crosses in which it is segregating . we use the same general settings as for the simulations comparing the proposed method to the naive approach , with four taxa and the true partition being either a|bcd or ab|cd , but here we vary the total sample size among 300 , 450 , and 600 individuals , and we vary the percentage phenotype variance explained by the qtl from 2.5 to 15% . the top of each figure indicates the power ( the chance that maxm exceeded its threshold and the true partition was contained in the 95% credible set ) ; the middle indicates the exact power ( the chance that maxm exceeded its threshold and that the credible set contained only the true partition ) ; the bottom indicates the false positive rate . the other curves are for the various choices of a minimal set of three crosses , with the curves in blue , red , and green corresponding to cases in which three , two , and one of the crosses are segregating the qtl , respectively . restricting the analysis to the five partitions induced by the tree has little effect on power ( compare figure s1 and figure s2 ) , but improves the chance that the credible set includes only the true partition ( compare figure s3 and figure s4 ) , and results in a somewhat lower false - positive rate ( compare figure s5 and figure s6 ) . the performance of the proposed method with different possible choices of minimal crosses is largely predicted by the number of crosses that are segregating a qtl : the solid curves of a given color ( which indicates the number of crosses segregating a qtl ) are largely coincident , but there are some differences ( red curves in figure 4 , middle right ) . ( for other sample sizes and for the analyses restricted to the partitions induced by the tree in figure 1 , see figure s9 , figure s10 , figure s11 , figure s12 , figure s13 , figure s14 , figure s15 , and figure s16 . ) detailed results on the estimated power ( top ) , exact power ( middle ) , and false positive rates ( bottom ) , for individual choices of crosses , in the case of four taxa with a total sample size of 450 , and with the qtl being responsible for 10% of the phenotypic variance in crosses in which it is segregating . in the case that the true partition is ab|cd , there are some differences among the choices of three crosses when two of the three are segregating the qtl , in terms of the chance that the 95% credible set contains only the true partition ( figure 5 , middle ) . for example , the use of the crosses a b , a c , and b d gives exact power of 50% , while the use of a b , a c , and a d gives , we need to consider the sign of the qtl effect in different crosses for the true partition and the best alternative partition ; these are shown in table 1 . if the true partition is ab|cd , with c and d having an allele that results in an increase in the phenotype , the a b cross does not segregate a qtl , while each of a c , b d , and a d have a segregating qtl with the latter taxon in each cross increasing the phenotype . with the crosses a b , a c , and a d , the best alternative partition after ab|cd would be a|bcd , in which a c and a d are also segregating the qtl , but a b should also be segregating the qtl , and note that for both partitions ab|cd and a|bcd , the qtl has effect in the same direction in the a c and a d crosses . on the other hand , with the crosses a b , a c , and b d ( which was seen to have better performance ) , in the only alternative partition with two crosses segregating a qtl , ad|bc , the two crosses should have qtl effects in opposite directions ( the a and d alleles both result in a decrease in phenotype ) , and so this should be easy to distinguish from the ab|cd partition . we display two sets of curves for the proposed method : for the dashed curves , the power indicates that maxm exceeded the threshold and the true partition was contained within the 95% credible set ; the dotted curves are more stringent and require that the credible set contained only the true partition . estimated receiver operating characteristic ( roc ) curves for the naive method ( solid curves ) , the proposed method , with power indicating that the true partition is contained within the 95% credible set ( dashed curves ) , and the proposed method , with power indicating that the 95% credible set contains only the true partition ( dotted curves ) , in the case of four taxa , with each of the six possible intercrosses having a sample size of 75 , and a qtl responsible for 10% of the phenotypic variance in the crosses in which it is segregating . we use the same general settings as for the simulations comparing the proposed method to the naive approach , with four taxa and the true partition being either a|bcd or ab|cd , but here we vary the total sample size among 300 , 450 , and 600 individuals , and we vary the percentage phenotype variance explained by the qtl from 2.5 to 15% . the top of each figure indicates the power ( the chance that maxm exceeded its threshold and the true partition was contained in the 95% credible set ) ; the middle indicates the exact power ( the chance that maxm exceeded its threshold and that the credible set contained only the true partition ) ; the bottom indicates the false positive rate . the other curves are for the various choices of a minimal set of three crosses , with the curves in blue , red , and green corresponding to cases in which three , two , and one of the crosses are segregating the qtl , respectively . restricting the analysis to the five partitions induced by the tree has little effect on power ( compare figure s1 and figure s2 ) , but improves the chance that the credible set includes only the true partition ( compare figure s3 and figure s4 ) , and results in a somewhat lower false - positive rate ( compare figure s5 and figure s6 ) . the performance of the proposed method with different possible choices of minimal crosses is largely predicted by the number of crosses that are segregating a qtl : the solid curves of a given color ( which indicates the number of crosses segregating a qtl ) are largely coincident , but there are some differences ( red curves in figure 4 , middle right ) . ( for other sample sizes and for the analyses restricted to the partitions induced by the tree in figure 1 , see figure s9 , figure s10 , figure s11 , figure s12 , figure s13 , figure s14 , figure s15 , and figure s16 . ) detailed results on the estimated power ( top ) , exact power ( middle ) , and false positive rates ( bottom ) , for individual choices of crosses , in the case of four taxa with a total sample size of 450 , and with the qtl being responsible for 10% of the phenotypic variance in crosses in which it is segregating . in the case that the true partition is ab|cd , there are some differences among the choices of three crosses when two of the three are segregating the qtl , in terms of the chance that the 95% credible set contains only the true partition ( figure 5 , middle ) . for example , the use of the crosses a b , a c , and b d gives exact power of 50% , while the use of a b , a c , and a d gives , we need to consider the sign of the qtl effect in different crosses for the true partition and the best alternative partition ; these are shown in table 1 . if the true partition is ab|cd , with c and d having an allele that results in an increase in the phenotype , the a b cross does not segregate a qtl , while each of a c , b d , and a d have a segregating qtl with the latter taxon in each cross increasing the phenotype . with the crosses a b , a c , and a d , the best alternative partition after ab|cd would be a|bcd , in which a c and a d are also segregating the qtl , but a b should also be segregating the qtl , and note that for both partitions ab|cd and a|bcd , the qtl has effect in the same direction in the a c and a d crosses . on the other hand , with the crosses a b , a c , and b d ( which was seen to have better performance ) , in the only alternative partition with two crosses segregating a qtl , ad|bc , the two crosses should have qtl effects in opposite directions ( the a and d alleles both result in a decrease in phenotype ) , and so this should be easy to distinguish from the ab|cd partition . these data concern four intercrosses among five inbred mouse strains , cast / ei ( c ) , dba/2 ( d ) , i / lnj ( i ) , pera / ei ( p ) , and 129s1/svimj ( s ) . for chromosome 1 , significant evidence for a qtl is seen in the crosses c s and d p but not in c d or i p. by the naive approach , we would infer the partition cd|ips , and this is the partition that li et al . our proposed method does give this partition the highest posterior probability ( 57% ) , but also gives reasonable weight to the alternative ps|cdi ( posterior probability 39% ) , in which case the qtl would also be segregating in the i p cross . however , by the proposed method , cs|dip has a posterior probability of only 20% , while the partition c|sdip ( in which the qtl would also be present in the c s cross ) has a posterior probability of 80% . if we assume that there is no qtl segregating in i p , we would infer the partition ds|cip , while if we take the evidence for a qtl in i p as sufficient , we would infer the partition cp|dis , and this is the partition that li et al . the latter is the partition with the highest posterior probability ( 78% ) , while the former has posterior probability 7% , and a third partition , c|dips , in which case the qtl is segregating in neither i p nor d p , has posterior probability 16% . for chromosome 5 , we see a qtl only in cross i p , and so by the naive approach we would infer the partition i|cdps ; this partition does have the highest posterior probability ( 83% ) and was the partition that li et al . but if we do allow that there is a qtl , two other partitions are contained within the 95% credible set : dp|cis , with posterior probability 9% , in which case the qtl is also segregating in the cross c d , and is|cdp , with posterior probability 6% , in which case the qtl is also segregating in the cross c s. for chromosome 6 , we have significant evidence for a qtl only in cross c d ( the other three crosses have maximum lod scores of 1.51.9 on chromosome 6 ) , and so the naive method would give the partition cs|dip , which has posterior probability < 0.01% and is not contained in the 95% credible set . the partitions with highest posterior are c|dips ( 47% ) , with the qtl also segregating in c s , and ci|dps ( 45% ) , with the qtl also segregating in c s and i p. the 95% credible set also contains a third partition , ds|cip , with posterior probability 7% . for chromosome 11 , there was significant evidence for a qtl only in the cross i p , although the cross d p has a maximum lod score of 3.16 ( corresponding to a genome - scan - adjusted p - value of 0.093 ) . the partition with highest posterior probability is di|cps ( posterior probability 60% ) , in which case the qtl is also segregating in c d. the 95% credible set also contains the partition ps|cdi ( posterior probability 21% ) , in which case the qtl is segregating in c s but not c d. as with chromosome 5 , the maximum lod score across partitions ( 4.70 ) does not meet our 5% significance threshold , and so by our proposed method we would not infer the presence of a qtl . in the presence of multiple qtl , the phenotypes of the strains can not be trusted for inferring the qtl alleles , and in the current application , the six qtl partition the five strains in diverse ways . ( 2005 ) , for the combined use of multiple crosses to more precisely map a qtl and , subsequently , with an inferred partition ( or partitions ) of strains in hand , to inform the analysis of the haplotypes of the strains ( see , for example , burgess - herbert et al . this variation includes both statistical uncertainty associated with phylogenetic inference and real phylogenetic discordance across the genome , which results from incomplete lineage sorting and introgression in recently diverged taxa ( pamilo and nei 1988 ; maddison 1997 ; pollard et al .

differential quantification of complex mixtures using high - performance liquid chromatography coupled to electrospray ionization and tandem mass spectrometry ( hplc esi ms / ms ) can help researchers study biological variation at the molecular level and gain insights into the molecular machinery of cellular activity and disease progression . researchers reveal biological variation most often by comparing two or more populations , typically collecting data for thousands of distinct molecules in each population , for example , genes , peptides , or metabolites , and then statistically analyzing the differences among populations . here a population comprises biological or technical replicates having a biological state in common , for example , healthy or diseased . ms / ms workflows aimed at discovering biological variation fall into two categories : labeled and label - free quantification strategies . labeled quantification strategies are popular because they allow researchers to analyze multiple peptide mixtures ( often derived from protein samples ) in a single hplc ( while we use peptide as a representative analyte , the set of analytes is not limited to peptides . ) researchers compute relative abundance ( fold changes ) of resulting ion intensities between the concurrently analyzed samples and determine which peptides ( or inferred proteins ) are differentially abundant . thus , labeling strategies do no scale to the level required for large - scale comparative studies , for example , a clinical study with tens , hundreds , or even thousands of biological samples . because labeled strategies do not scale , researchers are increasingly turning to label - free quantification strategies , either intensity - based ( ms1 ) or spectral counting ( ms2 ) . spectral counting is straightforward but is biased toward peptides derived from high abundance proteins because spectral counting requires multiple ms2 scans matched to each protein to obtain statistically valid results . intensity - based quantification is less straightforward than spectral counting , requiring the area under the curve computation using ms1 scans . however , intensity - based quantification is well - suited to study lower abundance peptides , which are often more interesting than higher abundance peptides . intensity - based strategies require repeatability and reproducibility , which are inherently problematic in hplc ms / ms workflows and lead to excessive false - positive and false - negative results . researchers will eventually discard the false - positive peptides via hypothesis - driven experiments , such as selected reaction monitoring ( srm ) , but false - negative results are detrimental because these candidates are never pursued . as a result , poor repeatability and reproducibility cause researchers to miss possible insights and draw incorrect inferences . ms / ms chromatographic data stem from extraneous variability , which includes systematic bias ( sample variability and instrument variability ) and complex variability . sources of sample variability include incomplete enzymatic digestion and pipetting errors made during sample preparation . instrument variability can stem from physical changes in the liquid chromatography , mass spectrometry ( ms ) hardware , or environment , including hplc performance degradation , ms calibration drift , and volatiles in the lab air that affect ionization efficiency . instrument variability is global in nature because each change similarly affects each ion s intensity in a run . it stems from signal distortion due to transient stochastic events that occur during an hplc esi ms / ms run . for example , variability in esi performance due to the mobile phase composition or flow rate fluctuations distorts the measured ion signal . it is complex because each event affects only a narrow temporal window within an hplc esi ms / ms run , where window widths vary and can overlap among runs when analyzing separate samples . normalization strategies in hplc ms / ms workflows attempt to remove systematic biases from the data before statistical inference . traditionally , normalization strategies use a combination of a global scaling function and a peptide selection method . global scaling functions include median scale , mean scale , quantile , ranking , and least - squares fitting using linear or polynomial regression . unfortunately , these global scaling functions often require a complete matrix on which to compute , specifically , no missing data . while it is possible to impute missing values , it is recommended to do so after normalization . thus , the selection of peptides for inclusion in the global scaling function is critical . peptide selection methods include : ( 1 ) common within sample ( cws ) ; ( 2 ) top l order statistics ( los ) ; ( 3 ) percentage of peptides present ( ppp ) ; and ( 4 ) peptides with rank invariant peptide ( rip ) . webb - robertson et al . developed a useful application named statistical procedure for the analyses of lc ms proteomics normalization strategies ( spans ) , which recommends the best global scaling function and peptide selection method combinations based on rigorous statistical tests . unfortunately , spans only includes global scaling functions and , regardless of the peptide selection method , global scaling functions can not capture and mitigate complex variability . although largely unexplored , complex variability during an hplc esi ms / ms run seems inevitable , even when researchers follow strict protocols . the national cancer institute s clinical proteomic tumor analysis consortium ( cptac ) studies provide an example . cptac established standard operating procedures to enable interlaboratory comparisons of proteomic studies , particularly in the context of cancer biomarker discovery . in their sixth study , they used their standard operating procedures to produce publicly available , community reference data sets generated from a yeast proteome digest with 48 spiked - in proteins ( ups1 standard from sigma aldrich ) . rudnick et al . found irregularities attributed to electrospray instability in one of the technical replicates ( sample c , replicate 2 ) from this data set generated by the instrument aliased ltq - xl - orbitrapp@65 . the chromatogram s distinctive sawtooth pattern ( supplemental figure 1 in the supporting information ) is a textbook example of complex variability . while rudnick et al . reported modestly diminished peptide identification performance for this replicate analysis , we suspected that the complex variability also diminished intensity - based peptide quantification performance . further investigating this examining the extracted chromatograms plots ( xcs ) , each representing all peptide signals in a run ( experimental procedures ) , reveals a trough in replicate 2 ( rep 2 ) ( figure 1a ) corresponding to the same time frame as the observed electrospray instability in the tic generated from the raw data ( supplemental figure 1 in the supporting information ) . unfortunately , global scaling functions fail to mitigate complex variability ( figure 1b ) . furthermore , global normalization can have unintended consequences and can adversely affect regions where no complex variability exists . figure 1b shows that two regions of the xc for rep 2 now have more extraneous variability than before normalization , potentially disguising true biological variation . complex variability in technical replicates . extracted chromatograms ( experimental procedures ) from three hplc esi ms / ms technical replicates ( cptac study 6 , ltq - xl - orbitrapp@86 ) show that even well - controlled hplc ( a ) extracted chromatogram for sample c s replicate 2 s un - normalized data ( exp c rep 2 , long - dashed red line ) contains a distinctive trough during the same time frame as the electrospray instability in its corresponding tic ( supplemental figure 1 in the supporting information ) . ( b ) same data normalized by median scale result in extracted chromatograms where the distinctive trough remains . extracted chromatograms for replicates 1 and 3 ( exp c rep 1 , solid blue line , and exp c rep 3 , short - dashed orange line ) only slightly diverge . however , replicate 2 s ( exp c rep 2 , long - dashed red line ) extracted chromatograms show that the complex variability is now exaggerated , showing the adverse effects of median scale normalization . ( c ) same data normalized by pin result in similar extracted chromatograms for each of the three replicates . pin removes the trough in replicate 2 ( exp c rep 2 , long - dashed red line ) . this demonstrates pin s ability to mitigate complex variability . in response to the shortcomings of current normalization strategies exemplified in this data from cptac , we developed proximity - based intensity normalization ( pin ) , which mitigates complex variability and systematic bias in ms1 chromatographic data . here we describe the underpinnings of this strategy and demonstrate that pin improves repeatability and reproducibility , allowing researchers to better detect biological variation from biological ms data . we collected and processed salivary endogenous peptides as previously described . in brief , we collected all saliva samples according to protocols approved by the university of minnesota institutional review board . donors declared that they were healthy nonsmokers and were free of confounding conditions . clarified saliva was prepared from fresh whole saliva samples by centrifuging at 3000 g at 4 c for 15 min , followed by 16 100 g at 4 c for 1 min . the supernatant was mixed in a 10:1 ratio with denaturing buffer consisting of 4% sds , 100 mol / l dithiothreitol , and 100 mmol / l tris , ph 7.4 . the samples were boiled for 5 min , cooled to room temperature , then added to a centrifugal filter ( amicon ultra , 0.5 ml , 10 kda , millipore ) . two hundred microliters of buffered urea ( 8 mol / l urea with 100 mmol / l tris ph 8.5 ) was added to the sample , and the mixture was centrifuged at 14 000 g at room temperature for 40 min . an additional 200 l of buffered urea was added , and the sample was centrifuged at 14 000 g at room temperature for 40 min . the filters were discarded , and the collected peptides were alkylated , by the addition of iodoacetamide in buffered urea to 50 mmol / l in the dark for 20 min . mcx ( oasis 3 cc , 60 mg , waters ) cleanup was performed by diluting the samples to 3 ml with 2% formic acid and h2o to ph 3 . the mcx columns were equilibrated with 2 ml of 1:1 methanol : water followed by the addition of the entire sample , then washed with 3 ml of 0.1% formic acid and 2 ml of methanol ; peptides were eluted with 1 ml of 95% methanol/5% ammonium hydroxide . the eluted peptides were dried in a speed - vacuum , redissolved in water , and quantified by a modified bca assay ( thermo scientific , waltham , ma ) using trypsin - digested saliva as a standard . three micrograms of peptides were further purified and concentrated using the stage - tip protocol . fresh saliva was collected from a single donor and processed for isolation of endogenous peptides . sufficient sample quantity was loaded in a single autosampler vial to run three replicate injections in succession . fresh saliva was collected from a single donor and divided into three portions . each aliquot was processed for endogenous peptide isolation with the identical protocol , placed into individual autosampler vials and analyzed in succession . fresh saliva was collected from a single donor and processed for isolation of endogenous peptides and aliquoted with increasing amounts ( 0.5 , 1.0 , 1.5 , 2.0 , 2.5 , and 3 g ) into individual vials . ms / ms on an ltq - orbitrap xl mass spectrometer ( thermo scientific , waltham , ma ) equipped with an eksigent ( eksigent technologies , redwood city , ca ) 1dlc nanoflow system and a microas autosampler . an in - house , pulled tip capillary column with a 100 m inner diameter was packed to 13 cm with magic c18aq 5 m , 200 pore particles ( michrom bioresources ) . peptide mixtures were dissolved in an aqueous solution containing 2% acn with 0.1% formic acid and then separated by a 240% acn gradient in 0.1% formic acid over 60 min at 250 nl / min . full - scan mass spectra were acquired in the orbitrap at 60 000 resolution at m / z 400 , followed by tandem mass spectrometry ( ms / ms ) in the ltq of the five most intense ions from the full scan . national cancer institute s cptac network s study 6 data set ltq - xl - orbitrapp@65 , which was downloaded from tranche , is now available at https://cptac-data-portal.georgetown.edu/cptac/public . because data acquisition for this study was performed in profile mode , we converted the .raw files to mzxml files using msconvert version 3.0.3364 specifying centroid = true . peptide signals were extracted using an in - house software application . in brief , the software takes in a list of mzxml files and processes the file sequentially . second , after all scans were processed , extracted ion chromatograms ( xics ) were constructed from the deisotoped peaks . the peptide signals m / z values and retention times were adopted from the xic s apex peak . in lieu of computing an xic s area under the curve , a peptide signal s intensity was computed by summing its xic peak intensities . we arbitrarily selected 2 min from the xics apex peak . ) finally , corresponding peptide signals were grouped across multiple analyses based on m / z and retention time tolerances . as a quality measure , we required at least two ms2 scans ( among all sequentially analyzed files ) corresponding to each peptide signal . this software and thermo .raw files used for analyses are available from the authors upon request . all ms / ms data were analyzed using sequest version 27 , rev 12 ( thermo scientific ) . first , we downloaded the yeast uniprot fasta database ( ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/proteomes/yeast.fasta.gz , february 3 , 2012 ) and the crap contaminant fasta database ( downloaded from ftp://ftp.thegpm.org/fasta/crap , february 28 , 2012 ) . the crap ups entries were replaced with entries from an updated ups fasta database ( http://www.sigmaaldrich.com/life-science/proteomics/mass-spectrometry/ups1-and-ups2-proteomic.html , february 22 , 2012 ) . the yeast and crap fasta databases were concatenated and designated as the forward database . each protein sequence was then reversed with a perl script ( matrix science , boston , ma ) , designated as the decoy database , and concatenated to the forward database . for the c versus e experiment , sequest parameters included a fragment ion mass tolerance of 1 da , oxidation of methionine as a variable modification , and 2 da mass tolerance . scaffold version 3.6.1 ( proteome software , portland , or ) was used to validate ms / ms - based peptide and protein identifications . peptide identifications were accepted if they met minimum criteria 7 ppm precursor mass tolerance , one tryptic terminus minimum , six amino acid minimum length , and 90.0% probability as specified by the peptide prophet algorithm . protein identifications were accepted if they could be established at > 80.0% probability ( protein prophet ) and contained at least one identified peptide . proteins with similar peptides that could not be differentiated based on ms / ms analysis alone were grouped to satisfy the principles of parsimony . the resulting false discovery rate ( fdr ) within scaffold was 1.4% at the protein level and 0.1% at the peptide level ( supplemental files 1 and 2 in the supporting information ) . unless otherwise specified , all statistical analyses were performed using the r statistical package , version 2.14 - 0 2011 - 10 - 31 , r.app 1.41 or 3.02 2013 - 09 - 25 . two sample t tests were conducted using the r function t.test and the default confidence level was 0.95 . prior to multivariate analyses , for example , pooled estimate of variance , data were first log - transformed to obtain a normal distribution prior to computing variance . we generated xcs by first determining each peptide s xic and then summing their recorded intensities within each scan . the resulting summed intensities ( y axis ) were plotted over time ( x axis ) using r s lowess function ( locally weighted polynomial smoother ) ; the smoothing span parameter set to 0.07 for figure 4 and 0.05 for figure 6 . linear regression normalization was performed by applying least - squares regression on minus versus average ( ma ) scatter plots using a pairwise iterative algorithm . first , the algorithm selected peptide signals using the cws method and then ma - transformed peptide signal intensities for each pair of runs using eqs 1 and 2 in supplementary note 2 in the supporting information . fitted data were generated in r with the function lm , for example , lm(ma ) , and subtracted from observed ratios with eq 3 . the data then were deconvoluted using eq 4 in supplementary note 2 in the supporting information . we performed the iteration process twice because the difference between the mean of all intensity ratios from the previous iteration was < 0.005 , as previously described . author : loess normalization was performed in r using the normalizecyclicloess function found in the limma package . the algorithm first selected peptide signals using the cws method ; next , the algorithm selected log2 transformed intensities ( as required by normalizecyclicloess ) prior to submitting them to normalizecyclicloess using default parameters . quantile normalization was performed in r using the normalizequantile function found in the limma package . the algorithm selected peptide signals using the cws method prior to normalizedquantile analysis with default parameters . the algorithm first selected signals using the cws method prior to computing the median of peptide signal intensity ratios , which was used as a normalization factor . median scale was performed by scaling peptide signal intensities values within each run by the median of peptide signals selected using the cws method . while we mathematically define pin s neighborhood construction ( see results and discussion ) , in practice we define a peptide signal s neighborhood boundary using a retention time window around the peptide signal s retention time ( figure 2 ) a static retention time window is centered at the peptide signal s retention time , plus and minus a specified time period , for example , 2 min . a dynamic retention time window is based on the width of a peptide signal s xic . once the neighborhood boundaries are established , the neighborhood is then populated with all peptide signal xic peaks within the retention time window . a peptide signal s intensity is then normalized by computing the proportion of signal within the neighborhood . the proportion is computed by dividing the peptide signal s intensity by the sum of neighborhood xic peak intensities . for all analyses discussed herein , neighborhood boundaries were determined by a dynamic retention time window corresponding to the peptide signal s xic width . pin s neighborhood construction . xics for three peptide signals ( a c ) are shown in three dimensions . here peptide signal b s neighborhood boundaries ( rt window depicted by the horizontal red lines ) are determined by its xic width . peptide signal b s neighborhood construction includes all xic peaks within its retention time window ( peaks in red and blue ) . in this case , the neighborhood includes a portion of peptide signals a s xic and a portion of peptide signal b s xic . in pin , we take a nontraditional approach . we observe that complex variability , such as esi instability , affects bounded temporal regions within chromatographic data , as opposed to systematic bias which affects the entire hplc therefore , to mitigate complex variability , pin normalizes each peptide signal s intensity by computing its proportion of intensity relative to its neighboring peptide signals ( figure 2 ) . mathematically , we define a peptide signal s neighborhood aswhere n is the set of neighboring peptide signals , n is a peptide signal , rtmin is the index of the peptide signal corresponding to the neighborhood s lower retention time boundary , and rtmax is the index of the peptide signal corresponding to the neighborhood s upper retention time boundary . with the neighborhood defined , a peptide signal s normalized intensity , that is , its proportion of neighborhood intensity , iswhere nj is the intensity of peptide signal j and ni is the intensity of peptide signal i falling within the neighborhood retention time boundaries . the premise for this new approach is that we view biological samples and hplc esi mathematically , a composition is defined as ... x of d parts is a d x 1 vector ( x1 , x2 , ... , xd ) of positive components whose sum is 1.(20 ) because the components quantities must sum to 1 , compositional data are an example of sum - constrained data . while the concept of sum - constrained data may seem foreign to most , sum - constrained data are actually quite prevalent . for example , simple percentages and parts per million ( ppm ) are sum - constrained measurements . percentages are sum - constrained because the total is constrained to 100 , and ppm measurements are sum - constrained because the total is constrained to one million . in analyzing compositional data , statistical analyses must be done with care because nave analysis of compositional data can lead to incorrect inferences . the only abundance information that remains for a single component is relative to the other components making up the whole . in other words , prior to statistical analyses , compositional data must meet two conditions : ( 1 ) the components of interest must be relatively small parts of the whole ; ( 2 ) components within the whole must remain relatively constant in size and composition . when these conditions are met , univariate statistical tests , such as a student s t test , on compositional data should not lead to incorrect inferences . the rationale for our treatment of biological samples as compositions is two - fold . first , biological systems , whether at the molecular , cellular , or organ level , are dynamic and interactive . for example , within a proteome , the presence , absence , or change in abundance of one or more proteins can affect the presence , absence , or change in abundance of one or more other proteins in the system . therefore , the abundance of a particular protein , relative to other proteins is important . as a result , researchers reveal biological variation by finding differences in a biological sample s composition . second , sample collection and preparation impose constraints on the number of proteins available for measurement . for example , aliquoting a portion of a sample , perhaps based on a bradford assay , puts a cap on the amount of protein used for comparison . as a result the rationale for our treatment of chromatographic data as compositional stems from the fact that constraints are imposed within hplc esi for example , during esi , coeluting peptides compete for a finite number of charges and a limited space on droplets . the finite number of charges and the limited space are constraints . as a result , mass spectral intensities of ionized peptides depend on their coeluting peptides , and thus we deem the resulting chromatographic data are compositional . therefore , when we treat chromatographic data resulting from the analysis of proteomic samples as compositional , we can detect statistically significant differences in proportions across populations . this compositional data meets the two prerequisite conditions for its statistical analysis : ( 1 ) the amount of a single component ( peptide ) is small relative to the whole , which remains true in its corresponding chromatographic data , and ( 2 ) in the vast majority of biological systems , the core proteome and its digested peptides , accounts for more than 90% of the measured protein mass in a sample ; that is , only 10% is compositionally different between similar biological samples . thus , when we view complex biological samples and chromatographic data as compositional , we can use simple univariate statistics ( such as student s t test ) to reveal biological variation . this reasoning stems from the fact that when using the same ( or very similar ) lc column , peptides elute in approximately the same order . as a result , retention time windows within resulting chromatograms will contain approximately the same set of peptide signals when analyzing different samples with overall similar composition . thus , this region becomes a subcomposition , which , for mathematical purposes , is just another composition . we can do so even when complex variability is present because complex variability similarly affects peptide signal intensities within close temporal proximity . finally , when we view regions ( also called neighborhoods ) of chromatographic data as compositional , we can detect statistically significant differences in peptide signal proportions across populations . this is true because the data again meet the two prerequisite conditions for comparisons of compositional data : ( 1 ) provided that neighborhood boundaries are properly set , a peptide signal s neighborhood population is large enough so that the intensity of a peptide signal remains small relative to the sum of its neighbors intensities and ( 2 ) provided that similar lc ( column type and gradient duration ) is used , neighborhoods will contain sufficiently overlapping populations . with underlying assumptions as a basis , we assessed our pin strategy s ability to mitigate systematic bias and complex variability and reveal statistically significant biological variation . as an initial evaluation , we applied pin to the cptac data described in the introduction data that alluded to the capabilities of global scaling functions and motivated our development of a new method . use of pin on this data produced the ideal result expected from normalization ( figure 1c ) : nearly identical xcs between replicates , even for the run containing the complex variability due to electrospray instability . encouraged by these results , we further evaluated pin vis - - vis median scale and other current normalization strategies . we applied several different peptide selection strategies and global scaling functions to archived data sets from four experiments : instrument variability , sample variability , serial dilution ( as a proxy for loading amount differences ) , and cptac study 6 . the results of these experiments , when taken together , demonstrate pin s superior mitigation of systematic bias and complex variability while retaining biological variability . to assess reduction in instrument variability , we generated three replicates by analyzing a single aliquot of salivary endogenous peptides using an autosampler and hplc esi ( see experimental procedures and supplemental tables 3 and 4 in the supporting information . ) first , we assessed reduction in variability between un - normalized data , data normalized by five global scaling functions , and data normalized by pin . we used two commonly employed metrics : pooled estimate of variance ( pev ) and median standard deviation coefficient of variance ( cv ) . although we evaluated numerous scaling functions , we only report pin and the five best performing normalization strategies determined by pev and cv reduction . for the global scaling functions , we employed the cws peptide selection strategy prior to normalization . for pin variability experiment , pin outperformed the five best current normalization strategies by reducing pev by 73% and cv by 46% , compared with an average of 13 and 19% respectively for the global scaling functions . four experiments show pin s superior performance in reducing variance using two commonly used measurements : pooled estimate of variance ( pev ) and coefficient of variation ( cv ) . ( a , c ) pin ( right - most column in each figure ) versus five common normalization strategies in reducing pev for each of the four experiments . ( b , d ) pin ( again , right - most column in each figure ) versus the same five common normalization strategies in reducing cv for each of the four experiments . to assess reduction in the variability resulting from sample handling , we followed the same protocol as the instrument variability experiment , except we prepared three aliquots of salivary endogenous peptides in parallel and analyzed each aliquot using an autosampler and hplc esi ( see the experimental procedures and supplemental tables 5 and 6 in the supporting information . ) again , we employed cv and pev and compared pin s results to the top five performing normalization strategies . pin continued to outperform these normalization strategies by reducing pev by 71% and cv by 41% compared with an average of 11 and 8% , respectively , for global scaling functions . to assess reduction in variability resulting from loading amount differences , we conducted serial dilution experiments using a complex mixture of salivary endogenous peptides and bradykinin as a spiked - in standard . ( see the experimental procedures and supplemental tables 7 and 8 in the supporting information . ) traditionally , researchers have conducted serial dilution experiments to determine peptide abundances in a concentrated sample or produce calibration curves . we used serial dilution experiments unconventionally , as a proxy for loading amount differences we prepared six aliquots of this mixture by combining increasing amounts ( 0.5 , 1.0 , 1.5 , 2.0 , 2.5 , and 3.0 g ) of salivary endogenous peptides with an equal amount of bradykinin ( 50 fmol ) . we again employ pev and cv and compared pin to the top five performing normalization strategies . pin reduced pev by 75% and cv by 55% compared with an average of 34 and 23% , respectively , for global scaling functions . to assess reduction in variability in the face of biological variation , we used data from the cptac study 6 data set for instrument aliased ltq - xl - orbitrapp@65 . in brief , cptac study 6 evaluated mixtures of yeast with sigma ups1 spiked in at five different levels ( a e ) , each level three times greater than the previous level . each sample was then analyzed three times by hplc esi ms / ms . we selected samples c and e because sample c contained complex variability and sample e contained nine times the amount of ups1 ( experimental procedures , supplemental tables 9 and 10 in the supporting information ) . using the c versus e data set , pin again outperformed global scaling function pin reduced pev by 61% and cv by 19% while global scaling functions , on average , pev by 9% and surprisingly increased cv by 14% ( figure 3d , fourth row ) . in this case , global scaling functions had a negative effect rather than a positive effect in normalizing intensities . to assess pin s ability to mitigate systematic bias again , we used the serial dilution data set as a proxy for loading amount differences . we chose to visualize loading amount differences because it was a classic example of systematic bias ( figure 4 and supplementary figure 2 in the supporting information ) . when we plotted the un - normalized peptide signal intensities , we observed divergent regression lines , indicative of systematic bias due to loading amount differences ( figure 4a ) . as described in supplemental note 1 in the supporting information , data with no systematic bias would result in regression lines lying on the horizontal line positioned at 0 on the y axis . we then plotted the same data after normalization using median scale as the global scaling function ( figure 4b ) . we observed that the regression lines have slightly converged and were repositioned below and above y = 0 , indicating improvement in systematic bias . finally , we plotted the data after normalization using pin ( figure 4c ) . we observed that the regression lines on the right end of the plot were then positioned on or very near y = 0 . however , we also noted that the lines remained below the horizontal flat line on the left end of the plot . unlike median scale , the regression lines converged , making the regression lines nearly indistinguishable . from these observations , we concluded that using pin performed well to mitigate the systematic bias , although a small amount remains . furthermore , we concluded that pin outperformed median scale normalization in making systematic bias consistent between runs . serial dilution - pin versus median scale minus versus average ( ma ) pots . ( a ) ma plot visually demonstrates that pin outperforms median - scale normalization in mitigating systematic bias . ( a ) ma plot of un - normalized data for six different loading amounts reveals systematic bias ( regression lines diverge from y = 0 ) . ( b ) ma plot of median scale data for six different loading amounts demonstrates a slight improvement in systematic bias ( regression lines begin to converge around y = 0 ) . ( c ) ma plot for pin normalized data for six different loading amounts demonstrates a substantial improvement in systematic bias ( regression lines converge around y = 0 ) . typically , in a serial dilution experiment , the data are normalized using an internal or spiked in peptide with the goal of determining absolute abundance and relative abundance , rather than composition . the standard metric for absolute abundance is the coefficient of correlation ( r ) , with the goal of perfect correlation ( r = 1.0 ) . the standard metric for relative abundance is fold change , with the goal of monotonically increasing fold changes corresponding perfectly to the original amounts loaded . here we expected fold changes of 0.5 , 1.0 , 1.5 , 2.0 , 2.5 , and 3.0 corresponding to the saliva peptide load amounts of 0.5 , 1.0 , 1.5 , 2.0 , 2.5 , and 3.0 g , respectively . we first plotted the un - normalized intensity for a single salivary peptide ( gpgifpppppqp ) , indicative of other salivary peptides found in each of the six serial dilution runs and computed the r and fold changes ( figure 5a , e ) . we noted that r = 0.81 , and fold changes were compressed and not monotonically increasing . we then plotted the spike - in normalized intensity ( with 500 fmol bradykinin peptide ) of the same peptide and computed the r and fold changes ( figure 5b , e ) . the measured correlation improved to r = 0.98 , and pin versus spiked - in standard normalization for a single peptide ( gpgifpppppqp ) intensity with six different sample loading amounts . ( a ) un - normalized intensity plot shows mediocre correlation ( r = 0.82 ) . ( b ) spiked - in ( bradykinin ) normalized intensity plot shows improved correlation ( r = 0.98 ) but does not remove systematic bias stemming from loading amount differences ( slope is very high ) . ( c ) pin normalized intensity plot shows decreased correlation ( r = 0.81 ) but removes systematic bias stemming from loading amount differences ( slope = 0.01 ) . ( d ) pin normalized data followed by original loading amount scaling shows improved correlation ( r = 0.99 ) , which is better than spiked - in standard normalization . ( e ) table showing fold changes for each intensity plot ( a d ) using the 1.0 g sample loading amount as the common denominator , while pin compresses the fold changes . however , when we use the serial dilution experiment as a proxy for loading amount differences , then , our goal should not be to achieve perfect correlation to absolute amounts ; rather , our goal is to find no compositional differences ( slope = 0.0 ) . this stems from the fact that even though overall loading amounts varied between these samples , within each sample the proportion of any given peptide to the whole did not change because overall composition did not change . furthermore , relative fold changes should all be equal to 1 , indicating no changes in the composition . we plotted the pin - normalized intensities for the same peptide and computed the slope and fold changes ( figure 5c , e ) . we observed that normalizing with pin achieves a slope = 0.01 , and fold changes are near 1 , indicating little change in biological variation . because we knew the loading amounts , we estimated the absolute abundance of peptides initially loaded onto the hplc column by scaling pin normalized intensities by the run s loading amount . scaling pin normalized data by the loading amount showed r = 0.99 ( figure 5d ) and monotonically increasing fold changes ( figure 5e ) . scaled pin outperformed spike - in normalization for estimating absolute peptide abundance and in estimating fold changes . we next turned our attention from a single peptide fold change to the analysis of composite xcs representing all peptide signals in a run . when we plotted the un - normalized xcs , we observed a clear separation of monotonically increasing xcs , indicating the presence of systematic bias ( figure 6a ) . however , upon closer inspection , we found that the xcs for the 2.0 g and 3.0 g samples were not in the expected order . we then plotted the spike - in normalized xc and observed that the 3.0 g xc shifted its position but still lay below the 2.0 g xc ( figure 6b ) . next we plotted the xcs for the pin normalized data and observed a convergence of the xcs ( figure 6c ) . furthermore , we observed an undulation in the xcs , consistent across the different runs . finally , we plotted the pin normalized data scaled by loading amounts and observed that the xcs became monotonically increasing and were in the correct order for nearly all time points ( figure 6d ) , with the exception of the lowest loading amounts . peptide signal extracted chromatograms pin versus spiked - in standard normalization for six different loading amounts . ( the 2.0 and 3.0 g extracted chromatograms are not in the correct order . ) ( b ) spiked - in ( bradykinin ) normalized extracted chromatogram plots show that the 2.0 g extracted chromatogram is now above the 1.0 g extracted chromatogram but is still below the 2.0 g extracted chromatogram . ( c ) pin normalized extracted chromatogram shows convergence , indicating the removal of systematic bias . ( d ) scaling pin normalized data by the original loading amounts plot shows extracted chromatograms monotonically increasing and in the correct order . finally , we assessed pin vis - - vis common normalization strategies in the context of detecting of biological variation . again , we used the cptac study 6 and selected samples c and e. here we used a student s t test to determine the number of differentially abundant ( or proportional ) ups1 and yeast proteins and peptides found . we used spans to recommend and perform the optimal peptide select strategy and global scaling function combinations . spans performed correctly , finding no global scaling functions were appropriate for the cptac c versus e data set . these results confirmed our previous findings that global scaling functions can not capture and mitigate complex variability . nonetheless , spans was run using three normalization strategies : ( 1 ) los ( 5% ) peptide selection strategy with mean global scaling ; ( 2 ) rip peptide selection strategy with mean global scaling ; and ( 3 ) rip peptide selection strategy with median global scaling ( supplemental table 11 in the supporting information ) . the up designation indicates proteins present lower abundance ( or proportion for pin ) in sample e compared with sample c. the down designation indicates lower abundance ( or proportion in pin ) in sample e compared with sample c. ( a ) ups1 proteins statistically different between samples c and e. ( b ) yeast proteins that are statistically different between samples c and e. ( c ) number of true positives , false - positives , and false - negatives for ups1 and yeast proteins . pin outperformed median and mean global scaling functions in finding statistically significant ups1 proteins ( figure 7a ) . with pin , we found 36 statistically different ups1 proteins going up ( true positives ) and only 1 ups1 protein going down in e versus c ( false - positive ) . global scaling functions performed abysmally , finding fewer ups1 proteins as statistically different compared with the un - normalized data . pin found 20% more ( 6 ) ups1 proteins as statistically different that were false negatives in the un - normalized data ( figure 7c ) . one important point is that when we treat chromatographic data as compositional data , we also measure statistically significant decreases in background yeast proteins in sample c versus e. we assert these are true positives , reflecting physical realities of these samples . our reasoning is that when viewing the proteome as compositional , then it follows that as the proportion of ups1 proteins goes up , the proportion of yeast proteins must go down . it is also well known that increasingly abundant peptides can suppress ionization of coeluting peptides , leading to decreased proportional intensities of some yeast peptides in response to increasing load of ups1 peptides . this may be even more prevalent an affect within adding relatively large amounts of the ups1 standard into the yeast background . therefore , with pin , we expect to find a larger number of yeast peptides with reduced proportion to the whole when compared with other scaling functions ( figure 7b , c ) . to our knowledge , we are the first to demonstrate that biological variation can be revealed in proteomic chromatographic data by viewing it as compositional . here we also introduce pin , a new local method that normalizes a peptide signal s intensity by computing its proportional intensity relative to its neighborhood . we show that pin dominates competing normalization strategies when measuring reduction in variability and finding biological variation , even when complex variability is present . the only related work is very recent work by lyutvinskiy et al . , which describes a new instrumental response correction method , a local normalization method , for significant variations in the eluent and analyte composition for improving accuracy of predictive models . we compared and contrasted pin with their method and found some similarities and some striking differences . both methods employ temporally bounded neighborhoods to normalize each peptide signal . however , the composition of the neighborhoods is decidedly different in the two methods . while pin takes an unbiased approach to populating its neighborhood by including all peptide signal xic peaks within a retention time window , their method takes a biased approach by populating their neighborhoods using only peptides confidently identified from ms2 spectra . furthermore , the method for correction is not clearly defined , making it difficult to evaluate . in one instance , they describe an abundance alignment method using unchanged peptides as internal standards . in another instance , they report using the square root of the median of all peptides within the retention time window . unfortunately , their report did not evaluate performance vis - - vis common normalization strategies , making it difficult to compare to our pin strategy . they did report that their normalization strategy improved their predictive model , but it is unclear to us whether the improvement stemmed from the local nature of their strategy or if other common normalization strategies would have produced similar results . regardless of the differences , we both agree on the central need to account for complex variability in intensity - based quantification . a key benefit of pin over common normalization strategies is its simultaneous mitigation of multiple types of measurement error , including systematic bias and complex variability . this benefit stems from treating chromatographic data as compositions and temporal regions within the data as subcompositions . thus , any bias affecting the composition as a whole will also affect subcompositions . as a result , when pin mitigates complex variability within the subcomposition , it inherently also mitigates systematic bias . a second benefit of pin over common normalization strategies is that pin requires no a priori knowledge of the type or source of measurement error . for example , a pipetting error doubling the amount of the sample does not necessarily correspond to a linear response in corresponding ion intensities in the resulting data . therefore , simply normalizing by a single global scaling factor , such as the median of measured ion intensities , is not accurate . computing the appropriate global scaling factor to mitigate systematic bias involves a priori knowledge of both absolute loading amounts and absolute ion counts . however , because pin treats chromatographic data as compositional , the only information about peptide abundance and ion counts is relative information ; thus , pin requires no knowledge of absolute loading amounts or absolute ion counts . a third benefit of pin over common normalization strategies utilizing global scaling functions is that pin does require a complete matrix on which to compute and thus implicitly handles the missing value problem . then , to compare a single peptide signal s pin normalized intensities ( proportions ) across runs , its neighborhoods must be compositionally similar . if needed , missing values can still be imputed after normalization but prior to statistical inference , as is recommended . of course , as with any method , pin s performance depends on some factors . first , pin relies on high - resolution instrumentation and accurate mass measurements ( < 10 ppm error ) to extract and quantify relevant chromatographic information . second , as with other methods , the peptide elution order for analyzed mixtures must be similar ; pin requires similar order to form similar neighborhoods . this means mixtures analyzed using different types of chromatographic systems may not be easily compared . third , although pin tends to compress the dynamic range of fold changes , the end results are unaffected because it is fold change statistical significance ( not numerical value ) that counts for determining biological variation . however , if the original loading amount is known , for example , in a serial dilution experiment , that information can be used to scale results and accurately compute fold changes . fourth , with any normalization method , overfitting and underfitting data is a concern . with pin , we first construct neighborhoods using a temporal window , and the width of this window primarily controls data fitting quality . we conducted several experiments ( results not shown ) varying the window width and found that varying the window width between 2 to 5 min as well setting window size to the temporal width of a true peptide signal s xic made little difference in the results . however , setting the window < 2 min and > 5 min tended to overfit and underfit the data , respectively . we expect that optimal window width will correlate somewhat with chromatographic characteristics , primarily gradient duration . therefore , we utilize the width of the peptide signal s xic as input to a function dynamically computing each peptide signal s temporal boundaries . despite pin s improvements in reducing variability and the number of false - negatives reported , ms - based results must still be confirmed via hypothesis - driven experiments , such as a western blot or targeted ms . targeted ms , for example , srm , is a powerful tool gaining popularity over traditional biochemical analyses due to the ever - increasing scale of today s high - throughput experiments . such experiments require as input a transition list or an inclusion list containing feature values ( m / z retention time pairs , which are simply peptide signals ) . because pin operates on a list of peptide signals , each with an associated ms / ms spectrum containing potential srm transitions , the software implementing pin thus , pin is well - suited to drive targeted ms experiments . given that common normalization strategies can not capture and correct systematic bias and complex variability inherently present in hplc ms / ms workflows , we expect pin to dramatically improve intensity - based quantification from hplc , although we studied complex protein digests and endogenous peptide mixtures , we believe pin will be widely applicable to many omics fields using hplc ms / ms to analyze complex mixtures , for example , lipidomics , glycomics , and metabolomics . the upshot will , we expect , be reproducibility- and repeatability - improved , and otherwise falsely reported or missed statistically significant biological variation will be discovered .

researchers are increasingly turning to label - free ms1 intensity - based quantification strategies within hplc esi ms / ms workflows to reveal biological variation at the molecule level . unfortunately , hplc esi ms / ms workflows using these strategies produce results with poor repeatability and reproducibility , primarily due to systematic bias and complex variability . while current global normalization strategies can mitigate systematic bias , they fail when faced with complex variability stemming from transient stochastic events during hplc esi ms / ms analysis . to address these problems , we developed a novel local normalization method , proximity - based intensity normalization ( pin ) , based on the analysis of compositional data . we evaluated pin against common normalization strategies . pin outperforms them in dramatically reducing variance and in identifying 20% more proteins with statistically significant abundance differences that other strategies missed . our results show the pin enables the discovery of statistically significant biological variation that otherwise is falsely reported or missed .

Introduction Experimental Procedures Results and Discussion

differential quantification of complex mixtures using high - performance liquid chromatography coupled to electrospray ionization and tandem mass spectrometry ( hplc esi ms / ms ) can help researchers study biological variation at the molecular level and gain insights into the molecular machinery of cellular activity and disease progression . researchers reveal biological variation most often by comparing two or more populations , typically collecting data for thousands of distinct molecules in each population , for example , genes , peptides , or metabolites , and then statistically analyzing the differences among populations . ms / ms workflows aimed at discovering biological variation fall into two categories : labeled and label - free quantification strategies . because labeled strategies do not scale , researchers are increasingly turning to label - free quantification strategies , either intensity - based ( ms1 ) or spectral counting ( ms2 ) . intensity - based quantification is less straightforward than spectral counting , requiring the area under the curve computation using ms1 scans . however , intensity - based quantification is well - suited to study lower abundance peptides , which are often more interesting than higher abundance peptides . intensity - based strategies require repeatability and reproducibility , which are inherently problematic in hplc ms / ms workflows and lead to excessive false - positive and false - negative results . researchers will eventually discard the false - positive peptides via hypothesis - driven experiments , such as selected reaction monitoring ( srm ) , but false - negative results are detrimental because these candidates are never pursued . as a result , poor repeatability and reproducibility cause researchers to miss possible insights and draw incorrect inferences . ms / ms chromatographic data stem from extraneous variability , which includes systematic bias ( sample variability and instrument variability ) and complex variability . it stems from signal distortion due to transient stochastic events that occur during an hplc esi ms / ms run . for example , variability in esi performance due to the mobile phase composition or flow rate fluctuations distorts the measured ion signal . it is complex because each event affects only a narrow temporal window within an hplc esi ms / ms run , where window widths vary and can overlap among runs when analyzing separate samples . normalization strategies in hplc ms / ms workflows attempt to remove systematic biases from the data before statistical inference . traditionally , normalization strategies use a combination of a global scaling function and a peptide selection method . unfortunately , these global scaling functions often require a complete matrix on which to compute , specifically , no missing data . developed a useful application named statistical procedure for the analyses of lc ms proteomics normalization strategies ( spans ) , which recommends the best global scaling function and peptide selection method combinations based on rigorous statistical tests . unfortunately , spans only includes global scaling functions and , regardless of the peptide selection method , global scaling functions can not capture and mitigate complex variability . although largely unexplored , complex variability during an hplc esi ms / ms run seems inevitable , even when researchers follow strict protocols . the chromatogram s distinctive sawtooth pattern ( supplemental figure 1 in the supporting information ) is a textbook example of complex variability . reported modestly diminished peptide identification performance for this replicate analysis , we suspected that the complex variability also diminished intensity - based peptide quantification performance . unfortunately , global scaling functions fail to mitigate complex variability ( figure 1b ) . furthermore , global normalization can have unintended consequences and can adversely affect regions where no complex variability exists . extracted chromatograms ( experimental procedures ) from three hplc esi ms / ms technical replicates ( cptac study 6 , ltq - xl - orbitrapp@86 ) show that even well - controlled hplc ( a ) extracted chromatogram for sample c s replicate 2 s un - normalized data ( exp c rep 2 , long - dashed red line ) contains a distinctive trough during the same time frame as the electrospray instability in its corresponding tic ( supplemental figure 1 in the supporting information ) . however , replicate 2 s ( exp c rep 2 , long - dashed red line ) extracted chromatograms show that the complex variability is now exaggerated , showing the adverse effects of median scale normalization . in response to the shortcomings of current normalization strategies exemplified in this data from cptac , we developed proximity - based intensity normalization ( pin ) , which mitigates complex variability and systematic bias in ms1 chromatographic data . here we describe the underpinnings of this strategy and demonstrate that pin improves repeatability and reproducibility , allowing researchers to better detect biological variation from biological ms data . ms / ms on an ltq - orbitrap xl mass spectrometer ( thermo scientific , waltham , ma ) equipped with an eksigent ( eksigent technologies , redwood city , ca ) 1dlc nanoflow system and a microas autosampler . full - scan mass spectra were acquired in the orbitrap at 60 000 resolution at m / z 400 , followed by tandem mass spectrometry ( ms / ms ) in the ltq of the five most intense ions from the full scan . finally , corresponding peptide signals were grouped across multiple analyses based on m / z and retention time tolerances . as a quality measure , we required at least two ms2 scans ( among all sequentially analyzed files ) corresponding to each peptide signal . all ms / ms data were analyzed using sequest version 27 , rev 12 ( thermo scientific ) . scaffold version 3.6.1 ( proteome software , portland , or ) was used to validate ms / ms - based peptide and protein identifications . proteins with similar peptides that could not be differentiated based on ms / ms analysis alone were grouped to satisfy the principles of parsimony . fitted data were generated in r with the function lm , for example , lm(ma ) , and subtracted from observed ratios with eq 3 . while we mathematically define pin s neighborhood construction ( see results and discussion ) , in practice we define a peptide signal s neighborhood boundary using a retention time window around the peptide signal s retention time ( figure 2 ) a static retention time window is centered at the peptide signal s retention time , plus and minus a specified time period , for example , 2 min . a dynamic retention time window is based on the width of a peptide signal s xic . we observe that complex variability , such as esi instability , affects bounded temporal regions within chromatographic data , as opposed to systematic bias which affects the entire hplc therefore , to mitigate complex variability , pin normalizes each peptide signal s intensity by computing its proportion of intensity relative to its neighboring peptide signals ( figure 2 ) . mathematically , we define a peptide signal s neighborhood aswhere n is the set of neighboring peptide signals , n is a peptide signal , rtmin is the index of the peptide signal corresponding to the neighborhood s lower retention time boundary , and rtmax is the index of the peptide signal corresponding to the neighborhood s upper retention time boundary . the premise for this new approach is that we view biological samples and hplc esi mathematically , a composition is defined as ... x of d parts is a d x 1 vector ( x1 , x2 , ... , xd ) of positive components whose sum is 1. (20 ) because the components quantities must sum to 1 , compositional data are an example of sum - constrained data . in analyzing compositional data , statistical analyses must be done with care because nave analysis of compositional data can lead to incorrect inferences . for example , within a proteome , the presence , absence , or change in abundance of one or more proteins can affect the presence , absence , or change in abundance of one or more other proteins in the system . as a result , researchers reveal biological variation by finding differences in a biological sample s composition . for example , aliquoting a portion of a sample , perhaps based on a bradford assay , puts a cap on the amount of protein used for comparison . as a result the rationale for our treatment of chromatographic data as compositional stems from the fact that constraints are imposed within hplc esi for example , during esi , coeluting peptides compete for a finite number of charges and a limited space on droplets . therefore , when we treat chromatographic data resulting from the analysis of proteomic samples as compositional , we can detect statistically significant differences in proportions across populations . thus , when we view complex biological samples and chromatographic data as compositional , we can use simple univariate statistics ( such as student s t test ) to reveal biological variation . finally , when we view regions ( also called neighborhoods ) of chromatographic data as compositional , we can detect statistically significant differences in peptide signal proportions across populations . this is true because the data again meet the two prerequisite conditions for comparisons of compositional data : ( 1 ) provided that neighborhood boundaries are properly set , a peptide signal s neighborhood population is large enough so that the intensity of a peptide signal remains small relative to the sum of its neighbors intensities and ( 2 ) provided that similar lc ( column type and gradient duration ) is used , neighborhoods will contain sufficiently overlapping populations . with underlying assumptions as a basis , we assessed our pin strategy s ability to mitigate systematic bias and complex variability and reveal statistically significant biological variation . use of pin on this data produced the ideal result expected from normalization ( figure 1c ) : nearly identical xcs between replicates , even for the run containing the complex variability due to electrospray instability . encouraged by these results , we further evaluated pin vis - - vis median scale and other current normalization strategies . the results of these experiments , when taken together , demonstrate pin s superior mitigation of systematic bias and complex variability while retaining biological variability . to assess reduction in instrument variability , we generated three replicates by analyzing a single aliquot of salivary endogenous peptides using an autosampler and hplc esi ( see experimental procedures and supplemental tables 3 and 4 in the supporting information . ) although we evaluated numerous scaling functions , we only report pin and the five best performing normalization strategies determined by pev and cv reduction . for pin variability experiment , pin outperformed the five best current normalization strategies by reducing pev by 73% and cv by 46% , compared with an average of 13 and 19% respectively for the global scaling functions . ( a , c ) pin ( right - most column in each figure ) versus five common normalization strategies in reducing pev for each of the four experiments . ( b , d ) pin ( again , right - most column in each figure ) versus the same five common normalization strategies in reducing cv for each of the four experiments . to assess reduction in the variability resulting from sample handling , we followed the same protocol as the instrument variability experiment , except we prepared three aliquots of salivary endogenous peptides in parallel and analyzed each aliquot using an autosampler and hplc esi ( see the experimental procedures and supplemental tables 5 and 6 in the supporting information . ) again , we employed cv and pev and compared pin s results to the top five performing normalization strategies . to assess reduction in variability in the face of biological variation , we used data from the cptac study 6 data set for instrument aliased ltq - xl - orbitrapp@65 . each sample was then analyzed three times by hplc esi ms / ms . to assess pin s ability to mitigate systematic bias again , we used the serial dilution data set as a proxy for loading amount differences . we chose to visualize loading amount differences because it was a classic example of systematic bias ( figure 4 and supplementary figure 2 in the supporting information ) . when we plotted the un - normalized peptide signal intensities , we observed divergent regression lines , indicative of systematic bias due to loading amount differences ( figure 4a ) . as described in supplemental note 1 in the supporting information , data with no systematic bias would result in regression lines lying on the horizontal line positioned at 0 on the y axis . we observed that the regression lines on the right end of the plot were then positioned on or very near y = 0 . however , we also noted that the lines remained below the horizontal flat line on the left end of the plot . from these observations , we concluded that using pin performed well to mitigate the systematic bias , although a small amount remains . furthermore , we concluded that pin outperformed median scale normalization in making systematic bias consistent between runs . ( a ) ma plot visually demonstrates that pin outperforms median - scale normalization in mitigating systematic bias . ( b ) spiked - in ( bradykinin ) normalized intensity plot shows improved correlation ( r = 0.98 ) but does not remove systematic bias stemming from loading amount differences ( slope is very high ) . ( c ) pin normalized intensity plot shows decreased correlation ( r = 0.81 ) but removes systematic bias stemming from loading amount differences ( slope = 0.01 ) . we next turned our attention from a single peptide fold change to the analysis of composite xcs representing all peptide signals in a run . when we plotted the un - normalized xcs , we observed a clear separation of monotonically increasing xcs , indicating the presence of systematic bias ( figure 6a ) . finally , we plotted the pin normalized data scaled by loading amounts and observed that the xcs became monotonically increasing and were in the correct order for nearly all time points ( figure 6d ) , with the exception of the lowest loading amounts . ( d ) scaling pin normalized data by the original loading amounts plot shows extracted chromatograms monotonically increasing and in the correct order . finally , we assessed pin vis - - vis common normalization strategies in the context of detecting of biological variation . one important point is that when we treat chromatographic data as compositional data , we also measure statistically significant decreases in background yeast proteins in sample c versus e. we assert these are true positives , reflecting physical realities of these samples . therefore , with pin , we expect to find a larger number of yeast peptides with reduced proportion to the whole when compared with other scaling functions ( figure 7b , c ) . to our knowledge , we are the first to demonstrate that biological variation can be revealed in proteomic chromatographic data by viewing it as compositional . we show that pin dominates competing normalization strategies when measuring reduction in variability and finding biological variation , even when complex variability is present . , which describes a new instrumental response correction method , a local normalization method , for significant variations in the eluent and analyte composition for improving accuracy of predictive models . unfortunately , their report did not evaluate performance vis - - vis common normalization strategies , making it difficult to compare to our pin strategy . they did report that their normalization strategy improved their predictive model , but it is unclear to us whether the improvement stemmed from the local nature of their strategy or if other common normalization strategies would have produced similar results . regardless of the differences , we both agree on the central need to account for complex variability in intensity - based quantification . a key benefit of pin over common normalization strategies is its simultaneous mitigation of multiple types of measurement error , including systematic bias and complex variability . as a result , when pin mitigates complex variability within the subcomposition , it inherently also mitigates systematic bias . a second benefit of pin over common normalization strategies is that pin requires no a priori knowledge of the type or source of measurement error . computing the appropriate global scaling factor to mitigate systematic bias involves a priori knowledge of both absolute loading amounts and absolute ion counts . a third benefit of pin over common normalization strategies utilizing global scaling functions is that pin does require a complete matrix on which to compute and thus implicitly handles the missing value problem . of course , as with any method , pin s performance depends on some factors . third , although pin tends to compress the dynamic range of fold changes , the end results are unaffected because it is fold change statistical significance ( not numerical value ) that counts for determining biological variation . fourth , with any normalization method , overfitting and underfitting data is a concern . with pin , we first construct neighborhoods using a temporal window , and the width of this window primarily controls data fitting quality . because pin operates on a list of peptide signals , each with an associated ms / ms spectrum containing potential srm transitions , the software implementing pin thus , pin is well - suited to drive targeted ms experiments . given that common normalization strategies can not capture and correct systematic bias and complex variability inherently present in hplc ms / ms workflows , we expect pin to dramatically improve intensity - based quantification from hplc , although we studied complex protein digests and endogenous peptide mixtures , we believe pin will be widely applicable to many omics fields using hplc ms / ms to analyze complex mixtures , for example , lipidomics , glycomics , and metabolomics . the upshot will , we expect , be reproducibility- and repeatability - improved , and otherwise falsely reported or missed statistically significant biological variation will be discovered .

differential quantification of complex mixtures using high - performance liquid chromatography coupled to electrospray ionization and tandem mass spectrometry ( hplc esi ms / ms ) can help researchers study biological variation at the molecular level and gain insights into the molecular machinery of cellular activity and disease progression . researchers reveal biological variation most often by comparing two or more populations , typically collecting data for thousands of distinct molecules in each population , for example , genes , peptides , or metabolites , and then statistically analyzing the differences among populations . ms / ms workflows aimed at discovering biological variation fall into two categories : labeled and label - free quantification strategies . labeled quantification strategies are popular because they allow researchers to analyze multiple peptide mixtures ( often derived from protein samples ) in a single hplc ( while we use peptide as a representative analyte , the set of analytes is not limited to peptides . ) researchers compute relative abundance ( fold changes ) of resulting ion intensities between the concurrently analyzed samples and determine which peptides ( or inferred proteins ) are differentially abundant . thus , labeling strategies do no scale to the level required for large - scale comparative studies , for example , a clinical study with tens , hundreds , or even thousands of biological samples . however , intensity - based quantification is well - suited to study lower abundance peptides , which are often more interesting than higher abundance peptides . intensity - based strategies require repeatability and reproducibility , which are inherently problematic in hplc ms / ms workflows and lead to excessive false - positive and false - negative results . instrument variability can stem from physical changes in the liquid chromatography , mass spectrometry ( ms ) hardware , or environment , including hplc performance degradation , ms calibration drift , and volatiles in the lab air that affect ionization efficiency . peptide selection methods include : ( 1 ) common within sample ( cws ) ; ( 2 ) top l order statistics ( los ) ; ( 3 ) percentage of peptides present ( ppp ) ; and ( 4 ) peptides with rank invariant peptide ( rip ) . developed a useful application named statistical procedure for the analyses of lc ms proteomics normalization strategies ( spans ) , which recommends the best global scaling function and peptide selection method combinations based on rigorous statistical tests . unfortunately , spans only includes global scaling functions and , regardless of the peptide selection method , global scaling functions can not capture and mitigate complex variability . cptac established standard operating procedures to enable interlaboratory comparisons of proteomic studies , particularly in the context of cancer biomarker discovery . in their sixth study , they used their standard operating procedures to produce publicly available , community reference data sets generated from a yeast proteome digest with 48 spiked - in proteins ( ups1 standard from sigma aldrich ) . reported modestly diminished peptide identification performance for this replicate analysis , we suspected that the complex variability also diminished intensity - based peptide quantification performance . further investigating this examining the extracted chromatograms plots ( xcs ) , each representing all peptide signals in a run ( experimental procedures ) , reveals a trough in replicate 2 ( rep 2 ) ( figure 1a ) corresponding to the same time frame as the observed electrospray instability in the tic generated from the raw data ( supplemental figure 1 in the supporting information ) . extracted chromatograms ( experimental procedures ) from three hplc esi ms / ms technical replicates ( cptac study 6 , ltq - xl - orbitrapp@86 ) show that even well - controlled hplc ( a ) extracted chromatogram for sample c s replicate 2 s un - normalized data ( exp c rep 2 , long - dashed red line ) contains a distinctive trough during the same time frame as the electrospray instability in its corresponding tic ( supplemental figure 1 in the supporting information ) . in response to the shortcomings of current normalization strategies exemplified in this data from cptac , we developed proximity - based intensity normalization ( pin ) , which mitigates complex variability and systematic bias in ms1 chromatographic data . here we describe the underpinnings of this strategy and demonstrate that pin improves repeatability and reproducibility , allowing researchers to better detect biological variation from biological ms data . the mcx columns were equilibrated with 2 ml of 1:1 methanol : water followed by the addition of the entire sample , then washed with 3 ml of 0.1% formic acid and 2 ml of methanol ; peptides were eluted with 1 ml of 95% methanol/5% ammonium hydroxide . fresh saliva was collected from a single donor and processed for isolation of endogenous peptides and aliquoted with increasing amounts ( 0.5 , 1.0 , 1.5 , 2.0 , 2.5 , and 3 g ) into individual vials . first , we downloaded the yeast uniprot fasta database ( ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/proteomes/yeast.fasta.gz , february 3 , 2012 ) and the crap contaminant fasta database ( downloaded from ftp://ftp.thegpm.org/fasta/crap , february 28 , 2012 ) . first , the algorithm selected peptide signals using the cws method and then ma - transformed peptide signal intensities for each pair of runs using eqs 1 and 2 in supplementary note 2 in the supporting information . the algorithm first selected peptide signals using the cws method ; next , the algorithm selected log2 transformed intensities ( as required by normalizecyclicloess ) prior to submitting them to normalizecyclicloess using default parameters . the algorithm first selected signals using the cws method prior to computing the median of peptide signal intensity ratios , which was used as a normalization factor . while we mathematically define pin s neighborhood construction ( see results and discussion ) , in practice we define a peptide signal s neighborhood boundary using a retention time window around the peptide signal s retention time ( figure 2 ) a static retention time window is centered at the peptide signal s retention time , plus and minus a specified time period , for example , 2 min . we observe that complex variability , such as esi instability , affects bounded temporal regions within chromatographic data , as opposed to systematic bias which affects the entire hplc therefore , to mitigate complex variability , pin normalizes each peptide signal s intensity by computing its proportion of intensity relative to its neighboring peptide signals ( figure 2 ) . mathematically , we define a peptide signal s neighborhood aswhere n is the set of neighboring peptide signals , n is a peptide signal , rtmin is the index of the peptide signal corresponding to the neighborhood s lower retention time boundary , and rtmax is the index of the peptide signal corresponding to the neighborhood s upper retention time boundary . with the neighborhood defined , a peptide signal s normalized intensity , that is , its proportion of neighborhood intensity , iswhere nj is the intensity of peptide signal j and ni is the intensity of peptide signal i falling within the neighborhood retention time boundaries . the premise for this new approach is that we view biological samples and hplc esi mathematically , a composition is defined as ... x of d parts is a d x 1 vector ( x1 , x2 , ... , xd ) of positive components whose sum is 1. in other words , prior to statistical analyses , compositional data must meet two conditions : ( 1 ) the components of interest must be relatively small parts of the whole ; ( 2 ) components within the whole must remain relatively constant in size and composition . for example , within a proteome , the presence , absence , or change in abundance of one or more proteins can affect the presence , absence , or change in abundance of one or more other proteins in the system . as a result the rationale for our treatment of chromatographic data as compositional stems from the fact that constraints are imposed within hplc esi for example , during esi , coeluting peptides compete for a finite number of charges and a limited space on droplets . therefore , when we treat chromatographic data resulting from the analysis of proteomic samples as compositional , we can detect statistically significant differences in proportions across populations . this compositional data meets the two prerequisite conditions for its statistical analysis : ( 1 ) the amount of a single component ( peptide ) is small relative to the whole , which remains true in its corresponding chromatographic data , and ( 2 ) in the vast majority of biological systems , the core proteome and its digested peptides , accounts for more than 90% of the measured protein mass in a sample ; that is , only 10% is compositionally different between similar biological samples . thus , when we view complex biological samples and chromatographic data as compositional , we can use simple univariate statistics ( such as student s t test ) to reveal biological variation . finally , when we view regions ( also called neighborhoods ) of chromatographic data as compositional , we can detect statistically significant differences in peptide signal proportions across populations . this is true because the data again meet the two prerequisite conditions for comparisons of compositional data : ( 1 ) provided that neighborhood boundaries are properly set , a peptide signal s neighborhood population is large enough so that the intensity of a peptide signal remains small relative to the sum of its neighbors intensities and ( 2 ) provided that similar lc ( column type and gradient duration ) is used , neighborhoods will contain sufficiently overlapping populations . as an initial evaluation , we applied pin to the cptac data described in the introduction data that alluded to the capabilities of global scaling functions and motivated our development of a new method . to assess reduction in instrument variability , we generated three replicates by analyzing a single aliquot of salivary endogenous peptides using an autosampler and hplc esi ( see experimental procedures and supplemental tables 3 and 4 in the supporting information . ) for pin variability experiment , pin outperformed the five best current normalization strategies by reducing pev by 73% and cv by 46% , compared with an average of 13 and 19% respectively for the global scaling functions . to assess reduction in the variability resulting from sample handling , we followed the same protocol as the instrument variability experiment , except we prepared three aliquots of salivary endogenous peptides in parallel and analyzed each aliquot using an autosampler and hplc esi ( see the experimental procedures and supplemental tables 5 and 6 in the supporting information . ) we used serial dilution experiments unconventionally , as a proxy for loading amount differences we prepared six aliquots of this mixture by combining increasing amounts ( 0.5 , 1.0 , 1.5 , 2.0 , 2.5 , and 3.0 g ) of salivary endogenous peptides with an equal amount of bradykinin ( 50 fmol ) . to assess reduction in variability in the face of biological variation , we used data from the cptac study 6 data set for instrument aliased ltq - xl - orbitrapp@65 . in brief , cptac study 6 evaluated mixtures of yeast with sigma ups1 spiked in at five different levels ( a e ) , each level three times greater than the previous level . using the c versus e data set , pin again outperformed global scaling function pin reduced pev by 61% and cv by 19% while global scaling functions , on average , pev by 9% and surprisingly increased cv by 14% ( figure 3d , fourth row ) . when we plotted the un - normalized peptide signal intensities , we observed divergent regression lines , indicative of systematic bias due to loading amount differences ( figure 4a ) . we observed that the regression lines on the right end of the plot were then positioned on or very near y = 0 . the standard metric for absolute abundance is the coefficient of correlation ( r ) , with the goal of perfect correlation ( r = 1.0 ) . here we expected fold changes of 0.5 , 1.0 , 1.5 , 2.0 , 2.5 , and 3.0 corresponding to the saliva peptide load amounts of 0.5 , 1.0 , 1.5 , 2.0 , 2.5 , and 3.0 g , respectively . we first plotted the un - normalized intensity for a single salivary peptide ( gpgifpppppqp ) , indicative of other salivary peptides found in each of the six serial dilution runs and computed the r and fold changes ( figure 5a , e ) . we then plotted the spike - in normalized intensity ( with 500 fmol bradykinin peptide ) of the same peptide and computed the r and fold changes ( figure 5b , e ) . the measured correlation improved to r = 0.98 , and pin versus spiked - in standard normalization for a single peptide ( gpgifpppppqp ) intensity with six different sample loading amounts . however , when we use the serial dilution experiment as a proxy for loading amount differences , then , our goal should not be to achieve perfect correlation to absolute amounts ; rather , our goal is to find no compositional differences ( slope = 0.0 ) . because we knew the loading amounts , we estimated the absolute abundance of peptides initially loaded onto the hplc column by scaling pin normalized intensities by the run s loading amount . when we plotted the un - normalized xcs , we observed a clear separation of monotonically increasing xcs , indicating the presence of systematic bias ( figure 6a ) . however , upon closer inspection , we found that the xcs for the 2.0 g and 3.0 g samples were not in the expected order . we then plotted the spike - in normalized xc and observed that the 3.0 g xc shifted its position but still lay below the 2.0 g xc ( figure 6b ) . finally , we plotted the pin normalized data scaled by loading amounts and observed that the xcs became monotonically increasing and were in the correct order for nearly all time points ( figure 6d ) , with the exception of the lowest loading amounts . ( b ) spiked - in ( bradykinin ) normalized extracted chromatogram plots show that the 2.0 g extracted chromatogram is now above the 1.0 g extracted chromatogram but is still below the 2.0 g extracted chromatogram . finally , we assessed pin vis - - vis common normalization strategies in the context of detecting of biological variation . again , we used the cptac study 6 and selected samples c and e. here we used a student s t test to determine the number of differentially abundant ( or proportional ) ups1 and yeast proteins and peptides found . nonetheless , spans was run using three normalization strategies : ( 1 ) los ( 5% ) peptide selection strategy with mean global scaling ; ( 2 ) rip peptide selection strategy with mean global scaling ; and ( 3 ) rip peptide selection strategy with median global scaling ( supplemental table 11 in the supporting information ) . the up designation indicates proteins present lower abundance ( or proportion for pin ) in sample e compared with sample c. the down designation indicates lower abundance ( or proportion in pin ) in sample e compared with sample c. ( a ) ups1 proteins statistically different between samples c and e. ( b ) yeast proteins that are statistically different between samples c and e. ( c ) number of true positives , false - positives , and false - negatives for ups1 and yeast proteins . one important point is that when we treat chromatographic data as compositional data , we also measure statistically significant decreases in background yeast proteins in sample c versus e. we assert these are true positives , reflecting physical realities of these samples . our reasoning is that when viewing the proteome as compositional , then it follows that as the proportion of ups1 proteins goes up , the proportion of yeast proteins must go down . therefore , with pin , we expect to find a larger number of yeast peptides with reduced proportion to the whole when compared with other scaling functions ( figure 7b , c ) . to our knowledge , we are the first to demonstrate that biological variation can be revealed in proteomic chromatographic data by viewing it as compositional . we conducted several experiments ( results not shown ) varying the window width and found that varying the window width between 2 to 5 min as well setting window size to the temporal width of a true peptide signal s xic made little difference in the results . therefore , we utilize the width of the peptide signal s xic as input to a function dynamically computing each peptide signal s temporal boundaries . given that common normalization strategies can not capture and correct systematic bias and complex variability inherently present in hplc ms / ms workflows , we expect pin to dramatically improve intensity - based quantification from hplc , although we studied complex protein digests and endogenous peptide mixtures , we believe pin will be widely applicable to many omics fields using hplc ms / ms to analyze complex mixtures , for example , lipidomics , glycomics , and metabolomics .

the identity of a human being should be preserved even after death , as there are consequences , often financial , that lead to the court of law , which demands that the identity of the deceased be established before a verdict is passed . forensic science based evidence is accepted in a judicial setting by the court and plays a major role in the identification of individuals who can not be identified visually or by other simple means . however , it is difficult to perform after marked post - mortem changes such as decomposition and skeletonization have taken place due to environmental factors such as humidity , temperature , and exposure to microorganisms . nevertheless , a post - mortem is obligatory in terms of the law and social norms . the identification process involves anthropological analysis for sexing skeletal material , which provides relatively fast and accurate data that help the police investigator narrow down his field of search to a limited geographic area or within a gender . gender has been determined from pelvis , skull , and long bones , with assessment of epiphysis and metaphysis in unknown skeletons . the distance between the basion and the prosthion , the circumference of the head , the length of the supraorbital ridge , mastoid process , and mandibular ramus , the shape and length of palate , the circumference of occipital condyle , the sizes of teeth , the length and height of head , the distance between the basion and nasion , the height of the mandibular symphysis , the foramen magnum , the sphenoidal sinus , the sella turcica , and the frontal sinuses have also been used recently for gender determination in unknown remains . it has been reported that maxillary sinuses remain intact despite the skull and other bones getting badly disfigured in victims who are incinerated . jovinic stated that maxillary sinuses reach their mature sizes at about the age of 20 years . during adulthood , their shapes and sizes the size of the maxillary sinus can be affected due to environmental factors , genetic diseases , or post infections . as the image represents a contiguous series of cross - sections and three - dimensional information and the machine is available in most of the hospitals , ct has been applied in the study of fossil skulls . ct scan can be used for determination of gender by measurement of maxillary sinus when other methods are inconclusive , though this method is not error - free . the aim of the study was to determine the size [ mediolateral ( ml ) , superoinferior ( si ) , and anteroposterior ( ap ) linear dimensions ] and the volume of the maxillary sinuses using ct scanning , and investigate whether these parameters can be used to determine the gender of an individual for forensic identification . the study was designed to measure the ml , si , and ap dimensions along with the volumes ( v ) of both the right and the left maxillary sinuses of an individual using ct . the study was carried out on a small regional population of patients presenting to the imaging center of institute of dental studies and technologies , modinagar , western uttar pradesh , india , between december 2009 and august 2011 . patients were selected on the basis of strict inclusion and exclusion criteria that favored intact maxillary sinuses without any pathological , physiological , or surgical deformity . thirty patients ( 15 male and 15 female ) were included in the study , who were between 20 and 50 years of age , had retained all their permanent teeth , and were referred to the imaging center to have a ct scan of the paranasal sinuses ( ct pns ) for various reasons , in which no pathological findings were detected on ct images . patients who were found to have maxillary sinus pathology on ct scan , facial deformities involving the maxilla , clinical facial asymmetries , or had previously undergone a surgery of the maxillary sinus were excluded from the study . non - contrast coronal ct scan was performed on all patients to visualize the maxillary sinuses using ge ct / e dual slice ct scanner ( ge healthcare technologies , waukesha , wi , usa ) . prior to the scan , the patient was instructed to remove all metallic objects including hair pins , jewelry , and so on from the head and neck region and positioned on the ct table in prone position . the patient 's neck was hyper - extended with the chin resting on a pad . for stabilization , sections of 3 mm thickness were planned on the preliminary scout view extending from the anterior margin of the frontal sinus to the posterior margin of the sphenoid sinus , with a reconstruction matrix size of 512 512 at 120 kv , 100 ma . coronal ct was performed after instructing the patient to remain steady during the entire procedure . for the purpose of standardization , all scans were performed by the same operator on the same machine using similar exposure parameters . the ct image stack thus acquired was transferred to the advantage workstation 5 ( ge healthcare technologies ) for post processing . the ml and si measurements were made where the maxillary sinus was in its widest position , with the help of the on - screen linear measurement tool on the ct workstation . the linear distance between the two points was annotated on the screen [ figure 1 ] . to measure the ap dimension of the maxillary sinus , the first and the last appearance of the sinus was noted in the sequential coronal ct sections , and the number of sections between them was determined . finally , the number of sections obtained was multiplied by 3 ( thickness of a single section ) to find the ap dimension of the sinus . linear measurement of mediolateral and superoinferior dimensions of maxillary sinus maxillary sinus volume ( v ) was calculated using the paint on slices tool on the workstation . to define a volume , the outline of the sinus was traced manually on each slice of the image stack using the on - screen mouse pointer in the coronal plane [ figure 2 ] . once the tracing was complete , the workstation automatically segmented the entire volume of the sinus from the surrounding structures and the segmented portion could be visualized and manipulated in 3d [ figure 3 ] . tool at this point , switching to the histogram view on the workstation [ figure 4 ] automatically reflected the volume of the sinus in cubic centimeters ( cc ) . the entire procedure was repeated for the right and the left maxillary sinuses separately for every patient . workstation showing the sinus volume the t - test for independent samples was used to compare these values in two groups . discriminative analysis was used to detect the gender by using the data obtained from ct scans . the patients were distributed into three age groups starting from 20 to 50 years with a class interval of 10 years . the mean , along with the standard deviation was calculated for all the dimensions of the right and the left maxillary sinuses , namely ml , si , and ap , for both males and females [ table 1 ] . distribution of maxillary sinus dimensions measured on ct and their standard deviation for the right maxillary sinus , the mean value of the ml dimension was found to be 27.53 4.26 mm in males and 25.12 6.75 mm in females . the mean of si dimension was 38.21 5.77 mm in males and 33.34 6.57 mm in females . also , the mean of ap dimension was 42.60 3.79 mm in males and 36.00 4.09 mm in females . for the left maxillary sinus , the mean value of the ml dimension was found to be 27.01 5.04 mm in males and 23.22 6.21 mm in females . the mean value of the si dimension was 36.99 4.45 mm in males and 33.11 6.71 mm in females . also , the mean value of ap was 40.80 2.73 mm in males and 37.20 2.96 mm in females . a statistically significant ( p < 0.05 ) difference was found in the right si dimension , left si dimension , and the left ap dimension of the left maxillary sinuses between males and females . a significant ( p < 0.01 ) difference was found in the right ap dimension of the maxillary sinus between males and females . the other maxillary sinus dimensions showed a pattern of being larger in males than in females ; however , the difference was statistically insignificant . a comparison was made between the dimensions of the right and left maxillary sinus within males and females separately [ table 2 ] . on non - statistical comparison in males , the right maxillary sinus gave an impression of being slightly larger than the left maxillary sinus in its overall dimensions . similarly , in females , the right maxillary sinus was marginally larger in dimensions than the left maxillary sinus , with the exception of the ap dimension . aberrantly , the ap dimension of right maxillary sinus was found to be less than that of the left maxillary sinus in females . however , these intra - gender findings were statistically insignificant ( p > 0.05 ) . distribution of right and left maxillary sinus dimensions in males ( n=15 ) and females ( n=15 ) the mean with standard deviation was calculated for the volume of the right ( vr ) and the left ( vl ) maxillary sinuses for both males and females [ table 3 ] . for the right maxillary sinus , the mean volume was found to be 16.63 4.54 cc in males and 11.61 5.15 cc in females . for the left maxillary sinus , the mean volume was found to be 15.19 3.94 cc in males and 10.95 4.98 cc in females . distribution of maxillary sinus volume measured on ct and its standard deviation the volume of the maxillary sinuses in males was overall larger than in females . a significant ( p < 0.01 ) statistical difference was found in the right maxillary sinus volume between males and females . a significant ( p < 0.05 ) difference was found in the left maxillary sinus volume between males and females . a comparison was made between the volume of the right and left maxillary sinus within males and females separately [ table 4 ] . overall , both in males and females , the right maxillary sinus volume was found to be larger than the left maxillary sinus volume . however , this intra - gender volume difference was statistically insignificant ( p > 0.05 ) . distribution of right and left maxillary sinus volume in males and females a discriminative analysis was performed using the spss software to determine whether these linear measurements and volume could be used for gender determination . the following formula could be used for gender determination from measurements of the right maxillary sinus : gender = 5.116 - 0.159mlr - 0.014 sir + 0.171 apr + 0.218 vr where mlr is the ml dimension of the right maxillary sinus , sir is the si dimension of the right maxillary sinus , and apr is the ap dimension of the right maxillary sinus . the accuracy of gender predicted using the linear measurements and volume of the right maxillary sinus was found to be 80.0% in both males and females . the following formula can be used for gender determination from measurements of the left maxillary sinus : gender = 0.720 - 0.258 mll - 0.146 sil + 0.120 apl + 0.586 vl where mll is the ml dimension of the left maxillary sinus , sil is the si dimension of the left maxillary sinus , and apl is the ap dimension of the left maxillary sinus . the accuracy of gender predicted from the linear measurements and volume of the left maxillary sinus was found to be 66.7% in males and 80.0% in females , with an overall gender prediction accuracy of 73.3% . the following formula can be used for gender determination from measurements of the right and the left maxillary sinuses together : gender = 4.033 - 0.101 mlr - 0.21 sir + 0.397 apr + 0.118 vr - 0.23 mll - 0.014 sil - 0.417 apl + 0.358 vl the accuracy of gender predicted from the linear measurements and volume of the right and the left maxillary sinuses together was found to be 80.0% in males and 86.7% in females . gender determination from measurements of right and left maxillary sinuses when the variables in the formulae derived above are substituted with the desired values , a numeric value is obtained for the gender . when this value is positive , the gender is predicted to be male . a negative value is predicted to be female . it was observed that the accuracy of gender prediction from the measurements of the right maxillary sinus ( 80.0% ) was more than that of the left maxillary sinus ( 73.3% ) . when the measurements of the right and the left maxillary sinuses were accounted together for predicting the gender , the accuracy rate increased to 83.3% . the mean , along with the standard deviation was calculated for all the dimensions of the right and the left maxillary sinuses , namely ml , si , and ap , for both males and females [ table 1 ] . distribution of maxillary sinus dimensions measured on ct and their standard deviation for the right maxillary sinus , the mean value of the ml dimension was found to be 27.53 4.26 mm in males and 25.12 6.75 mm in females . the mean of si dimension was 38.21 5.77 mm in males and 33.34 6.57 mm in females . also , the mean of ap dimension was 42.60 3.79 mm in males and 36.00 4.09 mm in females . for the left maxillary sinus , the mean value of the ml dimension was found to be 27.01 5.04 mm in males and 23.22 6.21 mm in females . the mean value of the si dimension was 36.99 4.45 mm in males and 33.11 6.71 mm in females . also , the mean value of ap was 40.80 2.73 mm in males and 37.20 2.96 mm in females . a statistically significant ( p < 0.05 ) difference was found in the right si dimension , left si dimension , and the left ap dimension of the left maxillary sinuses between males and females . a significant ( p < 0.01 ) difference was found in the right ap dimension of the maxillary sinus between males and females . the other maxillary sinus dimensions showed a pattern of being larger in males than in females ; however , the difference was statistically insignificant . a comparison was made between the dimensions of the right and left maxillary sinus within males and females separately [ table 2 ] . on non - statistical comparison in males , the right maxillary sinus gave an impression of being slightly larger than the left maxillary sinus in its overall dimensions . similarly , in females , the right maxillary sinus was marginally larger in dimensions than the left maxillary sinus , with the exception of the ap dimension . aberrantly , the ap dimension of right maxillary sinus was found to be less than that of the left maxillary sinus in females . however , these intra - gender findings were statistically insignificant ( p > 0.05 ) . distribution of right and left maxillary sinus dimensions in males ( n=15 ) and females ( n=15 ) the mean with standard deviation was calculated for the volume of the right ( vr ) and the left ( vl ) maxillary sinuses for both males and females [ table 3 ] . for the right maxillary sinus , the mean volume was found to be 16.63 4.54 cc in males and 11.61 5.15 cc in females . for the left maxillary sinus , the mean volume was found to be 15.19 3.94 cc in males and 10.95 4.98 cc in females . distribution of maxillary sinus volume measured on ct and its standard deviation the volume of the maxillary sinuses in males was overall larger than in females . a significant ( p < 0.01 ) statistical difference was found in the right maxillary sinus volume between males and females . a significant ( p < 0.05 ) difference was found in the left maxillary sinus volume between males and females . a comparison was made between the volume of the right and left maxillary sinus within males and females separately [ table 4 ] . overall , both in males and females , the right maxillary sinus volume was found to be larger than the left maxillary sinus volume . however , this intra - gender volume difference was statistically insignificant ( p > 0.05 ) . a discriminative analysis was performed using the spss software to determine whether these linear measurements and volume could be used for gender determination . the following formula could be used for gender determination from measurements of the right maxillary sinus : gender = 5.116 - 0.159mlr - 0.014 sir + 0.171 apr + 0.218 vr where mlr is the ml dimension of the right maxillary sinus , sir is the si dimension of the right maxillary sinus , and apr is the ap dimension of the right maxillary sinus . the accuracy of gender predicted using the linear measurements and volume of the right maxillary sinus was found to be 80.0% in both males and females . . the following formula can be used for gender determination from measurements of the left maxillary sinus : gender = 0.720 - 0.258 mll - 0.146 sil + 0.120 apl + 0.586 vl where mll is the ml dimension of the left maxillary sinus , sil is the si dimension of the left maxillary sinus , and apl is the ap dimension of the left maxillary sinus . the accuracy of gender predicted from the linear measurements and volume of the left maxillary sinus was found to be 66.7% in males and 80.0% in females , with an overall gender prediction accuracy of 73.3% . the following formula can be used for gender determination from measurements of the right and the left maxillary sinuses together : gender = 4.033 - 0.101 mlr - 0.21 sir + 0.397 apr + 0.118 vr - 0.23 mll - 0.014 sil - 0.417 apl + 0.358 vl the accuracy of gender predicted from the linear measurements and volume of the right and the left maxillary sinuses together was found to be 80.0% in males and 86.7% in females . gender determination from measurements of right and left maxillary sinuses when the variables in the formulae derived above are substituted with the desired values , a numeric value is obtained for the gender . a negative value is predicted to be female . the accuracy of gender prediction , however , was variable . it was observed that the accuracy of gender prediction from the measurements of the right maxillary sinus ( 80.0% ) was more than that of the left maxillary sinus ( 73.3% ) . when the measurements of the right and the left maxillary sinuses were accounted together for predicting the gender , determination of gender is an important aspect of forensic investigation as it narrows down the investigator 's field of search . various methods which have been discussed below have been used to identify the gender of the human remains from a crime scene or a site of mass disaster . it has been reported that 100% accuracy can be achieved in gender determination from skeleton , 98.0% from both pelvis and skull , 95.0% from pelvis only or the pelvis and long bones , 90.095.0% from both the skull and the long bones , and 80.090.0% from the long bones only . morphometric parameters such as mesio - distal and bucco - lingual dimensions of the right permanent teeth were shown to predict the gender with an accuracy of 87.0% . other parameters such as odontometric differences , root length , and crown diameter have been used to determine the gender with varying degrees of accuracy . the circumference and area of the foramen magnum were used to differentiate the gender with an accuracy of 67.0% and 69.3% , respectively . in their study of the linear measurement of palatal bone and skull base showed significant sexual dimorphism , with reliability rates of 63.0% and 65.0% , respectively . a study based on radius and ulna for gender determination showed an accuracy for forearm ranging between 76.0% and 86.0% , whereas osman showed a sex determination accuracy as high as 96.0% . eshak in his study of gender determination showed that metacarpals , proximal phalanges , and distal phalanges are sexually dimorphic with an accuracy of 80.0% , 76.6% , and 80.0% , respectively . three - dimensional volume - rendering reconstructed image of metacarpal gave more accurate result ( 92.0% ) . measurements of hand length and phalanges of the fingers and dimensions of the palm have been used for gender determination with varying degrees of accuracy . various methods involving the soft tissues , such as cheiloscopy ( lip prints ) , radiographic methods for gender determination such as distance between the crest of alveolar ridge and the superior margin of mental foramina on orthopantomogram of edentulous individuals , and radiograph of the calcaneus , have been used . torwalt and robert were able to predict the gender using width of the fourth rib and sternal area on chest radiographs with an accuracy of 95.8% for males and 90.3% for females . sex could be determined with an accuracy of 95.6% by making cephalometric plots on lateral teleradiography with an orthodontic software . hsiao et al . , attempted to develop a method to determine sex from lateral cephalometric and discriminant functional analysis and determined sex with 100% accuracy in a random sample of 100 taiwanese adults . the studies discussed above for gender determination used plain radiographs , which included lateral cephalograms , and panoramic view for mental foramen , gonial angle , and mandibular ramus , along with ankle and hand it seems that plain radiographs have a higher average degree of accuracy in predicting the gender when compared to ct used in our study . however , plain radiographs , along with other conventional methods discussed above can only be used when the bones , skull , or the skeleton are found intact . when human skeleton is found in fragmented or incomplete state , it is necessary to look for denser bones , such as the maxillary sinus , which has a higher possibility of remaining intact by resisting decomposition , fracture , and incineration . ct has been reported to be a robust method in the estimation of different dimensions of the maxillary sinus . previous studies have shown that the dimensions of maxillary sinuses from measurements of human skulls were similar to those obtained by ct scans and the consistency of measurements of the paranasal sinuses using ct images have been evaluated in the last decade . it may , therefore , be reasonably assumed that ct is a reliable method for measuring the dimensions of the maxillary sinuses . in the literature , the mean values of maxillary sinus measurements were 32 , 25 , and 35 mm in length , width , and height , respectively . our study measured the ml , si , and ap dimensions of the maxillary sinus on ct in 30 patients including both males ( n = 15 ) and females ( n = 15 ) . maxillary sinus showed an average size of 27.53 38.21 42.60 mm in males and 25.12 33.34 36 mm in females . the left maxillary sinus showed an average size of 27.01 36.99 40.80 mm in males and 23.22 33.11 37.20 mm in females . we found that the overall size of the maxillary sinus was larger in males than in females . these findings were in agreement with those of authors like fernandes , teke et al . , and sahlstrand - johnason et al . , who have separately reported that the overall size of the maxillary sinus is larger in males than in females . also , the right maxillary sinus gave an impression of being larger than the left maxillary sinus in overall dimensions in both males and females , but the difference was found to be statistically insignificant . the volume of the maxillary sinus has been previously measured using ct . sahlstrand - johnson et al . , reported that the volume of the maxillary sinus can also be accurately estimated by using a simple formula : ( ml dimension si dimension ap dimension ) divided by 2 . such an estimation of volume is debatable and was not considered for use in this study ; however , this tool might be beneficial in clinical practice for approximate estimation of the maxillary sinus volume , where volume measurement applications are not available or precision is not critical . in a study about gender determination , it was found that the volume of maxillary sinuses was larger and wider in males than in females in europe but narrower in males than in females in zululand . our study used the ct workstation to measure the volume of the right and left maxillary sinuses in males and females by tracing the outline of the sinus manually on each slice of the ct image stack using the on - screen pointer in the coronal plane . the software module on the workstation then automatically calculated the maxillary sinus volume in cubic centimeters . we found that for the right maxillary sinus , the average volume was 16.63 cc in males and 11.61 cc in females . for the left maxillary sinus , the volume was calculated to be 15.19 cc in males and 10.95 cc in females . therefore , it could be concluded that males have significantly larger maxillary sinus volumes than females . also , the volume of the right maxillary sinus is slightly larger than the left maxillary sinus in both males and females . sahlstrand - johnson et al . , measured the dimensions of 120 maxillary sinuses from head cts and found the volume to be larger in males than in females with a mean value of 15.7 5.3 cm . these authors reported differences in the volume of the maxillary sinus between males and females , and this finding is in accordance with the findings in our study . but they did not find any significant difference between the volumes of the right and the left maxillary sinuses , which is in contrast to our study , as we found that the volume of the right maxillary sinus was larger than that of the left maxillary sinus , though the difference was statistically insignificant . teke et al . , employed ct to measure the length , width , and height of the maxillary sinus and predicted the gender with an accuracy of 69.4% in females and 69.2% in males and an overall mean accuracy of 69.3% . uthman et al . , studied the accuracy and reliability of maxillary sinus dimensions measurement in gender classification through the use of reconstructed helical ct images and reported an accuracy of correctly sexing 74.4% of male and 73.3% of female sinuses . amin and hassan investigated the possibility of estimation of sex from some radiologic measurements of the maxillary sinus using multi - detector ct among a known cross - section of egyptian population , which gave a predictive accuracy of 70.8% in males and 62.5% in females . improvising on the ct - based studies , which did not include the volume of the maxillary sinuses , our study measured the volume of the maxillary sinus along with its three other linear dimensions ( ml , si , ap ) to predict the gender of an individual . it was observed that if the volume is also included as a variable in the discriminative analysis along with the other three dimensions , the accuracy of gender prediction increases to 80.0% in males and 86.7% in females , with an overall accuracy rate of 83.3% . the maxillary sinuses remain intact although the skull and other bones may be badly disfigured in victims who are incinerated , and therefore , maxillary sinuses can be used for identification . ct measurements of maxillary sinuses may be useful to support gender determination in forensic medicine . this study , building upon similar previous studies , demonstrates that the length , width , and height of the maxillary sinus , along with its volume can be used to predict the gender of an individual with a fair degree of accuracy . the accuracy rate in this study is comparable , if not higher than many other methods that have been used to predict the gender of an individual from skeletal remains . however , the result of the study does not prove this method to be infallible . further studies with larger sample size are required to make this procedure conclusive and achieve standardization .

purpose : identification of human body or remains after death is a forensic procedure , which is difficult to perform and is mandatory by law and in compliance with social norms . sexing the recovered human remains is an integral part of the identification process . maxillary sinus can be used for gender determination as it remains intact even when the skull and other bones may be badly damaged in casualties where the body is incinerated . computed tomography ( ct ) provides an excellent method for examining maxillary sinuses.materials and methods : ct images were used to measure the mediolateral , superoinferior , and anteroposterior dimensions and the volume of the maxillary sinuses in 30 patients ( 15 males and 15 females ) to investigate whether these parameters could be used to determine the gender of an individual for forensic identification . the t - test for independent samples was used to compare these values in males and females and the data were subjected to discriminative analysis using spss software.results:our method was able to predict the gender with an accuracy of 80.0% in males and 86.7% in females , with an overall accuracy rate of 83.3%.conclusion : the accuracy rate in this study was comparable , if not higher than many other methods that have been used to predict the gender of an individual from skeletal remains . the length , width , height , and volume of the maxillary sinuses together with other bones could be used for gender determination with a fair degree of accuracy when the whole skeleton is not available .

Introduction Materials and Methods Results Maxillary sinus dimensions Maxillary sinus volume Gender determination Discussion Conclusion

the distance between the basion and the prosthion , the circumference of the head , the length of the supraorbital ridge , mastoid process , and mandibular ramus , the shape and length of palate , the circumference of occipital condyle , the sizes of teeth , the length and height of head , the distance between the basion and nasion , the height of the mandibular symphysis , the foramen magnum , the sphenoidal sinus , the sella turcica , and the frontal sinuses have also been used recently for gender determination in unknown remains . it has been reported that maxillary sinuses remain intact despite the skull and other bones getting badly disfigured in victims who are incinerated . during adulthood , their shapes and sizes the size of the maxillary sinus can be affected due to environmental factors , genetic diseases , or post infections . ct scan can be used for determination of gender by measurement of maxillary sinus when other methods are inconclusive , though this method is not error - free . the aim of the study was to determine the size [ mediolateral ( ml ) , superoinferior ( si ) , and anteroposterior ( ap ) linear dimensions ] and the volume of the maxillary sinuses using ct scanning , and investigate whether these parameters can be used to determine the gender of an individual for forensic identification . the study was designed to measure the ml , si , and ap dimensions along with the volumes ( v ) of both the right and the left maxillary sinuses of an individual using ct . thirty patients ( 15 male and 15 female ) were included in the study , who were between 20 and 50 years of age , had retained all their permanent teeth , and were referred to the imaging center to have a ct scan of the paranasal sinuses ( ct pns ) for various reasons , in which no pathological findings were detected on ct images . the ml and si measurements were made where the maxillary sinus was in its widest position , with the help of the on - screen linear measurement tool on the ct workstation . to measure the ap dimension of the maxillary sinus , the first and the last appearance of the sinus was noted in the sequential coronal ct sections , and the number of sections between them was determined . once the tracing was complete , the workstation automatically segmented the entire volume of the sinus from the surrounding structures and the segmented portion could be visualized and manipulated in 3d [ figure 3 ] . workstation showing the sinus volume the t - test for independent samples was used to compare these values in two groups . discriminative analysis was used to detect the gender by using the data obtained from ct scans . the mean , along with the standard deviation was calculated for all the dimensions of the right and the left maxillary sinuses , namely ml , si , and ap , for both males and females [ table 1 ] . distribution of maxillary sinus dimensions measured on ct and their standard deviation for the right maxillary sinus , the mean value of the ml dimension was found to be 27.53 4.26 mm in males and 25.12 6.75 mm in females . for the left maxillary sinus , the mean value of the ml dimension was found to be 27.01 5.04 mm in males and 23.22 6.21 mm in females . the mean value of the si dimension was 36.99 4.45 mm in males and 33.11 6.71 mm in females . a statistically significant ( p < 0.05 ) difference was found in the right si dimension , left si dimension , and the left ap dimension of the left maxillary sinuses between males and females . a significant ( p < 0.01 ) difference was found in the right ap dimension of the maxillary sinus between males and females . similarly , in females , the right maxillary sinus was marginally larger in dimensions than the left maxillary sinus , with the exception of the ap dimension . distribution of right and left maxillary sinus dimensions in males ( n=15 ) and females ( n=15 ) the mean with standard deviation was calculated for the volume of the right ( vr ) and the left ( vl ) maxillary sinuses for both males and females [ table 3 ] . distribution of maxillary sinus volume measured on ct and its standard deviation the volume of the maxillary sinuses in males was overall larger than in females . a comparison was made between the volume of the right and left maxillary sinus within males and females separately [ table 4 ] . overall , both in males and females , the right maxillary sinus volume was found to be larger than the left maxillary sinus volume . distribution of right and left maxillary sinus volume in males and females a discriminative analysis was performed using the spss software to determine whether these linear measurements and volume could be used for gender determination . the following formula could be used for gender determination from measurements of the right maxillary sinus : gender = 5.116 - 0.159mlr - 0.014 sir + 0.171 apr + 0.218 vr where mlr is the ml dimension of the right maxillary sinus , sir is the si dimension of the right maxillary sinus , and apr is the ap dimension of the right maxillary sinus . the accuracy of gender predicted using the linear measurements and volume of the right maxillary sinus was found to be 80.0% in both males and females . the following formula can be used for gender determination from measurements of the left maxillary sinus : gender = 0.720 - 0.258 mll - 0.146 sil + 0.120 apl + 0.586 vl where mll is the ml dimension of the left maxillary sinus , sil is the si dimension of the left maxillary sinus , and apl is the ap dimension of the left maxillary sinus . the accuracy of gender predicted from the linear measurements and volume of the left maxillary sinus was found to be 66.7% in males and 80.0% in females , with an overall gender prediction accuracy of 73.3% . the following formula can be used for gender determination from measurements of the right and the left maxillary sinuses together : gender = 4.033 - 0.101 mlr - 0.21 sir + 0.397 apr + 0.118 vr - 0.23 mll - 0.014 sil - 0.417 apl + 0.358 vl the accuracy of gender predicted from the linear measurements and volume of the right and the left maxillary sinuses together was found to be 80.0% in males and 86.7% in females . it was observed that the accuracy of gender prediction from the measurements of the right maxillary sinus ( 80.0% ) was more than that of the left maxillary sinus ( 73.3% ) . when the measurements of the right and the left maxillary sinuses were accounted together for predicting the gender , the accuracy rate increased to 83.3% . the mean , along with the standard deviation was calculated for all the dimensions of the right and the left maxillary sinuses , namely ml , si , and ap , for both males and females [ table 1 ] . distribution of maxillary sinus dimensions measured on ct and their standard deviation for the right maxillary sinus , the mean value of the ml dimension was found to be 27.53 4.26 mm in males and 25.12 6.75 mm in females . for the left maxillary sinus , the mean value of the ml dimension was found to be 27.01 5.04 mm in males and 23.22 6.21 mm in females . the mean value of the si dimension was 36.99 4.45 mm in males and 33.11 6.71 mm in females . a statistically significant ( p < 0.05 ) difference was found in the right si dimension , left si dimension , and the left ap dimension of the left maxillary sinuses between males and females . a significant ( p < 0.01 ) difference was found in the right ap dimension of the maxillary sinus between males and females . a comparison was made between the dimensions of the right and left maxillary sinus within males and females separately [ table 2 ] . similarly , in females , the right maxillary sinus was marginally larger in dimensions than the left maxillary sinus , with the exception of the ap dimension . distribution of right and left maxillary sinus dimensions in males ( n=15 ) and females ( n=15 ) the mean with standard deviation was calculated for the volume of the right ( vr ) and the left ( vl ) maxillary sinuses for both males and females [ table 3 ] . distribution of maxillary sinus volume measured on ct and its standard deviation the volume of the maxillary sinuses in males was overall larger than in females . a comparison was made between the volume of the right and left maxillary sinus within males and females separately [ table 4 ] . overall , both in males and females , the right maxillary sinus volume was found to be larger than the left maxillary sinus volume . a discriminative analysis was performed using the spss software to determine whether these linear measurements and volume could be used for gender determination . the following formula could be used for gender determination from measurements of the right maxillary sinus : gender = 5.116 - 0.159mlr - 0.014 sir + 0.171 apr + 0.218 vr where mlr is the ml dimension of the right maxillary sinus , sir is the si dimension of the right maxillary sinus , and apr is the ap dimension of the right maxillary sinus . the accuracy of gender predicted using the linear measurements and volume of the right maxillary sinus was found to be 80.0% in both males and females . the following formula can be used for gender determination from measurements of the left maxillary sinus : gender = 0.720 - 0.258 mll - 0.146 sil + 0.120 apl + 0.586 vl where mll is the ml dimension of the left maxillary sinus , sil is the si dimension of the left maxillary sinus , and apl is the ap dimension of the left maxillary sinus . the accuracy of gender predicted from the linear measurements and volume of the left maxillary sinus was found to be 66.7% in males and 80.0% in females , with an overall gender prediction accuracy of 73.3% . the following formula can be used for gender determination from measurements of the right and the left maxillary sinuses together : gender = 4.033 - 0.101 mlr - 0.21 sir + 0.397 apr + 0.118 vr - 0.23 mll - 0.014 sil - 0.417 apl + 0.358 vl the accuracy of gender predicted from the linear measurements and volume of the right and the left maxillary sinuses together was found to be 80.0% in males and 86.7% in females . gender determination from measurements of right and left maxillary sinuses when the variables in the formulae derived above are substituted with the desired values , a numeric value is obtained for the gender . it was observed that the accuracy of gender prediction from the measurements of the right maxillary sinus ( 80.0% ) was more than that of the left maxillary sinus ( 73.3% ) . when the measurements of the right and the left maxillary sinuses were accounted together for predicting the gender , determination of gender is an important aspect of forensic investigation as it narrows down the investigator 's field of search . various methods which have been discussed below have been used to identify the gender of the human remains from a crime scene or a site of mass disaster . it has been reported that 100% accuracy can be achieved in gender determination from skeleton , 98.0% from both pelvis and skull , 95.0% from pelvis only or the pelvis and long bones , 90.095.0% from both the skull and the long bones , and 80.090.0% from the long bones only . morphometric parameters such as mesio - distal and bucco - lingual dimensions of the right permanent teeth were shown to predict the gender with an accuracy of 87.0% . other parameters such as odontometric differences , root length , and crown diameter have been used to determine the gender with varying degrees of accuracy . the circumference and area of the foramen magnum were used to differentiate the gender with an accuracy of 67.0% and 69.3% , respectively . a study based on radius and ulna for gender determination showed an accuracy for forearm ranging between 76.0% and 86.0% , whereas osman showed a sex determination accuracy as high as 96.0% . eshak in his study of gender determination showed that metacarpals , proximal phalanges , and distal phalanges are sexually dimorphic with an accuracy of 80.0% , 76.6% , and 80.0% , respectively . measurements of hand length and phalanges of the fingers and dimensions of the palm have been used for gender determination with varying degrees of accuracy . various methods involving the soft tissues , such as cheiloscopy ( lip prints ) , radiographic methods for gender determination such as distance between the crest of alveolar ridge and the superior margin of mental foramina on orthopantomogram of edentulous individuals , and radiograph of the calcaneus , have been used . torwalt and robert were able to predict the gender using width of the fourth rib and sternal area on chest radiographs with an accuracy of 95.8% for males and 90.3% for females . sex could be determined with an accuracy of 95.6% by making cephalometric plots on lateral teleradiography with an orthodontic software . the studies discussed above for gender determination used plain radiographs , which included lateral cephalograms , and panoramic view for mental foramen , gonial angle , and mandibular ramus , along with ankle and hand it seems that plain radiographs have a higher average degree of accuracy in predicting the gender when compared to ct used in our study . when human skeleton is found in fragmented or incomplete state , it is necessary to look for denser bones , such as the maxillary sinus , which has a higher possibility of remaining intact by resisting decomposition , fracture , and incineration . previous studies have shown that the dimensions of maxillary sinuses from measurements of human skulls were similar to those obtained by ct scans and the consistency of measurements of the paranasal sinuses using ct images have been evaluated in the last decade . it may , therefore , be reasonably assumed that ct is a reliable method for measuring the dimensions of the maxillary sinuses . in the literature , the mean values of maxillary sinus measurements were 32 , 25 , and 35 mm in length , width , and height , respectively . our study measured the ml , si , and ap dimensions of the maxillary sinus on ct in 30 patients including both males ( n = 15 ) and females ( n = 15 ) . the left maxillary sinus showed an average size of 27.01 36.99 40.80 mm in males and 23.22 33.11 37.20 mm in females . we found that the overall size of the maxillary sinus was larger in males than in females . , who have separately reported that the overall size of the maxillary sinus is larger in males than in females . the volume of the maxillary sinus has been previously measured using ct . , reported that the volume of the maxillary sinus can also be accurately estimated by using a simple formula : ( ml dimension si dimension ap dimension ) divided by 2 . such an estimation of volume is debatable and was not considered for use in this study ; however , this tool might be beneficial in clinical practice for approximate estimation of the maxillary sinus volume , where volume measurement applications are not available or precision is not critical . in a study about gender determination , it was found that the volume of maxillary sinuses was larger and wider in males than in females in europe but narrower in males than in females in zululand . our study used the ct workstation to measure the volume of the right and left maxillary sinuses in males and females by tracing the outline of the sinus manually on each slice of the ct image stack using the on - screen pointer in the coronal plane . for the left maxillary sinus , the volume was calculated to be 15.19 cc in males and 10.95 cc in females . also , the volume of the right maxillary sinus is slightly larger than the left maxillary sinus in both males and females . , measured the dimensions of 120 maxillary sinuses from head cts and found the volume to be larger in males than in females with a mean value of 15.7 5.3 cm . these authors reported differences in the volume of the maxillary sinus between males and females , and this finding is in accordance with the findings in our study . but they did not find any significant difference between the volumes of the right and the left maxillary sinuses , which is in contrast to our study , as we found that the volume of the right maxillary sinus was larger than that of the left maxillary sinus , though the difference was statistically insignificant . , employed ct to measure the length , width , and height of the maxillary sinus and predicted the gender with an accuracy of 69.4% in females and 69.2% in males and an overall mean accuracy of 69.3% . , studied the accuracy and reliability of maxillary sinus dimensions measurement in gender classification through the use of reconstructed helical ct images and reported an accuracy of correctly sexing 74.4% of male and 73.3% of female sinuses . amin and hassan investigated the possibility of estimation of sex from some radiologic measurements of the maxillary sinus using multi - detector ct among a known cross - section of egyptian population , which gave a predictive accuracy of 70.8% in males and 62.5% in females . improvising on the ct - based studies , which did not include the volume of the maxillary sinuses , our study measured the volume of the maxillary sinus along with its three other linear dimensions ( ml , si , ap ) to predict the gender of an individual . it was observed that if the volume is also included as a variable in the discriminative analysis along with the other three dimensions , the accuracy of gender prediction increases to 80.0% in males and 86.7% in females , with an overall accuracy rate of 83.3% . the maxillary sinuses remain intact although the skull and other bones may be badly disfigured in victims who are incinerated , and therefore , maxillary sinuses can be used for identification . this study , building upon similar previous studies , demonstrates that the length , width , and height of the maxillary sinus , along with its volume can be used to predict the gender of an individual with a fair degree of accuracy . the accuracy rate in this study is comparable , if not higher than many other methods that have been used to predict the gender of an individual from skeletal remains .

the identity of a human being should be preserved even after death , as there are consequences , often financial , that lead to the court of law , which demands that the identity of the deceased be established before a verdict is passed . forensic science based evidence is accepted in a judicial setting by the court and plays a major role in the identification of individuals who can not be identified visually or by other simple means . the distance between the basion and the prosthion , the circumference of the head , the length of the supraorbital ridge , mastoid process , and mandibular ramus , the shape and length of palate , the circumference of occipital condyle , the sizes of teeth , the length and height of head , the distance between the basion and nasion , the height of the mandibular symphysis , the foramen magnum , the sphenoidal sinus , the sella turcica , and the frontal sinuses have also been used recently for gender determination in unknown remains . during adulthood , their shapes and sizes the size of the maxillary sinus can be affected due to environmental factors , genetic diseases , or post infections . as the image represents a contiguous series of cross - sections and three - dimensional information and the machine is available in most of the hospitals , ct has been applied in the study of fossil skulls . the aim of the study was to determine the size [ mediolateral ( ml ) , superoinferior ( si ) , and anteroposterior ( ap ) linear dimensions ] and the volume of the maxillary sinuses using ct scanning , and investigate whether these parameters can be used to determine the gender of an individual for forensic identification . the study was designed to measure the ml , si , and ap dimensions along with the volumes ( v ) of both the right and the left maxillary sinuses of an individual using ct . the study was carried out on a small regional population of patients presenting to the imaging center of institute of dental studies and technologies , modinagar , western uttar pradesh , india , between december 2009 and august 2011 . thirty patients ( 15 male and 15 female ) were included in the study , who were between 20 and 50 years of age , had retained all their permanent teeth , and were referred to the imaging center to have a ct scan of the paranasal sinuses ( ct pns ) for various reasons , in which no pathological findings were detected on ct images . patients who were found to have maxillary sinus pathology on ct scan , facial deformities involving the maxilla , clinical facial asymmetries , or had previously undergone a surgery of the maxillary sinus were excluded from the study . prior to the scan , the patient was instructed to remove all metallic objects including hair pins , jewelry , and so on from the head and neck region and positioned on the ct table in prone position . for stabilization , sections of 3 mm thickness were planned on the preliminary scout view extending from the anterior margin of the frontal sinus to the posterior margin of the sphenoid sinus , with a reconstruction matrix size of 512 512 at 120 kv , 100 ma . the ml and si measurements were made where the maxillary sinus was in its widest position , with the help of the on - screen linear measurement tool on the ct workstation . to measure the ap dimension of the maxillary sinus , the first and the last appearance of the sinus was noted in the sequential coronal ct sections , and the number of sections between them was determined . finally , the number of sections obtained was multiplied by 3 ( thickness of a single section ) to find the ap dimension of the sinus . to define a volume , the outline of the sinus was traced manually on each slice of the image stack using the on - screen mouse pointer in the coronal plane [ figure 2 ] . once the tracing was complete , the workstation automatically segmented the entire volume of the sinus from the surrounding structures and the segmented portion could be visualized and manipulated in 3d [ figure 3 ] . tool at this point , switching to the histogram view on the workstation [ figure 4 ] automatically reflected the volume of the sinus in cubic centimeters ( cc ) . the mean , along with the standard deviation was calculated for all the dimensions of the right and the left maxillary sinuses , namely ml , si , and ap , for both males and females [ table 1 ] . distribution of maxillary sinus dimensions measured on ct and their standard deviation for the right maxillary sinus , the mean value of the ml dimension was found to be 27.53 4.26 mm in males and 25.12 6.75 mm in females . for the left maxillary sinus , the mean value of the ml dimension was found to be 27.01 5.04 mm in males and 23.22 6.21 mm in females . a statistically significant ( p < 0.05 ) difference was found in the right si dimension , left si dimension , and the left ap dimension of the left maxillary sinuses between males and females . a significant ( p < 0.01 ) difference was found in the right ap dimension of the maxillary sinus between males and females . the other maxillary sinus dimensions showed a pattern of being larger in males than in females ; however , the difference was statistically insignificant . a comparison was made between the dimensions of the right and left maxillary sinus within males and females separately [ table 2 ] . similarly , in females , the right maxillary sinus was marginally larger in dimensions than the left maxillary sinus , with the exception of the ap dimension . aberrantly , the ap dimension of right maxillary sinus was found to be less than that of the left maxillary sinus in females . distribution of right and left maxillary sinus dimensions in males ( n=15 ) and females ( n=15 ) the mean with standard deviation was calculated for the volume of the right ( vr ) and the left ( vl ) maxillary sinuses for both males and females [ table 3 ] . for the right maxillary sinus , the mean volume was found to be 16.63 4.54 cc in males and 11.61 5.15 cc in females . for the left maxillary sinus , the mean volume was found to be 15.19 3.94 cc in males and 10.95 4.98 cc in females . distribution of maxillary sinus volume measured on ct and its standard deviation the volume of the maxillary sinuses in males was overall larger than in females . overall , both in males and females , the right maxillary sinus volume was found to be larger than the left maxillary sinus volume . distribution of right and left maxillary sinus volume in males and females a discriminative analysis was performed using the spss software to determine whether these linear measurements and volume could be used for gender determination . the following formula could be used for gender determination from measurements of the right maxillary sinus : gender = 5.116 - 0.159mlr - 0.014 sir + 0.171 apr + 0.218 vr where mlr is the ml dimension of the right maxillary sinus , sir is the si dimension of the right maxillary sinus , and apr is the ap dimension of the right maxillary sinus . the accuracy of gender predicted using the linear measurements and volume of the right maxillary sinus was found to be 80.0% in both males and females . the following formula can be used for gender determination from measurements of the left maxillary sinus : gender = 0.720 - 0.258 mll - 0.146 sil + 0.120 apl + 0.586 vl where mll is the ml dimension of the left maxillary sinus , sil is the si dimension of the left maxillary sinus , and apl is the ap dimension of the left maxillary sinus . the accuracy of gender predicted from the linear measurements and volume of the left maxillary sinus was found to be 66.7% in males and 80.0% in females , with an overall gender prediction accuracy of 73.3% . the following formula can be used for gender determination from measurements of the right and the left maxillary sinuses together : gender = 4.033 - 0.101 mlr - 0.21 sir + 0.397 apr + 0.118 vr - 0.23 mll - 0.014 sil - 0.417 apl + 0.358 vl the accuracy of gender predicted from the linear measurements and volume of the right and the left maxillary sinuses together was found to be 80.0% in males and 86.7% in females . it was observed that the accuracy of gender prediction from the measurements of the right maxillary sinus ( 80.0% ) was more than that of the left maxillary sinus ( 73.3% ) . when the measurements of the right and the left maxillary sinuses were accounted together for predicting the gender , the accuracy rate increased to 83.3% . the mean , along with the standard deviation was calculated for all the dimensions of the right and the left maxillary sinuses , namely ml , si , and ap , for both males and females [ table 1 ] . distribution of maxillary sinus dimensions measured on ct and their standard deviation for the right maxillary sinus , the mean value of the ml dimension was found to be 27.53 4.26 mm in males and 25.12 6.75 mm in females . for the left maxillary sinus , the mean value of the ml dimension was found to be 27.01 5.04 mm in males and 23.22 6.21 mm in females . a statistically significant ( p < 0.05 ) difference was found in the right si dimension , left si dimension , and the left ap dimension of the left maxillary sinuses between males and females . a significant ( p < 0.01 ) difference was found in the right ap dimension of the maxillary sinus between males and females . similarly , in females , the right maxillary sinus was marginally larger in dimensions than the left maxillary sinus , with the exception of the ap dimension . aberrantly , the ap dimension of right maxillary sinus was found to be less than that of the left maxillary sinus in females . distribution of right and left maxillary sinus dimensions in males ( n=15 ) and females ( n=15 ) the mean with standard deviation was calculated for the volume of the right ( vr ) and the left ( vl ) maxillary sinuses for both males and females [ table 3 ] . for the right maxillary sinus , the mean volume was found to be 16.63 4.54 cc in males and 11.61 5.15 cc in females . for the left maxillary sinus , the mean volume was found to be 15.19 3.94 cc in males and 10.95 4.98 cc in females . distribution of maxillary sinus volume measured on ct and its standard deviation the volume of the maxillary sinuses in males was overall larger than in females . overall , both in males and females , the right maxillary sinus volume was found to be larger than the left maxillary sinus volume . the following formula could be used for gender determination from measurements of the right maxillary sinus : gender = 5.116 - 0.159mlr - 0.014 sir + 0.171 apr + 0.218 vr where mlr is the ml dimension of the right maxillary sinus , sir is the si dimension of the right maxillary sinus , and apr is the ap dimension of the right maxillary sinus . the accuracy of gender predicted using the linear measurements and volume of the right maxillary sinus was found to be 80.0% in both males and females . the following formula can be used for gender determination from measurements of the left maxillary sinus : gender = 0.720 - 0.258 mll - 0.146 sil + 0.120 apl + 0.586 vl where mll is the ml dimension of the left maxillary sinus , sil is the si dimension of the left maxillary sinus , and apl is the ap dimension of the left maxillary sinus . the accuracy of gender predicted from the linear measurements and volume of the left maxillary sinus was found to be 66.7% in males and 80.0% in females , with an overall gender prediction accuracy of 73.3% . the following formula can be used for gender determination from measurements of the right and the left maxillary sinuses together : gender = 4.033 - 0.101 mlr - 0.21 sir + 0.397 apr + 0.118 vr - 0.23 mll - 0.014 sil - 0.417 apl + 0.358 vl the accuracy of gender predicted from the linear measurements and volume of the right and the left maxillary sinuses together was found to be 80.0% in males and 86.7% in females . it was observed that the accuracy of gender prediction from the measurements of the right maxillary sinus ( 80.0% ) was more than that of the left maxillary sinus ( 73.3% ) . when the measurements of the right and the left maxillary sinuses were accounted together for predicting the gender , determination of gender is an important aspect of forensic investigation as it narrows down the investigator 's field of search . various methods which have been discussed below have been used to identify the gender of the human remains from a crime scene or a site of mass disaster . it has been reported that 100% accuracy can be achieved in gender determination from skeleton , 98.0% from both pelvis and skull , 95.0% from pelvis only or the pelvis and long bones , 90.095.0% from both the skull and the long bones , and 80.090.0% from the long bones only . morphometric parameters such as mesio - distal and bucco - lingual dimensions of the right permanent teeth were shown to predict the gender with an accuracy of 87.0% . the circumference and area of the foramen magnum were used to differentiate the gender with an accuracy of 67.0% and 69.3% , respectively . in their study of the linear measurement of palatal bone and skull base showed significant sexual dimorphism , with reliability rates of 63.0% and 65.0% , respectively . eshak in his study of gender determination showed that metacarpals , proximal phalanges , and distal phalanges are sexually dimorphic with an accuracy of 80.0% , 76.6% , and 80.0% , respectively . various methods involving the soft tissues , such as cheiloscopy ( lip prints ) , radiographic methods for gender determination such as distance between the crest of alveolar ridge and the superior margin of mental foramina on orthopantomogram of edentulous individuals , and radiograph of the calcaneus , have been used . torwalt and robert were able to predict the gender using width of the fourth rib and sternal area on chest radiographs with an accuracy of 95.8% for males and 90.3% for females . , attempted to develop a method to determine sex from lateral cephalometric and discriminant functional analysis and determined sex with 100% accuracy in a random sample of 100 taiwanese adults . the studies discussed above for gender determination used plain radiographs , which included lateral cephalograms , and panoramic view for mental foramen , gonial angle , and mandibular ramus , along with ankle and hand it seems that plain radiographs have a higher average degree of accuracy in predicting the gender when compared to ct used in our study . when human skeleton is found in fragmented or incomplete state , it is necessary to look for denser bones , such as the maxillary sinus , which has a higher possibility of remaining intact by resisting decomposition , fracture , and incineration . previous studies have shown that the dimensions of maxillary sinuses from measurements of human skulls were similar to those obtained by ct scans and the consistency of measurements of the paranasal sinuses using ct images have been evaluated in the last decade . our study measured the ml , si , and ap dimensions of the maxillary sinus on ct in 30 patients including both males ( n = 15 ) and females ( n = 15 ) . also , the right maxillary sinus gave an impression of being larger than the left maxillary sinus in overall dimensions in both males and females , but the difference was found to be statistically insignificant . such an estimation of volume is debatable and was not considered for use in this study ; however , this tool might be beneficial in clinical practice for approximate estimation of the maxillary sinus volume , where volume measurement applications are not available or precision is not critical . in a study about gender determination , it was found that the volume of maxillary sinuses was larger and wider in males than in females in europe but narrower in males than in females in zululand . our study used the ct workstation to measure the volume of the right and left maxillary sinuses in males and females by tracing the outline of the sinus manually on each slice of the ct image stack using the on - screen pointer in the coronal plane . we found that for the right maxillary sinus , the average volume was 16.63 cc in males and 11.61 cc in females . , measured the dimensions of 120 maxillary sinuses from head cts and found the volume to be larger in males than in females with a mean value of 15.7 5.3 cm . but they did not find any significant difference between the volumes of the right and the left maxillary sinuses , which is in contrast to our study , as we found that the volume of the right maxillary sinus was larger than that of the left maxillary sinus , though the difference was statistically insignificant . , employed ct to measure the length , width , and height of the maxillary sinus and predicted the gender with an accuracy of 69.4% in females and 69.2% in males and an overall mean accuracy of 69.3% . , studied the accuracy and reliability of maxillary sinus dimensions measurement in gender classification through the use of reconstructed helical ct images and reported an accuracy of correctly sexing 74.4% of male and 73.3% of female sinuses . amin and hassan investigated the possibility of estimation of sex from some radiologic measurements of the maxillary sinus using multi - detector ct among a known cross - section of egyptian population , which gave a predictive accuracy of 70.8% in males and 62.5% in females . improvising on the ct - based studies , which did not include the volume of the maxillary sinuses , our study measured the volume of the maxillary sinus along with its three other linear dimensions ( ml , si , ap ) to predict the gender of an individual . it was observed that if the volume is also included as a variable in the discriminative analysis along with the other three dimensions , the accuracy of gender prediction increases to 80.0% in males and 86.7% in females , with an overall accuracy rate of 83.3% . this study , building upon similar previous studies , demonstrates that the length , width , and height of the maxillary sinus , along with its volume can be used to predict the gender of an individual with a fair degree of accuracy .

the occurrence of hypothyroidism in patients with diabetes mellitus has attracted attention since joslin et al . revealed this association . several studies have reported a higher prevalence of thyroid dysfunction in diabetes [ 24 ] . this association may be related to autoimmunity as well as other factors associated with either type 1 or type 2 diabetes and suggests that persons with diabetes be screened for hypothyroidism [ 2 , 57 ] . the executive summary of standards of medical care in diabetes ( 2014 ) published by the american diabetes association ( ada ) as well as the clinical practice guidelines for hypothyroidism in adults ( 2012 ) copublished by the american association of clinical endocrinologists ( aace ) and the american thyroid association ( ata ) suggest screening patients with type 1 diabetes for thyroid diseases . age may be a factor associated with the higher prevalence of hypothyroidism among patients with type 2 diabetes [ 4 , 10 ] . in elderly patients , however , it may be difficult to determine if an abnormal thyroid stimulating hormone ( tsh ) pattern is due to actual thyroid disease or an unrelated cause or possibly is secondary to drug interference . although some studies investigated this , it was not always taken into consideration that the upper reference range for tsh in elderly individuals may be higher than in the general population [ 1012 ] . it is now clear that screening for thyroid dysfunction in elderly subjects with diabetes should take into account that an increase in serum tsh levels is expected . previously , an increase in tsh levels in elderly persons was attributed to elevated levels of circulating thyroid antibodies in this specific population . however several recent studies demonstrated that , even after excluding those with known thyroid diseases and circulating antithyroid antibodies , the tsh distribution curve remained shifted to the right ; the 97.5th percentile tsh values rose as the population aged [ 9 , 1317 ] . when considering screening for thyroid diseases in patients with diabetes , the diverse types of drugs used by a significant number of these patients ought to be taken into account , as many reportedly interfere with tsh levels , especially metformin and sulfonylureas [ 1822 ] . the major aim of this study was to evaluate the prevalence of previously undiagnosed cases of hypothyroidism in the elderly with diabetes by applying specific ri for serum tsh . a cross - sectional study of 1160 subjects over 60 years of age was performed , of whom 751 had diabetes . all participants underwent routine thyroid function tests , especially as thyroid function or autoimmunity tests had not been performed for all patients previously . the mean time since diabetes diagnosis was 10.2 years ( range : 2.614.1 years ) . only patients with diabetes who had been diagnosed at least 2 years priorly were included ; diagnosis was according to the ada criteria . the blood counts as well as renal and liver functions of all patients were normal . subjects normally resided in the metropolitan area of rio de janeiro and self - identified as belonging to middle and upper social classes . as for race , 68% were white , 20% were mulatto , and 12% were black . urinary iodine assessment was not performed , since salt iodization in brazil is determined by federal law and a previous study showed sufficient iodine intake in the rio de janeiro population . a national health surveillance agency review of salt from samples used in the city a year before the beginning of our study confirmed appropriate iodine levels . exclusion criteria were patients under treatment for thyroid diseases or with a history of thyroid disease , use of medications known to interfere with measurement of tsh or free thyroxine ( ft4 ) in the previous three months ( except for diabetes treatments ) , use of contrast media and other medications containing iodine in the last six months , illness , accident , or surgery in the last six months , and chronic disease except for hypertension and dyslipidaemia . there were 409 patients without diabetes who attended a clinical laboratory to collect routine tests for which function and/or autoimmunity thyroid evaluation had not been requested and that had normal blood count , kidney , and liver functions . all of them fulfilled the same exclusion criteria of patients with diabetes and were not under antidiabetes drugs for any other reason . this study was approved by the institutional review board of hospital clementino fraga filho , universidade federal do rio de janeiro . patients with diabetes were divided into subgroups according to the medications they used as follows : subgroup mtf : metformin ( 5003000 mg / day ) ; subgroup su : sulfonylureas ( glibenclamide 515 mg / day or glimepiride 14 mg / day ) ; subgroup dpp4 : dipeptidyl peptidase 4 ( dpp4 ) inhibitors ( vildagliptin or sitagliptin , 50100 mg / day ) ; subgroup tzd : thiazolidinedione ( pioglitazone , 1545 mg / day ) ; subgroup ins : insulin analogues glargine or detemir 1342 ui , plus analogues lispro or aspart 614 ui / day ; subgroup mtf+ : metformin in combination with one or more other medications ( sulfonylurea , dpp4 inhibitor , thiazolidinedione , or insulin analogues ) ; the mtf+ group used lower doses of some medications compared to the doses when they were used as the sole medication : mtf : 5001500 ; su ( glibenclamide : 510 mg / day , glimepiride : 1 - 2 mg / day ) ; tzd : 1530 mg / day ; dpp4 and ins doses did not differ from the previous subgroups . all patients with diabetes had been on the same medication for at least one year . other antidiabetes drugs were not evaluated in the study , as there was an insufficient number of patients to allow statistical analysis . body mass index ( bmi ) was obtained by dividing the body weight ( kg ) over the square of the height ( m ) . tsh , ft4 , antithyroperoxidase antibodies ( tpoab ) , fasting glucose ( fg ) , and glycated hemoglobin ( hba1c ) were measured in all patients . serum tsh , ft4 , and tpoab were measured by electrochemiluminescence immunoassays using the roche modular analytics e170 instrument ( roche diagnostics australia pty ltd . , serum tsh concentrations were measured by an immunometric method , with an intra - assay percent coefficient of variation ( % cv ) of 3.0% at concentrations of 0.040 0.001 the tsh reference interval ( ri ) established in our previous study of the population in the same region was 0.4 to 5.8 mu / l for subjects aged between 60 and 79 years and 0.4 to 6.7 mu / l for those aged 80 years and older . the % cv was 1.4% at concentrations of 9.0 0.1 pmol / l ( 0.7 0.01 ng / dl ) , 1.8% at 16.7 0.3 pmol / l ( 1.3 0.02 ng / dl ) , and 2.0% at 34.7 1.3 pmol / l ( 2.7 0.1 ng / dl ) . the ri for age groups , 60 years and older , was in the range of 9.021.9 pmol / l ( 0.71.7 ng / dl ) . the intra - assay % cv for tpoab was 6.3% at concentrations of 21.3 1.34 iu / ml , 5.1% at 51.2 2.6 iu / ml , and 2.7% at 473 12.7 iu / ml ; a score lower than 34 iu / ml indicated the absence of thyroid disease according to the assay 's manufacturer . fg was measured by an enzymatic reference method with hexokinase using roche / hitachi cobas c , gluc3 ( roche diagnostics gmbh , mannheim , germany ) . the lower limit of assay detection is 0.1 mmol / l ( 2.0 mg / dl ) , with an intra - assay % cv of 0.8% at concentrations of 5.2 0.04 mmol / l ( 93.2 0.7 mg / dl ) , 0.7% at 13.4 0.11 mmol / l ( 241.0 2.0 mg / dl ) , and 0.7% at 36.1 0.28 mmol / l ( 651.0 5.0 mg / dl ) . according to the ada , the normal value is less than 5.6 mmol / l ( 100.0 mg / dl ) . hba1c was measured by high - performance liquid chromatography ( hplc ) on the bio - rad variant ii hemoglobin a1c system ( bio - rad laboratories , inc . the hba1c reportable range is between 3.1 and 18.5 percent ( % ) in national glycohemoglobin standardization program ( ngsp ) units and between 10 and 179 mmol hba1c / mol hb ( mmol / mol ) in international federation of clinical chemistry and laboratory medicine ( ifcc ) units . the master equation for designated comparison method is as follows : ifcc = ( 10.93 ngsp ) 23.50 ( http://www.ngsp.org/convert1.asp ) . according to the manufacturer , the intra - assay cv was 0.9% for concentrations in healthy subjects and 0.59% for measurements obtained from individuals with diabetes . a patient with diabetes with hba1c levels below 7.0% ( 53 mmol / mol ifcc ) mu / l in patients aged 6079 years and above 6.7 mu / l in patients aged 80 years or older , with ft4 levels ranging between 9.0 and 21.9 pmol / l ( 0.7 and 1.7 ng / dl ) ; overt hypothyroidism was diagnosed with the same values of tsh and with serum ft4 below 9.0 pmol / l ( 0.7 ng / dl ) . autoimmunity was considered present when tpoab titres were greater than 34 iu / ml . general data were analyzed using the graphpad prism software , version 6.0 ( graphpad software , inc . , kolmogorov - smirnov tests were performed to assess the normal distribution . log 10 transformations of nonnormal variables were performed for analysis . to test if patients with diabetes and persons without diabetes ages were matched , the unpaired t - test with welch 's correction was used , as age had normal distribution . descriptive analyses of serum tsh , fg , and hba1c were reported as medians and 25 and 75 percentiles , because they were not normally distributed . descriptive analysis of serum ft4 was reported as mean with standard deviation , as it was normally distributed . two tailed mann - whitney and kruskal - wallis tests were used to compare the nonparametric tsh distributions in different subpopulations . comparison between two tsh subgroups was performed by an independent test of dunn multiple comparisons . for all parameters , p < 0.05 was considered statistically significant . to describe the relationship between tsh ( mu / l ) and mtf dose ( mg / day ) , a statistical model was used assessing the possible significance of mtf effect in the tsh variability and providing estimated values for tsh according to different doses . the class of generalized linear models ( glm ) likelihood - ratio tests were used to assess the significance of parameters estimates , and half - normal plots with simulation envelopes were used to assess goodness - of - fit of the models . the analysis was performed using r software ( r core team , 2014 ) , and the r package hnp was used to the half - normal plots . in addition , predicted and average curves were compared and residuals were analyzed . data of parameters not normally distributed are shown as follows : median , 2575 percentiles ( 95% ci ) . those normally distributed are shown as mean sd . there were no statistically significant differences in the mean of age between patients with diabetes and persons without diabetes , so that both groups were matching for study according to the age . the baseline demographic , clinical , and general laboratory data are presented in table 1 . data of tsh , ft4 , and autoimmunity in euthyroid patients are in table 2 , as well as the occurrence of hypothyroidism and autoimmunity in hypothyroid patients . a lower rate of circulating antibody among hypothyroid patients was observed in the subgroup of pioglitazone [ 25% ( 1/4 ) ] ; however , the small number in the group did not justify a statistical analysis . in relation to hormonal data between antidiabetic drugs mtf+ subgroup is shown separately , since we had interest in checking whether this subgroup behaved as the subgroup only on mtf or as the other subgroups , since the mtf doses used were lower . table 3 shows the results of euthyroid patients and table 4 those of hypothyroid patients . when we compared clinical and subclinical hypothyroidism between patients with diabetes as a whole group and persons without diabetes , the results in patients with clinical hypothyroidism were as follows : tsh values : 17.5 , 13.017.3 ( 95% ci 12.216.4 ) mu / l in patients with diabetes and 15.6 , 13.416.8 ( 95% ci 11.916.1 ) mu / l in persons without diabetes ( p = ns ) ; ft4 values : 7.1 0.3 ( 95% ci 6.67.6 ) pmol / l [ 0.55 0.02 ( 95% ci 0.510.59 ) ng / dl ] and 7.7 0.1 ( 95% ci 6.87.9 ) pmol / l [ 0.6 0.01 ( 95% ci 0.530.61 ) ng / dl ] , respectively ( p = ns ) . for patients with subclinical hypothyroidism tsh values were 12.3 , 10.414.0 ( 95% ci 11.214.8 ) mu / l in patients with diabetes and 10.9 , 9.413.8 , ( 95% ci 10.713.7 ) mu / l in persons without diabetes , with a marginal significance ( p = 0.049 ) ; and ft4 values were 14.2 0.39 ( 95% ci 11.512.0 ) pmol / l [ 1.1 0.03 ( 95% ci 0.890.93 ) ng / dl ] and 14.2 0.3 ( 95% ci 11.814.2 ) pmol / l [ 1.1 0.02 ( 95% ci 0.921.1 ) ng / dl ] , respectively , in addition to the data cited , it is noteworthy that tsh in mtf users was also lower than in persons without diabetes ( p = 0.032 ) . ins compared to all other subgroups together had highest levels of fg , hba1c , and bmi . these parameters were as follows ( mtf others , resp . ) : fg : 8.4 , 6.610.6 ( 95% ci , 7.310.4 ) 7.3 , 5.69.3 ( 95% ci 6.58.8 ) , p = 0.004 ; hba1c : 7.6 , 6.49.0 , ( 95% ci 5.810.7 ) 7.1 , 6.28.9 ( 95% ci 6.89.2 ) , p = 0.0381 ; and bmi : 30.9 6.3 28.4 5.2 , p = 0.0061 . with regard to the relationship between tsh levels and mtf dose the selected model assumes an inverse distribution for the square root of tsh in the random component of the glm , an identity link function , and a systematic component ( linear predictor ) with intercept and terms until third order to the dose of metformin . symbolically , we have the estimated tsh with mtf dose varying from 500 to 3000 mg / day . parameters estimates and standard errors , according to likelihood - ratio tests , were all significant . the half - normal plot related to this model indicates a good fit , since less than 5% of the points fell out of the simulation envelope . even more , the residuals have mean , first , and third quartiles close to zero : 0.0017 , 0.2540 , and 0.1398 , respectively . a graphical representation of the model is presented in figure 1 , showing that the two curves are close . the overall conclusion is that there was a significant reduction of tsh as the mtf dosage increased . it was also possible to obtain a confidence interval for the estimated tsh , based on the asymptotic properties of the maximum likelihood estimators . figure 2 shows the 95% confidence interval . regarding a possible influence of mtf+ subgroup on the tsh , this was not observed in the same model , and therefore , the curves were not performed . in order to assess whether an age - specific tsh ri to older patients had a clinical impact with respect to the diagnosis of hypothyroidism in patients with diabetes , we compared the amount of hypothyroidism diagnosis in these patients when using tsh adjusted for age with the amount that would be diagnosed if using the ri defined by the kit manufacturer . in the first case 6.4% ( 45/751 ) this diagnosis was increased to 14.6% ( 110/751 ) , that is , more than twice , if the ri were adopted without discrimination by age . in this study , we aimed to evaluate the prevalence of undiagnosed hypothyroidism in patients with diabetes over 60 years of age by adopting age - specific ri . using this method , we observed that the diagnosis of hypothyroidism was slightly but significantly higher in patients with diabetes than in persons without diabetes , albeit less frequent than reported in some previous studies . a pubmed search revealed that most studies on the prevalence of hypothyroidism in patients with diabetes were conducted before recent revelations in the field about tsh values in the elderly . this rate is quite variable , ranging from 5.7% to 27.7% in previous studies [ 2 , 3 , 5 , 10 , 12 ] . in the patients with diabetes population we studied , the difference among the various studies could be attributed to which ri were used for the diagnosis of hypothyroidism , to the heterogeneity of patients ' ages , including in the same group those younger than 60 years and those older than 60 years , and to the characteristics of each population . there were higher rates of positive tpoab in patients with diabetes ( 13.8% ) , especially in hypothyroidism patients ( 64.4% ) . of note , the percentage of patients with diabetes with hypothyroidism and tpoab positivity was higher than that observed in other studies , which ranged from 14.6% to 43% [ 6 , 7 , 10 ] . autoimmunity directed against the thyroid can vary depending on factors such as patients ' ages , environmental factors , and the laboratory methods used . as this study focused on a select population of elderly subjects , the involvement of environmental factors including differences in iodine diet content , stress , and drugs could be other factors to be considered . although tsh levels were equivalent when comparing patients with diabetes to persons without diabetes overall , patients with diabetes who were given mtf had slightly but significantly lower tsh compared to persons without diabetes as well as to patients with diabetes treated with other antidiabetes agents . mtf is the most widely used drug to treat diabetes and is the first choice medication recommended for the treatment of type 2 diabetes by ada . some reports suggest that all subjects treated with mtf likely have lower tsh levels [ 30 , 31 ] , while others report no significant lowering of tsh by the drug , except in those subjects with subclinical hypothyroidism or who are on levothyroxine treatment [ 19 , 32 ] . the reason mtf may reduce tsh may be due to its reported capacity to cross the blood brain barrier . it directly acts in several regions of the brain and can concentrate in the pituitaries and hypothalamuses of rats and can also suppress amp - activated protein kinase activity ( ampk ) [ 33 , 34 ] . as hypothalamic triiodothyronine administration also decreases this enzyme , it is postulated that mtf could decrease tsh in the same manner as thyroid hormone , but to a lesser degree . this idea is further supported by a study that suggests that mtf may have an impact on thyrotrope function in hypothyroid patients , lowering tsh in polycystic ovary syndrome , which was attributed to a probable effect increasing thyroid hormone action in the pituitary . we observed an inverse correlation between doses of mtf and tsh . when evaluating patients being given a combination of mtf and one of the other drugs , lower tsh levels were not significant . a possible explanation is that the mtf doses in mtf+ subgroup were smaller than those on mtf as a sole medication . this could be the reason tsh have not been affected by mtf in combination with other antidiabetic drugs . we question whether the relation between tsh and mtf doses could explain the different results observed regarding the effect of this medication on tsh levels when comparing different series . at this point , these results can not be considered definitive but are preliminary data that offer new perspectives regarding the relationship between mtf and tsh , requiring confirmation with a larger number of cases . it has been suggested that the effect of mtf on tsh could be secondary to weight reduction . in the model adopted , we did not compare the impacts of each medication on patients ' weights , but the bmi of each treatment subgroup was compared with the others . at the time of data collection , all subjects except those on ins treatment had similar bmi . therefore , weight did not appear to be a significant factor in the effect of mtf on tsh levels compared to other medications . investigators also pointed to a goitrogenic effect as well as a decrease in iodine uptake by the thyroid gland with second - generation su gliclazide . in our study , these effects were also not observed in another study of second - generation su glyburide . regarding dpp4 , no changes were observed in tsh between this subgroup and either persons without diabetes or subjects in the other diabetes treatment groups . a search of the pubmed database using the terms dpp4 inhibitors , thyroid , tsh , hypothyroidism , and hyperthyroidism did not yield any studies related to tsh changes with dpp4 inhibitors . the tsh levels of patients who used pioglitazone ( tzd ) did not differ from other subgroups . pioglitazone is a tzd , a class of drugs that act as an exogenous peroxisome proliferator - activated receptor- ( ppar ) agonist . besides controlling glucose metabolism and fatty acid storage , ppar pioglitazone is also capable of lowering the expression and release of certain cytokines in thyroid cells . we observed that tpoab levels were lower in tzd subgroup of patients with diabetes ; however we could not make conclusions regarding the effect of pioglitazone on autoimmunity because of the small number of patients . future studies with ppar agonists in autoimmune thyroid diseases are necessary to determine any such effect . we postulate that this may be due to selection bias because this group also had the highest bmi and hba1c levels . previous studies showed a positive correlation between tsh , even within its normal range , and body weight / bmi and hba1c [ 12 , 4042 ] . one study linked increased weight with the susceptibility to harbor thyroid autoimmunity . we did not consider this aspect , as tpoab values in patients using ins ( the subgroup with higher bmis ) were not different when compared to the other subgroups . considering the most appropriate evaluation of the prevalence of undiagnosed hypothyroidism among patients with diabetes , especially the elderly , our results possible bias in this study is that of other drug interference , since polymedicine is prevalent in elderly patients ; besides that , thyroid function was only measured once and thus cases of subclinical thyroid dysfunction could be due to nondetected nonthyroidal illness during the selection . we attempted to minimize some of these possible biases , by excluding those subjects who used medications that are known to interfere with tsh levels . the authors suggest that an annual tsh measurement in dm patients over 60 years would be appropriate for hypothyroidism screening . if tsh levels do begin to increase at any time in relation to the specific ri for age , ft4 and tpoab levels should be measured . in conclusion , in patients with diabetes aged 60 years or older , hypothyroidism was more prevalent than in persons without diabetes of the same age , justifying the suggestion of annual screening in these patients . use of tsh ri appropriate for the elderly can avoid the misdiagnosis of hypothyroidism . in this study we found an interesting association between the mtf dose when used as a single medication and tsh levels , encouraging that further studies are conducted on this issue .

background . studies have suggested that hypothyroidism is more frequent in the elderly with diabetes mellitus . however , an adaptation of tsh levels to age should be considered in this assessment . some antidiabetes drugs reportedly interfere with tsh levels . the objectives of this study were to evaluate the prevalence of undiagnosed hypothyroidism in patients with diabetes and the influence of antidiabetes drugs . material and methods . 1160 subjects , 60 years and older ( 751 with diabetes ) , were studied ; results were compared according to diabetes treatment and with persons without diabetes . tsh , ft4 , antithyroperoxidase , fasting glucose , and hba1c were measured . results and discussion . 6.4% of patients with diabetes had hypothyroidism , a higher prevalence compared with persons without diabetes ( 5.1% ) , but lower than observed in many studies . the use of age - specific tsh reference interval ( ri ) could explain this difference . patients taking metformin ( mtf ) had tsh ( showed in medians ) slightly lower ( 2.8 mu / l ) than those not on mtf ( 3.3 mu / l ) , p < 0.05 . mtf doses influenced tsh levels . conclusions . the use of specific tsh ri could avoid the misdiagnosis of hypothyroidism in elderly with diabetes . patients in use of mtf as single drug had lower tsh than those using other medications and persons without diabetes .

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

the occurrence of hypothyroidism in patients with diabetes mellitus has attracted attention since joslin et al . several studies have reported a higher prevalence of thyroid dysfunction in diabetes [ 24 ] . this association may be related to autoimmunity as well as other factors associated with either type 1 or type 2 diabetes and suggests that persons with diabetes be screened for hypothyroidism [ 2 , 57 ] . the executive summary of standards of medical care in diabetes ( 2014 ) published by the american diabetes association ( ada ) as well as the clinical practice guidelines for hypothyroidism in adults ( 2012 ) copublished by the american association of clinical endocrinologists ( aace ) and the american thyroid association ( ata ) suggest screening patients with type 1 diabetes for thyroid diseases . age may be a factor associated with the higher prevalence of hypothyroidism among patients with type 2 diabetes [ 4 , 10 ] . in elderly patients , however , it may be difficult to determine if an abnormal thyroid stimulating hormone ( tsh ) pattern is due to actual thyroid disease or an unrelated cause or possibly is secondary to drug interference . although some studies investigated this , it was not always taken into consideration that the upper reference range for tsh in elderly individuals may be higher than in the general population [ 1012 ] . it is now clear that screening for thyroid dysfunction in elderly subjects with diabetes should take into account that an increase in serum tsh levels is expected . previously , an increase in tsh levels in elderly persons was attributed to elevated levels of circulating thyroid antibodies in this specific population . when considering screening for thyroid diseases in patients with diabetes , the diverse types of drugs used by a significant number of these patients ought to be taken into account , as many reportedly interfere with tsh levels , especially metformin and sulfonylureas [ 1822 ] . the major aim of this study was to evaluate the prevalence of previously undiagnosed cases of hypothyroidism in the elderly with diabetes by applying specific ri for serum tsh . a cross - sectional study of 1160 subjects over 60 years of age was performed , of whom 751 had diabetes . only patients with diabetes who had been diagnosed at least 2 years priorly were included ; diagnosis was according to the ada criteria . exclusion criteria were patients under treatment for thyroid diseases or with a history of thyroid disease , use of medications known to interfere with measurement of tsh or free thyroxine ( ft4 ) in the previous three months ( except for diabetes treatments ) , use of contrast media and other medications containing iodine in the last six months , illness , accident , or surgery in the last six months , and chronic disease except for hypertension and dyslipidaemia . there were 409 patients without diabetes who attended a clinical laboratory to collect routine tests for which function and/or autoimmunity thyroid evaluation had not been requested and that had normal blood count , kidney , and liver functions . all of them fulfilled the same exclusion criteria of patients with diabetes and were not under antidiabetes drugs for any other reason . patients with diabetes were divided into subgroups according to the medications they used as follows : subgroup mtf : metformin ( 5003000 mg / day ) ; subgroup su : sulfonylureas ( glibenclamide 515 mg / day or glimepiride 14 mg / day ) ; subgroup dpp4 : dipeptidyl peptidase 4 ( dpp4 ) inhibitors ( vildagliptin or sitagliptin , 50100 mg / day ) ; subgroup tzd : thiazolidinedione ( pioglitazone , 1545 mg / day ) ; subgroup ins : insulin analogues glargine or detemir 1342 ui , plus analogues lispro or aspart 614 ui / day ; subgroup mtf+ : metformin in combination with one or more other medications ( sulfonylurea , dpp4 inhibitor , thiazolidinedione , or insulin analogues ) ; the mtf+ group used lower doses of some medications compared to the doses when they were used as the sole medication : mtf : 5001500 ; su ( glibenclamide : 510 mg / day , glimepiride : 1 - 2 mg / day ) ; tzd : 1530 mg / day ; dpp4 and ins doses did not differ from the previous subgroups . all patients with diabetes had been on the same medication for at least one year . other antidiabetes drugs were not evaluated in the study , as there was an insufficient number of patients to allow statistical analysis . tsh , ft4 , antithyroperoxidase antibodies ( tpoab ) , fasting glucose ( fg ) , and glycated hemoglobin ( hba1c ) were measured in all patients . serum tsh , ft4 , and tpoab were measured by electrochemiluminescence immunoassays using the roche modular analytics e170 instrument ( roche diagnostics australia pty ltd . , serum tsh concentrations were measured by an immunometric method , with an intra - assay percent coefficient of variation ( % cv ) of 3.0% at concentrations of 0.040 0.001 the tsh reference interval ( ri ) established in our previous study of the population in the same region was 0.4 to 5.8 mu / l for subjects aged between 60 and 79 years and 0.4 to 6.7 mu / l for those aged 80 years and older . the % cv was 1.4% at concentrations of 9.0 0.1 pmol / l ( 0.7 0.01 ng / dl ) , 1.8% at 16.7 0.3 pmol / l ( 1.3 0.02 ng / dl ) , and 2.0% at 34.7 1.3 pmol / l ( 2.7 0.1 ng / dl ) . the ri for age groups , 60 years and older , was in the range of 9.021.9 pmol / l ( 0.71.7 ng / dl ) . the intra - assay % cv for tpoab was 6.3% at concentrations of 21.3 1.34 iu / ml , 5.1% at 51.2 2.6 iu / ml , and 2.7% at 473 12.7 iu / ml ; a score lower than 34 iu / ml indicated the absence of thyroid disease according to the assay 's manufacturer . the lower limit of assay detection is 0.1 mmol / l ( 2.0 mg / dl ) , with an intra - assay % cv of 0.8% at concentrations of 5.2 0.04 mmol / l ( 93.2 0.7 mg / dl ) , 0.7% at 13.4 0.11 mmol / l ( 241.0 2.0 mg / dl ) , and 0.7% at 36.1 0.28 mmol / l ( 651.0 5.0 mg / dl ) . according to the ada , the normal value is less than 5.6 mmol / l ( 100.0 mg / dl ) . according to the manufacturer , the intra - assay cv was 0.9% for concentrations in healthy subjects and 0.59% for measurements obtained from individuals with diabetes . a patient with diabetes with hba1c levels below 7.0% ( 53 mmol / mol ifcc ) mu / l in patients aged 6079 years and above 6.7 mu / l in patients aged 80 years or older , with ft4 levels ranging between 9.0 and 21.9 pmol / l ( 0.7 and 1.7 ng / dl ) ; overt hypothyroidism was diagnosed with the same values of tsh and with serum ft4 below 9.0 pmol / l ( 0.7 ng / dl ) . to test if patients with diabetes and persons without diabetes ages were matched , the unpaired t - test with welch 's correction was used , as age had normal distribution . descriptive analyses of serum tsh , fg , and hba1c were reported as medians and 25 and 75 percentiles , because they were not normally distributed . for all parameters , p < 0.05 was considered statistically significant . to describe the relationship between tsh ( mu / l ) and mtf dose ( mg / day ) , a statistical model was used assessing the possible significance of mtf effect in the tsh variability and providing estimated values for tsh according to different doses . the class of generalized linear models ( glm ) likelihood - ratio tests were used to assess the significance of parameters estimates , and half - normal plots with simulation envelopes were used to assess goodness - of - fit of the models . the analysis was performed using r software ( r core team , 2014 ) , and the r package hnp was used to the half - normal plots . there were no statistically significant differences in the mean of age between patients with diabetes and persons without diabetes , so that both groups were matching for study according to the age . data of tsh , ft4 , and autoimmunity in euthyroid patients are in table 2 , as well as the occurrence of hypothyroidism and autoimmunity in hypothyroid patients . a lower rate of circulating antibody among hypothyroid patients was observed in the subgroup of pioglitazone [ 25% ( 1/4 ) ] ; however , the small number in the group did not justify a statistical analysis . in relation to hormonal data between antidiabetic drugs mtf+ subgroup is shown separately , since we had interest in checking whether this subgroup behaved as the subgroup only on mtf or as the other subgroups , since the mtf doses used were lower . when we compared clinical and subclinical hypothyroidism between patients with diabetes as a whole group and persons without diabetes , the results in patients with clinical hypothyroidism were as follows : tsh values : 17.5 , 13.017.3 ( 95% ci 12.216.4 ) mu / l in patients with diabetes and 15.6 , 13.416.8 ( 95% ci 11.916.1 ) mu / l in persons without diabetes ( p = ns ) ; ft4 values : 7.1 0.3 ( 95% ci 6.67.6 ) pmol / l [ 0.55 0.02 ( 95% ci 0.510.59 ) ng / dl ] and 7.7 0.1 ( 95% ci 6.87.9 ) pmol / l [ 0.6 0.01 ( 95% ci 0.530.61 ) ng / dl ] , respectively ( p = ns ) . for patients with subclinical hypothyroidism tsh values were 12.3 , 10.414.0 ( 95% ci 11.214.8 ) mu / l in patients with diabetes and 10.9 , 9.413.8 , ( 95% ci 10.713.7 ) mu / l in persons without diabetes , with a marginal significance ( p = 0.049 ) ; and ft4 values were 14.2 0.39 ( 95% ci 11.512.0 ) pmol / l [ 1.1 0.03 ( 95% ci 0.890.93 ) ng / dl ] and 14.2 0.3 ( 95% ci 11.814.2 ) pmol / l [ 1.1 0.02 ( 95% ci 0.921.1 ) ng / dl ] , respectively , in addition to the data cited , it is noteworthy that tsh in mtf users was also lower than in persons without diabetes ( p = 0.032 ) . ins compared to all other subgroups together had highest levels of fg , hba1c , and bmi . : fg : 8.4 , 6.610.6 ( 95% ci , 7.310.4 ) 7.3 , 5.69.3 ( 95% ci 6.58.8 ) , p = 0.004 ; hba1c : 7.6 , 6.49.0 , ( 95% ci 5.810.7 ) 7.1 , 6.28.9 ( 95% ci 6.89.2 ) , p = 0.0381 ; and bmi : 30.9 6.3 28.4 5.2 , p = 0.0061 . with regard to the relationship between tsh levels and mtf dose the selected model assumes an inverse distribution for the square root of tsh in the random component of the glm , an identity link function , and a systematic component ( linear predictor ) with intercept and terms until third order to the dose of metformin . parameters estimates and standard errors , according to likelihood - ratio tests , were all significant . the overall conclusion is that there was a significant reduction of tsh as the mtf dosage increased . regarding a possible influence of mtf+ subgroup on the tsh , this was not observed in the same model , and therefore , the curves were not performed . in order to assess whether an age - specific tsh ri to older patients had a clinical impact with respect to the diagnosis of hypothyroidism in patients with diabetes , we compared the amount of hypothyroidism diagnosis in these patients when using tsh adjusted for age with the amount that would be diagnosed if using the ri defined by the kit manufacturer . in the first case 6.4% ( 45/751 ) this diagnosis was increased to 14.6% ( 110/751 ) , that is , more than twice , if the ri were adopted without discrimination by age . in this study , we aimed to evaluate the prevalence of undiagnosed hypothyroidism in patients with diabetes over 60 years of age by adopting age - specific ri . using this method , we observed that the diagnosis of hypothyroidism was slightly but significantly higher in patients with diabetes than in persons without diabetes , albeit less frequent than reported in some previous studies . a pubmed search revealed that most studies on the prevalence of hypothyroidism in patients with diabetes were conducted before recent revelations in the field about tsh values in the elderly . in the patients with diabetes population we studied , the difference among the various studies could be attributed to which ri were used for the diagnosis of hypothyroidism , to the heterogeneity of patients ' ages , including in the same group those younger than 60 years and those older than 60 years , and to the characteristics of each population . there were higher rates of positive tpoab in patients with diabetes ( 13.8% ) , especially in hypothyroidism patients ( 64.4% ) . of note , the percentage of patients with diabetes with hypothyroidism and tpoab positivity was higher than that observed in other studies , which ranged from 14.6% to 43% [ 6 , 7 , 10 ] . autoimmunity directed against the thyroid can vary depending on factors such as patients ' ages , environmental factors , and the laboratory methods used . as this study focused on a select population of elderly subjects , the involvement of environmental factors including differences in iodine diet content , stress , and drugs could be other factors to be considered . although tsh levels were equivalent when comparing patients with diabetes to persons without diabetes overall , patients with diabetes who were given mtf had slightly but significantly lower tsh compared to persons without diabetes as well as to patients with diabetes treated with other antidiabetes agents . some reports suggest that all subjects treated with mtf likely have lower tsh levels [ 30 , 31 ] , while others report no significant lowering of tsh by the drug , except in those subjects with subclinical hypothyroidism or who are on levothyroxine treatment [ 19 , 32 ] . as hypothalamic triiodothyronine administration also decreases this enzyme , it is postulated that mtf could decrease tsh in the same manner as thyroid hormone , but to a lesser degree . when evaluating patients being given a combination of mtf and one of the other drugs , lower tsh levels were not significant . a possible explanation is that the mtf doses in mtf+ subgroup were smaller than those on mtf as a sole medication . we question whether the relation between tsh and mtf doses could explain the different results observed regarding the effect of this medication on tsh levels when comparing different series . at this point , these results can not be considered definitive but are preliminary data that offer new perspectives regarding the relationship between mtf and tsh , requiring confirmation with a larger number of cases . it has been suggested that the effect of mtf on tsh could be secondary to weight reduction . in the model adopted , we did not compare the impacts of each medication on patients ' weights , but the bmi of each treatment subgroup was compared with the others . therefore , weight did not appear to be a significant factor in the effect of mtf on tsh levels compared to other medications . regarding dpp4 , no changes were observed in tsh between this subgroup and either persons without diabetes or subjects in the other diabetes treatment groups . a search of the pubmed database using the terms dpp4 inhibitors , thyroid , tsh , hypothyroidism , and hyperthyroidism did not yield any studies related to tsh changes with dpp4 inhibitors . the tsh levels of patients who used pioglitazone ( tzd ) did not differ from other subgroups . we observed that tpoab levels were lower in tzd subgroup of patients with diabetes ; however we could not make conclusions regarding the effect of pioglitazone on autoimmunity because of the small number of patients . previous studies showed a positive correlation between tsh , even within its normal range , and body weight / bmi and hba1c [ 12 , 4042 ] . considering the most appropriate evaluation of the prevalence of undiagnosed hypothyroidism among patients with diabetes , especially the elderly , our results possible bias in this study is that of other drug interference , since polymedicine is prevalent in elderly patients ; besides that , thyroid function was only measured once and thus cases of subclinical thyroid dysfunction could be due to nondetected nonthyroidal illness during the selection . we attempted to minimize some of these possible biases , by excluding those subjects who used medications that are known to interfere with tsh levels . the authors suggest that an annual tsh measurement in dm patients over 60 years would be appropriate for hypothyroidism screening . if tsh levels do begin to increase at any time in relation to the specific ri for age , ft4 and tpoab levels should be measured . in conclusion , in patients with diabetes aged 60 years or older , hypothyroidism was more prevalent than in persons without diabetes of the same age , justifying the suggestion of annual screening in these patients . use of tsh ri appropriate for the elderly can avoid the misdiagnosis of hypothyroidism . in this study we found an interesting association between the mtf dose when used as a single medication and tsh levels , encouraging that further studies are conducted on this issue .

several studies have reported a higher prevalence of thyroid dysfunction in diabetes [ 24 ] . this association may be related to autoimmunity as well as other factors associated with either type 1 or type 2 diabetes and suggests that persons with diabetes be screened for hypothyroidism [ 2 , 57 ] . the executive summary of standards of medical care in diabetes ( 2014 ) published by the american diabetes association ( ada ) as well as the clinical practice guidelines for hypothyroidism in adults ( 2012 ) copublished by the american association of clinical endocrinologists ( aace ) and the american thyroid association ( ata ) suggest screening patients with type 1 diabetes for thyroid diseases . age may be a factor associated with the higher prevalence of hypothyroidism among patients with type 2 diabetes [ 4 , 10 ] . in elderly patients , however , it may be difficult to determine if an abnormal thyroid stimulating hormone ( tsh ) pattern is due to actual thyroid disease or an unrelated cause or possibly is secondary to drug interference . although some studies investigated this , it was not always taken into consideration that the upper reference range for tsh in elderly individuals may be higher than in the general population [ 1012 ] . it is now clear that screening for thyroid dysfunction in elderly subjects with diabetes should take into account that an increase in serum tsh levels is expected . previously , an increase in tsh levels in elderly persons was attributed to elevated levels of circulating thyroid antibodies in this specific population . however several recent studies demonstrated that , even after excluding those with known thyroid diseases and circulating antithyroid antibodies , the tsh distribution curve remained shifted to the right ; the 97.5th percentile tsh values rose as the population aged [ 9 , 1317 ] . when considering screening for thyroid diseases in patients with diabetes , the diverse types of drugs used by a significant number of these patients ought to be taken into account , as many reportedly interfere with tsh levels , especially metformin and sulfonylureas [ 1822 ] . the major aim of this study was to evaluate the prevalence of previously undiagnosed cases of hypothyroidism in the elderly with diabetes by applying specific ri for serum tsh . a cross - sectional study of 1160 subjects over 60 years of age was performed , of whom 751 had diabetes . only patients with diabetes who had been diagnosed at least 2 years priorly were included ; diagnosis was according to the ada criteria . subjects normally resided in the metropolitan area of rio de janeiro and self - identified as belonging to middle and upper social classes . a national health surveillance agency review of salt from samples used in the city a year before the beginning of our study confirmed appropriate iodine levels . exclusion criteria were patients under treatment for thyroid diseases or with a history of thyroid disease , use of medications known to interfere with measurement of tsh or free thyroxine ( ft4 ) in the previous three months ( except for diabetes treatments ) , use of contrast media and other medications containing iodine in the last six months , illness , accident , or surgery in the last six months , and chronic disease except for hypertension and dyslipidaemia . there were 409 patients without diabetes who attended a clinical laboratory to collect routine tests for which function and/or autoimmunity thyroid evaluation had not been requested and that had normal blood count , kidney , and liver functions . patients with diabetes were divided into subgroups according to the medications they used as follows : subgroup mtf : metformin ( 5003000 mg / day ) ; subgroup su : sulfonylureas ( glibenclamide 515 mg / day or glimepiride 14 mg / day ) ; subgroup dpp4 : dipeptidyl peptidase 4 ( dpp4 ) inhibitors ( vildagliptin or sitagliptin , 50100 mg / day ) ; subgroup tzd : thiazolidinedione ( pioglitazone , 1545 mg / day ) ; subgroup ins : insulin analogues glargine or detemir 1342 ui , plus analogues lispro or aspart 614 ui / day ; subgroup mtf+ : metformin in combination with one or more other medications ( sulfonylurea , dpp4 inhibitor , thiazolidinedione , or insulin analogues ) ; the mtf+ group used lower doses of some medications compared to the doses when they were used as the sole medication : mtf : 5001500 ; su ( glibenclamide : 510 mg / day , glimepiride : 1 - 2 mg / day ) ; tzd : 1530 mg / day ; dpp4 and ins doses did not differ from the previous subgroups . body mass index ( bmi ) was obtained by dividing the body weight ( kg ) over the square of the height ( m ) . , serum tsh concentrations were measured by an immunometric method , with an intra - assay percent coefficient of variation ( % cv ) of 3.0% at concentrations of 0.040 0.001 the tsh reference interval ( ri ) established in our previous study of the population in the same region was 0.4 to 5.8 mu / l for subjects aged between 60 and 79 years and 0.4 to 6.7 mu / l for those aged 80 years and older . the % cv was 1.4% at concentrations of 9.0 0.1 pmol / l ( 0.7 0.01 ng / dl ) , 1.8% at 16.7 0.3 pmol / l ( 1.3 0.02 ng / dl ) , and 2.0% at 34.7 1.3 pmol / l ( 2.7 0.1 ng / dl ) . the ri for age groups , 60 years and older , was in the range of 9.021.9 pmol / l ( 0.71.7 ng / dl ) . the intra - assay % cv for tpoab was 6.3% at concentrations of 21.3 1.34 iu / ml , 5.1% at 51.2 2.6 iu / ml , and 2.7% at 473 12.7 iu / ml ; a score lower than 34 iu / ml indicated the absence of thyroid disease according to the assay 's manufacturer . the lower limit of assay detection is 0.1 mmol / l ( 2.0 mg / dl ) , with an intra - assay % cv of 0.8% at concentrations of 5.2 0.04 mmol / l ( 93.2 0.7 mg / dl ) , 0.7% at 13.4 0.11 mmol / l ( 241.0 2.0 mg / dl ) , and 0.7% at 36.1 0.28 mmol / l ( 651.0 5.0 mg / dl ) . hba1c was measured by high - performance liquid chromatography ( hplc ) on the bio - rad variant ii hemoglobin a1c system ( bio - rad laboratories , inc . the hba1c reportable range is between 3.1 and 18.5 percent ( % ) in national glycohemoglobin standardization program ( ngsp ) units and between 10 and 179 mmol hba1c / mol hb ( mmol / mol ) in international federation of clinical chemistry and laboratory medicine ( ifcc ) units . according to the manufacturer , the intra - assay cv was 0.9% for concentrations in healthy subjects and 0.59% for measurements obtained from individuals with diabetes . a patient with diabetes with hba1c levels below 7.0% ( 53 mmol / mol ifcc ) mu / l in patients aged 6079 years and above 6.7 mu / l in patients aged 80 years or older , with ft4 levels ranging between 9.0 and 21.9 pmol / l ( 0.7 and 1.7 ng / dl ) ; overt hypothyroidism was diagnosed with the same values of tsh and with serum ft4 below 9.0 pmol / l ( 0.7 ng / dl ) . to test if patients with diabetes and persons without diabetes ages were matched , the unpaired t - test with welch 's correction was used , as age had normal distribution . descriptive analyses of serum tsh , fg , and hba1c were reported as medians and 25 and 75 percentiles , because they were not normally distributed . to describe the relationship between tsh ( mu / l ) and mtf dose ( mg / day ) , a statistical model was used assessing the possible significance of mtf effect in the tsh variability and providing estimated values for tsh according to different doses . the class of generalized linear models ( glm ) likelihood - ratio tests were used to assess the significance of parameters estimates , and half - normal plots with simulation envelopes were used to assess goodness - of - fit of the models . there were no statistically significant differences in the mean of age between patients with diabetes and persons without diabetes , so that both groups were matching for study according to the age . data of tsh , ft4 , and autoimmunity in euthyroid patients are in table 2 , as well as the occurrence of hypothyroidism and autoimmunity in hypothyroid patients . a lower rate of circulating antibody among hypothyroid patients was observed in the subgroup of pioglitazone [ 25% ( 1/4 ) ] ; however , the small number in the group did not justify a statistical analysis . in relation to hormonal data between antidiabetic drugs mtf+ subgroup is shown separately , since we had interest in checking whether this subgroup behaved as the subgroup only on mtf or as the other subgroups , since the mtf doses used were lower . table 3 shows the results of euthyroid patients and table 4 those of hypothyroid patients . when we compared clinical and subclinical hypothyroidism between patients with diabetes as a whole group and persons without diabetes , the results in patients with clinical hypothyroidism were as follows : tsh values : 17.5 , 13.017.3 ( 95% ci 12.216.4 ) mu / l in patients with diabetes and 15.6 , 13.416.8 ( 95% ci 11.916.1 ) mu / l in persons without diabetes ( p = ns ) ; ft4 values : 7.1 0.3 ( 95% ci 6.67.6 ) pmol / l [ 0.55 0.02 ( 95% ci 0.510.59 ) ng / dl ] and 7.7 0.1 ( 95% ci 6.87.9 ) pmol / l [ 0.6 0.01 ( 95% ci 0.530.61 ) ng / dl ] , respectively ( p = ns ) . for patients with subclinical hypothyroidism tsh values were 12.3 , 10.414.0 ( 95% ci 11.214.8 ) mu / l in patients with diabetes and 10.9 , 9.413.8 , ( 95% ci 10.713.7 ) mu / l in persons without diabetes , with a marginal significance ( p = 0.049 ) ; and ft4 values were 14.2 0.39 ( 95% ci 11.512.0 ) pmol / l [ 1.1 0.03 ( 95% ci 0.890.93 ) ng / dl ] and 14.2 0.3 ( 95% ci 11.814.2 ) pmol / l [ 1.1 0.02 ( 95% ci 0.921.1 ) ng / dl ] , respectively , in addition to the data cited , it is noteworthy that tsh in mtf users was also lower than in persons without diabetes ( p = 0.032 ) . ins compared to all other subgroups together had highest levels of fg , hba1c , and bmi . : fg : 8.4 , 6.610.6 ( 95% ci , 7.310.4 ) 7.3 , 5.69.3 ( 95% ci 6.58.8 ) , p = 0.004 ; hba1c : 7.6 , 6.49.0 , ( 95% ci 5.810.7 ) 7.1 , 6.28.9 ( 95% ci 6.89.2 ) , p = 0.0381 ; and bmi : 30.9 6.3 28.4 5.2 , p = 0.0061 . with regard to the relationship between tsh levels and mtf dose the selected model assumes an inverse distribution for the square root of tsh in the random component of the glm , an identity link function , and a systematic component ( linear predictor ) with intercept and terms until third order to the dose of metformin . symbolically , we have the estimated tsh with mtf dose varying from 500 to 3000 mg / day . even more , the residuals have mean , first , and third quartiles close to zero : 0.0017 , 0.2540 , and 0.1398 , respectively . the overall conclusion is that there was a significant reduction of tsh as the mtf dosage increased . it was also possible to obtain a confidence interval for the estimated tsh , based on the asymptotic properties of the maximum likelihood estimators . regarding a possible influence of mtf+ subgroup on the tsh , this was not observed in the same model , and therefore , the curves were not performed . in order to assess whether an age - specific tsh ri to older patients had a clinical impact with respect to the diagnosis of hypothyroidism in patients with diabetes , we compared the amount of hypothyroidism diagnosis in these patients when using tsh adjusted for age with the amount that would be diagnosed if using the ri defined by the kit manufacturer . in the first case 6.4% ( 45/751 ) this diagnosis was increased to 14.6% ( 110/751 ) , that is , more than twice , if the ri were adopted without discrimination by age . in this study , we aimed to evaluate the prevalence of undiagnosed hypothyroidism in patients with diabetes over 60 years of age by adopting age - specific ri . using this method , we observed that the diagnosis of hypothyroidism was slightly but significantly higher in patients with diabetes than in persons without diabetes , albeit less frequent than reported in some previous studies . a pubmed search revealed that most studies on the prevalence of hypothyroidism in patients with diabetes were conducted before recent revelations in the field about tsh values in the elderly . in the patients with diabetes population we studied , the difference among the various studies could be attributed to which ri were used for the diagnosis of hypothyroidism , to the heterogeneity of patients ' ages , including in the same group those younger than 60 years and those older than 60 years , and to the characteristics of each population . there were higher rates of positive tpoab in patients with diabetes ( 13.8% ) , especially in hypothyroidism patients ( 64.4% ) . of note , the percentage of patients with diabetes with hypothyroidism and tpoab positivity was higher than that observed in other studies , which ranged from 14.6% to 43% [ 6 , 7 , 10 ] . as this study focused on a select population of elderly subjects , the involvement of environmental factors including differences in iodine diet content , stress , and drugs could be other factors to be considered . although tsh levels were equivalent when comparing patients with diabetes to persons without diabetes overall , patients with diabetes who were given mtf had slightly but significantly lower tsh compared to persons without diabetes as well as to patients with diabetes treated with other antidiabetes agents . mtf is the most widely used drug to treat diabetes and is the first choice medication recommended for the treatment of type 2 diabetes by ada . some reports suggest that all subjects treated with mtf likely have lower tsh levels [ 30 , 31 ] , while others report no significant lowering of tsh by the drug , except in those subjects with subclinical hypothyroidism or who are on levothyroxine treatment [ 19 , 32 ] . it directly acts in several regions of the brain and can concentrate in the pituitaries and hypothalamuses of rats and can also suppress amp - activated protein kinase activity ( ampk ) [ 33 , 34 ] . this idea is further supported by a study that suggests that mtf may have an impact on thyrotrope function in hypothyroid patients , lowering tsh in polycystic ovary syndrome , which was attributed to a probable effect increasing thyroid hormone action in the pituitary . when evaluating patients being given a combination of mtf and one of the other drugs , lower tsh levels were not significant . we question whether the relation between tsh and mtf doses could explain the different results observed regarding the effect of this medication on tsh levels when comparing different series . at this point , these results can not be considered definitive but are preliminary data that offer new perspectives regarding the relationship between mtf and tsh , requiring confirmation with a larger number of cases . in the model adopted , we did not compare the impacts of each medication on patients ' weights , but the bmi of each treatment subgroup was compared with the others . therefore , weight did not appear to be a significant factor in the effect of mtf on tsh levels compared to other medications . regarding dpp4 , no changes were observed in tsh between this subgroup and either persons without diabetes or subjects in the other diabetes treatment groups . a search of the pubmed database using the terms dpp4 inhibitors , thyroid , tsh , hypothyroidism , and hyperthyroidism did not yield any studies related to tsh changes with dpp4 inhibitors . besides controlling glucose metabolism and fatty acid storage , ppar pioglitazone is also capable of lowering the expression and release of certain cytokines in thyroid cells . we observed that tpoab levels were lower in tzd subgroup of patients with diabetes ; however we could not make conclusions regarding the effect of pioglitazone on autoimmunity because of the small number of patients . we did not consider this aspect , as tpoab values in patients using ins ( the subgroup with higher bmis ) were not different when compared to the other subgroups . considering the most appropriate evaluation of the prevalence of undiagnosed hypothyroidism among patients with diabetes , especially the elderly , our results possible bias in this study is that of other drug interference , since polymedicine is prevalent in elderly patients ; besides that , thyroid function was only measured once and thus cases of subclinical thyroid dysfunction could be due to nondetected nonthyroidal illness during the selection . in conclusion , in patients with diabetes aged 60 years or older , hypothyroidism was more prevalent than in persons without diabetes of the same age , justifying the suggestion of annual screening in these patients . in this study we found an interesting association between the mtf dose when used as a single medication and tsh levels , encouraging that further studies are conducted on this issue .

the cluster of pathologies known as metabolic syndrome , including obesity , insulin resistance ( ir),type 2 diabetes , and cardiovascular disease ( cvd ) , has become one of the most serious threats to human health . the dramatic increase in the incidence of obesity in most parts of the world has contributed to the emergence of this disease cluster , particularly insulin resistance and type 2 diabetes . ir is associated with a number of diseases including obesity , metabolic syndrome , type 2 diabetes , lipodystrophies , polycystic ovary syndrome , and chronic infection . the main characteristics of ir are disinhibited lipolysis in adipose tissue , impaired uptake of glucose by muscle , and disinhibited gluconeogenesis . ir most often precedes the onset of overt type 2 diabetes and is compensated initially by hyperinsulinemia . but this chronic secretion of large amounts of insulin to overcome tissue insensitivity can itself finally lead to beta cell failure and occurrence of hyperglycemia . in ir , visceral adipose tissue is resistant to the antilipolytic effect of insulin and consequently releases excessive amounts of ffa . a major contributor to the development of ir is an overabundance of circulating fatty acids . insulin - resistant people with obesity and/or type 2 diabetes have been identified a defect in mitochondrial oxidative phosphorylation that relates to the accumulation of triglycerides and related lipid molecules in muscle . in type 2 diabetes , elevated blood glucose levels are clearly an important secondary cause of dyslipidemia in susceptible patients , and poor control of glycaemia can sometimes result in profound dyslipidaemia , including hypertriglyceridaemia and low high density lipoprotein ( hdl ) blood levels . sustained hyperglycemia in type 2 diabetes induces macrovascular and microvascular complications . in a recent report of world health organization and international diabetes federation it was found that about 80% diabetic morbidity and mortality is caused by diabetic cardiomyopathy which is closely related with diabetic dyslipidemia . the low density lipoprotein ( ldl ) blood level in a patient with diabetes may be somewhat misleading ; a patient with diabetes may have an increased proportion of small dense ldl particles and an increase in atherogenic risk , compared with a nondiabetic patient with similar ldl blood level . moreover , patients with small , dense ldl will also typically have lower hdl and elevated triglyceride blood levels , which may further increase atherosclerosis risk . plant derivatives with purported hypoglycemic properties have been used in folk medicine and traditional healing systems around the world . many modern pharmaceuticals used in conventional medicine today also have natural plant origins . among them , metformin was derived from the flowering plant galega officinalis ( goat 's rue or french lilac ) , which was a common traditional remedy for diabetes . oleander ( nerium oleander , no ) of the dogbane ( apocynaceae ) family grows along the whole mediterranean coast starting in southern portugal in the west , in syria , and in streambeds of the western and southern anatolia . central nervous system depressant activity and dose - dependent cardiotonic effect of no were exhibited in studies . ishikawa et al . were reported that postprandial rise in blood glucose when maltose and sucrose were loaded in nondiabetic healthy rats was reduced by hot water extract of nerium indicum leaves . recently ligands for the peroxisome proliferator - activated receptors ( ppars ) which play a key role in glucose and lipid metabolism are defined as new insulin sensitizing drugs and hypolipidemic fibrates . in the present study , we investigated the effects of no distillate on hyperglycemia and dyslipidemia and its activities on liver and adipose tissue ppars in type 2 diabetic rats . september period from mediterranean region of turkey , identified , and authenticated at the department of biology . firstly collected plant was washed , fresh shoots were chopped , and adequate distilled water was added . after the liquid started to evaporate , container lid was covered , and vapor was separated to other clean glass containers by causing it to come in contact with a surface cooled with cold water . no distillate was lyophilized in small glass bottle ( 20 ml ) by using lyophilizator . lyophilized no distillates were dissolved at concentrations of 7.5 , 75 , and 750 g / ml in distilled water . eighty male sprague dawley rats ( 1012 weeks ) were allocated to metabolic cages individually in an automatic ambient humidity ( 50 5% ) , temperature ( 22 2c ) , and light - dark ( 12 : 12 ) controlled room . animals were obtained from the experimental research center of akdeniz university , faculty of medicine , antalya , turkey and divided into eight groups . commercially available rat normal pellet diet and water were given ad libitum to all animals prior to dietary manipulation . the experimental protocol was approved by the ethics committee in animal experimentation of selcuk university , turkey . after 2 weeks feeding of high fat diet , diabetes was induced in fasted animals by a single intra - peritoneal injection of streptozotocin ( stz ) ( 35 mg / kg bw ) dissolved in citrate buffer ( ph 4.5 ) when only buffer was received by control groups . in one week after stz injection , rats with 16.65 mmol / l ( 300 mg / dl ) nonfasting blood glucose level were considered to be type 2 diabetic . healthy control ( c ) and healthy control administered the highest level of no ( cno-10 ) group had normal pellet diet although high fat group ( hf ) and other all type 2 diabetic groups had high fat diet which 58% of the metabolic energy is provided from animal fat . nutritional substances of normal pellet diet were dry matter 89% , crude protein 21% , metabolic energy 2850 kcal / kg , crude fiber 5% , methionine and cystein 0.75% , calcium 1.02.0% , phosphor 0.51.0% , and sodium 0.5% ( optima feeds , turkey ) . after induction of diabetes , animals were randomly allocated to five groups in which one of them did not had any treatment ( d ) ; other groups had active substances once a day by gavage for 12 weeks of the treatment period . the experimental groups according to diets and administrations applied to animals were represented in table 2 . although fasting blood samples were taken from tail vein of all rats at 15 day intervals through the experiment , the results in tables 3 and 4 represent only the last sampling . about 0.5 ml whole blood from each animal serum samples were analyzed immediately for fasting blood glucose ( fbg ) , total cholesterol ( total - c ) , hdl , ldl , triglyceride , alkaline phosphatase ( alp ) , aspartate transaminase ( ast ) , and alanine transaminase ( alt ) by using commercially available colorimetric diagnostic kits ( il test , instrumentation laboratory , milano , italy ) by autoanalyzer ( ilab , 300 , milano , italy ) . after sampling the animals in all test groups was euthanized under general anesthesia with thiopental sodium ( 50 mg / kg bw ) subsequent to the final sampling time . liver and white adipose tissue samples from subcutan adipose tissue were taken and kept under required conditions . data for homa - ir ( homa - ir : fasting insulin ( u / ml ) fasting glucose ( mmol / l)/22.5 ) , beta cell functions as homa- ( homa-(% ) : ( 20 fasting insulin ( u / ml)/{fasting glucose ( mmol / l ) 3.5 } ) , atherogenic index ( ai : ( [ total - c ] [ hdl])/[hdl ] ) , hdl % in total - c , and triglyceride to hdl ratio of the all studied groups were calculated . serum insulin ( drg , millipore , ma , usa ) and leptin ( r&d , mn , usa ) levels were analyzed by elisa according to kit procedures . total rna is extracted , and oligo - dt primed first - strand cdna is synthesized . a reverse transcription polymerase chain reaction ( rt - pcr ) is performed using a thermal cycler system , specific primers for ppar- , ppar- , and -actin ( roche diagnostics , rotkreuz , switzerland ) are used . were performed one - way anova followed by a multiple comparison test ( postdoc duncan 's test ) using spss 17.0 ( spss , chicago , usa ) . differences were considered significant at p less than 0.05 . also kruskall wallis and mann whitney - u test ( use these when the data is not normally distributed ) were used to determine statistically differences of in vitro data between groups . table 3 shows values of body weight , fbg , hba1c , total - c , hdl , ldl , and triglyceride , ai , hdl% in total - c , triglyceride to hdl ratio of the all studied groups . data for insulin , leptin , homa - ir , homa- , alp , ast , and alt were presented in table 4 . no gastrointestinal disorders were observed during or after no treatment . although being diabetic , the considerable elevation in body weight of no treated groups was estimated compared to d and g ( p < 0.0001 ) . fbg levels were significantly decreased by using no when compared to d and g ( p < 0.0001 ) . in parallel with the improvement of fbg as shown in table 3 , all other diabetic groups were hyperglycemic and had significantly higher hba1c than healthy groups and diabetic no groups ( p < 0.0001 ) . although having high fat diet , no-1 and no-10 displayed similar total - c concentrations compared to c ( p > 0.05 , table 3 ) . total - c was numerically lower in cno-10 and higher in hf than c. the increased values of hdl were found in no-0.1 and no-10 compared to c ( p < 0.0001 , table 3 ) . the reducing effect of all no regimens and g on ldl concentration was noticeable and the values were similar to healthy groups ( p > 0.05 ) . ldl levels in type 2 diabetic no-1 and no-10 groups were significantly lower than d ( p < 0.05 ) . hdl percentage in total - c of no-0.1 and no-1 groups was similar to c ( p > 0.05 ) and the highest hdl percentage was estimated in no-10 among healthy and diabetic groups except cno-10 . the similarity in terms of ai of no groups to healthy groups was noticeable ( p > 0.05 , table 3 ) . the reducing effect of no-10 on ai was significant when compared to d , g , no-0.1 , and no-1 ( p < 0.001 ) . there were significant reductions in triglyceride - hdl ratio of all no regimens compared to d ( p < 0.0001 , table 3 ) . these reducing effects of no on the ratio were noticeable and the results were similar to c ( p > 0.05 ) . triglyceride - hdl ratio was numerically lower in cno-10 and was higher in hf than c. similar results in g and d were noted in terms of triglyceride - hdl ratio . when we assessed insulin levels , the antihyperglycemic effect of no was seen on data that no significantly decreased insulin concentration compared to d ( p < 0.0001 , table 4 ) . although insulin levels in all no groups were numerically higher than other healthy control groups , the results were statistically similar with those groups ( p > 0.05).insulin level was decreased by g and data were not different with all no groups and healthy groups ( p > 0.05 ) . there was 26.72% fall in insulin level of cno-10 compared to c. leptin levels of all no groups were not significantly different compared to healthy control groups ( p > 0.05 ) . almost twofold increases were estimated in other diabetic groups when compared to all no treated groups ( p < 0.01 , table 4 ) calculated homa - ir in no groups and in g was significantly lower than d ( p < 0.0001 , table 4 ) . insulin sensitivity has been improved by no treatment . also numerically the lowest insulin resistance was found in cno-10 . homa- impaired dramatically in groups d and g. improved beta cell function was observed in all no groups and was similar to healthy group 's values ( p > 0.05 , table 4 ) . the highest alp activity was found in d ( p < 0.0001 , table 4 ) . all no treatment dosages decreased alp activity when compared to d ( p < 0.0001 ) . the alp activity of g was similar to all no regimens ( p > 0.05 ) . the elevated alt activities , compared with c , were observed in d and g ( p < 0.05 , table 4 ) . the primer sequences , pcr protocol , and product sizes are presented in table 5 . agarose gel electrophoresis of pcr products are shown in figures 1(a ) and 2(a ) . the densities of each band evaluated by diana v1.6 and aida 2.4.3 analysis programs ( raytest imaging system , germany ) and the ratio of liver and adipose tissues ppar- , - , - mrna expressions to -actin are given in figures 1(b ) , 1(c ) , 1(d),2(b ) , 2(c ) , and 2(d ) , respectively . in liver , ppar- mrna expression decreased in d compared to c ( p < 0.05 ) , whereas its expression increased in no-10 treated group ( p < 0.01 ) . ppar- mrna expression increased in no-0.1 ( p < 0.001 ) treated group compared to d. ppar- mrna expression increased in no treated groups as a dose dependent manner but the increase in no-10 group is not significant compared to c ( p > 0.05 ) . in adipose tissue , ppar- and - mrna expression increased in no-10 group compared to c and diabetic rats ( p < 0.01 ) . the increase in ppar- mrna expression is more prominent in no-10 treated group compared to c and diabetic group ( p < 0.001 ) . during the experiment , administration of combined drug and insulin was not in question . studies , most notably the dcct , have defined quantitatively the relationship between glycated hb and average glycemia . the increase in vascular disease in patients with diabetes is thought to be due to the deleterious effects of metabolic abnormalities , such as hyperglycemia , insulin resistance , dyslipidemia , and advanced glycation end products [ 18 , 19 ] . in diabetes , hba1c levels predict the risk of microvascular complications and glycemic control to a hba1c of less than 7% will reduce microvascular complications and could decrease risk for macrovascular disease as well . the most important result in terms of the fbg is that no administered groups exhibited a level which was close to the data from c and that this condition was confirmed with the hba1c levels obtained from the above - mentioned groups . no treatment significantly reduced hba1c levels that were similar to healthy groups when glibenclamide had no significant effect over that . no treatment reduced hba1c 15.97 , 15.94 , and 19.54% in no-0.1 , no-1 , and no-10 , respectively , when compared to d. all no regimens also decreased fbg compared to other diabetic groups at the end of trial . therefore , no may be beneficial to reduce microvascular and macrovascular risk of type 2 diabetes . homa - ir is an independent predictor of cvd in type 2 diabetes and the improvement of ir might have beneficial effects not only on glucose control but also on cvd in patients with type 2 diabetes mellitus . no administration resulted in significant lowering of both insulin level and homa - ir compared to d as observed in g. improved beta cell function ( homa- ) in all no dosages was established but the improvement was noticeable in no-0.1 . homa- was numerically higher in cno-10 than c. the evaluated data suggest that no ameliorates glycemic control by insulin secretagogue and sensitizing effects . dyslipidemia , as associated with diabetic metabolism and the metabolic syndrome , is characterized by a so - called proatherogenic blood lipid profile , comprising low levels of hdls , increased ldls , and serum triglycerides associated with vldls . in fact , hypertriglyceridemia is considered to represent an important risk factor for atherosclerosis and subsequent cardiovascular complications in type 2 diabetic patients . the atherogenic index has recently been proposed as a marker of plasma atherogenicity because it is increased in people at higher risk for coronary heart disease and is inversely correlated with ldl particle size . the increased risk of coronary heart disease in patients with the metabolic syndrome suggests that the insulin - resistant state is atherogenic without concomitant elevations in plasma glucose and glycosylated hemoglobin [ 26 , 27 ] . the present data showed that all no administration numerically decreased ai but the highest no administration level significantly lowered atherogenicity in blood compared to other diabetic groups . a positive correlation between mean homa - ir and ai ( p < 0.05 , r = 0.71 ) and also between mean hba1c and ai ( p < 0.01 , r = 0.86 ) suggests that improving blood glucose level and insulin receptor sensitivity may support to decline atherogenicity and cardiovascular complications in type 2 diabetes by using no distillate . moreover , the highly significant positive correlation between homa - ir and triglyceride levels ( p < 0.0001 , r = 0.74 ) and inclination of the reduction of triglyceride by no administration indicates no may have a good beneficial effect on glucose and lipid metabolism as inferred from homa - ir results . the atherogenic link between high triglycerides and hdl is due to the higher plasma concentration of triglyceride - rich , vldl that generates small , dense ldl during lipid exchange and lipolysis . these ldl particles accumulate in the circulation and form small , dense hdl particles , which undergo accelerated catabolism , thus closing the atherogenic circle [ 28 , 29 ] . the significant similarity of c levels especially in total - c of no-1 and no-10 administration and ldl concentrations in all no regimens of diabetics support lipid lowering effects of no . diabetic dyslipidemia includes an overall increase in atherogenic particles identifiable , by measuring apolipoprotein b , and a predominance of small dense ldl particles ( phenotype b ) . a high ratio of triglycerides to hdl - cholesterol correlates with ldl phenotype b , small hdl particles , and ir [ 3032 ] and found to be a powerful independent indicator of extensive coronary disease . according to the above explanations , the ratio of triglycerides to hdl results in this paper indicates that no treatment may prevent the extensive coronary disease due to decreasing atherogenic particles since the ratio found to be statistically very similar to the ratio of c. also , in healthy rats , no made reduction in triglycerides to hdl ratio . high hdl levels in blood are considered to be cardioprotective , since the apo - a - containing hdl particles that help transport cholesterol to the liver from peripheral tissues , as well as away from macrophages associated with cholesterol deposits within the vascular wall . however , this cardioprotective effect may not be solely due to cholesterol transport . for example , hdl may have direct antioxidant and anti - inflammatory effects on the vessel wall . the reduction in triglyceride levels together with the higher concentrations of hdl especially in no-0.1 and no-10 groups compared to c were remarkable effects of no over dyslipidemia of type 2 diabetic rats . the ratio of serum hdl cholesterol to total - c used a marker of cardiovascular risk . all no treatment levels were associated with increase in hdl percentage in total - c but a highly significant arise was found in no-10 compared to other diabetics although the lower percentages of hdl in total - c of diabetic groups were estimated especially in g and d. numerically increase in hdl percent in total - c of cno-10 compared to c also indicates the beneficial effect of no over the lipid metabolism of healthy individuals.all these positive results in respect to lipid lowering effects of no may indicate reducing risk factor for atherosclerosis and subsequent cardiovascular complications in type 2 diabetes . leptin is a hormone secreted predominantly by adipose tissue and is a signal of sufficiency of energy . leptin effects are mediated by its action on the hypothalamus and directly on target tissues ( muscle , gonads , beta - cells , and liver ) . in normal conditions of weight maintenance , leptin concentrations are positively correlated with total body fat mass . in short - term food deprivation , serum leptin levels decrease and soluble leptin receptors are thought to be important for transport to or over the blood brain barrier ( bbb ) , and it is the saturation of this transport or impairment of leptin receptor signal transduction that may be the cause of leptin resistance . so that , the significant elevated levels of leptin in d and g , except no treated ones , reflect leptin resistance and/or insulin resistant state . the effect of leptin on inducing insulin resistance was exhibited by positive correlation between mean homa - ir and leptin ( p < 0.05 , r = 0.76 ) in this research . no positively affected leptin levels and was found to be similar to healthy control level . this remarkable result indicates that no may help to compensate the energy metabolism by improving leptin and insulin levels , and homa - ir , homa- in type 2 diabetes . triglycerides are an important cause of leptin resistance as mediated by impaired transport across the bbb . in this research , positive correlation was found between mean triglyceride and leptin levels ( p < 0.05 , r = 0.70 ) . this data may indicate that high triglyceride levels resulted in high blood leptin levels in d and g groups . a number of studies have reported that alt , ast , and/or ggt levels independently predict incidents of type 2 diabetes , metabolic syndrome , and cvd . in addition , these markers have been shown to be associated with indirect measures of insulin resistance including fasting insulin levels and homa - ir [ 37 , 38 ] since alt was associated with insulin resistance independently and an inexpensive way to improve the identification of subjects with insulin resistance . the reduction of ast activity in no-10 was noticeable when compared to d. alt activity was significantly higher in d than c group but was similar in no groups compared with c ( p > 0.05 ) . also , mean alt activity was positively correlated with homa - ir ( p < 0.05 , r = 0.80 ) . although alp activities were not different between type 2 diabetic and nondiabetic patients , the increased alp activity has been reported in ketotic and nonketotic diabetic rats primarily due to an increase in intestinal and bone / liver alp isoenzyme . all no regimens exhibited the beneficial effect on reducing alp activity in type 2 diabetic rats . the results of liver enzymes by using no dosages show that no had no detrimental effect on the liver function . in rodents , adipose tissue ppar- mrna and protein levels are reduced after an overnight fast [ 42 , 43 ] in stz - induced diabetes , which is consistent with the stimulatory effect of insulin on ppar- expression . in addition , chronic feeding with high fat diets was shown to increase ppar- expression in adipose tissue , whereas fasting stimulates especially liver ppar- expression in rodents [ 42 , 43 ] . the overexpressions of ppar- and - in adipose tissue may indicate that the effect of no-10 group dosage on reducing insulin resistance and indirectly reducing the risk of atherosclerosis is being through the storage of fatty acids to adipocytes and regulating adipocyte differentiation . significantly increased ppar- expression in liver and adipose tissue of no-10 group suggests that no-10 dosage may have important effects on regulating fatty acid oxidation system , lipoprotein synthesis , and inflammatory responses . single agents that promote both ppar- and ppar- agonism could theoretically offer significant benefits in improving dyslipidaemia and reducing hyperglycaemia and thus reduce these cardiovascular risk factors associated with type 2 diabetes and metabolic syndrome . in addition , such a therapy could reduce the underlying insulin resistance and help to break the cycle of altered glucose and lipid metabolism that promotes type 2 diabetes . it has been considered that increasing fatty acid oxidation in liver by overexpression of liver ppar- led not to use too much fat from adipose tissues for energy and also storage of fatty acids in adipocytes by overexpression of adipose tissue ppar- of no treated animals improved insulin action . this progress prevented body weight loss of no treated animals due to an antilipolitic effect of insulin when compared to uncontrolled diabetic ones . homa - ir and homa- results also support this improvement . also the pulling down effect of no-10 dosage on body weight might be associated with overexpressions of ppar- and - in adipose tissue of healthy rats since overexpression of ppar- specifically in adipose tissue decreases fatty acid levels and protects against obesity . moreover ppar- and ppar- are both active participants in energy burning ; ppar- plays a critical role in fatty acid oxidation and is thus responsible for energy expenditure ; ppar- also enhances fatty acid catabolism and energy uncoupling in skeletal muscle and adipose tissue as well , as recently shown in the liver . the considerable beneficial effects of especially no-10 group treatment ( no distillate administration at a dose of 375 g/0.51 ml of distilled water / d ) on glucose metabolism , insulin resistance , insulinotropic activity , leptin , dyslipidemia , liver enzymes , and ppars may point out the insulin - like effect of no and offer new approaches to treatment strategies that target both fat and glucose metabolism ultimately leadind to a reduction in both the chronic microvascular complications of type 2 diabetes and the risk of macrovascular events such as cvd . in addition , this concentration of no may prevent and control elevated glucose and blood lipid levels of the remainder of the population .

diabetes was induced by intraperitoneal injection of streptozotocin ( 35 mg / kg bw ) in all rats of five groups after being fed for 2 weeks high - fat diet . type 2 diabetic nerium - oleander- ( no- ) administered groups received the no distillate at a dose of 3.75 , 37.5 , and 375 g/0.5 ml of distilled water ( no-0.1 , no-1 , no-10 , resp . ) ; positive control group had 0.6 mg glibenclamide / kg bw / d by gavage daily for 12 weeks . type 2 diabetic negative control group had no treatment . no distillate administration reduced fasting blood glucose , hba1c , insulin resistance , total cholesterol , low density lipoprotein , atherogenic index , triglyceride - hdl ratio , insulin , and leptin levels . improved beta cell function and hdl concentration were observed by no usage . hdl percentage in total cholesterol of all no groups was similar to healthy control . no-10 distillate enhanced mrna expressions of peroxisome proliferator - activated - receptor- ( ppar- ) , , and in adipose tissue and ppar- in liver . the findings from both in vivo and in vitro studies suggest that the considerable beneficial effect of no distillate administration at a dose of 375 g/0.5 ml of distilled water may offer new approaches to treatment strategies that target both fat and glucose metabolism in type 2 diabetes .

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

the cluster of pathologies known as metabolic syndrome , including obesity , insulin resistance ( ir),type 2 diabetes , and cardiovascular disease ( cvd ) , has become one of the most serious threats to human health . the dramatic increase in the incidence of obesity in most parts of the world has contributed to the emergence of this disease cluster , particularly insulin resistance and type 2 diabetes . ir is associated with a number of diseases including obesity , metabolic syndrome , type 2 diabetes , lipodystrophies , polycystic ovary syndrome , and chronic infection . the main characteristics of ir are disinhibited lipolysis in adipose tissue , impaired uptake of glucose by muscle , and disinhibited gluconeogenesis . ir most often precedes the onset of overt type 2 diabetes and is compensated initially by hyperinsulinemia . in ir , visceral adipose tissue is resistant to the antilipolytic effect of insulin and consequently releases excessive amounts of ffa . insulin - resistant people with obesity and/or type 2 diabetes have been identified a defect in mitochondrial oxidative phosphorylation that relates to the accumulation of triglycerides and related lipid molecules in muscle . in type 2 diabetes , elevated blood glucose levels are clearly an important secondary cause of dyslipidemia in susceptible patients , and poor control of glycaemia can sometimes result in profound dyslipidaemia , including hypertriglyceridaemia and low high density lipoprotein ( hdl ) blood levels . sustained hyperglycemia in type 2 diabetes induces macrovascular and microvascular complications . the low density lipoprotein ( ldl ) blood level in a patient with diabetes may be somewhat misleading ; a patient with diabetes may have an increased proportion of small dense ldl particles and an increase in atherogenic risk , compared with a nondiabetic patient with similar ldl blood level . among them , metformin was derived from the flowering plant galega officinalis ( goat 's rue or french lilac ) , which was a common traditional remedy for diabetes . oleander ( nerium oleander , no ) of the dogbane ( apocynaceae ) family grows along the whole mediterranean coast starting in southern portugal in the west , in syria , and in streambeds of the western and southern anatolia . central nervous system depressant activity and dose - dependent cardiotonic effect of no were exhibited in studies . were reported that postprandial rise in blood glucose when maltose and sucrose were loaded in nondiabetic healthy rats was reduced by hot water extract of nerium indicum leaves . recently ligands for the peroxisome proliferator - activated receptors ( ppars ) which play a key role in glucose and lipid metabolism are defined as new insulin sensitizing drugs and hypolipidemic fibrates . in the present study , we investigated the effects of no distillate on hyperglycemia and dyslipidemia and its activities on liver and adipose tissue ppars in type 2 diabetic rats . firstly collected plant was washed , fresh shoots were chopped , and adequate distilled water was added . no distillate was lyophilized in small glass bottle ( 20 ml ) by using lyophilizator . lyophilized no distillates were dissolved at concentrations of 7.5 , 75 , and 750 g / ml in distilled water . eighty male sprague dawley rats ( 1012 weeks ) were allocated to metabolic cages individually in an automatic ambient humidity ( 50 5% ) , temperature ( 22 2c ) , and light - dark ( 12 : 12 ) controlled room . after 2 weeks feeding of high fat diet , diabetes was induced in fasted animals by a single intra - peritoneal injection of streptozotocin ( stz ) ( 35 mg / kg bw ) dissolved in citrate buffer ( ph 4.5 ) when only buffer was received by control groups . in one week after stz injection , rats with 16.65 mmol / l ( 300 mg / dl ) nonfasting blood glucose level were considered to be type 2 diabetic . healthy control ( c ) and healthy control administered the highest level of no ( cno-10 ) group had normal pellet diet although high fat group ( hf ) and other all type 2 diabetic groups had high fat diet which 58% of the metabolic energy is provided from animal fat . nutritional substances of normal pellet diet were dry matter 89% , crude protein 21% , metabolic energy 2850 kcal / kg , crude fiber 5% , methionine and cystein 0.75% , calcium 1.02.0% , phosphor 0.51.0% , and sodium 0.5% ( optima feeds , turkey ) . after induction of diabetes , animals were randomly allocated to five groups in which one of them did not had any treatment ( d ) ; other groups had active substances once a day by gavage for 12 weeks of the treatment period . although fasting blood samples were taken from tail vein of all rats at 15 day intervals through the experiment , the results in tables 3 and 4 represent only the last sampling . about 0.5 ml whole blood from each animal serum samples were analyzed immediately for fasting blood glucose ( fbg ) , total cholesterol ( total - c ) , hdl , ldl , triglyceride , alkaline phosphatase ( alp ) , aspartate transaminase ( ast ) , and alanine transaminase ( alt ) by using commercially available colorimetric diagnostic kits ( il test , instrumentation laboratory , milano , italy ) by autoanalyzer ( ilab , 300 , milano , italy ) . after sampling the animals in all test groups was euthanized under general anesthesia with thiopental sodium ( 50 mg / kg bw ) subsequent to the final sampling time . liver and white adipose tissue samples from subcutan adipose tissue were taken and kept under required conditions . data for homa - ir ( homa - ir : fasting insulin ( u / ml ) fasting glucose ( mmol / l)/22.5 ) , beta cell functions as homa- ( homa-(% ) : ( 20 fasting insulin ( u / ml)/{fasting glucose ( mmol / l ) 3.5 } ) , atherogenic index ( ai : ( [ total - c ] [ hdl])/[hdl ] ) , hdl % in total - c , and triglyceride to hdl ratio of the all studied groups were calculated . serum insulin ( drg , millipore , ma , usa ) and leptin ( r&d , mn , usa ) levels were analyzed by elisa according to kit procedures . table 3 shows values of body weight , fbg , hba1c , total - c , hdl , ldl , and triglyceride , ai , hdl% in total - c , triglyceride to hdl ratio of the all studied groups . data for insulin , leptin , homa - ir , homa- , alp , ast , and alt were presented in table 4 . no gastrointestinal disorders were observed during or after no treatment . although being diabetic , the considerable elevation in body weight of no treated groups was estimated compared to d and g ( p < 0.0001 ) . although having high fat diet , no-1 and no-10 displayed similar total - c concentrations compared to c ( p > 0.05 , table 3 ) . the reducing effect of all no regimens and g on ldl concentration was noticeable and the values were similar to healthy groups ( p > 0.05 ) . ldl levels in type 2 diabetic no-1 and no-10 groups were significantly lower than d ( p < 0.05 ) . hdl percentage in total - c of no-0.1 and no-1 groups was similar to c ( p > 0.05 ) and the highest hdl percentage was estimated in no-10 among healthy and diabetic groups except cno-10 . the similarity in terms of ai of no groups to healthy groups was noticeable ( p > 0.05 , table 3 ) . the reducing effect of no-10 on ai was significant when compared to d , g , no-0.1 , and no-1 ( p < 0.001 ) . there were significant reductions in triglyceride - hdl ratio of all no regimens compared to d ( p < 0.0001 , table 3 ) . these reducing effects of no on the ratio were noticeable and the results were similar to c ( p > 0.05 ) . triglyceride - hdl ratio was numerically lower in cno-10 and was higher in hf than c. similar results in g and d were noted in terms of triglyceride - hdl ratio . when we assessed insulin levels , the antihyperglycemic effect of no was seen on data that no significantly decreased insulin concentration compared to d ( p < 0.0001 , table 4 ) . although insulin levels in all no groups were numerically higher than other healthy control groups , the results were statistically similar with those groups ( p > 0.05).insulin level was decreased by g and data were not different with all no groups and healthy groups ( p > 0.05 ) . there was 26.72% fall in insulin level of cno-10 compared to c. leptin levels of all no groups were not significantly different compared to healthy control groups ( p > 0.05 ) . almost twofold increases were estimated in other diabetic groups when compared to all no treated groups ( p < 0.01 , table 4 ) calculated homa - ir in no groups and in g was significantly lower than d ( p < 0.0001 , table 4 ) . insulin sensitivity has been improved by no treatment . homa- impaired dramatically in groups d and g. improved beta cell function was observed in all no groups and was similar to healthy group 's values ( p > 0.05 , table 4 ) . all no treatment dosages decreased alp activity when compared to d ( p < 0.0001 ) . the alp activity of g was similar to all no regimens ( p > 0.05 ) . the densities of each band evaluated by diana v1.6 and aida 2.4.3 analysis programs ( raytest imaging system , germany ) and the ratio of liver and adipose tissues ppar- , - , - mrna expressions to -actin are given in figures 1(b ) , 1(c ) , 1(d),2(b ) , 2(c ) , and 2(d ) , respectively . in liver , ppar- mrna expression decreased in d compared to c ( p < 0.05 ) , whereas its expression increased in no-10 treated group ( p < 0.01 ) . ppar- mrna expression increased in no-0.1 ( p < 0.001 ) treated group compared to d. ppar- mrna expression increased in no treated groups as a dose dependent manner but the increase in no-10 group is not significant compared to c ( p > 0.05 ) . in adipose tissue , ppar- and - mrna expression increased in no-10 group compared to c and diabetic rats ( p < 0.01 ) . the increase in vascular disease in patients with diabetes is thought to be due to the deleterious effects of metabolic abnormalities , such as hyperglycemia , insulin resistance , dyslipidemia , and advanced glycation end products [ 18 , 19 ] . no treatment significantly reduced hba1c levels that were similar to healthy groups when glibenclamide had no significant effect over that . no treatment reduced hba1c 15.97 , 15.94 , and 19.54% in no-0.1 , no-1 , and no-10 , respectively , when compared to d. all no regimens also decreased fbg compared to other diabetic groups at the end of trial . therefore , no may be beneficial to reduce microvascular and macrovascular risk of type 2 diabetes . homa - ir is an independent predictor of cvd in type 2 diabetes and the improvement of ir might have beneficial effects not only on glucose control but also on cvd in patients with type 2 diabetes mellitus . no administration resulted in significant lowering of both insulin level and homa - ir compared to d as observed in g. improved beta cell function ( homa- ) in all no dosages was established but the improvement was noticeable in no-0.1 . in fact , hypertriglyceridemia is considered to represent an important risk factor for atherosclerosis and subsequent cardiovascular complications in type 2 diabetic patients . the present data showed that all no administration numerically decreased ai but the highest no administration level significantly lowered atherogenicity in blood compared to other diabetic groups . a positive correlation between mean homa - ir and ai ( p < 0.05 , r = 0.71 ) and also between mean hba1c and ai ( p < 0.01 , r = 0.86 ) suggests that improving blood glucose level and insulin receptor sensitivity may support to decline atherogenicity and cardiovascular complications in type 2 diabetes by using no distillate . moreover , the highly significant positive correlation between homa - ir and triglyceride levels ( p < 0.0001 , r = 0.74 ) and inclination of the reduction of triglyceride by no administration indicates no may have a good beneficial effect on glucose and lipid metabolism as inferred from homa - ir results . the atherogenic link between high triglycerides and hdl is due to the higher plasma concentration of triglyceride - rich , vldl that generates small , dense ldl during lipid exchange and lipolysis . the significant similarity of c levels especially in total - c of no-1 and no-10 administration and ldl concentrations in all no regimens of diabetics support lipid lowering effects of no . according to the above explanations , the ratio of triglycerides to hdl results in this paper indicates that no treatment may prevent the extensive coronary disease due to decreasing atherogenic particles since the ratio found to be statistically very similar to the ratio of c. also , in healthy rats , no made reduction in triglycerides to hdl ratio . the reduction in triglyceride levels together with the higher concentrations of hdl especially in no-0.1 and no-10 groups compared to c were remarkable effects of no over dyslipidemia of type 2 diabetic rats . all no treatment levels were associated with increase in hdl percentage in total - c but a highly significant arise was found in no-10 compared to other diabetics although the lower percentages of hdl in total - c of diabetic groups were estimated especially in g and d. numerically increase in hdl percent in total - c of cno-10 compared to c also indicates the beneficial effect of no over the lipid metabolism of healthy individuals.all these positive results in respect to lipid lowering effects of no may indicate reducing risk factor for atherosclerosis and subsequent cardiovascular complications in type 2 diabetes . leptin is a hormone secreted predominantly by adipose tissue and is a signal of sufficiency of energy . in short - term food deprivation , serum leptin levels decrease and soluble leptin receptors are thought to be important for transport to or over the blood brain barrier ( bbb ) , and it is the saturation of this transport or impairment of leptin receptor signal transduction that may be the cause of leptin resistance . the effect of leptin on inducing insulin resistance was exhibited by positive correlation between mean homa - ir and leptin ( p < 0.05 , r = 0.76 ) in this research . no positively affected leptin levels and was found to be similar to healthy control level . this remarkable result indicates that no may help to compensate the energy metabolism by improving leptin and insulin levels , and homa - ir , homa- in type 2 diabetes . in this research , positive correlation was found between mean triglyceride and leptin levels ( p < 0.05 , r = 0.70 ) . a number of studies have reported that alt , ast , and/or ggt levels independently predict incidents of type 2 diabetes , metabolic syndrome , and cvd . the reduction of ast activity in no-10 was noticeable when compared to d. alt activity was significantly higher in d than c group but was similar in no groups compared with c ( p > 0.05 ) . although alp activities were not different between type 2 diabetic and nondiabetic patients , the increased alp activity has been reported in ketotic and nonketotic diabetic rats primarily due to an increase in intestinal and bone / liver alp isoenzyme . all no regimens exhibited the beneficial effect on reducing alp activity in type 2 diabetic rats . the results of liver enzymes by using no dosages show that no had no detrimental effect on the liver function . in rodents , adipose tissue ppar- mrna and protein levels are reduced after an overnight fast [ 42 , 43 ] in stz - induced diabetes , which is consistent with the stimulatory effect of insulin on ppar- expression . in addition , chronic feeding with high fat diets was shown to increase ppar- expression in adipose tissue , whereas fasting stimulates especially liver ppar- expression in rodents [ 42 , 43 ] . the overexpressions of ppar- and - in adipose tissue may indicate that the effect of no-10 group dosage on reducing insulin resistance and indirectly reducing the risk of atherosclerosis is being through the storage of fatty acids to adipocytes and regulating adipocyte differentiation . significantly increased ppar- expression in liver and adipose tissue of no-10 group suggests that no-10 dosage may have important effects on regulating fatty acid oxidation system , lipoprotein synthesis , and inflammatory responses . single agents that promote both ppar- and ppar- agonism could theoretically offer significant benefits in improving dyslipidaemia and reducing hyperglycaemia and thus reduce these cardiovascular risk factors associated with type 2 diabetes and metabolic syndrome . in addition , such a therapy could reduce the underlying insulin resistance and help to break the cycle of altered glucose and lipid metabolism that promotes type 2 diabetes . it has been considered that increasing fatty acid oxidation in liver by overexpression of liver ppar- led not to use too much fat from adipose tissues for energy and also storage of fatty acids in adipocytes by overexpression of adipose tissue ppar- of no treated animals improved insulin action . this progress prevented body weight loss of no treated animals due to an antilipolitic effect of insulin when compared to uncontrolled diabetic ones . also the pulling down effect of no-10 dosage on body weight might be associated with overexpressions of ppar- and - in adipose tissue of healthy rats since overexpression of ppar- specifically in adipose tissue decreases fatty acid levels and protects against obesity . moreover ppar- and ppar- are both active participants in energy burning ; ppar- plays a critical role in fatty acid oxidation and is thus responsible for energy expenditure ; ppar- also enhances fatty acid catabolism and energy uncoupling in skeletal muscle and adipose tissue as well , as recently shown in the liver . the considerable beneficial effects of especially no-10 group treatment ( no distillate administration at a dose of 375 g/0.51 ml of distilled water / d ) on glucose metabolism , insulin resistance , insulinotropic activity , leptin , dyslipidemia , liver enzymes , and ppars may point out the insulin - like effect of no and offer new approaches to treatment strategies that target both fat and glucose metabolism ultimately leadind to a reduction in both the chronic microvascular complications of type 2 diabetes and the risk of macrovascular events such as cvd .

the cluster of pathologies known as metabolic syndrome , including obesity , insulin resistance ( ir),type 2 diabetes , and cardiovascular disease ( cvd ) , has become one of the most serious threats to human health . the dramatic increase in the incidence of obesity in most parts of the world has contributed to the emergence of this disease cluster , particularly insulin resistance and type 2 diabetes . ir is associated with a number of diseases including obesity , metabolic syndrome , type 2 diabetes , lipodystrophies , polycystic ovary syndrome , and chronic infection . the main characteristics of ir are disinhibited lipolysis in adipose tissue , impaired uptake of glucose by muscle , and disinhibited gluconeogenesis . but this chronic secretion of large amounts of insulin to overcome tissue insensitivity can itself finally lead to beta cell failure and occurrence of hyperglycemia . in ir , visceral adipose tissue is resistant to the antilipolytic effect of insulin and consequently releases excessive amounts of ffa . insulin - resistant people with obesity and/or type 2 diabetes have been identified a defect in mitochondrial oxidative phosphorylation that relates to the accumulation of triglycerides and related lipid molecules in muscle . in type 2 diabetes , elevated blood glucose levels are clearly an important secondary cause of dyslipidemia in susceptible patients , and poor control of glycaemia can sometimes result in profound dyslipidaemia , including hypertriglyceridaemia and low high density lipoprotein ( hdl ) blood levels . in a recent report of world health organization and international diabetes federation it was found that about 80% diabetic morbidity and mortality is caused by diabetic cardiomyopathy which is closely related with diabetic dyslipidemia . the low density lipoprotein ( ldl ) blood level in a patient with diabetes may be somewhat misleading ; a patient with diabetes may have an increased proportion of small dense ldl particles and an increase in atherogenic risk , compared with a nondiabetic patient with similar ldl blood level . plant derivatives with purported hypoglycemic properties have been used in folk medicine and traditional healing systems around the world . among them , metformin was derived from the flowering plant galega officinalis ( goat 's rue or french lilac ) , which was a common traditional remedy for diabetes . oleander ( nerium oleander , no ) of the dogbane ( apocynaceae ) family grows along the whole mediterranean coast starting in southern portugal in the west , in syria , and in streambeds of the western and southern anatolia . were reported that postprandial rise in blood glucose when maltose and sucrose were loaded in nondiabetic healthy rats was reduced by hot water extract of nerium indicum leaves . recently ligands for the peroxisome proliferator - activated receptors ( ppars ) which play a key role in glucose and lipid metabolism are defined as new insulin sensitizing drugs and hypolipidemic fibrates . in the present study , we investigated the effects of no distillate on hyperglycemia and dyslipidemia and its activities on liver and adipose tissue ppars in type 2 diabetic rats . september period from mediterranean region of turkey , identified , and authenticated at the department of biology . after 2 weeks feeding of high fat diet , diabetes was induced in fasted animals by a single intra - peritoneal injection of streptozotocin ( stz ) ( 35 mg / kg bw ) dissolved in citrate buffer ( ph 4.5 ) when only buffer was received by control groups . in one week after stz injection , rats with 16.65 mmol / l ( 300 mg / dl ) nonfasting blood glucose level were considered to be type 2 diabetic . healthy control ( c ) and healthy control administered the highest level of no ( cno-10 ) group had normal pellet diet although high fat group ( hf ) and other all type 2 diabetic groups had high fat diet which 58% of the metabolic energy is provided from animal fat . nutritional substances of normal pellet diet were dry matter 89% , crude protein 21% , metabolic energy 2850 kcal / kg , crude fiber 5% , methionine and cystein 0.75% , calcium 1.02.0% , phosphor 0.51.0% , and sodium 0.5% ( optima feeds , turkey ) . after induction of diabetes , animals were randomly allocated to five groups in which one of them did not had any treatment ( d ) ; other groups had active substances once a day by gavage for 12 weeks of the treatment period . about 0.5 ml whole blood from each animal serum samples were analyzed immediately for fasting blood glucose ( fbg ) , total cholesterol ( total - c ) , hdl , ldl , triglyceride , alkaline phosphatase ( alp ) , aspartate transaminase ( ast ) , and alanine transaminase ( alt ) by using commercially available colorimetric diagnostic kits ( il test , instrumentation laboratory , milano , italy ) by autoanalyzer ( ilab , 300 , milano , italy ) . data for homa - ir ( homa - ir : fasting insulin ( u / ml ) fasting glucose ( mmol / l)/22.5 ) , beta cell functions as homa- ( homa-(% ) : ( 20 fasting insulin ( u / ml)/{fasting glucose ( mmol / l ) 3.5 } ) , atherogenic index ( ai : ( [ total - c ] [ hdl])/[hdl ] ) , hdl % in total - c , and triglyceride to hdl ratio of the all studied groups were calculated . table 3 shows values of body weight , fbg , hba1c , total - c , hdl , ldl , and triglyceride , ai , hdl% in total - c , triglyceride to hdl ratio of the all studied groups . data for insulin , leptin , homa - ir , homa- , alp , ast , and alt were presented in table 4 . although being diabetic , the considerable elevation in body weight of no treated groups was estimated compared to d and g ( p < 0.0001 ) . in parallel with the improvement of fbg as shown in table 3 , all other diabetic groups were hyperglycemic and had significantly higher hba1c than healthy groups and diabetic no groups ( p < 0.0001 ) . although having high fat diet , no-1 and no-10 displayed similar total - c concentrations compared to c ( p > 0.05 , table 3 ) . total - c was numerically lower in cno-10 and higher in hf than c. the increased values of hdl were found in no-0.1 and no-10 compared to c ( p < 0.0001 , table 3 ) . the reducing effect of all no regimens and g on ldl concentration was noticeable and the values were similar to healthy groups ( p > 0.05 ) . ldl levels in type 2 diabetic no-1 and no-10 groups were significantly lower than d ( p < 0.05 ) . hdl percentage in total - c of no-0.1 and no-1 groups was similar to c ( p > 0.05 ) and the highest hdl percentage was estimated in no-10 among healthy and diabetic groups except cno-10 . the similarity in terms of ai of no groups to healthy groups was noticeable ( p > 0.05 , table 3 ) . the reducing effect of no-10 on ai was significant when compared to d , g , no-0.1 , and no-1 ( p < 0.001 ) . there were significant reductions in triglyceride - hdl ratio of all no regimens compared to d ( p < 0.0001 , table 3 ) . triglyceride - hdl ratio was numerically lower in cno-10 and was higher in hf than c. similar results in g and d were noted in terms of triglyceride - hdl ratio . when we assessed insulin levels , the antihyperglycemic effect of no was seen on data that no significantly decreased insulin concentration compared to d ( p < 0.0001 , table 4 ) . although insulin levels in all no groups were numerically higher than other healthy control groups , the results were statistically similar with those groups ( p > 0.05).insulin level was decreased by g and data were not different with all no groups and healthy groups ( p > 0.05 ) . there was 26.72% fall in insulin level of cno-10 compared to c. leptin levels of all no groups were not significantly different compared to healthy control groups ( p > 0.05 ) . almost twofold increases were estimated in other diabetic groups when compared to all no treated groups ( p < 0.01 , table 4 ) calculated homa - ir in no groups and in g was significantly lower than d ( p < 0.0001 , table 4 ) . homa- impaired dramatically in groups d and g. improved beta cell function was observed in all no groups and was similar to healthy group 's values ( p > 0.05 , table 4 ) . the elevated alt activities , compared with c , were observed in d and g ( p < 0.05 , table 4 ) . the densities of each band evaluated by diana v1.6 and aida 2.4.3 analysis programs ( raytest imaging system , germany ) and the ratio of liver and adipose tissues ppar- , - , - mrna expressions to -actin are given in figures 1(b ) , 1(c ) , 1(d),2(b ) , 2(c ) , and 2(d ) , respectively . in liver , ppar- mrna expression decreased in d compared to c ( p < 0.05 ) , whereas its expression increased in no-10 treated group ( p < 0.01 ) . ppar- mrna expression increased in no-0.1 ( p < 0.001 ) treated group compared to d. ppar- mrna expression increased in no treated groups as a dose dependent manner but the increase in no-10 group is not significant compared to c ( p > 0.05 ) . in adipose tissue , ppar- and - mrna expression increased in no-10 group compared to c and diabetic rats ( p < 0.01 ) . the increase in ppar- mrna expression is more prominent in no-10 treated group compared to c and diabetic group ( p < 0.001 ) . the increase in vascular disease in patients with diabetes is thought to be due to the deleterious effects of metabolic abnormalities , such as hyperglycemia , insulin resistance , dyslipidemia , and advanced glycation end products [ 18 , 19 ] . in diabetes , hba1c levels predict the risk of microvascular complications and glycemic control to a hba1c of less than 7% will reduce microvascular complications and could decrease risk for macrovascular disease as well . the most important result in terms of the fbg is that no administered groups exhibited a level which was close to the data from c and that this condition was confirmed with the hba1c levels obtained from the above - mentioned groups . no treatment reduced hba1c 15.97 , 15.94 , and 19.54% in no-0.1 , no-1 , and no-10 , respectively , when compared to d. all no regimens also decreased fbg compared to other diabetic groups at the end of trial . homa - ir is an independent predictor of cvd in type 2 diabetes and the improvement of ir might have beneficial effects not only on glucose control but also on cvd in patients with type 2 diabetes mellitus . no administration resulted in significant lowering of both insulin level and homa - ir compared to d as observed in g. improved beta cell function ( homa- ) in all no dosages was established but the improvement was noticeable in no-0.1 . homa- was numerically higher in cno-10 than c. the evaluated data suggest that no ameliorates glycemic control by insulin secretagogue and sensitizing effects . dyslipidemia , as associated with diabetic metabolism and the metabolic syndrome , is characterized by a so - called proatherogenic blood lipid profile , comprising low levels of hdls , increased ldls , and serum triglycerides associated with vldls . the increased risk of coronary heart disease in patients with the metabolic syndrome suggests that the insulin - resistant state is atherogenic without concomitant elevations in plasma glucose and glycosylated hemoglobin [ 26 , 27 ] . the present data showed that all no administration numerically decreased ai but the highest no administration level significantly lowered atherogenicity in blood compared to other diabetic groups . a positive correlation between mean homa - ir and ai ( p < 0.05 , r = 0.71 ) and also between mean hba1c and ai ( p < 0.01 , r = 0.86 ) suggests that improving blood glucose level and insulin receptor sensitivity may support to decline atherogenicity and cardiovascular complications in type 2 diabetes by using no distillate . moreover , the highly significant positive correlation between homa - ir and triglyceride levels ( p < 0.0001 , r = 0.74 ) and inclination of the reduction of triglyceride by no administration indicates no may have a good beneficial effect on glucose and lipid metabolism as inferred from homa - ir results . the atherogenic link between high triglycerides and hdl is due to the higher plasma concentration of triglyceride - rich , vldl that generates small , dense ldl during lipid exchange and lipolysis . the significant similarity of c levels especially in total - c of no-1 and no-10 administration and ldl concentrations in all no regimens of diabetics support lipid lowering effects of no . a high ratio of triglycerides to hdl - cholesterol correlates with ldl phenotype b , small hdl particles , and ir [ 3032 ] and found to be a powerful independent indicator of extensive coronary disease . according to the above explanations , the ratio of triglycerides to hdl results in this paper indicates that no treatment may prevent the extensive coronary disease due to decreasing atherogenic particles since the ratio found to be statistically very similar to the ratio of c. also , in healthy rats , no made reduction in triglycerides to hdl ratio . high hdl levels in blood are considered to be cardioprotective , since the apo - a - containing hdl particles that help transport cholesterol to the liver from peripheral tissues , as well as away from macrophages associated with cholesterol deposits within the vascular wall . the reduction in triglyceride levels together with the higher concentrations of hdl especially in no-0.1 and no-10 groups compared to c were remarkable effects of no over dyslipidemia of type 2 diabetic rats . all no treatment levels were associated with increase in hdl percentage in total - c but a highly significant arise was found in no-10 compared to other diabetics although the lower percentages of hdl in total - c of diabetic groups were estimated especially in g and d. numerically increase in hdl percent in total - c of cno-10 compared to c also indicates the beneficial effect of no over the lipid metabolism of healthy individuals.all these positive results in respect to lipid lowering effects of no may indicate reducing risk factor for atherosclerosis and subsequent cardiovascular complications in type 2 diabetes . leptin effects are mediated by its action on the hypothalamus and directly on target tissues ( muscle , gonads , beta - cells , and liver ) . in short - term food deprivation , serum leptin levels decrease and soluble leptin receptors are thought to be important for transport to or over the blood brain barrier ( bbb ) , and it is the saturation of this transport or impairment of leptin receptor signal transduction that may be the cause of leptin resistance . so that , the significant elevated levels of leptin in d and g , except no treated ones , reflect leptin resistance and/or insulin resistant state . the effect of leptin on inducing insulin resistance was exhibited by positive correlation between mean homa - ir and leptin ( p < 0.05 , r = 0.76 ) in this research . this remarkable result indicates that no may help to compensate the energy metabolism by improving leptin and insulin levels , and homa - ir , homa- in type 2 diabetes . a number of studies have reported that alt , ast , and/or ggt levels independently predict incidents of type 2 diabetes , metabolic syndrome , and cvd . in addition , these markers have been shown to be associated with indirect measures of insulin resistance including fasting insulin levels and homa - ir [ 37 , 38 ] since alt was associated with insulin resistance independently and an inexpensive way to improve the identification of subjects with insulin resistance . the reduction of ast activity in no-10 was noticeable when compared to d. alt activity was significantly higher in d than c group but was similar in no groups compared with c ( p > 0.05 ) . although alp activities were not different between type 2 diabetic and nondiabetic patients , the increased alp activity has been reported in ketotic and nonketotic diabetic rats primarily due to an increase in intestinal and bone / liver alp isoenzyme . in rodents , adipose tissue ppar- mrna and protein levels are reduced after an overnight fast [ 42 , 43 ] in stz - induced diabetes , which is consistent with the stimulatory effect of insulin on ppar- expression . the overexpressions of ppar- and - in adipose tissue may indicate that the effect of no-10 group dosage on reducing insulin resistance and indirectly reducing the risk of atherosclerosis is being through the storage of fatty acids to adipocytes and regulating adipocyte differentiation . significantly increased ppar- expression in liver and adipose tissue of no-10 group suggests that no-10 dosage may have important effects on regulating fatty acid oxidation system , lipoprotein synthesis , and inflammatory responses . single agents that promote both ppar- and ppar- agonism could theoretically offer significant benefits in improving dyslipidaemia and reducing hyperglycaemia and thus reduce these cardiovascular risk factors associated with type 2 diabetes and metabolic syndrome . it has been considered that increasing fatty acid oxidation in liver by overexpression of liver ppar- led not to use too much fat from adipose tissues for energy and also storage of fatty acids in adipocytes by overexpression of adipose tissue ppar- of no treated animals improved insulin action . also the pulling down effect of no-10 dosage on body weight might be associated with overexpressions of ppar- and - in adipose tissue of healthy rats since overexpression of ppar- specifically in adipose tissue decreases fatty acid levels and protects against obesity . moreover ppar- and ppar- are both active participants in energy burning ; ppar- plays a critical role in fatty acid oxidation and is thus responsible for energy expenditure ; ppar- also enhances fatty acid catabolism and energy uncoupling in skeletal muscle and adipose tissue as well , as recently shown in the liver . the considerable beneficial effects of especially no-10 group treatment ( no distillate administration at a dose of 375 g/0.51 ml of distilled water / d ) on glucose metabolism , insulin resistance , insulinotropic activity , leptin , dyslipidemia , liver enzymes , and ppars may point out the insulin - like effect of no and offer new approaches to treatment strategies that target both fat and glucose metabolism ultimately leadind to a reduction in both the chronic microvascular complications of type 2 diabetes and the risk of macrovascular events such as cvd .

guinea pig anti - insulin , guinea pig anti - glucagon , guinea pig anti - pancreatic polypeptide , and rabbit anti - somatostatin were obtained from dako , carpinteria , ca . secondary antibodies used were fluorescein isothiocyanate conjugated donkey anti - rabbit igg ; fluorescein isothiocyanate conjugated donkey anti - guinea pig igg ; and cy3-conjugated donkey anti - mouse igg ( jackson immunoresearch , west grove , pa ) . goat anti - mouse igg conjugated to alexa fluor 488 was from invitrogen , carlsbad , ca . 2 ) : p1 , 5-cactgagcaggacatcctccga-3 ; p2 , 5-catcctcaaagcagtggatcca-3 ; p3 , 5-cttcctcaacaagaaagacctct-3 ; p4 , 5-ggtgagcggtttttgctttcaaa-3 ; p5 , 5-caagtggttcacagacacatcta-3 ; p6 , 5-ccttggatgtgagccacagct-3. oligos used for insulin expression assays are as follows : 5-cagcaagcaggtcattgttt-3 and 5-gggaccacaaagatgctgtt-3. for -cell mass assay , pancreata were weighed and cut as 20-m frozen sections . one - tenth of the sections confocal images ( covering 1/5 of all stained pancreatic areas ) were randomly captured using a 5 objective lens and analyzed with bioquant software ( 24 ) to calculate the area ratio between -cells and the whole pancreas to calculate islet mass . intraperitoneal ptx injection ( at 1 g/100 g body wt ) , intraperitoneal glucose tolerance test ( ipgtt ) , insulin sensitivity assays , and serum insulin assays followed published procedures ( 25,26 ) . islet isolation and perifusion followed an established protocol ( 27 ) . for camp assays , islets were incubated in ringer 's solution ( with 2 mmol / l glucose ) with 0.1 mmol / l isobutylmethylxanthine ( ibmx ) for 2 h and then used for assaying camp levels with the camp biotrak enzyme immunoassay system ( ge healthcare ) . for perifusion , islets of similar size and shape were used . hand - picked islet cells were isolated and placed in a 1-ml perifusion chamber , equilibrated in 5.6 mmol / l glucose for 30 min and then challenged with 16.7 mmol / l glucose ( 16.7 mmol / l glucose + 100 mol / l ibmx ) , 300 mol / l tolbutamide , and 20 mmol / l kcl . the perifusion fractions were collected in 3-min intervals at 1 ml / min flow rate and assayed for insulin by radioimmunoassay . , islets were first isolated from six animals and fixed ( 0.25% gluteraldehyde in pbs ) for sectioning and imaging . for quantification of vesicles docked on the cell membrane , images were captured using tem at magnification 10,00015,000 . the number of docked vesicles was counted before genotype identification , with vesicles whose outer surface was within 10 nm of the plasma membrane as docked granules . at least 50 randomly captured microscopic fields ( from different -cells ) of each genotype were analyzed before identifying their genotype . for calcium imaging , a series of images were acquired from isolated islets under glucose levels of 2 , 4 , 6 , 8 , 10 , and 15 mmol / l . images were background subtracted , and the mean fluo-4/fura - red intensity ratio was calculated across the whole islet ( 28 ) . this ratio was then normalized to the ratio calculated at 4 mmol / l glucose stimulation . total internal reflection fluorescence microscopy ( tirfm)-based live cell imaging followed published procedures ( 29,30 ) . briefly , islets were isolated and dispersed in calcium - free krebs - ringer buffer ( krb ) containing 1 mmol / l egta and cultured on high refractive index cover glass ( olympus ) in rpmi medium . -cells were then infected with recombinant adenovirus adex1ca insulin - green fluorescent protein ( gfp ) ( 29 ) for observation in krb containing 2.2 mmol / l glucose ( 37c ) . the olympus tirfm system was used with a high - aperture objective lens to observe the fluorescence of gfp with a charge - coupled device camera at 300-ms intervals using metamorph version 7.1 ( universal imaging ) . diiodomethane sulfur immersion oil ( n = 1.81 , cargille laboratories ) was used to make contact between the objective lens and the high refractive index cover glass . light propagates through the cover glass at an angle of 65 and undergoes total internal reflection at the glass cell interface . the refractive indexes for the glass ( n = 1.8 at 488 nm ) and cells ( n = 1.37 ) predict an evanescent field decline of threefold within 44 nm from the interface and of 10-fold within 100 nm . most analyses , including tracking ( single projection of different images ) and area calculations were performed using metamorph software , added with manual event selection . in this evanescent field setting , a granule would have a vertical distance of 9.6 nm from the plasma membrane and qualify as a morphologically docked granule ( granule distance from plasma membrane < 10 nm in electron microscopic studies ) ( 31 ) . we immunostained endogenous insulin granules in fixed pancreatic -cells . then , we manually counted bright spots as the docked granules . we examined go protein expression in both embryonic and adult pancreata using a monoclonal antibody that recognized both go1 and go2 . robust go production is detected in all hormone - expressing cells in all stages examined , including e11.5 , e17.5 , and 3-month - old adults ( fig . 1 ) . we do not detect go in exocrine acinar or pancreatic duct cells ( fig . 1 and data not shown ) . further rt - pcr analyses showed that both go1 and go2 mrna could be detected in adult islet cells , suggesting that both isoforms might be involved in islet cell function ( fig . expression patterns at three mouse stages , e11.5 ( a ) , e17.5 ( b e ) , and 3-month - old adult ( f i ) , are shown . three panels : go , hormone ( green ) , and a merged image . note that all hormone - expressing cells express go . ( a high - quality digital representation of this figure is available in the online issue . ) go is not required for islet cell differentiation . a : a diagram showing the go ( f ) allele . only some exons are shown ( from 5 to 8) . . arrows p1p6 indicate the oligonucleotides used for detecting go mrnas in b , which shows rt - pcr detection of go mrna in 4-month - old adult islet cells . rt reactions with insulin - specific oligos were used as controls ( with or without reverse transcription ) . ( p3 + p4 ) detects go2 mrna ( cre refers to go ; pdx1 ) . c : body weights of f / f and f / f;cre animals at 12 weeks of age . d : total insulin content in 4- and 8-week - old mice ( p28 and p56 ) . ( a high - quality color representation of this figure is available in the online issue . ) we used a go conditional allele , in which two loxp sites flanked the fifth and sixth exons of go , common to go1 and go2 ( go , fig . deletion of the flanked exons produces a truncated mrna that only codes for the nh2-terminal 156 amino acids , which lacks all motifs that bind to adenylyl cyclase , phospholipase cs ( plcs ) , and the subunits . we expect that this above manipulation results in a null go allele ( go ) . indeed , go animals display identical phenotypes as previously reported for null mutants ( data not shown ) , whereas go mice showed a similar phenotype as wild - type littermates . furthermore , the truncated protein did not perturb insulin secretion in a cultured -cell line ( supplementary fig . 1 , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1719/dc1 ) . go;pdx1 ( f / f;cre ) adult animals were derived from standard genetic crosses . pdx1 animals express cre in all undifferentiated pancreatic progenitors and inactivate go in all pancreatic progenitor cells of f / f;cre mice . in addition , no cre toxicity in pdx1 animals has been observed ( 22 ) . rt - pcr assays showed that the mrna sequence corresponding to the fifth and sixth exons of go was no longer detectable in islets of 4-month - old f / f;cre animals ( fig . the f / f;cre animals were no different in body weight from their control littermates ( go or f / f ) at all ages examined : 6 , 9 , 12 , and 20 weeks ( fig . 2c ) . additionally , no structural or behavioral ( aggression , feeding , moving , and mating ) defects were obvious in these animals . at postnatal day 1 ( p1 ) , the insulin contents in f / f;cre and f / f pancreata were not significantly different ( supplementary fig . 2a , available in an online appendix ) , suggesting that go is not required for -cell differentiation . by p28 , the insulin content in f / f;cre animals was reduced by 20% over that of control littermates ( fig . 2d ) . at p56 ( 8 weeks ) , the insulin content of f / f;cre animals consistent with this finding , the -cell mass was reduced in f / f;cre animals at p56 as well ( fig . we analyzed islet morphology and expression of several genes that are required for endocrine islet cell differentiation and function , including mafa , mafb , myt1 , nkx6.1 , and pdx1 ( 25 ) by immunofluorescence . these data suggest that go is not required for islet neogenesis , even though it is expressed in early ngn3-expressing endocrine progenitor cells ( 32 ) . gi and go inactivation by ptx uncouples the inhibitory effects of some neural hormones , such as adrenaline , on insulin secretion ( 12 ) . because both gi and go are expressed in islet cells and they both can be adp - ribosylated by ptx ( 14,33,34 ) , it is not clear which g - protein is mediating the ptx effect on insulin secretion . the fasting blood glucose levels in f / f;cre and f / f animals were similar ( fig . however , ipgtt showed that f / f;cre animals have significantly improved glucose clearance over control littermates ( fig . consistent with this observation , the fasting serum insulin levels are similar between f / f and f / f;cre animals . fifteen minutes after glucose challenge , the serum insulin levels in f / f control animals increased by 2-fold but increased up to 10-fold in f / f;cre mice ( fig . 3b ) . because the insulin sensitivity in f / f;cre and control animals was similar ( fig . 3c ) , the above findings demonstrate that losing go potentiates insulin secretion from -cells . we next tested whether gi proteins function to repress insulin secretion in the absence of go . if they do , we expect that ptx treatment of f / f;cre animals would further potentiate insulin secretion . ptx injection into f / f animals resulted in a significant increase in glucose tolerance . 3d ) , suggesting that although gi proteins are expressed in islet cells and may be adp - ribosylated by ptx , go is the major mediator of ptx 's effect on insulin secretion . pancreas - specific go inactivation enhances glucose - clearing capability in adult animals . a : ipgtt results in 6- and 12-week - old animals . go;pdx1 ( f / f;cre ) and go ( f / f ) animals were used . b : serum insulin levels 0 and 15 min after intraperitoneal glucose injection in 12-week - old f / f;cre and f / f males . note the fold increases in both control ( gray bars ) and f / f;cre ( black bars ) animals after glucose stimulation . c : insulin sensitivity of 12-week - old f / f;cre and f / f animals . the y - axis is presented as the ratio of glucose levels of the assay point over that of the 0-min value . islet perifusion assays were used to directly test how go inactivation affects insulin secretion in vitro . islets from 2-month - old animals were assayed for insulin secretion in response to glucose , ibmx , tolbutamide , and kcl stimulation . glucose induces insulin secretion through metabolism to alter the atp / adp ratio and other metabolites . ibmx inhibits camp phosphodiesterase to upregulate the levels of camp , which activates protein kinase a and/or gefii to facilitate insulin vesicle exocytosis ( 35,36 ) . tolbutamide , a katp channel blocker , depolarizes -cell membrane potential , as does kcl . in response to these stimuli , the insulin secretion in the f / f;cre islets was substantially increased compared with that of control littermates at every time point examined ( fig . the biggest increase was in response to glucose , increasing as much as 369% ( fig . this secretion increase was lower than what was detected during in vivo glucose challenge ( fig . 3b ) , likely due to the synergetic effect of multiple hormones that regulate insulin secretion through go in vivo , but not in vitro . importantly , these data suggest that go regulates insulin secretion through a mechanism that is shared by all these stimuli , most likely in steps that are distal to ca mobilization , as suggested for in vitro based studies ( 10 ) . go nullizygous islets secrete more insulin in response to multiple stimulations . a : perifusion assay results . the camp concentration is normalized against the od280 of islet extract ( as an assay of protein content ) . indeed , inactivation of go did not significantly affect camp production in isolated islets ( fig . , similar increases in intracellular free calcium ( [ ca]i ) concentration were seen in both f / f;cre mutant and control islets after elevated glucose stimulation ( fig . synchronous bursting and spiking activities were observed at 8 , 10 , and 15 mmol / l glucose , respectively ( fig . the mean fold increase in [ ca]i was also similar for various levels of glucose stimulation for both mutant and wild - type islets ( fig . this result suggests that go has little effect on -cell electrical activity and suggests that go regulates insulin secretion downstream of elevated [ ca]i in vivo . a : representative time courses of [ ca]i activity estimated from the normalized fluo4/furared intensity ratio in both f / f ( left ) and f / f;cre islets ( right ) 10 min after glucose challenge . shown are data from representative islets . note that 2 mmol / l glucose does not elicit ca mobilization , whereas 8 mmol / l glucose induces synchronous bursting of ca activity in both sets of islets ; 15 mmol / l glucose induces continuous spiking in both sets of islets . b : fold increases in [ ca]i estimated from the normalized fluo4/furared intensity ratio for control islets ( black ) and mutant islets ( gray ) under varying glucose stimulation . we next examined how go affects insulin secretion . because insulin vesicle docking on the cell membrane is necessary for insulin secretion , we used tem to investigate whether vesicle distribution in -cells is affected by loss of go . the size of each vesicle in -cells did not vary between f / f;cre and f / f control islets ( fig . the density of granules in the f / f;cre and control -cell remained unchanged as well ( fig . however , the number of secretory vesicles in direct contact with the cell membrane increased by about 100% in f / f;cre -cells as compared with that of controls ( fig . 6c , d , and f ) . because tem only allows us to examine vesicle docking on a thin section with limited depth , we used tirfm to verify the above findings . tirfm uses evanescent light waves to selectively illuminate the -cell surface at a 100-nm depth . thus , this technique allows us to exclusively visualize the granules that localize in the proximity of the cell membrane on a wide cell surface area . isolated islet cells were fixed and stained with insulin antibodies and subjected to tirfm ( fig . consistent with the above tem - based finding , we observed a significant ( p < 0.01 ) increase in the number of insulin vesicles close to plasma membrane in go mutants ( 257/m ) over that of the control cells ( 190/m ) ( fig . note that the fold increase of docked vesicles revealed by tirfm ( a 35% increase ) is lower than that observed from tem - based analysis ( 100% increase ) . this is an expected result because tem identifies the vesicles that directly contact the plasma membrane , which is only a small portion of the vesicles that localize within 100 nm of the plasma membrane visualized through tirfm . additionally , our vesicle density count with em and tirfm displayed a twofold difference ( fig . 6e and h ) . this discrepancy could be due to the unequal vesicle distribution within the cytoplasmic compartment and cell membrane . alternatively , it is possible that tirfm only visualizes high - insulin - content vesicles ( due to antibody staining related issues ) , whereas em allows us to visualize all vesicles . note that different -cells have different vesicle density or membrane - associated vesicles ( c and d ) . e : vesicle density in -cells , presented as number of vesicles on two - dimensional views . data are presented as number of vesicles over length of intercellular junctions ( p < 0.01 ) . g : tirfm images showing the presence of insulin vesicles on the surface of fixed and insulin ab - stained -cells of control ( f / f ) and go deleted ( f / f;cre ) animals . vesicles within the circled areas were counted and presented in h. tirfm visualizes vesicle movement in vivo in real time . we therefore recorded the vesicular dynamics close to the -cell membrane in wild - type and go mutant animals . enhanced green fluorescent protein ( egfp ) fusion protein , which was previously shown to be packaged in normal insulin vesicles and to not interfere with insulin trafficking . as a result , the egfp - marked insulin vesicles could be followed in real time ( 29,37 ) . islet cells were stimulated with 22 mmol / l glucose ( see research design and methods ) . 4 ) , go mutant -cells release significantly more vesicles than control -cells ( fig . it is possible that go inactivation could either shorten vesicle residence time on the plasma membrane before fusion or expedite transportation of vesicles from cytoplasm to plasma membrane . in order to differentiate between these possibilities , we counted the fusion events from predocked vesicles and newly arrived vesicles ( newcomers or vesicles that appear close to cell membranes after the start of recording ) during stimulation . membrane - docked vesicles in go mutant -cells showed a trend of increased readiness for release ( fig . 7b ) . specifically , upon glucose stimulation , 23.1% of predocked insulin vesicles were released within 10 min in control -cells , whereas 35.7% of predocked vesicles were released within the same time frame in -cells without go ( fig . , the fusion events contributed by newly arrived vesicles did not display a significant difference between the control and mutant -cells ( fig . overall , these data suggest that one of the possible go functions is to facilitate vesicle docking and , to a lesser extent , to increase the readiness of vesicle fusion to the plasma membrane ( fig . a : the numbers of vesicle plasma membrane fusion events at several time points with 22 mmol / l glucose stimulation . the events are presented as fusions from predocked vesicles and fusions from newly arrived vesicles , respectively . b : the percent of predocked vesicles that are released within 10 min of glucose stimulation . c : the number of newly arrived vesicles that are released within 10 min of glucose induction . d : a simple model summarizing where go could exert its function in the vesicle - secretion process , including vesicle docking and possibly priming . we examined go protein expression in both embryonic and adult pancreata using a monoclonal antibody that recognized both go1 and go2 . robust go production is detected in all hormone - expressing cells in all stages examined , including e11.5 , e17.5 , and 3-month - old adults ( fig . 1 ) . we do not detect go in exocrine acinar or pancreatic duct cells ( fig . 1 and data not shown ) . further rt - pcr analyses showed that both go1 and go2 mrna could be detected in adult islet cells , suggesting that both isoforms might be involved in islet cell function ( fig . expression patterns at three mouse stages , e11.5 ( a ) , e17.5 ( b e ) , and 3-month - old adult ( f i ) , are shown . three panels : go , hormone ( green ) , and a merged image . note that all hormone - expressing cells express go . ( a high - quality digital representation of this figure is available in the online issue . ) go is not required for islet cell differentiation . a : a diagram showing the go ( f ) allele . only some exons are shown ( from 5 to 8) . . arrows p1p6 indicate the oligonucleotides used for detecting go mrnas in b , which shows rt - pcr detection of go mrna in 4-month - old adult islet cells . rt reactions with insulin - specific oligos were used as controls ( with or without reverse transcription ) . ( p3 + p4 ) detects go2 mrna ( cre refers to go ; pdx1 ) . c : body weights of f / f and f / f;cre animals at 12 weeks of age . d : total insulin content in 4- and 8-week - old mice ( p28 and p56 ) . ( a high - quality color representation of this figure is available in the online issue . ) we used a go conditional allele , in which two loxp sites flanked the fifth and sixth exons of go , common to go1 and go2 ( go , fig . deletion of the flanked exons produces a truncated mrna that only codes for the nh2-terminal 156 amino acids , which lacks all motifs that bind to adenylyl cyclase , phospholipase cs ( plcs ) , and the subunits . we expect that this above manipulation results in a null go allele ( go ) . indeed , go animals display identical phenotypes as previously reported for null mutants ( data not shown ) , whereas go mice showed a similar phenotype as wild - type littermates . furthermore , the truncated protein did not perturb insulin secretion in a cultured -cell line ( supplementary fig . 1 , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1719/dc1 ) . go;pdx1 ( f / f;cre ) adult animals were derived from standard genetic crosses . pdx1 animals express cre in all undifferentiated pancreatic progenitors and inactivate go in all pancreatic progenitor cells of f / f;cre mice . in addition , no cre toxicity in pdx1 animals has been observed ( 22 ) . rt - pcr assays showed that the mrna sequence corresponding to the fifth and sixth exons of go was no longer detectable in islets of 4-month - old f / f;cre animals ( fig . the f / f;cre animals were no different in body weight from their control littermates ( go or f / f ) at all ages examined : 6 , 9 , 12 , and 20 weeks ( fig . 2c ) . additionally , no structural or behavioral ( aggression , feeding , moving , and mating ) defects were obvious in these animals . at postnatal day 1 ( p1 ) , the insulin contents in f / f;cre and f / f pancreata were not significantly different ( supplementary fig . 2a , available in an online appendix ) , suggesting that go is not required for -cell differentiation . by p28 , the insulin content in f / f;cre animals was reduced by 20% over that of control littermates ( fig . 2d ) . at p56 ( 8 weeks ) , the insulin content of f / f;cre animals consistent with this finding , the -cell mass was reduced in f / f;cre animals at p56 as well ( fig . we analyzed islet morphology and expression of several genes that are required for endocrine islet cell differentiation and function , including mafa , mafb , myt1 , nkx6.1 , and pdx1 ( 25 ) by immunofluorescence . these data suggest that go is not required for islet neogenesis , even though it is expressed in early ngn3-expressing endocrine progenitor cells ( 32 ) . gi and go inactivation by ptx uncouples the inhibitory effects of some neural hormones , such as adrenaline , on insulin secretion ( 12 ) . because both gi and go are expressed in islet cells and they both can be adp - ribosylated by ptx ( 14,33,34 ) , it is not clear which g - protein is mediating the ptx effect on insulin secretion . the fasting blood glucose levels in f / f;cre and f / f animals were similar ( fig . however , ipgtt showed that f / f;cre animals have significantly improved glucose clearance over control littermates ( fig . consistent with this observation , the fasting serum insulin levels are similar between f / f and f / f;cre animals . fifteen minutes after glucose challenge , the serum insulin levels in f / f control animals increased by 2-fold but increased up to 10-fold in f / f;cre mice ( fig . 3b ) . because the insulin sensitivity in f / f;cre and control animals was similar ( fig . 3c ) , the above findings demonstrate that losing go potentiates insulin secretion from -cells . we next tested whether gi proteins function to repress insulin secretion in the absence of go . if they do , we expect that ptx treatment of f / f;cre animals would further potentiate insulin secretion . ptx injection into f / f animals resulted in a significant increase in glucose tolerance . whereas ptx injection into f / f;cre animals had no significant effect ( fig . 3d ) , suggesting that although gi proteins are expressed in islet cells and may be adp - ribosylated by ptx , go is the major mediator of ptx 's effect on insulin secretion . pancreas - specific go inactivation enhances glucose - clearing capability in adult animals . a : ipgtt results in 6- and 12-week - old animals . go;pdx1 ( f / f;cre ) and go ( f / f ) animals were used . b : serum insulin levels 0 and 15 min after intraperitoneal glucose injection in 12-week - old f / f;cre and f / f males . note the fold increases in both control ( gray bars ) and f / f;cre ( black bars ) animals after glucose stimulation . c : insulin sensitivity of 12-week - old f / f;cre and f / f animals . the y - axis is presented as the ratio of glucose levels of the assay point over that of the 0-min value . islet perifusion assays were used to directly test how go inactivation affects insulin secretion in vitro . islets from 2-month - old animals were assayed for insulin secretion in response to glucose , ibmx , tolbutamide , and kcl stimulation . glucose induces insulin secretion through metabolism to alter the atp / adp ratio and other metabolites . ibmx inhibits camp phosphodiesterase to upregulate the levels of camp , which activates protein kinase a and/or gefii to facilitate insulin vesicle exocytosis ( 35,36 ) . tolbutamide , a katp channel blocker , depolarizes -cell membrane potential , as does kcl . in response to these stimuli , the insulin secretion in the f / f;cre islets was substantially increased compared with that of control littermates at every time point examined ( fig . the biggest increase was in response to glucose , increasing as much as 369% ( fig . this secretion increase was lower than what was detected during in vivo glucose challenge ( fig . 3b ) , likely due to the synergetic effect of multiple hormones that regulate insulin secretion through go in vivo , but not in vitro . importantly , these data suggest that go regulates insulin secretion through a mechanism that is shared by all these stimuli , most likely in steps that are distal to ca mobilization , as suggested for in vitro based studies ( 10 ) . go nullizygous islets secrete more insulin in response to multiple stimulations . a : perifusion assay results . the camp concentration is normalized against the od280 of islet extract ( as an assay of protein content ) . indeed , inactivation of go did not significantly affect camp production in isolated islets ( fig . , similar increases in intracellular free calcium ( [ ca]i ) concentration were seen in both f / f;cre mutant and control islets after elevated glucose stimulation ( fig . synchronous bursting and spiking activities were observed at 8 , 10 , and 15 mmol / l glucose , respectively ( fig . the mean fold increase in [ ca]i was also similar for various levels of glucose stimulation for both mutant and wild - type islets ( fig . this result suggests that go has little effect on -cell electrical activity and suggests that go regulates insulin secretion downstream of elevated [ ca]i in vivo . a : representative time courses of [ ca]i activity estimated from the normalized fluo4/furared intensity ratio in both f / f ( left ) and f / f;cre islets ( right ) 10 min after glucose challenge . shown are data from representative islets . note that 2 mmol / l glucose does not elicit ca mobilization , whereas 8 mmol / l glucose induces synchronous bursting of ca activity in both sets of islets ; 15 mmol / l glucose induces continuous spiking in both sets of islets . b : fold increases in [ ca]i estimated from the normalized fluo4/furared intensity ratio for control islets ( black ) and mutant islets ( gray ) under varying glucose stimulation . we next examined how go affects insulin secretion . because insulin vesicle docking on the cell membrane is necessary for insulin secretion , we used tem to investigate whether vesicle distribution in -cells is affected by loss of go . the size of each vesicle in -cells did not vary between f / f;cre and f / f control islets ( fig . the density of granules in the f / f;cre and control -cell remained unchanged as well ( fig . however , the number of secretory vesicles in direct contact with the cell membrane increased by about 100% in f / f;cre -cells as compared with that of controls ( fig . 6c , d , and f ) . because tem only allows us to examine vesicle docking on a thin section with limited depth , we used tirfm to verify the above findings . tirfm uses evanescent light waves to selectively illuminate the -cell surface at a 100-nm depth . thus , this technique allows us to exclusively visualize the granules that localize in the proximity of the cell membrane on a wide cell surface area . isolated islet cells were fixed and stained with insulin antibodies and subjected to tirfm ( fig . consistent with the above tem - based finding , we observed a significant ( p < 0.01 ) increase in the number of insulin vesicles close to plasma membrane in go mutants ( 257/m ) over that of the control cells ( 190/m ) ( fig . note that the fold increase of docked vesicles revealed by tirfm ( a 35% increase ) is lower than that observed from tem - based analysis ( 100% increase ) . this is an expected result because tem identifies the vesicles that directly contact the plasma membrane , which is only a small portion of the vesicles that localize within 100 nm of the plasma membrane visualized through tirfm . additionally , our vesicle density count with em and tirfm displayed a twofold difference ( fig . . this discrepancy could be due to the unequal vesicle distribution within the cytoplasmic compartment and cell membrane . alternatively , it is possible that tirfm only visualizes high - insulin - content vesicles ( due to antibody staining related issues ) , whereas em allows us to visualize all vesicles . note that different -cells have different vesicle density or membrane - associated vesicles ( c and d ) . e : vesicle density in -cells , presented as number of vesicles on two - dimensional views . data are presented as number of vesicles over length of intercellular junctions ( p < 0.01 ) . g : tirfm images showing the presence of insulin vesicles on the surface of fixed and insulin ab - stained -cells of control ( f / f ) and go deleted ( f / f;cre ) animals . we therefore recorded the vesicular dynamics close to the -cell membrane in wild - type and go mutant animals . enhanced green fluorescent protein ( egfp ) fusion protein , which was previously shown to be packaged in normal insulin vesicles and to not interfere with insulin trafficking . as a result , the egfp - marked insulin vesicles could be followed in real time ( 29,37 ) . islet cells were stimulated with 22 mmol / l glucose ( see research design and methods ) . 4 ) , go mutant -cells release significantly more vesicles than control -cells ( fig . it is possible that go inactivation could either shorten vesicle residence time on the plasma membrane before fusion or expedite transportation of vesicles from cytoplasm to plasma membrane . in order to differentiate between these possibilities , we counted the fusion events from predocked vesicles and newly arrived vesicles ( newcomers or vesicles that appear close to cell membranes after the start of recording ) during stimulation . membrane - docked vesicles in go mutant -cells showed a trend of increased readiness for release ( fig . 7b ) . specifically , upon glucose stimulation , 23.1% of predocked insulin vesicles were released within 10 min in control -cells , whereas 35.7% of predocked vesicles were released within the same time frame in -cells without go ( fig . , the fusion events contributed by newly arrived vesicles did not display a significant difference between the control and mutant -cells ( fig . overall , these data suggest that one of the possible go functions is to facilitate vesicle docking and , to a lesser extent , to increase the readiness of vesicle fusion to the plasma membrane ( fig . a : the numbers of vesicle plasma membrane fusion events at several time points with 22 mmol / l glucose stimulation . the events are presented as fusions from predocked vesicles and fusions from newly arrived vesicles , respectively . b : the percent of predocked vesicles that are released within 10 min of glucose stimulation . c : the number of newly arrived vesicles that are released within 10 min of glucose induction . d : a simple model summarizing where go could exert its function in the vesicle - secretion process , including vesicle docking and possibly priming . although the role of go in insulin secretion has been implicated for one - half century from ptx - based g - protein uncoupling studies ( 1113 ) , the nonspecificity of ptx ( which inactivates both gi and go ) has made it impossible to investigate how go functions in vivo . our findings suggest that go might regulate insulin granule dynamics distal to ca mobilization in vivo , a conclusion drawn from cell culture based studies ( 3842 ) . each -cell contains more than 10,000 vesicles ( 43,44 ) , yet only a small portion of these vesicles can be readily released within the first phase of glucose induction ( < 10 min in all studied species ) ( 2,7 ) . subsequently , insulin vesicles are transported from cytoplasm to the plasma membrane for docking , priming , and fusion to sustain the second phase of release . thus , vesicle docking , although not the rate - limiting step for insulin secretion , likely plays an essential role in regulating insulin secretion . consistent with this hypothesis , adult -cells that have lost the transcription factor gene foxa2 have more insulin vesicles docked on the cell membrane , and this phenotype is accompanied by excessive glucose - stimulated insulin secretion ( 45 ) . thus , understanding vesicle trafficking could provide key insights into the mechanisms that regulate insulin release in response to nutritional , neuronal , and hormonal stimuli . both our tem- and tirfm - based studies show that loss of go results in more vesicles docking to the plasma membrane at the resting state . furthermore , the docked vesicles in go nullizygous -cells appear more likely to fuse with the plasma membrane than docked vesicles in control cells . these data , combined with the finding that go inactivation does not significantly alter the transport of vesicles to plasma membrane , suggest that go could delay vesicle docking and possibly repress vesicle priming . further supporting this notion is our finding that go does not appear to affect calcium flux , which seems to contradict some previously published findings ( 10 ) . it is likely that only specific g - protein ( g)-coupled receptor ligand coupling could affect channel activity via go , which can not be activated in our in vitro assay . alternatively , the in vitro assays may not be sensitive enough to detect the subtle channel activity alteration with or without go . for example , go could regulate the resting ca levels in -cells , which would be consistent with the finding that resting ca level affects the pool size of readily releasable granules ( 46 ) . it would be interesting to analyze whether hormones , such as galanin , somatostatin , or adrenaline , can regulate specific channel activities in the presence or absence of go and how this might affect the resting ca levels in isolated islets . how go modulates the vesicle docking / priming process is not known . because there are high levels of go protein in neuronal and neuroendocrine cells , it was proposed that one function of go was to act as a reservoir for the g subunits within cells . when stimulated , go will dissociate from the g to release g as an effector to regulate cell function . first , expressing a g binding protein , the ph domain of the g - protein linked receptor kinase 2 stimulates insulin secretion in response to secretagogues , similar to the consequences of g trimer formation ( 47 ) . second , introducing g proteins in neuronal cells mimics the effect of go protein activation , that is , dissociation of the g complex ( 48 ) . in line with this possibility , loss of go could reduce cellular g subunits , which results in dysregulated vesicle trafficking and secretion ( 17 ) . unfortunately , it is currently unknown which specific - or -subunit interacts with go and has thus prevented us from directly examining this possibility . alternatively , go proteins could directly interact with unknown effectors to regulate insulin secretion . solving this issue will likely require a comprehensive understanding of all the protein / effectors that specifically interact with go under normal physiological conditions . , our analysis suggests that go modulates insulin secretion by regulating vesicle docking on the -cell membrane . addressing the specific mechanism likely requires a comprehensive analysis of proteins that interact with go and how these proteins modulate vesicle trafficking , docking , priming , and fusion processes .

objectivepertussis toxin uncoupling based studies have shown that gi and go can inhibit insulin secretion in pancreatic -cells . yet it is unclear whether gi and go operate through identical mechanisms and how these g - protein mediated signals inhibit insulin secretion in vivo . our objective is to examine whether / how go regulates islet development and insulin secretion in -cells.research design and methodsimmunoassays were used to analyze the go expression in mouse pancreatic cells . go was specifically inactivated in pancreatic progenitor cells by pancreatic cell specific gene deletion . hormone expression and insulin secretion in response to different stimuli were assayed in vivo and in vitro . electron microscope and total internal reflection fluorescence based assays were used to evaluate how go regulates insulin vesicle docking and secretion in response to glucose stimulation.resultsislet cells differentiate properly in go/ mutant mice . go inactivation significantly enhances insulin secretion both in vivo and in isolation . go nullizygous -cells contain an increased number of insulin granules docked on the cell plasma membrane , although the total number of vesicles per -cell remains unchanged.conclusionsgo is not required for endocrine islet cell differentiation , but it regulates the number of insulin vesicles docked on the -cell membrane .

RESEARCH DESIGN AND METHODS RESULTS Go is expressed in all endocrine islet cells. Go is not required for endocrine islet cell differentiation. Go is the major mediator of PTX's effect on insulin secretion inhibition. Go regulates insulin secretion at steps shared by different secretagogues. Go inhibits insulin granule docking to the -cell plasma membrane. None DISCUSSION Supplementary Material

for quantification of vesicles docked on the cell membrane , images were captured using tem at magnification 10,00015,000 . the number of docked vesicles was counted before genotype identification , with vesicles whose outer surface was within 10 nm of the plasma membrane as docked granules . this ratio was then normalized to the ratio calculated at 4 mmol / l glucose stimulation . total internal reflection fluorescence microscopy ( tirfm)-based live cell imaging followed published procedures ( 29,30 ) . light propagates through the cover glass at an angle of 65 and undergoes total internal reflection at the glass cell interface . in this evanescent field setting , a granule would have a vertical distance of 9.6 nm from the plasma membrane and qualify as a morphologically docked granule ( granule distance from plasma membrane < 10 nm in electron microscopic studies ) ( 31 ) . we immunostained endogenous insulin granules in fixed pancreatic -cells . further rt - pcr analyses showed that both go1 and go2 mrna could be detected in adult islet cells , suggesting that both isoforms might be involved in islet cell function ( fig . go is not required for islet cell differentiation . a : a diagram showing the go ( f ) allele . rt reactions with insulin - specific oligos were used as controls ( with or without reverse transcription ) . furthermore , the truncated protein did not perturb insulin secretion in a cultured -cell line ( supplementary fig . pdx1 animals express cre in all undifferentiated pancreatic progenitors and inactivate go in all pancreatic progenitor cells of f / f;cre mice . rt - pcr assays showed that the mrna sequence corresponding to the fifth and sixth exons of go was no longer detectable in islets of 4-month - old f / f;cre animals ( fig . 2a , available in an online appendix ) , suggesting that go is not required for -cell differentiation . at p56 ( 8 weeks ) , the insulin content of f / f;cre animals consistent with this finding , the -cell mass was reduced in f / f;cre animals at p56 as well ( fig . we analyzed islet morphology and expression of several genes that are required for endocrine islet cell differentiation and function , including mafa , mafb , myt1 , nkx6.1 , and pdx1 ( 25 ) by immunofluorescence . these data suggest that go is not required for islet neogenesis , even though it is expressed in early ngn3-expressing endocrine progenitor cells ( 32 ) . gi and go inactivation by ptx uncouples the inhibitory effects of some neural hormones , such as adrenaline , on insulin secretion ( 12 ) . because both gi and go are expressed in islet cells and they both can be adp - ribosylated by ptx ( 14,33,34 ) , it is not clear which g - protein is mediating the ptx effect on insulin secretion . 3c ) , the above findings demonstrate that losing go potentiates insulin secretion from -cells . we next tested whether gi proteins function to repress insulin secretion in the absence of go . if they do , we expect that ptx treatment of f / f;cre animals would further potentiate insulin secretion . pancreas - specific go inactivation enhances glucose - clearing capability in adult animals . go;pdx1 ( f / f;cre ) and go ( f / f ) animals were used . note the fold increases in both control ( gray bars ) and f / f;cre ( black bars ) animals after glucose stimulation . islet perifusion assays were used to directly test how go inactivation affects insulin secretion in vitro . islets from 2-month - old animals were assayed for insulin secretion in response to glucose , ibmx , tolbutamide , and kcl stimulation . glucose induces insulin secretion through metabolism to alter the atp / adp ratio and other metabolites . ibmx inhibits camp phosphodiesterase to upregulate the levels of camp , which activates protein kinase a and/or gefii to facilitate insulin vesicle exocytosis ( 35,36 ) . tolbutamide , a katp channel blocker , depolarizes -cell membrane potential , as does kcl . in response to these stimuli , the insulin secretion in the f / f;cre islets was substantially increased compared with that of control littermates at every time point examined ( fig . the biggest increase was in response to glucose , increasing as much as 369% ( fig . 3b ) , likely due to the synergetic effect of multiple hormones that regulate insulin secretion through go in vivo , but not in vitro . importantly , these data suggest that go regulates insulin secretion through a mechanism that is shared by all these stimuli , most likely in steps that are distal to ca mobilization , as suggested for in vitro based studies ( 10 ) . go nullizygous islets secrete more insulin in response to multiple stimulations . the mean fold increase in [ ca]i was also similar for various levels of glucose stimulation for both mutant and wild - type islets ( fig . this result suggests that go has little effect on -cell electrical activity and suggests that go regulates insulin secretion downstream of elevated [ ca]i in vivo . b : fold increases in [ ca]i estimated from the normalized fluo4/furared intensity ratio for control islets ( black ) and mutant islets ( gray ) under varying glucose stimulation . we next examined how go affects insulin secretion . because insulin vesicle docking on the cell membrane is necessary for insulin secretion , we used tem to investigate whether vesicle distribution in -cells is affected by loss of go . however , the number of secretory vesicles in direct contact with the cell membrane increased by about 100% in f / f;cre -cells as compared with that of controls ( fig . because tem only allows us to examine vesicle docking on a thin section with limited depth , we used tirfm to verify the above findings . tirfm uses evanescent light waves to selectively illuminate the -cell surface at a 100-nm depth . thus , this technique allows us to exclusively visualize the granules that localize in the proximity of the cell membrane on a wide cell surface area . consistent with the above tem - based finding , we observed a significant ( p < 0.01 ) increase in the number of insulin vesicles close to plasma membrane in go mutants ( 257/m ) over that of the control cells ( 190/m ) ( fig . this is an expected result because tem identifies the vesicles that directly contact the plasma membrane , which is only a small portion of the vesicles that localize within 100 nm of the plasma membrane visualized through tirfm . e : vesicle density in -cells , presented as number of vesicles on two - dimensional views . data are presented as number of vesicles over length of intercellular junctions ( p < 0.01 ) . g : tirfm images showing the presence of insulin vesicles on the surface of fixed and insulin ab - stained -cells of control ( f / f ) and go deleted ( f / f;cre ) animals . vesicles within the circled areas were counted and presented in h. tirfm visualizes vesicle movement in vivo in real time . we therefore recorded the vesicular dynamics close to the -cell membrane in wild - type and go mutant animals . enhanced green fluorescent protein ( egfp ) fusion protein , which was previously shown to be packaged in normal insulin vesicles and to not interfere with insulin trafficking . islet cells were stimulated with 22 mmol / l glucose ( see research design and methods ) . it is possible that go inactivation could either shorten vesicle residence time on the plasma membrane before fusion or expedite transportation of vesicles from cytoplasm to plasma membrane . specifically , upon glucose stimulation , 23.1% of predocked insulin vesicles were released within 10 min in control -cells , whereas 35.7% of predocked vesicles were released within the same time frame in -cells without go ( fig . overall , these data suggest that one of the possible go functions is to facilitate vesicle docking and , to a lesser extent , to increase the readiness of vesicle fusion to the plasma membrane ( fig . a : the numbers of vesicle plasma membrane fusion events at several time points with 22 mmol / l glucose stimulation . b : the percent of predocked vesicles that are released within 10 min of glucose stimulation . c : the number of newly arrived vesicles that are released within 10 min of glucose induction . d : a simple model summarizing where go could exert its function in the vesicle - secretion process , including vesicle docking and possibly priming . further rt - pcr analyses showed that both go1 and go2 mrna could be detected in adult islet cells , suggesting that both isoforms might be involved in islet cell function ( fig . go is not required for islet cell differentiation . a : a diagram showing the go ( f ) allele . furthermore , the truncated protein did not perturb insulin secretion in a cultured -cell line ( supplementary fig . pdx1 animals express cre in all undifferentiated pancreatic progenitors and inactivate go in all pancreatic progenitor cells of f / f;cre mice . rt - pcr assays showed that the mrna sequence corresponding to the fifth and sixth exons of go was no longer detectable in islets of 4-month - old f / f;cre animals ( fig . 2a , available in an online appendix ) , suggesting that go is not required for -cell differentiation . we analyzed islet morphology and expression of several genes that are required for endocrine islet cell differentiation and function , including mafa , mafb , myt1 , nkx6.1 , and pdx1 ( 25 ) by immunofluorescence . these data suggest that go is not required for islet neogenesis , even though it is expressed in early ngn3-expressing endocrine progenitor cells ( 32 ) . gi and go inactivation by ptx uncouples the inhibitory effects of some neural hormones , such as adrenaline , on insulin secretion ( 12 ) . because both gi and go are expressed in islet cells and they both can be adp - ribosylated by ptx ( 14,33,34 ) , it is not clear which g - protein is mediating the ptx effect on insulin secretion . we next tested whether gi proteins function to repress insulin secretion in the absence of go . if they do , we expect that ptx treatment of f / f;cre animals would further potentiate insulin secretion . 3d ) , suggesting that although gi proteins are expressed in islet cells and may be adp - ribosylated by ptx , go is the major mediator of ptx 's effect on insulin secretion . go;pdx1 ( f / f;cre ) and go ( f / f ) animals were used . islet perifusion assays were used to directly test how go inactivation affects insulin secretion in vitro . islets from 2-month - old animals were assayed for insulin secretion in response to glucose , ibmx , tolbutamide , and kcl stimulation . glucose induces insulin secretion through metabolism to alter the atp / adp ratio and other metabolites . ibmx inhibits camp phosphodiesterase to upregulate the levels of camp , which activates protein kinase a and/or gefii to facilitate insulin vesicle exocytosis ( 35,36 ) . tolbutamide , a katp channel blocker , depolarizes -cell membrane potential , as does kcl . in response to these stimuli , the insulin secretion in the f / f;cre islets was substantially increased compared with that of control littermates at every time point examined ( fig . the biggest increase was in response to glucose , increasing as much as 369% ( fig . 3b ) , likely due to the synergetic effect of multiple hormones that regulate insulin secretion through go in vivo , but not in vitro . importantly , these data suggest that go regulates insulin secretion through a mechanism that is shared by all these stimuli , most likely in steps that are distal to ca mobilization , as suggested for in vitro based studies ( 10 ) . go nullizygous islets secrete more insulin in response to multiple stimulations . this result suggests that go has little effect on -cell electrical activity and suggests that go regulates insulin secretion downstream of elevated [ ca]i in vivo . we next examined how go affects insulin secretion . because insulin vesicle docking on the cell membrane is necessary for insulin secretion , we used tem to investigate whether vesicle distribution in -cells is affected by loss of go . the size of each vesicle in -cells did not vary between f / f;cre and f / f control islets ( fig . however , the number of secretory vesicles in direct contact with the cell membrane increased by about 100% in f / f;cre -cells as compared with that of controls ( fig . because tem only allows us to examine vesicle docking on a thin section with limited depth , we used tirfm to verify the above findings . tirfm uses evanescent light waves to selectively illuminate the -cell surface at a 100-nm depth . consistent with the above tem - based finding , we observed a significant ( p < 0.01 ) increase in the number of insulin vesicles close to plasma membrane in go mutants ( 257/m ) over that of the control cells ( 190/m ) ( fig . this is an expected result because tem identifies the vesicles that directly contact the plasma membrane , which is only a small portion of the vesicles that localize within 100 nm of the plasma membrane visualized through tirfm . alternatively , it is possible that tirfm only visualizes high - insulin - content vesicles ( due to antibody staining related issues ) , whereas em allows us to visualize all vesicles . e : vesicle density in -cells , presented as number of vesicles on two - dimensional views . data are presented as number of vesicles over length of intercellular junctions ( p < 0.01 ) . g : tirfm images showing the presence of insulin vesicles on the surface of fixed and insulin ab - stained -cells of control ( f / f ) and go deleted ( f / f;cre ) animals . we therefore recorded the vesicular dynamics close to the -cell membrane in wild - type and go mutant animals . as a result , the egfp - marked insulin vesicles could be followed in real time ( 29,37 ) . islet cells were stimulated with 22 mmol / l glucose ( see research design and methods ) . it is possible that go inactivation could either shorten vesicle residence time on the plasma membrane before fusion or expedite transportation of vesicles from cytoplasm to plasma membrane . specifically , upon glucose stimulation , 23.1% of predocked insulin vesicles were released within 10 min in control -cells , whereas 35.7% of predocked vesicles were released within the same time frame in -cells without go ( fig . overall , these data suggest that one of the possible go functions is to facilitate vesicle docking and , to a lesser extent , to increase the readiness of vesicle fusion to the plasma membrane ( fig . a : the numbers of vesicle plasma membrane fusion events at several time points with 22 mmol / l glucose stimulation . b : the percent of predocked vesicles that are released within 10 min of glucose stimulation . c : the number of newly arrived vesicles that are released within 10 min of glucose induction . d : a simple model summarizing where go could exert its function in the vesicle - secretion process , including vesicle docking and possibly priming . although the role of go in insulin secretion has been implicated for one - half century from ptx - based g - protein uncoupling studies ( 1113 ) , the nonspecificity of ptx ( which inactivates both gi and go ) has made it impossible to investigate how go functions in vivo . our findings suggest that go might regulate insulin granule dynamics distal to ca mobilization in vivo , a conclusion drawn from cell culture based studies ( 3842 ) . subsequently , insulin vesicles are transported from cytoplasm to the plasma membrane for docking , priming , and fusion to sustain the second phase of release . thus , vesicle docking , although not the rate - limiting step for insulin secretion , likely plays an essential role in regulating insulin secretion . consistent with this hypothesis , adult -cells that have lost the transcription factor gene foxa2 have more insulin vesicles docked on the cell membrane , and this phenotype is accompanied by excessive glucose - stimulated insulin secretion ( 45 ) . thus , understanding vesicle trafficking could provide key insights into the mechanisms that regulate insulin release in response to nutritional , neuronal , and hormonal stimuli . both our tem- and tirfm - based studies show that loss of go results in more vesicles docking to the plasma membrane at the resting state . furthermore , the docked vesicles in go nullizygous -cells appear more likely to fuse with the plasma membrane than docked vesicles in control cells . these data , combined with the finding that go inactivation does not significantly alter the transport of vesicles to plasma membrane , suggest that go could delay vesicle docking and possibly repress vesicle priming . it is likely that only specific g - protein ( g)-coupled receptor ligand coupling could affect channel activity via go , which can not be activated in our in vitro assay . alternatively , the in vitro assays may not be sensitive enough to detect the subtle channel activity alteration with or without go . for example , go could regulate the resting ca levels in -cells , which would be consistent with the finding that resting ca level affects the pool size of readily releasable granules ( 46 ) . it would be interesting to analyze whether hormones , such as galanin , somatostatin , or adrenaline , can regulate specific channel activities in the presence or absence of go and how this might affect the resting ca levels in isolated islets . how go modulates the vesicle docking / priming process is not known . because there are high levels of go protein in neuronal and neuroendocrine cells , it was proposed that one function of go was to act as a reservoir for the g subunits within cells . first , expressing a g binding protein , the ph domain of the g - protein linked receptor kinase 2 stimulates insulin secretion in response to secretagogues , similar to the consequences of g trimer formation ( 47 ) . in line with this possibility , loss of go could reduce cellular g subunits , which results in dysregulated vesicle trafficking and secretion ( 17 ) . unfortunately , it is currently unknown which specific - or -subunit interacts with go and has thus prevented us from directly examining this possibility . alternatively , go proteins could directly interact with unknown effectors to regulate insulin secretion . , our analysis suggests that go modulates insulin secretion by regulating vesicle docking on the -cell membrane . addressing the specific mechanism likely requires a comprehensive analysis of proteins that interact with go and how these proteins modulate vesicle trafficking , docking , priming , and fusion processes .

one - tenth of the sections confocal images ( covering 1/5 of all stained pancreatic areas ) were randomly captured using a 5 objective lens and analyzed with bioquant software ( 24 ) to calculate the area ratio between -cells and the whole pancreas to calculate islet mass . intraperitoneal ptx injection ( at 1 g/100 g body wt ) , intraperitoneal glucose tolerance test ( ipgtt ) , insulin sensitivity assays , and serum insulin assays followed published procedures ( 25,26 ) . for camp assays , islets were incubated in ringer 's solution ( with 2 mmol / l glucose ) with 0.1 mmol / l isobutylmethylxanthine ( ibmx ) for 2 h and then used for assaying camp levels with the camp biotrak enzyme immunoassay system ( ge healthcare ) . hand - picked islet cells were isolated and placed in a 1-ml perifusion chamber , equilibrated in 5.6 mmol / l glucose for 30 min and then challenged with 16.7 mmol / l glucose ( 16.7 mmol / l glucose + 100 mol / l ibmx ) , 300 mol / l tolbutamide , and 20 mmol / l kcl . the number of docked vesicles was counted before genotype identification , with vesicles whose outer surface was within 10 nm of the plasma membrane as docked granules . for calcium imaging , a series of images were acquired from isolated islets under glucose levels of 2 , 4 , 6 , 8 , 10 , and 15 mmol / l . briefly , islets were isolated and dispersed in calcium - free krebs - ringer buffer ( krb ) containing 1 mmol / l egta and cultured on high refractive index cover glass ( olympus ) in rpmi medium . the olympus tirfm system was used with a high - aperture objective lens to observe the fluorescence of gfp with a charge - coupled device camera at 300-ms intervals using metamorph version 7.1 ( universal imaging ) . diiodomethane sulfur immersion oil ( n = 1.81 , cargille laboratories ) was used to make contact between the objective lens and the high refractive index cover glass . the refractive indexes for the glass ( n = 1.8 at 488 nm ) and cells ( n = 1.37 ) predict an evanescent field decline of threefold within 44 nm from the interface and of 10-fold within 100 nm . in this evanescent field setting , a granule would have a vertical distance of 9.6 nm from the plasma membrane and qualify as a morphologically docked granule ( granule distance from plasma membrane < 10 nm in electron microscopic studies ) ( 31 ) . we examined go protein expression in both embryonic and adult pancreata using a monoclonal antibody that recognized both go1 and go2 . robust go production is detected in all hormone - expressing cells in all stages examined , including e11.5 , e17.5 , and 3-month - old adults ( fig . further rt - pcr analyses showed that both go1 and go2 mrna could be detected in adult islet cells , suggesting that both isoforms might be involved in islet cell function ( fig . expression patterns at three mouse stages , e11.5 ( a ) , e17.5 ( b e ) , and 3-month - old adult ( f i ) , are shown . arrows p1p6 indicate the oligonucleotides used for detecting go mrnas in b , which shows rt - pcr detection of go mrna in 4-month - old adult islet cells . d : total insulin content in 4- and 8-week - old mice ( p28 and p56 ) . we used a go conditional allele , in which two loxp sites flanked the fifth and sixth exons of go , common to go1 and go2 ( go , fig . deletion of the flanked exons produces a truncated mrna that only codes for the nh2-terminal 156 amino acids , which lacks all motifs that bind to adenylyl cyclase , phospholipase cs ( plcs ) , and the subunits . rt - pcr assays showed that the mrna sequence corresponding to the fifth and sixth exons of go was no longer detectable in islets of 4-month - old f / f;cre animals ( fig . the f / f;cre animals were no different in body weight from their control littermates ( go or f / f ) at all ages examined : 6 , 9 , 12 , and 20 weeks ( fig . at postnatal day 1 ( p1 ) , the insulin contents in f / f;cre and f / f pancreata were not significantly different ( supplementary fig . by p28 , the insulin content in f / f;cre animals was reduced by 20% over that of control littermates ( fig . at p56 ( 8 weeks ) , the insulin content of f / f;cre animals consistent with this finding , the -cell mass was reduced in f / f;cre animals at p56 as well ( fig . we analyzed islet morphology and expression of several genes that are required for endocrine islet cell differentiation and function , including mafa , mafb , myt1 , nkx6.1 , and pdx1 ( 25 ) by immunofluorescence . these data suggest that go is not required for islet neogenesis , even though it is expressed in early ngn3-expressing endocrine progenitor cells ( 32 ) . because both gi and go are expressed in islet cells and they both can be adp - ribosylated by ptx ( 14,33,34 ) , it is not clear which g - protein is mediating the ptx effect on insulin secretion . the fasting blood glucose levels in f / f;cre and f / f animals were similar ( fig . however , ipgtt showed that f / f;cre animals have significantly improved glucose clearance over control littermates ( fig . consistent with this observation , the fasting serum insulin levels are similar between f / f and f / f;cre animals . fifteen minutes after glucose challenge , the serum insulin levels in f / f control animals increased by 2-fold but increased up to 10-fold in f / f;cre mice ( fig . we next tested whether gi proteins function to repress insulin secretion in the absence of go . if they do , we expect that ptx treatment of f / f;cre animals would further potentiate insulin secretion . 3d ) , suggesting that although gi proteins are expressed in islet cells and may be adp - ribosylated by ptx , go is the major mediator of ptx 's effect on insulin secretion . b : serum insulin levels 0 and 15 min after intraperitoneal glucose injection in 12-week - old f / f;cre and f / f males . the y - axis is presented as the ratio of glucose levels of the assay point over that of the 0-min value . islets from 2-month - old animals were assayed for insulin secretion in response to glucose , ibmx , tolbutamide , and kcl stimulation . in response to these stimuli , the insulin secretion in the f / f;cre islets was substantially increased compared with that of control littermates at every time point examined ( fig . 3b ) , likely due to the synergetic effect of multiple hormones that regulate insulin secretion through go in vivo , but not in vitro . importantly , these data suggest that go regulates insulin secretion through a mechanism that is shared by all these stimuli , most likely in steps that are distal to ca mobilization , as suggested for in vitro based studies ( 10 ) . , similar increases in intracellular free calcium ( [ ca]i ) concentration were seen in both f / f;cre mutant and control islets after elevated glucose stimulation ( fig . the mean fold increase in [ ca]i was also similar for various levels of glucose stimulation for both mutant and wild - type islets ( fig . this result suggests that go has little effect on -cell electrical activity and suggests that go regulates insulin secretion downstream of elevated [ ca]i in vivo . a : representative time courses of [ ca]i activity estimated from the normalized fluo4/furared intensity ratio in both f / f ( left ) and f / f;cre islets ( right ) 10 min after glucose challenge . note that 2 mmol / l glucose does not elicit ca mobilization , whereas 8 mmol / l glucose induces synchronous bursting of ca activity in both sets of islets ; 15 mmol / l glucose induces continuous spiking in both sets of islets . because insulin vesicle docking on the cell membrane is necessary for insulin secretion , we used tem to investigate whether vesicle distribution in -cells is affected by loss of go . the density of granules in the f / f;cre and control -cell remained unchanged as well ( fig . however , the number of secretory vesicles in direct contact with the cell membrane increased by about 100% in f / f;cre -cells as compared with that of controls ( fig . thus , this technique allows us to exclusively visualize the granules that localize in the proximity of the cell membrane on a wide cell surface area . consistent with the above tem - based finding , we observed a significant ( p < 0.01 ) increase in the number of insulin vesicles close to plasma membrane in go mutants ( 257/m ) over that of the control cells ( 190/m ) ( fig . note that the fold increase of docked vesicles revealed by tirfm ( a 35% increase ) is lower than that observed from tem - based analysis ( 100% increase ) . this is an expected result because tem identifies the vesicles that directly contact the plasma membrane , which is only a small portion of the vesicles that localize within 100 nm of the plasma membrane visualized through tirfm . g : tirfm images showing the presence of insulin vesicles on the surface of fixed and insulin ab - stained -cells of control ( f / f ) and go deleted ( f / f;cre ) animals . we therefore recorded the vesicular dynamics close to the -cell membrane in wild - type and go mutant animals . in order to differentiate between these possibilities , we counted the fusion events from predocked vesicles and newly arrived vesicles ( newcomers or vesicles that appear close to cell membranes after the start of recording ) during stimulation . membrane - docked vesicles in go mutant -cells showed a trend of increased readiness for release ( fig . specifically , upon glucose stimulation , 23.1% of predocked insulin vesicles were released within 10 min in control -cells , whereas 35.7% of predocked vesicles were released within the same time frame in -cells without go ( fig . , the fusion events contributed by newly arrived vesicles did not display a significant difference between the control and mutant -cells ( fig . overall , these data suggest that one of the possible go functions is to facilitate vesicle docking and , to a lesser extent , to increase the readiness of vesicle fusion to the plasma membrane ( fig . we examined go protein expression in both embryonic and adult pancreata using a monoclonal antibody that recognized both go1 and go2 . robust go production is detected in all hormone - expressing cells in all stages examined , including e11.5 , e17.5 , and 3-month - old adults ( fig . further rt - pcr analyses showed that both go1 and go2 mrna could be detected in adult islet cells , suggesting that both isoforms might be involved in islet cell function ( fig . expression patterns at three mouse stages , e11.5 ( a ) , e17.5 ( b e ) , and 3-month - old adult ( f i ) , are shown . arrows p1p6 indicate the oligonucleotides used for detecting go mrnas in b , which shows rt - pcr detection of go mrna in 4-month - old adult islet cells . deletion of the flanked exons produces a truncated mrna that only codes for the nh2-terminal 156 amino acids , which lacks all motifs that bind to adenylyl cyclase , phospholipase cs ( plcs ) , and the subunits . rt - pcr assays showed that the mrna sequence corresponding to the fifth and sixth exons of go was no longer detectable in islets of 4-month - old f / f;cre animals ( fig . the f / f;cre animals were no different in body weight from their control littermates ( go or f / f ) at all ages examined : 6 , 9 , 12 , and 20 weeks ( fig . at postnatal day 1 ( p1 ) , the insulin contents in f / f;cre and f / f pancreata were not significantly different ( supplementary fig . by p28 , the insulin content in f / f;cre animals was reduced by 20% over that of control littermates ( fig . at p56 ( 8 weeks ) , the insulin content of f / f;cre animals consistent with this finding , the -cell mass was reduced in f / f;cre animals at p56 as well ( fig . we analyzed islet morphology and expression of several genes that are required for endocrine islet cell differentiation and function , including mafa , mafb , myt1 , nkx6.1 , and pdx1 ( 25 ) by immunofluorescence . these data suggest that go is not required for islet neogenesis , even though it is expressed in early ngn3-expressing endocrine progenitor cells ( 32 ) . because both gi and go are expressed in islet cells and they both can be adp - ribosylated by ptx ( 14,33,34 ) , it is not clear which g - protein is mediating the ptx effect on insulin secretion . fifteen minutes after glucose challenge , the serum insulin levels in f / f control animals increased by 2-fold but increased up to 10-fold in f / f;cre mice ( fig . 3d ) , suggesting that although gi proteins are expressed in islet cells and may be adp - ribosylated by ptx , go is the major mediator of ptx 's effect on insulin secretion . the y - axis is presented as the ratio of glucose levels of the assay point over that of the 0-min value . in response to these stimuli , the insulin secretion in the f / f;cre islets was substantially increased compared with that of control littermates at every time point examined ( fig . 3b ) , likely due to the synergetic effect of multiple hormones that regulate insulin secretion through go in vivo , but not in vitro . importantly , these data suggest that go regulates insulin secretion through a mechanism that is shared by all these stimuli , most likely in steps that are distal to ca mobilization , as suggested for in vitro based studies ( 10 ) . , similar increases in intracellular free calcium ( [ ca]i ) concentration were seen in both f / f;cre mutant and control islets after elevated glucose stimulation ( fig . the mean fold increase in [ ca]i was also similar for various levels of glucose stimulation for both mutant and wild - type islets ( fig . a : representative time courses of [ ca]i activity estimated from the normalized fluo4/furared intensity ratio in both f / f ( left ) and f / f;cre islets ( right ) 10 min after glucose challenge . note that 2 mmol / l glucose does not elicit ca mobilization , whereas 8 mmol / l glucose induces synchronous bursting of ca activity in both sets of islets ; 15 mmol / l glucose induces continuous spiking in both sets of islets . because insulin vesicle docking on the cell membrane is necessary for insulin secretion , we used tem to investigate whether vesicle distribution in -cells is affected by loss of go . however , the number of secretory vesicles in direct contact with the cell membrane increased by about 100% in f / f;cre -cells as compared with that of controls ( fig . thus , this technique allows us to exclusively visualize the granules that localize in the proximity of the cell membrane on a wide cell surface area . consistent with the above tem - based finding , we observed a significant ( p < 0.01 ) increase in the number of insulin vesicles close to plasma membrane in go mutants ( 257/m ) over that of the control cells ( 190/m ) ( fig . g : tirfm images showing the presence of insulin vesicles on the surface of fixed and insulin ab - stained -cells of control ( f / f ) and go deleted ( f / f;cre ) animals . we therefore recorded the vesicular dynamics close to the -cell membrane in wild - type and go mutant animals . in order to differentiate between these possibilities , we counted the fusion events from predocked vesicles and newly arrived vesicles ( newcomers or vesicles that appear close to cell membranes after the start of recording ) during stimulation . specifically , upon glucose stimulation , 23.1% of predocked insulin vesicles were released within 10 min in control -cells , whereas 35.7% of predocked vesicles were released within the same time frame in -cells without go ( fig . overall , these data suggest that one of the possible go functions is to facilitate vesicle docking and , to a lesser extent , to increase the readiness of vesicle fusion to the plasma membrane ( fig . although the role of go in insulin secretion has been implicated for one - half century from ptx - based g - protein uncoupling studies ( 1113 ) , the nonspecificity of ptx ( which inactivates both gi and go ) has made it impossible to investigate how go functions in vivo . our findings suggest that go might regulate insulin granule dynamics distal to ca mobilization in vivo , a conclusion drawn from cell culture based studies ( 3842 ) . each -cell contains more than 10,000 vesicles ( 43,44 ) , yet only a small portion of these vesicles can be readily released within the first phase of glucose induction ( < 10 min in all studied species ) ( 2,7 ) . consistent with this hypothesis , adult -cells that have lost the transcription factor gene foxa2 have more insulin vesicles docked on the cell membrane , and this phenotype is accompanied by excessive glucose - stimulated insulin secretion ( 45 ) . thus , understanding vesicle trafficking could provide key insights into the mechanisms that regulate insulin release in response to nutritional , neuronal , and hormonal stimuli . these data , combined with the finding that go inactivation does not significantly alter the transport of vesicles to plasma membrane , suggest that go could delay vesicle docking and possibly repress vesicle priming . it would be interesting to analyze whether hormones , such as galanin , somatostatin , or adrenaline , can regulate specific channel activities in the presence or absence of go and how this might affect the resting ca levels in isolated islets . first , expressing a g binding protein , the ph domain of the g - protein linked receptor kinase 2 stimulates insulin secretion in response to secretagogues , similar to the consequences of g trimer formation ( 47 ) . second , introducing g proteins in neuronal cells mimics the effect of go protein activation , that is , dissociation of the g complex ( 48 ) .

photoinduced charge transfer ( ct ) via symmetry breaking ( sb ) plays a crucial role in photosynthetic reaction centers in living systems . ct from one chromophore to another occurs upon photoexcitation , producing sb charge separation . of great potential interest , but less well explored , are sbct processes in organic photovoltaics ( opv ) and related solar - harvesting systems . among well - documented simple organic compounds exhibiting sb phenomena are 9,9-bianthryl derivatives ; however , they do not absorb visible light , making them of limited use in systems harvesting the solar energy . indeed , very few organic dyes that absorb in the visible region undergo sbct processes . herein , we investigate the photophysics of zinc dipyrrin complexes ( figure 1 ) that exhibit intense visible absorption in a range of organic solvents . these compounds have structural features related to 9,9-bianthryl ( i.e. , poorly coupled orthogonal chromophores ) that are conducive to photoinduced sbct processes in weakly polar to polar solvents . zinc dipyrrins and analogous compounds are attractive because , in addition to strong absorption in the visible region of the spectrum , their syntheses are easy and scalable . moreover , the large body of work on boron dipyrrins ( bdip ) can be used to guide ligand design . structures of homoleptic ( zdip1zdip4 ) and heteroleptic ( zdip2 and zdip3 ) zinc dipyrrin complexes . in opvs , the low dielectric constants of organic materials ( s 3 ) lead to high exciton binding energies , and thus , a large energy offset between donor and acceptor is required to promote charge generation at the donor / acceptor interface ( d / a ) . use of organic dyes that undergo sbct processes might be a potential solution to reduce the energy cost for exciton dissociation to free charges at d / a . the polar environment at d / a may be sufficient to induce sbct in the chromophore , leading to spontaneous formation of internal ct excitons . charge separation between electron donor and acceptor materials from these sbct excitons is expected to proceed with lower energetic requirements than for typical frenkel excitons found in organic materials . that being the case , the ability of zinc dipyrrin complexes to undergo sbct makes them a potential family of new materials for use in organic photovoltaics . fluorescent metallodipyrrins have attracted considerable attention due to their potential use as probes for sensing metal ions ( in particular , zn ) in living systems . however , in spite of the increasing number of reported metallodipyrrins , their photophysics are not well - understood . unlike the highly fluorescent bdips , homoleptic complexes mdn ( d = dipyrrin or -extended dipyrrin ligands ) generally exhibit low to moderate fluorescent quantum yields . in contrast , the heteroleptic complexes mdxn ( x is an ancillary ligand ) were shown to have quite high luminescent efficiencies ( up to 80% ) . several nonradiative deactivation pathways have been suggested for the photoinduced excited state of homoleptic zinc dipyrrins and -extended dipyrrins . lindsey and co - workers reported that rotation of the phenyl ring at the meso - position on the dipyrrin ligand is a source of nonradiative deactivation of the excited state . replacement of phenyl with the more bulky mesityl substituent to form zdip1 inhibits this rotation , leading to an improved quantum yield of 36% in toluene . interestingly , a recent study on related mesityl - substituted zinc dipyrrins ( zdip2 and zdip4 ) has reported that the quantum yields of the homoleptic complexes are strongly dependent on solvent polarity , decreasing from 2030% in toluene to 5% in dichloromethane . the authors proposed that this decrease in polar solvents is due to thermal promotion from a locally excited state on a single dipyrrin ligand to a nonemissive , charge - separated state ( i.e. , d d ) ; however , no additional photophysical data was provided to support this hypothesis . strong excitonic coupling between nonorthogonal ligands has also been suggested as another nonradiative deactivation pathway of zinc -extended dipyrrin complexes . however , excitonic coupling between the nearly orthogonal ligands in homoleptic zinc dipyrrin compounds such as zdip2 and zdip4 is negligible and thus can not be used to explain the decrease in luminescent efficiency compared to their heteroleptic counterparts . in this paper , we show that sbct in polar solvents is an effective nonradiative decay pathway for the electronic excited states of homoleptic zinc dipyrrins . in nonpolar solvents such as cyclohexane , these complexes do not undergo sbct and thus exhibit even higher quantum yields than their heteroleptic analogs . the synthesis and characterization by nmr , mass spectroscopy , and elemental analysis of the zinc dipyrrin complexes described here are given in the supporting information ( si ) for this paper . cyclic voltammetry ( cv ) and differential pulse voltammetry ( dpv ) were performed using an eg&g potentiostat / galvanostat model 283 . freshly distilled thf ( vwr ) was used as the solvent under inert atmosphere with 0.1 m tetra(n - butyl)ammonium hexafluorophosphate ( aldrich ) as the supporting electrolyte . a glassy carbon rod , a platinum wire , and a silver wire were used as the working electrode , counter electrode , and pseudoreference electrode , respectively . electrochemical reversibility was established using cv , while all redox potentials were determined using dpv and are reported relative to a ferrocenium / ferrocene ( fc / fc ) redox couple used as an internal standard . visible spectra were recorded on a hewlett - packard 4853 diode array spectrophotometer . steady - state emission experiments at room temperature and 77 k were performed using a photon technology international quantamaster model c-60se spectrofluorometer . fluorescence lifetime measurements in cyclohexane , toluene , and thf were performed using an ibh fluorocube instrument equipped with a 405 nm led excitation source with the irf value of 0.4 ns . fluorescence lifetime measurements in dichloromethane and acetonitrile were carried out using an excitation wavelength of 500 nm obtained from an optical parametric amplifier ( coherent opa 9450 ) pumped by a 250 khz ti : sapphire amplifier ( coherent rega 9050 ) . the emission was collected at 520 nm for s1 state and 645 nm for the ct state using a r3809u-50 hamamatsu pmt with a becker and hickl spc 630 detection module ( 22 ps time resolution ) . quantum efficiency measurements were carried out using a hamamatsu c9920 system equipped with a xenon lamp , calibrated integrating sphere , and model c10027 photonic multichannel analyzer . the error in the emission lifetime measurements is 5% and for the quantum yields is 10% . pump and probe pulses were obtained from the output of a ti : sapphire regenerative amplifier ( coherent legend , 1 khz , 4 mj , 35 fs ) . the excitation pulses centered at 500 nm were generated by pumping a type - ii opa ( spectra physics opa-800c ) with 10% of the amplifier 800 nm output and mixing the resulting opa signal output with the residual 800 nm pump in a type - ii bbo crystal . white light supercontinuum probe pulses spanning the visible ( 320950 nm ) were obtained by focusing a small amount of the amplifier output into a rotating caf2 disk . the supercontinuum probe was collimated and focused with a pair of off - axis parabolic mirrors into sample , whereas the pump was focused before the sample position using a 25 cm caf2 lens . to avoid any contribution to the observed dynamics from orientational relaxation , the polarization of the supercontinuum was set at the magic angle ( 54.7 ) with respect to the pump polarization . the cross - correlation between pump and probe in a thin 1 mm quartz substrate gave a fwhm of 180 fs for 500 nm excitation . the supercontinuum probe was dispersed using a spectrograph ( oriel ms127i ) onto a 256-pixel silicon diode array ( hamamatsu ) for multiplexed detection of the probe . samples containing zdip1 , zdip2 , or zdip3 dissolved in cyclohexane , toluene , dichloromethane , or acetonitrile were placed in a closed , capped 1 mm quartz cuvette . the concentration of each sample was adjusted to give an optical density between 0.11 and 0.18 at 500 nm . the solutions were deaerated by bubbling with n2 prior to analysis . during data collection , the samples were slowly oscillated perpendicular to the pump and probe to reduce photodamage to the sample by the pump . at early time delays , a strong nonresonant signal from the sample cell and solvent is observed . the solvent response is found to relax within 180 fs for cyclohexane , dichloromethane , and acetonitrile , while in toluene this signal was stronger and obscured the first 300 fs . to effectively remove this nonresonant signal , a second measurement of the neat solvent was performed in an identical cuvette under same excitation conditions . the transient signal resulting from the solvent was then subtracted from the zinc dipyrrin solution signal . the nonresonant solvent response did , however , give a useful measure of the temporal dispersion of the supercontinuum after propagating through the caf2 plate and sample . transient absorption measurements were performed with pump fluences varying between 70 and 300 j / cm . over this range , the signal was found to scale linearly with the pump energy . samples were prepared in a nitrogen glovebox with dry solvents , such that the maximum absorbance was approximately od = 1 . these samples were sealed in 1 1 cm quartz cuvettes with kontes valves to keep the solution air - free . the third harmonic of a 10 hz q - switched nd : yag laser ( spectra - physics quanta - ray pro - series , pulse width : 8 ns ) was used to pump an optical parametric oscillator ( spectra - physics quanta - ray mopo-700 ) , tunable in the visible region . the excitation wavelength for each sample was chosen such that od ( at excitation ) = 0.30.4 , and the laser power was attenuated to 3 mj / pulse using a half - wave plate and polarizer combination . for single - wavelength transient absorption kinetics measurements , probe light was provided by a 75 w arc lamp operated in either continuous or pulsed mode . single wavelengths were selected by a double monochromator with 1 mm slits , detected by a photomultiplier tube , and amplified and recorded with a transient digitizer . single - wavelength traces were acquired at approximately 5 nm increments over the range of 350595 nm , on a 2 s , 100 s , or 10 ms timebase window , averaging over 300 laser pulses . a reference wavelength ( 400 nm ) was acquired as every third trace , to take into account the photodegradation of the sample . kinetics traces at each wavelength were scaled on the basis of the intensity of the previous reference trace . decays were fit to a single or double exponential with a long - time offset using matlab ( version 2010b ) curve fitting software . to generate transient absorption profiles at various time points , the od at that time point was taken from the exponential fit of each single - wavelength kinetics trace . the x - ray crystal structures of zdip3 , zdip4 , zdip2 , and zdip3 were determined using a bruker apex ii ccd system equipped with a triumph curved - crystal monochromator and a mo k fine - focus tube ( = 0.710 73 ) . the frames were integrated with the bruker saint software package using a saint v7.68a algorithm . representative synthetic schemes for preparing homoleptic ( zdip1zdip4 ) and heteroleptic ( zdip2 , zdip3 ) complexes are presented in scheme 1 . mesityl substituents on the meso - position of the dipyrrin ligand were chosen to eliminate aryl rotation as a potential nonradiative deactivation pathway . dipyrrins dip1dip4 were prepared from the corresponding pyrrole and mesitylaldehyde and used directly in consecutive reactions with ddq and zn(oac)2 to form the homoleptic complexes zdip1zdip4 in total yields of 813% . the heteroleptic complexes were prepared from a reaction zn(-diketonate)2 and the corresponding dipyrrin . of the three ancillary ligands examined , pentane-2,4-dione ( acac ) , 1,1,1,5,5,5-hexafluoropentane-2,4-dione , and 2,2,6,6-tetramethyl-3,5-heptanedione ( dpm ) , pure heteroleptic complexes were successfully isolated only using the dpm ligand . as seen for other heteroleptic zn complexes , zdip2 and zdip3 disproportionate to their respective homoleptic complexes ( zdip2 and zdip3 ) in chloroform over the course of several hours , as observed by nmr measurements ( see si ) . however , zdip2 and zdip3 are stable in the solid state and can be sublimed under vacuum without disproportionation . single crystal x - ray analysis and high - resolution mass spectroscopy confirmed formation of zdip2 and zdip3. it should be noted that freshly prepared zdip2 and zdip3 solutions were used for each step of subsequent photophysical characterization to minimize the effects of disproportionation . single crystal x - ray analysis was performed on zdip3 , zdip4 , zdip2 , and zdip3 ; structures of representative homoleptic ( zdip3 ) and heteroleptic complexes ( zdip3 ) are shown in figure 2 . the structures of zdip3 and zdip4 are similar to those published for zdip1 and zdip2 . n bond lengths in the complexes range from 1.95 to 1.99 , while the zn o bond lengths in zdip2 and zdip3 vary between 1.95 and 1.97 . the zinc center in all the complexes adopts a distorted tetrahedral configuration with the two ligands held nearly perpendicular to each other . the dihedral angles between mean planes of the two dipyrrin ligands in zdip1 , zdip2 , zdip3 , and zdip4 are 85.0 , 88.3 , 76.7 , and 83.4 , respectively , whereas values for the related angles between the two ligands of zdip2 and zdip3 are 87.8 and 89.8 , respectively . to evaluate the degree of distortion of the ligands , dihedral angles between the planes encompassing different groups of atoms as shown in figure 2 were measured ( table 1 ) . compared to boron dipyrrin compounds , which are essentially flat , the dipyrrin framework in the zinc dipyrrins is considerably more flexible , as dihedral angles between planes 1 and 2 vary from 3 to 18. however , no clear correlation is apparent between the degree of alkylation and variation of the dihedral angles , indicating that the distortions are likely dictated by crystal packing forces . ortep diagrams of ( a ) zdip3 and ( b ) zdip3 at 50% probability level . planes containing different groups of atoms are indicated by the colored lines . in order to gain insight into the electronic structure of the zinc dipyrrin complexes , theoretical calculations were performed at the b3lyp / lacvp * * level of theory . for simplicity , unsubstituted homoleptic zdip and heteroleptic zdip are presented , as it was found that they possess the same essential electronic features as their alkylated / arylated analogs . structures of the optimized complexes , highest and next highest occupied molecular orbitals ( homo , homo1 ) , and lowest and next lowest unoccupied molecular orbital ( lumo , lumo+1 ) , along with the corresponding energies , are shown in figure 3 . the homo ( a2 symmetry in the d2d point group ) and homo1 ( b1 ) of zdip localize on both dipyrrin ligands , while the doubly degenerate lumos are localized on separate dipyrrin ligands ( figure 3 ) . frontier orbitals of zdip solely populate the dipyrrin ligand , excluding any participation of the -diketonate ligand . consistent with mo analysis , the calculated homo and lumo energies of the two complexes are similar . there is little - to - no contribution from atomic orbitals on zinc to the frontier orbitals in either complex . the minor difference in energy between the homo and homo1 in zdip ( 0.11 ev ) is indicative of very poor electronic coupling between the two orthogonal dipyrrin ligands in the ground state . the transition dipole moment of the metallodipyrrin fragment lies in the plane of the dipyrrin ligand , along the long axis of the ligand . this places the transition dipole moments of the two dipyrrins in zdips orthogonal to each other , suggesting that there should be little or no excitonic or electronic coupling between the two dipyrrin ligands . sham lumos ( mesh ) and energies for ( a ) zdip and ( b ) zdip and homos ( transparent ) for ( c ) zdip and ( d ) zdip. zdip has d2d symmetry and zdip has c2v symmetry . absorption spectra of ( a ) zdip2 in different solvents and ( b ) zdip2 and zdip2 in cyclohexane . the zinc dipyrrin complexes absorb strongly from 400 to 550 nm [ = ( 1.011.17 ) 10 m cm ] . representative absorption spectra of zdip2 and zdip2 in different solvents are presented in figure 4 , and the emission spectra in the same solvents are given for zdip2 in figure 5 . the photophysical properties of zdip1zdip4 and zdip2 and zdip3 at room temperature are summarized in table 2 and the si . the absorption spectra and the principal band in the emission spectra are nearly independent of solvent polarity , indicating little change in the dipole moment or polarizability upon excitation . all of the complexes display emission spectra with small stokes shifts indicative of a localized excited state . representative spectra for zdip2 and zdip2 are shown in figure 6 . the luminescent quantum yields ( f ) range between 0.08 and 0.66 and the fluorescence decays are single exponential with lifetimes ( ) that vary from 0.8 to 4.8 ns . the differences in f are due to significant variations in the rate constant for nonradiative decay ( knr ) , as the rate constants for radiative decay ( kr ) are similar among all the derivatives . the data reveals an interesting effect of alkylation on knr : zdip2 zdip1 < zdip4 zdip3 ( table 2 ) . < zdip3. comparing zdip1 and zdip2 shows that methylation at the -position of the dipyrrin only slightly decreases knr , suggesting that hindering excited state distortion toward a planar coordination geometry has little effect on nonradiative decay . methyl substitution at the -position , adjacent to the mesitylene substituted meso - carbon , leads to a marked enhancement in the observed knr values for these complexes . the effect is also seen when comparing zdip2 to zdip3 , where -methylation leads to a 6-fold increase in knr . the variation in knr for zdip1zdip4 is roughly correlated with the fwhm of emission spectra ( figure 6 , inset ) . the association of broader emission profiles with a faster knr suggests that structural distortion of the excited states increases internal conversion to the ground state . while one would have expected that libration of the mesityl group is more favorable in zdip1 and zdip2 , thus increasing knr relative to their more sterically constrained analogs , zdip3 and zdip4 , the opposite is true . a likely explanation for this is that steric interactions between the ortho - methyls on the mesityl group and the -methyls of the dipyrrin exacerbate out - of - plane distortions on the dipyrrin ligand in zdip3 and zdip4 , which is not expected to be the case for zdip1 and zdip2 . distortion of the dipyrrin from planarity will give both an increase in fwhm and knr , as observed here . while the absorption spectra for all the complexes are solvent - independent , the emission spectra of the homoleptic and heteroleptic derivatives exhibit distinct differences with respect to solvent polarity . representative emission spectra of zdip2 measured in different solvents are shown in figure 5a , data for luminescent quantum yields from zdip1zdip4 and zdip2 and zdip3 are presented in figure 5b , and other photophysical data are given in table 2 and the si . with increasing solvent polarity there is little change in the emission maxima ; however , the quantum yields for zdip1zdip4 sharply decrease and the excited state transients display multiexponential lifetimes with an increasing amplitude for a subnanosecond component . the subnanosecond component is faster than our instrument s response time ( < 22 ps ) . the multiexponential decay indicates that with increasing solvent polarity the majority of the s1 population is going to a second state and only partially relaxing to the ground state by the radiative process ( figure s23 and table s14 , si ) . for the least polar solvent , cyclohexane , single exponential decay is observed with lifetimes of 3.7 , 4.8 , and 1.4 ns for zdip1 , zdip2 , and zdip3 , respectively . the radiative rate constants for emission in cyclohexane fall in a narrow range of 0.110.14 ns . in strong contrast , the luminescent quantum yields for zdip2 and zdip3 decline modestly in polar solvents and have radiative rate constants similar to those observed for zdip1zdip3 in cyclohexane ( i.e. , for zdip2 krad = 0.150.19 ns and for zdip3 krad = 0.11 ) . for the mixed ligand complexes , we believe that the decrease in pl efficiency in polar solvents is due to their photoinstability . both zdip2 and zdip3 disproportionate to zn(acac)2 and zn(dipyrrin ) thermally ; the process is likely driven optically and accelerated in polar media . the pronounced decrease in pl efficiency for zdip1zdip4 indicates that the locally excited state in the homoleptic derivatives equilibrates and deactivates to a weakly or nonemissive state in polar solvents . moreover , a broad emission band emerges at low energy for zdip2zdip4 in polar solvents ( figure 5a and si ) . the emission band red - shifts and becomes more pronounced with increased solvent polarity . the luminescence quantum yield and emission lifetime data ( f < 0.001 , = 2.2 ns ) indicate a very small radiative rate constant for this transition . however , the emission band is distinctly different from the phosphorescence of zdip2 recorded at 77 k in 2-methyltetrahydofuran ( 2-methf ) ( figure 5a , inset ) . to determine if the broad emission at 650 nm originates from an excimer or aggregate , emission intensities of zdip2 at 508 and 650 nm were measured at a range of concentrations ( see the si ) . the intensities of the two bands vary linearly with concentration , suggesting that excimer or aggregate emission is not responsible for the weak red emission in these compounds . thus , the low - energy emission band is assigned to a charge transfer transition similar to that reported for meso - coupled boron dipyrrin compounds . it is interesting to note that , in contrast to zdip2zdip4 , no low - energy emission is detected from nonalkylated zdip1 in polar solvent ( see the si ) . likewise , no comparable emission feature is observed in zdip2 or zdip3 in the same solvents . ( a ) emission spectra of zdip2 in various solvents ( inset is the phosphorescence spectrum in 2-methf at 77 k ) . ( b ) quantum yield of zdip1zdip4 and zdip2 and zdip3 plotted vs solvent from nonpolar to most polar . ( inset ) full width at half - maximum ( fwhm ) of emission plotted vs nonradiative rate constant knr for zdip1zdip4 and zdip2 and zdip3. the sharp decrease in luminescent efficiency from zdip1zdip4 in polar solvents , along with the simultaneous appearance of an additional broad peak at longer wavelengths in the emission spectra of zdip2zdip4 , suggests a deactivation pathway to a weakly emissive state . this state most likely has ct character and is formed via a symmetry - breaking mechanism similar to that which occurs in other bichromophoric systems . further support for the hypothesis that the observed photophysical properties of zdip1zdip4 are associated with sbct is the weaker dependence on solvent polarity for the heteroleptic complexes zdip2 and zdip3 , where sbct is impossible . cyclic voltammetry ( cv ) and differential pulse voltammetry ( dpv ) for zdip1zdip4 were carried out in dry thf under n2 , and the results are presented in table 3 . all of the complexes exhibit two distinct reversible reduction peaks and one irreversible oxidation peak , with the exception of zdip4 , where two quasireversible oxidation peaks are observed . the redox potentials are cathodically shifted by around 200 mv upon addition of two alkyl groups per ligand ; however , the difference between the first oxidation and reduction potentials ( electrochemical gap , eredox ) remains relatively constant for all the derivatives . this data can be used to evaluate the thermodynamic requirements for zdip1zdip4 to undergo photoinduced sbct . stabilization of the ct state in zdip1zdip4 is required to enable sbct , since eredox is greater than the optical s1 gap ( e00 ) . the required stabilization energies vary from 0.19 to 0.28 ev ( see table 3 ) . thus , while nonpolar solvents disfavor sbct , polar solvents such as dichloromethane ( s = 8.9 ) or acetonitrile ( s = 37.5 ) provide a stabilization energy estimated to exceed 0.3 ev and can therefore promote sbct . electrochemical values ( 0.02 v ) were determined by differential pulsed voltammetry ( dpv ) vs fc / fc . the optical gap e00 is defined by the midpoint between absorption and emission spectra in thf . triplet energies were measured in 2-methyltetrahydrofuran at 77 k. spectroelectrochemical measurements were also performed in order to identify absorption transitions characteristic of the ct state . the absorption profile for the one - electron - reduced form of zdip1 is broader than the neutral complex and has enhanced transitions between 370 and 430 nm along with a distinct peak at 517 nm ( figure 7 ) . unfortunately , the irreversible nature of electrochemical oxidation in zdip1zdip3 precluded optical characterization of the cation . likewise , the low stability of the zdip4 cation prevented characterization using spectroelectrochemical methods . femtosecond and nano - to - microsecond transient absorption ( ta ) measurements were performed to confirm the presence of sbct and to study the kinetics of such processes . femtosecond ta values of zdip1 in cyclohexane , toluene , dichloromethane , and acetonitrile are presented in figure 8 . other femtosecond ta spectra of zdip2 and zdip3 in cyclohexane , toluene , and acetonitrile are shown in the si . in cyclohexane ( figure 8a ) , excitation at 500 nm populates the s1 state , as observed by a ground - state bleach from 430 to 500 nm ( compare to figure 4 ) , stimulated emission ( 520600 nm , compare figure 6 ) , and excited state absorption at 345 nm . the stimulated emission remains over the probing time ( 1.1 ns ) , resulting in a minimal shift of the bleach peak ( figure 8a ) . femtosecond transient absorption of zdip1 in ( a ) cyclohexane , ( b ) toluene , ( c ) dichloromethane , and ( d ) acetonitrile . excitation at 500 nm and pump fluence of 160 j / cm were used for all , except panel c , which was performed at 70 j / cm . in polar solvents such as dichloromethane ( figure 8c ) and acetonitrile ( figure 8d ) , stimulated emission and excited state absorption at 345 nm from s1 appear immediately following excitation , similar to what is observed in cyclohexane . however , over the course of 46 ps , the stimulated emission band disappears and new induced absorption bands at 370 and 517 nm grow in . in contrast to the ta spectrum in cyclohexane , the disappearance of the stimulated emission results in the shift of the bleach peak ( figure 8c , d ) . the induced absorption at 517 nm is similar to that of the zdip1 anion [ see figures 7 and s25 ( si ) for a detailed comparison ] . since the induced absorption peak at 370 evolves with similar kinetics to that at 517 nm , we assign the 370 nm peak to the new excited state as well . this state is assigned as the sbct species ; note that the absence of characteristic sbct absorptions in cyclohexane indicates that stabilization by polar solvents is required to favor sbct over the local excited state , in agreement with the electrochemical analysis . the increase in the ground - state bleach during the first 10 ps ( figure 8c , d ) is a direct consequence of sbct . this is because only one of the dip ligands in the complex is bleached upon initial photoexcitation , whereas the second ligand is subsequently bleached upon sbct ; thus , the bleach increases approximately by a factor of 2 . in a weakly polar solvent , toluene ( figure 8b ) , both s1 stimulated emission and induced absorption at 370 nm are observed over the probing time of 1.1 ns , suggesting that the kinetic evolution of the transients is different from what was observed in either polar solvents or cyclohexane . the induced absorption at 370 nm indicates sbct of zdip1 in toluene ; however , the induced absorption at 517 nm is hidden due to the overlap with the stimulated emission band . additionally , the stimulated emission persists over the probing time ( 1.1 ns ) , which is much longer than that in acetonitrile or dichloromethane . the luminescent efficiency of zdip1 in toluene is also much higher than that in acetonitrile and dichloromethane ( table 2 ) . these observations can be explained by the presence of an equilibrium between the local excited state s1 and ct states in toluene . we propose that solvation by weakly polar toluene lowers the energy of the ct state close to that of the locally excited state . a similar equilibrium between locally excited and ct states of 9,9-bianthryl was reported in weakly polar media . in more polar solvents , large solvation energies further stabilize the ct states , shifting the equilibrium to formation of the charge transfer species . on the basis of the femtosecond ta measurements , a simplified jablonski diagram is proposed to explain the obtained results ( figure 9 ) . global fitting of ta data from zdip1zdip3 in different solvents has been performed using the proposed scheme , and the results are presented in table 4 ( a detailed description of the fitting scheme and the species - associated spectra are shown in the si ) . sbct does not occur in nonpolar cyclohexane , and the kinetics of transient species was fitted to a monoexponential decay with the lifetimes of 4.5 , 4.8 , and 1.4 ns for zdip1 , zdip2 , and zdip3 , respectively . these decay times are in good agreement with fluorescence lifetimes of the respective compounds in cyclohexane ( table s14 , si ) . a simplified jablonski diagram illustrating the dependence of the symmetry - breaking charge transfer process on solvent polarity . krec is the total rate for recombination to either the triplet or ground state . ta data of zdip1 in toluene were fitted with a different model to account for an equilibrium between the local excited state s1 and the ct state . the forward and backward rates ( 1/kct and 1/kcr ) between the s1 and the ct states are 13 and 25 ps , respectively . since these rates are 2 orders of magnitude faster than both the charge recombination rate of the ct state ( 1/krec = 3.5 ns ) and the decay rate of the local excited state ( = 3.0 ns ) , our assumption about a fast equilibrium is indeed reasonable . in polar dichloromethane , the fitting yields rates of 5.5 ps and 3.3 ns for the formation ( 1/kct ) and the recombination ( 1/krec ) of the ct state of zdip1 , respectively . in acetonitrile , the rates for formation and recombination of the ct state are faster compared to those in dichloromethane ( 1/kct = 3.6 ps and 1/krec = 2.1 ns ) . generally , the rate for formation of the ct state in polar solvents ( 1/kct = 1.15.5 ps ) is 3 orders of magnitude faster than that for recombination ( 1/krec = 0.93.3 ns ) ( table 4 ) . interestingly , the femtosecond ta measurements of zdip2 and zdip3 in acetonitrile ( figure 10 and the si ) show faster rates for both formation and recombination of the ct state ( 1/kct = 1.1 , 1.0 ps and 1/krec = 0.9 , 1.4 ns for zdip2 and zdip3 , respectively ) compared to zdip1 . the two dipyrrin ligands of zdip2 and zdip3 are expected to have less torsional freedom around the zn center relative to zdip1 due to the presence of methyl groups at the -positions . this steric hindrance constrains the ligands to adopt a more orthogonal configuration . without these steric impediments , this interaction will lower the energy of the excited state , making the s1 state less energetically favorable for forming the sbct state . ( a ) femtosecond transient absorption of zdip2 with 500 nm excitation in acetonitrile . ( b ) comparison of dynamics for formation of the ct state ( monitored at 370 nm , 2 ) ( filled ) and ground state bleach ( open ) between zdip1 ( red squares ) and zdip2 ( blue circles ) in acetonitrile . while femtosecond ta measurements of zdip1zdip3 show that sbct occurs in weakly polar to polar solvents , questions about deactivation pathways of the ct state still remain open . schutz and schmidt reported that the ct state of 9,9-bianthryl recombined radiatively to the ground state or nonradiatively to triplet states in polar solvents ; the nonradiative ct s0 internal conversion was negligible . in contrast to 9,9-bianthryl , poor electronic coupling makes the ct state in zdip1zdip4 at best only weakly emissive , indicating nonradiative deactivation via either direct ct s0 internal conversion or ct t1 recombination . direct internal conversion does occur as femtosecond ta measurements show partial recovery of the ground state bleach with a concomitant decrease of the ct induced absorption ( figure 10b ) . on the other hand , energies for the triplet states of zdip2zdip4 measured at 77 k in 2-methf ( 1.75 , 1.74 , and 1.72 ev , respectively , see table 3 ) are lower than those for the ct states ( 1.92 , 1.90 , and 1.84 ev , respectively , as estimated from maxima of the ct emission peaks in dichloromethane ) . deactivation of the ct states was further probed by performing nano - to - microsecond transient absorption measurements on zdip1 in different solvents ; results are presented in figure 11 and the si . in all solvents , induced absorption peaks from the ct state are absent within the instrument response time ( approximately 20 ns ) , and new induced absorption bands appear at 350450 nm ( max = 420 nm ) and 550600 nm . to elucidate the origin of these new induced absorption features , femtosecond ta of zdip1 was measured in dichloromethane / methyl iodide ( ch2cl2/ch3i 1/4 ) ( figure 11c ) , a solvent mixture that is expected to accelerate intersystem crossing from the excited singlet state to the triplet state . the induced absorption in this mixture matches well with the microsecond ta spectrum of zdip1 in acetonitrile ( figure 11a ) . thus , the induced absorption features from 20 ns to milliseconds observed in acetonitrile are assigned exclusively to the triplet state . formation of the triplet state in cyclohexane was also observed ; however , the signal intensity is much weaker than that observed in more polar solvents ( figure 11b ) . we speculate that formation of the triplet state via s1 t1 intersystem crossing is less efficient in cyclohexane than via s1 ct t1 recombination , as observed in toluene and acetonitrile . by fitting the microsecond ta traces to a single exponential decay , triplet lifetimes ( 1/kt1 ) for zdip1 were determined to be 16 , 50 , and 33 s in cyclohexane , toluene , and acetonitrile , respectively . nano - to - millisecond transient absorption of zdip1 in ( a ) acetonitrile and ( b ) different solvents at 0.5 s and ( c ) femtosecond transient absorption of zdip1 in ch2cl2/ch3i ( 1/4 ) . homoleptic zinc dipyrrins zdip1zdip4 exhibit photophysical properties that are strongly influenced by solvent polarity . these solvent - dependent properties are shown to occur by deactivation of the locally excited state via a symmetry - breaking charge transfer process . transient absorption measurements revealed that sbct proceeds in polar solvents at a rate 2 orders of magnitude faster than charge recombination . this fast charge transfer rate ( 1.014 ps ) , in combination with slower charge recombination rate ( 1.03.5 ns ) , is a desirable property for materials used in opvs , as it allows sufficient time for charge separation at a donor / acceptor interface . the weakly emissive nature of the ct state of zinc dipyrrins is in contrast to the efficient ct emission observed for bianthryl and biperylenyl . in the latter complexes , significant electronic coupling exists between the two chromophoric units . for zdip1zdip4 , poor molecular orbital overlap and weak dipolar coupling between the two nearly orthogonal dipyrrin ligands , as seen in the computational studies and crystal structure ( figures 2 and 3 ) , can be used to explain the decreased emissivity . it is interesting to note that no low energy emission was detected from nonalkylated zinc dipyrrin zdip1 in polar solvent in contrast to zdip2zdip4 ( see the si ) . the sbct also sheds light on the origin of the low luminescent efficiencies exhibited by homoleptic zinc dipyrrin complexes in polar and weakly polar solvents , especially when compared to their heteroleptic counterparts . our results suggest that metal complexes containing a single dipyrrin ligand are promising candidates as highly fluorescent probes in a range of applications ; however , the instability to disproportionation is a drawback for the zn derivatives . finally , this study demonstrates that sbct can occur in systems where the chromophores remain weakly coupled in the excited state . it is interesting to note that other molecules that have been shown to undergo sbct exhibit some degree of electronic overlap between chromophoric units , as indicated using calculated frontier orbitals . additionally , the previous systems have a degree of rotational freedom around a -bond , leading to a twisted internal charge transfer mechanism , whereas the zdip1zdip4 complexes are capable of sbct , even when rotational motion is severely restricted between the orthogonal ligands . similarly , weak intermolecular couplings are ubiquitous in the neat solid , where the dielectric constant should be comparable to that of toluene ( s = 2.4 ) . thus , formation of ct states should be a relatively common occurrence upon photoexcitation of related chromophores in the solid state .

zinc dipyrrin complexes with two identical dipyrrin ligands absorb strongly at 450550 nm and exhibit high fluorescence quantum yields in nonpolar solvents ( e.g. , 0.160.66 in cyclohexane ) and weak to nonexistent emission in polar solvents ( i.e. , < 103 , in acetonitrile ) . the low quantum efficiencies in polar solvents are attributed to the formation of a nonemissive symmetry - breaking charge transfer ( sbct ) state , which is not formed in nonpolar solvents . analysis using ultrafast spectroscopy shows that in polar solvents the singlet excited state relaxes to the sbct state in 1.05.5 ps and then decays via recombination to the triplet or ground states in 0.93.3 ns . in the weakly polar solvent toluene , the equilibrium between a localized excited state and the charge transfer state is established in 1122 ps .

Introduction Experimental Section Results and Discussion Conclusion

herein , we investigate the photophysics of zinc dipyrrin complexes ( figure 1 ) that exhibit intense visible absorption in a range of organic solvents . these compounds have structural features related to 9,9-bianthryl ( i.e. , poorly coupled orthogonal chromophores ) that are conducive to photoinduced sbct processes in weakly polar to polar solvents . zinc dipyrrins and analogous compounds are attractive because , in addition to strong absorption in the visible region of the spectrum , their syntheses are easy and scalable . structures of homoleptic ( zdip1zdip4 ) and heteroleptic ( zdip2 and zdip3 ) zinc dipyrrin complexes . in opvs , the low dielectric constants of organic materials ( s 3 ) lead to high exciton binding energies , and thus , a large energy offset between donor and acceptor is required to promote charge generation at the donor / acceptor interface ( d / a ) . that being the case , the ability of zinc dipyrrin complexes to undergo sbct makes them a potential family of new materials for use in organic photovoltaics . unlike the highly fluorescent bdips , homoleptic complexes mdn ( d = dipyrrin or -extended dipyrrin ligands ) generally exhibit low to moderate fluorescent quantum yields . the authors proposed that this decrease in polar solvents is due to thermal promotion from a locally excited state on a single dipyrrin ligand to a nonemissive , charge - separated state ( i.e. in this paper , we show that sbct in polar solvents is an effective nonradiative decay pathway for the electronic excited states of homoleptic zinc dipyrrins . in nonpolar solvents such as cyclohexane , these complexes do not undergo sbct and thus exhibit even higher quantum yields than their heteroleptic analogs . the synthesis and characterization by nmr , mass spectroscopy , and elemental analysis of the zinc dipyrrin complexes described here are given in the supporting information ( si ) for this paper . fluorescence lifetime measurements in cyclohexane , toluene , and thf were performed using an ibh fluorocube instrument equipped with a 405 nm led excitation source with the irf value of 0.4 ns . the error in the emission lifetime measurements is 5% and for the quantum yields is 10% . to avoid any contribution to the observed dynamics from orientational relaxation , the polarization of the supercontinuum was set at the magic angle ( 54.7 ) with respect to the pump polarization . during data collection , the samples were slowly oscillated perpendicular to the pump and probe to reduce photodamage to the sample by the pump . the third harmonic of a 10 hz q - switched nd : yag laser ( spectra - physics quanta - ray pro - series , pulse width : 8 ns ) was used to pump an optical parametric oscillator ( spectra - physics quanta - ray mopo-700 ) , tunable in the visible region . however , zdip2 and zdip3 are stable in the solid state and can be sublimed under vacuum without disproportionation . compared to boron dipyrrin compounds , which are essentially flat , the dipyrrin framework in the zinc dipyrrins is considerably more flexible , as dihedral angles between planes 1 and 2 vary from 3 to 18. however , no clear correlation is apparent between the degree of alkylation and variation of the dihedral angles , indicating that the distortions are likely dictated by crystal packing forces . in order to gain insight into the electronic structure of the zinc dipyrrin complexes , theoretical calculations were performed at the b3lyp / lacvp * * level of theory . the homo ( a2 symmetry in the d2d point group ) and homo1 ( b1 ) of zdip localize on both dipyrrin ligands , while the doubly degenerate lumos are localized on separate dipyrrin ligands ( figure 3 ) . the zinc dipyrrin complexes absorb strongly from 400 to 550 nm [ = ( 1.011.17 ) 10 m cm ] . representative absorption spectra of zdip2 and zdip2 in different solvents are presented in figure 4 , and the emission spectra in the same solvents are given for zdip2 in figure 5 . the absorption spectra and the principal band in the emission spectra are nearly independent of solvent polarity , indicating little change in the dipole moment or polarizability upon excitation . all of the complexes display emission spectra with small stokes shifts indicative of a localized excited state . the luminescent quantum yields ( f ) range between 0.08 and 0.66 and the fluorescence decays are single exponential with lifetimes ( ) that vary from 0.8 to 4.8 ns . methyl substitution at the -position , adjacent to the mesitylene substituted meso - carbon , leads to a marked enhancement in the observed knr values for these complexes . a likely explanation for this is that steric interactions between the ortho - methyls on the mesityl group and the -methyls of the dipyrrin exacerbate out - of - plane distortions on the dipyrrin ligand in zdip3 and zdip4 , which is not expected to be the case for zdip1 and zdip2 . representative emission spectra of zdip2 measured in different solvents are shown in figure 5a , data for luminescent quantum yields from zdip1zdip4 and zdip2 and zdip3 are presented in figure 5b , and other photophysical data are given in table 2 and the si . with increasing solvent polarity there is little change in the emission maxima ; however , the quantum yields for zdip1zdip4 sharply decrease and the excited state transients display multiexponential lifetimes with an increasing amplitude for a subnanosecond component . the multiexponential decay indicates that with increasing solvent polarity the majority of the s1 population is going to a second state and only partially relaxing to the ground state by the radiative process ( figure s23 and table s14 , si ) . the radiative rate constants for emission in cyclohexane fall in a narrow range of 0.110.14 ns . in strong contrast , the luminescent quantum yields for zdip2 and zdip3 decline modestly in polar solvents and have radiative rate constants similar to those observed for zdip1zdip3 in cyclohexane ( i.e. for the mixed ligand complexes , we believe that the decrease in pl efficiency in polar solvents is due to their photoinstability . the pronounced decrease in pl efficiency for zdip1zdip4 indicates that the locally excited state in the homoleptic derivatives equilibrates and deactivates to a weakly or nonemissive state in polar solvents . moreover , a broad emission band emerges at low energy for zdip2zdip4 in polar solvents ( figure 5a and si ) . the intensities of the two bands vary linearly with concentration , suggesting that excimer or aggregate emission is not responsible for the weak red emission in these compounds . thus , the low - energy emission band is assigned to a charge transfer transition similar to that reported for meso - coupled boron dipyrrin compounds . it is interesting to note that , in contrast to zdip2zdip4 , no low - energy emission is detected from nonalkylated zdip1 in polar solvent ( see the si ) . the sharp decrease in luminescent efficiency from zdip1zdip4 in polar solvents , along with the simultaneous appearance of an additional broad peak at longer wavelengths in the emission spectra of zdip2zdip4 , suggests a deactivation pathway to a weakly emissive state . thus , while nonpolar solvents disfavor sbct , polar solvents such as dichloromethane ( s = 8.9 ) or acetonitrile ( s = 37.5 ) provide a stabilization energy estimated to exceed 0.3 ev and can therefore promote sbct . likewise , the low stability of the zdip4 cation prevented characterization using spectroelectrochemical methods . femtosecond ta values of zdip1 in cyclohexane , toluene , dichloromethane , and acetonitrile are presented in figure 8 . other femtosecond ta spectra of zdip2 and zdip3 in cyclohexane , toluene , and acetonitrile are shown in the si . in cyclohexane ( figure 8a ) , excitation at 500 nm populates the s1 state , as observed by a ground - state bleach from 430 to 500 nm ( compare to figure 4 ) , stimulated emission ( 520600 nm , compare figure 6 ) , and excited state absorption at 345 nm . excitation at 500 nm and pump fluence of 160 j / cm were used for all , except panel c , which was performed at 70 j / cm . in polar solvents such as dichloromethane ( figure 8c ) and acetonitrile ( figure 8d ) , stimulated emission and excited state absorption at 345 nm from s1 appear immediately following excitation , similar to what is observed in cyclohexane . in contrast to the ta spectrum in cyclohexane , the disappearance of the stimulated emission results in the shift of the bleach peak ( figure 8c , d ) . since the induced absorption peak at 370 evolves with similar kinetics to that at 517 nm , we assign the 370 nm peak to the new excited state as well . this state is assigned as the sbct species ; note that the absence of characteristic sbct absorptions in cyclohexane indicates that stabilization by polar solvents is required to favor sbct over the local excited state , in agreement with the electrochemical analysis . this is because only one of the dip ligands in the complex is bleached upon initial photoexcitation , whereas the second ligand is subsequently bleached upon sbct ; thus , the bleach increases approximately by a factor of 2 . in a weakly polar solvent , toluene ( figure 8b ) , both s1 stimulated emission and induced absorption at 370 nm are observed over the probing time of 1.1 ns , suggesting that the kinetic evolution of the transients is different from what was observed in either polar solvents or cyclohexane . additionally , the stimulated emission persists over the probing time ( 1.1 ns ) , which is much longer than that in acetonitrile or dichloromethane . these observations can be explained by the presence of an equilibrium between the local excited state s1 and ct states in toluene . we propose that solvation by weakly polar toluene lowers the energy of the ct state close to that of the locally excited state . a similar equilibrium between locally excited and ct states of 9,9-bianthryl was reported in weakly polar media . in more polar solvents , large solvation energies further stabilize the ct states , shifting the equilibrium to formation of the charge transfer species . global fitting of ta data from zdip1zdip3 in different solvents has been performed using the proposed scheme , and the results are presented in table 4 ( a detailed description of the fitting scheme and the species - associated spectra are shown in the si ) . sbct does not occur in nonpolar cyclohexane , and the kinetics of transient species was fitted to a monoexponential decay with the lifetimes of 4.5 , 4.8 , and 1.4 ns for zdip1 , zdip2 , and zdip3 , respectively . a simplified jablonski diagram illustrating the dependence of the symmetry - breaking charge transfer process on solvent polarity . krec is the total rate for recombination to either the triplet or ground state . ta data of zdip1 in toluene were fitted with a different model to account for an equilibrium between the local excited state s1 and the ct state . since these rates are 2 orders of magnitude faster than both the charge recombination rate of the ct state ( 1/krec = 3.5 ns ) and the decay rate of the local excited state ( = 3.0 ns ) , our assumption about a fast equilibrium is indeed reasonable . in polar dichloromethane , the fitting yields rates of 5.5 ps and 3.3 ns for the formation ( 1/kct ) and the recombination ( 1/krec ) of the ct state of zdip1 , respectively . in acetonitrile , the rates for formation and recombination of the ct state are faster compared to those in dichloromethane ( 1/kct = 3.6 ps and 1/krec = 2.1 ns ) . generally , the rate for formation of the ct state in polar solvents ( 1/kct = 1.15.5 ps ) is 3 orders of magnitude faster than that for recombination ( 1/krec = 0.93.3 ns ) ( table 4 ) . interestingly , the femtosecond ta measurements of zdip2 and zdip3 in acetonitrile ( figure 10 and the si ) show faster rates for both formation and recombination of the ct state ( 1/kct = 1.1 , 1.0 ps and 1/krec = 0.9 , 1.4 ns for zdip2 and zdip3 , respectively ) compared to zdip1 . the two dipyrrin ligands of zdip2 and zdip3 are expected to have less torsional freedom around the zn center relative to zdip1 due to the presence of methyl groups at the -positions . without these steric impediments , this interaction will lower the energy of the excited state , making the s1 state less energetically favorable for forming the sbct state . ( b ) comparison of dynamics for formation of the ct state ( monitored at 370 nm , 2 ) ( filled ) and ground state bleach ( open ) between zdip1 ( red squares ) and zdip2 ( blue circles ) in acetonitrile . while femtosecond ta measurements of zdip1zdip3 show that sbct occurs in weakly polar to polar solvents , questions about deactivation pathways of the ct state still remain open . schutz and schmidt reported that the ct state of 9,9-bianthryl recombined radiatively to the ground state or nonradiatively to triplet states in polar solvents ; the nonradiative ct s0 internal conversion was negligible . to elucidate the origin of these new induced absorption features , femtosecond ta of zdip1 was measured in dichloromethane / methyl iodide ( ch2cl2/ch3i 1/4 ) ( figure 11c ) , a solvent mixture that is expected to accelerate intersystem crossing from the excited singlet state to the triplet state . thus , the induced absorption features from 20 ns to milliseconds observed in acetonitrile are assigned exclusively to the triplet state . formation of the triplet state in cyclohexane was also observed ; however , the signal intensity is much weaker than that observed in more polar solvents ( figure 11b ) . we speculate that formation of the triplet state via s1 t1 intersystem crossing is less efficient in cyclohexane than via s1 ct t1 recombination , as observed in toluene and acetonitrile . these solvent - dependent properties are shown to occur by deactivation of the locally excited state via a symmetry - breaking charge transfer process . transient absorption measurements revealed that sbct proceeds in polar solvents at a rate 2 orders of magnitude faster than charge recombination . this fast charge transfer rate ( 1.014 ps ) , in combination with slower charge recombination rate ( 1.03.5 ns ) , is a desirable property for materials used in opvs , as it allows sufficient time for charge separation at a donor / acceptor interface . for zdip1zdip4 , poor molecular orbital overlap and weak dipolar coupling between the two nearly orthogonal dipyrrin ligands , as seen in the computational studies and crystal structure ( figures 2 and 3 ) , can be used to explain the decreased emissivity . it is interesting to note that no low energy emission was detected from nonalkylated zinc dipyrrin zdip1 in polar solvent in contrast to zdip2zdip4 ( see the si ) . the sbct also sheds light on the origin of the low luminescent efficiencies exhibited by homoleptic zinc dipyrrin complexes in polar and weakly polar solvents , especially when compared to their heteroleptic counterparts . finally , this study demonstrates that sbct can occur in systems where the chromophores remain weakly coupled in the excited state . additionally , the previous systems have a degree of rotational freedom around a -bond , leading to a twisted internal charge transfer mechanism , whereas the zdip1zdip4 complexes are capable of sbct , even when rotational motion is severely restricted between the orthogonal ligands .

herein , we investigate the photophysics of zinc dipyrrin complexes ( figure 1 ) that exhibit intense visible absorption in a range of organic solvents . in opvs , the low dielectric constants of organic materials ( s 3 ) lead to high exciton binding energies , and thus , a large energy offset between donor and acceptor is required to promote charge generation at the donor / acceptor interface ( d / a ) . lindsey and co - workers reported that rotation of the phenyl ring at the meso - position on the dipyrrin ligand is a source of nonradiative deactivation of the excited state . interestingly , a recent study on related mesityl - substituted zinc dipyrrins ( zdip2 and zdip4 ) has reported that the quantum yields of the homoleptic complexes are strongly dependent on solvent polarity , decreasing from 2030% in toluene to 5% in dichloromethane . however , excitonic coupling between the nearly orthogonal ligands in homoleptic zinc dipyrrin compounds such as zdip2 and zdip4 is negligible and thus can not be used to explain the decrease in luminescent efficiency compared to their heteroleptic counterparts . in this paper , we show that sbct in polar solvents is an effective nonradiative decay pathway for the electronic excited states of homoleptic zinc dipyrrins . the synthesis and characterization by nmr , mass spectroscopy , and elemental analysis of the zinc dipyrrin complexes described here are given in the supporting information ( si ) for this paper . the third harmonic of a 10 hz q - switched nd : yag laser ( spectra - physics quanta - ray pro - series , pulse width : 8 ns ) was used to pump an optical parametric oscillator ( spectra - physics quanta - ray mopo-700 ) , tunable in the visible region . the x - ray crystal structures of zdip3 , zdip4 , zdip2 , and zdip3 were determined using a bruker apex ii ccd system equipped with a triumph curved - crystal monochromator and a mo k fine - focus tube ( = 0.710 73 ) . dipyrrins dip1dip4 were prepared from the corresponding pyrrole and mesitylaldehyde and used directly in consecutive reactions with ddq and zn(oac)2 to form the homoleptic complexes zdip1zdip4 in total yields of 813% . of the three ancillary ligands examined , pentane-2,4-dione ( acac ) , 1,1,1,5,5,5-hexafluoropentane-2,4-dione , and 2,2,6,6-tetramethyl-3,5-heptanedione ( dpm ) , pure heteroleptic complexes were successfully isolated only using the dpm ligand . as seen for other heteroleptic zn complexes , zdip2 and zdip3 disproportionate to their respective homoleptic complexes ( zdip2 and zdip3 ) in chloroform over the course of several hours , as observed by nmr measurements ( see si ) . single crystal x - ray analysis was performed on zdip3 , zdip4 , zdip2 , and zdip3 ; structures of representative homoleptic ( zdip3 ) and heteroleptic complexes ( zdip3 ) are shown in figure 2 . the dihedral angles between mean planes of the two dipyrrin ligands in zdip1 , zdip2 , zdip3 , and zdip4 are 85.0 , 88.3 , 76.7 , and 83.4 , respectively , whereas values for the related angles between the two ligands of zdip2 and zdip3 are 87.8 and 89.8 , respectively . to evaluate the degree of distortion of the ligands , dihedral angles between the planes encompassing different groups of atoms as shown in figure 2 were measured ( table 1 ) . compared to boron dipyrrin compounds , which are essentially flat , the dipyrrin framework in the zinc dipyrrins is considerably more flexible , as dihedral angles between planes 1 and 2 vary from 3 to 18. however , no clear correlation is apparent between the degree of alkylation and variation of the dihedral angles , indicating that the distortions are likely dictated by crystal packing forces . in order to gain insight into the electronic structure of the zinc dipyrrin complexes , theoretical calculations were performed at the b3lyp / lacvp * * level of theory . structures of the optimized complexes , highest and next highest occupied molecular orbitals ( homo , homo1 ) , and lowest and next lowest unoccupied molecular orbital ( lumo , lumo+1 ) , along with the corresponding energies , are shown in figure 3 . the homo ( a2 symmetry in the d2d point group ) and homo1 ( b1 ) of zdip localize on both dipyrrin ligands , while the doubly degenerate lumos are localized on separate dipyrrin ligands ( figure 3 ) . the minor difference in energy between the homo and homo1 in zdip ( 0.11 ev ) is indicative of very poor electronic coupling between the two orthogonal dipyrrin ligands in the ground state . this places the transition dipole moments of the two dipyrrins in zdips orthogonal to each other , suggesting that there should be little or no excitonic or electronic coupling between the two dipyrrin ligands . representative absorption spectra of zdip2 and zdip2 in different solvents are presented in figure 4 , and the emission spectra in the same solvents are given for zdip2 in figure 5 . the absorption spectra and the principal band in the emission spectra are nearly independent of solvent polarity , indicating little change in the dipole moment or polarizability upon excitation . the differences in f are due to significant variations in the rate constant for nonradiative decay ( knr ) , as the rate constants for radiative decay ( kr ) are similar among all the derivatives . comparing zdip1 and zdip2 shows that methylation at the -position of the dipyrrin only slightly decreases knr , suggesting that hindering excited state distortion toward a planar coordination geometry has little effect on nonradiative decay . while one would have expected that libration of the mesityl group is more favorable in zdip1 and zdip2 , thus increasing knr relative to their more sterically constrained analogs , zdip3 and zdip4 , the opposite is true . a likely explanation for this is that steric interactions between the ortho - methyls on the mesityl group and the -methyls of the dipyrrin exacerbate out - of - plane distortions on the dipyrrin ligand in zdip3 and zdip4 , which is not expected to be the case for zdip1 and zdip2 . while the absorption spectra for all the complexes are solvent - independent , the emission spectra of the homoleptic and heteroleptic derivatives exhibit distinct differences with respect to solvent polarity . representative emission spectra of zdip2 measured in different solvents are shown in figure 5a , data for luminescent quantum yields from zdip1zdip4 and zdip2 and zdip3 are presented in figure 5b , and other photophysical data are given in table 2 and the si . with increasing solvent polarity there is little change in the emission maxima ; however , the quantum yields for zdip1zdip4 sharply decrease and the excited state transients display multiexponential lifetimes with an increasing amplitude for a subnanosecond component . the multiexponential decay indicates that with increasing solvent polarity the majority of the s1 population is going to a second state and only partially relaxing to the ground state by the radiative process ( figure s23 and table s14 , si ) . for the least polar solvent , cyclohexane , single exponential decay is observed with lifetimes of 3.7 , 4.8 , and 1.4 ns for zdip1 , zdip2 , and zdip3 , respectively . in strong contrast , the luminescent quantum yields for zdip2 and zdip3 decline modestly in polar solvents and have radiative rate constants similar to those observed for zdip1zdip3 in cyclohexane ( i.e. the pronounced decrease in pl efficiency for zdip1zdip4 indicates that the locally excited state in the homoleptic derivatives equilibrates and deactivates to a weakly or nonemissive state in polar solvents . however , the emission band is distinctly different from the phosphorescence of zdip2 recorded at 77 k in 2-methyltetrahydofuran ( 2-methf ) ( figure 5a , inset ) . the sharp decrease in luminescent efficiency from zdip1zdip4 in polar solvents , along with the simultaneous appearance of an additional broad peak at longer wavelengths in the emission spectra of zdip2zdip4 , suggests a deactivation pathway to a weakly emissive state . further support for the hypothesis that the observed photophysical properties of zdip1zdip4 are associated with sbct is the weaker dependence on solvent polarity for the heteroleptic complexes zdip2 and zdip3 , where sbct is impossible . cyclic voltammetry ( cv ) and differential pulse voltammetry ( dpv ) for zdip1zdip4 were carried out in dry thf under n2 , and the results are presented in table 3 . the redox potentials are cathodically shifted by around 200 mv upon addition of two alkyl groups per ligand ; however , the difference between the first oxidation and reduction potentials ( electrochemical gap , eredox ) remains relatively constant for all the derivatives . the absorption profile for the one - electron - reduced form of zdip1 is broader than the neutral complex and has enhanced transitions between 370 and 430 nm along with a distinct peak at 517 nm ( figure 7 ) . in cyclohexane ( figure 8a ) , excitation at 500 nm populates the s1 state , as observed by a ground - state bleach from 430 to 500 nm ( compare to figure 4 ) , stimulated emission ( 520600 nm , compare figure 6 ) , and excited state absorption at 345 nm . in contrast to the ta spectrum in cyclohexane , the disappearance of the stimulated emission results in the shift of the bleach peak ( figure 8c , d ) . this state is assigned as the sbct species ; note that the absence of characteristic sbct absorptions in cyclohexane indicates that stabilization by polar solvents is required to favor sbct over the local excited state , in agreement with the electrochemical analysis . in a weakly polar solvent , toluene ( figure 8b ) , both s1 stimulated emission and induced absorption at 370 nm are observed over the probing time of 1.1 ns , suggesting that the kinetic evolution of the transients is different from what was observed in either polar solvents or cyclohexane . global fitting of ta data from zdip1zdip3 in different solvents has been performed using the proposed scheme , and the results are presented in table 4 ( a detailed description of the fitting scheme and the species - associated spectra are shown in the si ) . sbct does not occur in nonpolar cyclohexane , and the kinetics of transient species was fitted to a monoexponential decay with the lifetimes of 4.5 , 4.8 , and 1.4 ns for zdip1 , zdip2 , and zdip3 , respectively . since these rates are 2 orders of magnitude faster than both the charge recombination rate of the ct state ( 1/krec = 3.5 ns ) and the decay rate of the local excited state ( = 3.0 ns ) , our assumption about a fast equilibrium is indeed reasonable . in polar dichloromethane , the fitting yields rates of 5.5 ps and 3.3 ns for the formation ( 1/kct ) and the recombination ( 1/krec ) of the ct state of zdip1 , respectively . in acetonitrile , the rates for formation and recombination of the ct state are faster compared to those in dichloromethane ( 1/kct = 3.6 ps and 1/krec = 2.1 ns ) . generally , the rate for formation of the ct state in polar solvents ( 1/kct = 1.15.5 ps ) is 3 orders of magnitude faster than that for recombination ( 1/krec = 0.93.3 ns ) ( table 4 ) . interestingly , the femtosecond ta measurements of zdip2 and zdip3 in acetonitrile ( figure 10 and the si ) show faster rates for both formation and recombination of the ct state ( 1/kct = 1.1 , 1.0 ps and 1/krec = 0.9 , 1.4 ns for zdip2 and zdip3 , respectively ) compared to zdip1 . ( b ) comparison of dynamics for formation of the ct state ( monitored at 370 nm , 2 ) ( filled ) and ground state bleach ( open ) between zdip1 ( red squares ) and zdip2 ( blue circles ) in acetonitrile . schutz and schmidt reported that the ct state of 9,9-bianthryl recombined radiatively to the ground state or nonradiatively to triplet states in polar solvents ; the nonradiative ct s0 internal conversion was negligible . in contrast to 9,9-bianthryl , poor electronic coupling makes the ct state in zdip1zdip4 at best only weakly emissive , indicating nonradiative deactivation via either direct ct s0 internal conversion or ct t1 recombination . on the other hand , energies for the triplet states of zdip2zdip4 measured at 77 k in 2-methf ( 1.75 , 1.74 , and 1.72 ev , respectively , see table 3 ) are lower than those for the ct states ( 1.92 , 1.90 , and 1.84 ev , respectively , as estimated from maxima of the ct emission peaks in dichloromethane ) . in all solvents , induced absorption peaks from the ct state are absent within the instrument response time ( approximately 20 ns ) , and new induced absorption bands appear at 350450 nm ( max = 420 nm ) and 550600 nm . to elucidate the origin of these new induced absorption features , femtosecond ta of zdip1 was measured in dichloromethane / methyl iodide ( ch2cl2/ch3i 1/4 ) ( figure 11c ) , a solvent mixture that is expected to accelerate intersystem crossing from the excited singlet state to the triplet state . we speculate that formation of the triplet state via s1 t1 intersystem crossing is less efficient in cyclohexane than via s1 ct t1 recombination , as observed in toluene and acetonitrile . by fitting the microsecond ta traces to a single exponential decay , triplet lifetimes ( 1/kt1 ) for zdip1 were determined to be 16 , 50 , and 33 s in cyclohexane , toluene , and acetonitrile , respectively . nano - to - millisecond transient absorption of zdip1 in ( a ) acetonitrile and ( b ) different solvents at 0.5 s and ( c ) femtosecond transient absorption of zdip1 in ch2cl2/ch3i ( 1/4 ) . the weakly emissive nature of the ct state of zinc dipyrrins is in contrast to the efficient ct emission observed for bianthryl and biperylenyl . for zdip1zdip4 , poor molecular orbital overlap and weak dipolar coupling between the two nearly orthogonal dipyrrin ligands , as seen in the computational studies and crystal structure ( figures 2 and 3 ) , can be used to explain the decreased emissivity . the sbct also sheds light on the origin of the low luminescent efficiencies exhibited by homoleptic zinc dipyrrin complexes in polar and weakly polar solvents , especially when compared to their heteroleptic counterparts . our results suggest that metal complexes containing a single dipyrrin ligand are promising candidates as highly fluorescent probes in a range of applications ; however , the instability to disproportionation is a drawback for the zn derivatives . additionally , the previous systems have a degree of rotational freedom around a -bond , leading to a twisted internal charge transfer mechanism , whereas the zdip1zdip4 complexes are capable of sbct , even when rotational motion is severely restricted between the orthogonal ligands .

smoking is the main preventable cause of early and untimely death and disabilities , which leads to approximately four million deaths every year.1 although smoking causes more mortality than other factors such as acquired immune deficiency disease ( aids ) , alcohol drinking , driving accidents , murder , suicide , and fire , but still about one third of the adult population use tobacco products worldwide.2 according to the centers for disease control and prevention ( cdc ) in the united states of america , 80% of adults , started smoking before the age of eighteen and about three thousand people are smoking regularly at this early stage of life.3 if no considerations are taken in order to stop this harmful trend , in the near future more than five million children will die because of future tendency to smoke . intervention and prevention in the age of less than 20 years old has a great importance.4 smoking in this age , causes more morbidity because of accumulation of narcotics over times , also intrigues other friends to smoke cigarettes . tobacco use at this stage of age provides the chance of being attracted to other drugs and still it is difficult for them to give up smoking though they have realized the dangers of cigarette smoking.5 therefore , planning preventive actions is an important step to be considered . national surveys in iran showed that the mean age of the first attempt to smoke was in the teenage group.6 similar situation exists in various countries such as the united states ( usa),7 and china.8 in spite of marked differences in the socio - cultural background of various countries , the knowledge , attitude and practice ( kap ) of youths about smoking is important about their smoking behavior . therefore , in this study , we aimed to determine the kap of a sample of iranian college students and to compare it with their american and chinese counterparts . we used a questionnaire , with the original edition prepared and edited by torabi et al . ( chancellor 's professor , applied health science department , indiana university , usa ) in 2002.9 for the purpose of accomplishing this survey , the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment , after consulting with dr . torabi , the necessary modifications were taken into account . to be assured about the accuracy and effectiveness of the translation , we used the back translation method , i.e. the persian - translated questionnaire was again translated in to english , and unsuitable translations were corrected . this english edition has been used for gathering information from american and chinese college students . according to the obtained information , the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . title of the noted study is consumption of alcohol , tobacco and other drugs in children and adults of indiana . the attitude scale of using tobacco which is used in this survey was first established by awaisu et al.10 the translated questionnaire contained 55 questions , nine of them being demographic ones . because of the increase in water - pipe smoking by young people , we added four related items to the practice questions , hence the number of practice questions increased to 21 , and the number of total questions increased to 59 . questions about awareness had multiple choices with only one right answer , but the applied scale in questions about attitude was based on likert system . practice questions contained 5 questions about smoking cigarette , cigar , chewing tobacco , pipe , and waterpipe . for all of these noted tobacco products , the duration of consumption in the previous month and year , as well as quitting smoking were considered . we included this group to be comparable to studies conducted in the us and china . the male - to female ratio and the proportion of junior to senior students were similar to the above mentioned surveys . the outcomes of the studies were analyzed statistically by version 13.0 spss for windows ( spss inc . , chicago , il ) in order to obtain a reliable measurement for each scale . for reliability testing of knowledge and attitude scales , cronbach 's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . regarding to the understanding of each student who consumed cigarette , the score of one was given to correct answers and zero to wrong or blank answers . so , in the first stage , regarding the knowledge of students for each answer sheet , the total scores of knowledge questions could be a number between 0 and 11 . after changing the obtained numbers to the percentage , one could achieve the percentage between 1 and 100 percent . the perception of each student involving in smoking cigarettes was achieved to add the whole numbers in the questionnaire . number five showed the hatred of students towards smoking cigarettes and decreased from five toward one . the scale of each answer could be between 18 and 90 . to change the number to the percentage scale , the outcome would reach between 20 and 100 percent . in order to determine the differences in terms of gender , the analysis of variance ( anova ) was used . we used a questionnaire , with the original edition prepared and edited by torabi et al . ( chancellor 's professor , applied health science department , indiana university , usa ) in 2002.9 for the purpose of accomplishing this survey , the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment , after consulting with dr . torabi , the necessary modifications were taken into account . to be assured about the accuracy and effectiveness of the translation , we used the back translation method , i.e. the persian - translated questionnaire was again translated in to english , and unsuitable translations were corrected . this english edition has been used for gathering information from american and chinese college students . according to the obtained information , the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . title of the noted study is consumption of alcohol , tobacco and other drugs in children and adults of indiana . the attitude scale of using tobacco which is used in this survey was first established by awaisu et al.10 the translated questionnaire contained 55 questions , nine of them being demographic ones . because of the increase in water - pipe smoking by young people , we added four related items to the practice questions , hence the number of practice questions increased to 21 , and the number of total questions increased to 59 . questions about awareness had multiple choices with only one right answer , but the applied scale in questions about attitude was based on likert system . practice questions contained 5 questions about smoking cigarette , cigar , chewing tobacco , pipe , and waterpipe . for all of these noted tobacco products , the duration of consumption in the previous month and year , as well as quitting smoking were considered . we included this group to be comparable to studies conducted in the us and china . the male - to female ratio and the proportion of junior to senior students were similar to the above mentioned surveys . the outcomes of the studies were analyzed statistically by version 13.0 spss for windows ( spss inc . , chicago , il ) in order to obtain a reliable measurement for each scale . for reliability testing of knowledge and attitude scales , cronbach 's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . regarding to the understanding of each student who consumed cigarette , the score of one was given to correct answers and zero to wrong or blank answers . so , in the first stage , regarding the knowledge of students for each answer sheet , the total scores of knowledge questions could be a number between 0 and 11 . after changing the obtained numbers to the percentage , one could achieve the percentage between 1 and 100 percent . the perception of each student involving in smoking cigarettes was achieved to add the whole numbers in the questionnaire . number five showed the hatred of students towards smoking cigarettes and decreased from five toward one . the scale of each answer could be between 18 and 90 . to change the number to the percentage scale , the outcome would reach between 20 and 100 percent . in order to determine the differences in terms of gender , the analysis of variance ( anova ) was used . we used a questionnaire , with the original edition prepared and edited by torabi et al . ( chancellor 's professor , applied health science department , indiana university , usa ) in 2002.9 for the purpose of accomplishing this survey , the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment , after consulting with dr . torabi , the necessary modifications were taken into account . to be assured about the accuracy and effectiveness of the translation , we used the back translation method , i.e. the persian - translated questionnaire was again translated in to english , and unsuitable translations were corrected . this english edition has been used for gathering information from american and chinese college students . according to the obtained information , the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . title of the noted study is consumption of alcohol , tobacco and other drugs in children and adults of indiana . the attitude scale of using tobacco which is used in this survey was first established by awaisu et al.10 the translated questionnaire contained 55 questions , nine of them being demographic ones . because of the increase in water - pipe smoking by young people , we added four related items to the practice questions , hence the number of practice questions increased to 21 , and the number of total questions increased to 59 . questions about awareness had multiple choices with only one right answer , but the applied scale in questions about attitude was based on likert system . practice questions contained 5 questions about smoking cigarette , cigar , chewing tobacco , pipe , and waterpipe . for all of these noted tobacco products , the duration of consumption in the previous month and year , as well as quitting smoking were considered . we included this group to be comparable to studies conducted in the us and china . the male - to female ratio and the proportion of junior to senior students were similar to the above mentioned surveys . the outcomes of the studies were analyzed statistically by version 13.0 spss for windows ( spss inc . , chicago , il ) in order to obtain a reliable measurement for each scale . for reliability testing of knowledge and attitude scales , cronbach 's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . regarding to the understanding of each student who consumed cigarette , the score of one was given to correct answers and zero to wrong or blank answers . so , in the first stage , regarding the knowledge of students for each answer sheet , the total scores of knowledge questions could be a number between 0 and 11 . after changing the obtained numbers to the percentage , one could achieve the percentage between 1 and 100 percent . the perception of each student involving in smoking cigarettes was achieved to add the whole numbers in the questionnaire . number five showed the hatred of students towards smoking cigarettes and decreased from five toward one . the scale of each answer could be between 18 and 90 . to change the number to the percentage scale , the outcome would reach between 20 and 100 percent . in order to determine the differences in terms of gender , the analysis of variance ( anova ) this knowledge test is not a norm reliability test and its reliability was acceptable since it relied strongly on the criteria . in iran , from 1200 responses , 958 were complete to be used in the survey , which 456 of them were the responses given by male students ( 48.5% ) and 51.5% by female students . in china , out of 1534 students participating in the survey , 39.7% were women and 60.3% were men . in the united states , from 597 participants , 62.1% were women and 37.9% were men . overall , these three surveys comprised a total number of 3089 students consisting of 47.5% women and 52.5% men . significant differences existed in the structure of the place of the residence in three countries . in iran , 73.7% of students were from urban areas , whereas 47% of american college students were from suburban areas . among chinese students , regarding to marital status , the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . when the iranian participants were asked about their health status , 92.7% of them responded that they were in a good health condition ; 5.9% were on average and 1.4% were of poor health conditions . in usa , 67% enjoyed a very good health condition and 30.5% of them were average . in china , 83.5% were of good health and 14.5% were among the average . when the iranian students were asked about their daily stress , 42.2% claimed that they have not much stress , 38.6% of them told they were fairly stressful and 14.5% of them were of the average , 10.4% had no stress and 8.8% of them suffered from sever stress . internal consistency of the knowledge and the attitude of the three groups students were compared , and the results are summarized in table 1 . in all three surveys it was shown that almost a lower level of knowledge exists ( about 70% ) , but concerning the attitude , in all three countries satisfactory attitude score was obtained . internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students ( both men and women ) . similarly , table 3 shows the average number of attitude regarding to tobacco use among students ( both men and women ) . the anova test showed that there were no significant differences between the sexes and the average knowledge . the result obtained for men was anova = 1.26412 , p = 0.628 , and anova = 1.26412 , p = 0.96 . but the average meaningful knowledge , however , existed between iran and the other countries . ( anova for all = 342.703 , p < 0.001 ) . the average number of knowledge regarding to tobacco use among students ( both men and women ) the average number of attitude regarding to tobacco use among students ( both men and women ) in iran in both men and women this attitude is more than the americans and chinese . anova test showed that among the sexes , the average of attitude could clearly be observed ( in men , anova = 141.30 , p < 0.001 and anova = 284.14 , p < 0.001 for women ) . there is also a meaningful scale of attitude existed between the countries studied in this survey ( anova = 394.86 , p < 0.001 ) . it was shown that 70% of smoking participants started smoking between the ages of 19 - 22 . the survey carried out in china shows that among the cigarette addicts , most of the smokers had started trying cigarettes even before the age of thirteen . this percentage ( 37.6% ) is much higher than in iran ( 20.1% ) and the us ( 17.9% ) . in iran this statistics seems to be more expressive ( chi - square = 157.7 , p 0.001 ) . the age of first cigarette smoking among students ( both men and women ) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students , american and iranian students put much more effort to stop smoking . in iran , from 1200 responses , 958 were complete to be used in the survey , which 456 of them were the responses given by male students ( 48.5% ) and 51.5% by female students . in china , out of 1534 students participating in the survey , 39.7% were women and 60.3% were men . in the united states , from 597 participants , 62.1% were women and 37.9% were men . overall , these three surveys comprised a total number of 3089 students consisting of 47.5% women and 52.5% men . significant differences existed in the structure of the place of the residence in three countries . in iran , 73.7% of students were from urban areas , whereas 47% of american college students were from suburban areas . among chinese students , regarding to marital status , the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . when the iranian participants were asked about their health status , 92.7% of them responded that they were in a good health condition ; 5.9% were on average and 1.4% were of poor health conditions . in usa , 67% enjoyed a very good health condition and 30.5% of them were average . in china , when the iranian students were asked about their daily stress , 42.2% claimed that they have not much stress , 38.6% of them told they were fairly stressful and 14.5% of them were of the average , 10.4% had no stress and 8.8% of them suffered from sever stress . internal consistency of the knowledge and the attitude of the three groups students were compared , and the results are summarized in table 1 . in all three surveys it was shown that almost a lower level of knowledge exists ( about 70% ) , but concerning the attitude , in all three countries satisfactory attitude score was obtained . internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students ( both men and women ) . similarly , table 3 shows the average number of attitude regarding to tobacco use among students ( both men and women ) . the anova test showed that there were no significant differences between the sexes and the average knowledge . the result obtained for men was anova = 1.26412 , p = 0.628 , and anova = 1.26412 , p = 0.96 . but the average meaningful knowledge , however , existed between iran and the other countries . the average number of knowledge regarding to tobacco use among students ( both men and women ) the average number of attitude regarding to tobacco use among students ( both men and women ) in iran in both men and women this attitude is more than the americans and chinese . anova test showed that among the sexes , the average of attitude could clearly be observed ( in men , anova = 141.30 , p < 0.001 and anova = 284.14 , p < 0.001 for women ) . there is also a meaningful scale of attitude existed between the countries studied in this survey ( anova = 394.86 , p < 0.001 ) . it was shown that 70% of smoking participants started smoking between the ages of 19 - 22 . the survey carried out in china shows that among the cigarette addicts , most of the smokers had started trying cigarettes even before the age of thirteen . this percentage ( 37.6% ) is much higher than in iran ( 20.1% ) and the us ( 17.9% ) . in iran , this percentage increases along higher age groups . in the us , this statistics seems to be more expressive ( chi - square = 157.7 , p 0.001 ) . the age of first cigarette smoking among students ( both men and women ) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students , american and iranian students put much more effort to stop smoking . in iran , from 1200 responses , 958 were complete to be used in the survey , which 456 of them were the responses given by male students ( 48.5% ) and 51.5% by female students . in china , out of 1534 students participating in the survey , 39.7% were women and 60.3% were men . in the united states , from 597 participants , 62.1% were women and 37.9% were men . overall , these three surveys comprised a total number of 3089 students consisting of 47.5% women and 52.5% men . significant differences existed in the structure of the place of the residence in three countries . in iran , 73.7% of students were from urban areas , whereas 47% of american college students were from suburban areas . among chinese students , regarding to marital status , the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . when the iranian participants were asked about their health status , 92.7% of them responded that they were in a good health condition ; 5.9% were on average and 1.4% were of poor health conditions . in usa , 67% enjoyed a very good health condition and 30.5% of them were average . in china , when the iranian students were asked about their daily stress , 42.2% claimed that they have not much stress , 38.6% of them told they were fairly stressful and 14.5% of them were of the average , 10.4% had no stress and 8.8% of them suffered from sever stress . internal consistency of the knowledge and the attitude of the three groups students were compared , and the results are summarized in table 1 . in all three surveys it was shown that almost a lower level of knowledge exists ( about 70% ) , but concerning the attitude , in all three countries satisfactory attitude score was obtained . internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students ( both men and women ) . similarly , table 3 shows the average number of attitude regarding to tobacco use among students ( both men and women ) . the anova test showed that there were no significant differences between the sexes and the average knowledge . the result obtained for men was anova = 1.26412 , p = 0.628 , and anova = 1.26412 , p = 0.96 . but the average meaningful knowledge , however , existed between iran and the other countries . the average number of knowledge regarding to tobacco use among students ( both men and women ) the average number of attitude regarding to tobacco use among students ( both men and women ) in iran in both men and women this attitude is more than the americans and chinese . anova test showed that among the sexes , the average of attitude could clearly be observed ( in men , anova = 141.30 , p < 0.001 and anova = 284.14 , p < 0.001 for women ) . there is also a meaningful scale of attitude existed between the countries studied in this survey ( anova = 394.86 , p < 0.001 ) . it was shown that 70% of smoking participants started smoking between the ages of 19 - 22 . the survey carried out in china shows that among the cigarette addicts , most of the smokers had started trying cigarettes even before the age of thirteen . this percentage ( 37.6% ) is much higher than in iran ( 20.1% ) and the us ( 17.9% ) . in iran , this percentage increases along higher age groups . in the us , this statistics seems to be more expressive ( chi - square = 157.7 , p 0.001 ) . the age of first cigarette smoking among students ( both men and women ) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students , american and iranian students put much more effort to stop smoking . this survey has discussed the tendency toward smoking addiction among the students and their perception of this behavior and its outcome among the students in iran , the usa , and china . in this study students the knowledge and perception of each sex in one country and also between the three countries was carefully compared and evaluated . one of the problems the researchers are faced in this kind of survey is that students of the countries are of different cultures.1112 so , we can not expect that the students studied in these three countries give a relatively appropriate result . in the case of representation , participants should be carefully chosen and the results must be studied with almost care . there is also another criticism raised in this form of survey . since the questionnaires used in the three countries are the same and they are originally written in english , when being translated to persian and chinese the cultural differences might cause some problems . the young people studied in iran were all medical students , so there is a great possibility that their understanding of case and also their recognition of the harmful effects of smoking cigarettes might be much better than the students of other majors . this questionnaire has already been used in america and china.78 in case of iran , we were trying to translate it in to persian in a way that somehow agrees with our culture , but the problem still exists . the original questionnaire is in american english prepared by an iranian professor ( dr torabi , chancellor 's professor , applied health science department , indiana university , usa).10 we have already coordinated with him concerning the translation of the questionnaire . one of the changes that we made in persian version was adding waterpipe smoking to the text . this survey showed that male students in all stages of age and in all three countries smoke more than women.13 among the smoking products , cigarette is of high consumption . overall , beside cigarettes , cigars and pipes have had more consumers during the last year in all countries.78 fortunately , chewing tobacco ( being normal in western countries ) is still unknown in our country.14 a very few people use this kind of tobacco . so , the whole survey shows that the number of male students smoking tobacco products is much higher than women . studying carefully the true responses of students to the questions concerning the knowledge , reveals that women are little more concerned . the survey also shows that american students are more knowledgeable compared to iranian and chinese students . ( knowledgeable college students are about 8.70% , in america , 6.97% in iran and finally 6.84% in china ) . these varieties might be the result of differences between the system of education and the policies that americans have been following.12 however , there are no noticeable differences regarding the meaningful perception in all three countries . regarding the answers given by the students about their attitudes and perception , the survey showed that women 's concern about smoking was more than men that is very important and meaningful according to the statistical analysis . the perception of iranian men and women was more than the same group of people in the united states of america and china.1112 the total percentage of perception among iranian college students is 87.85% , comparing to 65.60% in america and 64.4% in china . iranian women 's attitude toward smoking depends on the cultural condition and the government policies.14 that is why women in iran smoke much less ( especially in public ) compared to women in china . regarding the cultural condition in china women are much more comfortable toward smoking in both outdoors and indoors.15 any way in all countries the consumption of cigarettes is important and statistically meaningful ( p < 0.001 ) . so , among these countries giving up cigarette smoking is statistically significant ( p < 0.001 ) . the current survey was planned to provide more information about the motives that provide certain conditions to start smoking . regarding the proved dangers of smoking and the results gained in the survey , important and crucial steps have been taken by the policy makers of the country to educate effectively and show the disastrous outcome of smoking all over the country . also , it is important to mention the fact that these kinds of surveys can submit valuable information to the health trainers , health policy makers , and ministry of health . in this way , the health ministry authorities can effectively understand the importance of using tobacco products and especially its cultural aspects . therefore , it would enable them to think of a way which results in prevention or decrease of smoking in the country . furthermore , we suggest to add some credit hours about smoking in universities educational curriculum .

objectives : the purpose of this survey was to compare the knowledge , attitude and practice of iranian , american and chinese college students about tobacco use.methods:in this cross - sectional study , a questionnaire that was designed for the first time in comparative studies for the purpose of gathering information from american and chinese college students was used . the students were selected by convenience sampling.results:in iran , 958 participants ( 456 male and 485 female ) enrolled in the study . in addition , 1534 chinese participants ( 39.7% female and 60.3% male ) and 597 american participants ( 62.1% female and 37.9% male ) were included in the study . comparing the consumption of tobacco among college students of the three mentioned countries , it is shown that american women have more experiences in smoking cigarettes , cigars , pipes and chewing tobacco.conclusions:this survey determined the tendency toward smoking among the students and their perception of this behavior and its outcome among the students from iran , usa and china . it provided significant differences , however the culture dissimilarities in responding to such type of surveys should be taken into account .

INTRODUCTION METHODS None Questionnaire Study population Statistical analysis RESULTS None The Participants Qualifications: DISCUSSION CONCLUSION

smoking is the main preventable cause of early and untimely death and disabilities , which leads to approximately four million deaths every year.1 although smoking causes more mortality than other factors such as acquired immune deficiency disease ( aids ) , alcohol drinking , driving accidents , murder , suicide , and fire , but still about one third of the adult population use tobacco products worldwide.2 according to the centers for disease control and prevention ( cdc ) in the united states of america , 80% of adults , started smoking before the age of eighteen and about three thousand people are smoking regularly at this early stage of life.3 if no considerations are taken in order to stop this harmful trend , in the near future more than five million children will die because of future tendency to smoke . tobacco use at this stage of age provides the chance of being attracted to other drugs and still it is difficult for them to give up smoking though they have realized the dangers of cigarette smoking.5 therefore , planning preventive actions is an important step to be considered . national surveys in iran showed that the mean age of the first attempt to smoke was in the teenage group.6 similar situation exists in various countries such as the united states ( usa),7 and china.8 in spite of marked differences in the socio - cultural background of various countries , the knowledge , attitude and practice ( kap ) of youths about smoking is important about their smoking behavior . therefore , in this study , we aimed to determine the kap of a sample of iranian college students and to compare it with their american and chinese counterparts . ( chancellor 's professor , applied health science department , indiana university , usa ) in 2002.9 for the purpose of accomplishing this survey , the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment , after consulting with dr . torabi , the necessary modifications were taken into account . to be assured about the accuracy and effectiveness of the translation , we used the back translation method , i.e. this english edition has been used for gathering information from american and chinese college students . title of the noted study is consumption of alcohol , tobacco and other drugs in children and adults of indiana . the attitude scale of using tobacco which is used in this survey was first established by awaisu et al.10 the translated questionnaire contained 55 questions , nine of them being demographic ones . because of the increase in water - pipe smoking by young people , we added four related items to the practice questions , hence the number of practice questions increased to 21 , and the number of total questions increased to 59 . we included this group to be comparable to studies conducted in the us and china . the outcomes of the studies were analyzed statistically by version 13.0 spss for windows ( spss inc . for reliability testing of knowledge and attitude scales , cronbach 's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . so , in the first stage , regarding the knowledge of students for each answer sheet , the total scores of knowledge questions could be a number between 0 and 11 . the perception of each student involving in smoking cigarettes was achieved to add the whole numbers in the questionnaire . number five showed the hatred of students towards smoking cigarettes and decreased from five toward one . in order to determine the differences in terms of gender , the analysis of variance ( anova ) was used . ( chancellor 's professor , applied health science department , indiana university , usa ) in 2002.9 for the purpose of accomplishing this survey , the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment , after consulting with dr . torabi , the necessary modifications were taken into account . to be assured about the accuracy and effectiveness of the translation , we used the back translation method , i.e. this english edition has been used for gathering information from american and chinese college students . according to the obtained information , the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . title of the noted study is consumption of alcohol , tobacco and other drugs in children and adults of indiana . the attitude scale of using tobacco which is used in this survey was first established by awaisu et al.10 the translated questionnaire contained 55 questions , nine of them being demographic ones . because of the increase in water - pipe smoking by young people , we added four related items to the practice questions , hence the number of practice questions increased to 21 , and the number of total questions increased to 59 . practice questions contained 5 questions about smoking cigarette , cigar , chewing tobacco , pipe , and waterpipe . for all of these noted tobacco products , the duration of consumption in the previous month and year , as well as quitting smoking were considered . we included this group to be comparable to studies conducted in the us and china . the outcomes of the studies were analyzed statistically by version 13.0 spss for windows ( spss inc . for reliability testing of knowledge and attitude scales , cronbach 's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . so , in the first stage , regarding the knowledge of students for each answer sheet , the total scores of knowledge questions could be a number between 0 and 11 . the perception of each student involving in smoking cigarettes was achieved to add the whole numbers in the questionnaire . ( chancellor 's professor , applied health science department , indiana university , usa ) in 2002.9 for the purpose of accomplishing this survey , the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment , after consulting with dr . torabi , the necessary modifications were taken into account . to be assured about the accuracy and effectiveness of the translation , we used the back translation method , i.e. this english edition has been used for gathering information from american and chinese college students . according to the obtained information , the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . title of the noted study is consumption of alcohol , tobacco and other drugs in children and adults of indiana . the attitude scale of using tobacco which is used in this survey was first established by awaisu et al.10 the translated questionnaire contained 55 questions , nine of them being demographic ones . because of the increase in water - pipe smoking by young people , we added four related items to the practice questions , hence the number of practice questions increased to 21 , and the number of total questions increased to 59 . practice questions contained 5 questions about smoking cigarette , cigar , chewing tobacco , pipe , and waterpipe . for all of these noted tobacco products , the duration of consumption in the previous month and year , as well as quitting smoking were considered . we included this group to be comparable to studies conducted in the us and china . the outcomes of the studies were analyzed statistically by version 13.0 spss for windows ( spss inc . for reliability testing of knowledge and attitude scales , cronbach 's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . so , in the first stage , regarding the knowledge of students for each answer sheet , the total scores of knowledge questions could be a number between 0 and 11 . the perception of each student involving in smoking cigarettes was achieved to add the whole numbers in the questionnaire . number five showed the hatred of students towards smoking cigarettes and decreased from five toward one . in iran , from 1200 responses , 958 were complete to be used in the survey , which 456 of them were the responses given by male students ( 48.5% ) and 51.5% by female students . in china , out of 1534 students participating in the survey , 39.7% were women and 60.3% were men . in the united states , from 597 participants , 62.1% were women and 37.9% were men . significant differences existed in the structure of the place of the residence in three countries . in iran , 73.7% of students were from urban areas , whereas 47% of american college students were from suburban areas . among chinese students , regarding to marital status , the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . in china , 83.5% were of good health and 14.5% were among the average . when the iranian students were asked about their daily stress , 42.2% claimed that they have not much stress , 38.6% of them told they were fairly stressful and 14.5% of them were of the average , 10.4% had no stress and 8.8% of them suffered from sever stress . internal consistency of the knowledge and the attitude of the three groups students were compared , and the results are summarized in table 1 . internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students ( both men and women ) . the anova test showed that there were no significant differences between the sexes and the average knowledge . but the average meaningful knowledge , however , existed between iran and the other countries . the average number of knowledge regarding to tobacco use among students ( both men and women ) the average number of attitude regarding to tobacco use among students ( both men and women ) in iran in both men and women this attitude is more than the americans and chinese . anova test showed that among the sexes , the average of attitude could clearly be observed ( in men , anova = 141.30 , p < 0.001 and anova = 284.14 , p < 0.001 for women ) . there is also a meaningful scale of attitude existed between the countries studied in this survey ( anova = 394.86 , p < 0.001 ) . the survey carried out in china shows that among the cigarette addicts , most of the smokers had started trying cigarettes even before the age of thirteen . the age of first cigarette smoking among students ( both men and women ) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students , american and iranian students put much more effort to stop smoking . in iran , from 1200 responses , 958 were complete to be used in the survey , which 456 of them were the responses given by male students ( 48.5% ) and 51.5% by female students . in china , out of 1534 students participating in the survey , 39.7% were women and 60.3% were men . in the united states , from 597 participants , 62.1% were women and 37.9% were men . significant differences existed in the structure of the place of the residence in three countries . in iran , 73.7% of students were from urban areas , whereas 47% of american college students were from suburban areas . among chinese students , regarding to marital status , the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . in china , when the iranian students were asked about their daily stress , 42.2% claimed that they have not much stress , 38.6% of them told they were fairly stressful and 14.5% of them were of the average , 10.4% had no stress and 8.8% of them suffered from sever stress . internal consistency of the knowledge and the attitude of the three groups students were compared , and the results are summarized in table 1 . in all three surveys it was shown that almost a lower level of knowledge exists ( about 70% ) , but concerning the attitude , in all three countries satisfactory attitude score was obtained . similarly , table 3 shows the average number of attitude regarding to tobacco use among students ( both men and women ) . but the average meaningful knowledge , however , existed between iran and the other countries . the average number of knowledge regarding to tobacco use among students ( both men and women ) the average number of attitude regarding to tobacco use among students ( both men and women ) in iran in both men and women this attitude is more than the americans and chinese . anova test showed that among the sexes , the average of attitude could clearly be observed ( in men , anova = 141.30 , p < 0.001 and anova = 284.14 , p < 0.001 for women ) . there is also a meaningful scale of attitude existed between the countries studied in this survey ( anova = 394.86 , p < 0.001 ) . the survey carried out in china shows that among the cigarette addicts , most of the smokers had started trying cigarettes even before the age of thirteen . this percentage ( 37.6% ) is much higher than in iran ( 20.1% ) and the us ( 17.9% ) . in the us , this statistics seems to be more expressive ( chi - square = 157.7 , p 0.001 ) . the age of first cigarette smoking among students ( both men and women ) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students , american and iranian students put much more effort to stop smoking . in iran , from 1200 responses , 958 were complete to be used in the survey , which 456 of them were the responses given by male students ( 48.5% ) and 51.5% by female students . in china , out of 1534 students participating in the survey , 39.7% were women and 60.3% were men . in the united states , from 597 participants , 62.1% were women and 37.9% were men . significant differences existed in the structure of the place of the residence in three countries . in iran , 73.7% of students were from urban areas , whereas 47% of american college students were from suburban areas . in china , when the iranian students were asked about their daily stress , 42.2% claimed that they have not much stress , 38.6% of them told they were fairly stressful and 14.5% of them were of the average , 10.4% had no stress and 8.8% of them suffered from sever stress . internal consistency of the knowledge and the attitude of the three groups students were compared , and the results are summarized in table 1 . internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students ( both men and women ) . similarly , table 3 shows the average number of attitude regarding to tobacco use among students ( both men and women ) . the anova test showed that there were no significant differences between the sexes and the average knowledge . but the average meaningful knowledge , however , existed between iran and the other countries . the average number of knowledge regarding to tobacco use among students ( both men and women ) the average number of attitude regarding to tobacco use among students ( both men and women ) in iran in both men and women this attitude is more than the americans and chinese . anova test showed that among the sexes , the average of attitude could clearly be observed ( in men , anova = 141.30 , p < 0.001 and anova = 284.14 , p < 0.001 for women ) . there is also a meaningful scale of attitude existed between the countries studied in this survey ( anova = 394.86 , p < 0.001 ) . it was shown that 70% of smoking participants started smoking between the ages of 19 - 22 . the survey carried out in china shows that among the cigarette addicts , most of the smokers had started trying cigarettes even before the age of thirteen . this percentage ( 37.6% ) is much higher than in iran ( 20.1% ) and the us ( 17.9% ) . in iran , this percentage increases along higher age groups . the age of first cigarette smoking among students ( both men and women ) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students , american and iranian students put much more effort to stop smoking . this survey has discussed the tendency toward smoking addiction among the students and their perception of this behavior and its outcome among the students in iran , the usa , and china . in this study students the knowledge and perception of each sex in one country and also between the three countries was carefully compared and evaluated . one of the problems the researchers are faced in this kind of survey is that students of the countries are of different cultures.1112 so , we can not expect that the students studied in these three countries give a relatively appropriate result . in the case of representation , participants should be carefully chosen and the results must be studied with almost care . since the questionnaires used in the three countries are the same and they are originally written in english , when being translated to persian and chinese the cultural differences might cause some problems . the young people studied in iran were all medical students , so there is a great possibility that their understanding of case and also their recognition of the harmful effects of smoking cigarettes might be much better than the students of other majors . this questionnaire has already been used in america and china.78 in case of iran , we were trying to translate it in to persian in a way that somehow agrees with our culture , but the problem still exists . the original questionnaire is in american english prepared by an iranian professor ( dr torabi , chancellor 's professor , applied health science department , indiana university , usa).10 we have already coordinated with him concerning the translation of the questionnaire . one of the changes that we made in persian version was adding waterpipe smoking to the text . this survey showed that male students in all stages of age and in all three countries smoke more than women.13 among the smoking products , cigarette is of high consumption . overall , beside cigarettes , cigars and pipes have had more consumers during the last year in all countries.78 fortunately , chewing tobacco ( being normal in western countries ) is still unknown in our country.14 a very few people use this kind of tobacco . studying carefully the true responses of students to the questions concerning the knowledge , reveals that women are little more concerned . the survey also shows that american students are more knowledgeable compared to iranian and chinese students . regarding the answers given by the students about their attitudes and perception , the survey showed that women 's concern about smoking was more than men that is very important and meaningful according to the statistical analysis . the perception of iranian men and women was more than the same group of people in the united states of america and china.1112 the total percentage of perception among iranian college students is 87.85% , comparing to 65.60% in america and 64.4% in china . regarding the cultural condition in china women are much more comfortable toward smoking in both outdoors and indoors.15 any way in all countries the consumption of cigarettes is important and statistically meaningful ( p < 0.001 ) . the current survey was planned to provide more information about the motives that provide certain conditions to start smoking . regarding the proved dangers of smoking and the results gained in the survey , important and crucial steps have been taken by the policy makers of the country to educate effectively and show the disastrous outcome of smoking all over the country . also , it is important to mention the fact that these kinds of surveys can submit valuable information to the health trainers , health policy makers , and ministry of health . therefore , it would enable them to think of a way which results in prevention or decrease of smoking in the country .

smoking is the main preventable cause of early and untimely death and disabilities , which leads to approximately four million deaths every year.1 although smoking causes more mortality than other factors such as acquired immune deficiency disease ( aids ) , alcohol drinking , driving accidents , murder , suicide , and fire , but still about one third of the adult population use tobacco products worldwide.2 according to the centers for disease control and prevention ( cdc ) in the united states of america , 80% of adults , started smoking before the age of eighteen and about three thousand people are smoking regularly at this early stage of life.3 if no considerations are taken in order to stop this harmful trend , in the near future more than five million children will die because of future tendency to smoke . intervention and prevention in the age of less than 20 years old has a great importance.4 smoking in this age , causes more morbidity because of accumulation of narcotics over times , also intrigues other friends to smoke cigarettes . tobacco use at this stage of age provides the chance of being attracted to other drugs and still it is difficult for them to give up smoking though they have realized the dangers of cigarette smoking.5 therefore , planning preventive actions is an important step to be considered . national surveys in iran showed that the mean age of the first attempt to smoke was in the teenage group.6 similar situation exists in various countries such as the united states ( usa),7 and china.8 in spite of marked differences in the socio - cultural background of various countries , the knowledge , attitude and practice ( kap ) of youths about smoking is important about their smoking behavior . therefore , in this study , we aimed to determine the kap of a sample of iranian college students and to compare it with their american and chinese counterparts . ( chancellor 's professor , applied health science department , indiana university , usa ) in 2002.9 for the purpose of accomplishing this survey , the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment , after consulting with dr . to be assured about the accuracy and effectiveness of the translation , we used the back translation method , i.e. according to the obtained information , the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . title of the noted study is consumption of alcohol , tobacco and other drugs in children and adults of indiana . because of the increase in water - pipe smoking by young people , we added four related items to the practice questions , hence the number of practice questions increased to 21 , and the number of total questions increased to 59 . questions about awareness had multiple choices with only one right answer , but the applied scale in questions about attitude was based on likert system . for all of these noted tobacco products , the duration of consumption in the previous month and year , as well as quitting smoking were considered . we included this group to be comparable to studies conducted in the us and china . the male - to female ratio and the proportion of junior to senior students were similar to the above mentioned surveys . the outcomes of the studies were analyzed statistically by version 13.0 spss for windows ( spss inc . for reliability testing of knowledge and attitude scales , cronbach 's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . so , in the first stage , regarding the knowledge of students for each answer sheet , the total scores of knowledge questions could be a number between 0 and 11 . number five showed the hatred of students towards smoking cigarettes and decreased from five toward one . to change the number to the percentage scale , the outcome would reach between 20 and 100 percent . in order to determine the differences in terms of gender , the analysis of variance ( anova ) was used . ( chancellor 's professor , applied health science department , indiana university , usa ) in 2002.9 for the purpose of accomplishing this survey , the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment , after consulting with dr . to be assured about the accuracy and effectiveness of the translation , we used the back translation method , i.e. the persian - translated questionnaire was again translated in to english , and unsuitable translations were corrected . according to the obtained information , the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . title of the noted study is consumption of alcohol , tobacco and other drugs in children and adults of indiana . because of the increase in water - pipe smoking by young people , we added four related items to the practice questions , hence the number of practice questions increased to 21 , and the number of total questions increased to 59 . questions about awareness had multiple choices with only one right answer , but the applied scale in questions about attitude was based on likert system . for all of these noted tobacco products , the duration of consumption in the previous month and year , as well as quitting smoking were considered . we included this group to be comparable to studies conducted in the us and china . the male - to female ratio and the proportion of junior to senior students were similar to the above mentioned surveys . the outcomes of the studies were analyzed statistically by version 13.0 spss for windows ( spss inc . for reliability testing of knowledge and attitude scales , cronbach 's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . so , in the first stage , regarding the knowledge of students for each answer sheet , the total scores of knowledge questions could be a number between 0 and 11 . to change the number to the percentage scale , the outcome would reach between 20 and 100 percent . in order to determine the differences in terms of gender , the analysis of variance ( anova ) was used . ( chancellor 's professor , applied health science department , indiana university , usa ) in 2002.9 for the purpose of accomplishing this survey , the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment , after consulting with dr . to be assured about the accuracy and effectiveness of the translation , we used the back translation method , i.e. according to the obtained information , the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . title of the noted study is consumption of alcohol , tobacco and other drugs in children and adults of indiana . because of the increase in water - pipe smoking by young people , we added four related items to the practice questions , hence the number of practice questions increased to 21 , and the number of total questions increased to 59 . questions about awareness had multiple choices with only one right answer , but the applied scale in questions about attitude was based on likert system . for all of these noted tobacco products , the duration of consumption in the previous month and year , as well as quitting smoking were considered . we included this group to be comparable to studies conducted in the us and china . the male - to female ratio and the proportion of junior to senior students were similar to the above mentioned surveys . for reliability testing of knowledge and attitude scales , cronbach 's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . so , in the first stage , regarding the knowledge of students for each answer sheet , the total scores of knowledge questions could be a number between 0 and 11 . to change the number to the percentage scale , the outcome would reach between 20 and 100 percent . in order to determine the differences in terms of gender , the analysis of variance ( anova ) this knowledge test is not a norm reliability test and its reliability was acceptable since it relied strongly on the criteria . in iran , from 1200 responses , 958 were complete to be used in the survey , which 456 of them were the responses given by male students ( 48.5% ) and 51.5% by female students . in china , out of 1534 students participating in the survey , 39.7% were women and 60.3% were men . in the united states , from 597 participants , 62.1% were women and 37.9% were men . overall , these three surveys comprised a total number of 3089 students consisting of 47.5% women and 52.5% men . significant differences existed in the structure of the place of the residence in three countries . among chinese students , regarding to marital status , the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . when the iranian participants were asked about their health status , 92.7% of them responded that they were in a good health condition ; 5.9% were on average and 1.4% were of poor health conditions . in china , 83.5% were of good health and 14.5% were among the average . when the iranian students were asked about their daily stress , 42.2% claimed that they have not much stress , 38.6% of them told they were fairly stressful and 14.5% of them were of the average , 10.4% had no stress and 8.8% of them suffered from sever stress . internal consistency of the knowledge and the attitude of the three groups students were compared , and the results are summarized in table 1 . in all three surveys it was shown that almost a lower level of knowledge exists ( about 70% ) , but concerning the attitude , in all three countries satisfactory attitude score was obtained . internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students ( both men and women ) . similarly , table 3 shows the average number of attitude regarding to tobacco use among students ( both men and women ) . but the average meaningful knowledge , however , existed between iran and the other countries . the average number of knowledge regarding to tobacco use among students ( both men and women ) the average number of attitude regarding to tobacco use among students ( both men and women ) in iran in both men and women this attitude is more than the americans and chinese . anova test showed that among the sexes , the average of attitude could clearly be observed ( in men , anova = 141.30 , p < 0.001 and anova = 284.14 , p < 0.001 for women ) . there is also a meaningful scale of attitude existed between the countries studied in this survey ( anova = 394.86 , p < 0.001 ) . the survey carried out in china shows that among the cigarette addicts , most of the smokers had started trying cigarettes even before the age of thirteen . this percentage ( 37.6% ) is much higher than in iran ( 20.1% ) and the us ( 17.9% ) . the age of first cigarette smoking among students ( both men and women ) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students , american and iranian students put much more effort to stop smoking . in iran , from 1200 responses , 958 were complete to be used in the survey , which 456 of them were the responses given by male students ( 48.5% ) and 51.5% by female students . in china , out of 1534 students participating in the survey , 39.7% were women and 60.3% were men . overall , these three surveys comprised a total number of 3089 students consisting of 47.5% women and 52.5% men . significant differences existed in the structure of the place of the residence in three countries . among chinese students , regarding to marital status , the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . when the iranian participants were asked about their health status , 92.7% of them responded that they were in a good health condition ; 5.9% were on average and 1.4% were of poor health conditions . in china , when the iranian students were asked about their daily stress , 42.2% claimed that they have not much stress , 38.6% of them told they were fairly stressful and 14.5% of them were of the average , 10.4% had no stress and 8.8% of them suffered from sever stress . internal consistency of the knowledge and the attitude of the three groups students were compared , and the results are summarized in table 1 . in all three surveys it was shown that almost a lower level of knowledge exists ( about 70% ) , but concerning the attitude , in all three countries satisfactory attitude score was obtained . internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students ( both men and women ) . similarly , table 3 shows the average number of attitude regarding to tobacco use among students ( both men and women ) . the average number of knowledge regarding to tobacco use among students ( both men and women ) the average number of attitude regarding to tobacco use among students ( both men and women ) in iran in both men and women this attitude is more than the americans and chinese . anova test showed that among the sexes , the average of attitude could clearly be observed ( in men , anova = 141.30 , p < 0.001 and anova = 284.14 , p < 0.001 for women ) . there is also a meaningful scale of attitude existed between the countries studied in this survey ( anova = 394.86 , p < 0.001 ) . the survey carried out in china shows that among the cigarette addicts , most of the smokers had started trying cigarettes even before the age of thirteen . the age of first cigarette smoking among students ( both men and women ) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students , american and iranian students put much more effort to stop smoking . in iran , from 1200 responses , 958 were complete to be used in the survey , which 456 of them were the responses given by male students ( 48.5% ) and 51.5% by female students . in china , out of 1534 students participating in the survey , 39.7% were women and 60.3% were men . significant differences existed in the structure of the place of the residence in three countries . among chinese students , regarding to marital status , the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . when the iranian participants were asked about their health status , 92.7% of them responded that they were in a good health condition ; 5.9% were on average and 1.4% were of poor health conditions . in china , when the iranian students were asked about their daily stress , 42.2% claimed that they have not much stress , 38.6% of them told they were fairly stressful and 14.5% of them were of the average , 10.4% had no stress and 8.8% of them suffered from sever stress . internal consistency of the knowledge and the attitude of the three groups students were compared , and the results are summarized in table 1 . in all three surveys it was shown that almost a lower level of knowledge exists ( about 70% ) , but concerning the attitude , in all three countries satisfactory attitude score was obtained . internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students ( both men and women ) . similarly , table 3 shows the average number of attitude regarding to tobacco use among students ( both men and women ) . the average number of knowledge regarding to tobacco use among students ( both men and women ) the average number of attitude regarding to tobacco use among students ( both men and women ) in iran in both men and women this attitude is more than the americans and chinese . anova test showed that among the sexes , the average of attitude could clearly be observed ( in men , anova = 141.30 , p < 0.001 and anova = 284.14 , p < 0.001 for women ) . there is also a meaningful scale of attitude existed between the countries studied in this survey ( anova = 394.86 , p < 0.001 ) . the survey carried out in china shows that among the cigarette addicts , most of the smokers had started trying cigarettes even before the age of thirteen . the age of first cigarette smoking among students ( both men and women ) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students , american and iranian students put much more effort to stop smoking . this survey has discussed the tendency toward smoking addiction among the students and their perception of this behavior and its outcome among the students in iran , the usa , and china . in this study students the knowledge and perception of each sex in one country and also between the three countries was carefully compared and evaluated . one of the problems the researchers are faced in this kind of survey is that students of the countries are of different cultures.1112 so , we can not expect that the students studied in these three countries give a relatively appropriate result . the young people studied in iran were all medical students , so there is a great possibility that their understanding of case and also their recognition of the harmful effects of smoking cigarettes might be much better than the students of other majors . this questionnaire has already been used in america and china.78 in case of iran , we were trying to translate it in to persian in a way that somehow agrees with our culture , but the problem still exists . this survey showed that male students in all stages of age and in all three countries smoke more than women.13 among the smoking products , cigarette is of high consumption . overall , beside cigarettes , cigars and pipes have had more consumers during the last year in all countries.78 fortunately , chewing tobacco ( being normal in western countries ) is still unknown in our country.14 a very few people use this kind of tobacco . studying carefully the true responses of students to the questions concerning the knowledge , reveals that women are little more concerned . these varieties might be the result of differences between the system of education and the policies that americans have been following.12 however , there are no noticeable differences regarding the meaningful perception in all three countries . regarding the answers given by the students about their attitudes and perception , the survey showed that women 's concern about smoking was more than men that is very important and meaningful according to the statistical analysis . the perception of iranian men and women was more than the same group of people in the united states of america and china.1112 the total percentage of perception among iranian college students is 87.85% , comparing to 65.60% in america and 64.4% in china . iranian women 's attitude toward smoking depends on the cultural condition and the government policies.14 that is why women in iran smoke much less ( especially in public ) compared to women in china . regarding the cultural condition in china women are much more comfortable toward smoking in both outdoors and indoors.15 any way in all countries the consumption of cigarettes is important and statistically meaningful ( p < 0.001 ) . regarding the proved dangers of smoking and the results gained in the survey , important and crucial steps have been taken by the policy makers of the country to educate effectively and show the disastrous outcome of smoking all over the country .

the model used here is of a population consisting of three types of female and male individuals denoted by t0 , t1 and t2 . the population reproduces in discrete and non - overlapping generations and is assumed to be stationary in senses which are to be described . this paper concerns mainly the first result of bernstein which was a proof that mendel s coefficients of heredity ( to be defined in section 2 ) were a necessary outcome if one assumed stationarity from the first generation of offspring , plus random mating , and that the union of two of the types , t0 and t2 always produced offspring of type t1 . s.n . bernstein ( see seneta ( 2001 ) for a biographical sketch ) published condensed versions of his work in two short papers ( bernstein 1923a , b ) . the second considers the case when there are an arbitrary number of types of individuals and another ( non - mendelian ) form of heredity . sheynin ( 2004 ) provides translations into english of bernstein s publications in russian which concern the 1923 papers . the first of these ( bernstein , 1922 ) gives background to the 1923 papers and refers to the studies on evolution by charles darwin ( 1859 ) , experimental findings by mendel ( 1866 , 1965 ) and biometrical work of francis galton and karl pearson . the second paper ( bernstein , 1924 ) gives the solution in detail together with other models not considered here . a similar english version of the second paper ( bernstein , 1942 ) was provided by emma lehner of the university of california , berkeley . lehner states that the original paper of 1924 appeared in annales scientifiques de lukraine , vol . 1 ( 1924 ) . lehner s version contains only the first half of the bernstein ( 1924 ) paper . ballonoff ( 1974 ) reproduced bernstein s two papers of 1923 ( in the original french ) . later ( bernstein , 1976 ) he published an english translation of large parts of the 1924 paper which omitted some details of the proofs of theorems ( in this he transliterated the author s name as bernshtein , but in the references of this paper , in the interests of consistency , it is given as this was accompanied by a short introduction in the same periodical ( ballonoff , 1976 ) . the following quotation from ballonoff ( 1976 ) suggests that ballonoff completely missed the main point of the bernstein ( 1923a ) paper , despite the point being spelled out in the title of the paper . there are two major results of this paper , one already an accepted part of genetics theory , the other yet un - explored ! the accepted result is the < < hardy - weinberg > > law for the equilibrium of mendelian genetic systems . the other result to which ballonoff refers is outside the scope of this paper . as noted above , the main point of bernstein s research was to establish mendel s first law . lyubich has written a number of papers , including lyubich ( 1971 - translated into english by j. wiegold ) and lyubich ( 1973 ) , which were inspired by bernstein s work , culminating in a monograph ( lyubich , 1992 ) . by the 1920 s the importance of the short paper by hardy ( 1908 ) was recognised by geneticists , although the almost simultaneous independent presentation of the same idea , almost in passing by weinberg ( 1908 ) in a more ambitious paper on the inheritance of twinning in humans , had been overlooked till the 1940 s an interesting feature of bernstein s presentation is that he derives mendel s first law through hardy s formula without any reference to hardy . the only references in bernstein ( 1924 ) were to his own paper of 1922 and to the monograph of johannsen , given as 3 . bernstein s ( 1922 ) paper is fairly general relating mainly to his desire to enhance biology by providing mathematical underpinnings to it . in his english version of bernstein ( 1924 ) sheynin s references include : johannsen , wm ( 1926 ) elemente der exakten erblichkeitslehre . clearly there is a discrepancy in publication years between bernstein ( 1924 ) and johannsen ( 1926 ) . the english - language version of bernstein ( 1924 ) given by sheynin contains the following : here , i shall not dwell on those fundamental considerations which convinced me in that , when constructing a mathematical theory of evolution , we ought to base it upon laws of heredity obeying the principle of stationarity . i only note that the mendelian law , which determines the inheritance of most of the precisely studied elementary traits , satisfies this principle ( johannsen 1926 , p. 488 ) . the so - called mendelian law concerns three classes of individuals , two of them being pure races and the third one , a race of hybrids always born when two individuals belonging to pure races are crossing . we shall assume that bernstein had to hand , in essence , johannsen ( 1913 ) , that is the 2 edition [ 2 auflage ] . johannsen was familiar with the hardy - weinberg distribution of genotype frequencies and gives it in his book ( johannsen , 1913 , p.486 ) . our next section gives basic concepts and notation as well as a summary of the hardy - weinberg formulae . this is followed by bernstein s ( 1924 ) question and a reference to his proof without the details . we then demonstrate that stasis ( that is , constancy of type proportions over all generations , including the zeroth parental proportions ) is possible under non - random mating . a model of assortative mating which is a special case of non - random mating , together with a numerical example , is next . the final section suggests that johannsen supplied all the background which bernstein needed and makes some general comments . the concepts and methods which we use have been described disparagingly by ernst mayr as beanbag genetics . individuals are completely characterised by type of which there are three , namely t0 , t1 , t2 . although they are not emphasized here , genes g and determine type according to the following correspondence : gg t0 ; g t1 ; gg~t2 . the effectively infinite population is reproduced sexually in discrete and non - overlapping generations . taking account of gender there are 9 mating combinations as defined by the matrix : ( 1)[t0t0t0t1t0t2t1t0t1t1t1t2t2t0t2t1t2t2 ] a child which is one of the three types arises from each coupling and the aggregate of children form the new generation , later to become parents , in their turn . from his study of peas for each of the above couplings it gives the set of probabilities as to type of child . if we take the outputs , by column , of the 9 couplings , mendel s first law can be expressed in the form of the following matrix : ( 2)m=[11/201/21/4000001/211/21/21/211/2000001/41/201/21]where the order of columns is by mating couples : ( 3)[t0t0,t0t1,t0t2,t1t0,t1t1,t1t2,t2t0,t2t1,t2t2]and the rows are the proportions of offspring in the respective categories t0(gg ) , t1(g ) , t2(gg ) . the important point to realise about m is that it expresses probabilities relating to outcomes of single coupling events . to use the law to make predictions about populations requires a further step , namely a specification of the rule of formation of the aggregate of couples . this can be expressed in the form of a matrix of proportions of couples in the order given before . following bernstein ( 1924 ) , we use the symbols , , to denote the frequencies ( proportions ) of the respective types t0 , t1 , t2 , the same in each gender . then random mating is given by a vector whose form is , in accordance with the form ( 3 ) : ( 4)u={2,,,,2,,,,2 } then the composition of the population , that is the proportions of the three types , following one round of random mating under mendel s law is given by ( 5)t=(mu)={(+2)2,2(+2)(+2),(+2)2 } the vector t is known as the hardy - weinberg distribution after the originators weinberg ( 1908 ) and hardy ( 1908 ) . this can be expressed more compactly after introducing the following definitions : ( 6)g=+2 ; g=+2 the quantities g and , which are referred to as gene frequencies , give the proportions of genes of the two kinds in the population since they weight the type frequencies according to the number of genes , g or , in each type . then ( 7)t={g2,2gg,g2 } the important property of the hardy - weinberg formulation follows if we now subject the new array of type frequencies to another round of random mating . the coupling frequencies are given by the vector form : ( 8)(u*)={(g2)2,2(g2)gg,g2g2,2ggg2,(2gg)2,2ggg2,g2,g2,2g2,gg,(g2)2 } since g + = 1 , the type frequencies among offspring are : ( 9)(mu*)={g2,2gg,g2}that is , identical to t. so one round of random mating produces a set of type frequencies which are maintained indefinitely under random mating , often referred to as hardy - weinberg equilibrium . notice that the mating vector u * has the property that frequency of matings of type t1 t1 is 4 times that of either t0 t2 or t2 t0 , since the corresponding array is { g , 2g , }. we express this in self - evident notation to be formalized shortly as : f11=4f02 hardy ( 1908 ) pointed out that if the initial parental frequencies satisfy the relation ( 10)2=4then equilibrium frequencies will have been attained under random mating , even after the first round of mating . ( 10 ) , hardy s equation , points out reasons why the hardy - weinberg law is so important . firstly , a population can be characterised by a single gene frequency rather than a set of genotype frequencies , so it provides economy of description . but much more important is the stability behaviour , that is : there is no tendency for genetic variability to dissipate . mayo ( 2008 ) concludes the summary of his review article on the hardy - weinberg law with bernstein ( 1924 ) repeated the steps above leading to the derivation of the hardy - weinberg proportions . he then turned the problem around by asking , if the population maintains constant proportions of types , namely { , , } , after an initial round of mating , and assuming mating is random , whether this implies that the heredity coefficients are necessarily those given by m. he began with a general form denoted by m where m is given by [ pqrstuvwx1pp1qq1rr1ss1tt1uu1vv1ww1xxpqrstuvwx ] however this form is too general to work towards constancy of type proportions from the first generation and bernstein modified it to ( 11)m=[pq0qtu0ux1pp1qq11qq1tt1uu11uu1xxpq0qtu0ux ] that is he assumed that mating t0 t2 and the reciprocal mating t2 t0 always produce offspring of type t1 and the other reciprocal matings produce offspring identically . after one round of random mating the population structure is ( 12)t=mu bernstein ( 1924 ) then submits this population to a further round of random mating and he proves that m is the only set of heredity coefficients which reproduce t , this being the hardy - weinberg array ( 13)t=(mu)={(+2)2,2(+2)(+2),(+2)2}that is m m. thus ( 14)p=1 , p=0 , q=1/2 , q=0,t=1/4 , t=1/4 , u=0 , u=1/2,x=0 , x=1 the argument by which he shows this is most readily seen in bernstein ( 1942 ) . in this section we show that with two crucial assumptions , it is possible to derive mendel s heredity coefficients under non - random mating , and the assumption of stasis : that the proportions of types remain constant from an initial parental generation . secondly assume that the matings vector is any probability distribution of the form : ( 15)u={a , b , c , d,4c , d , c , d , e } thus the mating matrix ( 15 ) has the property that the frequency of mating t1 t1 is 4 times that of both t0 t2 and t2 t0 . this last echoes the condition ( 10 ) of hardy . applying m and gives offspring of types t0 and t2 : ( 16)t0=pa+2qb+4tc+2ud+xe ( 17)t2=pa+2qb+4tc+2ud+xe however the parental distribution is ( 18){a+b+c , b+4c+d , c+d+e } equating the coefficients of { a , b , c , d , e } in eqs . ( 16 ) and ( 17 ) with their coefficients in the parental distribution ( 18 ) yields ( 14 ) . the step of equating coefficients of { a , b , c , d , e } in eqs . ( 16 ) and ( 17 ) with their coefficients in the parental distribution ( 18 ) can be justified rigorously by a few steps of matrix theory from the fact that , for example , eq . ( 16 ) is to hold for fixed { p , q , t , u , x } but for all { a , b , c , d , e } for which eq . we now denote the proportions of couples in the various mating combinations by fij , ( i = 0 , 1 , 2 ; j = 0 , 1 , 2 ) , indicating the proportion of the mating ti tj . stark ( 1976a , b ) gives a mating system which can maintain a given departure from the hardy - weinberg form of genotype frequencies . in these citations mendel s first law was assumed to hold [ that is , the heredity coefficients are given by eq . the proportions of mating couples are given by ( 19)fij = fifj(1+mdidj / v)where fi is the genotypic frequency of ti , i = 0 , 1 , 2 . = f1f2 + 4f0f2+f0f1 ( 22)d0=(2f2+f1 ) , d1=f0f2 , d2=2f0+f1 the terms d0 , d1 and d2 are phenotypic values attributed to the respective types . the second is intermediate in value between the other two and separated from each by 1 . v is the variance of these values with respect to the distribution of type frequencies and m is the correlation between mates with respect to their phenotypic values which are standardised by dividing by their standard deviation . ( 19 ) satisfies f11 = 4f02 , since f02 = f0(f1)f2/v and f11 is 4 times that expression , so that , by the general result of the previous section on mating matrices of type ( 15 ) , genotype frequencies are maintained , verifying the earlier results of stark ( 1976a , 1976b ) . we give a numerical example of such a mating matrix which serves to illustrate various features of the model ( and which allows for considerable flexibility as demonstrated here by incorporating a t0 ) : ( 23)c=1625=[00.04160.06240.04160.24960.22080.06240.22080.1008]c is symmetric with elements adding to 1 and the middle element is 4 times the upper right hand ( and lower left hand ) element . summing rows and columns of c gives the distribution of types in females and males , namely { 13/125 , 64/125 , 48/125}. the important property of c is that , if mendelian heredity coefficients are applied to it , the offspring distribution is identical to the parental distribution . the offspring then become the next parents and so can continue the population in unchanged form . example ( 23 ) conforms to ( 19 ) with { f0 , f1 , f2 } = { 13/125 , 64/125 , 48/125 } , m = 1/4 , d0 = 32/25 , d1 = 7/25 , d2 = 18/25 , v = 256/625 . by contrast , if random mating is applied to frequencies { 13/125 , 64/125 , 48/125 } , the distribution of offspring is { 81/625 , 288/625 , 256/625 } and following a further round of random mating , this offspring distribution is reproduced . this is stationarity in the bernsteinian sense , imitating the conclusion of the hardy - weinberg law . note that , considering these proportions as genotypic frequencies , gene frequencies remain constant at g = 9/25 and = 16/25 under both random and assortative mating , that is stasis in this gene frequency , rather than genotype frequency , sense is achieved under both systems of mating . since the theme of bernstein ( 1923a ) is the main focus of this paper , it is appropriate to discuss further bernstein s achievements and limitations , in respect of that paper . we believe that this paper encapsulates bernstein s most notable contribution to genetics although he wrote much more which he considered important . in his celebrated and contentious paper fisher ( 1936 ) writes in 1930 , as a result of a study of the development of darwin s ideas , i pointed out that the modern genetical system , apart from such special features as dominance and linkage , could have been inferred by any abstract thinker in the middle of the nineteenth century if he were led to postulate that inheritance was particulate , that the germinal material was structural , and that the contributions of the two parents were equivalent . bernstein ( 1923a ) demonstrates that he not only anticipated fisher s assertion but showed how it could be realised mathematically . fisher believed that mendel had a clear view of his own first law during the course of his experiment . in relation to ( 2008 , p. ix ) write it is our contention that this controversy should end . while fisher ( 1936 ) continues to fascinate , bernstein ( 1923a ) and his other writings have been largely ignored . his two papers of 1923 and bernstein ( 1942 ) are listed in felsenstein ( 1981 ) and there is a pointer to holgate ( 1975 ) against the key word stationarity. there is no reference to bernstein in wright ( 1969 ) but many to fisher and j. b. s. haldane , as well as to other notable figures in the development of population genetics . the discipline of genetics in the soviet union experienced two periods of turbulence and isolation . the first was because of the first world war and the revolution and the second was when lysenko was given wide powers of control over teaching and research in biology and agriculture . between these two periods , for reasons related to communist ideology and politics , there was a resurgence of lamarckism . in relation to the former period , dobzhansky ( 1980 ) noted that , after a period of about seven years , acquaintance with the experimental work of the morgan school , and with the findings of other geneticists in europe and in the united states , became possible only in about 1921 . stark and seneta ( 2011 ) kolmogorov took a stand against lysenkoism in 1940 and how the publication of a new edition of bernstein s monograph on probability was stopped because it included material on mendelism . both weismann and johannsen were included in the list of people who were the targets of lysenko s vitriol . johannsen ( 1913 ) was the only source cited by bernstein ( 1924 ) , apart from one paper of his own . when introducing mendel s first law johannsen ( 1913 , p. 486 ) gave a table , here reproduced as table 1 , which perhaps motivated bernstein as to how to approach his proof . in effect the table is a derivation of the hardy - weinberg formula through functional iteration . it can be said that johannsen supplied all the information that bernstein needed for his task . dunn ( 1965 ) and grant ( 1975 ) and others pay tribute to johannsen s important role in the development of genetics . but bernstein had to work in isolation from some important developments in mathematical genetics , such as fisher ( 1918 ) , haldane ( 1919 ) and wright ( 1921 ) . in johannsen ( 1913 ) there are many references to galton , and pearson , as well as to darwin , but fewer to morgan . johannsen , although a great supporter of statistical method in biology , was one of the leaders of a group of biologists opposing the views of galton , karl pearson and weldon on inheritance ( guttorp and lindgren , 2009 ) . the conflict with pearson started with johannsen s ( 1903 ) paper ( see peters , 1959 for a version in english ) . however yule ( 1904 ) , also a member of the english biometric school , came to johannsen s defence , calling his results one of the most important contributions to genetics . it may be of some relevance that bernstein was an admirer in the times of which we speak of karl pearson s grammar of science , in a russian translation of the second edition of 1900 ( read , 1982 , p. 24 ) . johannsen ( 1913 , p. 711 ) cites weinberg ( 1908 , 1909a , b ) . hill ( 1984 , p. 12 ) notes that weinberg s paper of 1908 was a small part of his work in genetics . it is this paper , with hardy ( 1908 ) , which is nowadays associated with the discovery of the hardy - weinberg law . hardy s ( 1908 ) paper is cited on p. 704 of johannsen ( 1913 ) , although this page is not mentioned in the index , where only hardy , 486 occurs . there is no indication in bernstein ( 1924 ) that he had used the weinberg(1908 ) reference and no mention of hardy . dunn ( 1965 , p. 94 ) makes a remark which is important in the context of bernstein s place in genetics in the soviet union between the two world wars and beyond . he writes likewise , johannsen , in effect , cleared the air of the fear that acquired characteristics might , after all , be inherited . weismann s arguments had in the long run been less effective than johannsen s simple experimental demonstrations , at least with those biologists who wanted to advance the study of heredity . johannsen s conclusion that acquired modifications were not inherited was backed up a little later by castle and phillips ( 1909 ) , using an argument of quite a different kind . while bernstein s model is ingenious and is supported by intricate calculations which are best displayed in bernstein ( 1942 ) , he starts out by , in a sense , violating his main postulate , namely stationarity : he requires random mating in the first ( and subsequent ) generations which involves a change from the initial population , unless it is already in hardy - weinberg form . it is the assumption that stationarity is required only from the first generation onwards which causes considerable mathematical difficulty in arriving at mendel s coefficients of heredity . while this assumption does imitate the hardy - weinberg law in its formulation , it does not seem realistic in considering stability of population proportions starting from an arbitrary time point . we have , in contrast , shown relatively simply that mendel s set of heredity coefficients follow necessarily from ( 11 ) , and ( 15 ) which embodies the property f11 = 4f02 . this last equation reflects the situation which exists in the hardy - weinberg approach after one generation of mating . before 1900 there were several kinds of study and much speculation aimed at elucidating the phenomenon of inheritance . essentially , mendel s was a study of individual hereditary events which could be collated to form the basis of a theory . the basic one of these was the hardy - weinberg model ( weinberg ( 1908 ) , hardy ( 1908 ) ) . this is idealistic in that the original formulation and current usage was and is based on the assumption of random mating . it has been shown by stark ( 1980 , 2005 , 2006a , b , 2007 ) and li ( 1988 ) that random mating with mendelian coefficients is a sufficient , but not a necessary , condition for hardy - weinberg equilibrium . failure to appreciate this for example wikipedia ( 2011 ) states violations from the hardy - weinberg assumptions can cause deviations from the expectations ... random mating ... violations ... will not have hardy - weinberg proportions . the novelty of bernstein s approach is that it starts from a view of a population and posits several conditions to derive a model of inheritance for single reproductive events .

around 1923 the soon - to - be famous soviet mathematician and probabilist sergei n. bernstein started to construct an axiomatic foundation of a theory of heredity . he began from the premise of stationarity ( constancy of type proportions ) from the first generation of offspring . this led him to derive the mendelian coefficients of heredity . it appears that he had no direct influence on the subsequent development of population genetics . a basic assumption of bernstein was that parents coupled randomly to produce offspring . this paper shows that a simple model of non - random mating , which nevertheless embodies a feature of the hardy - weinberg law , can produce mendelian coefficients of heredity while maintaining the population distribution . how w. johannsen s monograph influenced bernstein is discussed .

Introduction Basic Concepts and Notation and Hardy-Weinberg Equilibrium Bernsteins Question Stasis Under Non-Random Mating Stasis Under Assortative and Random Mating Discussion

the model used here is of a population consisting of three types of female and male individuals denoted by t0 , t1 and t2 . the population reproduces in discrete and non - overlapping generations and is assumed to be stationary in senses which are to be described . this paper concerns mainly the first result of bernstein which was a proof that mendel s coefficients of heredity ( to be defined in section 2 ) were a necessary outcome if one assumed stationarity from the first generation of offspring , plus random mating , and that the union of two of the types , t0 and t2 always produced offspring of type t1 . the second considers the case when there are an arbitrary number of types of individuals and another ( non - mendelian ) form of heredity . sheynin ( 2004 ) provides translations into english of bernstein s publications in russian which concern the 1923 papers . the first of these ( bernstein , 1922 ) gives background to the 1923 papers and refers to the studies on evolution by charles darwin ( 1859 ) , experimental findings by mendel ( 1866 , 1965 ) and biometrical work of francis galton and karl pearson . a similar english version of the second paper ( bernstein , 1942 ) was provided by emma lehner of the university of california , berkeley . lehner s version contains only the first half of the bernstein ( 1924 ) paper . later ( bernstein , 1976 ) he published an english translation of large parts of the 1924 paper which omitted some details of the proofs of theorems ( in this he transliterated the author s name as bernshtein , but in the references of this paper , in the interests of consistency , it is given as this was accompanied by a short introduction in the same periodical ( ballonoff , 1976 ) . the following quotation from ballonoff ( 1976 ) suggests that ballonoff completely missed the main point of the bernstein ( 1923a ) paper , despite the point being spelled out in the title of the paper . there are two major results of this paper , one already an accepted part of genetics theory , the other yet un - explored ! the accepted result is the < < hardy - weinberg > > law for the equilibrium of mendelian genetic systems . lyubich has written a number of papers , including lyubich ( 1971 - translated into english by j. wiegold ) and lyubich ( 1973 ) , which were inspired by bernstein s work , culminating in a monograph ( lyubich , 1992 ) . by the 1920 s the importance of the short paper by hardy ( 1908 ) was recognised by geneticists , although the almost simultaneous independent presentation of the same idea , almost in passing by weinberg ( 1908 ) in a more ambitious paper on the inheritance of twinning in humans , had been overlooked till the 1940 s an interesting feature of bernstein s presentation is that he derives mendel s first law through hardy s formula without any reference to hardy . in his english version of bernstein ( 1924 ) sheynin s references include : johannsen , wm ( 1926 ) elemente der exakten erblichkeitslehre . the english - language version of bernstein ( 1924 ) given by sheynin contains the following : here , i shall not dwell on those fundamental considerations which convinced me in that , when constructing a mathematical theory of evolution , we ought to base it upon laws of heredity obeying the principle of stationarity . i only note that the mendelian law , which determines the inheritance of most of the precisely studied elementary traits , satisfies this principle ( johannsen 1926 , p. 488 ) . johannsen was familiar with the hardy - weinberg distribution of genotype frequencies and gives it in his book ( johannsen , 1913 , p.486 ) . our next section gives basic concepts and notation as well as a summary of the hardy - weinberg formulae . we then demonstrate that stasis ( that is , constancy of type proportions over all generations , including the zeroth parental proportions ) is possible under non - random mating . a model of assortative mating which is a special case of non - random mating , together with a numerical example , is next . taking account of gender there are 9 mating combinations as defined by the matrix : ( 1)[t0t0t0t1t0t2t1t0t1t1t1t2t2t0t2t1t2t2 ] a child which is one of the three types arises from each coupling and the aggregate of children form the new generation , later to become parents , in their turn . if we take the outputs , by column , of the 9 couplings , mendel s first law can be expressed in the form of the following matrix : ( 2)m=[11/201/21/4000001/211/21/21/211/2000001/41/201/21]where the order of columns is by mating couples : ( 3)[t0t0,t0t1,t0t2,t1t0,t1t1,t1t2,t2t0,t2t1,t2t2]and the rows are the proportions of offspring in the respective categories t0(gg ) , t1(g ) , t2(gg ) . to use the law to make predictions about populations requires a further step , namely a specification of the rule of formation of the aggregate of couples . this can be expressed in the form of a matrix of proportions of couples in the order given before . following bernstein ( 1924 ) , we use the symbols , , to denote the frequencies ( proportions ) of the respective types t0 , t1 , t2 , the same in each gender . then random mating is given by a vector whose form is , in accordance with the form ( 3 ) : ( 4)u={2,,,,2,,,,2 } then the composition of the population , that is the proportions of the three types , following one round of random mating under mendel s law is given by ( 5)t=(mu)={(+2)2,2(+2)(+2),(+2)2 } the vector t is known as the hardy - weinberg distribution after the originators weinberg ( 1908 ) and hardy ( 1908 ) . this can be expressed more compactly after introducing the following definitions : ( 6)g=+2 ; g=+2 the quantities g and , which are referred to as gene frequencies , give the proportions of genes of the two kinds in the population since they weight the type frequencies according to the number of genes , g or , in each type . then ( 7)t={g2,2gg,g2 } the important property of the hardy - weinberg formulation follows if we now subject the new array of type frequencies to another round of random mating . the coupling frequencies are given by the vector form : ( 8)(u*)={(g2)2,2(g2)gg,g2g2,2ggg2,(2gg)2,2ggg2,g2,g2,2g2,gg,(g2)2 } since g + = 1 , the type frequencies among offspring are : ( 9)(mu*)={g2,2gg,g2}that is , identical to t. so one round of random mating produces a set of type frequencies which are maintained indefinitely under random mating , often referred to as hardy - weinberg equilibrium . notice that the mating vector u * has the property that frequency of matings of type t1 t1 is 4 times that of either t0 t2 or t2 t0 , since the corresponding array is { g , 2g , }. we express this in self - evident notation to be formalized shortly as : f11=4f02 hardy ( 1908 ) pointed out that if the initial parental frequencies satisfy the relation ( 10)2=4then equilibrium frequencies will have been attained under random mating , even after the first round of mating . ( 10 ) , hardy s equation , points out reasons why the hardy - weinberg law is so important . mayo ( 2008 ) concludes the summary of his review article on the hardy - weinberg law with bernstein ( 1924 ) repeated the steps above leading to the derivation of the hardy - weinberg proportions . he then turned the problem around by asking , if the population maintains constant proportions of types , namely { , , } , after an initial round of mating , and assuming mating is random , whether this implies that the heredity coefficients are necessarily those given by m. he began with a general form denoted by m where m is given by [ pqrstuvwx1pp1qq1rr1ss1tt1uu1vv1ww1xxpqrstuvwx ] however this form is too general to work towards constancy of type proportions from the first generation and bernstein modified it to ( 11)m=[pq0qtu0ux1pp1qq11qq1tt1uu11uu1xxpq0qtu0ux ] that is he assumed that mating t0 t2 and the reciprocal mating t2 t0 always produce offspring of type t1 and the other reciprocal matings produce offspring identically . after one round of random mating the population structure is ( 12)t=mu bernstein ( 1924 ) then submits this population to a further round of random mating and he proves that m is the only set of heredity coefficients which reproduce t , this being the hardy - weinberg array ( 13)t=(mu)={(+2)2,2(+2)(+2),(+2)2}that is m m. thus ( 14)p=1 , p=0 , q=1/2 , q=0,t=1/4 , t=1/4 , u=0 , u=1/2,x=0 , x=1 the argument by which he shows this is most readily seen in bernstein ( 1942 ) . in this section we show that with two crucial assumptions , it is possible to derive mendel s heredity coefficients under non - random mating , and the assumption of stasis : that the proportions of types remain constant from an initial parental generation . secondly assume that the matings vector is any probability distribution of the form : ( 15)u={a , b , c , d,4c , d , c , d , e } thus the mating matrix ( 15 ) has the property that the frequency of mating t1 t1 is 4 times that of both t0 t2 and t2 t0 . applying m and gives offspring of types t0 and t2 : ( 16)t0=pa+2qb+4tc+2ud+xe ( 17)t2=pa+2qb+4tc+2ud+xe however the parental distribution is ( 18){a+b+c , b+4c+d , c+d+e } equating the coefficients of { a , b , c , d , e } in eqs . the step of equating coefficients of { a , b , c , d , e } in eqs . we now denote the proportions of couples in the various mating combinations by fij , ( i = 0 , 1 , 2 ; j = 0 , 1 , 2 ) , indicating the proportion of the mating ti tj . stark ( 1976a , b ) gives a mating system which can maintain a given departure from the hardy - weinberg form of genotype frequencies . v is the variance of these values with respect to the distribution of type frequencies and m is the correlation between mates with respect to their phenotypic values which are standardised by dividing by their standard deviation . ( 19 ) satisfies f11 = 4f02 , since f02 = f0(f1)f2/v and f11 is 4 times that expression , so that , by the general result of the previous section on mating matrices of type ( 15 ) , genotype frequencies are maintained , verifying the earlier results of stark ( 1976a , 1976b ) . we give a numerical example of such a mating matrix which serves to illustrate various features of the model ( and which allows for considerable flexibility as demonstrated here by incorporating a t0 ) : ( 23)c=1625=[00.04160.06240.04160.24960.22080.06240.22080.1008]c is symmetric with elements adding to 1 and the middle element is 4 times the upper right hand ( and lower left hand ) element . the offspring then become the next parents and so can continue the population in unchanged form . by contrast , if random mating is applied to frequencies { 13/125 , 64/125 , 48/125 } , the distribution of offspring is { 81/625 , 288/625 , 256/625 } and following a further round of random mating , this offspring distribution is reproduced . this is stationarity in the bernsteinian sense , imitating the conclusion of the hardy - weinberg law . note that , considering these proportions as genotypic frequencies , gene frequencies remain constant at g = 9/25 and = 16/25 under both random and assortative mating , that is stasis in this gene frequency , rather than genotype frequency , sense is achieved under both systems of mating . since the theme of bernstein ( 1923a ) is the main focus of this paper , it is appropriate to discuss further bernstein s achievements and limitations , in respect of that paper . we believe that this paper encapsulates bernstein s most notable contribution to genetics although he wrote much more which he considered important . in his celebrated and contentious paper fisher ( 1936 ) writes in 1930 , as a result of a study of the development of darwin s ideas , i pointed out that the modern genetical system , apart from such special features as dominance and linkage , could have been inferred by any abstract thinker in the middle of the nineteenth century if he were led to postulate that inheritance was particulate , that the germinal material was structural , and that the contributions of the two parents were equivalent . bernstein ( 1923a ) demonstrates that he not only anticipated fisher s assertion but showed how it could be realised mathematically . there is no reference to bernstein in wright ( 1969 ) but many to fisher and j. b. s. haldane , as well as to other notable figures in the development of population genetics . the first was because of the first world war and the revolution and the second was when lysenko was given wide powers of control over teaching and research in biology and agriculture . in relation to the former period , dobzhansky ( 1980 ) noted that , after a period of about seven years , acquaintance with the experimental work of the morgan school , and with the findings of other geneticists in europe and in the united states , became possible only in about 1921 . stark and seneta ( 2011 ) kolmogorov took a stand against lysenkoism in 1940 and how the publication of a new edition of bernstein s monograph on probability was stopped because it included material on mendelism . in effect the table is a derivation of the hardy - weinberg formula through functional iteration . dunn ( 1965 ) and grant ( 1975 ) and others pay tribute to johannsen s important role in the development of genetics . johannsen , although a great supporter of statistical method in biology , was one of the leaders of a group of biologists opposing the views of galton , karl pearson and weldon on inheritance ( guttorp and lindgren , 2009 ) . the conflict with pearson started with johannsen s ( 1903 ) paper ( see peters , 1959 for a version in english ) . however yule ( 1904 ) , also a member of the english biometric school , came to johannsen s defence , calling his results one of the most important contributions to genetics . it may be of some relevance that bernstein was an admirer in the times of which we speak of karl pearson s grammar of science , in a russian translation of the second edition of 1900 ( read , 1982 , p. 24 ) . it is this paper , with hardy ( 1908 ) , which is nowadays associated with the discovery of the hardy - weinberg law . there is no indication in bernstein ( 1924 ) that he had used the weinberg(1908 ) reference and no mention of hardy . dunn ( 1965 , p. 94 ) makes a remark which is important in the context of bernstein s place in genetics in the soviet union between the two world wars and beyond . he writes likewise , johannsen , in effect , cleared the air of the fear that acquired characteristics might , after all , be inherited . weismann s arguments had in the long run been less effective than johannsen s simple experimental demonstrations , at least with those biologists who wanted to advance the study of heredity . johannsen s conclusion that acquired modifications were not inherited was backed up a little later by castle and phillips ( 1909 ) , using an argument of quite a different kind . while bernstein s model is ingenious and is supported by intricate calculations which are best displayed in bernstein ( 1942 ) , he starts out by , in a sense , violating his main postulate , namely stationarity : he requires random mating in the first ( and subsequent ) generations which involves a change from the initial population , unless it is already in hardy - weinberg form . it is the assumption that stationarity is required only from the first generation onwards which causes considerable mathematical difficulty in arriving at mendel s coefficients of heredity . while this assumption does imitate the hardy - weinberg law in its formulation , it does not seem realistic in considering stability of population proportions starting from an arbitrary time point . this last equation reflects the situation which exists in the hardy - weinberg approach after one generation of mating . essentially , mendel s was a study of individual hereditary events which could be collated to form the basis of a theory . the basic one of these was the hardy - weinberg model ( weinberg ( 1908 ) , hardy ( 1908 ) ) . this is idealistic in that the original formulation and current usage was and is based on the assumption of random mating . it has been shown by stark ( 1980 , 2005 , 2006a , b , 2007 ) and li ( 1988 ) that random mating with mendelian coefficients is a sufficient , but not a necessary , condition for hardy - weinberg equilibrium . failure to appreciate this for example wikipedia ( 2011 ) states violations from the hardy - weinberg assumptions can cause deviations from the expectations ... random mating ... violations ... will not have hardy - weinberg proportions . the novelty of bernstein s approach is that it starts from a view of a population and posits several conditions to derive a model of inheritance for single reproductive events .

the model used here is of a population consisting of three types of female and male individuals denoted by t0 , t1 and t2 . this paper concerns mainly the first result of bernstein which was a proof that mendel s coefficients of heredity ( to be defined in section 2 ) were a necessary outcome if one assumed stationarity from the first generation of offspring , plus random mating , and that the union of two of the types , t0 and t2 always produced offspring of type t1 . the second considers the case when there are an arbitrary number of types of individuals and another ( non - mendelian ) form of heredity . the first of these ( bernstein , 1922 ) gives background to the 1923 papers and refers to the studies on evolution by charles darwin ( 1859 ) , experimental findings by mendel ( 1866 , 1965 ) and biometrical work of francis galton and karl pearson . the second paper ( bernstein , 1924 ) gives the solution in detail together with other models not considered here . a similar english version of the second paper ( bernstein , 1942 ) was provided by emma lehner of the university of california , berkeley . lehner states that the original paper of 1924 appeared in annales scientifiques de lukraine , vol . lehner s version contains only the first half of the bernstein ( 1924 ) paper . later ( bernstein , 1976 ) he published an english translation of large parts of the 1924 paper which omitted some details of the proofs of theorems ( in this he transliterated the author s name as bernshtein , but in the references of this paper , in the interests of consistency , it is given as this was accompanied by a short introduction in the same periodical ( ballonoff , 1976 ) . the following quotation from ballonoff ( 1976 ) suggests that ballonoff completely missed the main point of the bernstein ( 1923a ) paper , despite the point being spelled out in the title of the paper . there are two major results of this paper , one already an accepted part of genetics theory , the other yet un - explored ! the accepted result is the < < hardy - weinberg > > law for the equilibrium of mendelian genetic systems . lyubich has written a number of papers , including lyubich ( 1971 - translated into english by j. wiegold ) and lyubich ( 1973 ) , which were inspired by bernstein s work , culminating in a monograph ( lyubich , 1992 ) . by the 1920 s the importance of the short paper by hardy ( 1908 ) was recognised by geneticists , although the almost simultaneous independent presentation of the same idea , almost in passing by weinberg ( 1908 ) in a more ambitious paper on the inheritance of twinning in humans , had been overlooked till the 1940 s an interesting feature of bernstein s presentation is that he derives mendel s first law through hardy s formula without any reference to hardy . the only references in bernstein ( 1924 ) were to his own paper of 1922 and to the monograph of johannsen , given as 3 . the english - language version of bernstein ( 1924 ) given by sheynin contains the following : here , i shall not dwell on those fundamental considerations which convinced me in that , when constructing a mathematical theory of evolution , we ought to base it upon laws of heredity obeying the principle of stationarity . i only note that the mendelian law , which determines the inheritance of most of the precisely studied elementary traits , satisfies this principle ( johannsen 1926 , p. 488 ) . the so - called mendelian law concerns three classes of individuals , two of them being pure races and the third one , a race of hybrids always born when two individuals belonging to pure races are crossing . we shall assume that bernstein had to hand , in essence , johannsen ( 1913 ) , that is the 2 edition [ 2 auflage ] . johannsen was familiar with the hardy - weinberg distribution of genotype frequencies and gives it in his book ( johannsen , 1913 , p.486 ) . our next section gives basic concepts and notation as well as a summary of the hardy - weinberg formulae . we then demonstrate that stasis ( that is , constancy of type proportions over all generations , including the zeroth parental proportions ) is possible under non - random mating . a model of assortative mating which is a special case of non - random mating , together with a numerical example , is next . individuals are completely characterised by type of which there are three , namely t0 , t1 , t2 . although they are not emphasized here , genes g and determine type according to the following correspondence : gg t0 ; g t1 ; gg~t2 . taking account of gender there are 9 mating combinations as defined by the matrix : ( 1)[t0t0t0t1t0t2t1t0t1t1t1t2t2t0t2t1t2t2 ] a child which is one of the three types arises from each coupling and the aggregate of children form the new generation , later to become parents , in their turn . from his study of peas for each of the above couplings it gives the set of probabilities as to type of child . if we take the outputs , by column , of the 9 couplings , mendel s first law can be expressed in the form of the following matrix : ( 2)m=[11/201/21/4000001/211/21/21/211/2000001/41/201/21]where the order of columns is by mating couples : ( 3)[t0t0,t0t1,t0t2,t1t0,t1t1,t1t2,t2t0,t2t1,t2t2]and the rows are the proportions of offspring in the respective categories t0(gg ) , t1(g ) , t2(gg ) . the important point to realise about m is that it expresses probabilities relating to outcomes of single coupling events . to use the law to make predictions about populations requires a further step , namely a specification of the rule of formation of the aggregate of couples . this can be expressed in the form of a matrix of proportions of couples in the order given before . following bernstein ( 1924 ) , we use the symbols , , to denote the frequencies ( proportions ) of the respective types t0 , t1 , t2 , the same in each gender . then random mating is given by a vector whose form is , in accordance with the form ( 3 ) : ( 4)u={2,,,,2,,,,2 } then the composition of the population , that is the proportions of the three types , following one round of random mating under mendel s law is given by ( 5)t=(mu)={(+2)2,2(+2)(+2),(+2)2 } the vector t is known as the hardy - weinberg distribution after the originators weinberg ( 1908 ) and hardy ( 1908 ) . this can be expressed more compactly after introducing the following definitions : ( 6)g=+2 ; g=+2 the quantities g and , which are referred to as gene frequencies , give the proportions of genes of the two kinds in the population since they weight the type frequencies according to the number of genes , g or , in each type . then ( 7)t={g2,2gg,g2 } the important property of the hardy - weinberg formulation follows if we now subject the new array of type frequencies to another round of random mating . the coupling frequencies are given by the vector form : ( 8)(u*)={(g2)2,2(g2)gg,g2g2,2ggg2,(2gg)2,2ggg2,g2,g2,2g2,gg,(g2)2 } since g + = 1 , the type frequencies among offspring are : ( 9)(mu*)={g2,2gg,g2}that is , identical to t. so one round of random mating produces a set of type frequencies which are maintained indefinitely under random mating , often referred to as hardy - weinberg equilibrium . notice that the mating vector u * has the property that frequency of matings of type t1 t1 is 4 times that of either t0 t2 or t2 t0 , since the corresponding array is { g , 2g , }. we express this in self - evident notation to be formalized shortly as : f11=4f02 hardy ( 1908 ) pointed out that if the initial parental frequencies satisfy the relation ( 10)2=4then equilibrium frequencies will have been attained under random mating , even after the first round of mating . firstly , a population can be characterised by a single gene frequency rather than a set of genotype frequencies , so it provides economy of description . mayo ( 2008 ) concludes the summary of his review article on the hardy - weinberg law with bernstein ( 1924 ) repeated the steps above leading to the derivation of the hardy - weinberg proportions . he then turned the problem around by asking , if the population maintains constant proportions of types , namely { , , } , after an initial round of mating , and assuming mating is random , whether this implies that the heredity coefficients are necessarily those given by m. he began with a general form denoted by m where m is given by [ pqrstuvwx1pp1qq1rr1ss1tt1uu1vv1ww1xxpqrstuvwx ] however this form is too general to work towards constancy of type proportions from the first generation and bernstein modified it to ( 11)m=[pq0qtu0ux1pp1qq11qq1tt1uu11uu1xxpq0qtu0ux ] that is he assumed that mating t0 t2 and the reciprocal mating t2 t0 always produce offspring of type t1 and the other reciprocal matings produce offspring identically . after one round of random mating the population structure is ( 12)t=mu bernstein ( 1924 ) then submits this population to a further round of random mating and he proves that m is the only set of heredity coefficients which reproduce t , this being the hardy - weinberg array ( 13)t=(mu)={(+2)2,2(+2)(+2),(+2)2}that is m m. thus ( 14)p=1 , p=0 , q=1/2 , q=0,t=1/4 , t=1/4 , u=0 , u=1/2,x=0 , x=1 the argument by which he shows this is most readily seen in bernstein ( 1942 ) . in this section we show that with two crucial assumptions , it is possible to derive mendel s heredity coefficients under non - random mating , and the assumption of stasis : that the proportions of types remain constant from an initial parental generation . secondly assume that the matings vector is any probability distribution of the form : ( 15)u={a , b , c , d,4c , d , c , d , e } thus the mating matrix ( 15 ) has the property that the frequency of mating t1 t1 is 4 times that of both t0 t2 and t2 t0 . applying m and gives offspring of types t0 and t2 : ( 16)t0=pa+2qb+4tc+2ud+xe ( 17)t2=pa+2qb+4tc+2ud+xe however the parental distribution is ( 18){a+b+c , b+4c+d , c+d+e } equating the coefficients of { a , b , c , d , e } in eqs . ( 16 ) and ( 17 ) with their coefficients in the parental distribution ( 18 ) can be justified rigorously by a few steps of matrix theory from the fact that , for example , eq . we now denote the proportions of couples in the various mating combinations by fij , ( i = 0 , 1 , 2 ; j = 0 , 1 , 2 ) , indicating the proportion of the mating ti tj . = f1f2 + 4f0f2+f0f1 ( 22)d0=(2f2+f1 ) , d1=f0f2 , d2=2f0+f1 the terms d0 , d1 and d2 are phenotypic values attributed to the respective types . v is the variance of these values with respect to the distribution of type frequencies and m is the correlation between mates with respect to their phenotypic values which are standardised by dividing by their standard deviation . ( 19 ) satisfies f11 = 4f02 , since f02 = f0(f1)f2/v and f11 is 4 times that expression , so that , by the general result of the previous section on mating matrices of type ( 15 ) , genotype frequencies are maintained , verifying the earlier results of stark ( 1976a , 1976b ) . we give a numerical example of such a mating matrix which serves to illustrate various features of the model ( and which allows for considerable flexibility as demonstrated here by incorporating a t0 ) : ( 23)c=1625=[00.04160.06240.04160.24960.22080.06240.22080.1008]c is symmetric with elements adding to 1 and the middle element is 4 times the upper right hand ( and lower left hand ) element . summing rows and columns of c gives the distribution of types in females and males , namely { 13/125 , 64/125 , 48/125}. the important property of c is that , if mendelian heredity coefficients are applied to it , the offspring distribution is identical to the parental distribution . example ( 23 ) conforms to ( 19 ) with { f0 , f1 , f2 } = { 13/125 , 64/125 , 48/125 } , m = 1/4 , d0 = 32/25 , d1 = 7/25 , d2 = 18/25 , v = 256/625 . by contrast , if random mating is applied to frequencies { 13/125 , 64/125 , 48/125 } , the distribution of offspring is { 81/625 , 288/625 , 256/625 } and following a further round of random mating , this offspring distribution is reproduced . this is stationarity in the bernsteinian sense , imitating the conclusion of the hardy - weinberg law . note that , considering these proportions as genotypic frequencies , gene frequencies remain constant at g = 9/25 and = 16/25 under both random and assortative mating , that is stasis in this gene frequency , rather than genotype frequency , sense is achieved under both systems of mating . since the theme of bernstein ( 1923a ) is the main focus of this paper , it is appropriate to discuss further bernstein s achievements and limitations , in respect of that paper . in his celebrated and contentious paper fisher ( 1936 ) writes in 1930 , as a result of a study of the development of darwin s ideas , i pointed out that the modern genetical system , apart from such special features as dominance and linkage , could have been inferred by any abstract thinker in the middle of the nineteenth century if he were led to postulate that inheritance was particulate , that the germinal material was structural , and that the contributions of the two parents were equivalent . the first was because of the first world war and the revolution and the second was when lysenko was given wide powers of control over teaching and research in biology and agriculture . in relation to the former period , dobzhansky ( 1980 ) noted that , after a period of about seven years , acquaintance with the experimental work of the morgan school , and with the findings of other geneticists in europe and in the united states , became possible only in about 1921 . stark and seneta ( 2011 ) kolmogorov took a stand against lysenkoism in 1940 and how the publication of a new edition of bernstein s monograph on probability was stopped because it included material on mendelism . both weismann and johannsen were included in the list of people who were the targets of lysenko s vitriol . in effect the table is a derivation of the hardy - weinberg formula through functional iteration . johannsen , although a great supporter of statistical method in biology , was one of the leaders of a group of biologists opposing the views of galton , karl pearson and weldon on inheritance ( guttorp and lindgren , 2009 ) . however yule ( 1904 ) , also a member of the english biometric school , came to johannsen s defence , calling his results one of the most important contributions to genetics . it may be of some relevance that bernstein was an admirer in the times of which we speak of karl pearson s grammar of science , in a russian translation of the second edition of 1900 ( read , 1982 , p. 24 ) . hill ( 1984 , p. 12 ) notes that weinberg s paper of 1908 was a small part of his work in genetics . it is this paper , with hardy ( 1908 ) , which is nowadays associated with the discovery of the hardy - weinberg law . hardy s ( 1908 ) paper is cited on p. 704 of johannsen ( 1913 ) , although this page is not mentioned in the index , where only hardy , 486 occurs . dunn ( 1965 , p. 94 ) makes a remark which is important in the context of bernstein s place in genetics in the soviet union between the two world wars and beyond . weismann s arguments had in the long run been less effective than johannsen s simple experimental demonstrations , at least with those biologists who wanted to advance the study of heredity . while bernstein s model is ingenious and is supported by intricate calculations which are best displayed in bernstein ( 1942 ) , he starts out by , in a sense , violating his main postulate , namely stationarity : he requires random mating in the first ( and subsequent ) generations which involves a change from the initial population , unless it is already in hardy - weinberg form . while this assumption does imitate the hardy - weinberg law in its formulation , it does not seem realistic in considering stability of population proportions starting from an arbitrary time point . we have , in contrast , shown relatively simply that mendel s set of heredity coefficients follow necessarily from ( 11 ) , and ( 15 ) which embodies the property f11 = 4f02 . the basic one of these was the hardy - weinberg model ( weinberg ( 1908 ) , hardy ( 1908 ) ) .

targeted genetic engineering is driving progress in new areas of basic biological research , biotechnology , and gene therapy . site - specific endonucleases , including zinc - finger nucleases ( zfns ) , meganucleases , tal effector nucleases ( talens ) , and crispr / cas systems , have dramatically enhanced the speed and efficiency with which researchers can introduce targeted genetic modifications into cells and organisms . although site - specific nucleases are versatile and promote a broad range of genetic alterations , they rely on cellular dna repair mechanisms , such as error - prone non - homologous end joining ( nhej ) and homology - directed repair ( hdr ) , to induce custom alterations . the lack of availability of dna repair pathways within certain cell types , however , may reduce the utility of this technology . in particular , poor induction of hdr via nuclease - induced dna double - strand breaks ( dsbs ) or nicks has been shown to be a major limiting factor for achieving high rates of site - specific integration . additionally , off - target dsbs induced by site - specific nucleases are difficult to comprehensively characterize in the absence of an accompanying donor template and can be potentially toxic to cells and organisms . thus , there remains a continued need for the development of new tools capable of achieving highly precise targeted modifications with minimal toxicity . site - specific recombinases ( ssrs , e.g. , cre , flp , phic31 , and bxb1 ) are a potentially powerful alternative to site - specific nucleases for targeted genetic engineering . ssrs are highly specialized enzymes that promote high - fidelity dna rearrangements ( e.g. , integration , excision , or inversion ) between defined segments of dna . the strict target specificities demonstrated by many ssr systems , however , have limited their adoption in disciplines that require tools with highly flexible recognition capabilities . to overcome this , various protein engineering strategies have been used to alter ssr target specificity . while these approaches permit the design of ssr variants with new properties , they nevertheless typically lead to the emergence of relaxed specificity , an undesirable byproduct that limits the utility and safety of these enzymes . hybrid recombinases composed of catalytic domains derived from the resolvase / invertase family of serine recombinases ( e.g. , gin , hin , tn3 , and ) fused to custom - designed cys2-his2 zinc - finger or tal effector dna - binding domains represent a unique solution to this problem ( figure 1a ) . in particular , zinc - finger recombinases ( zfrs ) are a flexible class of chimeric proteins capable of introducing targeted modifications into mammalian cells . zfrs promote site - specific recombination between dna targets that consist of two inverted zinc - finger binding sites flanking a central 20-bp core sequence recognized by the recombinase catalytic domain ( figure 1a ) . unlike targeted nucleases and conventional ssr systems , zfr specificity is the cooperative product of modular site - specific dna recognition and sequence - dependent catalysis . as such , new zfrs with diverse targeting capabilities can be generated in a , we have demonstrated that tailored zfr variants can be rapidly assembled from a library of pre - selected gin recombinase catalytic domains ( referred to here as gin , , , , , and ) and zinc - finger modules . this customization strategy allows for the design of synthetic recombinases that have the capacity to recognize a broad range of user - defined dna targets and direct site - specific integration into endogenous genomic loci . structure of a zinc - finger recombinase ( zfr ) and its dimer interface . ( a ) top : zfr monomers ( left , red ; right , yellow ) consist of an activated serine recombinase catalytic domain fused to a cys2-his2 zinc - finger dna - binding domain . zinc - finger proteins ( zfps ) can be replaced with tal effector dna - binding domains . model shows the structure of an engineered zfr , generated from the crystal structures of the resolvase and aart zinc - finger protein ( pdb ids : 1gdt and 2i13 , respectively ) . abbreviations are as follows : n indicates a , t , c , or g ; r indicates g or a ; y indicates c or t ; w indicates a or t ; zfbs indicates zinc - finger binding site . ( b ) interactions at the gin recombinase dimer interface from two vantage points . left e helix colored red , right e helix colored yellow . key residues shown as sticks ( pdb i d : 3uj3 ) . despite their ability to specifically recognize dna segments up to 56 bp in length , we previously observed that custom - designed zfrs targeted integration with low specificity . one factor contributing to this is that the protein protein interactions that govern zfr - mediated recombination are not selective for the heterodimeric zfr species . indeed , expression of any two zfr monomers required for genomic targeting inevitably leads to the formation of two side - product zfr homodimers capable of inducing off - target genomic modifications . similar phenomena have been observed with zfns and talens , which rely on dimerization of the foki cleavage domain for dsb induction . to overcome this , numerous studies have utilized dimer interface redesign to generate enzyme variants with improved specificity . most notably , structure - guided and selection - based approaches have yielded obligate heterodimeric variants of the foki cleavage domain capable of enhancing zfn and talen cleavage specificity . in addition , mutagenesis of the cre recombinase dimer interface has led to the isolation of mutants with improved recombination specificity , presumably due to destabilization of cre dimer binding cooperativity . here , we employ rational design and directed evolution to redesign the serine recombinase dimerization interface and generate a new hybrid recombinase architecture that prevents formation of side - product recombinase homodimers by > 500-fold . we show that zfrs composed of these enhanced catalytic domains demonstrate substantially improved targeting specificity and efficiency , and enable the site - specific delivery of therapeutic genes into the human genome with low toxicity . in order to redesign the gin recombinase dimer interface and engineer zfrs that preferentially heterodimerize , we sought to identify the specific amino acid residues that govern recombinase dimerization . to accomplish this , we examined the crystal structures of the resolvase dimer and the activated , tetrameric configurations of the gin and sin recombinases . we focused our search on residues within the e helix a key mediator of dimer dimer interactions between recombinase subunits and identified five residues that likely associate with one another via hydrophobic interactions : met 100 , phe 103 , phe 104 , val 107 , and met 108 ( all numbers hereafter according to the gin recombinase ; figure 1b ) . in accordance with these structural observations , previous studies had revealed that introduction of cys residues at positions 100 , 103 , and 107 leads to spontaneous cross - linking of two recombinase monomers . on the basis of these data , we hypothesized that substitution of these residues with complementary charged amino acids would ( i ) disfavor association of homodimers by charge and steric repulsion and ( ii ) promote heterodimer formation through favorable electrostatic contacts . to evaluate the effect that charged substitutions within the dimer interface have on recombination , we created a collection of recombinase mutants based on the gin and catalytic domains that contained either arg ( gin ) or asp ( gin ) substitutions at positions 100 , 103 , and 107 and evaluated their ability to recombine dna as homodimers ( i.e. , arg - arg or asp - asp ) and heterodimers ( i.e. , arg - asp ) . we determined recombination by split gene reassembly , a previously described method that links recombinase activity to antibiotic resistance . notably , gin homodimers that contained substitutions at position 103 showed a > 10,000-fold reduction in recombination compared to wild - type enzymes ( figure s1 ) . the corresponding heterodimer pair demonstrated a > 100-fold increase in recombination compared to the inactivated recombinase mutants ; however , no heterodimeric pair recombined dna as efficiently as the wild - type enzyme ( figure s1 ) . furthermore , combining charge substitutions did not enhance the efficiency of heterodimer - mediated recombination , presumably due to suboptimal protein protein interactions between recombinase monomers ( figure s1 ) . in order to enhance zfr heterodimer - mediated recombination , we employed directed evolution to select new dimer interface residues that more effectively facilitate heterodimerization . we randomized position 103 and the residues surrounding this region ( i.e. , positions 100 , 104 , 107 , and 108 ) within the gin catalytic domain ( figure 1b ) and held the complementary gin f103r monomer constant , as preliminary analysis indicated that arg at position 103 was 2-fold more effective at preventing homodimerization than asp ( figure s1 ) . we selected recombinase variants by split gene reassembly using cells that already harbored the gin f103r mutant expression plasmid ( figure 2a ) . to ensure the formation of the intended heterodimeric species and reduce the possibility of homodimer - mediated survival , we fused the gin catalytic domain library and the gin f103r monomer to zinc - finger dna - binding domains with orthogonal specificities . after only four rounds of selection , the activity of the mutant zfr population increased by > 500-fold in comparison to the parental gin f103d mutant ( figure 2b ) . we sequenced individual recombinase variants from the fourth round of selection and observed a striking degree of sequence similarity at positions 103 and 107 , and significant diversity at positions 104 and 108 ( figure 2c ) . intriguingly , we found that only 5% of selected clones contained a negatively charged residue at any position targeted for randomization . in particular , 93% of selected clones contained the native phe residue at position 103 . a nearly identical library that contained a fixed asp substitution at gin position 103 yielded no enrichment following multiple rounds of selection in the presence of gin f103r ( data not shown ) . re - engineering the gin recombinase dimer interface ( a ) schematic representation of the split gene reassembly system used to evaluate heterodimer - mediated recombination . expression of active recombinase variants leads to restoration of the -lactamase coding sequence and host cell resistance to carbenicillin , an ampicillin analogue . base positions 3 and 2 of the left half - site are indicated . ( b ) selection of gin mutants that recombine dna when paired with gin f103r . asterisk indicates the selection step in which incubation time was decreased from 16 to 4 h. ( c ) mutation frequencies ( % ) at positions targeted for randomization in the gin catalytic domain . ( d ) recombination by selected gin mutants on a symmetrical dna target upon forced homodimerization ( red ) or on an asymmetrical target when paired with gin f103r ( orange ) . ( e ) recombination by various pairs of zfrs that contain the ykwt / r dimer interface , with wild - type control . ( f ) recombination specificity of the ykwt / r dimer interface compared to wild - type . virtually all selected gin recombinase monomers demonstrated high - activity ( > 25% recombination ) in the presence of the complementary gin f103r monomer ( figure 2d ) . despite the absence of negative selection pressure , we found that the majority of the selected variants also showed a reduction in recombination upon forced homodimerization on a symmetric dna target ( figure 2d ) . one selected mutant ( gin m100y , f104k , v107w , and m108 t ; hereafter referred to as ykwt ) demonstrated a 2000-fold enhancement in recombination on an asymmetric dna target when paired with gin f103r compared to when used as a homodimer on a symmetric target ( figure 2e ) . this obligate heterodimer also recombined dna 2-fold more efficiently than the counterpart zfr composed of the wild - type dimer interface ( figure 2e ) . in order to determine whether the redesigned dimer interface negatively impacted zfr catalytic specificity , we next evaluated obligate heterodimer - mediated recombination on dna targets containing mutations within the 20-bp core site recognized by the gin catalytic domain . substitutions were introduced at core positions 3 and 2 ( figure 2a ) , as variations at these sites are highly tolerated by evolved serine recombinases with relaxed target specificity . in comparison to zfrs that contained the wild - type dimer interface , zfrs composed of the obligate heterodimeric architecture displayed a marked decrease in recombination on a non - cognate target harboring cc substitutions at positions 3 and 2 ( figure 2f ) . taken together , these data indicate that the serine recombinase dimer interface can be effectively redesigned to favor heterodimerization , and that zfrs composed of these enhanced catalytic domains display improved recombination efficiency and specificity in bacterial cells . we next investigated whether the redesigned gin recombinase dimer interface could improve zfr specificity in mammalian cells . to test this ( l ) and right ( r ) monomers of a zfr pair designed to target a 44-bp sequence from a non - protein coding region of human chromosome 4 ( figure 3a ) . importantly , this zfr pair provides an opportunity to directly assess the effectiveness of the redesigned dimer interface , as the left left homodimer side product of this zfr pair previously exhibited substantial recombination activity on the full - length zfr target site in mammalian cells . we measured recombination using a transient reporter assay that correlates zfr - mediated recombination with reduced luciferase expression in mammalian cells ( figure 3a ) . we co - transfected human embryonic kidney ( hek ) 293 t cells with a luciferase reporter plasmid containing the full - length zfr target site and expression vectors for either the l or r zfr monomers . we then directly compared the fold reduction in luciferase expression to that of 293 t cells co - transfected with both l and r zfr monomers and reporter plasmid . impressively , we found that zfr heterodimer pairs that contained the redesigned dimer interface demonstrated substantially improved specificity in comparison to the native zfrs , reducing off - target homodimer - mediated recombination by > 200-fold in both possible configurations ( l / r and l / r , figure 3b ) . however , these obligate heterodimeric pairs recombined dna 2- to 5-fold less efficiently than the standard zfrs ( figure 3c ) . western blot analysis confirmed that the reduction in activity was not due to reduced levels of protein expression ( figure s2 ) . zfr - mediated recombination leads to excision of the sv40 promoter and reduced luciferase expression in mammalian cells . ( b ) relative contribution to recombination from left right heterodimers and left left and the contribution of each homodimer to recombination was calculated by measuring the fold - reduction in luciferase expression in 293 t cells transfected with either l- or r - only zfr monomers , and dividing by the value obtained from cells transfected with both l and r zfr monomers . ( c ) recombination efficiency of wild - type and enhanced zfr heterodimers with and without the d12 g substitution . ( d ) crystal structure of the resolvase ( gray surface ) in complex with dna ( orange sticks ) . regions important for recombinase activity and specificity are highlighted and labeled . to further improve the recombination efficiency of the obligate zfr heterodimers , we searched our archive of evolved gin recombinase catalytic domains and identified four mutations that were frequently observed among hyperactivated variants : d12 g , n14s , k50e , and m70v . analysis of the crystal structure of an activated mutant of the resolvase catalytic domain indicates that these residues lie near the active site serine and may enhance catalysis by optimally positioning dna for cleavage and strand exchange ( figure 3d ; only d12 g is shown ) . we introduced each mutation individually into both the l and r monomers of the obligate heterodimeric zfr architecture and evaluated their impact on site - specific recombination . two of the four substitutions ( d12 g and n14s ) enhanced the catalytic activity of the obligate heterodimers ( figure s3 ) . in particular , inclusion of d12 g led to an increase in recombination efficiency that exceeded the standard zfr heterodimer and was similar to flpe , an evolved , highly efficient site - specific recombinase routinely used for cell - line engineering ( figure 3c ) . comparison of the relative non - specific contribution of each zfr homodimer to recombination revealed that the d12g / ykwt and d12g / f103r substitutions ( hereafter referred to as enhanced zfrs ; ezfrs ) retained the ability to fully prevent recombination by illegitimate homodimers ( figure 3b ) . we next examined the portability of the ezfr architecture by introducing each of the enhancing mutations into three different zfr pairs designed to target unique 44-bp sequences present on human chromosomes 1 , 4 , and x. importantly , these zfr pairs are composed of distinct combinations of gin recombinase catalytic domains , each with evolved recognition specificities . as such , this analysis served to evaluate the compatibility of the redesigned dimer interface with our collection of re - engineered gin catalytic domains . in comparison to wild - type zfrs , the ezfr pairs targeting human chromosomes x and 4 demonstrated increased recombination efficiency on their intended dna targets , while the ezfr pair designed to target chromosome 1 showed reduced activity ( figure s4 ) . however , when analyzed on a panel of non - cognate target sites in the context of the luciferase reporter assay , these ezfrs showed improved recombination specificity on the majority of substrates evaluated ( 28 out of 32 ) ( figure s5 ) . taken together , these results demonstrate that dimer interface redesign improves the recombination specificity of custom - designed zfrs in mammalian cells but that context - dependent interactions between the recombinase dimer interface and target site might influence recombination efficiency . as the primary aim of this work was the improvement of zfr specificity in the context of targeted genome engineering , we next sought to evaluate whether the ezfr framework improved the specificity of targeted integration in mammalian cells . to this end , we co - transfected hek293 t cells with enhanced and standard zfr heterodimers designed to target the previously mentioned 44-bp sequence present on human chromosome 4 together with a 4.5-kb donor plasmid containing the cognate zfr target site and a puromycin - resistance gene ( figure 4a ) . side - product homodimers for this zfr pair have been observed to catalyze integration at the selected genomic target . thus , this zfr pair allows for readout of the effectiveness of the ezfr architecture for preventing homodimerization ( figure 4b ) . we evaluated zfr and ezfr - mediated integration by pcr , amplifying the 5 and 3 junctions between the donor plasmid and the chromosomal target 72 h after transfection . as anticipated , both ezfr configurations ( l / r and l / r ) catalyzed integration at the intended genomic locus ( figure 4b ) . in contrast to the standard zfrs , no site - specific integration was observed after transfection with individual l or r ezfr monomers ( figure 4b ) . we determined the rate of genome - wide integration of the ezfr heterodimers by puromycin - selection and found that each configuration displayed improved targeting efficiency , with the l / r configuration yielding a genome - wide integration rate near 0.8% ( figure 4b ) . these efficiencies are similar to those reported for phic31-mediated site - specific integration in hek293 cells . we next investigated the specificity of ezfr - mediated integration by pcr analysis of individually expanded puromycin - resistant clones . in total , 10 of 16 ( 63% ) and 4 of 16 ( 25% ) clones were positive for targeted integration by ezfrs containing the l / r and l / r heterodimeric configurations , respectively ( figure 4c ) . compared to the standard zfr architecture ( 2 of 17 clones ; 11% ) , the l / r ezfr heterodimers demonstrated a significant increase in targeted integration ( = 9.23 , p < 0.03 ) , while the l/ ezfr configuration did not ( = 0.99 , p > 0.8 ) . dna sequencing confirmed site - specific integration at the intended genomic locus . site - specific integration into the human genome by enhanced zfrs . ( a ) schematic representation of sequence and location of the zfr target site on human chromosome 4 . ( b ) bulk pcr analysis of hek293 cells transfected with an empty donor plasmid containing only a puromycin - resistance gene and various zfr pairs designed to target human chromosome 4 . ( c ) clonal pcr analysis of puromycin - resistant cells transfected with empty donor and ezfrs in both orientations . we next evaluated the toxicity of the ezfrs by measuring their impact on cell viability . surprisingly , we observed that the standard zfrs targeting human chromosome 4 induced high toxicity , leading to a 60% decrease in cell viability after 5 days at the highest concentrations tested ( figure 5a ) . in contrast , ezfrs showed no apparent toxicity and demonstrated a viability profile similar to the rare - cutting and non - toxic i - scei homing endonuclease ( figure 5a ) . furthermore , western blot analysis revealed no difference in expression between zfr and ezfr variants ( figures 5b and s2 ) , indicating that the improved safety profiles of the ezfrs are not attributable to reduced expression levels . together , these findings demonstrate that ezfrs promote targeted integration with improved specificity and demonstrate substantially lower toxicity than zfrs composed of the wild - type dimer interface . ( a ) cell viability of hek293 cells transfected with increasing amounts of expression vector of standard or ezfrs . ( b ) western blot of lysate from hek293 cells transfected with increasing amounts of expression vector of standard zfrs or ezfrs . samples were taken 48 h after transfection and probed with horseradish peroxidase - conjugated anti - ha and anti--actin ( loading control ) antibodies . a potential application of zfr technology is the site - specific integration of therapeutic genes into the human genome . to explore the feasibility of this goal using ezfrs , we constructed a 6.25-kb donor plasmid containing ( i ) the zfr target site from human chromosome 4 , ( ii ) a puromycin - resistance gene , and ( iii ) the cdna for one of two disease - associated genes : the human coagulation factor ix ( fix ) gene , whose deficiency leads to hemophilia b , and the human -galactosidase ( gla ) gene , which is necessary for lipid metabolism and whose mutation results in the metabolic disorder known as fabry s disease ( figure 4a ) . the phic31 integrase has previously been used to deliver the human fix gene into animal models , but these studies were based on random integration into pseudo - recognition sites . co - transfection of hek293 cells with donor plasmid and ezfrs with the l / r dimeric configuration led to efficient integration of each therapeutic factor into the intended target site on chromosome 4 , and puromycin selection revealed a genome - wide ezfr - mediated integration rate of 0.3 and 0.4% for the fix- and gla - harboring donor plasmids , respectively ( figure 6a ) . we evaluated ezfr - mediated integration specificity by pcr analysis of individual puromycin - resistant clones and found that 9 of 12 ( 75% ) and 8 of 10 ( 80% ) clones contained fix and gla cdna , respectively , at the intended genomic target site ( figure 6b ) , which was verified by dna sequencing . notably , the specificity of transgene insertion achieved by these ezfrs is similar to those reported for zfns , talens , and crispr / cas systems , indicating that ezfrs are effective tools for site - specific integration into the human genome . lastly , toward characterizing the full integration landscape of the ezfrs , we computationally identified four potential off - target sites that contain up to three mismatches compared to the intended genomic target , and evaluated off - target integration from genomic dna isolated from puromycin - resistant cells that were negative for targeted integration . we observed no transgene insertions at any of the four pseudo - integration sites ( figure s6 ) . these findings indicate that more comprehensive genome - wide approaches are required to determine the full scope of ezfr - mediated off - target modifications . targeted integration of the human coagulation factor ix and -galactosidase genes by enhanced zfrs . ( a ) bulk pcr analysis of hek293 cells transfected with ezfrs targeting human chromosome 4 and donor plasmids harboring either the human coagulation factor ix ( fix ) or -galactosidase genes ( gla ) . ( b ) clonal analysis of puromycin - resistant cells transfected with ezfrs and donor plasmids containing the fix or gla genes . advances in targeted genetic engineering are driving progress in many fields , including biotechnology and gene therapy . while site - specific nucleases have facilitated many of these achievements , their capacity for inducing off - target mutations and reliance on dna repair mechanisms could limit their effectiveness . in particular , the establishment of a new class of tools capable of specifically and safely delivering large payloads into the human genome would be broadly useful across diverse fields , including basic research , gene therapy and synthetic biology . hybrid recombinases based on the serine resolvase / invertase family of enzymes are a class of reagents capable of delivering genetic payloads into the human genome with potentially few side effects . however , the specificity of these enzymes has proven low , primarily due to the formation of side - product homodimers capable of catalyzing off - target modifications . in this study , we have combined rational design and directed evolution to redesign the serine recombinase dimer interface to prevent formation of these deleterious homodimers , leading to the generation of a new class of hybrid recombinases that preferentially heterodimerize and catalyze site - specific integration into endogenous genomic loci with high specificity . this work expands upon our previous studies that focused on establishing a collection of re - engineered site - specific recombinases capable of targeting a broad range of genomic target sites . these results , and in particular our finding that ezfrs specifically introduce the human coagulation factor ix and -galactosidase genes into the human genome with minimal toxicity , support the continued development of this technology for potential therapeutic applications . however , further studies are required to evaluate the activity and flexibility of these enzymes in primary cells and their potential to modify genomic safe - harbor regions with large multi - gene payloads . future efforts will also focus on establishing optimal delivery methods by evaluating zfr compatibility with integration - deficient lentiviral vectors or adeno - associated virus . in comparison to published results in similar cell lines , the ezfrs containing the l / r dimeric configuration directed site - specific integration with specificities comparable to zfns , talens and crispr / cas9 systems ; however , the efficiency of ezfr - mediated integration remained lower than those typically observed with site - specific nuclease technologies . one reason for this is that zfr - mediated recombination is reversible , and as such , insertion events may be excised shortly after integration . the design of integration - competent / excision - defective zfr variants thus represents one potential solution for enhancing zfr - mediated integration efficiency . as proof - of principle of this concept , craig and co - workers recently reported the isolation of excision - competent / integration - defective variants of the piggybac transposase . to our surprise , zfrs composed of the wild - type dimer interface induced high levels of cellular toxicity . because zfr - mediated recombination necessitates the formation of covalent protein dna linkages that may activate the nhej repair pathway , we suspect that the high levels of cell death induced by the wild - type zfr pair could be attributed to excessive amounts of cleavage at pseudo - recombination sites by zfr homodimers or zfr - mediated rearrangements spurred by the presence of excess homodimers . thus , the dramatic reduction in toxicity observed with ezfrs can likely be attributed to the ability of the re - engineered dimer interface to prevent recombination by side - product homodimers . the improved efficiency and specificity demonstrated by ezfrs , as well as their ability to promote site - specific integration in the absence of dsbs , suggests that these tools might also be used to modify model organisms refractory to current genome engineering methods . moreover , this new dimer interface is extensible and should be directly portable to a broad range of hybrid recombinases , including those based on tal effector dna - binding domains , and perhaps crispr / cas technology . although it remains unknown whether the previously described tal effector architecture supports our expanded collection of gin recombinase catalytic domains , the dimer interface substitutions described here should nonetheless improve the specificity of any tal effector recombinase heterodimer that consists solely of the wild - type gin catalytic domain . these enhanced recombinases may also find utility in synthetic biology by enabling implementation of complex computational tasks using orthogonal custom recombinases . finally , the recombinase dimer interface mutations described in this work may facilitate new advances in the understanding of site - specific recombination . in particular , studies focused on the residues targeted in this work may shed new light on the mechanisms that govern the conformational changes during target site cleavage , strand exchange , and religation . in summary , our findings provide a general means for improving the targeting efficiency , specificity , and safety of customizable recombinases and illustrate the potential of these enzymes for diverse genome engineering applications , including therapeutic gene transfer . the split gene reassembly vector ( pbla ) was derived from pbluescriptii sk ( ) ( stratagene ) and modified to contain a chloramphenicol resistance gene and an interrupted tem-1 -lactamase gene under the control of a lac promoter . briefly , a gfpuv stuffer was pcr amplified with the primers gfp - zfr--h1--p2-xbai - fwd and gfp - zfr--h1--p2-hindiii - rev and cloned into the spei and hindiii restriction sites of pbla to generate the pbla - zfr substrate used for selections . briefly , the simian vacuolating virus 40 ( sv40 ) promoter was pcr amplified from pgl3-prm ( promega ) with the primers sv40-zfr - bglii - fwd and sv40-zfr - hindiii - rev . pcr products were digested with bglii and hindiii and ligated into the same restriction sites of pgl3-prm to generate the luciferase reporter vectors pgl3-zfr-1 , 2 , 3 , ... , 9 . zfr donor plasmids ( pdonor ; previously pbabe - puromycin ) were constructed as previously described with the following exceptions : cdna for the human coagulation factor ix ( fix ) and -galactosidase ( gla ) genes ( genecopoeia ) were pcr amplified with the primers psti - cmv - donor - fwd and bamh1-zfr - donor - rev . pcr products were digested with psti and bamh1 and ligated into the same restriction sites of pdonor . correct construction of each plasmid was verified by sequence analysis ( tables s1 and s2 ) . all primer sequences are provided in table s3 . to construct the zfr library , residues 1115 of the gin catalytic domain were pcr amplified from pbla - gin--h1 with the primers puc18-prim-2 and gin - dimer - lib - rev . mutations were introduced at positions 100 , 103 , 104 , 107 , and 108 with the degenerate codon dnk ( d : a , t , or g ; n : a , t , c , or g ; and k : g or t ) , which encodes all amino acids except pro , his and gln . residues 115 through 144 of the gin catalytic domain and the h1 zinc - finger protein were pcr amplified from pbla - gin--h1with the primers gin - dimer - fwd and puc18-prim-1 and fused to the gin library by overlap pcr with the primers puc18-prim-1 and -2 . fusion pcr products were digested with saci and xbai and ligated into the same restriction sites of pbla . ligations were ethanol precipitated and transformed by electroporation into e. coli top10f ( invitrogen ) cells , which were modified to harbor the expression vector pprolar - gin--f103r - p2 ( clontech laboratories , inc . ) . after 1 h recovery in super optimal broth with catabolite suppression ( soc ) medium , cells were incubated with 100 ml of super broth ( sb ) with 30 g ml of chloramphenicol and cultured at 37 c with shaking . at 16 h , 30 ml of cells was harvested by centrifugation and plasmid dna was isolated by mini - prep ( invitrogen ) ; 3 g of plasmid dna was then used to transform e. coli top10f. after 1 h recovery in 5 ml soc , a portion of cells was plated on solid lysogeny broth ( lb ) with 30 g ml of chloramphenicol or 30 g ml of chloramphenicol and 100 g ml of carbenicillin , an ampicillin analogue . recombination was determined as the number of colonies on chloramphenicol / carbenicillin plates , divided by the number of colonies on chloramphenicol - only plates . colony number was determined by automated counting using the geldoc xr imaging system ( bio - rad ) . the remaining recovery culture was incubated with 100 ml of sb medium with 30 g ml of chloramphenicol and 100 g ml of carbenicillin . at 16 h , cells were harvested , and plasmid dna was purified by maxi - prep ( invitrogen ) . selected zfrs were isolated by saci and xbai digestion and ligated into unmodified pbla for further selection . after each round of selection , sequence analysis ( eton biosciences ) was performed on individual carbenicillin - resistant clones . recombination assays with individually selected zfrs was performed as described above . for zfr construction , the gin , , , , , and catalytic domains were pcr amplified from the previously described templates pbla - gin- , , , , , or as two fragments with the primers gin - hbs - d12g - koz and gin - ykwt - rev or gin - f103r - rev and gin - ykwt - fwd or gin - f103r - fwd and gin - agei - rev . pcr products were fused by overlap pcr with the primers gin - hbs - d12g - koz and gin - agei - rev and cloned into the hindiii and agei restriction sites of pbh to generate the new superzif - compatible sub - cloning vectors : pbh - d12g - gin-- , - , - , - , - , or -ykwt or f103r ( ykwt denotes the mutations m100y , f104k , v107w , and m108 t ) . previously constructed zinc - finger domains were ligated into the agei and spei restriction sites of the appropriate pbh - gin sub - cloning vector to generate pbh - ezfr - l - or - r-1 , 2 , 3 , 4 , or 5 ( ezfr : enhanced zfr ; l : left ezfr ; r : right ezfr ) . each ezfr gene was released from pbh by sfii digestion and ligated into pcdna 3.1 ( invitrogen ) to generate pcdna - ezfr - l- or - r-1 , 2 , 3 , 4 , or 5 . human embryonic kidney ( hek ) 293 and 293 t cells ( american type culture collection , atcc ) were maintained in dulbecco s modified eagle s medium ( dmem ) containing 10% ( v / v ) fetal bovine serum ( fbs ; gibco ) and 1% ( v / v ) antibiotic - antimycotic ( anti - anti ; gibco ) . hek293 t cells were seeded onto 96-well plates at a density of 4 10 cells / well and established in a humidified 5% co2 atmosphere at 37 c . at 24 h after seeding , cells were transfected with 2550 ng of pcdna - ezfr - l-1 through 6 , 2550 ng of pcdna - ezfr - r-1 through 6 , 25 ng of pgl3-zfr , and 1 ng of prl - cmv ( promega ) using lipofectamine 2000 ( invitrogen ) according to the manufacturer s instructions . for cells transfected with only one zfr monomer , empty pcdna , cells were lysed with passive lysis buffer ( promega ) , and luciferase expression was determined with the dual - luciferase reporter assay system ( promega ) using a veritas microplate luminometer ( turner biosystems ) . hek293 cells were seeded onto 24-well plates at a density of 1 10 cells / well and maintained in serum - containing media in a humidified 5% co2 atmosphere at 37 c . at 24 h after seeding , cells were transfected with 80 ng of pdonor , 10 ng of pcdna - ezfr - l-1 , 10 ng of pcdna - ezfr - r-1 , and 1 ng of pcmv - egfp ( clontech ) using lipofectamine 2000 according to the manufacturer s instructions . we note that ezfrs - l- and - r-1 target human chromosome 4 . at 24 h after transfection , transfection efficiency was determined by flow cytometry analysis of egfp expression ( facscan dual laser cytometer ; bd biosciences ; facsdiva software ) . at 72 h after transfection , cells were harvested , and genomic dna was isolated using quick extract dna extraction solution ( epicentre ) . zfr targets and gapdh were pcr amplified from bulk genomic dna by nested pcr with the following primer combinations : gapdh - external - fwd and gapdh - external - rev , gapdh - internal - fwd and gapdh - internal - rev ( control ) ; zfr-1-external - rev and cmv - external , zfr-1-internal - rev and cmv - internal ( forward integration ) ; zfr-1-external - fwd and cmv - external , zfr-1-internal - fwd and cmv - internal ( reverse integration ) . for pdonor vectors that harbored the human fix and gla genes , the following primers were used for internal pcr : zfr-1-internal - rev and fix - internal ( fix forward integration ) ; zfr-1-internal - fwd and fix - internal ( fix reverse integration ) ; zfr-1-internal - rev and gla - internal ( gla forward integration ) ; zfr-1-internal - fwd and gla - internal ( gla reverse integration ) . for colony counting assays , at 72 h post - transfection , cells were split into 6-well plates at a density of 1 10 cells / well and maintained in serum - containing media with or without 2 g ml of puromycin . at 1418 days , cells were stained with 0.2% crystal violet staining solution , and genome - wide integration rates were determined by counting the number of colonies formed in puromycin - containing media divided by the number of colonies formed in the absence of puromycin . colony counting was determined by automated counting using the geldoc xr system ( bio - rad ) . for clonal analysis , at 72 after post - transfection , 1 10 cells were split onto a 100-mm dish and maintained in serum - containing media with 2 g ml of puromycin . individual colonies were isolated with 10- 10-mm open - ended cloning cylinders ( millipore ) with sterile silicone grease ( millipore ) and expanded in 96-well plates in the presence of 2 g ml of puromycin . genomic dna was isolated and used as the template for pcr as described above . sequence analysis ( eton biosciences ) was performed across the 5 and 3 junctions for each amplicon . at 48 h post - transfection , zfr expression was analyzed by sds - page with a novex 420% tris - glycine gel ( invitrogen ) . samples were transferred onto a 0.2 m nitrocellulose membrane and incubated for 2 h in transfer buffer ( 25 mm tris - base , 0.2 m glycine , 20% methanol , ph 8.5 ) . membranes were washed with 1 tbs ( 50 mm tris - hcl , 150 mm nacl , 0.05% tween 20 , ph 7.5 ) and visualized by automated chemiluminescence visualization using the gel doc xr imaging system . zfr was detected by horseradish peroxidase conjugated anti - ha antibody ( roche ) . -actin was used as an internal loading control and was detected with peroxidase - conjugated anti--actin antibody ( sigma ) . hek293 cells were seeded onto 24-well plates at a density of 1 10 cells / well . at 24 h after seeding , cells were transfected with 25500 ng of pcdna - zfr - l-1 , 25500 ng of pcdna - zfr - r-1 , 80 ng of pdonor , and 10 ng of pcmv - egfp . at 30 h post - transfection , cells were collected , and egfp fluorescence was measured by flow cytometry ( facscan dual laser cytometer ; bd biosciences ; facsdiva software ) . for each sample , 50,000 live events were collected , and data were analyzed using flowjo ( tree star , inc . ) . at 5 days post - transfection , cells were again collected , and egfp fluorescence was measured via flow cytometry as before . zfr - mediated toxicity was calculated by dividing the number of total viable cells ( i.e. , egfp - positive cells ) measured at 5 days post - transfection by the number of egfp - positive cells at 30 h post - transfection .

despite recent advances in genome engineering made possible by the emergence of site - specific endonucleases , there remains a need for tools capable of specifically delivering genetic payloads into the human genome . hybrid recombinases based on activated catalytic domains derived from the resolvase / invertase family of serine recombinases fused to cys2-his2 zinc - finger or tal effector dna - binding domains are a class of reagents capable of achieving this . the utility of these enzymes , however , has been constrained by their low overall targeting specificity , largely due to the formation of side - product homodimers capable of inducing off - target modifications . here , we combine rational design and directed evolution to re - engineer the serine recombinase dimerization interface and generate a recombinase architecture that reduces formation of these undesirable homodimers by > 500-fold . we show that these enhanced recombinases demonstrate substantially improved targeting specificity in mammalian cells and achieve rates of site - specific integration similar to those previously reported for site - specific nucleases . additionally , we show that enhanced recombinases exhibit low toxicity and promote the delivery of the human coagulation factor ix and -galactosidase genes into endogenous genomic loci with high specificity . these results provide a general means for improving hybrid recombinase specificity by protein engineering and illustrate the potential of these enzymes for basic research and therapeutic applications .

Introduction Results Discussion Materials and Methods

site - specific endonucleases , including zinc - finger nucleases ( zfns ) , meganucleases , tal effector nucleases ( talens ) , and crispr / cas systems , have dramatically enhanced the speed and efficiency with which researchers can introduce targeted genetic modifications into cells and organisms . although site - specific nucleases are versatile and promote a broad range of genetic alterations , they rely on cellular dna repair mechanisms , such as error - prone non - homologous end joining ( nhej ) and homology - directed repair ( hdr ) , to induce custom alterations . the lack of availability of dna repair pathways within certain cell types , however , may reduce the utility of this technology . in particular , poor induction of hdr via nuclease - induced dna double - strand breaks ( dsbs ) or nicks has been shown to be a major limiting factor for achieving high rates of site - specific integration . additionally , off - target dsbs induced by site - specific nucleases are difficult to comprehensively characterize in the absence of an accompanying donor template and can be potentially toxic to cells and organisms . thus , there remains a continued need for the development of new tools capable of achieving highly precise targeted modifications with minimal toxicity . , cre , flp , phic31 , and bxb1 ) are a potentially powerful alternative to site - specific nucleases for targeted genetic engineering . while these approaches permit the design of ssr variants with new properties , they nevertheless typically lead to the emergence of relaxed specificity , an undesirable byproduct that limits the utility and safety of these enzymes . hybrid recombinases composed of catalytic domains derived from the resolvase / invertase family of serine recombinases ( e.g. , gin , hin , tn3 , and ) fused to custom - designed cys2-his2 zinc - finger or tal effector dna - binding domains represent a unique solution to this problem ( figure 1a ) . in particular , zinc - finger recombinases ( zfrs ) are a flexible class of chimeric proteins capable of introducing targeted modifications into mammalian cells . zfrs promote site - specific recombination between dna targets that consist of two inverted zinc - finger binding sites flanking a central 20-bp core sequence recognized by the recombinase catalytic domain ( figure 1a ) . as such , new zfrs with diverse targeting capabilities can be generated in a , we have demonstrated that tailored zfr variants can be rapidly assembled from a library of pre - selected gin recombinase catalytic domains ( referred to here as gin , , , , , and ) and zinc - finger modules . this customization strategy allows for the design of synthetic recombinases that have the capacity to recognize a broad range of user - defined dna targets and direct site - specific integration into endogenous genomic loci . structure of a zinc - finger recombinase ( zfr ) and its dimer interface . ( a ) top : zfr monomers ( left , red ; right , yellow ) consist of an activated serine recombinase catalytic domain fused to a cys2-his2 zinc - finger dna - binding domain . zinc - finger proteins ( zfps ) can be replaced with tal effector dna - binding domains . model shows the structure of an engineered zfr , generated from the crystal structures of the resolvase and aart zinc - finger protein ( pdb ids : 1gdt and 2i13 , respectively ) . abbreviations are as follows : n indicates a , t , c , or g ; r indicates g or a ; y indicates c or t ; w indicates a or t ; zfbs indicates zinc - finger binding site . indeed , expression of any two zfr monomers required for genomic targeting inevitably leads to the formation of two side - product zfr homodimers capable of inducing off - target genomic modifications . in addition , mutagenesis of the cre recombinase dimer interface has led to the isolation of mutants with improved recombination specificity , presumably due to destabilization of cre dimer binding cooperativity . here , we employ rational design and directed evolution to redesign the serine recombinase dimerization interface and generate a new hybrid recombinase architecture that prevents formation of side - product recombinase homodimers by > 500-fold . we show that zfrs composed of these enhanced catalytic domains demonstrate substantially improved targeting specificity and efficiency , and enable the site - specific delivery of therapeutic genes into the human genome with low toxicity . in order to redesign the gin recombinase dimer interface and engineer zfrs that preferentially heterodimerize , we sought to identify the specific amino acid residues that govern recombinase dimerization . to accomplish this , we examined the crystal structures of the resolvase dimer and the activated , tetrameric configurations of the gin and sin recombinases . on the basis of these data , we hypothesized that substitution of these residues with complementary charged amino acids would ( i ) disfavor association of homodimers by charge and steric repulsion and ( ii ) promote heterodimer formation through favorable electrostatic contacts . to evaluate the effect that charged substitutions within the dimer interface have on recombination , we created a collection of recombinase mutants based on the gin and catalytic domains that contained either arg ( gin ) or asp ( gin ) substitutions at positions 100 , 103 , and 107 and evaluated their ability to recombine dna as homodimers ( i.e. in order to enhance zfr heterodimer - mediated recombination , we employed directed evolution to select new dimer interface residues that more effectively facilitate heterodimerization . to ensure the formation of the intended heterodimeric species and reduce the possibility of homodimer - mediated survival , we fused the gin catalytic domain library and the gin f103r monomer to zinc - finger dna - binding domains with orthogonal specificities . after only four rounds of selection , the activity of the mutant zfr population increased by > 500-fold in comparison to the parental gin f103d mutant ( figure 2b ) . in order to determine whether the redesigned dimer interface negatively impacted zfr catalytic specificity , we next evaluated obligate heterodimer - mediated recombination on dna targets containing mutations within the 20-bp core site recognized by the gin catalytic domain . taken together , these data indicate that the serine recombinase dimer interface can be effectively redesigned to favor heterodimerization , and that zfrs composed of these enhanced catalytic domains display improved recombination efficiency and specificity in bacterial cells . we next investigated whether the redesigned gin recombinase dimer interface could improve zfr specificity in mammalian cells . importantly , this zfr pair provides an opportunity to directly assess the effectiveness of the redesigned dimer interface , as the left left homodimer side product of this zfr pair previously exhibited substantial recombination activity on the full - length zfr target site in mammalian cells . we measured recombination using a transient reporter assay that correlates zfr - mediated recombination with reduced luciferase expression in mammalian cells ( figure 3a ) . impressively , we found that zfr heterodimer pairs that contained the redesigned dimer interface demonstrated substantially improved specificity in comparison to the native zfrs , reducing off - target homodimer - mediated recombination by > 200-fold in both possible configurations ( l / r and l / r , figure 3b ) . zfr - mediated recombination leads to excision of the sv40 promoter and reduced luciferase expression in mammalian cells . to further improve the recombination efficiency of the obligate zfr heterodimers , we searched our archive of evolved gin recombinase catalytic domains and identified four mutations that were frequently observed among hyperactivated variants : d12 g , n14s , k50e , and m70v . analysis of the crystal structure of an activated mutant of the resolvase catalytic domain indicates that these residues lie near the active site serine and may enhance catalysis by optimally positioning dna for cleavage and strand exchange ( figure 3d ; only d12 g is shown ) . we introduced each mutation individually into both the l and r monomers of the obligate heterodimeric zfr architecture and evaluated their impact on site - specific recombination . in particular , inclusion of d12 g led to an increase in recombination efficiency that exceeded the standard zfr heterodimer and was similar to flpe , an evolved , highly efficient site - specific recombinase routinely used for cell - line engineering ( figure 3c ) . as such , this analysis served to evaluate the compatibility of the redesigned dimer interface with our collection of re - engineered gin catalytic domains . taken together , these results demonstrate that dimer interface redesign improves the recombination specificity of custom - designed zfrs in mammalian cells but that context - dependent interactions between the recombinase dimer interface and target site might influence recombination efficiency . as the primary aim of this work was the improvement of zfr specificity in the context of targeted genome engineering , we next sought to evaluate whether the ezfr framework improved the specificity of targeted integration in mammalian cells . side - product homodimers for this zfr pair have been observed to catalyze integration at the selected genomic target . in contrast to the standard zfrs , no site - specific integration was observed after transfection with individual l or r ezfr monomers ( figure 4b ) . we determined the rate of genome - wide integration of the ezfr heterodimers by puromycin - selection and found that each configuration displayed improved targeting efficiency , with the l / r configuration yielding a genome - wide integration rate near 0.8% ( figure 4b ) . these efficiencies are similar to those reported for phic31-mediated site - specific integration in hek293 cells . dna sequencing confirmed site - specific integration at the intended genomic locus . site - specific integration into the human genome by enhanced zfrs . in contrast , ezfrs showed no apparent toxicity and demonstrated a viability profile similar to the rare - cutting and non - toxic i - scei homing endonuclease ( figure 5a ) . together , these findings demonstrate that ezfrs promote targeted integration with improved specificity and demonstrate substantially lower toxicity than zfrs composed of the wild - type dimer interface . a potential application of zfr technology is the site - specific integration of therapeutic genes into the human genome . to explore the feasibility of this goal using ezfrs , we constructed a 6.25-kb donor plasmid containing ( i ) the zfr target site from human chromosome 4 , ( ii ) a puromycin - resistance gene , and ( iii ) the cdna for one of two disease - associated genes : the human coagulation factor ix ( fix ) gene , whose deficiency leads to hemophilia b , and the human -galactosidase ( gla ) gene , which is necessary for lipid metabolism and whose mutation results in the metabolic disorder known as fabry s disease ( figure 4a ) . notably , the specificity of transgene insertion achieved by these ezfrs is similar to those reported for zfns , talens , and crispr / cas systems , indicating that ezfrs are effective tools for site - specific integration into the human genome . lastly , toward characterizing the full integration landscape of the ezfrs , we computationally identified four potential off - target sites that contain up to three mismatches compared to the intended genomic target , and evaluated off - target integration from genomic dna isolated from puromycin - resistant cells that were negative for targeted integration . these findings indicate that more comprehensive genome - wide approaches are required to determine the full scope of ezfr - mediated off - target modifications . targeted integration of the human coagulation factor ix and -galactosidase genes by enhanced zfrs . ( a ) bulk pcr analysis of hek293 cells transfected with ezfrs targeting human chromosome 4 and donor plasmids harboring either the human coagulation factor ix ( fix ) or -galactosidase genes ( gla ) . while site - specific nucleases have facilitated many of these achievements , their capacity for inducing off - target mutations and reliance on dna repair mechanisms could limit their effectiveness . in particular , the establishment of a new class of tools capable of specifically and safely delivering large payloads into the human genome would be broadly useful across diverse fields , including basic research , gene therapy and synthetic biology . hybrid recombinases based on the serine resolvase / invertase family of enzymes are a class of reagents capable of delivering genetic payloads into the human genome with potentially few side effects . however , the specificity of these enzymes has proven low , primarily due to the formation of side - product homodimers capable of catalyzing off - target modifications . in this study , we have combined rational design and directed evolution to redesign the serine recombinase dimer interface to prevent formation of these deleterious homodimers , leading to the generation of a new class of hybrid recombinases that preferentially heterodimerize and catalyze site - specific integration into endogenous genomic loci with high specificity . this work expands upon our previous studies that focused on establishing a collection of re - engineered site - specific recombinases capable of targeting a broad range of genomic target sites . these results , and in particular our finding that ezfrs specifically introduce the human coagulation factor ix and -galactosidase genes into the human genome with minimal toxicity , support the continued development of this technology for potential therapeutic applications . however , further studies are required to evaluate the activity and flexibility of these enzymes in primary cells and their potential to modify genomic safe - harbor regions with large multi - gene payloads . in comparison to published results in similar cell lines , the ezfrs containing the l / r dimeric configuration directed site - specific integration with specificities comparable to zfns , talens and crispr / cas9 systems ; however , the efficiency of ezfr - mediated integration remained lower than those typically observed with site - specific nuclease technologies . because zfr - mediated recombination necessitates the formation of covalent protein dna linkages that may activate the nhej repair pathway , we suspect that the high levels of cell death induced by the wild - type zfr pair could be attributed to excessive amounts of cleavage at pseudo - recombination sites by zfr homodimers or zfr - mediated rearrangements spurred by the presence of excess homodimers . thus , the dramatic reduction in toxicity observed with ezfrs can likely be attributed to the ability of the re - engineered dimer interface to prevent recombination by side - product homodimers . the improved efficiency and specificity demonstrated by ezfrs , as well as their ability to promote site - specific integration in the absence of dsbs , suggests that these tools might also be used to modify model organisms refractory to current genome engineering methods . moreover , this new dimer interface is extensible and should be directly portable to a broad range of hybrid recombinases , including those based on tal effector dna - binding domains , and perhaps crispr / cas technology . although it remains unknown whether the previously described tal effector architecture supports our expanded collection of gin recombinase catalytic domains , the dimer interface substitutions described here should nonetheless improve the specificity of any tal effector recombinase heterodimer that consists solely of the wild - type gin catalytic domain . these enhanced recombinases may also find utility in synthetic biology by enabling implementation of complex computational tasks using orthogonal custom recombinases . finally , the recombinase dimer interface mutations described in this work may facilitate new advances in the understanding of site - specific recombination . in summary , our findings provide a general means for improving the targeting efficiency , specificity , and safety of customizable recombinases and illustrate the potential of these enzymes for diverse genome engineering applications , including therapeutic gene transfer . zfr donor plasmids ( pdonor ; previously pbabe - puromycin ) were constructed as previously described with the following exceptions : cdna for the human coagulation factor ix ( fix ) and -galactosidase ( gla ) genes ( genecopoeia ) were pcr amplified with the primers psti - cmv - donor - fwd and bamh1-zfr - donor - rev . residues 115 through 144 of the gin catalytic domain and the h1 zinc - finger protein were pcr amplified from pbla - gin--h1with the primers gin - dimer - fwd and puc18-prim-1 and fused to the gin library by overlap pcr with the primers puc18-prim-1 and -2 . previously constructed zinc - finger domains were ligated into the agei and spei restriction sites of the appropriate pbh - gin sub - cloning vector to generate pbh - ezfr - l - or - r-1 , 2 , 3 , 4 , or 5 ( ezfr : enhanced zfr ; l : left ezfr ; r : right ezfr ) .

site - specific endonucleases , including zinc - finger nucleases ( zfns ) , meganucleases , tal effector nucleases ( talens ) , and crispr / cas systems , have dramatically enhanced the speed and efficiency with which researchers can introduce targeted genetic modifications into cells and organisms . although site - specific nucleases are versatile and promote a broad range of genetic alterations , they rely on cellular dna repair mechanisms , such as error - prone non - homologous end joining ( nhej ) and homology - directed repair ( hdr ) , to induce custom alterations . as such , new zfrs with diverse targeting capabilities can be generated in a , we have demonstrated that tailored zfr variants can be rapidly assembled from a library of pre - selected gin recombinase catalytic domains ( referred to here as gin , , , , , and ) and zinc - finger modules . here , we employ rational design and directed evolution to redesign the serine recombinase dimerization interface and generate a new hybrid recombinase architecture that prevents formation of side - product recombinase homodimers by > 500-fold . we show that zfrs composed of these enhanced catalytic domains demonstrate substantially improved targeting specificity and efficiency , and enable the site - specific delivery of therapeutic genes into the human genome with low toxicity . we focused our search on residues within the e helix a key mediator of dimer dimer interactions between recombinase subunits and identified five residues that likely associate with one another via hydrophobic interactions : met 100 , phe 103 , phe 104 , val 107 , and met 108 ( all numbers hereafter according to the gin recombinase ; figure 1b ) . on the basis of these data , we hypothesized that substitution of these residues with complementary charged amino acids would ( i ) disfavor association of homodimers by charge and steric repulsion and ( ii ) promote heterodimer formation through favorable electrostatic contacts . to evaluate the effect that charged substitutions within the dimer interface have on recombination , we created a collection of recombinase mutants based on the gin and catalytic domains that contained either arg ( gin ) or asp ( gin ) substitutions at positions 100 , 103 , and 107 and evaluated their ability to recombine dna as homodimers ( i.e. , positions 100 , 104 , 107 , and 108 ) within the gin catalytic domain ( figure 1b ) and held the complementary gin f103r monomer constant , as preliminary analysis indicated that arg at position 103 was 2-fold more effective at preventing homodimerization than asp ( figure s1 ) . to ensure the formation of the intended heterodimeric species and reduce the possibility of homodimer - mediated survival , we fused the gin catalytic domain library and the gin f103r monomer to zinc - finger dna - binding domains with orthogonal specificities . despite the absence of negative selection pressure , we found that the majority of the selected variants also showed a reduction in recombination upon forced homodimerization on a symmetric dna target ( figure 2d ) . one selected mutant ( gin m100y , f104k , v107w , and m108 t ; hereafter referred to as ykwt ) demonstrated a 2000-fold enhancement in recombination on an asymmetric dna target when paired with gin f103r compared to when used as a homodimer on a symmetric target ( figure 2e ) . in order to determine whether the redesigned dimer interface negatively impacted zfr catalytic specificity , we next evaluated obligate heterodimer - mediated recombination on dna targets containing mutations within the 20-bp core site recognized by the gin catalytic domain . in comparison to zfrs that contained the wild - type dimer interface , zfrs composed of the obligate heterodimeric architecture displayed a marked decrease in recombination on a non - cognate target harboring cc substitutions at positions 3 and 2 ( figure 2f ) . taken together , these data indicate that the serine recombinase dimer interface can be effectively redesigned to favor heterodimerization , and that zfrs composed of these enhanced catalytic domains display improved recombination efficiency and specificity in bacterial cells . importantly , this zfr pair provides an opportunity to directly assess the effectiveness of the redesigned dimer interface , as the left left homodimer side product of this zfr pair previously exhibited substantial recombination activity on the full - length zfr target site in mammalian cells . impressively , we found that zfr heterodimer pairs that contained the redesigned dimer interface demonstrated substantially improved specificity in comparison to the native zfrs , reducing off - target homodimer - mediated recombination by > 200-fold in both possible configurations ( l / r and l / r , figure 3b ) . ( b ) relative contribution to recombination from left right heterodimers and left left and the contribution of each homodimer to recombination was calculated by measuring the fold - reduction in luciferase expression in 293 t cells transfected with either l- or r - only zfr monomers , and dividing by the value obtained from cells transfected with both l and r zfr monomers . to further improve the recombination efficiency of the obligate zfr heterodimers , we searched our archive of evolved gin recombinase catalytic domains and identified four mutations that were frequently observed among hyperactivated variants : d12 g , n14s , k50e , and m70v . analysis of the crystal structure of an activated mutant of the resolvase catalytic domain indicates that these residues lie near the active site serine and may enhance catalysis by optimally positioning dna for cleavage and strand exchange ( figure 3d ; only d12 g is shown ) . in particular , inclusion of d12 g led to an increase in recombination efficiency that exceeded the standard zfr heterodimer and was similar to flpe , an evolved , highly efficient site - specific recombinase routinely used for cell - line engineering ( figure 3c ) . comparison of the relative non - specific contribution of each zfr homodimer to recombination revealed that the d12g / ykwt and d12g / f103r substitutions ( hereafter referred to as enhanced zfrs ; ezfrs ) retained the ability to fully prevent recombination by illegitimate homodimers ( figure 3b ) . we next examined the portability of the ezfr architecture by introducing each of the enhancing mutations into three different zfr pairs designed to target unique 44-bp sequences present on human chromosomes 1 , 4 , and x. importantly , these zfr pairs are composed of distinct combinations of gin recombinase catalytic domains , each with evolved recognition specificities . in comparison to wild - type zfrs , the ezfr pairs targeting human chromosomes x and 4 demonstrated increased recombination efficiency on their intended dna targets , while the ezfr pair designed to target chromosome 1 showed reduced activity ( figure s4 ) . however , when analyzed on a panel of non - cognate target sites in the context of the luciferase reporter assay , these ezfrs showed improved recombination specificity on the majority of substrates evaluated ( 28 out of 32 ) ( figure s5 ) . taken together , these results demonstrate that dimer interface redesign improves the recombination specificity of custom - designed zfrs in mammalian cells but that context - dependent interactions between the recombinase dimer interface and target site might influence recombination efficiency . as the primary aim of this work was the improvement of zfr specificity in the context of targeted genome engineering , we next sought to evaluate whether the ezfr framework improved the specificity of targeted integration in mammalian cells . to this end , we co - transfected hek293 t cells with enhanced and standard zfr heterodimers designed to target the previously mentioned 44-bp sequence present on human chromosome 4 together with a 4.5-kb donor plasmid containing the cognate zfr target site and a puromycin - resistance gene ( figure 4a ) . we determined the rate of genome - wide integration of the ezfr heterodimers by puromycin - selection and found that each configuration displayed improved targeting efficiency , with the l / r configuration yielding a genome - wide integration rate near 0.8% ( figure 4b ) . in total , 10 of 16 ( 63% ) and 4 of 16 ( 25% ) clones were positive for targeted integration by ezfrs containing the l / r and l / r heterodimeric configurations , respectively ( figure 4c ) . compared to the standard zfr architecture ( 2 of 17 clones ; 11% ) , the l / r ezfr heterodimers demonstrated a significant increase in targeted integration ( = 9.23 , p < 0.03 ) , while the l/ ezfr configuration did not ( = 0.99 , p > 0.8 ) . surprisingly , we observed that the standard zfrs targeting human chromosome 4 induced high toxicity , leading to a 60% decrease in cell viability after 5 days at the highest concentrations tested ( figure 5a ) . to explore the feasibility of this goal using ezfrs , we constructed a 6.25-kb donor plasmid containing ( i ) the zfr target site from human chromosome 4 , ( ii ) a puromycin - resistance gene , and ( iii ) the cdna for one of two disease - associated genes : the human coagulation factor ix ( fix ) gene , whose deficiency leads to hemophilia b , and the human -galactosidase ( gla ) gene , which is necessary for lipid metabolism and whose mutation results in the metabolic disorder known as fabry s disease ( figure 4a ) . co - transfection of hek293 cells with donor plasmid and ezfrs with the l / r dimeric configuration led to efficient integration of each therapeutic factor into the intended target site on chromosome 4 , and puromycin selection revealed a genome - wide ezfr - mediated integration rate of 0.3 and 0.4% for the fix- and gla - harboring donor plasmids , respectively ( figure 6a ) . we evaluated ezfr - mediated integration specificity by pcr analysis of individual puromycin - resistant clones and found that 9 of 12 ( 75% ) and 8 of 10 ( 80% ) clones contained fix and gla cdna , respectively , at the intended genomic target site ( figure 6b ) , which was verified by dna sequencing . notably , the specificity of transgene insertion achieved by these ezfrs is similar to those reported for zfns , talens , and crispr / cas systems , indicating that ezfrs are effective tools for site - specific integration into the human genome . lastly , toward characterizing the full integration landscape of the ezfrs , we computationally identified four potential off - target sites that contain up to three mismatches compared to the intended genomic target , and evaluated off - target integration from genomic dna isolated from puromycin - resistant cells that were negative for targeted integration . in particular , the establishment of a new class of tools capable of specifically and safely delivering large payloads into the human genome would be broadly useful across diverse fields , including basic research , gene therapy and synthetic biology . in this study , we have combined rational design and directed evolution to redesign the serine recombinase dimer interface to prevent formation of these deleterious homodimers , leading to the generation of a new class of hybrid recombinases that preferentially heterodimerize and catalyze site - specific integration into endogenous genomic loci with high specificity . these results , and in particular our finding that ezfrs specifically introduce the human coagulation factor ix and -galactosidase genes into the human genome with minimal toxicity , support the continued development of this technology for potential therapeutic applications . in comparison to published results in similar cell lines , the ezfrs containing the l / r dimeric configuration directed site - specific integration with specificities comparable to zfns , talens and crispr / cas9 systems ; however , the efficiency of ezfr - mediated integration remained lower than those typically observed with site - specific nuclease technologies . because zfr - mediated recombination necessitates the formation of covalent protein dna linkages that may activate the nhej repair pathway , we suspect that the high levels of cell death induced by the wild - type zfr pair could be attributed to excessive amounts of cleavage at pseudo - recombination sites by zfr homodimers or zfr - mediated rearrangements spurred by the presence of excess homodimers . thus , the dramatic reduction in toxicity observed with ezfrs can likely be attributed to the ability of the re - engineered dimer interface to prevent recombination by side - product homodimers . the improved efficiency and specificity demonstrated by ezfrs , as well as their ability to promote site - specific integration in the absence of dsbs , suggests that these tools might also be used to modify model organisms refractory to current genome engineering methods . although it remains unknown whether the previously described tal effector architecture supports our expanded collection of gin recombinase catalytic domains , the dimer interface substitutions described here should nonetheless improve the specificity of any tal effector recombinase heterodimer that consists solely of the wild - type gin catalytic domain . in summary , our findings provide a general means for improving the targeting efficiency , specificity , and safety of customizable recombinases and illustrate the potential of these enzymes for diverse genome engineering applications , including therapeutic gene transfer . zfr donor plasmids ( pdonor ; previously pbabe - puromycin ) were constructed as previously described with the following exceptions : cdna for the human coagulation factor ix ( fix ) and -galactosidase ( gla ) genes ( genecopoeia ) were pcr amplified with the primers psti - cmv - donor - fwd and bamh1-zfr - donor - rev . residues 115 through 144 of the gin catalytic domain and the h1 zinc - finger protein were pcr amplified from pbla - gin--h1with the primers gin - dimer - fwd and puc18-prim-1 and fused to the gin library by overlap pcr with the primers puc18-prim-1 and -2 . for zfr construction , the gin , , , , , and catalytic domains were pcr amplified from the previously described templates pbla - gin- , , , , , or as two fragments with the primers gin - hbs - d12g - koz and gin - ykwt - rev or gin - f103r - rev and gin - ykwt - fwd or gin - f103r - fwd and gin - agei - rev . pcr products were fused by overlap pcr with the primers gin - hbs - d12g - koz and gin - agei - rev and cloned into the hindiii and agei restriction sites of pbh to generate the new superzif - compatible sub - cloning vectors : pbh - d12g - gin-- , - , - , - , - , or -ykwt or f103r ( ykwt denotes the mutations m100y , f104k , v107w , and m108 t ) . previously constructed zinc - finger domains were ligated into the agei and spei restriction sites of the appropriate pbh - gin sub - cloning vector to generate pbh - ezfr - l - or - r-1 , 2 , 3 , 4 , or 5 ( ezfr : enhanced zfr ; l : left ezfr ; r : right ezfr ) . at 24 h after seeding , cells were transfected with 2550 ng of pcdna - ezfr - l-1 through 6 , 2550 ng of pcdna - ezfr - r-1 through 6 , 25 ng of pgl3-zfr , and 1 ng of prl - cmv ( promega ) using lipofectamine 2000 ( invitrogen ) according to the manufacturer s instructions . zfr targets and gapdh were pcr amplified from bulk genomic dna by nested pcr with the following primer combinations : gapdh - external - fwd and gapdh - external - rev , gapdh - internal - fwd and gapdh - internal - rev ( control ) ; zfr-1-external - rev and cmv - external , zfr-1-internal - rev and cmv - internal ( forward integration ) ; zfr-1-external - fwd and cmv - external , zfr-1-internal - fwd and cmv - internal ( reverse integration ) . for pdonor vectors that harbored the human fix and gla genes , the following primers were used for internal pcr : zfr-1-internal - rev and fix - internal ( fix forward integration ) ; zfr-1-internal - fwd and fix - internal ( fix reverse integration ) ; zfr-1-internal - rev and gla - internal ( gla forward integration ) ; zfr-1-internal - fwd and gla - internal ( gla reverse integration ) .

worldwide , squamous cell carcinoma of the head and neck ( scchn ) is the sixth most common cancer and is diagnosed in more than 600,000 patients each year . a better understanding of its biology has been accompanied by the introduction of a large and rapidly expanding number of targeted agents into its management strategies . planned and ongoing trials in scchn involving targeted agents are summarized in tables 1 , 2 , 3 , and 4 . potential targets include the epidermal growth factor receptor ( egfr ) and the erbb family , the vascular endothelial growth factor ( vegf ) and its receptor ( vegfr ) , insulin - like growth factor 1 receptor ( igf-1r ) , insulin receptor ( ir ) , histone deacetylase ( hdac ) , mammalian target of rapamycin ( mtor ) , platelet - derived growth factor ( pdgfr ) , heat - shock protein 90 ( hsp90 ) , nuclear factor - kappa b ( nf-b ) , aurora a or b , phosphatidylinositol 3-kinase ( pik3ca ) . the epidermal growth factor receptor ( egfr ) belongs to the erbb family of receptors which includes four members : egfr / erbb1 , erbb2/her2/neu , erbb3 , and erbb4 . egfr consists of an extracellular n - terminal ligand - binding domain , a hydrophobic transmembrane domain , and a c - terminal intracellular domain , which includes the tyrosine kinase domain and the autophosphorylation sites . the epidermal growth factor receptor ( egfr ) is overexpressed in the vast majority of cases of squamous cell carcinoma of the head and neck ( scchn ) . a high egfr expression and an increased egfr gene copy number are associated with an unfavorable prognosis [ 57 ] . two of the potential egfr targeting strategies that are currently in use in the treatment of scchn are the monoclonal antibodies directed at the extracellular domain of the receptor and the small molecule and atp - competitive tyrosine kinase inhibitors ( tkis ) . cetuximab is the only targeted agent that got approval by the food and drug administration and the european medicines agency for the treatment of scchn [ 8 , 9 ] and is still under active investigation in this disease ( tables 1 and 2 ) . cetuximab is a chimeric human / murine igg1 antibody which binds with higher affinity to the egfr than the natural ligands egf and tnf- , thereby disrupting egfr signaling pathways . another mechanism that contributes to the antitumor activity of cetuximab is depletion of the targeted receptors from the cell surface . the availability of egfr on the cell surface is reduced , and the receptor is downregulated [ 10 , 11 ] . finally , cetuximab 's construction on an igg1 framework potentially allows the drug to mediate antibody - dependent cell - mediated cytotoxicity ( adcc ) via natural killer ( nk ) cells and macrophages . cetuximab is administered once a week at an initial loading of 400 mg / m followed by a weekly dose of 250 mg / m . the recommended dose was used in the cetuximab studies in scchn studies mentioned below , unless stated otherwise . bonner et al . [ 13 , 14 ] conducted a multinational , randomized study comparing radiotherapy alone with radiotherapy plus cetuximab in 424 patients with stages iii or iv , nonmetastatic , measurable squamous cell carcinoma ( scc ) of the oropharynx , hypopharynx , or larynx . investigators were required to select one of three radiotherapy - fractionation regimens before patient registration : 70.0 gy in 35 daily fractions of 2.0 gy / fraction , 5 fractions / week for 7 weeks , or two daily fractions of 1.2 gy / fraction up to 72.076.8 gy in 6064 fractions , 10 fractions / week for 66.5 weeks , or a concomitant boost regimen ( 72.0 gy in 42 fractions : 32.4 gy ; 1.8 gy / fraction , 5 fractions / week for 3.6 weeks followed by a morning dose of 21.6 gy in fractions of 1.8 gy , 5 fractions / week for 2.4 weeks and an afternoon dose of 18.0 gy in fractions of 1.5 gy , 5 fractions / week for 2.4 weeks ) . uninvolved nodal areas of the neck were treated with 50 to 54 gy , depending on the fractionation regimen used . gross nodal disease received the same dose as the primary tumor . in the group assigned to receive radiotherapy plus cetuximab , cetuximab was initiated one week before radiotherapy at a loading dose of 400 mg / m , followed by a weekly dose of 250 mg / m for the duration of radiotherapy . the median duration of locoregional control ( primary endpoint ) was 24.4 months in patients treated with cetuximab plus radiotherapy and 14.9 months in patients treated with radiotherapy alone ( hazard ratio ( hr ) for locoregional progression or death , 0.68 ; p = 0.005 ) . the one- , two- , and three - year rates of locoregional control achieved with radiotherapy plus cetuximab ( 63 , 50 , and 47% ) , were significantly higher than those achieved with radiotherapy alone ( 55 , 41 , and 34% , resp . ) . median overall survival ( os ) for patients treated with cetuximab and radiotherapy was 49.0 months versus 29.3 months in the radiotherapy - alone group ( hr for death : 0.73 ; p = 0.018 ) . , the addition of cetuximab did not lead to an increased incidence of radiation dermatitis . however , as there is only one randomized phase iii trial with cetuximab - based bioradiation as opposed to the abundance of data supporting cisplatin - based concurrent chemoradiation ( crt ) [ 15 , 16 ] , the latter continues to represent the standard of care for medically fit patients with locoregionally ( la ) scchn , who can tolerate platinum - based therapy . the addition of cetuximab to cisplatin - based crt does not further improve the outcome . in radiation therapy oncology group ( rtog ) 0522 , 895 evaluable patients with stage iii / iv nonmetastatic scchn were randomized between chemoradiation ( 72 gy in 42 fractions over 6 weeks plus cisplatin 100 mg / m on days 1 and 22 ) or the same regimen plus weekly cetuximab . at the time of the third interim analysis after 337 events and after a median followup of 2.4 years for the surviving patients , the conditional power of the trial becoming positive was below 10% , triggering early reporting . over 90% of the patients received the planned two doses of cisplatin in both arms . the 2-year progression - free survival ( pfs ) , ( primary endpoint ) was 64.3% with chemoradiation and 63.4% with chemoradiation plus cetuximab ( hr : 1.05 ; 95% confidence interval ( ci ) : 0.841.29 ; p = 0.67 ) . the 2-year os was 79.7 and 82.6% , respectively ( hr : 0.87 ; 95% ci : 0.661.15 ; p = 0.17 ) . the estimated 2-year locoregional relapse rate was 19.8 and 24.5% , respectively ( p = 0.92 ) . the 2-year distant metastasis rate was 12 and 7.6% , respectively ( p = 0.07 ) . overall , there was no difference regarding acute grade 3/4 acute toxicities between both arms . however , grade 3/4 mucositis ( 43 versus 33% ) and in - field dermatitis ( 25 versus 15% ) was more common with the addition of cetuximab . grade 3/4 dermatitis outside the radiation field occurred in 19% of the patients treated with cetuximab . the tremplin trial is a randomized phase ii study in patients with scc of the larynx or hypopharynx suitable for total laryngectomy . after three 3 weekly cycles of tpf ( docetaxel 75 mg / m and cisplatin 75 mg / m on day 1 followed by 5-fu 750 mg / m/day , days 15 ) , patients who obtained at least a partial response ( 82% of the patients ) were randomized to receive radiotherapy ( 70 gy in 35 fractions over 7 weeks ) either with cisplatin 100 mg / m on days 1 , 22 , and 43 or with weekly cetuximab . the treatment compliance was better in the cetuximab arm with 71% of the patients receiving all planned cetuximab administrations . forty - three percent of the patients received three cycles of cisplatin , and 83% received 2 cycles . there was no difference in grade 3/4 mucosal toxicity , but grade 3/4 in - field dermatitis was more frequently observed with cetuximab ( 57 versus 26% ; p < 0.001 ) . grade 1 renal dysfunction at last evaluation was observed in 22.4% of the patients treated with cisplatin . the larynx preservation rate 3 months after treatment ( primary endpoint ) was 95% with cisplatin versus 93% with cetuximab . the locoregional failure rate after a median followup of 36 months was 11.7% with cisplatin and 21.4% with cetuximab . however , more salvage laryngectomies were performed in the cetuximab arm , resulting in a similar ultimate locoregional failure rate in the two arms ( 10% versus 8.9% ) . the 2-year laryngoesophageal dysfunction - free survival was 79% with cisplatin versus 71% with cetuximab ( p = 0.3 ) . randomized 110 patients with la scchn , who had received 2 cycles of carboplatin , paclitaxel , and cetuximab as induction chemotherapy , between weekly cetuximab in combination with either 5-fu , hydroxyurea , and hyperfractionated week - on week - off radiotherapy ( 7274 gy ) ( cetuxfhx ) , or cisplatin , accelerated radiation with concomitant boost ( cetuxpx ) ( 72 gy ) . after a median followup of 21 months , 2-year os rates were 89.5% with cetuxfhx and 91.4% with cetuxpx arm . two - year pfs rates were 82.3% and 89.7% , respectively ( p = 0.18 ) . grade 3 mucositis was present in 91.1% ( cetuxfhx ) and 94.3% ( cetuxpx ) of patients ; grade 3 dermatitis in 82.1% and 50.9% , respectively . ninety - five percent of patients completed therapy , demonstrating that cetuximab can be incorporated safely in both crt platforms . argiris et al . enrolled 32 patients in a phase i study combining pemetrexed , cetuximab and radiotherapy in poor prognosis head and neck cancer . cohort a included patients who had not been previously irradiated , while cohort b included patients who had received prior irradiation . the maximum tolerated dose ( mtd ) of pemetrexed was 500 mg in cohort a and 350 mg / m in cohort b. grade 3/4 neutropenia was common ( 50% in cohort a and 33% in cohort b ) and febrile neutropenia was the most frequent doselimiting toxicity . grade 3/4 mucositis was observed in 8 of the 9 patients treated at the mtd in cohort a . in eastern cooperative oncology group ( ecog ) e2303 , 63 patients with resectable stage iii / iv scchn , were treated with 6-week cycles of paclitaxel , carboplatin at an auc of 2 and cetuximab , followed by crt ( weekly paclitaxel , 30 mg / m , carboplatin auc 1 , and cetuximab ) . if at week 14 , after a radiation dose of 50 gy , tumor was still present on biopsy , salvage surgery was performed . in case of a negative biopsy ( 91% of the patients ) , crt was continued to a total dose of 6872 gy . jordan et al . treated 152 t3-t4 scchn patients with three 3-week cycles of tpf ( docetaxel 75 mg / m on day 1 , cisplatin 35 mg / m on days 1 and 2 , and 5-fu 750 mg / m/day , as a continuous infusion , days 15 , with pegfilgrastim support ) followed by chemoradiation ( 63 gy in 35 fractions of 1.8 gy over 7 weeks and weekly cisplatin , 40 mg / m ) plus weekly cetuximab . the complete response rate in the 142 patients , who were evaluated after the completion of therapy , was 57% . ferris et al . treated 34 la scchn patients with three 3-weekly cycles of cisplatin 75 mg / m and docetaxel 75 mg / m plus weekly cetuximab followed by crt ( 70 gy in 2 gy fractions over 7 weeks , weekly cisplatin 30 mg / m ) plus weekly cetuximab , followed by weekly cetuximab maintenance for 6 months . mesia et al . studied the role of cetuximab maintenance therapy in patients with la scc of the oropharynx . patients in group a were treated concomitant radiotherapy , 69.9 gy in 28 days , plus weekly cetuximab . the locoregional control rate at 1 year ( primary endpoint ) was 56.8% with bioradiation and 60.5% with bioradiation followed by cetuximab maintenance . at 1 year , event - free survival rates were 55.6 and 60.9% , respectively , and os rates were 75.6 and 87% , respectively . in the extreme trial ( erbitux in first - line treatment of recurrent or metastatic head and neck cancer ) , 442 patients with previously untreated r / m scchn were randomized to receive cisplatin 100 mg / m or carboplatin at an area under the curve ( auc ) of 5 mg / mg / min as an 1-hour infusion , followed by 5-fu 1000 mg / m day for 4 days as a continuous infusion every 3 weeks for a maximum of 6 cycles , either alone or in combination with cetuximab . after a maximum of six cycles of chemotherapy , patients in the cetuximab group who had at least stable disease received cetuximab monotherapy until disease progression or unacceptable toxic effects , whichever occurred first . the median overall survival ( primary endpoint ) was 10.1 months ( 95% ci : 8.611.2 ) in the cetuximab group and 7.4 months ( 95% ci : 6.48.3 ) in the chemotherapy - alone group ( hr for death : 0.80 ; 95% ci : 0.640.99 ; p = 0.04 ) . the median followup was 19.1 months in the cetuximab group and 18.2 months in the chemotherapy - alone group . median pfs was 5.6 months and 3.3 months for the combined group and the chemotherapy alone group , respectively ( hr for progression : 0.54 ; 95% ci : 0.430.67 ; p < 0.001 ) . the overall response rate ( orr ) was 36 and 20% , respectively ( odds ratio ( or ) : 2.33 ; p < 0.001 ) , and the disease control rate ( dcr ) was 80 and 60% , respectively ( or : 2.88 ; p < 0.001 ) . among the 100 patients who received cetuximab as maintenance treatment , the median pfs was 12 weeks from the start of maintenance treatment . the safety profile of the study treatment was consistent with that expected for the agents used , with no significant difference in the incidence of grades 3 or 4 adverse events between treatment arms . however , there were 9 cases of sepsis in the cetuximab group , as compared with 1 case in the chemotherapy - alone group ( p = 0.02 ) , and there were 11 cases of hypomagnesemia in the cetuximab group , as compared with 3 cases in the chemotherapy - alone group ( p = 0.05 ) . there were four grade 3 and two grade 4 infusion - related reactions after cetuximab . ecog randomized 117 r / m scchn patients to receive cisplatin 100 m / m every 4 weeks plus either weekly cetuximab ( group a ) or placebo ( group b ) . median pfs was 4.2 months in arm a and 2.7 in arm b ( hr : 0.78 ; 95% ci : 0.541.12 ; p = 0.09 ) . median os was 9.2 months in arm a and 8.0 months in arm b ( p = 0.21 ) . hitt et al . treated 46 patients with weekly cetuximab and paclitaxel 80 mg / m as first - line regimen for recurrence of metastatic scchn . common grade 3/4 adverse events were acne - like rash ( 24% ) , asthenia ( 17% ) , and neutropenia ( 13% ) . in groupe d'oncologie radiothrapie tte et cou ( gortec ) 200803 , 54 patients who had not received prior chemotherapy for r / m scchn were treated with docetaxel 75 mg / m and cisplatin 75 mg / m every 3 weeks and weekly cetuximab , up to 4 cycles , followed by cetuximab maintenance in the absence of disease progression . the orr and dcr were 51.9 and 96.1% , respectively . at time of reporting , high egfrviii and amphiregulin expression levels identify scchn patients who are less likely to benefit from combination treatment with cetuximab and docetaxel . three phase ii trials examined the role of cetuximab in platinum - refractory or platinum - resistant disease [ 3033 ] . responses ( 1013% ) were observed irrespective of reintroducing the originally used platinum compound to which they had become refractory or resistant . the survival of around 6 months achieved with cetuximab in platinum - refractory disease was found similar to that seen in first - line therapy in r / m - scchn and represented an increase in survival of 2.5 months compared with platinum - refractory historical controls . based on these results and particularly considering the fact that about 50% of the patients showed disease control , cetuximab monotherapy seems to be an option for patients with r / m scchn who have progressed on platinum - based chemotherapy . randomized 61 patients , who had received 2 prior cytotoxic chemotherapy regimens for r / m scchn to receive cetuximab every 2 weeks at either 500 ( a ) or 750 mg / m ( b ) . cetuximab 500 mg / m every 2 weeks seemed to yield similar efficacy and tolerability as conventional weekly dosing for patients with r / m scchn . however , it is unclear how many of the patients in this study had platinum - refractory disease . there is no apparent efficacy advantage associated with dose escalation to 750 mg / m q2w . randomly allocated 286 eligible incurable scchn in a 2 : 1 ratio to receive either zalutumumab plus best supportive care ( zalutumumab group ) or best supportive care with optional methotrexate ( control group ) . eligible were patients with progressive disease according to recist confirmed by an independent review committee during or within 6 months after the failure of platinum - based chemotherapy ( at least two cycles of cisplatin [ 60 mg / m per cycle ] or carboplatin [ 250 mg / m per cycle ] with an interval between the cycles of < 4 weeks ) . also eligible were patients with platinum intolerance which was defined as discontinuation or dose reduction of platinum - based chemotherapy due to adverse or toxic effects , irrespective of response . patients were given an initial loading dose of 8 mg / kg followed up by two week doses of 4 mg / kg by intravenous infusion in 1 h. after the first three administrations , in patients with no rash or grade 1 rash , the dose was increased by 4 mg / kg every 2 weeks up to a maximum dose of 16 mg / kg . treatment was withheld from patients with grade 3 rash until rash resolved to grade 1 . seventy - two percent of the control patients received methotrexate from the initiation of the trial , and a further 6% started methotrexate during the trial . median os ( primary endpoint ) was 6.7 months ( 95% ci : 5.87.0 ) in the zalutumumab group and 5.2 months ( 95% ci : 4.16.4 ) in the control group ( hazard ratio ( hr ) for death , stratified by who performance status : 0.77 ; 97.06% ci : 0.571.05 ; unadjusted p = 0.0648 ) . progression - free survival was longer in the zalutumumab group than in the control group ( hr for progression or death , stratified by who performance status : 0.63 ; 95% ci : 0.470.84 ; p = 0.0012 ) . the most common grade 3/4 adverse events were rash ( 21% of patients in the zalutumumab group versus none in the control group ) , anemia ( 6% and 5% , resp . ) , and pneumonia ( 5% and 2% , resp . ) . grade 3/4 infections occurred in 15% of the patients in the zalutumumab group and 9% in the control group . its pharmacokinetic profile allows a convenient three - week administration . in the spectrum trial ( study of panitumumab efficacy in patients with recurrent and/or metastatic head and neck cancer ) , 657 patients were randomized between cisplatin 100 mg / m on day 1 , followed by 5-fluorourcacil 1000 mg / m/day for 4 days or the same chemotherapy plus panitumumab 9 mg / kg administered on day 1 . patients were allowed to switch from cisplatin to carboplatin ( auc 5 ) during treatment for specific cisplatin - related toxicities , such as grade 2 neurotoxicity or a drop in creatinine clearance to < 50 ml / min . the median os in the combined arm was 11.1 months compared to 9.0 months in the chemotherapy alone arm ( hr = 0.87 ; 95% ci : 0.731.05 ; p = 0.14 ) . however , there was a statistically significant difference in response rate ( 36% versus 25% ; p = 0.007 ) and pfs ( median 5.8 months versus 4.6 months ; hr = 0.78 ; 95% ci : 0.660.92 ; p = 0.004 ) in favour of the panitumumab - containing arm . although the spectrum trial failed to meet its primary endpoint , the results are nevertheless consistent with the outcome of the extreme trial . nimotuzumab is a humanized egfr targeting monoclonal antibody which was studied in multiple phase ii trials . a weekly fixed dose of 200 mg was established as recommended dose for use in combination with radiotherapy in la scchn . no grade 3/4 adverse events were reported in a pilot study of weekly nimotuzumab ( 200 mg ) , radiotherapy ( 66 gy in 33 fractions/2 gy per fraction over 6.5 weeks ) and weekly cisplatin ( 40 mg / m ) in 17 patients with la scchn . enrolled 106 patients with inoperable la scchn in a double blind , randomized phase ii trial . the primary endpoint of the trial was the complete response rate , which was 59.5% with nimotuzumab versus 34.2% with placebo ( p = 0.028 ) . patients included in group a were treated with radiation ( 6066 gy ) with or without nimotuzumab . at 48 months of followup , os was 34% with rt plus nimotuzumab versus 13% with radiotherapy alone . patients included in group b were treated with crt ( 6066 gy plus weekly cisplatin at a dose of 50 mg ) with or without weekly nimotuzumab . at 48 months of followup , os rate was 47% with crt plus nimotuzumab versus 21% with crt ( p = 0.01 ) . tyrosine kinase inhibitors which have been tested in scchn include gefitinib and erlotinib , which are reversible specific egfr tkis , lapatinib , a reversible dual egfr / her2 tki , afatinib , an irreversible dual egfr / her2 tki , and pf-00299804 , a potent irreversible pan - her tki . planned to randomize 330 patients to receive docetaxel 35 mg / m on days 1 , 8 , and 15 every 28 days either plus placebo or in combination with gefitinib 250 mg / day . the data monitoring committee recommended early stopping of enrollment after inclusion of 270 patients because there was < 5% chance to meet the primary endpoint ( overall survival ) . eligible were patients who were previously treated with chemotherapy for r / m scchn ( 73% of the patients ) and patients previously untreated for r / m scchn either with a poor performance status ( ecog 2 ) or in case of relapse within 6 months after chemotherapy given as part the primary treatment with curative intent . median os was 6.8 months with docetaxel plus placebo versus 6.2 months with docetaxel plus gefitinib ( hr 0.99 ; 95% ci : 0.751.31 ; p = 0.97 ) . the time to progression was significantly longer with the addition of gefitinib ( median 3.5 months versus 2.1 months ; hr 0.69 ; 95% ci : 0.490.99 ; p = 0.047 ) . in the imex trial , 486 r / m scchn patients were randomly assigned to oral gefitinib 250 mg / day , gefitinib 500 mg / day , or methotrexate 40 mg / m intravenously weekly . physicians and patients were blinded to the gefitinib dose . patients were stratified into two groups : group a ( n = 256 ) consisted of patients who had stable or progressive disease after at least two cycles of platinum - based chemotherapy for recurrent disease ; group b ( n = 230 ) consisted of patients who were considered unsuitable for platinum - containing chemotherapy . neither gefitinib 250 mg / day nor gefitinib 500 mg / day improved os compared with methotrexate ( hr : 1.22 ; 95% ci : 0.951.57 ; p = 0.12 ; hr : 1.12 ; 95% ci : 0.871.43 ; p = 0.39 , resp . ) . median os was 5.6 , 6.0 , and 6.7 months in the gefitinib 250 mg / day , gefitinib 500 mg / day , and methotrexate groups , respectively . in group a , os was significantly longer with methotrexate ( hr for death : gefitinib 250 mg versus methotrexate : 1.62 ; p = 0.01 ; gefitinib 500 mg versus methotrexate : 1.5 ; p = 0.02 ) . tumor hemorrhage - type events were more common with gefitinib ( 250 and 500 mg ) than with methotrexate ( 8.9% , 11.4% , and 1.9% , resp . ) . the most common adverse events were rash , diarrhea , cancer pain , nausea , and vomiting with gefitinib , and stomatitis , nausea , and constipation with methotrexate.the os with gefitinib in the imex trial was similar to what was observed in earlier uncontrolled phase ii studies with gefitinib or erlotinib [ 4346 ] . dose escalation of gefitinib adaptive to skin toxicity grade did not improve response rate in a phase ii trial conducted by perez et al . . argiris et al . planned to randomize 330 patients to receive docetaxel 35 mg / m on days 1 , 8 , and 15 every 28 days either plus placebo or in combination with gefitinib 250 mg / day . the data monitoring committee recommended early stopping of enrollment after inclusion of 270 patients because there was < 5% chance to meet the primary endpoint ( overall survival ) . eligible were patients who were previously treated with chemotherapy for r / m scchn ( 73% of the patients ) and patients previously untreated for r / m scchn either with a poor performance status ( ecog 2 ) or in case of relapse within 6 months after chemotherapy given as part the primary treatment with curative intent . median os was 6.8 months with docetaxel plus placebo versus 6.2 months with docetaxel plus gefitinib ( hr 0.99 ; 95% ci : 0.751.31 ; p = 0.97 ) . the time to progression was significantly longer with the addition of gefitinib ( median 3.5 months versus 2.1 months ; hr 0.69 ; 95% ci : 0.490.99 ; p = 0.047 ) . in the imex trial , 486 r / m scchn patients were randomly assigned to oral gefitinib 250 mg / day , gefitinib 500 mg / day , or methotrexate 40 mg / m intravenously weekly . patients were stratified into two groups : group a ( n = 256 ) consisted of patients who had stable or progressive disease after at least two cycles of platinum - based chemotherapy for recurrent disease ; group b ( n = 230 ) consisted of patients who were considered unsuitable for platinum - containing chemotherapy . neither gefitinib 250 mg / day nor gefitinib 500 mg / day improved os compared with methotrexate ( hr : 1.22 ; 95% ci : 0.951.57 ; p = 0.12 ; hr : 1.12 ; 95% ci : 0.871.43 ; p = 0.39 , resp . ) . median os was 5.6 , 6.0 , and 6.7 months in the gefitinib 250 mg / day , gefitinib 500 mg / day , and methotrexate groups , respectively . in group a , os was significantly longer with methotrexate ( hr for death : gefitinib 250 mg versus methotrexate : 1.62 ; p = 0.01 ; gefitinib 500 mg versus methotrexate : 1.5 ; p = 0.02 ) . tumor hemorrhage - type events were more common with gefitinib ( 250 and 500 mg ) than with methotrexate ( 8.9% , 11.4% , and 1.9% , resp . ) . the most common adverse events were rash , diarrhea , cancer pain , nausea , and vomiting with gefitinib , and stomatitis , nausea , and constipation with methotrexate . the os with gefitinib in the imex trial was similar to what was observed in earlier uncontrolled phase ii studies with gefitinib or erlotinib [ 4346 ] . dose escalation of gefitinib adaptive to skin toxicity grade did not improve response rate in a phase ii trial conducted by perez et al . . randomized 34 patients with resectable scchn to receive erlotinib daily for 2 to 8 weeks prior to surgery at standard ( 150 mg ) or high doses ( 200 mg in never / former smokers , and 300 mg in current smokers ) . response rates were documented in 18% and 29% of the patients in the low - and high - dose arms , respectively , suggesting that a subgroup of previously untreated scchn is highly sensitive to egfr tyrosine kinase inhibition.hayes et al . randomly assigned 128 patients with la scchn to receive either cisplatin 100 mg / m on days 1 , 22 , and 43 combined with 70 gy of radiotherapy ( arm a ) or the same treatment plus 150 mg of erlotinib starting one week before crt and continued until the completion of radiotherapy ( arm b ) . serious adverse events were observed in 33% and 32% of the patients in arm a and b , respectively . most common serious adverse events were nausea , vomiting , and dehydration and accounted for 30% of all saes reported , with no difference between arms . gregoire et al . enrolled 226 patients in a randomized phase ii trial testing the value of gefitinib during and/or after crt in la scchn . patients received either placebo during crt followed by adjuvant placebo , or gefitinib 250 mg or 500 mg / day during crt followed by placebo , gefitinib 250 mg or 500 mg / day during and after crt , or placebo during crt followed by adjuvant gefitinib at a dose of 250 mg or 500 mg / day . there was no difference in 2-year local disease control rate ( primary endpoint ) between the 7 treatment arms . evaluated the toxicity and recommended dose for adjuvant erlotinib after definitive crt for la scchn . no dose limiting toxicities were observed at a daily dose of 100 or 150 mg for 6 months . at the 150 mg dose , randomized 34 patients with resectable scchn to receive erlotinib daily for 2 to 8 weeks prior to surgery at standard ( 150 mg ) or high doses ( 200 mg in never / former smokers , and 300 mg in current smokers ) . response rates were documented in 18% and 29% of the patients in the low - and high - dose arms , respectively , suggesting that a subgroup of previously untreated scchn is highly sensitive to egfr tyrosine kinase inhibition . . randomly assigned 128 patients with la scchn to receive either cisplatin 100 mg / m on days 1 , 22 , and 43 combined with 70 gy of radiotherapy ( arm a ) or the same treatment plus 150 mg of erlotinib starting one week before crt and continued until the completion of radiotherapy ( arm b ) . serious adverse events were observed in 33% and 32% of the patients in arm a and b , respectively . most common serious adverse events were nausea , vomiting , and dehydration and accounted for 30% of all saes reported , with no difference between arms . gregoire et al . enrolled 226 patients in a randomized phase ii trial testing the value of gefitinib during and/or after crt in la scchn . patients received either placebo during crt followed by adjuvant placebo , or gefitinib 250 mg or 500 mg / day during crt followed by placebo , gefitinib 250 mg or 500 mg / day during and after crt , or placebo during crt followed by adjuvant gefitinib at a dose of 250 mg or 500 mg / day . there was no difference in 2-year local disease control rate ( primary endpoint ) between the 7 treatment arms . evaluated the toxicity and recommended dose for adjuvant erlotinib after definitive crt for la scchn . no dose limiting toxicities were observed at a daily dose of 100 or 150 mg for 6 months . at the 150 mg dose , encouraging preliminary results in r / m scchn after failure of a platinum - containing therapy were reported with afatinib , a dual egfr / her2 irreversible tyrosine kinase inhibitor , which was compared to single - agent cetuximab in a randomized phase ii study . enrolled 67 patients into a randomized , placebo - controlled , phase ii trial , exploring the potential benefit of adding lapatinib to crt in patients with la scchn . lapatinib ( 1500 mg / day ) or placebo were started 1 week before crt ( 70 gy in 35 fractions over 7 weeks plus cisplatin 100 mg / m on days 1 , 22 , and 43 ) and continued during and after crt until disease progression . the addition of lapatinib did not impair the timely administration of crt and did not lead to an increase in mucositis and radiation dermatitis . the complete response rate at 6 months after treatment ( primary endpoint ) was 36% with placebo and 53% with lapatinib . treated 107 patients with la scchn with either lapatinib or placebo for 26 weeks prior to crt . the overall response rate before crt in the 40 patients who received > 4 weeks of lapatinib was 17% . siu et al . enrolled 69 patients in a phase ii study with pf-00299804 at a dose of 45 mg qd , in previously untreated r / m scchn . grade 3 adverse events were diarrhea ( 16% ) , fatigue ( 9% ) , acneiform dermatitis ( 7% ) , and hand - foot reaction ( 4% ) . a meta - analysis conducted by kyzas et al . demonstrated that vegf protein overexpression , as detected with immunohistochemistry , is associated with a worse os in patients with scchn . bevacizumab is a humanized igg1 monoclonal antibody that binds selectively to all isoforms of human vegf and neutralizes the biologic activities of vegf by blocking the binding of vegf to its receptors on the surface of endothelial cells . combined pemetrexed 500 mg / m and bevacizumab 15 mg / kg given intravenously every 21 days until disease progression in 40 patients presenting with previously untreated r / m scchn . the median ttp ( primary endpoint ) was 5 months , and the median os was 11.3 months . in 37 evaluable patients , the orr was 30% , and the dcr was 86% . grade 3 bleeding events occurred in 6/40 patients ( 15% ) and was fatal in 2 ( 5% ) . bevacizumab , 10 mg / kg , administered on day 1 of each 2-week cycle , can be safely combined with fhx crt regimen , consisting of five 2-week cycles of hydroxyurea 500 mg orally bid , 5-fu 600 mg / m/day administered as a continuous infusion , and radiotherapy , 1.5 gy bid for 5 days followed by 9 days without therapy ( fhx ) , in patients with poor prognosis scchn . salama et al . conducted a randomized phase ii study evaluating the impact of adding bevacizumab ( b ) to the fhx regimen . eligible were patients with t1 - 3 , n0 - 1 and t4 , n0 - 1 scchn . two - year os was 89% in patients treated with fhx and 58% in patients treated with bfhx . these unexpected findings are not well understood and could be due to chance , given the small sample size . harari et al . demonstrated the safety and feasibility of combining crt ( 70 gy in 33 fractions and weekly cisplatin , 30 mg / m ) with bevacizumab weeks 3 , 1 , 4 , and 7 with dose escalation from 5 to 10 to 15 mg / kg in 10 patients with la scchn . sorafenib and sunitinib are oral inhibitors of multiple kinases including the receptor tyrosine kinases of vascular endothelial growth factor ( vegf ) receptor . elser et al . treated 27 patients with r / m scchn or nasopharyngeal carcinoma ( 7 patients ) , who had received 1 chemotherapy for recurrent or metastatic disease with sorafenib 400 mg twice daily on a continuous basis . the treatment was well tolerated with few grade 3 or 4 toxicities . however , anticancer activity was modest . the same regimen was evaluated in the southwest oncology group study s0420 , which enrolled 41 r / m scchn patients who had not received prior chemotherapy for r / m disease . however , the estimated median pfs was 4 months , and the estimated median os was 9 months . in the gortec 200601 study , 38 patients with r / m scchn received sunitinib 37.5 mg / day on a continuous basis . forty - five percent and 3% of the patients had received one and two prior chemotherapy regimens for r / m disease , respectively . local complications , including the appearance or worsening of tumor skin ulceration or tumor fistula , were recorded in 39.5% of the patients , and a fatal arterial bleeding in the head and neck region occurred in 10.5% of the patients . fountzilas et al . treated 17 r / m scchn patients with sunitinib 50 mg per day administrated in 4-week cycles followed by a rest period of 2 weeks as first - line treatment for r / m disease and observed no objective responses . disease control rate was 18% , median ttp was 2.3 months , and median os was 4 months . the same intermittent sunitinib regimen was used by choong et al . who observed an orr of 4.5% and a dcr of 27.3% . sabichi et al . combined paclitaxel , carboplatin auc 6 , administered every 3 weeks , and sorafenib 400 mg bid , days 219 , in patients with r / m scchn . vandetanib is an inhibitor of vascular endothelial growth factor receptor-2 ( vegfr-2 ) , egfr , and rearranged during transfection ( ret ) tyrosine kinases . it restores hnscc cells ' sensitivity to cisplatin and radiation in vitro and in vivo . as a single agent , it has antiproliferative effects on scchn cells in vitro and inhibits tumor growth in nude mice orthotopically injected with human scchn cells . vandetanib can be safely combined with radiotherapy ( 2.2 gy / d , 5 days / week up to a total dose of 66 gy ) or radiotherapy ( 2 gy / d , 5 days / week up to a total dose of 70 gy ) plus weekly 30 mg / m of cisplatin . heat - shock protein 90 ( hsp90 ) is a protein which chaperones multiple client oncoproteins involved in tumor progression . investigated the antitumor effect of the novel hsp90 inhibitor nvp - auy922 against oral scc ( oscc ) . nvp - auy922 inhibited the proliferation of oscc cells in vitro and induced a robust antitumor response and suppressed p - akt and vegf expression in an hsc-2 xenograft model . several sirtuin family members ( sirt1 - 7 ) function either as nicotinamide adenine dinucleotide ( nad)-dependent deacetylases or as adp - ribosyl transferases and are involved in carcinogenesis . . demonstrated that sirt3 is overexpressed in oscc in vitro and in vivo and that sirt3 downregulation inhibits oscc cell growth and proliferation and increased oscc cell sensitivity to radiation and cisplatin treatments in vitro . histone deacetylases are enzymes involved in remodeling of chromatin by deacetylating the lysine residues and play a pivotal role in epigenetic regulation of gene expression . hdac inhibitors have radio - enhancing properties [ 75 , 76 ] , increase the susceptibility of scc cell lines to cisplatin in vitro [ 77 , 78 ] , and inhibit tumor growth in xenograft models of scchn . . demonstrated that the histone deacetylase inhibitor vorinostat in combination with the egfr tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation , migration , and invasion as well as induction of apoptosis in scchn cells , including cells resistant to gefitinib . high expression of aurora a or b is associated with a worse outcome in scchn [ 8186 ] . combined a dual aurora a / aurora b inhibitor with cetuximab in scchn cell lines in vitro and observed at least an additive effect . preclinical data strongly support the testing of mammalian target of rapamycin ( mtor ) in scchn [ 8890 ] . activation of mtor is observed in the majority of scchn and is associated with a poor outcome . patel et al . found that inhibition of mtor diminished lymphangiogenesis in the primary tumors and prevented the dissemination of scchn cancer cells to the cervical lymph nodes in an orthotopic mouse model . temsirolimus enhances the growth - inhibiting effects of the combination of bevacizumab , cetuximab , and irradiation in head and neck cancer xenografts . aissat et al . demonstrated that rapamycin displays antiproliferative effects and induces apoptosis in scchn cell lines and that combination of rapamycin with paclitaxel or carboplatin displayed synergistic and additive effects . temsirolimus appeared to be a more potent radiosensitizer than cisplatin in mice bearing squamous cell carcinoma xenografts . in a pharmacodynamic evaluation of temsirolimus in scchn patients , ekshyyan et al . found a significant inhibition of the mtor pathways in tumor cells and in peripheral blood mononuclear cells . everolimus 10 mg / day , days 121 can be safely combined with cisplatin 20 mg / m , days 1 , 8 , and 15 of a 28-day cycle in patients with solid tumors . c - src is a nonreceptor cytoplasmic tyrosine kinase that regulates signals from cell surface molecules and that plays a key role in modulating multiple cellular functions by activating the signal transducer and activator of transcription ( stat ) family of transcription factors . although preclinical data provided a rationale for testing c - scr inhibitors in scchn , the outcome with single - agent c - scr inhibitors in patients with r / m scchn was disappointing . brooks et al . treated 15 r / m scchn patients with dasatinib , a potent inhibitor of src - family kinases epha2 , at a dose 100 mg twice daily . saracatinib is also an orally available src kinase inhibitor which was administered to 9 r / m scchn patients at a daily dose of 175 mg . eight patients had disease progression within the first eight - week cycle , and one patient was removed from the study after 11 days due to rapid clinical decline . the study was closed early due to lack of efficacy according to the early stopping rule . nuclear factor kappa b is overexpressed in scchn , and nf-b expression is associated with a poor prognosis . bortezomib is a small - molecule proteasome inhibitor which affects multiple signaling pathways including nf-b . chung et al . treated 25 r / m scchn patients with bortezomib 1.6 mg / m and docetaxel 40 mg / m on days 1 and 8 of a 21-day cycle and observed a orr and dcr of 5 and 52% , respectively . were observed in the phase i portion of the study which included 10 patients up to the maximum planned dose of 15 mg / kg of bevacizumab , 46 additional patients were treated at that dose level . forty - eight percent of the patients had received prior chemotherapy for recurrent / metastatic disease . the observed response rate was 15% with 4 complete responses allowing rejection of the null hypothesis that the response rate is 5% and the percentage of patients not progressing within two months is 30% . higher ratios of phosphorylated over total vegf receptor-2 and egfr in pretreatment biopsies were associated with complete response ( p = 0.036 and p = 0.036 , resp . ) and tumor shrinkage ( p = 0.007 and p = 0.008 , resp . ) in a subset of 11 subjects with available tissue . the feasibility and efficacy of adding bevacizumab and erlotinib to concurrent crt in patients with la scchn was evaluated in a phase ii trial conducted by the sarah cannon oncology research consortium including 60 previously untreated patients . the treatment consisted of induction chemotherapy with 6 weeks of paclitaxel , carboplatin , infusional 5-fluorouracil , and bevacizumab , which was followed by radiation therapy , weekly paclitaxel , bevacizumab , and erlotinib . after a median followup of 32 months , the estimated 3-year pfs and os rates are 71% and 82% , respectively . integrins promote and regulate endothelial cell proliferation , migration , invasion , and survival in tumors , securing vascularisation and vascular remodeling in tumors . advantage is a phase i / ii trial evaluating cilengitide in combination with cetuximab , cisplatin and 5-fluorouracil in patients with r / m scchn . no dlts were observed in the phase i part of the study which tested cilengitide ( 500 , 1000 , and 2000 mg ) twice weekly with standard doses of cetuximab , cisplatin and 5-fluorouracil . cilengitide 2000 mg was considered safe and was selected for the subsequent randomised phase ii part assessing pfs . the road from preclinical evidence to clinical use is long and bumpy , and a large number of targeted agents are still at the start of the race . some others reached the last stretch but stumbled on one of the last hurdles in phase ii , or even phase iii trials . thus far , only the egfr - directed monoclonal antibody cetuximab has made it to the finish and is currently approved for the treatment of locoregionally advanced and recurrent / metastatic scchn , by the regulatory agencies of the united states and europe .

the epidermal growth factor receptor ( egfr ) is overexpressed in the vast majority of cases of squamous cell carcinoma of the head and neck ( scchn ) . a high egfr expression is associated with an unfavorable prognosis . cetuximab is a chimeric human / murine igg1 antibody which binds with high affinity to the egfr . it is the only targeted agent which got approval for the treatment of scchn from the regulatory agencies of europe and the united states , both in locoregionally advanced disease , in association with radiation , and in recurrent / metastatic disease . the outcome of trials involving other egfr - directed monoclonal antibodies , that is , zalutumumab and panitumumab , was consistent with the results with cetuximab . however these trials failed to meet their primary endpoint . the results with egfr - directed tyrosine kinase inhibitors have been disappointing . other potential targets for treatment in scchn include the entire erbb family , the vascular endothelial growth factor ( vegf ) and its receptor ( vegfr ) , the insulin - like growth factor 1 receptor ( igf-1r ) , the insulin receptor ( ir ) , histone deacetylases ( hdac ) , the mammalian target of rapamycin ( mtor ) , the platelet - derived growth factor receptor ( pdgfr ) , heat - shock protein 90 ( hsp90 ) , nuclear factor - kappa b ( nf-b ) , aurora a or b , and phosphatidylinositol 3-kinase ( pik3ca ) .

1. Introduction 2. EGFR-Directed Therapies 3. VEGFR-Directed Therapies 4. Other Targets 5. Combination of Targeted Agents 6. Conclusion

worldwide , squamous cell carcinoma of the head and neck ( scchn ) is the sixth most common cancer and is diagnosed in more than 600,000 patients each year . planned and ongoing trials in scchn involving targeted agents are summarized in tables 1 , 2 , 3 , and 4 . potential targets include the epidermal growth factor receptor ( egfr ) and the erbb family , the vascular endothelial growth factor ( vegf ) and its receptor ( vegfr ) , insulin - like growth factor 1 receptor ( igf-1r ) , insulin receptor ( ir ) , histone deacetylase ( hdac ) , mammalian target of rapamycin ( mtor ) , platelet - derived growth factor ( pdgfr ) , heat - shock protein 90 ( hsp90 ) , nuclear factor - kappa b ( nf-b ) , aurora a or b , phosphatidylinositol 3-kinase ( pik3ca ) . the epidermal growth factor receptor ( egfr ) belongs to the erbb family of receptors which includes four members : egfr / erbb1 , erbb2/her2/neu , erbb3 , and erbb4 . egfr consists of an extracellular n - terminal ligand - binding domain , a hydrophobic transmembrane domain , and a c - terminal intracellular domain , which includes the tyrosine kinase domain and the autophosphorylation sites . the epidermal growth factor receptor ( egfr ) is overexpressed in the vast majority of cases of squamous cell carcinoma of the head and neck ( scchn ) . a high egfr expression and an increased egfr gene copy number are associated with an unfavorable prognosis [ 57 ] . two of the potential egfr targeting strategies that are currently in use in the treatment of scchn are the monoclonal antibodies directed at the extracellular domain of the receptor and the small molecule and atp - competitive tyrosine kinase inhibitors ( tkis ) . cetuximab is the only targeted agent that got approval by the food and drug administration and the european medicines agency for the treatment of scchn [ 8 , 9 ] and is still under active investigation in this disease ( tables 1 and 2 ) . cetuximab is a chimeric human / murine igg1 antibody which binds with higher affinity to the egfr than the natural ligands egf and tnf- , thereby disrupting egfr signaling pathways . another mechanism that contributes to the antitumor activity of cetuximab is depletion of the targeted receptors from the cell surface . the availability of egfr on the cell surface is reduced , and the receptor is downregulated [ 10 , 11 ] . the recommended dose was used in the cetuximab studies in scchn studies mentioned below , unless stated otherwise . [ 13 , 14 ] conducted a multinational , randomized study comparing radiotherapy alone with radiotherapy plus cetuximab in 424 patients with stages iii or iv , nonmetastatic , measurable squamous cell carcinoma ( scc ) of the oropharynx , hypopharynx , or larynx . the median duration of locoregional control ( primary endpoint ) was 24.4 months in patients treated with cetuximab plus radiotherapy and 14.9 months in patients treated with radiotherapy alone ( hazard ratio ( hr ) for locoregional progression or death , 0.68 ; p = 0.005 ) . the one- , two- , and three - year rates of locoregional control achieved with radiotherapy plus cetuximab ( 63 , 50 , and 47% ) , were significantly higher than those achieved with radiotherapy alone ( 55 , 41 , and 34% , resp . ) median overall survival ( os ) for patients treated with cetuximab and radiotherapy was 49.0 months versus 29.3 months in the radiotherapy - alone group ( hr for death : 0.73 ; p = 0.018 ) . however , as there is only one randomized phase iii trial with cetuximab - based bioradiation as opposed to the abundance of data supporting cisplatin - based concurrent chemoradiation ( crt ) [ 15 , 16 ] , the latter continues to represent the standard of care for medically fit patients with locoregionally ( la ) scchn , who can tolerate platinum - based therapy . at the time of the third interim analysis after 337 events and after a median followup of 2.4 years for the surviving patients , the conditional power of the trial becoming positive was below 10% , triggering early reporting . the 2-year progression - free survival ( pfs ) , ( primary endpoint ) was 64.3% with chemoradiation and 63.4% with chemoradiation plus cetuximab ( hr : 1.05 ; 95% confidence interval ( ci ) : 0.841.29 ; p = 0.67 ) . however , grade 3/4 mucositis ( 43 versus 33% ) and in - field dermatitis ( 25 versus 15% ) was more common with the addition of cetuximab . grade 3/4 dermatitis outside the radiation field occurred in 19% of the patients treated with cetuximab . the tremplin trial is a randomized phase ii study in patients with scc of the larynx or hypopharynx suitable for total laryngectomy . after three 3 weekly cycles of tpf ( docetaxel 75 mg / m and cisplatin 75 mg / m on day 1 followed by 5-fu 750 mg / m/day , days 15 ) , patients who obtained at least a partial response ( 82% of the patients ) were randomized to receive radiotherapy ( 70 gy in 35 fractions over 7 weeks ) either with cisplatin 100 mg / m on days 1 , 22 , and 43 or with weekly cetuximab . the treatment compliance was better in the cetuximab arm with 71% of the patients receiving all planned cetuximab administrations . randomized 110 patients with la scchn , who had received 2 cycles of carboplatin , paclitaxel , and cetuximab as induction chemotherapy , between weekly cetuximab in combination with either 5-fu , hydroxyurea , and hyperfractionated week - on week - off radiotherapy ( 7274 gy ) ( cetuxfhx ) , or cisplatin , accelerated radiation with concomitant boost ( cetuxpx ) ( 72 gy ) . in case of a negative biopsy ( 91% of the patients ) , crt was continued to a total dose of 6872 gy . in the extreme trial ( erbitux in first - line treatment of recurrent or metastatic head and neck cancer ) , 442 patients with previously untreated r / m scchn were randomized to receive cisplatin 100 mg / m or carboplatin at an area under the curve ( auc ) of 5 mg / mg / min as an 1-hour infusion , followed by 5-fu 1000 mg / m day for 4 days as a continuous infusion every 3 weeks for a maximum of 6 cycles , either alone or in combination with cetuximab . the median overall survival ( primary endpoint ) was 10.1 months ( 95% ci : 8.611.2 ) in the cetuximab group and 7.4 months ( 95% ci : 6.48.3 ) in the chemotherapy - alone group ( hr for death : 0.80 ; 95% ci : 0.640.99 ; p = 0.04 ) . median pfs was 5.6 months and 3.3 months for the combined group and the chemotherapy alone group , respectively ( hr for progression : 0.54 ; 95% ci : 0.430.67 ; p < 0.001 ) . the overall response rate ( orr ) was 36 and 20% , respectively ( odds ratio ( or ) : 2.33 ; p < 0.001 ) , and the disease control rate ( dcr ) was 80 and 60% , respectively ( or : 2.88 ; p < 0.001 ) . the safety profile of the study treatment was consistent with that expected for the agents used , with no significant difference in the incidence of grades 3 or 4 adverse events between treatment arms . however , there were 9 cases of sepsis in the cetuximab group , as compared with 1 case in the chemotherapy - alone group ( p = 0.02 ) , and there were 11 cases of hypomagnesemia in the cetuximab group , as compared with 3 cases in the chemotherapy - alone group ( p = 0.05 ) . common grade 3/4 adverse events were acne - like rash ( 24% ) , asthenia ( 17% ) , and neutropenia ( 13% ) . patients were given an initial loading dose of 8 mg / kg followed up by two week doses of 4 mg / kg by intravenous infusion in 1 h. after the first three administrations , in patients with no rash or grade 1 rash , the dose was increased by 4 mg / kg every 2 weeks up to a maximum dose of 16 mg / kg . seventy - two percent of the control patients received methotrexate from the initiation of the trial , and a further 6% started methotrexate during the trial . median os ( primary endpoint ) was 6.7 months ( 95% ci : 5.87.0 ) in the zalutumumab group and 5.2 months ( 95% ci : 4.16.4 ) in the control group ( hazard ratio ( hr ) for death , stratified by who performance status : 0.77 ; 97.06% ci : 0.571.05 ; unadjusted p = 0.0648 ) . in the spectrum trial ( study of panitumumab efficacy in patients with recurrent and/or metastatic head and neck cancer ) , 657 patients were randomized between cisplatin 100 mg / m on day 1 , followed by 5-fluorourcacil 1000 mg / m/day for 4 days or the same chemotherapy plus panitumumab 9 mg / kg administered on day 1 . however , there was a statistically significant difference in response rate ( 36% versus 25% ; p = 0.007 ) and pfs ( median 5.8 months versus 4.6 months ; hr = 0.78 ; 95% ci : 0.660.92 ; p = 0.004 ) in favour of the panitumumab - containing arm . although the spectrum trial failed to meet its primary endpoint , the results are nevertheless consistent with the outcome of the extreme trial . the primary endpoint of the trial was the complete response rate , which was 59.5% with nimotuzumab versus 34.2% with placebo ( p = 0.028 ) . tyrosine kinase inhibitors which have been tested in scchn include gefitinib and erlotinib , which are reversible specific egfr tkis , lapatinib , a reversible dual egfr / her2 tki , afatinib , an irreversible dual egfr / her2 tki , and pf-00299804 , a potent irreversible pan - her tki . the data monitoring committee recommended early stopping of enrollment after inclusion of 270 patients because there was < 5% chance to meet the primary endpoint ( overall survival ) . the most common adverse events were rash , diarrhea , cancer pain , nausea , and vomiting with gefitinib , and stomatitis , nausea , and constipation with methotrexate.the os with gefitinib in the imex trial was similar to what was observed in earlier uncontrolled phase ii studies with gefitinib or erlotinib [ 4346 ] . eligible were patients who were previously treated with chemotherapy for r / m scchn ( 73% of the patients ) and patients previously untreated for r / m scchn either with a poor performance status ( ecog 2 ) or in case of relapse within 6 months after chemotherapy given as part the primary treatment with curative intent . response rates were documented in 18% and 29% of the patients in the low - and high - dose arms , respectively , suggesting that a subgroup of previously untreated scchn is highly sensitive to egfr tyrosine kinase inhibition.hayes et al . response rates were documented in 18% and 29% of the patients in the low - and high - dose arms , respectively , suggesting that a subgroup of previously untreated scchn is highly sensitive to egfr tyrosine kinase inhibition . serious adverse events were observed in 33% and 32% of the patients in arm a and b , respectively . lapatinib ( 1500 mg / day ) or placebo were started 1 week before crt ( 70 gy in 35 fractions over 7 weeks plus cisplatin 100 mg / m on days 1 , 22 , and 43 ) and continued during and after crt until disease progression . grade 3 adverse events were diarrhea ( 16% ) , fatigue ( 9% ) , acneiform dermatitis ( 7% ) , and hand - foot reaction ( 4% ) . demonstrated that vegf protein overexpression , as detected with immunohistochemistry , is associated with a worse os in patients with scchn . the median ttp ( primary endpoint ) was 5 months , and the median os was 11.3 months . in 37 evaluable patients , the orr was 30% , and the dcr was 86% . bevacizumab , 10 mg / kg , administered on day 1 of each 2-week cycle , can be safely combined with fhx crt regimen , consisting of five 2-week cycles of hydroxyurea 500 mg orally bid , 5-fu 600 mg / m/day administered as a continuous infusion , and radiotherapy , 1.5 gy bid for 5 days followed by 9 days without therapy ( fhx ) , in patients with poor prognosis scchn . sorafenib and sunitinib are oral inhibitors of multiple kinases including the receptor tyrosine kinases of vascular endothelial growth factor ( vegf ) receptor . treated 27 patients with r / m scchn or nasopharyngeal carcinoma ( 7 patients ) , who had received 1 chemotherapy for recurrent or metastatic disease with sorafenib 400 mg twice daily on a continuous basis . however , the estimated median pfs was 4 months , and the estimated median os was 9 months . local complications , including the appearance or worsening of tumor skin ulceration or tumor fistula , were recorded in 39.5% of the patients , and a fatal arterial bleeding in the head and neck region occurred in 10.5% of the patients . combined paclitaxel , carboplatin auc 6 , administered every 3 weeks , and sorafenib 400 mg bid , days 219 , in patients with r / m scchn . vandetanib is an inhibitor of vascular endothelial growth factor receptor-2 ( vegfr-2 ) , egfr , and rearranged during transfection ( ret ) tyrosine kinases . heat - shock protein 90 ( hsp90 ) is a protein which chaperones multiple client oncoproteins involved in tumor progression . demonstrated that sirt3 is overexpressed in oscc in vitro and in vivo and that sirt3 downregulation inhibits oscc cell growth and proliferation and increased oscc cell sensitivity to radiation and cisplatin treatments in vitro . hdac inhibitors have radio - enhancing properties [ 75 , 76 ] , increase the susceptibility of scc cell lines to cisplatin in vitro [ 77 , 78 ] , and inhibit tumor growth in xenograft models of scchn . demonstrated that the histone deacetylase inhibitor vorinostat in combination with the egfr tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation , migration , and invasion as well as induction of apoptosis in scchn cells , including cells resistant to gefitinib . high expression of aurora a or b is associated with a worse outcome in scchn [ 8186 ] . combined a dual aurora a / aurora b inhibitor with cetuximab in scchn cell lines in vitro and observed at least an additive effect . preclinical data strongly support the testing of mammalian target of rapamycin ( mtor ) in scchn [ 8890 ] . activation of mtor is observed in the majority of scchn and is associated with a poor outcome . found that inhibition of mtor diminished lymphangiogenesis in the primary tumors and prevented the dissemination of scchn cancer cells to the cervical lymph nodes in an orthotopic mouse model . temsirolimus enhances the growth - inhibiting effects of the combination of bevacizumab , cetuximab , and irradiation in head and neck cancer xenografts . temsirolimus appeared to be a more potent radiosensitizer than cisplatin in mice bearing squamous cell carcinoma xenografts . found a significant inhibition of the mtor pathways in tumor cells and in peripheral blood mononuclear cells . c - src is a nonreceptor cytoplasmic tyrosine kinase that regulates signals from cell surface molecules and that plays a key role in modulating multiple cellular functions by activating the signal transducer and activator of transcription ( stat ) family of transcription factors . although preclinical data provided a rationale for testing c - scr inhibitors in scchn , the outcome with single - agent c - scr inhibitors in patients with r / m scchn was disappointing . eight patients had disease progression within the first eight - week cycle , and one patient was removed from the study after 11 days due to rapid clinical decline . nuclear factor kappa b is overexpressed in scchn , and nf-b expression is associated with a poor prognosis . were observed in the phase i portion of the study which included 10 patients up to the maximum planned dose of 15 mg / kg of bevacizumab , 46 additional patients were treated at that dose level . forty - eight percent of the patients had received prior chemotherapy for recurrent / metastatic disease . the treatment consisted of induction chemotherapy with 6 weeks of paclitaxel , carboplatin , infusional 5-fluorouracil , and bevacizumab , which was followed by radiation therapy , weekly paclitaxel , bevacizumab , and erlotinib . advantage is a phase i / ii trial evaluating cilengitide in combination with cetuximab , cisplatin and 5-fluorouracil in patients with r / m scchn . no dlts were observed in the phase i part of the study which tested cilengitide ( 500 , 1000 , and 2000 mg ) twice weekly with standard doses of cetuximab , cisplatin and 5-fluorouracil . the road from preclinical evidence to clinical use is long and bumpy , and a large number of targeted agents are still at the start of the race . thus far , only the egfr - directed monoclonal antibody cetuximab has made it to the finish and is currently approved for the treatment of locoregionally advanced and recurrent / metastatic scchn , by the regulatory agencies of the united states and europe .

potential targets include the epidermal growth factor receptor ( egfr ) and the erbb family , the vascular endothelial growth factor ( vegf ) and its receptor ( vegfr ) , insulin - like growth factor 1 receptor ( igf-1r ) , insulin receptor ( ir ) , histone deacetylase ( hdac ) , mammalian target of rapamycin ( mtor ) , platelet - derived growth factor ( pdgfr ) , heat - shock protein 90 ( hsp90 ) , nuclear factor - kappa b ( nf-b ) , aurora a or b , phosphatidylinositol 3-kinase ( pik3ca ) . egfr consists of an extracellular n - terminal ligand - binding domain , a hydrophobic transmembrane domain , and a c - terminal intracellular domain , which includes the tyrosine kinase domain and the autophosphorylation sites . two of the potential egfr targeting strategies that are currently in use in the treatment of scchn are the monoclonal antibodies directed at the extracellular domain of the receptor and the small molecule and atp - competitive tyrosine kinase inhibitors ( tkis ) . [ 13 , 14 ] conducted a multinational , randomized study comparing radiotherapy alone with radiotherapy plus cetuximab in 424 patients with stages iii or iv , nonmetastatic , measurable squamous cell carcinoma ( scc ) of the oropharynx , hypopharynx , or larynx . investigators were required to select one of three radiotherapy - fractionation regimens before patient registration : 70.0 gy in 35 daily fractions of 2.0 gy / fraction , 5 fractions / week for 7 weeks , or two daily fractions of 1.2 gy / fraction up to 72.076.8 gy in 6064 fractions , 10 fractions / week for 66.5 weeks , or a concomitant boost regimen ( 72.0 gy in 42 fractions : 32.4 gy ; 1.8 gy / fraction , 5 fractions / week for 3.6 weeks followed by a morning dose of 21.6 gy in fractions of 1.8 gy , 5 fractions / week for 2.4 weeks and an afternoon dose of 18.0 gy in fractions of 1.5 gy , 5 fractions / week for 2.4 weeks ) . the median duration of locoregional control ( primary endpoint ) was 24.4 months in patients treated with cetuximab plus radiotherapy and 14.9 months in patients treated with radiotherapy alone ( hazard ratio ( hr ) for locoregional progression or death , 0.68 ; p = 0.005 ) . the one- , two- , and three - year rates of locoregional control achieved with radiotherapy plus cetuximab ( 63 , 50 , and 47% ) , were significantly higher than those achieved with radiotherapy alone ( 55 , 41 , and 34% , resp . ) median overall survival ( os ) for patients treated with cetuximab and radiotherapy was 49.0 months versus 29.3 months in the radiotherapy - alone group ( hr for death : 0.73 ; p = 0.018 ) . however , as there is only one randomized phase iii trial with cetuximab - based bioradiation as opposed to the abundance of data supporting cisplatin - based concurrent chemoradiation ( crt ) [ 15 , 16 ] , the latter continues to represent the standard of care for medically fit patients with locoregionally ( la ) scchn , who can tolerate platinum - based therapy . in radiation therapy oncology group ( rtog ) 0522 , 895 evaluable patients with stage iii / iv nonmetastatic scchn were randomized between chemoradiation ( 72 gy in 42 fractions over 6 weeks plus cisplatin 100 mg / m on days 1 and 22 ) or the same regimen plus weekly cetuximab . the 2-year progression - free survival ( pfs ) , ( primary endpoint ) was 64.3% with chemoradiation and 63.4% with chemoradiation plus cetuximab ( hr : 1.05 ; 95% confidence interval ( ci ) : 0.841.29 ; p = 0.67 ) . after three 3 weekly cycles of tpf ( docetaxel 75 mg / m and cisplatin 75 mg / m on day 1 followed by 5-fu 750 mg / m/day , days 15 ) , patients who obtained at least a partial response ( 82% of the patients ) were randomized to receive radiotherapy ( 70 gy in 35 fractions over 7 weeks ) either with cisplatin 100 mg / m on days 1 , 22 , and 43 or with weekly cetuximab . randomized 110 patients with la scchn , who had received 2 cycles of carboplatin , paclitaxel , and cetuximab as induction chemotherapy , between weekly cetuximab in combination with either 5-fu , hydroxyurea , and hyperfractionated week - on week - off radiotherapy ( 7274 gy ) ( cetuxfhx ) , or cisplatin , accelerated radiation with concomitant boost ( cetuxpx ) ( 72 gy ) . the maximum tolerated dose ( mtd ) of pemetrexed was 500 mg in cohort a and 350 mg / m in cohort b. grade 3/4 neutropenia was common ( 50% in cohort a and 33% in cohort b ) and febrile neutropenia was the most frequent doselimiting toxicity . in eastern cooperative oncology group ( ecog ) e2303 , 63 patients with resectable stage iii / iv scchn , were treated with 6-week cycles of paclitaxel , carboplatin at an auc of 2 and cetuximab , followed by crt ( weekly paclitaxel , 30 mg / m , carboplatin auc 1 , and cetuximab ) . treated 152 t3-t4 scchn patients with three 3-week cycles of tpf ( docetaxel 75 mg / m on day 1 , cisplatin 35 mg / m on days 1 and 2 , and 5-fu 750 mg / m/day , as a continuous infusion , days 15 , with pegfilgrastim support ) followed by chemoradiation ( 63 gy in 35 fractions of 1.8 gy over 7 weeks and weekly cisplatin , 40 mg / m ) plus weekly cetuximab . treated 34 la scchn patients with three 3-weekly cycles of cisplatin 75 mg / m and docetaxel 75 mg / m plus weekly cetuximab followed by crt ( 70 gy in 2 gy fractions over 7 weeks , weekly cisplatin 30 mg / m ) plus weekly cetuximab , followed by weekly cetuximab maintenance for 6 months . in the extreme trial ( erbitux in first - line treatment of recurrent or metastatic head and neck cancer ) , 442 patients with previously untreated r / m scchn were randomized to receive cisplatin 100 mg / m or carboplatin at an area under the curve ( auc ) of 5 mg / mg / min as an 1-hour infusion , followed by 5-fu 1000 mg / m day for 4 days as a continuous infusion every 3 weeks for a maximum of 6 cycles , either alone or in combination with cetuximab . the median overall survival ( primary endpoint ) was 10.1 months ( 95% ci : 8.611.2 ) in the cetuximab group and 7.4 months ( 95% ci : 6.48.3 ) in the chemotherapy - alone group ( hr for death : 0.80 ; 95% ci : 0.640.99 ; p = 0.04 ) . median pfs was 5.6 months and 3.3 months for the combined group and the chemotherapy alone group , respectively ( hr for progression : 0.54 ; 95% ci : 0.430.67 ; p < 0.001 ) . the overall response rate ( orr ) was 36 and 20% , respectively ( odds ratio ( or ) : 2.33 ; p < 0.001 ) , and the disease control rate ( dcr ) was 80 and 60% , respectively ( or : 2.88 ; p < 0.001 ) . however , there were 9 cases of sepsis in the cetuximab group , as compared with 1 case in the chemotherapy - alone group ( p = 0.02 ) , and there were 11 cases of hypomagnesemia in the cetuximab group , as compared with 3 cases in the chemotherapy - alone group ( p = 0.05 ) . in groupe d'oncologie radiothrapie tte et cou ( gortec ) 200803 , 54 patients who had not received prior chemotherapy for r / m scchn were treated with docetaxel 75 mg / m and cisplatin 75 mg / m every 3 weeks and weekly cetuximab , up to 4 cycles , followed by cetuximab maintenance in the absence of disease progression . the survival of around 6 months achieved with cetuximab in platinum - refractory disease was found similar to that seen in first - line therapy in r / m - scchn and represented an increase in survival of 2.5 months compared with platinum - refractory historical controls . based on these results and particularly considering the fact that about 50% of the patients showed disease control , cetuximab monotherapy seems to be an option for patients with r / m scchn who have progressed on platinum - based chemotherapy . eligible were patients with progressive disease according to recist confirmed by an independent review committee during or within 6 months after the failure of platinum - based chemotherapy ( at least two cycles of cisplatin [ 60 mg / m per cycle ] or carboplatin [ 250 mg / m per cycle ] with an interval between the cycles of < 4 weeks ) . patients were given an initial loading dose of 8 mg / kg followed up by two week doses of 4 mg / kg by intravenous infusion in 1 h. after the first three administrations , in patients with no rash or grade 1 rash , the dose was increased by 4 mg / kg every 2 weeks up to a maximum dose of 16 mg / kg . median os ( primary endpoint ) was 6.7 months ( 95% ci : 5.87.0 ) in the zalutumumab group and 5.2 months ( 95% ci : 4.16.4 ) in the control group ( hazard ratio ( hr ) for death , stratified by who performance status : 0.77 ; 97.06% ci : 0.571.05 ; unadjusted p = 0.0648 ) . progression - free survival was longer in the zalutumumab group than in the control group ( hr for progression or death , stratified by who performance status : 0.63 ; 95% ci : 0.470.84 ; p = 0.0012 ) . in the spectrum trial ( study of panitumumab efficacy in patients with recurrent and/or metastatic head and neck cancer ) , 657 patients were randomized between cisplatin 100 mg / m on day 1 , followed by 5-fluorourcacil 1000 mg / m/day for 4 days or the same chemotherapy plus panitumumab 9 mg / kg administered on day 1 . the median os in the combined arm was 11.1 months compared to 9.0 months in the chemotherapy alone arm ( hr = 0.87 ; 95% ci : 0.731.05 ; p = 0.14 ) . however , there was a statistically significant difference in response rate ( 36% versus 25% ; p = 0.007 ) and pfs ( median 5.8 months versus 4.6 months ; hr = 0.78 ; 95% ci : 0.660.92 ; p = 0.004 ) in favour of the panitumumab - containing arm . tyrosine kinase inhibitors which have been tested in scchn include gefitinib and erlotinib , which are reversible specific egfr tkis , lapatinib , a reversible dual egfr / her2 tki , afatinib , an irreversible dual egfr / her2 tki , and pf-00299804 , a potent irreversible pan - her tki . eligible were patients who were previously treated with chemotherapy for r / m scchn ( 73% of the patients ) and patients previously untreated for r / m scchn either with a poor performance status ( ecog 2 ) or in case of relapse within 6 months after chemotherapy given as part the primary treatment with curative intent . neither gefitinib 250 mg / day nor gefitinib 500 mg / day improved os compared with methotrexate ( hr : 1.22 ; 95% ci : 0.951.57 ; p = 0.12 ; hr : 1.12 ; 95% ci : 0.871.43 ; p = 0.39 , resp . ) in group a , os was significantly longer with methotrexate ( hr for death : gefitinib 250 mg versus methotrexate : 1.62 ; p = 0.01 ; gefitinib 500 mg versus methotrexate : 1.5 ; p = 0.02 ) . the most common adverse events were rash , diarrhea , cancer pain , nausea , and vomiting with gefitinib , and stomatitis , nausea , and constipation with methotrexate.the os with gefitinib in the imex trial was similar to what was observed in earlier uncontrolled phase ii studies with gefitinib or erlotinib [ 4346 ] . eligible were patients who were previously treated with chemotherapy for r / m scchn ( 73% of the patients ) and patients previously untreated for r / m scchn either with a poor performance status ( ecog 2 ) or in case of relapse within 6 months after chemotherapy given as part the primary treatment with curative intent . patients were stratified into two groups : group a ( n = 256 ) consisted of patients who had stable or progressive disease after at least two cycles of platinum - based chemotherapy for recurrent disease ; group b ( n = 230 ) consisted of patients who were considered unsuitable for platinum - containing chemotherapy . neither gefitinib 250 mg / day nor gefitinib 500 mg / day improved os compared with methotrexate ( hr : 1.22 ; 95% ci : 0.951.57 ; p = 0.12 ; hr : 1.12 ; 95% ci : 0.871.43 ; p = 0.39 , resp . ) in group a , os was significantly longer with methotrexate ( hr for death : gefitinib 250 mg versus methotrexate : 1.62 ; p = 0.01 ; gefitinib 500 mg versus methotrexate : 1.5 ; p = 0.02 ) . response rates were documented in 18% and 29% of the patients in the low - and high - dose arms , respectively , suggesting that a subgroup of previously untreated scchn is highly sensitive to egfr tyrosine kinase inhibition.hayes et al . randomly assigned 128 patients with la scchn to receive either cisplatin 100 mg / m on days 1 , 22 , and 43 combined with 70 gy of radiotherapy ( arm a ) or the same treatment plus 150 mg of erlotinib starting one week before crt and continued until the completion of radiotherapy ( arm b ) . response rates were documented in 18% and 29% of the patients in the low - and high - dose arms , respectively , suggesting that a subgroup of previously untreated scchn is highly sensitive to egfr tyrosine kinase inhibition . randomly assigned 128 patients with la scchn to receive either cisplatin 100 mg / m on days 1 , 22 , and 43 combined with 70 gy of radiotherapy ( arm a ) or the same treatment plus 150 mg of erlotinib starting one week before crt and continued until the completion of radiotherapy ( arm b ) . at the 150 mg dose , encouraging preliminary results in r / m scchn after failure of a platinum - containing therapy were reported with afatinib , a dual egfr / her2 irreversible tyrosine kinase inhibitor , which was compared to single - agent cetuximab in a randomized phase ii study . bevacizumab , 10 mg / kg , administered on day 1 of each 2-week cycle , can be safely combined with fhx crt regimen , consisting of five 2-week cycles of hydroxyurea 500 mg orally bid , 5-fu 600 mg / m/day administered as a continuous infusion , and radiotherapy , 1.5 gy bid for 5 days followed by 9 days without therapy ( fhx ) , in patients with poor prognosis scchn . demonstrated the safety and feasibility of combining crt ( 70 gy in 33 fractions and weekly cisplatin , 30 mg / m ) with bevacizumab weeks 3 , 1 , 4 , and 7 with dose escalation from 5 to 10 to 15 mg / kg in 10 patients with la scchn . vandetanib can be safely combined with radiotherapy ( 2.2 gy / d , 5 days / week up to a total dose of 66 gy ) or radiotherapy ( 2 gy / d , 5 days / week up to a total dose of 70 gy ) plus weekly 30 mg / m of cisplatin . demonstrated that the histone deacetylase inhibitor vorinostat in combination with the egfr tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation , migration , and invasion as well as induction of apoptosis in scchn cells , including cells resistant to gefitinib . were observed in the phase i portion of the study which included 10 patients up to the maximum planned dose of 15 mg / kg of bevacizumab , 46 additional patients were treated at that dose level . the feasibility and efficacy of adding bevacizumab and erlotinib to concurrent crt in patients with la scchn was evaluated in a phase ii trial conducted by the sarah cannon oncology research consortium including 60 previously untreated patients . the treatment consisted of induction chemotherapy with 6 weeks of paclitaxel , carboplatin , infusional 5-fluorouracil , and bevacizumab , which was followed by radiation therapy , weekly paclitaxel , bevacizumab , and erlotinib . no dlts were observed in the phase i part of the study which tested cilengitide ( 500 , 1000 , and 2000 mg ) twice weekly with standard doses of cetuximab , cisplatin and 5-fluorouracil . thus far , only the egfr - directed monoclonal antibody cetuximab has made it to the finish and is currently approved for the treatment of locoregionally advanced and recurrent / metastatic scchn , by the regulatory agencies of the united states and europe .

the recent years have seen a renewal of peptides as candidate therapeutics for several reasons . first , recent advances in peptide chemistry and delivery have overcome the traditional limitations of peptides as drug candidates ( 1 ) . second , the shift of therapeutic strategies towards the network of protein interactions , particularly the search for protein protein interaction inhibitors , has pushed forward the limits of small chemical molecules , whereas advances in protein recombinant technologies provide evidence that larger therapeutics such as peptides or peptide derivatives could offer plausible alternatives ( 2 ) . another motivation also comes from the large reservoir of natural peptides that have diverse and specific biological activities , and among these , bacterial small proteins ( 3 ) and venom peptides ( 4 ) raise more interest . finally , peptides are also described as promising candidates for the treatment of central nervous system disorders ( 5 ) . to assist peptide lead identification and optimization , robust computational methods recent efforts from the community of computer scientists have tackled various aspects including the design of generic databases devoted to peptide protein interactions such as pepx ( 8) , the problem of protein peptide docking ( 9 ) , the search for peptidomimetics ( 10 ) and the development of fast peptide structure prediction methods ( e.g. pep - fold , bhageerath , pepstr , peplook , i - tasser , rosetta ) ( 1116 ) . in 2009 , we introduced the pep - fold service ( 11 ) for de novo peptide structure prediction . though this first rapid on - line version has been used by external users for structural characterization of peptides or protein fragments ( 17,18 ) and peptide or vaccine design ( 19,20 ) , the maximal length of 25 amino acids limits the number of applications . in addition , like the bhageerath ( 12 ) and pepstr ( 13 ) servers , pep - fold was only available for linear peptides , whereas there are many natural cyclic peptides with disulfide bonds such as conotoxins or cyclotides ( 21 ) and disulfide bonds increase peptide stability ( 22 ) . recently , the peplook procedure ( not available on - line ) brought some improvements in this direction ( 14 ) . here , we introduce an improved version of the service open to the community that ( i ) extends the length of linear peptides to 36 amino acids and ( ii ) accepts cyclic peptides using disulfide bonds defined by the user . the 3d prediction scheme is very similar to that reported in ( 11 ) and ( 23 ) . it is based on a hidden markov model derived structural alphabet ( sa ) ( 24 ) , i.e. a kind of generalized secondary structure extending the number of states from 3 ( helix , coil , strand ) to 27 in our case . the first step predicts sa letters from the amino acid sequence . from the amino acid sequence , a psi - blast profile is generated and is used as input of a svm that returns a probability profile of each sa letter at each position of the sequence . the second step performs the 3d assembly of the prototype fragments associated with the letters selected . it relies on the sopep coarse grained force field ( 25 ) , which uses a six bead representation ( full backbone except the -hydrogen and one bead for each side chain ) . the 3d generation is achieved by an enhanced greedy procedure ( 26 ) that builds the peptide residue by residue . this build - up procedure works using a rigid assembly scheme and thus does not explore the full conformational space but only a discrete subset . the third step generates all - atom conformations from the coarse grained models returned by the 100 simulations and performs a clustering procedure . figure 1.pep-fold 2012 flowchart . two major improvements have been brought to this scheme in the new version of the service . first , the selection of the sa letters from the profile has been revisited so as to remove the letters with too low probabilities . as a result , whereas the initial pep - fold version used eight letters at each position , the new version uses 5.5 letters on average . we also removed the secondary structure constraints predicted by psi - pred because the sa profile contains this information . a second major modification involves the use of tm scores ( 27 ) to cluster the full generated structures and then the sopep energies or the predicted tmscores of apollo ( 28)an extension of ( 29 ) for the prediction gdt_ts from structural features to rank the clusters and the conformations within the clusters . , the disulfide bonds are not simply constrained to a typical bond distance because we grow the peptide from any position of the structure and thus all oxidized cysteines are not known in the early steps of the assembly . rather , the interaction between two oxidized cysteines i and j is described by : ( 1 ) where denotes the distance between the side - chain centroids , = 3.36 is the distance where the energy is the lowest ( 15 kcal / mol ) , = 2.39 is the distance where is 0 , and e0 is the energy value for . the left side term is identical to sopep former term and the right side results in a sharper behaviour preventing energy at longer distances . finally , other minor modifications have been brought to the side chain positioning in the all - atom generation step it is now achieved using oscar - star ( 30)and the disulfide bond specifications are passed to the quick all - atom minimization based on gromacs ( 31 ) . as described in figure 1 , the service is now fully embedded in the mobyle framework ( 32 ) , providing automated form generation , command execution and result display . the input fields include the primary sequence ( only standard amino acids ) and the specification of constraints ( disulfide bonds , residue proximities ) . in addition to building models up to 36 amino acids , it is possible to treat sequences of 50 amino acids and specify the maximal 36-residue region subjected to 3d modelling . this facility is useful for peptides where the n- and c - terminal regions are known to be disordered and the user wishes to focus on the modelling of the structured core . to this end , our tests show that the prediction of the sa profile is best using the complete sequence rather than a truncated sequence . the output fields have also been modified as a result of the new clustering procedure . although our results suggest that sorting the clusters using sopep remains best , they can also be sorted according to the predicted tm scores ( 27 ) obtained by apollo ( 28 ) . finally , the use of the mobyle framework comes with facilities to visualize the best models using the openastex ( 33 ) or jmol ( 34 ) applets and perform other analyses such as the secondary structure , side - chain conformations , etc . the modifications brought to pep - fold have a limited impact on the linear peptides with 925 amino acids . a table of the results on 24 peptides is presented on the on - line documentation of the service . we see that the sharper sa letter selection does not prevent pep - fold to generate near - native conformations , with the best rigid core ( rc ) conformations deviating on average by only 1.5 from the nmr structures , versus 1.7 using the earlier pep - fold service . note that the pdb entry 1wz4 is excluded from analysis since its nmr structure displays a clash between the backbone oxygens of glu7 and asp11 . the updated pep - fold procedure also generates lowest - energy conformations deviating on average by 2.5 from the nmr rcs . the results on a set of linear peptides with 2536 amino acids are presented in table 1 . averaged over the 13 systems , pep - fold returns lowest - energy conformations deviating from the first nmr model by 4.8 and 3.4 using the full structure ( fs ) and the rc . looking at the five clusters of lowest energy , the average rmsd is 3.6 ( fs ) and 2.8 ( rc ) . overall , pep - fold generates near - native conformations ( rc - d < 4 ) for 11 among 13 structurally diverse proteins with secondary structure compositions varying from , 2 ( 2l0 g figure 2c ) , 2 , 2 , , to 3 . of particular interest is the high quality prediction of the topology for the 36-residue 2ki0 protein with a rc - d of 2.0 based on the sopep energy ( figure 2e ) which is stabilized by long - range interactions in the amino acid sequence . similarly , the updated pep - fold version returns the native 3 conformation of the 36-residue 1e0n protein ( figure 2a ) , while the earlier version , using the 27 amino acids which are not random coil in the nmr structure , predicted a -strand packed against two helices ( 11 ) . for the 28-residue 1psv and 31-residue 2gdl , the lowest - energy conformations differ by 7.4 and 4.8 rms from nmr using the rc . in figure 2b , interestingly , experimental and computational studies on homologous peptides have shown that the -hairpin is only marginally stable ( 35 ) . in contrast , for the 2gdl target ( figure 2 g ) which displays an -coil- topology , the predicted -strand at the c - terminal is not compatible with the -helix structure observed by nmr . pep - fold models are in cyan . from left to right , top to bottom : 1e0n ( a ) , 1psv ( b ) , 2l0 g ( c ) , 1n0a ( d ) , 2ki0 ( e ) , 1kwd ( f ) and 2gdl ( g ) lowest - energy models and 1wm8 ( h ) best model . table 1.results obtained for 13 linear peptides with 2536 residuespdb idtoplrcsopepbest5fs - drc - dfs - drc - drnk1by0a271:234.361.752.941.7411yyba271:206.491.473.41.4212kblb2293:4|6:274.683.914.683.9111fsdab2281:264.143.883.913.6611psvab2282:257.197.444.614.642k76ba304:293.152.752.11.8812gdlaca318:8|10:11|14:15|21:297.574.846.534.5732l0ga2325:324.533.322.11.6412bn6a2334:294.493.183.092.1411e0nb3351:252.162.161.681.6821wr3b3365:155.723.744.263.511wr4b3365:344.683.264.683.2612ki0bab365:11|13:363.561.992.662.431mean4.83.43.62.8pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model.the lowest - energy models for 1e0n , 1psv , 2l0 g , 2ki0 and 2gdl are shown on figure 2 . pep - fold models . pep - fold models are in cyan . from left to right , top to bottom : 1e0n ( a ) , 1psv ( b ) , 2l0 g ( c ) , 1n0a ( d ) , 2ki0 ( e ) , 1kwd ( f ) and 2gdl ( g ) lowest - energy models and 1wm8 ( h ) best model . results obtained for 13 linear peptides with 2536 residues pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model . the lowest - energy models for 1e0n , 1psv , 2l0 g , 2ki0 and 2gdl are shown on figure 2 . table 2 presents the results of 34 peptides containing one , two or three disulfide bonds using the peplook test set ( 14 ) except the peptides 2p7r , 1qvl and 1foz < 8 amino acids , and the peptide 1ixu free of any disulfide bond in the nmr structure . for each peptide , we describe the models of lowest energy and of lowest rmsd ( best ) with respect to the nmr structure . we also give the number of ss bonds formed after the coarse - grained and the all - atom procedures . on average , the updated pep - fold generates best models deviating by only 2.7 and 2.5 rms and lowest - energy models deviating by 4.2 and 3.7 rms using the fss and rcs , respectively . this indicates that sopep is not optimal yet for recognizing near - native from higher rmsd conformations . compared to the results of the earlier pep - fold version , which did not consider the disulfide bonds explicitly ( 11 ) , we reach an improvement of 1.3 rms . compared to peplook performances , the pep - fold best models are also closer by 1 rms to the nmr conformations . whereas the server leads to good results for peptides containing 1 and 2 disulfide bonds ( figure 2f and d for 1kwd and 1n0a ) with 61.2% of the disulfide bonds formed at the coarse - grained level ( table 1 ) , the performances are degraded for the nine peptides with three disulfide bonds ( figure 2h for 1wm8 ) . for these peptides , the lowest - energy conformations deviate on average by 5.4 rms from the nmr structures and a similar behaviour is observed with peplook ( 5.6 rms ) . this discrepancy between prediction and experiment comes in part from the non - optimization of the sopep force field for cyclic peptides , but mainly we observe that the structures with three disulfide bonds are very constrained , leading to large deviations at the local level between the conformations observed in the nmr structures and the structural alphabet conformations predicted from the sequence free of any disulfide bond constraints . similar results are obtained if we vary the number of sa letters from 5.5 to 8 during assembly . table 2.results obtained for 34 cyclic peptides with disulfide bondspdb idl#ssrcsopepbestfs - drc - dcgaafs - drc - dcgaa1im72113:9::15:213.93.910002.52.510011jbl1612:143.33.310011.81.8001n0a1717:150.70.7010.40.4001n0c2412:11::14:242.32.310010.40.4011nim2411:233.81.8003.42.8011gnb1422:9::12:144.84.150143.85001b451323.63.310012.32.2001etl2223.13.110011.61.6001hje1923.63.65012.22.25021hp91222:113.53.55012.22.25011ien1324.84.85002.52.5001im11421.31.2011.21.25001kcn1625.75.610003.52.85011kwd3023:292.92.1100221.3011mii1121.81.85001.61.6001oig1522:7::11:146.56.15014.84.75011r8t2421:224.53.410012.62.25011rpc2123:216.96.35014.44001ter21275.950132.85001v6r2621:235.15.110023.93.9001wqc1322:131.71.310021.21.110021x7k2221:174.22.5004.22.5001xgb1623.5300335022ajw1322.40.95021.31002i282822.12.110011.51.5002oq92428:249.2410013.53.35012efz1234.24.2012.12.166.712nx71332:136.96.933.314.1433.311mmc1037.16.166.724.64.633.301orx2231:194.84.833.312.92.7001sp72831:10::12:284.54.366.722.6233.301v5a28355.166.713.73.8001wm81936:167766.723.63.666.722it72832:284466.713.73.766.71mean4.23.761.22.72.529.5pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation.our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation ( 1 ) lies within 0.7 . the discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied ( 2.0 0.2 ) in all - atom structures.the lowest - energy models for 1n0a and 1kwd , and the best rmsd model for 1wm8 are shown on figure 2 . results obtained for 34 cyclic peptides with disulfide bonds pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation . our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation ( 1 ) lies within 0.7 . the discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied ( 2.0 0.2 ) in all - atom structures . the lowest - energy models for 1n0a and 1kwd , and the best rmsd model for 1wm8 are shown on figure 2 . the modifications brought to pep - fold have a limited impact on the linear peptides with 925 amino acids . a table of the results on 24 peptides is presented on the on - line documentation of the service . we see that the sharper sa letter selection does not prevent pep - fold to generate near - native conformations , with the best rigid core ( rc ) conformations deviating on average by only 1.5 from the nmr structures , versus 1.7 using the earlier pep - fold service . note that the pdb entry 1wz4 is excluded from analysis since its nmr structure displays a clash between the backbone oxygens of glu7 and asp11 . the updated pep - fold procedure also generates lowest - energy conformations deviating on average by 2.5 from the nmr rcs . the results on a set of linear peptides with 2536 amino acids are presented in table 1 . averaged over the 13 systems , pep - fold returns lowest - energy conformations deviating from the first nmr model by 4.8 and 3.4 using the full structure ( fs ) and the rc . looking at the five clusters of lowest energy , the average rmsd is 3.6 ( fs ) and 2.8 ( rc ) . overall , pep - fold generates near - native conformations ( rc - d < 4 ) for 11 among 13 structurally diverse proteins with secondary structure compositions varying from , 2 ( 2l0 g figure 2c ) , 2 , 2 , , to 3 . of particular interest is the high quality prediction of the topology for the 36-residue 2ki0 protein with a rc - d of 2.0 based on the sopep energy ( figure 2e ) which is stabilized by long - range interactions in the amino acid sequence . similarly , the updated pep - fold version returns the native 3 conformation of the 36-residue 1e0n protein ( figure 2a ) , while the earlier version , using the 27 amino acids which are not random coil in the nmr structure , predicted a -strand packed against two helices ( 11 ) . for the 28-residue 1psv and 31-residue 2gdl , the lowest - energy conformations differ by 7.4 and 4.8 rms from nmr using the rc . in figure 2b , interestingly , experimental and computational studies on homologous peptides have shown that the -hairpin is only marginally stable ( 35 ) . in contrast , for the 2gdl target ( figure 2 g ) which displays an -coil- topology , the predicted -strand at the c - terminal is not compatible with the -helix structure observed by nmr . pep - fold models are in cyan . from left to right , top to bottom : 1e0n ( a ) , 1psv ( b ) , 2l0 g ( c ) , 1n0a ( d ) , 2ki0 ( e ) , 1kwd ( f ) and 2gdl ( g ) lowest - energy models and 1wm8 ( h ) best model . table 1.results obtained for 13 linear peptides with 2536 residuespdb idtoplrcsopepbest5fs - drc - dfs - drc - drnk1by0a271:234.361.752.941.7411yyba271:206.491.473.41.4212kblb2293:4|6:274.683.914.683.9111fsdab2281:264.143.883.913.6611psvab2282:257.197.444.614.642k76ba304:293.152.752.11.8812gdlaca318:8|10:11|14:15|21:297.574.846.534.5732l0ga2325:324.533.322.11.6412bn6a2334:294.493.183.092.1411e0nb3351:252.162.161.681.6821wr3b3365:155.723.744.263.511wr4b3365:344.683.264.683.2612ki0bab365:11|13:363.561.992.662.431mean4.83.43.62.8pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model.the lowest - energy models for 1e0n , 1psv , 2l0 g , 2ki0 and 2gdl are shown on figure 2 . pep - fold models . pep - fold models are in cyan . from left to right , top to bottom : 1e0n ( a ) , 1psv ( b ) , 2l0 g ( c ) , 1n0a ( d ) , 2ki0 ( e ) , 1kwd ( f ) and 2gdl ( g ) lowest - energy models and 1wm8 ( h ) best model . results obtained for 13 linear peptides with 2536 residues pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model . the lowest - energy models for 1e0n , 1psv , 2l0 g , 2ki0 and 2gdl are shown on figure 2 . table 2 presents the results of 34 peptides containing one , two or three disulfide bonds using the peplook test set ( 14 ) except the peptides 2p7r , 1qvl and 1foz < 8 amino acids , and the peptide 1ixu free of any disulfide bond in the nmr structure . for each peptide , we describe the models of lowest energy and of lowest rmsd ( best ) with respect to the nmr structure . we also give the number of ss bonds formed after the coarse - grained and the all - atom procedures . on average , the updated pep - fold generates best models deviating by only 2.7 and 2.5 rms and lowest - energy models deviating by 4.2 and 3.7 rms using the fss and rcs , respectively . this indicates that sopep is not optimal yet for recognizing near - native from higher rmsd conformations . compared to the results of the earlier pep - fold version , which did not consider the disulfide bonds explicitly ( 11 ) , we reach an improvement of 1.3 rms . compared to peplook performances , the pep - fold best models are also closer by 1 rms to the nmr conformations . whereas the server leads to good results for peptides containing 1 and 2 disulfide bonds ( figure 2f and d for 1kwd and 1n0a ) with 61.2% of the disulfide bonds formed at the coarse - grained level ( table 1 ) , the performances are degraded for the nine peptides with three disulfide bonds ( figure 2h for 1wm8 ) . for these peptides , the lowest - energy conformations deviate on average by 5.4 rms from the nmr structures and a similar behaviour is observed with peplook ( 5.6 rms ) . this discrepancy between prediction and experiment comes in part from the non - optimization of the sopep force field for cyclic peptides , but mainly we observe that the structures with three disulfide bonds are very constrained , leading to large deviations at the local level between the conformations observed in the nmr structures and the structural alphabet conformations predicted from the sequence free of any disulfide bond constraints . similar results are obtained if we vary the number of sa letters from 5.5 to 8 during assembly . table 2.results obtained for 34 cyclic peptides with disulfide bondspdb idl#ssrcsopepbestfs - drc - dcgaafs - drc - dcgaa1im72113:9::15:213.93.910002.52.510011jbl1612:143.33.310011.81.8001n0a1717:150.70.7010.40.4001n0c2412:11::14:242.32.310010.40.4011nim2411:233.81.8003.42.8011gnb1422:9::12:144.84.150143.85001b451323.63.310012.32.2001etl2223.13.110011.61.6001hje1923.63.65012.22.25021hp91222:113.53.55012.22.25011ien1324.84.85002.52.5001im11421.31.2011.21.25001kcn1625.75.610003.52.85011kwd3023:292.92.1100221.3011mii1121.81.85001.61.6001oig1522:7::11:146.56.15014.84.75011r8t2421:224.53.410012.62.25011rpc2123:216.96.35014.44001ter21275.950132.85001v6r2621:235.15.110023.93.9001wqc1322:131.71.310021.21.110021x7k2221:174.22.5004.22.5001xgb1623.5300335022ajw1322.40.95021.31002i282822.12.110011.51.5002oq92428:249.2410013.53.35012efz1234.24.2012.12.166.712nx71332:136.96.933.314.1433.311mmc1037.16.166.724.64.633.301orx2231:194.84.833.312.92.7001sp72831:10::12:284.54.366.722.6233.301v5a28355.166.713.73.8001wm81936:167766.723.63.666.722it72832:284466.713.73.766.71mean4.23.761.22.72.529.5pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation.our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation ( 1 ) lies within 0.7 . the discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied ( 2.0 0.2 ) in all - atom structures.the lowest - energy models for 1n0a and 1kwd , and the best rmsd model for 1wm8 are shown on figure 2 . results obtained for 34 cyclic peptides with disulfide bonds pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation . our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation ( 1 ) lies within 0.7 . the discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied ( 2.0 0.2 ) in all - atom structures . the lowest - energy models for 1n0a and 1kwd , and the best rmsd model for 1wm8 are shown on figure 2 . the first goal was to extend the length of linear peptides so as to develop a server able to explore systems with higher thermodynamic stabilities , and with new topologies such as the and 3 folds . using a benchmark of 37 peptides with 936 amino acids , and a total of 100 simulations for each system , the updated pep - fold version fails to generate the native conformation for only one system , where a -strand is preferred over an -helix at the c - terminus . note that our results are reproducible and do not change if we use 200 simulations . preliminary simulations also indicate that pep - fold performs well for sequences upwards of 50 amino acids where the fs is modelled ( data not shown ) , but opening the service for this length is under consideration given the computer resources needed . this increase in peptide size from 25 to 50 amino acids comes from the reduction of the sa letters considered at each position . the structure simulations of even larger systems within the framework of a greedy procedure remain to be evaluated . the second goal was to provide structural predictions for cylic peptides with disulfide bonds . for systems with one and two disulfide bonds , pep - fold performs better than peplook . for systems with more disulfide bonds , such as natural toxins , improvements in the sa letter prediction and atomistic construction one solution for construction , under investigation , is to enforce disulfide bond formation in the gromacs procedure and to relax the models by short molecular dynamics simulations . in summary , the updated version of pep - fold provides a fast and convenient on - line approach for the de novo design of peptides up to 36 residues , and it also supports the possibility to tackle peptide engineering by the insertion of disulfide bonds , a classical way to increase stability . the typical computational time for a 36-residue peptide is 40 min using 40 cores . the server also supports , using the same formalism as disulfide bonds , the possibility to specify additional constraints such as residue proximities . clearly , our results open the door to further improvements such as the consideration of other covalent linking between side - chains and the modelling of ribosomal peptides at a genome scale can be considered . the institut national de la sant et de la recherche medicale recurrent funding , the centre national de la recherche scientifique recurrent funding , and the ia bioinformatics grant [ bipbip ] . funding for open access charge : institut national de la sant et de la recherche medicale umr - s 973 .

in the context of the renewed interest of peptides as therapeutics , it is important to have an on - line resource for 3d structure prediction of peptides with well - defined structures in aqueous solution . we present an updated version of pep - fold allowing the treatment of both linear and disulphide bonded cyclic peptides with 936 amino acids . the server makes possible to define disulphide bonds and any residue residue proximity under the guidance of the biologists . using a benchmark of 34 cyclic peptides with one , two and three disulphide bonds , the best pep - fold models deviate by an average rms of 2.75 from the full nmr structures . using a benchmark of 37 linear peptides , pep - fold locates lowest - energy conformations deviating by 3 rms from the nmr rigid cores . the evolution of pep - fold comes as a new on - line service to supersede the previous server . the server is available at : http://bioserv.rpbs.univ-paris-diderot.fr/pep-fold .

INTRODUCTION MATERIALS AND METHODS SERVICE OVERVIEW RESULTS Linear peptides with 936 amino acids Disulfide-bonded cyclic peptides DISCUSSION FUNDING

though this first rapid on - line version has been used by external users for structural characterization of peptides or protein fragments ( 17,18 ) and peptide or vaccine design ( 19,20 ) , the maximal length of 25 amino acids limits the number of applications . in addition , like the bhageerath ( 12 ) and pepstr ( 13 ) servers , pep - fold was only available for linear peptides , whereas there are many natural cyclic peptides with disulfide bonds such as conotoxins or cyclotides ( 21 ) and disulfide bonds increase peptide stability ( 22 ) . the modifications brought to pep - fold have a limited impact on the linear peptides with 925 amino acids . we see that the sharper sa letter selection does not prevent pep - fold to generate near - native conformations , with the best rigid core ( rc ) conformations deviating on average by only 1.5 from the nmr structures , versus 1.7 using the earlier pep - fold service . the updated pep - fold procedure also generates lowest - energy conformations deviating on average by 2.5 from the nmr rcs . averaged over the 13 systems , pep - fold returns lowest - energy conformations deviating from the first nmr model by 4.8 and 3.4 using the full structure ( fs ) and the rc . similarly , the updated pep - fold version returns the native 3 conformation of the 36-residue 1e0n protein ( figure 2a ) , while the earlier version , using the 27 amino acids which are not random coil in the nmr structure , predicted a -strand packed against two helices ( 11 ) . for the 28-residue 1psv and 31-residue 2gdl , the lowest - energy conformations differ by 7.4 and 4.8 rms from nmr using the rc . table 1.results obtained for 13 linear peptides with 2536 residuespdb idtoplrcsopepbest5fs - drc - dfs - drc - drnk1by0a271:234.361.752.941.7411yyba271:206.491.473.41.4212kblb2293:4|6:274.683.914.683.9111fsdab2281:264.143.883.913.6611psvab2282:257.197.444.614.642k76ba304:293.152.752.11.8812gdlaca318:8|10:11|14:15|21:297.574.846.534.5732l0ga2325:324.533.322.11.6412bn6a2334:294.493.183.092.1411e0nb3351:252.162.161.681.6821wr3b3365:155.723.744.263.511wr4b3365:344.683.264.683.2612ki0bab365:11|13:363.561.992.662.431mean4.83.43.62.8pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model.the lowest - energy models for 1e0n , 1psv , 2l0 g , 2ki0 and 2gdl are shown on figure 2 . results obtained for 13 linear peptides with 2536 residues pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model . table 2 presents the results of 34 peptides containing one , two or three disulfide bonds using the peplook test set ( 14 ) except the peptides 2p7r , 1qvl and 1foz < 8 amino acids , and the peptide 1ixu free of any disulfide bond in the nmr structure . on average , the updated pep - fold generates best models deviating by only 2.7 and 2.5 rms and lowest - energy models deviating by 4.2 and 3.7 rms using the fss and rcs , respectively . for these peptides , the lowest - energy conformations deviate on average by 5.4 rms from the nmr structures and a similar behaviour is observed with peplook ( 5.6 rms ) . this discrepancy between prediction and experiment comes in part from the non - optimization of the sopep force field for cyclic peptides , but mainly we observe that the structures with three disulfide bonds are very constrained , leading to large deviations at the local level between the conformations observed in the nmr structures and the structural alphabet conformations predicted from the sequence free of any disulfide bond constraints . table 2.results obtained for 34 cyclic peptides with disulfide bondspdb idl#ssrcsopepbestfs - drc - dcgaafs - drc - dcgaa1im72113:9::15:213.93.910002.52.510011jbl1612:143.33.310011.81.8001n0a1717:150.70.7010.40.4001n0c2412:11::14:242.32.310010.40.4011nim2411:233.81.8003.42.8011gnb1422:9::12:144.84.150143.85001b451323.63.310012.32.2001etl2223.13.110011.61.6001hje1923.63.65012.22.25021hp91222:113.53.55012.22.25011ien1324.84.85002.52.5001im11421.31.2011.21.25001kcn1625.75.610003.52.85011kwd3023:292.92.1100221.3011mii1121.81.85001.61.6001oig1522:7::11:146.56.15014.84.75011r8t2421:224.53.410012.62.25011rpc2123:216.96.35014.44001ter21275.950132.85001v6r2621:235.15.110023.93.9001wqc1322:131.71.310021.21.110021x7k2221:174.22.5004.22.5001xgb1623.5300335022ajw1322.40.95021.31002i282822.12.110011.51.5002oq92428:249.2410013.53.35012efz1234.24.2012.12.166.712nx71332:136.96.933.314.1433.311mmc1037.16.166.724.64.633.301orx2231:194.84.833.312.92.7001sp72831:10::12:284.54.366.722.6233.301v5a28355.166.713.73.8001wm81936:167766.723.63.666.722it72832:284466.713.73.766.71mean4.23.761.22.72.529.5pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation.our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation ( 1 ) lies within 0.7 . results obtained for 34 cyclic peptides with disulfide bonds pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation . we see that the sharper sa letter selection does not prevent pep - fold to generate near - native conformations , with the best rigid core ( rc ) conformations deviating on average by only 1.5 from the nmr structures , versus 1.7 using the earlier pep - fold service . the updated pep - fold procedure also generates lowest - energy conformations deviating on average by 2.5 from the nmr rcs . averaged over the 13 systems , pep - fold returns lowest - energy conformations deviating from the first nmr model by 4.8 and 3.4 using the full structure ( fs ) and the rc . similarly , the updated pep - fold version returns the native 3 conformation of the 36-residue 1e0n protein ( figure 2a ) , while the earlier version , using the 27 amino acids which are not random coil in the nmr structure , predicted a -strand packed against two helices ( 11 ) . for the 28-residue 1psv and 31-residue 2gdl , the lowest - energy conformations differ by 7.4 and 4.8 rms from nmr using the rc . table 1.results obtained for 13 linear peptides with 2536 residuespdb idtoplrcsopepbest5fs - drc - dfs - drc - drnk1by0a271:234.361.752.941.7411yyba271:206.491.473.41.4212kblb2293:4|6:274.683.914.683.9111fsdab2281:264.143.883.913.6611psvab2282:257.197.444.614.642k76ba304:293.152.752.11.8812gdlaca318:8|10:11|14:15|21:297.574.846.534.5732l0ga2325:324.533.322.11.6412bn6a2334:294.493.183.092.1411e0nb3351:252.162.161.681.6821wr3b3365:155.723.744.263.511wr4b3365:344.683.264.683.2612ki0bab365:11|13:363.561.992.662.431mean4.83.43.62.8pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model.the lowest - energy models for 1e0n , 1psv , 2l0 g , 2ki0 and 2gdl are shown on figure 2 . table 2 presents the results of 34 peptides containing one , two or three disulfide bonds using the peplook test set ( 14 ) except the peptides 2p7r , 1qvl and 1foz < 8 amino acids , and the peptide 1ixu free of any disulfide bond in the nmr structure . on average , the updated pep - fold generates best models deviating by only 2.7 and 2.5 rms and lowest - energy models deviating by 4.2 and 3.7 rms using the fss and rcs , respectively . for these peptides , the lowest - energy conformations deviate on average by 5.4 rms from the nmr structures and a similar behaviour is observed with peplook ( 5.6 rms ) . this discrepancy between prediction and experiment comes in part from the non - optimization of the sopep force field for cyclic peptides , but mainly we observe that the structures with three disulfide bonds are very constrained , leading to large deviations at the local level between the conformations observed in the nmr structures and the structural alphabet conformations predicted from the sequence free of any disulfide bond constraints . table 2.results obtained for 34 cyclic peptides with disulfide bondspdb idl#ssrcsopepbestfs - drc - dcgaafs - drc - dcgaa1im72113:9::15:213.93.910002.52.510011jbl1612:143.33.310011.81.8001n0a1717:150.70.7010.40.4001n0c2412:11::14:242.32.310010.40.4011nim2411:233.81.8003.42.8011gnb1422:9::12:144.84.150143.85001b451323.63.310012.32.2001etl2223.13.110011.61.6001hje1923.63.65012.22.25021hp91222:113.53.55012.22.25011ien1324.84.85002.52.5001im11421.31.2011.21.25001kcn1625.75.610003.52.85011kwd3023:292.92.1100221.3011mii1121.81.85001.61.6001oig1522:7::11:146.56.15014.84.75011r8t2421:224.53.410012.62.25011rpc2123:216.96.35014.44001ter21275.950132.85001v6r2621:235.15.110023.93.9001wqc1322:131.71.310021.21.110021x7k2221:174.22.5004.22.5001xgb1623.5300335022ajw1322.40.95021.31002i282822.12.110011.51.5002oq92428:249.2410013.53.35012efz1234.24.2012.12.166.712nx71332:136.96.933.314.1433.311mmc1037.16.166.724.64.633.301orx2231:194.84.833.312.92.7001sp72831:10::12:284.54.366.722.6233.301v5a28355.166.713.73.8001wm81936:167766.723.63.666.722it72832:284466.713.73.766.71mean4.23.761.22.72.529.5pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation.our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation ( 1 ) lies within 0.7 . the discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied ( 2.0 0.2 ) in all - atom structures.the lowest - energy models for 1n0a and 1kwd , and the best rmsd model for 1wm8 are shown on figure 2 . results obtained for 34 cyclic peptides with disulfide bonds pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation . using a benchmark of 37 peptides with 936 amino acids , and a total of 100 simulations for each system , the updated pep - fold version fails to generate the native conformation for only one system , where a -strand is preferred over an -helix at the c - terminus . in summary , the updated version of pep - fold provides a fast and convenient on - line approach for the de novo design of peptides up to 36 residues , and it also supports the possibility to tackle peptide engineering by the insertion of disulfide bonds , a classical way to increase stability .

second , the shift of therapeutic strategies towards the network of protein interactions , particularly the search for protein protein interaction inhibitors , has pushed forward the limits of small chemical molecules , whereas advances in protein recombinant technologies provide evidence that larger therapeutics such as peptides or peptide derivatives could offer plausible alternatives ( 2 ) . to assist peptide lead identification and optimization , robust computational methods recent efforts from the community of computer scientists have tackled various aspects including the design of generic databases devoted to peptide protein interactions such as pepx ( 8) , the problem of protein peptide docking ( 9 ) , the search for peptidomimetics ( 10 ) and the development of fast peptide structure prediction methods ( e.g. a second major modification involves the use of tm scores ( 27 ) to cluster the full generated structures and then the sopep energies or the predicted tmscores of apollo ( 28)an extension of ( 29 ) for the prediction gdt_ts from structural features to rank the clusters and the conformations within the clusters . finally , other minor modifications have been brought to the side chain positioning in the all - atom generation step it is now achieved using oscar - star ( 30)and the disulfide bond specifications are passed to the quick all - atom minimization based on gromacs ( 31 ) . of particular interest is the high quality prediction of the topology for the 36-residue 2ki0 protein with a rc - d of 2.0 based on the sopep energy ( figure 2e ) which is stabilized by long - range interactions in the amino acid sequence . similarly , the updated pep - fold version returns the native 3 conformation of the 36-residue 1e0n protein ( figure 2a ) , while the earlier version , using the 27 amino acids which are not random coil in the nmr structure , predicted a -strand packed against two helices ( 11 ) . table 1.results obtained for 13 linear peptides with 2536 residuespdb idtoplrcsopepbest5fs - drc - dfs - drc - drnk1by0a271:234.361.752.941.7411yyba271:206.491.473.41.4212kblb2293:4|6:274.683.914.683.9111fsdab2281:264.143.883.913.6611psvab2282:257.197.444.614.642k76ba304:293.152.752.11.8812gdlaca318:8|10:11|14:15|21:297.574.846.534.5732l0ga2325:324.533.322.11.6412bn6a2334:294.493.183.092.1411e0nb3351:252.162.161.681.6821wr3b3365:155.723.744.263.511wr4b3365:344.683.264.683.2612ki0bab365:11|13:363.561.992.662.431mean4.83.43.62.8pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model.the lowest - energy models for 1e0n , 1psv , 2l0 g , 2ki0 and 2gdl are shown on figure 2 . results obtained for 13 linear peptides with 2536 residues pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model . table 2 presents the results of 34 peptides containing one , two or three disulfide bonds using the peplook test set ( 14 ) except the peptides 2p7r , 1qvl and 1foz < 8 amino acids , and the peptide 1ixu free of any disulfide bond in the nmr structure . whereas the server leads to good results for peptides containing 1 and 2 disulfide bonds ( figure 2f and d for 1kwd and 1n0a ) with 61.2% of the disulfide bonds formed at the coarse - grained level ( table 1 ) , the performances are degraded for the nine peptides with three disulfide bonds ( figure 2h for 1wm8 ) . this discrepancy between prediction and experiment comes in part from the non - optimization of the sopep force field for cyclic peptides , but mainly we observe that the structures with three disulfide bonds are very constrained , leading to large deviations at the local level between the conformations observed in the nmr structures and the structural alphabet conformations predicted from the sequence free of any disulfide bond constraints . table 2.results obtained for 34 cyclic peptides with disulfide bondspdb idl#ssrcsopepbestfs - drc - dcgaafs - drc - dcgaa1im72113:9::15:213.93.910002.52.510011jbl1612:143.33.310011.81.8001n0a1717:150.70.7010.40.4001n0c2412:11::14:242.32.310010.40.4011nim2411:233.81.8003.42.8011gnb1422:9::12:144.84.150143.85001b451323.63.310012.32.2001etl2223.13.110011.61.6001hje1923.63.65012.22.25021hp91222:113.53.55012.22.25011ien1324.84.85002.52.5001im11421.31.2011.21.25001kcn1625.75.610003.52.85011kwd3023:292.92.1100221.3011mii1121.81.85001.61.6001oig1522:7::11:146.56.15014.84.75011r8t2421:224.53.410012.62.25011rpc2123:216.96.35014.44001ter21275.950132.85001v6r2621:235.15.110023.93.9001wqc1322:131.71.310021.21.110021x7k2221:174.22.5004.22.5001xgb1623.5300335022ajw1322.40.95021.31002i282822.12.110011.51.5002oq92428:249.2410013.53.35012efz1234.24.2012.12.166.712nx71332:136.96.933.314.1433.311mmc1037.16.166.724.64.633.301orx2231:194.84.833.312.92.7001sp72831:10::12:284.54.366.722.6233.301v5a28355.166.713.73.8001wm81936:167766.723.63.666.722it72832:284466.713.73.766.71mean4.23.761.22.72.529.5pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation.our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation ( 1 ) lies within 0.7 . the discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied ( 2.0 0.2 ) in all - atom structures.the lowest - energy models for 1n0a and 1kwd , and the best rmsd model for 1wm8 are shown on figure 2 . results obtained for 34 cyclic peptides with disulfide bonds pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation . overall , pep - fold generates near - native conformations ( rc - d < 4 ) for 11 among 13 structurally diverse proteins with secondary structure compositions varying from , 2 ( 2l0 g figure 2c ) , 2 , 2 , , to 3 . of particular interest is the high quality prediction of the topology for the 36-residue 2ki0 protein with a rc - d of 2.0 based on the sopep energy ( figure 2e ) which is stabilized by long - range interactions in the amino acid sequence . similarly , the updated pep - fold version returns the native 3 conformation of the 36-residue 1e0n protein ( figure 2a ) , while the earlier version , using the 27 amino acids which are not random coil in the nmr structure , predicted a -strand packed against two helices ( 11 ) . table 1.results obtained for 13 linear peptides with 2536 residuespdb idtoplrcsopepbest5fs - drc - dfs - drc - drnk1by0a271:234.361.752.941.7411yyba271:206.491.473.41.4212kblb2293:4|6:274.683.914.683.9111fsdab2281:264.143.883.913.6611psvab2282:257.197.444.614.642k76ba304:293.152.752.11.8812gdlaca318:8|10:11|14:15|21:297.574.846.534.5732l0ga2325:324.533.322.11.6412bn6a2334:294.493.183.092.1411e0nb3351:252.162.161.681.6821wr3b3365:155.723.744.263.511wr4b3365:344.683.264.683.2612ki0bab365:11|13:363.561.992.662.431mean4.83.43.62.8pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model.the lowest - energy models for 1e0n , 1psv , 2l0 g , 2ki0 and 2gdl are shown on figure 2 . results obtained for 13 linear peptides with 2536 residues pdb i d , pdb identifier ; top , secondary structure topology ( a for helix , b for strand , c for coil ) ; l , peptide length ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the best model among the five clusters of lowest energy ( best5 ) ; rnk , the rank of the cluster containing the best model . whereas the server leads to good results for peptides containing 1 and 2 disulfide bonds ( figure 2f and d for 1kwd and 1n0a ) with 61.2% of the disulfide bonds formed at the coarse - grained level ( table 1 ) , the performances are degraded for the nine peptides with three disulfide bonds ( figure 2h for 1wm8 ) . this discrepancy between prediction and experiment comes in part from the non - optimization of the sopep force field for cyclic peptides , but mainly we observe that the structures with three disulfide bonds are very constrained , leading to large deviations at the local level between the conformations observed in the nmr structures and the structural alphabet conformations predicted from the sequence free of any disulfide bond constraints . table 2.results obtained for 34 cyclic peptides with disulfide bondspdb idl#ssrcsopepbestfs - drc - dcgaafs - drc - dcgaa1im72113:9::15:213.93.910002.52.510011jbl1612:143.33.310011.81.8001n0a1717:150.70.7010.40.4001n0c2412:11::14:242.32.310010.40.4011nim2411:233.81.8003.42.8011gnb1422:9::12:144.84.150143.85001b451323.63.310012.32.2001etl2223.13.110011.61.6001hje1923.63.65012.22.25021hp91222:113.53.55012.22.25011ien1324.84.85002.52.5001im11421.31.2011.21.25001kcn1625.75.610003.52.85011kwd3023:292.92.1100221.3011mii1121.81.85001.61.6001oig1522:7::11:146.56.15014.84.75011r8t2421:224.53.410012.62.25011rpc2123:216.96.35014.44001ter21275.950132.85001v6r2621:235.15.110023.93.9001wqc1322:131.71.310021.21.110021x7k2221:174.22.5004.22.5001xgb1623.5300335022ajw1322.40.95021.31002i282822.12.110011.51.5002oq92428:249.2410013.53.35012efz1234.24.2012.12.166.712nx71332:136.96.933.314.1433.311mmc1037.16.166.724.64.633.301orx2231:194.84.833.312.92.7001sp72831:10::12:284.54.366.722.6233.301v5a28355.166.713.73.8001wm81936:167766.723.63.666.722it72832:284466.713.73.766.71mean4.23.761.22.72.529.5pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation.our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation ( 1 ) lies within 0.7 . the discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied ( 2.0 0.2 ) in all - atom structures.the lowest - energy models for 1n0a and 1kwd , and the best rmsd model for 1wm8 are shown on figure 2 . results obtained for 34 cyclic peptides with disulfide bonds pdb i d , pdb identifier ; l , peptide length ; ss , number of disulfide bond ; rc , the definition of the rigid core ( pdb positions start at 1 ) ; fs - d ( rc - d ) , full structure ( rigid core ) rms deviation ( ) for the model of lowest energy ( sopep ) and the model of lowest rmsd ( best ) ; cg , ss bonds formed in the coarse grained representation ; aa , ss bonds formed in the final all - atom representation . in summary , the updated version of pep - fold provides a fast and convenient on - line approach for the de novo design of peptides up to 36 residues , and it also supports the possibility to tackle peptide engineering by the insertion of disulfide bonds , a classical way to increase stability .

we defined broad consent as a process in which participants agree prospectively to have their samples , genomic data , and health information retained for use in any future research deemed appropriate by a biobank and/or relevant oversight bodies . studies of broad consent may use an opt - in or an opt - out model . categorical consent , by contrast , is a process in which participants agree prospectively to future use of their samples and data for particular types of research , usually by categories of disease ( e.g. , cardiac diseases , diabetes ) . refers to the transfer of biospecimens with their associated genotypic and/or phenotypic information , data derived from biospecimens , and/or health information to researchers at institutions that are not directly affiliated with the biobanks or to other biorepositories . we systematically searched the literature on broad consent and data sharing for biobank research using the following databases : medline via the pubmed interface , web of science , national reference center for bioethics literature databases ( ethxweb , genethx ) , and dissertation abstracts international . search strategies used subject heading terms appropriate for each database and key words relevant to biobanking , consent , and data sharing ( supplementary table s1 online ) . searches were limited to the literature published since 1990 to capture current views about biobanking . we also manually searched the reference lists of included studies and of recent narrative and systematic reviews addressing the topic . our initial searches were done between october and december 2013 and were updated in march 2015 . two reviewers ( n.a.s . and a colleague ) initially screened titles and abstracts , and two investigators ( n.a.g . and e.w.c . ) reviewed the full text of the included articles . articles were included if they reported empirical data with sufficient detail to enable use and aggregation of the data and results about individuals in the united states regarding one or more of the following : participant perceptions of broad consent or data sharing for biobank research , preferences for different consent models for biobank research , information about people 's opinions about participating in biobank research , or providing broad consent for biobank research . disagreements between reviewers were resolved by discussion that included a third reviewer ( a.h.m.a . ) to reach consensus . we identified and screened a total of 3,205 citations and abstracts through the electronic database searches and manual review of articles and bibliographies ( figure 1 ) . after reviewing titles and abstracts , we excluded 2,714 studies that did not meet our criteria . we assessed the full text of the 491 remaining studies and excluded another 440 articles because they ( i ) did not address biobanking , consent , or data sharing ( n = 403 ) ; ( ii ) were not conducted in the united states ( n = 206 ) ; or ( iii ) were not obtainable ( n = 1 ) . two investigators ( n.a.g . and e.w.c . ) assessed the quality of studies using questions adapted from published criteria for the quality assessment of survey and focus group studies . scoring criteria fell into the following broad domains : ( i ) description of the methods , ( ii ) participant recruitment from a representative pool and response rates , ( iii ) appropriateness of objective study questions , and ( iv ) data analysis lending to reproducible results . articles that adequately defined criteria in all four domains were rated as good . articles containing information that had adequate descriptions of the methods but did not fulfill the criteria for all of the other domains received a rating of fair . articles that failed to adequately define their methods , thus preventing an evaluation of representativeness , bias , or reproducibility , received a rating of poor . two investigators ( n.a.g . and e.w.c . ) also characterized the studies as conducted in urban , rural , or combined settings . data were extracted into summary tables ( supplementary table s3 online ) by outlining the study population and biobank focus , methods , quality assessment , urban / rural residency , and key outcomes related to consent and data sharing . we report the relevant findings based on the terminology , percentages , and number of significant digits as presented in the publications . we defined broad consent as a process in which participants agree prospectively to have their samples , genomic data , and health information retained for use in any future research deemed appropriate by a biobank and/or relevant oversight bodies . studies of broad consent may use an opt - in or an opt - out model . categorical consent , by contrast , is a process in which participants agree prospectively to future use of their samples and data for particular types of research , usually by categories of disease ( e.g. , cardiac diseases , diabetes ) . refers to the transfer of biospecimens with their associated genotypic and/or phenotypic information , data derived from biospecimens , and/or health information to researchers at institutions that are not directly affiliated with the biobanks or to other biorepositories . we systematically searched the literature on broad consent and data sharing for biobank research using the following databases : medline via the pubmed interface , web of science , national reference center for bioethics literature databases ( ethxweb , genethx ) , and dissertation abstracts international . search strategies used subject heading terms appropriate for each database and key words relevant to biobanking , consent , and data sharing ( supplementary table s1 online ) . searches were limited to the literature published since 1990 to capture current views about biobanking . we also manually searched the reference lists of included studies and of recent narrative and systematic reviews addressing the topic . our initial searches were done between october and december 2013 and were updated in march 2015 . two reviewers ( n.a.s . and a colleague ) initially screened titles and abstracts , and two investigators ( n.a.g . and e.w.c . ) reviewed the full text of the included articles . articles were included if they reported empirical data with sufficient detail to enable use and aggregation of the data and results about individuals in the united states regarding one or more of the following : participant perceptions of broad consent or data sharing for biobank research , preferences for different consent models for biobank research , information about people 's opinions about participating in biobank research , or providing broad consent for biobank research . disagreements between reviewers were resolved by discussion that included a third reviewer ( a.h.m.a . ) to reach consensus . we identified and screened a total of 3,205 citations and abstracts through the electronic database searches and manual review of articles and bibliographies ( figure 1 ) . after reviewing titles and abstracts , we excluded 2,714 studies that did not meet our criteria . we assessed the full text of the 491 remaining studies and excluded another 440 articles because they ( i ) did not address biobanking , consent , or data sharing ( n = 403 ) ; ( ii ) were not conducted in the united states ( n = 206 ) ; or ( iii ) were not obtainable ( n = 1 ) . two investigators ( n.a.g . and e.w.c . ) assessed the quality of studies using questions adapted from published criteria for the quality assessment of survey and focus group studies . scoring criteria fell into the following broad domains : ( i ) description of the methods , ( ii ) participant recruitment from a representative pool and response rates , ( iii ) appropriateness of objective study questions , and ( iv ) data analysis lending to reproducible results . articles containing information that had adequate descriptions of the methods but did not fulfill the criteria for all of the other domains received a rating of fair . articles that failed to adequately define their methods , thus preventing an evaluation of representativeness , bias , or reproducibility , received a rating of poor . two investigators ( n.a.g . and e.w.c . ) also characterized the studies as conducted in urban , rural , or combined settings . data were extracted into summary tables ( supplementary table s3 online ) by outlining the study population and biobank focus , methods , quality assessment , urban / rural residency , and key outcomes related to consent and data sharing . we report the relevant findings based on the terminology , percentages , and number of significant digits as presented in the publications . a total of 51 publications comprising 48 studies were included in this review . most studies involved surveys ( n = 23 ) , followed by focus groups ( n = 8) , mixed methods ( n = 14 ) , interviews ( n = 1 ) , and analyses of consent forms ( n = 2 ) ( supplementary table s3 online ) . two publications used a mixed - methods approach that included qualitative studies that informed the development and implementation of a survey . nineteen studies were of good quality , 27 of fair quality , and 2 of poor quality . roughly one - third of the studies ( n = 20 ) were written and published after the office of human research protections issued the anprm in july 2011 . the number of studies published per year from 2008 to 2014 ranged from five to seven , with no notable difference after the anprm was issued . some of the studies published after 2011 mention the anprm . although we examined studies published since 1990 , no studies that met our inclusion criteria were published before 2001 . of these , just over half ( 51.3% ) of participants identified as white , and native - american , alaska native , native hawaiian , and pacific islander participants made up 2.2% of the sample . twenty - eight studies were conducted primarily in urban settings , two were conducted in rural settings , nine were conducted in both urban and rural settings , and nine studies were conducted nationwide . three papers each reported two unique studies ; other studies were reported in multiple papers . for example , a retrospective analysis of signed informed consent forms found that 87.1% of 1,298 research participants at the nih authorized all future research . in a different large national study , the national health and nutrition examination survey ( nhanes ) , 84.8% of 4,480 overall participants recruited in 1999 and 2000 agreed to dna specimen collection for inclusion in a national repository for genetic research . many studies asked participants hypothetical questions about their willingness to provide broad consent for research . in indiana , 88.4% of 273 cancer patients agreed that they would be willing to permit their tissue sample to be used in research on any condition . after time for deliberation , 85% of 40 focus group participants in north carolina reported that they would agree to have blood and information stored indefinitely in a biorepository for future research . similarly , 78% of 49 focus group participants in chicago were interested in participating in a biobank , and the majority stated they would give broad consent . of 30 patients who were interviewed at a hawaiian cancer center , 77% endorsed broad consent . one representative nationwide survey found that 68% of 1,593 respondents were willing to give broad consent for research , although their enthusiasm waned if they had a moral objection to certain types of studies for which their samples might be used . two studies examined patients ' willingness to participate in biobanks managed by kaiser permanente : 69% of 500 kaiser patients in the northwest and 69% of 203 in colorado agreed to participate in a biobank . in a focus group study in boston , patients with breast cancer were generally positive about having their samples used for secondary studies that were not planned at the time they gave consent . scott et al . reported the results of a 1998 survey of blood donors that asked about their views regarding storage and use of the blood for research . of the 49,775 respondents , 60.3% said that testing stored blood for any research was acceptable with the donor 's permission , and 35.5% would not require permission for research use . these studies reported substantial acceptance for broad consent . asking participants for their preference among different types of consent broad , study - by - study , or categorical consent revealed more mixed support for broad consent . for example , 47% of 931 veterans preferred to give broad consent over other types of consent for all research approved by an oversight board . after adjusting for missing data , a national survey of 4,569 adults found that 52% preferred broad consent , whereas 48% preferred study - by - study consent . in a survey of 751 iowans , 42% preferred broad consent and 29% favored study - specific consent , compared with 25% who favored categorical consent . in another study of 315 cancer patients at two hospitals in atlanta , 92 and 97% were willing to allow their samples to be used for research on other diseases ; when asked to specify a preference , 56% preferred one - time broad consent and 11% preferred study - by - study consent over no consent or no preference . in a 2001 nationwide survey , 43% of 2,621 participants were willing to donate blood for genetic research and to allow it to be stored for future research . similarly , only 39.3% of 30 patients who had already donated samples preferred broad consent over consent for specific studies . by contrast , 77.7% of 1,276 people recruited through a crowd - sourced internet marketplace were willing to donate to biobanks , even after receiving disclosures about potentially objectionable research ; however , 40.8% of participants still felt that specific consent was necessary , even if it might inhibit research progress . a similar nationwide survey of 1,599 individuals conducted through a probability - based online panel of adults found a wide range of opinions , with broad consent and real - time study - by - study consent considered the several studies showed that participants preferred to give informed consent for each study rather than a broad consent , with preferences ranging from 42 to 72% : 42% of a national sample of 4,700 us adults ( which rose to 48% after adjusting for missing data ) , 43 to 50% of 931 veterans nationwide , and 60.7% of adults recruited in new york . of 393 parents , 72% reported that they would want to consent each time to allow their child 's dried bloodspots to be used for research . in focus groups of 92 native hawaiians , respondents repeatedly expressed desire to re - consent , although some stated that they would be content if they trusted the researcher or the biobank 's governance . in one study of 273 jewish individuals , 6075% believed that consent should be required regardless of whether the dna was collected in a research or clinical setting . in a focus group study of 178 alaska native participants , some indicated a preference to have consent options for a variety of specimen uses , storage duration , and destruction of the sample at the completion of the study . in the same group , some wanted re - contact each time , whereas others felt that a one - time consent was appropriate for new studies . in chicago , 239 postpartum women were asked about their willingness to enroll their children into a pediatric biobank : 48% of women would enroll their child , but 24% would not ; of the latter , 82% of the participants were african american . in another focus group study , 11 of 15 participants preferred tiered consent over other methods to exert the greatest level of control regarding how they wanted their data to be shared ; however , participants who were willing to provide broad consent also appreciated the option to opt in or opt out of dna data sharing . some studies reported that most respondents favored an opt - in approach , whereas others found that opt - out was acceptable or even preferred by the majority . a majority of participants67% of 751 survey respondents and 63% of 57 focus group participants who were asked about biobank participation in iowa preferred opt - in , whereas 18% of survey respondents and 25% of focus group participants in the same study preferred opt - out . in a study of 451 nonactive military veterans , 82% thought it would be acceptable for the proposed million veterans biobank to use an opt - in approach , and 75% thought that an opt - out approach was acceptable ; 80% said that they would take part if the biobank were opt - in as opposed to 69% who would participate if it were an opt - out approach . when asked to choose which option they would prefer , 29% of respondents chose the opt - in method , 14% chose opt - out , 50% said either would be acceptable , and 7% would not want to participate . in some cases , biobank participants were re - contacted to inquire about their thoughts regarding proposed changes to the biobank in which they participated . thirty - two biobank participants who attended focus groups in wisconsin regarding proposed minimal - risk protocol changes were comfortable with using an opt - out model for future studies because of the initial broad consent given at the beginning of the study and their trust in the institution . a study of 365 participants who were re - contacted about their ongoing participation in a biobank in seattle showed that 55% thought that opt - out would be acceptable , compared with 40% who thought it would be unacceptable . similarly , several studies explored perspectives on the acceptability of an opt - out biobank at vanderbilt university . first , 91% of 1,003 participants surveyed in the community thought leftover blood and tissues should be used for anonymous medical research under an opt - out model ; these preferences varied by population , with 76% of african americans supporting this model compared with 93% of whites . in later studies of community members , approval rates for the opt - out biobank were generally high ( around 90% or more ) in all demographic groups surveyed , including university employees , adult cohorts , and parents of pediatric patients . three studies explored community perspectives on using newborn screening blood spots for research through the michigan biotrust for health program . first , 77% of 393 parents agreed that parents should be able to opt out of having their child 's blood stored for research . second , 87 participants were asked to indicate a preference : 55% preferred an opt - out model , 29% preferred to opt - in , and 16% felt that either option was acceptable . finally , 39% of 856 college students reported that they would give broad consent to research with their newborn blood spots , whereas 39% would want to give consent for each use for research . in a nationwide telephone survey regarding the use of samples collected from newborns , 46% of 1,186 adults believed that researchers should re - consent participants when they turn 18 years old . some studies examined the differences in participants ' willingness to provide broad consent for samples that were de - identified or anonymous as compared with identifiable . respondents generally preferred to give consent if their samples were identifiable . in two studies involving 429 primarily native hawaiian participants , 78% of native hawaiians and 66% of whites indicated that they would require consent for research if the specimens were identifiable and collected in the clinical setting . for genetics research , 81% native hawaiians and 78% of whites indicated that they would require consent if the specimens were identifiable . in a us - wide telephone survey , 81% of 1,193 respondents stated that they would want to be informed about research being done with their sample if it were identifiable ; additionally , 57% said they would require permission to use their samples if they were identifiable . for example , 65.8% of 504 adults who participated in a telephone survey across the united states reported that they would require consent for samples collected in the clinic if they were identifiable , compared with 27.3% who reported they would require consent if samples were anonymized . in the research setting , fewer people thought consent was required for identifiable ( 29.0% ) or anonymized ( 12.1% ) samples . in a study utilizing a hypothetical biobank scenario , 43% of 565 government and medical employees in new mexico indicated that they would donate their sample for future genetic testing if it could not be traced to them . not all studies found that people were worried about identifiability . in one survey of 144 clinicians , 86% said that they would donate a dna sample to a hypothetical biobank in new york regardless of whether it was linked to or unlinked from their identity . in the study in new mexico , 36% of 565 respondents found it acceptable for broadly consented samples to be used by their local university , even if the samples were linked to them . characteristics associated with favoring broad consent included being male , white / caucasian , older , and more affluent . by contrast , asians , black non - hispanics , african americans , and others ( who represented 14.3% of the total ) were less likely than whites to believe that research without explicit permission was acceptable . one study of consent forms showed that 75.0% of african americans gave broad consent compared with 88.4% of whites ( p = 0.002 ) . similarly , in the nhanes data , 78.7% of african americans and 87.1% of whites consented to genetics research . one study reported that participants who were significantly more likely to prefer broad consent also believed that participating would make me feel like i was contributing to society ( odds ratio = 1.85 ; p = 0.001 ) , that the study would accelerate medical treatments and cures ( odds ratio = 2.20 ; p = 0.001 ) , and that participating in the cohort study would be easy ( odds ratio = 1.59 ; p < 0.001 ) . other investigators reported that the large majority ( 97.7% ) of respondents said yes or maybe to the idea that it is a gift to society when an individual takes part in medical research . many other studies cited the benefit of research to improve health as a reason to favor broad consent . most studies of data sharing were conducted with studies of consent preferences ; however , six studies were conducted with the primary goal of eliciting preferences on data sharing . participants were generally willing to have their samples and information shared with other academic institutions . willingness to share data with academic and medical researchers was acceptable for 92% of 4,659 us adults generally , and 80% of 931 us veterans specifically . more than 70% of 100 young adults in baltimore who were enrolled in a longitudinal study of prevention were willing to share results arising from their dna . nearly three fourths of 40 community members in a focus group study in north carolina were comfortable with academic researchers having access to their samples . many of 79 focus group participants in seattle endorsed the value of sharing , agreed that sharing locally and with close collaborators was acceptable , and were comfortable with nonprofit and public - interest organizations using data from their samples . in one focus group study of 48 primarily white and female participants in iowa , the majority cited positive reasons for donating their samples to help and to contribute to advancements in research , and that data sharing would not affect their decision to enroll in a biobank . in another focus group study of 100 african americans in north carolina , many recognized the benefits of data sharing but wanted the potential risks to be disclosed , and some wanted the data to be restricted . in another study of patients with inflammatory bowel diseases , 97.3% of 92 respondents were comfortable with sharing their biological sample with investigators in the united states , but 23.8% were uncomfortable with sharing with investigators outside the united states . some participants expressed concern over sharing their data and information with federal repositories . in one study , 18.5% of 4,050 vanderbilt university faculty and staff were more likely to want to participate in their institution 's biobank if the de - identified data were deposited into a national database ; however , 12.1% were less likely to want to participate . in two large nationwide surveys , 80% of 4,659 adults were willing to have their data shared with government researchers ; however , 75% of the same sample also were concerned about the government having [ their ] samples and information . similarly , another study found that 71% of 931 veterans were willing to grant database access to government researchers , but half were concerned about the government having [ their ] samples and information . other studies have shown that some people are concerned about government involvement in maintaining databases containing biomedical information . more than half of the 40 participants in a focus group study of north carolina community members were concerned about government researchers having access to their institution 's biorepository . despite concerns , 61% of 203 kaiser patients in colorado would still provide a sample even if the data would be submitted to a government database , and 82% of 500 kaiser patients in oregon agreed to have their information posted in a us government database . in a large metropolitan area in southwest florida , some of 95 focus group participants believed that biospecimens were already being collected from leftover tissue , and others suspected that tissues were already being shared with researchers in other countries who lack strict laws ' governing research . in a focus group study of 178 alaska native participants , some cited mistrust of the government and police having access to their samples and wanted transparency from the researchers about how their samples were used . the majority of participants were willing to share with pharmaceutical company researchers , but the percentage was generally less than the percentage willing to share with academic researchers . seventy - five percent of 4,659 us adults , 54% of 931 veterans , 55.2% of 1,599 adults responding to a nationwide survey , and 75.1% of members of the crohn 's and colitis foundation of america partners cohort were willing to share with pharmaceutical company researchers . focus group participants in florida voiced concern about providing blanket consent because they would not benefit financially from any resulting discoveries . factors associated with views about data sharing . with the exception of gender , few demographic data ( e.g. , about race / ethnicity , socioeconomic status , education , and urban / rural residency ) were available . even when demographic information was obtained , investigators did not always report how these variables correlated with respondents ' opinions . therefore , it was largely not possible to draw meaningful conclusions about the associations between sociodemographic factors and views on data sharing . the willingness of patients with cancer to share seemed to be shaped by their devotion to the institution at which they were receiving care . for example , patients with cancer in indiana who agreed to participate in a biobank were less likely to be willing to allow their tissue samples to be used by researchers who were not affiliated with the local researchers ( 89.7% ) , compared with 96.3% who were willing to share with local university researchers ( p < 0.01 ) . half of 100 patients with breast cancer at md anderson cancer center preferred to allow only their physician ( 24% ) or other researchers at their hospital ( 26% ) to use their de - identified genetic data for research ; fewer patients were willing to share their de - identified data with any cancer researcher ( 25% ) or any researcher ( 18% ) . in another study , 95% of 315 patients with cancer in atlanta were willing to allow researchers to share samples with other local researchers , but only 85% and 92% of participants at two different sites were willing to have their samples shared elsewhere in the united states ( p < 0.05 ) . a total of 51 publications comprising 48 studies were included in this review . most studies involved surveys ( n = 23 ) , followed by focus groups ( n = 8) , mixed methods ( n = 14 ) , interviews ( n = 1 ) , and analyses of consent forms ( n = 2 ) ( supplementary table s3 online ) . two publications used a mixed - methods approach that included qualitative studies that informed the development and implementation of a survey . nineteen studies were of good quality , 27 of fair quality , and 2 of poor quality . roughly one - third of the studies ( n = 20 ) were written and published after the office of human research protections issued the anprm in july 2011 . the number of studies published per year from 2008 to 2014 ranged from five to seven , with no notable difference after the anprm was issued . some of the studies published after 2011 mention the anprm . although we examined studies published since 1990 , no studies that met our inclusion criteria were published before 2001 . of these , just over half ( 51.3% ) of participants identified as white , and 13.6% were african american and 6.3% were hispanic / latino . native - american , alaska native , native hawaiian , and pacific islander participants made up 2.2% of the sample . many studies did not report other demographic data . only 21 studies reported socioeconomic status , and 43 reported educational level . twenty - eight studies were conducted primarily in urban settings , two were conducted in rural settings , nine were conducted in both urban and rural settings , and nine studies were conducted nationwide . three papers each reported two unique studies ; other studies were reported in multiple papers . for example , a retrospective analysis of signed informed consent forms found that 87.1% of 1,298 research participants at the nih authorized all future research . in a different large national study , the national health and nutrition examination survey ( nhanes ) , 84.8% of 4,480 overall participants recruited in 1999 and 2000 agreed to dna specimen collection for inclusion in a national repository for genetic research . many studies asked participants hypothetical questions about their willingness to provide broad consent for research . in indiana , 88.4% of 273 cancer patients agreed that they would be willing to permit their tissue sample to be used in research on any condition . after time for deliberation , 85% of 40 focus group participants in north carolina reported that they would agree to have blood and information stored indefinitely in a biorepository for future research . similarly , 78% of 49 focus group participants in chicago were interested in participating in a biobank , and the majority stated they would give broad consent . of 30 patients who were interviewed at a hawaiian cancer center , 77% endorsed broad consent . one representative nationwide survey found that 68% of 1,593 respondents were willing to give broad consent for research , although their enthusiasm waned if they had a moral objection to certain types of studies for which their samples might be used . two studies examined patients ' willingness to participate in biobanks managed by kaiser permanente : 69% of 500 kaiser patients in the northwest and 69% of 203 in colorado agreed to participate in a biobank . in a focus group study in boston , patients with breast cancer were generally positive about having their samples used for secondary studies that were not planned at the time they gave consent . scott et al . reported the results of a 1998 survey of blood donors that asked about their views regarding storage and use of the blood for research . of the 49,775 respondents , 60.3% said that testing stored blood for any research was acceptable with the donor 's permission , and 35.5% would not require permission for research use . these studies reported substantial acceptance for broad consent . asking participants for their preference among different types of consent broad , study - by - study , or categorical consent revealed more mixed support for broad consent . for example , 47% of 931 veterans preferred to give broad consent over other types of consent for all research approved by an oversight board . after adjusting for missing data , a national survey of 4,569 adults found that 52% preferred broad consent , whereas 48% preferred study - by - study consent . in a survey of 751 iowans , 42% preferred broad consent and 29% favored study - specific consent , compared with 25% who favored categorical consent . in another study of 315 cancer patients at two hospitals in atlanta , 92 and 97% were willing to allow their samples to be used for research on other diseases ; when asked to specify a preference , 56% preferred one - time broad consent and 11% preferred study - by - study consent over no consent or no preference . in a 2001 nationwide survey , 43% of 2,621 participants were willing to donate blood for genetic research and to allow it to be stored for future research . similarly , only 39.3% of 30 patients who had already donated samples preferred broad consent over consent for specific studies . by contrast , 77.7% of 1,276 people recruited through a crowd - sourced internet marketplace were willing to donate to biobanks , even after receiving disclosures about potentially objectionable research ; however , 40.8% of participants still felt that specific consent was necessary , even if it might inhibit research progress . a similar nationwide survey of 1,599 individuals conducted through a probability - based online panel of adults found a wide range of opinions , with broad consent and real - time study - by - study consent considered the several studies showed that participants preferred to give informed consent for each study rather than a broad consent , with preferences ranging from 42 to 72% : 42% of a national sample of 4,700 us adults ( which rose to 48% after adjusting for missing data ) , 43 to 50% of 931 veterans nationwide , and 60.7% of adults recruited in new york . of 393 parents , 72% reported that they would want to consent each time to allow their child 's dried bloodspots to be used for research . in focus groups of 92 native hawaiians , respondents repeatedly expressed desire to re - consent , although some stated that they would be content if they trusted the researcher or the biobank 's governance . in one study of 273 jewish individuals , 6075% believed that consent should be required regardless of whether the dna was collected in a research or clinical setting . in a focus group study of 178 alaska native participants , some indicated a preference to have consent options for a variety of specimen uses , storage duration , and destruction of the sample at the completion of the study . in the same group , some wanted re - contact each time , whereas others felt that a one - time consent was appropriate for new studies . in chicago , 239 postpartum women were asked about their willingness to enroll their children into a pediatric biobank : 48% of women would enroll their child , but 24% would not ; of the latter , 82% of the participants were african american . in another focus group study , 11 of 15 participants preferred tiered consent over other methods to exert the greatest level of control regarding how they wanted their data to be shared ; however , participants who were willing to provide broad consent also appreciated the option to opt in or opt out of dna data sharing . some studies reported that most respondents favored an opt - in approach , whereas others found that opt - out was acceptable or even preferred by the majority . a majority of participants67% of 751 survey respondents and 63% of 57 focus group participants who were asked about biobank participation in iowa preferred opt - in , whereas 18% of survey respondents and 25% of focus group participants in the same study preferred opt - out . in a study of 451 nonactive military veterans , 82% thought it would be acceptable for the proposed million veterans biobank to use an opt - in approach , and 75% thought that an opt - out approach was acceptable ; 80% said that they would take part if the biobank were opt - in as opposed to 69% who would participate if it were an opt - out approach . when asked to choose which option they would prefer , 29% of respondents chose the opt - in method , 14% chose opt - out , 50% said either would be acceptable , and 7% would not want to participate . in some cases , biobank participants were re - contacted to inquire about their thoughts regarding proposed changes to the biobank in which they participated . thirty - two biobank participants who attended focus groups in wisconsin regarding proposed minimal - risk protocol changes were comfortable with using an opt - out model for future studies because of the initial broad consent given at the beginning of the study and their trust in the institution . a study of 365 participants who were re - contacted about their ongoing participation in a biobank in seattle showed that 55% thought that opt - out would be acceptable , compared with 40% who thought it would be unacceptable . similarly , several studies explored perspectives on the acceptability of an opt - out biobank at vanderbilt university . first , 91% of 1,003 participants surveyed in the community thought leftover blood and tissues should be used for anonymous medical research under an opt - out model ; these preferences varied by population , with 76% of african americans supporting this model compared with 93% of whites . in later studies of community members , approval rates for the opt - out biobank were generally high ( around 90% or more ) in all demographic groups surveyed , including university employees , adult cohorts , and parents of pediatric patients . three studies explored community perspectives on using newborn screening blood spots for research through the michigan biotrust for health program . first , 77% of 393 parents agreed that parents should be able to opt out of having their child 's blood stored for research . second , 87 participants were asked to indicate a preference : 55% preferred an opt - out model , 29% preferred to opt - in , and 16% felt that either option was acceptable . finally , 39% of 856 college students reported that they would give broad consent to research with their newborn blood spots , whereas 39% would want to give consent for each use for research . in a nationwide telephone survey regarding the use of samples collected from newborns , 46% of 1,186 adults believed that researchers should re - consent participants when they turn 18 years old . some studies examined the differences in participants ' willingness to provide broad consent for samples that were de - identified or anonymous as compared with identifiable . respondents generally preferred to give consent if their samples were identifiable . in two studies involving 429 primarily native hawaiian participants , 78% of native hawaiians and 66% of whites indicated that they would require consent for research if the specimens were identifiable and collected in the clinical setting . for genetics research , 81% native hawaiians and 78% of whites indicated that they would require consent if the specimens were identifiable . in a us - wide telephone survey , 81% of 1,193 respondents stated that they would want to be informed about research being done with their sample if it were identifiable ; additionally , 57% said they would require permission to use their samples if they were identifiable . for example , 65.8% of 504 adults who participated in a telephone survey across the united states reported that they would require consent for samples collected in the clinic if they were identifiable , compared with 27.3% who reported they would require consent if samples were anonymized . in the research setting , fewer people thought consent was required for identifiable ( 29.0% ) or anonymized ( 12.1% ) samples . in a study utilizing a hypothetical biobank scenario , 43% of 565 government and medical employees in new mexico indicated that they would donate their sample for future genetic testing if it could not be traced to them . not all studies found that people were worried about identifiability . in one survey of 144 clinicians , 86% said that they would donate a dna sample to a hypothetical biobank in new york regardless of whether it was linked to or unlinked from their identity . in the study in new mexico , 36% of 565 respondents found it acceptable for broadly consented samples to be used by their local university , even if the samples were linked to them . characteristics associated with favoring broad consent included being male , white / caucasian , older , and more affluent . by contrast , asians , black non - hispanics , african americans , and others ( who represented 14.3% of the total ) were less likely than whites to believe that research without explicit permission was acceptable . one study of consent forms showed that 75.0% of african americans gave broad consent compared with 88.4% of whites ( p = 0.002 ) . similarly , in the nhanes data , 78.7% of african americans and 87.1% of whites consented to genetics research . one study reported that participants who were significantly more likely to prefer broad consent also believed that participating would make me feel like i was contributing to society ( odds ratio = 1.85 ; p = 0.001 ) , that the study would accelerate medical treatments and cures ( odds ratio = 2.20 ; p = 0.001 ) , and that participating in the cohort study would be easy ( odds ratio = 1.59 ; p < 0.001 ) . other investigators reported that the large majority ( 97.7% ) of respondents said yes or maybe to the idea that it is a gift to society when an individual takes part in medical research . many other studies cited the benefit of research to improve health as a reason to favor broad consent . we identified 23 studies of data sharing . the earliest publications about participants ' preferences on data sharing date to 2006 . most studies of data sharing were conducted with studies of consent preferences ; however , six studies were conducted with the primary goal of eliciting preferences on data sharing . participants were generally willing to have their samples and information shared with other academic institutions . willingness to share data with academic and medical researchers was acceptable for 92% of 4,659 us adults generally , and 80% of 931 us veterans specifically . more than 70% of 100 young adults in baltimore who were enrolled in a longitudinal study of prevention were willing to share results arising from their dna . nearly three fourths of 40 community members in a focus group study in north carolina were comfortable with academic researchers having access to their samples . many of 79 focus group participants in seattle endorsed the value of sharing , agreed that sharing locally and with close collaborators was acceptable , and were comfortable with nonprofit and public - interest organizations using data from their samples . in one focus group study of 48 primarily white and female participants in iowa , the majority cited positive reasons for donating their samples to help and to contribute to advancements in research , and that data sharing would not affect their decision to enroll in a biobank . in another focus group study of 100 african americans in north carolina , many recognized the benefits of data sharing but wanted the potential risks to be disclosed , and some wanted the data to be restricted . in another study of patients with inflammatory bowel diseases , 97.3% of 92 respondents were comfortable with sharing their biological sample with investigators in the united states , but 23.8% were uncomfortable with sharing with investigators outside the united states . some participants expressed concern over sharing their data and information with federal repositories . in one study , 18.5% of 4,050 vanderbilt university faculty and staff were more likely to want to participate in their institution 's biobank if the de - identified data were deposited into a national database ; however , 12.1% were less likely to want to participate . in two large nationwide surveys , 80% of 4,659 adults were willing to have their data shared with government researchers ; however , 75% of the same sample also were concerned about the government having [ their ] samples and information . similarly , another study found that 71% of 931 veterans were willing to grant database access to government researchers , but half were concerned about the government having [ their ] samples and information . other studies have shown that some people are concerned about government involvement in maintaining databases containing biomedical information . more than half of the 40 participants in a focus group study of north carolina community members were concerned about government researchers having access to their institution 's biorepository . despite concerns , 61% of 203 kaiser patients in colorado would still provide a sample even if the data would be submitted to a government database , and 82% of 500 kaiser patients in oregon agreed to have their information posted in a us government database . in a large metropolitan area in southwest florida , some of 95 focus group participants believed that biospecimens were already being collected from leftover tissue , and others suspected that tissues were already being shared with researchers in other countries who lack strict laws ' governing research . in a focus group study of 178 alaska native participants , some cited mistrust of the government and police having access to their samples and wanted transparency from the researchers about how their samples were used . the majority of participants were willing to share with pharmaceutical company researchers , but the percentage was generally less than the percentage willing to share with academic researchers . seventy - five percent of 4,659 us adults , 54% of 931 veterans , 55.2% of 1,599 adults responding to a nationwide survey , and 75.1% of members of the crohn 's and colitis foundation of america partners cohort were willing to share with pharmaceutical company researchers . focus group participants in florida voiced concern about providing blanket consent because they would not benefit financially from any resulting discoveries . factors associated with views about data sharing . with the exception of gender , few demographic data ( e.g. , about race / ethnicity , socioeconomic status , education , and urban / rural residency ) even when demographic information was obtained , investigators did not always report how these variables correlated with respondents ' opinions . therefore , it was largely not possible to draw meaningful conclusions about the associations between sociodemographic factors and views on data sharing . the willingness of patients with cancer to share seemed to be shaped by their devotion to the institution at which they were receiving care . for example , patients with cancer in indiana who agreed to participate in a biobank were less likely to be willing to allow their tissue samples to be used by researchers who were not affiliated with the local researchers ( 89.7% ) , compared with 96.3% who were willing to share with local university researchers ( p < 0.01 ) . half of 100 patients with breast cancer at md anderson cancer center preferred to allow only their physician ( 24% ) or other researchers at their hospital ( 26% ) to use their de - identified genetic data for research ; fewer patients were willing to share their de - identified data with any cancer researcher ( 25% ) or any researcher ( 18% ) . in another study , 95% of 315 patients with cancer in atlanta were willing to allow researchers to share samples with other local researchers , but only 85% and 92% of participants at two different sites were willing to have their samples shared elsewhere in the united states ( p < 0.05 ) . in 2013 , nih funded the emerge consortium to perform a broad population - based survey to assess public opinion about broad consent for research and data sharing . this systematic literature review , which ultimately contained 48 studies involving 35,969 participants , was conducted to identify gaps and issues that needed to be addressed in this survey . the most notable finding is that many people do not favor broad consent for either research itself or for research and subsequent wide data sharing . while the majority often expressed support for broad consent when that was the only choice offered , only a minority of respondents favored broad consent when other options , such as tiered or study - by - study consent , were offered . furthermore , earlier studies focused on the importance of obtaining consent for research , whereas later studies focused on the preferences for different consent options . willingness to give broad consent increased if data were de - identified . while individuals were generally willing for data or biospecimens to be shared with other academic researchers , individuals were less willing for their data to be shared in federal databases or with commercial enterprises . what is equally striking are the large gaps in what is known about factors that affect people 's decisions . while a few studies generally found that men were more likely to support broad consent , most investigators did not examine the impact of gender on attitudes . although data about race / ethnicity are incomplete , it seems that minorities often have more concerns about broad consent , although existing evidence suggests that these concerns can be ameliorated in some cases by discussion and education . much less is known about the impact of sociodemographic factors such as socioeconomic status , education , and whether people live in urban or rural environments on attitudes toward broad consent and data sharing . building on these findings , the emerge cerc survey developed a sampling strategy , experimental study design , and survey questions to ascertain more uniformly the views of individuals throughout society in order to identify and address concerns . first , we used broad search terms to capture the existing literature on broad consent and data sharing . thus , while we used multiple approaches ( e.g. , searching multiple sources , reviewing reference lists , and searching the unpublished , gray literature , such as dissertations and reports ) to comprehensively identify studies , we may not have identified all salient research . second , we adapted existing metrics of quality scores to our study . for many studies , we were unable to ascertain the appropriateness of study questions or an analysis plan , thus limiting our ability to thoroughly assess the quality of the studies . third , the studies that have been conducted to date have a number of limitations , which in turn limit the generalizability of this literature review . many of the surveys focus what people say they think , rather what they actually do , even though opinions may differ from action . definitions of consent were not always consistent and have changed over time , which not only limits our ability to compare studies but also may affect our evaluation of older studies given today 's ethical standards for biobanking governance . however , all studies were sufficiently focused on broad consent for research or for data sharing to permit some comparison . most of the surveys heavily oversampled whites , whereas the qualitative studies disproportionately involved minority participants . studies that incorporated an educational component may have influenced respondents compared with those studies that did not involve education around biobanking practices . this review also was limited to the united states , which is warranted given the different policy preferences in other countries . the ultimate goal of this literature review and the emerge cerc survey is to obtain a more comprehensive understanding of public opinion about broad consent for data sharing and use . the studies included here typically noted a general acceptance for broad consent and endorsement of data sharing , but with notable privacy and governance concerns , especially by minority participants . the policy question will be what to do if some people , particularly from certain demographics , express a desire for more granular control over the use of data obtained from them in light of the policy trend toward requiring individual consent for broad data use and sharing . at a minimum , it suggests the need to engage those who are skeptical , even if it is decided that the public good of research to improve health outweighs honoring individual objections in some cases or the risk that some people will choose not to participate .

purpose : in 2011 , an advanced notice of proposed rulemaking proposed that de - identified human data and specimens be included in biobanks only if patients provide consent . the national institutes of health genomic data sharing policy went into effect in 2015 , requiring broad consent from almost all research participants.genet med 18 7 , 663671.methods:we conducted a systematic literature review of attitudes toward biobanking , broad consent , and data sharing . bibliographic databases included medline , web of science , ethxweb , and genethx . study screening was conducted using distillersr.genet med 18 7 , 663671.results:the final 48 studies included surveys ( n = 23 ) , focus groups ( n = 8) , mixed methods ( n = 14 ) , interviews ( n = 1 ) , and consent form analyses ( n = 2 ) . study quality was characterized as good ( n = 19 ) , fair ( n = 27 ) , and poor ( n = 2 ) . although many participants objected , broad consent was often preferred over tiered or study - specific consent , particularly when broad consent was the only option , samples were de - identified , logistics of biobanks were communicated , and privacy was addressed . willingness for data to be shared was high , but it was lower among individuals from under - represented minorities , individuals with privacy and confidentiality concerns , and when pharmaceutical companies had access to data.genet med 18 7 , 663671.conclusions:additional research is needed to understand factors affecting willingness to give broad consent for biobank research and data sharing in order to address concerns to enhance acceptability.genet med 18 7 , 663671 .

Materials and Methods Definitions Literature search strategy Data extraction and analysis Results Article selection Participant demographics Studies of broad consent Studies of data sharing Discussion and Conclusion Disclosure Supplementary Material

we defined broad consent as a process in which participants agree prospectively to have their samples , genomic data , and health information retained for use in any future research deemed appropriate by a biobank and/or relevant oversight bodies . categorical consent , by contrast , is a process in which participants agree prospectively to future use of their samples and data for particular types of research , usually by categories of disease ( e.g. we systematically searched the literature on broad consent and data sharing for biobank research using the following databases : medline via the pubmed interface , web of science , national reference center for bioethics literature databases ( ethxweb , genethx ) , and dissertation abstracts international . search strategies used subject heading terms appropriate for each database and key words relevant to biobanking , consent , and data sharing ( supplementary table s1 online ) . and a colleague ) initially screened titles and abstracts , and two investigators ( n.a.g . articles were included if they reported empirical data with sufficient detail to enable use and aggregation of the data and results about individuals in the united states regarding one or more of the following : participant perceptions of broad consent or data sharing for biobank research , preferences for different consent models for biobank research , information about people 's opinions about participating in biobank research , or providing broad consent for biobank research . we identified and screened a total of 3,205 citations and abstracts through the electronic database searches and manual review of articles and bibliographies ( figure 1 ) . we assessed the full text of the 491 remaining studies and excluded another 440 articles because they ( i ) did not address biobanking , consent , or data sharing ( n = 403 ) ; ( ii ) were not conducted in the united states ( n = 206 ) ; or ( iii ) were not obtainable ( n = 1 ) . data were extracted into summary tables ( supplementary table s3 online ) by outlining the study population and biobank focus , methods , quality assessment , urban / rural residency , and key outcomes related to consent and data sharing . we defined broad consent as a process in which participants agree prospectively to have their samples , genomic data , and health information retained for use in any future research deemed appropriate by a biobank and/or relevant oversight bodies . categorical consent , by contrast , is a process in which participants agree prospectively to future use of their samples and data for particular types of research , usually by categories of disease ( e.g. we systematically searched the literature on broad consent and data sharing for biobank research using the following databases : medline via the pubmed interface , web of science , national reference center for bioethics literature databases ( ethxweb , genethx ) , and dissertation abstracts international . search strategies used subject heading terms appropriate for each database and key words relevant to biobanking , consent , and data sharing ( supplementary table s1 online ) . and a colleague ) initially screened titles and abstracts , and two investigators ( n.a.g . articles were included if they reported empirical data with sufficient detail to enable use and aggregation of the data and results about individuals in the united states regarding one or more of the following : participant perceptions of broad consent or data sharing for biobank research , preferences for different consent models for biobank research , information about people 's opinions about participating in biobank research , or providing broad consent for biobank research . we identified and screened a total of 3,205 citations and abstracts through the electronic database searches and manual review of articles and bibliographies ( figure 1 ) . we assessed the full text of the 491 remaining studies and excluded another 440 articles because they ( i ) did not address biobanking , consent , or data sharing ( n = 403 ) ; ( ii ) were not conducted in the united states ( n = 206 ) ; or ( iii ) were not obtainable ( n = 1 ) . data were extracted into summary tables ( supplementary table s3 online ) by outlining the study population and biobank focus , methods , quality assessment , urban / rural residency , and key outcomes related to consent and data sharing . a total of 51 publications comprising 48 studies were included in this review . most studies involved surveys ( n = 23 ) , followed by focus groups ( n = 8) , mixed methods ( n = 14 ) , interviews ( n = 1 ) , and analyses of consent forms ( n = 2 ) ( supplementary table s3 online ) . roughly one - third of the studies ( n = 20 ) were written and published after the office of human research protections issued the anprm in july 2011 . in a different large national study , the national health and nutrition examination survey ( nhanes ) , 84.8% of 4,480 overall participants recruited in 1999 and 2000 agreed to dna specimen collection for inclusion in a national repository for genetic research . many studies asked participants hypothetical questions about their willingness to provide broad consent for research . similarly , 78% of 49 focus group participants in chicago were interested in participating in a biobank , and the majority stated they would give broad consent . one representative nationwide survey found that 68% of 1,593 respondents were willing to give broad consent for research , although their enthusiasm waned if they had a moral objection to certain types of studies for which their samples might be used . two studies examined patients ' willingness to participate in biobanks managed by kaiser permanente : 69% of 500 kaiser patients in the northwest and 69% of 203 in colorado agreed to participate in a biobank . asking participants for their preference among different types of consent broad , study - by - study , or categorical consent revealed more mixed support for broad consent . for example , 47% of 931 veterans preferred to give broad consent over other types of consent for all research approved by an oversight board . after adjusting for missing data , a national survey of 4,569 adults found that 52% preferred broad consent , whereas 48% preferred study - by - study consent . in a survey of 751 iowans , 42% preferred broad consent and 29% favored study - specific consent , compared with 25% who favored categorical consent . in another study of 315 cancer patients at two hospitals in atlanta , 92 and 97% were willing to allow their samples to be used for research on other diseases ; when asked to specify a preference , 56% preferred one - time broad consent and 11% preferred study - by - study consent over no consent or no preference . similarly , only 39.3% of 30 patients who had already donated samples preferred broad consent over consent for specific studies . by contrast , 77.7% of 1,276 people recruited through a crowd - sourced internet marketplace were willing to donate to biobanks , even after receiving disclosures about potentially objectionable research ; however , 40.8% of participants still felt that specific consent was necessary , even if it might inhibit research progress . a similar nationwide survey of 1,599 individuals conducted through a probability - based online panel of adults found a wide range of opinions , with broad consent and real - time study - by - study consent considered the several studies showed that participants preferred to give informed consent for each study rather than a broad consent , with preferences ranging from 42 to 72% : 42% of a national sample of 4,700 us adults ( which rose to 48% after adjusting for missing data ) , 43 to 50% of 931 veterans nationwide , and 60.7% of adults recruited in new york . in chicago , 239 postpartum women were asked about their willingness to enroll their children into a pediatric biobank : 48% of women would enroll their child , but 24% would not ; of the latter , 82% of the participants were african american . in another focus group study , 11 of 15 participants preferred tiered consent over other methods to exert the greatest level of control regarding how they wanted their data to be shared ; however , participants who were willing to provide broad consent also appreciated the option to opt in or opt out of dna data sharing . thirty - two biobank participants who attended focus groups in wisconsin regarding proposed minimal - risk protocol changes were comfortable with using an opt - out model for future studies because of the initial broad consent given at the beginning of the study and their trust in the institution . finally , 39% of 856 college students reported that they would give broad consent to research with their newborn blood spots , whereas 39% would want to give consent for each use for research . some studies examined the differences in participants ' willingness to provide broad consent for samples that were de - identified or anonymous as compared with identifiable . respondents generally preferred to give consent if their samples were identifiable . for example , 65.8% of 504 adults who participated in a telephone survey across the united states reported that they would require consent for samples collected in the clinic if they were identifiable , compared with 27.3% who reported they would require consent if samples were anonymized . in the study in new mexico , 36% of 565 respondents found it acceptable for broadly consented samples to be used by their local university , even if the samples were linked to them . characteristics associated with favoring broad consent included being male , white / caucasian , older , and more affluent . one study reported that participants who were significantly more likely to prefer broad consent also believed that participating would make me feel like i was contributing to society ( odds ratio = 1.85 ; p = 0.001 ) , that the study would accelerate medical treatments and cures ( odds ratio = 2.20 ; p = 0.001 ) , and that participating in the cohort study would be easy ( odds ratio = 1.59 ; p < 0.001 ) . in one focus group study of 48 primarily white and female participants in iowa , the majority cited positive reasons for donating their samples to help and to contribute to advancements in research , and that data sharing would not affect their decision to enroll in a biobank . in another focus group study of 100 african americans in north carolina , many recognized the benefits of data sharing but wanted the potential risks to be disclosed , and some wanted the data to be restricted . in one study , 18.5% of 4,050 vanderbilt university faculty and staff were more likely to want to participate in their institution 's biobank if the de - identified data were deposited into a national database ; however , 12.1% were less likely to want to participate . in a focus group study of 178 alaska native participants , some cited mistrust of the government and police having access to their samples and wanted transparency from the researchers about how their samples were used . half of 100 patients with breast cancer at md anderson cancer center preferred to allow only their physician ( 24% ) or other researchers at their hospital ( 26% ) to use their de - identified genetic data for research ; fewer patients were willing to share their de - identified data with any cancer researcher ( 25% ) or any researcher ( 18% ) . a total of 51 publications comprising 48 studies were included in this review . most studies involved surveys ( n = 23 ) , followed by focus groups ( n = 8) , mixed methods ( n = 14 ) , interviews ( n = 1 ) , and analyses of consent forms ( n = 2 ) ( supplementary table s3 online ) . roughly one - third of the studies ( n = 20 ) were written and published after the office of human research protections issued the anprm in july 2011 . in a different large national study , the national health and nutrition examination survey ( nhanes ) , 84.8% of 4,480 overall participants recruited in 1999 and 2000 agreed to dna specimen collection for inclusion in a national repository for genetic research . many studies asked participants hypothetical questions about their willingness to provide broad consent for research . similarly , 78% of 49 focus group participants in chicago were interested in participating in a biobank , and the majority stated they would give broad consent . one representative nationwide survey found that 68% of 1,593 respondents were willing to give broad consent for research , although their enthusiasm waned if they had a moral objection to certain types of studies for which their samples might be used . for example , 47% of 931 veterans preferred to give broad consent over other types of consent for all research approved by an oversight board . after adjusting for missing data , a national survey of 4,569 adults found that 52% preferred broad consent , whereas 48% preferred study - by - study consent . in a survey of 751 iowans , 42% preferred broad consent and 29% favored study - specific consent , compared with 25% who favored categorical consent . in another study of 315 cancer patients at two hospitals in atlanta , 92 and 97% were willing to allow their samples to be used for research on other diseases ; when asked to specify a preference , 56% preferred one - time broad consent and 11% preferred study - by - study consent over no consent or no preference . in a 2001 nationwide survey , 43% of 2,621 participants were willing to donate blood for genetic research and to allow it to be stored for future research . similarly , only 39.3% of 30 patients who had already donated samples preferred broad consent over consent for specific studies . a similar nationwide survey of 1,599 individuals conducted through a probability - based online panel of adults found a wide range of opinions , with broad consent and real - time study - by - study consent considered the several studies showed that participants preferred to give informed consent for each study rather than a broad consent , with preferences ranging from 42 to 72% : 42% of a national sample of 4,700 us adults ( which rose to 48% after adjusting for missing data ) , 43 to 50% of 931 veterans nationwide , and 60.7% of adults recruited in new york . in focus groups of 92 native hawaiians , respondents repeatedly expressed desire to re - consent , although some stated that they would be content if they trusted the researcher or the biobank 's governance . in another focus group study , 11 of 15 participants preferred tiered consent over other methods to exert the greatest level of control regarding how they wanted their data to be shared ; however , participants who were willing to provide broad consent also appreciated the option to opt in or opt out of dna data sharing . finally , 39% of 856 college students reported that they would give broad consent to research with their newborn blood spots , whereas 39% would want to give consent for each use for research . some studies examined the differences in participants ' willingness to provide broad consent for samples that were de - identified or anonymous as compared with identifiable . respondents generally preferred to give consent if their samples were identifiable . for example , 65.8% of 504 adults who participated in a telephone survey across the united states reported that they would require consent for samples collected in the clinic if they were identifiable , compared with 27.3% who reported they would require consent if samples were anonymized . in the study in new mexico , 36% of 565 respondents found it acceptable for broadly consented samples to be used by their local university , even if the samples were linked to them . characteristics associated with favoring broad consent included being male , white / caucasian , older , and more affluent . one study reported that participants who were significantly more likely to prefer broad consent also believed that participating would make me feel like i was contributing to society ( odds ratio = 1.85 ; p = 0.001 ) , that the study would accelerate medical treatments and cures ( odds ratio = 2.20 ; p = 0.001 ) , and that participating in the cohort study would be easy ( odds ratio = 1.59 ; p < 0.001 ) . in one focus group study of 48 primarily white and female participants in iowa , the majority cited positive reasons for donating their samples to help and to contribute to advancements in research , and that data sharing would not affect their decision to enroll in a biobank . in another focus group study of 100 african americans in north carolina , many recognized the benefits of data sharing but wanted the potential risks to be disclosed , and some wanted the data to be restricted . in one study , 18.5% of 4,050 vanderbilt university faculty and staff were more likely to want to participate in their institution 's biobank if the de - identified data were deposited into a national database ; however , 12.1% were less likely to want to participate . similarly , another study found that 71% of 931 veterans were willing to grant database access to government researchers , but half were concerned about the government having [ their ] samples and information . despite concerns , 61% of 203 kaiser patients in colorado would still provide a sample even if the data would be submitted to a government database , and 82% of 500 kaiser patients in oregon agreed to have their information posted in a us government database . in a focus group study of 178 alaska native participants , some cited mistrust of the government and police having access to their samples and wanted transparency from the researchers about how their samples were used . for example , patients with cancer in indiana who agreed to participate in a biobank were less likely to be willing to allow their tissue samples to be used by researchers who were not affiliated with the local researchers ( 89.7% ) , compared with 96.3% who were willing to share with local university researchers ( p < 0.01 ) . half of 100 patients with breast cancer at md anderson cancer center preferred to allow only their physician ( 24% ) or other researchers at their hospital ( 26% ) to use their de - identified genetic data for research ; fewer patients were willing to share their de - identified data with any cancer researcher ( 25% ) or any researcher ( 18% ) . in 2013 , nih funded the emerge consortium to perform a broad population - based survey to assess public opinion about broad consent for research and data sharing . this systematic literature review , which ultimately contained 48 studies involving 35,969 participants , was conducted to identify gaps and issues that needed to be addressed in this survey . the most notable finding is that many people do not favor broad consent for either research itself or for research and subsequent wide data sharing . while the majority often expressed support for broad consent when that was the only choice offered , only a minority of respondents favored broad consent when other options , such as tiered or study - by - study consent , were offered . willingness to give broad consent increased if data were de - identified . while individuals were generally willing for data or biospecimens to be shared with other academic researchers , individuals were less willing for their data to be shared in federal databases or with commercial enterprises . much less is known about the impact of sociodemographic factors such as socioeconomic status , education , and whether people live in urban or rural environments on attitudes toward broad consent and data sharing . building on these findings , the emerge cerc survey developed a sampling strategy , experimental study design , and survey questions to ascertain more uniformly the views of individuals throughout society in order to identify and address concerns . first , we used broad search terms to capture the existing literature on broad consent and data sharing . however , all studies were sufficiently focused on broad consent for research or for data sharing to permit some comparison . the ultimate goal of this literature review and the emerge cerc survey is to obtain a more comprehensive understanding of public opinion about broad consent for data sharing and use . the studies included here typically noted a general acceptance for broad consent and endorsement of data sharing , but with notable privacy and governance concerns , especially by minority participants . the policy question will be what to do if some people , particularly from certain demographics , express a desire for more granular control over the use of data obtained from them in light of the policy trend toward requiring individual consent for broad data use and sharing .

refers to the transfer of biospecimens with their associated genotypic and/or phenotypic information , data derived from biospecimens , and/or health information to researchers at institutions that are not directly affiliated with the biobanks or to other biorepositories . we systematically searched the literature on broad consent and data sharing for biobank research using the following databases : medline via the pubmed interface , web of science , national reference center for bioethics literature databases ( ethxweb , genethx ) , and dissertation abstracts international . articles were included if they reported empirical data with sufficient detail to enable use and aggregation of the data and results about individuals in the united states regarding one or more of the following : participant perceptions of broad consent or data sharing for biobank research , preferences for different consent models for biobank research , information about people 's opinions about participating in biobank research , or providing broad consent for biobank research . we identified and screened a total of 3,205 citations and abstracts through the electronic database searches and manual review of articles and bibliographies ( figure 1 ) . we assessed the full text of the 491 remaining studies and excluded another 440 articles because they ( i ) did not address biobanking , consent , or data sharing ( n = 403 ) ; ( ii ) were not conducted in the united states ( n = 206 ) ; or ( iii ) were not obtainable ( n = 1 ) . scoring criteria fell into the following broad domains : ( i ) description of the methods , ( ii ) participant recruitment from a representative pool and response rates , ( iii ) appropriateness of objective study questions , and ( iv ) data analysis lending to reproducible results . articles containing information that had adequate descriptions of the methods but did not fulfill the criteria for all of the other domains received a rating of fair . data were extracted into summary tables ( supplementary table s3 online ) by outlining the study population and biobank focus , methods , quality assessment , urban / rural residency , and key outcomes related to consent and data sharing . we report the relevant findings based on the terminology , percentages , and number of significant digits as presented in the publications . refers to the transfer of biospecimens with their associated genotypic and/or phenotypic information , data derived from biospecimens , and/or health information to researchers at institutions that are not directly affiliated with the biobanks or to other biorepositories . we systematically searched the literature on broad consent and data sharing for biobank research using the following databases : medline via the pubmed interface , web of science , national reference center for bioethics literature databases ( ethxweb , genethx ) , and dissertation abstracts international . articles were included if they reported empirical data with sufficient detail to enable use and aggregation of the data and results about individuals in the united states regarding one or more of the following : participant perceptions of broad consent or data sharing for biobank research , preferences for different consent models for biobank research , information about people 's opinions about participating in biobank research , or providing broad consent for biobank research . we identified and screened a total of 3,205 citations and abstracts through the electronic database searches and manual review of articles and bibliographies ( figure 1 ) . we assessed the full text of the 491 remaining studies and excluded another 440 articles because they ( i ) did not address biobanking , consent , or data sharing ( n = 403 ) ; ( ii ) were not conducted in the united states ( n = 206 ) ; or ( iii ) were not obtainable ( n = 1 ) . scoring criteria fell into the following broad domains : ( i ) description of the methods , ( ii ) participant recruitment from a representative pool and response rates , ( iii ) appropriateness of objective study questions , and ( iv ) data analysis lending to reproducible results . data were extracted into summary tables ( supplementary table s3 online ) by outlining the study population and biobank focus , methods , quality assessment , urban / rural residency , and key outcomes related to consent and data sharing . we report the relevant findings based on the terminology , percentages , and number of significant digits as presented in the publications . most studies involved surveys ( n = 23 ) , followed by focus groups ( n = 8) , mixed methods ( n = 14 ) , interviews ( n = 1 ) , and analyses of consent forms ( n = 2 ) ( supplementary table s3 online ) . roughly one - third of the studies ( n = 20 ) were written and published after the office of human research protections issued the anprm in july 2011 . of these , just over half ( 51.3% ) of participants identified as white , and native - american , alaska native , native hawaiian , and pacific islander participants made up 2.2% of the sample . in a different large national study , the national health and nutrition examination survey ( nhanes ) , 84.8% of 4,480 overall participants recruited in 1999 and 2000 agreed to dna specimen collection for inclusion in a national repository for genetic research . in indiana , 88.4% of 273 cancer patients agreed that they would be willing to permit their tissue sample to be used in research on any condition . after time for deliberation , 85% of 40 focus group participants in north carolina reported that they would agree to have blood and information stored indefinitely in a biorepository for future research . two studies examined patients ' willingness to participate in biobanks managed by kaiser permanente : 69% of 500 kaiser patients in the northwest and 69% of 203 in colorado agreed to participate in a biobank . in a focus group study in boston , patients with breast cancer were generally positive about having their samples used for secondary studies that were not planned at the time they gave consent . in another study of 315 cancer patients at two hospitals in atlanta , 92 and 97% were willing to allow their samples to be used for research on other diseases ; when asked to specify a preference , 56% preferred one - time broad consent and 11% preferred study - by - study consent over no consent or no preference . a similar nationwide survey of 1,599 individuals conducted through a probability - based online panel of adults found a wide range of opinions , with broad consent and real - time study - by - study consent considered the several studies showed that participants preferred to give informed consent for each study rather than a broad consent , with preferences ranging from 42 to 72% : 42% of a national sample of 4,700 us adults ( which rose to 48% after adjusting for missing data ) , 43 to 50% of 931 veterans nationwide , and 60.7% of adults recruited in new york . in a focus group study of 178 alaska native participants , some indicated a preference to have consent options for a variety of specimen uses , storage duration , and destruction of the sample at the completion of the study . in chicago , 239 postpartum women were asked about their willingness to enroll their children into a pediatric biobank : 48% of women would enroll their child , but 24% would not ; of the latter , 82% of the participants were african american . in another focus group study , 11 of 15 participants preferred tiered consent over other methods to exert the greatest level of control regarding how they wanted their data to be shared ; however , participants who were willing to provide broad consent also appreciated the option to opt in or opt out of dna data sharing . a majority of participants67% of 751 survey respondents and 63% of 57 focus group participants who were asked about biobank participation in iowa preferred opt - in , whereas 18% of survey respondents and 25% of focus group participants in the same study preferred opt - out . in a study of 451 nonactive military veterans , 82% thought it would be acceptable for the proposed million veterans biobank to use an opt - in approach , and 75% thought that an opt - out approach was acceptable ; 80% said that they would take part if the biobank were opt - in as opposed to 69% who would participate if it were an opt - out approach . thirty - two biobank participants who attended focus groups in wisconsin regarding proposed minimal - risk protocol changes were comfortable with using an opt - out model for future studies because of the initial broad consent given at the beginning of the study and their trust in the institution . first , 91% of 1,003 participants surveyed in the community thought leftover blood and tissues should be used for anonymous medical research under an opt - out model ; these preferences varied by population , with 76% of african americans supporting this model compared with 93% of whites . in later studies of community members , approval rates for the opt - out biobank were generally high ( around 90% or more ) in all demographic groups surveyed , including university employees , adult cohorts , and parents of pediatric patients . for example , 65.8% of 504 adults who participated in a telephone survey across the united states reported that they would require consent for samples collected in the clinic if they were identifiable , compared with 27.3% who reported they would require consent if samples were anonymized . by contrast , asians , black non - hispanics , african americans , and others ( who represented 14.3% of the total ) were less likely than whites to believe that research without explicit permission was acceptable . one study reported that participants who were significantly more likely to prefer broad consent also believed that participating would make me feel like i was contributing to society ( odds ratio = 1.85 ; p = 0.001 ) , that the study would accelerate medical treatments and cures ( odds ratio = 2.20 ; p = 0.001 ) , and that participating in the cohort study would be easy ( odds ratio = 1.59 ; p < 0.001 ) . many of 79 focus group participants in seattle endorsed the value of sharing , agreed that sharing locally and with close collaborators was acceptable , and were comfortable with nonprofit and public - interest organizations using data from their samples . in one focus group study of 48 primarily white and female participants in iowa , the majority cited positive reasons for donating their samples to help and to contribute to advancements in research , and that data sharing would not affect their decision to enroll in a biobank . in another focus group study of 100 african americans in north carolina , many recognized the benefits of data sharing but wanted the potential risks to be disclosed , and some wanted the data to be restricted . in another study of patients with inflammatory bowel diseases , 97.3% of 92 respondents were comfortable with sharing their biological sample with investigators in the united states , but 23.8% were uncomfortable with sharing with investigators outside the united states . in one study , 18.5% of 4,050 vanderbilt university faculty and staff were more likely to want to participate in their institution 's biobank if the de - identified data were deposited into a national database ; however , 12.1% were less likely to want to participate . more than half of the 40 participants in a focus group study of north carolina community members were concerned about government researchers having access to their institution 's biorepository . in a large metropolitan area in southwest florida , some of 95 focus group participants believed that biospecimens were already being collected from leftover tissue , and others suspected that tissues were already being shared with researchers in other countries who lack strict laws ' governing research . in a focus group study of 178 alaska native participants , some cited mistrust of the government and police having access to their samples and wanted transparency from the researchers about how their samples were used . seventy - five percent of 4,659 us adults , 54% of 931 veterans , 55.2% of 1,599 adults responding to a nationwide survey , and 75.1% of members of the crohn 's and colitis foundation of america partners cohort were willing to share with pharmaceutical company researchers . for example , patients with cancer in indiana who agreed to participate in a biobank were less likely to be willing to allow their tissue samples to be used by researchers who were not affiliated with the local researchers ( 89.7% ) , compared with 96.3% who were willing to share with local university researchers ( p < 0.01 ) . half of 100 patients with breast cancer at md anderson cancer center preferred to allow only their physician ( 24% ) or other researchers at their hospital ( 26% ) to use their de - identified genetic data for research ; fewer patients were willing to share their de - identified data with any cancer researcher ( 25% ) or any researcher ( 18% ) . in another study , 95% of 315 patients with cancer in atlanta were willing to allow researchers to share samples with other local researchers , but only 85% and 92% of participants at two different sites were willing to have their samples shared elsewhere in the united states ( p < 0.05 ) . most studies involved surveys ( n = 23 ) , followed by focus groups ( n = 8) , mixed methods ( n = 14 ) , interviews ( n = 1 ) , and analyses of consent forms ( n = 2 ) ( supplementary table s3 online ) . two studies examined patients ' willingness to participate in biobanks managed by kaiser permanente : 69% of 500 kaiser patients in the northwest and 69% of 203 in colorado agreed to participate in a biobank . in a focus group study in boston , patients with breast cancer were generally positive about having their samples used for secondary studies that were not planned at the time they gave consent . in another study of 315 cancer patients at two hospitals in atlanta , 92 and 97% were willing to allow their samples to be used for research on other diseases ; when asked to specify a preference , 56% preferred one - time broad consent and 11% preferred study - by - study consent over no consent or no preference . a similar nationwide survey of 1,599 individuals conducted through a probability - based online panel of adults found a wide range of opinions , with broad consent and real - time study - by - study consent considered the several studies showed that participants preferred to give informed consent for each study rather than a broad consent , with preferences ranging from 42 to 72% : 42% of a national sample of 4,700 us adults ( which rose to 48% after adjusting for missing data ) , 43 to 50% of 931 veterans nationwide , and 60.7% of adults recruited in new york . in a focus group study of 178 alaska native participants , some indicated a preference to have consent options for a variety of specimen uses , storage duration , and destruction of the sample at the completion of the study . in another focus group study , 11 of 15 participants preferred tiered consent over other methods to exert the greatest level of control regarding how they wanted their data to be shared ; however , participants who were willing to provide broad consent also appreciated the option to opt in or opt out of dna data sharing . a majority of participants67% of 751 survey respondents and 63% of 57 focus group participants who were asked about biobank participation in iowa preferred opt - in , whereas 18% of survey respondents and 25% of focus group participants in the same study preferred opt - out . in a study of 451 nonactive military veterans , 82% thought it would be acceptable for the proposed million veterans biobank to use an opt - in approach , and 75% thought that an opt - out approach was acceptable ; 80% said that they would take part if the biobank were opt - in as opposed to 69% who would participate if it were an opt - out approach . thirty - two biobank participants who attended focus groups in wisconsin regarding proposed minimal - risk protocol changes were comfortable with using an opt - out model for future studies because of the initial broad consent given at the beginning of the study and their trust in the institution . first , 91% of 1,003 participants surveyed in the community thought leftover blood and tissues should be used for anonymous medical research under an opt - out model ; these preferences varied by population , with 76% of african americans supporting this model compared with 93% of whites . by contrast , asians , black non - hispanics , african americans , and others ( who represented 14.3% of the total ) were less likely than whites to believe that research without explicit permission was acceptable . one study reported that participants who were significantly more likely to prefer broad consent also believed that participating would make me feel like i was contributing to society ( odds ratio = 1.85 ; p = 0.001 ) , that the study would accelerate medical treatments and cures ( odds ratio = 2.20 ; p = 0.001 ) , and that participating in the cohort study would be easy ( odds ratio = 1.59 ; p < 0.001 ) . many of 79 focus group participants in seattle endorsed the value of sharing , agreed that sharing locally and with close collaborators was acceptable , and were comfortable with nonprofit and public - interest organizations using data from their samples . in one focus group study of 48 primarily white and female participants in iowa , the majority cited positive reasons for donating their samples to help and to contribute to advancements in research , and that data sharing would not affect their decision to enroll in a biobank . in another focus group study of 100 african americans in north carolina , many recognized the benefits of data sharing but wanted the potential risks to be disclosed , and some wanted the data to be restricted . in another study of patients with inflammatory bowel diseases , 97.3% of 92 respondents were comfortable with sharing their biological sample with investigators in the united states , but 23.8% were uncomfortable with sharing with investigators outside the united states . in one study , 18.5% of 4,050 vanderbilt university faculty and staff were more likely to want to participate in their institution 's biobank if the de - identified data were deposited into a national database ; however , 12.1% were less likely to want to participate . in a large metropolitan area in southwest florida , some of 95 focus group participants believed that biospecimens were already being collected from leftover tissue , and others suspected that tissues were already being shared with researchers in other countries who lack strict laws ' governing research . in a focus group study of 178 alaska native participants , some cited mistrust of the government and police having access to their samples and wanted transparency from the researchers about how their samples were used . seventy - five percent of 4,659 us adults , 54% of 931 veterans , 55.2% of 1,599 adults responding to a nationwide survey , and 75.1% of members of the crohn 's and colitis foundation of america partners cohort were willing to share with pharmaceutical company researchers . for example , patients with cancer in indiana who agreed to participate in a biobank were less likely to be willing to allow their tissue samples to be used by researchers who were not affiliated with the local researchers ( 89.7% ) , compared with 96.3% who were willing to share with local university researchers ( p < 0.01 ) . half of 100 patients with breast cancer at md anderson cancer center preferred to allow only their physician ( 24% ) or other researchers at their hospital ( 26% ) to use their de - identified genetic data for research ; fewer patients were willing to share their de - identified data with any cancer researcher ( 25% ) or any researcher ( 18% ) . in another study , 95% of 315 patients with cancer in atlanta were willing to allow researchers to share samples with other local researchers , but only 85% and 92% of participants at two different sites were willing to have their samples shared elsewhere in the united states ( p < 0.05 ) .

a recent study found that survival after inhospital cardiac arrest has improved markedly over the past decade , from 13.7% in 2000 to 22.3% in 2009 . while encouraging , it remains unknown whether this survival trend has occurred uniformly across most hospitals or driven by large improvements at a smaller number of hospitals . it is possible that some hospitals have achieved larger gains in survival over time compared with other hospitals . identifying topperforming sites that have achieved large gains in inhospital cardiac arrest survival and the associated hospital factors is the critical next step to inform ongoing quality improvement efforts for inhospital resuscitation . to address these gaps in knowledge , we used data from get with the guidelines ( gwtg)resuscitation , a large national registry of inhospital cardiac arrests , to characterize hospitallevel trends in inhospital cardiac arrest survival over the past decade . additionally , we determined whether some hospitals have achieved greater improvements in survival compared with others , and evaluated hospital factors associated with the extent of hospital rates of survival improvement . gwtgresuscitation , formerly known as the national registry for cardiopulmonary resuscitation , is a large , prospective , hospitalbased clinical registry of patients with inhospital cardiac arrest in the united states . briefly , cardiac arrest in the registry is defined as the absence of a palpable central pulse , apnea , and unresponsiveness . consecutive patients with a cardiac arrest , without donotresuscitate orders , and who received cardiopulmonary resuscitation ( cpr ) are identified and enrolled by specially trained personnel using an online , interactive case report form . multiple case finding methods are used to ensure that all cases within a hospital are captured . these include centralized collection of cardiac arrest flow sheets , review of hospital page system logs , and routine checks of code carts , pharmacy tracer drug records , and hospital billing charges for use of resuscitation medications . data are collected according to the utsteinstyle definitions , which are a template of uniform reporting guidelines developed by international experts . prior to data collection and entry , moreover , the data submission software performs internal data checks to ensure data completeness and to alert the data entry person for outlier responses . within gwtgresuscitation , we identified 122 746 patients at 590 hospitals during 20002010 who were 18 years of age or older and had an index cardiac arrest with an identifiable initial rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , or pulseless ventricular tachycardia ) ( figure 1 ) . from this sample , we excluded patients who were missing information on survival ( n=197 ) and calendar year of the arrest ( n=55 ) . we also excluded 43 hospitals ( 5348 patients with cardiac arrest ) that were missing information on hospital characteristics . finally , given that the estimate of survival improvement from hospitals with low cardiac arrest volume or few years of available data would be unreliable , we restricted our sample to only those hospitals that participated in the registry for 5 years and had a mean annual case volume of 10 cases in accordance with previous studies . as a result our final sample consisted of 93 342 patients from 231 hospitals . in an effort to better understand which hospital characteristics are associated with survival improvement , we merged data from the 2008 american hospital association annual survey with gwtgresuscitation to obtain information on hospital characteristics . these included information on a hospital 's geographic region ( north midatlantic , south atlantic , north central , south central , and mountain / pacific ) , location ( rural , urban ) , ownership ( nonprofit , public , private ) , teaching status ( fellowship program , residency program , or nonteaching ) , and bed number ( 250 , 250 to 499 , 500 ) . patientlevel data available from gwtgresuscitation included demographics ( age , sex , race ) , initial cardiac arrest rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , pulseless ventricular tachycardia ) , location of cardiac arrest ( intensive care unit , telemetry unit , nonmonitored unit ) , time of day ( work hours : 7 am to 10:59 pm versus after hours : 11 pm to 6:59 am ) and day of week ( weekday versus weekend ) of cardiac arrest , and use of a hospitalwide cardiopulmonary arrest alert ( ie , code blue ) . moreover , we used information on comorbid conditions ( congestive heart failure ; myocardial infarction ; diabetes mellitus ; renal , hepatic , or respiratory insufficiency ; neurological status prearrest [ as determined by admission cerebral performance category { cpc } scores ] ; baseline evidence of motor ; cognitive , or functional deficits [ central nervous system depression ] ; acute stroke ; pneumonia ; hypotension ; arrhythmia ; sepsis ; trauma ; metabolic or electrolyte abnormality ; cancer ) , and therapeutic interventions in place at the time of cardiac arrest ( use of mechanical ventilation , antiarrhythmic drugs , intravenous vasopressors , dialysis , pulmonary artery catheter , intraaortic balloon pump ) . the main independent variable was calendar year , and the primary outcome was hospital rate of survival to discharge . we first conducted bivariate analyses to evaluate unadjusted trends in patient and hospital characteristics over time by using the maentelhaenszel test for categorical variables and linear regression for continuous variables . to evaluate temporal trends in hospitallevel survival rates such models account for clustering of patients within a hospital and avoid overestimation of significance of statistical associations . the initial model included hospitallevel random intercepts and slopes by calendar year , which was modeled as a continuous variable . from this model , empirical bayesian estimates of hospitalspecific intercepts and slopes were obtained , which are shrunken toward the mean for smallvolume hospitals , reflecting the lack of information available for those hospitals and thus mitigating issues of outliers . slopes estimates were exponentiated to represent the relative change in odds of survival per year for each hospital . 1 ) were considered to have improved trends in survival , with the magnitude of the slope ( and or ) quantifying the extent of annual survival improvement . finally , we repeated these analyses including adjustment for patient and hospital characteristics , to obtain adjusted annual trends in hospital rates of survival . we examined hospital variation in survival trends using a cumulative frequency plot of the hospitalspecific or . next , we categorized hospitals into quartiles using the hospitalspecific estimates of survival improvement from the model just described . finally , to explore which hospital characteristics were associated with a higher rate of survival improvement , we included interaction terms between each hospital characteristic ( as described earlier in the hospital and patient variables section ) and calendar year in our multivariable model . data were complete for all covariates , except race ( 6.5% ) , admission cpc score ( 15% ) , hospital location of arrest ( 2.1% ) , and time of cardiac arrest ( 1.1% ) . missing data on covariates first , to exclude the possibility that hospitals with the largest temporal improvement in survival to discharge were not confounded by higher rates of neurological disability , we examined whether hospitals with the greatest survival gains were also the same hospitals with the largest temporal improvement in favorable neurological survival . information on discharge neurological status in gwtgresuscitation was collected using cpc scores . because data on discharge cpc scores were missing in 2241 ( 12% ) survivors , we used multiple imputation to assign discharge cpc scores for survivors for whom that information was missing . we defined favorable neurological survival as survival to discharge with a cpc of 1 ( ie , none or mild neurological disability ) and calculated adjusted hospitallevel rates of favorable neurological survival using the hierarchical model just described . we then examined pearson 's correlation between hospital trends in favorable neurological survival and survival to discharge . second , to ensure that our findings were not influenced by temporal changes in hospital discharge practice patterns ( eg , early discharge to hospice of patients with poor likelihood of survival ) , we repeated our analyses of hospitallevel trends after classifying patients who were discharged to hospice as having died . we then examined whether this substantially altered our classification of hospitals with the greatest improvement in survival . all statistical analyses were prespecified and conducted using sas version 9.1.3 ( sas institute ) , iveware ( university of michigan ) , and r version 2.6.0 ( free software foundation ) . the institutional review board at university of iowa approved the study and waived the requirement for informed consent . gwtgresuscitation , formerly known as the national registry for cardiopulmonary resuscitation , is a large , prospective , hospitalbased clinical registry of patients with inhospital cardiac arrest in the united states . briefly , cardiac arrest in the registry is defined as the absence of a palpable central pulse , apnea , and unresponsiveness . consecutive patients with a cardiac arrest , without donotresuscitate orders , and who received cardiopulmonary resuscitation ( cpr ) are identified and enrolled by specially trained personnel using an online , interactive case report form . multiple case finding methods are used to ensure that all cases within a hospital are captured . these include centralized collection of cardiac arrest flow sheets , review of hospital page system logs , and routine checks of code carts , pharmacy tracer drug records , and hospital billing charges for use of resuscitation medications . data are collected according to the utsteinstyle definitions , which are a template of uniform reporting guidelines developed by international experts . prior to data collection and entry , moreover , the data submission software performs internal data checks to ensure data completeness and to alert the data entry person for outlier responses . within gwtgresuscitation , we identified 122 746 patients at 590 hospitals during 20002010 who were 18 years of age or older and had an index cardiac arrest with an identifiable initial rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , or pulseless ventricular tachycardia ) ( figure 1 ) . from this sample , we excluded patients who were missing information on survival ( n=197 ) and calendar year of the arrest ( n=55 ) . we also excluded 43 hospitals ( 5348 patients with cardiac arrest ) that were missing information on hospital characteristics . finally , given that the estimate of survival improvement from hospitals with low cardiac arrest volume or few years of available data would be unreliable , we restricted our sample to only those hospitals that participated in the registry for 5 years and had a mean annual case volume of 10 cases in accordance with previous studies . as a result in an effort to better understand which hospital characteristics are associated with survival improvement , we merged data from the 2008 american hospital association annual survey with gwtgresuscitation to obtain information on hospital characteristics . these included information on a hospital 's geographic region ( north midatlantic , south atlantic , north central , south central , and mountain / pacific ) , location ( rural , urban ) , ownership ( nonprofit , public , private ) , teaching status ( fellowship program , residency program , or nonteaching ) , and bed number ( 250 , 250 to 499 , 500 ) . patientlevel data available from gwtgresuscitation included demographics ( age , sex , race ) , initial cardiac arrest rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , pulseless ventricular tachycardia ) , location of cardiac arrest ( intensive care unit , telemetry unit , nonmonitored unit ) , time of day ( work hours : 7 am to 10:59 pm versus after hours : 11 pm to 6:59 am ) and day of week ( weekday versus weekend ) of cardiac arrest , and use of a hospitalwide cardiopulmonary arrest alert ( ie , code blue ) . moreover , we used information on comorbid conditions ( congestive heart failure ; myocardial infarction ; diabetes mellitus ; renal , hepatic , or respiratory insufficiency ; neurological status prearrest [ as determined by admission cerebral performance category { cpc } scores ] ; baseline evidence of motor ; cognitive , or functional deficits [ central nervous system depression ] ; acute stroke ; pneumonia ; hypotension ; arrhythmia ; sepsis ; trauma ; metabolic or electrolyte abnormality ; cancer ) , and therapeutic interventions in place at the time of cardiac arrest ( use of mechanical ventilation , antiarrhythmic drugs , intravenous vasopressors , dialysis , pulmonary artery catheter , intraaortic balloon pump ) . the main independent variable was calendar year , and the primary outcome was hospital rate of survival to discharge . we first conducted bivariate analyses to evaluate unadjusted trends in patient and hospital characteristics over time by using the maentelhaenszel test for categorical variables and linear regression for continuous variables . to evaluate temporal trends in hospitallevel survival rates such models account for clustering of patients within a hospital and avoid overestimation of significance of statistical associations . the initial model included hospitallevel random intercepts and slopes by calendar year , which was modeled as a continuous variable . from this model , empirical bayesian estimates of hospitalspecific intercepts and slopes were obtained , which are shrunken toward the mean for smallvolume hospitals , reflecting the lack of information available for those hospitals and thus mitigating issues of outliers . slopes estimates were exponentiated to represent the relative change in odds of survival per year for each hospital . 1 ) were considered to have improved trends in survival , with the magnitude of the slope ( and or ) quantifying the extent of annual survival improvement . finally , we repeated these analyses including adjustment for patient and hospital characteristics , to obtain adjusted annual trends in hospital rates of survival . we examined hospital variation in survival trends using a cumulative frequency plot of the hospitalspecific or . next , we categorized hospitals into quartiles using the hospitalspecific estimates of survival improvement from the model just described . finally , to explore which hospital characteristics were associated with a higher rate of survival improvement , we included interaction terms between each hospital characteristic ( as described earlier in the hospital and patient variables section ) and calendar year in our multivariable model . data were complete for all covariates , except race ( 6.5% ) , admission cpc score ( 15% ) , hospital location of arrest ( 2.1% ) , and time of cardiac arrest ( 1.1% ) . missing data on covariates first , to exclude the possibility that hospitals with the largest temporal improvement in survival to discharge were not confounded by higher rates of neurological disability , we examined whether hospitals with the greatest survival gains were also the same hospitals with the largest temporal improvement in favorable neurological survival . information on discharge neurological status in gwtgresuscitation was collected using cpc scores . because data on discharge cpc scores were missing in 2241 ( 12% ) survivors , we used multiple imputation to assign discharge cpc scores for survivors for whom that information was missing . we defined favorable neurological survival as survival to discharge with a cpc of 1 ( ie , none or mild neurological disability ) and calculated adjusted hospitallevel rates of favorable neurological survival using the hierarchical model just described . we then examined pearson 's correlation between hospital trends in favorable neurological survival and survival to discharge . second , to ensure that our findings were not influenced by temporal changes in hospital discharge practice patterns ( eg , early discharge to hospice of patients with poor likelihood of survival ) , we repeated our analyses of hospitallevel trends after classifying patients who were discharged to hospice as having died . we then examined whether this substantially altered our classification of hospitals with the greatest improvement in survival . all statistical analyses were prespecified and conducted using sas version 9.1.3 ( sas institute ) , iveware ( university of michigan ) , and r version 2.6.0 ( free software foundation ) . the institutional review board at university of iowa approved the study and waived the requirement for informed consent . table 1 describes characteristics of the study population . the mean age ( sd ) was 65.9 ( 15.9 ) years . the initial cardiac arrest rhythm was asystole or pulseless electrical activity in nearly 4 in 5 patients . more than 80% of inhospital cardiac arrests occurred in a monitored setting ( intensive care unit [ 48.0% ] , telemetry unit [ 15.7% ] , or other closely monitored hospital locations [ 17.6% ] ) , and > 30% of arrests each occurred during nighttime and on the weekend . approximately , 80% of all patients had a witnessed arrest , and this was substantially higher for cardiac arrests in the intensive care unit ( 94.5% ) compared with those in monitored units ( 68.7% ) and nonmonitored units ( 48.3% ) . in general , patients had a high burden of comorbidities heart failure ( 18.1% ) , respiratory insufficiency ( 42.5% ) , renal insufficiency ( 33.4% ) , diabetes mellitus ( 30.4% ) , septicemia ( 15.9% ) , as well as use of mechanical ventilation ( 31.0% ) and intravenous vasopressors ( 27.4% ) before the cardiac arrest . characteristics of study patients * cns indicates central nervous system ; cpc , cerebral performance category ; ed , emergency department ; pea , pulseless electrical activity , vf , ventricular fibrillation ; vt , ventricular tachycardia . for illustrative purposes , trends in baseline characteristics are presented as 3 time periods ( 20002003 , 20042006 , and 20072010 ) . however , the p value for trend is for temporal changes in these characteristics by calendar year . data were missing for the following variables : race 6023 ( 6.5% ) , time of cardiac arrest 1031 ( 1.1% ) , hospital location 1981 ( 2.1% ) , witnessed arrest 13 ( 0.01% ) , and cpc category on admission 13 969 ( 14.9% ) . temporal trends in patient characteristics are also described in table 1 . between 2000 and 2010 , there was a significant decline in cardiac arrests due to ventricular fibrillation and pulseless ventricular tachycardia ( p<0.001 ) . significant temporal trends were also noted in demographics ( decrease in age , p<0.001 ) , comorbidities ( decrease in prevalence of heart failure and myocardial infarction and increase in prevalence of respiratory insufficiency , renal insufficiency , diabetes , septicemia , and malignancy ; p<0.001 for all comparisons ) , and acute illness severity ( increase in intensive care unit location , in patients receiving mechanical ventilators and vasopressors before the cardiac arrest ; p<0.001 for all comparisons ) . the median duration of hospital participation in gwtgresuscitation was 7 years , and 73 ( 31.6% ) hospitals participated for 8 years . most hospitals were either nonprofit ( 70.1% ) or government owned ( 16.9% ) with few forprofit hospitals ( 13% ) . most hospitals ( 44.2% ) were intermediate in size ( 200 to 499 beds ) , whereas 35.5% were small hospitals ( < 250 beds ) and 20.3% were large hospitals ( 500 beds ) . a majority of hospitals were academic teaching hospitals with a residency ( 30.3% ) or a residency and fellowship ( 23.8% ) program . characteristics of study hospitals ( n=231 ) * at baseline ( ie , during the first year of participation in gwtgresuscitation ) , the mean unadjusted hospital survival rate for inhospital cardiac arrest was 18.2% . survival rates improved at 218 ( 94% ) hospitals ( ie , hospitals with estimate of slope > 0 ) , with a mean 4% improvement in survival per year ( or 1.04 , 95% ci 1.03 to 1.05 , p<0.001 ; figure 2a and table 3 ) . hospitals in the top quartile had a mean yearoveryear survival improvement of 7% , while hospitals in the second and third hospital quartile had a mean yearoveryear survival improvement of 5% and 3% , respectively . the mean yearoveryear change in survival for hospitals in the lowest hospital quartile was 1% , suggesting little to no improvement in survival over time . unadjusted and adjusted relative rate of annual survival improvement , by hospital quartiles * hospitals were divided into quartiles using hospitalspecific odds ratios for annual survival improvement obtained from the multivariable hierarchical regression model . values in the table are the mean and the range of the hospitalspecific odds ratio for annual survival improvement . after adjustment for patient and hospital characteristics , the mean relative improvement in inhospital cardiac arrest survival rates was 7% per year ( adjusted or 1.07 , 95% ci 1.06 to 1.08 , p<0.001 ; table 3 ) . compared with a mean adjusted hospital survival rate of 18.1% during the 20002003 period , hospital survival rate increased to 21.4% in 20072010 , which translated into a 3.3% absolute improvement in survival during this period ( figure 3 ) . notably , there was marked variation in annual survival improvement across sites ( figure 2b ) . hospitals in the top quartile achieved a mean yearoveryear adjusted survival increase of 11% , whereas the hospitals in the bottom quartile experienced only a mean annual improvement of survival of 3% ( table 3 ) . change in inhospital cardiac arrest survival rates from 20002003 to 20072010 . the mean adjusted hospital survival rate increased from 18.1% in 20002003 ( range 9.1% to 29.8% ) to 21.4% in 20072010 ( range 13.9% to 32.2% ) . there was a significant interaction between academic status and rate of survival improvement across hospitals . compared with minor teaching hospitals ( or 1.04 , 95% ci 1.02 to 1.06 ) , hospital rate of survival improvement was greater at major teaching ( or 1.08 , 95% ci 1.06 to 1.10 ) and nonteaching hospitals ( or 1.07 , 95% ci 1.05 to 1.09 , p value for interaction=0.03 ; table 4 ) . however , other hospital characteristics , including bed size , geographic status , ownership status , and rural versus urban location , were not associated with a hospital 's rate of survival improvement for inhospital cardiac arrest . association between hospital structural characteristics and improvement in cardiac arrest survival in sensitivity analyses , there was a moderately strong positive correlation between hospital trends in favorable neurological survival and survival to discharge ( pearson 's correlation coefficient=0.62 ; p<0.0001 ) , which confirms that hospitals with the highest survival gains were largely the same hospitals with the greatest improvements in favorable neurological survival . moreover , after reclassifying 833 patients ( 4.5% of survivors ) who were discharged to hospice as having died , we still found an average 6% peryear improvement in survival . there was excellent agreement when we compared hospital classification in quartiles of survival improvement by using this approach with our primary analysis ( =0.80 , 95% ci 0.74 to 0.87 ) , which suggests that our findings were robust to hospital discharge patterns . at baseline ( ie , during the first year of participation in gwtgresuscitation ) , the mean unadjusted hospital survival rate for inhospital cardiac arrest was 18.2% . survival rates improved at 218 ( 94% ) hospitals ( ie , hospitals with estimate of slope > 0 ) , with a mean 4% improvement in survival per year ( or 1.04 , 95% ci 1.03 to 1.05 , p<0.001 ; figure 2a and table 3 ) . hospitals in the top quartile had a mean yearoveryear survival improvement of 7% , while hospitals in the second and third hospital quartile had a mean yearoveryear survival improvement of 5% and 3% , respectively . the mean yearoveryear change in survival for hospitals in the lowest hospital quartile was 1% , suggesting little to no improvement in survival over time . unadjusted and adjusted relative rate of annual survival improvement , by hospital quartiles * hospitals were divided into quartiles using hospitalspecific odds ratios for annual survival improvement obtained from the multivariable hierarchical regression model . values in the table are the mean and the range of the hospitalspecific odds ratio for annual survival improvement . after adjustment for patient and hospital characteristics , the mean relative improvement in inhospital cardiac arrest survival rates was 7% per year ( adjusted or 1.07 , 95% ci 1.06 to 1.08 , p<0.001 ; table 3 ) . compared with a mean adjusted hospital survival rate of 18.1% during the 20002003 period , hospital survival rate increased to 21.4% in 20072010 , which translated into a 3.3% absolute improvement in survival during this period ( figure 3 ) . notably , there was marked variation in annual survival improvement across sites ( figure 2b ) . hospitals in the top quartile achieved a mean yearoveryear adjusted survival increase of 11% , whereas the hospitals in the bottom quartile experienced only a mean annual improvement of survival of 3% ( table 3 ) . the mean adjusted hospital survival rate increased from 18.1% in 20002003 ( range 9.1% to 29.8% ) to 21.4% in 20072010 ( range 13.9% to 32.2% ) . there was a significant interaction between academic status and rate of survival improvement across hospitals . compared with minor teaching hospitals ( or 1.04 , 95% ci 1.02 to 1.06 ) , hospital rate of survival improvement was greater at major teaching ( or 1.08 , 95% ci 1.06 to 1.10 ) and nonteaching hospitals ( or 1.07 , 95% ci 1.05 to 1.09 , p value for interaction=0.03 ; table 4 ) . however , other hospital characteristics , including bed size , geographic status , ownership status , and rural versus urban location , were not associated with a hospital 's rate of survival improvement for inhospital cardiac arrest . association between hospital structural characteristics and improvement in cardiac arrest survival in sensitivity analyses , there was a moderately strong positive correlation between hospital trends in favorable neurological survival and survival to discharge ( pearson 's correlation coefficient=0.62 ; p<0.0001 ) , which confirms that hospitals with the highest survival gains were largely the same hospitals with the greatest improvements in favorable neurological survival . moreover , after reclassifying 833 patients ( 4.5% of survivors ) who were discharged to hospice as having died , we still found an average 6% peryear improvement in survival . there was excellent agreement when we compared hospital classification in quartiles of survival improvement by using this approach with our primary analysis ( =0.80 , 95% ci 0.74 to 0.87 ) , which suggests that our findings were robust to hospital discharge patterns . among hospitals participating in a large national quality improvement registry , we found hospitallevel survival rates for inhospital cardiac arrest have significantly improved during the past decade . importantly , there was marked variation in the extent of survival improvement at participating hospitals . we found that a quarter of hospitals achieved an average 11% relative improvement in survival per year , while another quarter of hospitals experienced little to no improvement . many of the individual hospital structural characteristics were unrelated to the extent of variation in hospitallevel trends , which suggests that unmeasured hospital processes of care are likely driving the survival improvement at topperforming sites . recently , we reported that inhospital cardiac arrest survival has improved during the past decade at the patient level . while it is possible that the improved survival trends that we observed are limited to hospitals committed to quality improvement due to participation in gwtgresuscitation , 2 subsequent studies have confirmed similar trends in other nationally representative databases . here , we extend the findings of our previous work by showing that survival improvement varied markedly across sites even after accounting for differences in patient characteristics between sites . given that treatment of inhospital cardiac arrest is time sensitive and requires a coordinated effort among a diverse group of providers , marked variation in survival improvement across sites likely reflects differences in how individual hospitals approach resuscitation care and organize quality improvement efforts . patients and their appropriate triage to telemetry or intensive care units , timely recognition of cardiac arrest , prompt mobilization of a resuscitation team to the patient bedside , rapid evaluation of the patient and initiation of resuscitative efforts , conduct of an organized and coordinated acute resuscitation response , and highquality postresuscitation care . hospitals that consider cardiac arrest to be a priority , examine their performance through ongoing data collection and feedback , identify areas of weakness , and invest resources and personnel to improve processes of care are likely to have achieved greater gains in cardiac arrest survival . in prior work in acute myocardial infarction , a number of institutional attributes organizational values and goals , senior leadership , staff engagement , communication , coordination , problem solving , and learning from previous mistakes were also found to distinguish topperforming hospitals from lowperforming hospitals . it is likely that similar organizational factors underlie hospitals ' ability to improve systems of care for resuscitation . among hospital structural characteristics , only teaching status was found to be significantly associated with extent of survival improvement . survival improvement was comparable at major teaching hospitals ( hospitals with residency and fellowship programs ) and nonteaching hospitals but was lower at minor teaching hospitals ( hospitals with only residency programs ) . this relationship may be due to the presence of lessexperienced trainees ( eg , residents ) who may be first responders during acute resuscitation and primary providers during postresuscitation care . the presence of advanced trainees ( eg , fellows ) at major teaching hospitals appears to mitigate this relationship , as survival improvement at major teaching hospitals was similar to that of nonteaching hospitals where experienced physicians provide patient care . although this may be a plausible explanation , we did not have data on composition of code teams or inpatient staffing patterns to confirm whether this is accurate . the general lack of association between hospital structural characteristics and survival improvement is not entirely unexpected . chan et al found wide variation ( 2% to 55% ) in rates of delayed defibrillation for cardiac arrest due to ventricular fibrillation and pulseless ventricular tachycardia . in that study , hospital performance on defibrillation times was unrelated to measured structural characteristics ( except bed size ) . based on these findings , we posit that hospital processes of care , rather than structure , are more important in achieving improved inhospital cardiac arrest survival over time . in fact , singlecenter studies have demonstrated the value of innovative process redesign such as debriefing after a cardiac arrest event , use of simulation or routine mock codes , and implementation of efforts to improve quality of cpr , as well as devices capable of providing audiovisual feedback during resuscitation , in improving resuscitation performance and outcomes . it is possible that topperforming hospitals have implemented similar and other novel strategies to realize their cardiac arrest survival gains . evaluating important resuscitationrelated processes of care as potential drivers of inhospital cardiac arrest survival improvement is a critical next step . currently , neither gwtgresuscitation nor other existing hospital databases collect information on these innovative resuscitation processes of care and treatment strategies . therefore , to better understand the organizational and process variables that underlie hospital resuscitation performance , detailed site surveys within large registries like gwtgresuscitation or studies using mixed methods ( qualitative and quantitative ) may be needed . in fact , bradley and colleagues used a similar approach to identify best practices associated with shorter doortoballoon times for primary percutaneous coronary intervention for stelevation myocardial infarction another timesensitive condition . this approach has been widely successful in significantly reducing doortoballoon times nationally and serves as powerful example to guide future quality improvement work for other timesensitive conditions such as inhospital cardiac arrest . first , it is possible that hospital cardiac arrest survival rates have improved because of a decrease in overall risk among patients who undergo resuscitation . although mean patient age has decreased over time , there has been a temporal increase in the prevalence of comorbidities ( sepsis , renal insufficiency , respiratory insufficiency , and malignancy ) , mechanical ventilation , and vasopressor use before arrest , suggesting that illness severity has increased over time . this finding was also noted in another study , which found an increase in the mean charlson comorbidity score among patients with an inhospital cardiac arrest ( 2.5 in 20002001 to 2.7 in 20082009 ; p<0.001 ) . second , although clinical practice guidelines support the use of therapeutic hypothermia for preventing neurological damage and improving survival following an outofhospital cardiac arrest , 2 recent studies from gwtgresuscitation found therapeutic hypothermia to be rarely used after inhospital cardiac arrest ( < 3% ) . therefore , it is unlikely that the observed variation in survival improvement between hospitals is explained by differences in use of therapeutic hypothermia . third , we assumed that improvement in survival occurred in a linear fashion in this study . although we did not test this assumption , our prior work using data from gwtgresuscitation was most consistent with a linear temporal trend in survival . important processes of care ( eg , quality of cpr , innovative resuscitation strategies , quality improvement programs , etc ) that may be associated with survival improvement were not available . second , we had only a single year of american hospital association data to define hospitallevel characteristics , and it is possible that structural characteristics of hospitals changed over time . third , although we adjusted for a number of patient and hospital factors in our multivariable models , the possibility of residual confounding remains . we addressed this by requiring that study hospitals be restricted to those that participated in gwtgresuscitation for at least 5 years . finally , hospitals participating in gwtgresuscitation are more likely to be committed hospitals that are engaged in quality improvement . moreover , we also excluded hospitals that were low volume ( annual cardiac arrest volume < 10 ) or participated infrequently ( < 5 years ) in the registry . for both these reasons , our findings may not be generalizable to all us hospitals . during the past decade , survival after inhospital cardiac arrest survival has improved at nearly all hospitals participating in a large national quality improvement registry . however , marked differences in the extent of survival improvement were observed . most structural hospital characteristics were not associated with the extent of hospital improvements in cardiac arrest survival . future studies are needed to identify hospital process of care ( best practices ) that have achieved the largest improvement in inhospital cardiac arrest survival . the american heart association , which sponsors the get with the guidelinesresuscitation , had no role in the design and conduct of the study ; the collection , management , analysis , and interpretation of the data ; the preparation , review , or approval of the manuscript ; or the decision to submit the manuscript for publication . in addition to study authors saket girotra and paul s. chan , the american heart association get with the guidelines resuscitation adult trask force include comilla sasson , md , ms and steven bradley , md , mph , university of colorado ; michael w. donnino , md , beth israel deaconess medical center ; dana p. edelson , md , ms , university of chicago ; robert t. faillace , md , scm , geisinger healthcare system ; romergryko geocadin , md , johns hopkins university school of medicine ; raina merchant , md , mshp , university of pennsylvania school of medicine ; vincent n. mosesso , jr . , md , university of pittsburgh school of medicine ; joseph p. ornato , md and mary ann peberdy , md , virginia commonwealth university .

backgroundduring the past decade , survival after inhospital cardiac arrest has improved markedly . it remains unknown whether the improvement in survival has occurred uniformly at all hospitals or was driven by large improvements at only a few hospitals.methods and resultswe identified 93 342 adults with an inhospital cardiac arrest at 231 hospitals in the get with the guidelinesresuscitation registry during 20002010 . using hierarchical regression models , we evaluated hospitallevel trends in survival to discharge . mean age was 66 years , 59% were men , and 21% were black . between 2000 and 2010 , there was a significant decrease in age , prevalence of heart failure and myocardial infarction , and cardiac arrests due to shockable rhythms ( p<0.001 for all ) and an increase in prevalence of sepsis , respiratory insufficiency , renal insufficiency , intensive care unit location , and mechanical ventilation before arrest ( p<0.001 for all ) . after adjustment for temporal trends in baseline characteristics , hospital rates of inhospital cardiac arrest survival improved by 7% per year ( odds ratio [ or ] 1.07 , 95% ci 1.06 to 1.08 , p<0.001 ) . improvement in survival varied markedly and ranged from 3% in the bottom hospital quartile to 11% in the top hospital quartile . compared with minor teaching hospitals ( or 1.04 , 95% ci 1.02 to 1.06 ) , hospital rate of survival improvement was greater at major teaching ( or 1.08 , 95% ci 1.06 to 1.10 ) and nonteaching hospitals ( or 1.07 , 95% ci 1.05 to 1.09 , p value for interaction=0.03).conclusionalthough inhospital cardiac arrest survival has improved during the past decade , the magnitude of improvement varied across hospitals . future studies are needed to identify hospital processes that have led to the largest improvement in survival .

Introduction Methods Study Design Study Population Hospital and Patient Variables Statistical Analyses Sensitivity Analyses Results HospitalLevel Trends in Survival Discussion Conclusion Role of the Sponsor Appendix Supplementary Material

a recent study found that survival after inhospital cardiac arrest has improved markedly over the past decade , from 13.7% in 2000 to 22.3% in 2009 . while encouraging , it remains unknown whether this survival trend has occurred uniformly across most hospitals or driven by large improvements at a smaller number of hospitals . identifying topperforming sites that have achieved large gains in inhospital cardiac arrest survival and the associated hospital factors is the critical next step to inform ongoing quality improvement efforts for inhospital resuscitation . to address these gaps in knowledge , we used data from get with the guidelines ( gwtg)resuscitation , a large national registry of inhospital cardiac arrests , to characterize hospitallevel trends in inhospital cardiac arrest survival over the past decade . additionally , we determined whether some hospitals have achieved greater improvements in survival compared with others , and evaluated hospital factors associated with the extent of hospital rates of survival improvement . within gwtgresuscitation , we identified 122 746 patients at 590 hospitals during 20002010 who were 18 years of age or older and had an index cardiac arrest with an identifiable initial rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , or pulseless ventricular tachycardia ) ( figure 1 ) . finally , given that the estimate of survival improvement from hospitals with low cardiac arrest volume or few years of available data would be unreliable , we restricted our sample to only those hospitals that participated in the registry for 5 years and had a mean annual case volume of 10 cases in accordance with previous studies . patientlevel data available from gwtgresuscitation included demographics ( age , sex , race ) , initial cardiac arrest rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , pulseless ventricular tachycardia ) , location of cardiac arrest ( intensive care unit , telemetry unit , nonmonitored unit ) , time of day ( work hours : 7 am to 10:59 pm versus after hours : 11 pm to 6:59 am ) and day of week ( weekday versus weekend ) of cardiac arrest , and use of a hospitalwide cardiopulmonary arrest alert ( ie , code blue ) . moreover , we used information on comorbid conditions ( congestive heart failure ; myocardial infarction ; diabetes mellitus ; renal , hepatic , or respiratory insufficiency ; neurological status prearrest [ as determined by admission cerebral performance category { cpc } scores ] ; baseline evidence of motor ; cognitive , or functional deficits [ central nervous system depression ] ; acute stroke ; pneumonia ; hypotension ; arrhythmia ; sepsis ; trauma ; metabolic or electrolyte abnormality ; cancer ) , and therapeutic interventions in place at the time of cardiac arrest ( use of mechanical ventilation , antiarrhythmic drugs , intravenous vasopressors , dialysis , pulmonary artery catheter , intraaortic balloon pump ) . the main independent variable was calendar year , and the primary outcome was hospital rate of survival to discharge . 1 ) were considered to have improved trends in survival , with the magnitude of the slope ( and or ) quantifying the extent of annual survival improvement . finally , we repeated these analyses including adjustment for patient and hospital characteristics , to obtain adjusted annual trends in hospital rates of survival . finally , to explore which hospital characteristics were associated with a higher rate of survival improvement , we included interaction terms between each hospital characteristic ( as described earlier in the hospital and patient variables section ) and calendar year in our multivariable model . data were complete for all covariates , except race ( 6.5% ) , admission cpc score ( 15% ) , hospital location of arrest ( 2.1% ) , and time of cardiac arrest ( 1.1% ) . missing data on covariates first , to exclude the possibility that hospitals with the largest temporal improvement in survival to discharge were not confounded by higher rates of neurological disability , we examined whether hospitals with the greatest survival gains were also the same hospitals with the largest temporal improvement in favorable neurological survival . we defined favorable neurological survival as survival to discharge with a cpc of 1 ( ie , none or mild neurological disability ) and calculated adjusted hospitallevel rates of favorable neurological survival using the hierarchical model just described . second , to ensure that our findings were not influenced by temporal changes in hospital discharge practice patterns ( eg , early discharge to hospice of patients with poor likelihood of survival ) , we repeated our analyses of hospitallevel trends after classifying patients who were discharged to hospice as having died . within gwtgresuscitation , we identified 122 746 patients at 590 hospitals during 20002010 who were 18 years of age or older and had an index cardiac arrest with an identifiable initial rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , or pulseless ventricular tachycardia ) ( figure 1 ) . finally , given that the estimate of survival improvement from hospitals with low cardiac arrest volume or few years of available data would be unreliable , we restricted our sample to only those hospitals that participated in the registry for 5 years and had a mean annual case volume of 10 cases in accordance with previous studies . patientlevel data available from gwtgresuscitation included demographics ( age , sex , race ) , initial cardiac arrest rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , pulseless ventricular tachycardia ) , location of cardiac arrest ( intensive care unit , telemetry unit , nonmonitored unit ) , time of day ( work hours : 7 am to 10:59 pm versus after hours : 11 pm to 6:59 am ) and day of week ( weekday versus weekend ) of cardiac arrest , and use of a hospitalwide cardiopulmonary arrest alert ( ie , code blue ) . moreover , we used information on comorbid conditions ( congestive heart failure ; myocardial infarction ; diabetes mellitus ; renal , hepatic , or respiratory insufficiency ; neurological status prearrest [ as determined by admission cerebral performance category { cpc } scores ] ; baseline evidence of motor ; cognitive , or functional deficits [ central nervous system depression ] ; acute stroke ; pneumonia ; hypotension ; arrhythmia ; sepsis ; trauma ; metabolic or electrolyte abnormality ; cancer ) , and therapeutic interventions in place at the time of cardiac arrest ( use of mechanical ventilation , antiarrhythmic drugs , intravenous vasopressors , dialysis , pulmonary artery catheter , intraaortic balloon pump ) . the main independent variable was calendar year , and the primary outcome was hospital rate of survival to discharge . 1 ) were considered to have improved trends in survival , with the magnitude of the slope ( and or ) quantifying the extent of annual survival improvement . finally , we repeated these analyses including adjustment for patient and hospital characteristics , to obtain adjusted annual trends in hospital rates of survival . finally , to explore which hospital characteristics were associated with a higher rate of survival improvement , we included interaction terms between each hospital characteristic ( as described earlier in the hospital and patient variables section ) and calendar year in our multivariable model . data were complete for all covariates , except race ( 6.5% ) , admission cpc score ( 15% ) , hospital location of arrest ( 2.1% ) , and time of cardiac arrest ( 1.1% ) . missing data on covariates first , to exclude the possibility that hospitals with the largest temporal improvement in survival to discharge were not confounded by higher rates of neurological disability , we examined whether hospitals with the greatest survival gains were also the same hospitals with the largest temporal improvement in favorable neurological survival . second , to ensure that our findings were not influenced by temporal changes in hospital discharge practice patterns ( eg , early discharge to hospice of patients with poor likelihood of survival ) , we repeated our analyses of hospitallevel trends after classifying patients who were discharged to hospice as having died . more than 80% of inhospital cardiac arrests occurred in a monitored setting ( intensive care unit [ 48.0% ] , telemetry unit [ 15.7% ] , or other closely monitored hospital locations [ 17.6% ] ) , and > 30% of arrests each occurred during nighttime and on the weekend . approximately , 80% of all patients had a witnessed arrest , and this was substantially higher for cardiac arrests in the intensive care unit ( 94.5% ) compared with those in monitored units ( 68.7% ) and nonmonitored units ( 48.3% ) . in general , patients had a high burden of comorbidities heart failure ( 18.1% ) , respiratory insufficiency ( 42.5% ) , renal insufficiency ( 33.4% ) , diabetes mellitus ( 30.4% ) , septicemia ( 15.9% ) , as well as use of mechanical ventilation ( 31.0% ) and intravenous vasopressors ( 27.4% ) before the cardiac arrest . for illustrative purposes , trends in baseline characteristics are presented as 3 time periods ( 20002003 , 20042006 , and 20072010 ) . however , the p value for trend is for temporal changes in these characteristics by calendar year . data were missing for the following variables : race 6023 ( 6.5% ) , time of cardiac arrest 1031 ( 1.1% ) , hospital location 1981 ( 2.1% ) , witnessed arrest 13 ( 0.01% ) , and cpc category on admission 13 969 ( 14.9% ) . between 2000 and 2010 , there was a significant decline in cardiac arrests due to ventricular fibrillation and pulseless ventricular tachycardia ( p<0.001 ) . significant temporal trends were also noted in demographics ( decrease in age , p<0.001 ) , comorbidities ( decrease in prevalence of heart failure and myocardial infarction and increase in prevalence of respiratory insufficiency , renal insufficiency , diabetes , septicemia , and malignancy ; p<0.001 for all comparisons ) , and acute illness severity ( increase in intensive care unit location , in patients receiving mechanical ventilators and vasopressors before the cardiac arrest ; p<0.001 for all comparisons ) . characteristics of study hospitals ( n=231 ) * at baseline ( ie , during the first year of participation in gwtgresuscitation ) , the mean unadjusted hospital survival rate for inhospital cardiac arrest was 18.2% . survival rates improved at 218 ( 94% ) hospitals ( ie , hospitals with estimate of slope > 0 ) , with a mean 4% improvement in survival per year ( or 1.04 , 95% ci 1.03 to 1.05 , p<0.001 ; figure 2a and table 3 ) . hospitals in the top quartile had a mean yearoveryear survival improvement of 7% , while hospitals in the second and third hospital quartile had a mean yearoveryear survival improvement of 5% and 3% , respectively . the mean yearoveryear change in survival for hospitals in the lowest hospital quartile was 1% , suggesting little to no improvement in survival over time . after adjustment for patient and hospital characteristics , the mean relative improvement in inhospital cardiac arrest survival rates was 7% per year ( adjusted or 1.07 , 95% ci 1.06 to 1.08 , p<0.001 ; table 3 ) . compared with a mean adjusted hospital survival rate of 18.1% during the 20002003 period , hospital survival rate increased to 21.4% in 20072010 , which translated into a 3.3% absolute improvement in survival during this period ( figure 3 ) . hospitals in the top quartile achieved a mean yearoveryear adjusted survival increase of 11% , whereas the hospitals in the bottom quartile experienced only a mean annual improvement of survival of 3% ( table 3 ) . there was a significant interaction between academic status and rate of survival improvement across hospitals . compared with minor teaching hospitals ( or 1.04 , 95% ci 1.02 to 1.06 ) , hospital rate of survival improvement was greater at major teaching ( or 1.08 , 95% ci 1.06 to 1.10 ) and nonteaching hospitals ( or 1.07 , 95% ci 1.05 to 1.09 , p value for interaction=0.03 ; table 4 ) . however , other hospital characteristics , including bed size , geographic status , ownership status , and rural versus urban location , were not associated with a hospital 's rate of survival improvement for inhospital cardiac arrest . association between hospital structural characteristics and improvement in cardiac arrest survival in sensitivity analyses , there was a moderately strong positive correlation between hospital trends in favorable neurological survival and survival to discharge ( pearson 's correlation coefficient=0.62 ; p<0.0001 ) , which confirms that hospitals with the highest survival gains were largely the same hospitals with the greatest improvements in favorable neurological survival . there was excellent agreement when we compared hospital classification in quartiles of survival improvement by using this approach with our primary analysis ( =0.80 , 95% ci 0.74 to 0.87 ) , which suggests that our findings were robust to hospital discharge patterns . at baseline ( ie , during the first year of participation in gwtgresuscitation ) , the mean unadjusted hospital survival rate for inhospital cardiac arrest was 18.2% . survival rates improved at 218 ( 94% ) hospitals ( ie , hospitals with estimate of slope > 0 ) , with a mean 4% improvement in survival per year ( or 1.04 , 95% ci 1.03 to 1.05 , p<0.001 ; figure 2a and table 3 ) . hospitals in the top quartile had a mean yearoveryear survival improvement of 7% , while hospitals in the second and third hospital quartile had a mean yearoveryear survival improvement of 5% and 3% , respectively . the mean yearoveryear change in survival for hospitals in the lowest hospital quartile was 1% , suggesting little to no improvement in survival over time . after adjustment for patient and hospital characteristics , the mean relative improvement in inhospital cardiac arrest survival rates was 7% per year ( adjusted or 1.07 , 95% ci 1.06 to 1.08 , p<0.001 ; table 3 ) . compared with a mean adjusted hospital survival rate of 18.1% during the 20002003 period , hospital survival rate increased to 21.4% in 20072010 , which translated into a 3.3% absolute improvement in survival during this period ( figure 3 ) . hospitals in the top quartile achieved a mean yearoveryear adjusted survival increase of 11% , whereas the hospitals in the bottom quartile experienced only a mean annual improvement of survival of 3% ( table 3 ) . there was a significant interaction between academic status and rate of survival improvement across hospitals . compared with minor teaching hospitals ( or 1.04 , 95% ci 1.02 to 1.06 ) , hospital rate of survival improvement was greater at major teaching ( or 1.08 , 95% ci 1.06 to 1.10 ) and nonteaching hospitals ( or 1.07 , 95% ci 1.05 to 1.09 , p value for interaction=0.03 ; table 4 ) . however , other hospital characteristics , including bed size , geographic status , ownership status , and rural versus urban location , were not associated with a hospital 's rate of survival improvement for inhospital cardiac arrest . association between hospital structural characteristics and improvement in cardiac arrest survival in sensitivity analyses , there was a moderately strong positive correlation between hospital trends in favorable neurological survival and survival to discharge ( pearson 's correlation coefficient=0.62 ; p<0.0001 ) , which confirms that hospitals with the highest survival gains were largely the same hospitals with the greatest improvements in favorable neurological survival . there was excellent agreement when we compared hospital classification in quartiles of survival improvement by using this approach with our primary analysis ( =0.80 , 95% ci 0.74 to 0.87 ) , which suggests that our findings were robust to hospital discharge patterns . among hospitals participating in a large national quality improvement registry , we found hospitallevel survival rates for inhospital cardiac arrest have significantly improved during the past decade . importantly , there was marked variation in the extent of survival improvement at participating hospitals . many of the individual hospital structural characteristics were unrelated to the extent of variation in hospitallevel trends , which suggests that unmeasured hospital processes of care are likely driving the survival improvement at topperforming sites . recently , we reported that inhospital cardiac arrest survival has improved during the past decade at the patient level . here , we extend the findings of our previous work by showing that survival improvement varied markedly across sites even after accounting for differences in patient characteristics between sites . given that treatment of inhospital cardiac arrest is time sensitive and requires a coordinated effort among a diverse group of providers , marked variation in survival improvement across sites likely reflects differences in how individual hospitals approach resuscitation care and organize quality improvement efforts . patients and their appropriate triage to telemetry or intensive care units , timely recognition of cardiac arrest , prompt mobilization of a resuscitation team to the patient bedside , rapid evaluation of the patient and initiation of resuscitative efforts , conduct of an organized and coordinated acute resuscitation response , and highquality postresuscitation care . survival improvement was comparable at major teaching hospitals ( hospitals with residency and fellowship programs ) and nonteaching hospitals but was lower at minor teaching hospitals ( hospitals with only residency programs ) . the presence of advanced trainees ( eg , fellows ) at major teaching hospitals appears to mitigate this relationship , as survival improvement at major teaching hospitals was similar to that of nonteaching hospitals where experienced physicians provide patient care . based on these findings , we posit that hospital processes of care , rather than structure , are more important in achieving improved inhospital cardiac arrest survival over time . evaluating important resuscitationrelated processes of care as potential drivers of inhospital cardiac arrest survival improvement is a critical next step . although mean patient age has decreased over time , there has been a temporal increase in the prevalence of comorbidities ( sepsis , renal insufficiency , respiratory insufficiency , and malignancy ) , mechanical ventilation , and vasopressor use before arrest , suggesting that illness severity has increased over time . this finding was also noted in another study , which found an increase in the mean charlson comorbidity score among patients with an inhospital cardiac arrest ( 2.5 in 20002001 to 2.7 in 20082009 ; p<0.001 ) . second , although clinical practice guidelines support the use of therapeutic hypothermia for preventing neurological damage and improving survival following an outofhospital cardiac arrest , 2 recent studies from gwtgresuscitation found therapeutic hypothermia to be rarely used after inhospital cardiac arrest ( < 3% ) . second , we had only a single year of american hospital association data to define hospitallevel characteristics , and it is possible that structural characteristics of hospitals changed over time . moreover , we also excluded hospitals that were low volume ( annual cardiac arrest volume < 10 ) or participated infrequently ( < 5 years ) in the registry . during the past decade , survival after inhospital cardiac arrest survival has improved at nearly all hospitals participating in a large national quality improvement registry . future studies are needed to identify hospital process of care ( best practices ) that have achieved the largest improvement in inhospital cardiac arrest survival . the american heart association , which sponsors the get with the guidelinesresuscitation , had no role in the design and conduct of the study ; the collection , management , analysis , and interpretation of the data ; the preparation , review , or approval of the manuscript ; or the decision to submit the manuscript for publication .

identifying topperforming sites that have achieved large gains in inhospital cardiac arrest survival and the associated hospital factors is the critical next step to inform ongoing quality improvement efforts for inhospital resuscitation . to address these gaps in knowledge , we used data from get with the guidelines ( gwtg)resuscitation , a large national registry of inhospital cardiac arrests , to characterize hospitallevel trends in inhospital cardiac arrest survival over the past decade . additionally , we determined whether some hospitals have achieved greater improvements in survival compared with others , and evaluated hospital factors associated with the extent of hospital rates of survival improvement . within gwtgresuscitation , we identified 122 746 patients at 590 hospitals during 20002010 who were 18 years of age or older and had an index cardiac arrest with an identifiable initial rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , or pulseless ventricular tachycardia ) ( figure 1 ) . finally , given that the estimate of survival improvement from hospitals with low cardiac arrest volume or few years of available data would be unreliable , we restricted our sample to only those hospitals that participated in the registry for 5 years and had a mean annual case volume of 10 cases in accordance with previous studies . in an effort to better understand which hospital characteristics are associated with survival improvement , we merged data from the 2008 american hospital association annual survey with gwtgresuscitation to obtain information on hospital characteristics . these included information on a hospital 's geographic region ( north midatlantic , south atlantic , north central , south central , and mountain / pacific ) , location ( rural , urban ) , ownership ( nonprofit , public , private ) , teaching status ( fellowship program , residency program , or nonteaching ) , and bed number ( 250 , 250 to 499 , 500 ) . patientlevel data available from gwtgresuscitation included demographics ( age , sex , race ) , initial cardiac arrest rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , pulseless ventricular tachycardia ) , location of cardiac arrest ( intensive care unit , telemetry unit , nonmonitored unit ) , time of day ( work hours : 7 am to 10:59 pm versus after hours : 11 pm to 6:59 am ) and day of week ( weekday versus weekend ) of cardiac arrest , and use of a hospitalwide cardiopulmonary arrest alert ( ie , code blue ) . moreover , we used information on comorbid conditions ( congestive heart failure ; myocardial infarction ; diabetes mellitus ; renal , hepatic , or respiratory insufficiency ; neurological status prearrest [ as determined by admission cerebral performance category { cpc } scores ] ; baseline evidence of motor ; cognitive , or functional deficits [ central nervous system depression ] ; acute stroke ; pneumonia ; hypotension ; arrhythmia ; sepsis ; trauma ; metabolic or electrolyte abnormality ; cancer ) , and therapeutic interventions in place at the time of cardiac arrest ( use of mechanical ventilation , antiarrhythmic drugs , intravenous vasopressors , dialysis , pulmonary artery catheter , intraaortic balloon pump ) . to evaluate temporal trends in hospitallevel survival rates such models account for clustering of patients within a hospital and avoid overestimation of significance of statistical associations . finally , to explore which hospital characteristics were associated with a higher rate of survival improvement , we included interaction terms between each hospital characteristic ( as described earlier in the hospital and patient variables section ) and calendar year in our multivariable model . data were complete for all covariates , except race ( 6.5% ) , admission cpc score ( 15% ) , hospital location of arrest ( 2.1% ) , and time of cardiac arrest ( 1.1% ) . missing data on covariates first , to exclude the possibility that hospitals with the largest temporal improvement in survival to discharge were not confounded by higher rates of neurological disability , we examined whether hospitals with the greatest survival gains were also the same hospitals with the largest temporal improvement in favorable neurological survival . we defined favorable neurological survival as survival to discharge with a cpc of 1 ( ie , none or mild neurological disability ) and calculated adjusted hospitallevel rates of favorable neurological survival using the hierarchical model just described . second , to ensure that our findings were not influenced by temporal changes in hospital discharge practice patterns ( eg , early discharge to hospice of patients with poor likelihood of survival ) , we repeated our analyses of hospitallevel trends after classifying patients who were discharged to hospice as having died . within gwtgresuscitation , we identified 122 746 patients at 590 hospitals during 20002010 who were 18 years of age or older and had an index cardiac arrest with an identifiable initial rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , or pulseless ventricular tachycardia ) ( figure 1 ) . finally , given that the estimate of survival improvement from hospitals with low cardiac arrest volume or few years of available data would be unreliable , we restricted our sample to only those hospitals that participated in the registry for 5 years and had a mean annual case volume of 10 cases in accordance with previous studies . as a result in an effort to better understand which hospital characteristics are associated with survival improvement , we merged data from the 2008 american hospital association annual survey with gwtgresuscitation to obtain information on hospital characteristics . these included information on a hospital 's geographic region ( north midatlantic , south atlantic , north central , south central , and mountain / pacific ) , location ( rural , urban ) , ownership ( nonprofit , public , private ) , teaching status ( fellowship program , residency program , or nonteaching ) , and bed number ( 250 , 250 to 499 , 500 ) . patientlevel data available from gwtgresuscitation included demographics ( age , sex , race ) , initial cardiac arrest rhythm ( asystole , pulseless electrical activity , ventricular fibrillation , pulseless ventricular tachycardia ) , location of cardiac arrest ( intensive care unit , telemetry unit , nonmonitored unit ) , time of day ( work hours : 7 am to 10:59 pm versus after hours : 11 pm to 6:59 am ) and day of week ( weekday versus weekend ) of cardiac arrest , and use of a hospitalwide cardiopulmonary arrest alert ( ie , code blue ) . moreover , we used information on comorbid conditions ( congestive heart failure ; myocardial infarction ; diabetes mellitus ; renal , hepatic , or respiratory insufficiency ; neurological status prearrest [ as determined by admission cerebral performance category { cpc } scores ] ; baseline evidence of motor ; cognitive , or functional deficits [ central nervous system depression ] ; acute stroke ; pneumonia ; hypotension ; arrhythmia ; sepsis ; trauma ; metabolic or electrolyte abnormality ; cancer ) , and therapeutic interventions in place at the time of cardiac arrest ( use of mechanical ventilation , antiarrhythmic drugs , intravenous vasopressors , dialysis , pulmonary artery catheter , intraaortic balloon pump ) . finally , to explore which hospital characteristics were associated with a higher rate of survival improvement , we included interaction terms between each hospital characteristic ( as described earlier in the hospital and patient variables section ) and calendar year in our multivariable model . data were complete for all covariates , except race ( 6.5% ) , admission cpc score ( 15% ) , hospital location of arrest ( 2.1% ) , and time of cardiac arrest ( 1.1% ) . missing data on covariates first , to exclude the possibility that hospitals with the largest temporal improvement in survival to discharge were not confounded by higher rates of neurological disability , we examined whether hospitals with the greatest survival gains were also the same hospitals with the largest temporal improvement in favorable neurological survival . we defined favorable neurological survival as survival to discharge with a cpc of 1 ( ie , none or mild neurological disability ) and calculated adjusted hospitallevel rates of favorable neurological survival using the hierarchical model just described . second , to ensure that our findings were not influenced by temporal changes in hospital discharge practice patterns ( eg , early discharge to hospice of patients with poor likelihood of survival ) , we repeated our analyses of hospitallevel trends after classifying patients who were discharged to hospice as having died . more than 80% of inhospital cardiac arrests occurred in a monitored setting ( intensive care unit [ 48.0% ] , telemetry unit [ 15.7% ] , or other closely monitored hospital locations [ 17.6% ] ) , and > 30% of arrests each occurred during nighttime and on the weekend . approximately , 80% of all patients had a witnessed arrest , and this was substantially higher for cardiac arrests in the intensive care unit ( 94.5% ) compared with those in monitored units ( 68.7% ) and nonmonitored units ( 48.3% ) . in general , patients had a high burden of comorbidities heart failure ( 18.1% ) , respiratory insufficiency ( 42.5% ) , renal insufficiency ( 33.4% ) , diabetes mellitus ( 30.4% ) , septicemia ( 15.9% ) , as well as use of mechanical ventilation ( 31.0% ) and intravenous vasopressors ( 27.4% ) before the cardiac arrest . characteristics of study patients * cns indicates central nervous system ; cpc , cerebral performance category ; ed , emergency department ; pea , pulseless electrical activity , vf , ventricular fibrillation ; vt , ventricular tachycardia . data were missing for the following variables : race 6023 ( 6.5% ) , time of cardiac arrest 1031 ( 1.1% ) , hospital location 1981 ( 2.1% ) , witnessed arrest 13 ( 0.01% ) , and cpc category on admission 13 969 ( 14.9% ) . between 2000 and 2010 , there was a significant decline in cardiac arrests due to ventricular fibrillation and pulseless ventricular tachycardia ( p<0.001 ) . significant temporal trends were also noted in demographics ( decrease in age , p<0.001 ) , comorbidities ( decrease in prevalence of heart failure and myocardial infarction and increase in prevalence of respiratory insufficiency , renal insufficiency , diabetes , septicemia , and malignancy ; p<0.001 for all comparisons ) , and acute illness severity ( increase in intensive care unit location , in patients receiving mechanical ventilators and vasopressors before the cardiac arrest ; p<0.001 for all comparisons ) . characteristics of study hospitals ( n=231 ) * at baseline ( ie , during the first year of participation in gwtgresuscitation ) , the mean unadjusted hospital survival rate for inhospital cardiac arrest was 18.2% . survival rates improved at 218 ( 94% ) hospitals ( ie , hospitals with estimate of slope > 0 ) , with a mean 4% improvement in survival per year ( or 1.04 , 95% ci 1.03 to 1.05 , p<0.001 ; figure 2a and table 3 ) . hospitals in the top quartile had a mean yearoveryear survival improvement of 7% , while hospitals in the second and third hospital quartile had a mean yearoveryear survival improvement of 5% and 3% , respectively . the mean yearoveryear change in survival for hospitals in the lowest hospital quartile was 1% , suggesting little to no improvement in survival over time . unadjusted and adjusted relative rate of annual survival improvement , by hospital quartiles * hospitals were divided into quartiles using hospitalspecific odds ratios for annual survival improvement obtained from the multivariable hierarchical regression model . after adjustment for patient and hospital characteristics , the mean relative improvement in inhospital cardiac arrest survival rates was 7% per year ( adjusted or 1.07 , 95% ci 1.06 to 1.08 , p<0.001 ; table 3 ) . compared with a mean adjusted hospital survival rate of 18.1% during the 20002003 period , hospital survival rate increased to 21.4% in 20072010 , which translated into a 3.3% absolute improvement in survival during this period ( figure 3 ) . hospitals in the top quartile achieved a mean yearoveryear adjusted survival increase of 11% , whereas the hospitals in the bottom quartile experienced only a mean annual improvement of survival of 3% ( table 3 ) . compared with minor teaching hospitals ( or 1.04 , 95% ci 1.02 to 1.06 ) , hospital rate of survival improvement was greater at major teaching ( or 1.08 , 95% ci 1.06 to 1.10 ) and nonteaching hospitals ( or 1.07 , 95% ci 1.05 to 1.09 , p value for interaction=0.03 ; table 4 ) . association between hospital structural characteristics and improvement in cardiac arrest survival in sensitivity analyses , there was a moderately strong positive correlation between hospital trends in favorable neurological survival and survival to discharge ( pearson 's correlation coefficient=0.62 ; p<0.0001 ) , which confirms that hospitals with the highest survival gains were largely the same hospitals with the greatest improvements in favorable neurological survival . there was excellent agreement when we compared hospital classification in quartiles of survival improvement by using this approach with our primary analysis ( =0.80 , 95% ci 0.74 to 0.87 ) , which suggests that our findings were robust to hospital discharge patterns . at baseline ( ie , during the first year of participation in gwtgresuscitation ) , the mean unadjusted hospital survival rate for inhospital cardiac arrest was 18.2% . survival rates improved at 218 ( 94% ) hospitals ( ie , hospitals with estimate of slope > 0 ) , with a mean 4% improvement in survival per year ( or 1.04 , 95% ci 1.03 to 1.05 , p<0.001 ; figure 2a and table 3 ) . hospitals in the top quartile had a mean yearoveryear survival improvement of 7% , while hospitals in the second and third hospital quartile had a mean yearoveryear survival improvement of 5% and 3% , respectively . the mean yearoveryear change in survival for hospitals in the lowest hospital quartile was 1% , suggesting little to no improvement in survival over time . unadjusted and adjusted relative rate of annual survival improvement , by hospital quartiles * hospitals were divided into quartiles using hospitalspecific odds ratios for annual survival improvement obtained from the multivariable hierarchical regression model . after adjustment for patient and hospital characteristics , the mean relative improvement in inhospital cardiac arrest survival rates was 7% per year ( adjusted or 1.07 , 95% ci 1.06 to 1.08 , p<0.001 ; table 3 ) . compared with a mean adjusted hospital survival rate of 18.1% during the 20002003 period , hospital survival rate increased to 21.4% in 20072010 , which translated into a 3.3% absolute improvement in survival during this period ( figure 3 ) . hospitals in the top quartile achieved a mean yearoveryear adjusted survival increase of 11% , whereas the hospitals in the bottom quartile experienced only a mean annual improvement of survival of 3% ( table 3 ) . compared with minor teaching hospitals ( or 1.04 , 95% ci 1.02 to 1.06 ) , hospital rate of survival improvement was greater at major teaching ( or 1.08 , 95% ci 1.06 to 1.10 ) and nonteaching hospitals ( or 1.07 , 95% ci 1.05 to 1.09 , p value for interaction=0.03 ; table 4 ) . association between hospital structural characteristics and improvement in cardiac arrest survival in sensitivity analyses , there was a moderately strong positive correlation between hospital trends in favorable neurological survival and survival to discharge ( pearson 's correlation coefficient=0.62 ; p<0.0001 ) , which confirms that hospitals with the highest survival gains were largely the same hospitals with the greatest improvements in favorable neurological survival . there was excellent agreement when we compared hospital classification in quartiles of survival improvement by using this approach with our primary analysis ( =0.80 , 95% ci 0.74 to 0.87 ) , which suggests that our findings were robust to hospital discharge patterns . many of the individual hospital structural characteristics were unrelated to the extent of variation in hospitallevel trends , which suggests that unmeasured hospital processes of care are likely driving the survival improvement at topperforming sites . given that treatment of inhospital cardiac arrest is time sensitive and requires a coordinated effort among a diverse group of providers , marked variation in survival improvement across sites likely reflects differences in how individual hospitals approach resuscitation care and organize quality improvement efforts . patients and their appropriate triage to telemetry or intensive care units , timely recognition of cardiac arrest , prompt mobilization of a resuscitation team to the patient bedside , rapid evaluation of the patient and initiation of resuscitative efforts , conduct of an organized and coordinated acute resuscitation response , and highquality postresuscitation care . hospitals that consider cardiac arrest to be a priority , examine their performance through ongoing data collection and feedback , identify areas of weakness , and invest resources and personnel to improve processes of care are likely to have achieved greater gains in cardiac arrest survival . the presence of advanced trainees ( eg , fellows ) at major teaching hospitals appears to mitigate this relationship , as survival improvement at major teaching hospitals was similar to that of nonteaching hospitals where experienced physicians provide patient care . in fact , singlecenter studies have demonstrated the value of innovative process redesign such as debriefing after a cardiac arrest event , use of simulation or routine mock codes , and implementation of efforts to improve quality of cpr , as well as devices capable of providing audiovisual feedback during resuscitation , in improving resuscitation performance and outcomes . although mean patient age has decreased over time , there has been a temporal increase in the prevalence of comorbidities ( sepsis , renal insufficiency , respiratory insufficiency , and malignancy ) , mechanical ventilation , and vasopressor use before arrest , suggesting that illness severity has increased over time . this finding was also noted in another study , which found an increase in the mean charlson comorbidity score among patients with an inhospital cardiac arrest ( 2.5 in 20002001 to 2.7 in 20082009 ; p<0.001 ) . second , although clinical practice guidelines support the use of therapeutic hypothermia for preventing neurological damage and improving survival following an outofhospital cardiac arrest , 2 recent studies from gwtgresuscitation found therapeutic hypothermia to be rarely used after inhospital cardiac arrest ( < 3% ) . the american heart association , which sponsors the get with the guidelinesresuscitation , had no role in the design and conduct of the study ; the collection , management , analysis , and interpretation of the data ; the preparation , review , or approval of the manuscript ; or the decision to submit the manuscript for publication . in addition to study authors saket girotra and paul s. chan , the american heart association get with the guidelines resuscitation adult trask force include comilla sasson , md , ms and steven bradley , md , mph , university of colorado ; michael w. donnino , md , beth israel deaconess medical center ; dana p. edelson , md , ms , university of chicago ; robert t. faillace , md , scm , geisinger healthcare system ; romergryko geocadin , md , johns hopkins university school of medicine ; raina merchant , md , mshp , university of pennsylvania school of medicine ; vincent n. mosesso , jr .

the p53 tumor suppressor protein plays an important role in preventing malignant development ( vousden and prives , 2009 ) , functioning primarily as a transcription factor to regulate the expression of a large number of genes that induce cellular responses such as cell - cycle arrest and apoptosis ( beckerman and prives , 2010 ) . while effective in preventing cancer development , these activities of p53 must also be tightly controlled to allow normal growth and development . numerous mechanisms through which p53 is regulated have been described , including the control of translation , protein stability , subcellular localization , and interaction with other components of the transcriptional machinery ( hollstein and hainaut , 2010 ) . in many cancers , the function of p53 is ablated through point mutations that lead to the expression of a mutant p53 protein ( joerger and fersht , 2007 ) . these tumor - associated point mutations occur predominantly in the central dna binding domain of p53 and result in a diminished ability of p53 to bind consensus sites in the promoters of p53-regulated genes . while some of these mutations result in amino - acid substitutions of residues within p53 that directly contact the dna ( contact mutants ) , other mutations result in the misfolding of the p53 protein . the p53 dna binding domain shows a low thermodynamic stability in vitro , and mutations in this region can lead to further instability , causing the protein to become denatured at 37c ( joerger and fersht , 2007 ) and a potential to form p53 protein aggregates within the cell ( xu et al . , the net effect of these tumor - associated point mutants is both the loss of wild - type p53 activity and a gain of function that contributes to the invasive behavior of cancers ( muller et al . , 2011 ) . the mechanisms underlying this gain of function are still under investigation but at least partially reflect the ability of the mutant p53 proteins to modulate the activity of other transcription factors such as p63 , p73 , and srebp ( freed - pastor and prives , 2012 ) . molecular chaperones are a group of proteins that assist in protein folding ( hartl et al . , 2011 ) . they not only prevent misfolding and aggregation of proteins but can also target misfolded proteins for degradation . probably the best - understood chaperones are the heat shock proteins hsp70 and hsp90 , which play a role in conformational maturation and help to target improperly folded proteins for ubiquitination and proteolysis , and the ring complex chaperonins , which enclose proteins within their structure for folding of newly synthesized peptides ( mayer , 2010 ) . chaperonins are double - ring oligomers , each ring enclosing a cavity where protein folding takes place through an energy - consuming process ( douglas et al . , 2011 ; the cytosolic group ii chaperonin cct ( also known as tric ) consists of a double ring , each one containing eight subunits ( cct in mammals and cct18 in yeast ) . like other chaperonins , cct has two main conformations that are controlled by atp hydrolysis . the open conformation recognizes unfolded peptides , and atp binding and hydrolysis induce the closed conformation , which results in the folding of the protein ( douglas et al . although the mechanism of substrate - cct recognition and binding remains under investigation , each of the subunits can recognize different polar and hydrophobic motifs within substrate proteins ( yam et al . potentially up to 15% of all newly synthesized polypeptides can associate with the cct complex , although only a few proteins have so far been shown to depend on this chaperonin for folding and function ( thulasiraman et al . cct plays an important role in the folding of newly synthesized proteins ( frydman et al . , 1994 ; yam et al . , 2008a ) but can also prevent the aggregation of proteins with polyglutamine regions ( kitamura et al . , 2006 ; tam et al . , 2006 ) and so potentially contributes to the suppression of misfolding diseases such as huntington , parkinson , and alzheimer . these activities are executed in conjunction with other chaperones or cochaperones ( siegers et al . , 1999 ) . both wild - type and mutant p53 have been shown to be regulated by binding to hsp70 and hsp90 ( blagosklonny et al . however , a role for the chaperonins in the control of p53 has not been investigated . we show here that p53 is a client of the cct complex and that failure to interact with this molecular chaperone can promote oncogenic functions of p53 in the absence of classic tumor - derived dna binding domain mutations . we carried out a proteomic analysis to identify proteins that interact with wild - type p53 and a tumor - derived point mutant , 175h . p53 itself , parc , and cullin-7 ( andrews et al . , 2006 ; nikolaev et al . , 2003 ) were most frequently identified in immunoprecipitation with wild - type p53 ( figure 1a ) , validating this approach . interestingly , peptides from seven of the eight cct subunits were also found in complex with p53 ( figure 1a ) , consistent with our recent study identifying cct subunits as part of a p53 interactome ( coffill et al . , 2012 ) . to further study the consequences of the interaction of p53 with cct subunits in cells , we utilized several cell lines that each expressed broadly equal endogenous levels of various cct subunits ( figure s1a available online ) . coprecipitation of endogenous wild - type p53 with endogenous cct and cct from hct116 cells ( figure 1b ) or u2os cells ( where p53 was stabilized by treatment of cells with the mdm2 inhibitor nutlin 3 ; figure 1c ) demonstrated the ability of these proteins to interact in cells . to compare the binding of wild - type and mutant p53 directly in the same cells , we turned to h1299 , a p53 null tumor cell line transiently transfected with p53-expression constructs ( figure 1d ) . these results indicated that wild - type p53 , 175h , and 273h all efficiently interacted with endogenous cct . similarly , wild - type p53 and 248w were coprecipitated with cct in hct116 cells ( figure s1b ) . the specificity of the binding was confirmed by showing that the coprecipitation of p53 was dependent on cct expression ( figure 1e ) . cct is a large , multisubunit structure ( 960 kda ) , and we used gel filtration to determine whether p53 could associate with the whole cct complex ( figure 2a ) . endogenous wild - type p53 largely comigrated with cct and cct in high - molecular - weight complexes , consistent with an interaction with the whole cct complex ( figure 2a ) . immunoprecipitation of pooled fractions confirmed that the majority of the cct was present in fractions 813 and coprecipitated with p53 ( figure 2a ) . similar results were observed in lysates from h1299 cells transiently transfected with wild - type p53 ( figure s2 ) . depletion of cct and cct resulted in the release of some p53 from the larger complexes , supporting the interaction of p53 with cct ( figure 2b ) , although the retention of some p53 in larger complexes following cct depletion likely reflects p53 s interaction with other proteins ( collavin et al . , quantification of p53 levels in each fraction indicated that approximately 12% of the total p53 was released from the larger complexes , suggesting this proportion of p53 is bound to cct under these conditions . cooperation between various chaperone complexes in protein folding has been described , and p53 is known to associate with several other chaperones , including hsp70 ( blagosklonny et al . , 1996 ; walerych et al . , 2009 ) . hsp70 contributes to the interaction between vhl and cct ( melville et al . , 2003 ) , and we also found that hsp70 slightly increased the binding of p53 to cct ( figure 2c ) . to test directly whether p53 interacts with the cct complex , we mixed bacterially expressed wild - type p53 with purified bovine cct . following separation by native electrophoresis , the high - molecular - weight band corresponding to cct was excised and subjected to sds electrophoresis , which revealed the presence of cct subunits and p53 ( figure 3a ) , confirming the presence of a stable cct : p53 complex . first , over 1,000 end - on views of the isolated apo - cct particles were processed , and the average image revealed the typical presence of the doughnut - shaped structure with an empty cavity surrounded by eight similar masses ( figure 3b ) . the averaging of almost 2,000 cct : p53 particles revealed a similar structure except for the presence of mass inside the cavity that interacted mainly with one cct subunit ( figure 3c ; figures s3a previous studies have shown that the atp - dependent conformational change leading to closure of the cct complex also results in its protection against proteinase k digestion ( meyer et al . , 2003 ) . we were also able to confirm a dose - dependent degradation of the cct complex following incubation with proteinase k , which was prevented by the addition of atp ( figure 3d ) . importantly , this protection from degradation also applied to the p53 protein bound to cct , as the closed conformation of the chaperonin was able to protect the trapped p53 from protease attack ( figure 3e ) . taken together , these data demonstrate that p53 interacts with the cct complex and can be trapped in the interior of the chaperonin cavity in an atp - hydrolysis - dependent manner . to determine which regions of p53 were important for the cct interaction deletions or point mutations within the dna binding domain or the c terminus of p53 did not affect binding to cct ( figures 4a and 4b ) . interestingly , several of the c - terminal truncations also removed the oligomerization domain and the nuclear import signals from p53 , suggesting that neither of these is required for cct binding in cells . as shown previously , cct localized predominantly to the cytoplasm , although some nuclear staining was also detected ( figures s4a s4c ) ( joly et al . both endogenous and transfected wild - type p53 were found predominantly ( although not exclusively ) in the nuclear fractions ( figures s4b and s4c ) . however , despite detecting both p53 and cct proteins in the nucleus , coimmunoprecipitation showed that the endogenous p53/cct interaction occurs in the cytosol ( figure s4b ) . while alterations within the dna binding and c - terminal domains of p53 did not affect the cct interaction , deletion of the n - terminal 43 amino acids of p53 resulted in the loss of interaction with cct ( figure 4b ) . the n terminus of p53 is responsible for many protein interactions , including binding to mdm2 and the components of the transcriptional machinery ( residues 1729 ; kussie et al . , 1996 ) . while mutation in the mdm2 binding and transactivation domains ( deleted in 1318 and 1928 , mutated in 22/23 ) slightly reduced but did not abolish cct binding , deletion within the n - terminal 13 amino acids of p53 ( 213 and 413 ) resulted in substantial loss of interaction with cct ( figure 4c ) . deletion of these residues also resulted in the loss of binding of p53 to cct ( figure s4d ) . the failure of these mutants to interact with cct proteins was not a result of lack of cytoplasmic localization , since 213 was clearly detected in both nuclear and cytoplasmic fractions ( figure s4c ) . importantly , this interaction was also seen in a reciprocal immunoprecipitation , where wild - type but not 413 p53 pulled down endogenous cct ( figure 4d ) . the loss of cct binding to 213 and 413 p53 was also confirmed using in vitro translation in reticulocyte lysates , which contain endogenous cct ( figure 4e ) . the n - terminal region of p53 shows moderate conservation between different species and contains polar and hydrophobic amino acids ( figure 4f ) , which may mediate the interaction with cct ( gmez - puertas et al . , 2004 ) . to assess the contribution of hydrophobic bonds to the p53-cct interaction , we examined the effect of chaotropic salts containing cations that can weaken hydrophobic bonds ( feldman et al . the disruption of the p53-cct interaction following incubation with licl and mgso4 suggests a possible contribution of hydrophobic interactions . taken together , these results show that the first 13 amino acids of p53 are necessary for the p53-cct interaction . to further assess whether cct impacts mdm2 binding by p53 , we examined the effect of cct depletion on the endogenous p53/mdm2 interaction ( figure s4f ) . importantly , the binding of mdm2 to p53 was not affected by depletion of cct and cct , an effect that is most clearly seen following stabilization of the p53 protein using the proteasome inhibitor mg132 ( figure s4f ) . furthermore , the cct nonbinding n - terminal deletion of p53 ( 413 ) retained the ability to bind mdm2 , an activity that was lost in the more extensive n - terminal truncation 39 ( figure s4 we also examined the impact of loss of cct binding on the interaction between p53 and hsp70 ( figure 4 g ) . again , deletion of the extreme n terminus of p53 ( 413 ) that abolished the cct interaction did not prevent binding to hsp70 , although a more extensive deletion ( 39 ) reduced hsp70 binding . taken together , these results show that cct binding is not required for p53 to bind mdm2 or hsp70 . the interaction of p53 with the cct complex suggests that this chaperonin may play a role in promoting the correct folding , and thus activity , of p53 . the conformation of p53 can be assessed using two antibodies : ab1620 , which recognizes a domain only present in the correctly folded protein and therefore indicates functional wild - type p53 ( milner et al . , 1987 ) , and ab240 , which recognizes a cryptic epitope ( amino acids 211220 ) only exposed in the unfolded or denatured conformation ( gannon et al . , 1990 ) . the relative amounts of p53 recognized by ab1620 and ab240 provide an indication of the extent of p53 folding . to determine whether cct contributes to p53 folding , we used small interfering rna ( sirna ) to deplete u2os or mcf7 cells ( that express endogenous wild - type p53 ) of cct and cct ( figure 5a ) . immunoprecipitation of p53 with the conformation - specific antibodies showed a clear decrease in ab1620-reactive p53 and an accumulation of ab240-reactive p53 in cells depleted of cct complex components ( figure 5a ) . a similar response was seen in a2780 cells ( figure s5a ) and in cells where p53 protein was induced in response to doxycycline treatment ( figure 5b ) . in these cells , both wild - type p53 and the 273h mutant , which retains predominantly wild - type conformation , became misfolded ( and ab240 reactive ) following cct depletion ( figure 5b ) . during the course of these studies , we noted that depletion of both cct subunits and frequently led to an increase in p53 protein levels in cells expressing endogenous wild - type p53 cells ( figure 5a ; figure s5b ) , although this was not evident in cells expressing transfected mutant p53 ( figure 1e ) . the increase in p53 protein levels following cct knockdown was accompanied by a slight increase in p53 half - life in both hct116 and u2os cells ( figure s5b ) , and depletion of cct in otherwise unstressed cells resulted in a reduction in extent of ubiquitination of endogenous p53 ( figure s5c ) . previous studies have shown that cct depletion can result in cell - cycle arrest ( grantham et al . , 2006 ; liu et al . , 2005 ) , indicating that the incorrect folding of cct client proteins is likely to promote a stress response that could indirectly signal to activate p53 . following transient expression of p53 , both wild - type and transactivation domain mutant 22/23 retained predominantly wild - type ( ab1620 ) conformation , while depletion of cct resulted in an increase in misfolded p53 detected by ab240 ( figure 5c ) . by contrast , p5343 , lacking the cct binding and transactivation domain , adopted a more unfolded conformation that was not further affected by cct depletion ( figure 5c ) . to more closely define the effect of cct binding , we examined the smaller n - terminal deletions of p53 . using standard extraction procedure ( lysing cells and immunoprecipitating at 4c ) , we found variable results with the cct nonbinding p53 mutants ( 213 and 413 ) , which showed a slightly higher ratio of ab240 reactivity in some assays but mainly ab1620 reactivity similar to wild - type p53 in others ( for example , compare control lanes in figures 5d and 5e ) . previous studies have shown that p53 has a low thermodynamic stability in vitro , which is further reduced by mutations within the dna binding domain ( bullock et al . we therefore considered whether the p53 mutants that are unable to bind to cct might show a greater sensitivity to temperature - induced denaturation . to test this , we examined the conformation of p53 in cell extracts following incubation at 37c ( figure 5d ) . as previously described ( bullock et al . , 2000 ) , the 175h mutant was highly unstable compared to the wild - type p53 protein , which became predominantly ab240 reactive only after 8 min at 37c . interestingly , both p53213 and p53413 were clearly less stable than the wild - type protein in this assay ( figure 5d ) . to extend these studies , we incubated p53-transfected cells at 39c for 2 or 5 hr immediately before lysis and carried out conformation - specific immunoprecipitation ( figure 5e ) . these studies again showed that p53213 was less stable than wild - type p53 , shifting significantly to the unfolded , ab240-reactive conformation after only 2 hr heat shock , at which time point the wild - type protein remained predominantly ab1620 reactive . to look directly at the conformation of p53 in cells , we used an immunofluorescence assay in which we compared the signal from total p53 ( as detected using a p53 polyclonal antibody ) with the signal using ab240 ( figure 5f ) . to validate the assay , we showed that heat shock induced the expected increase in ab240 reactivity in cells expressing wild - type p53 , consistent with the results shown in figure 5e . turning to p53 mutants , both wild - type and transactivation domain mutant 22/23 showed low ab240 reactivity in cells incubated at 37c , while the 175h mutant reacted strongly with this antibody ( figure 5 g ) , as expected . in this assay , deletion within the n - terminal 13 amino acids ( 213 and 213 , 22/23 ) resulted in a significant acquisition of ab240 reactivity . taken together , these results show that deletion of the cct binding domain in the n terminus of p53 results in a protein that is structurally less stable than wild - type and more sensitive to unfolding under temperature stress . wild - type p53 showed a shift to the misfolded ab240 reactive conformation following treatment of cells with an hsp70 inhibitor alone or in combination with heat shock ( figure s5d ) . a similar shift in the conformation of wild - type p53 following treatment with the hsp70 inhibitor was seen in cells using the immunofluorescence assay ( figure s5e ) . these results indicate that both cct complex and hsp70 can promote the folding of p53 and that impeding either chaperone impacts the conformation of p53 . p53 has two transactivation domains : ad1 , which is located to residues 1428 , and ad2 , which is located to residues 3861 ( candau et al . , 1997 ; the transcriptional activity of the n - terminal p53 mutants described here was therefore assessed using a p53-responsive promoter ( pg13 luciferase ; figure 6a ) . as expected , deletion of residues within ad1 ( p5339 and 1928 ) significantly impaired the transcriptional activity of p53 , which was largely retained in a mutant deleted of residues outside this region ( p532840 and p534149 ; figure 6a ) . however , the cct binding mutants p53213 and p53413 ( which would not be predicted to impinge on ad1 ) showed reduced activity , consistent with the misfolding of these proteins . as seen in earlier assays , the levels of transfected p53 protein were not increased following cct depletion ( figure 6b ) , indicating that ectopic overexpressed protein is less sensitive to the endogenous p53 degradation machinery . however , the activity of wild - type p53 and transcriptionally active mutants that retained the n - terminal cct binding domain ( p532840 , p534149 ) was reduced following knockdown of cct and cct subunits ( figure 6b ) , consistent with a loss of wild - type conformation . by contrast , the lower level of transcriptional activity retained by p53213 and p53413 was not further compromised by inhibition of cct complex ( figure 6b ) , supporting our observation that these mutants no longer interact with , and are therefore not subject to , regulation by cct . the modulation of p53 s transcriptional activity by cct was also apparent when examining the activation of endogenous p53-dependent target genes . p53-induced expression of both puma and p21 was clearly impeded by the depletion of cct , both at the protein and messenger rna ( mrna ) level ( figures 6c and 6d ; figures s6a and s6b ) . interestingly , however , mdm2 expression was not affected by cct depletion , suggesting that the ability of p53 to induce the expression of mdm2 is less dependent on p53 conformation . expression of p53413 also showed a decreased ability to activate expression of p21 and puma ( figure 6d ) . as seen with the p53 reporter assays , depletion of cct reduced the transcriptional activity of wild - type p53 but did not impact the lower activity of the 413 mutant ( figure 6d ) . interestingly , depletion of the cct complex did not result in a profound decrease of endogenous p53 activity , either under basal conditions or following activation of p53 by treatment of the cells with nutlin 3 or low levels of actinomycin d ( figures s6c and s6d ) . although depletion of cct resulted in an increase in endogenous p53 stability ( figure s5 ) , no corresponding increase in transcriptional activity was detected either . taken together , these results suggested that the response of endogenous p53 to cct knockdown is complex and that the outcome is a balance between increased protein levels ( due to reduced degradation of p53 as part of a general stress response ) and decreased conformational integrity ( due to defects in the protein folding process ) . tumor - derived p53 point mutations such as 175h and 273h acquire an ability to promote matrigel invasion , invasion into organotypic assays , and random migration ( adorno et al . , 2009 ; expression of wild - type p53 in these cells inhibits proliferation and enhances cell death and can not be used as a control for invasion . we therefore used the transcriptionally defective p53 22/23 mutant ( which shows impaired growth inhibitory activity ) , which did not result in enhanced invasion above that seen with the empty vector control ( figure 7a ) . interestingly transient expression of non - cct binding mutants p53213 22/23 , p53413 22/23 , or p5339 promoted invasion , albeit to a lesser extent than the tumor - derived 175h or 273h mutant ( figure 7a ; figure s7a ) . notably , loss of the n terminus in 175h- or 273h - expressing cells did not impact invasive capacity ( muller et al . , 2009 ) . further investigation of the ability of the non - cct binding mutants of p53 to promote activities associated with tumor - derived mutants , such as random motility or invasion into organotypic assays , required cell lines that stably express these p53 proteins . despite attempts to generate h1299 cell lines stably expressing p53 22/23 , p53213 22/23 , p53413 22/23 , and p5339 , only p5339-containing cells retained expression over time , although this was lower than 273h ( figure 7b ; figure s7b ) . these results are likely to reflect the retention of some residual growth inhibitory activity in the p53 22/23 mutant . compared to empty vector transfected h1299 cells ( ctrl ) , p5339 cells induced random motility and invasion into matrigel and organotypic assays to a similar or slightly lesser extent than 273h ( figures 7b7d ) . furthermore , despite the loss of expression of transiently transfected p53 22/23 and p53413 22/23 during the course of the experiment , both p53413 22/23 and 175h 22/23 cells showed some invasion into organotypic plugs ( figure s7b ) . finally , we examined whether depletion of the cct complex would have an effect on cell invasion using rpe cells ( expressing wild - type p53 ) and variants stably expressing 273h or 175h , mutants that promoted the invasive ability of these cells ( figure 7e ; figure s7c ) . depletion of cct complex by sirna in either 175h- or 273h - expressing cells resulted in decreased invasion ( figure 7e ; figure s7c ) that is likely to reflect the misfolding of other cct client proteins like actin and tubulin , as previously described ( matus et al . , , we were unable to achieve a complete depletion of cct complex proteins , indicating that only cells with partial knockdown continued proliferation , consistent with our observation that cct complex depletion resulted in a stress response that activated p53 ( figure 6 ) . interestingly , cells that survived with a partial depletion of cct showed a reproducible increase in invasion ( figure 7e ) , consistent with a role of cct in maintaining wild - type conformation and function of p53 . depletion of wild - type p53 in these cells resulted in enhanced invasion ( figure s7d ) , so it was not possible to demonstrate p53 dependence of the increased invasion in cct - depleted wild - type p53-expressing cells . importantly , however , depletion of both p53 and cct decreased invasion ( figure s7d ) , similar to the effect seen in mutant p53-expressing cells . these results show that despite a general inhibition of invasive behavior in response to cct depletion , wild - type p53-expressing cells show enhanced invasion that correlates with the misfolding of the p53 protein . correct protein folding is essential for function , and this process is assisted by molecular chaperones . the chaperonin cct assists in specialized folding of selected proteins that are not fully folded by the nonspecialized chaperones alone . in this study , we demonstrate that p53 is a substrate for cct and provide evidence that this chaperonin regulates p53 folding and activity . many previous studies have shown that tumor - derived p53 point mutants result in the failure of p53 to adopt the wild - type conformation , leading to the concept that p53 can either be in the wild - type or mutant ( i.e. , misfolded ) conformation . misfolding of p53 due to cancer - associated dna binding domain mutations , which enhances the thermoinstability of p53 ( bullock et al . , 1997 ) , results in the loss of wild - type dna binding activity and the acquisition of transforming functions , including the ability to promote invasion . our studies suggest that cct plays a specific role in maintaining a pool of wild - type , functional p53 in the cell and that failure of p53 to bind to cct results in the accumulation of misfolded p53 , which then acquires activities characteristic of tumor - derived mutant p53s . while the most straightforward model for the role of cct is to assist in the correct folding of newly synthesized p53 ( mayer , 2010 ) , it is also possible that cct binding to p53 prevents its aggregation , as has been shown for other proteins . cct would function to allow the correct folding of p53 that occurs either spontaneously or through a cooperation with other chaperones , for example with hsp70 . in either case , the consequence of loss or modulation of the cct - p53 interaction would be the accumulation of misfolded p53 with invasive capacity . based on these results , we suggest that the regulation of wild - type p53 folding may be part of the normal biology of p53 . regulation of the interaction of p53 with cct could determine the generation of folded or unfolded p53 , without the requirement for mutations in the dna binding domain . our study shows that the effect of loss of cct binding on p53 conformation is subtle , supporting in vitro studies in which wild - type p53 can spontaneously fold into an active conformation . nevertheless , the role of cct in protein folding becomes more apparent under stress , as shown in the heat shock experiments described here . it seems possible that the role of cct will also be evident under other physiological stress conditions , such as hypoxia , reactive oxygen species , and loss of cell adhesion . several recent studies have shown that cct activity can be controlled by phosphorylation by rsk and s6k ( abe et al . , 2009 ) or deacetylation by sirt1 ( liu et al . , 2010 ) . in addition , phosphorylation of serine 6 and 9 in p53 has also been identified ( meek and anderson , 2009 ) , so further studies will determine whether the p53/cct interaction can be actively modulated through changes in posttranslational modifications of either protein . active control of cct binding in this way could allow for the expression of wild - type p53 in either folded or unfolded conformations resulting in distinct activities . according to this model , dna binding domain point mutations that are selected in cancers would force p53 to constitutively adopt the unfolded conformation . the activity of mutant p53 would therefore be the inappropriate manifestation of a normal state of wild - type p53 rather than the acquisition of a completely new activity . the accumulation of unfolded p53 may lead to the formation of aggregates , which would not be reversible ( ano bom et al . , however , in vitro studies have shown that there is an initial reversible step in the denaturation of p53 ( wilcken et al . , 2012 ) , and in cells , temperature - sensitive p53 mutants can shift from mutant to wild - type forms ( friedlander et al . understanding when wild - type p53 adopts the denatured or misfolded conformation , how this is regulated , and the functional consequences of this conformational switching in vivo will be an interesting future challenge . h1299 , hct116 , u2os , mcf-7 , and rpe cells were cultured as described in the supplemental experimental procedures . generation of stable cells lines expressing mutant p53 175h and 273h was performed as described elsewhere ( muller et al . , 2009 ) . all p53 mutant constructs have been described previously or created by site - directed mutagenesis as detailed in the supplemental information . details of immunoprecipitation and in vitro binding assays are described in the supplemental information . samples were applied onto carbon - coated copper grids previously glow - discharged and stained with 2% uranyl acetate . micrographs were taken and individual particles and image classification were performed as described in the supplemental information . pg13 luciferase and renilla luciferase constructs were transfected in combination with different p53 constructs and analyzed as described in the supplemental information . matrigel assays were performed as described previously ( muller et al . , 2009 ) . invasion toward a gradient of 10% fetal calf serum and a mixture of growth factors was measure by confocal microscopy in serial sections of 10 m each , and quantification of invading cells was performed by imagej software . organotypic invasion assays and migration assays ( wound scratch ) are detailed in the supplemental information .

summaryp53 is a transcription factor that mediates tumor suppressor responses . correct folding of the p53 protein is essential for these activities , and point mutations that induce conformational instability of p53 are frequently found in cancers . these mutant p53s not only lose wild - type activity but can also acquire the ability to promote invasion and metastasis . we show that folding of wild - type p53 is promoted by an interaction with the chaperonin cct . depletion of this chaperone in cells results in the accumulation of misfolded p53 , leading to a reduction in p53-dependent gene expression . intriguingly , p53 proteins mutated to prevent the interaction with cct show conformational instability and acquire an ability to promote invasion and random motility that is similar to the activity of tumor - derived p53 mutants . our data therefore suggest that both growth suppression and cell invasion may be differentially regulated functions of wild - type p53 .

Introduction Results Discussion Experimental Procedures

the p53 tumor suppressor protein plays an important role in preventing malignant development ( vousden and prives , 2009 ) , functioning primarily as a transcription factor to regulate the expression of a large number of genes that induce cellular responses such as cell - cycle arrest and apoptosis ( beckerman and prives , 2010 ) . numerous mechanisms through which p53 is regulated have been described , including the control of translation , protein stability , subcellular localization , and interaction with other components of the transcriptional machinery ( hollstein and hainaut , 2010 ) . in many cancers , the function of p53 is ablated through point mutations that lead to the expression of a mutant p53 protein ( joerger and fersht , 2007 ) . these tumor - associated point mutations occur predominantly in the central dna binding domain of p53 and result in a diminished ability of p53 to bind consensus sites in the promoters of p53-regulated genes . while some of these mutations result in amino - acid substitutions of residues within p53 that directly contact the dna ( contact mutants ) , other mutations result in the misfolding of the p53 protein . the p53 dna binding domain shows a low thermodynamic stability in vitro , and mutations in this region can lead to further instability , causing the protein to become denatured at 37c ( joerger and fersht , 2007 ) and a potential to form p53 protein aggregates within the cell ( xu et al . , the net effect of these tumor - associated point mutants is both the loss of wild - type p53 activity and a gain of function that contributes to the invasive behavior of cancers ( muller et al . the mechanisms underlying this gain of function are still under investigation but at least partially reflect the ability of the mutant p53 proteins to modulate the activity of other transcription factors such as p63 , p73 , and srebp ( freed - pastor and prives , 2012 ) . they not only prevent misfolding and aggregation of proteins but can also target misfolded proteins for degradation . the open conformation recognizes unfolded peptides , and atp binding and hydrolysis induce the closed conformation , which results in the folding of the protein ( douglas et al . , 2008a ) but can also prevent the aggregation of proteins with polyglutamine regions ( kitamura et al . we show here that p53 is a client of the cct complex and that failure to interact with this molecular chaperone can promote oncogenic functions of p53 in the absence of classic tumor - derived dna binding domain mutations . we carried out a proteomic analysis to identify proteins that interact with wild - type p53 and a tumor - derived point mutant , 175h . , 2003 ) were most frequently identified in immunoprecipitation with wild - type p53 ( figure 1a ) , validating this approach . to further study the consequences of the interaction of p53 with cct subunits in cells , we utilized several cell lines that each expressed broadly equal endogenous levels of various cct subunits ( figure s1a available online ) . coprecipitation of endogenous wild - type p53 with endogenous cct and cct from hct116 cells ( figure 1b ) or u2os cells ( where p53 was stabilized by treatment of cells with the mdm2 inhibitor nutlin 3 ; figure 1c ) demonstrated the ability of these proteins to interact in cells . to compare the binding of wild - type and mutant p53 directly in the same cells , we turned to h1299 , a p53 null tumor cell line transiently transfected with p53-expression constructs ( figure 1d ) . these results indicated that wild - type p53 , 175h , and 273h all efficiently interacted with endogenous cct . similarly , wild - type p53 and 248w were coprecipitated with cct in hct116 cells ( figure s1b ) . cct is a large , multisubunit structure ( 960 kda ) , and we used gel filtration to determine whether p53 could associate with the whole cct complex ( figure 2a ) . endogenous wild - type p53 largely comigrated with cct and cct in high - molecular - weight complexes , consistent with an interaction with the whole cct complex ( figure 2a ) . similar results were observed in lysates from h1299 cells transiently transfected with wild - type p53 ( figure s2 ) . depletion of cct and cct resulted in the release of some p53 from the larger complexes , supporting the interaction of p53 with cct ( figure 2b ) , although the retention of some p53 in larger complexes following cct depletion likely reflects p53 s interaction with other proteins ( collavin et al . , quantification of p53 levels in each fraction indicated that approximately 12% of the total p53 was released from the larger complexes , suggesting this proportion of p53 is bound to cct under these conditions . to test directly whether p53 interacts with the cct complex , we mixed bacterially expressed wild - type p53 with purified bovine cct . the averaging of almost 2,000 cct : p53 particles revealed a similar structure except for the presence of mass inside the cavity that interacted mainly with one cct subunit ( figure 3c ; figures s3a previous studies have shown that the atp - dependent conformational change leading to closure of the cct complex also results in its protection against proteinase k digestion ( meyer et al . importantly , this protection from degradation also applied to the p53 protein bound to cct , as the closed conformation of the chaperonin was able to protect the trapped p53 from protease attack ( figure 3e ) . taken together , these data demonstrate that p53 interacts with the cct complex and can be trapped in the interior of the chaperonin cavity in an atp - hydrolysis - dependent manner . interestingly , several of the c - terminal truncations also removed the oligomerization domain and the nuclear import signals from p53 , suggesting that neither of these is required for cct binding in cells . both endogenous and transfected wild - type p53 were found predominantly ( although not exclusively ) in the nuclear fractions ( figures s4b and s4c ) . while alterations within the dna binding and c - terminal domains of p53 did not affect the cct interaction , deletion of the n - terminal 43 amino acids of p53 resulted in the loss of interaction with cct ( figure 4b ) . the n terminus of p53 is responsible for many protein interactions , including binding to mdm2 and the components of the transcriptional machinery ( residues 1729 ; kussie et al . while mutation in the mdm2 binding and transactivation domains ( deleted in 1318 and 1928 , mutated in 22/23 ) slightly reduced but did not abolish cct binding , deletion within the n - terminal 13 amino acids of p53 ( 213 and 413 ) resulted in substantial loss of interaction with cct ( figure 4c ) . the n - terminal region of p53 shows moderate conservation between different species and contains polar and hydrophobic amino acids ( figure 4f ) , which may mediate the interaction with cct ( gmez - puertas et al . taken together , these results show that the first 13 amino acids of p53 are necessary for the p53-cct interaction . importantly , the binding of mdm2 to p53 was not affected by depletion of cct and cct , an effect that is most clearly seen following stabilization of the p53 protein using the proteasome inhibitor mg132 ( figure s4f ) . furthermore , the cct nonbinding n - terminal deletion of p53 ( 413 ) retained the ability to bind mdm2 , an activity that was lost in the more extensive n - terminal truncation 39 ( figure s4 we also examined the impact of loss of cct binding on the interaction between p53 and hsp70 ( figure 4 g ) . the interaction of p53 with the cct complex suggests that this chaperonin may play a role in promoting the correct folding , and thus activity , of p53 . the conformation of p53 can be assessed using two antibodies : ab1620 , which recognizes a domain only present in the correctly folded protein and therefore indicates functional wild - type p53 ( milner et al . to determine whether cct contributes to p53 folding , we used small interfering rna ( sirna ) to deplete u2os or mcf7 cells ( that express endogenous wild - type p53 ) of cct and cct ( figure 5a ) . immunoprecipitation of p53 with the conformation - specific antibodies showed a clear decrease in ab1620-reactive p53 and an accumulation of ab240-reactive p53 in cells depleted of cct complex components ( figure 5a ) . in these cells , both wild - type p53 and the 273h mutant , which retains predominantly wild - type conformation , became misfolded ( and ab240 reactive ) following cct depletion ( figure 5b ) . during the course of these studies , we noted that depletion of both cct subunits and frequently led to an increase in p53 protein levels in cells expressing endogenous wild - type p53 cells ( figure 5a ; figure s5b ) , although this was not evident in cells expressing transfected mutant p53 ( figure 1e ) . the increase in p53 protein levels following cct knockdown was accompanied by a slight increase in p53 half - life in both hct116 and u2os cells ( figure s5b ) , and depletion of cct in otherwise unstressed cells resulted in a reduction in extent of ubiquitination of endogenous p53 ( figure s5c ) . following transient expression of p53 , both wild - type and transactivation domain mutant 22/23 retained predominantly wild - type ( ab1620 ) conformation , while depletion of cct resulted in an increase in misfolded p53 detected by ab240 ( figure 5c ) . using standard extraction procedure ( lysing cells and immunoprecipitating at 4c ) , we found variable results with the cct nonbinding p53 mutants ( 213 and 413 ) , which showed a slightly higher ratio of ab240 reactivity in some assays but mainly ab1620 reactivity similar to wild - type p53 in others ( for example , compare control lanes in figures 5d and 5e ) . , 2000 ) , the 175h mutant was highly unstable compared to the wild - type p53 protein , which became predominantly ab240 reactive only after 8 min at 37c . these studies again showed that p53213 was less stable than wild - type p53 , shifting significantly to the unfolded , ab240-reactive conformation after only 2 hr heat shock , at which time point the wild - type protein remained predominantly ab1620 reactive . to look directly at the conformation of p53 in cells , we used an immunofluorescence assay in which we compared the signal from total p53 ( as detected using a p53 polyclonal antibody ) with the signal using ab240 ( figure 5f ) . to validate the assay , we showed that heat shock induced the expected increase in ab240 reactivity in cells expressing wild - type p53 , consistent with the results shown in figure 5e . turning to p53 mutants , both wild - type and transactivation domain mutant 22/23 showed low ab240 reactivity in cells incubated at 37c , while the 175h mutant reacted strongly with this antibody ( figure 5 g ) , as expected . taken together , these results show that deletion of the cct binding domain in the n terminus of p53 results in a protein that is structurally less stable than wild - type and more sensitive to unfolding under temperature stress . wild - type p53 showed a shift to the misfolded ab240 reactive conformation following treatment of cells with an hsp70 inhibitor alone or in combination with heat shock ( figure s5d ) . a similar shift in the conformation of wild - type p53 following treatment with the hsp70 inhibitor was seen in cells using the immunofluorescence assay ( figure s5e ) . these results indicate that both cct complex and hsp70 can promote the folding of p53 and that impeding either chaperone impacts the conformation of p53 . , 1997 ; the transcriptional activity of the n - terminal p53 mutants described here was therefore assessed using a p53-responsive promoter ( pg13 luciferase ; figure 6a ) . as expected , deletion of residues within ad1 ( p5339 and 1928 ) significantly impaired the transcriptional activity of p53 , which was largely retained in a mutant deleted of residues outside this region ( p532840 and p534149 ; figure 6a ) . as seen in earlier assays , the levels of transfected p53 protein were not increased following cct depletion ( figure 6b ) , indicating that ectopic overexpressed protein is less sensitive to the endogenous p53 degradation machinery . however , the activity of wild - type p53 and transcriptionally active mutants that retained the n - terminal cct binding domain ( p532840 , p534149 ) was reduced following knockdown of cct and cct subunits ( figure 6b ) , consistent with a loss of wild - type conformation . interestingly , however , mdm2 expression was not affected by cct depletion , suggesting that the ability of p53 to induce the expression of mdm2 is less dependent on p53 conformation . as seen with the p53 reporter assays , depletion of cct reduced the transcriptional activity of wild - type p53 but did not impact the lower activity of the 413 mutant ( figure 6d ) . interestingly , depletion of the cct complex did not result in a profound decrease of endogenous p53 activity , either under basal conditions or following activation of p53 by treatment of the cells with nutlin 3 or low levels of actinomycin d ( figures s6c and s6d ) . taken together , these results suggested that the response of endogenous p53 to cct knockdown is complex and that the outcome is a balance between increased protein levels ( due to reduced degradation of p53 as part of a general stress response ) and decreased conformational integrity ( due to defects in the protein folding process ) . tumor - derived p53 point mutations such as 175h and 273h acquire an ability to promote matrigel invasion , invasion into organotypic assays , and random migration ( adorno et al . , 2009 ; expression of wild - type p53 in these cells inhibits proliferation and enhances cell death and can not be used as a control for invasion . interestingly transient expression of non - cct binding mutants p53213 22/23 , p53413 22/23 , or p5339 promoted invasion , albeit to a lesser extent than the tumor - derived 175h or 273h mutant ( figure 7a ; figure s7a ) . further investigation of the ability of the non - cct binding mutants of p53 to promote activities associated with tumor - derived mutants , such as random motility or invasion into organotypic assays , required cell lines that stably express these p53 proteins . finally , we examined whether depletion of the cct complex would have an effect on cell invasion using rpe cells ( expressing wild - type p53 ) and variants stably expressing 273h or 175h , mutants that promoted the invasive ability of these cells ( figure 7e ; figure s7c ) . interestingly , cells that survived with a partial depletion of cct showed a reproducible increase in invasion ( figure 7e ) , consistent with a role of cct in maintaining wild - type conformation and function of p53 . depletion of wild - type p53 in these cells resulted in enhanced invasion ( figure s7d ) , so it was not possible to demonstrate p53 dependence of the increased invasion in cct - depleted wild - type p53-expressing cells . importantly , however , depletion of both p53 and cct decreased invasion ( figure s7d ) , similar to the effect seen in mutant p53-expressing cells . these results show that despite a general inhibition of invasive behavior in response to cct depletion , wild - type p53-expressing cells show enhanced invasion that correlates with the misfolding of the p53 protein . correct protein folding is essential for function , and this process is assisted by molecular chaperones . the chaperonin cct assists in specialized folding of selected proteins that are not fully folded by the nonspecialized chaperones alone . many previous studies have shown that tumor - derived p53 point mutants result in the failure of p53 to adopt the wild - type conformation , leading to the concept that p53 can either be in the wild - type or mutant ( i.e. , 1997 ) , results in the loss of wild - type dna binding activity and the acquisition of transforming functions , including the ability to promote invasion . our studies suggest that cct plays a specific role in maintaining a pool of wild - type , functional p53 in the cell and that failure of p53 to bind to cct results in the accumulation of misfolded p53 , which then acquires activities characteristic of tumor - derived mutant p53s . while the most straightforward model for the role of cct is to assist in the correct folding of newly synthesized p53 ( mayer , 2010 ) , it is also possible that cct binding to p53 prevents its aggregation , as has been shown for other proteins . cct would function to allow the correct folding of p53 that occurs either spontaneously or through a cooperation with other chaperones , for example with hsp70 . in either case , the consequence of loss or modulation of the cct - p53 interaction would be the accumulation of misfolded p53 with invasive capacity . based on these results , we suggest that the regulation of wild - type p53 folding may be part of the normal biology of p53 . regulation of the interaction of p53 with cct could determine the generation of folded or unfolded p53 , without the requirement for mutations in the dna binding domain . our study shows that the effect of loss of cct binding on p53 conformation is subtle , supporting in vitro studies in which wild - type p53 can spontaneously fold into an active conformation . active control of cct binding in this way could allow for the expression of wild - type p53 in either folded or unfolded conformations resulting in distinct activities . according to this model , dna binding domain point mutations that are selected in cancers would force p53 to constitutively adopt the unfolded conformation . the activity of mutant p53 would therefore be the inappropriate manifestation of a normal state of wild - type p53 rather than the acquisition of a completely new activity . the accumulation of unfolded p53 may lead to the formation of aggregates , which would not be reversible ( ano bom et al . , 2012 ) , and in cells , temperature - sensitive p53 mutants can shift from mutant to wild - type forms ( friedlander et al . understanding when wild - type p53 adopts the denatured or misfolded conformation , how this is regulated , and the functional consequences of this conformational switching in vivo will be an interesting future challenge .

the p53 tumor suppressor protein plays an important role in preventing malignant development ( vousden and prives , 2009 ) , functioning primarily as a transcription factor to regulate the expression of a large number of genes that induce cellular responses such as cell - cycle arrest and apoptosis ( beckerman and prives , 2010 ) . numerous mechanisms through which p53 is regulated have been described , including the control of translation , protein stability , subcellular localization , and interaction with other components of the transcriptional machinery ( hollstein and hainaut , 2010 ) . these tumor - associated point mutations occur predominantly in the central dna binding domain of p53 and result in a diminished ability of p53 to bind consensus sites in the promoters of p53-regulated genes . while some of these mutations result in amino - acid substitutions of residues within p53 that directly contact the dna ( contact mutants ) , other mutations result in the misfolding of the p53 protein . the p53 dna binding domain shows a low thermodynamic stability in vitro , and mutations in this region can lead to further instability , causing the protein to become denatured at 37c ( joerger and fersht , 2007 ) and a potential to form p53 protein aggregates within the cell ( xu et al . , the net effect of these tumor - associated point mutants is both the loss of wild - type p53 activity and a gain of function that contributes to the invasive behavior of cancers ( muller et al . the mechanisms underlying this gain of function are still under investigation but at least partially reflect the ability of the mutant p53 proteins to modulate the activity of other transcription factors such as p63 , p73 , and srebp ( freed - pastor and prives , 2012 ) . probably the best - understood chaperones are the heat shock proteins hsp70 and hsp90 , which play a role in conformational maturation and help to target improperly folded proteins for ubiquitination and proteolysis , and the ring complex chaperonins , which enclose proteins within their structure for folding of newly synthesized peptides ( mayer , 2010 ) . although the mechanism of substrate - cct recognition and binding remains under investigation , each of the subunits can recognize different polar and hydrophobic motifs within substrate proteins ( yam et al . potentially up to 15% of all newly synthesized polypeptides can associate with the cct complex , although only a few proteins have so far been shown to depend on this chaperonin for folding and function ( thulasiraman et al . we show here that p53 is a client of the cct complex and that failure to interact with this molecular chaperone can promote oncogenic functions of p53 in the absence of classic tumor - derived dna binding domain mutations . we carried out a proteomic analysis to identify proteins that interact with wild - type p53 and a tumor - derived point mutant , 175h . interestingly , peptides from seven of the eight cct subunits were also found in complex with p53 ( figure 1a ) , consistent with our recent study identifying cct subunits as part of a p53 interactome ( coffill et al . to further study the consequences of the interaction of p53 with cct subunits in cells , we utilized several cell lines that each expressed broadly equal endogenous levels of various cct subunits ( figure s1a available online ) . coprecipitation of endogenous wild - type p53 with endogenous cct and cct from hct116 cells ( figure 1b ) or u2os cells ( where p53 was stabilized by treatment of cells with the mdm2 inhibitor nutlin 3 ; figure 1c ) demonstrated the ability of these proteins to interact in cells . to compare the binding of wild - type and mutant p53 directly in the same cells , we turned to h1299 , a p53 null tumor cell line transiently transfected with p53-expression constructs ( figure 1d ) . endogenous wild - type p53 largely comigrated with cct and cct in high - molecular - weight complexes , consistent with an interaction with the whole cct complex ( figure 2a ) . depletion of cct and cct resulted in the release of some p53 from the larger complexes , supporting the interaction of p53 with cct ( figure 2b ) , although the retention of some p53 in larger complexes following cct depletion likely reflects p53 s interaction with other proteins ( collavin et al . , quantification of p53 levels in each fraction indicated that approximately 12% of the total p53 was released from the larger complexes , suggesting this proportion of p53 is bound to cct under these conditions . following separation by native electrophoresis , the high - molecular - weight band corresponding to cct was excised and subjected to sds electrophoresis , which revealed the presence of cct subunits and p53 ( figure 3a ) , confirming the presence of a stable cct : p53 complex . first , over 1,000 end - on views of the isolated apo - cct particles were processed , and the average image revealed the typical presence of the doughnut - shaped structure with an empty cavity surrounded by eight similar masses ( figure 3b ) . the averaging of almost 2,000 cct : p53 particles revealed a similar structure except for the presence of mass inside the cavity that interacted mainly with one cct subunit ( figure 3c ; figures s3a previous studies have shown that the atp - dependent conformational change leading to closure of the cct complex also results in its protection against proteinase k digestion ( meyer et al . we were also able to confirm a dose - dependent degradation of the cct complex following incubation with proteinase k , which was prevented by the addition of atp ( figure 3d ) . importantly , this protection from degradation also applied to the p53 protein bound to cct , as the closed conformation of the chaperonin was able to protect the trapped p53 from protease attack ( figure 3e ) . taken together , these data demonstrate that p53 interacts with the cct complex and can be trapped in the interior of the chaperonin cavity in an atp - hydrolysis - dependent manner . to determine which regions of p53 were important for the cct interaction deletions or point mutations within the dna binding domain or the c terminus of p53 did not affect binding to cct ( figures 4a and 4b ) . interestingly , several of the c - terminal truncations also removed the oligomerization domain and the nuclear import signals from p53 , suggesting that neither of these is required for cct binding in cells . however , despite detecting both p53 and cct proteins in the nucleus , coimmunoprecipitation showed that the endogenous p53/cct interaction occurs in the cytosol ( figure s4b ) . while alterations within the dna binding and c - terminal domains of p53 did not affect the cct interaction , deletion of the n - terminal 43 amino acids of p53 resulted in the loss of interaction with cct ( figure 4b ) . the n terminus of p53 is responsible for many protein interactions , including binding to mdm2 and the components of the transcriptional machinery ( residues 1729 ; kussie et al . while mutation in the mdm2 binding and transactivation domains ( deleted in 1318 and 1928 , mutated in 22/23 ) slightly reduced but did not abolish cct binding , deletion within the n - terminal 13 amino acids of p53 ( 213 and 413 ) resulted in substantial loss of interaction with cct ( figure 4c ) . the n - terminal region of p53 shows moderate conservation between different species and contains polar and hydrophobic amino acids ( figure 4f ) , which may mediate the interaction with cct ( gmez - puertas et al . to assess the contribution of hydrophobic bonds to the p53-cct interaction , we examined the effect of chaotropic salts containing cations that can weaken hydrophobic bonds ( feldman et al . taken together , these results show that the first 13 amino acids of p53 are necessary for the p53-cct interaction . to further assess whether cct impacts mdm2 binding by p53 , we examined the effect of cct depletion on the endogenous p53/mdm2 interaction ( figure s4f ) . importantly , the binding of mdm2 to p53 was not affected by depletion of cct and cct , an effect that is most clearly seen following stabilization of the p53 protein using the proteasome inhibitor mg132 ( figure s4f ) . furthermore , the cct nonbinding n - terminal deletion of p53 ( 413 ) retained the ability to bind mdm2 , an activity that was lost in the more extensive n - terminal truncation 39 ( figure s4 we also examined the impact of loss of cct binding on the interaction between p53 and hsp70 ( figure 4 g ) . again , deletion of the extreme n terminus of p53 ( 413 ) that abolished the cct interaction did not prevent binding to hsp70 , although a more extensive deletion ( 39 ) reduced hsp70 binding . to determine whether cct contributes to p53 folding , we used small interfering rna ( sirna ) to deplete u2os or mcf7 cells ( that express endogenous wild - type p53 ) of cct and cct ( figure 5a ) . immunoprecipitation of p53 with the conformation - specific antibodies showed a clear decrease in ab1620-reactive p53 and an accumulation of ab240-reactive p53 in cells depleted of cct complex components ( figure 5a ) . in these cells , both wild - type p53 and the 273h mutant , which retains predominantly wild - type conformation , became misfolded ( and ab240 reactive ) following cct depletion ( figure 5b ) . during the course of these studies , we noted that depletion of both cct subunits and frequently led to an increase in p53 protein levels in cells expressing endogenous wild - type p53 cells ( figure 5a ; figure s5b ) , although this was not evident in cells expressing transfected mutant p53 ( figure 1e ) . the increase in p53 protein levels following cct knockdown was accompanied by a slight increase in p53 half - life in both hct116 and u2os cells ( figure s5b ) , and depletion of cct in otherwise unstressed cells resulted in a reduction in extent of ubiquitination of endogenous p53 ( figure s5c ) . following transient expression of p53 , both wild - type and transactivation domain mutant 22/23 retained predominantly wild - type ( ab1620 ) conformation , while depletion of cct resulted in an increase in misfolded p53 detected by ab240 ( figure 5c ) . using standard extraction procedure ( lysing cells and immunoprecipitating at 4c ) , we found variable results with the cct nonbinding p53 mutants ( 213 and 413 ) , which showed a slightly higher ratio of ab240 reactivity in some assays but mainly ab1620 reactivity similar to wild - type p53 in others ( for example , compare control lanes in figures 5d and 5e ) . these studies again showed that p53213 was less stable than wild - type p53 , shifting significantly to the unfolded , ab240-reactive conformation after only 2 hr heat shock , at which time point the wild - type protein remained predominantly ab1620 reactive . to look directly at the conformation of p53 in cells , we used an immunofluorescence assay in which we compared the signal from total p53 ( as detected using a p53 polyclonal antibody ) with the signal using ab240 ( figure 5f ) . to validate the assay , we showed that heat shock induced the expected increase in ab240 reactivity in cells expressing wild - type p53 , consistent with the results shown in figure 5e . taken together , these results show that deletion of the cct binding domain in the n terminus of p53 results in a protein that is structurally less stable than wild - type and more sensitive to unfolding under temperature stress . wild - type p53 showed a shift to the misfolded ab240 reactive conformation following treatment of cells with an hsp70 inhibitor alone or in combination with heat shock ( figure s5d ) . a similar shift in the conformation of wild - type p53 following treatment with the hsp70 inhibitor was seen in cells using the immunofluorescence assay ( figure s5e ) . as expected , deletion of residues within ad1 ( p5339 and 1928 ) significantly impaired the transcriptional activity of p53 , which was largely retained in a mutant deleted of residues outside this region ( p532840 and p534149 ; figure 6a ) . as seen in earlier assays , the levels of transfected p53 protein were not increased following cct depletion ( figure 6b ) , indicating that ectopic overexpressed protein is less sensitive to the endogenous p53 degradation machinery . however , the activity of wild - type p53 and transcriptionally active mutants that retained the n - terminal cct binding domain ( p532840 , p534149 ) was reduced following knockdown of cct and cct subunits ( figure 6b ) , consistent with a loss of wild - type conformation . by contrast , the lower level of transcriptional activity retained by p53213 and p53413 was not further compromised by inhibition of cct complex ( figure 6b ) , supporting our observation that these mutants no longer interact with , and are therefore not subject to , regulation by cct . p53-induced expression of both puma and p21 was clearly impeded by the depletion of cct , both at the protein and messenger rna ( mrna ) level ( figures 6c and 6d ; figures s6a and s6b ) . interestingly , however , mdm2 expression was not affected by cct depletion , suggesting that the ability of p53 to induce the expression of mdm2 is less dependent on p53 conformation . as seen with the p53 reporter assays , depletion of cct reduced the transcriptional activity of wild - type p53 but did not impact the lower activity of the 413 mutant ( figure 6d ) . interestingly , depletion of the cct complex did not result in a profound decrease of endogenous p53 activity , either under basal conditions or following activation of p53 by treatment of the cells with nutlin 3 or low levels of actinomycin d ( figures s6c and s6d ) . taken together , these results suggested that the response of endogenous p53 to cct knockdown is complex and that the outcome is a balance between increased protein levels ( due to reduced degradation of p53 as part of a general stress response ) and decreased conformational integrity ( due to defects in the protein folding process ) . interestingly transient expression of non - cct binding mutants p53213 22/23 , p53413 22/23 , or p5339 promoted invasion , albeit to a lesser extent than the tumor - derived 175h or 273h mutant ( figure 7a ; figure s7a ) . further investigation of the ability of the non - cct binding mutants of p53 to promote activities associated with tumor - derived mutants , such as random motility or invasion into organotypic assays , required cell lines that stably express these p53 proteins . furthermore , despite the loss of expression of transiently transfected p53 22/23 and p53413 22/23 during the course of the experiment , both p53413 22/23 and 175h 22/23 cells showed some invasion into organotypic plugs ( figure s7b ) . finally , we examined whether depletion of the cct complex would have an effect on cell invasion using rpe cells ( expressing wild - type p53 ) and variants stably expressing 273h or 175h , mutants that promoted the invasive ability of these cells ( figure 7e ; figure s7c ) . depletion of cct complex by sirna in either 175h- or 273h - expressing cells resulted in decreased invasion ( figure 7e ; figure s7c ) that is likely to reflect the misfolding of other cct client proteins like actin and tubulin , as previously described ( matus et al . , , we were unable to achieve a complete depletion of cct complex proteins , indicating that only cells with partial knockdown continued proliferation , consistent with our observation that cct complex depletion resulted in a stress response that activated p53 ( figure 6 ) . interestingly , cells that survived with a partial depletion of cct showed a reproducible increase in invasion ( figure 7e ) , consistent with a role of cct in maintaining wild - type conformation and function of p53 . depletion of wild - type p53 in these cells resulted in enhanced invasion ( figure s7d ) , so it was not possible to demonstrate p53 dependence of the increased invasion in cct - depleted wild - type p53-expressing cells . importantly , however , depletion of both p53 and cct decreased invasion ( figure s7d ) , similar to the effect seen in mutant p53-expressing cells . these results show that despite a general inhibition of invasive behavior in response to cct depletion , wild - type p53-expressing cells show enhanced invasion that correlates with the misfolding of the p53 protein . many previous studies have shown that tumor - derived p53 point mutants result in the failure of p53 to adopt the wild - type conformation , leading to the concept that p53 can either be in the wild - type or mutant ( i.e. our studies suggest that cct plays a specific role in maintaining a pool of wild - type , functional p53 in the cell and that failure of p53 to bind to cct results in the accumulation of misfolded p53 , which then acquires activities characteristic of tumor - derived mutant p53s . while the most straightforward model for the role of cct is to assist in the correct folding of newly synthesized p53 ( mayer , 2010 ) , it is also possible that cct binding to p53 prevents its aggregation , as has been shown for other proteins . in either case , the consequence of loss or modulation of the cct - p53 interaction would be the accumulation of misfolded p53 with invasive capacity . based on these results , we suggest that the regulation of wild - type p53 folding may be part of the normal biology of p53 . regulation of the interaction of p53 with cct could determine the generation of folded or unfolded p53 , without the requirement for mutations in the dna binding domain . our study shows that the effect of loss of cct binding on p53 conformation is subtle , supporting in vitro studies in which wild - type p53 can spontaneously fold into an active conformation . in addition , phosphorylation of serine 6 and 9 in p53 has also been identified ( meek and anderson , 2009 ) , so further studies will determine whether the p53/cct interaction can be actively modulated through changes in posttranslational modifications of either protein .

copd is a progressive disease characterized by persistent airflow limitation , chronic and progressive dyspnea , cough , and sputum production and is often complicated by exacerbations.1 copd affects approximately 24 million us adults , including 12.7 million diagnosed patients and 12 million undiagnosed , and is the third leading cause of death in the united states.2,3 the annual cost of copd in 2010 was estimated at $ 49.9 billion usd , with $ 29.5 billion attributed to direct health care costs , in which hospital care accounted for the largest share.4 treatment goals for patients with stable copd include symptom relief , prevention of disease progression , and prevention and treatment of exacerbations.1 while symptom relief is the primary target for pharmacologic therapy , prevention of exacerbations is also important because of the impact on lung function , health - related quality of life , hospitalization , and mortality . recent copd treatment guidelines emphasize the central role of bronchodilators , including 2-agonists and anticholinergics , with drug selection depending on patient response and tolerability . inhaled corticosteroids ( ics ) have a more limited role for patients unable to achieve symptom control with bronchodilators alone.1 roflumilast is a selective phosphodiesterase-4 inhibitor approved by the us food and drug administration in 2011 . it is indicated as a treatment to reduce the risk of copd exacerbations in patients with severe copd associated with chronic bronchitis and a history of exacerbations.5 no studies have compared roflumilast to other copd maintenance medications , and there is very little real - world data on utilization , clinical , and economic outcomes associated with the use of roflumilast . we hypothesized that there were no differences in the relevant clinical and economic outcomes between roflumilast and other copd maintenance medications . this was a longitudinal , retrospective cohort study using us managed - care administrative data from the lifelink pharmetrics plus health plan claims database , which includes medical and pharmaceutical claims for over 150 million unique patients from over 90 health plans across the united states . the prescription claims include national drug code , drug names , dose , strength , days supply , and quantity dispensed . it also includes demographic variables , insurance information , and plan enrollment / eligibility data . we cleaned the obtained raw data by removing the duplicates and observations with missing values for key data elements , such as age , diagnosis date , and prescription date . the data in the claims databases are de - identified and fully compliant with the health insurance portability and accountability act privacy regulations , exempting this study from an institutional review board approval requirement . selection of patients for the study is outlined in figure 1 . to be included in the analysis , patients had to have at least one medical claim with a primary diagnosis of copd ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 491.xx , 492.xx , or 496.xx ) between may 1 , 2010 and september 30 , 2012 ; have initiated therapy with roflumilast or a third copd maintenance medication between may 1 , 2011 and september 30 , 2012 ( cohort selection period ) ; and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date , defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . according to gold ( global initiative for chronic obstructive lung disease ) guidelines,1 roflumilast is recommended as the third maintenance agent for patients with severe copd . to ensure disease severity was as comparable as possible between the comparison group ( patients who did not receive roflumilast ) and the group of patients who received roflumilast , patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent patients were excluded if they were younger than 40 years of age , had at least one medical claim with a diagnosis code for cystic fibrosis ( icd-9-cm code 277.xx ) or respiratory tract cancer ( icd-9-cm codes 160.xx164.xx or 231.xx ) during the study period , were corticosteroid dependent ( defined as 182 day supply of oral corticosteroids in the year before or after the index date ) , had at least one medical claim with a diagnosis of asthma ( icd-9-cm codes 493.0 , 493.1 , or 493.9 ) during the 12-month preindex period ( ie , baseline period ) , or received roflumilast in combination with theophylline . study patients were separated into two cohorts : patients who initiated roflumilast during the cohort selection period ( roflumilast group ) and patients who initiated the third of three copd maintenance medications during the cohort selection period ( non - roflumilast group ) . patient demographics included age at the index date , sex , insurance type , and census region . clinical characteristics included comorbid conditions , reported as a prevalence per condition and used to calculate charlson comorbidity index;6 number of copd medications and days using copd medications during the 12-month preindex period ( termed as baseline period ) ; number of copd exacerbations ( defined in the following paragraph ) ; hospitalizations ; and emergency department ( ed ) visits during the baseline period . the primary outcome was the rate of moderate and severe copd exacerbations per patient per month ( pppm ) over the 3 months after the index date . severe exacerbation was defined as a hospital admission with a primary diagnosis of copd with acute exacerbation ( icd-9-cm codes 491.21 , 491.22 , 493.22 , or 492.8 ) or a primary diagnosis of respiratory failure ( icd-9-cm codes 518.81 , 518.82 , or 518.84 ) combined with a secondary diagnosis of copd with acute exacerbation or emphysema ( 491.0 , 491.1 , 491.21 , 491.22 , 491.8 , 491.9 , 492.0 , 492.8 , 493.22 , or 496).7 moderate exacerbation was defined as a short course ( 14 days ) of oral corticosteroids within 7 days of an ed visit or an office visit with a copd diagnosis ( icd-9-cm codes 491.xx , 492.xx , and 496.xx ) ; an ed visit with a primary diagnosis of copd with acute exacerbation or a primary diagnosis of respiratory failure combined with a secondary diagnosis of copd with acute exacerbation or emphysema , as defined earlier ; or an ambulatory claim with a diagnosis of copd or another qualifying pulmonary condition ( icd-9-cm codes 136.3 , 466466.19 , 480486 , 487.0 , 490 , 491.21 , 491.22 , 493.02 , 493.12 , 493.22 , 493.92 , 494.1 , 506.0506.3 , 507507.8 , 511.0511.1 , 512512.8 , 517.1 , 518.0 , 518.81 , 518.82 , 518.84 , 770.84).8 events occurring within 15 days of each other were counted as a single exacerbation . secondary outcomes included health care resource utilization ( hcru ) , including the number of inpatient hospitalization admissions , ed and office visits , and total health care costs . costs were inflated to 2012 values using the 2012 consumer price index provided by the bureau of labor statistics.9 bivariate comparisons of patient demographics and baseline characteristics between the roflumilast and non - roflumilast groups were performed using pearson chi - square tests for categorical variables and t - tests for continuous variables . wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates ( pppm ) , monthly hcru , and monthly costs were calculated for each patient , and difference - in - difference ( did ) linear regression models ( appendix ) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier . the did model was used to control for observed baseline differences between treatment and control groups ( eg , severity of disease ) , as well as the effects of all time - varying determinants of the outcomes ( trend change in the outcomes over time ) that are common to both study groups.11 it is worth noting that the did model is based on an assumption that in the absence of treatment the difference between control and treatment groups would be constant or fixed over time . analyses were performed using statistical analysis software version 9.2.3 ( sas institute , cary , nc ) . for all analyses , statistical significance was defined as a p - value < 0.05 . this was a longitudinal , retrospective cohort study using us managed - care administrative data from the lifelink pharmetrics plus health plan claims database , which includes medical and pharmaceutical claims for over 150 million unique patients from over 90 health plans across the united states . the prescription claims include national drug code , drug names , dose , strength , days supply , and quantity dispensed . it also includes demographic variables , insurance information , and plan enrollment / eligibility data . we cleaned the obtained raw data by removing the duplicates and observations with missing values for key data elements , such as age , diagnosis date , and prescription date . the data in the claims databases are de - identified and fully compliant with the health insurance portability and accountability act privacy regulations , exempting this study from an institutional review board approval requirement . selection of patients for the study is outlined in figure 1 . to be included in the analysis , patients had to have at least one medical claim with a primary diagnosis of copd ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 491.xx , 492.xx , or 496.xx ) between may 1 , 2010 and september 30 , 2012 ; have initiated therapy with roflumilast or a third copd maintenance medication between may 1 , 2011 and september 30 , 2012 ( cohort selection period ) ; and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date , defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . according to gold ( global initiative for chronic obstructive lung disease ) guidelines,1 roflumilast is recommended as the third maintenance agent for patients with severe copd . to ensure disease severity was as comparable as possible between the comparison group ( patients who did not receive roflumilast ) and the group of patients who received roflumilast , patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent patients were excluded if they were younger than 40 years of age , had at least one medical claim with a diagnosis code for cystic fibrosis ( icd-9-cm code 277.xx ) or respiratory tract cancer ( icd-9-cm codes 160.xx164.xx or 231.xx ) during the study period , were corticosteroid dependent ( defined as 182 day supply of oral corticosteroids in the year before or after the index date ) , had at least one medical claim with a diagnosis of asthma ( icd-9-cm codes 493.0 , 493.1 , or 493.9 ) during the 12-month preindex period ( ie , baseline period ) , or received roflumilast in combination with theophylline . study patients were separated into two cohorts : patients who initiated roflumilast during the cohort selection period ( roflumilast group ) and patients who initiated the third of three copd maintenance medications during the cohort selection period ( non - roflumilast group ) . patient demographics included age at the index date , sex , insurance type , and census region . clinical characteristics included comorbid conditions , reported as a prevalence per condition and used to calculate charlson comorbidity index;6 number of copd medications and days using copd medications during the 12-month preindex period ( termed as baseline period ) ; number of copd exacerbations ( defined in the following paragraph ) ; hospitalizations ; and emergency department ( ed ) visits during the baseline period . the primary outcome was the rate of moderate and severe copd exacerbations per patient per month ( pppm ) over the 3 months after the index date . severe exacerbation was defined as a hospital admission with a primary diagnosis of copd with acute exacerbation ( icd-9-cm codes 491.21 , 491.22 , 493.22 , or 492.8 ) or a primary diagnosis of respiratory failure ( icd-9-cm codes 518.81 , 518.82 , or 518.84 ) combined with a secondary diagnosis of copd with acute exacerbation or emphysema ( 491.0 , 491.1 , 491.21 , 491.22 , 491.8 , 491.9 , 492.0 , 492.8 , 493.22 , or 496).7 moderate exacerbation was defined as a short course ( 14 days ) of oral corticosteroids within 7 days of an ed visit or an office visit with a copd diagnosis ( icd-9-cm codes 491.xx , 492.xx , and 496.xx ) ; an ed visit with a primary diagnosis of copd with acute exacerbation or a primary diagnosis of respiratory failure combined with a secondary diagnosis of copd with acute exacerbation or emphysema , as defined earlier ; or an ambulatory claim with a diagnosis of copd or another qualifying pulmonary condition ( icd-9-cm codes 136.3 , 466466.19 , 480486 , 487.0 , 490 , 491.21 , 491.22 , 493.02 , 493.12 , 493.22 , 493.92 , 494.1 , 506.0506.3 , 507507.8 , 511.0511.1 , 512512.8 , 517.1 , 518.0 , 518.81 , 518.82 , 518.84 , 770.84).8 events occurring within 15 days of each other were counted as a single exacerbation . secondary outcomes included health care resource utilization ( hcru ) , including the number of inpatient hospitalization admissions , ed and office visits , and total health care costs . costs were inflated to 2012 values using the 2012 consumer price index provided by the bureau of labor statistics.9 bivariate comparisons of patient demographics and baseline characteristics between the roflumilast and non - roflumilast groups were performed using pearson chi - square tests for categorical variables and t - tests for continuous variables . wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates ( pppm ) , monthly hcru , and monthly costs were calculated for each patient , and difference - in - difference ( did ) linear regression models ( appendix ) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier . the did model was used to control for observed baseline differences between treatment and control groups ( eg , severity of disease ) , as well as the effects of all time - varying determinants of the outcomes ( trend change in the outcomes over time ) that are common to both study groups.11 it is worth noting that the did model is based on an assumption that in the absence of treatment the difference between control and treatment groups would be constant or fixed over time . analyses were performed using statistical analysis software version 9.2.3 ( sas institute , cary , nc ) . for all analyses , statistical significance was defined as a p - value < 0.05 . of 818,457 patients identified as having copd using icd-9 codes , 804,246 met at least one of the criteria for exclusion ( figure 2 ) . the major reasons for exclusion were absence of claims for copd maintenance medications during the cohort selection period ( n=501,556 ) and not receiving at least three maintenance copd medications during the cohort selection period ( n=110,320 ) . after these exclusions , there were 710 patients in the roflumilast group and 13,501 in the non - roflumilast group . patients in the roflumilast group were more likely to have comorbid congestive heart failure , had a higher number of monthly exacerbations , were receiving more copd medications at baseline , had more ed visits , and were more likely to be hospitalized during the 12-month preindex period . there were no statistically significant differences in age , sex , or charlson comorbidity score between groups . in the non - roflumilast group , the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist ( laba ) and ics ( 47.2% ) , followed by a combination of those two classes plus a long - acting muscarinic antagonist ( 46.1% ) . in the roflumilast group , 33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist , laba , and ics , 30.4% were receiving roflumilast monotherapy ( of note , these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast ) , and 18.9% were receiving a combination of roflumilast , laba , and ics . figure 3 shows the change in overall rate of exacerbation pppm in the roflumilast and non - roflumilast groups from baseline . during the follow - up period , the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( figure 3a ; p<0.001 for did ) . while the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group , however , there was a significant difference in the rate of moderate exacerbation pppm between groups , with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations ( figure 3c ; p=0.001 ) . after controlling for the key covariates ( age , sex , region , insurance , baseline number of copd drugs , and charlson comorbidity index ) using a did model approach , roflumilast patients still had a greater reduction in the rate of overall exacerbation ( =0.0160 , p=0.01 ) and moderate exacerbation ( =0.0149 , p=0.01 ) versus the non - roflumilast group . there was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates ; however , the difference was not statistically significant ( =0.0012 , p=0.63 ) . monthly number of office visits , ed visits , and inpatient hospitalization admissions at baseline and during follow - up are shown in table 2 . during both the baseline and follow - up periods , patients in the roflumilast group had more of each type of encounter than patients in the non - roflumilast group . both treatment groups had increased office visits , ed visits , and hospitalizations during the follow - up period compared to the baseline period . however , the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions ( 0.0020.152 vs 0.0050.123 , p=0.024 ) and office visits ( 0.0810.938 vs 0.1220.889 , p=0.01 ) , but not for the ed visits . after controlling for key covariates using did models , roflumilast was associated with fewer hospital admissions ( =0.003 , p=0.57 ) and office visits ( =0.46 , p=0.26 ) ; however , the differences were not statistically significant . table 3 shows total monthly health care costs in the roflumilast and non - roflumilast groups during the baseline and follow - up periods . during the baseline and follow - up periods , costs increased for both groups from the baseline to the follow - up period , with no significant difference in the change from baseline between groups . after controlling for the key covariates using a did model , the total cost increase was $ 116 more for the control of patients not treated with roflu - milast versus patients treated with roflumilast ( p=0.62 ) . of 818,457 patients identified as having copd using icd-9 codes , 804,246 met at least one of the criteria for exclusion ( figure 2 ) . the major reasons for exclusion were absence of claims for copd maintenance medications during the cohort selection period ( n=501,556 ) and not receiving at least three maintenance copd medications during the cohort selection period ( n=110,320 ) . after these exclusions , there were 710 patients in the roflumilast group and 13,501 in the non - roflumilast group . patients in the roflumilast group were more likely to have comorbid congestive heart failure , had a higher number of monthly exacerbations , were receiving more copd medications at baseline , had more ed visits , and were more likely to be hospitalized during the 12-month preindex period . there were no statistically significant differences in age , sex , or charlson comorbidity score between groups . in the non - roflumilast group , the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist ( laba ) and ics ( 47.2% ) , followed by a combination of those two classes plus a long - acting muscarinic antagonist ( 46.1% ) . in the roflumilast group , 33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist , laba , and ics , 30.4% were receiving roflumilast monotherapy ( of note , these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast ) , and 18.9% were receiving a combination of roflumilast , laba , and ics . figure 3 shows the change in overall rate of exacerbation pppm in the roflumilast and non - roflumilast groups from baseline . during the follow - up period , the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( figure 3a ; p<0.001 for did ) . while the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group , however , there was a significant difference in the rate of moderate exacerbation pppm between groups , with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations ( figure 3c ; p=0.001 ) . after controlling for the key covariates ( age , sex , region , insurance , baseline number of copd drugs , and charlson comorbidity index ) using a did model approach , roflumilast patients still had a greater reduction in the rate of overall exacerbation ( =0.0160 , p=0.01 ) and moderate exacerbation ( =0.0149 , p=0.01 ) versus the non - roflumilast group . there was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates ; however , the difference was not statistically significant ( =0.0012 , p=0.63 ) . monthly number of office visits , ed visits , and inpatient hospitalization admissions at baseline and during follow - up are shown in table 2 . during both the baseline and follow - up periods , patients in the roflumilast group had more of each type of encounter than patients in the non - roflumilast group . both treatment groups had increased office visits , ed visits , and hospitalizations during the follow - up period compared to the baseline period . however , the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions ( 0.0020.152 vs 0.0050.123 , p=0.024 ) and office visits ( 0.0810.938 vs 0.1220.889 , p=0.01 ) , but not for the ed visits . after controlling for key covariates using did models , roflumilast was associated with fewer hospital admissions ( =0.003 , p=0.57 ) and office visits ( =0.46 , p=0.26 ) ; however , the differences were not statistically significant . table 3 shows total monthly health care costs in the roflumilast and non - roflumilast groups during the baseline and follow - up periods . during the baseline and follow - up periods , costs increased for both groups from the baseline to the follow - up period , with no significant difference in the change from baseline between groups . after controlling for the key covariates using a did model , the total cost increase was $ 116 more for the control of patients not treated with roflu - milast versus patients treated with roflumilast ( p=0.62 ) . this study found that patients initiating roflumilast had more severe copd in that they had a higher number of monthly exacerbations , received more copd medications , and used more hcru than patients initiating other copd maintenance medications ( based on the observation during the baseline period ) . exacerbation rates were significantly improved for patients treated with roflumilast , with an 11% decrease in the rate of exacerbations in the roflumilast group and a 16% increase in the non - roflumilast group ( a relative 27% reduction ) . after controlling for baseline differences using a did model approach , the significant differences in exacerbation rates between groups remained . after controlling for covariates using did models , changes in hospital admissions and total costs did not differ significantly between the roflumilast and non - roflumilast groups . the cost analysis in our study focused on the overall cost ( cost component shown in table s1 ) . in order to further understand how roflumilast impacts different cost components , future studies should examine the copd- or exacerbation - related costs by component , especially when the roflumilast was used as an add - on treatment following the gold guidance . randomized controlled trials evaluating the efficacy and safety of roflumilast in patients with copd found significant between - group differences in moderate or severe exacerbation rates ( a 17% reduction , p=0.0003 ) ; however , these trials were placebo - controlled and 6 months12 or 1 year13 in duration . significant differences in the rate of moderate , but not severe , exacerbations were initially found in two pivotal 1-year trials,12,13 which may be due to a lack of statistical power given the relatively small sample sizes of clinical trial data . similarly , in the current study , there may not have been sufficient power to detect a significant difference in the change in severe exacerbation rates between groups . relatively , the cohort of patients who received roflumilast was large in this retrospective cohort study ; however , there may be large treatment variations from patients in real - world settings compared to the strict treatment regimens of a clinical trial . additionally , severe exacerbations are relatively rare events , which may further reduce the power of the analysis for this outcome . as gold recommended , roflumilast can be used an add - on treatment for patients with copd in stages 3/4 ( forced expiratory volume in 1 second ( fev1 ) < 50% predicted after bronchodilatation ) and frequent exacerbations despite a long - acting bronchodilator treatment . it was found that the copd drugs usage before initiating roflumilast varies in our study . it may be informative to explore the subset of patients who strictly followed the gold guidance ( ie , those receiving ics and a laba or other long - acting bronchodilators prior to the use of roflumilast ) using the real - world data . future studies are warranted to assess the outcomes of those patients who have used ics and long - acting bronchodilator within certain window prior to initiating roflumilast using a large sample . previous studies examined the resource utilization and health care costs associated with exacerbations among copd patients.1417 in a us managed - care population initiating copd maintenance medications , abudagga et al found that exacerbation rates continued to be high , resulting in an estimated annual cost of $ 25,747 in the overall population and $ 29,861 in the patients with history of one or more exacerbations.14 in this study , calculated total monthly costs during the 3-month follow - up period were $ 2,472 and $ 2,851 for non - roflumilast and roflumilast group , respectively , which is higher than the abudagga study14 and likely due to the selection of patients with more severe copd . darnell et al reported an annual 3.07 clinic visits , 0.15 ed visits , and 0.41 hospitalizations per person per year , among copd patients , calculated using a large administrative database.15 the resource utilization reported here was similar or slightly higher than data in the darnell et al study15 and again , this discrepancy may be due to the selection of patients with more severe copd . of note , our study examined the economic burden associated with copd medications from the payer s perspective ; indirect costs , such as productivity loss , were not included , and thus , cost data may be conservative estimates . copd exacerbations result in increases in resource uti - lization , especially for severe exacerbations.9,18,19 consistent with previous studies , the current work found that reduced exacerbation rates among copd patients were associated with reduced all - cause resource utilization and costs from a managed - care payer s perspective . therefore , it is vital for physicians and payers to be aware that preventing copd exacerbations through appropriate treatments could minimize the need for hospitalization and other medical resource use . in addition , previous research also suggested that reducing exacerbation frequency may improve the quality of life among patients with copd.20 strengths of this study include the use of a robust national commercial insurance database with a longitudinal data structure that allows for tracking of both inpatient and outpatient diagnoses and procedures as well as outpatient prescription records . the availability of prescription claims provided rich information on treatment patterns and medication use as an indicator of exacerbation frequency . the use of did models controlled for significant baseline differences in study populations , which is important as there is potential for selection bias due to the observational study design . our study has several limitations and it is important to interpret our results in the context of these limitations . first , some clinical details such as lung function parameters are not available in the database . the cohort selection criteria is based on the use of copd maintenance treatments ( ie , patients who initiated roflumilast or a third copd drug ) , which may identify more severe patients by the criteria itself . second , the definition of copd exacerbations was based entirely on claims data rather than patient symptoms . the study by stein et al suggested that algorithms based on icd-9-cm codes may undercount the acute exacerbation for copd.21 the exacerbation rates among patients receiving roflumilast were similar to those reported in randomized trials,12,13,22 which validate our results . third , the study sample for the roflumilast group was restricted to those with insurance covering roflumilast . because data were available only for commercially insured patients , the results may not be generalizable to older patients covered by medicare . however , the mean age of patients in our study was only slightly lower than that of patients in randomized trials ( 63 years vs 65 years).12,13 fourth , we used a did model that mimicked an experimental study and controlled for the baseline difference between study groups . our adjusted did regressions were based on an assumption that in the absence of treatment , the difference between control ( without roflumilast ) and treatment ( with roflumilast ) groups would be constant or fixed over time . however , in reality , it is possible that the roflumilast group was inherently more likely to experience a reduction in exacerbation rate compared to the non - roflumilast group due to having higher exacerbation rates at baseline . in this study , we observed a decreased overall exacerbation rate in the roflumilast group contrasted with an increased exacerbation rate in the non - roflumilast group . the magnitude of the difference suggests that the observed results can not be attributed exclusively to unobserved confounders . fifth , it is challenging to adjust for the difference in adherence in the patients without roflumilast . patients adherence to treatments , especially to the combination therapy , may change quickly over time because patients may discontinue some drugs after the symptoms improve . it will be important for future studies to control for the adherence if a specific comparator drug is chosen . sixth , our results may not be able to identify the seasonal effect as the follow - up duration covers only 3 months.23 however , both study groups have the same index period so the difference in the exacerbation outcomes between the two study groups may not be biased . finally , we did not have a large sample of the roflumilast - treated group as roflumilast is only recently approved by the us food and drug administration ( february 2011 ) . this may affect the statistical power and our study may not be sufficient to determine the long - term effects of roflumilast on exacerbations , hcru , and costs . patients with copd - initiating roflumilast treatment had greater reductions in exacerbation rates in the 3 months after the new therapy was added than patients initiating other copd maintenance medications . we also found a numerically smaller increase from baseline in the monthly rates of hospital admissions , office visits , and monthly total costs between patients treated with roflumilast versus patients not treated with roflumilast . future studies using alternative methods such as patient survey or chart review are warranted to further evaluate the impact of roflumilast on clinical outcomes , hcru , and costs within a longer follow - up period . monthly total health care costs and cost by component ( $ ) notes : the result data represent average cost per patient per month . the total cost is more than the sum of inpatient , outpatient , er , and copd drug costs because there are other types of cost components not shown in this table . abbreviations : baseline , 12-month preindex period ; copd , chronic obstructive pulmonary disease ; sd , standard deviation ; er , emergency room .

backgroundroflumilast is approved in the united states to reduce the risk of copd exacerbations in patients with severe copd . exacerbation rates , health care resource utilization ( hcru ) , and costs were compared between roflumilast patients and those receiving other copd maintenance drugs.methodslifelink health plan claims database was used to identify patients diagnosed with copd who initiated roflumilast ( roflumilast group ) or 3 other copd maintenance drugs ( non - roflumilast group ) from may 1 , 2011 to december 31 , 2012 . patients must have been enrolled for 12 months before ( baseline ) and 3 months after ( postindex ) the initiation date , 40 years old , not systemic corticosteroid dependent , and without asthma diagnosis at baseline . difference - in - difference models compared change from baseline in exacerbations , hcru ( office , emergency visits , and hospitalizations ) , and total costs between groups , adjusting for baseline differences.resultsa total of 14,211 patients ( roflumilast , n=710 ; non - roflumilast , n=13,501 ) were included . during follow - up , the rate of overall exacerbations per patient per month decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( p<0.001 ) . after controlling for baseline differences , roflumilast - treated patients experienced a greater reduction in exacerbations ( 0.0160 fewer exacerbations per month , p=0.01 ) , numerically greater reductions in hospital admissions ( 0.003 fewer per month , p=0.57 ) , office visits ( 0.46 fewer per month , p=0.26 ) , and total costs from baseline compared with non - roflumilast patients ( $ 116 less per month , p=0.62).conclusionin a real - world setting , patients initiating roflumilast experienced reductions in exacerbations versus patients treated with other copd medications .

Introduction Methods Study design and data source Patient selection Study measures Statistical analyses Results Patient sample COPD regimens exposed on the index date Exacerbations Health care resource utilization Health care costs Discussion Conclusion Supplementary material

copd is a progressive disease characterized by persistent airflow limitation , chronic and progressive dyspnea , cough , and sputum production and is often complicated by exacerbations.1 copd affects approximately 24 million us adults , including 12.7 million diagnosed patients and 12 million undiagnosed , and is the third leading cause of death in the united states.2,3 the annual cost of copd in 2010 was estimated at $ 49.9 billion usd , with $ 29.5 billion attributed to direct health care costs , in which hospital care accounted for the largest share.4 treatment goals for patients with stable copd include symptom relief , prevention of disease progression , and prevention and treatment of exacerbations.1 while symptom relief is the primary target for pharmacologic therapy , prevention of exacerbations is also important because of the impact on lung function , health - related quality of life , hospitalization , and mortality . it is indicated as a treatment to reduce the risk of copd exacerbations in patients with severe copd associated with chronic bronchitis and a history of exacerbations.5 no studies have compared roflumilast to other copd maintenance medications , and there is very little real - world data on utilization , clinical , and economic outcomes associated with the use of roflumilast . to be included in the analysis , patients had to have at least one medical claim with a primary diagnosis of copd ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 491.xx , 492.xx , or 496.xx ) between may 1 , 2010 and september 30 , 2012 ; have initiated therapy with roflumilast or a third copd maintenance medication between may 1 , 2011 and september 30 , 2012 ( cohort selection period ) ; and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date , defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . to ensure disease severity was as comparable as possible between the comparison group ( patients who did not receive roflumilast ) and the group of patients who received roflumilast , patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent patients were excluded if they were younger than 40 years of age , had at least one medical claim with a diagnosis code for cystic fibrosis ( icd-9-cm code 277.xx ) or respiratory tract cancer ( icd-9-cm codes 160.xx164.xx or 231.xx ) during the study period , were corticosteroid dependent ( defined as 182 day supply of oral corticosteroids in the year before or after the index date ) , had at least one medical claim with a diagnosis of asthma ( icd-9-cm codes 493.0 , 493.1 , or 493.9 ) during the 12-month preindex period ( ie , baseline period ) , or received roflumilast in combination with theophylline . study patients were separated into two cohorts : patients who initiated roflumilast during the cohort selection period ( roflumilast group ) and patients who initiated the third of three copd maintenance medications during the cohort selection period ( non - roflumilast group ) . the primary outcome was the rate of moderate and severe copd exacerbations per patient per month ( pppm ) over the 3 months after the index date . secondary outcomes included health care resource utilization ( hcru ) , including the number of inpatient hospitalization admissions , ed and office visits , and total health care costs . wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates ( pppm ) , monthly hcru , and monthly costs were calculated for each patient , and difference - in - difference ( did ) linear regression models ( appendix ) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier . to be included in the analysis , patients had to have at least one medical claim with a primary diagnosis of copd ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 491.xx , 492.xx , or 496.xx ) between may 1 , 2010 and september 30 , 2012 ; have initiated therapy with roflumilast or a third copd maintenance medication between may 1 , 2011 and september 30 , 2012 ( cohort selection period ) ; and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date , defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . to ensure disease severity was as comparable as possible between the comparison group ( patients who did not receive roflumilast ) and the group of patients who received roflumilast , patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent patients were excluded if they were younger than 40 years of age , had at least one medical claim with a diagnosis code for cystic fibrosis ( icd-9-cm code 277.xx ) or respiratory tract cancer ( icd-9-cm codes 160.xx164.xx or 231.xx ) during the study period , were corticosteroid dependent ( defined as 182 day supply of oral corticosteroids in the year before or after the index date ) , had at least one medical claim with a diagnosis of asthma ( icd-9-cm codes 493.0 , 493.1 , or 493.9 ) during the 12-month preindex period ( ie , baseline period ) , or received roflumilast in combination with theophylline . study patients were separated into two cohorts : patients who initiated roflumilast during the cohort selection period ( roflumilast group ) and patients who initiated the third of three copd maintenance medications during the cohort selection period ( non - roflumilast group ) . clinical characteristics included comorbid conditions , reported as a prevalence per condition and used to calculate charlson comorbidity index;6 number of copd medications and days using copd medications during the 12-month preindex period ( termed as baseline period ) ; number of copd exacerbations ( defined in the following paragraph ) ; hospitalizations ; and emergency department ( ed ) visits during the baseline period . the primary outcome was the rate of moderate and severe copd exacerbations per patient per month ( pppm ) over the 3 months after the index date . secondary outcomes included health care resource utilization ( hcru ) , including the number of inpatient hospitalization admissions , ed and office visits , and total health care costs . wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates ( pppm ) , monthly hcru , and monthly costs were calculated for each patient , and difference - in - difference ( did ) linear regression models ( appendix ) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier . after these exclusions , there were 710 patients in the roflumilast group and 13,501 in the non - roflumilast group . patients in the roflumilast group were more likely to have comorbid congestive heart failure , had a higher number of monthly exacerbations , were receiving more copd medications at baseline , had more ed visits , and were more likely to be hospitalized during the 12-month preindex period . in the non - roflumilast group , the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist ( laba ) and ics ( 47.2% ) , followed by a combination of those two classes plus a long - acting muscarinic antagonist ( 46.1% ) . in the roflumilast group , 33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist , laba , and ics , 30.4% were receiving roflumilast monotherapy ( of note , these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast ) , and 18.9% were receiving a combination of roflumilast , laba , and ics . during the follow - up period , the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( figure 3a ; p<0.001 for did ) . while the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group , however , there was a significant difference in the rate of moderate exacerbation pppm between groups , with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations ( figure 3c ; p=0.001 ) . after controlling for the key covariates ( age , sex , region , insurance , baseline number of copd drugs , and charlson comorbidity index ) using a did model approach , roflumilast patients still had a greater reduction in the rate of overall exacerbation ( =0.0160 , p=0.01 ) and moderate exacerbation ( =0.0149 , p=0.01 ) versus the non - roflumilast group . there was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates ; however , the difference was not statistically significant ( =0.0012 , p=0.63 ) . monthly number of office visits , ed visits , and inpatient hospitalization admissions at baseline and during follow - up are shown in table 2 . during both the baseline and follow - up periods , patients in the roflumilast group had more of each type of encounter than patients in the non - roflumilast group . both treatment groups had increased office visits , ed visits , and hospitalizations during the follow - up period compared to the baseline period . however , the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions ( 0.0020.152 vs 0.0050.123 , p=0.024 ) and office visits ( 0.0810.938 vs 0.1220.889 , p=0.01 ) , but not for the ed visits . after controlling for key covariates using did models , roflumilast was associated with fewer hospital admissions ( =0.003 , p=0.57 ) and office visits ( =0.46 , p=0.26 ) ; however , the differences were not statistically significant . table 3 shows total monthly health care costs in the roflumilast and non - roflumilast groups during the baseline and follow - up periods . during the baseline and follow - up periods , costs increased for both groups from the baseline to the follow - up period , with no significant difference in the change from baseline between groups . after controlling for the key covariates using a did model , the total cost increase was $ 116 more for the control of patients not treated with roflu - milast versus patients treated with roflumilast ( p=0.62 ) . after these exclusions , there were 710 patients in the roflumilast group and 13,501 in the non - roflumilast group . patients in the roflumilast group were more likely to have comorbid congestive heart failure , had a higher number of monthly exacerbations , were receiving more copd medications at baseline , had more ed visits , and were more likely to be hospitalized during the 12-month preindex period . in the non - roflumilast group , the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist ( laba ) and ics ( 47.2% ) , followed by a combination of those two classes plus a long - acting muscarinic antagonist ( 46.1% ) . in the roflumilast group , 33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist , laba , and ics , 30.4% were receiving roflumilast monotherapy ( of note , these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast ) , and 18.9% were receiving a combination of roflumilast , laba , and ics . figure 3 shows the change in overall rate of exacerbation pppm in the roflumilast and non - roflumilast groups from baseline . during the follow - up period , the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( figure 3a ; p<0.001 for did ) . while the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group , however , there was a significant difference in the rate of moderate exacerbation pppm between groups , with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations ( figure 3c ; p=0.001 ) . after controlling for the key covariates ( age , sex , region , insurance , baseline number of copd drugs , and charlson comorbidity index ) using a did model approach , roflumilast patients still had a greater reduction in the rate of overall exacerbation ( =0.0160 , p=0.01 ) and moderate exacerbation ( =0.0149 , p=0.01 ) versus the non - roflumilast group . there was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates ; however , the difference was not statistically significant ( =0.0012 , p=0.63 ) . monthly number of office visits , ed visits , and inpatient hospitalization admissions at baseline and during follow - up are shown in table 2 . during both the baseline and follow - up periods , patients in the roflumilast group had more of each type of encounter than patients in the non - roflumilast group . both treatment groups had increased office visits , ed visits , and hospitalizations during the follow - up period compared to the baseline period . however , the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions ( 0.0020.152 vs 0.0050.123 , p=0.024 ) and office visits ( 0.0810.938 vs 0.1220.889 , p=0.01 ) , but not for the ed visits . after controlling for key covariates using did models , roflumilast was associated with fewer hospital admissions ( =0.003 , p=0.57 ) and office visits ( =0.46 , p=0.26 ) ; however , the differences were not statistically significant . table 3 shows total monthly health care costs in the roflumilast and non - roflumilast groups during the baseline and follow - up periods . during the baseline and follow - up periods , costs increased for both groups from the baseline to the follow - up period , with no significant difference in the change from baseline between groups . after controlling for the key covariates using a did model , the total cost increase was $ 116 more for the control of patients not treated with roflu - milast versus patients treated with roflumilast ( p=0.62 ) . this study found that patients initiating roflumilast had more severe copd in that they had a higher number of monthly exacerbations , received more copd medications , and used more hcru than patients initiating other copd maintenance medications ( based on the observation during the baseline period ) . exacerbation rates were significantly improved for patients treated with roflumilast , with an 11% decrease in the rate of exacerbations in the roflumilast group and a 16% increase in the non - roflumilast group ( a relative 27% reduction ) . after controlling for baseline differences using a did model approach , the significant differences in exacerbation rates between groups remained . after controlling for covariates using did models , changes in hospital admissions and total costs did not differ significantly between the roflumilast and non - roflumilast groups . previous studies examined the resource utilization and health care costs associated with exacerbations among copd patients.1417 in a us managed - care population initiating copd maintenance medications , abudagga et al found that exacerbation rates continued to be high , resulting in an estimated annual cost of $ 25,747 in the overall population and $ 29,861 in the patients with history of one or more exacerbations.14 in this study , calculated total monthly costs during the 3-month follow - up period were $ 2,472 and $ 2,851 for non - roflumilast and roflumilast group , respectively , which is higher than the abudagga study14 and likely due to the selection of patients with more severe copd . darnell et al reported an annual 3.07 clinic visits , 0.15 ed visits , and 0.41 hospitalizations per person per year , among copd patients , calculated using a large administrative database.15 the resource utilization reported here was similar or slightly higher than data in the darnell et al study15 and again , this discrepancy may be due to the selection of patients with more severe copd . the cohort selection criteria is based on the use of copd maintenance treatments ( ie , patients who initiated roflumilast or a third copd drug ) , which may identify more severe patients by the criteria itself . however , in reality , it is possible that the roflumilast group was inherently more likely to experience a reduction in exacerbation rate compared to the non - roflumilast group due to having higher exacerbation rates at baseline . in this study , we observed a decreased overall exacerbation rate in the roflumilast group contrasted with an increased exacerbation rate in the non - roflumilast group . patients with copd - initiating roflumilast treatment had greater reductions in exacerbation rates in the 3 months after the new therapy was added than patients initiating other copd maintenance medications . we also found a numerically smaller increase from baseline in the monthly rates of hospital admissions , office visits , and monthly total costs between patients treated with roflumilast versus patients not treated with roflumilast .

copd is a progressive disease characterized by persistent airflow limitation , chronic and progressive dyspnea , cough , and sputum production and is often complicated by exacerbations.1 copd affects approximately 24 million us adults , including 12.7 million diagnosed patients and 12 million undiagnosed , and is the third leading cause of death in the united states.2,3 the annual cost of copd in 2010 was estimated at $ 49.9 billion usd , with $ 29.5 billion attributed to direct health care costs , in which hospital care accounted for the largest share.4 treatment goals for patients with stable copd include symptom relief , prevention of disease progression , and prevention and treatment of exacerbations.1 while symptom relief is the primary target for pharmacologic therapy , prevention of exacerbations is also important because of the impact on lung function , health - related quality of life , hospitalization , and mortality . it is indicated as a treatment to reduce the risk of copd exacerbations in patients with severe copd associated with chronic bronchitis and a history of exacerbations.5 no studies have compared roflumilast to other copd maintenance medications , and there is very little real - world data on utilization , clinical , and economic outcomes associated with the use of roflumilast . to be included in the analysis , patients had to have at least one medical claim with a primary diagnosis of copd ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 491.xx , 492.xx , or 496.xx ) between may 1 , 2010 and september 30 , 2012 ; have initiated therapy with roflumilast or a third copd maintenance medication between may 1 , 2011 and september 30 , 2012 ( cohort selection period ) ; and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date , defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . to ensure disease severity was as comparable as possible between the comparison group ( patients who did not receive roflumilast ) and the group of patients who received roflumilast , patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent patients were excluded if they were younger than 40 years of age , had at least one medical claim with a diagnosis code for cystic fibrosis ( icd-9-cm code 277.xx ) or respiratory tract cancer ( icd-9-cm codes 160.xx164.xx or 231.xx ) during the study period , were corticosteroid dependent ( defined as 182 day supply of oral corticosteroids in the year before or after the index date ) , had at least one medical claim with a diagnosis of asthma ( icd-9-cm codes 493.0 , 493.1 , or 493.9 ) during the 12-month preindex period ( ie , baseline period ) , or received roflumilast in combination with theophylline . severe exacerbation was defined as a hospital admission with a primary diagnosis of copd with acute exacerbation ( icd-9-cm codes 491.21 , 491.22 , 493.22 , or 492.8 ) or a primary diagnosis of respiratory failure ( icd-9-cm codes 518.81 , 518.82 , or 518.84 ) combined with a secondary diagnosis of copd with acute exacerbation or emphysema ( 491.0 , 491.1 , 491.21 , 491.22 , 491.8 , 491.9 , 492.0 , 492.8 , 493.22 , or 496).7 moderate exacerbation was defined as a short course ( 14 days ) of oral corticosteroids within 7 days of an ed visit or an office visit with a copd diagnosis ( icd-9-cm codes 491.xx , 492.xx , and 496.xx ) ; an ed visit with a primary diagnosis of copd with acute exacerbation or a primary diagnosis of respiratory failure combined with a secondary diagnosis of copd with acute exacerbation or emphysema , as defined earlier ; or an ambulatory claim with a diagnosis of copd or another qualifying pulmonary condition ( icd-9-cm codes 136.3 , 466466.19 , 480486 , 487.0 , 490 , 491.21 , 491.22 , 493.02 , 493.12 , 493.22 , 493.92 , 494.1 , 506.0506.3 , 507507.8 , 511.0511.1 , 512512.8 , 517.1 , 518.0 , 518.81 , 518.82 , 518.84 , 770.84).8 events occurring within 15 days of each other were counted as a single exacerbation . wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates ( pppm ) , monthly hcru , and monthly costs were calculated for each patient , and difference - in - difference ( did ) linear regression models ( appendix ) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier . the did model was used to control for observed baseline differences between treatment and control groups ( eg , severity of disease ) , as well as the effects of all time - varying determinants of the outcomes ( trend change in the outcomes over time ) that are common to both study groups.11 it is worth noting that the did model is based on an assumption that in the absence of treatment the difference between control and treatment groups would be constant or fixed over time . to be included in the analysis , patients had to have at least one medical claim with a primary diagnosis of copd ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 491.xx , 492.xx , or 496.xx ) between may 1 , 2010 and september 30 , 2012 ; have initiated therapy with roflumilast or a third copd maintenance medication between may 1 , 2011 and september 30 , 2012 ( cohort selection period ) ; and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date , defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . to ensure disease severity was as comparable as possible between the comparison group ( patients who did not receive roflumilast ) and the group of patients who received roflumilast , patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent patients were excluded if they were younger than 40 years of age , had at least one medical claim with a diagnosis code for cystic fibrosis ( icd-9-cm code 277.xx ) or respiratory tract cancer ( icd-9-cm codes 160.xx164.xx or 231.xx ) during the study period , were corticosteroid dependent ( defined as 182 day supply of oral corticosteroids in the year before or after the index date ) , had at least one medical claim with a diagnosis of asthma ( icd-9-cm codes 493.0 , 493.1 , or 493.9 ) during the 12-month preindex period ( ie , baseline period ) , or received roflumilast in combination with theophylline . severe exacerbation was defined as a hospital admission with a primary diagnosis of copd with acute exacerbation ( icd-9-cm codes 491.21 , 491.22 , 493.22 , or 492.8 ) or a primary diagnosis of respiratory failure ( icd-9-cm codes 518.81 , 518.82 , or 518.84 ) combined with a secondary diagnosis of copd with acute exacerbation or emphysema ( 491.0 , 491.1 , 491.21 , 491.22 , 491.8 , 491.9 , 492.0 , 492.8 , 493.22 , or 496).7 moderate exacerbation was defined as a short course ( 14 days ) of oral corticosteroids within 7 days of an ed visit or an office visit with a copd diagnosis ( icd-9-cm codes 491.xx , 492.xx , and 496.xx ) ; an ed visit with a primary diagnosis of copd with acute exacerbation or a primary diagnosis of respiratory failure combined with a secondary diagnosis of copd with acute exacerbation or emphysema , as defined earlier ; or an ambulatory claim with a diagnosis of copd or another qualifying pulmonary condition ( icd-9-cm codes 136.3 , 466466.19 , 480486 , 487.0 , 490 , 491.21 , 491.22 , 493.02 , 493.12 , 493.22 , 493.92 , 494.1 , 506.0506.3 , 507507.8 , 511.0511.1 , 512512.8 , 517.1 , 518.0 , 518.81 , 518.82 , 518.84 , 770.84).8 events occurring within 15 days of each other were counted as a single exacerbation . costs were inflated to 2012 values using the 2012 consumer price index provided by the bureau of labor statistics.9 bivariate comparisons of patient demographics and baseline characteristics between the roflumilast and non - roflumilast groups were performed using pearson chi - square tests for categorical variables and t - tests for continuous variables . wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates ( pppm ) , monthly hcru , and monthly costs were calculated for each patient , and difference - in - difference ( did ) linear regression models ( appendix ) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier . the did model was used to control for observed baseline differences between treatment and control groups ( eg , severity of disease ) , as well as the effects of all time - varying determinants of the outcomes ( trend change in the outcomes over time ) that are common to both study groups.11 it is worth noting that the did model is based on an assumption that in the absence of treatment the difference between control and treatment groups would be constant or fixed over time . patients in the roflumilast group were more likely to have comorbid congestive heart failure , had a higher number of monthly exacerbations , were receiving more copd medications at baseline , had more ed visits , and were more likely to be hospitalized during the 12-month preindex period . in the non - roflumilast group , the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist ( laba ) and ics ( 47.2% ) , followed by a combination of those two classes plus a long - acting muscarinic antagonist ( 46.1% ) . in the roflumilast group , 33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist , laba , and ics , 30.4% were receiving roflumilast monotherapy ( of note , these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast ) , and 18.9% were receiving a combination of roflumilast , laba , and ics . during the follow - up period , the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( figure 3a ; p<0.001 for did ) . while the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group , however , there was a significant difference in the rate of moderate exacerbation pppm between groups , with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations ( figure 3c ; p=0.001 ) . after controlling for the key covariates ( age , sex , region , insurance , baseline number of copd drugs , and charlson comorbidity index ) using a did model approach , roflumilast patients still had a greater reduction in the rate of overall exacerbation ( =0.0160 , p=0.01 ) and moderate exacerbation ( =0.0149 , p=0.01 ) versus the non - roflumilast group . there was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates ; however , the difference was not statistically significant ( =0.0012 , p=0.63 ) . however , the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions ( 0.0020.152 vs 0.0050.123 , p=0.024 ) and office visits ( 0.0810.938 vs 0.1220.889 , p=0.01 ) , but not for the ed visits . patients in the roflumilast group were more likely to have comorbid congestive heart failure , had a higher number of monthly exacerbations , were receiving more copd medications at baseline , had more ed visits , and were more likely to be hospitalized during the 12-month preindex period . in the non - roflumilast group , the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist ( laba ) and ics ( 47.2% ) , followed by a combination of those two classes plus a long - acting muscarinic antagonist ( 46.1% ) . in the roflumilast group , 33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist , laba , and ics , 30.4% were receiving roflumilast monotherapy ( of note , these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast ) , and 18.9% were receiving a combination of roflumilast , laba , and ics . during the follow - up period , the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group ( figure 3a ; p<0.001 for did ) . while the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group , however , there was a significant difference in the rate of moderate exacerbation pppm between groups , with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations ( figure 3c ; p=0.001 ) . after controlling for the key covariates ( age , sex , region , insurance , baseline number of copd drugs , and charlson comorbidity index ) using a did model approach , roflumilast patients still had a greater reduction in the rate of overall exacerbation ( =0.0160 , p=0.01 ) and moderate exacerbation ( =0.0149 , p=0.01 ) versus the non - roflumilast group . there was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates ; however , the difference was not statistically significant ( =0.0012 , p=0.63 ) . however , the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions ( 0.0020.152 vs 0.0050.123 , p=0.024 ) and office visits ( 0.0810.938 vs 0.1220.889 , p=0.01 ) , but not for the ed visits . this study found that patients initiating roflumilast had more severe copd in that they had a higher number of monthly exacerbations , received more copd medications , and used more hcru than patients initiating other copd maintenance medications ( based on the observation during the baseline period ) . exacerbation rates were significantly improved for patients treated with roflumilast , with an 11% decrease in the rate of exacerbations in the roflumilast group and a 16% increase in the non - roflumilast group ( a relative 27% reduction ) . in order to further understand how roflumilast impacts different cost components , future studies should examine the copd- or exacerbation - related costs by component , especially when the roflumilast was used as an add - on treatment following the gold guidance . randomized controlled trials evaluating the efficacy and safety of roflumilast in patients with copd found significant between - group differences in moderate or severe exacerbation rates ( a 17% reduction , p=0.0003 ) ; however , these trials were placebo - controlled and 6 months12 or 1 year13 in duration . as gold recommended , roflumilast can be used an add - on treatment for patients with copd in stages 3/4 ( forced expiratory volume in 1 second ( fev1 ) < 50% predicted after bronchodilatation ) and frequent exacerbations despite a long - acting bronchodilator treatment . previous studies examined the resource utilization and health care costs associated with exacerbations among copd patients.1417 in a us managed - care population initiating copd maintenance medications , abudagga et al found that exacerbation rates continued to be high , resulting in an estimated annual cost of $ 25,747 in the overall population and $ 29,861 in the patients with history of one or more exacerbations.14 in this study , calculated total monthly costs during the 3-month follow - up period were $ 2,472 and $ 2,851 for non - roflumilast and roflumilast group , respectively , which is higher than the abudagga study14 and likely due to the selection of patients with more severe copd . copd exacerbations result in increases in resource uti - lization , especially for severe exacerbations.9,18,19 consistent with previous studies , the current work found that reduced exacerbation rates among copd patients were associated with reduced all - cause resource utilization and costs from a managed - care payer s perspective . however , the mean age of patients in our study was only slightly lower than that of patients in randomized trials ( 63 years vs 65 years).12,13 fourth , we used a did model that mimicked an experimental study and controlled for the baseline difference between study groups .

only about 0.5% of all cancers occur among children under 15 y. in contrast to adult malignancies , where carcinomas predominate , childhood cancers are histologically very diverse . there are 12 major groups including leukemias , lymphomas , brain and spinal tumors , sympathetic nervous system tumors , retinoblastoma , kidney tumors , liver tumors , bone tumors , soft tissue sarcomas , gonadal and germ - cell tumors , epithelial tumors , and other unspecified neoplasms . diagnostic groups are defined in the second edition of the international classification of diseases ( icd ) for oncology . cancer formation is generally considered as a genetic disease involving alterations in dna structure ranging from gene mutations to gross chromosomal rearrangements . however , dysregulation of gene expression can also be acquired by epigenetic abnormalities , which are a hallmark of cancer evolution at all stages . in contrast to the rest of the genome where most cpgs are methylated , cpg islands in 5 cis - regulatory regions of genes are usually unmethylated . methylation of these cpg islands during development or disease processes is associated with gene silencing . inactivation of tumor suppressor genes by promoter hypermethylation provides an important mechanism for tumor initiation and progression . the genomic caretaker brca1 is necessary for faithful rejoining of broken dna ends and one mutated brca1 allele may already be sufficient to impair this process . the compromised genomic stability in brca1 germline mutation carriers may trigger the genetic changes necessary for neoplastic transformation in hereditary breast cancer patients . sporadic breast tumors with brca1 promoter methylation are mainly estrogen- and progesterone - receptor negative and display similar pathological features as hereditary tumors with brca1 mutations . the concordance rates in monozygotic ( mz ) and dizygotic pairs of twins allow one to estimate the heritability of complex phenotypic traits . mz twins arise from the same zygote , which then divides into two genetically identical embryos . mz twins discordant for monogenic disorders are generally thought to represent rare examples of somatic mosaicism due to genetic mutations after the twinning event , which are then propagated to subsets of cells from one twin . our results suggest that post - zygotic epimutations are another source of somatic mosaicism leading to different disease susceptibility in mz twins . the twin sisters were born in 1977 by spontaneous delivery , seven weeks before term . no intensive care or icterus treatment were necessary . until 1982 8 mo one sister was diagnosed with precursor b - cell lymphoblastic leukemia ( icd10:c910 ) . chemotherapy had to be discontinued because of intolerability . a first relapse of the leukemia occurred in 1984 . the second chemotherapy could be completed , but another relapse made bone marrow transplantation from her healthy twin sister necessary . following bone marrow transplantation at age 7 y she received human growth hormone . in 2002 at the age of 25 y she was diagnosed with thyroid carcinoma ( icd10:c730 ) . when re - examined at age 34 y , the affected twin and her sister did not show any clinical manifestation of cancer . neither family history nor clinical examination gave any hints for a dna repair syndrome or another hereditary disease . apart from the affected twin there were no other cases of cancer in four generations . considering her medical history , it is not unexpected that the affected twin differed from her sister in some features , including height ( 156 cm vs. 168 cm ) and occipitofrontal circumference ( 52 cm vs. 51 cm ) . monozygosity was confirmed by genotyping short tandem repeats . chromosome banding analysis ( at the 500 band level ) of fibroblast cultures revealed normal female karyotypes in both sisters . methylation analysis . because mz twins are genetically identical , epigenetic differences are one plausible explanation for discordant phenotypes . to test this hypothesis , we analyzed the methylation patterns of several representative tumor suppressor genes ( atm , brca1 , brca2 , mlh1 , rad51c , and tp53 ) . one of the studied genes , brca1 , showed constitutive promoter hypermethylation in normal body cells of the affected twin , but not of the healthy twin . it can exactly ( 2% ) quantify the methylation of individual cpg sites located in the 3050 bp 3 from the sequencing primer . our pyrosequencing assay measures the methylation levels of five adjacent cpg sites in the brca1 5 promoter . because the density of methylated cpgs in a cis - regulatory region rather than individual cpgs turn a gene on or off , the average methylation of all analyzed cpgs ( in two independent dna samples ) was used as an epigenetic marker for brca1 promoter methylation . by bisulfite pyrosequencing of exponentially growing fibroblasts , the affected twin showed an increased brca1 methylation level of 12% ( fig . 1 ) primary skin fibroblasts were used for epimutation screening , because they constitute a homogenous cell population with intact cell cycle and dna repair checkpoints . ten additionally analyzed two - cancer patients , who survived a childhood malignancy and then , unrelated to the first event , developed a secondary cancer , as well as 10 carefully matched one - cancer patients without a second malignancy all showed normal methylation levels ( range 03% ) in fibroblasts . induction of dna damage by -irradiation ( 1 gy ) of primary fibroblast cultures did not affect the brca1 methylation levels . in the affected twin , we found 10% methylation at 0 h , 10% at 1 h , 9% at 4 h , 11% at 12 h , and 10% at 24 h after irradiation . the control twin displayed 4% , 4% , 3% , 2% , and 4% methylation , respectively . box plots showing the distribution of brca1 methylation values in fibroblasts of 10 one - cancer and 10 two - cancer patients . the bottom of the box indicates the 25th percentile , the top the 75th percentile . the t bars extend from the boxes to 1.5 times the height of the box . lymphoblastoid cells of both the affected and the control twin exhibited equally low brca1 methylation levels ( 3% and 2% , respectively ) . however , because the affected twin was bone marrow - transplanted , her blood cells were also derived from the healthy twin s stem cells . the normal range of brca1 methylation in blood cells of a large number ( > 100 ) of controls was 05% . similar to fibroblasts , saliva dna of the affected twin showed a much higher brca1 methylation ( 9% ) than that of her sister ( 2% ) . in addition to cells from buccal mucosa and salivary epithelium , saliva may also contain blood cells . this may explain the somewhat lower methylation level of saliva dna compared with fibroblast dna in the affected twin . to distinguish between single cpg methylation errors , which are most likely stochastic errors without pathological consequences , and true epimutations ( allele methylation errors ) , which can be expected to interfere with gene regulation , it is necessary to study the methylation patterns of individual dna molecules . classic bisulfite plasmid sequencing has the added advantage that it allows one to look at a larger number of cpg sites . here we analyzed a brca1 amplicon with 13 cpg sites including the five sites targeted by the pyrosequencing assay ( fig . 2 ) . forty - seven clones ( individual dna molecules ) were recovered from primary fibroblasts of each twin . six ( 13% ) clones of the affected twin exhibited epimutations , indicating that most ( at least 70% ) cpg sites on these dna molecules were aberrantly methylated , typical for epigenetically silenced alleles . in contrast , all 47 alleles of the healthy twin displayed normal hypomethylated patterns . the brca1 epimutation rate was significantly ( test ; p = 0.01 ) higher in the affected twin , compared with her sister . both sisters showed approximately 2% stochastic ( single cpg ) methylation errors : 10 of 531 cpg sites in the affected twin ( excluding abnormal alleles ) and 14 of 609 in the healthy twin were methylated . collectively , our results suggest a constitutively increased methylation level at the brca1 promoter in normal body cells of the affected twin . approximately 25% cells ( skin fibroblasts , cells from buccal mucosa and salivary epithelium ) derived from different embryonic lineages are endowed with one hypermethylated copy of the brca1 tumor suppressor gene , representing a first hit according to knudson s two - hit hypothesis of tumor development . methylation patterns of the brca1 promoter in fibroblasts of the affected twin and her healthy sister . each line represents an individual allele ( dna molecule ) analyzed by bisulfite plasmid sequencing . missing dots indicate cpg sites that could not be analyzed because of poor sequence quality . six of 47 analyzed alleles ( indicated by arrows ) in the affected twin represent epimutations with the majority of cpgs being aberrantly methylated , whereas all 47 alleles in the healthy twin are hypomethylated . because constitutional epimutations in different tumor suppressor genes have been linked to sequence variants in the 5 cis - regulatory region , we sequenced a 3.4 kb upstream fragment including the brca1 promoter and exon 1 . however , we did not find any genetic defect / sequence variant in the affected twin ( data not shown ) . in addition to brca1 , we performed an epimutation screening by bisulfite pyrosequencing for several other tumor suppressor genes ( atm , brca2 , mlh1 , rad51c , and tp53 ) in fibroblasts of the twin pair and the 20 one- or two - cancer patients . however , apart from the brca1 epimutation in the affected twin , all analyzed samples and promoters displayed normal ( < 5% ) methylation values ( data not shown ) , indicating that constitutive epimutations in tumor suppressor genes are rare events in childhood - cancer patients . customized antibody microarrays were used to compare the basal protein levels ( without induction of dna damage ) of brca1 and several other genomic caretakers ( atm , brca2 , mlh1 , rad51 , and tp53 ) in exponentially growing primary fibroblasts of the affected twin vs. her healthy sister . triplicate measurements ( technical replicates ) of the protein levels were performed on protein samples from three independent cell cultures each ( biological replicates ) . the brca1 protein expression ratio between the affected and the healthy twin ( z ratio normalized with log10 transformation and z scores ) was 0.75 ( fig . atm expression was slightly increased ( 1.25 ) in the affected twin , whereas actb ( positive control ) , brca2 , mlh1 , rad51 , and tp53 all showed comparable protein levels in both twins . the brca1 protein levels were also quantified in fibroblast cells at 1 h and 4 h after 1 gy -irradiation ( fig . the amount of brca1 protein increased 1.8-fold at 1 h after irradiation , compared with untreated cells , and reached basal levels at 4 h ( the z ratio of treated vs. untreated cells was 0.9 ) . in the affected twin , brca1 protein expression was induced 2.7-fold at 1 h and also back to basal levels ( z ratio of 1.1 ) at 4 h. figure 3 . ( a ) constitutive protein expression of actb ( control ) , atm , brca1 , brca2 , mlh1 , rad51 , and tp53 in untreated fibroblasts of the affected vs. the healthy twin . relative protein expression ( z ratio normalized by log10 transformation and z scores ) was measured by antibody microarrays using three biological replicates . ( b ) differential induction of brca1 protein in fibroblasts of the healthy twin ( gray bars ) and the affected twin ( black bars ) at 1 h and 4 h after 1 gy -irradiation . relative protein expression in treated vs. untreated cells of each twin was measured by antibody microarrays using three biological replicates . microarray analysis revealed a 181 kb heterozygous deletion containing the rspo3 gene on chromosome 6q22.33 and a 90 kb heterozygous deletion containing the open reading frame c5orf13 on chromosome 5q22.1 in primary fibroblasts of the affected twin . we did not see similar deletions in > 40 additionally studied childhood cancer patients and 100 normal healthy individuals ( from the gutenberg heart study ) . we did not find evidence for a microdeletion or duplication affecting the brca1 cis - regulatory region in the affected twin . by quantitative real - time pcr ( qpcr ) we confirmed the presence of the heterozygous rspo3 and c5orf13 deletions in a mosaic state ( fig . all qpcr experiments were performed on three independent dna samples ( biological replicates ) of the affected twin and a mixture of control dnas , respectively . using rfc3 as reference for a normal diploid gene ( with two copies ) , qpcr revealed a copy number of two for both rspo3 and c5orf13 in the controls . in contrast , the affected twin displayed copy numbers around 1.5 , indicating loss of one rspo3 and one c5orf13 copy in approximately 50% of cells . expression analysis with geneatlas microarrays using four independent rna samples ( from different cell cultures of the affected and the healthy twin ) demonstrated reduced ( approximately 50% ) rspo3 mrna levels in fibroblasts of the affected twin ( fig . 4b ) , whereas c5orf13 levels were comparable in both twins ( data not shown ) . ( a ) c5orf13 and rspo3 copy numbers in fibroblast cells of the two - cancer twin , compared with healthy controls , as determined by qpcr ( using the rfc3 gene copy number as reference ) . standard deviations represent three independent dna samples ( biological replicates ) of the affected twin and a mixture of control dnas . ( b ) rspo3 mrna expression ratio between the affected twin and her healthy sister , as determined by geneatlas microarrays . standard deviations represent rna samples from four different cell cultures of the affected and the healthy twin , respectively . traditionally , phenotypic discordances between mz twin pairs have been attributed to environmental factors ; however , accumulating experimental evidence also suggests a role for genetic and , more importantly , epigenetic differences between co - twins . microarray - based karyotype analyses revealed somatic mosaicism for copy number variations ( cnvs ) in a number ( estimated 10% ) of both phenotypically discordant ( for parkinson disease , parkinsonism or lewy body dementia ) and concordant mz twin pairs . other molecular karyotype and genome sequence studies could not detect any cnv differences between mz twins that were discordant for cleft lip palate or multiple sclerosis , suggesting that post - zygotic genomic alterations are at least not a common cause of phenotypic discordance . several recent studies clearly demonstrated the existence of genome - wide epigenetic differences between mz twins . this spontaneous epimutation rate can be modified by environmental factors , providing a link between lifestyle and phenotypic differences . in this light , stochastic and/or environmentally induced epimutations represent a major source of phenotypic variation and discordance for complex diseases between mz twins . the accumulation of epigenetic differences may also explain the phenotypic divergence of mz twins , as they age . here we studied a mz twin pair discordant for childhood cancer and a second primary cancer . microarray analysis and qpcr revealed mosaic heterozygous deletions on chromosome 5q22.1 ( c5orf13 ) and 6q22.33 ( rspo3 ) in the affected twin . it contains a conserved pest domain , which is important for targeting proteins for degradation by the ubiquitin / proteasome . r - spondin family members are modulators of the wnt/-catenin signaling pathway which plays a crucial role in cell growth , development and disease pathogenesis . rspo3 is frequently upregulated in human breast cancers and correlated with several tumor parameters , suggesting that it can function as a breast cancer oncogene . although c5orf13 and rspo3 haploinsufficiency may contribute to the discordant cancer phenotype , they are unlikely to be the primary cause . we propose that epigenetic changes , specifically hypermethylation of one brca1 allele constituted the first hit leading to inactivation of an important tumor suppressor gene and tumor initiation . germline mutations in brca1 are associated with hereditary breast cancer , ovarian cancer and some other malignancies ; however , so far they are generally not considered as a risk factor for childhood cancer . our index patient developed precursor b - cell lymphoblastic leukemia at the age of 4 y and 8 mo . leukemia , expression of the bcr - abl fusion protein leads to posttranscriptional downregulation of brca1 . one study reported partial hypermethylation and reduced expression of brca1 in primary and therapy - related acute myeloid leukemia , whereas others did not find abnormal brca1 methylation in leukemia samples . in the mz twin pair studied here , only one sister exhibited a brca1 epimutation in a mosaic state in multiple tissues / cell types , whereas the unaffected twin did not . this epimutation most likely originated by a methylation error during early embryogenesis after the twinning event . it affects a substantial subset of cells derived from different embryonic cell lineages , resulting in somatic mosaicism . genome - wide demethylation waves in the early embryo erase most germline methylation patterns , followed by de novo methylation and establishment of somatic methylation patterns . most constitutive epimutations may occur during this vulnerable time window , where epigenetic genome reprogramming occurs , and represent stochastic and/or environmentally induced errors in the establishment or maintenance of an epigenetic state . similar to our case , discordant methylation patterns at the kcnq1ot1 locus ( imprinting control region 2 on chromosome 11p15 ) have been described in a number of mz twin pairs discordant for beckwith - wiedemann syndrome , which is characterized by tumor predisposition and multiple congenital abnormalities . constitutive h19 epimutations ( imprinting control region 1 on chromosome 11p12 ) were found in several patients with sporadic wilms tumor without features of beckwith - wiedemann syndrome . somatic gene mutations and mosaicism ( with or without involvement of the germline ) are an important etiological mechanism for monogenic disorders with high de novo mutations rates such as duchenne muscular dystrophy , neurofibromatosis type 1 and retinoblastoma . in contrast , constitutive epimutations , characterized by soma - wide methylation abnormalities in functionally relevant cis - regulatory regions of disease genes , are an understudied phenomenon . constitutive epimutations in the dna mismatch repair gene mlh1 have been identified in a small number of mutation - negative cases of hereditary non - polyposis colorectal cancer ( hnpcc ) . in rare familial cases with dominant transmission of mosaic mlh1 methylation , the constitutional epimutation was linked to a single nucleotide variant in the 5 utr of mlh1 . similarly , constitutional epimutations in the dna mismatch repair gene msh2 , another rare cause of hnpcc , were linked to 3 end deletions of epcam , a gene directly upstream of msh2 . constitutive epimutations in the dapk1 gene that predispose to b cell chronic lymphocytic leukemia are caused by a point mutation upstream of the dapk1 promoter . neither sequencing nor microarray analysis of the 5 cis - regulatory region of brca1 revealed any detectable genetic differences between the discordant mz twin pair studied here . although we can not exclude a causative genetic defect elsewhere , our findings are consistent with the view that the affected twin exhibits a true brca1 epimutation . our study revealed the existence of a constitutive brca1 epimutation only in the affected sibling of a mz twin pair . compared with her healthy sister , the two - cancer twin showed reduced basal brca1 protein levels ( in untreated fibroblasts ) and a higher induction of protein expression by dna damage . upregulation of brca1 in irradiated cells of both the affected and the healthy twin was not mediated by demethylation of the brca1 promoter . in the affected twin the percentage of aberrantly methylated alleles remained constant ( at about 10% ) after dna damage and in the healthy twin the brca1 promoter was already completely demethylated in untreated cells . it is well known that the methylation of cpgs in 5 promoters that are usually protected from methylation in somatic tissues can suppress gene expression during development , differentiation and disease processes . however , other mechanisms , i.e. , gene body methylation , may be more important for regulating the cell - context specific efficiency of transcription . the basal atm protein levels were also increased in the affected twin , although both twins showed equally low promoter methylation levels ( around 1% ) of the atm gene . we speculate that atm is constitutively upregulated by a compensation mechanism to counterbalance the reduced brca1 levels . brca1 is regulated by an atm - dependent mechanism and , on the other hand , essential for the recruitment of previously activated atm to the sites of dna damage . although we can not exclude the formal possibility of a chance coincidence or a late ( i.e. , therapy - related ) somatic event , it is plausible to assume that epigenetic silencing of one copy of the brca1 tumor suppressor gene in 25% body cells and differences in the dna damage response contributed to the discordant cancer phenotype in the affected twin . preliminary evidence suggests that brca1 promoter hypermethylation can also be found in blood cells of a subset of breast cancer patients . collectively , these results suggest that constitutive brca1 epimutations occur in normal tissues and may be associated with an increased cancer risk . our pyrosequencing assay may prove useful for high - throughput screening of larger patient populations . the dna methylation analysis performed in this study was limited to a number of key tumor suppressor genes . considering the enormous epigenetic variability due to stochastic methylation errors or extrinsic variations during post - zygotic genome reprogramming , we can not rule out the possibility that epimutations in the affected twin in genes other than those studied are responsible for the discordant phenotype . nevertheless , 10% soma - wide promoter methylation in an essential tumor suppressor gene such as brca1 can be expected to contribute to a tumor susceptibility phenotype . the study was approved by the ethics committee of the medical association of rhineland - palatinate [ ( nr . 837.440.03(4102 ) ] . with the help of the german childhood cancer registry , we recruited a discordant mz twin pair . one twin suffered from childhood leukemia and later on thyroid carcinoma , whereas her twin sister was completely healthy ( without malignancy ) . in addition , we recruited 10 two - cancer patients as well as 10 carefully matched one - cancer patients ( same sex , same primary cancer , equal age at first diagnosis ) who did not develop a second malignancy . primary fibroblasts from skin biopsies were cultured in minimal essential medium with earle s salts ( invitrogen , karlsruhe , germany ) , supplemented with 10% fetal bovine serum , vitamins and antibiotics . the cell cultures of the twin sisters were always passaged in parallel and harvested in a subconfluent stage . to induce dna damage , subconfluent cultures were -irradiated with a dose of 1 gy using a gammacell 2000 ( cs ) irradiator . cells were harvested at 1 h , 4 h , 12 h , and 24 h after irradiation . lymphoblastoid cell lines were established by epstein - barr virus transformation of peripheral blood lymphocytes . dna sequence analysis . for sanger sequencing of the 5 utr and exon 1 of the brca1 gene , a 3.4 kb fragment on chromosome 17 ( 41.277.11241.280.530 bps , ensembl release 64 ) was divided into 11 amplicons ( table s1 ) . for amplicons 15 and 8 , the pcr reaction mixture ( 25 l ) consisted of 2.5 l 10x pcr buffer , 20 mm mgcl2 , 0.5 l 10 mm dntp mix , 1 l ( 10 pmol ) of each forward and reverse primer , 0.2 l ( 1 u ) faststart taq dna polymerase ( roche diagnostics , mannheim , germany ) , 18.8 l pcr - grade water , and 1 l ( ~100 ng ) template dna . for amplicons 6 , 7 , 8 , 10 , and 11 , the pcr mixture ( 25 l ) contained 25 mm ammonium sulfate , 750 mm tris - hcl , 0.1% tween 20 , 240 m dntps , 2.4 mm magnesium sulfate , 2.4x pcrx enhancer solution ( invitrogen ) , 0.4 m of each primer , 1 unit of platinum taq ( invitrogen ) , and 1 l ( ~100 ng ) template dna . all pcr amplifications were performed with an initial denaturation step at 95c for 5 min , 40 cycles of 95c for 30 sec , primer - specific annealing temperature ( table s1 ) for 30 sec , 72c for 45 sec , and a final extension step at 72c for 7 min . sequencing of the resulting pcr products was done on an abi 3130 x l automated sequencer . genomic dna from fibroblasts and ebv - transformed lymphoblasts was extracted using the qiagen mini dna kit ( qiagen , hilden , germany ) . saliva dna was extracted using the oragene dna extraction kit ( dna genotek , kanata , ontario , canada ) . bisulfite conversion of genomic dna was performed with the epitect bisulfite kit ( qiagen ) . bisulfite pyrosequencing was performed on a pyromarktmq96 md pyrosequencing system with the pyromark gold q96 cdt reagent kit ( qiagen ) . an assay quantifying the methylation levels of five representative cpg sites in the brca1 promoter was designed using the pyromark assay design 2.0 software ( qiagen ) . a 232 bp amplicon ( hsa17 : 41,277,29241,277,523 bps ; ensembl release 60 ) was pcr amplified from bisulfite - treated dna using forward primer 5-atttagagtagagggtgaagg-3 and biotinylated reverse primer 5-tctatccctcccatcctctaatt-3. the reaction mixture consisted of 2.5 l 10x pcr buffer , 20 mm mgcl2 , 0.5 l 10 mm dntp mix , 1 l ( 10 pmol ) of each primer , 0.2 l ( 1 u ) faststart taq dna polymerase ( roche diagnostics , mannheim , germany ) , 18.8 l pcr - grade water , and 1 l ( ~100 ng ) template dna . pcr amplifications were performed with an initial denaturation step at 95c for 5 min , 35 cycles of 95c for 30 sec , 55c for 30 sec , and 72c for 45 sec , and a final extension step at 72c for 5 min . sequencing was performed with primer 5-ttgagaaattttatagtttgtttt-3. the pyro q - cpg software ( qiagen ) was used for data analysis . for classical bisulfite plasmid sequencing brca1 pcr products were cloned into pcr2.1-topo vector using t4-dna ligase , the ta cloning kit and one shot top10 chemically competent escherichia coli ( invitrogen ) . plasmid dna of individual clones was isolated with the zr plasmid miniprep classic kit ( zymo research , freiburg , germany ) . insert - containing clones were sequenced using dye terminator cycle sequencing with m13 primers on an abi 3130xl automated sequencer ( life technologies , frankfurt , germany ) . cells were resuspended two times in 500 l of 10 mm hepes , 1.5 mm mgcl2 , 10 mm kcl , ph 7.9 and incubated on ice for 10 min . the pellet was resuspended in 100 l of 20 mm hepes , 0.42 m nacl , 1.5 mm mgcl2 , 0.2 mm edta , 25% glycerol , ph 7.9 and homogenized . after incubation on ice for 30 min and centrifugation for 30 min at maximum speed at 4c , the supernatant containing the nuclear protein extract was separated from the cytoplasmic pellet and stored at -80c . the protein concentration was measured according to bradford , using roti quant ( roth , karlsruhe , germany ) . customized antibody microarrays for quantification of brca1 ( antibody # sc-1553 , santa cruz biotechnology , heidelberg , germany ) and several other dna repair - associated proteins ( atm , brca2 , mlh1 , rad51 , and tp53 ) were prepared by spotting triplicate drops , each containing approximately 0.5 pg antibody onto nitrocellulose - coated slides ( oncyte , nitrocellulose 16 multi - pad slides , grace bio - labs , bend , or , usa ) , using a sciflexarrayer 3 non - contact spotter ( scienion , berlin , germany ) . antibodies against -actin ( actb ) ( # a5441 , sigma - aldrich , munich , germany ) served as positive , spotting buffer as negative control . nuclear proteins were labeled with an amine reactive fluorine dye , which forms a covalent amide bond between the primary amines of proteins . two micrograms of protein and 0.12 l fluorescent dye ( dylight 649 nhs ester , pierce , rockford , usa ) were incubated for 1 h at room temperature in the dark . then , excess fluorescent dye was inactivated by adding 100 mm glycine to the reaction . prior to use , antibody microarrays were covered with 16-pad fast frame hybridization chambers ( whatman , maidstone , uk ) . unspecific binding sites were blocked for 1 h at 4c with 120 l pbs containing 4% non - fat dry milk per subarray , followed by three washes with 120 l pbs each for 10 min . labeled protein samples were incubated on subarrays overnight at 4c . afterwards , the slides were washed two times for 15 min with pbs , 5% tween 20 and two times for 15 min with hplc - grade water . finally , the slides were dried in a speedvac and scanned with a high - resolution confocal affymetrix array scanner 428 tm ( affymetrix , high wycombe , uk ) . slide images were analyzed using the tm4 spotfinder ( version 3.1.1 ) software ( dana faber cancer institute , boston , usa ) . background subtraction was performed according to the formula : spot intensity = mean intensity sp - ( sum bkg sum top25bkg)/(number of pixelsp - number of pixel top25bkg ) , where sp represents any spot , bkg the corresponding background and top25bkg the top 25% of background pixel . three biological replicates ( using protein samples from different cell cultures of the affected and the healthy twin ) were performed for each experiment . molecular karyotype analysis . high - resolution screening for microdeletions and duplications was performed with the affymetrix genechip genome wide human snp array 6.0 and the genechip genome wide snp sty assay kit 5.0/6.0 , following the protocol developed by the manufacturer . copy numbers were determined using the affymetrix genotyping console 4.0 and chromosome analysis suite 1.0.1 . quantitative real - time pcr with the universal probe library set 04683633001 ( roche diagnostics ) was used to validate copy number changes of c5orf13 ( upl probe # 24 ) and rspo3 ( # 23 ) . pcr was performed on an abi 7500 fast real - time pcr system ( life technologies ) with one cycle of 95c for 10 min and 45 cycles of 95c for 30 sec and 60c for 60 sec . copy number calculation was performed with the -ct method , using the rfc3 ( upl probe # 32 ) gene copy number as reference . genome - wide expression analyses were performed with rna samples from four fibroblast cell cultures of each twin using the geneatlas personal microarray system ( affymetrix , santa clara , ca , usa ) . data were analyzed with the affymetrix expression console program .

we describe monozygotic twins discordant for childhood leukemia and secondary thyroid carcinoma . we used bisulfite pyrosequencing to compare the constitutive promoter methylation of brca1 and several other tumor suppressor genes in primary fibroblasts . the affected twin displayed an increased brca1 methylation ( 12% ) , compared with her sister ( 3% ) . subsequent bisulfite plasmid sequencing demonstrated that 13% ( 6 of 47 ) brca1 alleles were fully methylated in the affected twin , whereas her sister displayed only single cpg errors without functional implications . this between - twin methylation difference was also found in irradiated fibroblasts and untreated saliva cells . the brca1 epimutation may have originated by an early somatic event in the affected twin : approximately 25% of her body cells derived from different embryonic cell lineages carry one epigenetically inactivated brca1 allele . this epimutation was associated with reduced basal protein levels and a higher induction of brca1 after dna damage . in addition , we performed a genome - wide microarray analysis of both sisters and found several copy number variations , i.e. , heterozygous deletion and reduced expression of the rspo3 gene in the affected twin . this monozygotic twin pair represents an impressive example of epigenetic somatic mosaicism , suggesting a role for constitutive epimutations , maybe along with de novo genetic alterations in recurrent tumor development .

Introduction Results Discussion Materials and Methods Supplementary Material

diagnostic groups are defined in the second edition of the international classification of diseases ( icd ) for oncology . cancer formation is generally considered as a genetic disease involving alterations in dna structure ranging from gene mutations to gross chromosomal rearrangements . methylation of these cpg islands during development or disease processes is associated with gene silencing . inactivation of tumor suppressor genes by promoter hypermethylation provides an important mechanism for tumor initiation and progression . sporadic breast tumors with brca1 promoter methylation are mainly estrogen- and progesterone - receptor negative and display similar pathological features as hereditary tumors with brca1 mutations . mz twins discordant for monogenic disorders are generally thought to represent rare examples of somatic mosaicism due to genetic mutations after the twinning event , which are then propagated to subsets of cells from one twin . when re - examined at age 34 y , the affected twin and her sister did not show any clinical manifestation of cancer . apart from the affected twin there were no other cases of cancer in four generations . considering her medical history , it is not unexpected that the affected twin differed from her sister in some features , including height ( 156 cm vs. 168 cm ) and occipitofrontal circumference ( 52 cm vs. 51 cm ) . to test this hypothesis , we analyzed the methylation patterns of several representative tumor suppressor genes ( atm , brca1 , brca2 , mlh1 , rad51c , and tp53 ) . one of the studied genes , brca1 , showed constitutive promoter hypermethylation in normal body cells of the affected twin , but not of the healthy twin . it can exactly ( 2% ) quantify the methylation of individual cpg sites located in the 3050 bp 3 from the sequencing primer . our pyrosequencing assay measures the methylation levels of five adjacent cpg sites in the brca1 5 promoter . because the density of methylated cpgs in a cis - regulatory region rather than individual cpgs turn a gene on or off , the average methylation of all analyzed cpgs ( in two independent dna samples ) was used as an epigenetic marker for brca1 promoter methylation . by bisulfite pyrosequencing of exponentially growing fibroblasts , the affected twin showed an increased brca1 methylation level of 12% ( fig . induction of dna damage by -irradiation ( 1 gy ) of primary fibroblast cultures did not affect the brca1 methylation levels . in the affected twin , we found 10% methylation at 0 h , 10% at 1 h , 9% at 4 h , 11% at 12 h , and 10% at 24 h after irradiation . box plots showing the distribution of brca1 methylation values in fibroblasts of 10 one - cancer and 10 two - cancer patients . lymphoblastoid cells of both the affected and the control twin exhibited equally low brca1 methylation levels ( 3% and 2% , respectively ) . however , because the affected twin was bone marrow - transplanted , her blood cells were also derived from the healthy twin s stem cells . the normal range of brca1 methylation in blood cells of a large number ( > 100 ) of controls was 05% . similar to fibroblasts , saliva dna of the affected twin showed a much higher brca1 methylation ( 9% ) than that of her sister ( 2% ) . this may explain the somewhat lower methylation level of saliva dna compared with fibroblast dna in the affected twin . to distinguish between single cpg methylation errors , which are most likely stochastic errors without pathological consequences , and true epimutations ( allele methylation errors ) , which can be expected to interfere with gene regulation , it is necessary to study the methylation patterns of individual dna molecules . classic bisulfite plasmid sequencing has the added advantage that it allows one to look at a larger number of cpg sites . six ( 13% ) clones of the affected twin exhibited epimutations , indicating that most ( at least 70% ) cpg sites on these dna molecules were aberrantly methylated , typical for epigenetically silenced alleles . in contrast , all 47 alleles of the healthy twin displayed normal hypomethylated patterns . the brca1 epimutation rate was significantly ( test ; p = 0.01 ) higher in the affected twin , compared with her sister . both sisters showed approximately 2% stochastic ( single cpg ) methylation errors : 10 of 531 cpg sites in the affected twin ( excluding abnormal alleles ) and 14 of 609 in the healthy twin were methylated . collectively , our results suggest a constitutively increased methylation level at the brca1 promoter in normal body cells of the affected twin . approximately 25% cells ( skin fibroblasts , cells from buccal mucosa and salivary epithelium ) derived from different embryonic lineages are endowed with one hypermethylated copy of the brca1 tumor suppressor gene , representing a first hit according to knudson s two - hit hypothesis of tumor development . methylation patterns of the brca1 promoter in fibroblasts of the affected twin and her healthy sister . each line represents an individual allele ( dna molecule ) analyzed by bisulfite plasmid sequencing . six of 47 analyzed alleles ( indicated by arrows ) in the affected twin represent epimutations with the majority of cpgs being aberrantly methylated , whereas all 47 alleles in the healthy twin are hypomethylated . because constitutional epimutations in different tumor suppressor genes have been linked to sequence variants in the 5 cis - regulatory region , we sequenced a 3.4 kb upstream fragment including the brca1 promoter and exon 1 . however , we did not find any genetic defect / sequence variant in the affected twin ( data not shown ) . in addition to brca1 , we performed an epimutation screening by bisulfite pyrosequencing for several other tumor suppressor genes ( atm , brca2 , mlh1 , rad51c , and tp53 ) in fibroblasts of the twin pair and the 20 one- or two - cancer patients . however , apart from the brca1 epimutation in the affected twin , all analyzed samples and promoters displayed normal ( < 5% ) methylation values ( data not shown ) , indicating that constitutive epimutations in tumor suppressor genes are rare events in childhood - cancer patients . customized antibody microarrays were used to compare the basal protein levels ( without induction of dna damage ) of brca1 and several other genomic caretakers ( atm , brca2 , mlh1 , rad51 , and tp53 ) in exponentially growing primary fibroblasts of the affected twin vs. her healthy sister . triplicate measurements ( technical replicates ) of the protein levels were performed on protein samples from three independent cell cultures each ( biological replicates ) . the brca1 protein expression ratio between the affected and the healthy twin ( z ratio normalized with log10 transformation and z scores ) was 0.75 ( fig . atm expression was slightly increased ( 1.25 ) in the affected twin , whereas actb ( positive control ) , brca2 , mlh1 , rad51 , and tp53 all showed comparable protein levels in both twins . the brca1 protein levels were also quantified in fibroblast cells at 1 h and 4 h after 1 gy -irradiation ( fig . the amount of brca1 protein increased 1.8-fold at 1 h after irradiation , compared with untreated cells , and reached basal levels at 4 h ( the z ratio of treated vs. untreated cells was 0.9 ) . in the affected twin , brca1 protein expression was induced 2.7-fold at 1 h and also back to basal levels ( z ratio of 1.1 ) at 4 h. figure 3 . ( a ) constitutive protein expression of actb ( control ) , atm , brca1 , brca2 , mlh1 , rad51 , and tp53 in untreated fibroblasts of the affected vs. the healthy twin . ( b ) differential induction of brca1 protein in fibroblasts of the healthy twin ( gray bars ) and the affected twin ( black bars ) at 1 h and 4 h after 1 gy -irradiation . microarray analysis revealed a 181 kb heterozygous deletion containing the rspo3 gene on chromosome 6q22.33 and a 90 kb heterozygous deletion containing the open reading frame c5orf13 on chromosome 5q22.1 in primary fibroblasts of the affected twin . we did not find evidence for a microdeletion or duplication affecting the brca1 cis - regulatory region in the affected twin . all qpcr experiments were performed on three independent dna samples ( biological replicates ) of the affected twin and a mixture of control dnas , respectively . using rfc3 as reference for a normal diploid gene ( with two copies ) , qpcr revealed a copy number of two for both rspo3 and c5orf13 in the controls . in contrast , the affected twin displayed copy numbers around 1.5 , indicating loss of one rspo3 and one c5orf13 copy in approximately 50% of cells . expression analysis with geneatlas microarrays using four independent rna samples ( from different cell cultures of the affected and the healthy twin ) demonstrated reduced ( approximately 50% ) rspo3 mrna levels in fibroblasts of the affected twin ( fig . 4b ) , whereas c5orf13 levels were comparable in both twins ( data not shown ) . ( a ) c5orf13 and rspo3 copy numbers in fibroblast cells of the two - cancer twin , compared with healthy controls , as determined by qpcr ( using the rfc3 gene copy number as reference ) . standard deviations represent three independent dna samples ( biological replicates ) of the affected twin and a mixture of control dnas . ( b ) rspo3 mrna expression ratio between the affected twin and her healthy sister , as determined by geneatlas microarrays . standard deviations represent rna samples from four different cell cultures of the affected and the healthy twin , respectively . traditionally , phenotypic discordances between mz twin pairs have been attributed to environmental factors ; however , accumulating experimental evidence also suggests a role for genetic and , more importantly , epigenetic differences between co - twins . microarray - based karyotype analyses revealed somatic mosaicism for copy number variations ( cnvs ) in a number ( estimated 10% ) of both phenotypically discordant ( for parkinson disease , parkinsonism or lewy body dementia ) and concordant mz twin pairs . other molecular karyotype and genome sequence studies could not detect any cnv differences between mz twins that were discordant for cleft lip palate or multiple sclerosis , suggesting that post - zygotic genomic alterations are at least not a common cause of phenotypic discordance . several recent studies clearly demonstrated the existence of genome - wide epigenetic differences between mz twins . here we studied a mz twin pair discordant for childhood cancer and a second primary cancer . microarray analysis and qpcr revealed mosaic heterozygous deletions on chromosome 5q22.1 ( c5orf13 ) and 6q22.33 ( rspo3 ) in the affected twin . r - spondin family members are modulators of the wnt/-catenin signaling pathway which plays a crucial role in cell growth , development and disease pathogenesis . we propose that epigenetic changes , specifically hypermethylation of one brca1 allele constituted the first hit leading to inactivation of an important tumor suppressor gene and tumor initiation . germline mutations in brca1 are associated with hereditary breast cancer , ovarian cancer and some other malignancies ; however , so far they are generally not considered as a risk factor for childhood cancer . leukemia , expression of the bcr - abl fusion protein leads to posttranscriptional downregulation of brca1 . one study reported partial hypermethylation and reduced expression of brca1 in primary and therapy - related acute myeloid leukemia , whereas others did not find abnormal brca1 methylation in leukemia samples . in the mz twin pair studied here , only one sister exhibited a brca1 epimutation in a mosaic state in multiple tissues / cell types , whereas the unaffected twin did not . this epimutation most likely originated by a methylation error during early embryogenesis after the twinning event . it affects a substantial subset of cells derived from different embryonic cell lineages , resulting in somatic mosaicism . genome - wide demethylation waves in the early embryo erase most germline methylation patterns , followed by de novo methylation and establishment of somatic methylation patterns . most constitutive epimutations may occur during this vulnerable time window , where epigenetic genome reprogramming occurs , and represent stochastic and/or environmentally induced errors in the establishment or maintenance of an epigenetic state . somatic gene mutations and mosaicism ( with or without involvement of the germline ) are an important etiological mechanism for monogenic disorders with high de novo mutations rates such as duchenne muscular dystrophy , neurofibromatosis type 1 and retinoblastoma . in contrast , constitutive epimutations , characterized by soma - wide methylation abnormalities in functionally relevant cis - regulatory regions of disease genes , are an understudied phenomenon . constitutive epimutations in the dna mismatch repair gene mlh1 have been identified in a small number of mutation - negative cases of hereditary non - polyposis colorectal cancer ( hnpcc ) . in rare familial cases with dominant transmission of mosaic mlh1 methylation , the constitutional epimutation was linked to a single nucleotide variant in the 5 utr of mlh1 . constitutive epimutations in the dapk1 gene that predispose to b cell chronic lymphocytic leukemia are caused by a point mutation upstream of the dapk1 promoter . neither sequencing nor microarray analysis of the 5 cis - regulatory region of brca1 revealed any detectable genetic differences between the discordant mz twin pair studied here . although we can not exclude a causative genetic defect elsewhere , our findings are consistent with the view that the affected twin exhibits a true brca1 epimutation . our study revealed the existence of a constitutive brca1 epimutation only in the affected sibling of a mz twin pair . compared with her healthy sister , the two - cancer twin showed reduced basal brca1 protein levels ( in untreated fibroblasts ) and a higher induction of protein expression by dna damage . upregulation of brca1 in irradiated cells of both the affected and the healthy twin was not mediated by demethylation of the brca1 promoter . in the affected twin the percentage of aberrantly methylated alleles remained constant ( at about 10% ) after dna damage and in the healthy twin the brca1 promoter was already completely demethylated in untreated cells . however , other mechanisms , i.e. the basal atm protein levels were also increased in the affected twin , although both twins showed equally low promoter methylation levels ( around 1% ) of the atm gene . brca1 is regulated by an atm - dependent mechanism and , on the other hand , essential for the recruitment of previously activated atm to the sites of dna damage . , therapy - related ) somatic event , it is plausible to assume that epigenetic silencing of one copy of the brca1 tumor suppressor gene in 25% body cells and differences in the dna damage response contributed to the discordant cancer phenotype in the affected twin . collectively , these results suggest that constitutive brca1 epimutations occur in normal tissues and may be associated with an increased cancer risk . the dna methylation analysis performed in this study was limited to a number of key tumor suppressor genes . considering the enormous epigenetic variability due to stochastic methylation errors or extrinsic variations during post - zygotic genome reprogramming , we can not rule out the possibility that epimutations in the affected twin in genes other than those studied are responsible for the discordant phenotype . nevertheless , 10% soma - wide promoter methylation in an essential tumor suppressor gene such as brca1 can be expected to contribute to a tumor susceptibility phenotype . with the help of the german childhood cancer registry , we recruited a discordant mz twin pair . one twin suffered from childhood leukemia and later on thyroid carcinoma , whereas her twin sister was completely healthy ( without malignancy ) . in addition , we recruited 10 two - cancer patients as well as 10 carefully matched one - cancer patients ( same sex , same primary cancer , equal age at first diagnosis ) who did not develop a second malignancy . primary fibroblasts from skin biopsies were cultured in minimal essential medium with earle s salts ( invitrogen , karlsruhe , germany ) , supplemented with 10% fetal bovine serum , vitamins and antibiotics . for sanger sequencing of the 5 utr and exon 1 of the brca1 gene , a 3.4 kb fragment on chromosome 17 ( 41.277.11241.280.530 bps , ensembl release 64 ) was divided into 11 amplicons ( table s1 ) . an assay quantifying the methylation levels of five representative cpg sites in the brca1 promoter was designed using the pyromark assay design 2.0 software ( qiagen ) . for classical bisulfite plasmid sequencing brca1 pcr products were cloned into pcr2.1-topo vector using t4-dna ligase , the ta cloning kit and one shot top10 chemically competent escherichia coli ( invitrogen ) . customized antibody microarrays for quantification of brca1 ( antibody # sc-1553 , santa cruz biotechnology , heidelberg , germany ) and several other dna repair - associated proteins ( atm , brca2 , mlh1 , rad51 , and tp53 ) were prepared by spotting triplicate drops , each containing approximately 0.5 pg antibody onto nitrocellulose - coated slides ( oncyte , nitrocellulose 16 multi - pad slides , grace bio - labs , bend , or , usa ) , using a sciflexarrayer 3 non - contact spotter ( scienion , berlin , germany ) . two micrograms of protein and 0.12 l fluorescent dye ( dylight 649 nhs ester , pierce , rockford , usa ) were incubated for 1 h at room temperature in the dark . background subtraction was performed according to the formula : spot intensity = mean intensity sp - ( sum bkg sum top25bkg)/(number of pixelsp - number of pixel top25bkg ) , where sp represents any spot , bkg the corresponding background and top25bkg the top 25% of background pixel . three biological replicates ( using protein samples from different cell cultures of the affected and the healthy twin ) were performed for each experiment . genome - wide expression analyses were performed with rna samples from four fibroblast cell cultures of each twin using the geneatlas personal microarray system ( affymetrix , santa clara , ca , usa ) .

there are 12 major groups including leukemias , lymphomas , brain and spinal tumors , sympathetic nervous system tumors , retinoblastoma , kidney tumors , liver tumors , bone tumors , soft tissue sarcomas , gonadal and germ - cell tumors , epithelial tumors , and other unspecified neoplasms . diagnostic groups are defined in the second edition of the international classification of diseases ( icd ) for oncology . however , dysregulation of gene expression can also be acquired by epigenetic abnormalities , which are a hallmark of cancer evolution at all stages . in contrast to the rest of the genome where most cpgs are methylated , cpg islands in 5 cis - regulatory regions of genes are usually unmethylated . methylation of these cpg islands during development or disease processes is associated with gene silencing . the compromised genomic stability in brca1 germline mutation carriers may trigger the genetic changes necessary for neoplastic transformation in hereditary breast cancer patients . sporadic breast tumors with brca1 promoter methylation are mainly estrogen- and progesterone - receptor negative and display similar pathological features as hereditary tumors with brca1 mutations . the concordance rates in monozygotic ( mz ) and dizygotic pairs of twins allow one to estimate the heritability of complex phenotypic traits . mz twins discordant for monogenic disorders are generally thought to represent rare examples of somatic mosaicism due to genetic mutations after the twinning event , which are then propagated to subsets of cells from one twin . our results suggest that post - zygotic epimutations are another source of somatic mosaicism leading to different disease susceptibility in mz twins . until 1982 8 mo one sister was diagnosed with precursor b - cell lymphoblastic leukemia ( icd10:c910 ) . in 2002 at the age of 25 y she was diagnosed with thyroid carcinoma ( icd10:c730 ) . when re - examined at age 34 y , the affected twin and her sister did not show any clinical manifestation of cancer . apart from the affected twin there were no other cases of cancer in four generations . considering her medical history , it is not unexpected that the affected twin differed from her sister in some features , including height ( 156 cm vs. 168 cm ) and occipitofrontal circumference ( 52 cm vs. 51 cm ) . to test this hypothesis , we analyzed the methylation patterns of several representative tumor suppressor genes ( atm , brca1 , brca2 , mlh1 , rad51c , and tp53 ) . one of the studied genes , brca1 , showed constitutive promoter hypermethylation in normal body cells of the affected twin , but not of the healthy twin . it can exactly ( 2% ) quantify the methylation of individual cpg sites located in the 3050 bp 3 from the sequencing primer . our pyrosequencing assay measures the methylation levels of five adjacent cpg sites in the brca1 5 promoter . because the density of methylated cpgs in a cis - regulatory region rather than individual cpgs turn a gene on or off , the average methylation of all analyzed cpgs ( in two independent dna samples ) was used as an epigenetic marker for brca1 promoter methylation . by bisulfite pyrosequencing of exponentially growing fibroblasts , the affected twin showed an increased brca1 methylation level of 12% ( fig . 1 ) primary skin fibroblasts were used for epimutation screening , because they constitute a homogenous cell population with intact cell cycle and dna repair checkpoints . ten additionally analyzed two - cancer patients , who survived a childhood malignancy and then , unrelated to the first event , developed a secondary cancer , as well as 10 carefully matched one - cancer patients without a second malignancy all showed normal methylation levels ( range 03% ) in fibroblasts . induction of dna damage by -irradiation ( 1 gy ) of primary fibroblast cultures did not affect the brca1 methylation levels . in the affected twin , we found 10% methylation at 0 h , 10% at 1 h , 9% at 4 h , 11% at 12 h , and 10% at 24 h after irradiation . box plots showing the distribution of brca1 methylation values in fibroblasts of 10 one - cancer and 10 two - cancer patients . the bottom of the box indicates the 25th percentile , the top the 75th percentile . lymphoblastoid cells of both the affected and the control twin exhibited equally low brca1 methylation levels ( 3% and 2% , respectively ) . however , because the affected twin was bone marrow - transplanted , her blood cells were also derived from the healthy twin s stem cells . the normal range of brca1 methylation in blood cells of a large number ( > 100 ) of controls was 05% . similar to fibroblasts , saliva dna of the affected twin showed a much higher brca1 methylation ( 9% ) than that of her sister ( 2% ) . this may explain the somewhat lower methylation level of saliva dna compared with fibroblast dna in the affected twin . to distinguish between single cpg methylation errors , which are most likely stochastic errors without pathological consequences , and true epimutations ( allele methylation errors ) , which can be expected to interfere with gene regulation , it is necessary to study the methylation patterns of individual dna molecules . here we analyzed a brca1 amplicon with 13 cpg sites including the five sites targeted by the pyrosequencing assay ( fig . six ( 13% ) clones of the affected twin exhibited epimutations , indicating that most ( at least 70% ) cpg sites on these dna molecules were aberrantly methylated , typical for epigenetically silenced alleles . the brca1 epimutation rate was significantly ( test ; p = 0.01 ) higher in the affected twin , compared with her sister . both sisters showed approximately 2% stochastic ( single cpg ) methylation errors : 10 of 531 cpg sites in the affected twin ( excluding abnormal alleles ) and 14 of 609 in the healthy twin were methylated . collectively , our results suggest a constitutively increased methylation level at the brca1 promoter in normal body cells of the affected twin . approximately 25% cells ( skin fibroblasts , cells from buccal mucosa and salivary epithelium ) derived from different embryonic lineages are endowed with one hypermethylated copy of the brca1 tumor suppressor gene , representing a first hit according to knudson s two - hit hypothesis of tumor development . methylation patterns of the brca1 promoter in fibroblasts of the affected twin and her healthy sister . six of 47 analyzed alleles ( indicated by arrows ) in the affected twin represent epimutations with the majority of cpgs being aberrantly methylated , whereas all 47 alleles in the healthy twin are hypomethylated . because constitutional epimutations in different tumor suppressor genes have been linked to sequence variants in the 5 cis - regulatory region , we sequenced a 3.4 kb upstream fragment including the brca1 promoter and exon 1 . however , we did not find any genetic defect / sequence variant in the affected twin ( data not shown ) . in addition to brca1 , we performed an epimutation screening by bisulfite pyrosequencing for several other tumor suppressor genes ( atm , brca2 , mlh1 , rad51c , and tp53 ) in fibroblasts of the twin pair and the 20 one- or two - cancer patients . however , apart from the brca1 epimutation in the affected twin , all analyzed samples and promoters displayed normal ( < 5% ) methylation values ( data not shown ) , indicating that constitutive epimutations in tumor suppressor genes are rare events in childhood - cancer patients . customized antibody microarrays were used to compare the basal protein levels ( without induction of dna damage ) of brca1 and several other genomic caretakers ( atm , brca2 , mlh1 , rad51 , and tp53 ) in exponentially growing primary fibroblasts of the affected twin vs. her healthy sister . the brca1 protein expression ratio between the affected and the healthy twin ( z ratio normalized with log10 transformation and z scores ) was 0.75 ( fig . atm expression was slightly increased ( 1.25 ) in the affected twin , whereas actb ( positive control ) , brca2 , mlh1 , rad51 , and tp53 all showed comparable protein levels in both twins . the amount of brca1 protein increased 1.8-fold at 1 h after irradiation , compared with untreated cells , and reached basal levels at 4 h ( the z ratio of treated vs. untreated cells was 0.9 ) . in the affected twin , brca1 protein expression was induced 2.7-fold at 1 h and also back to basal levels ( z ratio of 1.1 ) at 4 h. figure 3 . ( a ) constitutive protein expression of actb ( control ) , atm , brca1 , brca2 , mlh1 , rad51 , and tp53 in untreated fibroblasts of the affected vs. the healthy twin . ( b ) differential induction of brca1 protein in fibroblasts of the healthy twin ( gray bars ) and the affected twin ( black bars ) at 1 h and 4 h after 1 gy -irradiation . we did not see similar deletions in > 40 additionally studied childhood cancer patients and 100 normal healthy individuals ( from the gutenberg heart study ) . we did not find evidence for a microdeletion or duplication affecting the brca1 cis - regulatory region in the affected twin . by quantitative real - time pcr ( qpcr ) we confirmed the presence of the heterozygous rspo3 and c5orf13 deletions in a mosaic state ( fig . expression analysis with geneatlas microarrays using four independent rna samples ( from different cell cultures of the affected and the healthy twin ) demonstrated reduced ( approximately 50% ) rspo3 mrna levels in fibroblasts of the affected twin ( fig . ( a ) c5orf13 and rspo3 copy numbers in fibroblast cells of the two - cancer twin , compared with healthy controls , as determined by qpcr ( using the rfc3 gene copy number as reference ) . traditionally , phenotypic discordances between mz twin pairs have been attributed to environmental factors ; however , accumulating experimental evidence also suggests a role for genetic and , more importantly , epigenetic differences between co - twins . microarray - based karyotype analyses revealed somatic mosaicism for copy number variations ( cnvs ) in a number ( estimated 10% ) of both phenotypically discordant ( for parkinson disease , parkinsonism or lewy body dementia ) and concordant mz twin pairs . other molecular karyotype and genome sequence studies could not detect any cnv differences between mz twins that were discordant for cleft lip palate or multiple sclerosis , suggesting that post - zygotic genomic alterations are at least not a common cause of phenotypic discordance . here we studied a mz twin pair discordant for childhood cancer and a second primary cancer . r - spondin family members are modulators of the wnt/-catenin signaling pathway which plays a crucial role in cell growth , development and disease pathogenesis . rspo3 is frequently upregulated in human breast cancers and correlated with several tumor parameters , suggesting that it can function as a breast cancer oncogene . we propose that epigenetic changes , specifically hypermethylation of one brca1 allele constituted the first hit leading to inactivation of an important tumor suppressor gene and tumor initiation . germline mutations in brca1 are associated with hereditary breast cancer , ovarian cancer and some other malignancies ; however , so far they are generally not considered as a risk factor for childhood cancer . our index patient developed precursor b - cell lymphoblastic leukemia at the age of 4 y and 8 mo . leukemia , expression of the bcr - abl fusion protein leads to posttranscriptional downregulation of brca1 . one study reported partial hypermethylation and reduced expression of brca1 in primary and therapy - related acute myeloid leukemia , whereas others did not find abnormal brca1 methylation in leukemia samples . in the mz twin pair studied here , only one sister exhibited a brca1 epimutation in a mosaic state in multiple tissues / cell types , whereas the unaffected twin did not . genome - wide demethylation waves in the early embryo erase most germline methylation patterns , followed by de novo methylation and establishment of somatic methylation patterns . similar to our case , discordant methylation patterns at the kcnq1ot1 locus ( imprinting control region 2 on chromosome 11p15 ) have been described in a number of mz twin pairs discordant for beckwith - wiedemann syndrome , which is characterized by tumor predisposition and multiple congenital abnormalities . constitutive h19 epimutations ( imprinting control region 1 on chromosome 11p12 ) were found in several patients with sporadic wilms tumor without features of beckwith - wiedemann syndrome . somatic gene mutations and mosaicism ( with or without involvement of the germline ) are an important etiological mechanism for monogenic disorders with high de novo mutations rates such as duchenne muscular dystrophy , neurofibromatosis type 1 and retinoblastoma . in contrast , constitutive epimutations , characterized by soma - wide methylation abnormalities in functionally relevant cis - regulatory regions of disease genes , are an understudied phenomenon . constitutive epimutations in the dna mismatch repair gene mlh1 have been identified in a small number of mutation - negative cases of hereditary non - polyposis colorectal cancer ( hnpcc ) . in rare familial cases with dominant transmission of mosaic mlh1 methylation , the constitutional epimutation was linked to a single nucleotide variant in the 5 utr of mlh1 . similarly , constitutional epimutations in the dna mismatch repair gene msh2 , another rare cause of hnpcc , were linked to 3 end deletions of epcam , a gene directly upstream of msh2 . constitutive epimutations in the dapk1 gene that predispose to b cell chronic lymphocytic leukemia are caused by a point mutation upstream of the dapk1 promoter . neither sequencing nor microarray analysis of the 5 cis - regulatory region of brca1 revealed any detectable genetic differences between the discordant mz twin pair studied here . our study revealed the existence of a constitutive brca1 epimutation only in the affected sibling of a mz twin pair . compared with her healthy sister , the two - cancer twin showed reduced basal brca1 protein levels ( in untreated fibroblasts ) and a higher induction of protein expression by dna damage . upregulation of brca1 in irradiated cells of both the affected and the healthy twin was not mediated by demethylation of the brca1 promoter . in the affected twin the percentage of aberrantly methylated alleles remained constant ( at about 10% ) after dna damage and in the healthy twin the brca1 promoter was already completely demethylated in untreated cells . it is well known that the methylation of cpgs in 5 promoters that are usually protected from methylation in somatic tissues can suppress gene expression during development , differentiation and disease processes . , gene body methylation , may be more important for regulating the cell - context specific efficiency of transcription . the basal atm protein levels were also increased in the affected twin , although both twins showed equally low promoter methylation levels ( around 1% ) of the atm gene . brca1 is regulated by an atm - dependent mechanism and , on the other hand , essential for the recruitment of previously activated atm to the sites of dna damage . , therapy - related ) somatic event , it is plausible to assume that epigenetic silencing of one copy of the brca1 tumor suppressor gene in 25% body cells and differences in the dna damage response contributed to the discordant cancer phenotype in the affected twin . preliminary evidence suggests that brca1 promoter hypermethylation can also be found in blood cells of a subset of breast cancer patients . collectively , these results suggest that constitutive brca1 epimutations occur in normal tissues and may be associated with an increased cancer risk . considering the enormous epigenetic variability due to stochastic methylation errors or extrinsic variations during post - zygotic genome reprogramming , we can not rule out the possibility that epimutations in the affected twin in genes other than those studied are responsible for the discordant phenotype . nevertheless , 10% soma - wide promoter methylation in an essential tumor suppressor gene such as brca1 can be expected to contribute to a tumor susceptibility phenotype . in addition , we recruited 10 two - cancer patients as well as 10 carefully matched one - cancer patients ( same sex , same primary cancer , equal age at first diagnosis ) who did not develop a second malignancy . for sanger sequencing of the 5 utr and exon 1 of the brca1 gene , a 3.4 kb fragment on chromosome 17 ( 41.277.11241.280.530 bps , ensembl release 64 ) was divided into 11 amplicons ( table s1 ) . for amplicons 15 and 8 , the pcr reaction mixture ( 25 l ) consisted of 2.5 l 10x pcr buffer , 20 mm mgcl2 , 0.5 l 10 mm dntp mix , 1 l ( 10 pmol ) of each forward and reverse primer , 0.2 l ( 1 u ) faststart taq dna polymerase ( roche diagnostics , mannheim , germany ) , 18.8 l pcr - grade water , and 1 l ( ~100 ng ) template dna . for amplicons 6 , 7 , 8 , 10 , and 11 , the pcr mixture ( 25 l ) contained 25 mm ammonium sulfate , 750 mm tris - hcl , 0.1% tween 20 , 240 m dntps , 2.4 mm magnesium sulfate , 2.4x pcrx enhancer solution ( invitrogen ) , 0.4 m of each primer , 1 unit of platinum taq ( invitrogen ) , and 1 l ( ~100 ng ) template dna . customized antibody microarrays for quantification of brca1 ( antibody # sc-1553 , santa cruz biotechnology , heidelberg , germany ) and several other dna repair - associated proteins ( atm , brca2 , mlh1 , rad51 , and tp53 ) were prepared by spotting triplicate drops , each containing approximately 0.5 pg antibody onto nitrocellulose - coated slides ( oncyte , nitrocellulose 16 multi - pad slides , grace bio - labs , bend , or , usa ) , using a sciflexarrayer 3 non - contact spotter ( scienion , berlin , germany ) . high - resolution screening for microdeletions and duplications was performed with the affymetrix genechip genome wide human snp array 6.0 and the genechip genome wide snp sty assay kit 5.0/6.0 , following the protocol developed by the manufacturer .

neurodegeneration is known as the progressive loss of structure or function of neurons , including death of neurons . many neurodegenerative diseases including alzheimer 's disease , parkinson 's disease , and huntington 's disease occur as a result of neurodegenerative processes . regenerative therapy could be a promising approach as the regeneration of lost or altered cellular functions , which could significantly reverse functional decline to an extent that raises the patient 's survival rate and physiological function . as a representative of the neurodegenerative disorders , alzheimer 's disease ( ad ) is pathologically characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions . neurofibrillary tangles , and senile plaques involve the basal forebrain cholinergic system , amygdala , hippocampus , and cortical areas . neural loss results in gross atrophy of the affected regions , including degeneration in the temporal lobe and parietal lobe , and parts of the frontal cortex and cingulate gyrus . the national institute of neurological and communicative disorders and stroke ( nincds ) established the most commonly used criteria for ad . according to these criteria , the presence of cognitive impairments ( learning & memory , language , perceptual skills , orientation ) , and a suspected dementia syndrome confirmed by neuropsychological testing are contributed to the clinical diagnosis of ad . the observable symptoms are often mistakenly thought to be age - related concerns , or manifestations of stress . in the early stage , the most common symptom is unable to acquire new memories , observed as difficulty in recalling recently observed events . when ad is suspected , the diagnosis is usually confirmed with behavioral assessments and cognitive tests , often followed by a brain scan if available . nowadays , specific treatments for particular symptoms of ad are available . however , specific disease - modifying treatments aimed at preventing or reversing the basic pathophysiologic processes of ad still remain under investigation . in this literature , the recent research advances in stem cell treatments of ad are reviewed from an experiment to a clinical research . currently , there is no proven cure for ad . in terms of drug therapy , no drug treatment can reverse , stop , or even slow this inexorable neurodegenerative process . also , non - drug treatments , including gene therapy and behavioral interventions , can only bring temporary symptomatic relief and not result in halting the progression of these diseases . in recent decades , neurogenesis has been proved to exist in restricted regions of the adult brain in many kinds of species including humans . this continuing neurogenesis in the subventricular zone , olfactory bulb , and hippocampal dentate gyrus is supported by the identification of neural stem cells ( nscs ) , suggesting that the adult central nervous system ( cns ) may be amenable to the cell intervention . a variety of animal models have been developed to examine the etiology of ad and to determine the responses of endogenous and/or transplanted stem cells to the pathological microenvironment of the brain . under many conditions , adult neurogenesis is impaired , and the dysfunctional neurogenesis , both decreased and increased , has been reported for ad transgenic models . increasing research evidences in recent decades may promise a bright future for stem cell based neuroreplacement therapies for ad . the adult - born neurons in the diseased brain seem to be a good candidate for those lost or apoptotic neurons . because stem cells can be genetically modified in vitro and have high migratory capacity after transplantation into the brain , they can be an efficient way to delivery neurotrophic factors or enhance gene expression that can modify the course of the disease . since neural progenitor / stem cells have been proven to exist in the adult cns and to be involved in the neurogenesis process , the activation of endogenous neural progenitor / stem cells populations that can migrate to the injured regions , proliferate and functionally integrate into the existing circuit represents a significant strategy to promote neural regeneration in the diseased brain . this activation within the brain is to protect the remaining tissues and prevent secondary neuron loss through the production of neurotrophic and neuroprotective factors , such as brain derived neuronal factor ( bdnf ) and vascular endothelial growth factor . a recent research has demonstrated that the self - repair in the adult brain can be augmented by the infusion of growth factors to activate endogenous neural precursor cells that contribute to new tissue formation and functional recovery after stroke . currently , it is widely accepted that neurogenesis in the hippocampus are involved in learning and memory formation . the hippocampus - dependent learning tasks can significantly increase the proliferation of endogenous neuronal progenitors , survival of new neurons and , the task performance by animals correlates positively with the amount of adult born neurons . alterations in the microenvironment where the endogenous nscs reside play an important role in nsc activation . in ad brain , for example , immune responses include the activation of microglia and astrocytes around the senile plaques area and t - lymphocyte infiltration into the damaged brain . these cells produce cytokines and other molecules that promote or inhibit the neurogenic function of the nscs . therefore , it is possible for damaged cells to be replaced from endogenous nsc pools . studies have also suggested that the capacity of endogenous nscs to compensate for lost cells is limited . in spite of the activated proliferation , obviously , most of the new migrated neurons in and near the injured area die before differentiating into functional neurons , possibly because of a lack of factors and stimulation to support their survival and differentiation ; thus , only 0.2% of the dead neurons are replaced . however , it has not been determined whether these effects depend directly on the promotion of neuronal regeneration by nscs , or whether accompanying events , such as enhanced glial regeneration and other types of trophic support , are more important . moreover , a key issue in the field of neuronal regeneration is that newly generated neurons need to make the appropriate connections , although the details of this process are still largely unknown . further studies are needed to clarify how newly generated neurons are associated with neurological improvement and to elucidate the comprehensive mechanism regulating the endogenous regeneration system . the success of isolation and easy gene - engineered modification of embryonic stem cells ( escs ) and nscs in vitro profoundly provides researchers a promising tool to replace the injured neurons in the ad brain . therefore , the cell transplantation strategy has given rise to hopes for clinical application of these in vitro produced neuronal cells in the cell replacement procedures for ad . since chronic inflammation is a characteristic property of ad brain , transplantation of neuronal precursor cells ( npcs ) has been proven to particularly inhibit ongoing inflammatory reactivity . researchers have tested that the intrahippocampal transplantation of npcs is effective in attenuating inflammatory responses and plays a neuroprotection role in beta - amyloid 42 ( a-42 ) peptide - injected rat hippocampus , indicating effects of npcs transplantation in ad models are consistent with cellular actions to attenuate inflammatory reactivity . the progressive degeneration of cholinergic neurons occurs in the forebrain cholinergic projection system especially in the nucleus basalis of meynert . moghadam et al used escs - derived npcs to treat ibotenic acid - induced ad models in order to investigate the production of cholinergic neurons derived from engrafted cells . after transplantation , not only a significant behavioral improvement in memory deficits was observed , but also the majority ( about 70% ) of the npcs retained neuronal phenotype and about 40% of them had a cholinergic cell phenotype with no tumor formation . similarly , in the a-induced ad model , the transplantation of escs - derived npcs into the injured hippocampus can also improve the memory dysfunction of the ad models . furthermore , the transplanted cells demonstrate characteristics of proper synapse formation between host and grafted neural cells . recently , another group reported the in vivo functional integration of the human escs - derived nscs after cell transplantation . at 6 months after transplantation , human axons identified with the human - specific middle - weight neurofilament protein antibody were found inside the stratum radiatum . alongside these projections , small patches of human synaptophysin immunoreactivity were detected , suggesting the formation of presynaptic terminals . hippocampal grafts placed in the dentate gyrus were projected to both the ipsilateral and contralateral pyramidal cell layers , while axons of donor neurons placed in the motor cortex extended via the external and internal capsules into the cervical spinal cord and via the corpus callosum into the contralateral cortex . their data indicate that neurons derived from human pluripotent stem cells ( pscs ) are endowed with a remarkable potential to establish orthotopic long - range projections in the adult mammalian brain . meanwhile , in another mouse ad model , the transplantation of nscs was reported to improve cognition function mediated by the neurotrophic factor bdnf . the differentiation of transplanted nscs in response to local cues in the brain suggests that environmental cues have the ability to direct the fate of stem cells to become the specific , terminally differentiated cells that are required to restore functions . alternatively , stem cells are differentiated prior to transplantation and then directed to the correct areas through surgery . however , care should be taken with the characterization of these cells in regard to their multipotentiality and genetic stability with increasing passages in culture , as transformed cells may contribute to the formation of tumors . efforts to investigate the pathophysiology of human ad are hampered by the lack of genuine in - vitro models . stem cells generated by induced direct reprogramming of adult somatic cells using are termed as ipscs , offering paradigm shifting opportunities by providing specific / personalized models for studying ad , and personalized renewable source of cells for practical autologous cell therapies and regenerative medicine applications , that avoid immune rejection . the possibility of using ipscs as a tool for development of such ad patient specific model systems , however , remains at best challenging . in 2006 , takahashi et al discovered that four transcription factors could reprogram mouse fibroblasts to a pluripotent state . ever since then ipscs have created excitement among researchers , as ipcs are more available , easier to make , and less ethically conflicted than escs . this has been widely regarded as a milestone advance in stem cell research , as it may allow researchers to obtain pscs without the controversial use of embryos . because ipscs are developed from an adult somatic cell , it is believed that ipscs can avoid immunogenic responses . ipscs are a specific cell type compromised by disease , even lost in patients , that can be recreated in culture . furthermore , ipscs have the potential to provide an unlimited source for any desired cell type . ultimately , aside from being an exciting research tool to probe embryogenesis and disease pathogenesis , ipscs are so - called disease modeling for drug screening , identifying novel drugs to treat diseases and patient - tailored cell therapy . most ( 9095% ) of ad patients are sporadic populations ( sad ) while 510% patients are diagnosed as having early - onset ad , half of whom are familiar ad ( fad ) . fad is caused by a mutation in at least one of three genes : presenilin 1/2 and amyloid precursor protein ( app ) . obviously , ipscs technology contributes to capture the genomes of ad patients and to generate live cellular models of both the fad and sad . these models allow us to identify the earliest events of ad and to investigate aspects of ad pathogenesis that are not replicated in animal models . yagi 's group recently pioneered to generate ipscs from fibroblasts of fad patients with mutations in presenilin 1 ( a246e ) and presenilin 2 ( n141i ) , and characterized the differentiation of these cells into neurons . their remarkable data showed that fad - ipsc - differentiated neurons increased a42 secretion , recapitulating the molecular pathogenesis of mutant presenilins . furthermore , a42 secreted from these neurons sharply responded to -secretase inhibitors and modulators , indicating the potential for identification and validation of candidate drugs . this finding significantly demonstrates that the fad - ipsc - derived neuron is a valid model of ad and provides an innovative strategy for the study of age - related neurodegenerative diseases . marchetto et al in their study of rett syndrome using ipscs , reported the in vitro differentiation of ipscs into neurons that contained glutamatergic synapses and were capable of generating spontaneous synaptic activity . the spontaneous synaptic activity observed in the differentiated neurons hinted that ipsc technology can be used to study not only human neurons but also patient - specific neural networks . previous research has reported the marked differences in differentiation propensity between psc lines , even between ipsc lines generated from the same individual . and furthermore , differentiation variability has become an important issue . this issue becomes more complex if this novel method is to investigate a disease with unclear developmental changes . thus , it is unclear if ipscs and ipsc - derived npcs from ad patients would generate neurons differently than control cells , such as embryonic stem cells and neural stem cells . despite some successes in animal models , ipscs technology is not yet ready for human trials . current ipscs protocols can not efficiently eliminate undifferentiated cells and , tend to be oncogenic and form teratomas , just like escs . additionally , most patient - specific ipscs have been generated using integrating vectors , which may not get silenced efficiently or can disrupt endogenous genes that are a potential impediment in human ipscs therapy . many mouse ipscs harbor epigenetic abnormalities are reported in recent studies , which may develop genetic mutation on prolonged culture and continue to retain epigenetic memory of their donor cells . in terms of the ipscs technique , ( 1)ethical hurdles : undoubtedly , it turns out that ipscs are not entirely problem - free . researchers working with ipscs still must countenance certain ethical concerns , and they may also face newly discovered scientific hurdles . ( 2)scientific hurdles : whether ipscs are truly equivalent to escs ? some studies have raised the possibility of significant differences . lanza group compared differentiated cells derived from a series of ipscs lines and escs lines , and found thatboth cells differentiated to form blood cells , vascular cells or retinal cells . however , the ipscs did so at a significantly lower rate and had a higher rate of cell death . another study raised a question about whether ipscs can serve as tools for modeling disease . researchers compared escs with ipscs that carried the mutation for the mental impairment disorder fragile x syndrome . some scientists hope that the retroviruses which these problems arise from can be used to generate ipscs . since neural progenitor / stem cells have been proven to exist in the adult cns and to be involved in the neurogenesis process , the activation of endogenous neural progenitor / stem cells populations that can migrate to the injured regions , proliferate and functionally integrate into the existing circuit represents a significant strategy to promote neural regeneration in the diseased brain . this activation within the brain is to protect the remaining tissues and prevent secondary neuron loss through the production of neurotrophic and neuroprotective factors , such as brain derived neuronal factor ( bdnf ) and vascular endothelial growth factor . a recent research has demonstrated that the self - repair in the adult brain can be augmented by the infusion of growth factors to activate endogenous neural precursor cells that contribute to new tissue formation and functional recovery after stroke . currently , it is widely accepted that neurogenesis in the hippocampus are involved in learning and memory formation . the hippocampus - dependent learning tasks can significantly increase the proliferation of endogenous neuronal progenitors , survival of new neurons and , the task performance by animals correlates positively with the amount of adult born neurons . alterations in the microenvironment where the endogenous nscs reside play an important role in nsc activation . in ad brain , for example , immune responses include the activation of microglia and astrocytes around the senile plaques area and t - lymphocyte infiltration into the damaged brain . these cells produce cytokines and other molecules that promote or inhibit the neurogenic function of the nscs . therefore , it is possible for damaged cells to be replaced from endogenous nsc pools . studies have also suggested that the capacity of endogenous nscs to compensate for lost cells is limited . in spite of the activated proliferation , obviously , most of the new migrated neurons in and near the injured area die before differentiating into functional neurons , possibly because of a lack of factors and stimulation to support their survival and differentiation ; thus , only 0.2% of the dead neurons are replaced . however , it has not been determined whether these effects depend directly on the promotion of neuronal regeneration by nscs , or whether accompanying events , such as enhanced glial regeneration and other types of trophic support , are more important . moreover , a key issue in the field of neuronal regeneration is that newly generated neurons need to make the appropriate connections , although the details of this process are still largely unknown . further studies are needed to clarify how newly generated neurons are associated with neurological improvement and to elucidate the comprehensive mechanism regulating the endogenous regeneration system . the success of isolation and easy gene - engineered modification of embryonic stem cells ( escs ) and nscs in vitro profoundly provides researchers a promising tool to replace the injured neurons in the ad brain . therefore , the cell transplantation strategy has given rise to hopes for clinical application of these in vitro produced neuronal cells in the cell replacement procedures for ad . since chronic inflammation is a characteristic property of ad brain , transplantation of neuronal precursor cells ( npcs ) has been proven to particularly inhibit ongoing inflammatory reactivity . researchers have tested that the intrahippocampal transplantation of npcs is effective in attenuating inflammatory responses and plays a neuroprotection role in beta - amyloid 42 ( a-42 ) peptide - injected rat hippocampus , indicating effects of npcs transplantation in ad models are consistent with cellular actions to attenuate inflammatory reactivity . the progressive degeneration of cholinergic neurons occurs in the forebrain cholinergic projection system especially in the nucleus basalis of meynert . moghadam et al used escs - derived npcs to treat ibotenic acid - induced ad models in order to investigate the production of cholinergic neurons derived from engrafted cells . after transplantation , not only a significant behavioral improvement in memory deficits was observed , but also the majority ( about 70% ) of the npcs retained neuronal phenotype and about 40% of them had a cholinergic cell phenotype with no tumor formation . similarly , in the a-induced ad model , the transplantation of escs - derived npcs into the injured hippocampus can also improve the memory dysfunction of the ad models . furthermore , the transplanted cells demonstrate characteristics of proper synapse formation between host and grafted neural cells . recently , another group reported the in vivo functional integration of the human escs - derived nscs after cell transplantation . at 6 months after transplantation , human axons identified with the human - specific middle - weight neurofilament protein antibody were found inside the stratum radiatum . alongside these projections , small patches of human synaptophysin immunoreactivity were detected , suggesting the formation of presynaptic terminals . hippocampal grafts placed in the dentate gyrus were projected to both the ipsilateral and contralateral pyramidal cell layers , while axons of donor neurons placed in the motor cortex extended via the external and internal capsules into the cervical spinal cord and via the corpus callosum into the contralateral cortex . their data indicate that neurons derived from human pluripotent stem cells ( pscs ) are endowed with a remarkable potential to establish orthotopic long - range projections in the adult mammalian brain . meanwhile , in another mouse ad model , the transplantation of nscs was reported to improve cognition function mediated by the neurotrophic factor bdnf . the differentiation of transplanted nscs in response to local cues in the brain suggests that environmental cues have the ability to direct the fate of stem cells to become the specific , terminally differentiated cells that are required to restore functions . alternatively , stem cells are differentiated prior to transplantation and then directed to the correct areas through surgery . however , care should be taken with the characterization of these cells in regard to their multipotentiality and genetic stability with increasing passages in culture , as transformed cells may contribute to the formation of tumors . efforts to investigate the pathophysiology of human ad are hampered by the lack of genuine in - vitro models . stem cells generated by induced direct reprogramming of adult somatic cells using are termed as ipscs , offering paradigm shifting opportunities by providing specific / personalized models for studying ad , and personalized renewable source of cells for practical autologous cell therapies and regenerative medicine applications , that avoid immune rejection . the possibility of using ipscs as a tool for development of such ad patient specific model systems , however , remains at best challenging . in 2006 , takahashi et al discovered that four transcription factors could reprogram mouse fibroblasts to a pluripotent state . ever since then ipscs have created excitement among researchers , as ipcs are more available , easier to make , and less ethically conflicted than escs . this has been widely regarded as a milestone advance in stem cell research , as it may allow researchers to obtain pscs without the controversial use of embryos . because ipscs are developed from an adult somatic cell , it is believed that ipscs can avoid immunogenic responses . ipscs are a specific cell type compromised by disease , even lost in patients , that can be recreated in culture . furthermore , ipscs have the potential to provide an unlimited source for any desired cell type . ultimately , aside from being an exciting research tool to probe embryogenesis and disease pathogenesis , ipscs are so - called disease modeling for drug screening , identifying novel drugs to treat diseases and patient - tailored cell therapy . most ( 9095% ) of ad patients are sporadic populations ( sad ) while 510% patients are diagnosed as having early - onset ad , half of whom are familiar ad ( fad ) . fad is caused by a mutation in at least one of three genes : presenilin 1/2 and amyloid precursor protein ( app ) . obviously , ipscs technology contributes to capture the genomes of ad patients and to generate live cellular models of both the fad and sad . these models allow us to identify the earliest events of ad and to investigate aspects of ad pathogenesis that are not replicated in animal models . yagi 's group recently pioneered to generate ipscs from fibroblasts of fad patients with mutations in presenilin 1 ( a246e ) and presenilin 2 ( n141i ) , and characterized the differentiation of these cells into neurons . their remarkable data showed that fad - ipsc - differentiated neurons increased a42 secretion , recapitulating the molecular pathogenesis of mutant presenilins . furthermore , a42 secreted from these neurons sharply responded to -secretase inhibitors and modulators , indicating the potential for identification and validation of candidate drugs . this finding significantly demonstrates that the fad - ipsc - derived neuron is a valid model of ad and provides an innovative strategy for the study of age - related neurodegenerative diseases . marchetto et al in their study of rett syndrome using ipscs , reported the in vitro differentiation of ipscs into neurons that contained glutamatergic synapses and were capable of generating spontaneous synaptic activity . the spontaneous synaptic activity observed in the differentiated neurons hinted that ipsc technology can be used to study not only human neurons but also patient - specific neural networks . previous research has reported the marked differences in differentiation propensity between psc lines , even between ipsc lines generated from the same individual . and this issue becomes more complex if this novel method is to investigate a disease with unclear developmental changes . thus , it is unclear if ipscs and ipsc - derived npcs from ad patients would generate neurons differently than control cells , such as embryonic stem cells and neural stem cells . despite some successes in animal models , ipscs technology is not yet ready for human trials . current ipscs protocols can not efficiently eliminate undifferentiated cells and , tend to be oncogenic and form teratomas , just like escs . additionally , most patient - specific ipscs have been generated using integrating vectors , which may not get silenced efficiently or can disrupt endogenous genes that are a potential impediment in human ipscs therapy . many mouse ipscs harbor epigenetic abnormalities are reported in recent studies , which may develop genetic mutation on prolonged culture and continue to retain epigenetic memory of their donor cells . in terms of the ipscs technique , ( 1)ethical hurdles : undoubtedly , it turns out that ipscs are not entirely problem - free . researchers working with ipscs still must countenance certain ethical concerns , and they may also face newly discovered scientific hurdles . lanza group compared differentiated cells derived from a series of ipscs lines and escs lines , and found thatboth cells differentiated to form blood cells , vascular cells or retinal cells . however , the ipscs did so at a significantly lower rate and had a higher rate of cell death . another study raised a question about whether ipscs can serve as tools for modeling disease . researchers compared escs with ipscs that carried the mutation for the mental impairment disorder fragile x syndrome . some scientists hope that the retroviruses which these problems arise from can be used to generate ipscs . although stem cell - based replacement strategies carried out in animal models have shown promising results , there are still many hurdles to overcome before these approaches can be translated into the ad patients . one major challenge is the development of a safe method to deliver stem cells to the injury region . in addition , the stage of differentiation of those cells needs careful consideration : fully differentiated cells are associated with a smaller efficiency due to poor viability , while undifferentiated cells present a higher risk of undirected differentiation and uncontrolled proliferation . adult neurogenesis is important for cellular therapy and physiopathology of the cns , as for development and pharmacology of the adult brain . hence , the role , contribution and significance of newborn neurons in the adult brain remain to be fully elucidated and understood . one of the main limitations in determining and understanding the role of newborn neurons in the adult brain is the use of bromodeoxyuridine ( brdu ) for studying neurogenesis . the microenvironment in which stem cells are placed also needs consideration , as local soluble factors are likely to affect differentiation events in the tissue . environmental factors need to be included : pro - inflammatory cytokines are associated with a negative effect on neural differentiation , while anti - inflammatory cytokines may have the opposite effect . the inflammatory status of the brain and the possible activation of inflammatory responses therefore need to be considered with cell replacement strategies . in all , developing our understanding of the processes controlling the activation , migration and differentiation of stem cells will be a critical step towards the usage of stem cells for new regenerative therapies . it has been a century since the first description of ad . in the past decades , tremendous advances in understanding of the molecular pathogenesis of ad have been springing out . however , no proven effective treatment is able to delay the onset or slow the progression of ad . now , many new therapies directly targeting the mechanisms underlying ad are now in the pipeline . a combination of psychosocial , behavioral , and pharmacologic strategies aims at slowing the process of ad and preserving quality of life for as long as possible . until medical research discovers definitive disease modifying treatments for patients with ad , we must continue to maximize all available resources to provide the best possible individualized patient - centered and family care . despite a better understanding of the pathology of cognitive impairments and clinical features of ad , which have aided diagnosis and management of the disease , further work is required to improve screening and decrease the burden of care on healthcare systems and families . furthermore , more research is needed to elucidate the mechanisms of the disease to enable new drug targets to be developed based on biological models , which may provide novel treatments for the prevention and management of ad . the situation for neuronal replacement aiming at functional restoration in ad is extremely complex because the stem cells have to be pre - differentiated in vitro to many different types of neuroblasts for subsequent implantation in a large number of brain areas . however , to give long - lasting symptomatic benefit , a cholinergic cell replacement approach will require intact target cells and host neurons that the new cholinergic neurons can act on . stem cell - based cell replacement strategies are very far from clinical application in ad . but the neuroreplacement strategy will undoubtedly become more feasible as we advance our understanding of the pathogenesis of ad and foster creativity in research aiming to elucidate the physiological role of nscs in the adult brain . also , the nscs indeed exist in the complexity and intricacy of the architecture of the human brain , the mechanisms and therapeutic potential of nscs just need to be further explored .

alzheimer 's disease , a progressive neurodegenerative illness , is the most common form of dementia . so far , there is neither an effective prevention nor a cure for alzheimer 's disease . in recent decades , stem cell therapy has been one of the most promising treatments for alzheimer 's disease patients . this article aims to summarize the current progress in the stem cell treatments for alzheimer 's disease from an experiment to a clinical research .

INTRODUCTION STEM CELL STRATEGY Endogenous neural progenitor/stem cells Exogenous neural progenitor/stem cells Induced-PSCs (iPSCs) PROSPECTIVE QUESTIONS CONCLUSIONS

neurodegeneration is known as the progressive loss of structure or function of neurons , including death of neurons . many neurodegenerative diseases including alzheimer 's disease , parkinson 's disease , and huntington 's disease occur as a result of neurodegenerative processes . as a representative of the neurodegenerative disorders , alzheimer 's disease ( ad ) is pathologically characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions . neural loss results in gross atrophy of the affected regions , including degeneration in the temporal lobe and parietal lobe , and parts of the frontal cortex and cingulate gyrus . the national institute of neurological and communicative disorders and stroke ( nincds ) established the most commonly used criteria for ad . the observable symptoms are often mistakenly thought to be age - related concerns , or manifestations of stress . in the early stage , the most common symptom is unable to acquire new memories , observed as difficulty in recalling recently observed events . when ad is suspected , the diagnosis is usually confirmed with behavioral assessments and cognitive tests , often followed by a brain scan if available . nowadays , specific treatments for particular symptoms of ad are available . in this literature , the recent research advances in stem cell treatments of ad are reviewed from an experiment to a clinical research . currently , there is no proven cure for ad . in terms of drug therapy , no drug treatment can reverse , stop , or even slow this inexorable neurodegenerative process . in recent decades , neurogenesis has been proved to exist in restricted regions of the adult brain in many kinds of species including humans . this continuing neurogenesis in the subventricular zone , olfactory bulb , and hippocampal dentate gyrus is supported by the identification of neural stem cells ( nscs ) , suggesting that the adult central nervous system ( cns ) may be amenable to the cell intervention . a variety of animal models have been developed to examine the etiology of ad and to determine the responses of endogenous and/or transplanted stem cells to the pathological microenvironment of the brain . under many conditions , adult neurogenesis is impaired , and the dysfunctional neurogenesis , both decreased and increased , has been reported for ad transgenic models . increasing research evidences in recent decades may promise a bright future for stem cell based neuroreplacement therapies for ad . the adult - born neurons in the diseased brain seem to be a good candidate for those lost or apoptotic neurons . because stem cells can be genetically modified in vitro and have high migratory capacity after transplantation into the brain , they can be an efficient way to delivery neurotrophic factors or enhance gene expression that can modify the course of the disease . since neural progenitor / stem cells have been proven to exist in the adult cns and to be involved in the neurogenesis process , the activation of endogenous neural progenitor / stem cells populations that can migrate to the injured regions , proliferate and functionally integrate into the existing circuit represents a significant strategy to promote neural regeneration in the diseased brain . this activation within the brain is to protect the remaining tissues and prevent secondary neuron loss through the production of neurotrophic and neuroprotective factors , such as brain derived neuronal factor ( bdnf ) and vascular endothelial growth factor . a recent research has demonstrated that the self - repair in the adult brain can be augmented by the infusion of growth factors to activate endogenous neural precursor cells that contribute to new tissue formation and functional recovery after stroke . currently , it is widely accepted that neurogenesis in the hippocampus are involved in learning and memory formation . alterations in the microenvironment where the endogenous nscs reside play an important role in nsc activation . these cells produce cytokines and other molecules that promote or inhibit the neurogenic function of the nscs . studies have also suggested that the capacity of endogenous nscs to compensate for lost cells is limited . in spite of the activated proliferation , obviously , most of the new migrated neurons in and near the injured area die before differentiating into functional neurons , possibly because of a lack of factors and stimulation to support their survival and differentiation ; thus , only 0.2% of the dead neurons are replaced . moreover , a key issue in the field of neuronal regeneration is that newly generated neurons need to make the appropriate connections , although the details of this process are still largely unknown . the success of isolation and easy gene - engineered modification of embryonic stem cells ( escs ) and nscs in vitro profoundly provides researchers a promising tool to replace the injured neurons in the ad brain . therefore , the cell transplantation strategy has given rise to hopes for clinical application of these in vitro produced neuronal cells in the cell replacement procedures for ad . since chronic inflammation is a characteristic property of ad brain , transplantation of neuronal precursor cells ( npcs ) has been proven to particularly inhibit ongoing inflammatory reactivity . the progressive degeneration of cholinergic neurons occurs in the forebrain cholinergic projection system especially in the nucleus basalis of meynert . after transplantation , not only a significant behavioral improvement in memory deficits was observed , but also the majority ( about 70% ) of the npcs retained neuronal phenotype and about 40% of them had a cholinergic cell phenotype with no tumor formation . similarly , in the a-induced ad model , the transplantation of escs - derived npcs into the injured hippocampus can also improve the memory dysfunction of the ad models . recently , another group reported the in vivo functional integration of the human escs - derived nscs after cell transplantation . hippocampal grafts placed in the dentate gyrus were projected to both the ipsilateral and contralateral pyramidal cell layers , while axons of donor neurons placed in the motor cortex extended via the external and internal capsules into the cervical spinal cord and via the corpus callosum into the contralateral cortex . their data indicate that neurons derived from human pluripotent stem cells ( pscs ) are endowed with a remarkable potential to establish orthotopic long - range projections in the adult mammalian brain . the differentiation of transplanted nscs in response to local cues in the brain suggests that environmental cues have the ability to direct the fate of stem cells to become the specific , terminally differentiated cells that are required to restore functions . alternatively , stem cells are differentiated prior to transplantation and then directed to the correct areas through surgery . however , care should be taken with the characterization of these cells in regard to their multipotentiality and genetic stability with increasing passages in culture , as transformed cells may contribute to the formation of tumors . stem cells generated by induced direct reprogramming of adult somatic cells using are termed as ipscs , offering paradigm shifting opportunities by providing specific / personalized models for studying ad , and personalized renewable source of cells for practical autologous cell therapies and regenerative medicine applications , that avoid immune rejection . in 2006 , takahashi et al discovered that four transcription factors could reprogram mouse fibroblasts to a pluripotent state . ever since then ipscs have created excitement among researchers , as ipcs are more available , easier to make , and less ethically conflicted than escs . this has been widely regarded as a milestone advance in stem cell research , as it may allow researchers to obtain pscs without the controversial use of embryos . because ipscs are developed from an adult somatic cell , it is believed that ipscs can avoid immunogenic responses . ipscs are a specific cell type compromised by disease , even lost in patients , that can be recreated in culture . ultimately , aside from being an exciting research tool to probe embryogenesis and disease pathogenesis , ipscs are so - called disease modeling for drug screening , identifying novel drugs to treat diseases and patient - tailored cell therapy . fad is caused by a mutation in at least one of three genes : presenilin 1/2 and amyloid precursor protein ( app ) . these models allow us to identify the earliest events of ad and to investigate aspects of ad pathogenesis that are not replicated in animal models . yagi 's group recently pioneered to generate ipscs from fibroblasts of fad patients with mutations in presenilin 1 ( a246e ) and presenilin 2 ( n141i ) , and characterized the differentiation of these cells into neurons . their remarkable data showed that fad - ipsc - differentiated neurons increased a42 secretion , recapitulating the molecular pathogenesis of mutant presenilins . the spontaneous synaptic activity observed in the differentiated neurons hinted that ipsc technology can be used to study not only human neurons but also patient - specific neural networks . thus , it is unclear if ipscs and ipsc - derived npcs from ad patients would generate neurons differently than control cells , such as embryonic stem cells and neural stem cells . despite some successes in animal models , ipscs technology is not yet ready for human trials . additionally , most patient - specific ipscs have been generated using integrating vectors , which may not get silenced efficiently or can disrupt endogenous genes that are a potential impediment in human ipscs therapy . many mouse ipscs harbor epigenetic abnormalities are reported in recent studies , which may develop genetic mutation on prolonged culture and continue to retain epigenetic memory of their donor cells . in terms of the ipscs technique , ( 1)ethical hurdles : undoubtedly , it turns out that ipscs are not entirely problem - free . researchers working with ipscs still must countenance certain ethical concerns , and they may also face newly discovered scientific hurdles . lanza group compared differentiated cells derived from a series of ipscs lines and escs lines , and found thatboth cells differentiated to form blood cells , vascular cells or retinal cells . however , the ipscs did so at a significantly lower rate and had a higher rate of cell death . since neural progenitor / stem cells have been proven to exist in the adult cns and to be involved in the neurogenesis process , the activation of endogenous neural progenitor / stem cells populations that can migrate to the injured regions , proliferate and functionally integrate into the existing circuit represents a significant strategy to promote neural regeneration in the diseased brain . this activation within the brain is to protect the remaining tissues and prevent secondary neuron loss through the production of neurotrophic and neuroprotective factors , such as brain derived neuronal factor ( bdnf ) and vascular endothelial growth factor . a recent research has demonstrated that the self - repair in the adult brain can be augmented by the infusion of growth factors to activate endogenous neural precursor cells that contribute to new tissue formation and functional recovery after stroke . currently , it is widely accepted that neurogenesis in the hippocampus are involved in learning and memory formation . the hippocampus - dependent learning tasks can significantly increase the proliferation of endogenous neuronal progenitors , survival of new neurons and , the task performance by animals correlates positively with the amount of adult born neurons . alterations in the microenvironment where the endogenous nscs reside play an important role in nsc activation . these cells produce cytokines and other molecules that promote or inhibit the neurogenic function of the nscs . in spite of the activated proliferation , obviously , most of the new migrated neurons in and near the injured area die before differentiating into functional neurons , possibly because of a lack of factors and stimulation to support their survival and differentiation ; thus , only 0.2% of the dead neurons are replaced . moreover , a key issue in the field of neuronal regeneration is that newly generated neurons need to make the appropriate connections , although the details of this process are still largely unknown . further studies are needed to clarify how newly generated neurons are associated with neurological improvement and to elucidate the comprehensive mechanism regulating the endogenous regeneration system . the success of isolation and easy gene - engineered modification of embryonic stem cells ( escs ) and nscs in vitro profoundly provides researchers a promising tool to replace the injured neurons in the ad brain . therefore , the cell transplantation strategy has given rise to hopes for clinical application of these in vitro produced neuronal cells in the cell replacement procedures for ad . since chronic inflammation is a characteristic property of ad brain , transplantation of neuronal precursor cells ( npcs ) has been proven to particularly inhibit ongoing inflammatory reactivity . researchers have tested that the intrahippocampal transplantation of npcs is effective in attenuating inflammatory responses and plays a neuroprotection role in beta - amyloid 42 ( a-42 ) peptide - injected rat hippocampus , indicating effects of npcs transplantation in ad models are consistent with cellular actions to attenuate inflammatory reactivity . the progressive degeneration of cholinergic neurons occurs in the forebrain cholinergic projection system especially in the nucleus basalis of meynert . after transplantation , not only a significant behavioral improvement in memory deficits was observed , but also the majority ( about 70% ) of the npcs retained neuronal phenotype and about 40% of them had a cholinergic cell phenotype with no tumor formation . similarly , in the a-induced ad model , the transplantation of escs - derived npcs into the injured hippocampus can also improve the memory dysfunction of the ad models . recently , another group reported the in vivo functional integration of the human escs - derived nscs after cell transplantation . at 6 months after transplantation , human axons identified with the human - specific middle - weight neurofilament protein antibody were found inside the stratum radiatum . hippocampal grafts placed in the dentate gyrus were projected to both the ipsilateral and contralateral pyramidal cell layers , while axons of donor neurons placed in the motor cortex extended via the external and internal capsules into the cervical spinal cord and via the corpus callosum into the contralateral cortex . their data indicate that neurons derived from human pluripotent stem cells ( pscs ) are endowed with a remarkable potential to establish orthotopic long - range projections in the adult mammalian brain . the differentiation of transplanted nscs in response to local cues in the brain suggests that environmental cues have the ability to direct the fate of stem cells to become the specific , terminally differentiated cells that are required to restore functions . alternatively , stem cells are differentiated prior to transplantation and then directed to the correct areas through surgery . however , care should be taken with the characterization of these cells in regard to their multipotentiality and genetic stability with increasing passages in culture , as transformed cells may contribute to the formation of tumors . in 2006 , takahashi et al discovered that four transcription factors could reprogram mouse fibroblasts to a pluripotent state . this has been widely regarded as a milestone advance in stem cell research , as it may allow researchers to obtain pscs without the controversial use of embryos . because ipscs are developed from an adult somatic cell , it is believed that ipscs can avoid immunogenic responses . ipscs are a specific cell type compromised by disease , even lost in patients , that can be recreated in culture . furthermore , ipscs have the potential to provide an unlimited source for any desired cell type . ultimately , aside from being an exciting research tool to probe embryogenesis and disease pathogenesis , ipscs are so - called disease modeling for drug screening , identifying novel drugs to treat diseases and patient - tailored cell therapy . fad is caused by a mutation in at least one of three genes : presenilin 1/2 and amyloid precursor protein ( app ) . obviously , ipscs technology contributes to capture the genomes of ad patients and to generate live cellular models of both the fad and sad . these models allow us to identify the earliest events of ad and to investigate aspects of ad pathogenesis that are not replicated in animal models . yagi 's group recently pioneered to generate ipscs from fibroblasts of fad patients with mutations in presenilin 1 ( a246e ) and presenilin 2 ( n141i ) , and characterized the differentiation of these cells into neurons . their remarkable data showed that fad - ipsc - differentiated neurons increased a42 secretion , recapitulating the molecular pathogenesis of mutant presenilins . the spontaneous synaptic activity observed in the differentiated neurons hinted that ipsc technology can be used to study not only human neurons but also patient - specific neural networks . previous research has reported the marked differences in differentiation propensity between psc lines , even between ipsc lines generated from the same individual . current ipscs protocols can not efficiently eliminate undifferentiated cells and , tend to be oncogenic and form teratomas , just like escs . many mouse ipscs harbor epigenetic abnormalities are reported in recent studies , which may develop genetic mutation on prolonged culture and continue to retain epigenetic memory of their donor cells . in terms of the ipscs technique , ( 1)ethical hurdles : undoubtedly , it turns out that ipscs are not entirely problem - free . although stem cell - based replacement strategies carried out in animal models have shown promising results , there are still many hurdles to overcome before these approaches can be translated into the ad patients . one major challenge is the development of a safe method to deliver stem cells to the injury region . adult neurogenesis is important for cellular therapy and physiopathology of the cns , as for development and pharmacology of the adult brain . hence , the role , contribution and significance of newborn neurons in the adult brain remain to be fully elucidated and understood . one of the main limitations in determining and understanding the role of newborn neurons in the adult brain is the use of bromodeoxyuridine ( brdu ) for studying neurogenesis . the microenvironment in which stem cells are placed also needs consideration , as local soluble factors are likely to affect differentiation events in the tissue . the inflammatory status of the brain and the possible activation of inflammatory responses therefore need to be considered with cell replacement strategies . in all , developing our understanding of the processes controlling the activation , migration and differentiation of stem cells will be a critical step towards the usage of stem cells for new regenerative therapies . it has been a century since the first description of ad . in the past decades , tremendous advances in understanding of the molecular pathogenesis of ad have been springing out . now , many new therapies directly targeting the mechanisms underlying ad are now in the pipeline . until medical research discovers definitive disease modifying treatments for patients with ad , we must continue to maximize all available resources to provide the best possible individualized patient - centered and family care . despite a better understanding of the pathology of cognitive impairments and clinical features of ad , which have aided diagnosis and management of the disease , further work is required to improve screening and decrease the burden of care on healthcare systems and families . furthermore , more research is needed to elucidate the mechanisms of the disease to enable new drug targets to be developed based on biological models , which may provide novel treatments for the prevention and management of ad . the situation for neuronal replacement aiming at functional restoration in ad is extremely complex because the stem cells have to be pre - differentiated in vitro to many different types of neuroblasts for subsequent implantation in a large number of brain areas . however , to give long - lasting symptomatic benefit , a cholinergic cell replacement approach will require intact target cells and host neurons that the new cholinergic neurons can act on . stem cell - based cell replacement strategies are very far from clinical application in ad . but the neuroreplacement strategy will undoubtedly become more feasible as we advance our understanding of the pathogenesis of ad and foster creativity in research aiming to elucidate the physiological role of nscs in the adult brain . also , the nscs indeed exist in the complexity and intricacy of the architecture of the human brain , the mechanisms and therapeutic potential of nscs just need to be further explored .

regenerative therapy could be a promising approach as the regeneration of lost or altered cellular functions , which could significantly reverse functional decline to an extent that raises the patient 's survival rate and physiological function . as a representative of the neurodegenerative disorders , alzheimer 's disease ( ad ) is pathologically characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions . neural loss results in gross atrophy of the affected regions , including degeneration in the temporal lobe and parietal lobe , and parts of the frontal cortex and cingulate gyrus . according to these criteria , the presence of cognitive impairments ( learning & memory , language , perceptual skills , orientation ) , and a suspected dementia syndrome confirmed by neuropsychological testing are contributed to the clinical diagnosis of ad . in the early stage , the most common symptom is unable to acquire new memories , observed as difficulty in recalling recently observed events . however , specific disease - modifying treatments aimed at preventing or reversing the basic pathophysiologic processes of ad still remain under investigation . in this literature , the recent research advances in stem cell treatments of ad are reviewed from an experiment to a clinical research . in terms of drug therapy , no drug treatment can reverse , stop , or even slow this inexorable neurodegenerative process . also , non - drug treatments , including gene therapy and behavioral interventions , can only bring temporary symptomatic relief and not result in halting the progression of these diseases . in recent decades , neurogenesis has been proved to exist in restricted regions of the adult brain in many kinds of species including humans . this continuing neurogenesis in the subventricular zone , olfactory bulb , and hippocampal dentate gyrus is supported by the identification of neural stem cells ( nscs ) , suggesting that the adult central nervous system ( cns ) may be amenable to the cell intervention . a variety of animal models have been developed to examine the etiology of ad and to determine the responses of endogenous and/or transplanted stem cells to the pathological microenvironment of the brain . under many conditions , adult neurogenesis is impaired , and the dysfunctional neurogenesis , both decreased and increased , has been reported for ad transgenic models . the adult - born neurons in the diseased brain seem to be a good candidate for those lost or apoptotic neurons . because stem cells can be genetically modified in vitro and have high migratory capacity after transplantation into the brain , they can be an efficient way to delivery neurotrophic factors or enhance gene expression that can modify the course of the disease . since neural progenitor / stem cells have been proven to exist in the adult cns and to be involved in the neurogenesis process , the activation of endogenous neural progenitor / stem cells populations that can migrate to the injured regions , proliferate and functionally integrate into the existing circuit represents a significant strategy to promote neural regeneration in the diseased brain . this activation within the brain is to protect the remaining tissues and prevent secondary neuron loss through the production of neurotrophic and neuroprotective factors , such as brain derived neuronal factor ( bdnf ) and vascular endothelial growth factor . a recent research has demonstrated that the self - repair in the adult brain can be augmented by the infusion of growth factors to activate endogenous neural precursor cells that contribute to new tissue formation and functional recovery after stroke . currently , it is widely accepted that neurogenesis in the hippocampus are involved in learning and memory formation . the hippocampus - dependent learning tasks can significantly increase the proliferation of endogenous neuronal progenitors , survival of new neurons and , the task performance by animals correlates positively with the amount of adult born neurons . alterations in the microenvironment where the endogenous nscs reside play an important role in nsc activation . in ad brain , for example , immune responses include the activation of microglia and astrocytes around the senile plaques area and t - lymphocyte infiltration into the damaged brain . in spite of the activated proliferation , obviously , most of the new migrated neurons in and near the injured area die before differentiating into functional neurons , possibly because of a lack of factors and stimulation to support their survival and differentiation ; thus , only 0.2% of the dead neurons are replaced . however , it has not been determined whether these effects depend directly on the promotion of neuronal regeneration by nscs , or whether accompanying events , such as enhanced glial regeneration and other types of trophic support , are more important . moreover , a key issue in the field of neuronal regeneration is that newly generated neurons need to make the appropriate connections , although the details of this process are still largely unknown . further studies are needed to clarify how newly generated neurons are associated with neurological improvement and to elucidate the comprehensive mechanism regulating the endogenous regeneration system . the success of isolation and easy gene - engineered modification of embryonic stem cells ( escs ) and nscs in vitro profoundly provides researchers a promising tool to replace the injured neurons in the ad brain . therefore , the cell transplantation strategy has given rise to hopes for clinical application of these in vitro produced neuronal cells in the cell replacement procedures for ad . since chronic inflammation is a characteristic property of ad brain , transplantation of neuronal precursor cells ( npcs ) has been proven to particularly inhibit ongoing inflammatory reactivity . researchers have tested that the intrahippocampal transplantation of npcs is effective in attenuating inflammatory responses and plays a neuroprotection role in beta - amyloid 42 ( a-42 ) peptide - injected rat hippocampus , indicating effects of npcs transplantation in ad models are consistent with cellular actions to attenuate inflammatory reactivity . moghadam et al used escs - derived npcs to treat ibotenic acid - induced ad models in order to investigate the production of cholinergic neurons derived from engrafted cells . after transplantation , not only a significant behavioral improvement in memory deficits was observed , but also the majority ( about 70% ) of the npcs retained neuronal phenotype and about 40% of them had a cholinergic cell phenotype with no tumor formation . similarly , in the a-induced ad model , the transplantation of escs - derived npcs into the injured hippocampus can also improve the memory dysfunction of the ad models . furthermore , the transplanted cells demonstrate characteristics of proper synapse formation between host and grafted neural cells . recently , another group reported the in vivo functional integration of the human escs - derived nscs after cell transplantation . at 6 months after transplantation , human axons identified with the human - specific middle - weight neurofilament protein antibody were found inside the stratum radiatum . hippocampal grafts placed in the dentate gyrus were projected to both the ipsilateral and contralateral pyramidal cell layers , while axons of donor neurons placed in the motor cortex extended via the external and internal capsules into the cervical spinal cord and via the corpus callosum into the contralateral cortex . their data indicate that neurons derived from human pluripotent stem cells ( pscs ) are endowed with a remarkable potential to establish orthotopic long - range projections in the adult mammalian brain . meanwhile , in another mouse ad model , the transplantation of nscs was reported to improve cognition function mediated by the neurotrophic factor bdnf . the differentiation of transplanted nscs in response to local cues in the brain suggests that environmental cues have the ability to direct the fate of stem cells to become the specific , terminally differentiated cells that are required to restore functions . however , care should be taken with the characterization of these cells in regard to their multipotentiality and genetic stability with increasing passages in culture , as transformed cells may contribute to the formation of tumors . efforts to investigate the pathophysiology of human ad are hampered by the lack of genuine in - vitro models . stem cells generated by induced direct reprogramming of adult somatic cells using are termed as ipscs , offering paradigm shifting opportunities by providing specific / personalized models for studying ad , and personalized renewable source of cells for practical autologous cell therapies and regenerative medicine applications , that avoid immune rejection . ultimately , aside from being an exciting research tool to probe embryogenesis and disease pathogenesis , ipscs are so - called disease modeling for drug screening , identifying novel drugs to treat diseases and patient - tailored cell therapy . obviously , ipscs technology contributes to capture the genomes of ad patients and to generate live cellular models of both the fad and sad . these models allow us to identify the earliest events of ad and to investigate aspects of ad pathogenesis that are not replicated in animal models . yagi 's group recently pioneered to generate ipscs from fibroblasts of fad patients with mutations in presenilin 1 ( a246e ) and presenilin 2 ( n141i ) , and characterized the differentiation of these cells into neurons . their remarkable data showed that fad - ipsc - differentiated neurons increased a42 secretion , recapitulating the molecular pathogenesis of mutant presenilins . this finding significantly demonstrates that the fad - ipsc - derived neuron is a valid model of ad and provides an innovative strategy for the study of age - related neurodegenerative diseases . marchetto et al in their study of rett syndrome using ipscs , reported the in vitro differentiation of ipscs into neurons that contained glutamatergic synapses and were capable of generating spontaneous synaptic activity . the spontaneous synaptic activity observed in the differentiated neurons hinted that ipsc technology can be used to study not only human neurons but also patient - specific neural networks . thus , it is unclear if ipscs and ipsc - derived npcs from ad patients would generate neurons differently than control cells , such as embryonic stem cells and neural stem cells . lanza group compared differentiated cells derived from a series of ipscs lines and escs lines , and found thatboth cells differentiated to form blood cells , vascular cells or retinal cells . since neural progenitor / stem cells have been proven to exist in the adult cns and to be involved in the neurogenesis process , the activation of endogenous neural progenitor / stem cells populations that can migrate to the injured regions , proliferate and functionally integrate into the existing circuit represents a significant strategy to promote neural regeneration in the diseased brain . this activation within the brain is to protect the remaining tissues and prevent secondary neuron loss through the production of neurotrophic and neuroprotective factors , such as brain derived neuronal factor ( bdnf ) and vascular endothelial growth factor . a recent research has demonstrated that the self - repair in the adult brain can be augmented by the infusion of growth factors to activate endogenous neural precursor cells that contribute to new tissue formation and functional recovery after stroke . the hippocampus - dependent learning tasks can significantly increase the proliferation of endogenous neuronal progenitors , survival of new neurons and , the task performance by animals correlates positively with the amount of adult born neurons . alterations in the microenvironment where the endogenous nscs reside play an important role in nsc activation . in ad brain , for example , immune responses include the activation of microglia and astrocytes around the senile plaques area and t - lymphocyte infiltration into the damaged brain . in spite of the activated proliferation , obviously , most of the new migrated neurons in and near the injured area die before differentiating into functional neurons , possibly because of a lack of factors and stimulation to support their survival and differentiation ; thus , only 0.2% of the dead neurons are replaced . however , it has not been determined whether these effects depend directly on the promotion of neuronal regeneration by nscs , or whether accompanying events , such as enhanced glial regeneration and other types of trophic support , are more important . moreover , a key issue in the field of neuronal regeneration is that newly generated neurons need to make the appropriate connections , although the details of this process are still largely unknown . further studies are needed to clarify how newly generated neurons are associated with neurological improvement and to elucidate the comprehensive mechanism regulating the endogenous regeneration system . the success of isolation and easy gene - engineered modification of embryonic stem cells ( escs ) and nscs in vitro profoundly provides researchers a promising tool to replace the injured neurons in the ad brain . therefore , the cell transplantation strategy has given rise to hopes for clinical application of these in vitro produced neuronal cells in the cell replacement procedures for ad . since chronic inflammation is a characteristic property of ad brain , transplantation of neuronal precursor cells ( npcs ) has been proven to particularly inhibit ongoing inflammatory reactivity . researchers have tested that the intrahippocampal transplantation of npcs is effective in attenuating inflammatory responses and plays a neuroprotection role in beta - amyloid 42 ( a-42 ) peptide - injected rat hippocampus , indicating effects of npcs transplantation in ad models are consistent with cellular actions to attenuate inflammatory reactivity . moghadam et al used escs - derived npcs to treat ibotenic acid - induced ad models in order to investigate the production of cholinergic neurons derived from engrafted cells . after transplantation , not only a significant behavioral improvement in memory deficits was observed , but also the majority ( about 70% ) of the npcs retained neuronal phenotype and about 40% of them had a cholinergic cell phenotype with no tumor formation . similarly , in the a-induced ad model , the transplantation of escs - derived npcs into the injured hippocampus can also improve the memory dysfunction of the ad models . furthermore , the transplanted cells demonstrate characteristics of proper synapse formation between host and grafted neural cells . recently , another group reported the in vivo functional integration of the human escs - derived nscs after cell transplantation . at 6 months after transplantation , human axons identified with the human - specific middle - weight neurofilament protein antibody were found inside the stratum radiatum . hippocampal grafts placed in the dentate gyrus were projected to both the ipsilateral and contralateral pyramidal cell layers , while axons of donor neurons placed in the motor cortex extended via the external and internal capsules into the cervical spinal cord and via the corpus callosum into the contralateral cortex . their data indicate that neurons derived from human pluripotent stem cells ( pscs ) are endowed with a remarkable potential to establish orthotopic long - range projections in the adult mammalian brain . meanwhile , in another mouse ad model , the transplantation of nscs was reported to improve cognition function mediated by the neurotrophic factor bdnf . the differentiation of transplanted nscs in response to local cues in the brain suggests that environmental cues have the ability to direct the fate of stem cells to become the specific , terminally differentiated cells that are required to restore functions . however , care should be taken with the characterization of these cells in regard to their multipotentiality and genetic stability with increasing passages in culture , as transformed cells may contribute to the formation of tumors . stem cells generated by induced direct reprogramming of adult somatic cells using are termed as ipscs , offering paradigm shifting opportunities by providing specific / personalized models for studying ad , and personalized renewable source of cells for practical autologous cell therapies and regenerative medicine applications , that avoid immune rejection . ultimately , aside from being an exciting research tool to probe embryogenesis and disease pathogenesis , ipscs are so - called disease modeling for drug screening , identifying novel drugs to treat diseases and patient - tailored cell therapy . obviously , ipscs technology contributes to capture the genomes of ad patients and to generate live cellular models of both the fad and sad . these models allow us to identify the earliest events of ad and to investigate aspects of ad pathogenesis that are not replicated in animal models . yagi 's group recently pioneered to generate ipscs from fibroblasts of fad patients with mutations in presenilin 1 ( a246e ) and presenilin 2 ( n141i ) , and characterized the differentiation of these cells into neurons . their remarkable data showed that fad - ipsc - differentiated neurons increased a42 secretion , recapitulating the molecular pathogenesis of mutant presenilins . this finding significantly demonstrates that the fad - ipsc - derived neuron is a valid model of ad and provides an innovative strategy for the study of age - related neurodegenerative diseases . marchetto et al in their study of rett syndrome using ipscs , reported the in vitro differentiation of ipscs into neurons that contained glutamatergic synapses and were capable of generating spontaneous synaptic activity . the spontaneous synaptic activity observed in the differentiated neurons hinted that ipsc technology can be used to study not only human neurons but also patient - specific neural networks . thus , it is unclear if ipscs and ipsc - derived npcs from ad patients would generate neurons differently than control cells , such as embryonic stem cells and neural stem cells . lanza group compared differentiated cells derived from a series of ipscs lines and escs lines , and found thatboth cells differentiated to form blood cells , vascular cells or retinal cells . although stem cell - based replacement strategies carried out in animal models have shown promising results , there are still many hurdles to overcome before these approaches can be translated into the ad patients . in addition , the stage of differentiation of those cells needs careful consideration : fully differentiated cells are associated with a smaller efficiency due to poor viability , while undifferentiated cells present a higher risk of undirected differentiation and uncontrolled proliferation . adult neurogenesis is important for cellular therapy and physiopathology of the cns , as for development and pharmacology of the adult brain . hence , the role , contribution and significance of newborn neurons in the adult brain remain to be fully elucidated and understood . one of the main limitations in determining and understanding the role of newborn neurons in the adult brain is the use of bromodeoxyuridine ( brdu ) for studying neurogenesis . environmental factors need to be included : pro - inflammatory cytokines are associated with a negative effect on neural differentiation , while anti - inflammatory cytokines may have the opposite effect . the inflammatory status of the brain and the possible activation of inflammatory responses therefore need to be considered with cell replacement strategies . in all , developing our understanding of the processes controlling the activation , migration and differentiation of stem cells will be a critical step towards the usage of stem cells for new regenerative therapies . in the past decades , tremendous advances in understanding of the molecular pathogenesis of ad have been springing out . a combination of psychosocial , behavioral , and pharmacologic strategies aims at slowing the process of ad and preserving quality of life for as long as possible . despite a better understanding of the pathology of cognitive impairments and clinical features of ad , which have aided diagnosis and management of the disease , further work is required to improve screening and decrease the burden of care on healthcare systems and families . furthermore , more research is needed to elucidate the mechanisms of the disease to enable new drug targets to be developed based on biological models , which may provide novel treatments for the prevention and management of ad . the situation for neuronal replacement aiming at functional restoration in ad is extremely complex because the stem cells have to be pre - differentiated in vitro to many different types of neuroblasts for subsequent implantation in a large number of brain areas . but the neuroreplacement strategy will undoubtedly become more feasible as we advance our understanding of the pathogenesis of ad and foster creativity in research aiming to elucidate the physiological role of nscs in the adult brain . also , the nscs indeed exist in the complexity and intricacy of the architecture of the human brain , the mechanisms and therapeutic potential of nscs just need to be further explored .

vascular dementia sometimes can precede or accompany alzheimer 's disease , and in these cases the development of alzheimer 's disease becomes more dramatic . the dementia of ischaemic type and alzheimer 's disease is synergistic or additive in the earliest stages of alzheimer 's disease , although the interactive mechanisms are not known [ 1 , 2 ] . both types of dementia are neurodegenerative diseases and for both the dysfunction and degeneration of cholinergic projective systems in the cortex and the hippocampus from the forebrain nuclei are critical [ 38 ] . a number of studies on animal models demonstrated a possible trigger role of cholinergic projective neurons in brain ischaemia . the early activation of cholinergic projective neurons was found to occur simultaneously with glutamatergic activation in the cortex and the hippocampus [ 912 ] . correlations between the development of cholinergic dysfunctions and the destruction of pyramidal neurons in the hippocampus [ 6 , 13 , 14 ] , and also damage to the cognitive functions of animals [ 48 , 10 ] , led to the presumption that a dysfunction in cholinergic afferents plays a major role in the development of ischaemic pathologies [ 9 , 12 , 14 , 15 ] . modern electrophysiology accumulated numerous data that interneurons of the cortex and the hippocampus actively participate in the modulation of neuronal activity including the hippocampal pyramidal neurons [ 1618 ] . it was revealed that the cholinergic effects on the interneurons of the cortex and the hippocampus was substantially mediated through nicotinic receptors ( nachrs ) [ 1621 ] . on the other hand the role of the cholinergic interneurons in behavioural , and neurodegenerative mechanisms is still unknown . our investigations on the light and heavy synaptosomal fractions of the cortex and the hippocampus allowed the study of the major cholinergic projection systems of the cortex and the hippocampus and their minor intrinsic systems of cholinergic interneurons . according to immunochemical data , both the cortex and the hippocampus the first major sources are the neuronal projections from the forebrain nuclei basalis magnocellularis into the cortex ( precursor of the meynert nucleus in primates and humans ) and projections from the forebrain medial septal nuclei and vertical limb nuclei of the diagonal band of broca into the hippocampus . the synaptosomes are presynaptic parts of synapses with their junction complexes ; these shall be termed presynapses in the present study . we previously showed that for both the cortex and the hippocampus the cholinergic presynapses from different sources are isolated in different synaptosomal fractions during preparation in the sucrose density gradient . the presynapses of cholinergic projections from the forebrain nuclei are accumulated mainly in the light synaptosomal fractions whereas the presynapses of cholinergic interneurons are accumulated mainly in the heavy synaptosomal fractions [ 2931 ] . it is probable that the heavy synaptosomal fraction of the hippocampus may also accumulate a small part of the cholinergic projective presynapses ( lateral projection pathway into the hippocampus ) . our studies on the cortical synaptosomal fractions of cats allowed suggest the involvement of the cholinergic interneurons in cognitive functions . in the studies on the cortical and hippocampal synaptosomal fractions of rat we revealed that during the first three hours of chronic brain ischaemia the cholinergic projective neurons were reactive , as were the interneurons of the cortex and the hippocampus as well . at present , the molecular , genetic and neurochemical mechanisms of cognitive functions are widely investigated in different behavioural models and widely discussed as well . some among these data induce to revise generally conception that memory formation involves an irreversible passage via labile phases , such as working and short - term memory to the stable form of long - term memory . thus it was shown , that several drugs inhibited short - term memory without altering long - term memory and that working , short - term , and long - term memory were differentially regulated in the various brain regions by the various neurotransmitter systems , including cholinergic one [ 3437 ] . the authors concluded that different types of memory had the separate mechanisms , various neurotransmitter systems , and regions . the basis of our investigation is importance of the cholinergic systems in human and animal cognition , and also the existence of general mechanisms in the development of dementias of different aetiologies . in the present study , the cholinergic synaptic organization of different forms of learning and memory in rats with normal and chronic ischaemic brains a marker of cholinergic neurons , enzyme of acetylcholine synthesis choline acetyltransferase ( chat ; ec 2.3.1.6 ) was used for estimation of the cholinergic systems . chat activity and also protein contents ( total synaptic parameters ) were measured in subfractions of the synaptic membranes and the synaptoplasm isolated from light and heavy synaptosomal fractions of the cortex and the hippocampus . thus , the participation of projective and intrinsic cholinergic systems of the cortex and hippocampus in mechanisms of learning and memory under normal and ischaemic conditions was researched . in addition , the regulation of learning performance under prolonged action of selective agonist of 42 subtype of nachr metanicotine ( rjr ) and selective antagonist of non7 subtypes of nachr mecamlamine was studied . outbred white adult male rats ( 220270 g ) were supplied from the animal 's nursery light mountains ( russia ) and then kept in the vivarium of our institute of general pathology and pathophysiology . the rats were housed in a temperature - controlled room ( 2024c ) with free access to food and water and kept on a 12 h light / dark cycle according to the national institutes of health animal care and the principles of laboratory animal care guidelines and the study was approved by the ethical committees of the institutes . chronic rat ischaemia was induced by permanent occlusion of the common carotid arteries ( two - vessel occlusion , 2vo ) by ligation . the bilateral common carotid arteries were tied with silk threads whilst the rats were under an appropriate level of pentobarbital anaesthesia . the common carotid arteries were separated from the cervical sympathetic and vagal nerves through a ventral cervical incision . the sham - operated animals ( control groups ) underwent a similar surgery but vessel ligation was excluded . behaviour was studied in spatial contextual ( noncued ) or spatial cued models of learning and memory in the morris water maze following standard procedures . the experimental apparatus consisted of a circular water pool ( diameter , 120 cm ; height , 60 cm ) filled with milk - clouded water at 24c to a depth of 40 cm . a plexiglas hidden platform ( 10 10 cm ) was submerged 2 cm below the water surface and was placed at the midpoint of one quadrant . the rats were trained during three daily sessions in the contextual ( sham - operated/2vo rats ) or the cued learning models . in both learning models the rats were given four daily attempts to find the hidden platform in a 60 s time interval and the estimated swim time for platform achievement ( latency time ) was recorded . rats which failed to find the platform within 60 s were considered unable to solve the task and were softly guided there by the investigator with 60 s scored . the other rats remained on the platform for 30 s and were returned to their home cage during the intertrial interval ( 60 s ) . in the contextual model the location of the hidden platform remained the same throughout the training period . the pool was located in a test room containing no prominent visual marks . at the start of all trials , the rats were placed in the pool at one of four starting positions . in the cued model a prominent visual mark ( cue ) was placed on the maze wall over the hidden platform to help the animal locate the platform . in this model the rats had the same starting position but the hidden platform with its cue was moved to four different positions during the session . the following forms of cognitive functions were observed and investigated : the inherited abilities ( the first noncasual attempts at decision making in the task , that is , 1s1 trial in the cued model and 1s2 trial in the contextual model ) ; working memory in the first session ( 1s24 and 1s3 - 4 averaged out over the following trials , resp . ) ; learning in the second and the third sessions ( 2s24 and 3s24 averaged trials , resp . ) ; and long - term memory on the days after the first and the second sessions of training ( 2s1 and 3s1 trials , resp . ) . all behavioural experiments were carried out by investigators who had no knowledge of the experimental groups . metanicotine ( rjr 2304 , tocris ) , a selective agonist of 42 subtype of nachr and mecamilamine ( sigma ) , a selective antagonist of non-7 subtypes of nachr , were used . the preparations were subchronically administered ( i.p . ) three times daily in doses of 26 and 3.9 nmoles / kg , respectively . both the sham - operated and the ischaemic rats received the first injection of the preparations immediately after the end of narcosis ( 1.53 hours after surgical intervention ) . some of animals were decapitated for biochemical analysis 3 - 4 days after the third session of training . it means , the rats which trained from 6 day after the surgery were decapitated at 11 or 12 days after the surgery and so on . the biochemical group included the control/2vo animals trained in the contextual model ( contextual biochemical subgroup ) or the cued model ( cued biochemical subgroup ) . briefly , the brain , cortex and hippocampus were removed , separated and homogenized . from each sample the light and heavy synaptosomal fractions were isolated , with further separation of the subfractions of the synaptic membranes and the synaptoplasm , following preparative and the disruptive procedures and the discontinued gradients of sucrose density as described previously [ 30 , 39 ] . the fractions of the synaptosomes were obtained from the rough mitochondrial fraction by centrifugation using a bucket rotor ( 84,000 g 120 min , 24c ) in layers between 1.01.2 m sucrose densities ( the light synaptosomes ) and between 1.21.4 m sucrose densities ( the heavy synaptosomes ) . the synaptosomes were disrupted by combined shock procedures : the synaptosome pellets were suspended in hypo - osmotic solution containing 6 mm tris - ncl buffer , ph 8.1 ( 100 mg tissue / ml ) and they were then exposed by freeze - thawing . the synaptoplasm subfractions were obtained as supernatants by centrifugation from the disrupted synaptosomal fractions ( 14,000 g 30 min , 24c ) . the pellets were suspended in the hypo - osmotic solution and stratified on discontinued gradients again . the synaptic membrane subfractions were obtained by centrifugation using the bucket rotor ( 130,000 g 120 min , 24c ) in layers between 0.61.2 m sucrose densities . the clean synaptic membrane subfractions were free from glial , mitochondrial and synaptic vesicle contamination . the activity of chat in subfractions of synaptic membranes and synaptoplasm of the cortex and the hippocampus was determined by the radiometric method of fonnum and the protein contents were determined by the method of lowry et al . . accordingly , the membrane - bound mchat activity and m - protein contents were determined in the synaptic membrane subfractions , and the water - soluble cchat activity and c - protein contents were estimated in synaptoplasm subfractions . moreover , this is the reason why changes in mchat activity could be exposed and changes in cchat activity could be masked in the small presynapses , whereas changes in cchat activity , but not in mchat activity , could be exposed in the large presynapses . therefore , estimations of mchat and cchat activities ( as well as m- and c - protein contents ) could give additional information on the characteristics of changes caused by ischaemia . the reactive mixture contained a final concentration of 0.2 mm acetyl coasa ( fluka ) and [ 1-c]-acetyl coasa ( amersham pharmacia bioscience ) with spa 5 mci / mmol , 300 mm nacl , 3 mm mgcl2 , 0.2 mm physostigmine salicylate ( sigma ) , 10 mm choline chloride ( serva ) , 0.5% triton x-100 ( serva ) , 0.5 mg / ml albumin from bull serum ( koch - light ) , 10 mm sodium phosphate buffer/1 mm edta - na2 , ph 7.8 and the subfraction samples ( near 3.5 mg of protein ) at a common volume of 0.050.1 ml . the reactive mixture was incubated in a water shaker at 37c for 3060 min . the reaction was stopped by adding 2 ml of ice - cold stop solution ( 0.2 mm acetylcholine in 10 mm sodium phosphate buffer/1 mm edta - na2 , ph 7.8 ) and by placing the mixture in an ice bath . then , a 1 ml solution of sodium tetraphenylborate ( sigma ) in butyl acetate ( 15 mg / ml ) was added and quickly subjected to intensive mixing in a shaker ( 500 turns / min , 4 min , room temperature ) . the organic phase was separated from the inorganic phase by centrifugation ( 1000 g 15 min , 24c ) . the organic phase with acetylcholine ( 0.50.7 ml ) was placed into scintillation liquid for organic solutions and the radioactively synthesized acetylcholine ( dpm ) was quantified with a beta counter . reactive solution ( biuret reagent ) was prepared at the day of experiment by mixing 0.5 ml of 1% cupric sulfate with 0.5 ml of 2% sodium potassium tartrate , followed by the addition of 50 ml of 2% sodium carbonate in 0.1 n naoh . bovine serum albumin ( bsa ) powder was dissolved in distilled water and diluted to a concentration of 1000 g / ml . a series of dilutions of the basic bsa solution ( 50 , 100 , 200 , 400 and 500 g / ml ) was made by mixed thoroughly of the aliquots of basic bsa solution and water with repeated pipeting . samples were within the bsa standard range ( 120 g in assay volume ) . reaction was started by intensive mixed of 0.02/0.04 ml of bsa or subfractions samples with 1 ml of the reactive solution . the mixture was then allowed to incubate at room temperature for 1015 min prior to the addition of 0.1 ml per tube of 1.0 n folin & ciocalteu 's reagent . color was allowed to develop for 2 hours in dark at room temperature and the absorbance of the reduced folin reagent measured at 750 nm and blanked on the water only control . after then the reaction was found to be stable for up to an hour at room temperature and kept in refrigerator at 58c for up to 1 - 2 days . the behavioural results were expressed in terms of time taken to swim to the hidden platform ( s ) and the biochemical results were expressed in terms of chat activity ( nmoles acetylcholine / min ) or protein content ( mg ) in 1 g wet weight of cortex and hippocampus tissue , respectively . the data were calculated using the nonparametric fisher 's exact test and the r - criterion of the pearson 's correlative test in microsoft excel with a glance of adjusting formula for small number of observations . the period of 610 days of chronic brain ischaemia led to a strong decline in training efficiency in the morris water maze . learning and long - term memory were impaired in both the contextual and cued models ( figure 1 ) . learning in 2s24 and 3s24 were impaired in a similar manner in both of the behavioural models , whereas impairment of the long - term memory had the specificity in each model . in the contextual model ( figure 1 , top ) , impairment of long - term memory developed gradually and only 3s1 was significantly impaired . in the cued model ( figure 2 , bottom ) , long - memory 2s1 was impaired and 3s1 was the same as the control . it can be noted that although the investigated cognitive functions were impaired , they were still performed in ischaemic rats . from all of the investigated animals ( n = 27 ) , only two rats could not solve the tasks in our experimental conditions ( in the contextual model ) . as a rule , the prolongation of solving tasks and/or the delay in learning ( successful solving of the task only occurred in the third session ) was observed . the period of 1114 days of chronic ischaemia resulted in significant changes in chat activity and protein content in the investigated subfractions of the synaptosomes , both of the cortex and the hippocampus ( figure 2-all rats ) . in the cortical light synaptosomal fraction , mchat activity and m - protein content were increased , and these changes were positively correlated amongst themselves ( r = + 0.770 , n = 18 , total control and 2vo groups data , p < .001 ; in the control group r = + 0.788 , n = 9 , p < .02 ) . the cchat activity did not significantly vary or correlate with mchat activity but it was positively correlated with increasing c - protein content ( r = + 0.669 , n = 9 , p < .05 ; in the control group r = + 0.305 , n = 9 , this indicated a reorganization of the synaptic pool in more than one synaptic population of the cholinergic projective neurons in the cortex . but mchat activation was accompanied by a reinforcement in the positive correlation between its values and the m - protein content ( r = + 0.694 , n = 9 , p < .05 ; in the control group r = + 0.291 , n = 9 , p > .05 ) . also , a reinforcement of the positive correlation between the values of cchat activity and c - protein content was observed ( r = + 0.835 , n = 9 , p < .01 ; in the control group r = + 0.633 , n = 9 , p > .05 ) . at the same time , the correlation between the activity of mchat and the activity of cchat became weaker than in the control ( r = + 0.579 , n = 9 , p > .05 ; in the control group r = + 0.754 , n = 9 , p < .02 ) . this indicated a reorganization of the synaptic pool in more than one synaptic population of the cholinergic interneurons in the cortex . in the hippocampal light synaptosomal fraction , however , significant correlations between the values of mchat and cchat activity , and between the values of chat activity and protein content , were absent . this indicated a reorganization of the synaptic pool in more than one synaptic population in the hippocampus , in both the cholinergic systems and some noncholinergic systems . in the hippocampal heavy synaptosomal fraction , m - protein content decreased and c - protein content increased . changes in the m - protein content did not correlate with chat activity and thus reflected reorganization of noncholinergic presynapses in the hippocampus . the activities of mchat and cchat did not differ from the controls although at the same time a positive correlation arose between their values ( r = + 0.802 , n = 8 , p < .02 ; in the control group r = 0.300 , n = 9 , p > .05 ) . also , the positive correlations between values of c - protein content and mchat activity ( r = + 0.927 , n = 8 , p < .01 ; in the control group r = + 0.265 , n = 9 , p > .05 ) and cchat activity ( r = + 0.844 , n = 8 , p < .01 ; in the control group r = + 0.091 , n = 9 , this indicated a reorganization of the presynapses of the cholinergic interneurons / lateral pathway projective neurons in the hippocampus . it seems that the unchanged values of chat activity reflected parallel processes of activation and inactivation of chat in different synaptic populations of this fraction . also , the biochemical data of the contextual biochemical subgroup were compared with the cued biochemical subgroup . analysis of the biochemical parameters in the control biochemical subgroups did not reveal significant changes between the subgroups ( table 1 ) . only the values of mchat activity were lower in the cortical heavy and in the hippocampal light synaptosomal subfractions in the cued biochemical subgroup of rats as compared with the contextual one . analysis of the biochemical parameters in the 2vo biochemical subgroups confirmed our observations about the reorganization of the synaptic pool . in the cortical light synaptosomal fraction in the contextual biochemical subgroup ( figure 2-contextual ) , independent correlations were detected between the values of mchat activity and m - protein content ( r = + 0.765 , n = 10 , total control and 2vo contextual biochemical subgroups data , p < .01 ; in the control contextual biochemical subgroup r = + 0.774 , n = 5 , p > .05 ) and between the values of cchat activity and c - protein content ( r = + 0.987 , n = 5 , p < .01 ; in the control contextual biochemical subgroup r = + 0.441 , n = 5 , p > .05 ) . in the cued subgroup ( figure 2-cued ) , a correlation was only detected between mchat activity and m - protein content ( r = + 0.783 , n = 8 , total control and 2vo cued biochemical subgroups data , p < .05 ; in the control cued biochemical subgroup r = + 0.622 , n = 4 , p > .05 ) and a correlation was revealed between mchat and cchat activities ( r = + 0.995 , n = 4 , p < .01 ; in the control biochemical cued biochemical subgroups r = 0.404 , n = 4 , p > .05 ) . in the cortical heavy synaptosomal fraction in the contextual biochemical subgroup , independent correlations were detected between mchat activity and m - protein content ( r = + 0.981 , n = 5 , p < .01 ) and between the values of cchat activity and c - protein content ( r = + 0.966 , n = 5 , p < .01 ) , whereas there was no correlation between the activities of mchat and cchat among themselves ( r = + 0.652 , n = 5 , p > .05 ) . in the cued biochemical subgroup , an increase in mchat activity was detected ( a tendency ) whereas a decrease in m - protein content was revealed . the decrease in m - protein content allows to suppose the changes in noncholinergic presynapses . in the hippocampal light synaptosomal fraction in the contextual biochemical subgroup , an independent decrease in mchat activity and an increase in cchat activity only , and in the cued biochemical subgroup an increase in cchat activity only , and a decrease in m - protein content and an increase in c - protein content were detected . in the hippocampal heavy synaptosomal fraction in the contextual biochemical subgroup , positive correlations were detected between the increased values of c - protein content and mchat activity ( r = + 0.922 , n = 5 , p < .01 ) and cchat activitiy ( r = + 0.919 , n = 5 , p < .05 ) , and between mchat and cchat activities ( r = + 0.910 , n = 5 , p < .05 ; in the control contextual biochemical subgroup r = 0.266 , n = 5 , p > .05 ) . however , in the cued biochemical subgroup , a decrease in mchat activity and m - protein content was revealed and these changes did not correlate among themselves . this indicated a reorganization of the presynapses of the cholinergic interneurons / lateral pathway projective neurons and noncholinergic neurons in the hippocampus . differentiation of the rats into biochemical subgroups , tested in the contextual and cued models , permit to compare the behavioural performance and chat activity in these rats . under the normal conditions , each form had individual cholinergic composition ( table 2 : sham - contextual , figure 3-i : sham ) . the inherited ability 1s2 cholinergic composition included large presynapses of projective cortical neurons ( positive correlation with cchat activity ) and presynapses of hippocampal interneurons / lateral pathway projective neurons ( negative correlation with mchat and cchat activities ) . the same cholinergic structures associated with the long - term memory 3s1 , but with inverse symbols of r - criterions . the long - term memory 2s1 had composition other than 3s1 which involved small presynapses of the projective hippocampal neurons ( negative correlation with mchat activity ) and some populations of hippocampal interneurons ( positive correlations with mchat and cchat activities ) . the composition of the working memory 1s3 - 4 involved small presynapses ( positive correlation with mchat activity ) and large presynapses ( negative correlation with cchat activity ) of the projective hippocampal neurons . the composition of learning 2s24 and 3s24 was identical and involved small presynapses of the projective cortical neurons ( positive correlation with mchat activity in both forms of learning ) and the hippocampal interneurons / lateral pathway projective neurons ( positive correlations with mchat activity ) . the similar analysis in the cued biochemical subgroup revealed other individual cholinergic compositions of learning and memory ( table 2 : sham - cued , figure 3-ii : sham ) . according to our data , working memory 1s24 composition involved small presynapses of both cortical cholinergic systems ( negative correlation with mchat activity ) and of the hippocampal interneurons / lateral pathway projective neurons ( positive correlation with mchat activity ) . the composition of learning 2s24 and 3s24 was identical and comprised large presynapses of cortical interneurons and projective hippocampal neurons ( positive correlations with cchat activity in both cases ) . the long - term memory composition in 2s1 involved large presynapses of the cortical interneurons and small presynapses of projective hippocampal neurons ( in both cases , there were positive correlations with cchat or mchat activities ) . and the long - term memory composition in 3s1 involved large presynapses of the cortical projective neurons ( positive correlations with cchat activity ) . chronic brain ischaemia had considerable effects on the cholinergic organization of the investigated cognitive functions . in the contextual model ( table 2 : 2vo - contextual , figure 3-i : 2vo ) , inherited abilities 1s2 , learning 2s24 and long - term memory 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the cortical cholinergic populations . the composition of working memory 1s3 - 4 only kept negative connections with the large presynapses of projective hippocampal neurons . the positive connections of learning 3s24 with the small presynapses of the hippocampal interneurons / lateral pathway projective neurons inversed to negative ones . the long - term memory composition 2s1 consisted of only new , positive connections with large presynapses of the projective hippocampal neurons . in the cued model ( table 2 : 2vo - cued , figure 3-ii : 2vo ) , long - term memory 2s1 and 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the hippocampal cholinergic influences . the working memory 1s24 lost its negative connections with the small presynapses of cortical projective neurons and its negative connections with the small presynapses of cortical interneurons inversed to positive ones . the composition of learning 2s24 included a reversal to negative connections with the large presynapses of cortical interneurons , new negative connections with presynapses of cortical projective neurons and new negative connections with the small presynapses of cortical interneurons . the learning composition 3s24 kept its connections with the large presynapses of cortical interneurons and added new negative connections with presynapses of the cortical projective neurons . so , under 2vo conditions as in the contextual and in the cued biochemical subgroup quantity of the cholinergic connections with the cognitive functions significantly reduced and some new links arose . each form of cognition as resulting had 2vo cholinergic synaptic composition organized differently from normal ones . we attempted to analyze the dependence of impairment of the investigated cognitive functions in 2vo conditions from the reorganization of key cholinergic systems . it seems , in the contextual biochemical subgroup only preservation of the inherited abilities 1s2 from damage could be explaned by preservation of the key cholinergic populations , revealed in the normal conditions ( figure 4 , middle row ) . but long - term memory 3s1 had the same cholinergic composition with inverse symbols of r - criterions . in this case it would be expected that 3s1 would also be protected ; however , this did not take place . moreover , according to the cholinergic organization under normal conditions , working memory 1s3 - 4 would be considerably impaired , whereas long - term memory 2s1 and learning 2s24 and 3s24 would be considerably improved ; however , these did not occur either . on the other hand , the new cholinergic composition in 2vo conditions had accordance between reinforcement of the negative influence of the hippocampal interneurons / lateral pathway projective neurons on learning 3s24 and impairment of this function ( figure 4 , bottom row ) . but reinforcement of new negative and positive influences of projective hippocampal neurons was not reflected in the performance of either working memory 1s3 - 4 or long - term memory 2s1 . in the cued biochemical subgroup , only the preservation of long - term memory 3s1 could be explained by the resistance to ischaemia of the key synaptic population revealed in normal conditions ( figure 5 , middle row ) . at the same time , working memory 1s24 would be considerably impaired , learning 2s24 and 3s24 would be equally improved or otherwise unchanged and long - term memory 2s1 would be unchanged , but these were not observed . on the other hand , the absence of 1s24 impairment could also be explained by the new , weakly expressed positive cholinergic influence ( tendency ) of the large presynapses of cortical interneurons ( figure 4 , bottom row ) . then the distinctions in learning 2s24 and 3s24 performance in 2vo conditions would be explained by their new cholinergic compositions if we suppose more considerable influence of the large presynapses of the cortical interneurons on these functions in comparison with the influence of the other new key synaptic populations . the impairment of 2s1 also would be explained by the reduction of the link with this key synaptic population under the normal conditions . so , it seems that performance of the cognitive functions as in the contextual and in the cued model under 2vo conditions , as a rule , did not depend on their cholinergic organization , revealed in normal conditions . contrary , new cholinergic organization , revealed in 2vo conditions showed more significant correlations with changes in behavioral performance . whereas , the contextual and cued learning 2s24 and 3s24 revealed cholinergic synaptic compositions identical for normal conditions and different ones , revealed in 2vo conditions , it was investigated prolonged action on the learning performance of the selective agonist of 42 subtype of nachr rjr and the selective antagonist of non-7 subtypes of nachr mecamilamine . in the contextual model under normal conditions , both the agonist rjr and the antagonist mecamilamine did not influence on learning as 2s24 and 3s24 performance . it seems , this fact indicate that non-7 subtypes of nachr did not participate in the regulation of ones ( figure 6 , contextual learning ) . under the 2vo conditions , effects of rjr on both learning performance evidently , the 42 subtype did not participate in the regulation of the contextual learning , as before , while some non-7 and non-42 subtypes participated with negative influences on learning 3s24 . in the cued model under normal conditions , the agonist rjr impaired learning 2s24 and did not affect learning 3s24 ( figure 6 , cued learning ) . the negative effect of the agonist on learning in 2s24 was significantly greater than that of the antagonist ( p < .05 ) . the difference between the agonistic and antagonistic actions on learning in 3s24 at were also significant ( p < .05 ) . it follows that the 42 subtype ( negative influence ) and some non-7 and non-42 subtypes of nachr ( positive but weak influence ) participated in the regulation of learning in 2s24 . at the same time , the 42 subtype did not participate in the regulation of learning 3s24 , while some non-7 and non-42 subtypes participated with negative influences . under the 2vo conditions , rjr did not correct the impaired functions 2s24 and 3s24 , and mecamilamine did not correct learning 2s24 but resulted in normal learning 3s24 performance . it seems , non-7 subtypes of were removed from the receptor composition of learning 2s24 . at the same time , the importance of some non-7 and non-42 subtypes of nachr was reinforced or new connections arose in the receptor organization of learning 3s24 ( negative influence ) . so , in the contextual learning 2s24 and 3s24 , nachr were absent in normal and were acquired in 2vo receptor composition ( 3s24 ) . then , the cued learning 2s24 and 3s24 , with identical cholinergic synaptic compositions in the norm , had differences in receptor compositions . moreover , the cued learning 2s24 and 3s24 had also differences in 2vo receptor compositions via another means . this research showed that the chronic 2vo brain ischaemia model was an efficient model of neurodegenerative disorders , which was the first purpose of our investigation . the period of 610 days of 2vo ischaemia provoked typical attributes of vascular dementias such as impairment of learning and long - term memory in both spatial - contextual and spatial - cued models of behaviour in the morris water maze . the inherited abilities and working memory remained intact , and damage to the cued long - term memory was transient in this ischaemic period . a considerable reorganization of the synaptic pool of all investigated cholinergic systems in the cortex and the hippocampus was revealed in these same 2vo rats 1114 days after the surgery . a decrease in mchat or cchat activity in one synaptosomal fraction and an increase in another were obtained as result of the 2vo influence , but not of the training , whereas the biochemical parameters did not reveal similar changes between the control contextual and cued biochemical subgroups except one . it is possible that decrease in mchat activity in the hippocampal heavy synaptosomal fraction in the cued biochemical subgroup was result of the training ( see table 1 and figure 2-cued ) . the decrease in mchat and cchat activity reflected cholinergic hypofunction or a degeneration of the cholinergic presynapses . neurodegeneration was observed in different brain ischaemia models starting from the second day up to half a year of ischaemia [ 45 , 46 ] . dysfunction of chat in the projective fibres in the hippocampus ( representing 8090% of the total activity of this enzyme , and see table 1 ) was described at 714 days of ischaemia [ 6 , 13 , 14 , 47 , 48 ] . it was shown in vitro that the activity of mchat was selectively suppressed when the exchange of acetylcholine was damaged by inhibition of the vesicular acetylcholine transporter or the high affinity transport of choline . the function of the vesicular acetylcholine transporter depends on the proton gradient , which in turn is disturbed due to falling atp levels ( inhibition of the proton atpase ) or acidosis . we did not reveal a decrease in protein content correlated with chat activity in our research . but we did suppose that the correlation between chat activity and protein content could be masked because of the complex opposing processes that took place in some of the synaptosomal fractions . at the same time , according to data in the literature , sprouting and destruction with the swelling of neurons and their terminals predominates in late brain ischaemia or postischaemic reoxygenation ( in days and months ) [ 3 , 48 , 53 , 54 ] . in our research , activation of chat was also observed in the majority of the synaptic subfractions , and it could have reflected cholinergic hyperfunction or synaptogenesis ( sprouting ) . it is known that synaptic hyperfunction is accompanied with an enhanced structuring of proteins from the synaptoplasm . under these conditions , the m - protein content will increase and the c - protein content will decrease . therefore , the correlated increase between mchat activity and m - protein content will be reflected as cholinergic hyperfunction and synaptogenesis ( cortical light synaptosomes in the biochemical total group and both subgroups ) , whereas the correlated increase between cchat / mchat activity and the c - protein contect will only reflect synaptogenesis ( cortical light synaptosomes in the biochemical total group and the contextual subgroup , hippocampal heavy synaptosomes in the total group and the cued subgroup ) . selective activation of mchat in vitro was shown under conditions of impaired ionic balance such as an accumulation of [ ca]i and [ zn]i [ 55 , 56 ] . it was revealed that [ zn]i precedes [ ca]i accumulation and [ ca]i in turn results in the functional hyperactivation and swelling of synapses [ 53 , 57 , 58 ] . according to data in vitro activation of cchat reflect cholinergic hyperfunction under normal ionic and metabolic conditions [ 49 , 59 , 60 ] . therefore , we suppose that the activation of cchat reflected synaptogenesis , in agreement with other researchers . thus , chronic brain ischaemia for 1114 days resulted in a complex reorganization of the cortical and hippocampal synaptic pool which involved synaptogenesis or hyperfunctions in the unbalanced ionic conditions of one of the cholinergic synaptic populations and degeneration or dysfunction of the others . we supposed that all of the cholinergic processes revealed under 2vo conditions were present in both cholinergic subgroups of the rats but with different intensities ( see figure 2-all rats , contextual and cued ) . we also supposed that the variety of cholinergic reactions was revealed by the phenotypical variety of the outbred rats and it was useful for understanding some of the principles of the organization of different forms of cognition . the second purpose of our research was a comparative analysis of the behavioural and biochemical parameters for identification of the cholinergic composition of the investigated cognitive functions under normal and 2vo conditions . in the first place it is necessary to note that the results of this research confirmed and expanded the knowledge about cholinergic mechanisms of cognitive functions under normal brain conditions . our data showed the active involvement of cholinergic projective systems and also regional ones of the cortex and the hippocampus in cognitive processes . cholinergic synaptic connections with the investigated cognitive functions revealed under normal conditions indicate that each form of cognition has an individual cholinergic synaptic and probably receptor compositions . this conclusion is conformed to the results of investigations , obtained in the morris water maze and some other behavioural models [ 3437 ] . then , the data showed the participation of the cholinergic systems not only in mechanisms of learning and working memory , which was repeatedly observed in previous studies [ 35 , 45 , 61 ] , but also in mechanisms of the inherited abilities and long - term memory . our inherited abilities in the contextual task in the morris water maze was firstly detected by r g morris and u frey as a distinct type of memory and termed as it seems the problem of future discussions . our data , concerning individual cholinergic organisation of function support contextual inherited abilities as a distinct form of cognition . our data concerning the same cholinergic structures associated with inherited abilities 1s2 and long - term memory 3s1 also testify to possible tight interaction between these two forms of cognition . the involvement of cholinergic projective systems in mechanisms of the long - term memory is usually denied [ 6467 ] , and was only discussed in a few studies [ 36 , 68 , 69 ] . our data confirmed that synaptic populations of cholinergic projective neurons and of the interneurons of the cortex and the hippocampus can have positive and negative connections with cognitive functions . a negative dependence of cognitive functions on cholinergic cortical efficiency was also revealed earlier in cats using a similar methodology for researching the cholinergic synaptic organization of cognitive functions . therefore , our data demonstrate that the cholinergic mechanisms of learning and memory are more complex than currently perceived . it is evident that this can complicate the detection of cholinergic effects on some cognitive functions by means of nonselective influences on cholinergic efficiency . for example , according to our data in the contextual model , the nonselective pharmacological cholinergic means as well as use of different methods of degeneration of the cholinergic projective systems would certainly have revealed the participation of the cholinergic projective systems in learning 2s24 and 3s24 , but it would probably have concealed a cholinergic participation in the mechanisms of the inherited abilities , the long - term and the working memory . such results would correspond to the data in the literature [ 62 , 63 , 68 , 70 ] . from the numerous data in the literature , preservation of the cholinergic projective systems is critical for the success of cognitive processes , and it was thought that cholinergic dysfunction or degeneration results in the impairment of memory in neurodegenerative diseases of different aetiologies . therefore , we analyzed the connections between reorganization of the cholinergic synaptic pool and impairment of learning and memory under 2vo conditions . the comparative analysis showed that the connections between the functional and cholinergic parameters revealed under normal conditions were practically lost in the ischemic rats . in our research , only impairment of cued long - term memory 2s1 was really dependent on degeneration of the key synaptic population of the cholinergic cortical interneurons , and also , probably , the intact cued memory 3s1 by the unchanged key synaptic population of the cholinergic cortical projective neurons . at the same time , our data also showed different cholinergic compositions of the cognitive functions under 2vo and under normal conditions in as the spatial contextual and the spatial cued models . under 2vo conditions , most connections of the investigated functions with cholinergic synaptic populations revealed under normal conditions disappeared and new connections with other cholinergic synaptic populations arose . the quantity of cholinergic synaptic populations , involving in mechanisms of the investigated cognitive functions , was considerable reduced . furthermore , cholinergic connections in general disappeared from the mechanisms of the following forms of cognition : inherited abilities 1s2 , learning 2s24 and long - term memory 3s1 in the contextual model , and long - term memory 2s1 and 3s1 in the cued model . moreover , brain region specializations of both the contextual and the cued functions were changed . cortical cholinergic influences had been completely removed from the contextual functions and hippocampal ones from the cued functions . all considerable differences between cholinergic organisation of the cognitive functions in the normal and 2vo conditions stated above are clear demonstrated in figure 3 . it is important that a consistency between the performances of cognitive functions and their new key cholinergic synaptic populations was found in the majority of the remaining cholinergic - dependent functions under 2vo conditions ( from four to six functions ) . thus , according to our data , we suggest that the normal cholinergic synaptic connections in learning and memory were progressively reduced and changed during chronic ischaemia . it is known that anticholinesterase drugs are only effective in the early stages of alzheimer 's disease ( early and mild alzheimer 's disease ) . it can be noted that the new cholinergic connections with the cognitive functions were not necessarily a consequence of degeneration or dysfunctions in the key cholinergic synaptic populations ( it was evident for contextual learning in 3s24 and long - term memory in 2s1 , that is , these new links could arise by other , indirect reasons ) . the dependence on the inclusion of cholinergic links in the realization of cognitive functions from the functional background of neuronal environments was recently revealed . this corresponds with the theory by d. a. sakharov about the nonsynaptic transfer of chemical information [ 71 , 72 ] . according to this theory , any change in any functional system results in a change in all systems as a result of the change in neuroactive compounds of intercellular environments ( the matrix ) . the changes in the matrix determine the activation of one or another neuronal ensemble which finally determines the behavioural act . from all of these viewpoints , it seems that the main value for cognitive functions is its receptor composition and its change in neurodegenerative pathology . our data concerning the different consequences on the learning performance under normal and 2vo conditions by the action of rjr and mecamilamine on the same subtypes of nachr testify to this version . it seems that the reasons for changes in the cholinergic organization of cognitive functions in an ischaemic pathology can be any neurodegenerative or , on the contrary , reparative process ( sprouting ) of cholinergic and noncholinergic synaptic populations . in spite of the brain reparative potentials , the cholinergic and the whole neurochemical organization of cognitive functions under the chronic actions of pathological factors will be formed as optimally as possible under the new conditions . a new organization of cognitive functions will be constructed on neuronal elements which are stable against pathological influences . this new organization can provide an optimum realization of some cognitive functions but not of others . in any case , the study of new key neurochemical links in the organization of cognitive functions may be promising . the plasticity of neurochemical links in the individual organization of certain types of cognition can be used in the future for alternative corrections of vascular and other degenerative dementia .

the purpose of this research was a comparative analysis of cholinergic synaptic organization following learning and memory in normal and chronic cerebral ischaemic rats in the morris water maze model . choline acetyltransferase and protein content were determined in subpopulations of presynapses of light and heavy synaptosomal fractions of the cortex and the hippocampus , and the cholinergic projective and intrinsic systems of the brain structures were taken into consideration . we found a strong involvement of cholinergic systems , both projective and intrinsic , in all forms of cognition . each form of cognition had an individual cholinergic molecular profile and the cholinergic synaptic compositions in the ischaemic rat brains differed significantly from normal ones . our data demonstrated that under ischaemic conditions , instead of damaged connections new key synaptic relationships , which were stable against pathological influences and able to restore damaged cognitive functions , arose . the plasticity of neurochemical links in the individual organization of certain types of cognition gave a new input into brain pathology and can be used in the future for alternative corrections of vascular and other degenerative dementias .

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

both types of dementia are neurodegenerative diseases and for both the dysfunction and degeneration of cholinergic projective systems in the cortex and the hippocampus from the forebrain nuclei are critical [ 38 ] . the early activation of cholinergic projective neurons was found to occur simultaneously with glutamatergic activation in the cortex and the hippocampus [ 912 ] . correlations between the development of cholinergic dysfunctions and the destruction of pyramidal neurons in the hippocampus [ 6 , 13 , 14 ] , and also damage to the cognitive functions of animals [ 48 , 10 ] , led to the presumption that a dysfunction in cholinergic afferents plays a major role in the development of ischaemic pathologies [ 9 , 12 , 14 , 15 ] . modern electrophysiology accumulated numerous data that interneurons of the cortex and the hippocampus actively participate in the modulation of neuronal activity including the hippocampal pyramidal neurons [ 1618 ] . it was revealed that the cholinergic effects on the interneurons of the cortex and the hippocampus was substantially mediated through nicotinic receptors ( nachrs ) [ 1621 ] . on the other hand the role of the cholinergic interneurons in behavioural , and neurodegenerative mechanisms is still unknown . our investigations on the light and heavy synaptosomal fractions of the cortex and the hippocampus allowed the study of the major cholinergic projection systems of the cortex and the hippocampus and their minor intrinsic systems of cholinergic interneurons . according to immunochemical data , both the cortex and the hippocampus the first major sources are the neuronal projections from the forebrain nuclei basalis magnocellularis into the cortex ( precursor of the meynert nucleus in primates and humans ) and projections from the forebrain medial septal nuclei and vertical limb nuclei of the diagonal band of broca into the hippocampus . we previously showed that for both the cortex and the hippocampus the cholinergic presynapses from different sources are isolated in different synaptosomal fractions during preparation in the sucrose density gradient . the presynapses of cholinergic projections from the forebrain nuclei are accumulated mainly in the light synaptosomal fractions whereas the presynapses of cholinergic interneurons are accumulated mainly in the heavy synaptosomal fractions [ 2931 ] . it is probable that the heavy synaptosomal fraction of the hippocampus may also accumulate a small part of the cholinergic projective presynapses ( lateral projection pathway into the hippocampus ) . our studies on the cortical synaptosomal fractions of cats allowed suggest the involvement of the cholinergic interneurons in cognitive functions . in the studies on the cortical and hippocampal synaptosomal fractions of rat we revealed that during the first three hours of chronic brain ischaemia the cholinergic projective neurons were reactive , as were the interneurons of the cortex and the hippocampus as well . the basis of our investigation is importance of the cholinergic systems in human and animal cognition , and also the existence of general mechanisms in the development of dementias of different aetiologies . in the present study , the cholinergic synaptic organization of different forms of learning and memory in rats with normal and chronic ischaemic brains a marker of cholinergic neurons , enzyme of acetylcholine synthesis choline acetyltransferase ( chat ; ec 2.3.1.6 ) was used for estimation of the cholinergic systems . chat activity and also protein contents ( total synaptic parameters ) were measured in subfractions of the synaptic membranes and the synaptoplasm isolated from light and heavy synaptosomal fractions of the cortex and the hippocampus . thus , the participation of projective and intrinsic cholinergic systems of the cortex and hippocampus in mechanisms of learning and memory under normal and ischaemic conditions was researched . behaviour was studied in spatial contextual ( noncued ) or spatial cued models of learning and memory in the morris water maze following standard procedures . the following forms of cognitive functions were observed and investigated : the inherited abilities ( the first noncasual attempts at decision making in the task , that is , 1s1 trial in the cued model and 1s2 trial in the contextual model ) ; working memory in the first session ( 1s24 and 1s3 - 4 averaged out over the following trials , resp . ) both the sham - operated and the ischaemic rats received the first injection of the preparations immediately after the end of narcosis ( 1.53 hours after surgical intervention ) . from each sample the light and heavy synaptosomal fractions were isolated , with further separation of the subfractions of the synaptic membranes and the synaptoplasm , following preparative and the disruptive procedures and the discontinued gradients of sucrose density as described previously [ 30 , 39 ] . the activity of chat in subfractions of synaptic membranes and synaptoplasm of the cortex and the hippocampus was determined by the radiometric method of fonnum and the protein contents were determined by the method of lowry et al . accordingly , the membrane - bound mchat activity and m - protein contents were determined in the synaptic membrane subfractions , and the water - soluble cchat activity and c - protein contents were estimated in synaptoplasm subfractions . the period of 610 days of chronic brain ischaemia led to a strong decline in training efficiency in the morris water maze . the period of 1114 days of chronic ischaemia resulted in significant changes in chat activity and protein content in the investigated subfractions of the synaptosomes , both of the cortex and the hippocampus ( figure 2-all rats ) . in the cortical light synaptosomal fraction , mchat activity and m - protein content were increased , and these changes were positively correlated amongst themselves ( r = + 0.770 , n = 18 , total control and 2vo groups data , p < .001 ; in the control group r = + 0.788 , n = 9 , p < .02 ) . the cchat activity did not significantly vary or correlate with mchat activity but it was positively correlated with increasing c - protein content ( r = + 0.669 , n = 9 , p < .05 ; in the control group r = + 0.305 , n = 9 , this indicated a reorganization of the synaptic pool in more than one synaptic population of the cholinergic projective neurons in the cortex . but mchat activation was accompanied by a reinforcement in the positive correlation between its values and the m - protein content ( r = + 0.694 , n = 9 , p < .05 ; in the control group r = + 0.291 , n = 9 , p > .05 ) . this indicated a reorganization of the synaptic pool in more than one synaptic population of the cholinergic interneurons in the cortex . in the hippocampal light synaptosomal fraction , however , significant correlations between the values of mchat and cchat activity , and between the values of chat activity and protein content , were absent . this indicated a reorganization of the synaptic pool in more than one synaptic population in the hippocampus , in both the cholinergic systems and some noncholinergic systems . also , the positive correlations between values of c - protein content and mchat activity ( r = + 0.927 , n = 8 , p < .01 ; in the control group r = + 0.265 , n = 9 , p > .05 ) and cchat activity ( r = + 0.844 , n = 8 , p < .01 ; in the control group r = + 0.091 , n = 9 , this indicated a reorganization of the presynapses of the cholinergic interneurons / lateral pathway projective neurons in the hippocampus . in the hippocampal light synaptosomal fraction in the contextual biochemical subgroup , an independent decrease in mchat activity and an increase in cchat activity only , and in the cued biochemical subgroup an increase in cchat activity only , and a decrease in m - protein content and an increase in c - protein content were detected . in the hippocampal heavy synaptosomal fraction in the contextual biochemical subgroup , positive correlations were detected between the increased values of c - protein content and mchat activity ( r = + 0.922 , n = 5 , p < .01 ) and cchat activitiy ( r = + 0.919 , n = 5 , p < .05 ) , and between mchat and cchat activities ( r = + 0.910 , n = 5 , p < .05 ; in the control contextual biochemical subgroup r = 0.266 , n = 5 , p > .05 ) . this indicated a reorganization of the presynapses of the cholinergic interneurons / lateral pathway projective neurons and noncholinergic neurons in the hippocampus . the composition of learning 2s24 and 3s24 was identical and involved small presynapses of the projective cortical neurons ( positive correlation with mchat activity in both forms of learning ) and the hippocampal interneurons / lateral pathway projective neurons ( positive correlations with mchat activity ) . the similar analysis in the cued biochemical subgroup revealed other individual cholinergic compositions of learning and memory ( table 2 : sham - cued , figure 3-ii : sham ) . according to our data , working memory 1s24 composition involved small presynapses of both cortical cholinergic systems ( negative correlation with mchat activity ) and of the hippocampal interneurons / lateral pathway projective neurons ( positive correlation with mchat activity ) . chronic brain ischaemia had considerable effects on the cholinergic organization of the investigated cognitive functions . in the contextual model ( table 2 : 2vo - contextual , figure 3-i : 2vo ) , inherited abilities 1s2 , learning 2s24 and long - term memory 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the cortical cholinergic populations . in the cued model ( table 2 : 2vo - cued , figure 3-ii : 2vo ) , long - term memory 2s1 and 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the hippocampal cholinergic influences . so , under 2vo conditions as in the contextual and in the cued biochemical subgroup quantity of the cholinergic connections with the cognitive functions significantly reduced and some new links arose . each form of cognition as resulting had 2vo cholinergic synaptic composition organized differently from normal ones . we attempted to analyze the dependence of impairment of the investigated cognitive functions in 2vo conditions from the reorganization of key cholinergic systems . it seems , in the contextual biochemical subgroup only preservation of the inherited abilities 1s2 from damage could be explaned by preservation of the key cholinergic populations , revealed in the normal conditions ( figure 4 , middle row ) . in the cued biochemical subgroup , only the preservation of long - term memory 3s1 could be explained by the resistance to ischaemia of the key synaptic population revealed in normal conditions ( figure 5 , middle row ) . then the distinctions in learning 2s24 and 3s24 performance in 2vo conditions would be explained by their new cholinergic compositions if we suppose more considerable influence of the large presynapses of the cortical interneurons on these functions in comparison with the influence of the other new key synaptic populations . so , it seems that performance of the cognitive functions as in the contextual and in the cued model under 2vo conditions , as a rule , did not depend on their cholinergic organization , revealed in normal conditions . whereas , the contextual and cued learning 2s24 and 3s24 revealed cholinergic synaptic compositions identical for normal conditions and different ones , revealed in 2vo conditions , it was investigated prolonged action on the learning performance of the selective agonist of 42 subtype of nachr rjr and the selective antagonist of non-7 subtypes of nachr mecamilamine . in the contextual model under normal conditions , both the agonist rjr and the antagonist mecamilamine did not influence on learning as 2s24 and 3s24 performance . so , in the contextual learning 2s24 and 3s24 , nachr were absent in normal and were acquired in 2vo receptor composition ( 3s24 ) . then , the cued learning 2s24 and 3s24 , with identical cholinergic synaptic compositions in the norm , had differences in receptor compositions . the period of 610 days of 2vo ischaemia provoked typical attributes of vascular dementias such as impairment of learning and long - term memory in both spatial - contextual and spatial - cued models of behaviour in the morris water maze . a considerable reorganization of the synaptic pool of all investigated cholinergic systems in the cortex and the hippocampus was revealed in these same 2vo rats 1114 days after the surgery . it is possible that decrease in mchat activity in the hippocampal heavy synaptosomal fraction in the cued biochemical subgroup was result of the training ( see table 1 and figure 2-cued ) . dysfunction of chat in the projective fibres in the hippocampus ( representing 8090% of the total activity of this enzyme , and see table 1 ) was described at 714 days of ischaemia [ 6 , 13 , 14 , 47 , 48 ] . but we did suppose that the correlation between chat activity and protein content could be masked because of the complex opposing processes that took place in some of the synaptosomal fractions . thus , chronic brain ischaemia for 1114 days resulted in a complex reorganization of the cortical and hippocampal synaptic pool which involved synaptogenesis or hyperfunctions in the unbalanced ionic conditions of one of the cholinergic synaptic populations and degeneration or dysfunction of the others . we also supposed that the variety of cholinergic reactions was revealed by the phenotypical variety of the outbred rats and it was useful for understanding some of the principles of the organization of different forms of cognition . the second purpose of our research was a comparative analysis of the behavioural and biochemical parameters for identification of the cholinergic composition of the investigated cognitive functions under normal and 2vo conditions . in the first place it is necessary to note that the results of this research confirmed and expanded the knowledge about cholinergic mechanisms of cognitive functions under normal brain conditions . our data showed the active involvement of cholinergic projective systems and also regional ones of the cortex and the hippocampus in cognitive processes . cholinergic synaptic connections with the investigated cognitive functions revealed under normal conditions indicate that each form of cognition has an individual cholinergic synaptic and probably receptor compositions . this conclusion is conformed to the results of investigations , obtained in the morris water maze and some other behavioural models [ 3437 ] . then , the data showed the participation of the cholinergic systems not only in mechanisms of learning and working memory , which was repeatedly observed in previous studies [ 35 , 45 , 61 ] , but also in mechanisms of the inherited abilities and long - term memory . our inherited abilities in the contextual task in the morris water maze was firstly detected by r g morris and u frey as a distinct type of memory and termed as it seems the problem of future discussions . our data , concerning individual cholinergic organisation of function support contextual inherited abilities as a distinct form of cognition . the involvement of cholinergic projective systems in mechanisms of the long - term memory is usually denied [ 6467 ] , and was only discussed in a few studies [ 36 , 68 , 69 ] . our data confirmed that synaptic populations of cholinergic projective neurons and of the interneurons of the cortex and the hippocampus can have positive and negative connections with cognitive functions . a negative dependence of cognitive functions on cholinergic cortical efficiency was also revealed earlier in cats using a similar methodology for researching the cholinergic synaptic organization of cognitive functions . therefore , our data demonstrate that the cholinergic mechanisms of learning and memory are more complex than currently perceived . for example , according to our data in the contextual model , the nonselective pharmacological cholinergic means as well as use of different methods of degeneration of the cholinergic projective systems would certainly have revealed the participation of the cholinergic projective systems in learning 2s24 and 3s24 , but it would probably have concealed a cholinergic participation in the mechanisms of the inherited abilities , the long - term and the working memory . from the numerous data in the literature , preservation of the cholinergic projective systems is critical for the success of cognitive processes , and it was thought that cholinergic dysfunction or degeneration results in the impairment of memory in neurodegenerative diseases of different aetiologies . therefore , we analyzed the connections between reorganization of the cholinergic synaptic pool and impairment of learning and memory under 2vo conditions . in our research , only impairment of cued long - term memory 2s1 was really dependent on degeneration of the key synaptic population of the cholinergic cortical interneurons , and also , probably , the intact cued memory 3s1 by the unchanged key synaptic population of the cholinergic cortical projective neurons . at the same time , our data also showed different cholinergic compositions of the cognitive functions under 2vo and under normal conditions in as the spatial contextual and the spatial cued models . the quantity of cholinergic synaptic populations , involving in mechanisms of the investigated cognitive functions , was considerable reduced . furthermore , cholinergic connections in general disappeared from the mechanisms of the following forms of cognition : inherited abilities 1s2 , learning 2s24 and long - term memory 3s1 in the contextual model , and long - term memory 2s1 and 3s1 in the cued model . all considerable differences between cholinergic organisation of the cognitive functions in the normal and 2vo conditions stated above are clear demonstrated in figure 3 . it is important that a consistency between the performances of cognitive functions and their new key cholinergic synaptic populations was found in the majority of the remaining cholinergic - dependent functions under 2vo conditions ( from four to six functions ) . thus , according to our data , we suggest that the normal cholinergic synaptic connections in learning and memory were progressively reduced and changed during chronic ischaemia . it can be noted that the new cholinergic connections with the cognitive functions were not necessarily a consequence of degeneration or dysfunctions in the key cholinergic synaptic populations ( it was evident for contextual learning in 3s24 and long - term memory in 2s1 , that is , these new links could arise by other , indirect reasons ) . the dependence on the inclusion of cholinergic links in the realization of cognitive functions from the functional background of neuronal environments was recently revealed . it seems that the reasons for changes in the cholinergic organization of cognitive functions in an ischaemic pathology can be any neurodegenerative or , on the contrary , reparative process ( sprouting ) of cholinergic and noncholinergic synaptic populations . in spite of the brain reparative potentials , the cholinergic and the whole neurochemical organization of cognitive functions under the chronic actions of pathological factors will be formed as optimally as possible under the new conditions . a new organization of cognitive functions will be constructed on neuronal elements which are stable against pathological influences . in any case , the study of new key neurochemical links in the organization of cognitive functions may be promising . the plasticity of neurochemical links in the individual organization of certain types of cognition can be used in the future for alternative corrections of vascular and other degenerative dementia .

the dementia of ischaemic type and alzheimer 's disease is synergistic or additive in the earliest stages of alzheimer 's disease , although the interactive mechanisms are not known [ 1 , 2 ] . both types of dementia are neurodegenerative diseases and for both the dysfunction and degeneration of cholinergic projective systems in the cortex and the hippocampus from the forebrain nuclei are critical [ 38 ] . correlations between the development of cholinergic dysfunctions and the destruction of pyramidal neurons in the hippocampus [ 6 , 13 , 14 ] , and also damage to the cognitive functions of animals [ 48 , 10 ] , led to the presumption that a dysfunction in cholinergic afferents plays a major role in the development of ischaemic pathologies [ 9 , 12 , 14 , 15 ] . our investigations on the light and heavy synaptosomal fractions of the cortex and the hippocampus allowed the study of the major cholinergic projection systems of the cortex and the hippocampus and their minor intrinsic systems of cholinergic interneurons . according to immunochemical data , both the cortex and the hippocampus the first major sources are the neuronal projections from the forebrain nuclei basalis magnocellularis into the cortex ( precursor of the meynert nucleus in primates and humans ) and projections from the forebrain medial septal nuclei and vertical limb nuclei of the diagonal band of broca into the hippocampus . we previously showed that for both the cortex and the hippocampus the cholinergic presynapses from different sources are isolated in different synaptosomal fractions during preparation in the sucrose density gradient . in the studies on the cortical and hippocampal synaptosomal fractions of rat we revealed that during the first three hours of chronic brain ischaemia the cholinergic projective neurons were reactive , as were the interneurons of the cortex and the hippocampus as well . thus it was shown , that several drugs inhibited short - term memory without altering long - term memory and that working , short - term , and long - term memory were differentially regulated in the various brain regions by the various neurotransmitter systems , including cholinergic one [ 3437 ] . in the present study , the cholinergic synaptic organization of different forms of learning and memory in rats with normal and chronic ischaemic brains a marker of cholinergic neurons , enzyme of acetylcholine synthesis choline acetyltransferase ( chat ; ec 2.3.1.6 ) was used for estimation of the cholinergic systems . chat activity and also protein contents ( total synaptic parameters ) were measured in subfractions of the synaptic membranes and the synaptoplasm isolated from light and heavy synaptosomal fractions of the cortex and the hippocampus . in addition , the regulation of learning performance under prolonged action of selective agonist of 42 subtype of nachr metanicotine ( rjr ) and selective antagonist of non7 subtypes of nachr mecamlamine was studied . the rats were housed in a temperature - controlled room ( 2024c ) with free access to food and water and kept on a 12 h light / dark cycle according to the national institutes of health animal care and the principles of laboratory animal care guidelines and the study was approved by the ethical committees of the institutes . the following forms of cognitive functions were observed and investigated : the inherited abilities ( the first noncasual attempts at decision making in the task , that is , 1s1 trial in the cued model and 1s2 trial in the contextual model ) ; working memory in the first session ( 1s24 and 1s3 - 4 averaged out over the following trials , resp . ) from each sample the light and heavy synaptosomal fractions were isolated , with further separation of the subfractions of the synaptic membranes and the synaptoplasm , following preparative and the disruptive procedures and the discontinued gradients of sucrose density as described previously [ 30 , 39 ] . the fractions of the synaptosomes were obtained from the rough mitochondrial fraction by centrifugation using a bucket rotor ( 84,000 g 120 min , 24c ) in layers between 1.01.2 m sucrose densities ( the light synaptosomes ) and between 1.21.4 m sucrose densities ( the heavy synaptosomes ) . accordingly , the membrane - bound mchat activity and m - protein contents were determined in the synaptic membrane subfractions , and the water - soluble cchat activity and c - protein contents were estimated in synaptoplasm subfractions . moreover , this is the reason why changes in mchat activity could be exposed and changes in cchat activity could be masked in the small presynapses , whereas changes in cchat activity , but not in mchat activity , could be exposed in the large presynapses . the behavioural results were expressed in terms of time taken to swim to the hidden platform ( s ) and the biochemical results were expressed in terms of chat activity ( nmoles acetylcholine / min ) or protein content ( mg ) in 1 g wet weight of cortex and hippocampus tissue , respectively . the period of 1114 days of chronic ischaemia resulted in significant changes in chat activity and protein content in the investigated subfractions of the synaptosomes , both of the cortex and the hippocampus ( figure 2-all rats ) . in the cortical light synaptosomal fraction , mchat activity and m - protein content were increased , and these changes were positively correlated amongst themselves ( r = + 0.770 , n = 18 , total control and 2vo groups data , p < .001 ; in the control group r = + 0.788 , n = 9 , p < .02 ) . the cchat activity did not significantly vary or correlate with mchat activity but it was positively correlated with increasing c - protein content ( r = + 0.669 , n = 9 , p < .05 ; in the control group r = + 0.305 , n = 9 , this indicated a reorganization of the synaptic pool in more than one synaptic population of the cholinergic projective neurons in the cortex . but mchat activation was accompanied by a reinforcement in the positive correlation between its values and the m - protein content ( r = + 0.694 , n = 9 , p < .05 ; in the control group r = + 0.291 , n = 9 , p > .05 ) . also , a reinforcement of the positive correlation between the values of cchat activity and c - protein content was observed ( r = + 0.835 , n = 9 , p < .01 ; in the control group r = + 0.633 , n = 9 , p > .05 ) . at the same time , the correlation between the activity of mchat and the activity of cchat became weaker than in the control ( r = + 0.579 , n = 9 , p > .05 ; in the control group r = + 0.754 , n = 9 , p < .02 ) . in the hippocampal light synaptosomal fraction , however , significant correlations between the values of mchat and cchat activity , and between the values of chat activity and protein content , were absent . the activities of mchat and cchat did not differ from the controls although at the same time a positive correlation arose between their values ( r = + 0.802 , n = 8 , p < .02 ; in the control group r = 0.300 , n = 9 , p > .05 ) . also , the positive correlations between values of c - protein content and mchat activity ( r = + 0.927 , n = 8 , p < .01 ; in the control group r = + 0.265 , n = 9 , p > .05 ) and cchat activity ( r = + 0.844 , n = 8 , p < .01 ; in the control group r = + 0.091 , n = 9 , this indicated a reorganization of the presynapses of the cholinergic interneurons / lateral pathway projective neurons in the hippocampus . only the values of mchat activity were lower in the cortical heavy and in the hippocampal light synaptosomal subfractions in the cued biochemical subgroup of rats as compared with the contextual one . in the cortical light synaptosomal fraction in the contextual biochemical subgroup ( figure 2-contextual ) , independent correlations were detected between the values of mchat activity and m - protein content ( r = + 0.765 , n = 10 , total control and 2vo contextual biochemical subgroups data , p < .01 ; in the control contextual biochemical subgroup r = + 0.774 , n = 5 , p > .05 ) and between the values of cchat activity and c - protein content ( r = + 0.987 , n = 5 , p < .01 ; in the control contextual biochemical subgroup r = + 0.441 , n = 5 , p > .05 ) . in the cued subgroup ( figure 2-cued ) , a correlation was only detected between mchat activity and m - protein content ( r = + 0.783 , n = 8 , total control and 2vo cued biochemical subgroups data , p < .05 ; in the control cued biochemical subgroup r = + 0.622 , n = 4 , p > .05 ) and a correlation was revealed between mchat and cchat activities ( r = + 0.995 , n = 4 , p < .01 ; in the control biochemical cued biochemical subgroups r = 0.404 , n = 4 , p > .05 ) . in the cortical heavy synaptosomal fraction in the contextual biochemical subgroup , independent correlations were detected between mchat activity and m - protein content ( r = + 0.981 , n = 5 , p < .01 ) and between the values of cchat activity and c - protein content ( r = + 0.966 , n = 5 , p < .01 ) , whereas there was no correlation between the activities of mchat and cchat among themselves ( r = + 0.652 , n = 5 , p > .05 ) . in the hippocampal light synaptosomal fraction in the contextual biochemical subgroup , an independent decrease in mchat activity and an increase in cchat activity only , and in the cued biochemical subgroup an increase in cchat activity only , and a decrease in m - protein content and an increase in c - protein content were detected . in the hippocampal heavy synaptosomal fraction in the contextual biochemical subgroup , positive correlations were detected between the increased values of c - protein content and mchat activity ( r = + 0.922 , n = 5 , p < .01 ) and cchat activitiy ( r = + 0.919 , n = 5 , p < .05 ) , and between mchat and cchat activities ( r = + 0.910 , n = 5 , p < .05 ; in the control contextual biochemical subgroup r = 0.266 , n = 5 , p > .05 ) . the long - term memory 2s1 had composition other than 3s1 which involved small presynapses of the projective hippocampal neurons ( negative correlation with mchat activity ) and some populations of hippocampal interneurons ( positive correlations with mchat and cchat activities ) . the composition of learning 2s24 and 3s24 was identical and involved small presynapses of the projective cortical neurons ( positive correlation with mchat activity in both forms of learning ) and the hippocampal interneurons / lateral pathway projective neurons ( positive correlations with mchat activity ) . the long - term memory composition in 2s1 involved large presynapses of the cortical interneurons and small presynapses of projective hippocampal neurons ( in both cases , there were positive correlations with cchat or mchat activities ) . in the contextual model ( table 2 : 2vo - contextual , figure 3-i : 2vo ) , inherited abilities 1s2 , learning 2s24 and long - term memory 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the cortical cholinergic populations . in the cued model ( table 2 : 2vo - cued , figure 3-ii : 2vo ) , long - term memory 2s1 and 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the hippocampal cholinergic influences . it seems , in the contextual biochemical subgroup only preservation of the inherited abilities 1s2 from damage could be explaned by preservation of the key cholinergic populations , revealed in the normal conditions ( figure 4 , middle row ) . on the other hand , the new cholinergic composition in 2vo conditions had accordance between reinforcement of the negative influence of the hippocampal interneurons / lateral pathway projective neurons on learning 3s24 and impairment of this function ( figure 4 , bottom row ) . in the cued biochemical subgroup , only the preservation of long - term memory 3s1 could be explained by the resistance to ischaemia of the key synaptic population revealed in normal conditions ( figure 5 , middle row ) . on the other hand , the absence of 1s24 impairment could also be explained by the new , weakly expressed positive cholinergic influence ( tendency ) of the large presynapses of cortical interneurons ( figure 4 , bottom row ) . then the distinctions in learning 2s24 and 3s24 performance in 2vo conditions would be explained by their new cholinergic compositions if we suppose more considerable influence of the large presynapses of the cortical interneurons on these functions in comparison with the influence of the other new key synaptic populations . so , it seems that performance of the cognitive functions as in the contextual and in the cued model under 2vo conditions , as a rule , did not depend on their cholinergic organization , revealed in normal conditions . whereas , the contextual and cued learning 2s24 and 3s24 revealed cholinergic synaptic compositions identical for normal conditions and different ones , revealed in 2vo conditions , it was investigated prolonged action on the learning performance of the selective agonist of 42 subtype of nachr rjr and the selective antagonist of non-7 subtypes of nachr mecamilamine . under the 2vo conditions , effects of rjr on both learning performance evidently , the 42 subtype did not participate in the regulation of the contextual learning , as before , while some non-7 and non-42 subtypes participated with negative influences on learning 3s24 . a decrease in mchat or cchat activity in one synaptosomal fraction and an increase in another were obtained as result of the 2vo influence , but not of the training , whereas the biochemical parameters did not reveal similar changes between the control contextual and cued biochemical subgroups except one . it is possible that decrease in mchat activity in the hippocampal heavy synaptosomal fraction in the cued biochemical subgroup was result of the training ( see table 1 and figure 2-cued ) . dysfunction of chat in the projective fibres in the hippocampus ( representing 8090% of the total activity of this enzyme , and see table 1 ) was described at 714 days of ischaemia [ 6 , 13 , 14 , 47 , 48 ] . it was shown in vitro that the activity of mchat was selectively suppressed when the exchange of acetylcholine was damaged by inhibition of the vesicular acetylcholine transporter or the high affinity transport of choline . at the same time , according to data in the literature , sprouting and destruction with the swelling of neurons and their terminals predominates in late brain ischaemia or postischaemic reoxygenation ( in days and months ) [ 3 , 48 , 53 , 54 ] . therefore , the correlated increase between mchat activity and m - protein content will be reflected as cholinergic hyperfunction and synaptogenesis ( cortical light synaptosomes in the biochemical total group and both subgroups ) , whereas the correlated increase between cchat / mchat activity and the c - protein contect will only reflect synaptogenesis ( cortical light synaptosomes in the biochemical total group and the contextual subgroup , hippocampal heavy synaptosomes in the total group and the cued subgroup ) . thus , chronic brain ischaemia for 1114 days resulted in a complex reorganization of the cortical and hippocampal synaptic pool which involved synaptogenesis or hyperfunctions in the unbalanced ionic conditions of one of the cholinergic synaptic populations and degeneration or dysfunction of the others . then , the data showed the participation of the cholinergic systems not only in mechanisms of learning and working memory , which was repeatedly observed in previous studies [ 35 , 45 , 61 ] , but also in mechanisms of the inherited abilities and long - term memory . our inherited abilities in the contextual task in the morris water maze was firstly detected by r g morris and u frey as a distinct type of memory and termed as it seems the problem of future discussions . for example , according to our data in the contextual model , the nonselective pharmacological cholinergic means as well as use of different methods of degeneration of the cholinergic projective systems would certainly have revealed the participation of the cholinergic projective systems in learning 2s24 and 3s24 , but it would probably have concealed a cholinergic participation in the mechanisms of the inherited abilities , the long - term and the working memory . from the numerous data in the literature , preservation of the cholinergic projective systems is critical for the success of cognitive processes , and it was thought that cholinergic dysfunction or degeneration results in the impairment of memory in neurodegenerative diseases of different aetiologies . in our research , only impairment of cued long - term memory 2s1 was really dependent on degeneration of the key synaptic population of the cholinergic cortical interneurons , and also , probably , the intact cued memory 3s1 by the unchanged key synaptic population of the cholinergic cortical projective neurons . furthermore , cholinergic connections in general disappeared from the mechanisms of the following forms of cognition : inherited abilities 1s2 , learning 2s24 and long - term memory 3s1 in the contextual model , and long - term memory 2s1 and 3s1 in the cued model . it is important that a consistency between the performances of cognitive functions and their new key cholinergic synaptic populations was found in the majority of the remaining cholinergic - dependent functions under 2vo conditions ( from four to six functions ) . it can be noted that the new cholinergic connections with the cognitive functions were not necessarily a consequence of degeneration or dysfunctions in the key cholinergic synaptic populations ( it was evident for contextual learning in 3s24 and long - term memory in 2s1 , that is , these new links could arise by other , indirect reasons ) . it seems that the reasons for changes in the cholinergic organization of cognitive functions in an ischaemic pathology can be any neurodegenerative or , on the contrary , reparative process ( sprouting ) of cholinergic and noncholinergic synaptic populations . in spite of the brain reparative potentials , the cholinergic and the whole neurochemical organization of cognitive functions under the chronic actions of pathological factors will be formed as optimally as possible under the new conditions .

naturally produced biopolymers in living organisms play crucial roles in materials discovery and development . they have inspired scientists to synthesize novel biomaterials through mimicking mother nature , or they can further serve as templates and building blocks to prepare new generations of biocompatible , bioregenerative , or biodegradable materials for biomedical applications . for instance , dna has been used to rationally design plasmonic nanostructures , to build nanoscaffolds for incorporating multiple - affinity ligands , and to self - assemble into numerous prescribed 3d shapes . cellular membranes have also been widely imitated by phospholipids and polysaccharides to form liposome or micelles for drug and imaging agent delivery . leukocyte membranes have also been used to coat silicon nanoparticles ( nps ) to yield hybrid nps that achieve cell - like functions , including avoiding clearance by the immune system . multimodal imaging combines different modalities together to provide complementary information and achieve synergistic advantages over any single modality alone . it has emerged as a very promising strategy for preclinical research and clinical applications . one major challenge of multimodal imaging is to develop an efficient platform to load various components with individual contrast properties together while maintaining compact size , good biocompatibility and targeting capability . exogenous inorganic nps - based reporters have attracted considerable interests , such as iron oxide nps for magnetic resonance imaging ( mri ) and quantum dots for fluorescence imaging . compared with inorganic nps , organic nps generally exhibit good biocompatibilities , biodistribution and clearance , although most of them only appear to possess optical imaging properties . some biomolecules based nps such as liposomes have been widely used for loading contrast agents and drugs . therefore , such biomolecules need complicated and time - consuming processes to prebuild various contrast properties or require chemical modifications to integrate different reporting moieties into one entity , which we term as a passive platform . for example , organic ligands are generally incorporated into a nanoplatform before chelating to radioactive or magnetic metal ions for positron emission tomography ( pet ) and magnetic resonance imaging ( mri ) . melanin , an amorphous , irregular functional biopolymer and a ubiquitous natural pigment that presents in many organisms including human skin , is a typical biomarker for disease imaging including melanoma detection and parkinson diseases diagnosis . in this study , we report the successful transferring of this biomarker into an imaging nanoplatform . by mimicking natural melanin , water - soluble melanin nanoparticle ( mnp ) has been synthesized and used as the active platform for multimodal imaging of tumors . we demonstrate that mnp can not only offer its native optical properties for photoacoustic imaging ( pai ) , but also actively chelate to metal ions ( cu , fe ) for pet and mri with a high loading capacity and stability utilizing its intrinsic chelating function . furthermore , ultrasmall size mnps ( 4.5 nm ) can be easily prepared and surface - modified . overall , these unique properties significantly simplify the process of preparation of multimodal imaging probes and make mnp a highly promising nanomaterial for biomedical applications . figure 1 schematically illustrates the procedure to prepare ultrasmall water - soluble mnp with multimodal imaging properties . to change the intrinsic poor water - solubility of melanin , pristine melanin granule was first dissolved in a 0.1 n naoh and then neutralized under the assistance of sonication to decrease interchain aggregation . ultrasmall mnp in high water monodispersity and homogeneity with a size of 4.5 0.5 nm , which was termed as plain water - soluble mnp ( pws - mnp ) , were successfully obtained ( figure 2a , b and figure s1a , supporting information ) . pws - mnp exhibited excellent water - solubility of 40 mg / ml and stability , which can be attributed to the highly negative potential of approximately 22.2 mv on the np surface that efficiently blocks the np aggregation through electrostatic repulsion ( figure s1b ) . furthermore , pws - mnp can be stored as lyophilized powder for over six months and effectively redissolved in water allowing long - term usage ( figure 2a ) . the ft - ir spectra of pristine melanin granule and pws - mnp were similar to each other , indicating no significant change of molecular structure ( figure s2a ) . the h nmr spectrum of pws - mnp in d2o showed no obvious signal belonging to the hydrogen atom on the arylene groups , suggesting most of the conjugated backbones were buried in the np ( figure s2b ) . the molecular weight of a pws - mnp was calculated from the nanoparticle size and its density ( 1.3 g / cm ) , which is about 40 kda . the melanin granules were first dissolved in 0.1 n naoh aqueous solution , and then neutralized under sonication to obtain melanin nanoparticles in high water monodispersity and homogeneity . after peg surface - modification , rgd was further attached to the mnp for tumor targeting . then fe and/or cu were chelated to the obtained mnps for pai / mri / pet multimodal imaging . characterization of physical properties of mnps . ( a ) from left to right : pictures of ( 1 ) pristine melanin granule in h2o , ( 2 ) melanin neutralized without sonication in h2o , ( 3 ) freeze - dried pws - mnp , ( 4 ) freeze - dried pws - mnp redissolved in pbs ( ph = 7.4 ) , ( 5 ) freeze - dried peg - mnp , ( 6 ) freeze - dried peg - mnp redissolved in pbs ( ph = 7.4 ) . ( b ) tem of pws - mnp ( left ) and peg - mnp ( right ) , scale bar = 20 nm . ( c ) the plot of the relationship between the number of metal ions attached on one mnp with feed ratio ( wions : wmnp ) . ( d ) stability study of metal ion - chelated mnps in pbs ( ph = 7.4 ) . in vitro and in vivo study of pai of mnps . ( a ) the photoacoustic signal produced by peg - mnps at concentrations of 0.625 , 1.25 , 2.5 , 5.0 , 10 , and 20 m , and it was observed to be linearly dependent on its concentration ( r = 0.995 ) . mice were injected subcutaneously ( region enveloped by blue dotted line ) with peg - mnp at concentrations of 0 , 5 , 10 ( from left to right in top row ) , and 20 , 40 , 80 ( from left to right in bottom row ) m . one vertical slice in the photoacoustic image ( red ) was overlaid on the corresponding slice in the ultrasound image ( gray ) . the linear regression is calculated on the five most concentrated inclusions ( r = 0.998 ) . ( d ) the overlaying of ultrasonic ( gray ) and photoacoustic ( red ) imagings of u87 mg tumor ( region enveloped by yellow dotted line ) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice ( n = 3 ) and their subtraction imagings . ( e ) quantitative analysis of enhanced pa signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h , compared with at 0 h. to retain the water - solubility of pws - mnps for further biomodification and metal ion - chelating , polyethylene glycol ( peg ) chains were introduced to the mnp . nh2-peg5000-nh2 was used because the amine groups can react with dihydroxyindole / indolequinone groups in melanin . the number of peg chains per mnp was determined to be 19 ( figure s3a and s3b ) . the diameter of the peg - functionalized mnp ( peg - mnp ) became large and reached 7.0 nm ( figure 2b and figure s1a ) . moreover , the surface potential of peg - mnp decreased to 6.1 mv ( figure s1b ) because of introduction of peg and positive nh2 groups on the mnp surface . the similar absorption spectrum of peg - mnp to pws - mnp demonstrated that the peg - modification did not influence the absorption properties of melanin ( figure s3c ) . lastly , for demonstrating that mnp can be used as a platform for biomodification , peg - mnp was further modified with biomolecules such as cyclic arg - gly - asp - d - phe - cys [ c(rgdfc ) ] peptide ( abbreviated as rgd ) , which can target tumor v3 integrin . the number of rgd attached to the mnp was calculated to be about 8 per mnp and the size of rgd - functionalized peg - mnp ( rgd - peg - mnp ) increased a little to 9.6 nm ( figure s4 ) . in vitro and in vivo study of mri of fe - chelated mnps . ( a ) t1 relaxation rates ( 1/t1 , s ) as a function of fe - rgd - peg - mnp ( mm ) in agarose gel ( 1.0 t , 25 c ) . ( b ) mri detection of fe - rgd - peg - mnps in living mice . mice were injected subcutaneously ( region enveloped by red dotted line ) with fe - rgd - peg - mnps at concentrations of 0 , 1.25 , 2.5 ( from left to right in upper layer ) , and 5 , 10 , 20 ( from left to right in bottom layer ) m . ( c ) quantitative analysis of enhanced mr signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h , compared with at 0 h. ( d ) mri images of u87 mg tumors ( region enveloped by yellow dotted line ) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice ( n = 3 ) ( tr : 700 ms , te : 5.2 ms ) . top row shows black and white images , and bottom row shows the pseudocolored images . to investigate the possibility of mnp as a platform for pet and mri , its chelating properties to cu ( cu for pet ) and fe ( for mri ) were studied . after adding metal ions ( 0.2 ml of 10 mm fecl3 or cucl2 ) into mnp aqueous solutions ( 1 ml of 20 m for pws - mnp and peg - mnp ) , the precipitation of pws - mnp quickly appeared , while peg - mnp maintained good water - solubility ( figure s5 ) . the fe or cu - chelated mnp ( fe - peg - mnp , fe - rgd - peg - mnp , cu - peg - mnp and cu - rgd - peg - mnp ) exhibited high loading capacities . the maximum quantities of one mnp to chelate to cu and fe are about 100 and 90 ions , respectively , no matter whether rgd is attached to the mnp or not ( figure 2c ) . after fe - chelating , the mnp sizes increased to 8.9 nm and 10.7 nm for fe - peg - mnp and fe - rgd - peg - mnp respectively and their zeta - potential remained in the neutral region ( figure s4 and table s1 ) . the optical stabilities of peg - mnp and rgd - peg - mnp under increasing durations of light exposure were further tested . compared with those reported dyes for pai , which exhibit significant reduced absorption ( > 30% ) under light exposure , peg - mnp and rgd - peg - mnp showed intriguing photostability ( only 3% reduced absorption ) ( figure s6 ) , indicating their high capability for pai . further stability assay of fe or cu - chelated mnps in pbs solution showed that only about 3% cu and 7% fe were released from those mnps at the first 2 h , and there was no further release at longer incubation time points , indicating the high stability of the chelating platform ( figure 2d ) . the first 2 h released metal ions may derive from those that were absorbed on the mnps through weak electrostatic interaction . furthermore , the high viability of nih3t3 and u87 mg cells ( about 90110% as compared to the nontoxic control ) after 24 h of incubation with peg - functionalized mnps was found , indicating high biocompatibility and low cytotoxic effect of peg - functionalized mnps ( figure s7 ) . to investigate the possibility of mnps to be used as a photoacoustic agent , we first studied the detection sensitivity of peg - mnp in aqueous solution at increasing concentrations from 0.625 to 20 m . the peg - mnp with 0.625 m was detected , and the photoacoustic signals increased linearly with the increase of peg - mnp concentrations ( r = 0.995 ) ( figure 3a ) . the detection sensitivity of mnp in living body was further tested by subcutaneous injection of peg - mnp on the lower back of mice ( n = 3 ) at increasing concentrations of 5 to 80 m ( figure 3b ) . a linear correlation ( r = 0.998 ) between the mnp concentration and the corresponding photoacoustic signal 2.5 m of peg - mnp was found to give the equivalent photoacuoustic signal strength as the tissue background . to further investigate the in vivo pai properties , one group of u87 mg tumor mice were tail - vein injected with 250 l of rgd - peg - mnp at a concentration of 200 m . mice showed obvious increase of photoacoustic signal in tumors after injection with rgd - peg - mnp at 4 h than that of prescan ( figure 3d ) . the increased photoacoustic signal of rgd - peg - mnp ( figure 3e ) could be attributed to the enhanced permeability and retention ( epr ) effect and the tumor targeting ability of rgd - peg - mnp to v3 integrin . furthermore , instead of vevo lazr pai system , using inveon research workplace ( nexus 128 ) was able to obtain 3d pa imaging , which provided the more clearly enhanced blood vessel signals in tumor after mnp injection ( figure s8 ) . in vitro and in vivo study of pet of cu - labeled mnps . ( a ) uptake of cu - rgd - peg - mnp with and without blocking in u87 mg cells at 37 c for 1 , 2 and 4 h incubation . all results , expressed as percentage of cellular uptake , are mean of triplicate measurements sd . ( b ) representative decay - corrected coronal ( top ) and transaxial ( bottom ) small animal pet images ( left three images ) and the overlaying of ct ( gray ) and pet ( color ) images ( right three images ) of u87 mg tumors ( region enveloped by yellow dotted line ) acquired at 2 , 4 , and 24 h after tail vein injection of cu - rgd - peg - mnp . ( c ) biodistribution of cu - rgd - peg - mnp in mice ( n = 3 ) at 2 , 4 , and 24 h after injection . the radioactive signal from each organ was calculated using a region of interest drawn over the whole organ region . to study whether fe ( t1 contrast agent ) retains mr signal - enhancing property after loading into mnps , t1-weighted mri images of various concentrations of fe - rgd - peg - mnp in agarose gel was investigated ( figure s9a ) . with the increase of np concentration , mr signal was significantly enhanced , suggesting fe - rgd - peg - mnp generate a high magnetic field gradient on their surface . r1 value of fe - rgd - peg - mnp ( the slope of the fitted curve in figure 4a , using gd as standard ) was calculated to be 1.2 mm s. the magnetic sensitivity in living mice was then tested by subcutaneous injection of fe - rgd - peg - mnp on the lower back of mice ( n = 3 ) at increasing concentrations of 1.25 to 20 m . it was extrapolated that 1.25 m of fe - rgd - peg - mnp produced the equivalent mri signal intensity as the tissue background ( figure 4b ) . to demonstrate the use of mnp as the platform for mri of tumors , t1-weighted images were obtained from mice bearing u87 mg tumors ( n = 3 ) . u87 mg tumors displayed increased signals after 4 h mnp injection ( figure 4d ) . the relative mr signal intensity of tumor at 4 h increased 30% compared with at 0 h , demonstrating that mnp can be used as a platform for mri ( figure 4c ) . furthermore , further optimizing the mr imaging conditions can provide more clearly enhanced mri signal and mnp accumulation in tumor ( figure s9b ) . to investigate the pet imaging properties of mnp , cu was selected as a pet radiolabel for mnp because it can be readily chelated by melanin and the intermediate half - life of cu ( 12.7 h ) makes it suitable for radiolabeling of biomolecules and imaging . simple mixing of rgd - peg - mnp and peg - mnp with cu allowed successfully labeling the nps in the yield of 80% . the resulting mnps , cu - rgd - peg - mnp and cu - peg - mnp , displayed excellent stability in pbs solution ( figure s10 ) . similar to cu - chelated mnps , only 3% cu released from the mnps after 24 h of incubation . thus , cu - labeled mnps were easily and reliably produced and exhibited reasonable stability in vitro . in vivo multimodality imaging of tumor ( region enveloped by yellow dotted line ) bearing mice with pai and mri / pet respectively . ( b ) the overlaying of ultrasonic ( gray ) and photoacoustic ( red ) imaging of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp ( 200 l of 10 m ) in living mice and their subtraction imaging . ( c ) the overlaying of representative decay - corrected coronal ( top ) and transaxial ( bottom ) small animal ct ( gray ) and pet ( color ) images of u87 mg tumors acquired at 2 , 4 , and 24 h after tail vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp ( 250 l of 200 m ) . ( d ) mri images of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp ( 250 l of 200 m ) in living mouse . top row shows black and white images , and bottom row shows the pseudocolored images . uptake of cu - rgd - peg - mnp by u87 mg cells with or without blocking agent rgd at 1 , 2 , and 4 h are shown in figure 5a . cu - rgd - peg - mnp exhibited higher uptakes than blocking group at all the incubation time , with a value of 3.32 0.37% , 5.32 0.43% and 7.18 0.33% for cu - rgd - peg - mnp at 1 , 2 , and 4 h. in comparison , for cu - rgd - peg - mnp blocking group , much lower uptake of cu - rgd - peg - mnp was observed with a value of 2.00 0.15% , 3.16 0.20% and 3.48 0.29% at 1 , 2 , and 4 h , respectively , indicating the successful biomodification of mnps with rgd peptide and the specific targeting ability of rgd contribute to the uptake of cu - rgd - peg - mnp by u87 mg cells . the in vivo pet of mnps was performed in u87mg - tumor - bearing mice . cu - rgd - peg - mnp showed tumor accumulation and clear tumor contrast after 2 h postinjection ( figure 5b ) . quantification analysis revealed that the tumor uptake values of cu - rgd - peg - mnp gradually increased with time to 24 h , and they were 4.75 0.63 , 5.87 0.87 , and 5.93 0.89% id / g at 2 , 4 , and 24 h , respectively ( figure 5c ) . in addition to the tumor , moderate activity accumulation was observed in the liver ( e.g. , 15.78 2.55% id / g at 24 h for all mnps ) , and relative lower activity accumulation was also found in the kidneys ( e.g. , 5.34 0.62% id / g at 24 h for all mnps ) . these data indicated the mnp was cleared mainly through hepatobiliary system . to further investigate the possible targeting property of mnps , the cu radiolabeled rgd - peg - mnp and control cyclic arg - ala - asp - d - phe - cys [ c(radfc ) ] peptide ( abbreviated as rad , with nontargeting property for tumor v3 integrin ) functionalized peg - mnp ( cu - rad - peg - mnp ) for u87 mg tumor pet imaging the obvious stronger pet signal of cu - rgd - peg - mnp can be found in tumor at 4 h than that of cu - rad - peg - mnp ( p < 0.05 ) , indicating the good and specific targeting property of rgd - peg - mnp . to investigate the possibility of using mnp platform for multimodality imaging , mnp were mixed with fe and cu in sequence to form the multifunctional probes fe - rgd - peg - mnp and cu - fe - rgd - peg - mnp ( the amount of fe per mnp is 56 ) for pet / pai / mri . pet of mice bearing u87 mg tumors were then obtained first at 2 , 4 , and 24 h postinjection of cu - fe - rgd - peg - mnp . after 48 h , t1-weighted mri and pai of mice bearing u87 mg tumors were then obtained respectively at 4 h after another injection of large dose of fe - rgd - peg - mnp . in figure 6 , cu - fe - rgd - peg - mnp showed very similar pet , mri and pai properties on u87 mg tumor , compared with the corresponding single modality imaging from cu - rgd - peg - mnp , fe - rgd - peg - mnp , and rgd - peg - mnp respectively . these results showed that using mnp as the active platform to load cu and fe together can efficiently combine its native photoacoustic properties with radioactive and magnetic properties together for multimodality imaging . to change the lack of contrast properties of biomolecule - based nanoplatform for multimodality imaging , recently porphyrin were successfully introduced into phospholipid to provide the platform with desirable optical properties , while it still requires complicated and time - consuming chemical modifications and other reporting molecules to achieve multimodality imaging ability . we herein developed the functional biomarker , melanin , as a novel nanoplatform with its native optical property and multifunctions , which can simply and actively collecting optical , magnetic and radioactive properties together for multimodality imaging . melanin , the oxidation products of tyrosine , plays an important role in living organism . accompanied with the development of molecular imaging probes in the past decade , melanin has been used as an effective molecular target as well as endogenous contrast agent for pai because of its strong light absorption properties . besides , melanin has intrinsic strong chelating properties to many metal ions including cu and fe , which can be used to nuclear imaging and mri . consequently melanotic melanomas show hyperintensity on t1-weighted mri images . considering the attractive properties of the biomarker melanin , we and others have engineered cancer cells to biologically produce melanin for multimodality imaging ( pai / mri / pet ) of cancer . however , this method requires genetic modification of cells , which is time - consuming and may have limited clinical value . thus , water - soluble mnps are more appropriate to behave as a natural active platform to simplify the preparation procedure for multimodal applications . considering only trace amount of cu ions utilized for pet and its final decay to zn ions , which is necessary for life process , and the abundant amount of fe ions in living body , cu and fe ions used in our system are expected to be metabolized in living subjects . in comparison , although gd ion has higher t1 mri effect than fe ion , its potential toxicity is still a problem . concomitantly , the traditional nanoparticle - based platform needs complicated functionalization of ligand to chelate gd for mri and cu for pet . more interestingly , the new mnp system can serve as an active nanoplatform and easily bind with metal ions without the traditional needs of surface modification and introducing chelating groups , which significantly simplifies the preparation process and reduces the heterogeneity of the resulting multimodal nps . furthermore , the mnp is an organic and biodegradable material and showed relatively good tumor imaging properties . all of these properties make mnps highly promising for potential clinical translation . despite its important functions , developing melanin for molecular imaging therefore , preparing mnps is desired while still a challenge for well - dispersing in water , especially for those with size around 10 nm that can provide not only appropriate blood circulation time , but also high surface - to - volume ratio to chelate enough metal ions for efficient bioimaging . although the formation mechanism of polymeric melanin is not clear , its molecular structure is generally considered to be composed of dihydroxyindole / indolequinone segments with hydrophobic conjugated main chain having strong interaction and hydrophilic hydroxyl groups on the benzene rings . therefore , to realize melanin water - soluble at neutral environments , decreasing the interchain aggregation of conjugated main chains and lowering the formed melanin particle size to expose more hydrophilic hydroxy groups on the surface of melanin is a promising way . in our work , we first realized the synthesis of ultrasmall mnps in water with high monodispersity and homogeneity under the assistance of sonication . another problem that should be resolved is the metal ion - initiated cross - linking and the formation of mnp precipitation . recent reports showed that fe is a strong cross - linker for catechol groups , which is one of the components in melanin molecular structure . in our work , peg encapsulation is found can not only enhance the biocompatibility and the water - solubility , but also efficiently prevent the formation of metal ion - initiated precipitation . investigation on the in vivo subcutaneous phantom showed that the signals between mri and pai had good linear relationship ( figure s12 ) . in addition , the slope of enhanced pa signal with different concentrations was higher than that of mri signal , indicating the pai imaging of mnp was more sensitive than the mri imaging . moreover , because of their easy conjugation with targeting groups , the mnps modified with rgd exhibited the tumor targeting ability to the v3 integrin overexpressed on the surface of tumor vasculatures and the u87 mg tumor cells ( from pet data ) , which afforded rgd - conjugated mnps higher accumulation capability in tumor than those rad - modified mnps through enhanced permeability and retention ( epr ) effect . this further indicated that mnps can conveniently function as a good nanoplatform for targeted imaging . though it is difficult to compare the imaging effects of our platform with other type of nanoplatforms ( considering the lack of the triplemodality analogues ) , we demonstrated the unique ability of mnp nanoplatform to combine pet , photoacoustic and mr imaging modalities together to get complementary information for tumor imaging . for example , pet efficiently provides the in vivo pharmacokinetics and biodistribution of the nanoprobes and can also provide physiological information on disease with whole body imaging capability , but it can not image tissues at high spatial resolution ; pai provides functional and molecular information on the tumor with high spatial resolution . it is a cheap and convenient way to image tumor in real time but suffers from limited tissue penetration ability , which can be compensated by pet . mri provides high spatial resolution image and anatomical information on disease but generally lacks of molecular imaging capability . combination of pet / pai / mri thus allows us to image diseases at different depths with molecular and anatomical information . recent developments showed that multimodality imaging is promising not only for accurate tumor imaging but also for guiding tumor resection . for example , pet / mri , which combines the exquisite anatomical information by mri with the extreme sensitivity of pet , can be used for whole - body imaging and deep tumor localization . mri / fluorescence imaging ( fi ) is appropriate for guiding superficial tumor resection . in comparison , combining pet / mri / pai together is anticipated to help for guiding both superficial and deep tumor surgery . to our knowledge , our triple - modality mnp system can be first used for pet and mri to obtain the detailed information on tumor for surgical planning in presurgery . pai can then be used to localize the superficial and relatively deep tumors in surgery for helping the tumor resection . overall , a reliable method for preparation of water - soluble mnp has been developed in our work , which lays down a foundation for its future biomedical applications . it can be easily envisioned that mnp can serve as a nanoplatform not only for molecular imaging but also for theranostics . considering the abundant functionalities of melanin , such as binding drugs , mnp - based platform used for drug delivery and therapy are now being investigated . in conclusion , we report mnp as the natural biomarker - transferred active platform for multimodality imaging . mnp is of particular interest because such an endogenous agent with native photoacoustic signals and strong chelating properties with metal ions can act as an active platform to simplify the assembling of different imaging moieties . mnp can be easily modified with biomolecules for targeted tumor multimodality imaging , and it showed good in vivo tumor imaging properties . we expect this work will stimulate further studies of multifunctional endogenous material as nanoplatforms for potential imaging and therapeutic applications . the following reagents were acquired and used as received : melanin ( sigma - aldrich ) , sodium hydroxide ( sigma - aldrich ) , hydrochloric acid ( 37 wt % , sigma - aldrich ) , nh4oh solution ( 28 wt % , sigma - aldrich ) , amine - peg5000-amine ( nh2-peg5000-nh2 , 5 kda , laysan bio ) , dimethylthiazolyl - diphenyltetrazolium ( mtt ; biotium ) , phosphate buffered saline ( pbs , gibco ) , and agarose ( invitrogen ) . tyrosine - derived synthetic melanin ( 20 mg ) was first dissolved in 10 ml of 0.1 n naoh aqueous solution under vigorous stirring . after dissolving , hcl aqueous solution ( 0.1 n ) was swiftly dropped into the obtained basic melanin solution to adjust the ph to 7.0 under sonication with output power = 10 w for 1 min . the neutralized solution was further centrifuged with a centrifugal - filter ( amicon centrifugal filter device , mwco = 30 kda ) and washed with deionized water and repeated several times to remove the produced nacl . the aqueous solvent was removed by freeze - drying to obtain 15 mg black solid of pws - mnp . nh4oh solution ( 28 wt % ) was added to 5 ml of pws - mnp aqueous solution ( 1 mg / ml of water ) to adjust the ph of the solution to 9 . this mixed solution was added dropwise into nh2-peg5000-nh2 ( 5 , 10 , 25 , and 50 mg ) aqueous solution with ph = 9 . after vigorous stirring for 12 h , peg - modified mnp was retrieved by centrifugation with a centrifugal - filter ( amicon centrifugal filter device , mwco = 30 kda ) and washed with deionized water several times by redispersion / centrifugation processes to remove the unreacted nh2-peg5000-nh2 . the aqueous solvent was removed by freeze - drying and the obtained peg - mnp was weighed to preliminary calculate the quantity of the peg attached on mnps . because of the existence of one nh2 group per peg chain on the surface of mnp , we then accurately determined the nh2 group on mnp with fluorescamine by spectrofluorometer to calculate the amount of peg on the nanoparticles ( using ethamine as the standard ) . the cross - linker solution was prepared freshly . the 4-(n - maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo - n - hydroxysuccinimide ester sodium salt ( sulfo - smcc ) ( 1.2 mg ) the water - soluble peg - mnp [ 1 mg in 1 ml of pbs ( ph = 7.2 ) ] was incubated with the above cross - linker solution for 2 h at room temperature . the resultant thiol - active mnp ran through a pd-10 column prewashed with pbs ( ph = 7.2 , 10 mm ) to remove the excessive sulfo - smcc and byproducts . the purified mnp was concentrated to the final volume of 0.5 ml with a centrifugal - filter ( mwco = 30 kda ) . the crgdfc stock solution ( 120 l of 5 mm in the degassed water ) was added to the above mnp solution with stirring . the uncoupled rgd peptide was removed through a pd-10 column and collected to analyze its quantity through hplc . the resultant product , rgd - peg - mnp , were concentrated by a centrifugal - filter ( mwco = 30 kda ) and stored at 4 c for one month without losing targeting activity . the final rgd - peg - mnp were reconstituted in pbs and filtered through a 0.22 m filter for cell and animal experiments . the mnp ( 1 mg in 1 ml h2o ) was labeled with fe or cu by addition of 20 l of fresh fecl3 ( 10 mg / ml ) in pbs ( ph = 7.4 ) or 20 l of cucl2 ( 10 mg / ml ) in buffer solution of ph = 5.5 followed by a 1 h incubation at 40 c ( the chelating mechanisms of mnps were shown in figure s13 ) . the products were washed out by pbs and passed through a 0.22-m millipore filter into a sterile vial for in vitro and animal experiments . the fe and cu concentrations of mnps were measured by inductively coupled plasma - mass spectrometry ( icp - ms ) analysis . the stability of metal ion - chelated mnps were studied by incubating those mnps in pbs ( ph = 7.4 ) at 37 c . those mnps were placed in dialysis tube ( mwco 10k ) with magnetic stirring , dialysis against 10 ml pbs . at a certain time , dialysate was removed for icp - ms analysis and replaced with fresh pbs solution . for icp - ms analysis , 100 l of the detected sample was first heated to evaporate the water solvent and then digested with 0.5 ml of concentrated nitric acid ( 70% w / w ) under heating . after the solvent was evaporated , the residue was then dissolved in 7 ml of dilute nitric acid ( 2% w / w ) for final icp - ms analysis . ft - ir spectra were measured in a transmission mode on a bio - rad ft - ir spectrophotometer ( model fts135 ) under ambient conditions . samples of pristine melanin granules and functionalized mnps were ground with kbr and then compressed into pellets . transmission electron microscopy ( tem ) images were recorded on a jeol 2010 transmission electron microscope at an accelerating voltage of 100 kv . the tem specimens were made by placing a drop of the nanoparticle aqueous solution on a carbon - coated copper grid . the hydrodynamic sizes of the mnps were determined by dynamic light scattering ( dls ) using a 90 plus particle size analyzer ( malvern , zetasizer nano zs90 ) . zeta potentials were measured using a zeta potential analyzer ( malvern , zetasizer nano zs90 ) . the h nmr spectra were recorded at 20 c on a 400 mhz nmr spectrometer ( bruker ) , using d2o as solvent . the mnps with or without fe were further radiolabeled with cu by addition of 11.5 mci of cucl2 in 0.1 n naoac ( ph 5.5 ) buffer followed by 1 h incubation at 40 c . the radiolabeled mnps were then purified by a pd-10 column ( ge healthcare , piscataway , nj , usa ) . the product was washed out by pbs and passed through a 0.22-m millipore filter into a sterile vial for in vitro and animal experiments . the investigation of the radiolabeling stability of mnps is similar to the metal ion - chelated mnps except that the detector icp - ms was replaced by perkinelmer 1470 automatic gamma - counter for counting radioactivity . in vitro cytotoxicity of mnps was determined in nih-3t3 and u87 mg cells by the mtt assay . nih-3t3 and u87 mg cells were incubated on 96-well plate in dmem medium containing 10% fbs and 1% penicillin / streptomycin at 37 c in 5% co2 humidified atmosphere for 24 h and 0.5 10 cells were seeded per well . cells were then cultured in the medium supplemented with indicated doses of different mnps for 24 h. the final concentrations of mnps in the culture medium were fixed at 3.125 , 6.25 , 12.5 , and 25 m in the experiment . addition of 10 l of mtt ( 0.5 mg / ml ) solution to each well and incubation for 3 h at 37 c was followed to produce formazan crystals . then , the supernatant was removed and the products were lysed with 200 l of dmso . the absorbance value was recorded at 590 nm using a microplate reader . the absorbance of the untreated cells was used as a control and its absorbance was as the reference value for calculating 100% cellular viability . u87 mg cells ( 1 10 per well ) were seeded in 12-well tissue culture plates and allowed to attach overnight . the cells were washed twice with serum - free dmem and incubated with the cu - labeled mnps ( 2 ci per well , final concentration approximately 6 nm ) in 400 l of serum - free dmem at 37 c . the specific binding of the probes with u87 mg cells was determined by coincubation with rgd ( 30 g per well ) . after 1 , 2 , and 4 h , the cells were washed three times with cold pbs and lysed with the addition of 200 l of 0.2 m naoh . the radioactivity of all fractions was counted using a perkinelmer 1470 automatic gamma - counter . the uptake ( counts per minute ) was expressed as the percentage of added radioactivity . for studying the pai properties of mnps , different concentrations of mnps aqueous solutions ranging from 0.625 to 20 m were filled into polyethylene capillaries and then the capillaries were laid on the surface of solidified agarose gel . the capillaries were further covered with thin 1% agarose gel to make the surface smooth . for the particle s sensitivity in living body , mnps aqueous solutions with different concentrations from 5 m to 80 m were mixed with matrigel at 0 c and then subcutaneously injected on the lower back of mice . toronto , canada ) with a laser at excitation wavelength of 680 nm and a focal depth of 10 mm was used to acquire photoacoustic and ultrasound images . mri experiments were performed at 25 c in a magnetic resonance ( mr ) scanner ( siemens 1.0 t ) . to simulate the biological environment , agarose gel , prepared in 300 l of the pcr tube using secondary distilled water as the solvent for dissolving the agarose , was used to demonstrate the magnetic signal . the bottom of the tube was first covered with a layer of 1% agarose gel . when agarose gel was cooled , the mixtures of mnps and aqueous solution of agarose ( ratio 1:1 by volume ) with iron concentrations at 62.5 , 125 , 250 , 500 , and 1000 m fe ( amount to 1.25 , 2.5 , 5 , 10 , 20 m mnp ) , were then filled into the intermediate portion of the pcr tube respectively while the sample was hot . after cooling , another 1% agarose gel was covered on the top layer of the cube . the tubes were placed into the mr scanner and a number of mr sequences were run , spin echo for r1 determination ( tr : 503000 ms ; te : 5.5 ms , flip angle 30 ; fov : 3.5 cm , matrix : 256 256 ; slice thickness : 1 mm ) . the luminance values of the resulting image were obtained through the imagej software processing , thereby calculating the r1 value . for measurement the mnps detection sensitivity in living subject , fe - chelated mnps aqueous solution with different concentrations from 1.25 m to 20 m were mixed with matrigel at 0 c and then subcutaneous injected on the lower back of mice . tr : 700 ms , te : 5.2 ms ; fov : 3.5 cm , matrix : 256 256 ; slice thickness : 1 mm . all animal experiments were performed in compliance with the guidelines for the care and use of research animals established by the stanford university animal studies committee . female athymic nude mice ( nu / nu ) in 46 weeks old were obtained from the charles river laboratories ( boston , ma , usa ) and kept under sterile conditions . u87 mg cells suspended in 100 l of pbs were inoculated subcutaneously in the shoulder of nude mice . when the tumors reached 0.50.8 cm in diameter , the tumor bearing mice were subjected to in vivo multimodality imaging ( pai , mri and pet ) and biodistribution studies . mice bearing tumor ( u87 mg ) were anesthetized with 2% isoflurane in oxygen and placed with lateral position . mri was performed using the same instrument , protocols and conditions as in the phantom mri study . pai was carried out using the same vevo lazr pai system as the in vitro study . quantification analysis of pa and mr signals was performed on the pai and mri images . because the enhanced signals of mri and pai in tumors were dispersed heterogeneously , the enhanced signal regions in tumors on the pai and mri images were used as rois to analysis the signal change with injection time ( comparing the signal intensity at 4 h injection with 0 h injection ) . small animal pet imaging of tumor - bearing mice was performed on a siemens inveon micropet - ct . mice bearing u87-mg tumors were injected with cu - labeled mnps ( 110.0 5.0 ci ) via the tail vein . at different times after injection ( 2 , 4 , and 24 h ) , the mice were anesthetized with 2% isoflurane and placed prone near the center of the fov of the scanner . all the small animal pet images were reconstructed using irw4.0 software by two - dimensional ordered - subsets expectation maximization ( osem ) algorithm . the radioactivity uptake in the tumor and normal tissues was calculated using a region of interest ( roi ) drawn over the whole organ region and expressed as a percentage of the injected radioactive dose per gram of tissue ( % id / g ) .

developing multifunctional and easily prepared nanoplatforms with integrated different modalities is highly challenging for molecular imaging . here , we report the successful transfer of an important molecular target , melanin , into a novel multimodality imaging nanoplatform . melanin is abundantly expressed in melanotic melanomas and thus has been actively studied as a target for melanoma imaging . in our work , the multifunctional biopolymer nanoplatform based on ultrasmall ( < 10 nm ) water - soluble melanin nanoparticle ( mnp ) was developed and showed unique photoacoustic property and natural binding ability with metal ions ( for example , 64cu2 + , fe3 + ) . therefore , mnp can serve not only as a photoacoustic contrast agent , but also as a nanoplatform for positron emission tomography ( pet ) and magnetic resonance imaging ( mri ) . traditional passive nanoplatforms require complicated and time - consuming processes for prebuilding reporting moieties or chemical modifications using active groups to integrate different contrast properties into one entity . in comparison , utilizing functional biomarker melanin can greatly simplify the building process . we further conjugated v3 integrins , cyclic c(rgdfc ) peptide , to mnps to allow for u87 mg tumor accumulation due to its targeting property combined with the enhanced permeability and retention ( epr ) effect . the multimodal properties of mnps demonstrate the high potential of endogenous materials with multifunctions as nanoplatforms for molecular theranostics and clinical translation .

Introduction Results Discussion Conclusion Materials and Methods

for instance , dna has been used to rationally design plasmonic nanostructures , to build nanoscaffolds for incorporating multiple - affinity ligands , and to self - assemble into numerous prescribed 3d shapes . exogenous inorganic nps - based reporters have attracted considerable interests , such as iron oxide nps for magnetic resonance imaging ( mri ) and quantum dots for fluorescence imaging . therefore , such biomolecules need complicated and time - consuming processes to prebuild various contrast properties or require chemical modifications to integrate different reporting moieties into one entity , which we term as a passive platform . for example , organic ligands are generally incorporated into a nanoplatform before chelating to radioactive or magnetic metal ions for positron emission tomography ( pet ) and magnetic resonance imaging ( mri ) . in this study , we report the successful transferring of this biomarker into an imaging nanoplatform . by mimicking natural melanin , water - soluble melanin nanoparticle ( mnp ) has been synthesized and used as the active platform for multimodal imaging of tumors . we demonstrate that mnp can not only offer its native optical properties for photoacoustic imaging ( pai ) , but also actively chelate to metal ions ( cu , fe ) for pet and mri with a high loading capacity and stability utilizing its intrinsic chelating function . furthermore , ultrasmall size mnps ( 4.5 nm ) can be easily prepared and surface - modified . figure 1 schematically illustrates the procedure to prepare ultrasmall water - soluble mnp with multimodal imaging properties . to change the intrinsic poor water - solubility of melanin , pristine melanin granule was first dissolved in a 0.1 n naoh and then neutralized under the assistance of sonication to decrease interchain aggregation . ultrasmall mnp in high water monodispersity and homogeneity with a size of 4.5 0.5 nm , which was termed as plain water - soluble mnp ( pws - mnp ) , were successfully obtained ( figure 2a , b and figure s1a , supporting information ) . pws - mnp exhibited excellent water - solubility of 40 mg / ml and stability , which can be attributed to the highly negative potential of approximately 22.2 mv on the np surface that efficiently blocks the np aggregation through electrostatic repulsion ( figure s1b ) . characterization of physical properties of mnps . ( d ) the overlaying of ultrasonic ( gray ) and photoacoustic ( red ) imagings of u87 mg tumor ( region enveloped by yellow dotted line ) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice ( n = 3 ) and their subtraction imagings . ( e ) quantitative analysis of enhanced pa signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h , compared with at 0 h. to retain the water - solubility of pws - mnps for further biomodification and metal ion - chelating , polyethylene glycol ( peg ) chains were introduced to the mnp . moreover , the surface potential of peg - mnp decreased to 6.1 mv ( figure s1b ) because of introduction of peg and positive nh2 groups on the mnp surface . lastly , for demonstrating that mnp can be used as a platform for biomodification , peg - mnp was further modified with biomolecules such as cyclic arg - gly - asp - d - phe - cys [ c(rgdfc ) ] peptide ( abbreviated as rgd ) , which can target tumor v3 integrin . ( c ) quantitative analysis of enhanced mr signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h , compared with at 0 h. ( d ) mri images of u87 mg tumors ( region enveloped by yellow dotted line ) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice ( n = 3 ) ( tr : 700 ms , te : 5.2 ms ) . to investigate the possibility of mnp as a platform for pet and mri , its chelating properties to cu ( cu for pet ) and fe ( for mri ) were studied . after adding metal ions ( 0.2 ml of 10 mm fecl3 or cucl2 ) into mnp aqueous solutions ( 1 ml of 20 m for pws - mnp and peg - mnp ) , the precipitation of pws - mnp quickly appeared , while peg - mnp maintained good water - solubility ( figure s5 ) . furthermore , the high viability of nih3t3 and u87 mg cells ( about 90110% as compared to the nontoxic control ) after 24 h of incubation with peg - functionalized mnps was found , indicating high biocompatibility and low cytotoxic effect of peg - functionalized mnps ( figure s7 ) . to investigate the possibility of mnps to be used as a photoacoustic agent , we first studied the detection sensitivity of peg - mnp in aqueous solution at increasing concentrations from 0.625 to 20 m . to further investigate the in vivo pai properties , one group of u87 mg tumor mice were tail - vein injected with 250 l of rgd - peg - mnp at a concentration of 200 m . the increased photoacoustic signal of rgd - peg - mnp ( figure 3e ) could be attributed to the enhanced permeability and retention ( epr ) effect and the tumor targeting ability of rgd - peg - mnp to v3 integrin . ( b ) representative decay - corrected coronal ( top ) and transaxial ( bottom ) small animal pet images ( left three images ) and the overlaying of ct ( gray ) and pet ( color ) images ( right three images ) of u87 mg tumors ( region enveloped by yellow dotted line ) acquired at 2 , 4 , and 24 h after tail vein injection of cu - rgd - peg - mnp . to demonstrate the use of mnp as the platform for mri of tumors , t1-weighted images were obtained from mice bearing u87 mg tumors ( n = 3 ) . the relative mr signal intensity of tumor at 4 h increased 30% compared with at 0 h , demonstrating that mnp can be used as a platform for mri ( figure 4c ) . to investigate the pet imaging properties of mnp , cu was selected as a pet radiolabel for mnp because it can be readily chelated by melanin and the intermediate half - life of cu ( 12.7 h ) makes it suitable for radiolabeling of biomolecules and imaging . ( b ) the overlaying of ultrasonic ( gray ) and photoacoustic ( red ) imaging of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp ( 200 l of 10 m ) in living mice and their subtraction imaging . ( c ) the overlaying of representative decay - corrected coronal ( top ) and transaxial ( bottom ) small animal ct ( gray ) and pet ( color ) images of u87 mg tumors acquired at 2 , 4 , and 24 h after tail vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp ( 250 l of 200 m ) . ( d ) mri images of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp ( 250 l of 200 m ) in living mouse . cu - rgd - peg - mnp exhibited higher uptakes than blocking group at all the incubation time , with a value of 3.32 0.37% , 5.32 0.43% and 7.18 0.33% for cu - rgd - peg - mnp at 1 , 2 , and 4 h. in comparison , for cu - rgd - peg - mnp blocking group , much lower uptake of cu - rgd - peg - mnp was observed with a value of 2.00 0.15% , 3.16 0.20% and 3.48 0.29% at 1 , 2 , and 4 h , respectively , indicating the successful biomodification of mnps with rgd peptide and the specific targeting ability of rgd contribute to the uptake of cu - rgd - peg - mnp by u87 mg cells . to further investigate the possible targeting property of mnps , the cu radiolabeled rgd - peg - mnp and control cyclic arg - ala - asp - d - phe - cys [ c(radfc ) ] peptide ( abbreviated as rad , with nontargeting property for tumor v3 integrin ) functionalized peg - mnp ( cu - rad - peg - mnp ) for u87 mg tumor pet imaging the obvious stronger pet signal of cu - rgd - peg - mnp can be found in tumor at 4 h than that of cu - rad - peg - mnp ( p < 0.05 ) , indicating the good and specific targeting property of rgd - peg - mnp . to investigate the possibility of using mnp platform for multimodality imaging , mnp were mixed with fe and cu in sequence to form the multifunctional probes fe - rgd - peg - mnp and cu - fe - rgd - peg - mnp ( the amount of fe per mnp is 56 ) for pet / pai / mri . in figure 6 , cu - fe - rgd - peg - mnp showed very similar pet , mri and pai properties on u87 mg tumor , compared with the corresponding single modality imaging from cu - rgd - peg - mnp , fe - rgd - peg - mnp , and rgd - peg - mnp respectively . these results showed that using mnp as the active platform to load cu and fe together can efficiently combine its native photoacoustic properties with radioactive and magnetic properties together for multimodality imaging . to change the lack of contrast properties of biomolecule - based nanoplatform for multimodality imaging , recently porphyrin were successfully introduced into phospholipid to provide the platform with desirable optical properties , while it still requires complicated and time - consuming chemical modifications and other reporting molecules to achieve multimodality imaging ability . we herein developed the functional biomarker , melanin , as a novel nanoplatform with its native optical property and multifunctions , which can simply and actively collecting optical , magnetic and radioactive properties together for multimodality imaging . melanin , the oxidation products of tyrosine , plays an important role in living organism . accompanied with the development of molecular imaging probes in the past decade , melanin has been used as an effective molecular target as well as endogenous contrast agent for pai because of its strong light absorption properties . besides , melanin has intrinsic strong chelating properties to many metal ions including cu and fe , which can be used to nuclear imaging and mri . considering the attractive properties of the biomarker melanin , we and others have engineered cancer cells to biologically produce melanin for multimodality imaging ( pai / mri / pet ) of cancer . however , this method requires genetic modification of cells , which is time - consuming and may have limited clinical value . thus , water - soluble mnps are more appropriate to behave as a natural active platform to simplify the preparation procedure for multimodal applications . in comparison , although gd ion has higher t1 mri effect than fe ion , its potential toxicity is still a problem . more interestingly , the new mnp system can serve as an active nanoplatform and easily bind with metal ions without the traditional needs of surface modification and introducing chelating groups , which significantly simplifies the preparation process and reduces the heterogeneity of the resulting multimodal nps . furthermore , the mnp is an organic and biodegradable material and showed relatively good tumor imaging properties . despite its important functions , developing melanin for molecular imaging therefore , preparing mnps is desired while still a challenge for well - dispersing in water , especially for those with size around 10 nm that can provide not only appropriate blood circulation time , but also high surface - to - volume ratio to chelate enough metal ions for efficient bioimaging . therefore , to realize melanin water - soluble at neutral environments , decreasing the interchain aggregation of conjugated main chains and lowering the formed melanin particle size to expose more hydrophilic hydroxy groups on the surface of melanin is a promising way . in our work , we first realized the synthesis of ultrasmall mnps in water with high monodispersity and homogeneity under the assistance of sonication . in our work , peg encapsulation is found can not only enhance the biocompatibility and the water - solubility , but also efficiently prevent the formation of metal ion - initiated precipitation . moreover , because of their easy conjugation with targeting groups , the mnps modified with rgd exhibited the tumor targeting ability to the v3 integrin overexpressed on the surface of tumor vasculatures and the u87 mg tumor cells ( from pet data ) , which afforded rgd - conjugated mnps higher accumulation capability in tumor than those rad - modified mnps through enhanced permeability and retention ( epr ) effect . for example , pet efficiently provides the in vivo pharmacokinetics and biodistribution of the nanoprobes and can also provide physiological information on disease with whole body imaging capability , but it can not image tissues at high spatial resolution ; pai provides functional and molecular information on the tumor with high spatial resolution . recent developments showed that multimodality imaging is promising not only for accurate tumor imaging but also for guiding tumor resection . for example , pet / mri , which combines the exquisite anatomical information by mri with the extreme sensitivity of pet , can be used for whole - body imaging and deep tumor localization . overall , a reliable method for preparation of water - soluble mnp has been developed in our work , which lays down a foundation for its future biomedical applications . it can be easily envisioned that mnp can serve as a nanoplatform not only for molecular imaging but also for theranostics . considering the abundant functionalities of melanin , such as binding drugs , mnp - based platform used for drug delivery and therapy are now being investigated . in conclusion , we report mnp as the natural biomarker - transferred active platform for multimodality imaging . mnp is of particular interest because such an endogenous agent with native photoacoustic signals and strong chelating properties with metal ions can act as an active platform to simplify the assembling of different imaging moieties . mnp can be easily modified with biomolecules for targeted tumor multimodality imaging , and it showed good in vivo tumor imaging properties . we expect this work will stimulate further studies of multifunctional endogenous material as nanoplatforms for potential imaging and therapeutic applications . the 4-(n - maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo - n - hydroxysuccinimide ester sodium salt ( sulfo - smcc ) ( 1.2 mg ) the water - soluble peg - mnp [ 1 mg in 1 ml of pbs ( ph = 7.2 ) ] was incubated with the above cross - linker solution for 2 h at room temperature . the mnp ( 1 mg in 1 ml h2o ) was labeled with fe or cu by addition of 20 l of fresh fecl3 ( 10 mg / ml ) in pbs ( ph = 7.4 ) or 20 l of cucl2 ( 10 mg / ml ) in buffer solution of ph = 5.5 followed by a 1 h incubation at 40 c ( the chelating mechanisms of mnps were shown in figure s13 ) . in vitro cytotoxicity of mnps was determined in nih-3t3 and u87 mg cells by the mtt assay . the cells were washed twice with serum - free dmem and incubated with the cu - labeled mnps ( 2 ci per well , final concentration approximately 6 nm ) in 400 l of serum - free dmem at 37 c . after 1 , 2 , and 4 h , the cells were washed three times with cold pbs and lysed with the addition of 200 l of 0.2 m naoh . for studying the pai properties of mnps , different concentrations of mnps aqueous solutions ranging from 0.625 to 20 m were filled into polyethylene capillaries and then the capillaries were laid on the surface of solidified agarose gel . when agarose gel was cooled , the mixtures of mnps and aqueous solution of agarose ( ratio 1:1 by volume ) with iron concentrations at 62.5 , 125 , 250 , 500 , and 1000 m fe ( amount to 1.25 , 2.5 , 5 , 10 , 20 m mnp ) , were then filled into the intermediate portion of the pcr tube respectively while the sample was hot . when the tumors reached 0.50.8 cm in diameter , the tumor bearing mice were subjected to in vivo multimodality imaging ( pai , mri and pet ) and biodistribution studies . because the enhanced signals of mri and pai in tumors were dispersed heterogeneously , the enhanced signal regions in tumors on the pai and mri images were used as rois to analysis the signal change with injection time ( comparing the signal intensity at 4 h injection with 0 h injection ) .

we demonstrate that mnp can not only offer its native optical properties for photoacoustic imaging ( pai ) , but also actively chelate to metal ions ( cu , fe ) for pet and mri with a high loading capacity and stability utilizing its intrinsic chelating function . to change the intrinsic poor water - solubility of melanin , pristine melanin granule was first dissolved in a 0.1 n naoh and then neutralized under the assistance of sonication to decrease interchain aggregation . ultrasmall mnp in high water monodispersity and homogeneity with a size of 4.5 0.5 nm , which was termed as plain water - soluble mnp ( pws - mnp ) , were successfully obtained ( figure 2a , b and figure s1a , supporting information ) . pws - mnp exhibited excellent water - solubility of 40 mg / ml and stability , which can be attributed to the highly negative potential of approximately 22.2 mv on the np surface that efficiently blocks the np aggregation through electrostatic repulsion ( figure s1b ) . the h nmr spectrum of pws - mnp in d2o showed no obvious signal belonging to the hydrogen atom on the arylene groups , suggesting most of the conjugated backbones were buried in the np ( figure s2b ) . ( a ) from left to right : pictures of ( 1 ) pristine melanin granule in h2o , ( 2 ) melanin neutralized without sonication in h2o , ( 3 ) freeze - dried pws - mnp , ( 4 ) freeze - dried pws - mnp redissolved in pbs ( ph = 7.4 ) , ( 5 ) freeze - dried peg - mnp , ( 6 ) freeze - dried peg - mnp redissolved in pbs ( ph = 7.4 ) . ( a ) the photoacoustic signal produced by peg - mnps at concentrations of 0.625 , 1.25 , 2.5 , 5.0 , 10 , and 20 m , and it was observed to be linearly dependent on its concentration ( r = 0.995 ) . ( d ) the overlaying of ultrasonic ( gray ) and photoacoustic ( red ) imagings of u87 mg tumor ( region enveloped by yellow dotted line ) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice ( n = 3 ) and their subtraction imagings . ( e ) quantitative analysis of enhanced pa signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h , compared with at 0 h. to retain the water - solubility of pws - mnps for further biomodification and metal ion - chelating , polyethylene glycol ( peg ) chains were introduced to the mnp . lastly , for demonstrating that mnp can be used as a platform for biomodification , peg - mnp was further modified with biomolecules such as cyclic arg - gly - asp - d - phe - cys [ c(rgdfc ) ] peptide ( abbreviated as rgd ) , which can target tumor v3 integrin . the number of rgd attached to the mnp was calculated to be about 8 per mnp and the size of rgd - functionalized peg - mnp ( rgd - peg - mnp ) increased a little to 9.6 nm ( figure s4 ) . mice were injected subcutaneously ( region enveloped by red dotted line ) with fe - rgd - peg - mnps at concentrations of 0 , 1.25 , 2.5 ( from left to right in upper layer ) , and 5 , 10 , 20 ( from left to right in bottom layer ) m . ( c ) quantitative analysis of enhanced mr signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h , compared with at 0 h. ( d ) mri images of u87 mg tumors ( region enveloped by yellow dotted line ) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice ( n = 3 ) ( tr : 700 ms , te : 5.2 ms ) . after adding metal ions ( 0.2 ml of 10 mm fecl3 or cucl2 ) into mnp aqueous solutions ( 1 ml of 20 m for pws - mnp and peg - mnp ) , the precipitation of pws - mnp quickly appeared , while peg - mnp maintained good water - solubility ( figure s5 ) . the fe or cu - chelated mnp ( fe - peg - mnp , fe - rgd - peg - mnp , cu - peg - mnp and cu - rgd - peg - mnp ) exhibited high loading capacities . after fe - chelating , the mnp sizes increased to 8.9 nm and 10.7 nm for fe - peg - mnp and fe - rgd - peg - mnp respectively and their zeta - potential remained in the neutral region ( figure s4 and table s1 ) . compared with those reported dyes for pai , which exhibit significant reduced absorption ( > 30% ) under light exposure , peg - mnp and rgd - peg - mnp showed intriguing photostability ( only 3% reduced absorption ) ( figure s6 ) , indicating their high capability for pai . further stability assay of fe or cu - chelated mnps in pbs solution showed that only about 3% cu and 7% fe were released from those mnps at the first 2 h , and there was no further release at longer incubation time points , indicating the high stability of the chelating platform ( figure 2d ) . furthermore , the high viability of nih3t3 and u87 mg cells ( about 90110% as compared to the nontoxic control ) after 24 h of incubation with peg - functionalized mnps was found , indicating high biocompatibility and low cytotoxic effect of peg - functionalized mnps ( figure s7 ) . to investigate the possibility of mnps to be used as a photoacoustic agent , we first studied the detection sensitivity of peg - mnp in aqueous solution at increasing concentrations from 0.625 to 20 m . the detection sensitivity of mnp in living body was further tested by subcutaneous injection of peg - mnp on the lower back of mice ( n = 3 ) at increasing concentrations of 5 to 80 m ( figure 3b ) . a linear correlation ( r = 0.998 ) between the mnp concentration and the corresponding photoacoustic signal 2.5 m of peg - mnp was found to give the equivalent photoacuoustic signal strength as the tissue background . to further investigate the in vivo pai properties , one group of u87 mg tumor mice were tail - vein injected with 250 l of rgd - peg - mnp at a concentration of 200 m . the increased photoacoustic signal of rgd - peg - mnp ( figure 3e ) could be attributed to the enhanced permeability and retention ( epr ) effect and the tumor targeting ability of rgd - peg - mnp to v3 integrin . ( b ) representative decay - corrected coronal ( top ) and transaxial ( bottom ) small animal pet images ( left three images ) and the overlaying of ct ( gray ) and pet ( color ) images ( right three images ) of u87 mg tumors ( region enveloped by yellow dotted line ) acquired at 2 , 4 , and 24 h after tail vein injection of cu - rgd - peg - mnp . to study whether fe ( t1 contrast agent ) retains mr signal - enhancing property after loading into mnps , t1-weighted mri images of various concentrations of fe - rgd - peg - mnp in agarose gel was investigated ( figure s9a ) . r1 value of fe - rgd - peg - mnp ( the slope of the fitted curve in figure 4a , using gd as standard ) was calculated to be 1.2 mm s. the magnetic sensitivity in living mice was then tested by subcutaneous injection of fe - rgd - peg - mnp on the lower back of mice ( n = 3 ) at increasing concentrations of 1.25 to 20 m . to investigate the pet imaging properties of mnp , cu was selected as a pet radiolabel for mnp because it can be readily chelated by melanin and the intermediate half - life of cu ( 12.7 h ) makes it suitable for radiolabeling of biomolecules and imaging . ( b ) the overlaying of ultrasonic ( gray ) and photoacoustic ( red ) imaging of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp ( 200 l of 10 m ) in living mice and their subtraction imaging . ( c ) the overlaying of representative decay - corrected coronal ( top ) and transaxial ( bottom ) small animal ct ( gray ) and pet ( color ) images of u87 mg tumors acquired at 2 , 4 , and 24 h after tail vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp ( 250 l of 200 m ) . ( d ) mri images of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp ( 250 l of 200 m ) in living mouse . cu - rgd - peg - mnp exhibited higher uptakes than blocking group at all the incubation time , with a value of 3.32 0.37% , 5.32 0.43% and 7.18 0.33% for cu - rgd - peg - mnp at 1 , 2 , and 4 h. in comparison , for cu - rgd - peg - mnp blocking group , much lower uptake of cu - rgd - peg - mnp was observed with a value of 2.00 0.15% , 3.16 0.20% and 3.48 0.29% at 1 , 2 , and 4 h , respectively , indicating the successful biomodification of mnps with rgd peptide and the specific targeting ability of rgd contribute to the uptake of cu - rgd - peg - mnp by u87 mg cells . quantification analysis revealed that the tumor uptake values of cu - rgd - peg - mnp gradually increased with time to 24 h , and they were 4.75 0.63 , 5.87 0.87 , and 5.93 0.89% id / g at 2 , 4 , and 24 h , respectively ( figure 5c ) . to further investigate the possible targeting property of mnps , the cu radiolabeled rgd - peg - mnp and control cyclic arg - ala - asp - d - phe - cys [ c(radfc ) ] peptide ( abbreviated as rad , with nontargeting property for tumor v3 integrin ) functionalized peg - mnp ( cu - rad - peg - mnp ) for u87 mg tumor pet imaging the obvious stronger pet signal of cu - rgd - peg - mnp can be found in tumor at 4 h than that of cu - rad - peg - mnp ( p < 0.05 ) , indicating the good and specific targeting property of rgd - peg - mnp . to investigate the possibility of using mnp platform for multimodality imaging , mnp were mixed with fe and cu in sequence to form the multifunctional probes fe - rgd - peg - mnp and cu - fe - rgd - peg - mnp ( the amount of fe per mnp is 56 ) for pet / pai / mri . in figure 6 , cu - fe - rgd - peg - mnp showed very similar pet , mri and pai properties on u87 mg tumor , compared with the corresponding single modality imaging from cu - rgd - peg - mnp , fe - rgd - peg - mnp , and rgd - peg - mnp respectively . to change the lack of contrast properties of biomolecule - based nanoplatform for multimodality imaging , recently porphyrin were successfully introduced into phospholipid to provide the platform with desirable optical properties , while it still requires complicated and time - consuming chemical modifications and other reporting molecules to achieve multimodality imaging ability . we herein developed the functional biomarker , melanin , as a novel nanoplatform with its native optical property and multifunctions , which can simply and actively collecting optical , magnetic and radioactive properties together for multimodality imaging . considering the attractive properties of the biomarker melanin , we and others have engineered cancer cells to biologically produce melanin for multimodality imaging ( pai / mri / pet ) of cancer . considering only trace amount of cu ions utilized for pet and its final decay to zn ions , which is necessary for life process , and the abundant amount of fe ions in living body , cu and fe ions used in our system are expected to be metabolized in living subjects . more interestingly , the new mnp system can serve as an active nanoplatform and easily bind with metal ions without the traditional needs of surface modification and introducing chelating groups , which significantly simplifies the preparation process and reduces the heterogeneity of the resulting multimodal nps . despite its important functions , developing melanin for molecular imaging therefore , preparing mnps is desired while still a challenge for well - dispersing in water , especially for those with size around 10 nm that can provide not only appropriate blood circulation time , but also high surface - to - volume ratio to chelate enough metal ions for efficient bioimaging . although the formation mechanism of polymeric melanin is not clear , its molecular structure is generally considered to be composed of dihydroxyindole / indolequinone segments with hydrophobic conjugated main chain having strong interaction and hydrophilic hydroxyl groups on the benzene rings . therefore , to realize melanin water - soluble at neutral environments , decreasing the interchain aggregation of conjugated main chains and lowering the formed melanin particle size to expose more hydrophilic hydroxy groups on the surface of melanin is a promising way . moreover , because of their easy conjugation with targeting groups , the mnps modified with rgd exhibited the tumor targeting ability to the v3 integrin overexpressed on the surface of tumor vasculatures and the u87 mg tumor cells ( from pet data ) , which afforded rgd - conjugated mnps higher accumulation capability in tumor than those rad - modified mnps through enhanced permeability and retention ( epr ) effect . though it is difficult to compare the imaging effects of our platform with other type of nanoplatforms ( considering the lack of the triplemodality analogues ) , we demonstrated the unique ability of mnp nanoplatform to combine pet , photoacoustic and mr imaging modalities together to get complementary information for tumor imaging . for example , pet efficiently provides the in vivo pharmacokinetics and biodistribution of the nanoprobes and can also provide physiological information on disease with whole body imaging capability , but it can not image tissues at high spatial resolution ; pai provides functional and molecular information on the tumor with high spatial resolution . because of the existence of one nh2 group per peg chain on the surface of mnp , we then accurately determined the nh2 group on mnp with fluorescamine by spectrofluorometer to calculate the amount of peg on the nanoparticles ( using ethamine as the standard ) . the 4-(n - maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo - n - hydroxysuccinimide ester sodium salt ( sulfo - smcc ) ( 1.2 mg ) the water - soluble peg - mnp [ 1 mg in 1 ml of pbs ( ph = 7.2 ) ] was incubated with the above cross - linker solution for 2 h at room temperature . the mnp ( 1 mg in 1 ml h2o ) was labeled with fe or cu by addition of 20 l of fresh fecl3 ( 10 mg / ml ) in pbs ( ph = 7.4 ) or 20 l of cucl2 ( 10 mg / ml ) in buffer solution of ph = 5.5 followed by a 1 h incubation at 40 c ( the chelating mechanisms of mnps were shown in figure s13 ) . for studying the pai properties of mnps , different concentrations of mnps aqueous solutions ranging from 0.625 to 20 m were filled into polyethylene capillaries and then the capillaries were laid on the surface of solidified agarose gel . when agarose gel was cooled , the mixtures of mnps and aqueous solution of agarose ( ratio 1:1 by volume ) with iron concentrations at 62.5 , 125 , 250 , 500 , and 1000 m fe ( amount to 1.25 , 2.5 , 5 , 10 , 20 m mnp ) , were then filled into the intermediate portion of the pcr tube respectively while the sample was hot . because the enhanced signals of mri and pai in tumors were dispersed heterogeneously , the enhanced signal regions in tumors on the pai and mri images were used as rois to analysis the signal change with injection time ( comparing the signal intensity at 4 h injection with 0 h injection ) .

numerous reports have documented opposing effects of the gs - coupled d1r and gi / o - coupled d2r on biological processes such as adenylyl cyclase activity and camp formation ( hyttel , 1978 ; onali et al . , 1985 ) and however , it has also been demonstrated that the concomitant activation of both d1r and d2r is essential to achieve certain other behavioral , biochemical , and electrophysiological effects . for instance , past studies have shown coactivation of the d1r and d2r as being necessary for maximal stereotyped and locomotor behavior ( walters et al . , 1987 ; white et al . , 1988 ; deboer and abercrombie , 1996 ) , and more recently it was reported that to evoke neural and behavioral phenotypes of cocaine sensitization , both d1r and d2r stimulation is required ( capper - loup et al . , 2002 ) . similarly , at a cellular level , combined administration of specific d1r and d2r agonists was shown to potentiate immediate early gene expression ( lahoste et al . , 1993 ; keefe and gerfen , 1995 ) . for example , while the systemic administration of a dopamine d1r agonist induced the expression of c - fos and zif268 in the dopamine - depleted striatum , and a d2r agonist decreased zif268 expression , both agonists together significantly enhanced gene expression compared to the d1r agonist alone ( keefe and gerfen , 1995 ) . electrophysiological evidence has also demonstrated that simultaneous administration of d1r and d2r agonists could synergistically inhibit the neuronal activity of nac neurons ( white and wang , 1986 ; hu et al . , 1990 ) , and restoration of long - term depression of synaptic transmission following dopamine depletion occurred by dopamine administration or coadministration of specific d1r and d2r agonists but not by either selective agonist alone ( calabresi et al . , 1992 ) . although the mechanisms for the d1r and d2r interaction have been suggested to occur by receptor activation on discrete neurons ( gerfen et al . , 1995 ; lahoste et al . , 2000 ) , or changes in d2r - mediated cholinergic interneuron activity with subsequent impact on d1r - expressing striatonigral neuronal transmission ( di chiara et al . , however , an interaction between the receptors occurring within the same cell may have been involved in some instances . msn connections in neonatal striatal cultures have been reported to exhibit modulation by both d1r and d2r agonists ( geldwert et al . , 2006 ) and the selective activation of d1r or d2r in single striatal neurons differentially modulated tetrodotoxin - sensitive sodium channels ( aizman et al . , 2000 the synergistic potentiation of the arachidonic acid release by coadministration of d1r - selective and d2r - selective agonists has also been observed in cells expressing both receptors ( piomelli et al . , 1991 ) . given the functional linkage between d1r and d2r , neuroanatomical studies began to address the question of dopamine receptor segregation and many of these studies have now shown , both at the mrna level and the protein level , that d1r and d2r coexist in a fraction of striatal msns . in situ hybridization studies examining d1r and d2r mrna in serial rat brain striatal sections have shown overlap of d1r and d2r transcript expression in the same neurons ( meador - woodruff et al . , 1991 ; lester et al . , 1993 ) and double in situ hybridization has been used to report colocalization of d1r and d2r mrna in some striatal neurons in primate ( aubert et al . , 2000 ) . d1r / d2r mrna coexistence was also reported using single cell reverse transcription pcr in neonatal neurons ( surmeier et al . , 1992 , 1996 ) and immunolabeling has been a widely used tool for visualizing striatal d1r / d2r coexpression at the protein level in both neonatal striatal cultures ( shetreat et al . , 1996 ; ; deng et al . , 2006 ; hasbi et al . , 2009 ; perreault et al . , it had been noted that while neurons expressing solely mrna for the d1r or d2r were abundant in sp or enk mrna respectively , neurons containing mrna for both d1r and d2r expressed both sp and enk mrnas ( surmeier et al . , 1996 ) . ( 2010 ) , using highly validated antibodies , it was shown that the d1r and d2r were expressed exclusively in neurons containing both dyn and enk . although a small minority of dyn / enk neurons did not express d1r or d2r , these findings indicated that sp / dyn enk coexpression could be a useful marker for identifying neurons coexpressing d1r and d2r . interestingly , unlike the controversy surrounding the localization of d1r and d2r , sp / dyn enk coexpression in striatal neurons has been widely accepted , and overlap of sp / dyn and enk mrna or protein in a proportion of striatal projection neurons has been reported for a number of species ( penny et al . , 1986 ; gerfen and young , 1988 ; anderson and reiner , 1990 ; besson et al . , 1990 ; enhanced green fluorescent protein ( egfp)-tagged promoter elements of d1r and d2r in bacterial artificial chromosome ( bac ) transgenic mice is one such method , and has been used to quantify the proportion of striatal neurons expressing the receptors within the striatonigral and striatopallidal pathways ( valjent et al . , 2009 ) . ( 2008 ) it was reported that while the majority of d1r and d2r were segregated to these discrete pathways , an estimated 17% of nac shell msns exhibited receptor colocalization with ~6% d1r / d2r coexpression in cp . although there has been doubt as to the reliability of bac transgenic mice in providing an accurate representation of dopamine receptor expression due to unlabeled neurons ( shuen et al . , 2008 ) , another group was able to show that 100% of msns in these mice expressed d1r , d2r or both ( matamales et al . , 2009 ) and that the proportion of neurons showing coexpression were similar to that observed in the bertran - gonzalez study . fluorescence activated cell sorting ( facs ) , another relatively novel method , is used for sorting a heterogeneous mixture of biological cells and is based upon the fluorescent characteristics of each cell . essentially , this method employs the fluorescent properties of the neurons in the bac transgenic drd1a - egfp or drd2-egfp mice to purify d1r - expressing and d2r - expressing msns , and is often used to assess specific characteristics of these two neuronal subtypes . the quality of the purification process can then be verified by assessing mrna content of the each sample . using this process , it was recently shown that while purified d1r - positive neurons from whole striatum displayed enrichment of d1r mrna , d2r mrna was also evident , albeit in small amounts ( lobo et al . , 2010 ) . similarly , d2r - positive neurons also displayed evidence of minimal d1r mrna being present . in addition , using bac transgenic mice with cre recombinase being driven by the d1r or d2r promoters , and using double immunofluoresence to stain for cre and enk , this group also showed that a fraction of striatal d1r - expressing neurons were also positive for enk . in recent years , technological advances have provided novel approaches for assessing dopamine receptor colocalization . enhanced green fluorescent protein ( egfp)-tagged promoter elements of d1r and d2r in bacterial artificial chromosome ( bac ) transgenic mice is one such method , and has been used to quantify the proportion of striatal neurons expressing the receptors within the striatonigral and striatopallidal pathways ( valjent et al . , 2009 ) . ( 2008 ) it was reported that while the majority of d1r and d2r were segregated to these discrete pathways , an estimated 17% of nac shell msns exhibited receptor colocalization with ~6% d1r / d2r coexpression in cp . although there has been doubt as to the reliability of bac transgenic mice in providing an accurate representation of dopamine receptor expression due to unlabeled neurons ( shuen et al . , 2008 ) , another group was able to show that 100% of msns in these mice expressed d1r , d2r or both ( matamales et al . , 2009 ) and that the proportion of neurons showing coexpression were similar to that observed in the bertran - gonzalez study . fluorescence activated cell sorting ( facs ) , another relatively novel method , is used for sorting a heterogeneous mixture of biological cells and is based upon the fluorescent characteristics of each cell . essentially , this method employs the fluorescent properties of the neurons in the bac transgenic drd1a - egfp or drd2-egfp mice to purify d1r - expressing and d2r - expressing msns , and is often used to assess specific characteristics of these two neuronal subtypes . the quality of the purification process can then be verified by assessing mrna content of the each sample . using this process , it was recently shown that while purified d1r - positive neurons from whole striatum displayed enrichment of d1r mrna , d2r mrna was also evident , albeit in small amounts ( lobo et al . , 2010 ) . similarly , d2r - positive neurons also displayed evidence of minimal d1r mrna being present . in addition , using bac transgenic mice with cre recombinase being driven by the d1r or d2r promoters , and using double immunofluoresence to stain for cre and enk , this group also showed that a fraction of striatal d1r - expressing neurons were also positive for enk . although the existence of d1r- and d2r - coexpressing msns is now generally accepted , little is yet known about the functional relevance of these neurons , most likely as a result of the methodological difficulties attempting to isolate them . however , it has been shown that while all msns express mrna for the glua1 and glua2 subunits of the ampa receptor ( chen et al . , 1998 ; stefani et al . , 1998 ; vorobjev et al . , 2000 ) , only sp / enk neurons preferentially express mrna for glua3 ( stefani et al . , 1998 ) a finding which the authors posited as being suggestive of distinctive postsynaptic glutamatergic mechanisms in the neurons . a role in neurotransmission is supported by evidence documenting that striatal projection neurons coexpressing d1r and d2r or sp and enk terminate in a number of regions including gp and epn ( gpi ) , as well as substantia nigra and ventral tegmental area ( deng et al . , 2006 ; wang et al . , 2006 , 2007 ) , two regions rich in dopamine cell bodies . in addition , a region - specific distribution of d1r / d2r - dyn / enk neurons throughout the rat basal ganglia and mesolimbic pathway has been reported , being present in nac ( figure 2 ) and cp , as well as gp and ventral pallidum and epn ( perreault et al . , 2010 ) . specifically , it was estimated that the fraction of d1r - expressing neurons also expressing the d2r in nac core and shell was ~25% and ~35% respectively , whereas in cp the percentage was much lower being only ~7% . in gp and vp , while the total number of d1r neurons was quite low compared to other regions , a high percentage of these neurons ( ~60% and ~30% ) also contained d2r . similarly , in epn the fraction of d1r containing neurons also expressing the d2r was relatively high ( ~50% ) . the dopamine d1 and d2 receptor are colocalized with dynorphin and enkephalin in rat nucleus accumbens shell . ( a , b ) confocal images showing d1r and d2r colocalization with dynorphin ( dyn ) or enkephalin ( enk ; white arrows ) . ( c , d ) neurons coexpressing dyn and enk also expressed the d1r or the d2r ( white arrows ) . in basal ganglia , the coexpression of d1r and d2r has been shown to occur both within neuronal cell bodies and selectively at presynaptic , but not postsynaptic terminals , as evidenced by d1r and d2r coexpression with the presynaptic marker synaptophysin , but not the postsynaptic marker psd-95 ( perreault et al . , 2010 ) . presynaptic colocalization of d1r and d2r on a fraction of varicosities has also been reported in neonatal striatal cultures ( wong et al . , 1999 ; geldwert et al . , 2006 ; mizuno et al . , 2007 ) and electrophysiology studies have demonstrated that presynaptic dopamine receptors on msn terminals could modulate gabaergic inhibitory postsynaptic currents ( ipscs ; delgado et al . , 2000 ; interestingly , in one of these studies it was shown that while the d2r agonist quinpirole predominantly mediated inhibition of autaptic connections in msns , the d1r agonist skf 38393 mediated either inhibition or facilitation ( geldwert et al . , 2006 ) . although the authors did not directly address the seemingly discrepant effects of the d1r agonist on gaba transmission , it has been shown that skf 38393 activates two biologically different signaling pathways , the camp pathway and phospholipase c ( plc)-phosphoinositide ( pi ) pathway ( undie and friedman , 1990 ; undie et al . , 1994 ) . although it has been suggested that these two pathways are linked directly to d1r ( undie et al . , 1994 ) , it is now known that in msns that coexpress d1r and d2r , the receptors can form a heteromeric complex , the dopamine d1d2 receptor heteromer , and it is this complex that directly activates the plc - pi pathway ( lee et al . , 2004 ; rashid et al . , 2007 ) . dopamine receptors exist as receptor homomers and can additionally form heteromeric receptor complexes that often exhibit pharmacological and cell signaling properties distinct from their constituent receptors ( lee et al . , 2004 ; rashid et al . , 2007 ; a physical interaction between the d1r and d2r was first shown by coimmunoprecipitation from rat and human striatum ( lee et al . , 2004 ) , and was the first evidence of a dopamine heteromeric receptor complex in brain . these findings were later confirmed by fluorescence resonance energy transfer ( fret ) a tool commonly used for the identification of receptor oligomers . although first performed in cells ( so et al . , 2005 ; dziedzicka - wasylewska et al . , 2006 ) , quantitative fret in situ has now been utilized to verify the presence of dopamine d1d2 receptor heteromers in neonatal cultured rat striatal neurons ( hasbi et al . , 2009 ) . similarly , this method has now been employed to show , in vivo , that in adult rat nac , cp ( hasbi et al . , 2009 ; 2010 ) and gp ( figure 3 ) the d1r and d2r existed in close < 50100 proximity indicative of d1d2 receptor heteromerization . however , the propensity for striatal neurons to exhibit d1d2 heteromers was region - dependent , with the majority ( > 90% ) of d1r / d2r - coexpressing neuronal cell bodies in nac core and shell showing robust d1d2 heteromer formation , but only ~25% of d1r / d2r neurons expressing the d1d2 heteromer in cp ( perreault et al . , 2010 ) this suggests that d1r and d2r can coexist as homomers in the same cell without forming heteromers . it was also shown in the neuropil of both nac and cp the presence of d1d2 heteromers at presynaptic terminals , a finding that suggests a possible involvement of synaptic d1d2 receptor heteromer in gaba release in both regions . fluorescence resonance energy transfer ( fret ) , using alexa 488 and alexa 350 was used to identify interactions between endogenous d1r and d2r in neurons of rat globus pallidus . an interaction between the d1r and d2r was evident , with a relatively high mean fret efficiency ( efficiency of energy transfer between the donor and acceptor fluorophores ) of ~22% . the receptor antibody - linked fluorophores were calculated to be in close proximity with a relative distance of 57 nm ( 5070 ) indicative of d1d2 receptor heteromer formation . the role of the d1d2 heteromer in vivo is just beginning to be elucidated , however several lines of evidence have emerged implicating the complex as a potential therapeutic target in disorders involving elevated dopamine transmission , such as schizophrenia and drug addiction . specifically , it has been postulated that abnormal regulation of calcium signaling may constitute the central dysfunction that is responsible for generating the psychopathology of schizophrenia ( lidow , 2003 ) . although the d1r and d2r have not been shown individually to be directly coupled to calcium signaling , coactivation of both receptors within the dopamine d1d2 receptor heteromer by concurrent administration of d1r and d2r agonists , or the selective d1d2 heteromer agonist skf 83959 , resulted in a novel gq - plc - linked increase in intracellular calcium release ( lee et al . in addition , dopamine d1d2 heteromer activation by skf 83959 also induced striatal calcium calmodulin kinase ii ( camkii ) phosphorylation ( rashid et al . , 2007 ; ng et al . , 2010 ) and brain - derived neurotrophic factor ( bdnf ) expression ( hasbi et al . , 2009 ) two proteins that have been linked to schizophrenia ( weickert et al . , 2003 ; issa et al . , 2010 ; jindal et al . , 2010 ; novak and seeman , 2010 ; wong et al . , 2010 ; carlino et al . , , an upregulation of striatal d1d2 heteromeric activity was seen following repeated amphetamine treatment in rats and in human gp of patients with schizophrenia ( perreault et al . , 2010 ) . given that amphetamine sensitization is often employed as an animal model for schizophrenia ( featherstone et al . , 2007 ) , but no common biochemical marker linking the two has previously been documented , it was inferred that the sensitized state of the d1d2 heteromer may provide the first neuropharmacological correlate between increased dopamine neurotransmission and its functional consequence . as antipsychotics invariably also target the d1d2 heteromer , these findings strongly suggest that further research examining the d1d2 receptor heteromer as a potential drug target in schizophrenia is warranted . in addition to an involvement in schizophrenia , camkii and bdnf have also been shown to play a major role in cocaine addiction ( anderson et al . , 2008 ; , 2010 ; wang et al . , 2010 ) . in one particularly compelling study , it was shown that cocaine reinstatement in rats depended upon an interaction between nac dopamine and glutamate systems that was mediated by camkii ( anderson et al . , 2008 ) . more specifically , cocaine reinstatement increased activation of nac shell camkii , which subsequently led to the phosphorylation of the glua1 subunit of the ampa receptor at ser , and increased cell - surface expression of glua1-containing ampa receptors . interestingly , in contrast to the effects of acute selective activation of the d1d2 heteromer in nac , more prolonged selective activation has been reported to reduce total camkii levels in this region and additionally reduce phosphorylation of glua1 at ser ( perreault et al . , 2010 ) , a finding effectively linking the d1d2 heteromer to the reward pathways of the brain . dopamine receptors exist as receptor homomers and can additionally form heteromeric receptor complexes that often exhibit pharmacological and cell signaling properties distinct from their constituent receptors ( lee et al . , 2004 ; rashid et al . , 2007 ; a physical interaction between the d1r and d2r was first shown by coimmunoprecipitation from rat and human striatum ( lee et al . , 2004 ) , and was the first evidence of a dopamine heteromeric receptor complex in brain . these findings were later confirmed by fluorescence resonance energy transfer ( fret ) a tool commonly used for the identification of receptor oligomers . dziedzicka - wasylewska et al . , 2006 ) , quantitative fret in situ has now been utilized to verify the presence of dopamine d1d2 receptor heteromers in neonatal cultured rat striatal neurons ( hasbi et al . , 2009 ) . similarly , this method has now been employed to show , in vivo , that in adult rat nac , cp ( hasbi et al . , 2009 ; perreault et al . , 2010 ) and gp ( figure 3 ) the d1r and d2r existed in close < 50100 proximity indicative of d1d2 receptor heteromerization . however , the propensity for striatal neurons to exhibit d1d2 heteromers was region - dependent , with the majority ( > 90% ) of d1r / d2r - coexpressing neuronal cell bodies in nac core and shell showing robust d1d2 heteromer formation , but only ~25% of d1r / d2r neurons expressing the d1d2 heteromer in cp ( perreault et al . this suggests that d1r and d2r can coexist as homomers in the same cell without forming heteromers . it was also shown in the neuropil of both nac and cp the presence of d1d2 heteromers at presynaptic terminals , a finding that suggests a possible involvement of synaptic d1d2 receptor heteromer in gaba release in both regions . fluorescence resonance energy transfer ( fret ) , using alexa 488 and alexa 350 was used to identify interactions between endogenous d1r and d2r in neurons of rat globus pallidus . an interaction between the d1r and d2r was evident , with a relatively high mean fret efficiency ( efficiency of energy transfer between the donor and acceptor fluorophores ) of ~22% . the receptor antibody - linked fluorophores were calculated to be in close proximity with a relative distance of 57 nm ( 5070 ) indicative of d1d2 receptor heteromer formation . the role of the d1d2 heteromer in vivo is just beginning to be elucidated , however several lines of evidence have emerged implicating the complex as a potential therapeutic target in disorders involving elevated dopamine transmission , such as schizophrenia and drug addiction . specifically , it has been postulated that abnormal regulation of calcium signaling may constitute the central dysfunction that is responsible for generating the psychopathology of schizophrenia ( lidow , 2003 ) . although the d1r and d2r have not been shown individually to be directly coupled to calcium signaling , coactivation of both receptors within the dopamine d1d2 receptor heteromer by concurrent administration of d1r and d2r agonists , or the selective d1d2 heteromer agonist skf 83959 , resulted in a novel gq - plc - linked increase in intracellular calcium release ( lee et al . , 2004 ; in addition , dopamine d1d2 heteromer activation by skf 83959 also induced striatal calcium calmodulin kinase ii ( camkii ) phosphorylation ( rashid et al . , 2007 ; ng et al . , 2010 ) and brain - derived neurotrophic factor ( bdnf ) expression ( hasbi et al . , 2009 ) two proteins that have been linked to schizophrenia ( weickert et al . , 2003 ; issa et al . , 2010 ; jindal et al . , 2010 ; novak and seeman , 2010 ; wong et al . , 2010 ; carlino et al . , 2011 ) . finally , of particular relevance , an upregulation of striatal d1d2 heteromeric activity was seen following repeated amphetamine treatment in rats and in human gp of patients with schizophrenia ( perreault et al . , 2010 ) . given that amphetamine sensitization is often employed as an animal model for schizophrenia ( featherstone et al . , 2007 ) , but no common biochemical marker linking the two has previously been documented , it was inferred that the sensitized state of the d1d2 heteromer may provide the first neuropharmacological correlate between increased dopamine neurotransmission and its functional consequence . as antipsychotics invariably also target the d1d2 heteromer , these findings strongly suggest that further research examining the d1d2 receptor heteromer as a potential drug target in schizophrenia is warranted . in addition to an involvement in schizophrenia , camkii and bdnf have also been shown to play a major role in cocaine addiction ( anderson et al . , 2008 ; lobo et al . , 2010 ; mcginty et al . , 2010 ; wang et al . , 2010 ) . in one particularly compelling study , it was shown that cocaine reinstatement in rats depended upon an interaction between nac dopamine and glutamate systems that was mediated by camkii ( anderson et al . , 2008 ) . more specifically , cocaine reinstatement increased activation of nac shell camkii , which subsequently led to the phosphorylation of the glua1 subunit of the ampa receptor at ser , and increased cell - surface expression of glua1-containing ampa receptors . interestingly , in contrast to the effects of acute selective activation of the d1d2 heteromer in nac , more prolonged selective activation has been reported to reduce total camkii levels in this region and additionally reduce phosphorylation of glua1 at ser ( perreault et al . , 2010 ) , a finding effectively linking the d1d2 heteromer to the reward pathways of the brain . it is now known that the agonist skf 83959 directly activates calcium signaling via the dopamine d1d2 receptor heteromer , but does not activate the d1r camp pathway or the gi - coupled d2r ( rashid et al . , 2007 ) . therefore findings from studies assessing the effects of skf 83959 would not likely have been derived from functional interactions between d1r or d2r homomers within the same neuron , or between the d1r - expressing striatonigral and d2r - expressing striatopallidal populations of msns . however , for many years it was believed that the effects of the agonist skf 83959 were mediated through its actions solely at the d1r . reports had demonstrated that skf 83959 had a substantial affinity for the d1r ( andringa et al . , 1999 ; neumeyer et al . , 2003 ) , however the physiological actions of skf 83959 at the d1r were the subject of much debate as the drug exhibited antagonistic properties on adenylyl cyclase activity ( arnt et al . 2003 ) yet mimicked certain behavioral characteristics of d1r agonism such as grooming or the induction of circling behavior ( deveney and waddington , 1995 ; gnanalingham et al . , 1995 ; waddington et al . , 1995 ; zhen et al . , 2005 ; perreault et al . , however , skf 83959 was also linked to plc activation and pi hydrolysis in brain ( panchalingam and undie , 2001 ; jin et al . , 2003 ; zhen et al . , 2005 ; rashid et al . , 2007 ) , a pathway that had also been observed to be induced with a number of other d1r agonists in brain tissue ( undie et al . , 1994 ; desai et al . , 2005 ) but , interestingly , not in cells expressing only the d1r ( lin et al . , 1995 ) . given the ability of d1r agonists to directly induce pi hydrolysis in brain , but not in cells expressing solely the d1r , this suggests that d1r agonists that stimulate this pathway do so through agonist activity at the d1d2 receptor heteromer . for example , the d1r agonists skf 81297 and skf 38393 are still often employed as selective d1r agonists , despite evidence demonstrating their ability to induce behavioral and neurochemical effects characteristic of dopamine d1d2 heteromer activation , such as grooming and activation of plc leading to intracellular calcium release ( molloy and waddington , 1987 ; undie and friedman , 1990 ; undie et al . , 1994 ; furthermore , the d5 receptor has also been shown to induce a calcium signal through plc activation or extracellular calcium influx ( so et al . , 2009 ) , further confounding the pharmacological profile of these agonists . as such , the interpretation of both past and future results should take into account that activation of individual dopamine receptors by these compounds most likely occurs in tandem with d1d2 receptor heteromer activation on the physiologically relevant subset neurons coexpressing both the d1r and d2r . the appropriate selection of dopaminergic drugs is therefore essential to effectively isolate the specific complex , or signaling pathway , under investigation . for the most part , studies agree that the d1r and d2r are predominantly segregated to discrete populations of msns in striatum and , additionally , that functional cross - talk between the d1r and d2r is critical in mediating some of the physiological effects of dopamine , such as the synergistic effects of d1r / d2r activation on immediate early gene expression . however , there is also an abundance of evidence showing that a proportion of msns in striatum coexpresses the d1r and d2r , and the physiological importance of these neurons is now beginning to become apparent . specifically , unlike d1r - only and d2r - only striatal msns , d1r / d2r - coexpressing striatal projection neurons have been reported to terminate in regions within both the striatonigral and striatopallidal pathways , as well as regions containing dopamine neuronal cell bodies . these findings , as well as the demonstration of d1r / d2r coexpression at presynaptic terminals , indicate that receptor coexpression may have a unique physiological function at a local level , via msn msn synaptic connections , as well as distal effects through their efferent projections ( figure 4 ) . this would likely contribute to the regulation of thalamic neurotransmission , perhaps with the purpose of maintaining homeostatic balance between the direct and indirect pathways . it is possible that this may occur , at least in part , through the regulation of postsynaptic glutamate transmission in the basal ganglia ( stefani et al . , 1998 ) , as these neurons exhibit a unique expression phenotype of the ampa glua subunits . in addition , these d1r / d2r msns also express the dopamine d1d2 receptor heteromer , a novel receptor complex that links dopamine receptor activation directly to calcium signaling and bdnf production in vivo . together , the evidence indicates that msns coexpressing the d1r and d2r in the basal ganglia embody a physiologically relevant , and functionally active , subset of neurons . we propose that d1r / d2r - coexpressing msns represent a third major dopamine receptor neuronal pathway , in addition to the striatonigral and striatopallidal pathways , with the potential to provide a novel approach to drug discovery in basal ganglia disorders . model of d1 and d2 receptor - coexpressing projections in the basal ganglia circuitry . previously reported ( solid lines ; deng et al . , 2006 ; wang et al . , 2006 , 2007 ) projection sites of d1r / d2r or sp / enk - coexpressing neurons and putative projections of d1r / d2r - dyn / enk neurons ( dashed lines ) , based on the reported regional distribution of neuronal cell bodies and presynaptic d1r and d2r colocalization ( perreault et al . , 2010 ) . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

dopaminergic signaling within the basal ganglia has classically been thought to occur within two distinct neuronal pathways ; the direct striatonigral pathway which contains the dopamine d1 receptor and the neuropeptides dynorphin ( dyn ) and substance p , and the indirect striatopallidal pathway which expresses the dopamine d2 receptor and enkephalin ( enk ) . a number of studies have also shown , however , that d1 and d2 receptors can co - exist within the same medium spiny neuron and emerging evidence indicates that these d1/d2-coexpressing neurons , which also express dyn and enk , may comprise a third neuronal pathway , with representation in both the striatonigral and striatopallidal projections of the basal ganglia . furthermore , within these coexpressing neurons it has been shown that the dopamine d1 and d2 receptor can form a novel and pharmacologically distinct receptor complex , the dopamine d1d2 receptor heteromer , with unique signaling properties . this is indicative of a functionally unique role for these neurons in brain . the aim of this review is to discuss the evidence in support of a novel third pathway coexpressing the d1 and d2 receptor , to discuss the potential relevance of this pathway to basal ganglia signaling , and to address its potential value , and that of the dopamine d1d2 receptor heteromer , in the search for new therapeutic strategies for disorders involving dopamine neurotransmission .

Dopamine D1R and D2R are Functionally Linked Neuroanatomical Evidence for D1R and D2R Colocalization Drd1a-EGFP and Drd2-EGFP BAC transgenic mice Functional Relevance of D1R/D2R-Coexpressing Neurons The dopamine D1D2 receptor heteromer A Cautionary Note on the Use of Dopamine Receptor Agonists Concluding Remarks Conflict of Interest Statement

, 1985 ) and however , it has also been demonstrated that the concomitant activation of both d1r and d2r is essential to achieve certain other behavioral , biochemical , and electrophysiological effects . for instance , past studies have shown coactivation of the d1r and d2r as being necessary for maximal stereotyped and locomotor behavior ( walters et al . for example , while the systemic administration of a dopamine d1r agonist induced the expression of c - fos and zif268 in the dopamine - depleted striatum , and a d2r agonist decreased zif268 expression , both agonists together significantly enhanced gene expression compared to the d1r agonist alone ( keefe and gerfen , 1995 ) . , however , an interaction between the receptors occurring within the same cell may have been involved in some instances . , 2006 ) and the selective activation of d1r or d2r in single striatal neurons differentially modulated tetrodotoxin - sensitive sodium channels ( aizman et al . given the functional linkage between d1r and d2r , neuroanatomical studies began to address the question of dopamine receptor segregation and many of these studies have now shown , both at the mrna level and the protein level , that d1r and d2r coexist in a fraction of striatal msns . , 1993 ) and double in situ hybridization has been used to report colocalization of d1r and d2r mrna in some striatal neurons in primate ( aubert et al . , 1992 , 1996 ) and immunolabeling has been a widely used tool for visualizing striatal d1r / d2r coexpression at the protein level in both neonatal striatal cultures ( shetreat et al . ( 2010 ) , using highly validated antibodies , it was shown that the d1r and d2r were expressed exclusively in neurons containing both dyn and enk . interestingly , unlike the controversy surrounding the localization of d1r and d2r , sp / dyn enk coexpression in striatal neurons has been widely accepted , and overlap of sp / dyn and enk mrna or protein in a proportion of striatal projection neurons has been reported for a number of species ( penny et al . , 1990 ; enhanced green fluorescent protein ( egfp)-tagged promoter elements of d1r and d2r in bacterial artificial chromosome ( bac ) transgenic mice is one such method , and has been used to quantify the proportion of striatal neurons expressing the receptors within the striatonigral and striatopallidal pathways ( valjent et al . , 2009 ) and that the proportion of neurons showing coexpression were similar to that observed in the bertran - gonzalez study . essentially , this method employs the fluorescent properties of the neurons in the bac transgenic drd1a - egfp or drd2-egfp mice to purify d1r - expressing and d2r - expressing msns , and is often used to assess specific characteristics of these two neuronal subtypes . in addition , using bac transgenic mice with cre recombinase being driven by the d1r or d2r promoters , and using double immunofluoresence to stain for cre and enk , this group also showed that a fraction of striatal d1r - expressing neurons were also positive for enk . enhanced green fluorescent protein ( egfp)-tagged promoter elements of d1r and d2r in bacterial artificial chromosome ( bac ) transgenic mice is one such method , and has been used to quantify the proportion of striatal neurons expressing the receptors within the striatonigral and striatopallidal pathways ( valjent et al . , 2009 ) and that the proportion of neurons showing coexpression were similar to that observed in the bertran - gonzalez study . essentially , this method employs the fluorescent properties of the neurons in the bac transgenic drd1a - egfp or drd2-egfp mice to purify d1r - expressing and d2r - expressing msns , and is often used to assess specific characteristics of these two neuronal subtypes . in addition , using bac transgenic mice with cre recombinase being driven by the d1r or d2r promoters , and using double immunofluoresence to stain for cre and enk , this group also showed that a fraction of striatal d1r - expressing neurons were also positive for enk . although the existence of d1r- and d2r - coexpressing msns is now generally accepted , little is yet known about the functional relevance of these neurons , most likely as a result of the methodological difficulties attempting to isolate them . however , it has been shown that while all msns express mrna for the glua1 and glua2 subunits of the ampa receptor ( chen et al . , 1998 ) a finding which the authors posited as being suggestive of distinctive postsynaptic glutamatergic mechanisms in the neurons . a role in neurotransmission is supported by evidence documenting that striatal projection neurons coexpressing d1r and d2r or sp and enk terminate in a number of regions including gp and epn ( gpi ) , as well as substantia nigra and ventral tegmental area ( deng et al . in addition , a region - specific distribution of d1r / d2r - dyn / enk neurons throughout the rat basal ganglia and mesolimbic pathway has been reported , being present in nac ( figure 2 ) and cp , as well as gp and ventral pallidum and epn ( perreault et al . in gp and vp , while the total number of d1r neurons was quite low compared to other regions , a high percentage of these neurons ( ~60% and ~30% ) also contained d2r . the dopamine d1 and d2 receptor are colocalized with dynorphin and enkephalin in rat nucleus accumbens shell . ( a , b ) confocal images showing d1r and d2r colocalization with dynorphin ( dyn ) or enkephalin ( enk ; white arrows ) . ( c , d ) neurons coexpressing dyn and enk also expressed the d1r or the d2r ( white arrows ) . in basal ganglia , the coexpression of d1r and d2r has been shown to occur both within neuronal cell bodies and selectively at presynaptic , but not postsynaptic terminals , as evidenced by d1r and d2r coexpression with the presynaptic marker synaptophysin , but not the postsynaptic marker psd-95 ( perreault et al . , 2007 ) and electrophysiology studies have demonstrated that presynaptic dopamine receptors on msn terminals could modulate gabaergic inhibitory postsynaptic currents ( ipscs ; delgado et al . , 2000 ; interestingly , in one of these studies it was shown that while the d2r agonist quinpirole predominantly mediated inhibition of autaptic connections in msns , the d1r agonist skf 38393 mediated either inhibition or facilitation ( geldwert et al . although the authors did not directly address the seemingly discrepant effects of the d1r agonist on gaba transmission , it has been shown that skf 38393 activates two biologically different signaling pathways , the camp pathway and phospholipase c ( plc)-phosphoinositide ( pi ) pathway ( undie and friedman , 1990 ; undie et al . although it has been suggested that these two pathways are linked directly to d1r ( undie et al . , 1994 ) , it is now known that in msns that coexpress d1r and d2r , the receptors can form a heteromeric complex , the dopamine d1d2 receptor heteromer , and it is this complex that directly activates the plc - pi pathway ( lee et al . , 2004 ) , and was the first evidence of a dopamine heteromeric receptor complex in brain . similarly , this method has now been employed to show , in vivo , that in adult rat nac , cp ( hasbi et al . , 2009 ; 2010 ) and gp ( figure 3 ) the d1r and d2r existed in close < 50100 proximity indicative of d1d2 receptor heteromerization . however , the propensity for striatal neurons to exhibit d1d2 heteromers was region - dependent , with the majority ( > 90% ) of d1r / d2r - coexpressing neuronal cell bodies in nac core and shell showing robust d1d2 heteromer formation , but only ~25% of d1r / d2r neurons expressing the d1d2 heteromer in cp ( perreault et al . , 2010 ) this suggests that d1r and d2r can coexist as homomers in the same cell without forming heteromers . it was also shown in the neuropil of both nac and cp the presence of d1d2 heteromers at presynaptic terminals , a finding that suggests a possible involvement of synaptic d1d2 receptor heteromer in gaba release in both regions . the receptor antibody - linked fluorophores were calculated to be in close proximity with a relative distance of 57 nm ( 5070 ) indicative of d1d2 receptor heteromer formation . the role of the d1d2 heteromer in vivo is just beginning to be elucidated , however several lines of evidence have emerged implicating the complex as a potential therapeutic target in disorders involving elevated dopamine transmission , such as schizophrenia and drug addiction . specifically , it has been postulated that abnormal regulation of calcium signaling may constitute the central dysfunction that is responsible for generating the psychopathology of schizophrenia ( lidow , 2003 ) . although the d1r and d2r have not been shown individually to be directly coupled to calcium signaling , coactivation of both receptors within the dopamine d1d2 receptor heteromer by concurrent administration of d1r and d2r agonists , or the selective d1d2 heteromer agonist skf 83959 , resulted in a novel gq - plc - linked increase in intracellular calcium release ( lee et al . , 2007 ) , but no common biochemical marker linking the two has previously been documented , it was inferred that the sensitized state of the d1d2 heteromer may provide the first neuropharmacological correlate between increased dopamine neurotransmission and its functional consequence . as antipsychotics invariably also target the d1d2 heteromer , these findings strongly suggest that further research examining the d1d2 receptor heteromer as a potential drug target in schizophrenia is warranted . in addition to an involvement in schizophrenia , camkii and bdnf have also been shown to play a major role in cocaine addiction ( anderson et al . more specifically , cocaine reinstatement increased activation of nac shell camkii , which subsequently led to the phosphorylation of the glua1 subunit of the ampa receptor at ser , and increased cell - surface expression of glua1-containing ampa receptors . interestingly , in contrast to the effects of acute selective activation of the d1d2 heteromer in nac , more prolonged selective activation has been reported to reduce total camkii levels in this region and additionally reduce phosphorylation of glua1 at ser ( perreault et al . , 2004 ) , and was the first evidence of a dopamine heteromeric receptor complex in brain . , 2006 ) , quantitative fret in situ has now been utilized to verify the presence of dopamine d1d2 receptor heteromers in neonatal cultured rat striatal neurons ( hasbi et al . similarly , this method has now been employed to show , in vivo , that in adult rat nac , cp ( hasbi et al . , 2010 ) and gp ( figure 3 ) the d1r and d2r existed in close < 50100 proximity indicative of d1d2 receptor heteromerization . however , the propensity for striatal neurons to exhibit d1d2 heteromers was region - dependent , with the majority ( > 90% ) of d1r / d2r - coexpressing neuronal cell bodies in nac core and shell showing robust d1d2 heteromer formation , but only ~25% of d1r / d2r neurons expressing the d1d2 heteromer in cp ( perreault et al . this suggests that d1r and d2r can coexist as homomers in the same cell without forming heteromers . it was also shown in the neuropil of both nac and cp the presence of d1d2 heteromers at presynaptic terminals , a finding that suggests a possible involvement of synaptic d1d2 receptor heteromer in gaba release in both regions . the receptor antibody - linked fluorophores were calculated to be in close proximity with a relative distance of 57 nm ( 5070 ) indicative of d1d2 receptor heteromer formation . the role of the d1d2 heteromer in vivo is just beginning to be elucidated , however several lines of evidence have emerged implicating the complex as a potential therapeutic target in disorders involving elevated dopamine transmission , such as schizophrenia and drug addiction . although the d1r and d2r have not been shown individually to be directly coupled to calcium signaling , coactivation of both receptors within the dopamine d1d2 receptor heteromer by concurrent administration of d1r and d2r agonists , or the selective d1d2 heteromer agonist skf 83959 , resulted in a novel gq - plc - linked increase in intracellular calcium release ( lee et al . , 2007 ) , but no common biochemical marker linking the two has previously been documented , it was inferred that the sensitized state of the d1d2 heteromer may provide the first neuropharmacological correlate between increased dopamine neurotransmission and its functional consequence . as antipsychotics invariably also target the d1d2 heteromer , these findings strongly suggest that further research examining the d1d2 receptor heteromer as a potential drug target in schizophrenia is warranted . more specifically , cocaine reinstatement increased activation of nac shell camkii , which subsequently led to the phosphorylation of the glua1 subunit of the ampa receptor at ser , and increased cell - surface expression of glua1-containing ampa receptors . interestingly , in contrast to the effects of acute selective activation of the d1d2 heteromer in nac , more prolonged selective activation has been reported to reduce total camkii levels in this region and additionally reduce phosphorylation of glua1 at ser ( perreault et al . it is now known that the agonist skf 83959 directly activates calcium signaling via the dopamine d1d2 receptor heteromer , but does not activate the d1r camp pathway or the gi - coupled d2r ( rashid et al . therefore findings from studies assessing the effects of skf 83959 would not likely have been derived from functional interactions between d1r or d2r homomers within the same neuron , or between the d1r - expressing striatonigral and d2r - expressing striatopallidal populations of msns . however , for many years it was believed that the effects of the agonist skf 83959 were mediated through its actions solely at the d1r . , however , skf 83959 was also linked to plc activation and pi hydrolysis in brain ( panchalingam and undie , 2001 ; jin et al . , 2007 ) , a pathway that had also been observed to be induced with a number of other d1r agonists in brain tissue ( undie et al . given the ability of d1r agonists to directly induce pi hydrolysis in brain , but not in cells expressing solely the d1r , this suggests that d1r agonists that stimulate this pathway do so through agonist activity at the d1d2 receptor heteromer . for example , the d1r agonists skf 81297 and skf 38393 are still often employed as selective d1r agonists , despite evidence demonstrating their ability to induce behavioral and neurochemical effects characteristic of dopamine d1d2 heteromer activation , such as grooming and activation of plc leading to intracellular calcium release ( molloy and waddington , 1987 ; undie and friedman , 1990 ; undie et al . , 1994 ; furthermore , the d5 receptor has also been shown to induce a calcium signal through plc activation or extracellular calcium influx ( so et al . as such , the interpretation of both past and future results should take into account that activation of individual dopamine receptors by these compounds most likely occurs in tandem with d1d2 receptor heteromer activation on the physiologically relevant subset neurons coexpressing both the d1r and d2r . the appropriate selection of dopaminergic drugs is therefore essential to effectively isolate the specific complex , or signaling pathway , under investigation . for the most part , studies agree that the d1r and d2r are predominantly segregated to discrete populations of msns in striatum and , additionally , that functional cross - talk between the d1r and d2r is critical in mediating some of the physiological effects of dopamine , such as the synergistic effects of d1r / d2r activation on immediate early gene expression . however , there is also an abundance of evidence showing that a proportion of msns in striatum coexpresses the d1r and d2r , and the physiological importance of these neurons is now beginning to become apparent . specifically , unlike d1r - only and d2r - only striatal msns , d1r / d2r - coexpressing striatal projection neurons have been reported to terminate in regions within both the striatonigral and striatopallidal pathways , as well as regions containing dopamine neuronal cell bodies . it is possible that this may occur , at least in part , through the regulation of postsynaptic glutamate transmission in the basal ganglia ( stefani et al . , 1998 ) , as these neurons exhibit a unique expression phenotype of the ampa glua subunits . in addition , these d1r / d2r msns also express the dopamine d1d2 receptor heteromer , a novel receptor complex that links dopamine receptor activation directly to calcium signaling and bdnf production in vivo . together , the evidence indicates that msns coexpressing the d1r and d2r in the basal ganglia embody a physiologically relevant , and functionally active , subset of neurons . we propose that d1r / d2r - coexpressing msns represent a third major dopamine receptor neuronal pathway , in addition to the striatonigral and striatopallidal pathways , with the potential to provide a novel approach to drug discovery in basal ganglia disorders . model of d1 and d2 receptor - coexpressing projections in the basal ganglia circuitry . , 2006 , 2007 ) projection sites of d1r / d2r or sp / enk - coexpressing neurons and putative projections of d1r / d2r - dyn / enk neurons ( dashed lines ) , based on the reported regional distribution of neuronal cell bodies and presynaptic d1r and d2r colocalization ( perreault et al . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

numerous reports have documented opposing effects of the gs - coupled d1r and gi / o - coupled d2r on biological processes such as adenylyl cyclase activity and camp formation ( hyttel , 1978 ; onali et al . , 1985 ) and however , it has also been demonstrated that the concomitant activation of both d1r and d2r is essential to achieve certain other behavioral , biochemical , and electrophysiological effects . , 1988 ; deboer and abercrombie , 1996 ) , and more recently it was reported that to evoke neural and behavioral phenotypes of cocaine sensitization , both d1r and d2r stimulation is required ( capper - loup et al . for example , while the systemic administration of a dopamine d1r agonist induced the expression of c - fos and zif268 in the dopamine - depleted striatum , and a d2r agonist decreased zif268 expression , both agonists together significantly enhanced gene expression compared to the d1r agonist alone ( keefe and gerfen , 1995 ) . , 1990 ) , and restoration of long - term depression of synaptic transmission following dopamine depletion occurred by dopamine administration or coadministration of specific d1r and d2r agonists but not by either selective agonist alone ( calabresi et al . , 2006 ) and the selective activation of d1r or d2r in single striatal neurons differentially modulated tetrodotoxin - sensitive sodium channels ( aizman et al . , 2000 the synergistic potentiation of the arachidonic acid release by coadministration of d1r - selective and d2r - selective agonists has also been observed in cells expressing both receptors ( piomelli et al . given the functional linkage between d1r and d2r , neuroanatomical studies began to address the question of dopamine receptor segregation and many of these studies have now shown , both at the mrna level and the protein level , that d1r and d2r coexist in a fraction of striatal msns . in situ hybridization studies examining d1r and d2r mrna in serial rat brain striatal sections have shown overlap of d1r and d2r transcript expression in the same neurons ( meador - woodruff et al . , 1993 ) and double in situ hybridization has been used to report colocalization of d1r and d2r mrna in some striatal neurons in primate ( aubert et al . , 1992 , 1996 ) and immunolabeling has been a widely used tool for visualizing striatal d1r / d2r coexpression at the protein level in both neonatal striatal cultures ( shetreat et al . , it had been noted that while neurons expressing solely mrna for the d1r or d2r were abundant in sp or enk mrna respectively , neurons containing mrna for both d1r and d2r expressed both sp and enk mrnas ( surmeier et al . interestingly , unlike the controversy surrounding the localization of d1r and d2r , sp / dyn enk coexpression in striatal neurons has been widely accepted , and overlap of sp / dyn and enk mrna or protein in a proportion of striatal projection neurons has been reported for a number of species ( penny et al . , 1990 ; enhanced green fluorescent protein ( egfp)-tagged promoter elements of d1r and d2r in bacterial artificial chromosome ( bac ) transgenic mice is one such method , and has been used to quantify the proportion of striatal neurons expressing the receptors within the striatonigral and striatopallidal pathways ( valjent et al . ( 2008 ) it was reported that while the majority of d1r and d2r were segregated to these discrete pathways , an estimated 17% of nac shell msns exhibited receptor colocalization with ~6% d1r / d2r coexpression in cp . , 2008 ) , another group was able to show that 100% of msns in these mice expressed d1r , d2r or both ( matamales et al . , 2009 ) and that the proportion of neurons showing coexpression were similar to that observed in the bertran - gonzalez study . essentially , this method employs the fluorescent properties of the neurons in the bac transgenic drd1a - egfp or drd2-egfp mice to purify d1r - expressing and d2r - expressing msns , and is often used to assess specific characteristics of these two neuronal subtypes . using this process , it was recently shown that while purified d1r - positive neurons from whole striatum displayed enrichment of d1r mrna , d2r mrna was also evident , albeit in small amounts ( lobo et al . in addition , using bac transgenic mice with cre recombinase being driven by the d1r or d2r promoters , and using double immunofluoresence to stain for cre and enk , this group also showed that a fraction of striatal d1r - expressing neurons were also positive for enk . enhanced green fluorescent protein ( egfp)-tagged promoter elements of d1r and d2r in bacterial artificial chromosome ( bac ) transgenic mice is one such method , and has been used to quantify the proportion of striatal neurons expressing the receptors within the striatonigral and striatopallidal pathways ( valjent et al . ( 2008 ) it was reported that while the majority of d1r and d2r were segregated to these discrete pathways , an estimated 17% of nac shell msns exhibited receptor colocalization with ~6% d1r / d2r coexpression in cp . , 2008 ) , another group was able to show that 100% of msns in these mice expressed d1r , d2r or both ( matamales et al . essentially , this method employs the fluorescent properties of the neurons in the bac transgenic drd1a - egfp or drd2-egfp mice to purify d1r - expressing and d2r - expressing msns , and is often used to assess specific characteristics of these two neuronal subtypes . using this process , it was recently shown that while purified d1r - positive neurons from whole striatum displayed enrichment of d1r mrna , d2r mrna was also evident , albeit in small amounts ( lobo et al . in addition , using bac transgenic mice with cre recombinase being driven by the d1r or d2r promoters , and using double immunofluoresence to stain for cre and enk , this group also showed that a fraction of striatal d1r - expressing neurons were also positive for enk . although the existence of d1r- and d2r - coexpressing msns is now generally accepted , little is yet known about the functional relevance of these neurons , most likely as a result of the methodological difficulties attempting to isolate them . a role in neurotransmission is supported by evidence documenting that striatal projection neurons coexpressing d1r and d2r or sp and enk terminate in a number of regions including gp and epn ( gpi ) , as well as substantia nigra and ventral tegmental area ( deng et al . in addition , a region - specific distribution of d1r / d2r - dyn / enk neurons throughout the rat basal ganglia and mesolimbic pathway has been reported , being present in nac ( figure 2 ) and cp , as well as gp and ventral pallidum and epn ( perreault et al . specifically , it was estimated that the fraction of d1r - expressing neurons also expressing the d2r in nac core and shell was ~25% and ~35% respectively , whereas in cp the percentage was much lower being only ~7% . in gp and vp , while the total number of d1r neurons was quite low compared to other regions , a high percentage of these neurons ( ~60% and ~30% ) also contained d2r . in basal ganglia , the coexpression of d1r and d2r has been shown to occur both within neuronal cell bodies and selectively at presynaptic , but not postsynaptic terminals , as evidenced by d1r and d2r coexpression with the presynaptic marker synaptophysin , but not the postsynaptic marker psd-95 ( perreault et al . , 2000 ; interestingly , in one of these studies it was shown that while the d2r agonist quinpirole predominantly mediated inhibition of autaptic connections in msns , the d1r agonist skf 38393 mediated either inhibition or facilitation ( geldwert et al . although the authors did not directly address the seemingly discrepant effects of the d1r agonist on gaba transmission , it has been shown that skf 38393 activates two biologically different signaling pathways , the camp pathway and phospholipase c ( plc)-phosphoinositide ( pi ) pathway ( undie and friedman , 1990 ; undie et al . , 1994 ) , it is now known that in msns that coexpress d1r and d2r , the receptors can form a heteromeric complex , the dopamine d1d2 receptor heteromer , and it is this complex that directly activates the plc - pi pathway ( lee et al . , 2006 ) , quantitative fret in situ has now been utilized to verify the presence of dopamine d1d2 receptor heteromers in neonatal cultured rat striatal neurons ( hasbi et al . however , the propensity for striatal neurons to exhibit d1d2 heteromers was region - dependent , with the majority ( > 90% ) of d1r / d2r - coexpressing neuronal cell bodies in nac core and shell showing robust d1d2 heteromer formation , but only ~25% of d1r / d2r neurons expressing the d1d2 heteromer in cp ( perreault et al . it was also shown in the neuropil of both nac and cp the presence of d1d2 heteromers at presynaptic terminals , a finding that suggests a possible involvement of synaptic d1d2 receptor heteromer in gaba release in both regions . an interaction between the d1r and d2r was evident , with a relatively high mean fret efficiency ( efficiency of energy transfer between the donor and acceptor fluorophores ) of ~22% . the role of the d1d2 heteromer in vivo is just beginning to be elucidated , however several lines of evidence have emerged implicating the complex as a potential therapeutic target in disorders involving elevated dopamine transmission , such as schizophrenia and drug addiction . specifically , it has been postulated that abnormal regulation of calcium signaling may constitute the central dysfunction that is responsible for generating the psychopathology of schizophrenia ( lidow , 2003 ) . although the d1r and d2r have not been shown individually to be directly coupled to calcium signaling , coactivation of both receptors within the dopamine d1d2 receptor heteromer by concurrent administration of d1r and d2r agonists , or the selective d1d2 heteromer agonist skf 83959 , resulted in a novel gq - plc - linked increase in intracellular calcium release ( lee et al . , , an upregulation of striatal d1d2 heteromeric activity was seen following repeated amphetamine treatment in rats and in human gp of patients with schizophrenia ( perreault et al . , 2007 ) , but no common biochemical marker linking the two has previously been documented , it was inferred that the sensitized state of the d1d2 heteromer may provide the first neuropharmacological correlate between increased dopamine neurotransmission and its functional consequence . as antipsychotics invariably also target the d1d2 heteromer , these findings strongly suggest that further research examining the d1d2 receptor heteromer as a potential drug target in schizophrenia is warranted . more specifically , cocaine reinstatement increased activation of nac shell camkii , which subsequently led to the phosphorylation of the glua1 subunit of the ampa receptor at ser , and increased cell - surface expression of glua1-containing ampa receptors . interestingly , in contrast to the effects of acute selective activation of the d1d2 heteromer in nac , more prolonged selective activation has been reported to reduce total camkii levels in this region and additionally reduce phosphorylation of glua1 at ser ( perreault et al . , 2006 ) , quantitative fret in situ has now been utilized to verify the presence of dopamine d1d2 receptor heteromers in neonatal cultured rat striatal neurons ( hasbi et al . however , the propensity for striatal neurons to exhibit d1d2 heteromers was region - dependent , with the majority ( > 90% ) of d1r / d2r - coexpressing neuronal cell bodies in nac core and shell showing robust d1d2 heteromer formation , but only ~25% of d1r / d2r neurons expressing the d1d2 heteromer in cp ( perreault et al . it was also shown in the neuropil of both nac and cp the presence of d1d2 heteromers at presynaptic terminals , a finding that suggests a possible involvement of synaptic d1d2 receptor heteromer in gaba release in both regions . the role of the d1d2 heteromer in vivo is just beginning to be elucidated , however several lines of evidence have emerged implicating the complex as a potential therapeutic target in disorders involving elevated dopamine transmission , such as schizophrenia and drug addiction . specifically , it has been postulated that abnormal regulation of calcium signaling may constitute the central dysfunction that is responsible for generating the psychopathology of schizophrenia ( lidow , 2003 ) . although the d1r and d2r have not been shown individually to be directly coupled to calcium signaling , coactivation of both receptors within the dopamine d1d2 receptor heteromer by concurrent administration of d1r and d2r agonists , or the selective d1d2 heteromer agonist skf 83959 , resulted in a novel gq - plc - linked increase in intracellular calcium release ( lee et al . finally , of particular relevance , an upregulation of striatal d1d2 heteromeric activity was seen following repeated amphetamine treatment in rats and in human gp of patients with schizophrenia ( perreault et al . , 2007 ) , but no common biochemical marker linking the two has previously been documented , it was inferred that the sensitized state of the d1d2 heteromer may provide the first neuropharmacological correlate between increased dopamine neurotransmission and its functional consequence . as antipsychotics invariably also target the d1d2 heteromer , these findings strongly suggest that further research examining the d1d2 receptor heteromer as a potential drug target in schizophrenia is warranted . more specifically , cocaine reinstatement increased activation of nac shell camkii , which subsequently led to the phosphorylation of the glua1 subunit of the ampa receptor at ser , and increased cell - surface expression of glua1-containing ampa receptors . interestingly , in contrast to the effects of acute selective activation of the d1d2 heteromer in nac , more prolonged selective activation has been reported to reduce total camkii levels in this region and additionally reduce phosphorylation of glua1 at ser ( perreault et al . it is now known that the agonist skf 83959 directly activates calcium signaling via the dopamine d1d2 receptor heteromer , but does not activate the d1r camp pathway or the gi - coupled d2r ( rashid et al . therefore findings from studies assessing the effects of skf 83959 would not likely have been derived from functional interactions between d1r or d2r homomers within the same neuron , or between the d1r - expressing striatonigral and d2r - expressing striatopallidal populations of msns . , 2003 ) , however the physiological actions of skf 83959 at the d1r were the subject of much debate as the drug exhibited antagonistic properties on adenylyl cyclase activity ( arnt et al . given the ability of d1r agonists to directly induce pi hydrolysis in brain , but not in cells expressing solely the d1r , this suggests that d1r agonists that stimulate this pathway do so through agonist activity at the d1d2 receptor heteromer . for example , the d1r agonists skf 81297 and skf 38393 are still often employed as selective d1r agonists , despite evidence demonstrating their ability to induce behavioral and neurochemical effects characteristic of dopamine d1d2 heteromer activation , such as grooming and activation of plc leading to intracellular calcium release ( molloy and waddington , 1987 ; undie and friedman , 1990 ; undie et al . as such , the interpretation of both past and future results should take into account that activation of individual dopamine receptors by these compounds most likely occurs in tandem with d1d2 receptor heteromer activation on the physiologically relevant subset neurons coexpressing both the d1r and d2r . for the most part , studies agree that the d1r and d2r are predominantly segregated to discrete populations of msns in striatum and , additionally , that functional cross - talk between the d1r and d2r is critical in mediating some of the physiological effects of dopamine , such as the synergistic effects of d1r / d2r activation on immediate early gene expression . however , there is also an abundance of evidence showing that a proportion of msns in striatum coexpresses the d1r and d2r , and the physiological importance of these neurons is now beginning to become apparent . specifically , unlike d1r - only and d2r - only striatal msns , d1r / d2r - coexpressing striatal projection neurons have been reported to terminate in regions within both the striatonigral and striatopallidal pathways , as well as regions containing dopamine neuronal cell bodies . these findings , as well as the demonstration of d1r / d2r coexpression at presynaptic terminals , indicate that receptor coexpression may have a unique physiological function at a local level , via msn msn synaptic connections , as well as distal effects through their efferent projections ( figure 4 ) . this would likely contribute to the regulation of thalamic neurotransmission , perhaps with the purpose of maintaining homeostatic balance between the direct and indirect pathways . together , the evidence indicates that msns coexpressing the d1r and d2r in the basal ganglia embody a physiologically relevant , and functionally active , subset of neurons . we propose that d1r / d2r - coexpressing msns represent a third major dopamine receptor neuronal pathway , in addition to the striatonigral and striatopallidal pathways , with the potential to provide a novel approach to drug discovery in basal ganglia disorders . , 2006 , 2007 ) projection sites of d1r / d2r or sp / enk - coexpressing neurons and putative projections of d1r / d2r - dyn / enk neurons ( dashed lines ) , based on the reported regional distribution of neuronal cell bodies and presynaptic d1r and d2r colocalization ( perreault et al .

the use of oral medications offers many advantages , because these medications act directly on the airways and require administration of lower doses , with no gastric changes . 2 using the proper inhaler technique ensures sufficient deposition of drug particles in the distal airways , optimizing drug effectiveness and reducing possible side effects . one of the determinants of the effectiveness of inhaled medications is the ability of the patient to adhere to good inhaler techniques . 3 for some patients , that can be difficult , and the prescription of medication should therefore always be accompanied by appropriate inhaler technique training delivered by a health professional . thus , it is possible to reduce the number of inhaler technique errors and minimize the clinical consequences of poor drug delivery . the first therapeutic aerosol devices were developed in the 1950s 4 and consisted of nebulizers and atomizers containing anticholinergics for treatment of asthma . 5 despite the long time since development and the wide use of these devices , inhaler technique errors continue to be common among respiratory patients , 6 reducing the benefits of inhaled medications . in chile , 7 observed that only 12.5% of the mothers of hospitalized infants have a correct inhaler technique . however , it is unknown whether this trend is reflected in adult patients , given that the elderly are more likely to make inhaler technique errors . 8 this promotes the assessment of inhaler technique by age group , because tailoring education to the needs of each patient could significantly improve disease management . the purposes of the present study were to assess inhaler technique in pediatric and adult patients with asthma ; to determine the most common errors in each group of patients ; and to compare the results between the two groups . this was a descriptive cross - sectional study conducted in the region of valparaso , chile , between march and may of 2014 . the sample consisted of male and female patients with a diagnosis of asthma based on spirometry , in accordance with the global initiative for asthma criteria . 9 the ages of the participants ranged from 5 to 90 years , and the sampling was non - probabilistic ( purposive ) . patients had to meet the following inclusion criteria : being enrolled in and attending follow - up visits as part of an asthma program in clinics in the region of valparaso , regardless of smoking status ; having received a prescription for a bronchodilator and having been instructed on the proper use of their inhaler ( practical demonstration by a nurse , physician , or kinesiologist at each follow - up appointment ) ; and being able to self - administer inhaled medication . we excluded patients who had a respiratory comorbidity or any concomitant condition that could directly affect inhaler technique ( prostration , oxygen dependence , or altered cognitive status ) . all participants gave written informed consent , and the study was approved by the research ethics committee of the university of santo toms at via del mar school of kinesiology . for comparative purposes , the patients were divided into two groups : pediatric patients ( 5 - 18 years ) ; and adult patients ( 19 - 90 years ) . the volunteers were recruited during their follow - up appointments at the health facilities . on that occasion , they were scheduled to undergo assessment one week later . on the assessment day , they were asked to use their inhaler as usual . the medication administered was albuterol ( 100 g ; fesema(r ) ; laboratorio etex , santiago , chile ) , used as rescue medication and delivered with an mdi . inhaler technique was assessed using a protocol described by melani , 10 as shown in table 1 . this protocol documents the performance of ten essential inhaler technique steps by means of closed dichotomous response options ( well performed / poorly performed ) . all assessments were made by two investigators with ten years of experience in the follow - up of asthma patients . after assessment , all patients were given supplemental instruction on inhaler technique by a health professional , in the form of a demonstration . insert the inhaler into the spacer5 . hold the inhaler upright with the mouthpiece at the bottom during use6 . hold breath for 10 seconds on the basis of a study of inhaler technique in pediatric patients , 11 which reported an 89.1% completion rate for " hold breath for 10 seconds " , we calculated that , in order to achieve an alpha of 5% , a statistical power of 80% , and an estimation error of 6% , a sample size of at least 104 patients was required . allowing for a loss of 10% , we determined that the minimum sample size needed was 115 patients . for data analysis , we used descriptive statistics , calculating the number of errors per patient and the percentage of completion for each step of the protocol . differences between the percentages of errors made by each group were determined by the equivalence test for two proportions . we excluded seven patients , for the following reasons : prostration ( n = 2 ) ; oxygen - dependence ( n = 2 ) ; alzheimer 's disease ( n = 2 ) ; and sequelae of pulmonary tuberculosis ( n = 1 ) . the final sample therefore consisted of 263 patients : 135 pediatric patients and 128 adult patients . the general characteristics of the participants are shown in table 2 . in the pediatric group , the most well - represented age group was the 13- to 18-year group , with 63 patients , whereas the 61- to 75-year group was predominant , with 51 patients , in the adult group . characteristic age , years n male gender , % mean tobacco consumption , % age , years fev1 fvc fev1/fvc pediatric patients 5 - 6 887.56.0 0.582 20100 972 90.07 - 8 1376.97.0 0.581 1598 870 80.09 - 10 2147.69.0 0.581 799 770 90.011 - 12 3050.012.0 0.579 1499 1071 50.013 - 18 6347.614.0 0.579 19101 872 730.0adult patients 19 - 30 1258.323 0.778 10101 1871 825.031 - 45 650.034.0 0.682 9102 1569 1133.346 - 60 2548.051.0 0.680 1099 1970 728.061 - 75 5149.067.0 0.781 1298 1871 929.476 - 90 3452.979.0 0.579 1595 1569 914.7total263100.0 anumber of patients in each age group . the most common errors in the pediatric group were failing to execute a 10-s breath - hold after inhalation ( in 8.1% ) and failing to continue to inhale after actuation ( in 6.1% ) . in the adult group , 53.1% failed to exhale before using the inhaler , whereas 46% failed to execute a 10-s breath - hold after inhalation . table 3.frequency and percentages of inhaler technique errors observed in the pediatric and adult groups . type of error pediatric group adult group n % n % failing to exhale before using the inhaler53.76853.1failing to hold breath for 10 s118.1 * 5946.0*failing to take only 1 puff at a time43.03728.0failing to continue to inhale after actuation of the inhaler86.13526.5failing to actuate the inhaler in the first half of inhalation43.03022.7failing to shake the inhaler before use00.02518.9failing to inhale gently and deeply while actuating the inhaler43.01410.6failing to insert the inhaler into the spacer10.7118.6failing to hold the inhaler upright with the mouthpiece at the bottom during use00.021.5anumber of patients who made the error . p < 0.001 ( equivalence test for two proportions ) . number of patients who made the error . 0.001 ( equivalence test for two proportions ) . table 4 shows the frequency of correct and incorrect inhaler technique , by patient age group . in the 61- to 75- and 76- to 90-year age groups , the frequency of incorrect technique was greatest ( 48 and 35 patients , respectively ) . significant differences were found in the frequency of incorrect technique between the pediatric and adult groups . age ( years ) correct technique incorrect technique pediatric patientsnn5 - 6 537 - 8 1219 - 10 14711 - 12 191113 - 18 4914%73.426.6 adult patients 19 - 30 9331 - 45 0646 - 60 12461 - 75 34876 - 90 135%9.490.6 * p < 0.05 ( equivalence test for two proportions ) . the results of the present study show that most of the pediatric patients used correct inhaler techniques . the most common errors were failing to execute a 10-s breath - hold after inhalation ( in 8.1% ) and failing to continue inhaling after actuation of the device ( in 6.1% ) . among the adult patients , the most common errors were failing to exhale before using the inhaler ( in 53.1% ) and failing to execute a 10-s breath - hold after inhalation ( in 46% ) . 11 stated that poor inhaler technique in older patients might be due to cognitive impairment and their inability to retain the instructions received from the medical team . it is important to point out that , although the protocol used in our study is a guide for correct inhaler technique in adult patients , we found that pediatric patients appear to have better inhaler technique . 12 studies of inhaler technique have established that the most common errors are , in order of incidence , as follows : poor coordination between actuation and inhalation ; an insufficient breath - hold after inhalation ; excessive inhalation flow ; failing to shake the canister vigorously before use ; failing to continue to inhale after actuation ; pressing the canister down several times during the course of a single inhalation ; exhaling during actuation ; and failing to hold the inhaler upright . 13 other authors have observed that the rate of inhaler technique errors decreases when devices other than mdis are used . 14 however , drug delivery effectiveness is similar when inhaler technique is correct , 15 regardless of the device used . 6 10 under this criterion , the results of our study allow us to state that the most frequent error made by the pediatric patients ( made by 13 subjects ) would moderately affect drug deposition in the lung , whereas the most frequent error made by the adult patients ( made by 90 subjects ) would slightly affect this deposition . as for specific consequences , the poor inhaler technique observed in the two groups of patients can affect drug delivery to the distal airways and prevent drug deposition on the respiratory epithelium . the clinical implication of these results is that , in patients with poor inhaler technique , there is a waste of inhaled medication . consequently , there would be an increase in the economic costs associated with the disease , an increase in the risk of side effects , and a reduction in treatment effectiveness . 18 all participants in our investigation regularly attend their follow - up appointments , and , at each such opportunity , they are instructed in the proper use of their medications . these mistakes are considered either unintentional ( patients not noticing that their inhaler technique is poor ) or intentional ( patients knowingly using the incorrect inhaler technique ) . 18 in addition , there is clear evidence that inhalers are underused among asthma patients . 19 in this aspect , one of the limitations of our study was that we did not delve into the causes of the observed errors , which would have made it possible to provide each patient with specific supplemental instruction on the proper administration of the medication . in our sample , we found that approximately 30% of the adult patients were smokers . although this proportion is lower than observed among adults in chile ( 40.6% ) , 20 it is significant , given that smoking affects asthma control . 21 therefore , tobacco consumption in these patients would further increase the difficulty in controlling the disease . recent studies suggest that better results would be obtained by tailoring inhaler prescription to suit the characteristics and functional capabilities of each patient . 22 it has been observed that even individuals with correct inhaler technique can make errors if they are reassessed over time , 23 which makes it mandatory to provide patients with ongoing education in the administration of inhaled medications . many times there are factors that hinder this learning process , such as limited duration of appointments , a lack of knowledge on the part of health care personnel about the correct steps of the inhaler technique , and the technical language used in teaching the technique . 16 therefore , it is necessary to use new methods to provide patients with supplemental instruction on correct inhaler use , such as videos or illustrative leaflets that can promote the retention of information by patients . 24 in addition , it is necessary that supplemental instruction on inhaler use protocols be properly provided to health personnel and included in asthma clinical guidelines , which rarely address the administration of inhaled medications . 25 in conclusion , we found that most pediatric asthma patients appear to have correct inhaler technique . however , the same does not seem to be true for approximately 90% of adult patients , among whom the most common error was failing to exhale before using the inhaler . we suggest that asthma patients , especially those who are older , should be given supplemental instruction on inhaler technique through the use of new methods , so that they can administer their medications properly . la tcnica inhalatoria es un conjunto de procedimientos mediante el cual se administra un frmaco al sistema respiratorio . se caracteriza por ser utilizada como primera lnea para tratar las enfermedades pulmonares . su correcta ejecucin garantiza un mayor depsito del frmaco en la va area distal , optimizando sus efectos teraputicos y disminuyendo los efectos secundarios . los objetivos de este estudio son describir la ejecucin de la tcnica inhalatoria en un grupo de pacientes asmticos peditricos versus un grupo de pacientes asmticos adultos , definir los errores ms comunes en cada grupo de pacientes y comparar los resultados entre ambos grupos . se evalu la tcnica inhalatoria segn un protocolo de diez pasos en 135 pacientes asmticos peditricos y 128 pacientes asmticos adultos . se encontr que el error ms comn en los pacientes peditricos fue no realizar una apnea de 10 s despus de la inhalacin , mientras que en los pacientes adultos el principal error fue no exhalar completamente antes de aplicar el inhalador . se determin que los pacientes asmticos peditricos cumplen con la mayora de los pasos para una correcta tcnica inhalatoria , lo que no se observa en los pacientes adultos . la tcnica inhalatoria ( ti ) es un conjunto de procedimientos mediante el cual se administra un frmaco al sistema respiratorio . se caracteriza por ser utilizada como primera lnea para tratar las enfermedades respiratorias , siendo el inhalador de dosis medida ( idm ) uno de los dispositivos comnmente utilizados por los pacientes . 1 el uso de medicamentos inhalados ofrece muchas ventajas , ya que ellos actan directamente en la va area y requieren menor dosis en su administracin , con ausencia de alteraciones a nivel gstrico . 2 la correcta ejecucin de la ti permite un mayor depsito de partculas en la va area distal , mejorando la eficiencia del frmaco y disminuyendo los posibles efectos adversos . uno de los factores determinantes de la eficiencia del medicamento inhalado es la manera en la que el paciente realiza la ti . 3 para algunos pacientes puede resultar difcil la ejecucin de sta , por lo que al momento de prescribir el medicamento debe acompaarse siempre de un adecuado entrenamiento en la ti por parte de un profesional . de esta manera se logra reducir el nmero de errores cometidos durante la ejecucin de la ti y aminorar las consecuencias clnicas de una mala administracin . los primeros aerosoles teraputicos fueron creados durante la dcada de 1950 , 4 que consistieron en nebulizadores y atomizadores con frmacos anticolinrgicos para tratar el asma . 5 a pesar del tiempo transcurrido desde su creacin y su amplia utilizacin , los errores en la ti siguen siendo comunes entre los pacientes respiratorios , 6 menoscabando los beneficios del medicamento inhalado . en chile , sols et al . 7 observaron que slo un 12,5% de las madres de lactantes hospitalizados ejecuta correctamente la ti . sin embargo , se desconoce si esta tendencia se mantiene en los pacientes adultos , considerando que en la tercera edad existe un riesgo ms alto de cometer errores en la ti . 8 este hecho motiva a investigar de qu manera realizan la ti los pacientes segn grupo etario , considerando que al adaptar la educacin a las necesidades de cada paciente podra mejorar considerablemente el manejo de la enfermedad . los objetivos del presente estudio son describir la ejecucin de la ti en un grupo de pacientes asmticos peditricos versus un grupo de pacientes asmticos adultos , definir los errores ms comunes en cada grupo de pacientes y comparar los resultados entre ambos grupos . estudio de tipo descriptivo transversal , realizado en la regin de valparaso , chile , entre los meses de marzo y mayo del ao 2014 . la muestra estuvo constituida por pacientes asmticos de ambos sexos diagnosticados mediante espirometra segn los criterios establecidos por la global initiative for asthma ( gina ) . 9 las edades de los participantes estuvieron comprendidas entre 5 y 90 aos , quienes fueron seleccionados de forma no probabilstica ( intencional ) . los pacientes debieron cumplir los siguientes criterios de inclusin : pacientes asmticos inscritos y controlados en consultorios pertenecientes a la regin de valparaso , fumadores o no fumadores , con indicacin mdica de broncodilatador e instruidos previamente en el uso correcto de su inhalador ( demostracin prctica en cada control por parte de la enfermera , mdico o kinesilogo ) , capaces de auto realizarse la ti . se excluyeron pacientes que tuviesen comorbilidad respiratoria o alguna condicin asociada que interfiera directamente con la ejecucin de la ti ( postracin , dependencia de oxgeno , estado cognitivo alterado ) . todos los participantes firmaron un consentimiento informado aprobado por el comit de tica perteneciente a la escuela de kinesiologa de la universidad santo toms , sede via del mar . para efectos comparativos se dividieron en dos grupos : pacientes peditricos ( 5 - 18 aos ) y pacientes adultos ( 19 - 90 aos ) . los voluntarios fueron reclutados cuando asistieron a sus controles en sus respectivos centros de salud . en esa oportunidad se les cit a evaluacin una semana despus . el da de evaluacin se les solicit que ejecutaran la ti de la manera habitual . todos los voluntarios utilizaron su espaciador personal con vlvulas ( adecuado segn la edad del paciente ) . el medicamento administrado correspondi al idm que usaban de rescate ( salbutamol , 100 g ; fesema ; laboratorio etex , santiago , chile ) . durante la evaluacin , se observ la ejecucin de la ti segn el protocolo descrito por melani , como se muestra en la tabla 1 . este protocolo registra la realizacin de los diez pasos fundamentales de la ti mediante preguntas cerradas con respuesta dicotmica ( bien ejecutado / mal ejecutado ) . todas las observaciones fueron realizadas por dos evaluadores , con diez aos de experiencia en control de pacientes asmticos . realizar una apnea de 10 segundos posterior a la evaluacin , todos los pacientes fueron reforzados en la correcta ejecucin de la ti , mediante una demostracin por parte del profesional de salud . basados en un estudio de ti en pacientes peditricos 11 en el que se reporta un 89,1% de cumplimiento para el tem " realizar apnea de 10 segundos " , con un alfa del 5% , una potencia estadstica del 80% y un error de estimacin del 6% , se calcul un tamao muestral necesario para el presente estudio de al menos 104 pacientes . considerando un 10% de prdida , se estim un tamao mnimo muestral de 115 pacientes . para el anlisis de los datos se utiliz estadstica descriptiva , calculando el nmero de errores por cada paciente y el porcentaje de cumplimiento de cada paso del protocolo . las diferencias entre los porcentajes de errores cometidos por cada grupo se obtuvieron mediante el test de comparacin de dos proporciones . el nmero total de pacientes seleccionados fue de 270 . se excluyeron dos pacientes postrados , dos pacientes oxgeno dependientes , dos pacientes con diagnstico de alzheimer y un paciente con secuelas de tuberculosis pulmonar . las caractersticas generales de los participantes se observan en la tabla 2 . en el grupo peditrico el mayor nmero de sujetos ( n = 63 ) corresponde a pacientes con edades entre 13 y 18 aos . en el grupo de pacientes adultos el mayor nmero de sujetos ( n = 51 ) se encuentra entre los 61 y 75 aos . caractersticas edad , aos n sexo masculino , % promedios consumo de tabaco , % edad , aos vef1 cvf vef1/cvf pacientes peditricos 5 - 6 887,56,0 0,582 20100 972 90,07 - 8 1376,97,0 0,581 1598 870 80,09 - 10 2147,69,0 0,581 799 770 90,011 - 12 3050,012,0 0,579 1499 1071 50,013 - 18 6347,614,0 0,579 19101 872 730,0pacientes adultos 19 - 30 1258,323 0,778 10101 1871 825,031 - 45 650,034,0 0,682 9102 1569 1133,346 - 60 2548,051,0 0,680 1099 1970 728,061 - 75 5149,067,0 0,781 1298 1871 929,476 - 90 3452,979,0 0,579 1595 1569 914,7total263100,0 anmero de pacientes segn rango de edad . valores expresados en media de . porcentaje del valor predicho . en la tabla 3 , se observan los tipos de errores cometidos por el grupo de pacientes peditricos y el grupo de pacientes adultos . los errores ms comunes en el grupo de pacientes peditricos fueron no realizar la apnea de 10 segundos despus de inhalar ( en 8,1% ) y no continuar inhalando despus de pulsar el idm ( en 6,1% ) . en el caso de los pacientes adultos se observa que un 53,1% no exhala antes de aplicar su inhalador , mientras que un 46% no realiza una apnea de 10 segundos posterior a la inhalacin . tabla 3.frecuencia y porcentajes de los errores en la tcnica inhalatoria observados en el grupo de pacientes peditricos y en el grupo de pacientes adultos . tipo de error grupo peditrico grupo adulto n % n % no exhalar antes de aplicar el inhalador53,76853,1no realizar apnea de 10 segundos118,1 * 5946,0*no administrar slo 1 puff a la vez43,03728,0no continuar inhalando despus de activar el inhalador86,13526,5no activar el inhalador en la primera mitad de la inhalacin43,03022,7no agitar el inhalador antes de usarlo00,02518,9no inhalar suave y profundamente mientras activa el inhalador43,01410,6no posicionar correctamente la aerocmara10,7118,6no sostener el inhalador vertical , con la boquilla hacia abajo durante el uso00,021,5anmero de pacientes que cometieron el error sealado . * p < 0,001 ( test de comparacin de dos proporciones ) . en la tabla 4 se observa la frecuencia de entre los rangos de 61 a 75 y de 76 a 90 aos de edad se observan la mayor cantidad de maniobras incorrectas de inhalacin ( 48 y 35 maniobras , respectivamente ) . se verificaron diferencias significativas entre el porcentaje de ejecuciones incorrectas de los grupos peditrico y adulto . edad ( aos ) ejecucin correcta ejecucin incorrecta pacientes peditricosnn5 - 6 537 - 8 1219 - 10 14711 - 12 191113 - 18 4914%73,426,6 * pacientes adultos 19 - 30 9331 - 45 0646 - 60 12461 - 75 34876 - 90 135%9,490,6 * * p < 0,05 ( test de comparacin de dos proporciones ) . los resultados obtenidos del estudio muestran que la mayora de los pacientes peditricos ejecutan la ti de manera correcta . los errores ms comunes fueron no realizar la apnea de 10 segundos ( en 8,1% ) y no continuar inhalando despus de activar el dispositivo ( en 6,1% ) . entre los pacientes adultos , los errores ms comunes fueron no exhalar antes de activar el inhalador ( en 53,1% ) y no realizar una apnea de 10 segundos despus de la inhalacin ( en 46% ) . crompton et al.relatan que la deficiente calidad de la ti observada en los pacientes mayores puede estar determinada por un deterioro cognitivo y su incapacidad para retener las instrucciones recibidas desde el equipo mdico . es importante indicar que el protocolo utilizado en nuestro estudio est orientando a la correcta inhalacin del paciente adulto , y a pesar de ello se observa que los pacientes peditricos son los que mejor ejecutan la ti . 12 estudios sobre la ti establecen que los errores ms comunes por orden de incidencia son : mala coordinacin entre pulsacin del dispositivo e inspiracin ; periodos de apnea tras la maniobra demasiados cortos ; flujo inspiratorio excesivo ; no agitar bien el cartucho antes de usarlo ; interrumpir la inhalacin despus de la activacin ; presionar el cartucho varias veces durante una nica maniobra respiratoria ; exhalar durante el disparo y no colocar el inhalador en posicin vertical . 13 otros estudios han observado que la tasa de errores en la ti disminuye al utilizar un dispositivo distinto al idm . 14 sin embargo , la eficacia en la entrega del medicamento es similar cuando la ti se ejecuta correctamente , 15 independiente del dispositivo utilizado . una mala ejecucin de la ti trae consigo consecuencias clnicas que van desde pequeas hasta crticas . 6 10 bajo este criterio , los resultados observados en nuestro estudio permiten afirmar que la mayor frecuencia ( 13 sujetos ) de los errores cometidos por el grupo de pacientes peditricos incidira moderadamente en el depsito del medicamento en el pulmn , mientras que en los pacientes adultos la mayor frecuencia ( 90 sujetos ) de los errores cometidos incidira levemente en este depsito . respecto a las consecuencias especficas , la mala ti observada en ambos grupos de pacientes puede afectar el ingreso del medicamento a la va area distal e impedir su adherencia al epitelio respiratorio . 16 las implicancias clnicas de estos resultados indican que en los pacientes con mala ti se produce un desperdicio del medicamento inhalado . en consecuencia aumentara el costo econmico asociado a la enfermedad , incrementara el riesgo de sufrir efectos secundarios y disminuira la efectividad del tratamiento . la educacin de los pacientes respiratorios es un factor crtico en el correcto uso de sus frmacos . 17 los programas de educacin al paciente asmtico mejoran sustancialmente la adherencia y la ti . 18 todos los participantes de nuestra investigacin asisten regularmente a sus controles de salud y en cada oportunidad se les enfatiza el uso correcto de sus medicamentos . a pesar de ello , los resultados muestran que persisten errores en la utilizacin del inhalador . estas equivocaciones se consideran involuntarias , cuando el paciente no advierte que la ti est mal ejecutada , o intencionales , cuando el paciente conoce la ti correcta pero no la ejecuta por omisin . 18 adems , existe evidencia de una clara subutilizacin de estos dispositivos entre los pacientes asmticos . 19 en ese aspecto , una de las limitaciones de nuestro estudio fue no profundizar en las causas de los errores observados , lo que hubiese permitido reforzar de manera ms especfica en cada paciente la correcta administracin del medicamento . a pesar que esta cifra es inferior al porcentaje de poblacin adulta fumadora en chile ( 40,6% ) , 20 es un porcentaje importante de pacientes considerando que el hbito tabquico interfiere en el control del asma . 21 por lo anterior , el consumo de tabaco en estos pacientes agregara una mayor dificultad al control de la enfermedad . estudios recientes sugieren que se obtendran mejores resultados si la prescripcin de inhaladores se ajustara a cada paciente segn sus caractersticas y capacidades funcionales . 22 se ha observado que incluso personas que realizan correctamente la ti pueden cometer errores nuevamente al ser reevaluadas en una segunda instancia , 23 lo que obliga a educar constantemente a nuestros pacientes en la administracin de sus medicamentos inhalados . existen factores que muchas veces dificultan este proceso de aprendizaje , entre ellos el escaso tiempo que existe para atender a cada paciente , el desconocimiento de los pasos correctos de la ti por parte del personal de salud y el lenguaje tcnico que se utiliza para ensear . 16 por lo anterior , es necesario utilizar nuevas metodologas para reforzar el correcto uso de los inhaladores , por ejemplo videos o folletos ilustrativos que promuevan la retencin de informacin en el paciente , 24 adems de reforzar el protocolo correctamente en el personal de salud y en las guas clnicas de asma que escasamente hacen referencia a la administracin de medicamentos inhalados . 25 como conclusin se determin que la mayora de los pacientes asmticos peditricos realiza la ti de manera correcta , mientras que aproximadamente el 90% de los pacientes adultos la ejecuta de manera incorrecta , siendo el error ms comn no exhalar antes de aplicar el inhalador . se sugiere reforzar en los pacientes asmticos , especialmente en los de mayor edad , la ti a travs de nuevos mtodos para lograr una correcta administracin de sus medicamentos .

objective : inhaler technique comprises a set of procedures for drug delivery to the respiratory system . the oral inhalation of medications is the first - line treatment for lung diseases . using the proper inhaler technique ensures sufficient drug deposition in the distal airways , optimizing therapeutic effects and reducing side effects . the purposes of this study were to assess inhaler technique in pediatric and adult patients with asthma ; to determine the most common errors in each group of patients ; and to compare the results between the two groups . methods : this was a descriptive cross - sectional study . using a ten - step protocol , we assessed inhaler technique in 135 pediatric asthma patients and 128 adult asthma patients . results : the most common error among the pediatric patients was failing to execute a 10-s breath - hold after inhalation , whereas the most common error among the adult patients was failing to exhale fully before using the inhaler . conclusions : pediatric asthma patients appear to perform most of the inhaler technique steps correctly . however , the same does not seem to be true for adult patients .

INTRODUCTION METHODS RESULTS DISCUSSION Objetivo: Mtodos: Resultados: Conclusiones: INTRODUCCIN MTODOS RESULTADOS DISCUSIN

2 using the proper inhaler technique ensures sufficient deposition of drug particles in the distal airways , optimizing drug effectiveness and reducing possible side effects . one of the determinants of the effectiveness of inhaled medications is the ability of the patient to adhere to good inhaler techniques . 3 for some patients , that can be difficult , and the prescription of medication should therefore always be accompanied by appropriate inhaler technique training delivered by a health professional . thus , it is possible to reduce the number of inhaler technique errors and minimize the clinical consequences of poor drug delivery . the first therapeutic aerosol devices were developed in the 1950s 4 and consisted of nebulizers and atomizers containing anticholinergics for treatment of asthma . 5 despite the long time since development and the wide use of these devices , inhaler technique errors continue to be common among respiratory patients , 6 reducing the benefits of inhaled medications . in chile , 7 observed that only 12.5% of the mothers of hospitalized infants have a correct inhaler technique . however , it is unknown whether this trend is reflected in adult patients , given that the elderly are more likely to make inhaler technique errors . 8 this promotes the assessment of inhaler technique by age group , because tailoring education to the needs of each patient could significantly improve disease management . the purposes of the present study were to assess inhaler technique in pediatric and adult patients with asthma ; to determine the most common errors in each group of patients ; and to compare the results between the two groups . this was a descriptive cross - sectional study conducted in the region of valparaso , chile , between march and may of 2014 . the sample consisted of male and female patients with a diagnosis of asthma based on spirometry , in accordance with the global initiative for asthma criteria . 9 the ages of the participants ranged from 5 to 90 years , and the sampling was non - probabilistic ( purposive ) . patients had to meet the following inclusion criteria : being enrolled in and attending follow - up visits as part of an asthma program in clinics in the region of valparaso , regardless of smoking status ; having received a prescription for a bronchodilator and having been instructed on the proper use of their inhaler ( practical demonstration by a nurse , physician , or kinesiologist at each follow - up appointment ) ; and being able to self - administer inhaled medication . we excluded patients who had a respiratory comorbidity or any concomitant condition that could directly affect inhaler technique ( prostration , oxygen dependence , or altered cognitive status ) . all participants gave written informed consent , and the study was approved by the research ethics committee of the university of santo toms at via del mar school of kinesiology . for comparative purposes , the patients were divided into two groups : pediatric patients ( 5 - 18 years ) ; and adult patients ( 19 - 90 years ) . on that occasion , they were scheduled to undergo assessment one week later . on the assessment day , they were asked to use their inhaler as usual . inhaler technique was assessed using a protocol described by melani , 10 as shown in table 1 . this protocol documents the performance of ten essential inhaler technique steps by means of closed dichotomous response options ( well performed / poorly performed ) . all assessments were made by two investigators with ten years of experience in the follow - up of asthma patients . after assessment , all patients were given supplemental instruction on inhaler technique by a health professional , in the form of a demonstration . insert the inhaler into the spacer5 . hold the inhaler upright with the mouthpiece at the bottom during use6 . hold breath for 10 seconds on the basis of a study of inhaler technique in pediatric patients , 11 which reported an 89.1% completion rate for " hold breath for 10 seconds " , we calculated that , in order to achieve an alpha of 5% , a statistical power of 80% , and an estimation error of 6% , a sample size of at least 104 patients was required . allowing for a loss of 10% , we determined that the minimum sample size needed was 115 patients . for data analysis , we used descriptive statistics , calculating the number of errors per patient and the percentage of completion for each step of the protocol . differences between the percentages of errors made by each group were determined by the equivalence test for two proportions . we excluded seven patients , for the following reasons : prostration ( n = 2 ) ; oxygen - dependence ( n = 2 ) ; alzheimer 's disease ( n = 2 ) ; and sequelae of pulmonary tuberculosis ( n = 1 ) . the final sample therefore consisted of 263 patients : 135 pediatric patients and 128 adult patients . the general characteristics of the participants are shown in table 2 . in the pediatric group , the most well - represented age group was the 13- to 18-year group , with 63 patients , whereas the 61- to 75-year group was predominant , with 51 patients , in the adult group . characteristic age , years n male gender , % mean tobacco consumption , % age , years fev1 fvc fev1/fvc pediatric patients 5 - 6 887.56.0 0.582 20100 972 90.07 - 8 1376.97.0 0.581 1598 870 80.09 - 10 2147.69.0 0.581 799 770 90.011 - 12 3050.012.0 0.579 1499 1071 50.013 - 18 6347.614.0 0.579 19101 872 730.0adult patients 19 - 30 1258.323 0.778 10101 1871 825.031 - 45 650.034.0 0.682 9102 1569 1133.346 - 60 2548.051.0 0.680 1099 1970 728.061 - 75 5149.067.0 0.781 1298 1871 929.476 - 90 3452.979.0 0.579 1595 1569 914.7total263100.0 anumber of patients in each age group . the most common errors in the pediatric group were failing to execute a 10-s breath - hold after inhalation ( in 8.1% ) and failing to continue to inhale after actuation ( in 6.1% ) . in the adult group , 53.1% failed to exhale before using the inhaler , whereas 46% failed to execute a 10-s breath - hold after inhalation . table 3.frequency and percentages of inhaler technique errors observed in the pediatric and adult groups . type of error pediatric group adult group n % n % failing to exhale before using the inhaler53.76853.1failing to hold breath for 10 s118.1 * 5946.0*failing to take only 1 puff at a time43.03728.0failing to continue to inhale after actuation of the inhaler86.13526.5failing to actuate the inhaler in the first half of inhalation43.03022.7failing to shake the inhaler before use00.02518.9failing to inhale gently and deeply while actuating the inhaler43.01410.6failing to insert the inhaler into the spacer10.7118.6failing to hold the inhaler upright with the mouthpiece at the bottom during use00.021.5anumber of patients who made the error . p < 0.001 ( equivalence test for two proportions ) . number of patients who made the error . table 4 shows the frequency of correct and incorrect inhaler technique , by patient age group . in the 61- to 75- and 76- to 90-year age groups , the frequency of incorrect technique was greatest ( 48 and 35 patients , respectively ) . significant differences were found in the frequency of incorrect technique between the pediatric and adult groups . age ( years ) correct technique incorrect technique pediatric patientsnn5 - 6 537 - 8 1219 - 10 14711 - 12 191113 - 18 4914%73.426.6 adult patients 19 - 30 9331 - 45 0646 - 60 12461 - 75 34876 - 90 135%9.490.6 * p < 0.05 ( equivalence test for two proportions ) . the results of the present study show that most of the pediatric patients used correct inhaler techniques . the most common errors were failing to execute a 10-s breath - hold after inhalation ( in 8.1% ) and failing to continue inhaling after actuation of the device ( in 6.1% ) . among the adult patients , the most common errors were failing to exhale before using the inhaler ( in 53.1% ) and failing to execute a 10-s breath - hold after inhalation ( in 46% ) . 11 stated that poor inhaler technique in older patients might be due to cognitive impairment and their inability to retain the instructions received from the medical team . it is important to point out that , although the protocol used in our study is a guide for correct inhaler technique in adult patients , we found that pediatric patients appear to have better inhaler technique . 12 studies of inhaler technique have established that the most common errors are , in order of incidence , as follows : poor coordination between actuation and inhalation ; an insufficient breath - hold after inhalation ; excessive inhalation flow ; failing to shake the canister vigorously before use ; failing to continue to inhale after actuation ; pressing the canister down several times during the course of a single inhalation ; exhaling during actuation ; and failing to hold the inhaler upright . 13 other authors have observed that the rate of inhaler technique errors decreases when devices other than mdis are used . 14 however , drug delivery effectiveness is similar when inhaler technique is correct , 15 regardless of the device used . 6 10 under this criterion , the results of our study allow us to state that the most frequent error made by the pediatric patients ( made by 13 subjects ) would moderately affect drug deposition in the lung , whereas the most frequent error made by the adult patients ( made by 90 subjects ) would slightly affect this deposition . as for specific consequences , the poor inhaler technique observed in the two groups of patients can affect drug delivery to the distal airways and prevent drug deposition on the respiratory epithelium . the clinical implication of these results is that , in patients with poor inhaler technique , there is a waste of inhaled medication . consequently , there would be an increase in the economic costs associated with the disease , an increase in the risk of side effects , and a reduction in treatment effectiveness . 18 all participants in our investigation regularly attend their follow - up appointments , and , at each such opportunity , they are instructed in the proper use of their medications . these mistakes are considered either unintentional ( patients not noticing that their inhaler technique is poor ) or intentional ( patients knowingly using the incorrect inhaler technique ) . 18 in addition , there is clear evidence that inhalers are underused among asthma patients . 19 in this aspect , one of the limitations of our study was that we did not delve into the causes of the observed errors , which would have made it possible to provide each patient with specific supplemental instruction on the proper administration of the medication . in our sample , we found that approximately 30% of the adult patients were smokers . although this proportion is lower than observed among adults in chile ( 40.6% ) , 20 it is significant , given that smoking affects asthma control . 22 it has been observed that even individuals with correct inhaler technique can make errors if they are reassessed over time , 23 which makes it mandatory to provide patients with ongoing education in the administration of inhaled medications . many times there are factors that hinder this learning process , such as limited duration of appointments , a lack of knowledge on the part of health care personnel about the correct steps of the inhaler technique , and the technical language used in teaching the technique . 16 therefore , it is necessary to use new methods to provide patients with supplemental instruction on correct inhaler use , such as videos or illustrative leaflets that can promote the retention of information by patients . 25 in conclusion , we found that most pediatric asthma patients appear to have correct inhaler technique . however , the same does not seem to be true for approximately 90% of adult patients , among whom the most common error was failing to exhale before using the inhaler . we suggest that asthma patients , especially those who are older , should be given supplemental instruction on inhaler technique through the use of new methods , so that they can administer their medications properly . se caracteriza por ser utilizada como primera lnea para tratar las enfermedades pulmonares . los objetivos de este estudio son describir la ejecucin de la tcnica inhalatoria en un grupo de pacientes asmticos peditricos versus un grupo de pacientes asmticos adultos , definir los errores ms comunes en cada grupo de pacientes y comparar los resultados entre ambos grupos . uno de los factores determinantes de la eficiencia del medicamento inhalado es la manera en la que el paciente realiza la ti . de esta manera se logra reducir el nmero de errores cometidos durante la ejecucin de la ti y aminorar las consecuencias clnicas de una mala administracin . en chile , sols et al . sin embargo , se desconoce si esta tendencia se mantiene en los pacientes adultos , considerando que en la tercera edad existe un riesgo ms alto de cometer errores en la ti . para efectos comparativos se dividieron en dos grupos : pacientes peditricos ( 5 - 18 aos ) y pacientes adultos ( 19 - 90 aos ) . en esa oportunidad se les cit a evaluacin una semana despus . el medicamento administrado correspondi al idm que usaban de rescate ( salbutamol , 100 g ; fesema ; laboratorio etex , santiago , chile ) . basados en un estudio de ti en pacientes peditricos 11 en el que se reporta un 89,1% de cumplimiento para el tem " realizar apnea de 10 segundos " , con un alfa del 5% , una potencia estadstica del 80% y un error de estimacin del 6% , se calcul un tamao muestral necesario para el presente estudio de al menos 104 pacientes . para el anlisis de los datos se utiliz estadstica descriptiva , calculando el nmero de errores por cada paciente y el porcentaje de cumplimiento de cada paso del protocolo . en el grupo peditrico el mayor nmero de sujetos ( n = 63 ) corresponde a pacientes con edades entre 13 y 18 aos . en el grupo de pacientes adultos el mayor nmero de sujetos ( n = 51 ) se encuentra entre los 61 y 75 aos . valores expresados en media de . porcentaje del valor predicho . en la tabla 3 , se observan los tipos de errores cometidos por el grupo de pacientes peditricos y el grupo de pacientes adultos . en el caso de los pacientes adultos se observa que un 53,1% no exhala antes de aplicar su inhalador , mientras que un 46% no realiza una apnea de 10 segundos posterior a la inhalacin . tabla 3.frecuencia y porcentajes de los errores en la tcnica inhalatoria observados en el grupo de pacientes peditricos y en el grupo de pacientes adultos . en la tabla 4 se observa la frecuencia de entre los rangos de 61 a 75 y de 76 a 90 aos de edad se observan la mayor cantidad de maniobras incorrectas de inhalacin ( 48 y 35 maniobras , respectivamente ) . se verificaron diferencias significativas entre el porcentaje de ejecuciones incorrectas de los grupos peditrico y adulto . es importante indicar que el protocolo utilizado en nuestro estudio est orientando a la correcta inhalacin del paciente adulto , y a pesar de ello se observa que los pacientes peditricos son los que mejor ejecutan la ti . una mala ejecucin de la ti trae consigo consecuencias clnicas que van desde pequeas hasta crticas . 6 10 bajo este criterio , los resultados observados en nuestro estudio permiten afirmar que la mayor frecuencia ( 13 sujetos ) de los errores cometidos por el grupo de pacientes peditricos incidira moderadamente en el depsito del medicamento en el pulmn , mientras que en los pacientes adultos la mayor frecuencia ( 90 sujetos ) de los errores cometidos incidira levemente en este depsito . a pesar de ello , los resultados muestran que persisten errores en la utilizacin del inhalador . 19 en ese aspecto , una de las limitaciones de nuestro estudio fue no profundizar en las causas de los errores observados , lo que hubiese permitido reforzar de manera ms especfica en cada paciente la correcta administracin del medicamento . existen factores que muchas veces dificultan este proceso de aprendizaje , entre ellos el escaso tiempo que existe para atender a cada paciente , el desconocimiento de los pasos correctos de la ti por parte del personal de salud y el lenguaje tcnico que se utiliza para ensear .

2 using the proper inhaler technique ensures sufficient deposition of drug particles in the distal airways , optimizing drug effectiveness and reducing possible side effects . one of the determinants of the effectiveness of inhaled medications is the ability of the patient to adhere to good inhaler techniques . 3 for some patients , that can be difficult , and the prescription of medication should therefore always be accompanied by appropriate inhaler technique training delivered by a health professional . thus , it is possible to reduce the number of inhaler technique errors and minimize the clinical consequences of poor drug delivery . the first therapeutic aerosol devices were developed in the 1950s 4 and consisted of nebulizers and atomizers containing anticholinergics for treatment of asthma . 5 despite the long time since development and the wide use of these devices , inhaler technique errors continue to be common among respiratory patients , 6 reducing the benefits of inhaled medications . in chile , 7 observed that only 12.5% of the mothers of hospitalized infants have a correct inhaler technique . however , it is unknown whether this trend is reflected in adult patients , given that the elderly are more likely to make inhaler technique errors . 8 this promotes the assessment of inhaler technique by age group , because tailoring education to the needs of each patient could significantly improve disease management . the purposes of the present study were to assess inhaler technique in pediatric and adult patients with asthma ; to determine the most common errors in each group of patients ; and to compare the results between the two groups . this was a descriptive cross - sectional study conducted in the region of valparaso , chile , between march and may of 2014 . the sample consisted of male and female patients with a diagnosis of asthma based on spirometry , in accordance with the global initiative for asthma criteria . 9 the ages of the participants ranged from 5 to 90 years , and the sampling was non - probabilistic ( purposive ) . patients had to meet the following inclusion criteria : being enrolled in and attending follow - up visits as part of an asthma program in clinics in the region of valparaso , regardless of smoking status ; having received a prescription for a bronchodilator and having been instructed on the proper use of their inhaler ( practical demonstration by a nurse , physician , or kinesiologist at each follow - up appointment ) ; and being able to self - administer inhaled medication . we excluded patients who had a respiratory comorbidity or any concomitant condition that could directly affect inhaler technique ( prostration , oxygen dependence , or altered cognitive status ) . for comparative purposes , the patients were divided into two groups : pediatric patients ( 5 - 18 years ) ; and adult patients ( 19 - 90 years ) . the medication administered was albuterol ( 100 g ; fesema(r ) ; laboratorio etex , santiago , chile ) , used as rescue medication and delivered with an mdi . inhaler technique was assessed using a protocol described by melani , 10 as shown in table 1 . this protocol documents the performance of ten essential inhaler technique steps by means of closed dichotomous response options ( well performed / poorly performed ) . all assessments were made by two investigators with ten years of experience in the follow - up of asthma patients . after assessment , all patients were given supplemental instruction on inhaler technique by a health professional , in the form of a demonstration . hold breath for 10 seconds on the basis of a study of inhaler technique in pediatric patients , 11 which reported an 89.1% completion rate for " hold breath for 10 seconds " , we calculated that , in order to achieve an alpha of 5% , a statistical power of 80% , and an estimation error of 6% , a sample size of at least 104 patients was required . allowing for a loss of 10% , we determined that the minimum sample size needed was 115 patients . for data analysis , we used descriptive statistics , calculating the number of errors per patient and the percentage of completion for each step of the protocol . we excluded seven patients , for the following reasons : prostration ( n = 2 ) ; oxygen - dependence ( n = 2 ) ; alzheimer 's disease ( n = 2 ) ; and sequelae of pulmonary tuberculosis ( n = 1 ) . in the pediatric group , the most well - represented age group was the 13- to 18-year group , with 63 patients , whereas the 61- to 75-year group was predominant , with 51 patients , in the adult group . characteristic age , years n male gender , % mean tobacco consumption , % age , years fev1 fvc fev1/fvc pediatric patients 5 - 6 887.56.0 0.582 20100 972 90.07 - 8 1376.97.0 0.581 1598 870 80.09 - 10 2147.69.0 0.581 799 770 90.011 - 12 3050.012.0 0.579 1499 1071 50.013 - 18 6347.614.0 0.579 19101 872 730.0adult patients 19 - 30 1258.323 0.778 10101 1871 825.031 - 45 650.034.0 0.682 9102 1569 1133.346 - 60 2548.051.0 0.680 1099 1970 728.061 - 75 5149.067.0 0.781 1298 1871 929.476 - 90 3452.979.0 0.579 1595 1569 914.7total263100.0 anumber of patients in each age group . the most common errors in the pediatric group were failing to execute a 10-s breath - hold after inhalation ( in 8.1% ) and failing to continue to inhale after actuation ( in 6.1% ) . in the adult group , 53.1% failed to exhale before using the inhaler , whereas 46% failed to execute a 10-s breath - hold after inhalation . table 3.frequency and percentages of inhaler technique errors observed in the pediatric and adult groups . type of error pediatric group adult group n % n % failing to exhale before using the inhaler53.76853.1failing to hold breath for 10 s118.1 * 5946.0*failing to take only 1 puff at a time43.03728.0failing to continue to inhale after actuation of the inhaler86.13526.5failing to actuate the inhaler in the first half of inhalation43.03022.7failing to shake the inhaler before use00.02518.9failing to inhale gently and deeply while actuating the inhaler43.01410.6failing to insert the inhaler into the spacer10.7118.6failing to hold the inhaler upright with the mouthpiece at the bottom during use00.021.5anumber of patients who made the error . in the 61- to 75- and 76- to 90-year age groups , the frequency of incorrect technique was greatest ( 48 and 35 patients , respectively ) . significant differences were found in the frequency of incorrect technique between the pediatric and adult groups . age ( years ) correct technique incorrect technique pediatric patientsnn5 - 6 537 - 8 1219 - 10 14711 - 12 191113 - 18 4914%73.426.6 adult patients 19 - 30 9331 - 45 0646 - 60 12461 - 75 34876 - 90 135%9.490.6 * p < 0.05 ( equivalence test for two proportions ) . the results of the present study show that most of the pediatric patients used correct inhaler techniques . the most common errors were failing to execute a 10-s breath - hold after inhalation ( in 8.1% ) and failing to continue inhaling after actuation of the device ( in 6.1% ) . among the adult patients , the most common errors were failing to exhale before using the inhaler ( in 53.1% ) and failing to execute a 10-s breath - hold after inhalation ( in 46% ) . 11 stated that poor inhaler technique in older patients might be due to cognitive impairment and their inability to retain the instructions received from the medical team . it is important to point out that , although the protocol used in our study is a guide for correct inhaler technique in adult patients , we found that pediatric patients appear to have better inhaler technique . 12 studies of inhaler technique have established that the most common errors are , in order of incidence , as follows : poor coordination between actuation and inhalation ; an insufficient breath - hold after inhalation ; excessive inhalation flow ; failing to shake the canister vigorously before use ; failing to continue to inhale after actuation ; pressing the canister down several times during the course of a single inhalation ; exhaling during actuation ; and failing to hold the inhaler upright . 14 however , drug delivery effectiveness is similar when inhaler technique is correct , 15 regardless of the device used . 6 10 under this criterion , the results of our study allow us to state that the most frequent error made by the pediatric patients ( made by 13 subjects ) would moderately affect drug deposition in the lung , whereas the most frequent error made by the adult patients ( made by 90 subjects ) would slightly affect this deposition . as for specific consequences , the poor inhaler technique observed in the two groups of patients can affect drug delivery to the distal airways and prevent drug deposition on the respiratory epithelium . the clinical implication of these results is that , in patients with poor inhaler technique , there is a waste of inhaled medication . consequently , there would be an increase in the economic costs associated with the disease , an increase in the risk of side effects , and a reduction in treatment effectiveness . 19 in this aspect , one of the limitations of our study was that we did not delve into the causes of the observed errors , which would have made it possible to provide each patient with specific supplemental instruction on the proper administration of the medication . although this proportion is lower than observed among adults in chile ( 40.6% ) , 20 it is significant , given that smoking affects asthma control . many times there are factors that hinder this learning process , such as limited duration of appointments , a lack of knowledge on the part of health care personnel about the correct steps of the inhaler technique , and the technical language used in teaching the technique . 16 therefore , it is necessary to use new methods to provide patients with supplemental instruction on correct inhaler use , such as videos or illustrative leaflets that can promote the retention of information by patients . 24 in addition , it is necessary that supplemental instruction on inhaler use protocols be properly provided to health personnel and included in asthma clinical guidelines , which rarely address the administration of inhaled medications . 25 in conclusion , we found that most pediatric asthma patients appear to have correct inhaler technique . however , the same does not seem to be true for approximately 90% of adult patients , among whom the most common error was failing to exhale before using the inhaler . we suggest that asthma patients , especially those who are older , should be given supplemental instruction on inhaler technique through the use of new methods , so that they can administer their medications properly . los objetivos de este estudio son describir la ejecucin de la tcnica inhalatoria en un grupo de pacientes asmticos peditricos versus un grupo de pacientes asmticos adultos , definir los errores ms comunes en cada grupo de pacientes y comparar los resultados entre ambos grupos . se encontr que el error ms comn en los pacientes peditricos fue no realizar una apnea de 10 s despus de la inhalacin , mientras que en los pacientes adultos el principal error fue no exhalar completamente antes de aplicar el inhalador . 1 el uso de medicamentos inhalados ofrece muchas ventajas , ya que ellos actan directamente en la va area y requieren menor dosis en su administracin , con ausencia de alteraciones a nivel gstrico . 5 a pesar del tiempo transcurrido desde su creacin y su amplia utilizacin , los errores en la ti siguen siendo comunes entre los pacientes respiratorios , 6 menoscabando los beneficios del medicamento inhalado . los objetivos del presente estudio son describir la ejecucin de la ti en un grupo de pacientes asmticos peditricos versus un grupo de pacientes asmticos adultos , definir los errores ms comunes en cada grupo de pacientes y comparar los resultados entre ambos grupos . los pacientes debieron cumplir los siguientes criterios de inclusin : pacientes asmticos inscritos y controlados en consultorios pertenecientes a la regin de valparaso , fumadores o no fumadores , con indicacin mdica de broncodilatador e instruidos previamente en el uso correcto de su inhalador ( demostracin prctica en cada control por parte de la enfermera , mdico o kinesilogo ) , capaces de auto realizarse la ti . basados en un estudio de ti en pacientes peditricos 11 en el que se reporta un 89,1% de cumplimiento para el tem " realizar apnea de 10 segundos " , con un alfa del 5% , una potencia estadstica del 80% y un error de estimacin del 6% , se calcul un tamao muestral necesario para el presente estudio de al menos 104 pacientes . caractersticas edad , aos n sexo masculino , % promedios consumo de tabaco , % edad , aos vef1 cvf vef1/cvf pacientes peditricos 5 - 6 887,56,0 0,582 20100 972 90,07 - 8 1376,97,0 0,581 1598 870 80,09 - 10 2147,69,0 0,581 799 770 90,011 - 12 3050,012,0 0,579 1499 1071 50,013 - 18 6347,614,0 0,579 19101 872 730,0pacientes adultos 19 - 30 1258,323 0,778 10101 1871 825,031 - 45 650,034,0 0,682 9102 1569 1133,346 - 60 2548,051,0 0,680 1099 1970 728,061 - 75 5149,067,0 0,781 1298 1871 929,476 - 90 3452,979,0 0,579 1595 1569 914,7total263100,0 anmero de pacientes segn rango de edad . tipo de error grupo peditrico grupo adulto n % n % no exhalar antes de aplicar el inhalador53,76853,1no realizar apnea de 10 segundos118,1 * 5946,0*no administrar slo 1 puff a la vez43,03728,0no continuar inhalando despus de activar el inhalador86,13526,5no activar el inhalador en la primera mitad de la inhalacin43,03022,7no agitar el inhalador antes de usarlo00,02518,9no inhalar suave y profundamente mientras activa el inhalador43,01410,6no posicionar correctamente la aerocmara10,7118,6no sostener el inhalador vertical , con la boquilla hacia abajo durante el uso00,021,5anmero de pacientes que cometieron el error sealado . edad ( aos ) ejecucin correcta ejecucin incorrecta pacientes peditricosnn5 - 6 537 - 8 1219 - 10 14711 - 12 191113 - 18 4914%73,426,6 * pacientes adultos 19 - 30 9331 - 45 0646 - 60 12461 - 75 34876 - 90 135%9,490,6 * * p < 0,05 ( test de comparacin de dos proporciones ) . 6 10 bajo este criterio , los resultados observados en nuestro estudio permiten afirmar que la mayor frecuencia ( 13 sujetos ) de los errores cometidos por el grupo de pacientes peditricos incidira moderadamente en el depsito del medicamento en el pulmn , mientras que en los pacientes adultos la mayor frecuencia ( 90 sujetos ) de los errores cometidos incidira levemente en este depsito . 16 por lo anterior , es necesario utilizar nuevas metodologas para reforzar el correcto uso de los inhaladores , por ejemplo videos o folletos ilustrativos que promuevan la retencin de informacin en el paciente , 24 adems de reforzar el protocolo correctamente en el personal de salud y en las guas clnicas de asma que escasamente hacen referencia a la administracin de medicamentos inhalados .

a man is as old as his arteries was a favorite axiom of william osler ( 18491919 ) , sometimes called the father of modern medicine , and to some extent accurately represents the effect of vascular dysfunction on various aging processes . to date , it has been recognized that arterial dysfunction , such as increased arterial stiffness , is closely associated with the pathogenesis of cardiovascular disease , which in turn increases mortality by increasing the risk of events such as myocardial infarction and stroke [ 24 ] . a higher physical activity level as well as regular exercise may be effective at diminishing the risk of coronary heart disease [ 5 , 6 ] and stroke [ 7 , 8 ] . from the standpoint of exercise physiology , exercise is categorized as aerobic and resistance exercise . briefly , aerobic exercise is a physical exercise of relatively low intensity that depends primarily on the aerobic energy - generating process , for example , running and leg cycling . in contrast , resistance exercise is also physical exercise of relatively moderate and higher intensity that uses a resistance to the force of muscular contraction , in other words , strength training . although aerobic exercise may improve arterial function , it has also been reported that aerobic exercise is insufficient to inhibit the loss in muscular strength that comes with advancing age [ 12 , 13 ] . resistance exercise is recommended to prevent sarcopenia , age - induced muscular degeneration which often entails reduced activities of daily living ( adls ) . according to the guideline of american college of sports medicine ( acsm ) , a mechanical load greater than 70% of the one - repetition maximum load ( 1 rm ) can produce morphological and functional muscular adaptations . however , these higher - load exercises are frequently associated with orthopedic complications [ 15 , 16 ] . in addition , it has been reported that high - intensity resistance training ( > 80% of 1 rm ) reduces central artery compliance . these findings suggest that such a high - intensity resistance exercise should be prescribed carefully , particularly for aged people and patients with cardiovascular disease . recently , several studies have demonstrated that low - intensity resistance exercise with blood flow restriction ( bfr ) [ 1723 ] and bfr walking dramatically leads to muscle hypertrophy and strength gain and that it results in adaptations equal to those of high - intensity resistance training . although the effect of resistance exercise with bfr and bfr walking on vascular function is still unclear , there is a possibility that this exercise modality can be an important therapeutic prescription not only for sarcopenia but also for vascular dysfunction because of the lower exercise intensity compared to high - intensity resistance training . in this review , we would like to focus on the impact of such exercise on vascular function in comparison with the effects of aerobic and resistance training alone and in combination . in human studies , as it is almost impossible to evaluate large arterial function directly , various noninvasive methodologies have been used to evaluate arterial function in humans . in this section , we introduced several methodologies , which have investigated the impact of bfr exercise and training . generally , arterial compliance can be measured by a combination of ultrasound imaging of any artery , for example , carotid artery , with simultaneous applanation of tonometrically obtained arterial pressure from the contralateral artery , permits noninvasive determination of arterial compliance . this methodology can be applied to any artery , which can measure pulse wave , for example , radial and femoral arteries . in addition to arterial compliance , -stiffness index provides an indicator of arterial compliance adjusted for distending pressure . arterial compliance decreases with advancing age [ 2729 ] , and these reductions are associated with isolated systolic hypertension , accompanied with left ventricular hypertrophy . indeed , several studies demonstrated that decreased arterial compliance and/or increased arterial stiffness have been identified as independent risk factors of cardiovascular disease [ 27 , 3136 ] . although ankle and brachial blood pressure indicates similar value in healthy humans , under continued occlusion and/or stenosis in lower limbs induced by arteriosclerosis , ankle blood pressure would decrease compared with brachial blood pressure . therefore , ankle - brachial blood pressure index ( abi ) is a typical indicator for screening peripheral arterial disease ( pad ) . indeed , diagnostic accuracy for stenosis above 50% in leg arteries in pad patients showed excellent values , that is , sensitivity is 90% and specificity is 98% , respectively [ 3739 ] . however , abi is simple , inexpensive , and noninvasive methodology , and it is also reported that abi is a good predictive factor for coronary arterial disease , suggesting that this indicator can be useful test for arterial dysfunction . arterial stiffness is defined by a decrease in aortic distensibility . in human clinical studies , the measurement of the pulse wave velocity ( pwv ) has been broadly used and generally accepted as the gold standard to evaluate aortic distensibility . pwv is calculated by dividing the distance between any two different arteries , for example , carotid and femoral arteries , by the traveled time in the pulse wave from one site to the other site in arteries [ 42 , 43 ] . pwv is inversely related to caliber of a blood vessel and blood viscosity and is proportional to vessel wall thickness and distensibility , indicating that higher speed of pwv reflects lower aortic distensibility . clinically , the pwv is closely associated with pathogenesis in cardiovascular disease [ 3236 , 4446 ] . as endothelial dysfunction precedes arteriosclerosis , assessment of endothelium - mediated vasodilator function has also been widely used to evaluate endothelial function [ 47 , 48 ] . in general , flow - mediated dilation ( fmd ) can be described by any vasodilatation of an artery following an increase in luminal blood flow and internal - wall shear stress induced by reactive hyperemia . the principles , assessment , and evaluation are stated in some excellent reviews [ 49 , 50 ] , briefly , after several minutes of arterial cuff occlusion at proximal or distal portion in any artery , for example , brachial and popliteal artery , immediate cuff deflation can lead to increase shear stress induced by reactive hyperemia and activate endothelial nitric oxide ( no ) synthase ( enos ) . this activation leads to a shear - stress - mediated augmented no production in endothelial cells . fmd is calculated as the difference between the maximum diameter and during reactive hyperemia and baseline diameter , and it is expressed as the relative change ( % ) . since previous studies have shown that brachial artery fmd is emerging as an independent predictor of future cardiac events [ 5153 ] , fmd can be a good predictor indicating that this assessment can be a good noninvasive marker of local no bioavailability in the endothelial cell , which is an important factor and predictor in protecting against cardiovascular disease . aerobic capacity determined by maximal oxygen uptake ( vo2max ) is strongly associated with the risk of cardiovascular disease [ 54 , 55 ] , and an inverse relationship has been observed between vo2max and pwv . since aerobic capacity is improved by regular aerobic training , to date , numerous studies have demonstrated the influence of regular aerobic training on vascular function in athletes , sedentary subjects , and aged people . decreased arterial stiffness has been observed in cyclists , middle- or long - distance runners , and triathletes compared with sedentary subjects . it was also reported that collegiate middle- or long - distance runners had lower arterial stiffness [ 58 , 59 ] and middle - aged and older athletes had more distensibility in their central arteries compared with age - matched sedentary people [ 11 , 60 ] . moreover , in healthy young men and postmenopausal women , regular aerobic exercise reduced arterial stiffness . in addition to these cross - sectional studies , intervention studies of regular aerobic training also revealed an improvement in arterial stiffness and endothelial function . despite differences in the training period from 4 to 16 weeks , regular aerobic training improved arterial stiffness in young healthy subjects [ 61 , 63 ] and in middle- and older - aged people . it was also reported that 16 weeks of regular aerobic training improved pwv in middle- and older - aged people and pre- and stage 1 hypertensive patients . moreover higashi et al . revealed that regular aerobic exercise reduced resting blood pressure and improved endothelium - dependent vasodilatation in essential hypertensive patients . similar results were observed in healthy subjects , diabetic patients , and those with coronary arterial disease , respectively . interestingly , daily physical activity level was also associated with abi , intima - media thickness , and pwv [ 72 , 73 ] . to the best of our knowledge , one study demonstrated that endurance athletes showed a higher pww compared to recreational active control subjects . although this mechanism is unclear , it is assumed that repeated and excessive training stress due to lower resting heart rate , resulting in increased stroke volume , imposed on the elastic component , may cause mechanical fatigue of the arterial wall . however , this is clearly insufficient evidence to support that aerobic exercise impairs vascular function because the results emerged from extremely trained athletes and cross - sectional study . taken together , continued aerobic exercise training may improve vascular function assessed by various methodologies even though with one exception . during resistance exercise , for example , weight lifting , both systolic and diastolic blood pressures increase dramatically [ 75 , 76 ] , whereas only systolic blood pressure increases during aerobic exercise , without an accompanying increase in diastolic blood pressure [ 77 , 78 ] . thus , in the classical paradigm , resistance training would not be recommended for aged people , and in particular , for patients with cardiovascular disease . however , recent studies have revealed that aerobic training alone can not maintain muscle volume and strength , which are needed to prevent sarcopenia [ 12 , 13 ] , the degenerative loss of skeletal muscle with advanced age , often leading to a bed - ridden lifestyle with reduced activities of daily living ( adls ) . resistance exercise is widely recommended to protect against metabolic syndrome , because such exercise can increase muscle strength and volume and may have a positive effect on glucose metabolism , blood lipids , and basal metabolic rate [ 13 , 14 ] . its value for older - aged subjects at risk of sarcopenia or vascular disease thus seems potentially significant . however , because of conventional wisdom regarding the dangers of resistance training , studies about the impact of resistance training on vascular function are ten years behind where they might be today . . revealed that aortic distensibility in resistance - trained men was lower than that in age - matched control subjects . it was also reported that age - related arterial stiffness is more pronounced in strength - trained men than in age - matched sedentary subjects . however , results of more recent studies , which investigated effects of resistance exercise and training on vascular function , seem to be controversial . greater arterial stiffness was also observed in strength - exercised athletes than in sedentary people [ 58 , 59 , 80 ] . it was also reported that greater age - related reduction in arterial compliance in resistance - trained men was observed compared to sedentary men . in contrast , arterial stiffness assessed by central / peripheral pwv did not differ between highly resistance trained and sedentary men . it was also reported that ischemic reperfusion injury induced by 20-min cuff occlusion significantly reduced brachial artery fmd in sedentary young men but unchanged in resistance - trained adults . phillips et al . demonstrated that resistance and endurance trained subjects showed a similar responses in fmd to acute impairment of endothelial dysfunction induced by single weight lifting . moreover , fahs et al . demonstrated that a significant inverse association between muscular strength and central pww , independent of aerobic fitness , suggested that resistance training , which can improve muscular strength , might improve aortic stiffness . in addition to these cross - sectional studies , an intervention study found that four months ' resistance training reduced arterial compliance in young men . in contrast , it was reported that resistance training did not decrease arterial compliance in young men , in premenopausal women , and in elderly men . in addition , resistance training did not alter resting and postexercise aortic blood pressure and wave reflection in middle - aged women . it has been suggested that continued resistance exercise did not affect endothelial function assessed by fmd in young men and postmenopausal women . however , shorter period of resistance training , that is , four weeks , improved peak forearm blood flow induced by reactive hyperemia but increased arterial stiffness in pre- and stage-1 hypertensives . . demonstrated that fmd responses were not altered with resistance training , however , brachial artery vessel diameter increases and postocclusion blood flow increases with this training modality . these results may have an important role in clinical application because resistance training can be a stimulator that may enhance resistance vessel function . similarly , slow movement resistance training increased basal limb blood flow as well as resistance training at normal speed , and weight training increased forearm blood flow as well as aerobic exercise in healthy males . a recent finding also indicated that resting forearm blood flow and peak blood flow in response to reactive hyperemia significantly increased by resistance training in overweight and obese women . from the point view of comparison in different populations , it was reported that decreased augmentation index was observed in old women , but not in old men . heffernan et al . indicated that resistance training leads to increase in microvascular endothelial function in african - american and white men , while it increased brachial stiffness in only african - american . additionally , resistance training improved endothelium - dependent vasodilatation as well as aerobic training and aerobic plus resistance training in patients with recent myocardial infarction , indicating that these improvements were independent of exercise type . recently , the effects of combined exercise , that is , aerobic and resistance training on vascular function have been elucidated [ 100 , 101 ] . combined training consisting of high - intensity resistance training , followed by 30-min aerobic leg cycling , demonstrated a slight increase in carotid arterial compliance ( p = 0.06 ) in young men ; furthermore , moderate - intensity combined circuit resistance exercise and endurance exercise improved arterial stiffness in postmenopausal women . however , from the viewpoint of elderly health , it may not be necessary to use higher loads , for example , > 80% 1 rm . thus , recent studies have investigated the influence of moderate - intensity resistance exercise on vascular function . it was reported that three months ' moderate - intensity resistance exercise training did not alter brachial - ankle ( ba ) pwv , arterial compliance , or arterial stiffness . moreover , 1-year resistance training intervention in overweight women significantly improved fmd , and low - intensity resistance training with short interset rest period improved bapwv and fmd in young subjects . similarly , moderate - intensity resistance training for short period , that is , four weeks training , improved fmd responses in end - stage heart failure patients . although the physiological mechanisms underlying the vascular function , such as arterial stiffness , compliance , and endothelial function assessed by fmd , associated with resistance training are unclear , it is well known that higher - intensity resistance training may be a potent stimulator to increase sympathetic nervous system activity [ 108 , 109 ] . augmented sympathetic nerve activity may act to increase arterial stiffness by providing chronic restraint on the arterial wall via greater sympathetic adrenergic vasoconstrictor tone . during resistance exercise , for example , weight lifting , these acute elevated blood pressures during resistance exercise may alter the arterial structure , and/or arterial load - bearing properties , resulting in arterial stiffness increase and/or impaired reactive hyperemic blood flow with repeated exposure . one interesting and supporting finding was that upper body resistance training group increased arterial stiffness but unchanged with lower limb resistance training group and sedentary control subjects . in their study , norepinephrine concentration ( ne ) significantly increased in only upper body training group after 10-week training . as it is established that ne release is strongly related to the changes in absolute levels of sympathetic nerve activity , elevated ne concentration suggested that resting sympathetic nerve activity increased by 10-week upper body resistance training , resulted in that increased arterial stiffness caused by alteration arterial structure , and/or arterial load - bearing properties . given these numerous previous studies , it is still unclear and in controversy whether resistance training increases arterial stiffness and/or diminish endothelial function . it might be possible that effect of resistance exercise on vascular function may be affected by various factors , such as exercise intensity , different populations , and exercise modality . according to the guidelines of the acsm , resistance training at 70% or greater of 1-rm is recommended in order to achieve muscle hypertrophy and increased strength . however , it is difficult for certain individuals , such as the elderly and rehabilitating athletes , to use such a high - intensity load . in recent years , studies on one alternative , namely , low - intensity resistance training with bfr and bfr walking , have provided compelling data that such training leads to muscle hypertrophy and strength increases [ 17 , 1922 , 24 , 113117 ] and results in adaptations equal to those of high - intensity resistance training [ 21 , 22 ] . as the universal way of this exercise modality seems not to be established , applied cuff pressure and exercise intensity are varied . the underlying principle of this unorthodox technique is that occlusive cuff pressure is greater above individual 's systolic blood pressure and exercise intensity is 2030% of maximal voluntary contraction . during bfr exercise , cuff pressure occludes venous return and causes arterial blood flow to become turbulent , resulting in the enhanced metabolic stress and fast - twitch fiber recruitment in skeletal muscle . at the end of exercise , ischemic reperfusion induced by cuff deflation stimulates shear stress , followed by greater vasodilatation and/or enhanced blood flow . although the precise mechanism by which bfr exercise produces muscle hypertrophy is still unclear , it was reported that low - intensity exercise with bfr can increase that rate of muscle protein synthesis and stimulate mammalian target of rapamycin complex 1 ( mtorc1 ) and mapk - mediated anabolic signaling . however , a recent study revealed that reactive hyperemic blood flow is not a primary mechanism for bfr exercise - induced mtorc1 signaling and muscle protein synthesis . another potential factor to induce muscle hypertrophy may be an intramuscular metabolic stress such as depletion of phosphocreatine , an increase in inorganic phosphate , and a decrease in muscle ph . we recently investigated the effects of short - term resistance training with bfr on muscle mass and strength , and found that elevated metabolic stress may be a crucial factor in obtaining successful results from resistance training with bfr . taken together , it is likely that bfr training can produce muscle hypertrophy without the effect of reactive hyperemic blood flow . however , since hyperemic blood flow per se may be induced by greater shear stress and this greater shear stress induces vasodilatation , which results in increased nitric oxide [ 49 , 50 ] , moreover , maximal dilation was observed after ischemic exercise , there might be a possibility that bfr exercise and training would have a beneficial effect on vascular function such as arterial compliance and endothelial function , indeed , observed . tables 1 and 2 summarize studies undertaken to investigate the effects of acute or chronic bfr exercise on vascular function such as arterial compliance , endothelial function , and related biomarkers . it has been difficult to obtain consensus due to the large number of variables such as age , gender , intensity and exercise modality , intervention period , applied cuff pressure and cuff width , and evaluation of vascular function , all of which may influence the training effect . we discuss the influence of bfr exercise and training on vascular function based on current findings in the following session . reported that acute knee extension and flexion with bfr increased arterial compliance without changes in vascular conductance and that this increase was similar to low - intensity knee extension without bfr . they suggested that this acute increase in arterial compliance may be attributed to augmented regional vasoactive substances , and decreased systemic sympathetic vasoconstrictor tone . additionally , several interventional studies have indicated the influences of bfr training on arterial compliance . kim et al . reported that arterial compliance assessed in the radial artery of young men did not change by low - intensity resistance training with bfr for 3 weeks . similarly , four - weeks bfr training did not alter arterial stiffness evaluated by the pwv between the femoral and tibial arteries in young male subjects . extended the training period from 3 to 6 weeks and observed no change in arterial compliance assessed by the same device that had been used in the previous study . interestingly , one recent study revealed that low - intensity ( 30% 1 rm ) upper - body bfr exercise training , that is , bench press did not diminish carotid artery compliance , while high - intensity bench press training without bfr decreased arterial compliance . the underlying physiological mechanism(s ) associated with unaltered arterial compliance , that is , four intervention studies , and with increased arterial compliance , that is , one cross - sectional study , is unclear . impaired arterial compliance induced by high - intensity resistance training without bfr may be attributed to acute elevated blood pressure via increasing sympathetic nerve activity system [ 108110 ] , resulting in alteration arterial structure and/or arterial load - bearing properties . these changes may be related to impaired arterial compliance . in the previous studies of bfr training , resting blood pressure did not change after several weeks intervention with bfr resistance training [ 127 , 129 ] . moreover , the resistance training with and without bfr - induced carotid arterial compliance changes was associated with changes in systolic blood pressure during training intervention , indicating that elevation blood pressure may play a role to induce arterial compliance changes during bfr training . conversely , postexercise hypotension did not occur after low - intensity resistance training with bfr , while high - intensity resistance training elicited greater postexercise hypotension . postexercise hypotension can be considered an important strategy to control resting blood pressure , especially in hypertensive patients [ 132 , 133 ] . therefore , it may be reasonable that bfr training did not change arterial compliance based on unobserved postexercise hypotension with acute bfr exercise . in addition to these resistance exercise and training with bfr , effect of chronic bfr walking on arterial compliance has been reported . ozaki et al . revealed that carotid arterial compliance significantly improved by 10 weeks of bfr walking in elderly people . the interesting considerations regarding their study are that the training period was longer than in previous studies ( 10 weeks versus 3 to 6 weeks ) , and the participants were elderly , with the majority being women ( 3 men and 10 women ) . this result might have great clinical relevance in understanding the potential application of bfr training . recently , age- and sex - related differences in cardiovascular responses both at rest and during exercise have been elegantly reviewed . for example , ( 1 ) reducing oxidative stress improves carotid arterial compliance in postmenopausal women but not older men , ( 2 ) habitual exercise abolished age - related differences in central arterial stiffness in older women , ( 3 ) vasoconstrictor responses to sympathetic stimulation is blunted in women compared to men ( see in detail ) . these gender differences in cardiovascular responses and adaptation to exercise training might affect vascular function in response to bfr exercise and training though only one study showed that carotid arterial compliance was improved by bfr training in elderly women . it is well known that regular aerobic training improves carotid arterial compliance , whereas high - intensity resistance training reduces arterial compliance . although the mechanism of improved arterial compliance in elderly women is still unclear , there are several possible physiological mechanisms to explain these changes . a previous study demonstrated that bfr training - induced improvement in carotid arterial compliance was approximately 30% . in the previous cross - sectional study , carotid arterial compliance in middle - aged and older men is 2035% higher than that in age - matched recreational and sedentary subjects . they also conducted interventional study and demonstrated that 13.5 weeks of aerobic training produced a 25% increase in carotid arterial compliance . similarly , carotid artery compliance in postmenopausal women increased about 40% following 3 months of home - based regular aerobic exercise . reported that 6 weeks of bfr walking training did not improve peak aerobic capacity , but it significantly increased muscular size and strength in elderly subjects ( 2 men and 9 women ) . thus , it might be significant that arterial compliance was improved in elderly women , probably without systemic aerobic capacity improvement . however , only study revealed these results , hence , future studies are needed whether these improvements are specific for elderly women . taken together , bfr exercise and training did not change arterial compliance , while only two studies showed that acute bfr exercise and chronic bfr walking improved arterial compliance [ 124 , 134 ] . however , due to insufficient evidence , at least what we can say is that bfr training including resistance exercise and walking may not worse arterial compliance , possibly , associated with unchanged resting blood pressure with bfr resistance training [ 127 , 129 ] and bfr walking . endothelium - dependent vasodilatation in conduit artery , for example , brachial and popliteal arteries , can be evaluated by fmd , which is a good in protecting against cardiovascular disease [ 5153 ] . bfr exercise can lead to reactive hyperemic blood flow and increased microvascular filtration capacity induced by ischemic reperfusion . thus , it might be possible that these physiological responses may lead to an enhanced vascular reactivity such as flow - mediated dilation ( fmd ) responses and/or improved basal limb blood flow . however , the effect of bfr exercise on vascular reactivity is still controversial . to date , three studies examined about acute effects of bfr walking and resistance training with bfr [ 140 , 141 ] on endothelial function assessed by fmd . although exercise modality and target conduit artery vary , two of three studies showed impaired fmd with bfr exercise / training and one study indicated unaltered fmd with four - week training with bfr . . demonstrated that fmd was impaired after bfr walking compared to walking without bfr . in their study , systemic cardiovascular responses , such as blood pressure , and total peripheral resistance significantly increased and stroke volume / pulse pressure ( an index of systemic arterial compliance ) significantly decreased during walking compared to those in the control condition while abi showed a similar value . in addition to this cross - sectional study , two studies on chronic bfr training showed different results in fmd responses . credeur et al . reported that four - weeks hand - grip training with bfr reduced fmd , whereas hunt et al . demonstrated that four - weeks hand - grip training with bfr did not alter fmd responses but induced transient adaptations to brachial artery structure , although the mechanism underlying impaired fmd responses after acute bfr walking and chronic bfr training is still unclear , it is assumed that reduced fmd is a product of diminished endothelial function . moreover , they speculated that oxidative stress induced by ischemic reperfusion injury might be the cause of the reduced fmd [ 139 , 140 ] . as increased oxidative stress is associated with endothelial dysfunction [ 142 , 143 ] , followed by arteriosclerosis , augmented oxidative stress plays an important role in the pathogenesis and development of cardiovascular disease . it has been reported that high - intensity resistance exercise , that is , > 70% 1 rm , elicits oxidative stress markers [ 145149 ] , whereas these responses to lower - intensity exercise have been inconsistent [ 150 , 151 ] . during bfr exercise , as far as we know , only two studies have examined the effect of bfr exercise on oxidative stress markers . measured serum interleukin-6 ( il-6 ) and lipid peroxides ( lp ) before and after low - intensity resistance exercise ( 20% 1 rm ) with moderate blood flow restriction and found no significant increases . although exhaustive exercise in humans has been shown to cause a sustained elevation of muscular xanthine oxidase activity as well as increases in serum il-6 and lp concentrations , it did not seem to produce an excess amount of oxygen - derived free radicals in takarada et al . it was also reported that protein carbonyls and blood glutathione , which are indicators of oxidative stress , did not increase during low - intensity ( 30% 1 rm ) bfr exercise , but did increase in moderate - resistance exercise ( up to 70% 1 rm ) . since both of previous studies did not measure oxidative stress markers [ 139 , 140 ] , it is unknown whether impaired fmd responses are associated with elevated oxidative stress . however , the study of credeur et al . used higher - intensity resistance load ( 60% 1 rm ) and applied the cuff for a longer occlusion time ( 20 min ) . thus , there is a possibility that increased oxidative stress may have reduced the fmd in the case of credeur 's study because it was reported that the region between 60 and 70% 1 rm may be a critical load marker [ 145151 ] , in terms of inducing oxidative stress during resistance training without bfr . another explanation associated with impaired fmd responses after bfr walking is that elevated blood pressure during 20-min bfr walking . the blood pressure of subjects during 20-min walking with blood flow restriction in renzi et al was > 20% higher than controls , whereas takano et al . observed only slightly higher blood pressure during knee extension with bfr than that without bfr ( 127 versus 113 mmhg ) . since acute elevations in arterial blood pressure are associated with the arterial structure and/or the arterial load - bearing properties of collagen and elastin , the rise in blood pressure during bfr walking might have caused the diminished fmd . collectively , although the influence of bfr walking and training with small muscle group on endothelial dysfunction assessed by fmd is still controversial , these contradictory results may be dependent on the variety of methodologies used , including exercise intensity and occlusion time during exercise . however , it should be still noted that cuff release - induced hyperemic blood flow may be expected , suggesting that it stimulates shear stress , followed by nitric oxide increase , simultaneously , bfr exercise and training should be carefully prescribed because it is supposed that systemic arterial compliance increase during bfr walking . as large muscle group dynamic exercise may alter not only local vasculature but also systemic cardiovascular responses to either acute or chronic exercise , small muscle group exercise may be useful as a physiological model to observe impact of exercise training on localized vasomotor control in skeletal muscles without marked changes in central hemodynamics during exercise . thus , to date , several studies have used handgrip exercise training to elucidate vascular function . accordingly , two studies of handgrip exercise training with bfr have been investigated , furthermore , studies of calf raise with own body weight and planter flexion exercise training with bfr were also carried out . generally , handgrip exercise training increases forearm blood flow and/or improves fmd with one exceptional unchanged study . enhanced brachial fmd responses as well as endothelium - dependent vasodilatation were observed after isometric handgrip training in old men , in elderly hypertensives [ 156 , 157 ] , and chronic heart failure subjects [ 158 , 159 ] . similarly , forearm blood flow was increased with regular handgrip exercise training in young men [ 160 , 161 ] and in middle aged . in contrast , improved blood flow was not observed in patients with chronic heart failure . moreover , mcgowan et al . reported that isometric handgrip training did not alter fmd with normal blood pressure . one interesting finding was that four - week handgrip exercise training improved peak vasodilator capacity induced by 10-minute ischemic stimulus without influencing endothelium vasodilator system . these results suggested that evaluation for vascular function should be considered from many aspects , such as endothelium - dependent dilation , basal blood flow , and peak vasodilator capacity . in addition to these previous studies of handgrip exercise training without bfr , two studies of handgrip exercise training with bfr have been elucidated , resulted in impaired and unchanged fmd responses . as above stated , exercise intensity , which was used in one previous study , was higher and longer , that is , 60% mvc dynamic handgrip exercise training for 20 min , compared to another previous study with bfr ( tables 1 and 2 ) . it is possible that these higher exercise intensity and longer duration may cause oxidative stress , resulted in diminished fmd responses . interesting findings were that handgrip exercise training with bfr did not improve fmd responses but caused brachial artery structural modifications , that is , increased resting and maximal diameters . based on the principle of fmd calculation , changes in baseline diameter can influence the magnitude of fmd responses . moreover , it should be taken a consideration about time course alterations in vessel structure and function . animal studies showed that prolonged training induces structural changes , namely , arterial remodeling [ 165 , 166 ] , while short - term training improves vascular function via enhanced no bioavailability [ 167170 ] . indeed , in human studies , brachial artery fmd increases after just one week of handgrip exercise training and decreased after two weeks , whereas conduit artery vasodilator capacity showed a progressive increase for eight weeks during training intervention . in one of the bfr study , fmd did not change after four weeks training , however , peak blood flow after ischemia and baseline diameter significantly increased . it may be speculated that four - week bfr handgrip exercise training might cause improvement in functional changes within a few weeks , followed by structural changes after four weeks , resulting in without changes fmd responses normalized by increased basal vessel diameter . it is well known that coagulation factors and fibrinolysis are closely associated with the risk of cardiovascular diseases such as ischemic heart disease , particularly acute coronary syndrome . several previous studies have demonstrated the influence of bfr training on coagulation factors and fibrinolysis . however , to date , no study has observed a significant increase in either acute [ 120 , 174 , 175 ] or chronic bfr exercise . it was reported that thrombin generation is associated with exercise - induced metabolites [ 176 , 177 ] . indeed , bfr exercise produced greater metabolites ; hence , there is a possibility that increased metabolites might produce coagulation factors and fibrinolysis . in contrast , hilberg et al . [ 178 , 179 ] reported that thrombin antithrombin iii complex ( tat ) and d - dimer increased after 6090 min prolonged exercise but did not increase after high - intensity exercise , from which blood lactate concentrations were significantly increased , suggesting that the tat and d - dimer may be dependent on exercise time . thus , it is unlikely that accumulated metabolites during bfr would be related to coagulation factors and fibrinolysis . another possible explanation is that expanded blood flow after cuff release would exclude thrombosis , so that the possibility of thrombosis during bfr could be ruled out . more important findings were that the incidence of serious side effects related to thrombosis was lower in 7 cases ( 0.055% ) based on the national survey for more than 12,000 people in japan . it has been pointed out that endothelial dysfunction may be improved through angiogenesis induced by vascular endothelial growth factors . one study revealed that acute bfr exercise significantly increased vascular endothelial growth factor ( vegf ) compared to control exercise . in fact , vegf secretion and production are activated under hypoxia [ 182 , 183 ] and/or under decreases in local muscle oxygen tension during exercise . increased vegf may produce angiogenesis , followed by enhanced no bioavailability , which might be a beneficial effect for endothelial function . however , since no studies have examined the effects of chronic bfr training on vegf , it is uncertain whether chronic bfr training can increase vefg , resulting in an improvement of endothelial function . in this review , we focused on what is currently known ( tables 1 and 2 ) and hypothesised changes ( figure 1 ) of the influences of bfr exercise and training on vascular function . it is well established that aerobic exercise can improve vascular function but with insufficient muscle hypertrophy , while it is possible that high - intensity resistance training can produce muscle hypertrophy but with impaired vascular function . higher load of resistance training should be considered carefully to apply aged people , disease patients , and rehabilitaining athletes . bfr exercise and training might be a novel therapeutic modality because it combines lower exercise intensity than higher - intensity resistance training from the point view of during exercise with enhanced reactive hyperemic blood flow after cuff release . although accumulating evidence has revealed that bfr training leads to muscle hypertrophy and strength increase as well as high - intensity resistance training , little attention has been given to the impact of bfr training on vascular function . at least the majority of the previous studies have demonstrated that bfr training did not impair arterial compliance . conversely , the effect of bfr training and bfr walking on endothelial function assessed by fmd is not consistent . available evidence suggests that acute bfr training did not increase oxidative stress markers or coagulation factors . also , the effect of bfr training on vascular function may be influenced by various factors , such as , age , sex , exercise type , intensity , applied cuff pressure , and intervention period , not to mention the different evaluative methods for vascular function . however , the majority of bfr exercise and training use lower intensity , that is , 2040% 1 rm , compared to high - intensity resistance training , for example , > 80% 1 rm . therefore , bfr exercise and training seems to become novel method because bfr exercise and training can obtain muscle hypertrophy as well as high - intensity resistance training even if bfr exercise and training does not affect vascular function . future studies should be conducted with the aim of elucidating the mechanisms of the influence of bfr training on vascular function , with careful selection of the influencing parameters mentioned above and the potential therapeutic benefits for the elderly and for the rehabilitation of patients with cardiovascular disease and physical injury .

it is established that regular aerobic training improves vascular function , for example , endothelium - dependent vasodilatation and arterial stiffness or compliance and thereby constitutes a preventative measure against cardiovascular disease . in contrast , high - intensity resistance training impairs vascular function , while the influence of moderate - intensity resistance training on vascular function is still controversial . however , aerobic training is insufficient to inhibit loss in muscular strength with advancing age ; thus , resistance training is recommended to prevent sarcopenia . recently , several lines of study have provided compelling data showing that exercise and training with blood flow restriction ( bfr ) leads to muscle hypertrophy and strength increase . as such , bfr training might be a novel means of overcoming the contradiction between aerobic and high - intensity resistance training . although it is not enough evidence to obtain consensus about impact of bfr training on vascular function , available evidences suggested that bfr training did not change coagulation factors and arterial compliance though with inconsistence results in endothelial function . this paper is a review of the literature on the impact of bfr exercise and training on vascular function , such as endothelial function , arterial compliance , or other potential factors in comparison with those of aerobic and resistance training .

1. Introduction 2. Evaluation for Vascular Function 3. Impact of Aerobic Exercise and Training on Vascular Function 4. Impact of Resistance Exercise and Training on Vascular Function 5. Impact of Blood Flow Restricted Exercise and Training on Vascular Function 6. Summary and Future Perspectives

to date , it has been recognized that arterial dysfunction , such as increased arterial stiffness , is closely associated with the pathogenesis of cardiovascular disease , which in turn increases mortality by increasing the risk of events such as myocardial infarction and stroke [ 24 ] . briefly , aerobic exercise is a physical exercise of relatively low intensity that depends primarily on the aerobic energy - generating process , for example , running and leg cycling . although aerobic exercise may improve arterial function , it has also been reported that aerobic exercise is insufficient to inhibit the loss in muscular strength that comes with advancing age [ 12 , 13 ] . resistance exercise is recommended to prevent sarcopenia , age - induced muscular degeneration which often entails reduced activities of daily living ( adls ) . in addition , it has been reported that high - intensity resistance training ( > 80% of 1 rm ) reduces central artery compliance . these findings suggest that such a high - intensity resistance exercise should be prescribed carefully , particularly for aged people and patients with cardiovascular disease . recently , several studies have demonstrated that low - intensity resistance exercise with blood flow restriction ( bfr ) [ 1723 ] and bfr walking dramatically leads to muscle hypertrophy and strength gain and that it results in adaptations equal to those of high - intensity resistance training . although the effect of resistance exercise with bfr and bfr walking on vascular function is still unclear , there is a possibility that this exercise modality can be an important therapeutic prescription not only for sarcopenia but also for vascular dysfunction because of the lower exercise intensity compared to high - intensity resistance training . in this review , we would like to focus on the impact of such exercise on vascular function in comparison with the effects of aerobic and resistance training alone and in combination . in this section , we introduced several methodologies , which have investigated the impact of bfr exercise and training . generally , arterial compliance can be measured by a combination of ultrasound imaging of any artery , for example , carotid artery , with simultaneous applanation of tonometrically obtained arterial pressure from the contralateral artery , permits noninvasive determination of arterial compliance . indeed , several studies demonstrated that decreased arterial compliance and/or increased arterial stiffness have been identified as independent risk factors of cardiovascular disease [ 27 , 3136 ] . however , abi is simple , inexpensive , and noninvasive methodology , and it is also reported that abi is a good predictive factor for coronary arterial disease , suggesting that this indicator can be useful test for arterial dysfunction . pwv is calculated by dividing the distance between any two different arteries , for example , carotid and femoral arteries , by the traveled time in the pulse wave from one site to the other site in arteries [ 42 , 43 ] . the principles , assessment , and evaluation are stated in some excellent reviews [ 49 , 50 ] , briefly , after several minutes of arterial cuff occlusion at proximal or distal portion in any artery , for example , brachial and popliteal artery , immediate cuff deflation can lead to increase shear stress induced by reactive hyperemia and activate endothelial nitric oxide ( no ) synthase ( enos ) . since aerobic capacity is improved by regular aerobic training , to date , numerous studies have demonstrated the influence of regular aerobic training on vascular function in athletes , sedentary subjects , and aged people . in addition to these cross - sectional studies , intervention studies of regular aerobic training also revealed an improvement in arterial stiffness and endothelial function . revealed that regular aerobic exercise reduced resting blood pressure and improved endothelium - dependent vasodilatation in essential hypertensive patients . however , this is clearly insufficient evidence to support that aerobic exercise impairs vascular function because the results emerged from extremely trained athletes and cross - sectional study . thus , in the classical paradigm , resistance training would not be recommended for aged people , and in particular , for patients with cardiovascular disease . however , recent studies have revealed that aerobic training alone can not maintain muscle volume and strength , which are needed to prevent sarcopenia [ 12 , 13 ] , the degenerative loss of skeletal muscle with advanced age , often leading to a bed - ridden lifestyle with reduced activities of daily living ( adls ) . however , because of conventional wisdom regarding the dangers of resistance training , studies about the impact of resistance training on vascular function are ten years behind where they might be today . however , results of more recent studies , which investigated effects of resistance exercise and training on vascular function , seem to be controversial . in contrast , arterial stiffness assessed by central / peripheral pwv did not differ between highly resistance trained and sedentary men . demonstrated that a significant inverse association between muscular strength and central pww , independent of aerobic fitness , suggested that resistance training , which can improve muscular strength , might improve aortic stiffness . in contrast , it was reported that resistance training did not decrease arterial compliance in young men , in premenopausal women , and in elderly men . in addition , resistance training did not alter resting and postexercise aortic blood pressure and wave reflection in middle - aged women . however , shorter period of resistance training , that is , four weeks , improved peak forearm blood flow induced by reactive hyperemia but increased arterial stiffness in pre- and stage-1 hypertensives . demonstrated that fmd responses were not altered with resistance training , however , brachial artery vessel diameter increases and postocclusion blood flow increases with this training modality . indicated that resistance training leads to increase in microvascular endothelial function in african - american and white men , while it increased brachial stiffness in only african - american . additionally , resistance training improved endothelium - dependent vasodilatation as well as aerobic training and aerobic plus resistance training in patients with recent myocardial infarction , indicating that these improvements were independent of exercise type . recently , the effects of combined exercise , that is , aerobic and resistance training on vascular function have been elucidated [ 100 , 101 ] . combined training consisting of high - intensity resistance training , followed by 30-min aerobic leg cycling , demonstrated a slight increase in carotid arterial compliance ( p = 0.06 ) in young men ; furthermore , moderate - intensity combined circuit resistance exercise and endurance exercise improved arterial stiffness in postmenopausal women . however , from the viewpoint of elderly health , it may not be necessary to use higher loads , for example , > 80% 1 rm . thus , recent studies have investigated the influence of moderate - intensity resistance exercise on vascular function . it was reported that three months ' moderate - intensity resistance exercise training did not alter brachial - ankle ( ba ) pwv , arterial compliance , or arterial stiffness . moreover , 1-year resistance training intervention in overweight women significantly improved fmd , and low - intensity resistance training with short interset rest period improved bapwv and fmd in young subjects . similarly , moderate - intensity resistance training for short period , that is , four weeks training , improved fmd responses in end - stage heart failure patients . although the physiological mechanisms underlying the vascular function , such as arterial stiffness , compliance , and endothelial function assessed by fmd , associated with resistance training are unclear , it is well known that higher - intensity resistance training may be a potent stimulator to increase sympathetic nervous system activity [ 108 , 109 ] . during resistance exercise , for example , weight lifting , these acute elevated blood pressures during resistance exercise may alter the arterial structure , and/or arterial load - bearing properties , resulting in arterial stiffness increase and/or impaired reactive hyperemic blood flow with repeated exposure . as it is established that ne release is strongly related to the changes in absolute levels of sympathetic nerve activity , elevated ne concentration suggested that resting sympathetic nerve activity increased by 10-week upper body resistance training , resulted in that increased arterial stiffness caused by alteration arterial structure , and/or arterial load - bearing properties . given these numerous previous studies , it is still unclear and in controversy whether resistance training increases arterial stiffness and/or diminish endothelial function . it might be possible that effect of resistance exercise on vascular function may be affected by various factors , such as exercise intensity , different populations , and exercise modality . according to the guidelines of the acsm , resistance training at 70% or greater of 1-rm is recommended in order to achieve muscle hypertrophy and increased strength . however , it is difficult for certain individuals , such as the elderly and rehabilitating athletes , to use such a high - intensity load . in recent years , studies on one alternative , namely , low - intensity resistance training with bfr and bfr walking , have provided compelling data that such training leads to muscle hypertrophy and strength increases [ 17 , 1922 , 24 , 113117 ] and results in adaptations equal to those of high - intensity resistance training [ 21 , 22 ] . although the precise mechanism by which bfr exercise produces muscle hypertrophy is still unclear , it was reported that low - intensity exercise with bfr can increase that rate of muscle protein synthesis and stimulate mammalian target of rapamycin complex 1 ( mtorc1 ) and mapk - mediated anabolic signaling . however , a recent study revealed that reactive hyperemic blood flow is not a primary mechanism for bfr exercise - induced mtorc1 signaling and muscle protein synthesis . we recently investigated the effects of short - term resistance training with bfr on muscle mass and strength , and found that elevated metabolic stress may be a crucial factor in obtaining successful results from resistance training with bfr . taken together , it is likely that bfr training can produce muscle hypertrophy without the effect of reactive hyperemic blood flow . however , since hyperemic blood flow per se may be induced by greater shear stress and this greater shear stress induces vasodilatation , which results in increased nitric oxide [ 49 , 50 ] , moreover , maximal dilation was observed after ischemic exercise , there might be a possibility that bfr exercise and training would have a beneficial effect on vascular function such as arterial compliance and endothelial function , indeed , observed . tables 1 and 2 summarize studies undertaken to investigate the effects of acute or chronic bfr exercise on vascular function such as arterial compliance , endothelial function , and related biomarkers . it has been difficult to obtain consensus due to the large number of variables such as age , gender , intensity and exercise modality , intervention period , applied cuff pressure and cuff width , and evaluation of vascular function , all of which may influence the training effect . we discuss the influence of bfr exercise and training on vascular function based on current findings in the following session . additionally , several interventional studies have indicated the influences of bfr training on arterial compliance . reported that arterial compliance assessed in the radial artery of young men did not change by low - intensity resistance training with bfr for 3 weeks . similarly , four - weeks bfr training did not alter arterial stiffness evaluated by the pwv between the femoral and tibial arteries in young male subjects . interestingly , one recent study revealed that low - intensity ( 30% 1 rm ) upper - body bfr exercise training , that is , bench press did not diminish carotid artery compliance , while high - intensity bench press training without bfr decreased arterial compliance . impaired arterial compliance induced by high - intensity resistance training without bfr may be attributed to acute elevated blood pressure via increasing sympathetic nerve activity system [ 108110 ] , resulting in alteration arterial structure and/or arterial load - bearing properties . in the previous studies of bfr training , resting blood pressure did not change after several weeks intervention with bfr resistance training [ 127 , 129 ] . moreover , the resistance training with and without bfr - induced carotid arterial compliance changes was associated with changes in systolic blood pressure during training intervention , indicating that elevation blood pressure may play a role to induce arterial compliance changes during bfr training . conversely , postexercise hypotension did not occur after low - intensity resistance training with bfr , while high - intensity resistance training elicited greater postexercise hypotension . therefore , it may be reasonable that bfr training did not change arterial compliance based on unobserved postexercise hypotension with acute bfr exercise . in addition to these resistance exercise and training with bfr , effect of chronic bfr walking on arterial compliance has been reported . for example , ( 1 ) reducing oxidative stress improves carotid arterial compliance in postmenopausal women but not older men , ( 2 ) habitual exercise abolished age - related differences in central arterial stiffness in older women , ( 3 ) vasoconstrictor responses to sympathetic stimulation is blunted in women compared to men ( see in detail ) . these gender differences in cardiovascular responses and adaptation to exercise training might affect vascular function in response to bfr exercise and training though only one study showed that carotid arterial compliance was improved by bfr training in elderly women . it is well known that regular aerobic training improves carotid arterial compliance , whereas high - intensity resistance training reduces arterial compliance . a previous study demonstrated that bfr training - induced improvement in carotid arterial compliance was approximately 30% . reported that 6 weeks of bfr walking training did not improve peak aerobic capacity , but it significantly increased muscular size and strength in elderly subjects ( 2 men and 9 women ) . taken together , bfr exercise and training did not change arterial compliance , while only two studies showed that acute bfr exercise and chronic bfr walking improved arterial compliance [ 124 , 134 ] . however , due to insufficient evidence , at least what we can say is that bfr training including resistance exercise and walking may not worse arterial compliance , possibly , associated with unchanged resting blood pressure with bfr resistance training [ 127 , 129 ] and bfr walking . endothelium - dependent vasodilatation in conduit artery , for example , brachial and popliteal arteries , can be evaluated by fmd , which is a good in protecting against cardiovascular disease [ 5153 ] . thus , it might be possible that these physiological responses may lead to an enhanced vascular reactivity such as flow - mediated dilation ( fmd ) responses and/or improved basal limb blood flow . however , the effect of bfr exercise on vascular reactivity is still controversial . to date , three studies examined about acute effects of bfr walking and resistance training with bfr [ 140 , 141 ] on endothelial function assessed by fmd . demonstrated that four - weeks hand - grip training with bfr did not alter fmd responses but induced transient adaptations to brachial artery structure , although the mechanism underlying impaired fmd responses after acute bfr walking and chronic bfr training is still unclear , it is assumed that reduced fmd is a product of diminished endothelial function . measured serum interleukin-6 ( il-6 ) and lipid peroxides ( lp ) before and after low - intensity resistance exercise ( 20% 1 rm ) with moderate blood flow restriction and found no significant increases . it was also reported that protein carbonyls and blood glutathione , which are indicators of oxidative stress , did not increase during low - intensity ( 30% 1 rm ) bfr exercise , but did increase in moderate - resistance exercise ( up to 70% 1 rm ) . thus , there is a possibility that increased oxidative stress may have reduced the fmd in the case of credeur 's study because it was reported that the region between 60 and 70% 1 rm may be a critical load marker [ 145151 ] , in terms of inducing oxidative stress during resistance training without bfr . the blood pressure of subjects during 20-min walking with blood flow restriction in renzi et al was > 20% higher than controls , whereas takano et al . collectively , although the influence of bfr walking and training with small muscle group on endothelial dysfunction assessed by fmd is still controversial , these contradictory results may be dependent on the variety of methodologies used , including exercise intensity and occlusion time during exercise . however , it should be still noted that cuff release - induced hyperemic blood flow may be expected , suggesting that it stimulates shear stress , followed by nitric oxide increase , simultaneously , bfr exercise and training should be carefully prescribed because it is supposed that systemic arterial compliance increase during bfr walking . thus , to date , several studies have used handgrip exercise training to elucidate vascular function . these results suggested that evaluation for vascular function should be considered from many aspects , such as endothelium - dependent dilation , basal blood flow , and peak vasodilator capacity . animal studies showed that prolonged training induces structural changes , namely , arterial remodeling [ 165 , 166 ] , while short - term training improves vascular function via enhanced no bioavailability [ 167170 ] . in one of the bfr study , fmd did not change after four weeks training , however , peak blood flow after ischemia and baseline diameter significantly increased . it is well known that coagulation factors and fibrinolysis are closely associated with the risk of cardiovascular diseases such as ischemic heart disease , particularly acute coronary syndrome . several previous studies have demonstrated the influence of bfr training on coagulation factors and fibrinolysis . indeed , bfr exercise produced greater metabolites ; hence , there is a possibility that increased metabolites might produce coagulation factors and fibrinolysis . thus , it is unlikely that accumulated metabolites during bfr would be related to coagulation factors and fibrinolysis . however , since no studies have examined the effects of chronic bfr training on vegf , it is uncertain whether chronic bfr training can increase vefg , resulting in an improvement of endothelial function . in this review , we focused on what is currently known ( tables 1 and 2 ) and hypothesised changes ( figure 1 ) of the influences of bfr exercise and training on vascular function . it is well established that aerobic exercise can improve vascular function but with insufficient muscle hypertrophy , while it is possible that high - intensity resistance training can produce muscle hypertrophy but with impaired vascular function . bfr exercise and training might be a novel therapeutic modality because it combines lower exercise intensity than higher - intensity resistance training from the point view of during exercise with enhanced reactive hyperemic blood flow after cuff release . although accumulating evidence has revealed that bfr training leads to muscle hypertrophy and strength increase as well as high - intensity resistance training , little attention has been given to the impact of bfr training on vascular function . at least the majority of the previous studies have demonstrated that bfr training did not impair arterial compliance . conversely , the effect of bfr training and bfr walking on endothelial function assessed by fmd is not consistent . available evidence suggests that acute bfr training did not increase oxidative stress markers or coagulation factors . also , the effect of bfr training on vascular function may be influenced by various factors , such as , age , sex , exercise type , intensity , applied cuff pressure , and intervention period , not to mention the different evaluative methods for vascular function . however , the majority of bfr exercise and training use lower intensity , that is , 2040% 1 rm , compared to high - intensity resistance training , for example , > 80% 1 rm . therefore , bfr exercise and training seems to become novel method because bfr exercise and training can obtain muscle hypertrophy as well as high - intensity resistance training even if bfr exercise and training does not affect vascular function . future studies should be conducted with the aim of elucidating the mechanisms of the influence of bfr training on vascular function , with careful selection of the influencing parameters mentioned above and the potential therapeutic benefits for the elderly and for the rehabilitation of patients with cardiovascular disease and physical injury .

to date , it has been recognized that arterial dysfunction , such as increased arterial stiffness , is closely associated with the pathogenesis of cardiovascular disease , which in turn increases mortality by increasing the risk of events such as myocardial infarction and stroke [ 24 ] . a higher physical activity level as well as regular exercise may be effective at diminishing the risk of coronary heart disease [ 5 , 6 ] and stroke [ 7 , 8 ] . in contrast , resistance exercise is also physical exercise of relatively moderate and higher intensity that uses a resistance to the force of muscular contraction , in other words , strength training . according to the guideline of american college of sports medicine ( acsm ) , a mechanical load greater than 70% of the one - repetition maximum load ( 1 rm ) can produce morphological and functional muscular adaptations . recently , several studies have demonstrated that low - intensity resistance exercise with blood flow restriction ( bfr ) [ 1723 ] and bfr walking dramatically leads to muscle hypertrophy and strength gain and that it results in adaptations equal to those of high - intensity resistance training . although the effect of resistance exercise with bfr and bfr walking on vascular function is still unclear , there is a possibility that this exercise modality can be an important therapeutic prescription not only for sarcopenia but also for vascular dysfunction because of the lower exercise intensity compared to high - intensity resistance training . in this review , we would like to focus on the impact of such exercise on vascular function in comparison with the effects of aerobic and resistance training alone and in combination . in human studies , as it is almost impossible to evaluate large arterial function directly , various noninvasive methodologies have been used to evaluate arterial function in humans . in this section , we introduced several methodologies , which have investigated the impact of bfr exercise and training . generally , arterial compliance can be measured by a combination of ultrasound imaging of any artery , for example , carotid artery , with simultaneous applanation of tonometrically obtained arterial pressure from the contralateral artery , permits noninvasive determination of arterial compliance . although ankle and brachial blood pressure indicates similar value in healthy humans , under continued occlusion and/or stenosis in lower limbs induced by arteriosclerosis , ankle blood pressure would decrease compared with brachial blood pressure . therefore , ankle - brachial blood pressure index ( abi ) is a typical indicator for screening peripheral arterial disease ( pad ) . indeed , diagnostic accuracy for stenosis above 50% in leg arteries in pad patients showed excellent values , that is , sensitivity is 90% and specificity is 98% , respectively [ 3739 ] . however , abi is simple , inexpensive , and noninvasive methodology , and it is also reported that abi is a good predictive factor for coronary arterial disease , suggesting that this indicator can be useful test for arterial dysfunction . in human clinical studies , the measurement of the pulse wave velocity ( pwv ) has been broadly used and generally accepted as the gold standard to evaluate aortic distensibility . pwv is inversely related to caliber of a blood vessel and blood viscosity and is proportional to vessel wall thickness and distensibility , indicating that higher speed of pwv reflects lower aortic distensibility . in general , flow - mediated dilation ( fmd ) can be described by any vasodilatation of an artery following an increase in luminal blood flow and internal - wall shear stress induced by reactive hyperemia . the principles , assessment , and evaluation are stated in some excellent reviews [ 49 , 50 ] , briefly , after several minutes of arterial cuff occlusion at proximal or distal portion in any artery , for example , brachial and popliteal artery , immediate cuff deflation can lead to increase shear stress induced by reactive hyperemia and activate endothelial nitric oxide ( no ) synthase ( enos ) . fmd is calculated as the difference between the maximum diameter and during reactive hyperemia and baseline diameter , and it is expressed as the relative change ( % ) . since previous studies have shown that brachial artery fmd is emerging as an independent predictor of future cardiac events [ 5153 ] , fmd can be a good predictor indicating that this assessment can be a good noninvasive marker of local no bioavailability in the endothelial cell , which is an important factor and predictor in protecting against cardiovascular disease . aerobic capacity determined by maximal oxygen uptake ( vo2max ) is strongly associated with the risk of cardiovascular disease [ 54 , 55 ] , and an inverse relationship has been observed between vo2max and pwv . since aerobic capacity is improved by regular aerobic training , to date , numerous studies have demonstrated the influence of regular aerobic training on vascular function in athletes , sedentary subjects , and aged people . it was also reported that collegiate middle- or long - distance runners had lower arterial stiffness [ 58 , 59 ] and middle - aged and older athletes had more distensibility in their central arteries compared with age - matched sedentary people [ 11 , 60 ] . despite differences in the training period from 4 to 16 weeks , regular aerobic training improved arterial stiffness in young healthy subjects [ 61 , 63 ] and in middle- and older - aged people . it was also reported that 16 weeks of regular aerobic training improved pwv in middle- and older - aged people and pre- and stage 1 hypertensive patients . to the best of our knowledge , one study demonstrated that endurance athletes showed a higher pww compared to recreational active control subjects . although this mechanism is unclear , it is assumed that repeated and excessive training stress due to lower resting heart rate , resulting in increased stroke volume , imposed on the elastic component , may cause mechanical fatigue of the arterial wall . during resistance exercise , for example , weight lifting , both systolic and diastolic blood pressures increase dramatically [ 75 , 76 ] , whereas only systolic blood pressure increases during aerobic exercise , without an accompanying increase in diastolic blood pressure [ 77 , 78 ] . thus , in the classical paradigm , resistance training would not be recommended for aged people , and in particular , for patients with cardiovascular disease . however , recent studies have revealed that aerobic training alone can not maintain muscle volume and strength , which are needed to prevent sarcopenia [ 12 , 13 ] , the degenerative loss of skeletal muscle with advanced age , often leading to a bed - ridden lifestyle with reduced activities of daily living ( adls ) . resistance exercise is widely recommended to protect against metabolic syndrome , because such exercise can increase muscle strength and volume and may have a positive effect on glucose metabolism , blood lipids , and basal metabolic rate [ 13 , 14 ] . however , because of conventional wisdom regarding the dangers of resistance training , studies about the impact of resistance training on vascular function are ten years behind where they might be today . however , results of more recent studies , which investigated effects of resistance exercise and training on vascular function , seem to be controversial . it was also reported that ischemic reperfusion injury induced by 20-min cuff occlusion significantly reduced brachial artery fmd in sedentary young men but unchanged in resistance - trained adults . demonstrated that a significant inverse association between muscular strength and central pww , independent of aerobic fitness , suggested that resistance training , which can improve muscular strength , might improve aortic stiffness . however , shorter period of resistance training , that is , four weeks , improved peak forearm blood flow induced by reactive hyperemia but increased arterial stiffness in pre- and stage-1 hypertensives . similarly , slow movement resistance training increased basal limb blood flow as well as resistance training at normal speed , and weight training increased forearm blood flow as well as aerobic exercise in healthy males . from the point view of comparison in different populations , it was reported that decreased augmentation index was observed in old women , but not in old men . indicated that resistance training leads to increase in microvascular endothelial function in african - american and white men , while it increased brachial stiffness in only african - american . additionally , resistance training improved endothelium - dependent vasodilatation as well as aerobic training and aerobic plus resistance training in patients with recent myocardial infarction , indicating that these improvements were independent of exercise type . recently , the effects of combined exercise , that is , aerobic and resistance training on vascular function have been elucidated [ 100 , 101 ] . combined training consisting of high - intensity resistance training , followed by 30-min aerobic leg cycling , demonstrated a slight increase in carotid arterial compliance ( p = 0.06 ) in young men ; furthermore , moderate - intensity combined circuit resistance exercise and endurance exercise improved arterial stiffness in postmenopausal women . moreover , 1-year resistance training intervention in overweight women significantly improved fmd , and low - intensity resistance training with short interset rest period improved bapwv and fmd in young subjects . although the physiological mechanisms underlying the vascular function , such as arterial stiffness , compliance , and endothelial function assessed by fmd , associated with resistance training are unclear , it is well known that higher - intensity resistance training may be a potent stimulator to increase sympathetic nervous system activity [ 108 , 109 ] . during resistance exercise , for example , weight lifting , these acute elevated blood pressures during resistance exercise may alter the arterial structure , and/or arterial load - bearing properties , resulting in arterial stiffness increase and/or impaired reactive hyperemic blood flow with repeated exposure . as it is established that ne release is strongly related to the changes in absolute levels of sympathetic nerve activity , elevated ne concentration suggested that resting sympathetic nerve activity increased by 10-week upper body resistance training , resulted in that increased arterial stiffness caused by alteration arterial structure , and/or arterial load - bearing properties . in recent years , studies on one alternative , namely , low - intensity resistance training with bfr and bfr walking , have provided compelling data that such training leads to muscle hypertrophy and strength increases [ 17 , 1922 , 24 , 113117 ] and results in adaptations equal to those of high - intensity resistance training [ 21 , 22 ] . although the precise mechanism by which bfr exercise produces muscle hypertrophy is still unclear , it was reported that low - intensity exercise with bfr can increase that rate of muscle protein synthesis and stimulate mammalian target of rapamycin complex 1 ( mtorc1 ) and mapk - mediated anabolic signaling . we recently investigated the effects of short - term resistance training with bfr on muscle mass and strength , and found that elevated metabolic stress may be a crucial factor in obtaining successful results from resistance training with bfr . however , since hyperemic blood flow per se may be induced by greater shear stress and this greater shear stress induces vasodilatation , which results in increased nitric oxide [ 49 , 50 ] , moreover , maximal dilation was observed after ischemic exercise , there might be a possibility that bfr exercise and training would have a beneficial effect on vascular function such as arterial compliance and endothelial function , indeed , observed . tables 1 and 2 summarize studies undertaken to investigate the effects of acute or chronic bfr exercise on vascular function such as arterial compliance , endothelial function , and related biomarkers . it has been difficult to obtain consensus due to the large number of variables such as age , gender , intensity and exercise modality , intervention period , applied cuff pressure and cuff width , and evaluation of vascular function , all of which may influence the training effect . we discuss the influence of bfr exercise and training on vascular function based on current findings in the following session . similarly , four - weeks bfr training did not alter arterial stiffness evaluated by the pwv between the femoral and tibial arteries in young male subjects . the underlying physiological mechanism(s ) associated with unaltered arterial compliance , that is , four intervention studies , and with increased arterial compliance , that is , one cross - sectional study , is unclear . impaired arterial compliance induced by high - intensity resistance training without bfr may be attributed to acute elevated blood pressure via increasing sympathetic nerve activity system [ 108110 ] , resulting in alteration arterial structure and/or arterial load - bearing properties . moreover , the resistance training with and without bfr - induced carotid arterial compliance changes was associated with changes in systolic blood pressure during training intervention , indicating that elevation blood pressure may play a role to induce arterial compliance changes during bfr training . the interesting considerations regarding their study are that the training period was longer than in previous studies ( 10 weeks versus 3 to 6 weeks ) , and the participants were elderly , with the majority being women ( 3 men and 10 women ) . for example , ( 1 ) reducing oxidative stress improves carotid arterial compliance in postmenopausal women but not older men , ( 2 ) habitual exercise abolished age - related differences in central arterial stiffness in older women , ( 3 ) vasoconstrictor responses to sympathetic stimulation is blunted in women compared to men ( see in detail ) . these gender differences in cardiovascular responses and adaptation to exercise training might affect vascular function in response to bfr exercise and training though only one study showed that carotid arterial compliance was improved by bfr training in elderly women . in the previous cross - sectional study , carotid arterial compliance in middle - aged and older men is 2035% higher than that in age - matched recreational and sedentary subjects . taken together , bfr exercise and training did not change arterial compliance , while only two studies showed that acute bfr exercise and chronic bfr walking improved arterial compliance [ 124 , 134 ] . however , due to insufficient evidence , at least what we can say is that bfr training including resistance exercise and walking may not worse arterial compliance , possibly , associated with unchanged resting blood pressure with bfr resistance training [ 127 , 129 ] and bfr walking . endothelium - dependent vasodilatation in conduit artery , for example , brachial and popliteal arteries , can be evaluated by fmd , which is a good in protecting against cardiovascular disease [ 5153 ] . in their study , systemic cardiovascular responses , such as blood pressure , and total peripheral resistance significantly increased and stroke volume / pulse pressure ( an index of systemic arterial compliance ) significantly decreased during walking compared to those in the control condition while abi showed a similar value . demonstrated that four - weeks hand - grip training with bfr did not alter fmd responses but induced transient adaptations to brachial artery structure , although the mechanism underlying impaired fmd responses after acute bfr walking and chronic bfr training is still unclear , it is assumed that reduced fmd is a product of diminished endothelial function . as increased oxidative stress is associated with endothelial dysfunction [ 142 , 143 ] , followed by arteriosclerosis , augmented oxidative stress plays an important role in the pathogenesis and development of cardiovascular disease . although exhaustive exercise in humans has been shown to cause a sustained elevation of muscular xanthine oxidase activity as well as increases in serum il-6 and lp concentrations , it did not seem to produce an excess amount of oxygen - derived free radicals in takarada et al . it was also reported that protein carbonyls and blood glutathione , which are indicators of oxidative stress , did not increase during low - intensity ( 30% 1 rm ) bfr exercise , but did increase in moderate - resistance exercise ( up to 70% 1 rm ) . thus , there is a possibility that increased oxidative stress may have reduced the fmd in the case of credeur 's study because it was reported that the region between 60 and 70% 1 rm may be a critical load marker [ 145151 ] , in terms of inducing oxidative stress during resistance training without bfr . since acute elevations in arterial blood pressure are associated with the arterial structure and/or the arterial load - bearing properties of collagen and elastin , the rise in blood pressure during bfr walking might have caused the diminished fmd . collectively , although the influence of bfr walking and training with small muscle group on endothelial dysfunction assessed by fmd is still controversial , these contradictory results may be dependent on the variety of methodologies used , including exercise intensity and occlusion time during exercise . however , it should be still noted that cuff release - induced hyperemic blood flow may be expected , suggesting that it stimulates shear stress , followed by nitric oxide increase , simultaneously , bfr exercise and training should be carefully prescribed because it is supposed that systemic arterial compliance increase during bfr walking . as large muscle group dynamic exercise may alter not only local vasculature but also systemic cardiovascular responses to either acute or chronic exercise , small muscle group exercise may be useful as a physiological model to observe impact of exercise training on localized vasomotor control in skeletal muscles without marked changes in central hemodynamics during exercise . accordingly , two studies of handgrip exercise training with bfr have been investigated , furthermore , studies of calf raise with own body weight and planter flexion exercise training with bfr were also carried out . enhanced brachial fmd responses as well as endothelium - dependent vasodilatation were observed after isometric handgrip training in old men , in elderly hypertensives [ 156 , 157 ] , and chronic heart failure subjects [ 158 , 159 ] . in addition to these previous studies of handgrip exercise training without bfr , two studies of handgrip exercise training with bfr have been elucidated , resulted in impaired and unchanged fmd responses . as above stated , exercise intensity , which was used in one previous study , was higher and longer , that is , 60% mvc dynamic handgrip exercise training for 20 min , compared to another previous study with bfr ( tables 1 and 2 ) . indeed , in human studies , brachial artery fmd increases after just one week of handgrip exercise training and decreased after two weeks , whereas conduit artery vasodilator capacity showed a progressive increase for eight weeks during training intervention . [ 178 , 179 ] reported that thrombin antithrombin iii complex ( tat ) and d - dimer increased after 6090 min prolonged exercise but did not increase after high - intensity exercise , from which blood lactate concentrations were significantly increased , suggesting that the tat and d - dimer may be dependent on exercise time . more important findings were that the incidence of serious side effects related to thrombosis was lower in 7 cases ( 0.055% ) based on the national survey for more than 12,000 people in japan . however , since no studies have examined the effects of chronic bfr training on vegf , it is uncertain whether chronic bfr training can increase vefg , resulting in an improvement of endothelial function . in this review , we focused on what is currently known ( tables 1 and 2 ) and hypothesised changes ( figure 1 ) of the influences of bfr exercise and training on vascular function . also , the effect of bfr training on vascular function may be influenced by various factors , such as , age , sex , exercise type , intensity , applied cuff pressure , and intervention period , not to mention the different evaluative methods for vascular function . therefore , bfr exercise and training seems to become novel method because bfr exercise and training can obtain muscle hypertrophy as well as high - intensity resistance training even if bfr exercise and training does not affect vascular function . future studies should be conducted with the aim of elucidating the mechanisms of the influence of bfr training on vascular function , with careful selection of the influencing parameters mentioned above and the potential therapeutic benefits for the elderly and for the rehabilitation of patients with cardiovascular disease and physical injury .

tooth loss is one of the common causes of reduced quality of life in adults . dental implants have become a widely accepted treatment option for patients with both partially and complete edentulous posterior mandibles . rehabilitation of edentulous posterior mandibular regions with severe ridge atrophy using implants is subject to anatomical , surgical and biological difficulties and poses a challenge to dental teams . osseointegrated dental implants are often placed in the posterior mandible , mostly to support fixed restorative prostheses . in many cases , the bone has so severely atrophied that sufficiently long fixtures can not be placed without encroaching on the inferior alveolar nerve ( ian ) . in this situation , restorative options include the use of short fixtures , onlay bone grafting to increase ridge height , and more complicated and detailed imaging analysis to allow positioning of implants alongside , rather than into , the nerve canal during the procedure . another option is to move the ian laterally from its canal by either nerve lateralization ( ianl ) . with nerve lateralization , the ian is exposed and traction is used to deflect it laterally while the implants are placed . the ian is then left to fall back into position , against the fixtures . with this procedure , there is no interference with the incisive nerve . with nerve transposition , a corticotomy is done around the mental foramen and the incisive nerve is transacted , such that the mental foramen is repositioned more posteriorly . in the posterior mandible , the bone quality may not be as good as it is in the anterior mandible . in particular , if shorter implants are used to ensure that there is no encroachment on the nerve canal , initial implant stability will be unicortical . in addition , there is a risk to the ian as the operator tries to maximise implant length on the basis of measured available bone height . the advantages of iant include the ability to place longer fixtures and to engage 2 cortices for initial stability . kan et al . pointed out that the amount of bone superior to the mandibular canal ( mc ) is often insufficient for the placement of fixtures of the ideal length . in addition , the existing bone that is superior to the mc is often of poorer quality than its cortical counterpart . these factors and the fact that shorter implants have been associated with higher failure rates have led to the development of methods of ian repositioning that allow placement of longer fixtures ; with these methods , the inferior cortex of the mandible is engaged , which leads to greater initial stability . therefore , the purpose of the present paper was to review the literature concerning the indications , contraindications , advantages , disadvantages and the surgical techniques of the lateralization and transposition of the inferior alveolar nerve , followed by the implants placement in an edentulous atrophic posterior mandible . ian lateralization ( ianl ) and transposition ( iant ) definition ianl and iant are surgical procedures that reposition the ian for the purpose of implant placement without bone augmentation . this procedure raises the risk of neuropathies , such as paresthesia , hypoesthesia , and anaesthesia of the ian . ianl is defined as the lateral reflection of the ian without incisive nerve traction ( preservation of the incisive nerve and lateralization of inferior alveolar neurovascular bundle posterior to the mental foramen ) ; exposure and traction are used to deflect the ian laterally while the implants are placed . the ian is then left to fall back in against the fixtures . during the iant procedure , a corticotomy is done around the mental foramen and the incisive nerve is transacted ( incisive neurovascular bundle is sacrificed ) , to allow transposition of both the mental foramen and the ian such that the mental foramen is repositioned more posteriorly . the first case of ian repositioning was reported by alling ( 1977 ) to rehabilitate patients with severe atrophy for dentures . in 1987 , jenson and nock carried out iant for the placement of dental implants in posterior mandibular regions . according to subjective criteria , sensory function of the mental nerve returned to normal five weeks post - operative . there is limited information about implants survival rate . in 1992 , rosenquist performed the first case series study using iant followed by 26 implants placement on 10 patients . neurosensory function of the mental nerve was evaluated objectively using the two - point discrimination ( 2-pd ) method . at one year follow - up , he reported an implant survival rate of 96% and implant survival rate of 93.6% for this procedure ; therefore , this technique was accepted as the best - practice treatment modality for reconstruction of the dentoalveolar system with dental implants in the posterior mandible . as a result , this technique is constantly undergoing improvement . iant is a new procedure that needs further refinements in terms of technique and instrumentation to decrease complications . the review included studies , case reports , clinical trials on human subjects that were published in english between january 1997 and july 2014 , and included a minimum of 6 months of follow - up . letters and phd theses were excluded , as well as abstracts and reviews and studies on animals . the information source was the medline ( ncbi pubmed and pmc ) database and other scientific electronic sources . according to the prisma guidelines , an electronic search was conducted using the medline ( ncbi pubmed and pmc ) database to locate articles concerning ian lateralization or transposition and implant placement in an edentulous atrophic posterior mandible . the search terms used were : inferior alveolar nerve lateralization , inferior alveolar nerve repositioning , inferior alveolar nerve transposition , implants in atrophic posterior mandible + repositioning , inferior alveolar nerve transposition + mental , implants in atrophic posterior mandible + lateralization , implants in atrophic posterior mandible + transposition , mandibular atrophy + repositioning , and inferior alveolar nerve + minimal bone height . due to the low number of relevant articles and to ensure the sensitivity of the systemic review process , articles were searched from january 1997 to july 2014 . titles derived from this broad search were independently screened by two authors based on the inclusion criteria . full reports were obtained for all the studies that were deemed eligible for inclusion in this paper . inclusion and exclusion criteria inclusion criteria for the selection were : articles regarding to ianl and iant procedures ; all articles in english ; clinical reports with minimum 6 months follow - up ; information regarding implant osseointegration and survival ; studies on adult ( between the ages 18 and 80 ) human beings , with no immunologic diseases , uncontrolled diabetes mellitus , osteoporosis , or other contraindicating systemic conditions . exclusion criteria for the selection were : clinical reports with no minimum 6 months of follow - up ; not enough information regarding the selected topic ; no information regarding implant osseointegration and survival ; studies of patients with immunologic diseases , uncontrolled diabetes mellitus , osteoporosis or other contraindicating systemic conditions ; studies of adolescents ( under 18 years of age ) and elderly people ( over 80 ) . article review and data extraction article review and data extraction was performed according to a prisma flow diagram ( figure 1 ) . the search displayed 876 results from the ncbi pmc and pubmed databases , and 3 results from other sources ( dental-tribune.com , acta.tums.ac.ir , hindawi.com/journals ) . a total of 38 titles and abstracts were selected according to relevancy after the removal of duplications . another exclusion was made based upon follow - up time ( n = 6 ) and information regarding implant osseointegration and survival ( n = 4 ) . studies of adult human beings between 18 and 80 years of age with minimal residual bone above the mc , in which ianl and iant + implant placement had been performed , were selected . data collection process data was independently extracted from reports in the form of variables according to the aim and themes of the present review as listed below . the review aims to achieve the following : to describe the purpose ( indications and contraindications ) of ianl and iant . to describe the current surgical techniques used for ianl and iant with simultaneous implant placement . risk of bias assessment risk of bias ( e.g. , lack of information or selective reports on variables of interest ) was assessed at the study level . the risks were indicated as lack of precise information of interest in each individual study that can blind the reader from particular information about the examined samples . the cochrane collaboration tool for assessing risk of bias was used to assess bias across the studies that could affect cumulative evidence . the search displayed 876 results from the ncbi pmc and pubmed databases , and 3 results from other sources ( dental-tribune.com , acta.tums.ac.ir , hindawi.com/journals ) . a total of 38 titles and abstracts were selected according to relevancy after duplications removal . exclusion was made according to information amount regarding selected topic ( n = 12 ) . twenty - six full - text articles were in the end , assessed for eligibility . during the eligibility stage , articles that did not meet the inclusion and exclusion criteria where filtered as follows : no minimum 6 months of follow - up ( n = 6 ) and no information regarding implant osseointegration and survival ( n = 4 ) . in the end , 16 articles that included data on 160 patients were utilised for the systematic review ( figure 1 ) . nine were related to iant , 4 to ianl and 3 to both iant and ianl ( table 1 ) . description of studies included in the review risk of bias within studies the cochrane collaboration bias summary for potential bias was used to assess the quality of studies and identify papers with intrinsic flaws in method and design . after analysing the risk of bias ( table 2 ) , we found that six authors [ 5,13 - 17 ] selected the patients randomly . in four studies [ 1 - 3,5 ] , the surgical procedure was not performed by the authors ; and in two studies , the method of implant osseointegration evaluation was not mentioned . assesment of the risks of bias results of individual studies results of individual studies of implant treatment using are shown in table 3 . results of individual studies ianl = inferior alveolar nerve lateralization ; iant = inferior alveolar nerve transposition . from the results of individual study data ( table 3 ) , we can conclude that the most popular surgical technique for ian repositioning was iant . it was calculated that 51.44% ( 107/208 ) of all operations performed utilised iant , and 48.56% ( 101/208 ) utilised ianl . number of implants placed after iant was 211 , while number of implants placed after ianl was 95 . in the study by lorean et al . ( in which 68 ianl and 11 iant were performed ) , the authors failed to mention how many implants were placed during each procedure . it was 88% ; in ferrigno et al . , it was 95.7% ; and kan et al . presented a 93.8% implant survival rate.the lowest implant survival rates were found in kan et al . with 33.33% and karlis et al . with the most popular method of implant osseointegration evaluation was panoramic radiograph ( 12/16 studies ) . in two studies , both of which were performed by the same author ( proussaefs ) , the method of implant osseointegration evaluation was a perio - test unit ( siemens , bensheim , germany ) ; while in two other studies ( lorean et al . , kan et al . ) , the implant osseointegration evaluation method was not mentioned . the purpose ( indications and contraindications ) of ianl and iant the study of number selected articles [ 9,22 - 25 ] revealed the following indications of ianl and iant : the major reason for using this technique is to prevent ian injury during implant placement in edentulous posterior atrophic mandibles . class iv , v , or vi of cawood and howell with extrusion of the antagonist tooth and reduced prosthetic free space . class v or vi of cawood and howell with presence of interforaminal teeth ( patients were not candidates for interforaminal implant - prosthetic methods ) . class v or vi of cawood and howell if the patient desires a fast implant - prosthetic rehabilitation with predictable outcomes . in orthognathic surgeries , such as lower border shaving and total mandibular subapical osteotomy . in the pre - prosthetic surgery . in the anastomosis and repairing of a disrupted ian . preservation of ian in cancer surgery in the posterior mandible . when placement of short implants is not a viable option ( in case of severely atrophic mandibles when the residual bone above mc ranges between 0.5 and 1.5 mm ) . less than 10 - 11 mm bone height above the canal , when the quality of the spongy bone does not provide sufficient stability for implant placement . contraindications if the mandible presents advanced resorption of the alveolar process . if the patient has poor general health , including systemic diseases that may worsen the patient s health condition after the ian reposition procedure . limitations in accessing the surgical site . people who become easily stressed out and are over sensitive even towards the smallest surgical complications . such patients do not have tolerance and compatibility skills and , therefore , are not good candidates for nerve transposition surgery . the current surgical techniques utilised for lateralization and transposition of ian with simultaneous implant placement the preoperative work - up included an assessment of the ian using appropriate diagnostic records , such as a panoramic radiograph , a computed tomography ( ct ) scan , casts , diagnostic wax - up , and surgical templates . during preoperative consultation with the patients , the risk of postoperative neurosensory disturbances ( nd ) that can result following the ian repositioning is discussed . this possibility gives many patients pause to consider the ramifications of the procedure . to help the patient decide whether this would be tolerable , the clinician can perform a preoperative block with a long - acting local anaesthetic , such as marcaine , which reproduces symptoms lasting 8 to 16 hours that are similar to the postoperative anaesthesia the patient may experience . two ian repositioning techniques have been developed : lateralization and transposition ( table 4 ) . both procedures include an ian block plus local infiltrating anaesthesia with vasoconstrictors . intravenous sedation is recommended because of the procedure s technique - sensitive nature and the need for patient cooperation . a crestal incision with anterior- and posterior releasing incisions is made , and a labial mucoperiosteal flap is reflected that exposes the alveolar ridge and buccal cortex . the incision should extend at least 1 cm beyond the anticipated site of the osteotomy . care is taken during flap reflection to preserve the integrity of the periosteum and the neurovascular bundle where it exits the mental foramen and enters the soft tissue . to increase the flaps relaxation and improve exposure the flaps are raised , one lingual to access the alveolar crest , and another vestibular for subperiosteal dissection of the ian , until sufficient surgical field is obtained . ct is used to locate the approximate area of the mental foramen , after which blunt dissection is used to identify and isolate the mental nerve . from this point inferior alveolar nerve ( ian ) replacement division into two distinct surgical procedures , i.e. transposition and lateralization ; the steps are outlined below prf = platelet - rich fibrin . ian transposition ( iant ) an osteotomy is performed at the mental foramen , drilling around the orifice to obtain a ring of external cortical bone . a window also may be made about 5 mm ahead of the foramen , in order to avoid damaging the nerve over its anterior loop . an en bloc osteotomy is then made at the external cortical level , or a posterior window is performed in the external cortical layer along the intrabony trajectory of the nerve ( figure 2 ) . a round drill is used to create the window , replacing it with a diamond drill while working close to the mc , to minimise the risk of nerve damage . bone is removed using a round bur number 700 or 701 , a straight handpiece and copious normal saline for irrigation or a piezosurgery device . in order to secure complete mobilisation of the ian , the incisor branch , located about 5 mm from the mental foramen , a = before transposition ; b = after transposition ( courtesy of dr dainius razukevicius , kauno implantologijos centras kaunas , lithuania ) . then , with the nerve fully lateralized , the dental implants are placed under direct visualisation - in this case bicortically , taking advantage of the mandibular basal layer . once the implants have been positioned , the vestibular cortical layer is replaced in those cases where an osteotomy has been performed , or the nerve is passively positioned against the implants in those cases where cortical drilling has been carried out . in either case , the emergence of the nerve becomes more distal . ian lateralization ( ianl ) in this case , neither dissection of the terminal branches of the ian nor sectioning of the incisor branch is needed . the technique involves the preparation of a cortical bone window ( via osteotomy or drilling ) that is located posterior to the mental foramen ( figure 3 and figure 4 ) . intraoperative photography showing the inferior alveolar neurovascular bundle lateralization ( courtesy of dr dainius razukevicius , kauno implantologijos centras kaunas , lithuania ) . schematic drawing showing the inferior alveolar neurovascular bundle transposition ( a ) and lateralization ( b ) . bone is removed using a round bur number 700 or 701 , a straight handpiece and copious normal saline for irrigation or a piezosurgery device . after removing the cortical bone , a curette may be used for removal of spongy bone and the cortical layer of the canal in cases where the cortical layer surrounding the canal is not dense or thick . a special instrument ( hassani nerve protector , ali hassani , iran ) is required to protect the ian while the cortical layer is removed using surgical burs or a piezosurgery device . bone removal in close vicinity to the neurovascular bundle should be performed patiently and thoroughly . this is usually performed using special curettes parallel to the surface of the nerve bundles in an antero - posterior direction . the area should be thoroughly irrigated so that the nerve bundle can be clearly seen . the neurovascular bundle inside the canal is freed using special curettes and is moved laterally using a nerve hook . then , a 10 mm wide gauze cord or elastic band is passed below the nerve to retract it from the surgical site in order to decrease the risk of ischemic trauma to the nerve . the second purpose of retracting the nerve from the surgical site during the operation is to reduce the risk of ian damage . after carefully freeing the nerve , the latter is separated using a vessel loop - while applying gentle traction outwards as the implants are positioned , the implant should be long enough to pass the mc and engage the basal body below the canal to achieve sufficient primary stability . finally , the vessel loop is removed and the nerve is replaced , positioning a resorbable membrane between it and the bone window to avoid direct contact with the implants . the procedure is completed by suturing the mucoperiosteal flap . the decision whether to use iant or ianl depends on the amount of stretching that is needed in order to mobilize the ian . study , stretching the nerve by 10 - 17% of its original length may result in disruption of the nerve fibers internally . in situations where only small traction is needed , anl may be utilised . kan et al . retrospective study demonstrated that the nd for iant were 77.8% ( 7/9 ) and for the ianl 33.3% ( 4/12 ) . as a summary of both surgical techniques ( iant and ianl ) , it can be concluded that ianl produces less side effects than iant . the use of piezosurgery to avoid ian damage some studies recommend piezosurgery for bone removal in nerve transposition surgery . this device causes vibrations in the range of 20 - 200 m and cuts through the mineralized tissue completely and smoothly . if soft tissue or the neurovascular bundle comes in contact with this device , it stops functioning because it is designed to stop working when it contacts unmineralized tissue . this device is especially beneficial when a small osteotomy is going to be performed . among the disadvantages of this device are the long duration of time that it takes to remove bone . also , there remains controversy regarding the indications of piezosurgery devices , and some believe that the vibrations they generate may damage the nerve . repositioning the neurovascular bundle inside the mc before this phase , the surgeon should decide whether or not to place materials between the implant and the inferior alveolar neurovascular bundle . the preference is to place a collagen membrane or bone material between the implant and the inferior alveolar neurovascular bundle . a potential advantage of bone over a membrane is that , if proper healing occurs in the area , the contact area of implant and bone will increase . before releasing the inferior alveolar neurovascular bundle from the elastic band this way , the inferior alveolar neurovascular bundle will be in a vent that is medially adjacent to the implants and covered by the mucoperiosteal flap . alternatively , the inferior alveolar neurovascular bundle may be left to lie passively outside of the mc . the bone defect is then covered by several methods : repositioning the bony window that was removed or the bony window can be crushed and mixed with an allograft or xenograft . postoperative measures antibiotic and corticosteroid prophylaxis is recommended because of the extensiveness and duration of surgery . however , there is no consensus in this regard ; since inflammation can be among the causes of nerve dysfunction , corticosteroid therapy can be beneficial . after analysing the current literature concerning ianl and iant , several advantages and disadvantages of the procedures were found and are described below . the advantages of the ianl and iant longer implants can be placed in the same surgical step . possibility of placement of a greater number of implants , which improves the overall strength of the final prosthesis . possibility for simultaneous placement of implants during surgery , which allows a reduction in treatment time compared with other techniques as bone grafts that require a long waiting period before implant insertion together with additional surgeries . the option for immediate loading for the enhancement of masticatory function , dramatically improving the patient s quality of life . the evaluation values for implant survival rates are similar to those for standard implantation procedures . as a biomechanical advantage , ian transposition presents an increase in resistance to occlusal forces and promotes a good proportion between the implant and the prosthesis . in addition , with this procedure , all of the following are accomplished : restoration of the correct vertical dimension ; prevention of tissues atrophy ; replacement of dentures ; stabilisation of the anterior dentition ; temporomandibular joint and masticatory muscle balance . the disadvantages of the ianl and iant one of the disadvantages of this procedure is that it does not recover alveolar ridge anatomy . another negative point of this procedure is that it temporarily weakens the mandible due to removal of cortical bone ; which , in combination with implant placement , may lead to mandibular fracture at the operation site . according to peleg , cylindrical non - threaded implants are recommended after the ianl procedure , as threaded implants in close contact with the nerve may cause neurosensory problems . this recommendation may be a disadvantage because non - threaded implants have a lower surface area compared to threaded implants , which decreases the stability of the implant . since this surgical procedure is delicate , it is best performed under a general anesthesia to eliminate patient movement and to maximise access . the general anaesthetic management of patients with myotonic dystrophy ( dystrophia myotonica ) can , however , be challenging . the most common failures are due to bruxism and poor occlusal relations ; therefore , patients with bruxism or poor occlusal relations make , implant stability and survival to less favourable . . the main limitations of this review were that six authors [ 5,13 - 17 ] selected the patients randomly ; in four studies [ 1 - 3,5 ] , the surgical procedure was not performed by the authors and , in two studies , the method of implant osseointegration evaluation was not mentioned . inferior alveolar nerve repositioning is a technique that has been used for more than 20 years with good survival and survival rates . this is sometimes the only possible procedure to help patients to obtain a fixed prosthesis , especially in edentulous atrophic posterior mandibles . there are two main techniques for inferior alveolar nerve repositioning that are relatively safe and offer a high survival rate : inferior alveolar nerve lateralization and inferior alveolar nerve transposition . increased protection of the neurovascular bundle inferior alveolar nerve lateralization and transposition in combination with the installation of dental implants offer advantages , such as reducing the risk of inferior alveolar nerve damage . with careful pre - operative surgical and prosthetic planning , imaging , and extremely precise surgical technique , this procedure can be successfully used for implant placement in an edentulous atrophic posterior mandible .

abstractobjectivesthe purpose of this first part of a two - part series was to review the literature concerning the indications , contraindications , advantages , disadvantages and surgical techniques of the lateralization and transposition of the inferior alveolar nerve , followed by the placement of an implant in an edentulous atrophic posterior mandible.material and methodsa comprehensive review of the current literature was conducted according to the prisma guidelines by accessing the ncbi pubmed and pmc database , academic sites and books . the articles were searched from january 1997 to july 2014 and comprised english - language articles that included adult patients between 18 and 80 years old with minimal residual bone above the mandibular canal who had undergone inferior alveolar nerve ( ian ) repositioning with a minimum 6 months of follow-up.resultsa total of 16 studies were included in this review . nine were related to ian transposition , 4 to ian lateralization and 3 to both transposition and lateralization . implant treatment results and complications were presented.conclusionsinferior alveolar nerve lateralization and transposition in combination with the installation of dental implants is sometimes the only possible procedure to help patients to obtain a fixed prosthesis , in edentulous atrophic posterior mandibles . with careful pre - operative surgical and prosthetic planning , imaging , and extremely precise surgical technique , this procedure can be successfully used for implant placement in edentulous posterior mandibular segments .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS

tooth loss is one of the common causes of reduced quality of life in adults . dental implants have become a widely accepted treatment option for patients with both partially and complete edentulous posterior mandibles . rehabilitation of edentulous posterior mandibular regions with severe ridge atrophy using implants is subject to anatomical , surgical and biological difficulties and poses a challenge to dental teams . osseointegrated dental implants are often placed in the posterior mandible , mostly to support fixed restorative prostheses . in many cases , the bone has so severely atrophied that sufficiently long fixtures can not be placed without encroaching on the inferior alveolar nerve ( ian ) . in this situation , restorative options include the use of short fixtures , onlay bone grafting to increase ridge height , and more complicated and detailed imaging analysis to allow positioning of implants alongside , rather than into , the nerve canal during the procedure . another option is to move the ian laterally from its canal by either nerve lateralization ( ianl ) . with nerve lateralization , the ian is exposed and traction is used to deflect it laterally while the implants are placed . with this procedure , there is no interference with the incisive nerve . with nerve transposition , a corticotomy is done around the mental foramen and the incisive nerve is transacted , such that the mental foramen is repositioned more posteriorly . pointed out that the amount of bone superior to the mandibular canal ( mc ) is often insufficient for the placement of fixtures of the ideal length . in addition , the existing bone that is superior to the mc is often of poorer quality than its cortical counterpart . these factors and the fact that shorter implants have been associated with higher failure rates have led to the development of methods of ian repositioning that allow placement of longer fixtures ; with these methods , the inferior cortex of the mandible is engaged , which leads to greater initial stability . therefore , the purpose of the present paper was to review the literature concerning the indications , contraindications , advantages , disadvantages and the surgical techniques of the lateralization and transposition of the inferior alveolar nerve , followed by the implants placement in an edentulous atrophic posterior mandible . ian lateralization ( ianl ) and transposition ( iant ) definition ianl and iant are surgical procedures that reposition the ian for the purpose of implant placement without bone augmentation . this procedure raises the risk of neuropathies , such as paresthesia , hypoesthesia , and anaesthesia of the ian . ianl is defined as the lateral reflection of the ian without incisive nerve traction ( preservation of the incisive nerve and lateralization of inferior alveolar neurovascular bundle posterior to the mental foramen ) ; exposure and traction are used to deflect the ian laterally while the implants are placed . during the iant procedure , a corticotomy is done around the mental foramen and the incisive nerve is transacted ( incisive neurovascular bundle is sacrificed ) , to allow transposition of both the mental foramen and the ian such that the mental foramen is repositioned more posteriorly . in 1987 , jenson and nock carried out iant for the placement of dental implants in posterior mandibular regions . according to subjective criteria , sensory function of the mental nerve returned to normal five weeks post - operative . in 1992 , rosenquist performed the first case series study using iant followed by 26 implants placement on 10 patients . neurosensory function of the mental nerve was evaluated objectively using the two - point discrimination ( 2-pd ) method . at one year follow - up , he reported an implant survival rate of 96% and implant survival rate of 93.6% for this procedure ; therefore , this technique was accepted as the best - practice treatment modality for reconstruction of the dentoalveolar system with dental implants in the posterior mandible . as a result , this technique is constantly undergoing improvement . the review included studies , case reports , clinical trials on human subjects that were published in english between january 1997 and july 2014 , and included a minimum of 6 months of follow - up . the information source was the medline ( ncbi pubmed and pmc ) database and other scientific electronic sources . according to the prisma guidelines , an electronic search was conducted using the medline ( ncbi pubmed and pmc ) database to locate articles concerning ian lateralization or transposition and implant placement in an edentulous atrophic posterior mandible . the search terms used were : inferior alveolar nerve lateralization , inferior alveolar nerve repositioning , inferior alveolar nerve transposition , implants in atrophic posterior mandible + repositioning , inferior alveolar nerve transposition + mental , implants in atrophic posterior mandible + lateralization , implants in atrophic posterior mandible + transposition , mandibular atrophy + repositioning , and inferior alveolar nerve + minimal bone height . due to the low number of relevant articles and to ensure the sensitivity of the systemic review process , articles were searched from january 1997 to july 2014 . full reports were obtained for all the studies that were deemed eligible for inclusion in this paper . inclusion and exclusion criteria inclusion criteria for the selection were : articles regarding to ianl and iant procedures ; all articles in english ; clinical reports with minimum 6 months follow - up ; information regarding implant osseointegration and survival ; studies on adult ( between the ages 18 and 80 ) human beings , with no immunologic diseases , uncontrolled diabetes mellitus , osteoporosis , or other contraindicating systemic conditions . exclusion criteria for the selection were : clinical reports with no minimum 6 months of follow - up ; not enough information regarding the selected topic ; no information regarding implant osseointegration and survival ; studies of patients with immunologic diseases , uncontrolled diabetes mellitus , osteoporosis or other contraindicating systemic conditions ; studies of adolescents ( under 18 years of age ) and elderly people ( over 80 ) . the search displayed 876 results from the ncbi pmc and pubmed databases , and 3 results from other sources ( dental-tribune.com , acta.tums.ac.ir , hindawi.com/journals ) . a total of 38 titles and abstracts were selected according to relevancy after the removal of duplications . studies of adult human beings between 18 and 80 years of age with minimal residual bone above the mc , in which ianl and iant + implant placement had been performed , were selected . data collection process data was independently extracted from reports in the form of variables according to the aim and themes of the present review as listed below . to describe the current surgical techniques used for ianl and iant with simultaneous implant placement . the search displayed 876 results from the ncbi pmc and pubmed databases , and 3 results from other sources ( dental-tribune.com , acta.tums.ac.ir , hindawi.com/journals ) . a total of 38 titles and abstracts were selected according to relevancy after duplications removal . exclusion was made according to information amount regarding selected topic ( n = 12 ) . twenty - six full - text articles were in the end , assessed for eligibility . during the eligibility stage , articles that did not meet the inclusion and exclusion criteria where filtered as follows : no minimum 6 months of follow - up ( n = 6 ) and no information regarding implant osseointegration and survival ( n = 4 ) . in the end , 16 articles that included data on 160 patients were utilised for the systematic review ( figure 1 ) . nine were related to iant , 4 to ianl and 3 to both iant and ianl ( table 1 ) . description of studies included in the review risk of bias within studies the cochrane collaboration bias summary for potential bias was used to assess the quality of studies and identify papers with intrinsic flaws in method and design . in four studies [ 1 - 3,5 ] , the surgical procedure was not performed by the authors ; and in two studies , the method of implant osseointegration evaluation was not mentioned . assesment of the risks of bias results of individual studies results of individual studies of implant treatment using are shown in table 3 . results of individual studies ianl = inferior alveolar nerve lateralization ; iant = inferior alveolar nerve transposition . from the results of individual study data ( table 3 ) , we can conclude that the most popular surgical technique for ian repositioning was iant . it was calculated that 51.44% ( 107/208 ) of all operations performed utilised iant , and 48.56% ( 101/208 ) utilised ianl . with the most popular method of implant osseointegration evaluation was panoramic radiograph ( 12/16 studies ) . in two studies , both of which were performed by the same author ( proussaefs ) , the method of implant osseointegration evaluation was a perio - test unit ( siemens , bensheim , germany ) ; while in two other studies ( lorean et al . the purpose ( indications and contraindications ) of ianl and iant the study of number selected articles [ 9,22 - 25 ] revealed the following indications of ianl and iant : the major reason for using this technique is to prevent ian injury during implant placement in edentulous posterior atrophic mandibles . class iv , v , or vi of cawood and howell with extrusion of the antagonist tooth and reduced prosthetic free space . in the pre - prosthetic surgery . in the anastomosis and repairing of a disrupted ian . when placement of short implants is not a viable option ( in case of severely atrophic mandibles when the residual bone above mc ranges between 0.5 and 1.5 mm ) . less than 10 - 11 mm bone height above the canal , when the quality of the spongy bone does not provide sufficient stability for implant placement . contraindications if the mandible presents advanced resorption of the alveolar process . limitations in accessing the surgical site . the current surgical techniques utilised for lateralization and transposition of ian with simultaneous implant placement the preoperative work - up included an assessment of the ian using appropriate diagnostic records , such as a panoramic radiograph , a computed tomography ( ct ) scan , casts , diagnostic wax - up , and surgical templates . this possibility gives many patients pause to consider the ramifications of the procedure . to help the patient decide whether this would be tolerable , the clinician can perform a preoperative block with a long - acting local anaesthetic , such as marcaine , which reproduces symptoms lasting 8 to 16 hours that are similar to the postoperative anaesthesia the patient may experience . two ian repositioning techniques have been developed : lateralization and transposition ( table 4 ) . intravenous sedation is recommended because of the procedure s technique - sensitive nature and the need for patient cooperation . a crestal incision with anterior- and posterior releasing incisions is made , and a labial mucoperiosteal flap is reflected that exposes the alveolar ridge and buccal cortex . the incision should extend at least 1 cm beyond the anticipated site of the osteotomy . care is taken during flap reflection to preserve the integrity of the periosteum and the neurovascular bundle where it exits the mental foramen and enters the soft tissue . to increase the flaps relaxation and improve exposure the flaps are raised , one lingual to access the alveolar crest , and another vestibular for subperiosteal dissection of the ian , until sufficient surgical field is obtained . ct is used to locate the approximate area of the mental foramen , after which blunt dissection is used to identify and isolate the mental nerve . from this point inferior alveolar nerve ( ian ) replacement division into two distinct surgical procedures , i.e. transposition and lateralization ; the steps are outlined below prf = platelet - rich fibrin . ian transposition ( iant ) an osteotomy is performed at the mental foramen , drilling around the orifice to obtain a ring of external cortical bone . a window also may be made about 5 mm ahead of the foramen , in order to avoid damaging the nerve over its anterior loop . an en bloc osteotomy is then made at the external cortical level , or a posterior window is performed in the external cortical layer along the intrabony trajectory of the nerve ( figure 2 ) . a round drill is used to create the window , replacing it with a diamond drill while working close to the mc , to minimise the risk of nerve damage . in order to secure complete mobilisation of the ian , the incisor branch , located about 5 mm from the mental foramen , a = before transposition ; b = after transposition ( courtesy of dr dainius razukevicius , kauno implantologijos centras kaunas , lithuania ) . then , with the nerve fully lateralized , the dental implants are placed under direct visualisation - in this case bicortically , taking advantage of the mandibular basal layer . in either case , the emergence of the nerve becomes more distal . ian lateralization ( ianl ) in this case , neither dissection of the terminal branches of the ian nor sectioning of the incisor branch is needed . the technique involves the preparation of a cortical bone window ( via osteotomy or drilling ) that is located posterior to the mental foramen ( figure 3 and figure 4 ) . intraoperative photography showing the inferior alveolar neurovascular bundle lateralization ( courtesy of dr dainius razukevicius , kauno implantologijos centras kaunas , lithuania ) . schematic drawing showing the inferior alveolar neurovascular bundle transposition ( a ) and lateralization ( b ) . after removing the cortical bone , a curette may be used for removal of spongy bone and the cortical layer of the canal in cases where the cortical layer surrounding the canal is not dense or thick . bone removal in close vicinity to the neurovascular bundle should be performed patiently and thoroughly . this is usually performed using special curettes parallel to the surface of the nerve bundles in an antero - posterior direction . the area should be thoroughly irrigated so that the nerve bundle can be clearly seen . then , a 10 mm wide gauze cord or elastic band is passed below the nerve to retract it from the surgical site in order to decrease the risk of ischemic trauma to the nerve . the second purpose of retracting the nerve from the surgical site during the operation is to reduce the risk of ian damage . after carefully freeing the nerve , the latter is separated using a vessel loop - while applying gentle traction outwards as the implants are positioned , the implant should be long enough to pass the mc and engage the basal body below the canal to achieve sufficient primary stability . finally , the vessel loop is removed and the nerve is replaced , positioning a resorbable membrane between it and the bone window to avoid direct contact with the implants . study , stretching the nerve by 10 - 17% of its original length may result in disruption of the nerve fibers internally . as a summary of both surgical techniques ( iant and ianl ) , it can be concluded that ianl produces less side effects than iant . among the disadvantages of this device are the long duration of time that it takes to remove bone . also , there remains controversy regarding the indications of piezosurgery devices , and some believe that the vibrations they generate may damage the nerve . repositioning the neurovascular bundle inside the mc before this phase , the surgeon should decide whether or not to place materials between the implant and the inferior alveolar neurovascular bundle . the preference is to place a collagen membrane or bone material between the implant and the inferior alveolar neurovascular bundle . before releasing the inferior alveolar neurovascular bundle from the elastic band this way , the inferior alveolar neurovascular bundle will be in a vent that is medially adjacent to the implants and covered by the mucoperiosteal flap . alternatively , the inferior alveolar neurovascular bundle may be left to lie passively outside of the mc . postoperative measures antibiotic and corticosteroid prophylaxis is recommended because of the extensiveness and duration of surgery . however , there is no consensus in this regard ; since inflammation can be among the causes of nerve dysfunction , corticosteroid therapy can be beneficial . after analysing the current literature concerning ianl and iant , several advantages and disadvantages of the procedures were found and are described below . the advantages of the ianl and iant longer implants can be placed in the same surgical step . possibility of placement of a greater number of implants , which improves the overall strength of the final prosthesis . the evaluation values for implant survival rates are similar to those for standard implantation procedures . as a biomechanical advantage , ian transposition presents an increase in resistance to occlusal forces and promotes a good proportion between the implant and the prosthesis . in addition , with this procedure , all of the following are accomplished : restoration of the correct vertical dimension ; prevention of tissues atrophy ; replacement of dentures ; stabilisation of the anterior dentition ; temporomandibular joint and masticatory muscle balance . the disadvantages of the ianl and iant one of the disadvantages of this procedure is that it does not recover alveolar ridge anatomy . another negative point of this procedure is that it temporarily weakens the mandible due to removal of cortical bone ; which , in combination with implant placement , may lead to mandibular fracture at the operation site . according to peleg , cylindrical non - threaded implants are recommended after the ianl procedure , as threaded implants in close contact with the nerve may cause neurosensory problems . this recommendation may be a disadvantage because non - threaded implants have a lower surface area compared to threaded implants , which decreases the stability of the implant . the main limitations of this review were that six authors [ 5,13 - 17 ] selected the patients randomly ; in four studies [ 1 - 3,5 ] , the surgical procedure was not performed by the authors and , in two studies , the method of implant osseointegration evaluation was not mentioned . inferior alveolar nerve repositioning is a technique that has been used for more than 20 years with good survival and survival rates . this is sometimes the only possible procedure to help patients to obtain a fixed prosthesis , especially in edentulous atrophic posterior mandibles . there are two main techniques for inferior alveolar nerve repositioning that are relatively safe and offer a high survival rate : inferior alveolar nerve lateralization and inferior alveolar nerve transposition . increased protection of the neurovascular bundle inferior alveolar nerve lateralization and transposition in combination with the installation of dental implants offer advantages , such as reducing the risk of inferior alveolar nerve damage . with careful pre - operative surgical and prosthetic planning , imaging , and extremely precise surgical technique , this procedure can be successfully used for implant placement in an edentulous atrophic posterior mandible .

tooth loss is one of the common causes of reduced quality of life in adults . dental implants have become a widely accepted treatment option for patients with both partially and complete edentulous posterior mandibles . rehabilitation of edentulous posterior mandibular regions with severe ridge atrophy using implants is subject to anatomical , surgical and biological difficulties and poses a challenge to dental teams . in many cases , the bone has so severely atrophied that sufficiently long fixtures can not be placed without encroaching on the inferior alveolar nerve ( ian ) . in this situation , restorative options include the use of short fixtures , onlay bone grafting to increase ridge height , and more complicated and detailed imaging analysis to allow positioning of implants alongside , rather than into , the nerve canal during the procedure . another option is to move the ian laterally from its canal by either nerve lateralization ( ianl ) . with nerve lateralization , the ian is exposed and traction is used to deflect it laterally while the implants are placed . with nerve transposition , a corticotomy is done around the mental foramen and the incisive nerve is transacted , such that the mental foramen is repositioned more posteriorly . in the posterior mandible , the bone quality may not be as good as it is in the anterior mandible . in particular , if shorter implants are used to ensure that there is no encroachment on the nerve canal , initial implant stability will be unicortical . in addition , there is a risk to the ian as the operator tries to maximise implant length on the basis of measured available bone height . the advantages of iant include the ability to place longer fixtures and to engage 2 cortices for initial stability . pointed out that the amount of bone superior to the mandibular canal ( mc ) is often insufficient for the placement of fixtures of the ideal length . in addition , the existing bone that is superior to the mc is often of poorer quality than its cortical counterpart . these factors and the fact that shorter implants have been associated with higher failure rates have led to the development of methods of ian repositioning that allow placement of longer fixtures ; with these methods , the inferior cortex of the mandible is engaged , which leads to greater initial stability . therefore , the purpose of the present paper was to review the literature concerning the indications , contraindications , advantages , disadvantages and the surgical techniques of the lateralization and transposition of the inferior alveolar nerve , followed by the implants placement in an edentulous atrophic posterior mandible . ian lateralization ( ianl ) and transposition ( iant ) definition ianl and iant are surgical procedures that reposition the ian for the purpose of implant placement without bone augmentation . this procedure raises the risk of neuropathies , such as paresthesia , hypoesthesia , and anaesthesia of the ian . ianl is defined as the lateral reflection of the ian without incisive nerve traction ( preservation of the incisive nerve and lateralization of inferior alveolar neurovascular bundle posterior to the mental foramen ) ; exposure and traction are used to deflect the ian laterally while the implants are placed . during the iant procedure , a corticotomy is done around the mental foramen and the incisive nerve is transacted ( incisive neurovascular bundle is sacrificed ) , to allow transposition of both the mental foramen and the ian such that the mental foramen is repositioned more posteriorly . the first case of ian repositioning was reported by alling ( 1977 ) to rehabilitate patients with severe atrophy for dentures . in 1987 , jenson and nock carried out iant for the placement of dental implants in posterior mandibular regions . according to subjective criteria , sensory function of the mental nerve returned to normal five weeks post - operative . in 1992 , rosenquist performed the first case series study using iant followed by 26 implants placement on 10 patients . neurosensory function of the mental nerve was evaluated objectively using the two - point discrimination ( 2-pd ) method . at one year follow - up , he reported an implant survival rate of 96% and implant survival rate of 93.6% for this procedure ; therefore , this technique was accepted as the best - practice treatment modality for reconstruction of the dentoalveolar system with dental implants in the posterior mandible . the review included studies , case reports , clinical trials on human subjects that were published in english between january 1997 and july 2014 , and included a minimum of 6 months of follow - up . the information source was the medline ( ncbi pubmed and pmc ) database and other scientific electronic sources . according to the prisma guidelines , an electronic search was conducted using the medline ( ncbi pubmed and pmc ) database to locate articles concerning ian lateralization or transposition and implant placement in an edentulous atrophic posterior mandible . the search terms used were : inferior alveolar nerve lateralization , inferior alveolar nerve repositioning , inferior alveolar nerve transposition , implants in atrophic posterior mandible + repositioning , inferior alveolar nerve transposition + mental , implants in atrophic posterior mandible + lateralization , implants in atrophic posterior mandible + transposition , mandibular atrophy + repositioning , and inferior alveolar nerve + minimal bone height . due to the low number of relevant articles and to ensure the sensitivity of the systemic review process , articles were searched from january 1997 to july 2014 . inclusion and exclusion criteria inclusion criteria for the selection were : articles regarding to ianl and iant procedures ; all articles in english ; clinical reports with minimum 6 months follow - up ; information regarding implant osseointegration and survival ; studies on adult ( between the ages 18 and 80 ) human beings , with no immunologic diseases , uncontrolled diabetes mellitus , osteoporosis , or other contraindicating systemic conditions . exclusion criteria for the selection were : clinical reports with no minimum 6 months of follow - up ; not enough information regarding the selected topic ; no information regarding implant osseointegration and survival ; studies of patients with immunologic diseases , uncontrolled diabetes mellitus , osteoporosis or other contraindicating systemic conditions ; studies of adolescents ( under 18 years of age ) and elderly people ( over 80 ) . article review and data extraction article review and data extraction was performed according to a prisma flow diagram ( figure 1 ) . the search displayed 876 results from the ncbi pmc and pubmed databases , and 3 results from other sources ( dental-tribune.com , acta.tums.ac.ir , hindawi.com/journals ) . a total of 38 titles and abstracts were selected according to relevancy after the removal of duplications . another exclusion was made based upon follow - up time ( n = 6 ) and information regarding implant osseointegration and survival ( n = 4 ) . studies of adult human beings between 18 and 80 years of age with minimal residual bone above the mc , in which ianl and iant + implant placement had been performed , were selected . data collection process data was independently extracted from reports in the form of variables according to the aim and themes of the present review as listed below . the review aims to achieve the following : to describe the purpose ( indications and contraindications ) of ianl and iant . to describe the current surgical techniques used for ianl and iant with simultaneous implant placement . the risks were indicated as lack of precise information of interest in each individual study that can blind the reader from particular information about the examined samples . the cochrane collaboration tool for assessing risk of bias was used to assess bias across the studies that could affect cumulative evidence . the search displayed 876 results from the ncbi pmc and pubmed databases , and 3 results from other sources ( dental-tribune.com , acta.tums.ac.ir , hindawi.com/journals ) . twenty - six full - text articles were in the end , assessed for eligibility . during the eligibility stage , articles that did not meet the inclusion and exclusion criteria where filtered as follows : no minimum 6 months of follow - up ( n = 6 ) and no information regarding implant osseointegration and survival ( n = 4 ) . in the end , 16 articles that included data on 160 patients were utilised for the systematic review ( figure 1 ) . nine were related to iant , 4 to ianl and 3 to both iant and ianl ( table 1 ) . description of studies included in the review risk of bias within studies the cochrane collaboration bias summary for potential bias was used to assess the quality of studies and identify papers with intrinsic flaws in method and design . after analysing the risk of bias ( table 2 ) , we found that six authors [ 5,13 - 17 ] selected the patients randomly . in four studies [ 1 - 3,5 ] , the surgical procedure was not performed by the authors ; and in two studies , the method of implant osseointegration evaluation was not mentioned . assesment of the risks of bias results of individual studies results of individual studies of implant treatment using are shown in table 3 . from the results of individual study data ( table 3 ) , we can conclude that the most popular surgical technique for ian repositioning was iant . with the most popular method of implant osseointegration evaluation was panoramic radiograph ( 12/16 studies ) . in two studies , both of which were performed by the same author ( proussaefs ) , the method of implant osseointegration evaluation was a perio - test unit ( siemens , bensheim , germany ) ; while in two other studies ( lorean et al . the purpose ( indications and contraindications ) of ianl and iant the study of number selected articles [ 9,22 - 25 ] revealed the following indications of ianl and iant : the major reason for using this technique is to prevent ian injury during implant placement in edentulous posterior atrophic mandibles . class iv , v , or vi of cawood and howell with extrusion of the antagonist tooth and reduced prosthetic free space . class v or vi of cawood and howell with presence of interforaminal teeth ( patients were not candidates for interforaminal implant - prosthetic methods ) . class v or vi of cawood and howell if the patient desires a fast implant - prosthetic rehabilitation with predictable outcomes . preservation of ian in cancer surgery in the posterior mandible . when placement of short implants is not a viable option ( in case of severely atrophic mandibles when the residual bone above mc ranges between 0.5 and 1.5 mm ) . less than 10 - 11 mm bone height above the canal , when the quality of the spongy bone does not provide sufficient stability for implant placement . contraindications if the mandible presents advanced resorption of the alveolar process . if the patient has poor general health , including systemic diseases that may worsen the patient s health condition after the ian reposition procedure . such patients do not have tolerance and compatibility skills and , therefore , are not good candidates for nerve transposition surgery . the current surgical techniques utilised for lateralization and transposition of ian with simultaneous implant placement the preoperative work - up included an assessment of the ian using appropriate diagnostic records , such as a panoramic radiograph , a computed tomography ( ct ) scan , casts , diagnostic wax - up , and surgical templates . during preoperative consultation with the patients , the risk of postoperative neurosensory disturbances ( nd ) that can result following the ian repositioning is discussed . to help the patient decide whether this would be tolerable , the clinician can perform a preoperative block with a long - acting local anaesthetic , such as marcaine , which reproduces symptoms lasting 8 to 16 hours that are similar to the postoperative anaesthesia the patient may experience . a crestal incision with anterior- and posterior releasing incisions is made , and a labial mucoperiosteal flap is reflected that exposes the alveolar ridge and buccal cortex . care is taken during flap reflection to preserve the integrity of the periosteum and the neurovascular bundle where it exits the mental foramen and enters the soft tissue . to increase the flaps relaxation and improve exposure the flaps are raised , one lingual to access the alveolar crest , and another vestibular for subperiosteal dissection of the ian , until sufficient surgical field is obtained . ct is used to locate the approximate area of the mental foramen , after which blunt dissection is used to identify and isolate the mental nerve . ian transposition ( iant ) an osteotomy is performed at the mental foramen , drilling around the orifice to obtain a ring of external cortical bone . a window also may be made about 5 mm ahead of the foramen , in order to avoid damaging the nerve over its anterior loop . an en bloc osteotomy is then made at the external cortical level , or a posterior window is performed in the external cortical layer along the intrabony trajectory of the nerve ( figure 2 ) . a round drill is used to create the window , replacing it with a diamond drill while working close to the mc , to minimise the risk of nerve damage . in order to secure complete mobilisation of the ian , the incisor branch , located about 5 mm from the mental foramen , a = before transposition ; b = after transposition ( courtesy of dr dainius razukevicius , kauno implantologijos centras kaunas , lithuania ) . then , with the nerve fully lateralized , the dental implants are placed under direct visualisation - in this case bicortically , taking advantage of the mandibular basal layer . once the implants have been positioned , the vestibular cortical layer is replaced in those cases where an osteotomy has been performed , or the nerve is passively positioned against the implants in those cases where cortical drilling has been carried out . ian lateralization ( ianl ) in this case , neither dissection of the terminal branches of the ian nor sectioning of the incisor branch is needed . the technique involves the preparation of a cortical bone window ( via osteotomy or drilling ) that is located posterior to the mental foramen ( figure 3 and figure 4 ) . intraoperative photography showing the inferior alveolar neurovascular bundle lateralization ( courtesy of dr dainius razukevicius , kauno implantologijos centras kaunas , lithuania ) . schematic drawing showing the inferior alveolar neurovascular bundle transposition ( a ) and lateralization ( b ) . after removing the cortical bone , a curette may be used for removal of spongy bone and the cortical layer of the canal in cases where the cortical layer surrounding the canal is not dense or thick . then , a 10 mm wide gauze cord or elastic band is passed below the nerve to retract it from the surgical site in order to decrease the risk of ischemic trauma to the nerve . the second purpose of retracting the nerve from the surgical site during the operation is to reduce the risk of ian damage . after carefully freeing the nerve , the latter is separated using a vessel loop - while applying gentle traction outwards as the implants are positioned , the implant should be long enough to pass the mc and engage the basal body below the canal to achieve sufficient primary stability . finally , the vessel loop is removed and the nerve is replaced , positioning a resorbable membrane between it and the bone window to avoid direct contact with the implants . the decision whether to use iant or ianl depends on the amount of stretching that is needed in order to mobilize the ian . study , stretching the nerve by 10 - 17% of its original length may result in disruption of the nerve fibers internally . retrospective study demonstrated that the nd for iant were 77.8% ( 7/9 ) and for the ianl 33.3% ( 4/12 ) . as a summary of both surgical techniques ( iant and ianl ) , it can be concluded that ianl produces less side effects than iant . among the disadvantages of this device are the long duration of time that it takes to remove bone . also , there remains controversy regarding the indications of piezosurgery devices , and some believe that the vibrations they generate may damage the nerve . repositioning the neurovascular bundle inside the mc before this phase , the surgeon should decide whether or not to place materials between the implant and the inferior alveolar neurovascular bundle . a potential advantage of bone over a membrane is that , if proper healing occurs in the area , the contact area of implant and bone will increase . before releasing the inferior alveolar neurovascular bundle from the elastic band this way , the inferior alveolar neurovascular bundle will be in a vent that is medially adjacent to the implants and covered by the mucoperiosteal flap . however , there is no consensus in this regard ; since inflammation can be among the causes of nerve dysfunction , corticosteroid therapy can be beneficial . after analysing the current literature concerning ianl and iant , several advantages and disadvantages of the procedures were found and are described below . possibility of placement of a greater number of implants , which improves the overall strength of the final prosthesis . possibility for simultaneous placement of implants during surgery , which allows a reduction in treatment time compared with other techniques as bone grafts that require a long waiting period before implant insertion together with additional surgeries . the option for immediate loading for the enhancement of masticatory function , dramatically improving the patient s quality of life . in addition , with this procedure , all of the following are accomplished : restoration of the correct vertical dimension ; prevention of tissues atrophy ; replacement of dentures ; stabilisation of the anterior dentition ; temporomandibular joint and masticatory muscle balance . the disadvantages of the ianl and iant one of the disadvantages of this procedure is that it does not recover alveolar ridge anatomy . another negative point of this procedure is that it temporarily weakens the mandible due to removal of cortical bone ; which , in combination with implant placement , may lead to mandibular fracture at the operation site . according to peleg , cylindrical non - threaded implants are recommended after the ianl procedure , as threaded implants in close contact with the nerve may cause neurosensory problems . the general anaesthetic management of patients with myotonic dystrophy ( dystrophia myotonica ) can , however , be challenging . the main limitations of this review were that six authors [ 5,13 - 17 ] selected the patients randomly ; in four studies [ 1 - 3,5 ] , the surgical procedure was not performed by the authors and , in two studies , the method of implant osseointegration evaluation was not mentioned . increased protection of the neurovascular bundle inferior alveolar nerve lateralization and transposition in combination with the installation of dental implants offer advantages , such as reducing the risk of inferior alveolar nerve damage . with careful pre - operative surgical and prosthetic planning , imaging , and extremely precise surgical technique , this procedure can be successfully used for implant placement in an edentulous atrophic posterior mandible .

obesity is associated with a low - grade proinflammatory state resulting in an increase of circulating cytokines and inflammatory markers . inflammatory cytokines have been involved in the impairment of insulin signaling , thus providing molecular links between inflammation and insulin resistance . inflammation reportedly produces metabolic alterations in skeletal muscle with both inflammatory response and insulin resistance being associated with loss of muscle mass by decreased protein synthesis and increased proteolysis [ 35 ] . recently , our group has shown that leptin reverses muscle loss of ob / ob mice by inhibiting the activity of the transcriptional factor forkhead box class o3a ( foxo3a ) . leptin is an adipocyte - derived peptidic hormone that inhibits food intake and increases thermogenesis by acting through its hypothalamic receptors [ 8 , 9 ] . leptin - deficient ob / ob mice are obese , hyperphagic , exhibit type 2 diabetes , decreased body temperature and hypogonadotropic hypogonadism . leptin is a member of the long - chain helical cytokine family and its receptors , which belong to the class i cytokine receptors , are present in bone marrow and spleen as well as on peripheral monocytes and lymphocytes . leptin increases in response to acute infection and sepsis and it has been reported to exert a profound influence on the function and proliferation of t lymphocytes and natural killer cells , on the phagocytosis of macrophages / monocytes , and to have a direct effect on the secretion of anti- and proinflammatory cytokines . in this regard , impaired cellular and humoral immunity have been shown in leptin - deficient ob / ob mice as well as in leptin receptor - deficient db / db mice [ 14 , 15 ] . these studies reflect the molecular nature of leptin as a cytokine and are consistent with leptin signaling playing a pivotal role in the pathogenesis of obesity - associated inflammation and muscle loss . in the present paper , gastrocnemius muscle samples from wild type and ob / ob mice were analyzed for mrna presence of over 41,000 transcripts by microarray analysis to identify genes involved in inflammation and oxidative stress that are affected by leptin deficiency and leptin administration in ob / ob mice . it was shown that leptin increases the gastrocnemius weight and reduces the high expression levels of genes related to the obesity - associated low - grade inflammation in skeletal muscle of ob / ob mice . ten - week - old male genetically obese ob / ob mice ( c57bl/6j ) ( n = 15 ) and their lean control littermates wild type ( n = 5 ) supplied by harlan ( barcelona , spain ) were housed in a room with controlled temperature ( 22 2c ) and a 12:12 light - dark cycle ( lights on at 08:00 am ) . body weight of ob / ob mice was measured before randomization into control , leptin - treated ( 1 mg / kg / d ) and pair - fed groups ( n = 5 per group ) . the control and pair - fed groups received vehicle ( pbs ) , while leptin - treated mice were intraperitoneally administered with leptin ( bachem , bubendorf , switzerland ) twice daily at 08:00 am and 08:00 pm for 28 days . control and leptin - treated groups were provided with water and food ad libitum with a standard rodent chow ( 2014s teklad , harlan ) , while daily food intake of the pair - fed group was matched to the amount consumed by the leptin - treated group the day before in order to discriminate the inhibitory effect of leptin on appetite . animals were sacrificed on the 28th day of treatment by co2 inhalation 20 hours after the last pbs or leptin administration ( in order to avoid picking up effects reflecting an acute response ) and after 8 hours of fasting . all experimental procedures conformed to the european guidelines for the care and use of laboratory animals ( directive 86/609 ) and were approved by the ethical committee for animal experimentation of the university of navarra ( 080/05 ) . serum glucose was analyzed using a sensitive - automatic glucose sensor ( ascensia elite , bayer , barcelona , spain ) . free fatty acid ( ffa ) concentrations were measured by a colorimetric determination using the nefa c kit ( wako chemicals , neuss , germany ) . serum triglycerides ( tg ) concentrations were spectrophotometrically determined using a commercial kit ( infinity , thermo electron , melbourne , australia ) . insulin and leptin were determined using specific mouse elisa kits ( crystal chem inc . , intra- and interassay coefficients of variation for measurements of insulin and leptin were 3.5% and 6.3% , respectively , for the former , and 2.8% and 5.8% , for the latter . adiponectin concentrations were also assessed using a mouse elisa kit ( biovendor laboratory medicine , inc . , modrice , czech republic ) . insulin resistance was calculated using the homeostasis model assessment score ( homa ; fasting insulin ( u / ml ) fasting glucose ( mmol / l)/22.5 ) . an indirect measure of insulin sensitivity was calculated by using the quantitative insulin sensitivity check index ( quicki ; 1/[log(fasting insulin mu / ml ) + log(fasting glucose mg / dl ) ] . lipid peroxidation was analyzed by the measurement of thiobarbituric acid reactive substances ( tbars ) in serum and gastrocnemius as previously described by conti et al . with some modifications . since the best - known specific tbars is malondialdehyde ( mda ) , we used serum mda levels , a secondary product of lipid peroxidation , as an indicator of lipid peroxidation and oxidative stress . gastrocnemius samples ( 2030 mg ) were homogenized in 20 volumes of phosphate buffer ph 7.4 . serum , muscle homogenates ( 5 l ) or standard ( mda ) were mixed with 120 l of diethyl thiobarbituric acid ( detba ) 10 mm and vortexed for 5 seconds . the reaction mixture was then incubated at 95c for 60 minutes . after cooling to room temperature detba - mda adducts were extracted in 360 l n - butanol vortexing for 1 minute and centrifuged at 1,600 g for 10 minutes at room temperature . then , the chromophore of the detba - mda adduct was quantified in 200 l of the upper butanol phase by fluorescence emission at 535 nm with an excitation at 590 nm . mda equivalents ( tbars ) were quantified using a calibration curve prepared using mda standard working solutions and expressed as serum mda m and gastrocnemius mda m / mg protein . protein concentrations were determined using a bradford protein assay kit ( biorad , hercules , ca , usa ) . total rna was extracted from 2030 mg of gastrocnemius muscle samples by homogenization with an ultra - turrax t 25 basic ( ika werke gmbh , staufen , germany ) using trizol reagent ( invitrogen , barcelona , spain ) . rna was purified using the rneasy mini kit ( qiagen , barcelona , spain ) and treated with dnase i ( rnase - free dnase set , qiagen ) in order to remove any trace of genomic dna . gene expression analyses were conducted using the agilent whole mouse genome array ( g4121b , agilent technologies , santa clara , ca , usa ) containing ~41 , 000 mouse genes and transcripts . fluorescence - labeled cdna probes were prepared from 1 g of total rna from each sample ( 5 animals per group ) to be subsequently amino - allyl labeled and amplified using the amino allyl messageamp ii arna amplification kit ( ambion , austin , tx , usa ) . aliquots ( 1.2 g ) of amplified arna were fluorescently labeled using cy3/cy5 ( amersham biosciences , buckinghamshire , uk ) and then appropriately combined and hybridized to agilent microarrays . hybridizations were performed following a reference design , where control samples were pools of rna from all individual samples . two hybridizations with fluor reversal ( dye - swap ) were performed for each sample . after washing , microarray slides were scanned using a gene pix 4100a scanner ( axon instruments , union city , ca , usa ) and image quantization was performed using the software genepix pro 6.0 . gene expression data for all replicate experiments were analyzed using the genespring gx software version 7.3.1 ( agilent technologies ) . in addition , gene ontology database ( http://babelomics.bioinfo.cipf.es ) and the kegg website ( http://www.genome.ad.jp/kegg/pathway ) were used in conjunction with genespring ( http://www.agilent.com/chem/genespring ) to identify pathways and functional groups of genes . more information regarding the microarray experiments can be found at the embl - european bioinformatics institute ( http://www.ebi.ac.uk/aerep/login . arrayexpress accession number : e - mexp-1831 ) . to validate the microarray data , a number of representative differentially expressed genes were selected to be individually studied by real - time pcr ( 7300 real time pcr system , applied biosystems , foster city , ca , usa ) ( n = 5 per group ) as previously described . primers and probes were designed using the software primer express 2.0 ( applied biosystems ) and purchased from genosys ( sigma , madrid , spain ) ( table 1 ) . differences between groups were assessed by kruskal - wallis followed by mann whitney 's u test . as previously outlined , gene ontology groupings were used to identify pathways significantly affected by leptin deficiency as opposed to its administration . furthermore , statistical comparisons for microarray data to identify differentially expressed genes across different groups were performed using one - way anova and student 's t - tests as appropriate . all statistical analyses were performed by using the spss statistical program version 15.0 for windows ( spss , chicago , il , usa ) and statistical significance was defined as p < .05 . the morphological and biochemical characteristics of wild type and ob / ob mice are reported in table 2 . as expected , leptin treatment corrected the obese and diabetic phenotype of ob / ob mice . body weight was significantly higher ( p < .01 ) in the control ob / ob group as compared to wild type mice . leptin - treated mice exhibited a decreased body weight ( p < .01 ) as compared to control and pair - fed ob / ob animals . importantly , leptin treatment normalized body weight of ob / ob mice as compared to wild type ( p = .690 ) . in addition , the gastrocnemius of control ob / ob mice exhibited a lower ( p < .01 ) muscle weight than that of wild type mice and it was increased ( p < .01 ) by leptin administration in comparison with that of control and pair - fed ob / ob rodents . as depicted in table 2 , higher fasting glucose ( p < .05 ) and insulin ( p < .01 ) concentrations were observed in the control ob / ob mice compared to wild types . although no differences in glucose concentrations were observed in pair - fed as compared to leptin - treated ob / ob mice , higher serum insulin concentrations ( p < .05 ) were detected in the pair - fed animals than in the leptin - treated ob / ob group . furthermore , leptin administration normalized both the glucose and insulin levels in ob / ob mice compared to wild types . these data suggest that leptin increases the insulin sensitivity in peripheral tissues , as evidenced by the lower homa and higher quicki indices ( p < .01 ) in the leptin - treated in comparison with the control ob / ob animals . serum glycerol was markedly increased ( p < .05 ) in the control ob / ob mice , while ffa and tg levels remained unchanged as compared to wild type mice . interestingly , leptin not only decreased circulating concentrations of ffa ( p < .05 ) and glycerol ( p < .01 ) levels as compared to control ob / ob mice , but also ffa ( p < .01 ) , glycerol ( p < .01 ) and tg ( p < .05 ) concentrations as compared to pair - fed mice . leptin administration to ob / ob mice reduced serum glycerol concentrations ( p = .032 ) and tended to decrease ffa ( p = .095 ) as compared to wild types . furthermore , leptin treatment increased the low concentrations of adiponectin of ob / ob mice , but the differences fell out of statistical significance ( p = .095 ) . control ob / ob mice exhibited significantly higher serum tbars than wild type littermates ( p < .01 ) , which were significantly reduced after leptin administration as compared to the control ( p < .01 ) and pair - fed ( p < .05 ) ob / ob groups ( figure 1(a ) ) . in addition , leptin decreased ( p < .01 ) the high concentrations of mda measured in the gastrocnemius muscle of control ob / ob mice , while this effect was not observed in the pair - fed group ( figure 1(b ) ) . serum and gastrocnemius tbars levels were positively associated with body weight , ffa , insulin , and the homa index . oppositely , tbars levels were negatively associated with adiponectin and the quicki index both in serum and muscle . importantly , a high positive relation were found between serum and gastrocnemius concentrations of tbars ( = 0.63 , p = .003 ) ( table 3 ) . differential gene expression profiles in gastrocnemius muscle of wild type and ob / ob groups were compared by microarray analysis . only genes whose mrna levels were changed 1.5-fold or higher and identified as significantly changed by statistical analysis were designated as differentially expressed genes . applying these criteria , microarray data showed that 7,582 genes were differentially expressed by leptin deficiency and leptin administration in ob / ob mice . in particular , leptin deficiency altered the expression of 1,127 genes between wild type and control ob / ob mice . of these , 580 were upregulated and 547 were downregulated in ob / ob mice . leptin treatment modified the expression of 1,546 genes in ob / ob mice , upregulating 512 and repressing 1,034 . in addition , leptin repressed 736 genes that were upregulated in gastrocnemius muscle of control ob / ob and increased the transcript levels of 846 downregulated genes . functional enrichment analysis using geneontology and kegg databases revealed that the set of genes with altered expression levels induced by leptin deficiency and administration represents a broad spectrum of biological processes . however , for the purpose of the present paper we focused on the effects of leptin on the set of genes encoding proteins involved in oxidative stress and inflammation . table 4 shows that leptin deficiency and leptin administration altered the expression of a large number of genes involved in oxidative stress and inflammation . the biological processes mainly affected between control ob / ob mice and wild types included furthermore , several processes regulating proliferation , differentiation , and activity of lymphocytes were also significantly affected by leptin deficiency . importantly , comparison of leptin - treated and control ob / ob groups showed that leptin administration altered the expression of genes implicated in the positive regulation of lymphocyte activation ( p = .0187 ) , as well as genes involved in the chaperone cofactor dependent protein folding dna microarray analysis showed that 86 genes encoding proteins related to defense , stress , and inflammatory responses were altered in the gastrocnemius muscle of control ob / ob mice and modified by leptin administration . leptin reduced the mrna levels of various isoforms of the family of heat shock proteins ( hsps ) ( dnajc16 , dnaja4 , dnajb4 , hspa2 , hspa4 , and hspb7 ) , metallothioneins ( mt2 , mt4 ) , crystallins ( cryab , crybb1 ) and rna binding proteins ( rbms ) ( rbm9 , rbm22 ) in ob / ob mice ( table 5 ) . in addition , histocompatibility 2 , complement component factor b h2-bf and several genes of the acute - phase response or inflammatory processes , such as kallikrein 5 ( klk5 ) , and serine ( or cysteine ) proteinase inhibitor clade c member 1 ( serpinc1 ) and clade b member 1a ( serpinb1a ) , displayed an increased expression in ob / ob mice that was reduced by leptin administration . on the contrary , gene expression of cryl1 , hsp105 , rbm5 , and h2-aa were enhanced in ob / ob mice after treated with leptin . pair - feeding , which accounts for the decrease in food intake that is independent of the direct action of leptin , altered the expression of 1,960 genes , upregulating 984 while downregulating 976 genes . in the context of a food intake reduction as compared to the simple effect due to the caloric restriction , leptin administration further significantly altered the expression of genes involved in processes encompassing ( p = 2.99e ) , positive regulation of t cell activation ( p = .0003 ) and positive regulation of immune cell mediated cytotoxicity in particular , the gene array analysis provided evidence for elevated hspa4 , mt4 , crybb1 , and serpinb8 mrna levels in the pair - fed group as compared to the leptin - treated ob / ob mice ( table 6 ) . on the contrary , leptin increased the gene expression of h2-ab1 and h2-eb1 in ob / ob mice . to confirm the microarray data , the mrna expression of several representative transcripts was analyzed by real - time pcr ( figure 2 ) . in this sense , leptin administration reduced the mrna levels of the muscle atrophy - related transcription factor forkhead box o1 ( foxo1 ) and of the e3 ubiquitin - ligases muscle atrophy f - box ( mafbx ) and muscle ring finger 1 ( murf1 ) in leptin - treated ob / ob mice , while no effect of leptin was evidenced on the mrna levels of the transcriptional coactivator peroxisome proliferator - activated receptor- coactivator-1 ( pgc1 ) . obesity is accompanied by a chronic proinflammatory state associated not only with insulin resistance , but also with muscular atrophy [ 4 , 5 ] . our study provides evidence that leptin constitutes a negative regulator of oxidative stress and inflammation in the gastrocnemius , which is a representative skeletal muscle of the whole skeletal musculature . this statement is supported by findings reported herein : ( a ) leptin deficiency is accompanied by systemic and skeletal muscle oxidative stress , muscle inflammation , and reduced muscle mass ; ( b ) systemic and skeletal muscle oxidative stress , muscle atrophy and inflammation of ob / ob mice are reversed by leptin administration independently of the effects of food intake inhibition . therefore , leptin is able to prevent the muscle atrophy associated with obese and inflammatory states . skeletal muscle constitutes an important target for leptin playing a key role on the regulation of lipid and glucose metabolism . since obese ob / ob mice exhibit an increased oxidative stress and impaired immune response [ 14 , 15 ] and a reduced skeletal muscle mass compared with their lean littermates , we aimed to identify the genes related to inflammatory processes differentially altered by leptin in the gastrocnemius muscle of obese ob / ob mice . in particular , 86 transcripts encoding inflammation - related proteins were shown to be modified by exogenous leptin administration . however , it has to be taken into account that many of these genes are multifunctional and may have important functions in other biological processes . among them , leptin repressed the high expression levels of acute - phase reactants and several members of the hsp and rbm families . in addition , confirming a previous study of our group , leptin treatment increased the reduced muscle weight of gastrocnemius muscle of ob / ob mice . taken together , these data suggest that leptin may prevent the obesity - associated inflammatory state and the muscle mass loss related to inflammatory states in leptin - deficient ob / ob mice . leptin - deficient ob / ob and leptin receptor - deficient db / db mice display many abnormalities in the immune response similar to those observed in starved animals and malnourished humans [ 14 , 15 , 22 ] . in this respect , exogenous leptin replacement to ob / ob mice modulates t cell responses in mice and prevents starvation - induced immunosuppression , suggesting that lack of leptin is directly involved in these immune system abnormalities [ 23 , 24 ] . in agreement with these studies , our findings show that leptin deficiency and administration differentially regulate biological processes related to the immune response as well as the t and b cell differentiation and activation in gastrocnemius muscle of ob / ob mice . oxidative stress is defined as the imbalanced redox state in which prooxidants overwhelm the antioxidant capacity , resulting in an increased production of reactive oxygen species ( ros ) , ultimately leading to oxidative damage of cellular macromolecules . dismutation of o2 by superoxide dismutase ( sod ) produces hydrogen peroxide ( h2o2 ) , a more stable ros , which , in turn , is converted to water by catalase and glutathione peroxidase ( gpx ) . oxidative stress is increased in diabetes [ 26 , 27 ] with leptin administration reportedly improving insulin sensitivity in normal and diabetic rodents [ 2830 ] . leptin stimulates in vitro ros production by inflammatory cells and endothelial cells and the level of systemic oxidative stress in nonobese animals [ 33 , 34 ] , suggesting a prooxidative role of leptin . however , administration of recombinant leptin reduces the oxidative stress induced by a high - fat diet in mice . in this sense , findings of our study show a high oxidative stress in diabetic ob / ob mice , as reflected by increased tbars concentrations in serum and the gastrocemius muscle . these observations are in agreement with a large number of studies related to increased plasma tbars or mda in diabetic rats and humans . lipid peroxidation is a common index of free radical mediated injury and induction of antioxidant enzyme is a common cellular response . more importantly , leptin administration decreased serum and gastrocnemius tbars concentrations as compared to control ob / ob mice , with tbars levels in gastrocnemius muscle from pair - fed ob / ob animals remaining very similar to those of control ob / ob mice . in this sense , from a molecular perspective , our results further show that transcript levels of sod1 , gpx3 and glutathione s - transferase 1 gstp1 are downregulated in control ob / ob mice as compared to wild type controls being upregulated after leptin treatment . furthermore , leptin administration also upregulated gpx7 , glutathione s - transferase 5 ( gstm5 ) and glutathione s - transferase 2 ( gstt2 ) . on the contrary , the high expression of catalase ( cat ) was repressed by the exogenous injection of leptin to ob / ob mice . these findings are in line with previous observations showing the restoration of the defective antioxidant enzyme activity in plasma of ob / ob mice and humans with a leptin gene mutation . acute - phase reactants have been suggested to contribute to the maintenance of the chronic low - grade inflammation state involved in the progression of obesity and related diseases . interestingly , our study provides evidence that genes of the acute - phase response were altered in gastrocnemius muscle of ob / ob mice , which were counteracted by exogenous leptin administration . leptin reduced the elevated gene expression of tissue - type plasminogen activator ( plat ) and lipocalin-2 ( lcn2 ) , which are upregulated in many inflammatory conditions [ 42 , 43 ] , including human obesity . in addition , a pivotal role for oxidative stress in the pathogenesis of muscle wasting in disuse and in a variety of pathological conditions is now being widely recognized . a potential link between oxidative stress and muscle moreover , various inflammatory cytokines induce oxidative stress and muscle atrophy through the activation of the lysosomal [ 48 , 49 ] and the ubiquitin - proteolysis system . in this context , biological processes related to oxidative stress and inflammatory responses were altered in the gastrocnemius muscle of ob / ob mice and improved following leptin treatment . in spite of the usual upregulation of the e3 ubiquitin - ligases mafbx and murf1 in most conditions associated with atrophy , their gene expression levels in ob / ob were lower as compared to wild type animals , although no statistically significant differences were observed . contrarily to what would be expected , leptin administration prevented the increase of both mafbx and murf1 mrna expression levels induced by pair - feeding in ob / ob mice . a plausible explanation for this surprising finding may relate to the fact that in extreme conditions the energy homeostasis system is overriden whereby leptin is able to inhibit muscular protein degradation associated to food intake reduction . these data are in accordance with a previous study of our group evidencing that leptin replacement inhibits the ubiquitin proteolysis system activity in leptin - deficient mice . muscle atrophy is associated with increased expression of genes coding for rbm proteins which facilitate the translation , protection , and restoration of native rna conformations during oxidative stress . it has been suggested that the gene expression of rbm proteins may increase as a compensatory mechanism in response to loss of muscle proteins [ 51 , 52 ] . other proteins involved in oxidative stress are metallothioneins , endogenous antioxidants that have been shown to be overexpressed in muscle atrophy in rodents [ 5456 ] . in the present work , we have observed that administration of leptin inhibits the gene expression of several members of the rbm ( rbm9 , rbm22 ) and metallothioneins ( mt2 , mt4 ) families in the gastrocnemius of ob / ob mice , suggesting that leptin may modulate the inflammatory and oxidative stress responses and consequently , the muscle loss related to inflammatory states . genes involved in the chaperone system were also differentially expressed in ob / ob mice as compared to wild types and modified by leptin treatment . hsps represent a family of molecular chaperones induced in response to cellular stress , responsible for maintaining the structure of proteins and contributing to the repair of damaged or malformed proteins in highly oxidative and lipotoxic conditions . as a result in fact , hsps are repressed in many rat models of skeletal muscle atrophy [ 54 , 59 , 60 ] . hsp70 is constitutively expressed in skeletal muscle , but its levels are increased in response to oxidative stress with the induction of hsp70 expression by hyperthermia and during inactivity attenuating muscle atrophy [ 62 , 63 ] . in this regard , a recent study has shown that hsp70 prevents muscle atrophy induced by physical inactivity through inhibition of the muscle atrophy - related transcription factor foxo3a and the expression of mafbx and murf1 . among the hsps , hsp70 and b - crystallin in particular , are considered negative regulators of muscle cell apoptosis [ 65 , 66 ] and may inhibit the loss of nuclei taking place during muscle atrophy . in addition , ros induce the activity of foxo and gene expression of members of the ubiquitin - proteolysis system in myotubes . in this sense , our results provide evidence that leptin inhibits the increased gene expression of different members of the hsps ( hspb7 , dnajc16 , hspa4 , cryab , and crybb1 ) in the gastrocnemius muscle of ob / ob mice . taken together , the elevated expression of hsps in the control and pair - fed ob / ob groups suggests a high defense and stress response in these mice . moreover , induction of hsps may confer broader health benefits to patients who are insulin resistant or diabetic . in mammals , caloric restriction has been shown to upregulate hsp induction [ 70 , 71 ] , while expression of hsp72 has been found to be low in skeletal muscle of patients with insulin resistance or type 2 diabetes [ 72 , 73 ] . have recently shown that leptin downregulates hsp70 gene expression in chicken liver and hypothalamus but not in muscle , which was independent of food intake restriction . on the contrary , bonior et al . reported an increase in hsp60 gene expression in pancreatic cells by leptin . obesity is accompanied by a chronic proinflammatory state resulting in an increase in circulating cytokines and inflammatory markers . in this regard , inflammation produces metabolic alterations in skeletal muscle with both inflammatory response and insulin resistance being associated with muscle mass loss . findings of our study provide evidence that systemic and skeletal muscle oxidative stress , muscle atrophy and the elevated expression of genes involved in oxidative stress and inflammation of ob / ob mice are reversed by leptin administration . taken together , these data thereby support that leptin is able to prevent the muscle atrophy associated with obese and inflammatory states in ob / ob mice . most obese people develop muscle atrophy in spite of exhibiting high leptin circulating concentrations , which may be explained by the leptin resistance present in these patients . our paper sheds light on the relation between obesity and the loss of muscle mass associated to inflammatory states suggesting that leptin treatment may be an attractive therapeutic approach to prevent muscle loss associated with inflammatory diseases .

obese leptin - deficient ob / ob mice exhibit a low - grade chronic inflammation together with a low muscle mass . our aim was to analyze the changes in muscle expression levels of genes related to oxidative stress and inflammatory responses in leptin deficiency and to identify the effect of in vivo leptin administration . ob / ob mice were divided in three groups as follows : control ob / ob , leptin - treated ob / ob ( 1 mg / kg / d ) and leptin pair - fed ob / ob mice . gastrocnemius weight was lower in control ob / ob than in wild type mice ( p < .01 ) exhibiting an increase after leptin treatment compared to control and pair - fed ( p < .01 ) ob / ob animals . thiobarbituric acid reactive substances , markers of oxidative stress , were higher in serum ( p < .01 ) and gastrocnemius ( p = .05 ) of control ob / ob than in wild type mice and were significantly decreased ( p < .01 ) by leptin treatment . leptin deficiency altered the expression of 1,546 genes , while leptin treatment modified the regulation of 1,127 genes with 86 of them being involved in oxidative stress , immune defense and inflammatory response . leptin administration decreased the high expression of crybb1 , hspb3 , hspb7 , mt4 , cat , rbm9 , serpinc1 and serpinb1a observed in control ob / ob mice , indicating that it improves inflammation and muscle loss .

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

obesity is associated with a low - grade proinflammatory state resulting in an increase of circulating cytokines and inflammatory markers . inflammatory cytokines have been involved in the impairment of insulin signaling , thus providing molecular links between inflammation and insulin resistance . recently , our group has shown that leptin reverses muscle loss of ob / ob mice by inhibiting the activity of the transcriptional factor forkhead box class o3a ( foxo3a ) . leptin - deficient ob / ob mice are obese , hyperphagic , exhibit type 2 diabetes , decreased body temperature and hypogonadotropic hypogonadism . in this regard , impaired cellular and humoral immunity have been shown in leptin - deficient ob / ob mice as well as in leptin receptor - deficient db / db mice [ 14 , 15 ] . these studies reflect the molecular nature of leptin as a cytokine and are consistent with leptin signaling playing a pivotal role in the pathogenesis of obesity - associated inflammation and muscle loss . in the present paper , gastrocnemius muscle samples from wild type and ob / ob mice were analyzed for mrna presence of over 41,000 transcripts by microarray analysis to identify genes involved in inflammation and oxidative stress that are affected by leptin deficiency and leptin administration in ob / ob mice . it was shown that leptin increases the gastrocnemius weight and reduces the high expression levels of genes related to the obesity - associated low - grade inflammation in skeletal muscle of ob / ob mice . ten - week - old male genetically obese ob / ob mice ( c57bl/6j ) ( n = 15 ) and their lean control littermates wild type ( n = 5 ) supplied by harlan ( barcelona , spain ) were housed in a room with controlled temperature ( 22 2c ) and a 12:12 light - dark cycle ( lights on at 08:00 am ) . body weight of ob / ob mice was measured before randomization into control , leptin - treated ( 1 mg / kg / d ) and pair - fed groups ( n = 5 per group ) . the control and pair - fed groups received vehicle ( pbs ) , while leptin - treated mice were intraperitoneally administered with leptin ( bachem , bubendorf , switzerland ) twice daily at 08:00 am and 08:00 pm for 28 days . control and leptin - treated groups were provided with water and food ad libitum with a standard rodent chow ( 2014s teklad , harlan ) , while daily food intake of the pair - fed group was matched to the amount consumed by the leptin - treated group the day before in order to discriminate the inhibitory effect of leptin on appetite . animals were sacrificed on the 28th day of treatment by co2 inhalation 20 hours after the last pbs or leptin administration ( in order to avoid picking up effects reflecting an acute response ) and after 8 hours of fasting . all experimental procedures conformed to the european guidelines for the care and use of laboratory animals ( directive 86/609 ) and were approved by the ethical committee for animal experimentation of the university of navarra ( 080/05 ) . lipid peroxidation was analyzed by the measurement of thiobarbituric acid reactive substances ( tbars ) in serum and gastrocnemius as previously described by conti et al . aliquots ( 1.2 g ) of amplified arna were fluorescently labeled using cy3/cy5 ( amersham biosciences , buckinghamshire , uk ) and then appropriately combined and hybridized to agilent microarrays . in addition , gene ontology database ( http://babelomics.bioinfo.cipf.es ) and the kegg website ( http://www.genome.ad.jp/kegg/pathway ) were used in conjunction with genespring ( http://www.agilent.com/chem/genespring ) to identify pathways and functional groups of genes . as previously outlined , gene ontology groupings were used to identify pathways significantly affected by leptin deficiency as opposed to its administration . all statistical analyses were performed by using the spss statistical program version 15.0 for windows ( spss , chicago , il , usa ) and statistical significance was defined as p < .05 . the morphological and biochemical characteristics of wild type and ob / ob mice are reported in table 2 . as expected , leptin treatment corrected the obese and diabetic phenotype of ob / ob mice . body weight was significantly higher ( p < .01 ) in the control ob / ob group as compared to wild type mice . leptin - treated mice exhibited a decreased body weight ( p < .01 ) as compared to control and pair - fed ob / ob animals . importantly , leptin treatment normalized body weight of ob / ob mice as compared to wild type ( p = .690 ) . in addition , the gastrocnemius of control ob / ob mice exhibited a lower ( p < .01 ) muscle weight than that of wild type mice and it was increased ( p < .01 ) by leptin administration in comparison with that of control and pair - fed ob / ob rodents . as depicted in table 2 , higher fasting glucose ( p < .05 ) and insulin ( p < .01 ) concentrations were observed in the control ob / ob mice compared to wild types . although no differences in glucose concentrations were observed in pair - fed as compared to leptin - treated ob / ob mice , higher serum insulin concentrations ( p < .05 ) were detected in the pair - fed animals than in the leptin - treated ob / ob group . furthermore , leptin administration normalized both the glucose and insulin levels in ob / ob mice compared to wild types . these data suggest that leptin increases the insulin sensitivity in peripheral tissues , as evidenced by the lower homa and higher quicki indices ( p < .01 ) in the leptin - treated in comparison with the control ob / ob animals . serum glycerol was markedly increased ( p < .05 ) in the control ob / ob mice , while ffa and tg levels remained unchanged as compared to wild type mice . interestingly , leptin not only decreased circulating concentrations of ffa ( p < .05 ) and glycerol ( p < .01 ) levels as compared to control ob / ob mice , but also ffa ( p < .01 ) , glycerol ( p < .01 ) and tg ( p < .05 ) concentrations as compared to pair - fed mice . leptin administration to ob / ob mice reduced serum glycerol concentrations ( p = .032 ) and tended to decrease ffa ( p = .095 ) as compared to wild types . furthermore , leptin treatment increased the low concentrations of adiponectin of ob / ob mice , but the differences fell out of statistical significance ( p = .095 ) . control ob / ob mice exhibited significantly higher serum tbars than wild type littermates ( p < .01 ) , which were significantly reduced after leptin administration as compared to the control ( p < .01 ) and pair - fed ( p < .05 ) ob / ob groups ( figure 1(a ) ) . in addition , leptin decreased ( p < .01 ) the high concentrations of mda measured in the gastrocnemius muscle of control ob / ob mice , while this effect was not observed in the pair - fed group ( figure 1(b ) ) . oppositely , tbars levels were negatively associated with adiponectin and the quicki index both in serum and muscle . importantly , a high positive relation were found between serum and gastrocnemius concentrations of tbars ( = 0.63 , p = .003 ) ( table 3 ) . differential gene expression profiles in gastrocnemius muscle of wild type and ob / ob groups were compared by microarray analysis . applying these criteria , microarray data showed that 7,582 genes were differentially expressed by leptin deficiency and leptin administration in ob / ob mice . in particular , leptin deficiency altered the expression of 1,127 genes between wild type and control ob / ob mice . of these , 580 were upregulated and 547 were downregulated in ob / ob mice . leptin treatment modified the expression of 1,546 genes in ob / ob mice , upregulating 512 and repressing 1,034 . in addition , leptin repressed 736 genes that were upregulated in gastrocnemius muscle of control ob / ob and increased the transcript levels of 846 downregulated genes . functional enrichment analysis using geneontology and kegg databases revealed that the set of genes with altered expression levels induced by leptin deficiency and administration represents a broad spectrum of biological processes . however , for the purpose of the present paper we focused on the effects of leptin on the set of genes encoding proteins involved in oxidative stress and inflammation . table 4 shows that leptin deficiency and leptin administration altered the expression of a large number of genes involved in oxidative stress and inflammation . the biological processes mainly affected between control ob / ob mice and wild types included furthermore , several processes regulating proliferation , differentiation , and activity of lymphocytes were also significantly affected by leptin deficiency . importantly , comparison of leptin - treated and control ob / ob groups showed that leptin administration altered the expression of genes implicated in the positive regulation of lymphocyte activation ( p = .0187 ) , as well as genes involved in the chaperone cofactor dependent protein folding dna microarray analysis showed that 86 genes encoding proteins related to defense , stress , and inflammatory responses were altered in the gastrocnemius muscle of control ob / ob mice and modified by leptin administration . leptin reduced the mrna levels of various isoforms of the family of heat shock proteins ( hsps ) ( dnajc16 , dnaja4 , dnajb4 , hspa2 , hspa4 , and hspb7 ) , metallothioneins ( mt2 , mt4 ) , crystallins ( cryab , crybb1 ) and rna binding proteins ( rbms ) ( rbm9 , rbm22 ) in ob / ob mice ( table 5 ) . in addition , histocompatibility 2 , complement component factor b h2-bf and several genes of the acute - phase response or inflammatory processes , such as kallikrein 5 ( klk5 ) , and serine ( or cysteine ) proteinase inhibitor clade c member 1 ( serpinc1 ) and clade b member 1a ( serpinb1a ) , displayed an increased expression in ob / ob mice that was reduced by leptin administration . on the contrary , gene expression of cryl1 , hsp105 , rbm5 , and h2-aa were enhanced in ob / ob mice after treated with leptin . pair - feeding , which accounts for the decrease in food intake that is independent of the direct action of leptin , altered the expression of 1,960 genes , upregulating 984 while downregulating 976 genes . in the context of a food intake reduction as compared to the simple effect due to the caloric restriction , leptin administration further significantly altered the expression of genes involved in processes encompassing ( p = 2.99e ) , positive regulation of t cell activation ( p = .0003 ) and positive regulation of immune cell mediated cytotoxicity in particular , the gene array analysis provided evidence for elevated hspa4 , mt4 , crybb1 , and serpinb8 mrna levels in the pair - fed group as compared to the leptin - treated ob / ob mice ( table 6 ) . on the contrary , leptin increased the gene expression of h2-ab1 and h2-eb1 in ob / ob mice . in this sense , leptin administration reduced the mrna levels of the muscle atrophy - related transcription factor forkhead box o1 ( foxo1 ) and of the e3 ubiquitin - ligases muscle atrophy f - box ( mafbx ) and muscle ring finger 1 ( murf1 ) in leptin - treated ob / ob mice , while no effect of leptin was evidenced on the mrna levels of the transcriptional coactivator peroxisome proliferator - activated receptor- coactivator-1 ( pgc1 ) . our study provides evidence that leptin constitutes a negative regulator of oxidative stress and inflammation in the gastrocnemius , which is a representative skeletal muscle of the whole skeletal musculature . this statement is supported by findings reported herein : ( a ) leptin deficiency is accompanied by systemic and skeletal muscle oxidative stress , muscle inflammation , and reduced muscle mass ; ( b ) systemic and skeletal muscle oxidative stress , muscle atrophy and inflammation of ob / ob mice are reversed by leptin administration independently of the effects of food intake inhibition . therefore , leptin is able to prevent the muscle atrophy associated with obese and inflammatory states . since obese ob / ob mice exhibit an increased oxidative stress and impaired immune response [ 14 , 15 ] and a reduced skeletal muscle mass compared with their lean littermates , we aimed to identify the genes related to inflammatory processes differentially altered by leptin in the gastrocnemius muscle of obese ob / ob mice . among them , leptin repressed the high expression levels of acute - phase reactants and several members of the hsp and rbm families . in addition , confirming a previous study of our group , leptin treatment increased the reduced muscle weight of gastrocnemius muscle of ob / ob mice . taken together , these data suggest that leptin may prevent the obesity - associated inflammatory state and the muscle mass loss related to inflammatory states in leptin - deficient ob / ob mice . leptin - deficient ob / ob and leptin receptor - deficient db / db mice display many abnormalities in the immune response similar to those observed in starved animals and malnourished humans [ 14 , 15 , 22 ] . in this respect , exogenous leptin replacement to ob / ob mice modulates t cell responses in mice and prevents starvation - induced immunosuppression , suggesting that lack of leptin is directly involved in these immune system abnormalities [ 23 , 24 ] . in agreement with these studies , our findings show that leptin deficiency and administration differentially regulate biological processes related to the immune response as well as the t and b cell differentiation and activation in gastrocnemius muscle of ob / ob mice . oxidative stress is increased in diabetes [ 26 , 27 ] with leptin administration reportedly improving insulin sensitivity in normal and diabetic rodents [ 2830 ] . in this sense , findings of our study show a high oxidative stress in diabetic ob / ob mice , as reflected by increased tbars concentrations in serum and the gastrocemius muscle . more importantly , leptin administration decreased serum and gastrocnemius tbars concentrations as compared to control ob / ob mice , with tbars levels in gastrocnemius muscle from pair - fed ob / ob animals remaining very similar to those of control ob / ob mice . in this sense , from a molecular perspective , our results further show that transcript levels of sod1 , gpx3 and glutathione s - transferase 1 gstp1 are downregulated in control ob / ob mice as compared to wild type controls being upregulated after leptin treatment . furthermore , leptin administration also upregulated gpx7 , glutathione s - transferase 5 ( gstm5 ) and glutathione s - transferase 2 ( gstt2 ) . on the contrary , the high expression of catalase ( cat ) was repressed by the exogenous injection of leptin to ob / ob mice . these findings are in line with previous observations showing the restoration of the defective antioxidant enzyme activity in plasma of ob / ob mice and humans with a leptin gene mutation . acute - phase reactants have been suggested to contribute to the maintenance of the chronic low - grade inflammation state involved in the progression of obesity and related diseases . interestingly , our study provides evidence that genes of the acute - phase response were altered in gastrocnemius muscle of ob / ob mice , which were counteracted by exogenous leptin administration . leptin reduced the elevated gene expression of tissue - type plasminogen activator ( plat ) and lipocalin-2 ( lcn2 ) , which are upregulated in many inflammatory conditions [ 42 , 43 ] , including human obesity . a potential link between oxidative stress and muscle moreover , various inflammatory cytokines induce oxidative stress and muscle atrophy through the activation of the lysosomal [ 48 , 49 ] and the ubiquitin - proteolysis system . in this context , biological processes related to oxidative stress and inflammatory responses were altered in the gastrocnemius muscle of ob / ob mice and improved following leptin treatment . in spite of the usual upregulation of the e3 ubiquitin - ligases mafbx and murf1 in most conditions associated with atrophy , their gene expression levels in ob / ob were lower as compared to wild type animals , although no statistically significant differences were observed . contrarily to what would be expected , leptin administration prevented the increase of both mafbx and murf1 mrna expression levels induced by pair - feeding in ob / ob mice . these data are in accordance with a previous study of our group evidencing that leptin replacement inhibits the ubiquitin proteolysis system activity in leptin - deficient mice . muscle atrophy is associated with increased expression of genes coding for rbm proteins which facilitate the translation , protection , and restoration of native rna conformations during oxidative stress . other proteins involved in oxidative stress are metallothioneins , endogenous antioxidants that have been shown to be overexpressed in muscle atrophy in rodents [ 5456 ] . in the present work , we have observed that administration of leptin inhibits the gene expression of several members of the rbm ( rbm9 , rbm22 ) and metallothioneins ( mt2 , mt4 ) families in the gastrocnemius of ob / ob mice , suggesting that leptin may modulate the inflammatory and oxidative stress responses and consequently , the muscle loss related to inflammatory states . genes involved in the chaperone system were also differentially expressed in ob / ob mice as compared to wild types and modified by leptin treatment . in this regard , a recent study has shown that hsp70 prevents muscle atrophy induced by physical inactivity through inhibition of the muscle atrophy - related transcription factor foxo3a and the expression of mafbx and murf1 . in this sense , our results provide evidence that leptin inhibits the increased gene expression of different members of the hsps ( hspb7 , dnajc16 , hspa4 , cryab , and crybb1 ) in the gastrocnemius muscle of ob / ob mice . taken together , the elevated expression of hsps in the control and pair - fed ob / ob groups suggests a high defense and stress response in these mice . reported an increase in hsp60 gene expression in pancreatic cells by leptin . obesity is accompanied by a chronic proinflammatory state resulting in an increase in circulating cytokines and inflammatory markers . in this regard , inflammation produces metabolic alterations in skeletal muscle with both inflammatory response and insulin resistance being associated with muscle mass loss . findings of our study provide evidence that systemic and skeletal muscle oxidative stress , muscle atrophy and the elevated expression of genes involved in oxidative stress and inflammation of ob / ob mice are reversed by leptin administration . taken together , these data thereby support that leptin is able to prevent the muscle atrophy associated with obese and inflammatory states in ob / ob mice . our paper sheds light on the relation between obesity and the loss of muscle mass associated to inflammatory states suggesting that leptin treatment may be an attractive therapeutic approach to prevent muscle loss associated with inflammatory diseases .

inflammatory cytokines have been involved in the impairment of insulin signaling , thus providing molecular links between inflammation and insulin resistance . inflammation reportedly produces metabolic alterations in skeletal muscle with both inflammatory response and insulin resistance being associated with loss of muscle mass by decreased protein synthesis and increased proteolysis [ 35 ] . recently , our group has shown that leptin reverses muscle loss of ob / ob mice by inhibiting the activity of the transcriptional factor forkhead box class o3a ( foxo3a ) . leptin - deficient ob / ob mice are obese , hyperphagic , exhibit type 2 diabetes , decreased body temperature and hypogonadotropic hypogonadism . leptin is a member of the long - chain helical cytokine family and its receptors , which belong to the class i cytokine receptors , are present in bone marrow and spleen as well as on peripheral monocytes and lymphocytes . leptin increases in response to acute infection and sepsis and it has been reported to exert a profound influence on the function and proliferation of t lymphocytes and natural killer cells , on the phagocytosis of macrophages / monocytes , and to have a direct effect on the secretion of anti- and proinflammatory cytokines . in this regard , impaired cellular and humoral immunity have been shown in leptin - deficient ob / ob mice as well as in leptin receptor - deficient db / db mice [ 14 , 15 ] . these studies reflect the molecular nature of leptin as a cytokine and are consistent with leptin signaling playing a pivotal role in the pathogenesis of obesity - associated inflammation and muscle loss . in the present paper , gastrocnemius muscle samples from wild type and ob / ob mice were analyzed for mrna presence of over 41,000 transcripts by microarray analysis to identify genes involved in inflammation and oxidative stress that are affected by leptin deficiency and leptin administration in ob / ob mice . it was shown that leptin increases the gastrocnemius weight and reduces the high expression levels of genes related to the obesity - associated low - grade inflammation in skeletal muscle of ob / ob mice . ten - week - old male genetically obese ob / ob mice ( c57bl/6j ) ( n = 15 ) and their lean control littermates wild type ( n = 5 ) supplied by harlan ( barcelona , spain ) were housed in a room with controlled temperature ( 22 2c ) and a 12:12 light - dark cycle ( lights on at 08:00 am ) . body weight of ob / ob mice was measured before randomization into control , leptin - treated ( 1 mg / kg / d ) and pair - fed groups ( n = 5 per group ) . the control and pair - fed groups received vehicle ( pbs ) , while leptin - treated mice were intraperitoneally administered with leptin ( bachem , bubendorf , switzerland ) twice daily at 08:00 am and 08:00 pm for 28 days . control and leptin - treated groups were provided with water and food ad libitum with a standard rodent chow ( 2014s teklad , harlan ) , while daily food intake of the pair - fed group was matched to the amount consumed by the leptin - treated group the day before in order to discriminate the inhibitory effect of leptin on appetite . animals were sacrificed on the 28th day of treatment by co2 inhalation 20 hours after the last pbs or leptin administration ( in order to avoid picking up effects reflecting an acute response ) and after 8 hours of fasting . , intra- and interassay coefficients of variation for measurements of insulin and leptin were 3.5% and 6.3% , respectively , for the former , and 2.8% and 5.8% , for the latter . since the best - known specific tbars is malondialdehyde ( mda ) , we used serum mda levels , a secondary product of lipid peroxidation , as an indicator of lipid peroxidation and oxidative stress . then , the chromophore of the detba - mda adduct was quantified in 200 l of the upper butanol phase by fluorescence emission at 535 nm with an excitation at 590 nm . in addition , gene ontology database ( http://babelomics.bioinfo.cipf.es ) and the kegg website ( http://www.genome.ad.jp/kegg/pathway ) were used in conjunction with genespring ( http://www.agilent.com/chem/genespring ) to identify pathways and functional groups of genes . to validate the microarray data , a number of representative differentially expressed genes were selected to be individually studied by real - time pcr ( 7300 real time pcr system , applied biosystems , foster city , ca , usa ) ( n = 5 per group ) as previously described . as previously outlined , gene ontology groupings were used to identify pathways significantly affected by leptin deficiency as opposed to its administration . body weight was significantly higher ( p < .01 ) in the control ob / ob group as compared to wild type mice . leptin - treated mice exhibited a decreased body weight ( p < .01 ) as compared to control and pair - fed ob / ob animals . importantly , leptin treatment normalized body weight of ob / ob mice as compared to wild type ( p = .690 ) . in addition , the gastrocnemius of control ob / ob mice exhibited a lower ( p < .01 ) muscle weight than that of wild type mice and it was increased ( p < .01 ) by leptin administration in comparison with that of control and pair - fed ob / ob rodents . as depicted in table 2 , higher fasting glucose ( p < .05 ) and insulin ( p < .01 ) concentrations were observed in the control ob / ob mice compared to wild types . although no differences in glucose concentrations were observed in pair - fed as compared to leptin - treated ob / ob mice , higher serum insulin concentrations ( p < .05 ) were detected in the pair - fed animals than in the leptin - treated ob / ob group . these data suggest that leptin increases the insulin sensitivity in peripheral tissues , as evidenced by the lower homa and higher quicki indices ( p < .01 ) in the leptin - treated in comparison with the control ob / ob animals . serum glycerol was markedly increased ( p < .05 ) in the control ob / ob mice , while ffa and tg levels remained unchanged as compared to wild type mice . interestingly , leptin not only decreased circulating concentrations of ffa ( p < .05 ) and glycerol ( p < .01 ) levels as compared to control ob / ob mice , but also ffa ( p < .01 ) , glycerol ( p < .01 ) and tg ( p < .05 ) concentrations as compared to pair - fed mice . leptin administration to ob / ob mice reduced serum glycerol concentrations ( p = .032 ) and tended to decrease ffa ( p = .095 ) as compared to wild types . furthermore , leptin treatment increased the low concentrations of adiponectin of ob / ob mice , but the differences fell out of statistical significance ( p = .095 ) . control ob / ob mice exhibited significantly higher serum tbars than wild type littermates ( p < .01 ) , which were significantly reduced after leptin administration as compared to the control ( p < .01 ) and pair - fed ( p < .05 ) ob / ob groups ( figure 1(a ) ) . in addition , leptin decreased ( p < .01 ) the high concentrations of mda measured in the gastrocnemius muscle of control ob / ob mice , while this effect was not observed in the pair - fed group ( figure 1(b ) ) . serum and gastrocnemius tbars levels were positively associated with body weight , ffa , insulin , and the homa index . oppositely , tbars levels were negatively associated with adiponectin and the quicki index both in serum and muscle . importantly , a high positive relation were found between serum and gastrocnemius concentrations of tbars ( = 0.63 , p = .003 ) ( table 3 ) . differential gene expression profiles in gastrocnemius muscle of wild type and ob / ob groups were compared by microarray analysis . in particular , leptin deficiency altered the expression of 1,127 genes between wild type and control ob / ob mice . functional enrichment analysis using geneontology and kegg databases revealed that the set of genes with altered expression levels induced by leptin deficiency and administration represents a broad spectrum of biological processes . however , for the purpose of the present paper we focused on the effects of leptin on the set of genes encoding proteins involved in oxidative stress and inflammation . table 4 shows that leptin deficiency and leptin administration altered the expression of a large number of genes involved in oxidative stress and inflammation . the biological processes mainly affected between control ob / ob mice and wild types included furthermore , several processes regulating proliferation , differentiation , and activity of lymphocytes were also significantly affected by leptin deficiency . importantly , comparison of leptin - treated and control ob / ob groups showed that leptin administration altered the expression of genes implicated in the positive regulation of lymphocyte activation ( p = .0187 ) , as well as genes involved in the chaperone cofactor dependent protein folding dna microarray analysis showed that 86 genes encoding proteins related to defense , stress , and inflammatory responses were altered in the gastrocnemius muscle of control ob / ob mice and modified by leptin administration . leptin reduced the mrna levels of various isoforms of the family of heat shock proteins ( hsps ) ( dnajc16 , dnaja4 , dnajb4 , hspa2 , hspa4 , and hspb7 ) , metallothioneins ( mt2 , mt4 ) , crystallins ( cryab , crybb1 ) and rna binding proteins ( rbms ) ( rbm9 , rbm22 ) in ob / ob mice ( table 5 ) . in addition , histocompatibility 2 , complement component factor b h2-bf and several genes of the acute - phase response or inflammatory processes , such as kallikrein 5 ( klk5 ) , and serine ( or cysteine ) proteinase inhibitor clade c member 1 ( serpinc1 ) and clade b member 1a ( serpinb1a ) , displayed an increased expression in ob / ob mice that was reduced by leptin administration . on the contrary , gene expression of cryl1 , hsp105 , rbm5 , and h2-aa were enhanced in ob / ob mice after treated with leptin . pair - feeding , which accounts for the decrease in food intake that is independent of the direct action of leptin , altered the expression of 1,960 genes , upregulating 984 while downregulating 976 genes . in the context of a food intake reduction as compared to the simple effect due to the caloric restriction , leptin administration further significantly altered the expression of genes involved in processes encompassing ( p = 2.99e ) , positive regulation of t cell activation ( p = .0003 ) and positive regulation of immune cell mediated cytotoxicity in particular , the gene array analysis provided evidence for elevated hspa4 , mt4 , crybb1 , and serpinb8 mrna levels in the pair - fed group as compared to the leptin - treated ob / ob mice ( table 6 ) . in this sense , leptin administration reduced the mrna levels of the muscle atrophy - related transcription factor forkhead box o1 ( foxo1 ) and of the e3 ubiquitin - ligases muscle atrophy f - box ( mafbx ) and muscle ring finger 1 ( murf1 ) in leptin - treated ob / ob mice , while no effect of leptin was evidenced on the mrna levels of the transcriptional coactivator peroxisome proliferator - activated receptor- coactivator-1 ( pgc1 ) . our study provides evidence that leptin constitutes a negative regulator of oxidative stress and inflammation in the gastrocnemius , which is a representative skeletal muscle of the whole skeletal musculature . this statement is supported by findings reported herein : ( a ) leptin deficiency is accompanied by systemic and skeletal muscle oxidative stress , muscle inflammation , and reduced muscle mass ; ( b ) systemic and skeletal muscle oxidative stress , muscle atrophy and inflammation of ob / ob mice are reversed by leptin administration independently of the effects of food intake inhibition . since obese ob / ob mice exhibit an increased oxidative stress and impaired immune response [ 14 , 15 ] and a reduced skeletal muscle mass compared with their lean littermates , we aimed to identify the genes related to inflammatory processes differentially altered by leptin in the gastrocnemius muscle of obese ob / ob mice . taken together , these data suggest that leptin may prevent the obesity - associated inflammatory state and the muscle mass loss related to inflammatory states in leptin - deficient ob / ob mice . leptin - deficient ob / ob and leptin receptor - deficient db / db mice display many abnormalities in the immune response similar to those observed in starved animals and malnourished humans [ 14 , 15 , 22 ] . in this respect , exogenous leptin replacement to ob / ob mice modulates t cell responses in mice and prevents starvation - induced immunosuppression , suggesting that lack of leptin is directly involved in these immune system abnormalities [ 23 , 24 ] . in agreement with these studies , our findings show that leptin deficiency and administration differentially regulate biological processes related to the immune response as well as the t and b cell differentiation and activation in gastrocnemius muscle of ob / ob mice . oxidative stress is defined as the imbalanced redox state in which prooxidants overwhelm the antioxidant capacity , resulting in an increased production of reactive oxygen species ( ros ) , ultimately leading to oxidative damage of cellular macromolecules . dismutation of o2 by superoxide dismutase ( sod ) produces hydrogen peroxide ( h2o2 ) , a more stable ros , which , in turn , is converted to water by catalase and glutathione peroxidase ( gpx ) . leptin stimulates in vitro ros production by inflammatory cells and endothelial cells and the level of systemic oxidative stress in nonobese animals [ 33 , 34 ] , suggesting a prooxidative role of leptin . in this sense , findings of our study show a high oxidative stress in diabetic ob / ob mice , as reflected by increased tbars concentrations in serum and the gastrocemius muscle . more importantly , leptin administration decreased serum and gastrocnemius tbars concentrations as compared to control ob / ob mice , with tbars levels in gastrocnemius muscle from pair - fed ob / ob animals remaining very similar to those of control ob / ob mice . in this sense , from a molecular perspective , our results further show that transcript levels of sod1 , gpx3 and glutathione s - transferase 1 gstp1 are downregulated in control ob / ob mice as compared to wild type controls being upregulated after leptin treatment . acute - phase reactants have been suggested to contribute to the maintenance of the chronic low - grade inflammation state involved in the progression of obesity and related diseases . interestingly , our study provides evidence that genes of the acute - phase response were altered in gastrocnemius muscle of ob / ob mice , which were counteracted by exogenous leptin administration . leptin reduced the elevated gene expression of tissue - type plasminogen activator ( plat ) and lipocalin-2 ( lcn2 ) , which are upregulated in many inflammatory conditions [ 42 , 43 ] , including human obesity . in addition , a pivotal role for oxidative stress in the pathogenesis of muscle wasting in disuse and in a variety of pathological conditions is now being widely recognized . a potential link between oxidative stress and muscle moreover , various inflammatory cytokines induce oxidative stress and muscle atrophy through the activation of the lysosomal [ 48 , 49 ] and the ubiquitin - proteolysis system . in this context , biological processes related to oxidative stress and inflammatory responses were altered in the gastrocnemius muscle of ob / ob mice and improved following leptin treatment . in spite of the usual upregulation of the e3 ubiquitin - ligases mafbx and murf1 in most conditions associated with atrophy , their gene expression levels in ob / ob were lower as compared to wild type animals , although no statistically significant differences were observed . contrarily to what would be expected , leptin administration prevented the increase of both mafbx and murf1 mrna expression levels induced by pair - feeding in ob / ob mice . muscle atrophy is associated with increased expression of genes coding for rbm proteins which facilitate the translation , protection , and restoration of native rna conformations during oxidative stress . in the present work , we have observed that administration of leptin inhibits the gene expression of several members of the rbm ( rbm9 , rbm22 ) and metallothioneins ( mt2 , mt4 ) families in the gastrocnemius of ob / ob mice , suggesting that leptin may modulate the inflammatory and oxidative stress responses and consequently , the muscle loss related to inflammatory states . hsps represent a family of molecular chaperones induced in response to cellular stress , responsible for maintaining the structure of proteins and contributing to the repair of damaged or malformed proteins in highly oxidative and lipotoxic conditions . hsp70 is constitutively expressed in skeletal muscle , but its levels are increased in response to oxidative stress with the induction of hsp70 expression by hyperthermia and during inactivity attenuating muscle atrophy [ 62 , 63 ] . in this regard , a recent study has shown that hsp70 prevents muscle atrophy induced by physical inactivity through inhibition of the muscle atrophy - related transcription factor foxo3a and the expression of mafbx and murf1 . among the hsps , hsp70 and b - crystallin in particular , are considered negative regulators of muscle cell apoptosis [ 65 , 66 ] and may inhibit the loss of nuclei taking place during muscle atrophy . in addition , ros induce the activity of foxo and gene expression of members of the ubiquitin - proteolysis system in myotubes . in this sense , our results provide evidence that leptin inhibits the increased gene expression of different members of the hsps ( hspb7 , dnajc16 , hspa4 , cryab , and crybb1 ) in the gastrocnemius muscle of ob / ob mice . taken together , the elevated expression of hsps in the control and pair - fed ob / ob groups suggests a high defense and stress response in these mice . in mammals , caloric restriction has been shown to upregulate hsp induction [ 70 , 71 ] , while expression of hsp72 has been found to be low in skeletal muscle of patients with insulin resistance or type 2 diabetes [ 72 , 73 ] . findings of our study provide evidence that systemic and skeletal muscle oxidative stress , muscle atrophy and the elevated expression of genes involved in oxidative stress and inflammation of ob / ob mice are reversed by leptin administration . taken together , these data thereby support that leptin is able to prevent the muscle atrophy associated with obese and inflammatory states in ob / ob mice . our paper sheds light on the relation between obesity and the loss of muscle mass associated to inflammatory states suggesting that leptin treatment may be an attractive therapeutic approach to prevent muscle loss associated with inflammatory diseases .

although fish farming in iraq started in 1955 with a small pond in al - zaafaraniya , south of baghdad city , an advance was achieved in fish farming industry in iraq during the seventies and early eighties of the last century when many fish farms were established especially in the middle of iraq . however , such achievement was hindered due to consequences of the war situations during 19801988 and 1991 as well as the economic sanction imposed by the un against iraq on august 6 , 1990 . during the last few years , a great advance was achieved in fish farming in general and fish cages in particular due to the increasing demand on fish protein as well as the increasing investment in fish - culture industry in most provinces of iraq . according to the statistics , a total of 441 working fish farms are scattered in iraq . of these farms , a total of 72 working fish farms are situated in babylon province alone with a water area of 44.5% of the total water area of fish farms in iraq . under extensive fish culture and inadequate administrative and control measures , fish farms are vulnerable to great hazards due to the infection with parasites and other disease agents [ 2 , 4 , 5 ] . many parasite species can easily spread among fishes suffering from crowd and bad managements , especially those parasites with direct life cycles . in connection with the parasites of cultured fishes of babylon province , mhaisen et al . surveyed the literature on the parasitic fauna of fishes of al - furat fish farm ( previously known as babylon fish farm ) , which is the biggest fish farm in babylon province , and showed that the parasitic fauna of fishes of that farm included 60 valid parasite species ( 10 protozoans , three myxozoans , one trematode , 29 monogeneans , five cestodes , three nematodes , two acanthocephalans , six crustaceans , and one mollusc larva ) . such data are scattered in different local journals , unpublished theses , and few other sources . therefore , the present paper was aimed at gathering data from the literature concerning all fish farms of babylon province and providing a list of parasite species according to their major groups as well as a host - parasite list for cultured fishes of these farms and some other fish species found in such farms . such parasite list will help owners of fish farms and fish veterinarians to know what sort of parasites are found in their fish farms , which will help them later in taking appropriate measures for their control . a total of 50 references ( 33 published articles , 12 unpublished theses , two unpublished reports , one book , one conference abstract , and one review article ) dealing with the parasites of farm fishes of babylon province were used to prepare the present paper . data from such references was gathered to provide host - parasite and parasite - host lists . the systematic account of these parasites is based on some electronic sites [ 812 ] as well as some taxonomic references [ 1316 ] . the index - catalogue of parasites and disease agents of fishes of iraq was used to indicate the total number of fish hosts harbouring each parasite species in the whole waters of iraq . so far 25 chronologically arranged references [ 1842 ] were concerned with the parasitic fauna of al - furat fish farm . only seven references [ 4349 ] were concerned with the parasitic fauna of al - shark al - awsat fish farm . the literature concerned with fish parasites of other farms in babylon province included those from al - latifiya fish farm [ 6 , 5052 ] ; al - bajaa fish farm ; abdul - razzak al - janabi fish farm ; fawzi al - janabi fish farm and ali al - hayali fish farm ; three fish farms at al - iskandariya district : abdul - hadi al - matloob fish farm , hussain al - gaiem fish farm , and maki chinak fish farm ; technical institute of al - musaib fish farm ; and al - manahil ( al - bilad ) fish farm at al - iskandariya district . in addition , surveys were done from some unnamed fish ponds such as those at al - mahaweel district , al - musaib district , al - iskandariya district , and sadat al - hindiya district as well as some other unnamed farms in the province [ 6165 ] . surveying the literature concerning the parasites so far recorded from fish farms of babylon province showed the presence of 92 parasite species . these parasites included one mastigophoran , three apicomplexans , 13 ciliophorans , five myxozoans , five trematodes , 45 monogeneans , five cestodes , three nematodes , two acanthocephalans , nine arthropods , and one mollusc . the common carp was found to harbour 81 species of parasites , the grass carp 30 species , the silver carp 28 species , l. abu 13 species , c. auratus one species , and h. fossilis one species . the layout and names of the major taxonomic groups ( phyla and classes ) followed a checklist of an fao fisheries technical paper . these major groups represent the concerned phyla of the parasites , but due to the great numbers of parasite species of the phylum platyhelminthes , its three classes ( trematoda , monogenea , and cestoda ) were applied in addition to their phylum . the parasite - host list is arranged in the major groups ( phyla or classes ) of parasitic fauna according to kirjuina and vismanis . for each major group , a list of species together with their hosts and concerned references is given . to economize space , names of fish farms also the systematic account of all major groups is given down to the specific name of all parasites . for each parasite species , all records in farm fishes in babylon province are given together with the first record of each concerned parasite in iraq as well as the present number of all hosts so far known in iraq for each concerned species based on the index - catalogue of parasites and disease agents of fishes of iraq . the phylum mastigophora is represented in farm fishes of babylon province with only one parasite species of the genus ichthyobodo . the systematic account of this parasite , followed by parasite - host list , is given here . phylum mastigophora class kinetoplastidea order kinetoplastida family bodonidae ichthyobodo necator ( henneguy , 1884 ) pinto , 1928 class kinetoplastidea order kinetoplastida family bodonidae ichthyobodo necator ( henneguy , 1884 ) pinto , 1928 family bodonidae ichthyobodo necator ( henneguy , 1884 ) pinto , 1928 ichthyobodo necator ( henneguy , 1884 ) pinto , 1928 ichthyobodo necator ( henneguy , 1884 ) pinto , 1928 ichthyobodo necator ( henneguy , 1884 ) pinto , 1928 , was erroneously reported as costia necatrix from the skin and gills of c. carpio . the first record of c. necatrix in iraq was from body surface of h. fossilis from al - ashar canal at basrah . seven fish host species are so far known for this parasite ( as c. necatrix ) in iraq . the phylum apicomplexa , which is known as phylum myzozoa according to worms , is represented in farm fishes of babylon province with three species ; two of them belonged to the genus eimeria and one unspecified species to the genus haemogregarina . phylum apicomplexa class sporozoa order eucoccidiorida family eimeriidae eimeria dogieli ( dogiel , 1948 ) pellerdy , 1963 eimeria mylopharyngodoni chen , 1956 family haemogregarinidae haemogregarina sp . order eucoccidiorida family eimeriidae eimeria dogieli ( dogiel , 1948 ) pellerdy , 1963 eimeria mylopharyngodoni chen , 1956 family haemogregarinidae haemogregarina sp . family eimeriidae eimeria dogieli ( dogiel , 1948 ) pellerdy , 1963 eimeria mylopharyngodoni chen , 1956 family haemogregarinidae haemogregarina sp . eimeria dogieli ( dogiel , 1948 ) pellerdy , 1963 eimeria mylopharyngodoni chen , 1956 eimeria dogieli ( dogiel , 1948 ) pellerdy , 1963 eimeria mylopharyngodoni chen , 1956 family haemogregarinidae eimeria dogieli ( dogiel , 1948 ) pellerdy , 1963 , was recorded from the intestine of c. carpio . so far , this is the only record of e. dogieli from fishes of iraq . the specific name was misspelled as mylopharyngodon and no authority , site of infection , parasite description , and illustration were given for this parasite by hussain et al . . so this record is considered as questionable especially if we take in consideration that hussain et al . examined c. carpio externally while e. mylopharyngodoni is known to infect intestine , kidneys , and liver of fishes . three species of haemogregarina were so far recorded from blood of three fish species in basrah province only . so we think that the record of haemogregarina sp . from gills of c. carpio by hussain et al . with neither description nor a good illustration is considered as questionable . the phylum ciliophora is represented in farm fishes of babylon province with 13 species , three of which belonged to the genera chilodonella , ichthyophthirius , and tripartiella , five to the genus apiosoma , and four to the genus trichodina in addition to unspecified species of the genus trichodina . phylum ciliophora class kinetophragminophorea order cyrtophorida family chilodonellidae chilodonella cyprini ( moroff , 1902 ) strand , 1928 class oligohymenophorea order hymenostomatida family ichthyophthiriidae ichthyophthirius multifiliis fouquet , 1876 order petrichida family epistylididae apiosoma amoebae ( grenfell , 1887 ) lom , 1966 apiosoma cylindriformis ( chen , 1955 ) apiosoma minuta ( chen , 1961 ) lom , 1966 apiosoma piscicola blanchard , 1885 apiosoma poteriformis ( timofeev , 1962 ) lom , 1966 order mobilida family trichodinidae trichodina cottidarum dogiel , 1948 trichodina domerguei ( wallengren , 1897 ) trichodina gracilis polyanskii , 1955 trichodina nigra lom , 1960 trichodina sp . tripartiella amurensis ( chan , 1961 ) class kinetophragminophorea order cyrtophorida family chilodonellidae chilodonella cyprini ( moroff , 1902 ) strand , 1928 family chilodonellidae chilodonella cyprini ( moroff , 1902 ) strand , 1928 family chilodonellidae chilodonella cyprini ( moroff , 1902 ) strand , 1928 chilodonella cyprini ( moroff , 1902 ) strand , 1928 class oligohymenophorea order hymenostomatida family ichthyophthiriidae ichthyophthirius multifiliis fouquet , 1876 order petrichida family epistylididae apiosoma amoebae ( grenfell , 1887 ) lom , 1966 apiosoma cylindriformis ( chen , 1955 ) apiosoma minuta ( chen , 1961 ) lom , 1966 apiosoma piscicola blanchard , 1885 apiosoma poteriformis ( timofeev , 1962 ) lom , 1966 order mobilida family trichodinidae trichodina cottidarum dogiel , 1948 trichodina domerguei ( wallengren , 1897 ) trichodina gracilis polyanskii , 1955 trichodina nigra lom , 1960 trichodina sp . tripartiella amurensis ( chan , 1961 ) order hymenostomatida family ichthyophthiriidae ichthyophthirius multifiliis fouquet , 1876 family ichthyophthiriidae ichthyophthirius multifiliis fouquet , 1876 ichthyophthirius multifiliis fouquet , 1876 family epistylididae apiosoma amoebae ( grenfell , 1887 ) lom , 1966 apiosoma cylindriformis ( chen , 1955 ) apiosoma minuta ( chen , 1961 ) lom , 1966 apiosoma piscicola blanchard , 1885 apiosoma poteriformis ( timofeev , 1962 ) lom , 1966 apiosoma amoebae ( grenfell , 1887 ) lom , 1966 apiosoma cylindriformis ( chen , 1955 ) apiosoma minuta ( chen , 1961 ) lom , 1966 apiosoma piscicola blanchard , 1885 apiosoma poteriformis ( timofeev , 1962 ) lom , 1966 apiosoma amoebae ( grenfell , 1887 ) lom , 1966 apiosoma cylindriformis ( chen , 1955 ) apiosoma minuta ( chen , 1961 ) lom , 1966 apiosoma piscicola blanchard , 1885 apiosoma poteriformis ( timofeev , 1962 ) lom , 1966 family trichodinidae trichodina cottidarum dogiel , 1948 trichodina domerguei ( wallengren , 1897 ) trichodina gracilis polyanskii , 1955 trichodina nigra lom , 1960 trichodina sp . tripartiella amurensis ( chan , 1961 ) trichodina cottidarum dogiel , 1948 trichodina domerguei ( wallengren , 1897 ) trichodina gracilis polyanskii , 1955 trichodina nigra lom , 1960 trichodina sp . tripartiella amurensis ( chan , 1961 ) trichodina cottidarum dogiel , 1948 trichodina domerguei ( wallengren , 1897 ) trichodina gracilis polyanskii , 1955 trichodina nigra lom , 1960 tripartiella amurensis ( chan , 1961 ) chilodonella cyprini ( moroff , 1902 ) strand , 1928 , was recorded from skin , buccal cavity , and gills of c. idella [ 27 , 29 , 43 , 51 ] , skin , fins , buccal cavity , and gills of c. carpio [ 27 , 30 , 43 , 51 , 53 ] , and skin and gills of h. molitrix [ 27 , 43 , 51 ] . the first record of c. cyprini in iraq was from skin , buccal cavity , and gills of mystus pelusius from tigris river at baghdad . ichthyophthirius multifiliis fouquet , 1876 , was recorded from skin and gills of c. idella [ 27 , 29 , 43 , 51 , 56 ] , skin , fins , and gills of c. carpio [ 25 , 27 , 29 , 30 , 35 , 43 , 45 , 47 , 49 , 51 , 5356 , 59 , 60 , 65 ] , and skin and gills of h. molitrix [ 27 , 29 , 45 , 47 , 51 ] . the first record of i. multifiliis in iraq was from the skin and gills of chelon subviridis ( reported as mugil dussumieri ) from tigris river near baghdad . apiosoma amoebae ( grenfell , 1887 ) lom , 1966 , was reported from skin , buccal cavity , and gills of c. idella [ 20 , 24 , 27 ] , skin and buccal cavity of c. carpio [ 27 , 55 ] , and gills and buccal cavity of h. molitrix [ 20 , 27 ] . it is appropriate to mention here that a. amoebae was reported as glossatella amoebae [ 27 , 55 ] . the first record of a. amoebae in iraq was from the skin , buccal cavity , and gills of c. idella from babylon fish farm . apiosoma cylindriformis ( chen , 1955 ) was reported from gills of c. idella [ 20 , 24 , 27 ] , buccal cavity and gills of c. carpio , and gills of h. molitrix [ 20 , 21 , 27 ] . the first record of a. cylindriformis in iraq was from gills of c. idella and h. molitrix from babylon fish farm . apiosoma minuta ( chen , 1961 ) lom , 1966 , was recorded from skin of c. carpio [ 45 , 47 ] . so far , a. minuta has two fish host species in iraq as it was recently recorded from luciobarbus xanthopterus by al - salmany . apiosoma piscicola blanchard , 1885 , was recorded from skin , buccal cavity , and gills of c. idella [ 27 , 51 ] , skin , buccal cavity , and gills of c. carpio [ 27 , 29 , 51 , 55 ] , and buccal cavity and gills of h. molitrix [ 20 , 21 , 27 , 29 , 51 ] . a. piscicola was recorded for the first time in iraq from skin , buccal cavity , and gills of c. idella , c. carpio and h. molitrix from al - suwaira and al - latifiya fish farms . apiosoma poteriformis ( timofeev , 1962 ) lom , 1966 , was recorded from skin and gills of c. idella [ 20 , 24 , 27 ] and buccal cavity and gills of c. carpio . it is appropriate to mention here that a. poteriformis was reported as glossatella poteriformis by al - zubaidy . a. poteriformis was recorded for the first time in iraq from gills of c. idella from babylon fish farm . trichodina cottidarum dogiel , 1948 , was recorded from skin and gills of c. carpio [ 45 , 47 , 49 , 54 , 59 , 60 ] and skin and gills of h. molitrix [ 45 , 47 ] . t. cottidarum was recorded for the first time in iraq from gills of c. carpio from a manmade lake at baghdad city . trichodina domerguei ( wallengren , 1897 ) was recorded from skin , fins , buccal cavity , and gills of c. idella [ 24 , 27 , 29 , 43 , 51 , 56 ] , skin , fins , buccal cavity , and gills of c. carpio [ 25 , 27 , 29 , 30 , 35 , 43 , 51 , 53 , 55 , 56 , 59 ] , skin , buccal cavity , and gills of h. molitrix [ 20 , 27 , 51 ] , and skin and gills of l. abu [ 23 , 56 ] . the first record of t. domerguei in iraq was from skin , fins , and gills of eight freshwater fish species from tigris river , al - tharthar lake , and fish markets in baghdad city . so far , t. domerguei has 39 host species in iraq and , therefore , it is the most prevalent ciliate species among fishes of iraq . trichodina gracilis polyanskii , 1955 , was recorded from skin of c. carpio [ 45 , 47 ] . trichodina nigra lom , 1960 , was recorded from skin and gills of the three carp species : c. idella , c. carpio [ 27 , 29 , 35 , 49 , 55 , 59 , 60 ] , and h. molitrix [ 27 , 29 ] . the first record of t. nigra in iraq was from skin and gills of c. carpio and gills of h. molitrix from babylon fish farm . unidentified specimen of trichodina was recorded from l. abu with no mention to site of infection . in addition to 24 recognized trichodina species so far recorded from fishes of iraq , some unidentified species of trichodina were so far recorded from six fish species . tripartiella amurensis ( chan , 1961 ) was recorded from skin of c. carpio [ 45 , 47 ] . the phylum myxozoa is represented in farm fishes of babylon province with five species : one species belonged to the genus myxobilatus and four species to the genus myxobolus . phylum myxozoa class myxosporea order bivalvulida family sphaerosporidae myxobilatus legeri ( cpde , 1905 ) family myxobolidae myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski , 1940 myxobolus muelleri btschli , 1882 myxobolus oviformis thlohan , 1892 myxobolus pfeifferi thlohan , 1895 order bivalvulida family sphaerosporidae myxobilatus legeri ( cpde , 1905 ) family myxobolidae myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski , 1940 myxobolus muelleri btschli , 1882 myxobolus oviformis thlohan , 1892 myxobolus pfeifferi thlohan , 1895 family sphaerosporidae myxobilatus legeri ( cpde , 1905 ) family myxobolidae myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski , 1940 myxobolus muelleri btschli , 1882 myxobolus oviformis thlohan , 1892 myxobolus pfeifferi thlohan , 1895 family sphaerosporidae myxobilatus legeri ( cpde , 1905 ) myxobilatus legeri ( cpde , 1905 ) myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski , 1940 myxobolus muelleri btschli , 1882 myxobolus oviformis thlohan , 1892 myxobolus pfeifferi thlohan , 1895 myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski , 1940 myxobolus muelleri btschli , 1882 myxobolus oviformis thlohan , 1892 myxobolus pfeifferi thlohan , 1895 myxobilatus legeri ( cpde , 1905 ) , erroneously reported as myxobllatus legerl , with no given authority , description , and illustration , was recorded from skin and gills of c. carpio . myxobolus dogieli bykhovskaya - pavlovskaya & bykhovski , 1940 , was recorded from kidneys and gallbladder of l. abu . the first record of m. dogieli in iraq was mainly from the external surface of heart , liver , and ovaries of l. abu from tigris river at baiji town . myxobolus muelleri btschli , 1882 , was recorded from intestine and liver of c. carpio with the specific name spelled as mlleri . the first record of m. muelleri in iraq was from gills of luciobarbus xanthopterus , reported as b. xanthopterus . myxobolus oviformis thlohan , 1892 , was recorded from skin , intestine , and kidneys of c. carpio [ 27 , 54 , 59 ] . the first report of m. oviformis in iraq was from gill arches and heart of four fish species . myxobolus pfeifferi thlohan , 1895 , was recorded from gills , intestine , liver , kidneys , and gallbladder of c. idella , gills , gallbladder , intestine , kidneys , and liver of c. carpio [ 25 , 27 , 29 , 53 , 59 ] , gills , liver , and intestine of h. molitrix [ 27 , 29 ] , and gills , intestinal wall , and gonads of l. abu . the first report of m. pfeifferi in iraq was from gills of acanthobrama marmid from tigris river at mosul city . so far , m. pfeifferi is the prevalent myxozoan among fishes of iraq as it has 35 fish host species . the class trematoda of the phylum platyhelminthes is represented in farm fishes of babylon province with five species ; two species belonged to the genera apharyngostrigea and ascocotyle and three species to the genus diplostomum . phylum platyhelminthes class trematoda order diplostomida family strigeidae apharyngostrigea cornu ( zeder , 1800 ) family diplostomidae diplostomum indistinctum ( guberlet , 1923 ) hughes , 1929 diplostomum paraspathaceum schigin , 1965 diplostomum spathaceum ( rudolphi , 1819 ) olsson , 1876 order plagiorchiida family heterophyidae ascocotyle coleostoma ( looss , 1896 ) looss , 1899 order diplostomida family strigeidae apharyngostrigea cornu ( zeder , 1800 ) family diplostomidae diplostomum indistinctum ( guberlet , 1923 ) hughes , 1929 diplostomum paraspathaceum schigin , 1965 diplostomum spathaceum ( rudolphi , 1819 ) olsson , 1876 order plagiorchiida family heterophyidae ascocotyle coleostoma ( looss , 1896 ) looss , 1899 family strigeidae apharyngostrigea cornu ( zeder , 1800 ) family diplostomidae diplostomum indistinctum ( guberlet , 1923 ) hughes , 1929 diplostomum paraspathaceum schigin , 1965 diplostomum spathaceum ( rudolphi , 1819 ) olsson , 1876 apharyngostrigea cornu ( zeder , 1800 ) apharyngostrigea cornu ( zeder , 1800 ) diplostomum indistinctum ( guberlet , 1923 ) hughes , 1929 diplostomum paraspathaceum schigin , 1965 diplostomum spathaceum ( rudolphi , 1819 ) olsson , 1876 diplostomum indistinctum ( guberlet , 1923 ) hughes , 1929 diplostomum paraspathaceum schigin , 1965 diplostomum spathaceum ( rudolphi , 1819 ) olsson , 1876 family heterophyidae ascocotyle coleostoma ( looss , 1896 ) looss , 1899 ascocotyle coleostoma ( looss , 1896 ) looss , 1899 ascocotyle coleostoma ( looss , 1896 ) looss , 1899 apharyngostrigea cornu ( zeder , 1800 ) was recorded as metacercaria from mesentery , coelom and liver of c. carpio . no more records are so far known on the occurrence of a. cornu from fishes of iraq . the adult worm of this parasite was detected from the intestine of the purple heron ardea purpurea in bahr al - najaf depression . ascocotyle coleostoma ( looss , 1896 ) looss , 1899 , was recorded as metacercaria from gills of h. fossilis . this parasite was reported for the first time in iraq from gills of h. fossilis and l. abu from diyala river . the adult worm of a. coleostoma was detected from the grey heron a. cinerea in babylon ( now al - furat ) fish farm . diplostomum indistinctum ( guberlet , 1923 ) hughes , 1929 , was recorded as metacercaria from eyes of h. molitrix . the first occurrence of metacercariae of d. indistinctum was from eyes of luciobarbus esocinus , reported as b. esocinus from fish market in mosul city . no more records are so far known on the occurrence of this parasite from fishes of iraq . diplostomum paraspathaceum schigin , 1965 , was recorded as metacercaria from eyes of both c. idella and c. carpio . diplostomum spathaceum ( rudolphi , 1819 ) olsson , 1876 , was recorded as metacercaria from eyes of the three carp species : c. idella [ 43 , 56 ] , c. carpio [ 29 , 43 ] , and h. molitrix [ 43 , 56 ] . the first occurrence of metacercariae of d. spathaceum was from eyes of c. luteus , reported as b. luteus , cyprinion macrostomum , and c. carpio from dokan lake . thirty - four hosts are so far known for this parasite in fishes of iraq . adult worms of this parasite were found in the intestine of some fish - eating birds such as the silver gull larus argentatus in bahr al - najaf depression . the class monogenea of the phylum platyhelminthes is represented in farm fishes of babylon province with 45 species : 12 species of the genus gyrodactylus , 26 species of dactylogyrus , and one species of each of the genera pseudacolpenteron , diplozoon , eudiplozoon , paradiplozoon , and microcotyle in addition to some unidentified species of dactylogyrus and diplozoon . it is appropriate to mention here that this group is considered as monogenea by some electronic sites [ 912 ] but as monogenoidea in some references [ 14 , 66 ] . phylum platyhelminthes class monogenea order gyrodactylidea family gyrodactylidae gyrodactylus baicalensis bogolepova , 1950 gyrodactylus ctenopharngodontis ling in gusev , 1952 gyrodactylus elegans von nordmann , 1832 gyrodactylus kherulensis ergens , 1974 gyrodactylus macracanthus hukuda , 1940 gyrodactylus malmbergi ergens , 1961 gyrodactylus markevitschi kulakovskaya , 1952 gyrodactylus medius kathariner , 1895 gyrodactylus menschikowi gvosdev , 1950 gyrodactylus salaris malmberg , 1957 gyrodactylus sprostonae ling , 1962 gyrodactylus vicinus bychowsky , 1957 order dactylogyridea family dactylogyridae dactylogyrus achmerowi gusev , 1955 dactylogyrus amurensis akhmerov , 1952 dactylogyrus anchoratus ( dujardin , 1845 ) wagener , 1857 dactylogyrus arcuatus yamaguti , 1942 dactylogyrus barbioides gusev , ali , abdul - ameer , amin & molnr , 1993 dactylogyrus cornu linstow , 1878 dactylogyrus crassus kulwiec , 1927 dactylogyrus ctenopharyngodonis achmerow , 1952 dactylogyrus dogieli gusev , 1953 dactylogyrus ergensi molnr , 1964 dactylogyrus extensus mueller & van cleave , 1932 dactylogyrus gobii gvosdev , 1950 dactylogyrus hypophthalmichthys akhmerov , 1952 dactylogyrus inexpectatus izjumova , in gusev , 1955 dactylogyrus jamansajensis osmanov , 1958 dactylogyrus lamellatus akhmerov , 1952 dactylogyrus latituba gusev , 1955 dactylogyrus lopuchinae jukhimenko , 1981 dactylogyrus minutus kulwiec , 1927 dactylogyrus navicularis a. gusev , 1955 dactylogyrus phoxini malevitskaia , 1949 dactylogyrus propinquus bychowsky , 1931 dactylogyrus sahuensis ling in chen et al . , 1973 dactylogyrus simplex bychowsky , 1936 dactylogyrus skrjabini akhmerov , 1954 dactylogyrus vastator nybelin , 1924 dactylogyrus spp . pseudacolpenteron pavlovskii bychowsky & gussev , 1955 order mazocraeidea family diplozoidae diplozoon paradoxum nordmann , 1832 diplozoon sp . eudiplozoon nipponicum ( goto , 1891 ) paradiplozoon barbi ( reichenbach - klinke , 1951 ) family microcotylidae microcotyle donavini van beneden & hesse , 1863 order gyrodactylidea family gyrodactylidae gyrodactylus baicalensis bogolepova , 1950 gyrodactylus ctenopharngodontis ling in gusev , 1952 gyrodactylus elegans von nordmann , 1832 gyrodactylus kherulensis ergens , 1974 gyrodactylus macracanthus hukuda , 1940 gyrodactylus malmbergi ergens , 1961 gyrodactylus markevitschi kulakovskaya , 1952 gyrodactylus medius kathariner , 1895 gyrodactylus menschikowi gvosdev , 1950 gyrodactylus salaris malmberg , 1957 gyrodactylus sprostonae ling , 1962 gyrodactylus vicinus bychowsky , 1957 order dactylogyridea family dactylogyridae dactylogyrus achmerowi gusev , 1955 dactylogyrus amurensis akhmerov , 1952 dactylogyrus anchoratus ( dujardin , 1845 ) wagener , 1857 dactylogyrus arcuatus yamaguti , 1942 dactylogyrus barbioides gusev , ali , abdul - ameer , amin & molnr , 1993 dactylogyrus cornu linstow , 1878 dactylogyrus crassus kulwiec , 1927 dactylogyrus ctenopharyngodonis achmerow , 1952 dactylogyrus dogieli gusev , 1953 dactylogyrus ergensi molnr , 1964 dactylogyrus extensus mueller & van cleave , 1932 dactylogyrus gobii gvosdev , 1950 dactylogyrus hypophthalmichthys akhmerov , 1952 dactylogyrus inexpectatus izjumova , in gusev , 1955 dactylogyrus jamansajensis osmanov , 1958 dactylogyrus lamellatus akhmerov , 1952 dactylogyrus latituba gusev , 1955 dactylogyrus lopuchinae jukhimenko , 1981 dactylogyrus minutus kulwiec , 1927 dactylogyrus navicularis a. gusev , 1955 dactylogyrus phoxini malevitskaia , 1949 dactylogyrus propinquus bychowsky , 1931 dactylogyrus sahuensis ling in chen et al . , 1973 dactylogyrus simplex bychowsky , 1936 dactylogyrus skrjabini akhmerov , 1954 dactylogyrus vastator nybelin , 1924 dactylogyrus spp . pseudacolpenteron pavlovskii bychowsky & gussev , 1955 order mazocraeidea family diplozoidae diplozoon paradoxum nordmann , 1832 diplozoon sp . eudiplozoon nipponicum ( goto , 1891 ) paradiplozoon barbi ( reichenbach - klinke , 1951 ) family microcotylidae microcotyle donavini van beneden & hesse , 1863 family gyrodactylidae gyrodactylus baicalensis bogolepova , 1950 gyrodactylus ctenopharngodontis ling in gusev , 1952 gyrodactylus elegans von nordmann , 1832 gyrodactylus kherulensis ergens , 1974 gyrodactylus macracanthus hukuda , 1940 gyrodactylus malmbergi ergens , 1961 gyrodactylus markevitschi kulakovskaya , 1952 gyrodactylus medius kathariner , 1895 gyrodactylus menschikowi gvosdev , 1950 gyrodactylus salaris malmberg , 1957 gyrodactylus sprostonae ling , 1962 gyrodactylus vicinus bychowsky , 1957 family gyrodactylidae gyrodactylus baicalensis bogolepova , 1950 gyrodactylus ctenopharngodontis ling in gusev , 1952 gyrodactylus elegans von nordmann , 1832 gyrodactylus kherulensis ergens , 1974 gyrodactylus macracanthus hukuda , 1940 gyrodactylus malmbergi ergens , 1961 gyrodactylus markevitschi kulakovskaya , 1952 gyrodactylus medius kathariner , 1895 gyrodactylus menschikowi gvosdev , 1950 gyrodactylus salaris malmberg , 1957 gyrodactylus sprostonae ling , 1962 gyrodactylus vicinus bychowsky , 1957 gyrodactylus baicalensis bogolepova , 1950 gyrodactylus ctenopharngodontis ling in gusev , 1952 gyrodactylus elegans von nordmann , 1832 gyrodactylus kherulensis ergens , 1974 gyrodactylus macracanthus hukuda , 1940 gyrodactylus malmbergi ergens , 1961 gyrodactylus markevitschi kulakovskaya , 1952 gyrodactylus medius kathariner , 1895 gyrodactylus menschikowi gvosdev , 1950 gyrodactylus salaris malmberg , 1957 gyrodactylus sprostonae ling , 1962 gyrodactylus vicinus bychowsky , 1957 family dactylogyridae dactylogyrus achmerowi gusev , 1955 dactylogyrus amurensis akhmerov , 1952 dactylogyrus anchoratus ( dujardin , 1845 ) wagener , 1857 dactylogyrus arcuatus yamaguti , 1942 dactylogyrus barbioides gusev , ali , abdul - ameer , amin & molnr , 1993 dactylogyrus cornu linstow , 1878 dactylogyrus crassus kulwiec , 1927 dactylogyrus ctenopharyngodonis achmerow , 1952 dactylogyrus dogieli gusev , 1953 dactylogyrus ergensi molnr , 1964 dactylogyrus extensus mueller & van cleave , 1932 dactylogyrus gobii gvosdev , 1950 dactylogyrus hypophthalmichthys akhmerov , 1952 dactylogyrus inexpectatus izjumova , in gusev , 1955 dactylogyrus jamansajensis osmanov , 1958 dactylogyrus lamellatus akhmerov , 1952 dactylogyrus latituba gusev , 1955 dactylogyrus lopuchinae jukhimenko , 1981 dactylogyrus minutus kulwiec , 1927 dactylogyrus navicularis a. gusev , 1955 dactylogyrus phoxini malevitskaia , 1949 dactylogyrus propinquus bychowsky , 1931 dactylogyrus sahuensis ling in chen et al . , 1973 dactylogyrus simplex bychowsky , 1936 dactylogyrus skrjabini akhmerov , 1954 dactylogyrus vastator nybelin , 1924 dactylogyrus spp . pseudacolpenteron pavlovskii bychowsky & gussev , 1955 family dactylogyridae dactylogyrus achmerowi gusev , 1955 dactylogyrus amurensis akhmerov , 1952 dactylogyrus anchoratus ( dujardin , 1845 ) wagener , 1857 dactylogyrus arcuatus yamaguti , 1942 dactylogyrus barbioides gusev , ali , abdul - ameer , amin & molnr , 1993 dactylogyrus cornu linstow , 1878 dactylogyrus crassus kulwiec , 1927 dactylogyrus ctenopharyngodonis achmerow , 1952 dactylogyrus dogieli gusev , 1953 dactylogyrus ergensi molnr , 1964 dactylogyrus extensus mueller & van cleave , 1932 dactylogyrus gobii gvosdev , 1950 dactylogyrus hypophthalmichthys akhmerov , 1952 dactylogyrus inexpectatus izjumova , in gusev , 1955 dactylogyrus jamansajensis osmanov , 1958 dactylogyrus lamellatus akhmerov , 1952 dactylogyrus latituba gusev , 1955 dactylogyrus lopuchinae jukhimenko , 1981 dactylogyrus minutus kulwiec , 1927 dactylogyrus navicularis a. gusev , 1955 dactylogyrus phoxini malevitskaia , 1949 dactylogyrus propinquus bychowsky , 1931 dactylogyrus sahuensis ling in chen et al . , 1973 dactylogyrus simplex bychowsky , 1936 dactylogyrus skrjabini akhmerov , 1954 dactylogyrus vastator nybelin , 1924 dactylogyrus spp . pseudacolpenteron pavlovskii bychowsky & gussev , 1955 dactylogyrus achmerowi gusev , 1955 dactylogyrus amurensis akhmerov , 1952 dactylogyrus anchoratus ( dujardin , 1845 ) wagener , 1857 dactylogyrus arcuatus yamaguti , 1942 dactylogyrus barbioides gusev , ali , abdul - ameer , amin & molnr , 1993 dactylogyrus cornu linstow , 1878 dactylogyrus crassus kulwiec , 1927 dactylogyrus ctenopharyngodonis achmerow , 1952 dactylogyrus dogieli gusev , 1953 dactylogyrus ergensi molnr , 1964 dactylogyrus extensus mueller & van cleave , 1932 dactylogyrus gobii gvosdev , 1950 dactylogyrus hypophthalmichthys akhmerov , 1952 dactylogyrus inexpectatus izjumova , in gusev , 1955 dactylogyrus jamansajensis osmanov , 1958 dactylogyrus lamellatus akhmerov , 1952 dactylogyrus latituba gusev , 1955 dactylogyrus lopuchinae jukhimenko , 1981 dactylogyrus minutus kulwiec , 1927 dactylogyrus navicularis a. gusev , 1955 dactylogyrus phoxini malevitskaia , 1949 dactylogyrus propinquus bychowsky , 1931 dactylogyrus sahuensis ling in chen et al . , 1973 dactylogyrus simplex bychowsky , 1936 dactylogyrus skrjabini akhmerov , 1954 dactylogyrus vastator nybelin , 1924 pseudacolpenteron pavlovskii bychowsky & gussev , 1955 family diplozoidae diplozoon paradoxum nordmann , 1832 diplozoon sp . eudiplozoon nipponicum ( goto , 1891 ) paradiplozoon barbi ( reichenbach - klinke , 1951 ) family microcotylidae microcotyle donavini van beneden & hesse , 1863 diplozoon paradoxum nordmann , 1832 diplozoon sp . eudiplozoon nipponicum ( goto , 1891 ) paradiplozoon barbi ( reichenbach - klinke , 1951 ) diplozoon paradoxum nordmann , 1832 eudiplozoon nipponicum ( goto , 1891 ) paradiplozoon barbi ( reichenbach - klinke , 1951 ) family microcotylidae microcotyle donavini van beneden & hesse , 1863 microcotyle donavini van beneden & hesse , 1863 gyrodactylus baicalensis bogolepova , 1950 , was recorded from skin , fins , and gills of c. carpio [ 25 , 27 , 29 , 52 ] . the first report of g. baicalensis in iraq was from skin , buccal cavity , and gills of c. carpio from al - suwaira and al - latifiya fish farms . gyrodactylus ctenopharngodontis ling in gusev , 1952 , was recorded from skin , fins , buccal cavity , and gills of c. idella [ 24 , 27 ] . the first report of g. ctenopharngodontis in iraq was from gills of c. idella from babylon fish farm . no more hosts are so far recorded for this parasite in iraq . gyrodactylus elegans von nordmann , 1832 , was recorded from skin , fins , buccal cavity , and gills of both c. idella [ 27 , 29 , 52 ] and c. carpio [ 2527 , 29 , 30 , 32 , 33 , 43 , 50 , 52 , 53 , 55 , 56 , 60 ] as well as from gills of h. molitrix and from l. abu with no mention to site of infection . the first report of g. elegans in iraq was from c. carpio from al - zaafaraniya fish farm and l. abu from al - latifiya fish farm . gyrodactylus kherulensis ergens , 1974 , was recorded from skin and gills of c. idella and skin , fins , and gills of c. carpio [ 18 , 27 ] . the first report of g. kherulensis in iraq was from gills of c. carpio from babylon fish farm . gyrodactylus macracanthus hukuda , 1940 ( reported as g. paralatus gusev , 1955 ) , was recorded from skin and gills of c. carpio and skin , fins , and buccal cavity of h. molitrix . later on , it was reported from both hosts as g. paralatus also . according to gussev and pugachev et al . no more hosts are so far known for g. macracanthus or its synonym g. paralatus from fishes of iraq . gyrodactylus malmbergi ergens , 1961 , was recorded from skin , fins , and gills of c. carpio and from skin and gills of h. molitrix . this was its first report in iraq and no more hosts are so far known for g. malmbergi from fishes of iraq . gyrodactylus markevitschi kulakovskaya , 1952 , was recorded from skin and gills of c. carpio [ 27 , 45 , 47 , 60 ] . the first report of this parasite in iraq was from gills of capoeta trutta ( reported as varicorhinus trutta ) from tigris river at baiji town . gyrodactylus medius kathariner , 1895 , was recorded from skin and fins of c. carpio . the year of authority of this parasite was erroneously given as 1893 instead of 1895 by al - zubaidy as well as by three other references according to mhaisen and abdul - ameer . also the authorship of this parasite was given as katheriner instead of kathariner according to monodb . gyrodactylus menschikowi gvosdev , 1950 , was recorded from skin and gills of c. carpio . the first report of this parasite in iraq was from gills and skin of c. carpio and skin , fins , and gills of l. abu both from hilla river . gyrodactylus salaris malmberg , 1957 , was recorded from skin and gills of c. carpio [ 27 , 29 ] . the first report of this parasite in iraq was from gills and skin of c. carpio from al - furat fish farm . the year of authority of this parasite was reported as 1956 instead of 1957 by al - zubaidy and al - jadoaa . gyrodactylus vicinus bychowsky , 1957 , was recorded from skin , fins , and gills of c. carpio [ 27 , 35 ] . the first report of this parasite in iraq was from skin , fins , and gills of c. carpio from al - furat fish farm . now , it has three host species in iraq . finally , the unidentified gyrodactylus species reported from c. carpio [ 34 , 38 ] were the same 11 species which had been recorded in al - zubaidy . in iraq , so far 15 fish host species were reported for some unspecified gyrodactylus species . dactylogyrus achmerowi gusev , 1955 , was recorded from gills of c. carpio [ 19 , 27 , 29 , 30 , 32 , 33 , 36 , 54 , 55 , 60 ] . the first report of d. achmerowi in iraq was from gills of c. carpio from al - wahda fish hatchery at al - suwaira and babylon fish farm . dactylogyrus anchoratus ( dujardin , 1845 ) wagener , 1857 , was recorded from gills of c. carpio [ 45 , 47 ] . the first report and description of d. anchoratus in iraq were from gills of c. carpio from tigris river at al - zaafaraniya [ 85 , 86 ] . dactylogyrus arcuatus yamaguti , 1942 , was recorded from gills of c. idella and skin , buccal cavity , and gills of c. carpio [ 25 , 27 , 35 , 36 , 49 , 52 , 55 , 59 , 60 ] . the first report of d. arcuatus in iraq was from skin , buccal cavity , and gills of c. carpio from al - suwaira and al - latifiya fish farms . dactylogyrus barbioides gusev , ali , abdul - ameer , amin & molnr , 1993 , was recorded from gills of c. carpio . d. barbioides was described as a new species from gills of barbus grypus from tigris river near baiji town . dactylogyrus cornu linstow , 1878 , was recorded from gills of c. carpio [ 27 , 59 ] . d. cornu was recorded for the first time in iraq from gills of five fish species from diyala river . dactylogyrus crassus kulwiec , 1927 , was recorded from gills of c. carpio [ 45 , 47 , 55 , 60 ] . d. crassus was recorded for the first time in iraq from gills of c. carpio from al - shark al - awsat fish farm . it is appropriate to mention here that the specific name crassus was misspelled as carassus by al - rubaie et al . . dactylogyrus ctenopharyngodonis achmerow , 1952 , was recorded from gills of c. idella from al - shark al - awsat fish farm . this is the only report on the occurrence of d. ctenopharyngodonis in fishes of iraq . dactylogyrus dogieli gusev , 1953 , was recorded from gills of c. carpio in fish cages and an earthen pond in sadat al - hindiya . the first report of d. dogieli was from five fish species from euphrates river at al - musaib city and its full description and illustration were published later by al - sa'adi et al . . dactylogyrus ergensi molnr , 1964 , was recorded from gills of c. carpio from al - furat fish farm . dactylogyrus extensus mueller & van cleave , 1932 , was recorded from gills of c. idella , buccal cavity and gills of c. carpio [ 25 , 27 , 29 , 30 , 32 , 33 , 36 , 43 , 45 , 47 , 52 , 54 , 55 , 59 , 60 ] , and gills of h. molitrix [ 29 , 45 , 47 ] . the first report of d. extensus in iraq was from the buccal cavity and gills of c. carpio from al - suwaira and al - latifiya fish farms . d. solidus which was also recorded from the same host by salih et al . as well as by mhaisen & abul - eis and al - rubaie et al . d. extensus and its synonym d. solidus have so far 17 fish host species in iraq . dactylogyrus gobii gvosdev , 1950 , was recorded from skin and gills of c. carpio [ 45 , 47 , 60 ] . d. gobii was recorded for the first time in iraq from gills of c. carpio from al - shark al - awsat fish farm . dactylogyrus hypophthalmichthys akhmerov , 1952 , was recorded from skin , buccal cavity , and gills of h. molitrix [ 21 , 27 , 43 , 45 , 47 , 52 , 56 ] . it is reliable to state here that d. hypophthalmichthys was reported as neodactylogyrus hypophthalmichthys by asmar et al . . the first report of d. hypophthalmichthys in iraq was from the buccal cavity and gills of h. molitrix from al - suwaira and al - latifiya fish farms . h. molitrix is the only host so far known for d. hypophthalmichthys in iraq . dactylogyrus inexpectatus izjumova , in gusev , 1955 , was recorded from skin and gills of c. idella [ 27 , 29 , 52 ] , gills of c. carpio [ 27 , 29 ] , and gills of h. molitrix . the first report of d. inexpectatus in iraq was from skin and gills of c. idella from al - suwaira and al - latifiya fish farms . dactylogyrus jamansajensis osmanov , 1958 , was recorded from gills of c. carpio [ 45 , 47 ] . the first report of d. jamansajensis in iraq was from gills of c. luteus from manmade lakes , north of baghdad . dactylogyrus lamellatus akhmerov , 1952 , was recorded from skin , fins , buccal cavity , and gills of c. idella [ 24 , 27 , 43 , 52 , 56 ] and gills of c. carpio . the first report of d. lamellatus in iraq was from the skin , buccal cavity , and gills of c. idella from al - suwaira and al - latifiya fish farms . dactylogyrus latituba gusev , 1955 , was recorded from gills of both c. idella and c. carpio [ 25 , 27 ] as well as from the buccal cavity and gills of h. molitrix . the first report of d. latituba in iraq was from gills of c. luteus from manmade lakes , north of baghdad . dactylogyrus lopuchinae jukhimenko , 1981 , was recorded from gills of c. carpio [ 45 , 47 , 54 ] . d. lopuchinae was recorded for the first time in iraq from gills of c. carpio from al - shark al - awsat fish farm . dactylogyrus minutus kulwiec , 1927 , was recorded from skin , fins , and gills of c. carpio [ 27 , 30 , 32 , 33 , 45 , 47 , 55 , 56 , 59 , 60 ] . the first report on this parasite in iraq was from gills of c. carpio from tigris river at al - zaafaraniya , south of baghdad , and al - qadisia dam lake , while its description and illustration were given later by mhaisen et al . . dactylogyrus navicularis a. gusev , 1955 , was recorded from fins , buccal cavity , and gills of c. carpio [ 27 , 35 , 60 ] . the first report of d. navicularis in iraq was from the buccal cavity , fins , and gills of c. carpio from al - furat fish farm . c. carpio is the only host so far known for d. navicularis in iraq . dactylogyrus phoxini malevitskaia , 1949 , was recorded from skin and gills of c. carpio [ 45 , 47 , 60 ] . the first record of d. phoxini in iraq was in june 1995 from gills of c. carpio from tigris river at al - zaafaraniya but the report was published later by balasem et al . . dactylogyrus propinquus bychowsky , 1931 , was recorded from gills of c. carpio [ 27 , 35 , 49 ] . the first report of d. propinquus in iraq was from gills of c. carpio from al - furat fish farm . c. carpio is the only host so far known for d. propinquus in iraq . the first report of d. sahuensis in iraq was from fins and gills of c. carpio from al - furat fish farm . c. carpio is the only host so far known for d. sahuensis in iraq . dactylogyrus simplex bychowsky , 1936 , was recorded from c. carpio with no mention to site of infection . the first report of d. simplex in iraq was from gills of c. carpio from the new fish farm of the fish research center at al - zaafaraniya . dactylogyrus skrjabini akhmerov , 1954 , was recorded from buccal cavity and gills of c. carpio [ 27 , 45 , 47 ] and buccal cavity and gills of h. molitrix [ 21 , 27 , 52 ] . the first report of d. skrjabini in iraq was from buccal cavity and gills of h. molitrix from al - suwaira and al - latifiya fish farms . dactylogyrus vastator nybelin , 1924 , was recorded from gills of c. idella and skin and gills of c. carpio [ 26 , 27 , 30 , 32 , 33 , 35 , 36 , 43 , 49 , 52 , 53 , 56 , 59 , 60 , 64 ] . the first report of d. vastator from iraq was from skin and gills of c. macrostomum from tigris river at baghdad . so far , d. vastator was reported from 33 fish host species from north , middle , and south of iraq . unidentified dactylogyrus species were recorded from skin and gills of c. idella and from skin , buccal cavity , and gills of c. carpio [ 34 , 38 , 56 ] . some of these specimens were larval stages , while the unidentified dactylogyrus species of al - zubaidy et al . [ 34 , 38 ] were the same 15 species which were recorded in al - zubaidy . in iraq , so far nine fish host species were reported for some unspecified dactylogyrus species . pseudacolpenteron pavlovskii bychowsky & gussev , 1955 , was recorded from fins and gills of c. carpio [ 25 , 27 , 45 , 47 ] and gills of h. molitrix . the first report of p. pavlovskii from iraq was from skin and gills of c. carpio from babylon fish farm . this parasite was reported for the first time in iraq from gills of carasobarbus luteus , reported as barbus luteus , from al - husainia creek , karbala province . some other unidentified diplozoon species occurred as larvae in 12 fish host species in iraq . this parasite was recorded for the first time in iraq from gills of c. carpio from a manmade lake in baghdad as diplozoon nipponicum but then it was reported by its valid name e. nipponicum by all subsequent researchers . paradiplozoon barbi ( reichenbach - klinke , 1951 ) was recorded from gills of c. carpio as diplozoon barbi . this parasite was reported for the first time in iraq from gills of chondrostoma nasus , c. regium , and c. carpio from tigris river at baghdad as diplozoon barbi . also , all the subsequent records in the iraqi literature , except the checklists of mhaisen and abdul - ameer , referred to this parasite as d. barbi . according to khotenovsky , d. barbi is a synonym of p. barbi . microcotyle donavini van beneden & hesse , 1863 , was recorded from gills of l. abu from babylon fish farm . the class cestoda of the phylum platyhelminthes is represented in farm fishes of babylon province with five species : one species of each of the genera bothriocephalus , ligula , and neogryporhynchus as well as two species of proteocephalus . phylum platyhelminthes class cestoda order bothriocephalidea family bothriocephalidae bothriocephalus acheilognathi yamaguti , 1934 order diphyllobothriidea family diphyllobothriidae ligula intestinalis ( l. , 1758 ) bloch , 1782 order proteocephalidea family proteocephalidae proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 order cyclophyllidea family dilepididae neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 order bothriocephalidea family bothriocephalidae bothriocephalus acheilognathi yamaguti , 1934 order diphyllobothriidea family diphyllobothriidae ligula intestinalis ( l. , 1758 ) bloch , 1782 order proteocephalidea family proteocephalidae proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 order cyclophyllidea family dilepididae neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 order bothriocephalidea family bothriocephalidae bothriocephalus acheilognathi yamaguti , 1934 family bothriocephalidae bothriocephalus acheilognathi yamaguti , 1934 bothriocephalus acheilognathi yamaguti , 1934 order diphyllobothriidea family diphyllobothriidae ligula intestinalis ( l. , 1758 ) bloch , 1782 family diphyllobothriidae ligula intestinalis ( l. , 1758 ) bloch , 1782 ligula intestinalis ( l. , 1758 ) bloch , 1782 order proteocephalidea family proteocephalidae proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 family proteocephalidae proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 family dilepididae neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 bothriocephalus acheilognathi yamaguti , 1934 , was recorded from the intestine of both c. idella [ 24 , 56 ] and c. carpio [ 2527 , 52 , 53 , 62 ] . it is appropriate to mention here that this worm was reported by its synonym b. opsariichthydis by salih et al . and al - zubaidy . the first report of b. acheilognathi in iraq was from the intestine of c. carpio from different fish farms near baghdad . two other species of bothriocephalus , b. gowkongensis yeh , 1955 , and b. opsariichthydis yamaguti , 1934 , were also reported from iraq . according to molnr , both , b. acheilognathi and both of its above - named synonyms has so far a total of 21 host species in iraq . ligula intestinalis ( l. , 1758 ) bloch , 1782 , was recorded from the body cavity of both c. idella [ 24 , 27 , 29 ] and c. carpio . l. intestinalis was reported for the first time in iraq as a plerocercoid from the body cavity of leuciscus vorax ( reported as a. vorax ) from shatt al - arab river . , the adult stage of l. intestinalis was reported from the intestine of the moorhen gallinula chloropus chloropus from around baghdad . proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 , was recorded from the intestine of c. carpio . the first report of this parasite in iraq was from the intestine of l. vorax ( reported as a. vorax ) from al - tharthar lake . proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 , was recorded from intestine of c. carpio . the first report of this parasite in iraq was from the intestine of c. carpio from a fish farm near baghdad city . neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 , was recorded from the intestine of c. carpio by al - zubaidy as gryporhynchus cheilancristrotus . the first report of n. cheilancristrotus in iraq was from the intestine of l. abu from diyala river . the phylum nematoda is represented in farm fishes of babylon province with three species : unidentified larval species of the genus contracaecum as well as one species of each of the genera cucullanus and rhabdochona . phylum nematoda class secernentea order ascaridida family anisakidae contracaecum spp . family cucullanidae cucullanus cyprini yamaguti , 1941 order spirurida family rhabdochonidae rhabdochona hellichi ( srmek , 1901 ) order ascaridida family anisakidae contracaecum spp . family cucullanidae cucullanus cyprini yamaguti , 1941 order spirurida family rhabdochonidae rhabdochona hellichi ( srmek , 1901 ) family anisakidae contracaecum spp . family cucullanidae cucullanus cyprini yamaguti , 1941 cucullanus cyprini yamaguti , 1941 cucullanus cyprini yamaguti , 1941 family rhabdochonidae rhabdochona hellichi ( srmek , 1901 ) family rhabdochonidae rhabdochona hellichi ( srmek , 1901 ) rhabdochona hellichi ( srmek , 1901 ) unidentified larval species of contracaecum was recorded from the intestinal wall , body cavity , liver , spleen , heart , and gonads of c. carpio [ 27 , 29 ] and intestinal wall of l. abu [ 22 , 37 , 42 ] . larvae in iraq was from the body cavity and different viscera of 10 fish species from different inland waters of iraq . adult worms of contracaecum spp . were detected from six species of aquatic birds in iraq , egretta alba , e. garzetta , ardeola ralloides , botaurus stellaris , ardea purpurea , and ceryle rudis , from bahr al - najaf depression . the first report of this parasite in iraq was from the intestine of alburnus caeruleus and luciobarbus xanthopterus ( reported as b. xanthopterus ) from al - tharthar lake . rhabdochona hellichi ( srmek , 1901 ) , erroneously reported as r. bellichi , was recorded from intestine of the three carp species : c. idella , c. carpio , and h. molitrix . ali et al . reported this parasite ( also erroneously as r. bellichi ) from the intestine and coelom of l. xanthopterus ( reported as b. xanthopterus ) , h. fossilis , and mystus pelusius ( reported as m. halepensis ) . the phylum acanthocephala is represented in farm fishes of babylon province with two valid species of the genus neoechinorhynchus . phylum acanthocephala class eoacanthocephala order neoechinorhynchida family neoechinorhynchidae neoechinorhynchus iraqensis amin , al - sady , mhaisen & bassat , 2001 neoechinorhynchus rutili ( mller , 1780 ) hamann , 1892 class eoacanthocephala order neoechinorhynchida family neoechinorhynchidae neoechinorhynchus iraqensis amin , al - sady , mhaisen & bassat , 2001 neoechinorhynchus rutili ( mller , 1780 ) hamann , 1892 order neoechinorhynchida family neoechinorhynchidae neoechinorhynchus iraqensis amin , al - sady , mhaisen & bassat , 2001 neoechinorhynchus rutili ( mller , 1780 ) hamann , 1892 family neoechinorhynchidae neoechinorhynchus iraqensis amin , al - sady , mhaisen & bassat , 2001 neoechinorhynchus rutili ( mller , 1780 ) hamann , 1892 neoechinorhynchus iraqensis amin , al - sady , mhaisen & bassat , 2001 neoechinorhynchus rutili ( mller , 1780 ) hamann , 1892 neoechinorhynchus iraqensis amin , al - sady , mhaisen & bassat , 2001 , was recorded from intestine of both c. carpio and l. abu [ 22 , 50 ] . it is appropriate to mention here that this species was reported as n. agilis from c. carpio and l. abu by al - zubaidy and ali et al . the first report of n. iraqensis was as species de novo from the intestine of l. abu from the euphrates river at al - fallujah region , while its description was given later by amin et al . . n. iraqensis and the misidentified n. agilis have so far 24 fish host species in iraq . neoechinorhynchus rutili ( mller , 1780 ) hamann , 1892 , was recorded from the intestine of the three carp species : c. idella , c. carpio [ 27 , 29 ] , and h. molitrix . n. rutili was firstly recorded by herzog from l. xanthopterus ( reported as b. xanthopterus ) from tigris and diyala rivers near baghdad and from l. abu ( reported as mugil abu ) from citscher oasis near al - fallujah . the subphylum crustacea of the phylum arthropoda is represented in farm fishes of babylon province with nine species : one species of each of the genera argulus , dermoergasilus , paraergasilus , lamproglena , and lernaea and three species of ergasilus in addition to unidentified species of ergasilus . phylum arthropoda subphylum crustacea class maxillopoda order arguloida family argulidae argulus foliaceus ( l. , 1758 ) order cyclopoida family ergasilidae dermoergasilus varicoleus ho , jayarajan & radhakrishnan , 1992 ergasilus barbi rahemo , 1982 ergasilus mosulensis rahemo , 1982 ergasilus sieboldi von nordmann , 1832 ergasilus sp . paraergasilus inflatus ho , khamees & mhaisen , 1996 family lernaeidae lamproglena pulchella von nordmann , 1832 lernaea cyprinacea l. , 1758 class maxillopoda order arguloida family argulidae argulus foliaceus ( l. , 1758 ) order cyclopoida family ergasilidae dermoergasilus varicoleus ho , jayarajan & radhakrishnan , 1992 ergasilus barbi rahemo , 1982 ergasilus mosulensis rahemo , 1982 ergasilus sieboldi von nordmann , 1832 ergasilus sp . paraergasilus inflatus ho , khamees & mhaisen , 1996 family lernaeidae lamproglena pulchella von nordmann , 1832 lernaea cyprinacea l. , 1758 order arguloida family argulidae argulus foliaceus ( l. , 1758 ) order cyclopoida family ergasilidae dermoergasilus varicoleus ho , jayarajan & radhakrishnan , 1992 ergasilus barbi rahemo , 1982 ergasilus mosulensis rahemo , 1982 ergasilus sieboldi von nordmann , 1832 ergasilus sp . paraergasilus inflatus ho , khamees & mhaisen , 1996 family lernaeidae lamproglena pulchella von nordmann , 1832 lernaea cyprinacea l. , 1758 family argulidae argulus foliaceus ( l. , 1758 ) argulus foliaceus ( l. , 1758 ) family ergasilidae dermoergasilus varicoleus ho , jayarajan & radhakrishnan , 1992 ergasilus barbi rahemo , 1982 ergasilus mosulensis rahemo , 1982 ergasilus sieboldi von nordmann , 1832 ergasilus sp . paraergasilus inflatus ho , khamees & mhaisen , 1996 family lernaeidae lamproglena pulchella von nordmann , 1832 lernaea cyprinacea l. , 1758 dermoergasilus varicoleus ho , jayarajan & radhakrishnan , 1992 ergasilus barbi rahemo , 1982 ergasilus mosulensis rahemo , 1982 ergasilus sieboldi von nordmann , 1832 paraergasilus inflatus ho , khamees & mhaisen , 1996 lamproglena pulchella von nordmann , 1832 lernaea cyprinacea l. , 1758 argulus foliaceus ( l. , 1758 ) was recorded from gills of the three carp species : c. idella , c. carpio [ 25 , 27 ] , and h. molitrix as well as from fins of c. auratus . a. foliaceus was reported for the first time in iraq from skin of c. carpio from al - zaafaraniya fish - culture station and c. luteus ( reported as b. luteus ) from al - habbaniyah lake . dermoergasilus varicoleus ho , jayarajan & radhakrishnan , 1992 , was recorded from gills of l. abu [ 31 , 40 ] . this crustacean was reported for the first time in iraq from gills of l. abu from shatt al - arab river . this crustacean was firstly detected from gills of b. grypus from tigris river at mosul city by fattohy and its full description as a new species was achieved by rahemo . ergasilus mosulensis rahemo , 1982 , was recorded from gills of the three carp species , c. idella , c. carpio , and h. molitrix as well as gills of l. abu . this crustacean was firstly detected from gills of l. abu from tigris river at mosul city by fattohy and its full description as a new species was achieved by rahemo . ergasilus sieboldi von nordmann , 1832 , was recorded from gills , buccal cavity , and skin of the three carp species : c. idella [ 27 , 29 ] , c. carpio [ 25 , 27 , 29 , 51 , 55 ] , and h. molitrix [ 27 , 29 , 56 ] . the first report of e. sieboldi in iraq was from gills of l. vorax ( reported as a. vorax ) from al - habbaniyah lake . ergasilus sp . was recorded from l. abu with no mention to site of infection . in addition to 11 species of ergasilus so far recorded from fishes of iraq , some specimens of unidentified ergasilus species were also reported from 12 fish species in iraq . paraergasilus inflatus ho , khamees & mhaisen , 1996 , was recorded from gills of h. molitrix . this crustacean was reported as a new species from gill rakers of l. abu from shatt al - arab river . the first report of l. pulchella in iraq was from gills of chondrostoma regium and capoeta trutta ( reported as varicorhinus trutta ) from tigris river at mosul city . lernaea cyprinacea l. , 1758 , was recorded from skin , fins , and gills of the three carp species , c. idella [ 24 , 2729 , 43 , 51 ] , c. carpio [ 6 , 2531 , 39 , 41 , 43 , 44 , 48 , 51 , 5355 , 57 , 61 , 63 ] , and h. molitrix [ 21 , 2729 , 43 ] , as well as from gills of l. abu . the first report of the anchor worm l. cyprinacea in iraq was from skin , fins , buccal cavity , pharyngeal cavity , gills , and anus of seven freshwater fish species from al - zaafaraniya fish - culture station . l. cyprinacea is the commonest crustacean among fishes of iraq as it has so far 30 fish host species in iraq . the phylum mollusca is represented in farm fishes of babylon province with only one parasite species of the genus unio . the systematic account of this parasite , followed by parasite - host list , is given here . phylum mollusca class bivalvia order unionoida family unionidae unio pictorum ( linnaeus , 1758 ) order unionoida family unionidae unio pictorum ( linnaeus , 1758 ) family unionidae unio pictorum ( linnaeus , 1758 ) unio pictorum ( linnaeus , 1758 ) unio pictorum ( linnaeus , 1758 ) unio pictorum ( linnaeus , 1758 ) was recorded from gills of both c. carpio [ 27 , 29 ] and h. molitrix . the first report of the glochidial larvae of u. pictorum in iraq was from gills of eight freshwater fish species from diyala river . it is appropriate to mention here that the authority of u. pictorum was erroneously stated as zhadin , 1938 , in all the iraqi literature except al - salmany . the following host - parasite list for fish parasites in fish farms of babylon province is compiled . for each host , the scientific names of all recorded parasites are alphabetically enlisted under their major parasitic groups . to economize space , these can be obtained from the account of each concerned parasite species in the part of major groups of parasitic fauna within the results and discussion part . ciliophora : apiosoma amoebae , a. cylindriformis , a. minuta , a. piscicola , a. poteriformis , chilodonella cyprini , ichthyophthirius multifiliis , trichodina cottidarum , t. domerguei , t. gracilis , t. nigra , and tripartiella amurensis . myxozoa : myxobilatus legeri , myxobolus muelleri , m. oviformis , and m. pfeifferi . monogenea : dactylogyrus achmerowi , d. amurensis , d. anchoratus , d. arcuatus , d. barbioides , d. cornu , d. crassus , d. dogieli , d. ergensi , d. extensus , d. gobii , d. inexpectatus , d. jamansajensis , d. lamellatus , d. latituba , d. lopuchinae , d. minutus , d. navicularis , d. phoxini , d. propinquus , d. sahuensis , d. simplex , d. skrjabini , d. vastator , dactylogyrus spp . , diplozoon paradoxum , diplozoon sp . , eudiplozoon nipponicum , gyrodactylus baicalensis , g. elegans , g. kherulensis , g. macracanthus , g. malmbergi , g. markewitschi , g. medius , g. menschikowi , g. salaris , g. sprostonae , g. vicinus , paradiplozoon barbi , and pseudacolpenteron pavlovskii . cestoda : bothriocephalus acheilognathi , ligula intestinalis , neogryporhynchus cheilancristrotus , proteocephalus osculatus , and p. torulosus . crustacea : argulus foliaceus , ergasilus mosulensis , e. sieboldi , lamproglena pulchella , and lernaea cyprinacea . ciliophora : apiosoma amoebae , a. cylindriformis , a. minuta , a. piscicola , a. poteriformis , chilodonella cyprini , ichthyophthirius multifiliis , trichodina cottidarum , t. domerguei , t. gracilis , t. nigra , and tripartiella amurensis . monogenea : dactylogyrus achmerowi , d. amurensis , d. anchoratus , d. arcuatus , d. barbioides , d. cornu , d. crassus , d. dogieli , d. ergensi , d. extensus , d. gobii , d. inexpectatus , d. jamansajensis , d. lamellatus , d. latituba , d. lopuchinae , d. minutus , d. navicularis , d. phoxini , d. propinquus , d. sahuensis , d. simplex , d. skrjabini , d. vastator , dactylogyrus spp . , eudiplozoon nipponicum , gyrodactylus baicalensis , g. elegans , g. kherulensis , g. macracanthus , g. malmbergi , g. markewitschi , g. medius , g. menschikowi , g. salaris , g. sprostonae , g. vicinus , paradiplozoon barbi , and pseudacolpenteron pavlovskii . cestoda : bothriocephalus acheilognathi , ligula intestinalis , neogryporhynchus cheilancristrotus , proteocephalus osculatus , and p. torulosus . crustacea : argulus foliaceus , ergasilus mosulensis , e. sieboldi , lamproglena pulchella , and lernaea cyprinacea . ciliophora : apiosoma amoebae , a. cylindriformis , a. piscicola , a. poteriformis , chilodonella cyprini , ichthyophthirius multifiliis , trichodina domerguei , and t. nigra . monogenea : dactylogyrus arcuatus , d. ctenopharyngodonis , d. extensus , d. inexpectatus , d. lamellatus , d. latituba , d. vastator , dactylogyrus sp . crustacea : argulus foliaceus , ergasilus mosulensis , e. sieboldi , and lernaea cyprinacea . ciliophora : apiosoma amoebae , a. cylindriformis , a. piscicola , a. poteriformis , chilodonella cyprini , ichthyophthirius multifiliis , trichodina domerguei , and t. nigra . monogenea : dactylogyrus arcuatus , d. ctenopharyngodonis , d. extensus , d. inexpectatus , d. lamellatus , d. latituba , d. vastator , dactylogyrus sp . crustacea : argulus foliaceus , ergasilus mosulensis , e. sieboldi , and lernaea cyprinacea . ciliophora : apiosoma amoebae , a. cylindriformis , a. piscicola , chilodonella cyprini , ichthyophthirius multifiliis , trichodina cottidarum , t. domerguei , and t. nigra . monogenea : dactylogyrus extensus , d. hypophthalmichthys , d. inexpectatus , d. latituba , d. skrjabini , gyrodactylus elegans , g. macracanthus , g. malmbergi , and pseudacolpenteron pavlovskii . crustacea : argulus foliaceus , ergasilus mosulensis , e. sieboldi , lernaea cyprinacea , and paraergasilus inflatus . ciliophora : apiosoma amoebae , a. cylindriformis , a. piscicola , chilodonella cyprini , ichthyophthirius multifiliis , trichodina cottidarum , t. domerguei , and t. nigra . monogenea : dactylogyrus extensus , d. hypophthalmichthys , d. inexpectatus , d. latituba , d. skrjabini , gyrodactylus elegans , g. macracanthus , g. malmbergi , and pseudacolpenteron pavlovskii . crustacea : argulus foliaceus , ergasilus mosulensis , e. sieboldi , lernaea cyprinacea , and paraergasilus inflatus . crustacea : dermoergasilus varicoleus , ergasilus barbi , e. mosulensis , ergasilus sp . , and lernaea cyprinacea . crustacea : dermoergasilus varicoleus , ergasilus barbi , e. mosulensis , ergasilus sp . , and lernaea cyprinacea .

literature reviews of all references concerning the parasitic fauna of fishes in fish farms of babylon province , middle of iraq , showed that a total of 92 valid parasite species are so far known from the common carp ( cyprinus carpio ) , the grass carp ( ctenopharyngodon idella ) , and the silver carp ( hypophthalmichthys molitrix ) as well as from three freshwater fish species ( carassius auratus , liza abu , and heteropneustes fossilis ) which were found in some fish farms of the same province . the parasitic fauna included one mastigophoran , three apicomplexans , 13 ciliophorans , five myxozoans , five trematodes , 45 monogeneans , five cestodes , three nematodes , two acanthocephalans , nine arthropods , and one mollusc . the common carp was found to harbour 81 species of parasites , the grass carp 30 species , the silver carp 28 species , l. abu 13 species , c. auratus one species , and h. fossilis one species . a host - parasite list for each fish species was also provided .

1. Introduction 2. Sources and Methods 3. Parasitological Investigations Achieved on Fish Farms of Babylon Province 4. Results and Discussion 5. Major Groups of Parasitic Fauna: Parasite-Host List 6. Host-Parasite List

although fish farming in iraq started in 1955 with a small pond in al - zaafaraniya , south of baghdad city , an advance was achieved in fish farming industry in iraq during the seventies and early eighties of the last century when many fish farms were established especially in the middle of iraq . however , such achievement was hindered due to consequences of the war situations during 19801988 and 1991 as well as the economic sanction imposed by the un against iraq on august 6 , 1990 . during the last few years , a great advance was achieved in fish farming in general and fish cages in particular due to the increasing demand on fish protein as well as the increasing investment in fish - culture industry in most provinces of iraq . according to the statistics , a total of 441 working fish farms are scattered in iraq . of these farms , a total of 72 working fish farms are situated in babylon province alone with a water area of 44.5% of the total water area of fish farms in iraq . in connection with the parasites of cultured fishes of babylon province , mhaisen et al . surveyed the literature on the parasitic fauna of fishes of al - furat fish farm ( previously known as babylon fish farm ) , which is the biggest fish farm in babylon province , and showed that the parasitic fauna of fishes of that farm included 60 valid parasite species ( 10 protozoans , three myxozoans , one trematode , 29 monogeneans , five cestodes , three nematodes , two acanthocephalans , six crustaceans , and one mollusc larva ) . therefore , the present paper was aimed at gathering data from the literature concerning all fish farms of babylon province and providing a list of parasite species according to their major groups as well as a host - parasite list for cultured fishes of these farms and some other fish species found in such farms . such parasite list will help owners of fish farms and fish veterinarians to know what sort of parasites are found in their fish farms , which will help them later in taking appropriate measures for their control . a total of 50 references ( 33 published articles , 12 unpublished theses , two unpublished reports , one book , one conference abstract , and one review article ) dealing with the parasites of farm fishes of babylon province were used to prepare the present paper . data from such references was gathered to provide host - parasite and parasite - host lists . the index - catalogue of parasites and disease agents of fishes of iraq was used to indicate the total number of fish hosts harbouring each parasite species in the whole waters of iraq . so far 25 chronologically arranged references [ 1842 ] were concerned with the parasitic fauna of al - furat fish farm . only seven references [ 4349 ] were concerned with the parasitic fauna of al - shark al - awsat fish farm . the literature concerned with fish parasites of other farms in babylon province included those from al - latifiya fish farm [ 6 , 5052 ] ; al - bajaa fish farm ; abdul - razzak al - janabi fish farm ; fawzi al - janabi fish farm and ali al - hayali fish farm ; three fish farms at al - iskandariya district : abdul - hadi al - matloob fish farm , hussain al - gaiem fish farm , and maki chinak fish farm ; technical institute of al - musaib fish farm ; and al - manahil ( al - bilad ) fish farm at al - iskandariya district . in addition , surveys were done from some unnamed fish ponds such as those at al - mahaweel district , al - musaib district , al - iskandariya district , and sadat al - hindiya district as well as some other unnamed farms in the province [ 6165 ] . surveying the literature concerning the parasites so far recorded from fish farms of babylon province showed the presence of 92 parasite species . these parasites included one mastigophoran , three apicomplexans , 13 ciliophorans , five myxozoans , five trematodes , 45 monogeneans , five cestodes , three nematodes , two acanthocephalans , nine arthropods , and one mollusc . the common carp was found to harbour 81 species of parasites , the grass carp 30 species , the silver carp 28 species , l. abu 13 species , c. auratus one species , and h. fossilis one species . these major groups represent the concerned phyla of the parasites , but due to the great numbers of parasite species of the phylum platyhelminthes , its three classes ( trematoda , monogenea , and cestoda ) were applied in addition to their phylum . to economize space , names of fish farms also the systematic account of all major groups is given down to the specific name of all parasites . for each parasite species , all records in farm fishes in babylon province are given together with the first record of each concerned parasite in iraq as well as the present number of all hosts so far known in iraq for each concerned species based on the index - catalogue of parasites and disease agents of fishes of iraq . the phylum mastigophora is represented in farm fishes of babylon province with only one parasite species of the genus ichthyobodo . seven fish host species are so far known for this parasite ( as c. necatrix ) in iraq . the phylum apicomplexa , which is known as phylum myzozoa according to worms , is represented in farm fishes of babylon province with three species ; two of them belonged to the genus eimeria and one unspecified species to the genus haemogregarina . examined c. carpio externally while e. mylopharyngodoni is known to infect intestine , kidneys , and liver of fishes . three species of haemogregarina were so far recorded from blood of three fish species in basrah province only . the phylum ciliophora is represented in farm fishes of babylon province with 13 species , three of which belonged to the genera chilodonella , ichthyophthirius , and tripartiella , five to the genus apiosoma , and four to the genus trichodina in addition to unspecified species of the genus trichodina . the first record of a. amoebae in iraq was from the skin , buccal cavity , and gills of c. idella from babylon fish farm . a. piscicola was recorded for the first time in iraq from skin , buccal cavity , and gills of c. idella , c. carpio and h. molitrix from al - suwaira and al - latifiya fish farms . the first record of t. domerguei in iraq was from skin , fins , and gills of eight freshwater fish species from tigris river , al - tharthar lake , and fish markets in baghdad city . so far , t. domerguei has 39 host species in iraq and , therefore , it is the most prevalent ciliate species among fishes of iraq . trichodina nigra lom , 1960 , was recorded from skin and gills of the three carp species : c. idella , c. carpio [ 27 , 29 , 35 , 49 , 55 , 59 , 60 ] , and h. molitrix [ 27 , 29 ] . in addition to 24 recognized trichodina species so far recorded from fishes of iraq , some unidentified species of trichodina were so far recorded from six fish species . the phylum myxozoa is represented in farm fishes of babylon province with five species : one species belonged to the genus myxobilatus and four species to the genus myxobolus . the first record of m. dogieli in iraq was mainly from the external surface of heart , liver , and ovaries of l. abu from tigris river at baiji town . so far , m. pfeifferi is the prevalent myxozoan among fishes of iraq as it has 35 fish host species . the class trematoda of the phylum platyhelminthes is represented in farm fishes of babylon province with five species ; two species belonged to the genera apharyngostrigea and ascocotyle and three species to the genus diplostomum . no more records are so far known on the occurrence of a. cornu from fishes of iraq . the adult worm of this parasite was detected from the intestine of the purple heron ardea purpurea in bahr al - najaf depression . this parasite was reported for the first time in iraq from gills of h. fossilis and l. abu from diyala river . no more records are so far known on the occurrence of this parasite from fishes of iraq . diplostomum spathaceum ( rudolphi , 1819 ) olsson , 1876 , was recorded as metacercaria from eyes of the three carp species : c. idella [ 43 , 56 ] , c. carpio [ 29 , 43 ] , and h. molitrix [ 43 , 56 ] . thirty - four hosts are so far known for this parasite in fishes of iraq . adult worms of this parasite were found in the intestine of some fish - eating birds such as the silver gull larus argentatus in bahr al - najaf depression . the class monogenea of the phylum platyhelminthes is represented in farm fishes of babylon province with 45 species : 12 species of the genus gyrodactylus , 26 species of dactylogyrus , and one species of each of the genera pseudacolpenteron , diplozoon , eudiplozoon , paradiplozoon , and microcotyle in addition to some unidentified species of dactylogyrus and diplozoon . gyrodactylus elegans von nordmann , 1832 , was recorded from skin , fins , buccal cavity , and gills of both c. idella [ 27 , 29 , 52 ] and c. carpio [ 2527 , 29 , 30 , 32 , 33 , 43 , 50 , 52 , 53 , 55 , 56 , 60 ] as well as from gills of h. molitrix and from l. abu with no mention to site of infection . no more hosts are so far known for g. macracanthus or its synonym g. paralatus from fishes of iraq . this was its first report in iraq and no more hosts are so far known for g. malmbergi from fishes of iraq . dactylogyrus extensus mueller & van cleave , 1932 , was recorded from gills of c. idella , buccal cavity and gills of c. carpio [ 25 , 27 , 29 , 30 , 32 , 33 , 36 , 43 , 45 , 47 , 52 , 54 , 55 , 59 , 60 ] , and gills of h. molitrix [ 29 , 45 , 47 ] . d. solidus which was also recorded from the same host by salih et al . the first report of d. hypophthalmichthys in iraq was from the buccal cavity and gills of h. molitrix from al - suwaira and al - latifiya fish farms . the first report of d. lamellatus in iraq was from the skin , buccal cavity , and gills of c. idella from al - suwaira and al - latifiya fish farms . dactylogyrus latituba gusev , 1955 , was recorded from gills of both c. idella and c. carpio [ 25 , 27 ] as well as from the buccal cavity and gills of h. molitrix . c. carpio is the only host so far known for d. navicularis in iraq . c. carpio is the only host so far known for d. sahuensis in iraq . so far , d. vastator was reported from 33 fish host species from north , middle , and south of iraq . [ 34 , 38 ] were the same 15 species which were recorded in al - zubaidy . in iraq , so far nine fish host species were reported for some unspecified dactylogyrus species . the class cestoda of the phylum platyhelminthes is represented in farm fishes of babylon province with five species : one species of each of the genera bothriocephalus , ligula , and neogryporhynchus as well as two species of proteocephalus . according to molnr , both , b. acheilognathi and both of its above - named synonyms has so far a total of 21 host species in iraq . , the adult stage of l. intestinalis was reported from the intestine of the moorhen gallinula chloropus chloropus from around baghdad . the first report of n. cheilancristrotus in iraq was from the intestine of l. abu from diyala river . the phylum nematoda is represented in farm fishes of babylon province with three species : unidentified larval species of the genus contracaecum as well as one species of each of the genera cucullanus and rhabdochona . family cucullanidae cucullanus cyprini yamaguti , 1941 cucullanus cyprini yamaguti , 1941 cucullanus cyprini yamaguti , 1941 family rhabdochonidae rhabdochona hellichi ( srmek , 1901 ) family rhabdochonidae rhabdochona hellichi ( srmek , 1901 ) rhabdochona hellichi ( srmek , 1901 ) unidentified larval species of contracaecum was recorded from the intestinal wall , body cavity , liver , spleen , heart , and gonads of c. carpio [ 27 , 29 ] and intestinal wall of l. abu [ 22 , 37 , 42 ] . larvae in iraq was from the body cavity and different viscera of 10 fish species from different inland waters of iraq . were detected from six species of aquatic birds in iraq , egretta alba , e. garzetta , ardeola ralloides , botaurus stellaris , ardea purpurea , and ceryle rudis , from bahr al - najaf depression . rhabdochona hellichi ( srmek , 1901 ) , erroneously reported as r. bellichi , was recorded from intestine of the three carp species : c. idella , c. carpio , and h. molitrix . reported this parasite ( also erroneously as r. bellichi ) from the intestine and coelom of l. xanthopterus ( reported as b. xanthopterus ) , h. fossilis , and mystus pelusius ( reported as m. halepensis ) . the phylum acanthocephala is represented in farm fishes of babylon province with two valid species of the genus neoechinorhynchus . the first report of n. iraqensis was as species de novo from the intestine of l. abu from the euphrates river at al - fallujah region , while its description was given later by amin et al . neoechinorhynchus rutili ( mller , 1780 ) hamann , 1892 , was recorded from the intestine of the three carp species : c. idella , c. carpio [ 27 , 29 ] , and h. molitrix . the subphylum crustacea of the phylum arthropoda is represented in farm fishes of babylon province with nine species : one species of each of the genera argulus , dermoergasilus , paraergasilus , lamproglena , and lernaea and three species of ergasilus in addition to unidentified species of ergasilus . paraergasilus inflatus ho , khamees & mhaisen , 1996 family lernaeidae lamproglena pulchella von nordmann , 1832 lernaea cyprinacea l. , 1758 dermoergasilus varicoleus ho , jayarajan & radhakrishnan , 1992 ergasilus barbi rahemo , 1982 ergasilus mosulensis rahemo , 1982 ergasilus sieboldi von nordmann , 1832 paraergasilus inflatus ho , khamees & mhaisen , 1996 lamproglena pulchella von nordmann , 1832 lernaea cyprinacea l. , 1758 argulus foliaceus ( l. , 1758 ) was recorded from gills of the three carp species : c. idella , c. carpio [ 25 , 27 ] , and h. molitrix as well as from fins of c. auratus . ergasilus mosulensis rahemo , 1982 , was recorded from gills of the three carp species , c. idella , c. carpio , and h. molitrix as well as gills of l. abu . ergasilus sieboldi von nordmann , 1832 , was recorded from gills , buccal cavity , and skin of the three carp species : c. idella [ 27 , 29 ] , c. carpio [ 25 , 27 , 29 , 51 , 55 ] , and h. molitrix [ 27 , 29 , 56 ] . in addition to 11 species of ergasilus so far recorded from fishes of iraq , some specimens of unidentified ergasilus species were also reported from 12 fish species in iraq . lernaea cyprinacea l. , 1758 , was recorded from skin , fins , and gills of the three carp species , c. idella [ 24 , 2729 , 43 , 51 ] , c. carpio [ 6 , 2531 , 39 , 41 , 43 , 44 , 48 , 51 , 5355 , 57 , 61 , 63 ] , and h. molitrix [ 21 , 2729 , 43 ] , as well as from gills of l. abu . the first report of the anchor worm l. cyprinacea in iraq was from skin , fins , buccal cavity , pharyngeal cavity , gills , and anus of seven freshwater fish species from al - zaafaraniya fish - culture station . the phylum mollusca is represented in farm fishes of babylon province with only one parasite species of the genus unio . the first report of the glochidial larvae of u. pictorum in iraq was from gills of eight freshwater fish species from diyala river . the following host - parasite list for fish parasites in fish farms of babylon province is compiled . for each host , the scientific names of all recorded parasites are alphabetically enlisted under their major parasitic groups . to economize space , these can be obtained from the account of each concerned parasite species in the part of major groups of parasitic fauna within the results and discussion part .

although fish farming in iraq started in 1955 with a small pond in al - zaafaraniya , south of baghdad city , an advance was achieved in fish farming industry in iraq during the seventies and early eighties of the last century when many fish farms were established especially in the middle of iraq . during the last few years , a great advance was achieved in fish farming in general and fish cages in particular due to the increasing demand on fish protein as well as the increasing investment in fish - culture industry in most provinces of iraq . surveyed the literature on the parasitic fauna of fishes of al - furat fish farm ( previously known as babylon fish farm ) , which is the biggest fish farm in babylon province , and showed that the parasitic fauna of fishes of that farm included 60 valid parasite species ( 10 protozoans , three myxozoans , one trematode , 29 monogeneans , five cestodes , three nematodes , two acanthocephalans , six crustaceans , and one mollusc larva ) . therefore , the present paper was aimed at gathering data from the literature concerning all fish farms of babylon province and providing a list of parasite species according to their major groups as well as a host - parasite list for cultured fishes of these farms and some other fish species found in such farms . a total of 50 references ( 33 published articles , 12 unpublished theses , two unpublished reports , one book , one conference abstract , and one review article ) dealing with the parasites of farm fishes of babylon province were used to prepare the present paper . the index - catalogue of parasites and disease agents of fishes of iraq was used to indicate the total number of fish hosts harbouring each parasite species in the whole waters of iraq . the literature concerned with fish parasites of other farms in babylon province included those from al - latifiya fish farm [ 6 , 5052 ] ; al - bajaa fish farm ; abdul - razzak al - janabi fish farm ; fawzi al - janabi fish farm and ali al - hayali fish farm ; three fish farms at al - iskandariya district : abdul - hadi al - matloob fish farm , hussain al - gaiem fish farm , and maki chinak fish farm ; technical institute of al - musaib fish farm ; and al - manahil ( al - bilad ) fish farm at al - iskandariya district . in addition , surveys were done from some unnamed fish ponds such as those at al - mahaweel district , al - musaib district , al - iskandariya district , and sadat al - hindiya district as well as some other unnamed farms in the province [ 6165 ] . these parasites included one mastigophoran , three apicomplexans , 13 ciliophorans , five myxozoans , five trematodes , 45 monogeneans , five cestodes , three nematodes , two acanthocephalans , nine arthropods , and one mollusc . the common carp was found to harbour 81 species of parasites , the grass carp 30 species , the silver carp 28 species , l. abu 13 species , c. auratus one species , and h. fossilis one species . these major groups represent the concerned phyla of the parasites , but due to the great numbers of parasite species of the phylum platyhelminthes , its three classes ( trematoda , monogenea , and cestoda ) were applied in addition to their phylum . for each parasite species , all records in farm fishes in babylon province are given together with the first record of each concerned parasite in iraq as well as the present number of all hosts so far known in iraq for each concerned species based on the index - catalogue of parasites and disease agents of fishes of iraq . the phylum ciliophora is represented in farm fishes of babylon province with 13 species , three of which belonged to the genera chilodonella , ichthyophthirius , and tripartiella , five to the genus apiosoma , and four to the genus trichodina in addition to unspecified species of the genus trichodina . ichthyophthirius multifiliis fouquet , 1876 , was recorded from skin and gills of c. idella [ 27 , 29 , 43 , 51 , 56 ] , skin , fins , and gills of c. carpio [ 25 , 27 , 29 , 30 , 35 , 43 , 45 , 47 , 49 , 51 , 5356 , 59 , 60 , 65 ] , and skin and gills of h. molitrix [ 27 , 29 , 45 , 47 , 51 ] . apiosoma amoebae ( grenfell , 1887 ) lom , 1966 , was reported from skin , buccal cavity , and gills of c. idella [ 20 , 24 , 27 ] , skin and buccal cavity of c. carpio [ 27 , 55 ] , and gills and buccal cavity of h. molitrix [ 20 , 27 ] . apiosoma piscicola blanchard , 1885 , was recorded from skin , buccal cavity , and gills of c. idella [ 27 , 51 ] , skin , buccal cavity , and gills of c. carpio [ 27 , 29 , 51 , 55 ] , and buccal cavity and gills of h. molitrix [ 20 , 21 , 27 , 29 , 51 ] . a. piscicola was recorded for the first time in iraq from skin , buccal cavity , and gills of c. idella , c. carpio and h. molitrix from al - suwaira and al - latifiya fish farms . trichodina domerguei ( wallengren , 1897 ) was recorded from skin , fins , buccal cavity , and gills of c. idella [ 24 , 27 , 29 , 43 , 51 , 56 ] , skin , fins , buccal cavity , and gills of c. carpio [ 25 , 27 , 29 , 30 , 35 , 43 , 51 , 53 , 55 , 56 , 59 ] , skin , buccal cavity , and gills of h. molitrix [ 20 , 27 , 51 ] , and skin and gills of l. abu [ 23 , 56 ] . myxobolus pfeifferi thlohan , 1895 , was recorded from gills , intestine , liver , kidneys , and gallbladder of c. idella , gills , gallbladder , intestine , kidneys , and liver of c. carpio [ 25 , 27 , 29 , 53 , 59 ] , gills , liver , and intestine of h. molitrix [ 27 , 29 ] , and gills , intestinal wall , and gonads of l. abu . diplostomum spathaceum ( rudolphi , 1819 ) olsson , 1876 , was recorded as metacercaria from eyes of the three carp species : c. idella [ 43 , 56 ] , c. carpio [ 29 , 43 ] , and h. molitrix [ 43 , 56 ] . the class monogenea of the phylum platyhelminthes is represented in farm fishes of babylon province with 45 species : 12 species of the genus gyrodactylus , 26 species of dactylogyrus , and one species of each of the genera pseudacolpenteron , diplozoon , eudiplozoon , paradiplozoon , and microcotyle in addition to some unidentified species of dactylogyrus and diplozoon . gyrodactylus elegans von nordmann , 1832 , was recorded from skin , fins , buccal cavity , and gills of both c. idella [ 27 , 29 , 52 ] and c. carpio [ 2527 , 29 , 30 , 32 , 33 , 43 , 50 , 52 , 53 , 55 , 56 , 60 ] as well as from gills of h. molitrix and from l. abu with no mention to site of infection . dactylogyrus extensus mueller & van cleave , 1932 , was recorded from gills of c. idella , buccal cavity and gills of c. carpio [ 25 , 27 , 29 , 30 , 32 , 33 , 36 , 43 , 45 , 47 , 52 , 54 , 55 , 59 , 60 ] , and gills of h. molitrix [ 29 , 45 , 47 ] . the first report on this parasite in iraq was from gills of c. carpio from tigris river at al - zaafaraniya , south of baghdad , and al - qadisia dam lake , while its description and illustration were given later by mhaisen et al . dactylogyrus vastator nybelin , 1924 , was recorded from gills of c. idella and skin and gills of c. carpio [ 26 , 27 , 30 , 32 , 33 , 35 , 36 , 43 , 49 , 52 , 53 , 56 , 59 , 60 , 64 ] . this parasite was recorded for the first time in iraq from gills of c. carpio from a manmade lake in baghdad as diplozoon nipponicum but then it was reported by its valid name e. nipponicum by all subsequent researchers . this parasite was reported for the first time in iraq from gills of chondrostoma nasus , c. regium , and c. carpio from tigris river at baghdad as diplozoon barbi . the class cestoda of the phylum platyhelminthes is represented in farm fishes of babylon province with five species : one species of each of the genera bothriocephalus , ligula , and neogryporhynchus as well as two species of proteocephalus . phylum platyhelminthes class cestoda order bothriocephalidea family bothriocephalidae bothriocephalus acheilognathi yamaguti , 1934 order diphyllobothriidea family diphyllobothriidae ligula intestinalis ( l. , 1758 ) bloch , 1782 order proteocephalidea family proteocephalidae proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 order cyclophyllidea family dilepididae neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 order bothriocephalidea family bothriocephalidae bothriocephalus acheilognathi yamaguti , 1934 order diphyllobothriidea family diphyllobothriidae ligula intestinalis ( l. , 1758 ) bloch , 1782 order proteocephalidea family proteocephalidae proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 order cyclophyllidea family dilepididae neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 order bothriocephalidea family bothriocephalidae bothriocephalus acheilognathi yamaguti , 1934 family bothriocephalidae bothriocephalus acheilognathi yamaguti , 1934 bothriocephalus acheilognathi yamaguti , 1934 order diphyllobothriidea family diphyllobothriidae ligula intestinalis ( l. , 1758 ) bloch , 1782 family diphyllobothriidae ligula intestinalis ( l. , 1758 ) bloch , 1782 ligula intestinalis ( l. , 1758 ) bloch , 1782 order proteocephalidea family proteocephalidae proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 family proteocephalidae proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 proteocephalus osculatus ( goeze , 1782 ) nybelin , 1942 proteocephalus torulosus ( batsch , 1786 ) nufer , 1905 family dilepididae neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 neogryporhynchus cheilancristrotus ( wedl , 1855 ) baer & bona , 1960 bothriocephalus acheilognathi yamaguti , 1934 , was recorded from the intestine of both c. idella [ 24 , 56 ] and c. carpio [ 2527 , 52 , 53 , 62 ] . the phylum nematoda is represented in farm fishes of babylon province with three species : unidentified larval species of the genus contracaecum as well as one species of each of the genera cucullanus and rhabdochona . family cucullanidae cucullanus cyprini yamaguti , 1941 cucullanus cyprini yamaguti , 1941 cucullanus cyprini yamaguti , 1941 family rhabdochonidae rhabdochona hellichi ( srmek , 1901 ) family rhabdochonidae rhabdochona hellichi ( srmek , 1901 ) rhabdochona hellichi ( srmek , 1901 ) unidentified larval species of contracaecum was recorded from the intestinal wall , body cavity , liver , spleen , heart , and gonads of c. carpio [ 27 , 29 ] and intestinal wall of l. abu [ 22 , 37 , 42 ] . were detected from six species of aquatic birds in iraq , egretta alba , e. garzetta , ardeola ralloides , botaurus stellaris , ardea purpurea , and ceryle rudis , from bahr al - najaf depression . the first report of this parasite in iraq was from the intestine of alburnus caeruleus and luciobarbus xanthopterus ( reported as b. xanthopterus ) from al - tharthar lake . the subphylum crustacea of the phylum arthropoda is represented in farm fishes of babylon province with nine species : one species of each of the genera argulus , dermoergasilus , paraergasilus , lamproglena , and lernaea and three species of ergasilus in addition to unidentified species of ergasilus . lernaea cyprinacea l. , 1758 , was recorded from skin , fins , and gills of the three carp species , c. idella [ 24 , 2729 , 43 , 51 ] , c. carpio [ 6 , 2531 , 39 , 41 , 43 , 44 , 48 , 51 , 5355 , 57 , 61 , 63 ] , and h. molitrix [ 21 , 2729 , 43 ] , as well as from gills of l. abu . the first report of the anchor worm l. cyprinacea in iraq was from skin , fins , buccal cavity , pharyngeal cavity , gills , and anus of seven freshwater fish species from al - zaafaraniya fish - culture station . phylum mollusca class bivalvia order unionoida family unionidae unio pictorum ( linnaeus , 1758 ) order unionoida family unionidae unio pictorum ( linnaeus , 1758 ) family unionidae unio pictorum ( linnaeus , 1758 ) unio pictorum ( linnaeus , 1758 ) unio pictorum ( linnaeus , 1758 ) unio pictorum ( linnaeus , 1758 ) was recorded from gills of both c. carpio [ 27 , 29 ] and h. molitrix . monogenea : dactylogyrus achmerowi , d. amurensis , d. anchoratus , d. arcuatus , d. barbioides , d. cornu , d. crassus , d. dogieli , d. ergensi , d. extensus , d. gobii , d. inexpectatus , d. jamansajensis , d. lamellatus , d. latituba , d. lopuchinae , d. minutus , d. navicularis , d. phoxini , d. propinquus , d. sahuensis , d. simplex , d. skrjabini , d. vastator , dactylogyrus spp . , eudiplozoon nipponicum , gyrodactylus baicalensis , g. elegans , g. kherulensis , g. macracanthus , g. malmbergi , g. markewitschi , g. medius , g. menschikowi , g. salaris , g. sprostonae , g. vicinus , paradiplozoon barbi , and pseudacolpenteron pavlovskii . monogenea : dactylogyrus achmerowi , d. amurensis , d. anchoratus , d. arcuatus , d. barbioides , d. cornu , d. crassus , d. dogieli , d. ergensi , d. extensus , d. gobii , d. inexpectatus , d. jamansajensis , d. lamellatus , d. latituba , d. lopuchinae , d. minutus , d. navicularis , d. phoxini , d. propinquus , d. sahuensis , d. simplex , d. skrjabini , d. vastator , dactylogyrus spp . , eudiplozoon nipponicum , gyrodactylus baicalensis , g. elegans , g. kherulensis , g. macracanthus , g. malmbergi , g. markewitschi , g. medius , g. menschikowi , g. salaris , g. sprostonae , g. vicinus , paradiplozoon barbi , and pseudacolpenteron pavlovskii .

comparative modeling methods , also known as homology modeling or template - based modeling methods , are used widely to model protein structures . significant improvement in the accuracy of homology models stemming from various advancements has been described in other publications . however , even when using multiple templates , the resulting homology models can show significant deviation from the crystal structures , especially in certain local areas of the protein structure , depending on the sequence alignments . in order to be useful for functional analysis and drug design one of the outstanding problems in protein structure prediction is the lack of a consistent and reliable method for refinement of low resolution protein structural models to atomic level accuracy . however , limitations in the conformational search occur in the energy driven torsional monte carlo . these limitations can possibly be overcome by using the force driven molecular dynamics ( md ) methods that enable going over energy barriers . therefore , an md simulation offers an attractive force driven conformational search method that overcomes the pitfalls of monte carlo - based methods . all - atom md simulations , also known as cartesian md simulations , have shown limited success in protein structure refinement . however , in combination with knowledge based potentials and/or restraints using experimental data , structural refinement has been achieved using all - atom md simulations . mirjalili and feig have shown that the use of restraints to the starting structure during md gives better refinement than without . this is in agreement with the findings from the work of shaw and co - workers . feig et al also showed that the md ensemble averaged structures show better refinement than selecting one conformation from the ensemble . in this paper , we focus on the use of md methods for structure refinement without restraints . the generalized newton euler inverse mass operator ( gneimo ) method is an md simulation method based on the use of internal coordinates . the use of the gneimo torsional dynamics method combined with the temperature replica exchange ( rexmd ) method has been demonstrated for refinement of protein homology models without the use of knowledge based restraints . treating the high frequency degrees of freedom as rigid using hard holonomic constraints , along with temperature based replica exchange , leads to efficient conformational sampling in the low frequency torsional degrees of freedom . in this paper , we have applied the gneimo - rexmd method for the refinement of 30 proteins from the list of target proteins released by the critical assessment of techniques for protein structure refinement and prediction ( casp ) casp8 and casp9 . these 30 target proteins consist of both structure prediction and structure refinement categories from casp8 and casp9 . since the focus of this work was to examine the performance of gneimo - rexmd method for homology model refinement , we chose casp targets for which the crystal structures were available at the time of our study we have studied the extent of structure refinement that gneimo provides without using experimental data as restraints . our ultimate goal is to examine if the gneimo torsional md , in combination with torsional monte carlo method with and without experimental data is capable of protein structure refinement . briefly , gneimo is a constrained md method using internal coordinates , where the high frequency degrees of freedom are held rigid using holonomic constraints and the protein is modeled as a collection of user - defined rigid bodies known as clusters connected by flexible hinges . clusters can range in scale from single atoms to helices to whole domains of proteins , as chosen by the user . the equations of motion in internal coordinates are coupled , and the computational cost of solving the coupled equations of motion in internal coordinates scales as cubic power of the number of degrees of freedom . using the gneimo algorithm , however , the computational cost scales linearly with the number of degrees of freedom that enables the use of torsional md computationally feasible for protein simulations . other groups have used the gneimo algorithm for torsional md simulations and for nmr structure refinement . we have incorporated various advanced internal coordinate dynamics techniques to make the current implementation of gneimo a robust md technique for long time scale simulations . we have also demonstrated the use of gneimo for structure refinement of small proteins and for mapping the domain motion in proteins . in this paper we have used the gneimo torsional md that restricts internal motion to torsional angles for refining protein homology models . the gneimo - based protocol for protein structure refinement combines the gneimo torsional md method with the rexmd method for extended conformational search in torsional space . the gneimo - rexmd protocol used for refinement is an adaptation of the protocol previously derived for protein structure refinement . briefly , the gneimo constrained md simulations were carried out using the gneimo code using the amber99sb force field with the generalized born / surface area ( gb / sa ) obc implicit solvation model , an interior dielectric value of 1.5 for the solute , and exterior dielectric constant of 78.3 for the solvent . we used a solvent probe radius of 1.4 for the nonpolar solvation energy component of gb / sa . the nonbonded forces were switched off at a cutoff radius of 20 . gneimo md simulations were performed using all torsional degrees of freedom and at constant temperature using the nose - hoover method , a lobatto integrator , and an integration time step size of 5 fs . we added the temperature replica exchange md ( rexmd ) algorithm to the gneimo md method to enhance conformational sampling . gneimo all - torsion rexmd was then performed using 32 replicas over the 310415 k range of temperatures , and the temperature sorting was done based on the metropolis algorithm every 5 ps . we have studied refinement of casp targets from two categories : ( 1 ) the refinement category where a given decoy is to be refined and ( 2 ) the structure prediction category where only the sequence of amino acids in the target protein is provided . in this paper , we have studied 23 proteins of various sizes from the casp8 and casp9 refinement category and 7 proteins from the structure prediction category . the decoy structures for these 23 proteins were downloaded from the casp web site : www.predictioncenter.org . these structures were first subjected to all - atom conjugate gradient minimization using the sander program and the amber99sb force field . a total simulation time of 15100 ns ( for each replica ) for 32-replicas gneimo - rexmd simulations were performed for each target . the list of 23 target proteins from the structure refinement category ( tr ) and seven target proteins from structure prediction category ( t0 ) selected from the casp8 and casp9 is shown in table i. the casp10 targets were not released when we started this work . in addition , we wanted to validate the use of gneimo method for known targets and derive a protocol before we applied it to unknown targets . the starting decoys for the structure refinement targets were taken from the casp web site . for the targets from the structure prediction category , we derived homology models using the modeler method . the template structures for the modeler program were chosen by using the pdb sequence query search for sequences of 3080% identity to the target . we removed the structure of the target protein and its close homologues ( that were published after the respective casp competitions ) from the template structure search , to avoid any bias . one hundred models for each target were generated using modeler , and these models were then clustered into five groups . the best representative structure out of the five groups , as scored by procheck g - factor , was chosen to be the starting decoy for refinement for that target . minimization on each decoy was run using the sander utility in the amber suite and with the amber ff99sb force field , followed by gneimo - rexmd simulations . the third column in each block shows the scores for the best structure submitted to casp . we have calculated several metrics to assess the native likeness of the conformations sampled by gneimo - rexmd . we have calculated the root - mean - square deviation in coordinates ( rmsd ) of the backbone atoms to the x - ray and nmr structures . the rmsd was calculated using snapshots from the combined rexmd trajectory of all replicas . to determine whether the refinement in the model structure came from the secondary structure regions or the loop regions , we further calculated the rmsd of the backbone atoms in the secondary structure region ( as defined by the set of residues which are in helix or sheet in the native conformation ) and the rmsd of the backbone atoms for the whole structure including the loops and termini , both with respect to native structures . we used the package known as mdanalysis for the rmsd calculations . to measure the extent of refinement in the overall packing and fold of a protein , we compared the percent of native contacts made in the gneimo simulation trajectories with those in the native crystal and nmr structures . we calculated the n n matrices consisting of pairwise c(i)c(j ) atom distances for the native structures , and for the whole trajectories of gneimo - rexmd , where n is the number of residues in the protein , and i and j are residue indices . a pairwise c(i)c(j ) distance smaller than 8 was considered a contact and given an index value of 1 . hence , the calculation of the contact map results in the construction of an n n contact matrix consisting of 0 s ( atom pairs farther than the 8 distance cutoff or within the 4-residue neighbor cutoff in sequence ) and 1 s ( an atom pair within the 8 cutoff and more than 4 residues apart in sequence ) . we then considered each c pair in the simulation snapshots to be a contact , if the distance between both the atoms were within 0.5 of the same distance in the contact map of the native structure . we calculated the percentage native contacts as ( number of identical contacts between native and decoy / total number of contacts seen in the native structure ) . mdanalysis was used to calculate the pairwise distances needed for determination of percent native contacts . the gdt and tm scores are commonly used metrics in the casp assessments since they are more stringent than rmsd . gdt is defined as the average number of aligned c atoms that fit under a distance - to - native cutoff for four different cutoff distances . the most often used set of cutoff values is { 8 , 4 , 2 , 1 } ( ) and is referred to as gdt_ts . the tm - score was developed to correlate well with human - expert assessment of protein model quality and to address the limitations of other scores such as rmsd and gdt . the program maxcluster ( www.sbg.bio.ic.ac.uk/maxcluster ) was used to calculate the gdt_ts and tm - scores . the crystal and/or nmr structures used as reference structures for the calculation of the above - described metrics were downloaded from the pdb web site ( www.rcsb.org ) corresponding to each target as indicated by casp . if the native structure was deposited as an nmr ensemble , we used the top ranked nmr structure as the reference . table i lists the gdt , the tm score , and the rmsd for the c atoms of the best structures from the gneimo - rexmd trajectories for 23 different casp targets in the refinement category . the gdt and the tm scores for the best structure from gneimo improved in comparison to the starting decoy for 19 out of 23 proteins . the gdt scores showed an increase of up to 14.0 points while the increase in tm score is up to 0.13 . the extent of refinement by gneimo is comparable to the best structure submitted to casp for each target . the average increase in gdt score over all the 23 targets is 4.9 , 0.04 in tm score , and 0.52 in rmsd . this was a modest improvement for 19 out of 23 targets and in 4 cases there was no refinement from the starting model . to understand if there is a correlation between the extent of structure refinement and the secondary structure content , we calculated the secondary structure content of all the tr targets . we observed more than a 5.0 point improvement in the gdt scores for the tr429 , tr435 , tr453 , tr454 , tr464 , tr476 , tr530 , tr557 , tr574 , and tr624 targets , and these proteins showed at least 55% secondary structure content . some of the refinement targets for which there was little to no refinement by gneimo had less than 40% secondary structure content . tr462 , a target that showed little improvement with gneimo , is a two - domain protein connected by a linker region . although the overall refinement is 3.3 in gdt score , we observed refinement of 5.7 and 6.2 in the gdt scores for the individual domains . other targets such as tr576 , tr594 , tr606 , tr614 , and tr622 showed no improvement in gdt scores . tr614 has a loop that is 25 residues long , and tr606 has two loops that are 10 and 15 residues long that showed no improvement for gneimo simulations . figure s1 of the supporting information shows the extent of the refinement in the secondary structure regions in the target protein structures . apart from the refinement category , we also predicted the structures for the casp8 and casp9 targets in the structure prediction category denoted as the t0 targets . as described in the computational methods section , we first derived a homology model for each target using the modeler program starting from the amino acid sequence . we avoided using crystal structures that were published after the corresponding casp8 or casp9 assessment dates as templates for the homology models . starting from the homology models , we performed the gneimo - rexmd simulations for refinement of the model . table ii shows the extent of refinement ( gdt and tm scores ) yielded by just the refinement cycle using the gneimo simulations . all the structures predicted were within 4 rmsd from the respective crystal structures except for t0488 . the average improvements of 4.5 , 0.04 , and 0.7 in gdt , tm scores , and rmsd , respectively , were obtained using gneimo - rexmd refinement simulations . the rmsd of the structural models generated by modeler ranges from 1 to 9 . in every case , we observed substantial refinement in the structures compared to the starting decoy from modeler . figure 1 shows the contact map for targets with substantial refinement ( t0453 , tr429 , tr454 , tr530 ) and one target that did not show any refinement ( tr576 ) . these figures show the absolute distance between each residue in the gneimo refined model and the same residue in the corresponding crystal structure for various targets . the deeper the red color , the farther it is from the crystal structure . the cartoon picture of the backbone of the initial decoy , the gneimo refined , and the experimental structures are also shown for these targets . the white regions in the contact map are closer to the native structure , and the dark red regions are farther from the corresponding native structures . figure 1a for target t0453 shows substantial improvement in the packing of the loop structure with the rest of the protein structure upon gneimo refinement . the long - range inter - residue contacts for residues in the loop region between 30 and 40 with residues that are in the core of the protein between 40 and 60 ( shown in dark red in contact map for the starting decoy ) improve from 14 to 16 to 24 as seen in the contact map for the refined structure . this is an example where most of the refinement is in the packing of the loop with the secondary structure core of the rest of the protein . figure 1b for tr429 shows that residue ranges 2531 and 3642 form a -sheet that is missing in the starting decoy structure ( see dashed line rectangles in figure 1b ) . the three - strand -sheet motif that ranges from residues 2060 improves its packing to the rest of the protein , upon gneimo refinement . figure 1c shows that the two helices in the target tr454 are already formed in the initial decoy . refinement in this situation requires improved packing of these two helices shown in dashed rectangles in figure 1c . as seen in figure 1c , tr454 shows the long - range helix packing into a more nativelike structure , resulting in an rmsd of 2.47 in coordinates of the backbone atoms to the crystal structure . gneimo - rexmd refinement of the tr530 target leads to the proper folding of the n - terminal region , as shown in figure 1d ( dashed line rectangles ) . refinement is also seen in other long - range contacts throughout the molecule , due to slight improvements in local packing . ( a d ) refinement of casp targets with different types of secondary structure as shown in the figures and corresponding distance - to - native map . the distance to native map shows how far each residue is corresponding to the native structure . ( 1a ) example of refinement of loop structure ; ( 1b ) refinement of -sheet packing ; ( 1c ) packing of -helices ; ( 1d ) -sheet growth ; ( 1e ) example of a structure that was not refined by gneimo . tr576 has a substantial -sheet content as well as long loop regions , and the carboxy terminus region of tr576 needed substantial refinement . this structure was deemed to be of high difficulty by the casp assessment team , due to crystal contacts . the starting decoy is misfolded into a partial antiparallel -sheet while it is a parallel -sheet in the native structure . this could not be refined by gneimo possibly due to a high energy barrier in refolding this region . we did not use explicit water in these gneimo simulations , which may play an important role in stabilizing the loop structures . many of the targets studied here show better refinement with gneimo simulations than the best structure submitted for the casp assessment . it should be emphasized that we have compared the best structure by rmsd in coordinates generated by gneimo - rexmd simulations to the native structure and not utilized energy or scoring functions to pick the best structure . however , the gneimo method can be combined with any energy function or scoring function for picking the best structure . the chances of identifying the closest to native structure as the best scoring structure are improved if there is a substantial population of near - native conformations compared to the starting decoy generated during the gneimo - rexmd simulations . therefore , we calculated the fraction of the population from gneimo - rexmd simulations that are closer to the native structure compared to the starting decoy to assess the enrichment of nativelike structures in the gneimo - rexmd trajectories . figure 2 shows the population histogram with respect to gdt score and tm score for some targets , and figure s2 of the supporting information shows the same for all the targets . population distribution of the ensemble generated from the gneimo simulations compared to the cartesian simulations for various refinement casp targets . it is seen that , for tr429 and tr454 , a significant ( over 50% ) population shift occurs toward the native structure compared to the starting decoy ( denoted by the dotted line in figure 2 ) in both gdt and tm scores . structurally , both of these proteins are notable in that they have over 50% secondary structure content . both tr568 and tr624 show a small percentage ( about 1020% ) of the population getting refined . the targets tr576 , tr606 , tr614 , and tr622 show little to no population shift toward the native structure . these structures have less than 40% secondary structure content and have large loops , thus leading to poor refinement . thus , gneimo shows effective refinement of secondary structure regions and their packing with the failures occurring in the refinement of the loop regions . we are exploring the use of gneimo simulations with side chain replacement methods to improve the refinement of loop regions . in this section we compare the effectiveness of the conformational sampling of gneimo method to cartesian md method . however , we are not examining the effectiveness of energy functions that are used for selecting the best structure for cases where the experimental structure is unknown . our future goal is to combine gneimo with other refinement methods , namely those methods such as torsional monte carlo methods , that complement the torsional dynamics based conformational search of gneimo . figure 2 shows the comparison of the population of structures in the gneimo ensemble that get refined with respect to the starting decoy structures as measured by the gdt scores , compared to the corresponding populations in the cartesian md . the md simulations for both gneimo and cartesian md were done with the identical forcefield and gbsa solvation . the dotted line in the figure shows the position of the starting decoy . figure 2 shows that the relative proportion of gneimo ensemble that gets refined is more than the population of refined structures from cartesian simulations . this shows that the conformational sampling afforded by sampling torsional angles using the same forcefield is more effective in getting closer to the native structures than the cartesian all - atom simulations . shaw and co - workers have tested the ability of several microseconds of cartesian md simulations to refine homology models for 25 casp refinement targets , 21 of which are common to our study . they observed that the long cartesian md simulations lead to unraveling of the homology model away from the crystal or nmr structures . using the same forcefield we have shown in this paper that gneimo torsional dynamics method leads to more refinement than the cartesian md simulations . using an energy function that would preserve and funnel toward the native structure is a critical component for structure refinement . in the next section , we compare the all - atom amber energy function that we have used with gneimo md in this study to the knowledge based energy function in rosetta . scoring functions are important in selecting the most refined structure from the ensemble of conformations generated during the gneimo - rexmd simulations . in this paper , we have not addressed this issue . briefly , we have calculated the all - atom amber energies and energies from the rosetta energy function for all the conformations generated in the gneimo - rexmd trajectories of three target proteins . the rosetta energy function is based largely on the charmm energy function with additional knowledge based hydrogen bond terms . as shown in figure 3 , the rosetta energy function showed a more funnel like character for some of the targets , i.e. , the near - native structures showed the lowest energy . thus , use of the rosetta energy function could improve the selection of the best refined structure . however , for many other targets , both the amber and rosetta energy functions did not show a funnel like behavior . in the next stage of our study , we will examine many other knowledge based energy functions for rescoring the conformations generated by gneimo - rexmd simulations . also , we will explore the use of a force field derived from the rosetta potential energy function for driving the gneimo dynamics . potential energies of the conformations generated in the gneimo - rexmd simulation trajectories calculated using ( a ) amber99sb all atom forcefield and ( b ) rosetta energy function . an advantage of using the gneimo method is the time required to perform each sampling . by taking stable time steps of 10 fs , gneimo combined with rexmd is able to explore more regions of conformational space in the same number of processor cycles compared to cartesian simulations . while the targets in this study were run for up to 100 ns ( for all replicas combined ) , existing casp teams which replace cartesian md with gneimo could simulate about 1 order of magnitude longer in the same clock time . unlike torsional monte carlo method where the moves are random and scored by energy , the forces govern the moves in gneimo torsional md . performing conformational search in the torsional degrees of freedom appears to focus the search in the low frequency degrees of freedom . coupling gneimo torsional md with rexmd provides enough thermal energy to overcome barriers that can arise from the stiffness in the dynamic model from freezing high frequency degrees of freedom . giving such high thermal energy to all atoms in the cartesian all - atom dynamics can however result in an unraveling of structured regions in the starting decoy . the gneimo approach is also highly extensible . some monte carlo based methods that were used in casp restrict sampling of certain regions or attempt to rigidly dock individual domains of the same protein . the ability to perform dynamics of coarsened bodies is inherent within gneimo , and the generalized coordinate system can naturally incorporate constraints into the equations of motion to rigidify or free any desired degree of freedom . further , gneimo is not restricted to any specific force field but rather has a modular design with an extensible interface class for any force field that can be wrapped to fit a template . numerical integration methods for time propagation are also optional modules in gneimo , and methods , which rely on torsional monte carlo sampling ( like rosetta ) , can use gneimo to directly sample coordinates in phase space based on any definable coordinate system . in this paper , we report some of the promising developments on the application of torsional molecular dynamics method ( gneimo ) to structure refinement of casp target proteins . we have applied brute force torsional md without any restraints on any part of the structure from known structural information to refining homology models . the torsional md refinement yields results that are better compared to the all - atom md simulations performed under the same conditions . there is still much progress and issues that need to be addressed to improve gneimo as a refinement tool . as observed in table i , the extent of refinement by gneimo is the same whether the starting decoy is of low resolution ( greater than 5 ) or of high resolution ( less than 3 ) . one possibility is to test the extent of refinement using different clustering schemes in gneimo in combination with side chain refinement methods . the use of a force field tailored for protein structure prediction can also improve refinement with gneimo . if distance restraints are available from known experimental data , refinement can be improved at low resolution . presently , we are working on all these aspects to make gneimo a robust and generic refinement tool . we have shown that the gneimo - rexmd simulation technique leads to refinement of up to 1.3 for 30 casp target proteins starting from their homology models of variable resolution . gneimo method leads to refinement for 21 out of 23 refinement targets although the average refinement for 23 targets is 4.0 in gdt score , 0.04 in tm score , and 0.5 in rmsd in coordinates . significantly , we did observe that gneimo with rexmd simulations enable focused conformational sampling in the low frequency torsional space that is essential for structure refinement . further testing of this method , applying residue - based distance restraints obtained from experiments and testing a suitable energy function that provides identification of the native structure , is ongoing . additionally feig and co - workers have demonstrated that an ensemble average shows better refinement than rather than a single structure from md simulations . further testing of gneimo ensembles using an ensemble average could provide better refinement than seen in this study . enhancement in side chain sampling can be obtained by combining the gneimo simulations with side chain reassignment from a rotamer library . our ultimate goal is to combine gneimo torsional dynamics with a torsional monte carlo method and test the method in a future casp in the refinement category .

a longstanding challenge in using computational methods for protein structure prediction is the refinement of low - resolution structural models derived from comparative modeling methods into highly accurate atomistic models useful for detailed structural studies . previously , we have developed and demonstrated the utility of the internal coordinate molecular dynamics ( md ) technique , generalized newton euler inverse mass operator ( gneimo ) , for refinement of small proteins . using gneimo , the high - frequency degrees of freedom are frozen and the protein is modeled as a collection of rigid clusters connected by torsional hinges . this physical model allows larger integration time steps and focuses the conformational search in the low frequency torsional degrees of freedom . here , we have applied gneimo with temperature replica exchange to refine low - resolution protein models of 30 proteins taken from the continuous assessment of structure prediction ( casp ) competition . we have shown that gneimo torsional md method leads to refinement of up to 1.3 in the root - mean - square deviation in coordinates for 30 casp target proteins without using any experimental data as restraints in performing the gneimo simulations . this is in contrast with the unconstrained all - atom cartesian md method performed under the same conditions , where refinement requires the use of restraints during the simulations .

Introduction Computational Methods Results and Discussion Conclusions

comparative modeling methods , also known as homology modeling or template - based modeling methods , are used widely to model protein structures . however , even when using multiple templates , the resulting homology models can show significant deviation from the crystal structures , especially in certain local areas of the protein structure , depending on the sequence alignments . in order to be useful for functional analysis and drug design one of the outstanding problems in protein structure prediction is the lack of a consistent and reliable method for refinement of low resolution protein structural models to atomic level accuracy . however , limitations in the conformational search occur in the energy driven torsional monte carlo . these limitations can possibly be overcome by using the force driven molecular dynamics ( md ) methods that enable going over energy barriers . all - atom md simulations , also known as cartesian md simulations , have shown limited success in protein structure refinement . however , in combination with knowledge based potentials and/or restraints using experimental data , structural refinement has been achieved using all - atom md simulations . mirjalili and feig have shown that the use of restraints to the starting structure during md gives better refinement than without . this is in agreement with the findings from the work of shaw and co - workers . in this paper , we focus on the use of md methods for structure refinement without restraints . the generalized newton euler inverse mass operator ( gneimo ) method is an md simulation method based on the use of internal coordinates . the use of the gneimo torsional dynamics method combined with the temperature replica exchange ( rexmd ) method has been demonstrated for refinement of protein homology models without the use of knowledge based restraints . treating the high frequency degrees of freedom as rigid using hard holonomic constraints , along with temperature based replica exchange , leads to efficient conformational sampling in the low frequency torsional degrees of freedom . in this paper , we have applied the gneimo - rexmd method for the refinement of 30 proteins from the list of target proteins released by the critical assessment of techniques for protein structure refinement and prediction ( casp ) casp8 and casp9 . these 30 target proteins consist of both structure prediction and structure refinement categories from casp8 and casp9 . since the focus of this work was to examine the performance of gneimo - rexmd method for homology model refinement , we chose casp targets for which the crystal structures were available at the time of our study we have studied the extent of structure refinement that gneimo provides without using experimental data as restraints . our ultimate goal is to examine if the gneimo torsional md , in combination with torsional monte carlo method with and without experimental data is capable of protein structure refinement . briefly , gneimo is a constrained md method using internal coordinates , where the high frequency degrees of freedom are held rigid using holonomic constraints and the protein is modeled as a collection of user - defined rigid bodies known as clusters connected by flexible hinges . the equations of motion in internal coordinates are coupled , and the computational cost of solving the coupled equations of motion in internal coordinates scales as cubic power of the number of degrees of freedom . using the gneimo algorithm , however , the computational cost scales linearly with the number of degrees of freedom that enables the use of torsional md computationally feasible for protein simulations . we have also demonstrated the use of gneimo for structure refinement of small proteins and for mapping the domain motion in proteins . in this paper we have used the gneimo torsional md that restricts internal motion to torsional angles for refining protein homology models . the gneimo - based protocol for protein structure refinement combines the gneimo torsional md method with the rexmd method for extended conformational search in torsional space . the gneimo - rexmd protocol used for refinement is an adaptation of the protocol previously derived for protein structure refinement . briefly , the gneimo constrained md simulations were carried out using the gneimo code using the amber99sb force field with the generalized born / surface area ( gb / sa ) obc implicit solvation model , an interior dielectric value of 1.5 for the solute , and exterior dielectric constant of 78.3 for the solvent . gneimo md simulations were performed using all torsional degrees of freedom and at constant temperature using the nose - hoover method , a lobatto integrator , and an integration time step size of 5 fs . we added the temperature replica exchange md ( rexmd ) algorithm to the gneimo md method to enhance conformational sampling . gneimo all - torsion rexmd was then performed using 32 replicas over the 310415 k range of temperatures , and the temperature sorting was done based on the metropolis algorithm every 5 ps . we have studied refinement of casp targets from two categories : ( 1 ) the refinement category where a given decoy is to be refined and ( 2 ) the structure prediction category where only the sequence of amino acids in the target protein is provided . in this paper , we have studied 23 proteins of various sizes from the casp8 and casp9 refinement category and 7 proteins from the structure prediction category . these structures were first subjected to all - atom conjugate gradient minimization using the sander program and the amber99sb force field . the list of 23 target proteins from the structure refinement category ( tr ) and seven target proteins from structure prediction category ( t0 ) selected from the casp8 and casp9 is shown in table i. the casp10 targets were not released when we started this work . in addition , we wanted to validate the use of gneimo method for known targets and derive a protocol before we applied it to unknown targets . the starting decoys for the structure refinement targets were taken from the casp web site . for the targets from the structure prediction category , we derived homology models using the modeler method . we removed the structure of the target protein and its close homologues ( that were published after the respective casp competitions ) from the template structure search , to avoid any bias . minimization on each decoy was run using the sander utility in the amber suite and with the amber ff99sb force field , followed by gneimo - rexmd simulations . we have calculated several metrics to assess the native likeness of the conformations sampled by gneimo - rexmd . we have calculated the root - mean - square deviation in coordinates ( rmsd ) of the backbone atoms to the x - ray and nmr structures . to determine whether the refinement in the model structure came from the secondary structure regions or the loop regions , we further calculated the rmsd of the backbone atoms in the secondary structure region ( as defined by the set of residues which are in helix or sheet in the native conformation ) and the rmsd of the backbone atoms for the whole structure including the loops and termini , both with respect to native structures . to measure the extent of refinement in the overall packing and fold of a protein , we compared the percent of native contacts made in the gneimo simulation trajectories with those in the native crystal and nmr structures . we calculated the n n matrices consisting of pairwise c(i)c(j ) atom distances for the native structures , and for the whole trajectories of gneimo - rexmd , where n is the number of residues in the protein , and i and j are residue indices . hence , the calculation of the contact map results in the construction of an n n contact matrix consisting of 0 s ( atom pairs farther than the 8 distance cutoff or within the 4-residue neighbor cutoff in sequence ) and 1 s ( an atom pair within the 8 cutoff and more than 4 residues apart in sequence ) . we then considered each c pair in the simulation snapshots to be a contact , if the distance between both the atoms were within 0.5 of the same distance in the contact map of the native structure . table i lists the gdt , the tm score , and the rmsd for the c atoms of the best structures from the gneimo - rexmd trajectories for 23 different casp targets in the refinement category . to understand if there is a correlation between the extent of structure refinement and the secondary structure content , we calculated the secondary structure content of all the tr targets . although the overall refinement is 3.3 in gdt score , we observed refinement of 5.7 and 6.2 in the gdt scores for the individual domains . figure s1 of the supporting information shows the extent of the refinement in the secondary structure regions in the target protein structures . apart from the refinement category , we also predicted the structures for the casp8 and casp9 targets in the structure prediction category denoted as the t0 targets . as described in the computational methods section , we first derived a homology model for each target using the modeler program starting from the amino acid sequence . starting from the homology models , we performed the gneimo - rexmd simulations for refinement of the model . table ii shows the extent of refinement ( gdt and tm scores ) yielded by just the refinement cycle using the gneimo simulations . the rmsd of the structural models generated by modeler ranges from 1 to 9 . in every case , we observed substantial refinement in the structures compared to the starting decoy from modeler . these figures show the absolute distance between each residue in the gneimo refined model and the same residue in the corresponding crystal structure for various targets . the cartoon picture of the backbone of the initial decoy , the gneimo refined , and the experimental structures are also shown for these targets . the white regions in the contact map are closer to the native structure , and the dark red regions are farther from the corresponding native structures . figure 1a for target t0453 shows substantial improvement in the packing of the loop structure with the rest of the protein structure upon gneimo refinement . the long - range inter - residue contacts for residues in the loop region between 30 and 40 with residues that are in the core of the protein between 40 and 60 ( shown in dark red in contact map for the starting decoy ) improve from 14 to 16 to 24 as seen in the contact map for the refined structure . this is an example where most of the refinement is in the packing of the loop with the secondary structure core of the rest of the protein . gneimo - rexmd refinement of the tr530 target leads to the proper folding of the n - terminal region , as shown in figure 1d ( dashed line rectangles ) . it should be emphasized that we have compared the best structure by rmsd in coordinates generated by gneimo - rexmd simulations to the native structure and not utilized energy or scoring functions to pick the best structure . the chances of identifying the closest to native structure as the best scoring structure are improved if there is a substantial population of near - native conformations compared to the starting decoy generated during the gneimo - rexmd simulations . therefore , we calculated the fraction of the population from gneimo - rexmd simulations that are closer to the native structure compared to the starting decoy to assess the enrichment of nativelike structures in the gneimo - rexmd trajectories . figure 2 shows the population histogram with respect to gdt score and tm score for some targets , and figure s2 of the supporting information shows the same for all the targets . population distribution of the ensemble generated from the gneimo simulations compared to the cartesian simulations for various refinement casp targets . thus , gneimo shows effective refinement of secondary structure regions and their packing with the failures occurring in the refinement of the loop regions . we are exploring the use of gneimo simulations with side chain replacement methods to improve the refinement of loop regions . in this section we compare the effectiveness of the conformational sampling of gneimo method to cartesian md method . our future goal is to combine gneimo with other refinement methods , namely those methods such as torsional monte carlo methods , that complement the torsional dynamics based conformational search of gneimo . figure 2 shows the comparison of the population of structures in the gneimo ensemble that get refined with respect to the starting decoy structures as measured by the gdt scores , compared to the corresponding populations in the cartesian md . the md simulations for both gneimo and cartesian md were done with the identical forcefield and gbsa solvation . this shows that the conformational sampling afforded by sampling torsional angles using the same forcefield is more effective in getting closer to the native structures than the cartesian all - atom simulations . they observed that the long cartesian md simulations lead to unraveling of the homology model away from the crystal or nmr structures . using the same forcefield we have shown in this paper that gneimo torsional dynamics method leads to more refinement than the cartesian md simulations . in the next section , we compare the all - atom amber energy function that we have used with gneimo md in this study to the knowledge based energy function in rosetta . scoring functions are important in selecting the most refined structure from the ensemble of conformations generated during the gneimo - rexmd simulations . in this paper , we have not addressed this issue . briefly , we have calculated the all - atom amber energies and energies from the rosetta energy function for all the conformations generated in the gneimo - rexmd trajectories of three target proteins . as shown in figure 3 , the rosetta energy function showed a more funnel like character for some of the targets , i.e. thus , use of the rosetta energy function could improve the selection of the best refined structure . also , we will explore the use of a force field derived from the rosetta potential energy function for driving the gneimo dynamics . potential energies of the conformations generated in the gneimo - rexmd simulation trajectories calculated using ( a ) amber99sb all atom forcefield and ( b ) rosetta energy function . an advantage of using the gneimo method is the time required to perform each sampling . by taking stable time steps of 10 fs , gneimo combined with rexmd is able to explore more regions of conformational space in the same number of processor cycles compared to cartesian simulations . while the targets in this study were run for up to 100 ns ( for all replicas combined ) , existing casp teams which replace cartesian md with gneimo could simulate about 1 order of magnitude longer in the same clock time . unlike torsional monte carlo method where the moves are random and scored by energy , the forces govern the moves in gneimo torsional md . performing conformational search in the torsional degrees of freedom appears to focus the search in the low frequency degrees of freedom . coupling gneimo torsional md with rexmd provides enough thermal energy to overcome barriers that can arise from the stiffness in the dynamic model from freezing high frequency degrees of freedom . giving such high thermal energy to all atoms in the cartesian all - atom dynamics can however result in an unraveling of structured regions in the starting decoy . the ability to perform dynamics of coarsened bodies is inherent within gneimo , and the generalized coordinate system can naturally incorporate constraints into the equations of motion to rigidify or free any desired degree of freedom . numerical integration methods for time propagation are also optional modules in gneimo , and methods , which rely on torsional monte carlo sampling ( like rosetta ) , can use gneimo to directly sample coordinates in phase space based on any definable coordinate system . in this paper , we report some of the promising developments on the application of torsional molecular dynamics method ( gneimo ) to structure refinement of casp target proteins . we have applied brute force torsional md without any restraints on any part of the structure from known structural information to refining homology models . the torsional md refinement yields results that are better compared to the all - atom md simulations performed under the same conditions . as observed in table i , the extent of refinement by gneimo is the same whether the starting decoy is of low resolution ( greater than 5 ) or of high resolution ( less than 3 ) . the use of a force field tailored for protein structure prediction can also improve refinement with gneimo . we have shown that the gneimo - rexmd simulation technique leads to refinement of up to 1.3 for 30 casp target proteins starting from their homology models of variable resolution . gneimo method leads to refinement for 21 out of 23 refinement targets although the average refinement for 23 targets is 4.0 in gdt score , 0.04 in tm score , and 0.5 in rmsd in coordinates . significantly , we did observe that gneimo with rexmd simulations enable focused conformational sampling in the low frequency torsional space that is essential for structure refinement . enhancement in side chain sampling can be obtained by combining the gneimo simulations with side chain reassignment from a rotamer library . our ultimate goal is to combine gneimo torsional dynamics with a torsional monte carlo method and test the method in a future casp in the refinement category .

however , even when using multiple templates , the resulting homology models can show significant deviation from the crystal structures , especially in certain local areas of the protein structure , depending on the sequence alignments . in order to be useful for functional analysis and drug design one of the outstanding problems in protein structure prediction is the lack of a consistent and reliable method for refinement of low resolution protein structural models to atomic level accuracy . however , limitations in the conformational search occur in the energy driven torsional monte carlo . therefore , an md simulation offers an attractive force driven conformational search method that overcomes the pitfalls of monte carlo - based methods . however , in combination with knowledge based potentials and/or restraints using experimental data , structural refinement has been achieved using all - atom md simulations . in this paper , we focus on the use of md methods for structure refinement without restraints . the use of the gneimo torsional dynamics method combined with the temperature replica exchange ( rexmd ) method has been demonstrated for refinement of protein homology models without the use of knowledge based restraints . treating the high frequency degrees of freedom as rigid using hard holonomic constraints , along with temperature based replica exchange , leads to efficient conformational sampling in the low frequency torsional degrees of freedom . in this paper , we have applied the gneimo - rexmd method for the refinement of 30 proteins from the list of target proteins released by the critical assessment of techniques for protein structure refinement and prediction ( casp ) casp8 and casp9 . since the focus of this work was to examine the performance of gneimo - rexmd method for homology model refinement , we chose casp targets for which the crystal structures were available at the time of our study we have studied the extent of structure refinement that gneimo provides without using experimental data as restraints . our ultimate goal is to examine if the gneimo torsional md , in combination with torsional monte carlo method with and without experimental data is capable of protein structure refinement . the equations of motion in internal coordinates are coupled , and the computational cost of solving the coupled equations of motion in internal coordinates scales as cubic power of the number of degrees of freedom . using the gneimo algorithm , however , the computational cost scales linearly with the number of degrees of freedom that enables the use of torsional md computationally feasible for protein simulations . we have incorporated various advanced internal coordinate dynamics techniques to make the current implementation of gneimo a robust md technique for long time scale simulations . we have also demonstrated the use of gneimo for structure refinement of small proteins and for mapping the domain motion in proteins . in this paper we have used the gneimo torsional md that restricts internal motion to torsional angles for refining protein homology models . the gneimo - based protocol for protein structure refinement combines the gneimo torsional md method with the rexmd method for extended conformational search in torsional space . briefly , the gneimo constrained md simulations were carried out using the gneimo code using the amber99sb force field with the generalized born / surface area ( gb / sa ) obc implicit solvation model , an interior dielectric value of 1.5 for the solute , and exterior dielectric constant of 78.3 for the solvent . we used a solvent probe radius of 1.4 for the nonpolar solvation energy component of gb / sa . we have studied refinement of casp targets from two categories : ( 1 ) the refinement category where a given decoy is to be refined and ( 2 ) the structure prediction category where only the sequence of amino acids in the target protein is provided . in this paper , we have studied 23 proteins of various sizes from the casp8 and casp9 refinement category and 7 proteins from the structure prediction category . the list of 23 target proteins from the structure refinement category ( tr ) and seven target proteins from structure prediction category ( t0 ) selected from the casp8 and casp9 is shown in table i. the casp10 targets were not released when we started this work . in addition , we wanted to validate the use of gneimo method for known targets and derive a protocol before we applied it to unknown targets . for the targets from the structure prediction category , we derived homology models using the modeler method . we removed the structure of the target protein and its close homologues ( that were published after the respective casp competitions ) from the template structure search , to avoid any bias . the best representative structure out of the five groups , as scored by procheck g - factor , was chosen to be the starting decoy for refinement for that target . minimization on each decoy was run using the sander utility in the amber suite and with the amber ff99sb force field , followed by gneimo - rexmd simulations . we have calculated several metrics to assess the native likeness of the conformations sampled by gneimo - rexmd . we have calculated the root - mean - square deviation in coordinates ( rmsd ) of the backbone atoms to the x - ray and nmr structures . to determine whether the refinement in the model structure came from the secondary structure regions or the loop regions , we further calculated the rmsd of the backbone atoms in the secondary structure region ( as defined by the set of residues which are in helix or sheet in the native conformation ) and the rmsd of the backbone atoms for the whole structure including the loops and termini , both with respect to native structures . to measure the extent of refinement in the overall packing and fold of a protein , we compared the percent of native contacts made in the gneimo simulation trajectories with those in the native crystal and nmr structures . we calculated the n n matrices consisting of pairwise c(i)c(j ) atom distances for the native structures , and for the whole trajectories of gneimo - rexmd , where n is the number of residues in the protein , and i and j are residue indices . hence , the calculation of the contact map results in the construction of an n n contact matrix consisting of 0 s ( atom pairs farther than the 8 distance cutoff or within the 4-residue neighbor cutoff in sequence ) and 1 s ( an atom pair within the 8 cutoff and more than 4 residues apart in sequence ) . we then considered each c pair in the simulation snapshots to be a contact , if the distance between both the atoms were within 0.5 of the same distance in the contact map of the native structure . we calculated the percentage native contacts as ( number of identical contacts between native and decoy / total number of contacts seen in the native structure ) . the tm - score was developed to correlate well with human - expert assessment of protein model quality and to address the limitations of other scores such as rmsd and gdt . the crystal and/or nmr structures used as reference structures for the calculation of the above - described metrics were downloaded from the pdb web site ( www.rcsb.org ) corresponding to each target as indicated by casp . table i lists the gdt , the tm score , and the rmsd for the c atoms of the best structures from the gneimo - rexmd trajectories for 23 different casp targets in the refinement category . the gdt and the tm scores for the best structure from gneimo improved in comparison to the starting decoy for 19 out of 23 proteins . to understand if there is a correlation between the extent of structure refinement and the secondary structure content , we calculated the secondary structure content of all the tr targets . we observed more than a 5.0 point improvement in the gdt scores for the tr429 , tr435 , tr453 , tr454 , tr464 , tr476 , tr530 , tr557 , tr574 , and tr624 targets , and these proteins showed at least 55% secondary structure content . although the overall refinement is 3.3 in gdt score , we observed refinement of 5.7 and 6.2 in the gdt scores for the individual domains . figure s1 of the supporting information shows the extent of the refinement in the secondary structure regions in the target protein structures . apart from the refinement category , we also predicted the structures for the casp8 and casp9 targets in the structure prediction category denoted as the t0 targets . as described in the computational methods section , we first derived a homology model for each target using the modeler program starting from the amino acid sequence . starting from the homology models , we performed the gneimo - rexmd simulations for refinement of the model . table ii shows the extent of refinement ( gdt and tm scores ) yielded by just the refinement cycle using the gneimo simulations . the average improvements of 4.5 , 0.04 , and 0.7 in gdt , tm scores , and rmsd , respectively , were obtained using gneimo - rexmd refinement simulations . in every case , we observed substantial refinement in the structures compared to the starting decoy from modeler . figure 1 shows the contact map for targets with substantial refinement ( t0453 , tr429 , tr454 , tr530 ) and one target that did not show any refinement ( tr576 ) . these figures show the absolute distance between each residue in the gneimo refined model and the same residue in the corresponding crystal structure for various targets . the cartoon picture of the backbone of the initial decoy , the gneimo refined , and the experimental structures are also shown for these targets . the white regions in the contact map are closer to the native structure , and the dark red regions are farther from the corresponding native structures . figure 1a for target t0453 shows substantial improvement in the packing of the loop structure with the rest of the protein structure upon gneimo refinement . the long - range inter - residue contacts for residues in the loop region between 30 and 40 with residues that are in the core of the protein between 40 and 60 ( shown in dark red in contact map for the starting decoy ) improve from 14 to 16 to 24 as seen in the contact map for the refined structure . this is an example where most of the refinement is in the packing of the loop with the secondary structure core of the rest of the protein . as seen in figure 1c , tr454 shows the long - range helix packing into a more nativelike structure , resulting in an rmsd of 2.47 in coordinates of the backbone atoms to the crystal structure . gneimo - rexmd refinement of the tr530 target leads to the proper folding of the n - terminal region , as shown in figure 1d ( dashed line rectangles ) . ( a d ) refinement of casp targets with different types of secondary structure as shown in the figures and corresponding distance - to - native map . ( 1a ) example of refinement of loop structure ; ( 1b ) refinement of -sheet packing ; ( 1c ) packing of -helices ; ( 1d ) -sheet growth ; ( 1e ) example of a structure that was not refined by gneimo . many of the targets studied here show better refinement with gneimo simulations than the best structure submitted for the casp assessment . it should be emphasized that we have compared the best structure by rmsd in coordinates generated by gneimo - rexmd simulations to the native structure and not utilized energy or scoring functions to pick the best structure . the chances of identifying the closest to native structure as the best scoring structure are improved if there is a substantial population of near - native conformations compared to the starting decoy generated during the gneimo - rexmd simulations . therefore , we calculated the fraction of the population from gneimo - rexmd simulations that are closer to the native structure compared to the starting decoy to assess the enrichment of nativelike structures in the gneimo - rexmd trajectories . figure 2 shows the population histogram with respect to gdt score and tm score for some targets , and figure s2 of the supporting information shows the same for all the targets . population distribution of the ensemble generated from the gneimo simulations compared to the cartesian simulations for various refinement casp targets . it is seen that , for tr429 and tr454 , a significant ( over 50% ) population shift occurs toward the native structure compared to the starting decoy ( denoted by the dotted line in figure 2 ) in both gdt and tm scores . thus , gneimo shows effective refinement of secondary structure regions and their packing with the failures occurring in the refinement of the loop regions . in this section we compare the effectiveness of the conformational sampling of gneimo method to cartesian md method . however , we are not examining the effectiveness of energy functions that are used for selecting the best structure for cases where the experimental structure is unknown . our future goal is to combine gneimo with other refinement methods , namely those methods such as torsional monte carlo methods , that complement the torsional dynamics based conformational search of gneimo . figure 2 shows the comparison of the population of structures in the gneimo ensemble that get refined with respect to the starting decoy structures as measured by the gdt scores , compared to the corresponding populations in the cartesian md . this shows that the conformational sampling afforded by sampling torsional angles using the same forcefield is more effective in getting closer to the native structures than the cartesian all - atom simulations . shaw and co - workers have tested the ability of several microseconds of cartesian md simulations to refine homology models for 25 casp refinement targets , 21 of which are common to our study . using the same forcefield we have shown in this paper that gneimo torsional dynamics method leads to more refinement than the cartesian md simulations . in the next section , we compare the all - atom amber energy function that we have used with gneimo md in this study to the knowledge based energy function in rosetta . scoring functions are important in selecting the most refined structure from the ensemble of conformations generated during the gneimo - rexmd simulations . briefly , we have calculated the all - atom amber energies and energies from the rosetta energy function for all the conformations generated in the gneimo - rexmd trajectories of three target proteins . in the next stage of our study , we will examine many other knowledge based energy functions for rescoring the conformations generated by gneimo - rexmd simulations . also , we will explore the use of a force field derived from the rosetta potential energy function for driving the gneimo dynamics . potential energies of the conformations generated in the gneimo - rexmd simulation trajectories calculated using ( a ) amber99sb all atom forcefield and ( b ) rosetta energy function . by taking stable time steps of 10 fs , gneimo combined with rexmd is able to explore more regions of conformational space in the same number of processor cycles compared to cartesian simulations . while the targets in this study were run for up to 100 ns ( for all replicas combined ) , existing casp teams which replace cartesian md with gneimo could simulate about 1 order of magnitude longer in the same clock time . performing conformational search in the torsional degrees of freedom appears to focus the search in the low frequency degrees of freedom . giving such high thermal energy to all atoms in the cartesian all - atom dynamics can however result in an unraveling of structured regions in the starting decoy . the ability to perform dynamics of coarsened bodies is inherent within gneimo , and the generalized coordinate system can naturally incorporate constraints into the equations of motion to rigidify or free any desired degree of freedom . numerical integration methods for time propagation are also optional modules in gneimo , and methods , which rely on torsional monte carlo sampling ( like rosetta ) , can use gneimo to directly sample coordinates in phase space based on any definable coordinate system . in this paper , we report some of the promising developments on the application of torsional molecular dynamics method ( gneimo ) to structure refinement of casp target proteins . as observed in table i , the extent of refinement by gneimo is the same whether the starting decoy is of low resolution ( greater than 5 ) or of high resolution ( less than 3 ) . we have shown that the gneimo - rexmd simulation technique leads to refinement of up to 1.3 for 30 casp target proteins starting from their homology models of variable resolution . gneimo method leads to refinement for 21 out of 23 refinement targets although the average refinement for 23 targets is 4.0 in gdt score , 0.04 in tm score , and 0.5 in rmsd in coordinates . significantly , we did observe that gneimo with rexmd simulations enable focused conformational sampling in the low frequency torsional space that is essential for structure refinement . further testing of this method , applying residue - based distance restraints obtained from experiments and testing a suitable energy function that provides identification of the native structure , is ongoing . our ultimate goal is to combine gneimo torsional dynamics with a torsional monte carlo method and test the method in a future casp in the refinement category .

based on the report of world health organization ( who ) , road traffic injuries ( rtis ) stand for 25% of injury - related mortalities . traffic injuries constitute the largest proportion of unintentional injury deaths ( 33% ) in the world . road traffic injuries are the ninth leading cause of all deaths and leading contributor to the worldwide burden of disease . yearly traffic collisions cause 1.2 million deaths and 20 to 50 million disabilities in the world . who estimates that without proper measures , the incidence of traffic mortalities rise by 67% and in 2020 it will be the third cause of death among 5 to 45 year old people . low and middle - income countries have 85 percent of traffic mortalities in the world . only 14% of traffic mortalities occur in highly motorized countries ( hmc ) such as north america , australia , newzeland , japan and west europe although these countries have comprised about 60% of the world vehicles . previous studies showed that death caused by traffic injuries has been increased in africa , eastern mediterranean and asia in recent years . in addition , the rate of traffic mortality to whole mortality is 2.5 in the world and 1.9 in the region ( eastern mediterranean)while this ratio in iran is about 7.5 . while having one percent of the world population , one fortieth of road traffic accidents occur in iran . studies have indicated that iran has too many road traffic injuries ; for example , iran with rti fatality rate of 31 . 8 per 100 000 in the year 2007 had the highest rate in the region of eastern mediterranean . in iran , there are about one vehicle for every four individuals ; in comparison , this ratio is higher than that of eastern mediterranean countries but lower than those of industrialized and developed countries . moreover , rtis stand for the most prevalent cause of injury and the second leading cause of death in iran . a report by who showed that despite technological development , it is not clear why mortality rate in traffic injuries has a progressive trend in many countries . although new traffic laws had effects on reduction of fatal and non - fatal rates of road traffic injuries in iran , injuries from road traffic incidents have been increased in recent years . comparative studies show that an important portion of traffic mortalities occur in post - crash phase ( after crash , until transferring the injured victim to hospital ) in low and middle income countries . one noticeable part of these mortalities are preventable by proper pre - hospital interventions . one of the most important roles of pre - hospital services is life support and prevention of future disabilities in the post - crash phase . triage of an injured patient to an appropriate trauma center can have an impact on morbidity and mortality . studying the process and structure of pre - hospital service system provides knowledge and insight for assessment of these types of services . despite the importance of pre - hospital service in preventing mortalities and disabilities caused by road traffic injuries , there is lack of knowledge in these fields , especially cultural and contextual aspects . since the function of pre - hospital service is culture and context bound , qualitative approach was an appropriate method to design the study . also , since process of pre - hospital services is a phenomenon that consists human relationships , action - interactions , and emotions that are usually hidden and could not be assessed by quantitative approaches , qualitative method was selected as an approach . by qualitative approaches , we can observe phenomenon from the eyes of the participants , and the hidden aspects of phenomenon can be explored . determining the barriers of pre - hospital service in viewpoints of participants was another reason for selecting the qualitative approach . the aim of this study was to explore the barriers of pre - hospital service in traffic injuries in tehran , iran . is suitable when the existing theories are inadequate to describe the phenomenon . in content analysis , concepts are gained directly and inductively from the raw data . it is a method with the purpose of providing knowledge , new understanding , and representation of realities . the aim of content analysis is to attain a summarized and comprehensive description of the phenomenon . in this method , another advantage of this method is the possibility of observation of the phenomenon , as viewed by the participants . first , interviewer proposed some general questions to start the interviews such as please explain one of your assignments . then , based on the participants responses and trend of the interview , next questions were arranged . each interview lasted about 45 - 60 minutes . the time and location of the interviews interviews were recorded in the form of audio files ( mp3 ) and transcribed verbatim . in addition , researchers used other methods of data gathering such as field notes , focus groups and observation . for data gathering by observation , the researcher observed the scenes of crushing and recorded the observed events ( field note ) . the se notes consisted of the type of crushing , the way first aids were provided to the injured victim , the person who provides aids to the injured person , the way the crowded people in scene of crush interfere with care giving process , and the way the action of other organizations interferes with interventions of pre - hospital service providers . in addition , one session of focus group for enrichment of data was performed with pre - hospital managers and paramedics . eighteen participants were selected by purposive sampling and that those with at least 2 years experience in the field of pre - hospital services were considered to be enrolled in the study . the participants consisted of 14 paramedics , 1 dispatch operator , 2 pre - hospital service managers and 1 police officer . each interview was recorded and typed verbatim by the first researcher . for analyzing the data at first , the researcher read the texts several times ( intensive reading ) for getting a complete understanding of the respondents accounts ; then , the meaning units of phrases was identified and summarized . finally , after coding , based on common properties or dimensions , similar codes were classified in higher - level categories with a new and high abstract label ( table 1 ) . data collection continued until data saturation , meaning that adding further data does not offer any new information and new properties for each category . example of meaning units , condensed meaning units , codes , subcategories and categories for examining the rigor of data and results , guba and lincoln strategies were used . to insure the credibility of data , several strategies were used including : a ) prolonged engagement ( data gathering lasted about one year and during this period , researchers were oriented to culture , language and environment of the participants and atmosphere of the field ) , b ) peer review ( data and interpretation of data were checked by other researchers ) , and c ) member checking ( data rechecked by participants and our interpretations from data were reviewed and confirmed by them ) . in addition , to insure the dependability of data , triangulation strategy for data collection such as interview , observation , focus group and field notes were applied . by this strategy , they were also informed about their authority for withdrawal from the study at any stage of study . in addition , their anonymity as well as confidentiality was insured and the participants signed the informed consent form . ethics committee of tehran university and the department of social welfare and rehabilitation sciences approved the study . in addition , one abstract of the results of the study was mailed for participants interested in the findings . data was gathered through semi - structured interviews . first , interviewer proposed some general questions to start the interviews such as please explain one of your assignments . then , based on the participants responses and trend of the interview , next questions were arranged . each interview lasted about 45 - 60 minutes . the time and location of the interviews interviews were recorded in the form of audio files ( mp3 ) and transcribed verbatim . in addition , researchers used other methods of data gathering such as field notes , focus groups and observation . for data gathering by observation , the researcher observed the scenes of crushing and recorded the observed events ( field note ) . the se notes consisted of the type of crushing , the way first aids were provided to the injured victim , the person who provides aids to the injured person , the way the crowded people in scene of crush interfere with care giving process , and the way the action of other organizations interferes with interventions of pre - hospital service providers . in addition , one session of focus group for enrichment of data was performed with pre - hospital managers and paramedics . eighteen participants were selected by purposive sampling and that those with at least 2 years experience in the field of pre - hospital services were considered to be enrolled in the study . the participants consisted of 14 paramedics , 1 dispatch operator , 2 pre - hospital service managers and 1 police officer . each interview was recorded and typed verbatim by the first researcher . for analyzing the data at first , the researcher read the texts several times ( intensive reading ) for getting a complete understanding of the respondents accounts ; then , the meaning units of phrases was identified and summarized . finally , after coding , based on common properties or dimensions , similar codes were classified in higher - level categories with a new and high abstract label ( table 1 ) . data collection continued until data saturation , meaning that adding further data does not offer any new information and new properties for each category . for examining the rigor of data and results , guba and lincoln strategies were used . to insure the credibility of data , several strategies were used including : a ) prolonged engagement ( data gathering lasted about one year and during this period , researchers were oriented to culture , language and environment of the participants and atmosphere of the field ) , b ) peer review ( data and interpretation of data were checked by other researchers ) , and c ) member checking ( data rechecked by participants and our interpretations from data were reviewed and confirmed by them ) . in addition , to insure the dependability of data , triangulation strategy for data collection such as interview , observation , focus group and field notes were applied . by this strategy , data can be collected from many aspects and dependability of data will be increased . they were also informed about their authority for withdrawal from the study at any stage of study . in addition , their anonymity as well as confidentiality was insured and the participants signed the informed consent form . ethics committee of tehran university and the department of social welfare and rehabilitation sciences approved the study . in addition , one abstract of the results of the study was mailed for participants interested in the findings . based on the data , 13 categories were developed and classified into four main categories ( table 2 ) . main categories were classified into four branches : categories and subcategories of the barriers to pre - hospital services in road trafficinjuries a.barriers related to people consisting of concepts ( subcategory ) that have at least one common property . this property included barriers that were in a way related to people such as laypeople involvement , inadequate knowledge about first aids and mistake calls.b.barriers related to metropolitan infrastructures . this category consisted of concepts whose common properties were problems related to characteristics and structure of the city such as traffic , accessibility to streets and naming of the alleys.c.barriers related to profession . in this main category , there were concepts that explain barriers related to pre - hospital service providing profession . in this category , there are three subcategories : professional autonomy , workload and work related injuries.d.the fourth main category named barriers related to managerial issues consisting of four concepts that were common in property of managerial problems in the field of pre - hospital service . these concepts were named inadequate telecommunication technology , human resources , inadequate workload related privilege and lack of organizational coordination . in the next section , you can see categorization of main categories and related quotations : barriers related to people consisting of concepts ( subcategory ) that have at least one common property . this property included barriers that were in a way related to people such as laypeople involvement , inadequate knowledge about first aids and mistake calls . this category consisted of concepts whose common properties were problems related to characteristics and structure of the city such as traffic , accessibility to streets and naming of the alleys . barriers related to profession . in this main category , there were concepts that explain barriers related to pre - hospital service providing profession . in this category , there are three subcategories : professional autonomy , workload and work related injuries . the fourth main category named barriers related to managerial issues consisting of four concepts that were common in property of managerial problems in the field of pre - hospital service . these concepts were named inadequate telecommunication technology , human resources , inadequate workload related privilege and lack of organizational coordination . in the next section , you can see categorization of main categories and related quotations : a. barriers related to people : a. 1 laypeople -involvement : crowded area in the crash scenes is a problem that was brought up by the participants . overcrowding around the crash scenes may act as a barrier for proper and on - time pre - hospital interventions . one of the participants ( p2 ) stated : in many situations , when we arrive in the crash location , people crowding around the scene area barrier for timely interventions . a. 2- inadequate knowledge on first aids : usually , people directly perform interventions that may worsen the problems of the injured person . the cause of these inappropriate actions is usually inadequate knowledge about first aids interventions . as an example always people do not have enough skills for proper interventions in the accident scene and this problem may worsen the severity of injuries . a. 3- mistake calls : in many situations , people call the number of emergency medical system ( ems ) , instead of other organizations such as firefighter and the other one of the participants ( p3 ) noted : we have about 10000 calls , daily , in tehran that one half of them are mistake or irrelevant . b. barriers related to metropolitan infrastructure : b. 1- traffic : traffic jam is a serious problem and barrier for prompt interventions by pre - hospital service providers . one of the participants ( p11 ) stated : many of our people have not acquired acceptable traffic culture and knowledge and this problem is a barrier for on time arrival to the scene and transporting the injured victim to hospital . b. 2- accessibility to streets and alleys : problem with accessibility is one of the barriers mentioned by the participants . one ( p6 ) stated : in many situations , because the streets and alleys are too narrow or too crowded , we have to stop and leave the ambulance faraway from the crush location . this acts as one cause of delay and this is especially noticeable and problematic during transport of the injured victim to ambulance . b. 3- naming of alleys : in many locations , there is not a logical and systematic approach for naming the pathways . one participant ( p1 ) expressed that : non - systematic approach for naming of alleys causes problems and makes it complicated for pre - hospital service providers to find a location . for example , many alleys are named using numbers followed by an alley named using a special noun . c. barriers related to profession : c. 1- professional autonomy : participants frequently mentioned inadequate professional autonomy . one participant ( p5 ) announced that : the pre - hospital system does not consider our professional capacities , knowledge and experiences . when i consult with the physician in our center , the only order is to transport the patient to the hospital . c. 2- workload : based on the participants accounts , workload may act as a barrier for service providing . specially , this is considerable about payment system . according to participants , salaries do not suit their workload . one said ( p13 ) : in this part of the city , we have a high number of accidents and traffic injuries , but this is not considered by administrators of pre - hospital system . in fact , there is no difference between a high workload center and other centers . c. 3- work - related injuries : many injuries may occur during care giving by pre - hospital service providers . when we transport an injured victim to ambulance , injuries such as low back injuries and other problems may occur . these problems may affect the paramedics quality of life and even at the end , lead to losing the job . d. barriers related to managerial issues : d. 1- inadequate telecommunication technology : improper and inadequate telecommunication technology was one of the problems often announced by the participants . one ( p2 ) stated : in many situations , when our ambulance can not go so closed to collision scene , we have to communicate to our dispatch center using mobile phones or landline telephone . in these situations , d. 2- inadequate human resources : one of the barriers of delivering proper pre - hospital services in viewpoints of care providers was inadequate number of care providers . in addition , the absence of physician in ambulances was another problem implied by many participants . one of the participants ( p18 ) said : when i am bedside an injured woman , because of cultural and religious considerations , there are many limitations to give care to her . in these situations , having a woman technician in the ambulance may decrease such problems and promote the quality of care providing . d. 3- inappropriate workload related privilege : this problem was another issue noticed , by the participants . a participant ( p12 ) announced : this problem may decrease our motivation and the quality of pre - hospital care . in fact , the level of rewards in high and low workload centers is equal and this is one of the weaknesses of our pre - hospital care system . d. 4- lack of organizational coordination : problems on inter- and intra - organizational coordination are are issue mentioned by the participants . one participant ( p4 ) said : involvement of other service providers such as firefighters or police in the accident location can be disturbing as they may sometimes interfere with our activities . i think this problem is related to the lack of coordination between the involved organizations . this study was an attempt to identify the barriers of pre - hospital caregiving classified into four main categories : barriers related to people , to metropolitan infrastructure , to the profession and to managerial problems . they argued that administrational and organizational factors , also staff qualifications and competences , availability and distribution of resources , communication and transport , involved organizations and laypeople could act as barriers in the chain of care from crush incident to the hospital . causes such as , administration , communication and transportation , involved organizations ( named as lack of organizational coordination ) , and laypeople involvement , that have been brought up by haghparast et al . are confirmed in the current study . moreover , findings of the present study are in the same line with khorasani et al . laypeople involvement , lack of coordination , inadequate pre - hospital services , and shortcomings in infrastructure are found as barriers of pre - hospital service in road traffic injuries both by the current study and in that of khorasani et al . in the study of big deli et al . also traffic , crowding of laypeople in the scene of the crush and involvement of other organizations have been mentioned as factors that interfere with interventions of pre - hospital care providers in traffic accidents . one category of barriers identified by the present study is barriers related to people classified into three subcategories : laypeople involvement , inadequate knowledge about first aids , and mistake calls . based on this study findings , one aspect that differentiates pre - hospital care in road traffic injuries from other fields of pre - hospital care giving is crowding of people in the scene of the crush . based on the findings of the current study , this problem not only prevents prompt care provision to the injured victims , but also it may worsen the condition of the injured victim because of inappropriate interventions delivered by people in the scene of accident . another result of our study was that although laypeople involvement has been considered as a barrier , according to many participants , if care providers manage the scene , laypeople could be turned into a capacity to accelerate and facilitate pre - hospital interventions . findings presented by a few studies such as that of tannvik et al . have emphasized this matter . inadequate information and knowledge about emergency agencies and wrong calls were also combined in one category named the barrier related to people . knowledge providing and distribution of information on first aids and pre - hospital service processes among people may act as a factor to reduce their improper interventions in the scene of the crush . another category of barriers identified by this study is those of metropolitan infrastructure including traffic , accessibility of alleys , and naming of alleys and streets . in fact , this category of barriers , especially traffic , has a remarkable confounding role in pre - hospital service delivery . traffic is a barrier that has been highlighted in both this study and several other studies . to resolve these problems , there is a need for comprehensive assessment of the pre - hospital system and the ways other organizations ( for example firefighters , police , and so on ) may involve and collaborate in this system . our results also show that there are some profession - related issues that may act as barriers for pre - hospital care delivery . several previous studies have considered workload and work related injuries as barriers for care providing in the pre - hospital situations . however , professional autonomy is a concept that in the current study was emphasized by many of care providers ( paramedics ) as a factor that can decrease their motivation . in addition , use of woman paramedics in ambulance crew is an aspect of barriers that has not been mentioned by other similar studies . based on our research findings , it can be inferred that issues such as workload and work related injuries might strongly affect the circumstances and the manner of actions done by care providers . further , these factors can diminish the staff s motivation , as mentioned by most participants . lack of motivation , in turn , may act as a reason for worsening the quality of services . some issues that my decrease the motivation of care providers and quality of care and noted by participants were payments that do not correspond to workload , inadequate number of colleagues , and the rigidity of the physician orders . results of the present study showed that there are problems in the field of management . these factors were classified as barriers related to managerial problems , including inadequate telecommunication technology , inadequate human resources , inadequate workload based privilege , and lack of organizational coordination . study of nielsen et al . also showed that one of the problems in low and middle income countries was managerial issues . one of the aspects that can be mentioned as a barrier and was emphasized by participants was lack of women in ambulance crew . studies show in advanced countries , women are in arrangement of paramedics . another issue mentioned as barrier by participants was lack of physician in ambulance assignments . studies show that pre - hospital systems in high - income countries use physicians in ambulance crew . for example , when interviewer was in the pre - hospital context to interview with paramedics , they had to go for emergency assignment and this was a factor that interrupted the interview process . in these conditions interviewer had to continue the interview in the ambulance during the assignment or after returning the participants from their assignment . based on conditions , many interviews performed in ambulance crew and traffic and noises were factors that interfered with recording of interviews . this caused the interviewer to plan for another session of interview with the participant or recheck the previous interview with them . this study found that not only laypeople s involvement could not be seen merely as barrier but also it can be apotential in some situations and contexts , if care providers manage the scene of the crash . based on these findings , a fundamental change in management system may lessen the problems and solve the barriers like metropolitan infrastructure and managerial issues . according to our knowledge , some barriers such as laypeople involvement , inadequate knowledge about first aids and wrong calls can be reduced through public education , information campaigns and changing attitudes . because of socioeconomic and cultural context , future studies are suggested to explore issues related to laypeople s involvement and their interventions in the scene of collision , because based on the study findings , laypeople s involvement can not be seen merely as a barrier . in addition , future studies are suggested to assess the effect of women ( as care providers)and physicians in ambulance crew on quality of pre - hospital service provided specially for injured victims . considering the fact that the process of providing pre - hospital service in traffic accidents is not well established other qualitative approaches such as grounded theory method is recommended for further studies . findings of this study contribute to improvement of the pre - hospital service system and can be used by health policy makers . for example , laypeople education , improvement of organizational coordination , use of strategies to expand the professional autonomy of paramedics , and application of practicable strategies can increase the motivation of pre - hospital care providers . in addition , use of new and appropriate telecommunication technologies and recruitment of more women and physicians as the ambulance crew may be a part of their future programs to promote and improve the quality of pre - hospital care .

background : iran is one of the countries with considerable road traffic injuries . pre - hospital interventions have an important role in preventing mortalities and disabilities caused by traffic accidents . the present study aimed to explore the barriers of pre - hospital care in traffic injuries in tehran , iran.methods:a qualitative content analysis approach was conducted based on 21 semi - structured interviews with 18 participants . a purposeful sampling method was applied until reaching data saturation . interviews were transcribed verbatim , and then data condensing , labeling , coding and defining categories were performed by qualitative content analysis.results:four main barriers including 4 main categories and 13 subcategories emerged ; they included barriers related to people , barriers related to metropolitan infrastructure , barriers related to the profession and barriers related to managerial issues.conclusion:based on the findings of this study , pre - hospital service barriers in traffic accidents have many dimensions including cultural , structural and managerial domains . policy makers in health system can use these findings to promote the quality of pre - hospital services , especially in the field of traffic injuries .

I M Data Collection Participants Data Analysis Trustworthiness Ethical Considerations R D C

based on the report of world health organization ( who ) , road traffic injuries ( rtis ) stand for 25% of injury - related mortalities . traffic injuries constitute the largest proportion of unintentional injury deaths ( 33% ) in the world . road traffic injuries are the ninth leading cause of all deaths and leading contributor to the worldwide burden of disease . yearly traffic collisions cause 1.2 million deaths and 20 to 50 million disabilities in the world . who estimates that without proper measures , the incidence of traffic mortalities rise by 67% and in 2020 it will be the third cause of death among 5 to 45 year old people . low and middle - income countries have 85 percent of traffic mortalities in the world . only 14% of traffic mortalities occur in highly motorized countries ( hmc ) such as north america , australia , newzeland , japan and west europe although these countries have comprised about 60% of the world vehicles . previous studies showed that death caused by traffic injuries has been increased in africa , eastern mediterranean and asia in recent years . in addition , the rate of traffic mortality to whole mortality is 2.5 in the world and 1.9 in the region ( eastern mediterranean)while this ratio in iran is about 7.5 . while having one percent of the world population , one fortieth of road traffic accidents occur in iran . studies have indicated that iran has too many road traffic injuries ; for example , iran with rti fatality rate of 31 . 8 per 100 000 in the year 2007 had the highest rate in the region of eastern mediterranean . a report by who showed that despite technological development , it is not clear why mortality rate in traffic injuries has a progressive trend in many countries . although new traffic laws had effects on reduction of fatal and non - fatal rates of road traffic injuries in iran , injuries from road traffic incidents have been increased in recent years . comparative studies show that an important portion of traffic mortalities occur in post - crash phase ( after crash , until transferring the injured victim to hospital ) in low and middle income countries . one noticeable part of these mortalities are preventable by proper pre - hospital interventions . one of the most important roles of pre - hospital services is life support and prevention of future disabilities in the post - crash phase . triage of an injured patient to an appropriate trauma center can have an impact on morbidity and mortality . studying the process and structure of pre - hospital service system provides knowledge and insight for assessment of these types of services . despite the importance of pre - hospital service in preventing mortalities and disabilities caused by road traffic injuries , there is lack of knowledge in these fields , especially cultural and contextual aspects . since the function of pre - hospital service is culture and context bound , qualitative approach was an appropriate method to design the study . also , since process of pre - hospital services is a phenomenon that consists human relationships , action - interactions , and emotions that are usually hidden and could not be assessed by quantitative approaches , qualitative method was selected as an approach . by qualitative approaches , we can observe phenomenon from the eyes of the participants , and the hidden aspects of phenomenon can be explored . determining the barriers of pre - hospital service in viewpoints of participants was another reason for selecting the qualitative approach . the aim of this study was to explore the barriers of pre - hospital service in traffic injuries in tehran , iran . in content analysis , concepts are gained directly and inductively from the raw data . it is a method with the purpose of providing knowledge , new understanding , and representation of realities . the aim of content analysis is to attain a summarized and comprehensive description of the phenomenon . in this method , another advantage of this method is the possibility of observation of the phenomenon , as viewed by the participants . first , interviewer proposed some general questions to start the interviews such as please explain one of your assignments . then , based on the participants responses and trend of the interview , next questions were arranged . the time and location of the interviews interviews were recorded in the form of audio files ( mp3 ) and transcribed verbatim . the se notes consisted of the type of crushing , the way first aids were provided to the injured victim , the person who provides aids to the injured person , the way the crowded people in scene of crush interfere with care giving process , and the way the action of other organizations interferes with interventions of pre - hospital service providers . in addition , one session of focus group for enrichment of data was performed with pre - hospital managers and paramedics . eighteen participants were selected by purposive sampling and that those with at least 2 years experience in the field of pre - hospital services were considered to be enrolled in the study . the participants consisted of 14 paramedics , 1 dispatch operator , 2 pre - hospital service managers and 1 police officer . for analyzing the data at first , the researcher read the texts several times ( intensive reading ) for getting a complete understanding of the respondents accounts ; then , the meaning units of phrases was identified and summarized . finally , after coding , based on common properties or dimensions , similar codes were classified in higher - level categories with a new and high abstract label ( table 1 ) . data collection continued until data saturation , meaning that adding further data does not offer any new information and new properties for each category . to insure the credibility of data , several strategies were used including : a ) prolonged engagement ( data gathering lasted about one year and during this period , researchers were oriented to culture , language and environment of the participants and atmosphere of the field ) , b ) peer review ( data and interpretation of data were checked by other researchers ) , and c ) member checking ( data rechecked by participants and our interpretations from data were reviewed and confirmed by them ) . in addition , one abstract of the results of the study was mailed for participants interested in the findings . data was gathered through semi - structured interviews . first , interviewer proposed some general questions to start the interviews such as please explain one of your assignments . then , based on the participants responses and trend of the interview , next questions were arranged . the time and location of the interviews interviews were recorded in the form of audio files ( mp3 ) and transcribed verbatim . the se notes consisted of the type of crushing , the way first aids were provided to the injured victim , the person who provides aids to the injured person , the way the crowded people in scene of crush interfere with care giving process , and the way the action of other organizations interferes with interventions of pre - hospital service providers . in addition , one session of focus group for enrichment of data was performed with pre - hospital managers and paramedics . eighteen participants were selected by purposive sampling and that those with at least 2 years experience in the field of pre - hospital services were considered to be enrolled in the study . the participants consisted of 14 paramedics , 1 dispatch operator , 2 pre - hospital service managers and 1 police officer . for analyzing the data at first , the researcher read the texts several times ( intensive reading ) for getting a complete understanding of the respondents accounts ; then , the meaning units of phrases was identified and summarized . finally , after coding , based on common properties or dimensions , similar codes were classified in higher - level categories with a new and high abstract label ( table 1 ) . data collection continued until data saturation , meaning that adding further data does not offer any new information and new properties for each category . to insure the credibility of data , several strategies were used including : a ) prolonged engagement ( data gathering lasted about one year and during this period , researchers were oriented to culture , language and environment of the participants and atmosphere of the field ) , b ) peer review ( data and interpretation of data were checked by other researchers ) , and c ) member checking ( data rechecked by participants and our interpretations from data were reviewed and confirmed by them ) . in addition , one abstract of the results of the study was mailed for participants interested in the findings . based on the data , 13 categories were developed and classified into four main categories ( table 2 ) . main categories were classified into four branches : categories and subcategories of the barriers to pre - hospital services in road trafficinjuries a.barriers related to people consisting of concepts ( subcategory ) that have at least one common property . this property included barriers that were in a way related to people such as laypeople involvement , inadequate knowledge about first aids and mistake calls.b.barriers related to metropolitan infrastructures . this category consisted of concepts whose common properties were problems related to characteristics and structure of the city such as traffic , accessibility to streets and naming of the alleys.c.barriers related to profession . in this main category , there were concepts that explain barriers related to pre - hospital service providing profession . in this category , there are three subcategories : professional autonomy , workload and work related injuries.d.the fourth main category named barriers related to managerial issues consisting of four concepts that were common in property of managerial problems in the field of pre - hospital service . in the next section , you can see categorization of main categories and related quotations : barriers related to people consisting of concepts ( subcategory ) that have at least one common property . this property included barriers that were in a way related to people such as laypeople involvement , inadequate knowledge about first aids and mistake calls . this category consisted of concepts whose common properties were problems related to characteristics and structure of the city such as traffic , accessibility to streets and naming of the alleys . barriers related to profession . in this main category , there were concepts that explain barriers related to pre - hospital service providing profession . in this category , there are three subcategories : professional autonomy , workload and work related injuries . the fourth main category named barriers related to managerial issues consisting of four concepts that were common in property of managerial problems in the field of pre - hospital service . in the next section , you can see categorization of main categories and related quotations : a. barriers related to people : a. 1 laypeople -involvement : crowded area in the crash scenes is a problem that was brought up by the participants . overcrowding around the crash scenes may act as a barrier for proper and on - time pre - hospital interventions . one of the participants ( p2 ) stated : in many situations , when we arrive in the crash location , people crowding around the scene area barrier for timely interventions . 2- inadequate knowledge on first aids : usually , people directly perform interventions that may worsen the problems of the injured person . as an example always people do not have enough skills for proper interventions in the accident scene and this problem may worsen the severity of injuries . 3- mistake calls : in many situations , people call the number of emergency medical system ( ems ) , instead of other organizations such as firefighter and the other one of the participants ( p3 ) noted : we have about 10000 calls , daily , in tehran that one half of them are mistake or irrelevant . b. barriers related to metropolitan infrastructure : b. 1- traffic : traffic jam is a serious problem and barrier for prompt interventions by pre - hospital service providers . one of the participants ( p11 ) stated : many of our people have not acquired acceptable traffic culture and knowledge and this problem is a barrier for on time arrival to the scene and transporting the injured victim to hospital . 2- accessibility to streets and alleys : problem with accessibility is one of the barriers mentioned by the participants . this acts as one cause of delay and this is especially noticeable and problematic during transport of the injured victim to ambulance . one participant ( p1 ) expressed that : non - systematic approach for naming of alleys causes problems and makes it complicated for pre - hospital service providers to find a location . c. barriers related to profession : c. 1- professional autonomy : participants frequently mentioned inadequate professional autonomy . one participant ( p5 ) announced that : the pre - hospital system does not consider our professional capacities , knowledge and experiences . when i consult with the physician in our center , the only order is to transport the patient to the hospital . c. 2- workload : based on the participants accounts , workload may act as a barrier for service providing . one said ( p13 ) : in this part of the city , we have a high number of accidents and traffic injuries , but this is not considered by administrators of pre - hospital system . c. 3- work - related injuries : many injuries may occur during care giving by pre - hospital service providers . these problems may affect the paramedics quality of life and even at the end , lead to losing the job . d. barriers related to managerial issues : d. 1- inadequate telecommunication technology : improper and inadequate telecommunication technology was one of the problems often announced by the participants . one ( p2 ) stated : in many situations , when our ambulance can not go so closed to collision scene , we have to communicate to our dispatch center using mobile phones or landline telephone . in these situations , d. 2- inadequate human resources : one of the barriers of delivering proper pre - hospital services in viewpoints of care providers was inadequate number of care providers . one of the participants ( p18 ) said : when i am bedside an injured woman , because of cultural and religious considerations , there are many limitations to give care to her . in these situations , having a woman technician in the ambulance may decrease such problems and promote the quality of care providing . a participant ( p12 ) announced : this problem may decrease our motivation and the quality of pre - hospital care . in fact , the level of rewards in high and low workload centers is equal and this is one of the weaknesses of our pre - hospital care system . one participant ( p4 ) said : involvement of other service providers such as firefighters or police in the accident location can be disturbing as they may sometimes interfere with our activities . i think this problem is related to the lack of coordination between the involved organizations . this study was an attempt to identify the barriers of pre - hospital caregiving classified into four main categories : barriers related to people , to metropolitan infrastructure , to the profession and to managerial problems . they argued that administrational and organizational factors , also staff qualifications and competences , availability and distribution of resources , communication and transport , involved organizations and laypeople could act as barriers in the chain of care from crush incident to the hospital . causes such as , administration , communication and transportation , involved organizations ( named as lack of organizational coordination ) , and laypeople involvement , that have been brought up by haghparast et al . are confirmed in the current study . moreover , findings of the present study are in the same line with khorasani et al . laypeople involvement , lack of coordination , inadequate pre - hospital services , and shortcomings in infrastructure are found as barriers of pre - hospital service in road traffic injuries both by the current study and in that of khorasani et al . in the study of big deli et al . also traffic , crowding of laypeople in the scene of the crush and involvement of other organizations have been mentioned as factors that interfere with interventions of pre - hospital care providers in traffic accidents . one category of barriers identified by the present study is barriers related to people classified into three subcategories : laypeople involvement , inadequate knowledge about first aids , and mistake calls . based on this study findings , one aspect that differentiates pre - hospital care in road traffic injuries from other fields of pre - hospital care giving is crowding of people in the scene of the crush . based on the findings of the current study , this problem not only prevents prompt care provision to the injured victims , but also it may worsen the condition of the injured victim because of inappropriate interventions delivered by people in the scene of accident . another result of our study was that although laypeople involvement has been considered as a barrier , according to many participants , if care providers manage the scene , laypeople could be turned into a capacity to accelerate and facilitate pre - hospital interventions . inadequate information and knowledge about emergency agencies and wrong calls were also combined in one category named the barrier related to people . knowledge providing and distribution of information on first aids and pre - hospital service processes among people may act as a factor to reduce their improper interventions in the scene of the crush . another category of barriers identified by this study is those of metropolitan infrastructure including traffic , accessibility of alleys , and naming of alleys and streets . in fact , this category of barriers , especially traffic , has a remarkable confounding role in pre - hospital service delivery . traffic is a barrier that has been highlighted in both this study and several other studies . to resolve these problems , there is a need for comprehensive assessment of the pre - hospital system and the ways other organizations ( for example firefighters , police , and so on ) may involve and collaborate in this system . our results also show that there are some profession - related issues that may act as barriers for pre - hospital care delivery . several previous studies have considered workload and work related injuries as barriers for care providing in the pre - hospital situations . however , professional autonomy is a concept that in the current study was emphasized by many of care providers ( paramedics ) as a factor that can decrease their motivation . based on our research findings , it can be inferred that issues such as workload and work related injuries might strongly affect the circumstances and the manner of actions done by care providers . lack of motivation , in turn , may act as a reason for worsening the quality of services . some issues that my decrease the motivation of care providers and quality of care and noted by participants were payments that do not correspond to workload , inadequate number of colleagues , and the rigidity of the physician orders . results of the present study showed that there are problems in the field of management . these factors were classified as barriers related to managerial problems , including inadequate telecommunication technology , inadequate human resources , inadequate workload based privilege , and lack of organizational coordination . also showed that one of the problems in low and middle income countries was managerial issues . one of the aspects that can be mentioned as a barrier and was emphasized by participants was lack of women in ambulance crew . studies show that pre - hospital systems in high - income countries use physicians in ambulance crew . for example , when interviewer was in the pre - hospital context to interview with paramedics , they had to go for emergency assignment and this was a factor that interrupted the interview process . in these conditions interviewer had to continue the interview in the ambulance during the assignment or after returning the participants from their assignment . based on conditions , many interviews performed in ambulance crew and traffic and noises were factors that interfered with recording of interviews . this study found that not only laypeople s involvement could not be seen merely as barrier but also it can be apotential in some situations and contexts , if care providers manage the scene of the crash . based on these findings , a fundamental change in management system may lessen the problems and solve the barriers like metropolitan infrastructure and managerial issues . because of socioeconomic and cultural context , future studies are suggested to explore issues related to laypeople s involvement and their interventions in the scene of collision , because based on the study findings , laypeople s involvement can not be seen merely as a barrier . in addition , future studies are suggested to assess the effect of women ( as care providers)and physicians in ambulance crew on quality of pre - hospital service provided specially for injured victims . considering the fact that the process of providing pre - hospital service in traffic accidents is not well established other qualitative approaches such as grounded theory method is recommended for further studies . findings of this study contribute to improvement of the pre - hospital service system and can be used by health policy makers . for example , laypeople education , improvement of organizational coordination , use of strategies to expand the professional autonomy of paramedics , and application of practicable strategies can increase the motivation of pre - hospital care providers . in addition , use of new and appropriate telecommunication technologies and recruitment of more women and physicians as the ambulance crew may be a part of their future programs to promote and improve the quality of pre - hospital care .

based on the report of world health organization ( who ) , road traffic injuries ( rtis ) stand for 25% of injury - related mortalities . traffic injuries constitute the largest proportion of unintentional injury deaths ( 33% ) in the world . who estimates that without proper measures , the incidence of traffic mortalities rise by 67% and in 2020 it will be the third cause of death among 5 to 45 year old people . low and middle - income countries have 85 percent of traffic mortalities in the world . only 14% of traffic mortalities occur in highly motorized countries ( hmc ) such as north america , australia , newzeland , japan and west europe although these countries have comprised about 60% of the world vehicles . previous studies showed that death caused by traffic injuries has been increased in africa , eastern mediterranean and asia in recent years . in addition , the rate of traffic mortality to whole mortality is 2.5 in the world and 1.9 in the region ( eastern mediterranean)while this ratio in iran is about 7.5 . while having one percent of the world population , one fortieth of road traffic accidents occur in iran . studies have indicated that iran has too many road traffic injuries ; for example , iran with rti fatality rate of 31 . 8 per 100 000 in the year 2007 had the highest rate in the region of eastern mediterranean . in iran , there are about one vehicle for every four individuals ; in comparison , this ratio is higher than that of eastern mediterranean countries but lower than those of industrialized and developed countries . moreover , rtis stand for the most prevalent cause of injury and the second leading cause of death in iran . a report by who showed that despite technological development , it is not clear why mortality rate in traffic injuries has a progressive trend in many countries . although new traffic laws had effects on reduction of fatal and non - fatal rates of road traffic injuries in iran , injuries from road traffic incidents have been increased in recent years . comparative studies show that an important portion of traffic mortalities occur in post - crash phase ( after crash , until transferring the injured victim to hospital ) in low and middle income countries . one of the most important roles of pre - hospital services is life support and prevention of future disabilities in the post - crash phase . studying the process and structure of pre - hospital service system provides knowledge and insight for assessment of these types of services . despite the importance of pre - hospital service in preventing mortalities and disabilities caused by road traffic injuries , there is lack of knowledge in these fields , especially cultural and contextual aspects . since the function of pre - hospital service is culture and context bound , qualitative approach was an appropriate method to design the study . also , since process of pre - hospital services is a phenomenon that consists human relationships , action - interactions , and emotions that are usually hidden and could not be assessed by quantitative approaches , qualitative method was selected as an approach . by qualitative approaches , we can observe phenomenon from the eyes of the participants , and the hidden aspects of phenomenon can be explored . determining the barriers of pre - hospital service in viewpoints of participants was another reason for selecting the qualitative approach . the aim of this study was to explore the barriers of pre - hospital service in traffic injuries in tehran , iran . in content analysis , concepts are gained directly and inductively from the raw data . it is a method with the purpose of providing knowledge , new understanding , and representation of realities . the aim of content analysis is to attain a summarized and comprehensive description of the phenomenon . in this method , another advantage of this method is the possibility of observation of the phenomenon , as viewed by the participants . first , interviewer proposed some general questions to start the interviews such as please explain one of your assignments . then , based on the participants responses and trend of the interview , next questions were arranged . the time and location of the interviews interviews were recorded in the form of audio files ( mp3 ) and transcribed verbatim . in addition , researchers used other methods of data gathering such as field notes , focus groups and observation . for data gathering by observation , the researcher observed the scenes of crushing and recorded the observed events ( field note ) . the se notes consisted of the type of crushing , the way first aids were provided to the injured victim , the person who provides aids to the injured person , the way the crowded people in scene of crush interfere with care giving process , and the way the action of other organizations interferes with interventions of pre - hospital service providers . in addition , one session of focus group for enrichment of data was performed with pre - hospital managers and paramedics . eighteen participants were selected by purposive sampling and that those with at least 2 years experience in the field of pre - hospital services were considered to be enrolled in the study . the participants consisted of 14 paramedics , 1 dispatch operator , 2 pre - hospital service managers and 1 police officer . for analyzing the data at first , the researcher read the texts several times ( intensive reading ) for getting a complete understanding of the respondents accounts ; then , the meaning units of phrases was identified and summarized . finally , after coding , based on common properties or dimensions , similar codes were classified in higher - level categories with a new and high abstract label ( table 1 ) . example of meaning units , condensed meaning units , codes , subcategories and categories for examining the rigor of data and results , guba and lincoln strategies were used . to insure the credibility of data , several strategies were used including : a ) prolonged engagement ( data gathering lasted about one year and during this period , researchers were oriented to culture , language and environment of the participants and atmosphere of the field ) , b ) peer review ( data and interpretation of data were checked by other researchers ) , and c ) member checking ( data rechecked by participants and our interpretations from data were reviewed and confirmed by them ) . in addition , to insure the dependability of data , triangulation strategy for data collection such as interview , observation , focus group and field notes were applied . by this strategy , they were also informed about their authority for withdrawal from the study at any stage of study . in addition , one abstract of the results of the study was mailed for participants interested in the findings . first , interviewer proposed some general questions to start the interviews such as please explain one of your assignments . then , based on the participants responses and trend of the interview , next questions were arranged . the time and location of the interviews interviews were recorded in the form of audio files ( mp3 ) and transcribed verbatim . in addition , researchers used other methods of data gathering such as field notes , focus groups and observation . for data gathering by observation , the researcher observed the scenes of crushing and recorded the observed events ( field note ) . the se notes consisted of the type of crushing , the way first aids were provided to the injured victim , the person who provides aids to the injured person , the way the crowded people in scene of crush interfere with care giving process , and the way the action of other organizations interferes with interventions of pre - hospital service providers . in addition , one session of focus group for enrichment of data was performed with pre - hospital managers and paramedics . eighteen participants were selected by purposive sampling and that those with at least 2 years experience in the field of pre - hospital services were considered to be enrolled in the study . the participants consisted of 14 paramedics , 1 dispatch operator , 2 pre - hospital service managers and 1 police officer . for analyzing the data at first , the researcher read the texts several times ( intensive reading ) for getting a complete understanding of the respondents accounts ; then , the meaning units of phrases was identified and summarized . finally , after coding , based on common properties or dimensions , similar codes were classified in higher - level categories with a new and high abstract label ( table 1 ) . for examining the rigor of data and results , guba and lincoln strategies were used . to insure the credibility of data , several strategies were used including : a ) prolonged engagement ( data gathering lasted about one year and during this period , researchers were oriented to culture , language and environment of the participants and atmosphere of the field ) , b ) peer review ( data and interpretation of data were checked by other researchers ) , and c ) member checking ( data rechecked by participants and our interpretations from data were reviewed and confirmed by them ) . in addition , to insure the dependability of data , triangulation strategy for data collection such as interview , observation , focus group and field notes were applied . by this strategy , data can be collected from many aspects and dependability of data will be increased . in addition , one abstract of the results of the study was mailed for participants interested in the findings . based on the data , 13 categories were developed and classified into four main categories ( table 2 ) . main categories were classified into four branches : categories and subcategories of the barriers to pre - hospital services in road trafficinjuries a.barriers related to people consisting of concepts ( subcategory ) that have at least one common property . this property included barriers that were in a way related to people such as laypeople involvement , inadequate knowledge about first aids and mistake calls.b.barriers related to metropolitan infrastructures . this category consisted of concepts whose common properties were problems related to characteristics and structure of the city such as traffic , accessibility to streets and naming of the alleys.c.barriers related to profession . in this main category , there were concepts that explain barriers related to pre - hospital service providing profession . in this category , there are three subcategories : professional autonomy , workload and work related injuries.d.the fourth main category named barriers related to managerial issues consisting of four concepts that were common in property of managerial problems in the field of pre - hospital service . in the next section , you can see categorization of main categories and related quotations : barriers related to people consisting of concepts ( subcategory ) that have at least one common property . this property included barriers that were in a way related to people such as laypeople involvement , inadequate knowledge about first aids and mistake calls . this category consisted of concepts whose common properties were problems related to characteristics and structure of the city such as traffic , accessibility to streets and naming of the alleys . in this main category , there were concepts that explain barriers related to pre - hospital service providing profession . in this category , there are three subcategories : professional autonomy , workload and work related injuries . the fourth main category named barriers related to managerial issues consisting of four concepts that were common in property of managerial problems in the field of pre - hospital service . in the next section , you can see categorization of main categories and related quotations : a. barriers related to people : a. 1 laypeople -involvement : crowded area in the crash scenes is a problem that was brought up by the participants . overcrowding around the crash scenes may act as a barrier for proper and on - time pre - hospital interventions . one of the participants ( p2 ) stated : in many situations , when we arrive in the crash location , people crowding around the scene area barrier for timely interventions . 2- inadequate knowledge on first aids : usually , people directly perform interventions that may worsen the problems of the injured person . as an example always people do not have enough skills for proper interventions in the accident scene and this problem may worsen the severity of injuries . 3- mistake calls : in many situations , people call the number of emergency medical system ( ems ) , instead of other organizations such as firefighter and the other one of the participants ( p3 ) noted : we have about 10000 calls , daily , in tehran that one half of them are mistake or irrelevant . 1- traffic : traffic jam is a serious problem and barrier for prompt interventions by pre - hospital service providers . one of the participants ( p11 ) stated : many of our people have not acquired acceptable traffic culture and knowledge and this problem is a barrier for on time arrival to the scene and transporting the injured victim to hospital . 2- accessibility to streets and alleys : problem with accessibility is one of the barriers mentioned by the participants . one ( p6 ) stated : in many situations , because the streets and alleys are too narrow or too crowded , we have to stop and leave the ambulance faraway from the crush location . this acts as one cause of delay and this is especially noticeable and problematic during transport of the injured victim to ambulance . 3- naming of alleys : in many locations , there is not a logical and systematic approach for naming the pathways . one participant ( p1 ) expressed that : non - systematic approach for naming of alleys causes problems and makes it complicated for pre - hospital service providers to find a location . c. barriers related to profession : c. 1- professional autonomy : participants frequently mentioned inadequate professional autonomy . one participant ( p5 ) announced that : the pre - hospital system does not consider our professional capacities , knowledge and experiences . when i consult with the physician in our center , the only order is to transport the patient to the hospital . c. 2- workload : based on the participants accounts , workload may act as a barrier for service providing . one said ( p13 ) : in this part of the city , we have a high number of accidents and traffic injuries , but this is not considered by administrators of pre - hospital system . c. 3- work - related injuries : many injuries may occur during care giving by pre - hospital service providers . these problems may affect the paramedics quality of life and even at the end , lead to losing the job . d. barriers related to managerial issues : d. 1- inadequate telecommunication technology : improper and inadequate telecommunication technology was one of the problems often announced by the participants . in these situations , d. 2- inadequate human resources : one of the barriers of delivering proper pre - hospital services in viewpoints of care providers was inadequate number of care providers . one of the participants ( p18 ) said : when i am bedside an injured woman , because of cultural and religious considerations , there are many limitations to give care to her . a participant ( p12 ) announced : this problem may decrease our motivation and the quality of pre - hospital care . in fact , the level of rewards in high and low workload centers is equal and this is one of the weaknesses of our pre - hospital care system . one participant ( p4 ) said : involvement of other service providers such as firefighters or police in the accident location can be disturbing as they may sometimes interfere with our activities . this study was an attempt to identify the barriers of pre - hospital caregiving classified into four main categories : barriers related to people , to metropolitan infrastructure , to the profession and to managerial problems . they argued that administrational and organizational factors , also staff qualifications and competences , availability and distribution of resources , communication and transport , involved organizations and laypeople could act as barriers in the chain of care from crush incident to the hospital . causes such as , administration , communication and transportation , involved organizations ( named as lack of organizational coordination ) , and laypeople involvement , that have been brought up by haghparast et al . moreover , findings of the present study are in the same line with khorasani et al . laypeople involvement , lack of coordination , inadequate pre - hospital services , and shortcomings in infrastructure are found as barriers of pre - hospital service in road traffic injuries both by the current study and in that of khorasani et al . also traffic , crowding of laypeople in the scene of the crush and involvement of other organizations have been mentioned as factors that interfere with interventions of pre - hospital care providers in traffic accidents . one category of barriers identified by the present study is barriers related to people classified into three subcategories : laypeople involvement , inadequate knowledge about first aids , and mistake calls . based on this study findings , one aspect that differentiates pre - hospital care in road traffic injuries from other fields of pre - hospital care giving is crowding of people in the scene of the crush . based on the findings of the current study , this problem not only prevents prompt care provision to the injured victims , but also it may worsen the condition of the injured victim because of inappropriate interventions delivered by people in the scene of accident . another result of our study was that although laypeople involvement has been considered as a barrier , according to many participants , if care providers manage the scene , laypeople could be turned into a capacity to accelerate and facilitate pre - hospital interventions . inadequate information and knowledge about emergency agencies and wrong calls were also combined in one category named the barrier related to people . knowledge providing and distribution of information on first aids and pre - hospital service processes among people may act as a factor to reduce their improper interventions in the scene of the crush . another category of barriers identified by this study is those of metropolitan infrastructure including traffic , accessibility of alleys , and naming of alleys and streets . in fact , this category of barriers , especially traffic , has a remarkable confounding role in pre - hospital service delivery . to resolve these problems , there is a need for comprehensive assessment of the pre - hospital system and the ways other organizations ( for example firefighters , police , and so on ) may involve and collaborate in this system . our results also show that there are some profession - related issues that may act as barriers for pre - hospital care delivery . several previous studies have considered workload and work related injuries as barriers for care providing in the pre - hospital situations . however , professional autonomy is a concept that in the current study was emphasized by many of care providers ( paramedics ) as a factor that can decrease their motivation . based on our research findings , it can be inferred that issues such as workload and work related injuries might strongly affect the circumstances and the manner of actions done by care providers . some issues that my decrease the motivation of care providers and quality of care and noted by participants were payments that do not correspond to workload , inadequate number of colleagues , and the rigidity of the physician orders . results of the present study showed that there are problems in the field of management . one of the aspects that can be mentioned as a barrier and was emphasized by participants was lack of women in ambulance crew . studies show that pre - hospital systems in high - income countries use physicians in ambulance crew . for example , when interviewer was in the pre - hospital context to interview with paramedics , they had to go for emergency assignment and this was a factor that interrupted the interview process . this study found that not only laypeople s involvement could not be seen merely as barrier but also it can be apotential in some situations and contexts , if care providers manage the scene of the crash . based on these findings , a fundamental change in management system may lessen the problems and solve the barriers like metropolitan infrastructure and managerial issues . because of socioeconomic and cultural context , future studies are suggested to explore issues related to laypeople s involvement and their interventions in the scene of collision , because based on the study findings , laypeople s involvement can not be seen merely as a barrier . in addition , future studies are suggested to assess the effect of women ( as care providers)and physicians in ambulance crew on quality of pre - hospital service provided specially for injured victims . considering the fact that the process of providing pre - hospital service in traffic accidents is not well established other qualitative approaches such as grounded theory method is recommended for further studies . findings of this study contribute to improvement of the pre - hospital service system and can be used by health policy makers . for example , laypeople education , improvement of organizational coordination , use of strategies to expand the professional autonomy of paramedics , and application of practicable strategies can increase the motivation of pre - hospital care providers . in addition , use of new and appropriate telecommunication technologies and recruitment of more women and physicians as the ambulance crew may be a part of their future programs to promote and improve the quality of pre - hospital care .

esophageal cancer is one of the most common digestive malignancies with the 5-year relative survival rate less than 20% , ranking the fourth leading cause of cancer death among both men and women in china . like most solid tumors , pathological tumor node metastasis ( tnm ) stage is still a main prognostic indicator of esophageal cancer patient survival . however , the molecular heterogeneity and complexity of esophageal cancer make clinical outcomes difficult to predict and even patients within the same stage present wide variations in survival . therefore , it is urgent to develop novel biomarkers or models for survival risk prediction in esophageal cancer which would provide patients more effective therapies . long non - coding rnas ( lncrnas ) are mostly defined as rna transcripts exceeding 200 nucleotides ( nt ) in length apparently without protein coding capacity . accumulating evidence indicates that lncrnas play critical roles in a spectrum of biological processes via transcriptional , post - transcriptional and epigenetic mechanisms . dysregulation of lncrnas has been observed in various cancers , including breast cancer , colorectal cancer , lung cancer , prostate cancer as well as esophageal cancer [ 610 ] . it has been reported that dysregulated lncrnas are associated with cancer pathogenesis and function as oncogenic or tumor suppressive regulators in cancer development . they have been shown to affect cell proliferation , migration , and invasion through regulating the expression of genes involved in various tumorigenetic pathways . one of the recent techniques used for transcriptome analyses is rna sequencing ( rna - seq ) , a next - generation sequencing technique with high - sensitivity , high - throughput and the ability of detecting novel exons , splice sites , and transcripts . however , rna - seq based work focused on the prognostic power of lncrna signatures for survival risk of esophageal cancer patients is quite rare . in this study , we identified for the first time a rna - seq based lncrna signature as a predictor of survival risk of esophageal cancer patients using a cohort of more than 100 cases from the cancer genome atlas ( tcga ) database . cox regression analysis and risk score model method were utilized to develop an 8-lncrna signature which could distinguish patients with good and poor survival . a higher area under curve ( auc ) of the receiver operating characteristic ( roc ) curve confirmed good sensitivity and specificity of the prognostic model while multivariate cox regression analysis and stratified analysis indicated the independence of predictive capacity of the 8-lncrna prognostic signature from other clinicopathological factors . furthermore , our functional enrichment analysis suggested that the eight predictive lncrnas were probably involved in the progression of esophageal cancer through exerting their roles in esophageal cancer related biological processes and pathways , such as regulation of glucose metabolic process , positive regulation of mapk cascade , and amino acid and lipids metabolism . the preprocessed level 3 rna - seq data and corresponding clinical information of esophageal cancer patients were collected from the cancer genome atlas ( tcga ) database ( http://cancergenome.nih.gov/ ) ( as of september , 2016 ) . the patients meeting the following criteria were included in the study : ( 1 ) patients with complete information of lncrna expression profiles and clinical characteristics ( including age , gender , race , stage , survival status and survival time ) ( 2 ) the overall survival time was more than one month . according to the inclusion criteria , a total of 122 esophageal cancer patients were enrolled in the study ( table 1 ) . the gene expression profiling data of the 122 esophageal cancer samples and 11 normal samples were downloaded from the tcga database . the package of edger in r language was employed to identify the differentially expressed lncrnas between esophageal cancer and normal tissues with the |log2fc| > 2 and fdr < 0.01 set as the threshold . then the unsupervised hierarchical clustering was performed based on the expression of these altered lncrnas by the pheatmap package in r ( https://cran.r-project.org/web/packages/pheatmap/index.html , version 1.0.8 ) . the association between the expression of differentially expressed lncrnas and patient overall survival was evaluated by univariate cox proportional hazards regression analysis using the survival r package . only those lncrnas with p - value < 0.05 were considered as candidate variables and entered into a stepwise multivariate cox regression analysis tested by aic ( akaike information criterion , assessing the goodness of fit of a statistical model ) to identify the predictive model with the best explanatory and informative efficacy . then , a lncrna - related prognostic model was established to evaluate each patient s survival risk as follows : where k is the number of prognostic lncrnas , ci represents the coefficient of the ith lncrna in the multivariate cox regression analysis , vi is the expression value of the ith lncrna . the lncrnas with ci > 0 were defined as high - risk signatures while those with ci < 0 were defined as protective lncrnas . according to the predictive lncrna signature model the patients were then classified into high - risk or low - risk group using the median risk score as the cutoff value . overall survival curves were generated using the kaplan - meier method , and two - sided log - rank tests were employed to compare the differences in overall survival time between the high - risk and low - risk patient groups . the sensitivity and specificity of the lncrna prognostic model to predict clinical outcome were evaluated by calculating the area under curve ( auc ) of the receiver operating characteristic ( roc ) curve in the r package of survival roc . to determine whether predictive capacity of the lncrna signature was independent of other clinical factors ( including race , gender , stage , and age ) of esophageal cancer patients , multivariate cox regression analysis was carried out using overall survival as the dependent variable and lncrna signature and other conventional clinical factors as independent variables . for clinical features with p - value < 0.01 in cox regression analysis , stratification analysis was further performed to determine whether the lncrna signature exhibit prognostic value within the same clinical factor . to identify potential biological processes and pathways which the predictive lncrnas were involved in , first , the pearson correlation coefficients between the expression profiles of the eight prognostic lncrnas and protein - coding genes ( pcgs ) were calculated to determine the co - expression relationships of the lncrnas and pcgs . the pcgs with |pearson correlation coefficient| > 0.40 were considered to be lncrnas - related pcgs . gene ontology ( go ) biological process ( bp ) and kyoto encyclopedia of genes and genomes ( kegg ) pathway enrichment analysis were carried out for those pcgs using the database for annotation , visualization , and integrated discovery ( david , https://david.ncifcrf.gov/ , version 6.8 ) . the p - value < 0.05 was set as the cutoff criterion for both go and kegg functional analysis . the preprocessed level 3 rna - seq data and corresponding clinical information of esophageal cancer patients were collected from the cancer genome atlas ( tcga ) database ( http://cancergenome.nih.gov/ ) ( as of september , 2016 ) . the patients meeting the following criteria were included in the study : ( 1 ) patients with complete information of lncrna expression profiles and clinical characteristics ( including age , gender , race , stage , survival status and survival time ) ( 2 ) the overall survival time was more than one month . according to the inclusion criteria , a total of 122 esophageal cancer patients were enrolled in the study ( table 1 ) . the gene expression profiling data of the 122 esophageal cancer samples and 11 normal samples were downloaded from the tcga database . the package of edger in r language was employed to identify the differentially expressed lncrnas between esophageal cancer and normal tissues with the |log2fc| > 2 and fdr < 0.01 set as the threshold . then the unsupervised hierarchical clustering was performed based on the expression of these altered lncrnas by the pheatmap package in r ( https://cran.r-project.org/web/packages/pheatmap/index.html , version 1.0.8 ) . the association between the expression of differentially expressed lncrnas and patient overall survival was evaluated by univariate cox proportional hazards regression analysis using the survival r package . only those lncrnas with p - value < 0.05 were considered as candidate variables and entered into a stepwise multivariate cox regression analysis tested by aic ( akaike information criterion , assessing the goodness of fit of a statistical model ) to identify the predictive model with the best explanatory and informative efficacy . then , a lncrna - related prognostic model was established to evaluate each patient s survival risk as follows : where k is the number of prognostic lncrnas , ci represents the coefficient of the ith lncrna in the multivariate cox regression analysis , vi is the expression value of the ith lncrna . the lncrnas with ci > 0 were defined as high - risk signatures while those with ci < 0 were defined as protective lncrnas . according to the predictive lncrna signature model , the risk score of each of the 122 patients was calculated . the patients were then classified into high - risk or low - risk group using the median risk score as the cutoff value . overall survival curves were generated using the kaplan - meier method , and two - sided log - rank tests were employed to compare the differences in overall survival time between the high - risk and low - risk patient groups . the sensitivity and specificity of the lncrna prognostic model to predict clinical outcome were evaluated by calculating the area under curve ( auc ) of the receiver operating characteristic ( roc ) curve in the r package of survival roc . to determine whether predictive capacity of the lncrna signature was independent of other clinical factors ( including race , gender , stage , and age ) of esophageal cancer patients , multivariate cox regression analysis was carried out using overall survival as the dependent variable and lncrna signature and other conventional clinical factors as independent variables . for clinical features with p - value < 0.01 in cox regression analysis , stratification analysis was further performed to determine whether the lncrna signature exhibit prognostic value within the same clinical factor . to identify potential biological processes and pathways which the predictive lncrnas were involved in , functional enrichment analysis was performed . first , the pearson correlation coefficients between the expression profiles of the eight prognostic lncrnas and protein - coding genes ( pcgs ) were calculated to determine the co - expression relationships of the lncrnas and pcgs . the pcgs with |pearson correlation coefficient| > 0.40 were considered to be lncrnas - related pcgs . gene ontology ( go ) biological process ( bp ) and kyoto encyclopedia of genes and genomes ( kegg ) pathway enrichment analysis were carried out for those pcgs using the database for annotation , visualization , and integrated discovery ( david , https://david.ncifcrf.gov/ , version 6.8 ) . the p - value < 0.05 was set as the cutoff criterion for both go and kegg functional analysis . according to the cutoff criteria , a total of 265 differentially expressed ( including 112 upregulated and 153 downregulated ) lncrnas were identified between esophageal cancer tissues and normal tissues . the results of unsupervised hierarchical cluster analysis in figure 1 showed that the esophageal cancer samples could be clearly distinguished from the normal controls with the expression of differentially expressed lncrnas . to identify prognosis - related lncrnas , we first used univariate cox regression analysis to evaluate the associations between the expression level of each of the differentially expressed lncrnas and patients overall survival , and found that 13 lncrnas were significantly related to overall survival ( p<0.05 ) . then a stepwise multivariate cox regression analysis was performed and eight lncrnas therein ( as shown in table 2 ) were finally screened out to establish a predictive model . as previously described , the predictive model was defined as the linear combination of the expression levels of the eight lncrnas weighted by their relative coefficient in multivariate cox regression as follows : survival risk score = ( 0.0398 expression value of gs1 - 600g8.5 ) + ( 0.9990 expression value of linc00365 ) + ( 0.6216 expression value of ctd-2357a8.3 ) + ( 13.6225 expression value of rp11 - 705o24.1 ) + ( 1.7105 expression value of linc01554 ) + ( 0.8297 expression value of rp1 - 90j4.1 ) + ( 6.2336 expression value of rp11 - 327j17.1 ) + ( 1.2226 expression value of linc00176 ) . among these , gs1 - 600g8.5 , linc00365 , ctd-2357a8.3 , rp11 - 705o24.1 , linc01554 , and rp1 - 90j4.1 showed positive coefficients in cox regression analysis , indicating high - risk signatures for these six lncrnas since their high expression signified a shorter overall survival of patients . for the remaining two lncrnas , we observed negative coefficients in cox regression analysis , implying that these lncrnas could be regarded as protective lncrnas since patients with higher expression levels of these lncrnas tended to have longer overall survival compared with those with lower expression levels of these lncrnas . for each of the 122 patients in our study , we were able to calculate an 8-lncrna expression - based survival risk score ( referred to as srs ) and assigned them into a high - risk group or a low - risk group using the median risk score of 1.058 as the cutoff point . as a result , 61 patients were classified into the high - risk group since their srss were greater than the cutoff value , whereas the other 61 patients were assigned to the low - risk group with their srss less than the cutoff point ( figure 2a ) . the kaplan - meier overall survival curves of the two groups based on the eight lncrnas were notably different ( log - rank p=1.72 e-05 < 0.001 ) , showing overall survival in 9.89% and 50.5% at five years for patients with high - risk and low - risk srs , respectively ( figure 2b ) . the prognostic power of the 8-lncrna signature was evaluated by calculating the auc of roc curve . higher auc indicates better model performance and auc more than 0.80 is considered good performance . in our study , the roc curve analysis achieved auc of 0.845 , showing good sensitivity and specificity of the 8-lncrna signature model in predicting esophageal cancer patient survival risk ( figure 2c ) . multivariate cox regression analysis demonstrated that the 8-lncrna signature risk score maintained an independent predictive ability from other clinical factors ( hr=5.951 , 95% ci 2.57713.741 , p=2.95 e-05 , shown in table 3 ) . meanwhile , we also found that tnm stage was an independent predictor for overall survival of esophageal cancer patients . therefore , stratification analysis was further carried out to examine whether the 8-lncrna signature could provide predict value for patients within the same tnm stage . because the sample sizes in stage i and iv were too small to draw any reliable conclusions ( n=14 in stage i and n=6 in stage iv ) , stratification analysis was performed only in stage ii and iii patients . log - rank test for patients in stage ii demonstrated that the 8-lncrna signature could distinguish patients with significantly different survival ( p=0.015 , figure 3a ) . similar predictive value of the 8-lncrna signature was observed for stage iii patients ( p=0.003 , figure 3b ) . altogether , these results indicated that the prognostic capability of the 8-lncrna signature is independent of conventional clinical factors for survival prediction of esophageal cancer patients . we performed go and kegg functional enrichment analysis for the pcgs co - expressed with the lncrnas in the predictive signature to reveal the potential functions of the eight prognostic lncrnas . the results showed that co - expressed pcgs were enriched in 73 go bp terms , which mainly clustered in regulation of diverse biological processes ( such as go : 0010906~regulation of glucose metabolic process , go : 0010628~positive regulation of gene expression , go : 0043410~positive regulation of mapk cascade ) , transport of various substances ( including go : 0071805~potassium ion transmembrane transport , go : 0035879~plasma membrane lactate transport , go : 0098719~sodium ion import across plasma membrane ) and response to different stimulants ( such as go : 0042594~response to starvation , go : 0043627~response to estrogen , go : 0042493~response to drug ) ( the top 30 go bp terms were shown in table 4 ) . fourteen kegg pathways were enriched which mainly focused on digestive functions ( including hsa04971 : gastric acid secretion , hsa04974 : protein digestion and absorption , hsa04972 : pancreatic secretion ) and basic substance metabolism ( such as hsa01200 : carbon metabolism , hsa00280 : valine , leucine and isoleucine degradation , hsa01100 : metabolic pathways and hsa00071 : fatty acid degradation ) ( figure 4 ) . according to the cutoff criteria , a total of 265 differentially expressed ( including 112 upregulated and 153 downregulated ) lncrnas were identified between esophageal cancer tissues and normal tissues . the results of unsupervised hierarchical cluster analysis in figure 1 showed that the esophageal cancer samples could be clearly distinguished from the normal controls with the expression of differentially expressed lncrnas . to identify prognosis - related lncrnas , we first used univariate cox regression analysis to evaluate the associations between the expression level of each of the differentially expressed lncrnas and patients overall survival , and found that 13 lncrnas were significantly related to overall survival ( p<0.05 ) . then a stepwise multivariate cox regression analysis was performed and eight lncrnas therein ( as shown in table 2 ) were finally screened out to establish a predictive model . as previously described , the predictive model was defined as the linear combination of the expression levels of the eight lncrnas weighted by their relative coefficient in multivariate cox regression as follows : survival risk score = ( 0.0398 expression value of gs1 - 600g8.5 ) + ( 0.9990 expression value of linc00365 ) + ( 0.6216 expression value of ctd-2357a8.3 ) + ( 13.6225 expression value of rp11 - 705o24.1 ) + ( 1.7105 expression value of linc01554 ) + ( 0.8297 expression value of rp1 - 90j4.1 ) + ( 6.2336 expression value of rp11 - 327j17.1 ) + ( 1.2226 expression value of linc00176 ) . among these , gs1 - 600g8.5 , linc00365 , ctd-2357a8.3 , rp11 - 705o24.1 , linc01554 , and rp1 - 90j4.1 showed positive coefficients in cox regression analysis , indicating high - risk signatures for these six lncrnas since their high expression signified a shorter overall survival of patients . for the remaining two lncrnas , we observed negative coefficients in cox regression analysis , implying that these lncrnas could be regarded as protective lncrnas since patients with higher expression levels of these lncrnas tended to have longer overall survival compared with those with lower expression levels of these lncrnas . for each of the 122 patients in our study , we were able to calculate an 8-lncrna expression - based survival risk score ( referred to as srs ) and assigned them into a high - risk group or a low - risk group using the median risk score of 1.058 as the cutoff point . as a result , 61 patients were classified into the high - risk group since their srss were greater than the cutoff value , whereas the other 61 patients were assigned to the low - risk group with their srss less than the cutoff point ( figure 2a ) . the kaplan - meier overall survival curves of the two groups based on the eight lncrnas were notably different ( log - rank p=1.72 e-05 < 0.001 ) , showing overall survival in 9.89% and 50.5% at five years for patients with high - risk and low - risk srs , respectively ( figure 2b ) . the prognostic power of the 8-lncrna signature was evaluated by calculating the auc of roc curve . higher auc indicates better model performance and auc more than 0.80 is considered good performance . in our study , the roc curve analysis achieved auc of 0.845 , showing good sensitivity and specificity of the 8-lncrna signature model in predicting esophageal cancer patient survival risk ( figure 2c ) . multivariate cox regression analysis demonstrated that the 8-lncrna signature risk score maintained an independent predictive ability from other clinical factors ( hr=5.951 , 95% ci 2.57713.741 , p=2.95 e-05 , shown in table 3 ) . meanwhile , we also found that tnm stage was an independent predictor for overall survival of esophageal cancer patients . therefore , stratification analysis was further carried out to examine whether the 8-lncrna signature could provide predict value for patients within the same tnm stage . because the sample sizes in stage i and iv were too small to draw any reliable conclusions ( n=14 in stage i and n=6 in stage iv ) , stratification analysis was performed only in stage ii and iii patients . log - rank test for patients in stage ii demonstrated that the 8-lncrna signature could distinguish patients with significantly different survival ( p=0.015 , figure 3a ) . similar predictive value of the 8-lncrna signature was observed for stage iii patients ( p=0.003 , figure 3b ) . altogether , these results indicated that the prognostic capability of the 8-lncrna signature is independent of conventional clinical factors for survival prediction of esophageal cancer patients . we performed go and kegg functional enrichment analysis for the pcgs co - expressed with the lncrnas in the predictive signature to reveal the potential functions of the eight prognostic lncrnas . the results showed that co - expressed pcgs were enriched in 73 go bp terms , which mainly clustered in regulation of diverse biological processes ( such as go : 0010906~regulation of glucose metabolic process , go : 0010628~positive regulation of gene expression , go : 0043410~positive regulation of mapk cascade ) , transport of various substances ( including go : 0071805~potassium ion transmembrane transport , go : 0035879~plasma membrane lactate transport , go : 0098719~sodium ion import across plasma membrane ) and response to different stimulants ( such as go : 0042594~response to starvation , go : 0043627~response to estrogen , go : 0042493~response to drug ) ( the top 30 go bp terms were shown in table 4 ) . fourteen kegg pathways were enriched which mainly focused on digestive functions ( including hsa04971 : gastric acid secretion , hsa04974 : protein digestion and absorption , hsa04972 : pancreatic secretion ) and basic substance metabolism ( such as hsa01200 : carbon metabolism , hsa00280 : valine , leucine and isoleucine degradation , hsa01100 : metabolic pathways and hsa00071 : fatty acid degradation ) ( figure 4 ) . esophageal cancer is a global health threat with high morbidity and mortality . owing to the heterogeneity , conventional prognostic systems such as tnm staging system often show insufficient prediction for risk stratification and clinical outcome estimations . therefore , considerable efforts have been made in recent decades to develop novel prognostic signatures to promote the prediction of esophageal cancer patient survival [ 2022 ] . as a new focused class of ncrnas , lncrnas were indicated to participate in multiple biological processes including x chromosome inactivation , genomic imprinting , and tumor related alterations . accumulating reports have demonstrated the dysregulation of lncrnas and their potential as biomarkers in various cancers [ 2426 ] . li et al . have established a 3-lncrna signature associated with the patient survival in esophageal squamous cell carcinoma using microarray analysis . however , microarray technology has some undesired shortcomings , such as the bias due to the probe selection and the limitation of identifying only known transcripts . compared with microarrays , rna - seq technique has been developed as an emerging sequencing technology with advantages reducing these defects and standing out especially with the ability of finding novel transcripts . nevertheless , research investigating the impact of lncrnas in esophageal cancer patient survival , which were based on rna - seq technology , are quite deficient . in this study , we developed an 8-lncrna signature which was able to predict the clinical outcome of esophageal cancer . to the best of our knowledge , this is the first lncrna - related predictive model based on rna - seq technology using a cohort of more than 100 cases in esophageal cancer . the differentially expressed lncrnas were first screened out between esophageal cancer and normal tissues with the data downloaded from tcga database . then the expression profiles of these lncrnas of 122 esophageal cancer patients were analyzed by univariate and stepwise multiple cox proportional hazards regression analysis . then eight lncrnas were finally identified to establish a predictive model based on the linear combination of these lncrnas . a distinctive separation was observed in survival curves between patient groups with high - risk and low - risk scores using the predictive model . and roc analysis achieved an auc of 0.845 which demonstrated high sensitivity and specificity of the lncrna signature model . when taking other clinical factors together , multivariate cox regression analysis revealed that the 8-lncrna signature was independent of these conventional clinicopathological factors including race , gender , age , and tumor stage . further stratification analysis indicated favorable discrimination of the 8-lncrna signature in predicting different survival of patients in the same tnm stage . this might provide more references for clinical doctors to select better individualized and effective treatment for patients with different survival risk . the carcinogenesis of esophageal cancer is a multi - step process hallmarked by a series of genetic alterations . although growing attention has begun to focus on the study of lncrnas , the functions of most lncrnas are still unknown . computational annotation of lncrna functions through their co - expressed mrnas has been proven to be effective . in the present study , we performed go and kegg enrichment analysis for the co - expressed mrnas of the eight lncrnas to explore the functions of the predictive lncrnas . the results showed that the prognostic lncrnas were involved in significant biological processes such as regulation of glucose metabolic process , positive regulation of gene expression , positive regulation of mapk cascade and enriched in kegg pathways including gastric acid secretion , amino acid metabolism ( valine , leucine and isoleucine degradation ) , and lipids metabolism ( fatty acid degradation ) . studies have identified aberrant glucose metabolic processes in esophageal cancer , including high plasma and urine glucose levels , excessive glucose uptake and accumulation and alterations of glucose metabolism associated genes . higher gastric acid secretion has been observed in gastroesophageal junction adenocarcinoma , including barrett esophageal cancer compared to normal subjects . amino acid and lipids metabolism disorders , such as increasing leucine level and highly activated fatty acids metabolism , were also found to be responsible for the development of esophageal cancer . therefore , it is plausible to infer that the eight prognostic lncrnas participate in the progression of esophageal cancer through interacting with pcgs in these esophageal cancer - related biological pathways . this study identified a rna - seq based 8-lncrna signature which could predict the survival risk of esophageal cancer patients . the signature displayed independent prognostic capacity of conventional clinicopathological factors and could robustly predict survival outcomes of esophageal cancer patients within the same tnm stage . it could be used to identify patients with high - risk scores who will benefit from more effective and individualized therapy . it could not only serve as a novel potential biomarker for esophageal cancer patient survival risk stratification , but also provide us a better understanding of molecular mechanisms involved in the development of esophageal cancer . however , further clinical studies validating the predictive efficacy of the signature and experimental research investigating the functions of the prognostic lncrnas need to be conducted .

backgroundaccumulating evidence suggests the involvement of long non - coding rnas ( lncrnas ) as oncogenic or tumor suppressive regulators in the development of various cancers . in the present study , we aimed to identify a lncrna signature based on rna sequencing ( rna - seq ) data to predict survival in esophageal cancer.material/methodsthe rna - seq lncrna expression data and clinical information were downloaded from the cancer genome atlas ( tcga ) database . differentially expressed lncrnas were screened out between esophageal cancer and normal tissues . univariate and multivariate cox regression analysis were performed to establish a lncrna - related prognostic model . receiver operating characteristic ( roc ) analysis was conducted to test the sensitivity and specificity of the model . go ( gene ontology ) functional and kegg pathway enrichment analyses were performed for mrnas co - expressed with the lncrnas to explore the potential functions of the prognostic lncrnas.resultsa total of 265 differentially expressed lncrnas were identified between esophageal cancer and normal tissues . after univariate and multivariate cox regression analysis , eight lncrnas ( gs1 - 600g8.5 , linc00365 , ctd-2357a8.3 , rp11 - 705o24.1 , linc01554 , rp1 - 90j4.1 , rp11 - 327j17.1 , and linc00176 ) were finally screened out to establish a predictive model by which patients could be classified into high - risk and low - risk groups with significantly different overall survival . further analysis indicated independent prognostic capability of the 8-lncrna signature from other clinicopathological factors . roc curve analysis demonstrated good performance of the 8-lncrna signature . functional enrichment analysis showed that the prognostic lncrnas were mainly associated with esophageal cancer related biological processes such as regulation of glucose metabolic process and amino acid and lipids metabolism.conclusionsour study developed a novel candidate model providing additional and more powerful prognostic information beyond conventional clinicopathological factors for survival prediction of esophageal cancer patients . moreover , it also brings us new insights into the molecular mechanisms underlying esophageal cancer .

Background Material and Methods The esophageal cancer patient dataset Differentially expressed lncRNAs screening between esophageal cancer and normal tissues Survival analysis and definition of lncRNA related prognostic model Risk stratification and ROC curve Independence of survival prediction by the prognostic lncRNA signature from other clinical variables Functional enrichment analysis Results Differentially expressed lncRNAs between esophageal cancer and normal tissues Establishment of the 8-lncRNA signature associated with overall survival of esophageal cancer patients Risk stratification and ROC curve indicate good performance of the 8-lncRNA signature in predicting the overall survival of esophageal cancer patients Prognostic value of the 8-lncRNA signature is independent of conventional clinical factors Identification of the 8-lncRNA signature related biological processes and pathways Discussion Conclusions

long non - coding rnas ( lncrnas ) are mostly defined as rna transcripts exceeding 200 nucleotides ( nt ) in length apparently without protein coding capacity . it has been reported that dysregulated lncrnas are associated with cancer pathogenesis and function as oncogenic or tumor suppressive regulators in cancer development . one of the recent techniques used for transcriptome analyses is rna sequencing ( rna - seq ) , a next - generation sequencing technique with high - sensitivity , high - throughput and the ability of detecting novel exons , splice sites , and transcripts . however , rna - seq based work focused on the prognostic power of lncrna signatures for survival risk of esophageal cancer patients is quite rare . in this study , we identified for the first time a rna - seq based lncrna signature as a predictor of survival risk of esophageal cancer patients using a cohort of more than 100 cases from the cancer genome atlas ( tcga ) database . a higher area under curve ( auc ) of the receiver operating characteristic ( roc ) curve confirmed good sensitivity and specificity of the prognostic model while multivariate cox regression analysis and stratified analysis indicated the independence of predictive capacity of the 8-lncrna prognostic signature from other clinicopathological factors . furthermore , our functional enrichment analysis suggested that the eight predictive lncrnas were probably involved in the progression of esophageal cancer through exerting their roles in esophageal cancer related biological processes and pathways , such as regulation of glucose metabolic process , positive regulation of mapk cascade , and amino acid and lipids metabolism . the preprocessed level 3 rna - seq data and corresponding clinical information of esophageal cancer patients were collected from the cancer genome atlas ( tcga ) database ( http://cancergenome.nih.gov/ ) ( as of september , 2016 ) . the gene expression profiling data of the 122 esophageal cancer samples and 11 normal samples were downloaded from the tcga database . the package of edger in r language was employed to identify the differentially expressed lncrnas between esophageal cancer and normal tissues with the |log2fc| > 2 and fdr < 0.01 set as the threshold . only those lncrnas with p - value < 0.05 were considered as candidate variables and entered into a stepwise multivariate cox regression analysis tested by aic ( akaike information criterion , assessing the goodness of fit of a statistical model ) to identify the predictive model with the best explanatory and informative efficacy . then , a lncrna - related prognostic model was established to evaluate each patient s survival risk as follows : where k is the number of prognostic lncrnas , ci represents the coefficient of the ith lncrna in the multivariate cox regression analysis , vi is the expression value of the ith lncrna . according to the predictive lncrna signature model the patients were then classified into high - risk or low - risk group using the median risk score as the cutoff value . overall survival curves were generated using the kaplan - meier method , and two - sided log - rank tests were employed to compare the differences in overall survival time between the high - risk and low - risk patient groups . the sensitivity and specificity of the lncrna prognostic model to predict clinical outcome were evaluated by calculating the area under curve ( auc ) of the receiver operating characteristic ( roc ) curve in the r package of survival roc . to determine whether predictive capacity of the lncrna signature was independent of other clinical factors ( including race , gender , stage , and age ) of esophageal cancer patients , multivariate cox regression analysis was carried out using overall survival as the dependent variable and lncrna signature and other conventional clinical factors as independent variables . for clinical features with p - value < 0.01 in cox regression analysis , stratification analysis was further performed to determine whether the lncrna signature exhibit prognostic value within the same clinical factor . to identify potential biological processes and pathways which the predictive lncrnas were involved in , first , the pearson correlation coefficients between the expression profiles of the eight prognostic lncrnas and protein - coding genes ( pcgs ) were calculated to determine the co - expression relationships of the lncrnas and pcgs . the preprocessed level 3 rna - seq data and corresponding clinical information of esophageal cancer patients were collected from the cancer genome atlas ( tcga ) database ( http://cancergenome.nih.gov/ ) ( as of september , 2016 ) . the gene expression profiling data of the 122 esophageal cancer samples and 11 normal samples were downloaded from the tcga database . the package of edger in r language was employed to identify the differentially expressed lncrnas between esophageal cancer and normal tissues with the |log2fc| > 2 and fdr < 0.01 set as the threshold . only those lncrnas with p - value < 0.05 were considered as candidate variables and entered into a stepwise multivariate cox regression analysis tested by aic ( akaike information criterion , assessing the goodness of fit of a statistical model ) to identify the predictive model with the best explanatory and informative efficacy . then , a lncrna - related prognostic model was established to evaluate each patient s survival risk as follows : where k is the number of prognostic lncrnas , ci represents the coefficient of the ith lncrna in the multivariate cox regression analysis , vi is the expression value of the ith lncrna . the patients were then classified into high - risk or low - risk group using the median risk score as the cutoff value . overall survival curves were generated using the kaplan - meier method , and two - sided log - rank tests were employed to compare the differences in overall survival time between the high - risk and low - risk patient groups . the sensitivity and specificity of the lncrna prognostic model to predict clinical outcome were evaluated by calculating the area under curve ( auc ) of the receiver operating characteristic ( roc ) curve in the r package of survival roc . to determine whether predictive capacity of the lncrna signature was independent of other clinical factors ( including race , gender , stage , and age ) of esophageal cancer patients , multivariate cox regression analysis was carried out using overall survival as the dependent variable and lncrna signature and other conventional clinical factors as independent variables . for clinical features with p - value < 0.01 in cox regression analysis , stratification analysis was further performed to determine whether the lncrna signature exhibit prognostic value within the same clinical factor . to identify potential biological processes and pathways which the predictive lncrnas were involved in , functional enrichment analysis was performed . first , the pearson correlation coefficients between the expression profiles of the eight prognostic lncrnas and protein - coding genes ( pcgs ) were calculated to determine the co - expression relationships of the lncrnas and pcgs . according to the cutoff criteria , a total of 265 differentially expressed ( including 112 upregulated and 153 downregulated ) lncrnas were identified between esophageal cancer tissues and normal tissues . the results of unsupervised hierarchical cluster analysis in figure 1 showed that the esophageal cancer samples could be clearly distinguished from the normal controls with the expression of differentially expressed lncrnas . to identify prognosis - related lncrnas , we first used univariate cox regression analysis to evaluate the associations between the expression level of each of the differentially expressed lncrnas and patients overall survival , and found that 13 lncrnas were significantly related to overall survival ( p<0.05 ) . then a stepwise multivariate cox regression analysis was performed and eight lncrnas therein ( as shown in table 2 ) were finally screened out to establish a predictive model . as previously described , the predictive model was defined as the linear combination of the expression levels of the eight lncrnas weighted by their relative coefficient in multivariate cox regression as follows : survival risk score = ( 0.0398 expression value of gs1 - 600g8.5 ) + ( 0.9990 expression value of linc00365 ) + ( 0.6216 expression value of ctd-2357a8.3 ) + ( 13.6225 expression value of rp11 - 705o24.1 ) + ( 1.7105 expression value of linc01554 ) + ( 0.8297 expression value of rp1 - 90j4.1 ) + ( 6.2336 expression value of rp11 - 327j17.1 ) + ( 1.2226 expression value of linc00176 ) . among these , gs1 - 600g8.5 , linc00365 , ctd-2357a8.3 , rp11 - 705o24.1 , linc01554 , and rp1 - 90j4.1 showed positive coefficients in cox regression analysis , indicating high - risk signatures for these six lncrnas since their high expression signified a shorter overall survival of patients . for the remaining two lncrnas , we observed negative coefficients in cox regression analysis , implying that these lncrnas could be regarded as protective lncrnas since patients with higher expression levels of these lncrnas tended to have longer overall survival compared with those with lower expression levels of these lncrnas . for each of the 122 patients in our study , we were able to calculate an 8-lncrna expression - based survival risk score ( referred to as srs ) and assigned them into a high - risk group or a low - risk group using the median risk score of 1.058 as the cutoff point . the kaplan - meier overall survival curves of the two groups based on the eight lncrnas were notably different ( log - rank p=1.72 e-05 < 0.001 ) , showing overall survival in 9.89% and 50.5% at five years for patients with high - risk and low - risk srs , respectively ( figure 2b ) . in our study , the roc curve analysis achieved auc of 0.845 , showing good sensitivity and specificity of the 8-lncrna signature model in predicting esophageal cancer patient survival risk ( figure 2c ) . multivariate cox regression analysis demonstrated that the 8-lncrna signature risk score maintained an independent predictive ability from other clinical factors ( hr=5.951 , 95% ci 2.57713.741 , p=2.95 e-05 , shown in table 3 ) . altogether , these results indicated that the prognostic capability of the 8-lncrna signature is independent of conventional clinical factors for survival prediction of esophageal cancer patients . we performed go and kegg functional enrichment analysis for the pcgs co - expressed with the lncrnas in the predictive signature to reveal the potential functions of the eight prognostic lncrnas . the results showed that co - expressed pcgs were enriched in 73 go bp terms , which mainly clustered in regulation of diverse biological processes ( such as go : 0010906~regulation of glucose metabolic process , go : 0010628~positive regulation of gene expression , go : 0043410~positive regulation of mapk cascade ) , transport of various substances ( including go : 0071805~potassium ion transmembrane transport , go : 0035879~plasma membrane lactate transport , go : 0098719~sodium ion import across plasma membrane ) and response to different stimulants ( such as go : 0042594~response to starvation , go : 0043627~response to estrogen , go : 0042493~response to drug ) ( the top 30 go bp terms were shown in table 4 ) . according to the cutoff criteria , a total of 265 differentially expressed ( including 112 upregulated and 153 downregulated ) lncrnas were identified between esophageal cancer tissues and normal tissues . the results of unsupervised hierarchical cluster analysis in figure 1 showed that the esophageal cancer samples could be clearly distinguished from the normal controls with the expression of differentially expressed lncrnas . to identify prognosis - related lncrnas , we first used univariate cox regression analysis to evaluate the associations between the expression level of each of the differentially expressed lncrnas and patients overall survival , and found that 13 lncrnas were significantly related to overall survival ( p<0.05 ) . then a stepwise multivariate cox regression analysis was performed and eight lncrnas therein ( as shown in table 2 ) were finally screened out to establish a predictive model . as previously described , the predictive model was defined as the linear combination of the expression levels of the eight lncrnas weighted by their relative coefficient in multivariate cox regression as follows : survival risk score = ( 0.0398 expression value of gs1 - 600g8.5 ) + ( 0.9990 expression value of linc00365 ) + ( 0.6216 expression value of ctd-2357a8.3 ) + ( 13.6225 expression value of rp11 - 705o24.1 ) + ( 1.7105 expression value of linc01554 ) + ( 0.8297 expression value of rp1 - 90j4.1 ) + ( 6.2336 expression value of rp11 - 327j17.1 ) + ( 1.2226 expression value of linc00176 ) . among these , gs1 - 600g8.5 , linc00365 , ctd-2357a8.3 , rp11 - 705o24.1 , linc01554 , and rp1 - 90j4.1 showed positive coefficients in cox regression analysis , indicating high - risk signatures for these six lncrnas since their high expression signified a shorter overall survival of patients . for the remaining two lncrnas , we observed negative coefficients in cox regression analysis , implying that these lncrnas could be regarded as protective lncrnas since patients with higher expression levels of these lncrnas tended to have longer overall survival compared with those with lower expression levels of these lncrnas . for each of the 122 patients in our study , we were able to calculate an 8-lncrna expression - based survival risk score ( referred to as srs ) and assigned them into a high - risk group or a low - risk group using the median risk score of 1.058 as the cutoff point . the kaplan - meier overall survival curves of the two groups based on the eight lncrnas were notably different ( log - rank p=1.72 e-05 < 0.001 ) , showing overall survival in 9.89% and 50.5% at five years for patients with high - risk and low - risk srs , respectively ( figure 2b ) . the prognostic power of the 8-lncrna signature was evaluated by calculating the auc of roc curve . in our study , the roc curve analysis achieved auc of 0.845 , showing good sensitivity and specificity of the 8-lncrna signature model in predicting esophageal cancer patient survival risk ( figure 2c ) . multivariate cox regression analysis demonstrated that the 8-lncrna signature risk score maintained an independent predictive ability from other clinical factors ( hr=5.951 , 95% ci 2.57713.741 , p=2.95 e-05 , shown in table 3 ) . meanwhile , we also found that tnm stage was an independent predictor for overall survival of esophageal cancer patients . altogether , these results indicated that the prognostic capability of the 8-lncrna signature is independent of conventional clinical factors for survival prediction of esophageal cancer patients . we performed go and kegg functional enrichment analysis for the pcgs co - expressed with the lncrnas in the predictive signature to reveal the potential functions of the eight prognostic lncrnas . the results showed that co - expressed pcgs were enriched in 73 go bp terms , which mainly clustered in regulation of diverse biological processes ( such as go : 0010906~regulation of glucose metabolic process , go : 0010628~positive regulation of gene expression , go : 0043410~positive regulation of mapk cascade ) , transport of various substances ( including go : 0071805~potassium ion transmembrane transport , go : 0035879~plasma membrane lactate transport , go : 0098719~sodium ion import across plasma membrane ) and response to different stimulants ( such as go : 0042594~response to starvation , go : 0043627~response to estrogen , go : 0042493~response to drug ) ( the top 30 go bp terms were shown in table 4 ) . nevertheless , research investigating the impact of lncrnas in esophageal cancer patient survival , which were based on rna - seq technology , are quite deficient . in this study , we developed an 8-lncrna signature which was able to predict the clinical outcome of esophageal cancer . to the best of our knowledge , this is the first lncrna - related predictive model based on rna - seq technology using a cohort of more than 100 cases in esophageal cancer . the differentially expressed lncrnas were first screened out between esophageal cancer and normal tissues with the data downloaded from tcga database . then eight lncrnas were finally identified to establish a predictive model based on the linear combination of these lncrnas . a distinctive separation was observed in survival curves between patient groups with high - risk and low - risk scores using the predictive model . when taking other clinical factors together , multivariate cox regression analysis revealed that the 8-lncrna signature was independent of these conventional clinicopathological factors including race , gender , age , and tumor stage . in the present study , we performed go and kegg enrichment analysis for the co - expressed mrnas of the eight lncrnas to explore the functions of the predictive lncrnas . the results showed that the prognostic lncrnas were involved in significant biological processes such as regulation of glucose metabolic process , positive regulation of gene expression , positive regulation of mapk cascade and enriched in kegg pathways including gastric acid secretion , amino acid metabolism ( valine , leucine and isoleucine degradation ) , and lipids metabolism ( fatty acid degradation ) . amino acid and lipids metabolism disorders , such as increasing leucine level and highly activated fatty acids metabolism , were also found to be responsible for the development of esophageal cancer . therefore , it is plausible to infer that the eight prognostic lncrnas participate in the progression of esophageal cancer through interacting with pcgs in these esophageal cancer - related biological pathways . this study identified a rna - seq based 8-lncrna signature which could predict the survival risk of esophageal cancer patients . the signature displayed independent prognostic capacity of conventional clinicopathological factors and could robustly predict survival outcomes of esophageal cancer patients within the same tnm stage . it could not only serve as a novel potential biomarker for esophageal cancer patient survival risk stratification , but also provide us a better understanding of molecular mechanisms involved in the development of esophageal cancer .

esophageal cancer is one of the most common digestive malignancies with the 5-year relative survival rate less than 20% , ranking the fourth leading cause of cancer death among both men and women in china . dysregulation of lncrnas has been observed in various cancers , including breast cancer , colorectal cancer , lung cancer , prostate cancer as well as esophageal cancer [ 610 ] . one of the recent techniques used for transcriptome analyses is rna sequencing ( rna - seq ) , a next - generation sequencing technique with high - sensitivity , high - throughput and the ability of detecting novel exons , splice sites , and transcripts . however , rna - seq based work focused on the prognostic power of lncrna signatures for survival risk of esophageal cancer patients is quite rare . in this study , we identified for the first time a rna - seq based lncrna signature as a predictor of survival risk of esophageal cancer patients using a cohort of more than 100 cases from the cancer genome atlas ( tcga ) database . a higher area under curve ( auc ) of the receiver operating characteristic ( roc ) curve confirmed good sensitivity and specificity of the prognostic model while multivariate cox regression analysis and stratified analysis indicated the independence of predictive capacity of the 8-lncrna prognostic signature from other clinicopathological factors . furthermore , our functional enrichment analysis suggested that the eight predictive lncrnas were probably involved in the progression of esophageal cancer through exerting their roles in esophageal cancer related biological processes and pathways , such as regulation of glucose metabolic process , positive regulation of mapk cascade , and amino acid and lipids metabolism . the preprocessed level 3 rna - seq data and corresponding clinical information of esophageal cancer patients were collected from the cancer genome atlas ( tcga ) database ( http://cancergenome.nih.gov/ ) ( as of september , 2016 ) . the patients meeting the following criteria were included in the study : ( 1 ) patients with complete information of lncrna expression profiles and clinical characteristics ( including age , gender , race , stage , survival status and survival time ) ( 2 ) the overall survival time was more than one month . the package of edger in r language was employed to identify the differentially expressed lncrnas between esophageal cancer and normal tissues with the |log2fc| > 2 and fdr < 0.01 set as the threshold . then the unsupervised hierarchical clustering was performed based on the expression of these altered lncrnas by the pheatmap package in r ( https://cran.r-project.org/web/packages/pheatmap/index.html , version 1.0.8 ) . the association between the expression of differentially expressed lncrnas and patient overall survival was evaluated by univariate cox proportional hazards regression analysis using the survival r package . only those lncrnas with p - value < 0.05 were considered as candidate variables and entered into a stepwise multivariate cox regression analysis tested by aic ( akaike information criterion , assessing the goodness of fit of a statistical model ) to identify the predictive model with the best explanatory and informative efficacy . then , a lncrna - related prognostic model was established to evaluate each patient s survival risk as follows : where k is the number of prognostic lncrnas , ci represents the coefficient of the ith lncrna in the multivariate cox regression analysis , vi is the expression value of the ith lncrna . overall survival curves were generated using the kaplan - meier method , and two - sided log - rank tests were employed to compare the differences in overall survival time between the high - risk and low - risk patient groups . the sensitivity and specificity of the lncrna prognostic model to predict clinical outcome were evaluated by calculating the area under curve ( auc ) of the receiver operating characteristic ( roc ) curve in the r package of survival roc . to determine whether predictive capacity of the lncrna signature was independent of other clinical factors ( including race , gender , stage , and age ) of esophageal cancer patients , multivariate cox regression analysis was carried out using overall survival as the dependent variable and lncrna signature and other conventional clinical factors as independent variables . for clinical features with p - value < 0.01 in cox regression analysis , stratification analysis was further performed to determine whether the lncrna signature exhibit prognostic value within the same clinical factor . to identify potential biological processes and pathways which the predictive lncrnas were involved in , first , the pearson correlation coefficients between the expression profiles of the eight prognostic lncrnas and protein - coding genes ( pcgs ) were calculated to determine the co - expression relationships of the lncrnas and pcgs . gene ontology ( go ) biological process ( bp ) and kyoto encyclopedia of genes and genomes ( kegg ) pathway enrichment analysis were carried out for those pcgs using the database for annotation , visualization , and integrated discovery ( david , https://david.ncifcrf.gov/ , version 6.8 ) . the preprocessed level 3 rna - seq data and corresponding clinical information of esophageal cancer patients were collected from the cancer genome atlas ( tcga ) database ( http://cancergenome.nih.gov/ ) ( as of september , 2016 ) . the patients meeting the following criteria were included in the study : ( 1 ) patients with complete information of lncrna expression profiles and clinical characteristics ( including age , gender , race , stage , survival status and survival time ) ( 2 ) the overall survival time was more than one month . the package of edger in r language was employed to identify the differentially expressed lncrnas between esophageal cancer and normal tissues with the |log2fc| > 2 and fdr < 0.01 set as the threshold . then the unsupervised hierarchical clustering was performed based on the expression of these altered lncrnas by the pheatmap package in r ( https://cran.r-project.org/web/packages/pheatmap/index.html , version 1.0.8 ) . only those lncrnas with p - value < 0.05 were considered as candidate variables and entered into a stepwise multivariate cox regression analysis tested by aic ( akaike information criterion , assessing the goodness of fit of a statistical model ) to identify the predictive model with the best explanatory and informative efficacy . then , a lncrna - related prognostic model was established to evaluate each patient s survival risk as follows : where k is the number of prognostic lncrnas , ci represents the coefficient of the ith lncrna in the multivariate cox regression analysis , vi is the expression value of the ith lncrna . the sensitivity and specificity of the lncrna prognostic model to predict clinical outcome were evaluated by calculating the area under curve ( auc ) of the receiver operating characteristic ( roc ) curve in the r package of survival roc . to determine whether predictive capacity of the lncrna signature was independent of other clinical factors ( including race , gender , stage , and age ) of esophageal cancer patients , multivariate cox regression analysis was carried out using overall survival as the dependent variable and lncrna signature and other conventional clinical factors as independent variables . for clinical features with p - value < 0.01 in cox regression analysis , stratification analysis was further performed to determine whether the lncrna signature exhibit prognostic value within the same clinical factor . first , the pearson correlation coefficients between the expression profiles of the eight prognostic lncrnas and protein - coding genes ( pcgs ) were calculated to determine the co - expression relationships of the lncrnas and pcgs . gene ontology ( go ) biological process ( bp ) and kyoto encyclopedia of genes and genomes ( kegg ) pathway enrichment analysis were carried out for those pcgs using the database for annotation , visualization , and integrated discovery ( david , https://david.ncifcrf.gov/ , version 6.8 ) . to identify prognosis - related lncrnas , we first used univariate cox regression analysis to evaluate the associations between the expression level of each of the differentially expressed lncrnas and patients overall survival , and found that 13 lncrnas were significantly related to overall survival ( p<0.05 ) . as previously described , the predictive model was defined as the linear combination of the expression levels of the eight lncrnas weighted by their relative coefficient in multivariate cox regression as follows : survival risk score = ( 0.0398 expression value of gs1 - 600g8.5 ) + ( 0.9990 expression value of linc00365 ) + ( 0.6216 expression value of ctd-2357a8.3 ) + ( 13.6225 expression value of rp11 - 705o24.1 ) + ( 1.7105 expression value of linc01554 ) + ( 0.8297 expression value of rp1 - 90j4.1 ) + ( 6.2336 expression value of rp11 - 327j17.1 ) + ( 1.2226 expression value of linc00176 ) . among these , gs1 - 600g8.5 , linc00365 , ctd-2357a8.3 , rp11 - 705o24.1 , linc01554 , and rp1 - 90j4.1 showed positive coefficients in cox regression analysis , indicating high - risk signatures for these six lncrnas since their high expression signified a shorter overall survival of patients . for the remaining two lncrnas , we observed negative coefficients in cox regression analysis , implying that these lncrnas could be regarded as protective lncrnas since patients with higher expression levels of these lncrnas tended to have longer overall survival compared with those with lower expression levels of these lncrnas . for each of the 122 patients in our study , we were able to calculate an 8-lncrna expression - based survival risk score ( referred to as srs ) and assigned them into a high - risk group or a low - risk group using the median risk score of 1.058 as the cutoff point . as a result , 61 patients were classified into the high - risk group since their srss were greater than the cutoff value , whereas the other 61 patients were assigned to the low - risk group with their srss less than the cutoff point ( figure 2a ) . the kaplan - meier overall survival curves of the two groups based on the eight lncrnas were notably different ( log - rank p=1.72 e-05 < 0.001 ) , showing overall survival in 9.89% and 50.5% at five years for patients with high - risk and low - risk srs , respectively ( figure 2b ) . in our study , the roc curve analysis achieved auc of 0.845 , showing good sensitivity and specificity of the 8-lncrna signature model in predicting esophageal cancer patient survival risk ( figure 2c ) . multivariate cox regression analysis demonstrated that the 8-lncrna signature risk score maintained an independent predictive ability from other clinical factors ( hr=5.951 , 95% ci 2.57713.741 , p=2.95 e-05 , shown in table 3 ) . we performed go and kegg functional enrichment analysis for the pcgs co - expressed with the lncrnas in the predictive signature to reveal the potential functions of the eight prognostic lncrnas . the results showed that co - expressed pcgs were enriched in 73 go bp terms , which mainly clustered in regulation of diverse biological processes ( such as go : 0010906~regulation of glucose metabolic process , go : 0010628~positive regulation of gene expression , go : 0043410~positive regulation of mapk cascade ) , transport of various substances ( including go : 0071805~potassium ion transmembrane transport , go : 0035879~plasma membrane lactate transport , go : 0098719~sodium ion import across plasma membrane ) and response to different stimulants ( such as go : 0042594~response to starvation , go : 0043627~response to estrogen , go : 0042493~response to drug ) ( the top 30 go bp terms were shown in table 4 ) . fourteen kegg pathways were enriched which mainly focused on digestive functions ( including hsa04971 : gastric acid secretion , hsa04974 : protein digestion and absorption , hsa04972 : pancreatic secretion ) and basic substance metabolism ( such as hsa01200 : carbon metabolism , hsa00280 : valine , leucine and isoleucine degradation , hsa01100 : metabolic pathways and hsa00071 : fatty acid degradation ) ( figure 4 ) . to identify prognosis - related lncrnas , we first used univariate cox regression analysis to evaluate the associations between the expression level of each of the differentially expressed lncrnas and patients overall survival , and found that 13 lncrnas were significantly related to overall survival ( p<0.05 ) . as previously described , the predictive model was defined as the linear combination of the expression levels of the eight lncrnas weighted by their relative coefficient in multivariate cox regression as follows : survival risk score = ( 0.0398 expression value of gs1 - 600g8.5 ) + ( 0.9990 expression value of linc00365 ) + ( 0.6216 expression value of ctd-2357a8.3 ) + ( 13.6225 expression value of rp11 - 705o24.1 ) + ( 1.7105 expression value of linc01554 ) + ( 0.8297 expression value of rp1 - 90j4.1 ) + ( 6.2336 expression value of rp11 - 327j17.1 ) + ( 1.2226 expression value of linc00176 ) . among these , gs1 - 600g8.5 , linc00365 , ctd-2357a8.3 , rp11 - 705o24.1 , linc01554 , and rp1 - 90j4.1 showed positive coefficients in cox regression analysis , indicating high - risk signatures for these six lncrnas since their high expression signified a shorter overall survival of patients . for the remaining two lncrnas , we observed negative coefficients in cox regression analysis , implying that these lncrnas could be regarded as protective lncrnas since patients with higher expression levels of these lncrnas tended to have longer overall survival compared with those with lower expression levels of these lncrnas . for each of the 122 patients in our study , we were able to calculate an 8-lncrna expression - based survival risk score ( referred to as srs ) and assigned them into a high - risk group or a low - risk group using the median risk score of 1.058 as the cutoff point . as a result , 61 patients were classified into the high - risk group since their srss were greater than the cutoff value , whereas the other 61 patients were assigned to the low - risk group with their srss less than the cutoff point ( figure 2a ) . the kaplan - meier overall survival curves of the two groups based on the eight lncrnas were notably different ( log - rank p=1.72 e-05 < 0.001 ) , showing overall survival in 9.89% and 50.5% at five years for patients with high - risk and low - risk srs , respectively ( figure 2b ) . in our study , the roc curve analysis achieved auc of 0.845 , showing good sensitivity and specificity of the 8-lncrna signature model in predicting esophageal cancer patient survival risk ( figure 2c ) . multivariate cox regression analysis demonstrated that the 8-lncrna signature risk score maintained an independent predictive ability from other clinical factors ( hr=5.951 , 95% ci 2.57713.741 , p=2.95 e-05 , shown in table 3 ) . we performed go and kegg functional enrichment analysis for the pcgs co - expressed with the lncrnas in the predictive signature to reveal the potential functions of the eight prognostic lncrnas . the results showed that co - expressed pcgs were enriched in 73 go bp terms , which mainly clustered in regulation of diverse biological processes ( such as go : 0010906~regulation of glucose metabolic process , go : 0010628~positive regulation of gene expression , go : 0043410~positive regulation of mapk cascade ) , transport of various substances ( including go : 0071805~potassium ion transmembrane transport , go : 0035879~plasma membrane lactate transport , go : 0098719~sodium ion import across plasma membrane ) and response to different stimulants ( such as go : 0042594~response to starvation , go : 0043627~response to estrogen , go : 0042493~response to drug ) ( the top 30 go bp terms were shown in table 4 ) . fourteen kegg pathways were enriched which mainly focused on digestive functions ( including hsa04971 : gastric acid secretion , hsa04974 : protein digestion and absorption , hsa04972 : pancreatic secretion ) and basic substance metabolism ( such as hsa01200 : carbon metabolism , hsa00280 : valine , leucine and isoleucine degradation , hsa01100 : metabolic pathways and hsa00071 : fatty acid degradation ) ( figure 4 ) . to the best of our knowledge , this is the first lncrna - related predictive model based on rna - seq technology using a cohort of more than 100 cases in esophageal cancer . in the present study , we performed go and kegg enrichment analysis for the co - expressed mrnas of the eight lncrnas to explore the functions of the predictive lncrnas . the results showed that the prognostic lncrnas were involved in significant biological processes such as regulation of glucose metabolic process , positive regulation of gene expression , positive regulation of mapk cascade and enriched in kegg pathways including gastric acid secretion , amino acid metabolism ( valine , leucine and isoleucine degradation ) , and lipids metabolism ( fatty acid degradation ) . studies have identified aberrant glucose metabolic processes in esophageal cancer , including high plasma and urine glucose levels , excessive glucose uptake and accumulation and alterations of glucose metabolism associated genes . therefore , it is plausible to infer that the eight prognostic lncrnas participate in the progression of esophageal cancer through interacting with pcgs in these esophageal cancer - related biological pathways . it could not only serve as a novel potential biomarker for esophageal cancer patient survival risk stratification , but also provide us a better understanding of molecular mechanisms involved in the development of esophageal cancer . however , further clinical studies validating the predictive efficacy of the signature and experimental research investigating the functions of the prognostic lncrnas need to be conducted .

the regulation of pancreas development from the gut endoderm is a complex process that involves a carefully balanced interplay between several common signaling pathways . studies in several model organisms indicate conservation of the main mechanisms of pancreas development from lower to higher vertebrates ( table 1 ) . gastrulation in the developing embryo ( e7.5 in mice ) results in the formation of three germ layers , namely the ectoderm , the endoderm , and the mesoderm . it appears that these germ layers do not further develop in isolation from each other , but signals emanate from one to pattern the other and vice versa ( 1 ) . several structures temporally participate in these early tissue interactions including the notochord ( 2 ) , the cardiac mesoderm , the septum transversum mesenchyme and the lateral plate mesoderm ( 3 ) , the aortic endothelial cells and the vitelline veins ( 4 ) , and the developing pancreatic mesenchyme ( 5 ) . time scale of pancreas development in mouse and human the major sequences of developmental processes are similar in mouse and human embryonic pancreas . the striking difference resides in the duration of each stage , which can vary by a factor of 5 to 15 . as far as the pancreas is concerned , specific growth and differentiation factors released by adjacent tissues control a set of transcription factors expression , resulting in the patterning of the ventral and dorsal prepancreatic endoderm . the initial interactions usually confer competence to respond to additional inductive signals that establish organ determination and specification at particular time points referred to as competence windows . although our present knowledge of extrinsic factors that control patterning , proliferation , and differentiation cues throughout the developing pancreas is not yet complete , there is strong evidence emanating from all vertebrate developmental models that complex spatio - temporal combinations of common signaling pathways are crucial for appropriate specification of pancreatic cell fates from the endoderm ( 69 ) . these involve , for instance , the hedgehog , wnt , retinoid , and notch pathways , as well as activin / bone morphogenetic protein ( bmp ) , fibroblast growth factor ( fgf ) , vascular endothelial growth factor ( vegf ) , epidermal growth factor ( egf ) , and hepatocyte growth factor ( hgf ) signal transductions . an overview of the roles played in pancreas development by the most relevant growth and differentiation factors is summarized in table 2 . a detailed sequential presentation is difficult to address because many signaling events are not clearly dissociated from each other , while others function redundantly or intermittently at several developmental stages . it is also worth noting that the developmental effects elicited by these common signals can vary significantly according to the cell type being stimulated or according to the stage of development when the signal occurs . this level of complexity is further enhanced by the interplay between transcription factors that lie downstream of these extracellular stimuli and are responsible for observed developmental features . overview of growth and differentiation factors that participate in vertebrate pancreas development cmes , cardiac mesoderm ; dmes , dorsal mesenchyme ; gcg , glucagon ; gdf , growth differentiation factor ; hb - egf , heparin - binding epidermal growth factor ; ihh , indian hedgehog ; lpm , lateral plate mesoderm ; pepi , pancreatic epithelium ; pmes , pancreatic mesenchyme ; pnep , pancreatic ngn3 endocrine progenitor . , considerable progress has been achieved in understanding pancreas embryogenesis thanks to the numerous genetic studies involved in transcription factors . most of them belong to the homeobox domain , the paired - box , or the basic helix - loop - helix families . the sequential expression of different transcription factors that control pancreas development has been extensively reviewed elsewhere ( 68,10 ) . 1 with special focus on 1 ) the early pancreatic epithelial cells , 2 ) the pancreatic endocrine progenitors , and 3 ) the endocrine subtypes selection . expression of different combinations of tfs determines the sequential lineage segregation in the developing pancreas . not only are the combinations of tfs important , but the expression level of a particular tf is known to guide differentiation as well . the most relevant tfs that are landmarks for the selection of each stage / lineage are shown . it is presently well recognized that attempts to generate pancreatic cells from pluripotent cells should first establish a definitive endoderm ( de ) population as occurs in vivo . wnt and nodal signals were identified as sufficient to induce de from mouse and human embryonic stem ( he s ) cells , but also from induced pluripotent stem ( ips ) cells . in this model , high concentrations of activin a ( acta ) are used to mimic the function of the endogenous endoderm inducer nodal ( 11 ) . beside the activation of this pathway , initial treatment with wnt3a or bmp4 was found to improve the efficiency of de induction by setting up a transient mesendoderm progenitor population , especially when cultures are performed in feeder cell - free conditions ( 12,13 ) . because active pi3k signaling is inhibitory for de differentiation from embryonic stem ( es ) cells , antagonists of this pathway ( ly294002 , wortmanin ) were also included in some protocols , resulting in an increased efficiency ( 14,15 ) . in addition , supplementation of sodium butyrate was found to support endoderm differentiation when combined with acta ( 16 ) , though it remains unclear how this chemical participates in the signaling network required for the definitive endoderm . the standard and modified protocols for de induction from es cells allow for the derivation of at least 6080% cells that express the characteristic markers foxa2 , sox17 , gsc , and cxcr4 ( table 3 ) but not the visceral endoderm marker sox7 ( 13,14,1620 ) . whether the quest for a universal -cell differentiation protocol is a desired myth or an achievable reality , it appears clear from the literature that combined wnt3a and acta treatments efficiently activate de phenotype in a vast majority of he s cell lines . in other words , the molecular events that lead de differentiation in vivo have been largely if not completely unraveled and implemented in vitro . despite the recently described differences in the response of he s cell lines to differentiation cues ( 21 ) , the variability and the low success rate in the outcome of initial pancreas differentiation protocols might first of all reflect the lack of such a universal developmentally based strategy that thoroughly reproduces in vivo events in the culture dish after the de stage . derivation of pdx1 progenitors from he s cells recent models of pancreas differentiation from he s cells integrate bmp antagonism and retinoid signaling early after definitive endoderm induction . cyclo , cyclopamine ; dapt , n-[n-(3,5-difluorophenacetyl)-l - alanyl]-s - phenylglycine t - butyl ester ( gamma secretase inhibitor ) ; ebs , embryoid bodies ; ex4 , exendin-4 ; fbs , fetal bovine serum ; its , insulin selenium transferring supplement ; na , nicotinamide ; nabut , sodium butyrate ; ng , noggin ; stz , streptozotocin ; 3d , three dimensional . when maintained in culture without added growth factors or when transplanted under the kidney capsule of immunocompromised mice , hes - derived definitive endoderm cells could spontaneously generate hepatocyte - like cells characterized by the expression of afp , albumin ( alb ) ( fig . this tendency for hepatic lineage commitment was also reported in several studies wherein hes - derived de cells were exposed to bmp and/or fgf , two growth factors implicated in embryonic liver induction and specification ( 13,20,22,23 ) . the contribution of these pathways was further demonstrated by the treatment with bmp antagonist noggin and fgf receptor antagonist su5402 , leading to the complete abrogation of hepatic gene expression from de progenies that otherwise was mainly composed of hepatocytes . as suggested from animal studies , these experiments also indicated that fgf did not function in the induction but essentially contributed to the amplification of liver progenitors initially induced by bmp signaling ( 20 ) . therefore , the molecular signals elicited by bmp and fgf are the main factors driving hepatocyte differentiation from hes - derived de cells . expression of pancreas - related transcription factors in hes - derived de progenies treated with noggin , ra , and cyclopamine . foxa2 and albumin expression in control cultures ( a ) and following treatment with noggin , ra , and cyclopamine ( b ) . d : several nkx6.1 cells are also detectable , the majority of which express lower levels of pdx1 ( not shown ) . ( a high - quality digital representation of this figure is available in the online issue . ) in vivo , bmp activity generated by the adjacent mesoderm derived structures and operating in the ventral endoderm is associated with hepatic induction during early embryonic development . on the contrary , ventral pancreas progenitors are located away from the midline endoderm with active bmp signaling , whereas bmp inhibition in the dorsal endoderm plays a crucial function in the acquisition of a pancreatic fate ( 3,24 ) . the requirement for bmp antagonism is reverted after pancreas induction since this organ requires bmp signaling later on for the maintenance of pdx1 expression and further differentiation . this underscores the existence of a tight competence window and the versatile functions of growth factors during development , which complicates their in vitro implementation for directed differentiation of es cells ( 24 ) . retinoic acid ( ra ) plays a crucial role shortly after gastrulation in endoderm patterning and in the induction of pdx1 expression by pancreas progenitors in several organisms from xenopus to humans , which indicates the conservation of its activity during evolution ( 2527 ) . this role for ra has already been evaluated during mouse and human es cell differentiation in vitro and has proven to be relevant for the induction of pancreatic gene expression , notably detectable levels of pdx1 ( 17,23,2832 ) . in line with the in vivo recognized functions of these two signaling pathways on pancreas induction , recent studies have combined bmp inhibition ( noggin supplementation ) with retinoid signaling immediately after the de induction stage and succeeded in producing large amounts ( up to 80% ) of pdx1 pancreas progenitor cells ( table 3 ) , ( 13,14,20 ) . concomitantly , the proportion of hepatocytes that could be detected in these conditions was significantly reduced ( fig . 2a and b ) , indicating an efficient blockade of their induction upon bmp antagonism ( 20,33 ) . this combination was efficient not only in different he s cell lines , but also in ips cells ( 14 ) , suggesting that the basic molecular events that should be monitored in order to design a universal pancreatic differentiation protocol are being progressively clarified . these minimally include the signals for the blockade of hepatic induction on the one hand and those required for pancreatic induction ( namely pdx1 expression ) on the other ( fig . the necessity for such a combination was recently highlighted by treatment of hes - derived de cells with noggin in the absence of ra ; this blocked the expression of liver markers , but did not generate pdx1 cells ( 20,23 ) . this requirement for combining ra treatment with bmp antagonism could also be seen from another angle . indeed , retinoid signaling was shown to activate bmp expression in several in vivo and in vitro systems including embryonal carcinoma cells and differentiating mouse embryoid bodies ( 3436 ) . it is not yet clear whether this inductive effect on bmp also occurs during he s cell differentiation . nevertheless , considering the in vivo function of bmp signaling in the midgut endoderm , it appears reasonable that both retinoid signaling and bmp antagonism were required in these studies for strong induction of a pancreatic fate from the de cells while preventing hepatic differentiation . however , it is likely that the effect of ra is context - dependent because when applied on hes - derived de cells cultured at low density , it resulted in the inhibition of smad 1/5/8 phosphorylation ( 23 ) . it remains to be elucidated whether this later effect represents an artifact of in vitro culture or a specific retinoid effect at the single cell level . four main pathways regulate the early stages of pancreas differentiation from definitive endoderm and control the acquisition of hepatic vs. pancreatic fate . hedgehog signaling is well characterized as a potent inhibitor of pancreatic initiation ( a ) that is expressed in the hepatic domain ( b ) under the influence of fgf signaling . retinoic acid is expressed in the developing pancreas and participates in the induction of pdx1 expression ( c ) . it is also suggested to contribute to hepatic gene expression to a certain degree ( d ) . an inhibitory effect of retinoic acid on smad1 - 5 - 8 phosphorylation ( e ) was demonstrated on hes - derived definitive endoderm seeded at low density , which contrasts with the early studies indicating activation of bmp signaling by retinoic acid . bmp is well described as an inhibitor of early pancreas development ( f ) in contrast to its requirement for hepatic initiation ( g ) . this potent inhibitory effect at early stages gets reverted afterward and pancreatic progenitors require bmp signaling ( f ) . the inhibition of bmp signaling in the pancreatic domain is under the control of noggin ( h ) . as for bmp pathway , fgf signaling via erk / mapk also controls early pancreas induction with high concentration being inhibitory ( i ) whereas low concentrations are required ( j ) . in late stages , fgf plays in the proliferation of pancreas progenitors ( j ) . on the contrary the markers displayed in the progenitors recall the current status of protein detection from differentiated he s cells . other studies made use of ra combined with fgf ligands ( fgf4 , fgf7 , fgf10 ) or of noggin combined with fgf2 and egf to induce pancreatic cells albeit at a lower efficiency than for combined noggin and ra ( table 3 ) ( 13,14,16,17,19,23 ) . our unpublished observations suggest that fgf signals favor hepatic gene expression in the absence of bmp antagonism , but the addition of fgf ligands together with bmp antagonism ( noggin ) and ra enhances pancreatic gene expression . furthermore , the induction of pdx1 cells by combined noggin and ra requires at least a basal level of fgf activity ( via the extracellular signal regulated kinase / mitogen - activated protein kinase [ erk / mapk ] pathway [ fig . 3 ] ) given that the addition of the mapk - inhibitor u1026 prevented pancreatic cell differentiation ( 20 ) . because ra is known to act synergistically with fgf signaling during endoderm patterning , supplemented fgf can be seen as an enhancing factor in protocols that already integrate noggin and ra ( 13,14,19 ) . this view might resolve the nature of factors x suggested as involved in the indirect effects of ra in xenopus dorsal endoderm ( 37 ) . it was recently suggested that in the presence of fgf7 , ra can efficiently induce pdx1 + progenitors from hes - derived de cells seeded at low density ( 550,000 cells / cm ) and that this effect is paralleled by inhibition of smad1/5/8 phosphorylation , therefore recapitulating bmp antagonism described above ( 23 ) . recent analysis of the signaling network during early liver and pancreas development in 36 somites - stage mouse embryos further indicated that inhibition of the activin signaling significantly increases the proportion of cells fated toward pdx1 expression . these findings therefore suggest that the addition of activin inhibitors to the above - mentioned cocktails might still boost the occurrence of pdx1 cells in he s cell cultures ( 13,24 ) . despite the fact that combining bmp antagonism with retinoid signaling efficiently generates pdx1 pancreatic progenitors from hes - derived definitive endoderm , the requirement for hedgehog antagonism with cyclopamine during pancreas induction from these cells has not been clarified by these studies . the initial report by d'amour et al . ( 18 ) indicated the necessity to antagonize hedgehog signaling that is indeed activated during es cell differentiation , and it was shown to also play a negative role ex vivo by limiting endocrine and exocrine genes expression in embryonic ( e12.5 ) mouse pancreatic explants ( 38,39 ) . however , additional studies using noggin , fgf ligands , and/or ra after de induction succeeded in generating pdx1 cells without the use of hedgehog inhibitors ( 14,17,19,23 ) . owing to the previously described increase in the expression of hedgehog ligands and effectors upon de induction , we added cyclopamine to the combination of noggin and ra in our experimental setup , but it remains unclear whether this was mandatory for the efficient differentiation of pdx1 cells that we observed ( 20,38,39 ) . while waiting for conclusive data on this issue , we speculate that supplementation of hedgehog antagonists would become obsolete in settings where combined noggin and ra treatment of de cells essentially induces a true pancreatic endoderm cell type , which is normally devoid of hedgehog activity . gli1 transcripts profiling in cells treated with noggin and ra combination would help to clarify this issue . pancreatic progenitor cells are characterized by their expression of a subset of transcription factors . for instance , the combined expression of pdx1 , foxa2 , sox9 , hnf6 , nkx6.1 , and ptf1a ( fig . the last two transcription factors are more specific to the pancreas , whereas pdx1 , foxa2 , sox9 , and hnf6 are also detected in the adjacent duodenum , stomach , and liver . following the induction of pancreas progenitor cells from hes - derived de cells , pdx1 co - expression with foxa2 , sox9 , and hnf6 was demonstrated in a context where hepatic ( afp , alb ) and intestinal ( cdx1 , cdx2 ) markers were not induced or only minimally induced ( fig . however , none of the above - mentioned studies have assessed the expression of all the six transcription factors by the pancreatic progenitors obtained from he s cells . more specifically , demonstration of ptf1a expression has remained elusive until now , possibly owing to the lack of an optimal antibody against this protein . the same holds true for ngn3 , which has been detected at the protein level in only few studies . in our experimental setup , ptf1a transcripts were detected only at low levels , whereas a significant emergence of nkx6.1-positive cells was denoted . nevertheless , when combined with the demonstration of ins and pdx1 co - expression at later stages , these findings point toward the pancreatic nature of the differentiated pdx1 cells . additional efforts are required to obtain their efficient differentiation toward pancreatic endocrine cells in vitro ( table 3 ) ( 14,20 ) . during pancreas development , the pdx1 progenitor cells are amplified thanks to signals emanating from the pancreatic mesenchyme . fgf10 controls this mesenchymal - to - epithelial interaction , and its over - expression beyond the competence window ( e11.5e13.5 in the mouse ) significantly induces progenitor cell proliferation and arrests their terminal differentiation ( 40,41 ) . this signaling operates via activation of the notch pathway , which temporally prevents progenitor differentiation toward the endocrine or exocrine fates ( 42 ) . in agreement with these , fgf10 or egf supplementation induces the proliferation of hes - derived pdx1 cells , suggesting the contribution of erk / mapk and akt activation in this effect ( 14,16,20 ) furthermore , before the proliferation step , establishment of the pdx1 progenitors from hes - derived definitive endoderm was also shown to be highly dependent on erk / mapk signaling and possibly mediated by fgf2 ( 20,43 ) . the initiation of endocrine differentiation from the pdx1 progenitors relies on the activation of ngn3 expression , which should be paralleled or preceded by downregulation of notch activity and further sustained by additional induction of pax6 ( fig . although the transcription factors cascade regulating pancreatic endocrine differentiation has been largely explored , to date the instructive extracellular signals that trigger their coordinated expression remain elusive . to this end , there is a growing list of proposed growth factors and chemicals whose effects will need to be extensively assessed in the endocrine commitment and maturation of pdx1 progenitors obtained in vitro from hes - derived de cells . these include but are not limited to wortmannin and ly294002 that are pi3k inhibitors ( 46 ) , vegf ( 4,47 ) , ra ( 26 ) , conophylline ( 48 ) , wnt , and hedgehog ligands ( 49,50 ) , and antagonists of the notch pathway such as gamma secretase inhibitors , notch ligands , and soluble notch . not only is the nature of these factors important , but their timely combination will also be of consideration given the demonstration of a competence window for endocrine patterning beyond which the endocrine differentiation arrest becomes irreversible ( 41 ) . for instance in our experimental setup ( table 3 ) , fgf10 was supplemented for controlling pdx1 cell proliferation together with a gamma secretase inhibitor ( compound e ) for notch antagonism in view of endocrine induction ( 20 ) . considering the above - mentioned crosstalk between fgf10 and notch activity in the proliferation of the pdx1 progenitors , such a protocol therefore included two phenomenas that were actually antagonistic . this issue might explain the very limited progression toward endocrine cells that we obtained , a hypothesis that will need further evaluation by dissociating fgf10 treatment from notch inhibition in this protocol . the next step after endocrine commitment will be the further endocrine differentiation and maturation by use of old and new players such as betacellulin , activin , exendin-4 , insulin - like growth factor , hgf , nicotinamide , bone morphogenetic protein , fgf , and ra ( 14,16,18,20,26,28 ) . similarly , three - dimensional cultures will merit further investigation in order to mimic the actual environment of the -cells in islets as was recently described in differentiating he s cells ( 16 ) . some of these factors have been used singly or in combination on pdx1 progenitors generated from he s cells . because of the complex nature of pancreatic endocrine differentiation , which is presently not yet elucidated , the generation of mature and functional -cells has not been efficiently achieved in vitro . the demonstration of human - specific cell functions in animal models following transplantation of pancreatic progenitors derived from he s cells ( 19,32,51 ) underscores this gap in our current knowledge . based on these data , it has been proposed that diabetic patients could be grafted with pancreatic progenitors derived from he s cells , eliminating the need for finding the efficient strategy for terminal differentiation in vitro . however , this approach would be hampered by teratoma formation from cells that were not fully differentiated at the time of transplantation ( see below ) . although the differentiation of specific cell types such as pancreatic cells can be established through their characterization with several markers , it remains obvious that no protocol currently offers a factual conversion of all cultured stem cells to the desired phenotype . therefore , the possibility exists that other cell types or even multi- or pluripotent cells might be transplanted to the diabetic recipients , which raises safety issues . indeed , despite the fact that functional -cells developed in vivo after transplantation of hes - derived pancreatic cell preparations , teratoma formation was also observed in at least 15% of grafts ( 19,51,52 ) . several tools have been investigated in order to select out the undifferentiated cells that express surface markers ( ssea4 , ssea3 , tra-160 , tra-181 ) or to specifically isolate the desired cell type ( cell - trapping ) that have been modified to express an antibiotic resistance gene , a fluorescent or a bioluminescent reporter under ck19 , pdx1 , or ins promoter ( 5355 ) . whereas these techniques would allow for the selection of differentiated -cells up to purity , transplantation of such cells is presently not approved given the genotoxic , the mutagenic , and the oncogenic risks associated with transgenic approaches . the in vitro differentiation of functional -cells from human pluripotent cells ( he s and ips ) represents a major challenge for diabetes cell therapy in the future . progress toward this goal already encompasses the efficient generation of definitive endoderm and pdx1-expressing pancreas progenitors . the recent successful studies indicate that the differentiation of pdx1 progenitors concomitantly requires at least two events after de establishment in order to mimic the in vivo situation : blockade of hepatic induction by bmp antagonism and induction of pancreas progenitors by retinoid signaling ( fig . optimization of this step might still prove beneficial in order to unravel the role of fgf and hedgehog modulation and in order to definitely establish cells that undoubtedly show combined expression of the major transcription factors recognized for the multipotent pancreas progenitor , namely pdx1 , ptf1a , and nkx6.1 . in addition , the recently described temporal shift in the requirement for bmp antagonism to a need for bmp signaling during mouse pancreas development will merit implementation in he s cell differentiation . despite the latest data indicating differentiation of the pdx1 progenitors into insulin / c - peptide cells , we believe that additional work is needed for an optimal in vitro control of the progression toward ngn3-expressing endocrine progenitor cells and for the maturation of glucose - sensing and insulin - secretion functions in the -like cells . a step forward toward these goals will certainly shorten the time frame for achieving our objective of using he s cells as alternative sources of functioning -cells in diabetes cell therapy .

recent studies with human embryonic stem ( he s ) cells have established new protocols for substantial generation of pancreatic progenitors from definitive endoderm . these findings add to the efficient derivation of definitive endoderm , which is controlled by wnt and nodal pathways , and delineate a step forward in the quest for alternative -cell sources . it also indicates that critical refining of the available strategies might help define a universal protocol for pancreatic differentiation applicable to several cell lines , therefore offering the possibility for transplantation of immune - matched or patient - specific hes derived -cells . we appraise here the fundamental role that bone morphogenetic protein , fibroblast growth factor , and retinoid signaling play during pancreas development , and describe a fundamental emergence of their combination in recent studies that generated pancreatic cells from he s cells . we finally enumerate some prospects that might improve further differentiation of the progenitor cells into functional -cells needed in diabetes cell therapy .

Overview of embryonic pancreas development. Efficient definitive endoderm differentiation from many human embryonic stem cell lines and protocols. Noggin and retinoids: key players in blocking in vitro liver induction and favoring pancreas specification from definitive endoderm. Amplification of early pancreas progenitors by FGF signaling. Prospects for further endocrine differentiation of hES cellderived pancreatic progenitors. Molecular beacons for cell selection. Concluding remarks.

the regulation of pancreas development from the gut endoderm is a complex process that involves a carefully balanced interplay between several common signaling pathways . studies in several model organisms indicate conservation of the main mechanisms of pancreas development from lower to higher vertebrates ( table 1 ) . gastrulation in the developing embryo ( e7.5 in mice ) results in the formation of three germ layers , namely the ectoderm , the endoderm , and the mesoderm . time scale of pancreas development in mouse and human the major sequences of developmental processes are similar in mouse and human embryonic pancreas . the striking difference resides in the duration of each stage , which can vary by a factor of 5 to 15 . as far as the pancreas is concerned , specific growth and differentiation factors released by adjacent tissues control a set of transcription factors expression , resulting in the patterning of the ventral and dorsal prepancreatic endoderm . although our present knowledge of extrinsic factors that control patterning , proliferation , and differentiation cues throughout the developing pancreas is not yet complete , there is strong evidence emanating from all vertebrate developmental models that complex spatio - temporal combinations of common signaling pathways are crucial for appropriate specification of pancreatic cell fates from the endoderm ( 69 ) . these involve , for instance , the hedgehog , wnt , retinoid , and notch pathways , as well as activin / bone morphogenetic protein ( bmp ) , fibroblast growth factor ( fgf ) , vascular endothelial growth factor ( vegf ) , epidermal growth factor ( egf ) , and hepatocyte growth factor ( hgf ) signal transductions . an overview of the roles played in pancreas development by the most relevant growth and differentiation factors is summarized in table 2 . it is also worth noting that the developmental effects elicited by these common signals can vary significantly according to the cell type being stimulated or according to the stage of development when the signal occurs . overview of growth and differentiation factors that participate in vertebrate pancreas development cmes , cardiac mesoderm ; dmes , dorsal mesenchyme ; gcg , glucagon ; gdf , growth differentiation factor ; hb - egf , heparin - binding epidermal growth factor ; ihh , indian hedgehog ; lpm , lateral plate mesoderm ; pepi , pancreatic epithelium ; pmes , pancreatic mesenchyme ; pnep , pancreatic ngn3 endocrine progenitor . , considerable progress has been achieved in understanding pancreas embryogenesis thanks to the numerous genetic studies involved in transcription factors . most of them belong to the homeobox domain , the paired - box , or the basic helix - loop - helix families . expression of different combinations of tfs determines the sequential lineage segregation in the developing pancreas . it is presently well recognized that attempts to generate pancreatic cells from pluripotent cells should first establish a definitive endoderm ( de ) population as occurs in vivo . wnt and nodal signals were identified as sufficient to induce de from mouse and human embryonic stem ( he s ) cells , but also from induced pluripotent stem ( ips ) cells . in this model , high concentrations of activin a ( acta ) are used to mimic the function of the endogenous endoderm inducer nodal ( 11 ) . because active pi3k signaling is inhibitory for de differentiation from embryonic stem ( es ) cells , antagonists of this pathway ( ly294002 , wortmanin ) were also included in some protocols , resulting in an increased efficiency ( 14,15 ) . in addition , supplementation of sodium butyrate was found to support endoderm differentiation when combined with acta ( 16 ) , though it remains unclear how this chemical participates in the signaling network required for the definitive endoderm . the standard and modified protocols for de induction from es cells allow for the derivation of at least 6080% cells that express the characteristic markers foxa2 , sox17 , gsc , and cxcr4 ( table 3 ) but not the visceral endoderm marker sox7 ( 13,14,1620 ) . whether the quest for a universal -cell differentiation protocol is a desired myth or an achievable reality , it appears clear from the literature that combined wnt3a and acta treatments efficiently activate de phenotype in a vast majority of he s cell lines . despite the recently described differences in the response of he s cell lines to differentiation cues ( 21 ) , the variability and the low success rate in the outcome of initial pancreas differentiation protocols might first of all reflect the lack of such a universal developmentally based strategy that thoroughly reproduces in vivo events in the culture dish after the de stage . derivation of pdx1 progenitors from he s cells recent models of pancreas differentiation from he s cells integrate bmp antagonism and retinoid signaling early after definitive endoderm induction . when maintained in culture without added growth factors or when transplanted under the kidney capsule of immunocompromised mice , hes - derived definitive endoderm cells could spontaneously generate hepatocyte - like cells characterized by the expression of afp , albumin ( alb ) ( fig . the contribution of these pathways was further demonstrated by the treatment with bmp antagonist noggin and fgf receptor antagonist su5402 , leading to the complete abrogation of hepatic gene expression from de progenies that otherwise was mainly composed of hepatocytes . as suggested from animal studies , these experiments also indicated that fgf did not function in the induction but essentially contributed to the amplification of liver progenitors initially induced by bmp signaling ( 20 ) . expression of pancreas - related transcription factors in hes - derived de progenies treated with noggin , ra , and cyclopamine . foxa2 and albumin expression in control cultures ( a ) and following treatment with noggin , ra , and cyclopamine ( b ) . ( a high - quality digital representation of this figure is available in the online issue . ) in vivo , bmp activity generated by the adjacent mesoderm derived structures and operating in the ventral endoderm is associated with hepatic induction during early embryonic development . the requirement for bmp antagonism is reverted after pancreas induction since this organ requires bmp signaling later on for the maintenance of pdx1 expression and further differentiation . this underscores the existence of a tight competence window and the versatile functions of growth factors during development , which complicates their in vitro implementation for directed differentiation of es cells ( 24 ) . retinoic acid ( ra ) plays a crucial role shortly after gastrulation in endoderm patterning and in the induction of pdx1 expression by pancreas progenitors in several organisms from xenopus to humans , which indicates the conservation of its activity during evolution ( 2527 ) . this role for ra has already been evaluated during mouse and human es cell differentiation in vitro and has proven to be relevant for the induction of pancreatic gene expression , notably detectable levels of pdx1 ( 17,23,2832 ) . in line with the in vivo recognized functions of these two signaling pathways on pancreas induction , recent studies have combined bmp inhibition ( noggin supplementation ) with retinoid signaling immediately after the de induction stage and succeeded in producing large amounts ( up to 80% ) of pdx1 pancreas progenitor cells ( table 3 ) , ( 13,14,20 ) . this combination was efficient not only in different he s cell lines , but also in ips cells ( 14 ) , suggesting that the basic molecular events that should be monitored in order to design a universal pancreatic differentiation protocol are being progressively clarified . these minimally include the signals for the blockade of hepatic induction on the one hand and those required for pancreatic induction ( namely pdx1 expression ) on the other ( fig . the necessity for such a combination was recently highlighted by treatment of hes - derived de cells with noggin in the absence of ra ; this blocked the expression of liver markers , but did not generate pdx1 cells ( 20,23 ) . it is not yet clear whether this inductive effect on bmp also occurs during he s cell differentiation . nevertheless , considering the in vivo function of bmp signaling in the midgut endoderm , it appears reasonable that both retinoid signaling and bmp antagonism were required in these studies for strong induction of a pancreatic fate from the de cells while preventing hepatic differentiation . however , it is likely that the effect of ra is context - dependent because when applied on hes - derived de cells cultured at low density , it resulted in the inhibition of smad 1/5/8 phosphorylation ( 23 ) . four main pathways regulate the early stages of pancreas differentiation from definitive endoderm and control the acquisition of hepatic vs. pancreatic fate . hedgehog signaling is well characterized as a potent inhibitor of pancreatic initiation ( a ) that is expressed in the hepatic domain ( b ) under the influence of fgf signaling . retinoic acid is expressed in the developing pancreas and participates in the induction of pdx1 expression ( c ) . an inhibitory effect of retinoic acid on smad1 - 5 - 8 phosphorylation ( e ) was demonstrated on hes - derived definitive endoderm seeded at low density , which contrasts with the early studies indicating activation of bmp signaling by retinoic acid . this potent inhibitory effect at early stages gets reverted afterward and pancreatic progenitors require bmp signaling ( f ) . in late stages , fgf plays in the proliferation of pancreas progenitors ( j ) . on the contrary the markers displayed in the progenitors recall the current status of protein detection from differentiated he s cells . other studies made use of ra combined with fgf ligands ( fgf4 , fgf7 , fgf10 ) or of noggin combined with fgf2 and egf to induce pancreatic cells albeit at a lower efficiency than for combined noggin and ra ( table 3 ) ( 13,14,16,17,19,23 ) . 3 ] ) given that the addition of the mapk - inhibitor u1026 prevented pancreatic cell differentiation ( 20 ) . this view might resolve the nature of factors x suggested as involved in the indirect effects of ra in xenopus dorsal endoderm ( 37 ) . it was recently suggested that in the presence of fgf7 , ra can efficiently induce pdx1 + progenitors from hes - derived de cells seeded at low density ( 550,000 cells / cm ) and that this effect is paralleled by inhibition of smad1/5/8 phosphorylation , therefore recapitulating bmp antagonism described above ( 23 ) . recent analysis of the signaling network during early liver and pancreas development in 36 somites - stage mouse embryos further indicated that inhibition of the activin signaling significantly increases the proportion of cells fated toward pdx1 expression . these findings therefore suggest that the addition of activin inhibitors to the above - mentioned cocktails might still boost the occurrence of pdx1 cells in he s cell cultures ( 13,24 ) . despite the fact that combining bmp antagonism with retinoid signaling efficiently generates pdx1 pancreatic progenitors from hes - derived definitive endoderm , the requirement for hedgehog antagonism with cyclopamine during pancreas induction from these cells has not been clarified by these studies . ( 18 ) indicated the necessity to antagonize hedgehog signaling that is indeed activated during es cell differentiation , and it was shown to also play a negative role ex vivo by limiting endocrine and exocrine genes expression in embryonic ( e12.5 ) mouse pancreatic explants ( 38,39 ) . owing to the previously described increase in the expression of hedgehog ligands and effectors upon de induction , we added cyclopamine to the combination of noggin and ra in our experimental setup , but it remains unclear whether this was mandatory for the efficient differentiation of pdx1 cells that we observed ( 20,38,39 ) . while waiting for conclusive data on this issue , we speculate that supplementation of hedgehog antagonists would become obsolete in settings where combined noggin and ra treatment of de cells essentially induces a true pancreatic endoderm cell type , which is normally devoid of hedgehog activity . pancreatic progenitor cells are characterized by their expression of a subset of transcription factors . for instance , the combined expression of pdx1 , foxa2 , sox9 , hnf6 , nkx6.1 , and ptf1a ( fig . the last two transcription factors are more specific to the pancreas , whereas pdx1 , foxa2 , sox9 , and hnf6 are also detected in the adjacent duodenum , stomach , and liver . following the induction of pancreas progenitor cells from hes - derived de cells , pdx1 co - expression with foxa2 , sox9 , and hnf6 was demonstrated in a context where hepatic ( afp , alb ) and intestinal ( cdx1 , cdx2 ) markers were not induced or only minimally induced ( fig . however , none of the above - mentioned studies have assessed the expression of all the six transcription factors by the pancreatic progenitors obtained from he s cells . the same holds true for ngn3 , which has been detected at the protein level in only few studies . in our experimental setup , ptf1a transcripts were detected only at low levels , whereas a significant emergence of nkx6.1-positive cells was denoted . nevertheless , when combined with the demonstration of ins and pdx1 co - expression at later stages , these findings point toward the pancreatic nature of the differentiated pdx1 cells . during pancreas development , the pdx1 progenitor cells are amplified thanks to signals emanating from the pancreatic mesenchyme . fgf10 controls this mesenchymal - to - epithelial interaction , and its over - expression beyond the competence window ( e11.5e13.5 in the mouse ) significantly induces progenitor cell proliferation and arrests their terminal differentiation ( 40,41 ) . this signaling operates via activation of the notch pathway , which temporally prevents progenitor differentiation toward the endocrine or exocrine fates ( 42 ) . in agreement with these , fgf10 or egf supplementation induces the proliferation of hes - derived pdx1 cells , suggesting the contribution of erk / mapk and akt activation in this effect ( 14,16,20 ) furthermore , before the proliferation step , establishment of the pdx1 progenitors from hes - derived definitive endoderm was also shown to be highly dependent on erk / mapk signaling and possibly mediated by fgf2 ( 20,43 ) . the initiation of endocrine differentiation from the pdx1 progenitors relies on the activation of ngn3 expression , which should be paralleled or preceded by downregulation of notch activity and further sustained by additional induction of pax6 ( fig . to this end , there is a growing list of proposed growth factors and chemicals whose effects will need to be extensively assessed in the endocrine commitment and maturation of pdx1 progenitors obtained in vitro from hes - derived de cells . these include but are not limited to wortmannin and ly294002 that are pi3k inhibitors ( 46 ) , vegf ( 4,47 ) , ra ( 26 ) , conophylline ( 48 ) , wnt , and hedgehog ligands ( 49,50 ) , and antagonists of the notch pathway such as gamma secretase inhibitors , notch ligands , and soluble notch . considering the above - mentioned crosstalk between fgf10 and notch activity in the proliferation of the pdx1 progenitors , such a protocol therefore included two phenomenas that were actually antagonistic . the next step after endocrine commitment will be the further endocrine differentiation and maturation by use of old and new players such as betacellulin , activin , exendin-4 , insulin - like growth factor , hgf , nicotinamide , bone morphogenetic protein , fgf , and ra ( 14,16,18,20,26,28 ) . similarly , three - dimensional cultures will merit further investigation in order to mimic the actual environment of the -cells in islets as was recently described in differentiating he s cells ( 16 ) . some of these factors have been used singly or in combination on pdx1 progenitors generated from he s cells . because of the complex nature of pancreatic endocrine differentiation , which is presently not yet elucidated , the generation of mature and functional -cells has not been efficiently achieved in vitro . the demonstration of human - specific cell functions in animal models following transplantation of pancreatic progenitors derived from he s cells ( 19,32,51 ) underscores this gap in our current knowledge . based on these data , it has been proposed that diabetic patients could be grafted with pancreatic progenitors derived from he s cells , eliminating the need for finding the efficient strategy for terminal differentiation in vitro . although the differentiation of specific cell types such as pancreatic cells can be established through their characterization with several markers , it remains obvious that no protocol currently offers a factual conversion of all cultured stem cells to the desired phenotype . therefore , the possibility exists that other cell types or even multi- or pluripotent cells might be transplanted to the diabetic recipients , which raises safety issues . indeed , despite the fact that functional -cells developed in vivo after transplantation of hes - derived pancreatic cell preparations , teratoma formation was also observed in at least 15% of grafts ( 19,51,52 ) . whereas these techniques would allow for the selection of differentiated -cells up to purity , transplantation of such cells is presently not approved given the genotoxic , the mutagenic , and the oncogenic risks associated with transgenic approaches . the in vitro differentiation of functional -cells from human pluripotent cells ( he s and ips ) represents a major challenge for diabetes cell therapy in the future . progress toward this goal already encompasses the efficient generation of definitive endoderm and pdx1-expressing pancreas progenitors . the recent successful studies indicate that the differentiation of pdx1 progenitors concomitantly requires at least two events after de establishment in order to mimic the in vivo situation : blockade of hepatic induction by bmp antagonism and induction of pancreas progenitors by retinoid signaling ( fig . optimization of this step might still prove beneficial in order to unravel the role of fgf and hedgehog modulation and in order to definitely establish cells that undoubtedly show combined expression of the major transcription factors recognized for the multipotent pancreas progenitor , namely pdx1 , ptf1a , and nkx6.1 . in addition , the recently described temporal shift in the requirement for bmp antagonism to a need for bmp signaling during mouse pancreas development will merit implementation in he s cell differentiation . despite the latest data indicating differentiation of the pdx1 progenitors into insulin / c - peptide cells , we believe that additional work is needed for an optimal in vitro control of the progression toward ngn3-expressing endocrine progenitor cells and for the maturation of glucose - sensing and insulin - secretion functions in the -like cells . a step forward toward these goals will certainly shorten the time frame for achieving our objective of using he s cells as alternative sources of functioning -cells in diabetes cell therapy .

studies in several model organisms indicate conservation of the main mechanisms of pancreas development from lower to higher vertebrates ( table 1 ) . gastrulation in the developing embryo ( e7.5 in mice ) results in the formation of three germ layers , namely the ectoderm , the endoderm , and the mesoderm . several structures temporally participate in these early tissue interactions including the notochord ( 2 ) , the cardiac mesoderm , the septum transversum mesenchyme and the lateral plate mesoderm ( 3 ) , the aortic endothelial cells and the vitelline veins ( 4 ) , and the developing pancreatic mesenchyme ( 5 ) . time scale of pancreas development in mouse and human the major sequences of developmental processes are similar in mouse and human embryonic pancreas . as far as the pancreas is concerned , specific growth and differentiation factors released by adjacent tissues control a set of transcription factors expression , resulting in the patterning of the ventral and dorsal prepancreatic endoderm . although our present knowledge of extrinsic factors that control patterning , proliferation , and differentiation cues throughout the developing pancreas is not yet complete , there is strong evidence emanating from all vertebrate developmental models that complex spatio - temporal combinations of common signaling pathways are crucial for appropriate specification of pancreatic cell fates from the endoderm ( 69 ) . these involve , for instance , the hedgehog , wnt , retinoid , and notch pathways , as well as activin / bone morphogenetic protein ( bmp ) , fibroblast growth factor ( fgf ) , vascular endothelial growth factor ( vegf ) , epidermal growth factor ( egf ) , and hepatocyte growth factor ( hgf ) signal transductions . an overview of the roles played in pancreas development by the most relevant growth and differentiation factors is summarized in table 2 . it is also worth noting that the developmental effects elicited by these common signals can vary significantly according to the cell type being stimulated or according to the stage of development when the signal occurs . this level of complexity is further enhanced by the interplay between transcription factors that lie downstream of these extracellular stimuli and are responsible for observed developmental features . overview of growth and differentiation factors that participate in vertebrate pancreas development cmes , cardiac mesoderm ; dmes , dorsal mesenchyme ; gcg , glucagon ; gdf , growth differentiation factor ; hb - egf , heparin - binding epidermal growth factor ; ihh , indian hedgehog ; lpm , lateral plate mesoderm ; pepi , pancreatic epithelium ; pmes , pancreatic mesenchyme ; pnep , pancreatic ngn3 endocrine progenitor . most of them belong to the homeobox domain , the paired - box , or the basic helix - loop - helix families . 1 with special focus on 1 ) the early pancreatic epithelial cells , 2 ) the pancreatic endocrine progenitors , and 3 ) the endocrine subtypes selection . wnt and nodal signals were identified as sufficient to induce de from mouse and human embryonic stem ( he s ) cells , but also from induced pluripotent stem ( ips ) cells . in this model , high concentrations of activin a ( acta ) are used to mimic the function of the endogenous endoderm inducer nodal ( 11 ) . beside the activation of this pathway , initial treatment with wnt3a or bmp4 was found to improve the efficiency of de induction by setting up a transient mesendoderm progenitor population , especially when cultures are performed in feeder cell - free conditions ( 12,13 ) . because active pi3k signaling is inhibitory for de differentiation from embryonic stem ( es ) cells , antagonists of this pathway ( ly294002 , wortmanin ) were also included in some protocols , resulting in an increased efficiency ( 14,15 ) . in addition , supplementation of sodium butyrate was found to support endoderm differentiation when combined with acta ( 16 ) , though it remains unclear how this chemical participates in the signaling network required for the definitive endoderm . the standard and modified protocols for de induction from es cells allow for the derivation of at least 6080% cells that express the characteristic markers foxa2 , sox17 , gsc , and cxcr4 ( table 3 ) but not the visceral endoderm marker sox7 ( 13,14,1620 ) . whether the quest for a universal -cell differentiation protocol is a desired myth or an achievable reality , it appears clear from the literature that combined wnt3a and acta treatments efficiently activate de phenotype in a vast majority of he s cell lines . despite the recently described differences in the response of he s cell lines to differentiation cues ( 21 ) , the variability and the low success rate in the outcome of initial pancreas differentiation protocols might first of all reflect the lack of such a universal developmentally based strategy that thoroughly reproduces in vivo events in the culture dish after the de stage . cyclo , cyclopamine ; dapt , n-[n-(3,5-difluorophenacetyl)-l - alanyl]-s - phenylglycine t - butyl ester ( gamma secretase inhibitor ) ; ebs , embryoid bodies ; ex4 , exendin-4 ; fbs , fetal bovine serum ; its , insulin selenium transferring supplement ; na , nicotinamide ; nabut , sodium butyrate ; ng , noggin ; stz , streptozotocin ; 3d , three dimensional . when maintained in culture without added growth factors or when transplanted under the kidney capsule of immunocompromised mice , hes - derived definitive endoderm cells could spontaneously generate hepatocyte - like cells characterized by the expression of afp , albumin ( alb ) ( fig . this tendency for hepatic lineage commitment was also reported in several studies wherein hes - derived de cells were exposed to bmp and/or fgf , two growth factors implicated in embryonic liver induction and specification ( 13,20,22,23 ) . the contribution of these pathways was further demonstrated by the treatment with bmp antagonist noggin and fgf receptor antagonist su5402 , leading to the complete abrogation of hepatic gene expression from de progenies that otherwise was mainly composed of hepatocytes . as suggested from animal studies , these experiments also indicated that fgf did not function in the induction but essentially contributed to the amplification of liver progenitors initially induced by bmp signaling ( 20 ) . expression of pancreas - related transcription factors in hes - derived de progenies treated with noggin , ra , and cyclopamine . in vivo , bmp activity generated by the adjacent mesoderm derived structures and operating in the ventral endoderm is associated with hepatic induction during early embryonic development . on the contrary , ventral pancreas progenitors are located away from the midline endoderm with active bmp signaling , whereas bmp inhibition in the dorsal endoderm plays a crucial function in the acquisition of a pancreatic fate ( 3,24 ) . this underscores the existence of a tight competence window and the versatile functions of growth factors during development , which complicates their in vitro implementation for directed differentiation of es cells ( 24 ) . retinoic acid ( ra ) plays a crucial role shortly after gastrulation in endoderm patterning and in the induction of pdx1 expression by pancreas progenitors in several organisms from xenopus to humans , which indicates the conservation of its activity during evolution ( 2527 ) . this role for ra has already been evaluated during mouse and human es cell differentiation in vitro and has proven to be relevant for the induction of pancreatic gene expression , notably detectable levels of pdx1 ( 17,23,2832 ) . in line with the in vivo recognized functions of these two signaling pathways on pancreas induction , recent studies have combined bmp inhibition ( noggin supplementation ) with retinoid signaling immediately after the de induction stage and succeeded in producing large amounts ( up to 80% ) of pdx1 pancreas progenitor cells ( table 3 ) , ( 13,14,20 ) . this combination was efficient not only in different he s cell lines , but also in ips cells ( 14 ) , suggesting that the basic molecular events that should be monitored in order to design a universal pancreatic differentiation protocol are being progressively clarified . these minimally include the signals for the blockade of hepatic induction on the one hand and those required for pancreatic induction ( namely pdx1 expression ) on the other ( fig . the necessity for such a combination was recently highlighted by treatment of hes - derived de cells with noggin in the absence of ra ; this blocked the expression of liver markers , but did not generate pdx1 cells ( 20,23 ) . indeed , retinoid signaling was shown to activate bmp expression in several in vivo and in vitro systems including embryonal carcinoma cells and differentiating mouse embryoid bodies ( 3436 ) . nevertheless , considering the in vivo function of bmp signaling in the midgut endoderm , it appears reasonable that both retinoid signaling and bmp antagonism were required in these studies for strong induction of a pancreatic fate from the de cells while preventing hepatic differentiation . however , it is likely that the effect of ra is context - dependent because when applied on hes - derived de cells cultured at low density , it resulted in the inhibition of smad 1/5/8 phosphorylation ( 23 ) . hedgehog signaling is well characterized as a potent inhibitor of pancreatic initiation ( a ) that is expressed in the hepatic domain ( b ) under the influence of fgf signaling . an inhibitory effect of retinoic acid on smad1 - 5 - 8 phosphorylation ( e ) was demonstrated on hes - derived definitive endoderm seeded at low density , which contrasts with the early studies indicating activation of bmp signaling by retinoic acid . the inhibition of bmp signaling in the pancreatic domain is under the control of noggin ( h ) . other studies made use of ra combined with fgf ligands ( fgf4 , fgf7 , fgf10 ) or of noggin combined with fgf2 and egf to induce pancreatic cells albeit at a lower efficiency than for combined noggin and ra ( table 3 ) ( 13,14,16,17,19,23 ) . our unpublished observations suggest that fgf signals favor hepatic gene expression in the absence of bmp antagonism , but the addition of fgf ligands together with bmp antagonism ( noggin ) and ra enhances pancreatic gene expression . furthermore , the induction of pdx1 cells by combined noggin and ra requires at least a basal level of fgf activity ( via the extracellular signal regulated kinase / mitogen - activated protein kinase [ erk / mapk ] pathway [ fig . because ra is known to act synergistically with fgf signaling during endoderm patterning , supplemented fgf can be seen as an enhancing factor in protocols that already integrate noggin and ra ( 13,14,19 ) . it was recently suggested that in the presence of fgf7 , ra can efficiently induce pdx1 + progenitors from hes - derived de cells seeded at low density ( 550,000 cells / cm ) and that this effect is paralleled by inhibition of smad1/5/8 phosphorylation , therefore recapitulating bmp antagonism described above ( 23 ) . recent analysis of the signaling network during early liver and pancreas development in 36 somites - stage mouse embryos further indicated that inhibition of the activin signaling significantly increases the proportion of cells fated toward pdx1 expression . these findings therefore suggest that the addition of activin inhibitors to the above - mentioned cocktails might still boost the occurrence of pdx1 cells in he s cell cultures ( 13,24 ) . despite the fact that combining bmp antagonism with retinoid signaling efficiently generates pdx1 pancreatic progenitors from hes - derived definitive endoderm , the requirement for hedgehog antagonism with cyclopamine during pancreas induction from these cells has not been clarified by these studies . ( 18 ) indicated the necessity to antagonize hedgehog signaling that is indeed activated during es cell differentiation , and it was shown to also play a negative role ex vivo by limiting endocrine and exocrine genes expression in embryonic ( e12.5 ) mouse pancreatic explants ( 38,39 ) . however , additional studies using noggin , fgf ligands , and/or ra after de induction succeeded in generating pdx1 cells without the use of hedgehog inhibitors ( 14,17,19,23 ) . owing to the previously described increase in the expression of hedgehog ligands and effectors upon de induction , we added cyclopamine to the combination of noggin and ra in our experimental setup , but it remains unclear whether this was mandatory for the efficient differentiation of pdx1 cells that we observed ( 20,38,39 ) . while waiting for conclusive data on this issue , we speculate that supplementation of hedgehog antagonists would become obsolete in settings where combined noggin and ra treatment of de cells essentially induces a true pancreatic endoderm cell type , which is normally devoid of hedgehog activity . following the induction of pancreas progenitor cells from hes - derived de cells , pdx1 co - expression with foxa2 , sox9 , and hnf6 was demonstrated in a context where hepatic ( afp , alb ) and intestinal ( cdx1 , cdx2 ) markers were not induced or only minimally induced ( fig . however , none of the above - mentioned studies have assessed the expression of all the six transcription factors by the pancreatic progenitors obtained from he s cells . nevertheless , when combined with the demonstration of ins and pdx1 co - expression at later stages , these findings point toward the pancreatic nature of the differentiated pdx1 cells . fgf10 controls this mesenchymal - to - epithelial interaction , and its over - expression beyond the competence window ( e11.5e13.5 in the mouse ) significantly induces progenitor cell proliferation and arrests their terminal differentiation ( 40,41 ) . this signaling operates via activation of the notch pathway , which temporally prevents progenitor differentiation toward the endocrine or exocrine fates ( 42 ) . in agreement with these , fgf10 or egf supplementation induces the proliferation of hes - derived pdx1 cells , suggesting the contribution of erk / mapk and akt activation in this effect ( 14,16,20 ) furthermore , before the proliferation step , establishment of the pdx1 progenitors from hes - derived definitive endoderm was also shown to be highly dependent on erk / mapk signaling and possibly mediated by fgf2 ( 20,43 ) . the initiation of endocrine differentiation from the pdx1 progenitors relies on the activation of ngn3 expression , which should be paralleled or preceded by downregulation of notch activity and further sustained by additional induction of pax6 ( fig . to this end , there is a growing list of proposed growth factors and chemicals whose effects will need to be extensively assessed in the endocrine commitment and maturation of pdx1 progenitors obtained in vitro from hes - derived de cells . these include but are not limited to wortmannin and ly294002 that are pi3k inhibitors ( 46 ) , vegf ( 4,47 ) , ra ( 26 ) , conophylline ( 48 ) , wnt , and hedgehog ligands ( 49,50 ) , and antagonists of the notch pathway such as gamma secretase inhibitors , notch ligands , and soluble notch . not only is the nature of these factors important , but their timely combination will also be of consideration given the demonstration of a competence window for endocrine patterning beyond which the endocrine differentiation arrest becomes irreversible ( 41 ) . considering the above - mentioned crosstalk between fgf10 and notch activity in the proliferation of the pdx1 progenitors , such a protocol therefore included two phenomenas that were actually antagonistic . the next step after endocrine commitment will be the further endocrine differentiation and maturation by use of old and new players such as betacellulin , activin , exendin-4 , insulin - like growth factor , hgf , nicotinamide , bone morphogenetic protein , fgf , and ra ( 14,16,18,20,26,28 ) . because of the complex nature of pancreatic endocrine differentiation , which is presently not yet elucidated , the generation of mature and functional -cells has not been efficiently achieved in vitro . the demonstration of human - specific cell functions in animal models following transplantation of pancreatic progenitors derived from he s cells ( 19,32,51 ) underscores this gap in our current knowledge . based on these data , it has been proposed that diabetic patients could be grafted with pancreatic progenitors derived from he s cells , eliminating the need for finding the efficient strategy for terminal differentiation in vitro . although the differentiation of specific cell types such as pancreatic cells can be established through their characterization with several markers , it remains obvious that no protocol currently offers a factual conversion of all cultured stem cells to the desired phenotype . indeed , despite the fact that functional -cells developed in vivo after transplantation of hes - derived pancreatic cell preparations , teratoma formation was also observed in at least 15% of grafts ( 19,51,52 ) . several tools have been investigated in order to select out the undifferentiated cells that express surface markers ( ssea4 , ssea3 , tra-160 , tra-181 ) or to specifically isolate the desired cell type ( cell - trapping ) that have been modified to express an antibiotic resistance gene , a fluorescent or a bioluminescent reporter under ck19 , pdx1 , or ins promoter ( 5355 ) . whereas these techniques would allow for the selection of differentiated -cells up to purity , transplantation of such cells is presently not approved given the genotoxic , the mutagenic , and the oncogenic risks associated with transgenic approaches . the recent successful studies indicate that the differentiation of pdx1 progenitors concomitantly requires at least two events after de establishment in order to mimic the in vivo situation : blockade of hepatic induction by bmp antagonism and induction of pancreas progenitors by retinoid signaling ( fig . optimization of this step might still prove beneficial in order to unravel the role of fgf and hedgehog modulation and in order to definitely establish cells that undoubtedly show combined expression of the major transcription factors recognized for the multipotent pancreas progenitor , namely pdx1 , ptf1a , and nkx6.1 . in addition , the recently described temporal shift in the requirement for bmp antagonism to a need for bmp signaling during mouse pancreas development will merit implementation in he s cell differentiation . despite the latest data indicating differentiation of the pdx1 progenitors into insulin / c - peptide cells , we believe that additional work is needed for an optimal in vitro control of the progression toward ngn3-expressing endocrine progenitor cells and for the maturation of glucose - sensing and insulin - secretion functions in the -like cells .

celem niniejszej pracy jest przedstawienie obecnej taktyki postpowania zarwno diagnostycznego , jak i terapeutycznego u chorych z pseudotorbielami oraz torbielowatymi guzami trzustki . praca zostaa napisana na podstawie materiau zebranego i przygotowanego w klinice autora oraz wspczesnych doniesie z zaprezentowanego pimiennictwa . w latach 20002012 w ii klinice chirurgii oglnej , gastroenterologicznej i nowotworw ukadu pokarmowego przebywao 179 chorych z torbielowatymi zmianami w obrbie trzustki , w tej grupie stwierdzono 12 przypadkw guzw torbielowatych oraz 167 zmian o charakterze rzekomych torbieli u 75 wykonano zabieg drenaowy na drodze operacyjnej , 48 chorych zakwalifikowano do endoskopowej cystogastrostomii lub cystoduodenostomii . w grupie pacjentw z guzem torbielowatym trzustki na 12 badanych u 6 ( 50% ) wykonano lecznicz resekcj guza z odpowiedni czci narzdu . the aim of this paper was to present current management , both diagnostic and therapeutic , of patients with pancreatic pseudocysts and cystic tumors . the article has been written based on the material collected and prepared in the author 's department taking into consideration contemporary approaches and opinions included in the references . in 20002012 , the second department of general , gastrointestinal and oncological surgery of the alimentary tract treated 179 patients with cystic lesions in the region of the pancreas . the diagnostic procedures included : abdominal ultrasound and doppler examinations , ct and in certain cases , also eus and intraoperative ultrasound ( ious ) . when a cystic tumor was suspected , contrast enhanced ct , mri or mrcp were conducted . patients with suspected or confirmed neoplastic tumors were included in a separate group of cystic lesions . the majority of the lesions adhered to the pancreas but their outer outlines could border on various parts of the gastrointestinal tract and the mesentery or could be localized in the extraperitoneal space . the exact specification of adherence to , e.g. head , body or tail of the pancreas , is quite troublesome . therefore , in our department , the description usually specifies to which aspect of the pancreas , proximal or distal , the cyst adheres . in the material presented herein , the proximal localization ( head , body ) was found in 118 cases and distal ( body , tail ) in 61 cases . most of the treated lesions ranged from 3 to over 25 cm . in 2000 , we introduced endoscopic treatment method of pseudocysts . if in transabdominal ultrasound examination , cysts with defined walls are detected , located near the stomach or duodenum and indenting their lumina , eus and videoendoscopy are conducted to determine the indications for endoscopic cystogastrostomy or cystoduodenostomy . when the adequate puncture site in a non - vascular region has been selected in eus , it is marked with dye , spot coagulation or , currently , an appropriate drainage set is placed , directly guided by eus . with the use of the lateral - viewing duodenoscope and diathermy , the gastric wall and adjacent cyst are punctured . subsequently , having conducted the aspiration attempt , the prosthesis is inserted which allows for free outflow of cystic contents ( fig . the efficacy of the procedure is evaluated in sonography . up to 2012 , a total of 48 patients were qualified to endoscopic drainage procedures . eus - guided pancreatic pseudocyst drainage . puncturing the pseudocyst and inserting a guidewire eus - guided cystogastrostomy . the pancreatic juice flows through cystogastrostomy ( prosthesis is inserted to the lumen of the pseudocyst ) when the initial diagnosis can not rule out a cystic neoplasm of the pancreas , the patient undergoes detailed imaging examinations , biochemical and serological tests as well as , in certain cases , fine - needle aspiration biopsy ( fnab ) . in the subject group of 179 patients with cystic lesions in the region of the pancreas , 12 presented with cystic tumors and 167 with pseudocysts , both acute and chronic . from among all 167 cases with symptomatic cysts , 23 patients ( 13.8% ) were monitored only ( involution of the lesions during the observation period ) and the remaining 144 patients were qualified to procedures . in 75 patients ( 44.9% ) , the resection of the cyst was possible in merely 4 cases , the remaining patients underwent drainage procedures . in 24 cases , cystogastrostomy ( jurasz procedure ) was conducted and in 39 cases , roux - en - y cystojejunostomy was performed . in the remaining patients , complex procedures took place , such as drainage of several cysts or total drainage of pseudocysts and pancreatic duct , also performed according to roux - en - y technique . during postoperative monitoring period , complications were observed in 16% of patients . furthermore , 21 patients ( 12.6% ) with thin - walled retention cysts secondary to acute pancreatitis ( ap ) , were treated by external us - guided drainage . in 8 cases ( 38% ) , 48 patients ( 28.7% ) were qualified to endoscopic cystogastrostomy or cystoduodenostomy if transabdominal ultrasound and subsequently , eus had confirmed such qualification . in all cases , it was attempted to introduce an appropriate stent ( prosthesis ) to the cysts through the wall of the gastrointestinal tract . the efficacy of the procedure was usually evaluated on the second day following the procedure by means of us examination . drainage was successful in 37 patients , 12 of whom required correction of drain placement and a repeated procedure was necessary . due to technical reasons , the placement of the drain was impossible in 11 patients ( 23% ) they underwent surgical drainage instead . during examination , all patients underwent imaging examinations , such as : us examination with doppler mode , ct and in 4 patients , mri and mrcp . the analysis of the contents ( cytology , level of markers , test for mucous content ) was positive in only 2 patients . in the remaining cases the analysis was not reliable . due to the results of imaging examinations , in 5 patients with slight lesions ( mainly from 2 to 3 cm ) in whom a benign serous cystic neoplasm was suspected ( based on us / ct ) , observation was recommended . in this group , only one patient underwent fnab ( unreliable attempt ) . evident growth of the tumorous lesion was not observed in the monitored patients within the observation period of 23 years . in further 6 patients with over 4 cm lesions that showed features of neoplastic cystic tumors , resection of the tumor with an adequate part of the pancreas was performed . in 5 patients , fnab was conducted ( the result was positive only in 2 cases , see above ) . in 1 patient , a malignant neoplasm was confirmed which infiltrated adjacent structures and gave metastases to the liver . this case was considered inoperative ( as assessed in diagnostic laparoscopy with tissue sample collection ) . in patients diagnosed with and treated due to pancreatic pseudocysts , transabdominal ultrasound is the main diagnostic tool , followed by ct and eus , in qualifying patients to endoscopic treatment . in us thin - walled cisterns do not qualify to surgical treatment ( anastomosis ) due to the difficulties to perform anastomosis and the possibility of secondary leak . in such cases , external percutaneous drainage should be considered or , in certain favorable conditions internal endoscopic drainage . the treatment of pancreatic cysts should depend on their origin , character , size , location and individual clinical symptoms . slight cysts measuring not more than 4 cm which appeared after acute pancreatitis receive conservative treatment . they are usually necrotic collections of aseptic contents which do not contain any large breakdown fragments . slight lesions , from 3 to 4 cm , which may be effectively controlled by means of periodical us examinations , do not receive any treatment provided that their neoplastic character has been ruled out . only large cysts are qualified to procedures usually those measuring more than 56 cm particularly when other characteristic clinical symptoms are present , such as pain , obstructive jaundice , obstruction in the upper gastrointestinal tract , decreased appetite or weight loss ( fig . 2 ) . a similar situation refers to cysts whose continuous growth is observed in us check - up examinations and complicated cysts . in accordance with current principles , uncomplicated cysts and fluid collections secondary to ap are not subject to treatment if the period of us observation and monitoring is shorter than 6 weeks . in the discussed period of time , from among 167 patients diagnosed with symptomatic cysts of the pancreas , 44.9% underwent surgeries , 12.6% were treated by means of external drainage and 28.7% by internal eus - guided endoscopic drainage . still , the largest group underwent surgeries in spite of the fact that endoscopic procedures introduced in 2000 have a greater and greater therapeutic value . such a procedure was performed in 48 patients , but in 11 of them ( 23% ) endoscopic drainage was not successful mainly due to technical problems connected with prosthesis placement , difficult angular access of the endoscope to the cyst , bleeding and suspicion of perforation . in such cases , surgical drainage is the method of choice . in the remaining 37 patients with pancreatic pseudocysts , it allows for a rapid return to everyday activities , patients do not need to follow a rigorous diet ( as it happens after a surgery ) and their physical activity is not restricted due to the postoperative wound . at present , thanks to the advancement of diagnostic techniques , we may accurately assess and distinguish neoplastic cystic tumors of the pancreas from ordinary cystic lesions . it is currently believed that even 10% of all cases of cystic lesions are related to a neoplasm and that these lesions account for 15% of all pancreatic neoplasms . it is believed that asymptomatic lesions constitute approximately 3575% of cystic tumors of the pancreas . the appearance of certain symptoms may be associated with growth of the tumor , increased internal pressure or malignant transformation . the most common reported symptoms include : pain in the abdomen , nausea , vomiting and increased circumference of the abdomen , weight loss , obstructive jaundice , constipation or diarrhea and weakness . patients with ipmn may be sometimes treated due to recurrent episodes of acute pancreatitis , particularly , in cases when the main pancreatic duct is involved . large tumors are most frequently found in patients with mucinous cystic neoplasms ( mcn ) and spn these lesions usually give symptoms of a midabdominal tumor . exacerbation of pain is associated with sudden hemorrhage to the tumor or results from its rupture ( spn ) . in advanced malignant lesions , apart from the above - listed symptoms the following may occur : bleeding from the gastrointestinal tract in the case of gastric infiltration , portal hypertension and hemobilia as well as , eventually , diabetes when the pancreas is considerably damaged . in the analyzed time period , after thorough imaging analysis , including doppler ultrasound , contrast enhanced ct as well as in some cases , mri , mrcp and eus , 6 patients were qualified to fnab . only in 2 cases did fnab and assessment of the aspirated fluid confirm the presence of a cystic neoplasm . similarly in the quoted references imaging examinations are decisive when treatment - related decisions are made . the assessment of the aspirate is usually less specific and the final verification of the type of the tumor is acquired following the analysis of the postoperative specimen . in our material , the postoperative verification confirmed the presence of 3 serous cystadenomas and 3 mucinous cystadenomas in 2 cases , the lesions had already been identified as cystadenocarcinomas . in one patient , a malignant spn tumor was detected with slight metastases to the liver inoperative lesion . in further 5 patients , slight ( 23 cm ) tumor - like lesions they were identified as serous tumors which do not require surgical treatment . in these patients , annual check - ups were recommended which in the period of analysis , did not show any growth or change of the inner structure . according to numerous authors , such lesions do not require immediate qualification to resection and in the majority of cases , they do not change their appearance during the monitoring period . in the next part , the material has been collected based on quoted recent publications concerning diagnosis , methods of differentiation and proposed types of treatment . they are usually solitary , with the size of 1.535 cm and constitute approximately 30% of all primary pancreatic cystic tumors . we mainly encounter two types of such lesions : microcystic cystadenomas and macrocystic or oligocystic cystadenomas . the microcystic form usually develops in women at the age of 5060 and its typical localization is the body and tail of the pancreas . it is usually asymptomatic and signals , such as pain in the abdomen , weight loss , feeling of distension in the epigastric region and peristalsis disorders appear when the lesion shows large growth . a typical form of the tumor is composed of a large number of small cysts lined with cuboidal epithelium producing glycogen . the size of the cysts ranges from 0.2 to 2.0 cm and the entire tumor measures 1.430 cm . the lesion may grow locally leading to almost complete damage of the healthy pancreatic tissue . in imaging examinations ( us , ct ) , such a cystic tumor frequently has the appearance of a honeycomb which is related to a large number of small cysts ( fig . the lesion is relatively well - circumscribed with a central scar or , sometimes , with a calcification . serous cystadenoma ( microcystic type ) . a typical serous tumor is composed of multiple small cysts and has a the cystic mass is situated in the pancreatic tail , about 5 cm in diameter ious . well - defined microcystic tumor , poorly vascularized , adhering to the splenic vessels ( arrow ) ( color doppler ) doppler us . serous cystadenoma in the head of the pancreas , poorly vascularized , 5.6 cm in diameter . structure with oligocystic components and central scar ( arrow ) contrast - enhanced ct shows a classic serous cystadenoma in the head of the pancreas ( 4.1 2.9 cm ) . the lesion has the appearance of a solid mass with numerous small cysts and septations honeycomb structure . the calcified central scar ( arrow ) the other form of the tumor , i.e. macrocystic or oligocystic cystadenoma , with its appearance resembles a mucinous cystic tumor and is difficult to differentiate based on imaging examinations . it develops equally frequently in men and women , usually , after the age of 40 . therefore , the basic symptoms of its presence , next to the feeling of discomfort in the epigastric region and sometimes , pain when the lesion grows , is jaundice the tumor is composed of other well - circumscribed cystic lesions , with their diameters usually greater than 2 cm . they are generally single cysts or there are a few poorly circumscribed ones with some internal septations . in the present - day imaging diagnosis it is a modern us examination with the use of contrast media which considerably broadens diagnostic possibilities of doppler us examination . ceus allows for a good visualization of the honeycomb appearance and of the septations inside a neoplastic cyst . when , in small cystic tumors , which do not qualify to surgical treatment , ceus is performed , there is no need for further examinations , such as mri , mrcp and even contrast enhanced ct . owing to the high resolution , one may visualize lesions composed of very slight cysts ( of 5 mm in diameter ) in a more detailed manner as compared to conventional ultrasound . moreover , eus examination enables precise visualization of the honeycomb structure , calcifications and internal septations . it appears that a confirmation of a microcystic structure is sufficient for differential diagnosis and there is no need for the analysis of the fluid collected by means of eus - guided fine - needle aspiration biopsy ( eus - fna ) . what is more , the collection of adequate amount of fluid from such slight structures is frequently not feasible . fluid aspiration and its examination seem necessary when a larger cyst with a suspected neoplastic character has been visualized . , the collected fluid does not show any traces of mucin or amylase , and the test for the contents of cell elements ( cytological examination ) is possible in less than 50% of cases . contrary to patients with marked malignant transformation , the level of tumor markers ( mainly cea ) is low , usually below 5 ng / ml . in the material presented herein , in the case of serous tumors , aspiration demonstrated correct level of the markers ( cea and ca 19 - 9 ) and cytological examination did not reveal any neoplastic cells . a great majority of pancreatic serous tumors are benign the possibility of their malignant transformation concerns less than 3% of cases . a surgical resection of such a tumor together with a fragment of the pancreas is indicated in symptomatic lesions , in tumors that grow during the period of monitoring and in cases in which malignant transformation can not be ruled out . in patients qualified to surgical treatment , ious appears to be useful both in terms of final verification of the diagnosis and in the assessment of the range of resection . according to the quoted literature , the size of the tumor at which surgery should be considered is over 45 cm ( fig . up to 4 cm , tumors tend to grow slowly ( on average , 0.12 cm / year ) . it was observed that tumors larger than 4 cm grow faster ( on average , 1.6 cm / year ) , symptoms begin to appear and malignant transformation becomes more possible . it may happen that in imaging scans , a tumorous lesion shows local infiltration on its surrounding structures but the possibility to diagnose carcinoma based on imaging methods , endoscopic techniques and biopsy are limited . serous cystadenoma situated in the pancreatic body . a symptomatic lesion , 5.5 cm in the diameter , qualified to surgical resection ious . postoperative specimen the debate whether serous tumors should be only observed or at once qualified to surgical treatment is still open . there are mixed opinions since the cost of check - ups is considerable and the sole awareness of patients of a potential threat to develop a cancerous disease is of great significance . thus , slight lesions with 23 cm in diameter should be observed ( fig . 6 ) . check - up examinations , including us and ct , ought to be performed at least every 12 years . in the remaining cases , resection of the tumor it might be performed with the application of minimally invasive techniques when the lesion is relatively small and when surgical treatment should proceed with no complications . in our department , the aforementioned principles are observed . contrast - enhanced ct a small hypodense cystic tumor in the pancreatic tail ( 2.5 2 cm ) . poorly vascularized , multiple small cysts with polycyclic border of the tumor it is currently believed that mucinous cystic tumors constitute the most common form of pancreatic cystic neoplasms ( 1045% ) . due to the fact that in numerous cases they resemble ordinary cysts , a proper differentiation between these two is essential . they are encountered almost only in women ( 95% ) , mainly middle - aged ones , and are usually localized in the body and tail of the pancreas . a mean size of the tumor during the first diagnosis equals > 5 cm and in certain cases , the lesion may grow even up to 2535 cm . such lesions are usually solitary and do not maintain any connection with the main pancreatic duct . the most frequently encountered form of such a tumor is a single large lesion of cystic character with or without internal septations and with a well - defined , usually thickened outer wall ( fig . 7 ) . the cyst is lined with columnar epithelium which produces mucus that forms its inner layer . the outer layer is made up of stroma which resembles ovarian structure . these cysts usually contain a certain amount of mucous substance and one may also find sanguineous contents after hemorrhages or necrotic changes . typical radiological features of the tumor include : septations , solid intramural nodules and peripheral calcifications . in ultrasound examination , a mucinous tumor is visualized as a hypoechoic , well - circumscribed mass with diverse , often irregular thickening of the wall and sometimes , with solid nodules or calcifications . ceus examination allows for a better identification of the lesions within the wall ( nodules ) and thickened septations , which is caused by increased vascularity of the altered structures attesting to malignant transformation of the tumor . from the histological viewpoint the visualization of the symptom triad , i.e. irregular cystic wall thickening , peripheral calcifications and intramural solid lesions , in us , ct or mri may correspond to malignant transformation even in 95% cases ( fig . only a slight per cent of mucinous cystic tumors present a microcystic form , usually a slight one , single with few septations . in such cases , the cystic lesion is situated in the head of the pancreas with low - grade dysplasia ( arrows ) us power doppler . cystic neoplasm with reduced fluid capacity , mainly growth of solid tissue in order to learn the morphology of cystic tumors , eus examination is performed . this imaging modality provides reliable information allowing for the characterization of the cyst and determining the possibilities of resection when features of malignancy are present . due to the extension of the method to include aspiration biopsy ( eus - fna ) , it is possible to find extracellular mucus in the cystic fluid , conduct cytological or , more rarely , histological analyses , make biochemical assessment , determine the level of tumor markers and perform molecular analysis . as for cytological analysis , the reasons may be technical difficulties to collect fluid ( too little amount ) or low contents of cell elements in the aspirate . from among numerous tumor markers , the most specific is carcinoembryonic antigen cea . with the cut - off value of 200 ng / ml , higher values obtained in the analysis of cystic fluid indicate the presence of a mucinous tumor . in the statistical analysis for this level , a similar situation may be observed in the analysis of amylase concentration in the fluid . for the cut - off value of 250 iu / l , in the case of this level , the sensitivity and specificity constituted 45% and 89% respectively . unfortunately , the papers published so far do not specify the level of cea marker above which the cancerous transformation might be confirmed . this is verified after thorough evaluation of all data , particularly , imaging examinations and possible findings of eus - fna . what is important is the fact that small mucinous tumors may manifest no symptoms in as many as 75% of patients . when symptoms appear , the tumor is usually large and potentially malignant . therefore , in all such cases rapid diagnosis and surgical resection of the tumor are essential . the basis for the qualification to surgical treatment is the outcome of imaging examinations , clinical examination and , to a lower degree , also data obtained from cystic fluid aspiration . when a mucinous tumor of the pancreas is identified , one should remember that such lesions are frequently malignant or of highgrade dysplasia and they qualify to surgical treatment . a total evaluation of the tumor 's character is possible only following its resection , after histopathological examination . in our material , the examination of a postoperative specimen verified the presence of malignant mucinous tumors in two patients . aspiration biopsy performed prior to the procedure revealed elevated cea level and presence of mucus . cytological examination gave positive outcomes in the analysis of fluid from the inside of the cystic tumor in only one patient . this type of tumor is usually encountered in elderly men , aged 6080 . in a large majority of patients , these lesions manifest no symptoms . the most common symptom is recurrent pain , similar to that in the course of chronic , exacerbating pancreatitis or episodes of acute pancreatitis , occurring mainly after meals . such mucus travels to the pancreatic duct where it contributes to large pressure rise , which causes significant dilatation of the duct . the features of the tumor 's image depend on its type and localization in the pancreas . we distinguish three types of ipmn : central type which encompasses the main pancreatic duct , peripheral type involving the branches of the ducts , and mixed - type . the central type is the most common ( even up to 75% of cases ) . the lesion looks like a papilloma or polyp originating from the epithelium of the pancreatic duct . the changes are the effect of highgrade dysplasia of a borderline type or even a cancerous lesion ( local or invasive ) . the peripheral type , which is encountered much more rarely , is mainly localized in the region of the uncinate process . one of the features of its morphological presentation is a set of cysts resembling a cluster of grapes . the cystic appearance of the lesion renders it similar not only to a mucinous tumor , but also to a pseudocyst . the grape - like presentation is caused by the dilatation of numerous peripheral pancreatic ducts filled with mucus . sometimes , but rarely , instead of the lesion described above , a single , considerably dilated peripheral duct is visualized which forms the structure of the cyst . at present , imaging examinations , such as ultrasound , but ct and mri in particular , are capable of relatively accurate visualization of the dilatation of the pancreatic duct , secondary cystic lesions and connection between the duct and altered , dilated cystic structure . the decisive examinations in the case of ipmn are : mrcp , eus and ercp . ercp allows for the identification of the intraductal lesions and collection of the pancreatic juice in order to determine the content of mucin , obtain a cytological picture as well as determine the level of tumor markers in the aspirate . this examination may simultaneously wash out and even temporarily decompress the duct from dense mucus . mrcp , on the other hand , is the method of choice in the diagnosis and assessment of ipmn progression . it may prove more accurate in identification of the peripheral types as compared to ercp . the function of imaging examinations is also to determine a potential infiltration of adjacent tissues , enlargement of the lymph nodes or confirmation of existing metastases . eus allows for visualizing the connection between the main pancreatic duct and dilated peripheral ducts . the examination is also performed to differentiate between hypoechoic mucus and internal growths of papillary or polypoid type . it accurately assesses the thickening of the ductal wall and in peripheral types the thickening of the cystic wall and presence of intramural nodules which are normally signs of malignant transformation . the risk of developing a malignancy rises with the increase in the diameter of the main pancreatic duct , in the case of visualizing a large amount of mucus crossing through the ampulla of vater and when obstructive jaundice and/or diabetes are clinically confirmed . eus examination is extended by eus - fna thanks to which we may conduct cytological tests , determine the concentration of tumor markers and amylase and perform fnab of enlarged peripancreatic lymph nodes . the risk of malignant transformation of a central - type ipmn involving the main pancreatic duct is estimated at 70% . in the case of the peripheral type , it is 25% . therefore , there are specific indications for a surgical resection of a central - type tumor , even for total pancreatectomy if the main duct is involved to a great extent . when the tumor is localized peripherally , the decision concerning surgical treatment should be made based on the general condition of the patient and on associated risks and benefits of such a procedure taking into account the presence of given symptoms . furthermore , the progression of the local tumor ( > 3 cm ) and other patient 's conditions should also be taken into consideration . in many cases intraoperative ultrasound examination helps to decide about the range of surgery and resection margin . it is also useful in differentiating between pancreatic cysts and cystic tumors if following the previous examinations , both imaging scans and others , there are still certain diagnostic doubts . so far , over 600 cases have been reported which constitutes approximately 1% of all cystic neoplasms of the pancreas . in over 90% , it develops in women in the 2 and 3 decades of life . usually wellcircumscribed , the tumor is composed of solid part , cystic lesions , papillary structures , areas of necrosis and hemorrhagic components which are a result of numerous extravasations into the tumorous tissue . the tumors may develop both in the head and in the tail of the pancreas . it has been reported to grow to 135 cm but the average size is 6 cm . the recommended imaging examinations in the diagnosis of spn are : us , ct and mri . the scans reveal a well - encapsulated , heterogeneous tumor with areas of cystic degeneration and hemorrhagic components . other examinations include tumor marker assessment , recommended markers are as follows : 1-antitrypsin , 1-antichymotrypsin , neuron - specific enolase ( nse ) and vimentin . spn shows a relatively low malignancy potential and the risk is estimated at 15% . in 1015% of patients , the neoplasm metastasizes to the liver , infiltrates vascular structures and adjacent organs ( fig . a surgical resection of the tumor with adequate portion of the pancreas ( proximal or distal resection ) is the treatment of choice . it is also recommended to surgically remove metastases and to perform lymphadenectomy . even in such cases , the prognosis is good and longterm survival depends on the specific biology of the spn tumor . the remaining cystic neoplasms are encountered rarely and thus , their separate description is not needed . the tumor localized in the tail of the pancreas , approximately 10 cm in diameter . the content of the lesion is solid with signs of necrosis or hemorrhage which are responsible for the cystic capacity . the tumor with malignant potential malignant transformation of spn . contrast - enhanced ct scans show a mixed solid and cystic mass in the pancreatic head ( 10 7.5 cm ) . thick , well - circumscribed capsule , hyperdense areas due to hemorrhage , and areas of necrosis they are usually solitary , with the size of 1.535 cm and constitute approximately 30% of all primary pancreatic cystic tumors . we mainly encounter two types of such lesions : microcystic cystadenomas and macrocystic or oligocystic cystadenomas . the microcystic form usually develops in women at the age of 5060 and its typical localization is the body and tail of the pancreas . it is usually asymptomatic and signals , such as pain in the abdomen , weight loss , feeling of distension in the epigastric region and peristalsis disorders appear when the lesion shows large growth . a typical form of the tumor is composed of a large number of small cysts lined with cuboidal epithelium producing glycogen . the size of the cysts ranges from 0.2 to 2.0 cm and the entire tumor measures 1.430 cm . the lesion may grow locally leading to almost complete damage of the healthy pancreatic tissue . in imaging examinations ( us , ct ) , such a cystic tumor frequently has the appearance of a honeycomb which is related to a large number of small cysts ( fig . the lesion is relatively well - circumscribed with a central scar or , sometimes , with a calcification . the cystic mass is situated in the pancreatic tail , about 5 cm in diameter ious . well - defined microcystic tumor , poorly vascularized , adhering to the splenic vessels ( arrow ) ( color doppler ) doppler us . serous cystadenoma in the head of the pancreas , poorly vascularized , 5.6 cm in diameter . structure with oligocystic components and central scar ( arrow ) contrast - enhanced ct shows a classic serous cystadenoma in the head of the pancreas ( 4.1 2.9 cm ) . the lesion has the appearance of a solid mass with numerous small cysts and septations honeycomb structure . the calcified central scar ( arrow ) the other form of the tumor , i.e. macrocystic or oligocystic cystadenoma , with its appearance resembles a mucinous cystic tumor and is difficult to differentiate based on imaging examinations . it develops equally frequently in men and women , usually , after the age of 40 . therefore , the basic symptoms of its presence , next to the feeling of discomfort in the epigastric region and sometimes , pain when the lesion grows , is jaundice the tumor is composed of other well - circumscribed cystic lesions , with their diameters usually greater than 2 cm . they are generally single cysts or there are a few poorly circumscribed ones with some internal septations . in the present - day imaging diagnosis it is a modern us examination with the use of contrast media which considerably broadens diagnostic possibilities of doppler us examination . ceus allows for a good visualization of the honeycomb appearance and of the septations inside a neoplastic cyst . when , in small cystic tumors , which do not qualify to surgical treatment , ceus is performed , there is no need for further examinations , such as mri , mrcp and even contrast enhanced ct . owing to the high resolution , one may visualize lesions composed of very slight cysts ( of 5 mm in diameter ) in a more detailed manner as compared to conventional ultrasound . moreover , eus examination enables precise visualization of the honeycomb structure , calcifications and internal septations . it appears that a confirmation of a microcystic structure is sufficient for differential diagnosis and there is no need for the analysis of the fluid collected by means of eus - guided fine - needle aspiration biopsy ( eus - fna ) . what is more , the collection of adequate amount of fluid from such slight structures is frequently not feasible . fluid aspiration and its examination seem necessary when a larger cyst with a suspected neoplastic character has been visualized . , the collected fluid does not show any traces of mucin or amylase , and the test for the contents of cell elements ( cytological examination ) is possible in less than 50% of cases . contrary to patients with marked malignant transformation , the level of tumor markers ( mainly cea ) is low , usually below 5 ng / ml . in the material presented herein , in the case of serous tumors , aspiration demonstrated correct level of the markers ( cea and ca 19 - 9 ) and cytological examination did not reveal any neoplastic cells . a great majority of pancreatic serous tumors are benign the possibility of their malignant transformation concerns less than 3% of cases . a surgical resection of such a tumor together with a fragment of the pancreas is indicated in symptomatic lesions , in tumors that grow during the period of monitoring and in cases in which malignant transformation can not be ruled out . in patients qualified to surgical treatment , ious appears to be useful both in terms of final verification of the diagnosis and in the assessment of the range of resection . according to the quoted literature , the size of the tumor at which surgery should be considered is over 45 cm ( fig . , tumors tend to grow slowly ( on average , 0.12 cm / year ) . it was observed that tumors larger than 4 cm grow faster ( on average , 1.6 cm / year ) , symptoms begin to appear and malignant transformation becomes more possible . it may happen that in imaging scans , a tumorous lesion shows local infiltration on its surrounding structures but the possibility to diagnose carcinoma based on imaging methods , endoscopic techniques and biopsy are limited . serous cystadenoma situated in the pancreatic body . a symptomatic lesion , 5.5 cm in the diameter , qualified to surgical resection ious . postoperative specimen the debate whether serous tumors should be only observed or at once qualified to surgical treatment is still open . there are mixed opinions since the cost of check - ups is considerable and the sole awareness of patients of a potential threat to develop a cancerous disease is of great significance . check - up examinations , including us and ct , ought to be performed at least every 12 years . in the remaining cases , resection of the tumor it might be performed with the application of minimally invasive techniques when the lesion is relatively small and when surgical treatment should proceed with no complications . in our department , the aforementioned principles are observed . contrast - enhanced ct a small hypodense cystic tumor in the pancreatic tail ( 2.5 2 cm ) . it is currently believed that mucinous cystic tumors constitute the most common form of pancreatic cystic neoplasms ( 1045% ) . due to the fact that in numerous cases they resemble ordinary cysts , a proper differentiation between these two is essential . they are encountered almost only in women ( 95% ) , mainly middle - aged ones , and are usually localized in the body and tail of the pancreas . a mean size of the tumor during the first diagnosis equals > 5 cm and in certain cases , the lesion may grow even up to 2535 cm . such lesions are usually solitary and do not maintain any connection with the main pancreatic duct . the most frequently encountered form of such a tumor is a single large lesion of cystic character with or without internal septations and with a well - defined , usually thickened outer wall ( fig . the cyst is lined with columnar epithelium which produces mucus that forms its inner layer . these cysts usually contain a certain amount of mucous substance and one may also find sanguineous contents after hemorrhages or necrotic changes . typical radiological features of the tumor include : septations , solid intramural nodules and peripheral calcifications . in ultrasound examination , a mucinous tumor is visualized as a hypoechoic , well - circumscribed mass with diverse , often irregular thickening of the wall and sometimes , with solid nodules or calcifications . ceus examination allows for a better identification of the lesions within the wall ( nodules ) and thickened septations , which is caused by increased vascularity of the altered structures attesting to malignant transformation of the tumor . from the histological viewpoint the visualization of the symptom triad , i.e. irregular cystic wall thickening , peripheral calcifications and intramural solid lesions , in us , ct or mri may correspond to malignant transformation even in 95% cases ( fig . only a slight per cent of mucinous cystic tumors present a microcystic form , usually a slight one , single with few septations . in such cases , the cystic lesion is situated in the head of the pancreas with low - grade dysplasia ( arrows ) us power doppler . cystic neoplasm with reduced fluid capacity , mainly growth of solid tissue in order to learn the morphology of cystic tumors , eus examination is performed . this imaging modality provides reliable information allowing for the characterization of the cyst and determining the possibilities of resection when features of malignancy are present . due to the extension of the method to include aspiration biopsy ( eus - fna ) , it is possible to find extracellular mucus in the cystic fluid , conduct cytological or , more rarely , histological analyses , make biochemical assessment , determine the level of tumor markers and perform molecular analysis . as for cytological analysis , the reasons may be technical difficulties to collect fluid ( too little amount ) or low contents of cell elements in the aspirate . from among numerous tumor markers , the cut - off value of 200 ng / ml , higher values obtained in the analysis of cystic fluid indicate the presence of a mucinous tumor . in the statistical analysis for this level , a similar situation may be observed in the analysis of amylase concentration in the fluid . for the cut - off value of 250 iu / l , in the case of this level , the sensitivity and specificity constituted 45% and 89% respectively . unfortunately , the papers published so far do not specify the level of cea marker above which the cancerous transformation might be confirmed . this is verified after thorough evaluation of all data , particularly , imaging examinations and possible findings of eus - fna . what is important is the fact that small mucinous tumors may manifest no symptoms in as many as 75% of patients . when symptoms appear , the tumor is usually large and potentially malignant . therefore , in all such cases rapid diagnosis and surgical resection of the tumor are essential . the basis for the qualification to surgical treatment is the outcome of imaging examinations , clinical examination and , to a lower degree , also data obtained from cystic fluid aspiration . when a mucinous tumor of the pancreas is identified , one should remember that such lesions are frequently malignant or of highgrade dysplasia and they qualify to surgical treatment . a total evaluation of the tumor 's character is possible only following its resection , after histopathological examination . in our material , the examination of a postoperative specimen verified the presence of malignant mucinous tumors in two patients . aspiration biopsy performed prior to the procedure revealed elevated cea level and presence of mucus . cytological examination gave positive outcomes in the analysis of fluid from the inside of the cystic tumor in only one patient . this type of tumor is usually encountered in elderly men , aged 6080 . in a large majority of patients , the most common symptom is recurrent pain , similar to that in the course of chronic , exacerbating pancreatitis or episodes of acute pancreatitis , occurring mainly after meals . such mucus travels to the pancreatic duct where it contributes to large pressure rise , which causes significant dilatation of the duct . the features of the tumor 's image depend on its type and localization in the pancreas . we distinguish three types of ipmn : central type which encompasses the main pancreatic duct , peripheral type involving the branches of the ducts , and mixed - type . the central type is the most common ( even up to 75% of cases ) . the lesion looks like a papilloma or polyp originating from the epithelium of the pancreatic duct . the changes are the effect of highgrade dysplasia of a borderline type or even a cancerous lesion ( local or invasive ) . the peripheral type , which is encountered much more rarely , is mainly localized in the region of the uncinate process . one of the features of its morphological presentation is a set of cysts resembling a cluster of grapes . the cystic appearance of the lesion renders it similar not only to a mucinous tumor , but also to a pseudocyst . the grape - like presentation is caused by the dilatation of numerous peripheral pancreatic ducts filled with mucus . sometimes , but rarely , instead of the lesion described above , a single , considerably dilated peripheral duct is visualized which forms the structure of the cyst . at present , imaging examinations , such as ultrasound , but ct and mri in particular , are capable of relatively accurate visualization of the dilatation of the pancreatic duct , secondary cystic lesions and connection between the duct and altered , dilated cystic structure . the decisive examinations in the case of ipmn are : mrcp , eus and ercp . ercp allows for the identification of the intraductal lesions and collection of the pancreatic juice in order to determine the content of mucin , obtain a cytological picture as well as determine the level of tumor markers in the aspirate . this examination may simultaneously wash out and even temporarily decompress the duct from dense mucus . mrcp , on the other hand , is the method of choice in the diagnosis and assessment of ipmn progression . it may prove more accurate in identification of the peripheral types as compared to ercp . the function of imaging examinations is also to determine a potential infiltration of adjacent tissues , enlargement of the lymph nodes or confirmation of existing metastases . eus allows for visualizing the connection between the main pancreatic duct and dilated peripheral ducts . the examination is also performed to differentiate between hypoechoic mucus and internal growths of papillary or polypoid type . it accurately assesses the thickening of the ductal wall and in peripheral types the thickening of the cystic wall and presence of intramural nodules which are normally signs of malignant transformation . the risk of developing a malignancy rises with the increase in the diameter of the main pancreatic duct , in the case of visualizing a large amount of mucus crossing through the ampulla of vater and when obstructive jaundice and/or diabetes are clinically confirmed . eus examination is extended by eus - fna thanks to which we may conduct cytological tests , determine the concentration of tumor markers and amylase and perform fnab of enlarged peripancreatic lymph nodes . the risk of malignant transformation of a central - type ipmn involving the main pancreatic duct is estimated at 70% . in the case of the peripheral type , it is 25% . therefore , there are specific indications for a surgical resection of a central - type tumor , even for total pancreatectomy if the main duct is involved to a great extent . when the tumor is localized peripherally , the decision concerning surgical treatment should be made based on the general condition of the patient and on associated risks and benefits of such a procedure taking into account the presence of given symptoms . furthermore , the progression of the local tumor ( > 3 cm ) and other patient 's conditions should also be taken into consideration . in many cases intraoperative ultrasound examination it is also useful in differentiating between pancreatic cysts and cystic tumors if following the previous examinations , both imaging scans and others , there are still certain diagnostic doubts . spn is a rare tumor . so far , over 600 cases have been reported which constitutes approximately 1% of all cystic neoplasms of the pancreas . in over 90% , it develops in women in the 2 and 3 decades of life . usually wellcircumscribed , the tumor is composed of solid part , cystic lesions , papillary structures , areas of necrosis and hemorrhagic components which are a result of numerous extravasations into the tumorous tissue . the tumors may develop both in the head and in the tail of the pancreas . it has been reported to grow to 135 cm but the average size is 6 cm . the recommended imaging examinations in the diagnosis of spn are : us , ct and mri . the scans reveal a well - encapsulated , heterogeneous tumor with areas of cystic degeneration and hemorrhagic components . other examinations include tumor marker assessment , recommended markers are as follows : 1-antitrypsin , 1-antichymotrypsin , neuron - specific enolase ( nse ) and vimentin . spn shows a relatively low malignancy potential and the risk is estimated at 15% . in 1015% of patients , the neoplasm metastasizes to the liver , infiltrates vascular structures and adjacent organs ( fig . a surgical resection of the tumor with adequate portion of the pancreas ( proximal or distal resection ) is the treatment of choice . it is also recommended to surgically remove metastases and to perform lymphadenectomy . even in such cases , the prognosis is good and longterm survival depends on the specific biology of the spn tumor . the remaining cystic neoplasms are encountered rarely and thus , their separate description is not needed . solid pseudopapillary neoplasm ( spn ) . a large symptomatic lesion , well - circumscribed , surrounded by a thick capsule . the tumor localized in the tail of the pancreas , approximately 10 cm in diameter . the content of the lesion is solid with signs of necrosis or hemorrhage which are responsible for the cystic capacity . the tumor with malignant potential malignant transformation of spn . contrast - enhanced ct scans show a mixed solid and cystic mass in the pancreatic head ( 10 7.5 cm ) . thick , well - circumscribed capsule , hyperdense areas due to hemorrhage , and areas of necrosis the procedural treatment is applied in the case of large ( > 4 cm ) , symptomatic cysts of the pancreas.endoscopic drainage constitutes an effective and safe method of minimally invasive treatment of pancreatic cysts and surgical procedures are an alternative when minimally invasive techniques are not possible.the differentiation of a neoplasm from a typical cyst is of fundamental significance for the selection of an adequate treatment method . the procedural treatment is applied in the case of large ( > 4 cm ) , symptomatic cysts of the pancreas . endoscopic drainage constitutes an effective and safe method of minimally invasive treatment of pancreatic cysts and surgical procedures are an alternative when minimally invasive techniques are not possible . the differentiation of a neoplasm from a typical cyst is of fundamental significance for the selection of an adequate treatment method . authors do not report any financial or personal links with other persons or organizations , which might affect negatively the content of this publication and/or claim authorship rights to this publication .

pseudocysts constitute the most basic cystic lesions of the pancreas . symptomatic cysts may be treated by means of both minimally invasive methods and surgery . currently , it is believed that approximately 5% of cystic lesions in the pancreas may in fact , be neoplastic cystic tumors . their presence is manifested by generally irregular multilocular structures , solid nodules inside the cyst or in the pancreatic duct , frequently vascularized , as well as fragmentary thickening of the cystic wall or septation.aimthe aim of this paper was to present current management , both diagnostic and therapeutic , in patients with pancreatic pseudocysts and cystic tumors . the article has been written based on the material collected and prepared in the author 's department as well as on the basis of current reports found in the quoted literature.material and methods , resultsin 20002012 , the second department of general , gastrointestinal and oncological surgery of the alimentary tract treated 179 patients with cystic lesions in the region of the pancreas . this group comprised 12 cases of cystic tumors and 167 pseudocysts . twenty - three patients ( 13.8% ) were monitored only and 144 received procedural treatment . out of the latter group , 75 patients underwent drainage procedures and 48 were qualified to endoscopic cystogastrostomy or cystoduodenostomy . the endoscopic procedure was unsuccessful in 11 cases ( 23% ) . in a group of patients with a pancreatic cystic tumor ( 12 patients ) , 6 of them ( 50% ) underwent therapeutic resection of the tumor with adequate fragment of the gland.conclusionsendoscopic drainage is an effective and safe method of minimally invasive treatment of pancreatic cysts . the patients who do not qualify to endoscopic procedures require surgical treatment . the differentiation of a neoplasm from a typical cyst is of fundamental significance for the selection of the treatment method .

Cel pracy Materia i metoda, wyniki Wnioski Aim of the paper Material and methods Results Discussion Serous cystadenoma Mucinous cystadenoma (cystadenocarcinoma) Intraductal papillary mucinous neoplasm (IPMN) Solid pseudopapillary neoplasm (SPN) Conclusions Conflict of interest

the aim of this paper was to present current management , both diagnostic and therapeutic , of patients with pancreatic pseudocysts and cystic tumors . the article has been written based on the material collected and prepared in the author 's department taking into consideration contemporary approaches and opinions included in the references . in 20002012 , the second department of general , gastrointestinal and oncological surgery of the alimentary tract treated 179 patients with cystic lesions in the region of the pancreas . patients with suspected or confirmed neoplastic tumors were included in a separate group of cystic lesions . therefore , in our department , the description usually specifies to which aspect of the pancreas , proximal or distal , the cyst adheres . in the material presented herein , the proximal localization ( head , body ) was found in 118 cases and distal ( body , tail ) in 61 cases . when the adequate puncture site in a non - vascular region has been selected in eus , it is marked with dye , spot coagulation or , currently , an appropriate drainage set is placed , directly guided by eus . up to 2012 , a total of 48 patients were qualified to endoscopic drainage procedures . the pancreatic juice flows through cystogastrostomy ( prosthesis is inserted to the lumen of the pseudocyst ) when the initial diagnosis can not rule out a cystic neoplasm of the pancreas , the patient undergoes detailed imaging examinations , biochemical and serological tests as well as , in certain cases , fine - needle aspiration biopsy ( fnab ) . in the subject group of 179 patients with cystic lesions in the region of the pancreas , 12 presented with cystic tumors and 167 with pseudocysts , both acute and chronic . from among all 167 cases with symptomatic cysts , 23 patients ( 13.8% ) were monitored only ( involution of the lesions during the observation period ) and the remaining 144 patients were qualified to procedures . in 75 patients ( 44.9% ) , the resection of the cyst was possible in merely 4 cases , the remaining patients underwent drainage procedures . in the remaining patients , complex procedures took place , such as drainage of several cysts or total drainage of pseudocysts and pancreatic duct , also performed according to roux - en - y technique . furthermore , 21 patients ( 12.6% ) with thin - walled retention cysts secondary to acute pancreatitis ( ap ) , were treated by external us - guided drainage . in 8 cases ( 38% ) , 48 patients ( 28.7% ) were qualified to endoscopic cystogastrostomy or cystoduodenostomy if transabdominal ultrasound and subsequently , eus had confirmed such qualification . the efficacy of the procedure was usually evaluated on the second day following the procedure by means of us examination . due to technical reasons , the placement of the drain was impossible in 11 patients ( 23% ) they underwent surgical drainage instead . due to the results of imaging examinations , in 5 patients with slight lesions ( mainly from 2 to 3 cm ) in whom a benign serous cystic neoplasm was suspected ( based on us / ct ) , observation was recommended . in further 6 patients with over 4 cm lesions that showed features of neoplastic cystic tumors , resection of the tumor with an adequate part of the pancreas was performed . in patients diagnosed with and treated due to pancreatic pseudocysts , transabdominal ultrasound is the main diagnostic tool , followed by ct and eus , in qualifying patients to endoscopic treatment . in us thin - walled cisterns do not qualify to surgical treatment ( anastomosis ) due to the difficulties to perform anastomosis and the possibility of secondary leak . the treatment of pancreatic cysts should depend on their origin , character , size , location and individual clinical symptoms . slight lesions , from 3 to 4 cm , which may be effectively controlled by means of periodical us examinations , do not receive any treatment provided that their neoplastic character has been ruled out . in the discussed period of time , from among 167 patients diagnosed with symptomatic cysts of the pancreas , 44.9% underwent surgeries , 12.6% were treated by means of external drainage and 28.7% by internal eus - guided endoscopic drainage . still , the largest group underwent surgeries in spite of the fact that endoscopic procedures introduced in 2000 have a greater and greater therapeutic value . such a procedure was performed in 48 patients , but in 11 of them ( 23% ) endoscopic drainage was not successful mainly due to technical problems connected with prosthesis placement , difficult angular access of the endoscope to the cyst , bleeding and suspicion of perforation . in the remaining 37 patients with pancreatic pseudocysts , it allows for a rapid return to everyday activities , patients do not need to follow a rigorous diet ( as it happens after a surgery ) and their physical activity is not restricted due to the postoperative wound . at present , thanks to the advancement of diagnostic techniques , we may accurately assess and distinguish neoplastic cystic tumors of the pancreas from ordinary cystic lesions . it is currently believed that even 10% of all cases of cystic lesions are related to a neoplasm and that these lesions account for 15% of all pancreatic neoplasms . it is believed that asymptomatic lesions constitute approximately 3575% of cystic tumors of the pancreas . patients with ipmn may be sometimes treated due to recurrent episodes of acute pancreatitis , particularly , in cases when the main pancreatic duct is involved . large tumors are most frequently found in patients with mucinous cystic neoplasms ( mcn ) and spn these lesions usually give symptoms of a midabdominal tumor . in advanced malignant lesions , apart from the above - listed symptoms the following may occur : bleeding from the gastrointestinal tract in the case of gastric infiltration , portal hypertension and hemobilia as well as , eventually , diabetes when the pancreas is considerably damaged . in the analyzed time period , after thorough imaging analysis , including doppler ultrasound , contrast enhanced ct as well as in some cases , mri , mrcp and eus , 6 patients were qualified to fnab . in the next part , the material has been collected based on quoted recent publications concerning diagnosis , methods of differentiation and proposed types of treatment . a typical form of the tumor is composed of a large number of small cysts lined with cuboidal epithelium producing glycogen . a typical serous tumor is composed of multiple small cysts and has a the cystic mass is situated in the pancreatic tail , about 5 cm in diameter ious . serous cystadenoma in the head of the pancreas , poorly vascularized , 5.6 cm in diameter . therefore , the basic symptoms of its presence , next to the feeling of discomfort in the epigastric region and sometimes , pain when the lesion grows , is jaundice the tumor is composed of other well - circumscribed cystic lesions , with their diameters usually greater than 2 cm . when , in small cystic tumors , which do not qualify to surgical treatment , ceus is performed , there is no need for further examinations , such as mri , mrcp and even contrast enhanced ct . it appears that a confirmation of a microcystic structure is sufficient for differential diagnosis and there is no need for the analysis of the fluid collected by means of eus - guided fine - needle aspiration biopsy ( eus - fna ) . in the material presented herein , in the case of serous tumors , aspiration demonstrated correct level of the markers ( cea and ca 19 - 9 ) and cytological examination did not reveal any neoplastic cells . a surgical resection of such a tumor together with a fragment of the pancreas is indicated in symptomatic lesions , in tumors that grow during the period of monitoring and in cases in which malignant transformation can not be ruled out . in patients qualified to surgical treatment , ious appears to be useful both in terms of final verification of the diagnosis and in the assessment of the range of resection . according to the quoted literature , the size of the tumor at which surgery should be considered is over 45 cm ( fig . in the remaining cases , resection of the tumor it might be performed with the application of minimally invasive techniques when the lesion is relatively small and when surgical treatment should proceed with no complications . poorly vascularized , multiple small cysts with polycyclic border of the tumor it is currently believed that mucinous cystic tumors constitute the most common form of pancreatic cystic neoplasms ( 1045% ) . they are encountered almost only in women ( 95% ) , mainly middle - aged ones , and are usually localized in the body and tail of the pancreas . irregular cystic wall thickening , peripheral calcifications and intramural solid lesions , in us , ct or mri may correspond to malignant transformation even in 95% cases ( fig . in such cases , the cystic lesion is situated in the head of the pancreas with low - grade dysplasia ( arrows ) us power doppler . due to the extension of the method to include aspiration biopsy ( eus - fna ) , it is possible to find extracellular mucus in the cystic fluid , conduct cytological or , more rarely , histological analyses , make biochemical assessment , determine the level of tumor markers and perform molecular analysis . as for cytological analysis , the reasons may be technical difficulties to collect fluid ( too little amount ) or low contents of cell elements in the aspirate . for the cut - off value of 250 iu / l , in the case of this level , the sensitivity and specificity constituted 45% and 89% respectively . therefore , in all such cases rapid diagnosis and surgical resection of the tumor are essential . when a mucinous tumor of the pancreas is identified , one should remember that such lesions are frequently malignant or of highgrade dysplasia and they qualify to surgical treatment . cytological examination gave positive outcomes in the analysis of fluid from the inside of the cystic tumor in only one patient . the features of the tumor 's image depend on its type and localization in the pancreas . the lesion looks like a papilloma or polyp originating from the epithelium of the pancreatic duct . the peripheral type , which is encountered much more rarely , is mainly localized in the region of the uncinate process . at present , imaging examinations , such as ultrasound , but ct and mri in particular , are capable of relatively accurate visualization of the dilatation of the pancreatic duct , secondary cystic lesions and connection between the duct and altered , dilated cystic structure . ercp allows for the identification of the intraductal lesions and collection of the pancreatic juice in order to determine the content of mucin , obtain a cytological picture as well as determine the level of tumor markers in the aspirate . mrcp , on the other hand , is the method of choice in the diagnosis and assessment of ipmn progression . it accurately assesses the thickening of the ductal wall and in peripheral types the thickening of the cystic wall and presence of intramural nodules which are normally signs of malignant transformation . the risk of developing a malignancy rises with the increase in the diameter of the main pancreatic duct , in the case of visualizing a large amount of mucus crossing through the ampulla of vater and when obstructive jaundice and/or diabetes are clinically confirmed . in the case of the peripheral type , it is 25% . when the tumor is localized peripherally , the decision concerning surgical treatment should be made based on the general condition of the patient and on associated risks and benefits of such a procedure taking into account the presence of given symptoms . it is also useful in differentiating between pancreatic cysts and cystic tumors if following the previous examinations , both imaging scans and others , there are still certain diagnostic doubts . usually wellcircumscribed , the tumor is composed of solid part , cystic lesions , papillary structures , areas of necrosis and hemorrhagic components which are a result of numerous extravasations into the tumorous tissue . a surgical resection of the tumor with adequate portion of the pancreas ( proximal or distal resection ) is the treatment of choice . the tumor localized in the tail of the pancreas , approximately 10 cm in diameter . a typical form of the tumor is composed of a large number of small cysts lined with cuboidal epithelium producing glycogen . in imaging examinations ( us , ct ) , such a cystic tumor frequently has the appearance of a honeycomb which is related to a large number of small cysts ( fig . serous cystadenoma in the head of the pancreas , poorly vascularized , 5.6 cm in diameter . therefore , the basic symptoms of its presence , next to the feeling of discomfort in the epigastric region and sometimes , pain when the lesion grows , is jaundice the tumor is composed of other well - circumscribed cystic lesions , with their diameters usually greater than 2 cm . when , in small cystic tumors , which do not qualify to surgical treatment , ceus is performed , there is no need for further examinations , such as mri , mrcp and even contrast enhanced ct . it appears that a confirmation of a microcystic structure is sufficient for differential diagnosis and there is no need for the analysis of the fluid collected by means of eus - guided fine - needle aspiration biopsy ( eus - fna ) . in the material presented herein , in the case of serous tumors , aspiration demonstrated correct level of the markers ( cea and ca 19 - 9 ) and cytological examination did not reveal any neoplastic cells . a surgical resection of such a tumor together with a fragment of the pancreas is indicated in symptomatic lesions , in tumors that grow during the period of monitoring and in cases in which malignant transformation can not be ruled out . in patients qualified to surgical treatment , ious appears to be useful both in terms of final verification of the diagnosis and in the assessment of the range of resection . according to the quoted literature , the size of the tumor at which surgery should be considered is over 45 cm ( fig . in the remaining cases , resection of the tumor it might be performed with the application of minimally invasive techniques when the lesion is relatively small and when surgical treatment should proceed with no complications . it is currently believed that mucinous cystic tumors constitute the most common form of pancreatic cystic neoplasms ( 1045% ) . they are encountered almost only in women ( 95% ) , mainly middle - aged ones , and are usually localized in the body and tail of the pancreas . in such cases , the cystic lesion is situated in the head of the pancreas with low - grade dysplasia ( arrows ) us power doppler . due to the extension of the method to include aspiration biopsy ( eus - fna ) , it is possible to find extracellular mucus in the cystic fluid , conduct cytological or , more rarely , histological analyses , make biochemical assessment , determine the level of tumor markers and perform molecular analysis . from among numerous tumor markers , the cut - off value of 200 ng / ml , higher values obtained in the analysis of cystic fluid indicate the presence of a mucinous tumor . for the cut - off value of 250 iu / l , in the case of this level , the sensitivity and specificity constituted 45% and 89% respectively . therefore , in all such cases rapid diagnosis and surgical resection of the tumor are essential . the basis for the qualification to surgical treatment is the outcome of imaging examinations , clinical examination and , to a lower degree , also data obtained from cystic fluid aspiration . when a mucinous tumor of the pancreas is identified , one should remember that such lesions are frequently malignant or of highgrade dysplasia and they qualify to surgical treatment . cytological examination gave positive outcomes in the analysis of fluid from the inside of the cystic tumor in only one patient . in a large majority of patients , the most common symptom is recurrent pain , similar to that in the course of chronic , exacerbating pancreatitis or episodes of acute pancreatitis , occurring mainly after meals . the features of the tumor 's image depend on its type and localization in the pancreas . the peripheral type , which is encountered much more rarely , is mainly localized in the region of the uncinate process . at present , imaging examinations , such as ultrasound , but ct and mri in particular , are capable of relatively accurate visualization of the dilatation of the pancreatic duct , secondary cystic lesions and connection between the duct and altered , dilated cystic structure . ercp allows for the identification of the intraductal lesions and collection of the pancreatic juice in order to determine the content of mucin , obtain a cytological picture as well as determine the level of tumor markers in the aspirate . it accurately assesses the thickening of the ductal wall and in peripheral types the thickening of the cystic wall and presence of intramural nodules which are normally signs of malignant transformation . the risk of developing a malignancy rises with the increase in the diameter of the main pancreatic duct , in the case of visualizing a large amount of mucus crossing through the ampulla of vater and when obstructive jaundice and/or diabetes are clinically confirmed . in the case of the peripheral type , it is 25% . when the tumor is localized peripherally , the decision concerning surgical treatment should be made based on the general condition of the patient and on associated risks and benefits of such a procedure taking into account the presence of given symptoms . in many cases intraoperative ultrasound examination it is also useful in differentiating between pancreatic cysts and cystic tumors if following the previous examinations , both imaging scans and others , there are still certain diagnostic doubts . usually wellcircumscribed , the tumor is composed of solid part , cystic lesions , papillary structures , areas of necrosis and hemorrhagic components which are a result of numerous extravasations into the tumorous tissue . a surgical resection of the tumor with adequate portion of the pancreas ( proximal or distal resection ) is the treatment of choice . even in such cases , the prognosis is good and longterm survival depends on the specific biology of the spn tumor . the tumor localized in the tail of the pancreas , approximately 10 cm in diameter . thick , well - circumscribed capsule , hyperdense areas due to hemorrhage , and areas of necrosis the procedural treatment is applied in the case of large ( > 4 cm ) , symptomatic cysts of the pancreas.endoscopic drainage constitutes an effective and safe method of minimally invasive treatment of pancreatic cysts and surgical procedures are an alternative when minimally invasive techniques are not possible.the differentiation of a neoplasm from a typical cyst is of fundamental significance for the selection of an adequate treatment method . the procedural treatment is applied in the case of large ( > 4 cm ) , symptomatic cysts of the pancreas . endoscopic drainage constitutes an effective and safe method of minimally invasive treatment of pancreatic cysts and surgical procedures are an alternative when minimally invasive techniques are not possible . the differentiation of a neoplasm from a typical cyst is of fundamental significance for the selection of an adequate treatment method .

the aim of this paper was to present current management , both diagnostic and therapeutic , of patients with pancreatic pseudocysts and cystic tumors . the article has been written based on the material collected and prepared in the author 's department taking into consideration contemporary approaches and opinions included in the references . in 20002012 , the second department of general , gastrointestinal and oncological surgery of the alimentary tract treated 179 patients with cystic lesions in the region of the pancreas . the majority of the lesions adhered to the pancreas but their outer outlines could border on various parts of the gastrointestinal tract and the mesentery or could be localized in the extraperitoneal space . therefore , in our department , the description usually specifies to which aspect of the pancreas , proximal or distal , the cyst adheres . in the material presented herein , the proximal localization ( head , body ) was found in 118 cases and distal ( body , tail ) in 61 cases . if in transabdominal ultrasound examination , cysts with defined walls are detected , located near the stomach or duodenum and indenting their lumina , eus and videoendoscopy are conducted to determine the indications for endoscopic cystogastrostomy or cystoduodenostomy . when the adequate puncture site in a non - vascular region has been selected in eus , it is marked with dye , spot coagulation or , currently , an appropriate drainage set is placed , directly guided by eus . with the use of the lateral - viewing duodenoscope and diathermy , the gastric wall and adjacent cyst are punctured . subsequently , having conducted the aspiration attempt , the prosthesis is inserted which allows for free outflow of cystic contents ( fig . the pancreatic juice flows through cystogastrostomy ( prosthesis is inserted to the lumen of the pseudocyst ) when the initial diagnosis can not rule out a cystic neoplasm of the pancreas , the patient undergoes detailed imaging examinations , biochemical and serological tests as well as , in certain cases , fine - needle aspiration biopsy ( fnab ) . in the subject group of 179 patients with cystic lesions in the region of the pancreas , 12 presented with cystic tumors and 167 with pseudocysts , both acute and chronic . from among all 167 cases with symptomatic cysts , 23 patients ( 13.8% ) were monitored only ( involution of the lesions during the observation period ) and the remaining 144 patients were qualified to procedures . in 75 patients ( 44.9% ) , the resection of the cyst was possible in merely 4 cases , the remaining patients underwent drainage procedures . in the remaining patients , complex procedures took place , such as drainage of several cysts or total drainage of pseudocysts and pancreatic duct , also performed according to roux - en - y technique . in 8 cases ( 38% ) , 48 patients ( 28.7% ) were qualified to endoscopic cystogastrostomy or cystoduodenostomy if transabdominal ultrasound and subsequently , eus had confirmed such qualification . due to technical reasons , the placement of the drain was impossible in 11 patients ( 23% ) they underwent surgical drainage instead . the analysis of the contents ( cytology , level of markers , test for mucous content ) was positive in only 2 patients . due to the results of imaging examinations , in 5 patients with slight lesions ( mainly from 2 to 3 cm ) in whom a benign serous cystic neoplasm was suspected ( based on us / ct ) , observation was recommended . evident growth of the tumorous lesion was not observed in the monitored patients within the observation period of 23 years . in further 6 patients with over 4 cm lesions that showed features of neoplastic cystic tumors , resection of the tumor with an adequate part of the pancreas was performed . in patients diagnosed with and treated due to pancreatic pseudocysts , transabdominal ultrasound is the main diagnostic tool , followed by ct and eus , in qualifying patients to endoscopic treatment . in the discussed period of time , from among 167 patients diagnosed with symptomatic cysts of the pancreas , 44.9% underwent surgeries , 12.6% were treated by means of external drainage and 28.7% by internal eus - guided endoscopic drainage . still , the largest group underwent surgeries in spite of the fact that endoscopic procedures introduced in 2000 have a greater and greater therapeutic value . such a procedure was performed in 48 patients , but in 11 of them ( 23% ) endoscopic drainage was not successful mainly due to technical problems connected with prosthesis placement , difficult angular access of the endoscope to the cyst , bleeding and suspicion of perforation . in the remaining 37 patients with pancreatic pseudocysts , it allows for a rapid return to everyday activities , patients do not need to follow a rigorous diet ( as it happens after a surgery ) and their physical activity is not restricted due to the postoperative wound . at present , thanks to the advancement of diagnostic techniques , we may accurately assess and distinguish neoplastic cystic tumors of the pancreas from ordinary cystic lesions . the most common reported symptoms include : pain in the abdomen , nausea , vomiting and increased circumference of the abdomen , weight loss , obstructive jaundice , constipation or diarrhea and weakness . patients with ipmn may be sometimes treated due to recurrent episodes of acute pancreatitis , particularly , in cases when the main pancreatic duct is involved . large tumors are most frequently found in patients with mucinous cystic neoplasms ( mcn ) and spn these lesions usually give symptoms of a midabdominal tumor . in advanced malignant lesions , apart from the above - listed symptoms the following may occur : bleeding from the gastrointestinal tract in the case of gastric infiltration , portal hypertension and hemobilia as well as , eventually , diabetes when the pancreas is considerably damaged . in the analyzed time period , after thorough imaging analysis , including doppler ultrasound , contrast enhanced ct as well as in some cases , mri , mrcp and eus , 6 patients were qualified to fnab . only in 2 cases did fnab and assessment of the aspirated fluid confirm the presence of a cystic neoplasm . the assessment of the aspirate is usually less specific and the final verification of the type of the tumor is acquired following the analysis of the postoperative specimen . in our material , the postoperative verification confirmed the presence of 3 serous cystadenomas and 3 mucinous cystadenomas in 2 cases , the lesions had already been identified as cystadenocarcinomas . in these patients , annual check - ups were recommended which in the period of analysis , did not show any growth or change of the inner structure . in the next part , the material has been collected based on quoted recent publications concerning diagnosis , methods of differentiation and proposed types of treatment . it is usually asymptomatic and signals , such as pain in the abdomen , weight loss , feeling of distension in the epigastric region and peristalsis disorders appear when the lesion shows large growth . in imaging examinations ( us , ct ) , such a cystic tumor frequently has the appearance of a honeycomb which is related to a large number of small cysts ( fig . structure with oligocystic components and central scar ( arrow ) contrast - enhanced ct shows a classic serous cystadenoma in the head of the pancreas ( 4.1 2.9 cm ) . therefore , the basic symptoms of its presence , next to the feeling of discomfort in the epigastric region and sometimes , pain when the lesion grows , is jaundice the tumor is composed of other well - circumscribed cystic lesions , with their diameters usually greater than 2 cm . in the present - day imaging diagnosis it is a modern us examination with the use of contrast media which considerably broadens diagnostic possibilities of doppler us examination . ceus allows for a good visualization of the honeycomb appearance and of the septations inside a neoplastic cyst . it appears that a confirmation of a microcystic structure is sufficient for differential diagnosis and there is no need for the analysis of the fluid collected by means of eus - guided fine - needle aspiration biopsy ( eus - fna ) . , the collected fluid does not show any traces of mucin or amylase , and the test for the contents of cell elements ( cytological examination ) is possible in less than 50% of cases . in the material presented herein , in the case of serous tumors , aspiration demonstrated correct level of the markers ( cea and ca 19 - 9 ) and cytological examination did not reveal any neoplastic cells . a surgical resection of such a tumor together with a fragment of the pancreas is indicated in symptomatic lesions , in tumors that grow during the period of monitoring and in cases in which malignant transformation can not be ruled out . in patients qualified to surgical treatment , ious appears to be useful both in terms of final verification of the diagnosis and in the assessment of the range of resection . there are mixed opinions since the cost of check - ups is considerable and the sole awareness of patients of a potential threat to develop a cancerous disease is of great significance . in the remaining cases , resection of the tumor it might be performed with the application of minimally invasive techniques when the lesion is relatively small and when surgical treatment should proceed with no complications . poorly vascularized , multiple small cysts with polycyclic border of the tumor it is currently believed that mucinous cystic tumors constitute the most common form of pancreatic cystic neoplasms ( 1045% ) . they are encountered almost only in women ( 95% ) , mainly middle - aged ones , and are usually localized in the body and tail of the pancreas . a mean size of the tumor during the first diagnosis equals > 5 cm and in certain cases , the lesion may grow even up to 2535 cm . ceus examination allows for a better identification of the lesions within the wall ( nodules ) and thickened septations , which is caused by increased vascularity of the altered structures attesting to malignant transformation of the tumor . in such cases , the cystic lesion is situated in the head of the pancreas with low - grade dysplasia ( arrows ) us power doppler . this imaging modality provides reliable information allowing for the characterization of the cyst and determining the possibilities of resection when features of malignancy are present . due to the extension of the method to include aspiration biopsy ( eus - fna ) , it is possible to find extracellular mucus in the cystic fluid , conduct cytological or , more rarely , histological analyses , make biochemical assessment , determine the level of tumor markers and perform molecular analysis . with the cut - off value of 200 ng / ml , higher values obtained in the analysis of cystic fluid indicate the presence of a mucinous tumor . for the cut - off value of 250 iu / l , in the case of this level , the sensitivity and specificity constituted 45% and 89% respectively . the basis for the qualification to surgical treatment is the outcome of imaging examinations , clinical examination and , to a lower degree , also data obtained from cystic fluid aspiration . cytological examination gave positive outcomes in the analysis of fluid from the inside of the cystic tumor in only one patient . the most common symptom is recurrent pain , similar to that in the course of chronic , exacerbating pancreatitis or episodes of acute pancreatitis , occurring mainly after meals . we distinguish three types of ipmn : central type which encompasses the main pancreatic duct , peripheral type involving the branches of the ducts , and mixed - type . at present , imaging examinations , such as ultrasound , but ct and mri in particular , are capable of relatively accurate visualization of the dilatation of the pancreatic duct , secondary cystic lesions and connection between the duct and altered , dilated cystic structure . ercp allows for the identification of the intraductal lesions and collection of the pancreatic juice in order to determine the content of mucin , obtain a cytological picture as well as determine the level of tumor markers in the aspirate . mrcp , on the other hand , is the method of choice in the diagnosis and assessment of ipmn progression . it accurately assesses the thickening of the ductal wall and in peripheral types the thickening of the cystic wall and presence of intramural nodules which are normally signs of malignant transformation . the risk of developing a malignancy rises with the increase in the diameter of the main pancreatic duct , in the case of visualizing a large amount of mucus crossing through the ampulla of vater and when obstructive jaundice and/or diabetes are clinically confirmed . eus examination is extended by eus - fna thanks to which we may conduct cytological tests , determine the concentration of tumor markers and amylase and perform fnab of enlarged peripancreatic lymph nodes . therefore , there are specific indications for a surgical resection of a central - type tumor , even for total pancreatectomy if the main duct is involved to a great extent . when the tumor is localized peripherally , the decision concerning surgical treatment should be made based on the general condition of the patient and on associated risks and benefits of such a procedure taking into account the presence of given symptoms . usually wellcircumscribed , the tumor is composed of solid part , cystic lesions , papillary structures , areas of necrosis and hemorrhagic components which are a result of numerous extravasations into the tumorous tissue . even in such cases , the prognosis is good and longterm survival depends on the specific biology of the spn tumor . structure with oligocystic components and central scar ( arrow ) contrast - enhanced ct shows a classic serous cystadenoma in the head of the pancreas ( 4.1 2.9 cm ) . therefore , the basic symptoms of its presence , next to the feeling of discomfort in the epigastric region and sometimes , pain when the lesion grows , is jaundice the tumor is composed of other well - circumscribed cystic lesions , with their diameters usually greater than 2 cm . in the present - day imaging diagnosis it is a modern us examination with the use of contrast media which considerably broadens diagnostic possibilities of doppler us examination . it appears that a confirmation of a microcystic structure is sufficient for differential diagnosis and there is no need for the analysis of the fluid collected by means of eus - guided fine - needle aspiration biopsy ( eus - fna ) . , the collected fluid does not show any traces of mucin or amylase , and the test for the contents of cell elements ( cytological examination ) is possible in less than 50% of cases . in the material presented herein , in the case of serous tumors , aspiration demonstrated correct level of the markers ( cea and ca 19 - 9 ) and cytological examination did not reveal any neoplastic cells . a surgical resection of such a tumor together with a fragment of the pancreas is indicated in symptomatic lesions , in tumors that grow during the period of monitoring and in cases in which malignant transformation can not be ruled out . in patients qualified to surgical treatment , ious appears to be useful both in terms of final verification of the diagnosis and in the assessment of the range of resection . there are mixed opinions since the cost of check - ups is considerable and the sole awareness of patients of a potential threat to develop a cancerous disease is of great significance . in the remaining cases , resection of the tumor it might be performed with the application of minimally invasive techniques when the lesion is relatively small and when surgical treatment should proceed with no complications . ceus examination allows for a better identification of the lesions within the wall ( nodules ) and thickened septations , which is caused by increased vascularity of the altered structures attesting to malignant transformation of the tumor . due to the extension of the method to include aspiration biopsy ( eus - fna ) , it is possible to find extracellular mucus in the cystic fluid , conduct cytological or , more rarely , histological analyses , make biochemical assessment , determine the level of tumor markers and perform molecular analysis . from among numerous tumor markers , the cut - off value of 200 ng / ml , higher values obtained in the analysis of cystic fluid indicate the presence of a mucinous tumor . for the cut - off value of 250 iu / l , in the case of this level , the sensitivity and specificity constituted 45% and 89% respectively . the basis for the qualification to surgical treatment is the outcome of imaging examinations , clinical examination and , to a lower degree , also data obtained from cystic fluid aspiration . in a large majority of patients , the most common symptom is recurrent pain , similar to that in the course of chronic , exacerbating pancreatitis or episodes of acute pancreatitis , occurring mainly after meals . we distinguish three types of ipmn : central type which encompasses the main pancreatic duct , peripheral type involving the branches of the ducts , and mixed - type . at present , imaging examinations , such as ultrasound , but ct and mri in particular , are capable of relatively accurate visualization of the dilatation of the pancreatic duct , secondary cystic lesions and connection between the duct and altered , dilated cystic structure . ercp allows for the identification of the intraductal lesions and collection of the pancreatic juice in order to determine the content of mucin , obtain a cytological picture as well as determine the level of tumor markers in the aspirate . it accurately assesses the thickening of the ductal wall and in peripheral types the thickening of the cystic wall and presence of intramural nodules which are normally signs of malignant transformation . the risk of developing a malignancy rises with the increase in the diameter of the main pancreatic duct , in the case of visualizing a large amount of mucus crossing through the ampulla of vater and when obstructive jaundice and/or diabetes are clinically confirmed . eus examination is extended by eus - fna thanks to which we may conduct cytological tests , determine the concentration of tumor markers and amylase and perform fnab of enlarged peripancreatic lymph nodes . therefore , there are specific indications for a surgical resection of a central - type tumor , even for total pancreatectomy if the main duct is involved to a great extent . when the tumor is localized peripherally , the decision concerning surgical treatment should be made based on the general condition of the patient and on associated risks and benefits of such a procedure taking into account the presence of given symptoms . usually wellcircumscribed , the tumor is composed of solid part , cystic lesions , papillary structures , areas of necrosis and hemorrhagic components which are a result of numerous extravasations into the tumorous tissue . thick , well - circumscribed capsule , hyperdense areas due to hemorrhage , and areas of necrosis the procedural treatment is applied in the case of large ( > 4 cm ) , symptomatic cysts of the pancreas.endoscopic drainage constitutes an effective and safe method of minimally invasive treatment of pancreatic cysts and surgical procedures are an alternative when minimally invasive techniques are not possible.the differentiation of a neoplasm from a typical cyst is of fundamental significance for the selection of an adequate treatment method .

5-ht3a receptor constructs and transfection of cdnas cdnas encoding human wild - type and mutant 5-ht3a receptor subunits were cloned into pgw1 ( 17 ) . point mutations were introduced using standard molecular biological techniques ( 31 ) , and all cdnas were sequenced to confirm fidelity . wild - type and mutant receptor subunit cdnas were co - transfected into tsa-201 or hek-293 cells , with a cdna encoding green fluorescent protein to identify transfected cells . transfection was performed by electroporation ( 400 v , 125 microfarads , infinite resistance ) using a bio - rad gene electropulser ii ( biorad ) or the calcium phosphate precipitation method . transfected cells were routinely cultured in dulbecco 's modified eagle 's medium supplemented with 10% ( v / v ) fetal bovine serum , 2 mm glutamine , 1 mm sodium pyruvate , 100 g ml streptomycin , and 100 units ml penicillin and maintained at 37 c for 2448 h in 95% air , 5% co2 at 100% humidity before use . electrophysiological recordings whole - cell and outside - out patch configurations were used to record macroscopic and single channel currents , respectively , from transfected cells . the recording chamber was routinely superfused ( 5 ml min ) with an extracellular solution ( e1 ) comprising ( in mm ) : nacl 140 , kcl 2.8 , mgcl2 2.0 , cacl2 1.0 , glucose 10 , hepes 10 ( ph 7.2 , adjusted with 1 m naoh ; final [ na]o = 146 mm ) . patch electrodes ( resistance = 28 megohms when measured in solution e1 ) were filled with an intracellular solution ( i1 ) containing ( in mm ) : cscl 140 , cacl2 0.1 , egta 1.1 , hepes 10 ( ph 7.2 , adjusted by 1 m csoh , final [ cs]i = 143 mm ) . in solution i2 , cscl was totally replaced by nacl ( ph 7.2 , adjusted by 1 m naoh , final [ na]i = 145 mm ) . the intracellular free calcium concentration ( [ ca]i ) for i1 and i2 was estimated to be 10 nm ( 35 ) . to determine the relative permeability of na versus cs ( pna / pcs ) , in solution e2 , additional nacl totally replaced kcl in the extracellular solution , and the concentrations of cacl2 and mgcl2 were each reduced to 0.1 mm to minimize the influence of divalent cations upon the reversal potential of the macroscopic current response to 5-ht ( e5-ht ) . the permeability of ca relative to cs ( pca / pcs ) was determined using an extracellular solution ( e3 ) containing ( in mm ) : cacl2 100 , l - histidine 5 , glucose 10 ( ph 7.2 ) ( 20 ) . to prevent changes in reference electrode potential during the superfusion of media with altered ionic composition , a bridge containing 3 m kcl in agar ( 4% w / v ) was employed . liquid junction potentials arising at the tip of the patch pipette were measured as described by fenwick et al . the membrane potential was stepped from 60 mv to 100 mv for 100 ms and subsequently ramped to + 60 mv within 1 s at the peak of the macroscopic current response to pressure - applied 5-ht ( 1 m ) . care was taken to ensure that the agonist - induced current recorded at 60 mv before and immediately following the voltage ramp did not change . subtraction of the leakage current recorded in the absence of 5-ht from the current recorded in the presence of the agonist yielded the current - voltage ( i - v ) relationship attributable to the 5-ht - evoked conductance increase . in the second method , peak current responses to pressure - applied 5-ht ( 10 m ) were recorded at steady holding potentials closely bracketing e5-ht , which was subsequently determined by interpolation . concentration - response relationships to 5-ht ( 0.3100 m ) were determined from the peak inward whole - cell current response recorded in the presence of solutions e1 and i1 at a holding potential of 60 mv . , san jose , ca ) by an equation of the form ( eq . 1)\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*}\frac{i}{i_{{\mathrm{max}}}}=\left(\frac{[a]}{[a]+[{\mathrm{ec}}_{50}]}\right)^{n_{h}}\end{equation*}\end{document } where i is the peak current elicited by 5-ht at concentration [ a ] , imax is the peak current evoked by a saturating concentration of 5-ht , ec50 is the concentration of 5-ht that evokes a half - maximal response , and nh is the hill coefficient . to combine the concentration - response data obtained from several cells , agonist - evoked currents were normalized and expressed as a percentage of the maximal inward current response produced by a saturating concentration of 5-ht obtained for each cell . single channel currents evoked by pressure application of 5-ht ( 10 m ) were recorded from excised outside - out membrane patches clamped at steady holding potentials within the range of 100 to + 100 mv as specified under results . the solutions used in such experiments were i1 and e1 , e2 , and e3 , as appropriate . additionally , in experiments that determined e5-ht and single channel conductance in mixtures of extracellular na and ca , nacl was held at a constant concentration of 95 mm in the presence of variable concentrations of cacl2 ( 103 10 m ) , sucrose ( 090 mm , as appropriate to maintain constant osmolarity ) , glucose ( 10 mm ) , and l - histidine ( 5 mm ; ph 7.2 ) . currents were recorded using an axopatch-1d amplifier ( axon instruments , union city , ca ) , low pass - filtered ( 5 khz , bessel characteristic ) , and recorded onto magnetic tape using a bio - logic dat recorder ( bio - logic , claix , france ) for subsequent offline analysis . data analysis permeability ratios ( relative to cs ) were determined from measurements of e5-ht and calculated ion activities ( i.e. the ionic concentration multiplied by ion activity coefficient ( ion ) ) . the latter were estimated ( following the guggenheim convention ) to be : cs 0.72 ( 140 mm cs ) na 0.76 ( 140 mm na ) , and ca 0.26 ( 100 mm ca ) . for varying concentrations of ca in the presence of 95 mm na , the mean molal activity coefficients for cacl2 in the presence of 100 mm nacl , as tabulated by butler ( 36 ) , allowed calculation of ca . sucrose had a negligible influence upon na over the range of sugar concentrations employed ( 37 ) . the permeability ratio pna / pcs was calculated from the goldman - hodgkin - katz ( voltage ) equation , ( eq . 2)\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*}\;e_{5{\mathrm{-}}{\mathrm{ht}}}=\frac{rt}{f}{\mathrm{ln}}\frac{(p_{{\mathrm{na}}}/p_{{\mathrm{cs}}})[{\mathrm{na}}^{+}]_{o}}{[{\mathrm{cs}}^{+}]_{i}}\end{equation*}\end{document } where r , t , and f have their usual meaning and [ na]o and [ cs]i are the calculated activities of extracellular na and internal cs ions , respectively . this equation ignores the very small error anticipated due to the presence of low concentrations of permeant divalent ions ( e.g. ca ) within the extra- or intracellular solutions . the permeability ratio , pca / pcs , was calculated from a modified goldman - hodgkin - katz ( voltage ) equation ( 38 ) which , with both na and ca present as permeant species in the extracellular medium but only cs and negligible ca present in the pipette ( ` intracellular ' ) solution , can be written as ( 20 ) ( eq . 3)\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*}\;e_{5{\mathrm{-}}{\mathrm{ht}}}=\frac{rt}{f}{\mathrm{ln}}\frac{p_{{\mathrm{na}}}/p_{{\mathrm{cs}}}[{\mathrm{na}}^{+}]_{o}+4(p^{^{\prime}}_{ca}/p_{{\mathrm{cs}}})[{\mathrm{ca}}^{2+}]_{o}}{[{\mathrm{cs}}^{+}]_{i}}\end{equation*}\end{document } where [ na]o and [ ca]o are the external activities of na and ca , [ cs]i is the internal activity of cs , and pca / pcs is a modified term relating the permeability of ca to cs . substituting for pca / pcs , the above can also be written as ( eq . 4)\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{exp}}^{e_{5{\mathrm{-}}{\mathrm{ht}}}f / rt}=\frac{p_{{\mathrm{na}}}[{\mathrm{na}}^{+}]_{o}}{p_{cs}[{\mathrm{cs}}^{+}]_{i}}+4\frac{p_{{\mathrm{ca}}}[{\mathrm{ca}}^{2+}]_{o}}{{\mathrm{p}}_{{\mathrm{cs}}}(1+{\mathrm{exp}}^{e_{5{\mathrm{-}}{\mathrm{ht}}}f / rt})[{\mathrm{cs}}^{+}]_{i}}\end{equation*}\end{document } when ca was the sole cation in the extracellular medium , the term pna / pcs was omitted . for simplicity , in the text we routinely refer to ion concentrations ( in square brackets ) rather than activities ( in parentheses ) , although the latter were used in all calculations of relative permeability and are presented in the figures where relevant . single channel currents were low pass - filtered offline at 1 khz , digitized at 10 khz via a digidata 1302a ( axon instruments ) interface . using the winedr v2 7.6 electrophysiology data recorder ( j. dempster , dept . of physiology and pharmacology , university of strathclyde , uk ) , single channel current amplitude histograms were constructed from sections of single channel activity in which unitary events predominated . a transition detection threshold of 3040% of the predominant unitary event amplitude events less than 1 ms in duration ( classified as incompletely resolved ) , obvious artifacts , and those corresponding to multiple openings were rejected . the resulting mean open state amplitude histogram was fitted using an iterative least squares algorithm with a single gaussian probability density function from which unitary event amplitude ( i ) was determined . the analysis was restricted to the dominant unitary current state , as events of differing amplitude were relatively rare . single channel conductance ( ) is routinely reported as the chord conductance , i.e. = i/(vm e5-ht ) , where vm is the holding potential ( including liquid junction potential correction ) and e5-ht is the reversal potential of the agonist - evoked macroscopic response determined as described above under the appropriate ionic conditions . statistical analysis was conducted using one - way analysis of variance with the post hoc dunnett or tukey test as appropriate . residues in the ma helix of the 5-ht3a receptor influence ca permeability we recently demonstrated that arginine residues 432 , 436 , and 440 within the ma helix motif of the tm34 loop at positions termed ma 4 , 0 , and 4 , respectively , which line putative cytoplasmic portals ( fig . 1c ) , are responsible for the sub - ps single channel conductance of the 5-ht3a receptor ( 2 , 22 , 31 ) . we began this study by testing the hypothesis that the same 5-ht3a ma helix arginine residues also serve as determinants of ionic selectivity . figure 1.domains of the 5-ht3 receptor that influence ion conduction and selectivity between mono- and divalent cations . a , homology model of the wild - type 5-ht3a receptor using the nicotinic ach receptor of torpedo marmorata as a template ( 32 ) . the functional modules are denoted : extracellular domain ( ec ) , poreforming transmembrane helices ( tm ) , and the portal - framing cytoplasmic ma helices ( ma ) . b , sequence alignment of the second transmembrane domain ( tm2 ) and flanking sequences of the human 5-ht3a and 5-ht3b subunits . the residue mutated to alanine in the mutant 5-ht3a receptor constructs is boxed . c , an alignment of amino acids within the ma helices of wild - type 5-ht3a and 5-ht3b subunits and the mutant 5-ht3a(qda ) construct used in this study , indicating the positions of the 4 , 0 , and 4 residues ( boxed ) . amino acids are numbered according to the human 5-ht3a subunit amino acid sequence ( 51 ) . domains of the 5-ht3 receptor that influence ion conduction and selectivity between mono- and divalent cations . a , homology model of the wild - type 5-ht3a receptor using the nicotinic ach receptor of torpedo marmorata as a template ( 32 ) . the functional modules are denoted : extracellular domain ( ec ) , poreforming transmembrane helices ( tm ) , and the portal - framing cytoplasmic ma helices ( ma ) . b , sequence alignment of the second transmembrane domain ( tm2 ) and flanking sequences of the human 5-ht3a and 5-ht3b subunits . c , an alignment of amino acids within the ma helices of wild - type 5-ht3a and 5-ht3b subunits and the mutant 5-ht3a(qda ) construct used in this study , indicating the positions of the 4 , 0 , and 4 residues ( boxed ) . amino acids are numbered according to the human 5-ht3a subunit amino acid sequence ( 51 ) . the relative permeability ratio , pna / pcs , of wild - type and mutant 5-ht3a receptors was calculated by determining e5-ht from voltage ramps ( 100 to + 60 mv ) that coincided with the peak of the macroscopic current response to pressure - applied 5-ht in the presence of extracellular and intracellular electrolytes e2 and i1 , respectively ( fig ramp currents generated in the absence of 5-ht were subtracted from ramp currents in the presence of 5-ht yielding an inwardly rectifying i - v relationship with an e5-ht of 2.1 0.7 mv , corresponding to a pna / pcs ratio of 1.02 0.03 ( n = 15 ; figs . 2b and 3a , table 1 ) for the wild - type receptor . in six cells from this population , e5-ht was redetermined following the exchange of solution e2 with e3 ( i.e. complete replacement of na by 100 mm ca ) , resulting in a negative shift in e5-ht to 6.1 1.3 mv accompanied by a pronounced depression in the amplitude of the 5-ht - evoked current and the loss of inward rectification in the i - v relationship , most noticeable at positive potentials ( fig . 2b ) . despite substantial inhibition of current amplitude by extracellular ca , pca / pcs ( 1.42 0.11 , n = 6 ; fig . 3a , table 1 ) was greater than pna / pcs ( p = 0.021 , paired t test ) . table 1the influence of mutations within the intracellular ma helix upon the permeabilities of ca and na relative to csrelative permeabilities ( i.e. pca / pcs and pna / pcs ) were calculated from determinations of e5-ht in extracellular solutions containing na ( solution e2 ) or ca ( solution e3 ) as the permeant cation . * , indicates significantly different from the appropriate wild - type permeability ratio , p < 0.01 . wt , wild - type ; qda , 5-ht3a(r432q , r436d , r440a ) ; qea , 5-ht3a ( r432q , r436e , r440a ) ; qaa , 5-ht3a(r432q , r436a , r440a ) ; qfa , ( 5-ht3a(r432q , r436f , r440a ) ; qra , 5-ht3a(r432q , r440a).5-ht3a constructs na extracellular ca extracellular e5-htpna / pcsne5-htpca / pcsnmvmv wt 5-ht3a 2.1 0.7 1.03 0.03 15 -6.1 1.3 1.42 0.11 6 r432q 6.0 0.9 1.20 0.04 17 -3.4 2.6 1.70 0.26 7 r436d -0.4 0.7 0.93 0.03 13 7.1 1.4 3.13 0.31 * 7 r440a 4.8 1.1 1.15 0.05 5 2.3 1.7 2.33 0.23 5 qda -1.3 1.0 0.90 0.04 10 9.4 1.4 3.65 0.30 * 10 qea -3.6 0.6 0.81 0.02 4 7.6 0.9 3.15 0.17 * 4 qaa -6.8 0.9 0.72 0.02 * 8 3.5 0.9 2.46 0.14 6 qfa -5.2 0.5 0.77 0.01 * 8 2.1 0.4 2.25 0.06 5 qra -2.0 1.6 0.88 0.05 7 -2.1 1.4 1.80 0.15 7 the influence of mutations within the intracellular ma helix upon the permeabilities of ca and na relative to cs relative permeabilities ( i.e. pca / pcs and pna / pcs ) were calculated from determinations of e5-ht in extracellular solutions containing na ( solution e2 ) or ca ( solution e3 ) as the permeant cation . statistical significance was determined by analysis of variance with dunnett 's post hoc test . * , indicates significantly different from the appropriate wild - type permeability ratio , p < 0.01 . wt , wild - type ; qda , 5-ht3a(r432q , r436d , r440a ) ; qea , 5-ht3a ( r432q , r436e , r440a ) ; qaa , 5-ht3a(r432q , r436a , r440a ) ; qfa , ( 5-ht3a(r432q , r436f , r440a ) ; qra , 5-ht3a(r432q , r440a ) . experiments performed on the 5-ht3a(qda ) mutant using the voltage ramp protocol yielded a pna / pcs ratio of 0.9 0.04 ( e5-ht = 1.3 1.0 mv , n = 13 ) , which is not significantly different from that of the wild - type 5-ht3a receptor ( fig . an essentially identical value for e5-ht ( 1.4 0.5 mv , n = 3 ) was found from measurements of 5-ht - induced peak current amplitudes at potentials closely bracketing that of current reversal . however , in contrast to the negative shift in e5-ht found for the wild - type 5-ht3a receptor upon replacement of solution e2 by e3 , that of the mutant displayed a robust positive shift to 9.4 1.4 mv ( n = 10 ) , reflecting a significant increase in pca / pcs to 3.65 0.34 ( n = 10 , figs . 2c and 3a , table 1 ) . despite the enhancement of pca / pcs at the 5-ht3a(qda ) receptor , inhibition of the macroscopic current responses by extracellular ca persisted , although the effect was less pronounced at positive potentials due to the outward , versus mild inward , rectification typically observed in the ca- and na - containing extracellular solutions , respectively ( fig . , we evaluated the contributions of the individual mutations r432q ( ma 4 ) , r436d ( ma 0 ) , and r440a ( ma 4 ) to the enhancement of pca/ pcs . the r436d charge reversal mutation produces an 6-fold increase in single channel conductance compared with the wild - type 5-ht3a receptor ( 22 ) . the 5-ht3a(r436d ) receptor also had a significantly enhanced relative permeability to ca ( pca / pcs = 3.13 0.31 , n = 7 , fig . by contrast , the introduction of aspartate did not significantly affect pna / pcs compared with wild - type 5-ht3a receptors ( fig . notably , the pca / pcs of the 5-ht3a(qda ) receptor was not significantly greater than that of the 5-ht3a(r436d ) receptor . the individual replacement of arg-432 by the equivalent 5-ht3b subunit residue glutamine had no significant effect on single channel conductance ( 22 ) . likewise , the permeability of ca relative to cs of the 5-ht3a(r432q ) mutant was similar to that of the wild - type 5-ht3a receptor , suggesting that this ma helix residue has little influence on ion selectivity ( fig . although the 5-ht3a(r440a ) mutant exhibited a trend toward an increased pca / pcs ratio relative to the wild - type receptor , the effect failed to reach statistical significance ( fig . the validity of the above estimates of pna / pcs and pca / pcs for the 5-ht3a(qda ) receptor construct relies upon the mutant retaining the near perfect cation versus anion selectivity of the wild - type receptor ( pna / pcl = 53 ) ( 30 ) . we tested this assumption rigorously by dilution experiments in which the nacl content of the extracellular solution e2 was reduced to 95 , 50 , and 20 mm by replacement with sucrose . under such conditions a plot of e5-ht as a function of the logarithm of extracellular na activity , ( na)o , was linear and yielded a slope of 59 mv / decade change in ( na)o ( fig . a similar dependence upon ( na)o was observed under simplified bi - ionic conditions in which na totally replaced cs within the intracellular solution . because only a slope of less than 58 mv / decade change in ( na)o is compatible with anion permeation ( 28 ) , we concluded that for the 5-ht3a(qda ) receptor construct pna / pcl = . the foregoing results indicate that the arginine residues present at positions 436 ( ma 0 ) and to a lesser extent 440 ( ma 4 ) of the human wild - type 5-ht3a receptor collectively suppress the permeability of ca , but not na , relative to cs , revealing that the large intracellular loop of a cys - loop receptor is an important determinant of divalent versus monovalent cation selectivity . figure 2.the reversal potential of 5-ht - evoked macroscopic currents at wild - type 5-ht3a and 5-ht3a(qda ) receptor constructs with na or ca as the charge carrier in the extracellular solution . a , voltage ramp ( left ) applied at the peak of the macroscopic inward current response to pressure - applied 5-ht ( 1 m)(right ) to determine the reversal potential ( e5-ht ) of the agonist - induced response mediated by the wild - type 5-ht3a receptor . b , current - voltage relationships recorded from a representative hek-293 cell expressing the wild - type 5-ht3a receptor with an intracellular cscl - based solution ( i1 ) as reference and nacl ( 146 mm)-based solution e2 or cacl2 ( 100 mm)-based solution e3 as the extracellular electrolyte . note the negative shift in e5-ht ( arrow ) upon complete iso - osmotic replacement of extracellular na by ca . c , current - voltage relationships recorded from a representative hek-293 cell expressing the 5-ht3a(qda ) receptor under the conditions described in b. note that complete iso - osmotic replacement of extracellular na by ca causes a positive shift in e5-ht ( arrow ) . the reversal potential of 5-ht - evoked macroscopic currents at wild - type 5-ht3a and 5-ht3a(qda ) receptor constructs with na or ca as the charge carrier in the extracellular solution . a , voltage ramp ( left ) applied at the peak of the macroscopic inward current response to pressure - applied 5-ht ( 1 m)(right ) to determine the reversal potential ( e5-ht ) of the agonist - induced response mediated by the wild - type 5-ht3a receptor b , current - voltage relationships recorded from a representative hek-293 cell expressing the wild - type 5-ht3a receptor with an intracellular cscl - based solution ( i1 ) as reference and nacl ( 146 mm)-based solution e2 or cacl2 ( 100 mm)-based solution e3 as the extracellular electrolyte . note the negative shift in e5-ht ( arrow ) upon complete iso - osmotic replacement of extracellular na by ca . c , current - voltage relationships recorded from a representative hek-293 cell expressing the 5-ht3a(qda ) receptor under the conditions described in b. note that complete iso - osmotic replacement of extracellular na by ca causes a positive shift in e5-ht ( arrow ) . the charge of the ma stretch 0 residue is the principal determinant of ca permeability we further evaluated the influence of charge at the ma 0 position by determining pca / pcs for constructs in which the residue was either negative ( 5-ht3a(r432q , r436e , r440a ) ; abbreviated as 5-ht3a(qea ) ) , neutral ( 5-ht3a(r432q , r436a , r440a ) and 5-ht3a(r432q , r436f , r440a ) ; abbreviated as 5-ht3a(qaa ) and 5-ht3a(qfa ) , respectively ) , or remained positive ( 5-ht3a(r432q , r440a ) ; abbreviated as 5-ht3a(qra ) ) . pca / pcs was significantly enhanced compared with the wild - type 5-ht3a receptor for the 5-ht3a(qea ) construct . the enhancement observed for the 5-ht3a(qea ) construct was not significantly different from that found for the 5-ht3a(qda ) receptor . although pca / pcs was not significantly different from the wild - type 5-ht3a receptor for the 5-ht3a(qaa ) , 5-ht3a(qfa ) , and 5-ht3a(qra ) constructs , the ratio tended to decrease progressively as the charge at position 436 changed from negative , through neutral , to positive ( fig . thus , charge inversion appears necessary to cause a shift in e5-ht sufficient to produce a statistically significant difference in pca / pcs values . finally , measurements of e5-ht with na as the predominant extracellular cation revealed no significant change in pna / pcs relative to the wild - type receptor , apart from the 5-ht3a(qaa ) and 5-ht3a(qfa ) constructs in which the ratio was modestly reduced to 0.72 0.02 and 0.77 0.01 , respectively ( fig . , the sign of the charge at position 436 clearly had no consistent influence upon pna/ pcs ( fig . , the results suggest that the reversal of charge at position 436 within the 5-ht3a(qda ) construct was the predominant cause of a greatly increased pca / pcs relative to the wild - type receptor . figure 3.comparison of the relative ion permeability of wild - type and mutant 5-ht3a receptor constructs . a , bar graph illustrating the relative permeability ( px / pcs ) of na ( black bars ) , or ca ( white bars ) relative to cs for the indicated receptor constructs . permeability ratios were determined with i1 as the intracellular reference solution and nacl ( 146 mm)-based solution e2 or cacl2 ( 100 mm)-based solution e3 as the extracellular electrolyte . mutant constructs in which pna / pcs or pca / pcs was significantly different from the appropriate wild - type control are indicated with an asterisk ( p < 0.05 ; analysis of variance followed by dunnett 's post hoc test ) . b , relationship between the reversal potential of the macroscopic current evoked by 5-ht ( e5-ht ) and the activity of na in the extracellular medium ( ( na)o ) for the 5-ht3a(qda ) receptor construct . responses were recorded with extracellular electrolytes based on solution e2 wherein the concentration of nacl was reduced by substitution with sucrose . intracellular solutions were either cscl - based i1 ( filled circles ) or a solution in which nacl totally replaced cscl ( open circles ) to produce bi - ionic recording conditions . the regression line fitted to the filled circles yielded a slope of 59 mv / decade change in ( na)o . data points are the mean of 36 independent determinations of e5-ht , and vertical bars indicate s.e . c , relationship between pna / pcs ( filled circles ) and pca / pcs ( open circles ) and the charge of the amino acid residue at position 436 of the ma helix . comparison of the relative ion permeability of wild - type and mutant 5-ht3a receptor constructs . a , bar graph illustrating the relative permeability ( px / pcs ) of na ( black bars ) , or ca ( white bars ) relative to cs for the indicated receptor constructs . permeability ratios were determined with i1 as the intracellular reference solution and nacl ( 146 mm)-based solution e2 or cacl2 ( 100 mm)-based solution e3 as the extracellular electrolyte . mutant constructs in which pna / pcs or pca / pcs was significantly different from the appropriate wild - type control are indicated with an asterisk ( p < 0.05 ; analysis of variance followed by dunnett 's post hoc test ) . b , relationship between the reversal potential of the macroscopic current evoked by 5-ht ( e5-ht ) and the activity of na in the extracellular medium ( ( na)o ) for the 5-ht3a(qda ) receptor construct . responses were recorded with extracellular electrolytes based on solution e2 wherein the concentration of nacl was reduced by substitution with sucrose . intracellular solutions were either cscl - based i1 ( filled circles ) or a solution in which nacl totally replaced cscl ( open circles ) to produce bi - ionic recording conditions . the regression line fitted to the filled circles yielded a slope of 59 mv / decade change in ( na)o . data points are the mean of 36 independent determinations of e5-ht , and vertical bars indicate s.e . c , relationship between pna / pcs ( filled circles ) and pca / pcs ( open circles ) and the charge of the amino acid residue at position 436 of the ma helix . the 5-ht3a(qda ) receptor conducts ca less efficiently than na determinations of relative permeability based on reversal potential measurements do not predict the relative ease with which ion channels conduct permeant ions ( 39 ) . for example , a binding site for ca within the permeation pathway may confer selectivity toward this divalent , yet retard its flux in comparison to monovalent cations . indeed , we previously demonstrated that extracellular divalent cations ( mg and ca ) reduce the single channel conductance of the wild - type 5-ht3a receptor as estimated using fluctuation analysis ( 20 ) . however , the latter technique tends to underestimate single channel conductance , and it is preferable to measure single channel conductances directly using excised patch recording ( 22 ) . this is impossible for the wild - type 5-ht3a receptor , with a conductance of < 1 ps , which is below the resolution of the patch - clamp technique ( 20 , 21 , 40 ) . in contrast , the 5-ht3a(qda ) construct has a single channel conductance that enables the direct resolution of single channel currents in outside - out patch recordings ( slope conductance of 37 ps at 74 mv ( 22 ) ) . with na as the predominant extracellular cation , the single channel current - voltage relationship ( i - v ) for the 5-ht3a ( qda ) receptor shows a very mild outward rectification over a wide range of holding potentials ( 100 to 100 mv ) and yields chord conductance levels of 46 and 40 ps at holding potentials of + 100 and 100 mv , respectively ( fig . the latter is slightly larger than previously reported by us ( 22 ) ; this minor difference is due to the reduced concentration of divalent cations in the extracellular medium e2 . inclusion of 1 mm ca and 2 mm mg ( medium e1 ) also yielded an essentially linear i - v relationship but with a slope conductance corresponding to our previous estimate ( 22 ) . in contrast to the mild outward rectification of the i - v relationship observed with na , when ca was the sole extracellular cation , 5-ht - evoked single channel events mediated by the 5-ht3a(qda ) receptor displayed marked outward rectification ( fig . 4 , a and b ) similar to that seen in whole - cell current recordings ( fig . negative holding potentials , single channel current amplitudes were greatly reduced in comparison to when na was the permeant cation . at the most negative potential studied ( 100 mv ) , where it can be assumed that single channel currents reflect inward movement of ca with little opposing outwardly directed monovalent cation flux , a single channel chord conductance of 5.7 ps was calculated , assuming an e5-ht of 9.4 mv obtained from macroscopic currents recorded under identical ionic conditions . the disparity between single channel current amplitudes recorded in na- and ca - containing solutions decreased as the holding potential was progressively shifted to more positive values ( fig . 4c , which plots unitary current amplitude against driving force ( i.e. holding potential minus e5-ht ) . indeed , at the most positive potentials examined , where an outwardly directed flux of cs predominates , single channel current amplitudes were indistinguishable between na- and ca - based extracellular solutions ( fig . the data indicate that the 5-ht3a(qda ) receptor construct , despite selecting for ca over na , conducts the former less efficiently , thus predicting that extracellular ca will ( at negative holding potentials ) reduce single channel conductance in mixtures of na and ca as we previously inferred for the human wild - type 5-ht3a receptor by fluctuation analysis ( 20 ) . figure 4.single channel currents mediated by the 5-ht3a(qda ) receptor construct with na or ca as the extracellular permeant ion species . single channel currents ( i ) were recorded from outside - out membrane patches in response to pressure - applied 5-ht ( 10 m ) at holding potentials ( vh ) ranging from 100 to + 100 mv . the patch pipette contained cscl - based solution i1 , and the patch was superfused with nacl ( 146 mm)-based solution e2 or cacl2 ( 100 mm)-based solution e3 . a , exemplar single channel currents with either na ( left ) or ca ( right ) as the extracellular permeant cation . b , current - voltage ( i - v ) plot for single channel currents recorded with nacl - based solution e2 ( filled circles ) or cacl2-based solution e3 ( open circles ) as the extracellular electrolyte . note the mild outward rectification with na as the permeant ion species in the extracellular medium ( compare dashed line fitted to data obtained at positive potentials to curve fitted to all data points ) . c , i - v plot depicted in b but with driving force ( i.e. vh the plot reveals that outwardly directed single channel current amplitudes , at the most positive vh values examined , are essentially identical in na- and ca - containing extracellular solutions when equivalent driving forces are considered . each data point in b and c is the mean of 310 determinations of single channel current amplitude derived from different outside - out membrane patches . the vertical bars , when exceeding the size of the symbols , indicate s.e . single channel currents mediated by the 5-ht3a(qda ) receptor construct with na or ca as the extracellular permeant ion species . single channel currents ( i ) were recorded from outside - out membrane patches in response to pressure - applied 5-ht ( 10 m ) at holding potentials ( vh ) ranging from 100 to + 100 mv . the patch pipette contained cscl - based solution i1 , and the patch was superfused with nacl ( 146 mm)-based solution e2 or cacl2 ( 100 mm)-based solution e3 . a , exemplar single channel currents with either na ( left ) or ca ( right ) as the extracellular permeant cation . b , current - voltage ( i - v ) plot for single channel currents recorded with nacl - based solution e2 ( filled circles ) or cacl2-based solution e3 ( open circles ) as the extracellular electrolyte . note the mild outward rectification with na as the permeant ion species in the extracellular medium ( compare dashed line fitted to data obtained at positive potentials to curve fitted to all data points ) . c , i - v plot depicted in b but with driving force ( i.e. vh the plot reveals that outwardly directed single channel current amplitudes , at the most positive vh values examined , are essentially identical in na- and ca - containing extracellular solutions when equivalent driving forces are considered . each data point in b and c is the mean of 310 determinations of single channel current amplitude derived from different outside - out membrane patches . the vertical bars , when exceeding the size of the symbols , indicate s.e . extracellular ca reduces the single channel conductance of the 5-ht3a(qda ) receptor in a concentration - dependent fashion we examined the influence of extracellular ca upon the single channel conductance of the 5-ht3a(qda ) receptor construct with an extracellular solution containing 95 mm na and varying concentrations of ca ( 10 nm to 30 mm ) . chord conductances were derived from single channel current amplitudes recorded at a holding potential of 80 mv , and measurements of e5-ht were made from macroscopic currents in the ionic mixtures . in this series of experiments , e5-ht was estimated by interpolation between the peak amplitude of inwardly and outwardly directed 5-ht - induced currents that closely bracketed the zero current potential . consistent with the relative permeability measurements made with high ca solution ( e3 , table 1 ) , the addition of increasing concentrations of ca was associated with a progressive shift in e5-ht to more positive values ( from 15.0 0.04 mv ( n = 4 ) with [ ca]o = 0.1 mm to 1.6 0.7 mv single channel conductance was reduced by 65% over the range [ ca]o = 0.1 mm ( 41.3 0.88 ps ) to [ ca]o = 30 mm ( 14.8 0.63 ps ; fig . 5 , b and c ) . however , it should be noted that ca will itself contribute to inward current amplitude , particularly at high [ ca]o . for this reason the calculation of an ic50 value for the suppressant effect of ca upon single channel conductance was inappropriate . a simple interpretation of such data is that ca reduces single channel conductance by binding to a site(s ) of comparatively low affinity within the permeation pathway and thereby occludes the flux of na . in agreement with such a violation of the independent movement of na and ca within the permeation pathway , [ na]o fixed at 95 mm ( and assuming pna / pcs = 0.9 ; see above ) , pca / pcs was calculated from determinations of e5-ht to be 0.5 and 1.8 in the presence of 10 and 30 mm extracellular ca , respectively , versus the value of 3.7 found with ca as the sole extracellular charge carrier . extracellular ca reduces single channel conductance of the 5-ht3a(qda ) receptor in a voltage - independent manner blockade by ca of macroscopic current responses mediated by the 5-ht3a receptor expressed in mammalian cell hosts is voltage - independent ( 20 , 21 , 34 , 40 , 41 ) . the readily resolvable single channel conductance of the 5-ht3a(qda ) receptor allowed direct measurements of the influence of extracellular ca upon single channel current amplitudes over a range of membrane potentials . 6 depicts the results of such studies where [ ca]o was varied between 0.3 and 10 mm in the presence of 95 mm na and single channel events evoked by pressure - applied 5-ht ( 10 m ) were recorded from outside - out membrane patches at holding potentials in the range of 40 to 100 mv . at all concentrations of extracellular ca studied the single channel i - v relationship was well fitted by a linear function and showed no evidence of a region of negative slope conductance or outward rectification ( fig . in addition , the single channel conductances derived from the slope of the i - v relationships ( i.e. 35.8 , 29.4 , 25.0 , and 20.0 ps in the presence of 0.3 , 1 , 3 , and 10 mm ca , respectively ) were in excellent agreement with the chord conductances reported in fig . 5c . thus , the reduction in single channel conductance by extracellular ca is voltage - independent over the range of negative potentials examined . a , reversal potential of the macroscopic current response to 5-ht ( e5-ht ) with [ na]o set to 95 mm and [ ca]o within the range of 0.1 to 30 mm . note that ca ion activity , ( ca)o , rather than concentration , [ ca]o , is plotted in a and c. data are the mean of 34 observations , and vertical bars indicate s.e . * * , p < 0.01 as determined by analysis of variance followed by dunnett 's post hoc test . b , single channel currents in response to 5-ht ( 10 m ) applied by pressure to outside - out membrane patches voltage - clamped at a holding potential ( vh ) of 80 mv . increasing [ ca]o depresses unitary current amplitude in a concentration - dependent manner . note that an increased driving force ( i.e. vh e5-ht ) when [ ca]o is equal to 10 and 30 mm ( corresponding to a ( ca)o of 3.12 and 8.56 mm , respectively ; see a ) partially masks the magnitude of the suppression of single channel current amplitudes by ca . c , concentration - response relationship illustrating the depressant effect of extracellular ca upon single channel conductance . data are the mean s.e . of measurements performed on 45 outside - out membrane patches under the conditions described in b. extracellular ca depresses the single channel conductance of the 5-ht3a(qda ) receptor construct . a , reversal potential of the macroscopic current response to 5-ht ( e5-ht ) with [ na]o set to 95 mm and [ ca]o within the range of 0.1 to 30 mm . note that ca ion activity , ( ca)o , rather than concentration , [ ca]o , is plotted in a and c. data are the mean of 34 observations , and vertical bars indicate s.e . * * , p < 0.01 as determined by analysis of variance followed by dunnett 's post hoc test . b , single channel currents in response to 5-ht ( 10 m ) applied by pressure to outside - out membrane patches voltage - clamped at a holding potential ( vh ) of 80 mv . increasing [ ca]o depresses unitary current amplitude in a concentration - dependent manner . note that an increased driving force ( i.e. vh e5-ht ) when [ ca]o is equal to 10 and 30 mm ( corresponding to a ( ca)o of 3.12 and 8.56 mm , respectively ; see a ) partially masks the magnitude of the suppression of single channel current amplitudes by ca . c , concentration - response relationship illustrating the depressant effect of extracellular ca upon single channel conductance . data are the mean s.e . of measurements performed on 45 outside - out membrane patches under the conditions described in b. extracellular na influences reduction of the single channel conductance of the 5-ht3a(qda ) receptor by extracellular ca and mg the single channel conductance of the 5-ht3a(qda ) receptor decreased from 41.1 to 26.5 ps when [ ca]o was elevated from 0.1 to 3 mm in an extracellular solution containing 95 mm na ( fig . 5c ) . however , only a modest decrease in single channel conductance ( i.e. from 40.8 to 35.7 ps ) occurred when solution e1 ( containing 1.0 mm ca and 2.0 mm mg in the presence of 140 mm na ) replaced solution e2 ( containing 0.1 mm ca and 0.1 mm mg in the presence of 140 mm na ) . the 5-ps decrease in conductance is far less than that predicted ( 11 ps ) from fig . 5c when [ ca]o was increased from 0.1 to 1.0 mm , even neglecting a likely contribution from mg ( 20 , 40 ) . the reason for this apparent discrepancy is an interaction between [ na]o and [ cation]o ( fig . where [ na]o was set at 140 , 95 , and 50 mm na ( osmolarity maintained with sucrose ) , single channel chord conductance recorded at a holding potential of 80 mv in the presence of 0.1 mm each ca and mg was unchanged between the 140 and 95 mm solutions but was significantly depressed in the 50 mm solution ( range 40.8 to 34.4 ps ; fig . 7 ) . however , in similar solutions containing 1 mm ca and 2 mm mg , single channel conductance decreased markedly in parallel with diminished [ na]o ( range , 35.7 to 18.3 ps ; fig . the data are consistent with competition between na and divalent cations for a binding site within the permeation pathway . figure 6.extracellular ca reduces the single channel conductance of the 5-ht3a(qda ) receptor construct in a manner independent of transmembrane potential . a , single channel currents recorded from outside - out membrane patches in response to pressure - applied 5-ht ( 10 m ) . the currents were recorded at holding potentials in the range of 100 to 40 mv in the presence of 0.3 , 1 , 3 , or 10 mm extracellular ca and 95 mm na . b , i - v relationships for single channel events recorded with [ na]o constant at 95 mm and [ ca]o present at 0.3 mm ( filled circles ) , 1 mm ( open circles ) , 3 mm ( filled inverted triangles ) , or 10 mm ( open inverted triangles ) . the relationship between single channel current amplitude and vh at each concentration of ca was fitted by linear regression analysis to yield slope conductance values of 35.8 , 29.4 , 25.0 , and 20.0 ps in the presence of 0.3 , 1 , 3 , and 10 mm ca , respectively . dashed lines represent an extrapolation of each i - v plot to the zero current level ( e5-ht ) . each data point is the mean of single channel current amplitudes derived from recordings performed on 35 outside - out membrane patches , and the vertical bars indicate s.e . extracellular ca reduces the single channel conductance of the 5-ht3a(qda ) receptor construct in a manner independent of transmembrane potential . a , single channel currents recorded from outside - out membrane patches in response to pressure - applied 5-ht ( 10 m ) . the currents were recorded at holding potentials in the range of 100 to 40 mv in the presence of 0.3 , 1 , 3 , or 10 mm extracellular ca and 95 mm na . b , i - v relationships for single channel events recorded with [ na]o constant at 95 mm and [ ca]o present at 0.3 mm ( filled circles ) , 1 mm ( open circles ) , 3 mm ( filled inverted triangles ) , or 10 mm ( open inverted triangles ) . the relationship between single channel current amplitude and vh at each concentration of ca was fitted by linear regression analysis to yield slope conductance values of 35.8 , 29.4 , 25.0 , and 20.0 ps in the presence of 0.3 , 1 , 3 , and 10 mm ca , respectively . dashed lines represent an extrapolation of each i - v plot to the zero current level ( e5-ht ) . each data point is the mean of single channel current amplitudes derived from recordings performed on 35 outside - out membrane patches , and the vertical bars indicate s.e . figure 7.the extracellular concentration of na influences depression of single channel conductance by extracellular divalent cations . bar graph depicting the single channel conductance of the 5-ht3a(qda ) receptor construct in the presence of 0.1 mm ca and 0.1 mg ( black bars ) or 1 mm ca and 2 mm mg ( white bars ) with [ na]o set at 140 , 95 , or 50 mm . increasing the extracellular concentration of ca and mg causes a significant reduction of single channel conductance at all concentrations of na examined , but the magnitude of the depression ( expressed as a percentage within the white bars ) increases as [ na]o is reduced . in the presence of 0.1 mm ca and 0.1 mm mg reducing [ na]o from 140 to 50 mm , but not to 95 mm , significantly decreases single channel conductance . with ca and mg present at 1 and 2 mm , respectively , reducing [ na]o to both 95 and 50 data are the mean s.e . of observations made from 310 outside - out membrane patches . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 ; as determined by analysis of variance followed by tukey 's post hoc test . the extracellular concentration of na influences depression of single channel conductance by extracellular divalent cations . bar graph depicting the single channel conductance of the 5-ht3a(qda ) receptor construct in the presence of 0.1 mm ca and 0.1 mg ( black bars ) or 1 mm ca and 2 mm mg ( white bars ) with [ na]o set at 140 , 95 , or 50 mm . increasing the extracellular concentration of ca and mg causes a significant reduction of single channel conductance at all concentrations of na examined , but the magnitude of the depression ( expressed as a percentage within the white bars ) increases as [ na]o is reduced . in the presence of 0.1 mm ca and 0.1 mm mg reducing [ na]o from 140 to 50 mm , but not to 95 mm , significantly decreases single channel conductance . with ca and mg present at 1 and 2 mm , respectively , reducing [ na]o to both 95 and 50 data are the mean s.e . of observations made from 310 outside - out membrane patches . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 ; as determined by analysis of variance followed by tukey 's post hoc test . effect of neutralizing the extracellular ring of charge of the 5-ht3a and 5-ht3a(qda ) receptor extracellular ca exerts multiple effects upon the wild - type 5-ht3a receptor including reductions in the apparent affinity of agonist binding ( 34 , 41 ) , depression of macroscopic and single channel current amplitudes ( 20 , 34 , 42 ) , and an acceleration of the kinetics of activation , deactivation , and desensitization ( 34 ) . hu and lovinger ( 34 ) demonstrated that neutralization of the extracellular ring of negative charge formed by the 20 aspartate residue ( fig . 1b ) at the extracellular vestibule of the mouse 5-ht3a receptor channel by the mutation d298a prevents modulation of macroscopic current kinetics and amplitude by ca . we have examined directly whether the ca - induced decrease in single channel conductance involves the homologous residue ( asp-293 ) of the human 5-ht3a(qda ) receptor construct and also have evaluated its effect upon the relative permeability to ca . we additionally determined the pca / pcs ratio for 5-ht3a receptors in which the d293a substitution was the only mutation present ( i.e. 5-ht3a(d293a ) ) . application of 1 m 5-ht to cells transfected with the 5-ht3a(qda d293a ) receptor cdna elicited only small inward current responses . hence , we constructed full concentration - response relationships to 5-ht for both the 5-ht3a(qda ) and 5-ht3a(qda d293a ) receptors to determine whether the d293a mutation was associated with a reduction in the agonist potency of 5-ht . such experiments demonstrated that relative to the 5-ht3a(qda ) receptor ( ec50 1.1 m ) , the 5-ht3a(qda d293a ) construct is less sensitive to 5-ht ( ec50 3.6 m ; supplemental fig . similarly , a comparison of wild - type 5-ht3a and 5-ht3a(d293a ) receptors revealed that the d293a mutation decreases the potency of 5-ht , as the ec50 value was shifted dextrally from 3 to 7 m ( supplemental fig . in subsequent experiments , 2 m 5-ht was employed to elicit macroscopic currents from cells expressing the 5-ht3a(d293a ) and 5-ht3a(qda d293a ) receptor constructs . in common with the 5-ht3a(qda ) construct , the 5-ht3a(qda d293a ) mutant maintained exclusive selectivity for cations because the slope of the relationship between the logarithm of ( na)o and e5-ht was determined to be 61 mv / decade change in ( na)o ( fig . 8a ) . using solutions e3 and i2 and the voltage ramp protocol , macroscopic currents mediated by the 5-ht3a(qda d293a ) construct in response to 5-ht demonstrated an e5-ht of 40.5 0.8 mv ( n = 6 ) , corresponding to a pca / pcs ratio of only 0.25 0.01 ( n = 6 ) compared with that of 3.7 for the 5-ht3a(qda ) receptor ( fig . ( n = 6 ) for the 5-ht3a(d293a ) mutant indicates a pca / pcs of 0.44 0.08 ( n = 6 ) , which is also significantly reduced versus the wild - type receptor ( 1.4 ) . the pca / pcs ratios of the 5-ht3a(d293a ) and 5-ht3a(qda d293a ) receptors were not significantly different . the pna / pcs ratio of either construct was unchanged by the d293a mutation ( fig . thus , for both the wild - type 5-ht3a and 5-ht3a(qda ) constructs , neutralization of the negatively charged 20 residue greatly reduces the pca / pcs ratio but has no effect upon pna / pcs . in addition to reducing the relative permeability of ca , as evidenced by a decreased sensitivity of e5-ht to changes in [ ca]o ( fig . 8c ) , the introduction of the d293a mutation into the 5-ht3a(qda ) receptor construct also decreased single channel conductance . in the presence of 0.1 mm ca and 95 mm na , the single channel chord conductance of the 5-ht3a(qda d293a ) receptor was 25.8 1.9 ps ( n = 4 ) at a holding potential of 80 mv in comparison with 41.3 ps for the 5-ht3a(qda ) construct . similarly , the single channel chord conductance ( at 80 mv ) of the 5-ht3a(qda d293a ) receptor determined with solution e2 ( 29.8 0.5 , n = 7 ) was significantly less than that of the 5-ht3a(qda ) construct ( 40.8 ps ) . however , no difference in single channel conductance was observed between the 5-ht3a(qda ) and 5-ht3a(qda d293a ) constructs over a range of positive holding potentials ( 60 to 100 mv ) , indicating that the d293a mutation preferentially suppresses inwardly directed cation flux ( fig . as found for the 5-ht3a(qda ) receptor , elevated [ ca]o ( 0.130 mm ) also depressed the single channel conductance of the 5-ht3a(qda d293a ) construct in a concentration - dependent manner ( fig . at all values of [ ca]o studied , the single channel chord conductance of the 5-ht3a(qda d293a ) construct was significantly less than that of the 5-ht3a(qda ) receptor . clearly , the alleviation of macroscopic current block by the d293a mutation ( 34 ) can not be attributed to a reduced influence of ca upon single channel conductance . figure 8.comparison of the relative ion permeability of wild - type , 5-ht3a(d293a ) , 5-ht3a(qda ) , and 5-ht3a(qda d293a ) receptor constructs with na and ca . a , the relationship between the reversal potential ( e5-ht ) of the macroscopic current response to pressure - applied 5-ht ( 10 m ) recorded from the 5-ht3a(qda d293a ) receptor construct and the activity of na in the extracellular medium ( ( na)o ) . responses were recorded with extracellular electrolytes based on solution e2 wherein the concentration of nacl was reduced by substitution with sucrose . intracellular solutions were either cscl - based i1 ( filled circles ) or a solution in which nacl totally replaced cscl ( open circles ) . the regression line fitted to the filled circles yielded a slope of 61 mv / decade change in ( na)o . data points are the mean of 36 independent determinations of e5-ht , and vertical bars indicate mean s.e . in a , c , d , and e , the dashed gray lines summarize the corresponding data for the 5-ht3a(qda ) receptor construct illustrated in detail in figs . 3 , 4 , and 5 . b , bar graph illustrating the permeability ( px / pcs ) of na ( black bars ) or ca ( white bars ) relative to cs for the indicated receptor constructs . e5-ht was determined with an intracellular cscl - based solution ( i1 ) as reference and nacl ( 146 mm)-based solution e2 or with cacl2 ( 100 mm)-based solution e3 as the extracellular electrolyte . mutant constructs in which pca / pcs was significantly different from the wild - type control are indicated with asterisks ( p < 0.01 ; analysis of variance followed by dunnett 's post hoc test ) . no significant differences in pna / pcs were found between the wild - type and mutant receptors . c , e5-ht for the 5-ht3a(qda d293a ) receptor construct with [ na]o set to 95 mm and [ ca]o within the range of 0.1 to 30 mm . note that ca ion activity ( ( ca)o ) rather than concentration ( [ ca]o ) is plotted . d , current - voltage ( i - v ) plot for single channel currents recorded from the 5-ht3a(qda d293a ) receptor construct with nacl - based solution e2 as the extracellular electrolyte and i1 as the intracellular solution . note that the additional d293a mutation causes an intensification of outward rectification and a reduced single channel conductance at negative potentials in comparison with the 5-ht3a(qda ) construct . e , concentration - response relationship and exemplar single channel currents illustrating the depressant effect of [ ca]o upon the single channel conductance of the 5-ht3a(qda d293a ) receptor construct . note that ca ion activity , rather than concentration , is plotted . data are the mean s.e . of measurements performed on 34 outside - out membrane patches at a holding potential of 80 mv under the ionic conditions described in c. comparison of the relative ion permeability of wild - type , 5-ht3a(d293a ) , 5-ht3a(qda ) , and 5-ht3a(qda d293a ) receptor constructs with na and ca . a , the relationship between the reversal potential ( e5-ht ) of the macroscopic current response to pressure - applied 5-ht ( 10 m ) recorded from the 5-ht3a(qda d293a ) receptor construct and the activity of na in the extracellular medium ( ( na)o ) . responses were recorded with extracellular electrolytes based on solution e2 wherein the concentration of nacl was reduced by substitution with sucrose . intracellular solutions were either cscl - based i1 ( filled circles ) or a solution in which nacl totally replaced cscl ( open circles ) . the regression line fitted to the filled circles yielded a slope of 61 mv / decade change in ( na)o . data points are the mean of 36 independent determinations of e5-ht , and vertical bars indicate mean s.e . in a , c , d , and e , the dashed gray lines summarize the corresponding data for the 5-ht3a(qda ) receptor construct illustrated in detail in figs . 3 , 4 , and 5 . b , bar graph illustrating the permeability ( px / pcs ) of na ( black bars ) or ca ( white bars ) relative to cs for the indicated receptor constructs . e5-ht was determined with an intracellular cscl - based solution ( i1 ) as reference and nacl ( 146 mm)-based solution e2 or with cacl2 ( 100 mm)-based solution e3 as the extracellular electrolyte . mutant constructs in which pca / pcs was significantly different from the wild - type control are indicated with asterisks ( p < 0.01 ; analysis of variance followed by dunnett 's post hoc test ) . no significant differences in pna / pcs were found between the wild - type and mutant receptors . c , e5-ht for the 5-ht3a(qda d293a ) receptor construct with [ na]o set to 95 mm and [ ca]o within the range of 0.1 to 30 mm . note that ca ion activity ( ( ca)o ) rather than concentration ( [ ca]o ) is plotted . d , current - voltage ( i - v ) plot for single channel currents recorded from the 5-ht3a(qda d293a ) receptor construct with nacl - based solution e2 as the extracellular electrolyte and i1 as the intracellular solution . note that the additional d293a mutation causes an intensification of outward rectification and a reduced single channel conductance at negative potentials in comparison with the 5-ht3a(qda ) construct . e , concentration - response relationship and exemplar single channel currents illustrating the depressant effect of [ ca]o upon the single channel conductance of the 5-ht3a(qda d293a ) receptor construct . note that ca ion activity , rather than concentration , is plotted . data are the mean s.e . of measurements performed on 34 outside - out membrane patches at a holding potential of 80 mv under the ionic conditions described in c. two decades of research have focused upon tm2 and flanking sequences as the primary determinants of ion selectivity in cys - loop receptors ( 2 , 28 , 43 ) . for the nicotinic ach receptor in particular , multiple sites within such domains ( i.e. the 1 , 13 , 16 , and 17 positions ; fig . 1b ) that affect the conduction of ca have been identified ( 28 , 43 ) . we have added to this information by demonstrating that residues within the ma helix of the cytoplasmic loop of the 5-ht3a receptor , a cousin of the nicotinic ach receptors , exert a strong influence on the permeability of ca relative to monovalent cations . the replacement of arginine residues 432 , 436 , and 440 of the human 5-ht3a receptor by those aligned in the human 5-ht3b subunit ( i.e. glutamine , aspartate , and alanine ) , generating the 5-ht3a(qda ) receptor construct , increased pca / pcs from a wild - type value of 1.4 to 3.7 . such an effect was selective because neither pna / pcs nor pna / pcl was perturbed by the mutation , the latter being inferred by comparison with values within the literature ( 20 , 29 , 30 ) . notably , the 5-ht3a(qda ) construct retained perfect selectivity toward cations versus anions , a finding that accords with the results of a recent study in which deletion of the entire intracellular loop of the 5-ht3a receptor had no measurable influence upon pna / pcl ( 44 ) . indeed , residues within and adjacent to tm2 govern charge selectivity in the 5-ht3a receptor ( 29 ) . in particular , the single amino acid substitution e1a at the intracellular border of tm2 ( fig . 1b ) is sufficient to generate a 5-ht3a receptor that does not discriminate between monovalent cations and anions ( pna / pcl = 0.89 ) ( 30 ) . in future studies , it would be of interest to generate the e1a mutation within the 5-ht3a(qda ) background to assess whether a role for the ma helix emerges when a primary determinant of charge selectivity is silenced . our results indicate that the single mutation r436d is sufficient to enhance pca / pcs to an extent comparable with that found for the 5-ht3a(qda ) construct . analysis of several amino acid substitutions involving neutralization , or inversion , of charge at this key locus suggests that an electrostatic repulsive effect contributes to a suppression of pca / pcs in the wild - type receptor . the latter is consistent with the results of modeling studies that place the highly basic ( pka = 12.48 ) guanidinium group of arg-436 within the intracellular permeation pathways ( portals ) located between adjacent ma - stretch -helices ( 26 , 32 ) . because of the small maximal width of the portals ( 26 ) , interactions between partially hydrated permeating ions and arg-436 are likely . previous studies of ca permeation through homomeric 5-ht3 receptor channels have , because of its very low single channel conductance , been restricted to the analysis of macroscopic currents . the latter are suppressed by extracellular divalent cations by mechanisms that potentially include a reduction in agonist binding affinity ( 34 , 41 ) , an acceleration of the kinetics of deactivation and desensitization ( 34 ) , and depression of macroscopic and single channel current amplitudes ( 20 , 34 ) , the latter being inferred by fluctuation analysis ( 20 ) . in the present study , we employed the 5-ht3a(qda ) receptor construct to examine directly the influence of extracellular ca upon single channel conductance . a solution ( e3 ) , in which ca was the sole extracellular cation , supported outwardly rectifying single channel currents but with a chord conductance at negative potentials ( 5.7 ps ) , which was greatly reduced in comparison with the value of 40.8 ps obtained with solution e2 , in which na was the permeant species . thus , although the permeability of ca relative to cs is greatly increased in the 5-ht3a(qda ) construct , the channel does not conduct ca efficiently . moreover , the addition of ca to na - containing extracellular solutions caused a concentration - dependent inhibition of the inwardly directed single channel current amplitude , in a manner reminiscent of that observed for single channel currents mediated by the nicotinic ach receptor of skeletal muscle ( 45 ) . such results are most parsimoniously explained by hypothesizing that the permeation pathway presents a low affinity binding site ( or sites ) for ca , which confers a degree of selectivity for ca over monovalent cations , yet with an off - rate sufficient to allow significant flux of both na and ca . an interaction between ca and na within the channel is supported by our experimental observations indicating the pca / pna ratio for the 5-ht3a(qda ) receptor construct varies with [ ca]o ( i.e. the principle of independence is violated ( 39 ) ) . consistent with this notion , the reduction in single channel current amplitude by extracellular divalent cations was more pronounced when [ na]o was reduced . we considered the d293a residue of the human 5-ht3a receptor subunit , which forms the 20 negatively charged ring within the extracellular vestibule of the channel ( 2 , 28 ) , to be a strong candidate for the putative ca binding site that limits the rate of charge transfer through the channel ( fig . firstly , mutation of the corresponding residue ( asp-298 ) of the mouse 5-ht3a subunit to alanine alleviates the block of macroscopic current responses by extracellular ca ( 34 ) . secondly , ca reduced the amplitude of single channel currents mediated by the 5-ht3a(qda ) receptor construct in a voltage - independent manner . the lack of observable voltage dependence suggests a binding site for ca away from the electrical field of the membrane . however , because ca is permeant , rather than a simple impermeant open channel blocker , it remains possible that ca does interact with a site(s ) within the field but that the increased driving force provided by hyperpolarization facilitates its dissociation and inward conduction . note that this scenario is quite different from that found for an impermeant cationic blocker , for which the dwell time within the channel would be increased by hyperpolarization because of a decreased probability of the molecule exiting the pore to the extracellular environment . the potency of 5-ht to activate the mouse 5-ht3a(d298a ) construct is less than for the murine wild - type receptor ( 34 ) , a result concordant with our observation that the d293a mutation introduced into either the human wild - type or 5-ht3a(qda ) construct causes a dextral shift in the 5-ht concentration - response relationship . the d293a mutation within the 5-ht3a(qda ) receptor background caused a reduction in the amplitude of inwardly , but not outwardly , directed single channel current events . such an effect is consistent with a simple electrostatic mechanism in which the negative charge of the five asp-293 residues acts to increase the local concentration of permeant cations within the extracellular vestibule . removal of such charges would thus reduce a focusing mechanism , consistent with the appearance of a enhanced outward rectification in the i - v relationship for the 5-ht3a(qda d293a ) receptor construct versus the 5-ht3a(qda ) receptor . very similar effects of neutralizing the extracellular ring of charge upon single channel conductance and rectification have been reported for nicotinic ach and anion - selective -aminobutyric acid type a and glycine receptors ( 28 ) . because of the very low conductance of the wild - type 5-ht3a receptor ( 20 , 21 ) and kinetic properties of the 5-ht3a(d293a ) receptor , which are unfavorable to fluctuation analysis ( 34),5 we could not estimate the effect of the d293a mutation upon single channel conductance in this construct . however , it is notable that the pattern of inward rectification of macroscopic currents mediated by the mouse 5-ht3a(d298a ) mutant resembles that of the murine wild - type receptor ( 34 ) . the latter differs from the present observations comparing the human 5-ht3a(qda ) and 5-ht3a(qda d293a ) receptors at the single channel level . an important difference in our studies is the removal of positive electrostatic potential in the intracellular vestibule by the mutations r432q , r436a , and r440a . such charges are present in the mouse 5-ht3a and 5-ht3a(d298a ) receptors studied by hu and lovinger ( 34 ) and would reduce the local concentration of permeant ions within the intracellular vestibule . it is possible that such an influence is dominant , masking any tendency toward linearization that might be anticipated from the 5-ht3a(d293a ) mutation . indeed , in our studies , the macroscopic current i - v relationship shifts from the pattern of inward rectification characteristic of the wild - type 5-ht3a receptor toward linearity in the 5-ht3a(qda ) construct . for both the wild - type 5-ht3a and the 5-ht3a(qda ) constructs , the introduction of the d293a mutation drastically reduced the pca / pcs ratio . once more , such an effect can probably be attributed to a reduction in a local negative electrostatic potential that particularly favors the accumulation of ca at the entrance to the pore , as suggested by molecular dynamics simulations performed on models of the 7 and skeletal muscle nicotinic ach receptors ( 46 , 47 ) . such simulations indicate monovalent cations to be stabilized at the 20 position , where the ion dwells during its passage through the channel axis . also , heteromeric 42 nicotinic ach receptors , harboring a larger complement of 2 subunits in which lysine occupies the 20 position , appear to have reduced selectivity toward ca ( 48 ) . however , the d293a residue does not contribute crucially to cation versus anion selectivity because the pna / pcl ratio for the 5-ht3a(qda d293a ) construct was best described as infinite . a previous study of mouse 5-ht3a receptors in which cysteine residues engineered into the tm2 domain were successfully probed with a negatively charged sulfydryl - modifying agent ( methane - thiosulfonate ethylsulfonate ( mtses ) ) applied extracellularly also indicates that the extracellular ring does not totally select against anions gaining access to deeper regions of the pore ( 49 ) . in addition , neutralization of the 20 charge was not required to invert the ion selectivity of the mouse 5-ht3a receptor ( 29 ) . finally , the recent crystal structure of a prokaryotic pentameric ligand - gated channel from erwinia chrysanthemi reveals that phenylalanine occupies the 20 position , yet the channel is cation - selective ( 50 ) . heteromeric 5-ht3 receptors expressed in tsa-201 cells assemble with the stoichiometry ( 5-ht3a)2(5-ht3b)3 as a subunit rosette in the order b - b - a - b - a , yielding 2 a - b , 2 b - a , and 1 b - b subunit interfaces ( 16 ) . hence , compared with homomeric receptors , the influence of arg-432 , arg-436 , and arg-440 will be removed from three of the cytoplasmic portals , irrespective of whether a clockwise or anticlockwise arrangement is assumed . yet , the inclusion of the 5-ht3b subunit within heteromeric 5-ht3 receptors reduces permeability to ca ( pca / pcs = 0.50.6 ) in comparison with homomeric receptors ( pca / pcs = 1.11.4 ) ( ref . this apparent paradox might be explained by the presence of a polar asparagine , rather than an acidic aspartate , at the 20 locus and a non - polar alanine , rather than an acidic glutamate , at the 1 position of the human 5-ht3b versus 5-ht3a subunit ( fig . the present work suggests that the 5-ht3b subunit 20 asparagine may not be optimal for a high pca / pcs , ratio and it is known that a e1a substitution in the 7 nicotinic ach receptor abolishes permeabilty to ca ( 28 ) . macroscopic current responses mediated by the mouse 5-ht3a(d298a ) receptor are insensitive to [ ca]o within the range of 0.1 to 10 mm ( 34 ) , but we observed that single channel currents recorded from the human 5-ht3a(qda d293a ) receptor were reduced in amplitude in the presence of extracellular ca . such an effect was concentration - dependent and occurred within the range observed for the 5-ht3a(qda ) receptor . hence , the asp-293 residue can not be a crucial site at which ca binds to reduce single channel conductance in the 5-ht3a(qda ) receptor construct . it remains to be explained why increasing [ ca]o does not affect the macroscopic current response of the mouse 5-ht3a(d298a ) receptor , yet clearly depresses single channel conductance in the human 5-ht3a(qda d293a ) construct . however , as clearly noted by hu and lovinger ( 34 ) , depression of single channel conductance can not be the sole mechanism by which ca depresses macroscopic currents , because the magnitude of the effect is dependent upon the concentration of 5-ht employed to activate the receptor . indeed , at saturating concentrations of 5-ht ( i.e. 30 m ) , increasing [ ca]o from 1.8 to 10 mm exerts only a modest depressant effect upon 5-ht - evoked currents ( 34 ) , which might indicate that the influence of ca derives , at least in part , via indirect , or direct , actions upon ligand binding ( 41 ) . in addition , ca accelerates desensitization of the murine wild - type 5-ht3a receptor , and the d293a mutation suppresses this effect . it is possible that the reduced relative permeability of the 5-ht3a(d293a ) mutant contributes to such resistance . in summary , we have demonstrated that structural elements in both the intracellular and extracellular regions of the human 5-ht3a control relative permeability to ca . our report represents the first description of the influence of the ma helix upon ion selectivity , a process that has conventionally been linked to specific amino acid residues within tm2 and immediate flanking sequences ( 28 ) . it is thus an oversimplification to refer to a single selectivity filter in the 5-ht3a receptor and , by inference , in other cys - loop receptors . instead , the extracellular vestibule , tm2 , and the ma helix act collectively to determine ion permeation .

cation - selective cysteine ( cys)-loop transmitter - gated ion channels provide an important pathway for ca2 + entry into neurones . we examined the influence on ca2 + permeation of amino acids located at intra- and extracellular ends of the conduction pathway of the human 5-hydroxytryptamine type 3a ( 5-ht3a ) receptor . mutation of cytoplasmic arginine residues 432 , 436 , and 440 to glutamine , aspartate , and alanine ( the aligned residues of the human 5-ht3b subunit ( yielding 5-ht3a(qda ) ) increased pca / pcs from 1.4 to 3.7 . the effect was attributable to the removal of an electrostatic influence of the arg-436 residue . despite its relatively high permeability to ca2 + , the single channel conductance of the 5-ht3a(qda ) receptor was depressed in a concentration - dependent and voltage - independent manner by extracellular ca2 + . a conserved aspartate , located toward the extracellular end of the conduction pathway and known to influence ionic selectivity , contributed to the inhibitory effect of ca2 + on macroscopic currents mediated by 5-ht3a receptors . we introduced a d293a mutation into the 5-ht3a(qda ) receptor ( yielding the 5-ht3a(qda d293a ) construct ) to determine whether the aspartate is required for the suppression of single channel conductance by ca2 + . the d293a mutation decreased the pca / pcs ratio to 0.25 and reduced inwardly directed single channel conductance from 41 to 30 ps but did not prevent suppression of single channel conductance by ca2 + . the d293a mutation also reduced pca / pcs when engineered into the wild - type 5-ht3a receptor . the data helped to identify key residues in the cytoplasmic domain ( arg-436 ) and extracellular vestibule ( asp-293 ) that markedly influence pca / pcs and additionally directly demonstrated a depression of single channel conductance by ca2 + .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

to determine the relative permeability of na versus cs ( pna / pcs ) , in solution e2 , additional nacl totally replaced kcl in the extracellular solution , and the concentrations of cacl2 and mgcl2 were each reduced to 0.1 mm to minimize the influence of divalent cations upon the reversal potential of the macroscopic current response to 5-ht ( e5-ht ) . residues in the ma helix of the 5-ht3a receptor influence ca permeability we recently demonstrated that arginine residues 432 , 436 , and 440 within the ma helix motif of the tm34 loop at positions termed ma 4 , 0 , and 4 , respectively , which line putative cytoplasmic portals ( fig . c , an alignment of amino acids within the ma helices of wild - type 5-ht3a and 5-ht3b subunits and the mutant 5-ht3a(qda ) construct used in this study , indicating the positions of the 4 , 0 , and 4 residues ( boxed ) . c , an alignment of amino acids within the ma helices of wild - type 5-ht3a and 5-ht3b subunits and the mutant 5-ht3a(qda ) construct used in this study , indicating the positions of the 4 , 0 , and 4 residues ( boxed ) . the relative permeability ratio , pna / pcs , of wild - type and mutant 5-ht3a receptors was calculated by determining e5-ht from voltage ramps ( 100 to + 60 mv ) that coincided with the peak of the macroscopic current response to pressure - applied 5-ht in the presence of extracellular and intracellular electrolytes e2 and i1 , respectively ( fig ramp currents generated in the absence of 5-ht were subtracted from ramp currents in the presence of 5-ht yielding an inwardly rectifying i - v relationship with an e5-ht of 2.1 0.7 mv , corresponding to a pna / pcs ratio of 1.02 0.03 ( n = 15 ; figs . experiments performed on the 5-ht3a(qda ) mutant using the voltage ramp protocol yielded a pna / pcs ratio of 0.9 0.04 ( e5-ht = 1.3 1.0 mv , n = 13 ) , which is not significantly different from that of the wild - type 5-ht3a receptor ( fig . however , in contrast to the negative shift in e5-ht found for the wild - type 5-ht3a receptor upon replacement of solution e2 by e3 , that of the mutant displayed a robust positive shift to 9.4 1.4 mv ( n = 10 ) , reflecting a significant increase in pca / pcs to 3.65 0.34 ( n = 10 , figs . despite the enhancement of pca / pcs at the 5-ht3a(qda ) receptor , inhibition of the macroscopic current responses by extracellular ca persisted , although the effect was less pronounced at positive potentials due to the outward , versus mild inward , rectification typically observed in the ca- and na - containing extracellular solutions , respectively ( fig . the r436d charge reversal mutation produces an 6-fold increase in single channel conductance compared with the wild - type 5-ht3a receptor ( 22 ) . notably , the pca / pcs of the 5-ht3a(qda ) receptor was not significantly greater than that of the 5-ht3a(r436d ) receptor . likewise , the permeability of ca relative to cs of the 5-ht3a(r432q ) mutant was similar to that of the wild - type 5-ht3a receptor , suggesting that this ma helix residue has little influence on ion selectivity ( fig . although the 5-ht3a(r440a ) mutant exhibited a trend toward an increased pca / pcs ratio relative to the wild - type receptor , the effect failed to reach statistical significance ( fig . the validity of the above estimates of pna / pcs and pca / pcs for the 5-ht3a(qda ) receptor construct relies upon the mutant retaining the near perfect cation versus anion selectivity of the wild - type receptor ( pna / pcl = 53 ) ( 30 ) . the foregoing results indicate that the arginine residues present at positions 436 ( ma 0 ) and to a lesser extent 440 ( ma 4 ) of the human wild - type 5-ht3a receptor collectively suppress the permeability of ca , but not na , relative to cs , revealing that the large intracellular loop of a cys - loop receptor is an important determinant of divalent versus monovalent cation selectivity . figure 2.the reversal potential of 5-ht - evoked macroscopic currents at wild - type 5-ht3a and 5-ht3a(qda ) receptor constructs with na or ca as the charge carrier in the extracellular solution . a , voltage ramp ( left ) applied at the peak of the macroscopic inward current response to pressure - applied 5-ht ( 1 m)(right ) to determine the reversal potential ( e5-ht ) of the agonist - induced response mediated by the wild - type 5-ht3a receptor . a , voltage ramp ( left ) applied at the peak of the macroscopic inward current response to pressure - applied 5-ht ( 1 m)(right ) to determine the reversal potential ( e5-ht ) of the agonist - induced response mediated by the wild - type 5-ht3a receptor b , current - voltage relationships recorded from a representative hek-293 cell expressing the wild - type 5-ht3a receptor with an intracellular cscl - based solution ( i1 ) as reference and nacl ( 146 mm)-based solution e2 or cacl2 ( 100 mm)-based solution e3 as the extracellular electrolyte . pca / pcs was significantly enhanced compared with the wild - type 5-ht3a receptor for the 5-ht3a(qea ) construct . although pca / pcs was not significantly different from the wild - type 5-ht3a receptor for the 5-ht3a(qaa ) , 5-ht3a(qfa ) , and 5-ht3a(qra ) constructs , the ratio tended to decrease progressively as the charge at position 436 changed from negative , through neutral , to positive ( fig . , the results suggest that the reversal of charge at position 436 within the 5-ht3a(qda ) construct was the predominant cause of a greatly increased pca / pcs relative to the wild - type receptor . b , relationship between the reversal potential of the macroscopic current evoked by 5-ht ( e5-ht ) and the activity of na in the extracellular medium ( ( na)o ) for the 5-ht3a(qda ) receptor construct . b , relationship between the reversal potential of the macroscopic current evoked by 5-ht ( e5-ht ) and the activity of na in the extracellular medium ( ( na)o ) for the 5-ht3a(qda ) receptor construct . indeed , we previously demonstrated that extracellular divalent cations ( mg and ca ) reduce the single channel conductance of the wild - type 5-ht3a receptor as estimated using fluctuation analysis ( 20 ) . this is impossible for the wild - type 5-ht3a receptor , with a conductance of < 1 ps , which is below the resolution of the patch - clamp technique ( 20 , 21 , 40 ) . in contrast , the 5-ht3a(qda ) construct has a single channel conductance that enables the direct resolution of single channel currents in outside - out patch recordings ( slope conductance of 37 ps at 74 mv ( 22 ) ) . the data indicate that the 5-ht3a(qda ) receptor construct , despite selecting for ca over na , conducts the former less efficiently , thus predicting that extracellular ca will ( at negative holding potentials ) reduce single channel conductance in mixtures of na and ca as we previously inferred for the human wild - type 5-ht3a receptor by fluctuation analysis ( 20 ) . extracellular ca reduces the single channel conductance of the 5-ht3a(qda ) receptor in a concentration - dependent fashion we examined the influence of extracellular ca upon the single channel conductance of the 5-ht3a(qda ) receptor construct with an extracellular solution containing 95 mm na and varying concentrations of ca ( 10 nm to 30 mm ) . extracellular ca reduces single channel conductance of the 5-ht3a(qda ) receptor in a voltage - independent manner blockade by ca of macroscopic current responses mediated by the 5-ht3a receptor expressed in mammalian cell hosts is voltage - independent ( 20 , 21 , 34 , 40 , 41 ) . the readily resolvable single channel conductance of the 5-ht3a(qda ) receptor allowed direct measurements of the influence of extracellular ca upon single channel current amplitudes over a range of membrane potentials . of measurements performed on 45 outside - out membrane patches under the conditions described in b. extracellular ca depresses the single channel conductance of the 5-ht3a(qda ) receptor construct . of measurements performed on 45 outside - out membrane patches under the conditions described in b. extracellular na influences reduction of the single channel conductance of the 5-ht3a(qda ) receptor by extracellular ca and mg the single channel conductance of the 5-ht3a(qda ) receptor decreased from 41.1 to 26.5 ps when [ ca]o was elevated from 0.1 to 3 mm in an extracellular solution containing 95 mm na ( fig . figure 6.extracellular ca reduces the single channel conductance of the 5-ht3a(qda ) receptor construct in a manner independent of transmembrane potential . extracellular ca reduces the single channel conductance of the 5-ht3a(qda ) receptor construct in a manner independent of transmembrane potential . figure 7.the extracellular concentration of na influences depression of single channel conductance by extracellular divalent cations . bar graph depicting the single channel conductance of the 5-ht3a(qda ) receptor construct in the presence of 0.1 mm ca and 0.1 mg ( black bars ) or 1 mm ca and 2 mm mg ( white bars ) with [ na]o set at 140 , 95 , or 50 mm . bar graph depicting the single channel conductance of the 5-ht3a(qda ) receptor construct in the presence of 0.1 mm ca and 0.1 mg ( black bars ) or 1 mm ca and 2 mm mg ( white bars ) with [ na]o set at 140 , 95 , or 50 mm . effect of neutralizing the extracellular ring of charge of the 5-ht3a and 5-ht3a(qda ) receptor extracellular ca exerts multiple effects upon the wild - type 5-ht3a receptor including reductions in the apparent affinity of agonist binding ( 34 , 41 ) , depression of macroscopic and single channel current amplitudes ( 20 , 34 , 42 ) , and an acceleration of the kinetics of activation , deactivation , and desensitization ( 34 ) . we have examined directly whether the ca - induced decrease in single channel conductance involves the homologous residue ( asp-293 ) of the human 5-ht3a(qda ) receptor construct and also have evaluated its effect upon the relative permeability to ca . hence , we constructed full concentration - response relationships to 5-ht for both the 5-ht3a(qda ) and 5-ht3a(qda d293a ) receptors to determine whether the d293a mutation was associated with a reduction in the agonist potency of 5-ht . such experiments demonstrated that relative to the 5-ht3a(qda ) receptor ( ec50 1.1 m ) , the 5-ht3a(qda d293a ) construct is less sensitive to 5-ht ( ec50 3.6 m ; supplemental fig . using solutions e3 and i2 and the voltage ramp protocol , macroscopic currents mediated by the 5-ht3a(qda d293a ) construct in response to 5-ht demonstrated an e5-ht of 40.5 0.8 mv ( n = 6 ) , corresponding to a pca / pcs ratio of only 0.25 0.01 ( n = 6 ) compared with that of 3.7 for the 5-ht3a(qda ) receptor ( fig . ( n = 6 ) for the 5-ht3a(d293a ) mutant indicates a pca / pcs of 0.44 0.08 ( n = 6 ) , which is also significantly reduced versus the wild - type receptor ( 1.4 ) . thus , for both the wild - type 5-ht3a and 5-ht3a(qda ) constructs , neutralization of the negatively charged 20 residue greatly reduces the pca / pcs ratio but has no effect upon pna / pcs . 8c ) , the introduction of the d293a mutation into the 5-ht3a(qda ) receptor construct also decreased single channel conductance . in the presence of 0.1 mm ca and 95 mm na , the single channel chord conductance of the 5-ht3a(qda d293a ) receptor was 25.8 1.9 ps ( n = 4 ) at a holding potential of 80 mv in comparison with 41.3 ps for the 5-ht3a(qda ) construct . similarly , the single channel chord conductance ( at 80 mv ) of the 5-ht3a(qda d293a ) receptor determined with solution e2 ( 29.8 0.5 , n = 7 ) was significantly less than that of the 5-ht3a(qda ) construct ( 40.8 ps ) . however , no difference in single channel conductance was observed between the 5-ht3a(qda ) and 5-ht3a(qda d293a ) constructs over a range of positive holding potentials ( 60 to 100 mv ) , indicating that the d293a mutation preferentially suppresses inwardly directed cation flux ( fig . as found for the 5-ht3a(qda ) receptor , elevated [ ca]o ( 0.130 mm ) also depressed the single channel conductance of the 5-ht3a(qda d293a ) construct in a concentration - dependent manner ( fig . at all values of [ ca]o studied , the single channel chord conductance of the 5-ht3a(qda d293a ) construct was significantly less than that of the 5-ht3a(qda ) receptor . figure 8.comparison of the relative ion permeability of wild - type , 5-ht3a(d293a ) , 5-ht3a(qda ) , and 5-ht3a(qda d293a ) receptor constructs with na and ca . e , concentration - response relationship and exemplar single channel currents illustrating the depressant effect of [ ca]o upon the single channel conductance of the 5-ht3a(qda d293a ) receptor construct . of measurements performed on 34 outside - out membrane patches at a holding potential of 80 mv under the ionic conditions described in c. comparison of the relative ion permeability of wild - type , 5-ht3a(d293a ) , 5-ht3a(qda ) , and 5-ht3a(qda d293a ) receptor constructs with na and ca . a , the relationship between the reversal potential ( e5-ht ) of the macroscopic current response to pressure - applied 5-ht ( 10 m ) recorded from the 5-ht3a(qda d293a ) receptor construct and the activity of na in the extracellular medium ( ( na)o ) . e , concentration - response relationship and exemplar single channel currents illustrating the depressant effect of [ ca]o upon the single channel conductance of the 5-ht3a(qda d293a ) receptor construct . the replacement of arginine residues 432 , 436 , and 440 of the human 5-ht3a receptor by those aligned in the human 5-ht3b subunit ( i.e. glutamine , aspartate , and alanine ) , generating the 5-ht3a(qda ) receptor construct , increased pca / pcs from a wild - type value of 1.4 to 3.7 . analysis of several amino acid substitutions involving neutralization , or inversion , of charge at this key locus suggests that an electrostatic repulsive effect contributes to a suppression of pca / pcs in the wild - type receptor . moreover , the addition of ca to na - containing extracellular solutions caused a concentration - dependent inhibition of the inwardly directed single channel current amplitude , in a manner reminiscent of that observed for single channel currents mediated by the nicotinic ach receptor of skeletal muscle ( 45 ) . secondly , ca reduced the amplitude of single channel currents mediated by the 5-ht3a(qda ) receptor construct in a voltage - independent manner . the potency of 5-ht to activate the mouse 5-ht3a(d298a ) construct is less than for the murine wild - type receptor ( 34 ) , a result concordant with our observation that the d293a mutation introduced into either the human wild - type or 5-ht3a(qda ) construct causes a dextral shift in the 5-ht concentration - response relationship . removal of such charges would thus reduce a focusing mechanism , consistent with the appearance of a enhanced outward rectification in the i - v relationship for the 5-ht3a(qda d293a ) receptor construct versus the 5-ht3a(qda ) receptor . because of the very low conductance of the wild - type 5-ht3a receptor ( 20 , 21 ) and kinetic properties of the 5-ht3a(d293a ) receptor , which are unfavorable to fluctuation analysis ( 34),5 we could not estimate the effect of the d293a mutation upon single channel conductance in this construct . indeed , in our studies , the macroscopic current i - v relationship shifts from the pattern of inward rectification characteristic of the wild - type 5-ht3a receptor toward linearity in the 5-ht3a(qda ) construct . for both the wild - type 5-ht3a and the 5-ht3a(qda ) constructs , the introduction of the d293a mutation drastically reduced the pca / pcs ratio . such an effect was concentration - dependent and occurred within the range observed for the 5-ht3a(qda ) receptor . it remains to be explained why increasing [ ca]o does not affect the macroscopic current response of the mouse 5-ht3a(d298a ) receptor , yet clearly depresses single channel conductance in the human 5-ht3a(qda d293a ) construct .

to determine the relative permeability of na versus cs ( pna / pcs ) , in solution e2 , additional nacl totally replaced kcl in the extracellular solution , and the concentrations of cacl2 and mgcl2 were each reduced to 0.1 mm to minimize the influence of divalent cations upon the reversal potential of the macroscopic current response to 5-ht ( e5-ht ) . additionally , in experiments that determined e5-ht and single channel conductance in mixtures of extracellular na and ca , nacl was held at a constant concentration of 95 mm in the presence of variable concentrations of cacl2 ( 103 10 m ) , sucrose ( 090 mm , as appropriate to maintain constant osmolarity ) , glucose ( 10 mm ) , and l - histidine ( 5 mm ; ph 7.2 ) . c , an alignment of amino acids within the ma helices of wild - type 5-ht3a and 5-ht3b subunits and the mutant 5-ht3a(qda ) construct used in this study , indicating the positions of the 4 , 0 , and 4 residues ( boxed ) . the relative permeability ratio , pna / pcs , of wild - type and mutant 5-ht3a receptors was calculated by determining e5-ht from voltage ramps ( 100 to + 60 mv ) that coincided with the peak of the macroscopic current response to pressure - applied 5-ht in the presence of extracellular and intracellular electrolytes e2 and i1 , respectively ( fig ramp currents generated in the absence of 5-ht were subtracted from ramp currents in the presence of 5-ht yielding an inwardly rectifying i - v relationship with an e5-ht of 2.1 0.7 mv , corresponding to a pna / pcs ratio of 1.02 0.03 ( n = 15 ; figs . complete replacement of na by 100 mm ca ) , resulting in a negative shift in e5-ht to 6.1 1.3 mv accompanied by a pronounced depression in the amplitude of the 5-ht - evoked current and the loss of inward rectification in the i - v relationship , most noticeable at positive potentials ( fig . wt , wild - type ; qda , 5-ht3a(r432q , r436d , r440a ) ; qea , 5-ht3a ( r432q , r436e , r440a ) ; qaa , 5-ht3a(r432q , r436a , r440a ) ; qfa , ( 5-ht3a(r432q , r436f , r440a ) ; qra , 5-ht3a(r432q , r440a).5-ht3a constructs na extracellular ca extracellular e5-htpna / pcsne5-htpca / pcsnmvmv wt 5-ht3a 2.1 0.7 1.03 0.03 15 -6.1 1.3 1.42 0.11 6 r432q 6.0 0.9 1.20 0.04 17 -3.4 2.6 1.70 0.26 7 r436d -0.4 0.7 0.93 0.03 13 7.1 1.4 3.13 0.31 * 7 r440a 4.8 1.1 1.15 0.05 5 2.3 1.7 2.33 0.23 5 qda -1.3 1.0 0.90 0.04 10 9.4 1.4 3.65 0.30 * 10 qea -3.6 0.6 0.81 0.02 4 7.6 0.9 3.15 0.17 * 4 qaa -6.8 0.9 0.72 0.02 * 8 3.5 0.9 2.46 0.14 6 qfa -5.2 0.5 0.77 0.01 * 8 2.1 0.4 2.25 0.06 5 qra -2.0 1.6 0.88 0.05 7 -2.1 1.4 1.80 0.15 7 the influence of mutations within the intracellular ma helix upon the permeabilities of ca and na relative to cs relative permeabilities ( i.e. wt , wild - type ; qda , 5-ht3a(r432q , r436d , r440a ) ; qea , 5-ht3a ( r432q , r436e , r440a ) ; qaa , 5-ht3a(r432q , r436a , r440a ) ; qfa , ( 5-ht3a(r432q , r436f , r440a ) ; qra , 5-ht3a(r432q , r440a ) . however , in contrast to the negative shift in e5-ht found for the wild - type 5-ht3a receptor upon replacement of solution e2 by e3 , that of the mutant displayed a robust positive shift to 9.4 1.4 mv ( n = 10 ) , reflecting a significant increase in pca / pcs to 3.65 0.34 ( n = 10 , figs . despite the enhancement of pca / pcs at the 5-ht3a(qda ) receptor , inhibition of the macroscopic current responses by extracellular ca persisted , although the effect was less pronounced at positive potentials due to the outward , versus mild inward , rectification typically observed in the ca- and na - containing extracellular solutions , respectively ( fig . likewise , the permeability of ca relative to cs of the 5-ht3a(r432q ) mutant was similar to that of the wild - type 5-ht3a receptor , suggesting that this ma helix residue has little influence on ion selectivity ( fig . the validity of the above estimates of pna / pcs and pca / pcs for the 5-ht3a(qda ) receptor construct relies upon the mutant retaining the near perfect cation versus anion selectivity of the wild - type receptor ( pna / pcl = 53 ) ( 30 ) . the foregoing results indicate that the arginine residues present at positions 436 ( ma 0 ) and to a lesser extent 440 ( ma 4 ) of the human wild - type 5-ht3a receptor collectively suppress the permeability of ca , but not na , relative to cs , revealing that the large intracellular loop of a cys - loop receptor is an important determinant of divalent versus monovalent cation selectivity . a , voltage ramp ( left ) applied at the peak of the macroscopic inward current response to pressure - applied 5-ht ( 1 m)(right ) to determine the reversal potential ( e5-ht ) of the agonist - induced response mediated by the wild - type 5-ht3a receptor . a , voltage ramp ( left ) applied at the peak of the macroscopic inward current response to pressure - applied 5-ht ( 1 m)(right ) to determine the reversal potential ( e5-ht ) of the agonist - induced response mediated by the wild - type 5-ht3a receptor b , current - voltage relationships recorded from a representative hek-293 cell expressing the wild - type 5-ht3a receptor with an intracellular cscl - based solution ( i1 ) as reference and nacl ( 146 mm)-based solution e2 or cacl2 ( 100 mm)-based solution e3 as the extracellular electrolyte . the charge of the ma stretch 0 residue is the principal determinant of ca permeability we further evaluated the influence of charge at the ma 0 position by determining pca / pcs for constructs in which the residue was either negative ( 5-ht3a(r432q , r436e , r440a ) ; abbreviated as 5-ht3a(qea ) ) , neutral ( 5-ht3a(r432q , r436a , r440a ) and 5-ht3a(r432q , r436f , r440a ) ; abbreviated as 5-ht3a(qaa ) and 5-ht3a(qfa ) , respectively ) , or remained positive ( 5-ht3a(r432q , r440a ) ; abbreviated as 5-ht3a(qra ) ) . although pca / pcs was not significantly different from the wild - type 5-ht3a receptor for the 5-ht3a(qaa ) , 5-ht3a(qfa ) , and 5-ht3a(qra ) constructs , the ratio tended to decrease progressively as the charge at position 436 changed from negative , through neutral , to positive ( fig . finally , measurements of e5-ht with na as the predominant extracellular cation revealed no significant change in pna / pcs relative to the wild - type receptor , apart from the 5-ht3a(qaa ) and 5-ht3a(qfa ) constructs in which the ratio was modestly reduced to 0.72 0.02 and 0.77 0.01 , respectively ( fig . with na as the predominant extracellular cation , the single channel current - voltage relationship ( i - v ) for the 5-ht3a ( qda ) receptor shows a very mild outward rectification over a wide range of holding potentials ( 100 to 100 mv ) and yields chord conductance levels of 46 and 40 ps at holding potentials of + 100 and 100 mv , respectively ( fig . extracellular ca reduces the single channel conductance of the 5-ht3a(qda ) receptor in a concentration - dependent fashion we examined the influence of extracellular ca upon the single channel conductance of the 5-ht3a(qda ) receptor construct with an extracellular solution containing 95 mm na and varying concentrations of ca ( 10 nm to 30 mm ) . consistent with the relative permeability measurements made with high ca solution ( e3 , table 1 ) , the addition of increasing concentrations of ca was associated with a progressive shift in e5-ht to more positive values ( from 15.0 0.04 mv ( n = 4 ) with [ ca]o = 0.1 mm to 1.6 0.7 mv single channel conductance was reduced by 65% over the range [ ca]o = 0.1 mm ( 41.3 0.88 ps ) to [ ca]o = 30 mm ( 14.8 0.63 ps ; fig . in agreement with such a violation of the independent movement of na and ca within the permeation pathway , [ na]o fixed at 95 mm ( and assuming pna / pcs = 0.9 ; see above ) , pca / pcs was calculated from determinations of e5-ht to be 0.5 and 1.8 in the presence of 10 and 30 mm extracellular ca , respectively , versus the value of 3.7 found with ca as the sole extracellular charge carrier . effect of neutralizing the extracellular ring of charge of the 5-ht3a and 5-ht3a(qda ) receptor extracellular ca exerts multiple effects upon the wild - type 5-ht3a receptor including reductions in the apparent affinity of agonist binding ( 34 , 41 ) , depression of macroscopic and single channel current amplitudes ( 20 , 34 , 42 ) , and an acceleration of the kinetics of activation , deactivation , and desensitization ( 34 ) . using solutions e3 and i2 and the voltage ramp protocol , macroscopic currents mediated by the 5-ht3a(qda d293a ) construct in response to 5-ht demonstrated an e5-ht of 40.5 0.8 mv ( n = 6 ) , corresponding to a pca / pcs ratio of only 0.25 0.01 ( n = 6 ) compared with that of 3.7 for the 5-ht3a(qda ) receptor ( fig . in the presence of 0.1 mm ca and 95 mm na , the single channel chord conductance of the 5-ht3a(qda d293a ) receptor was 25.8 1.9 ps ( n = 4 ) at a holding potential of 80 mv in comparison with 41.3 ps for the 5-ht3a(qda ) construct . similarly , the single channel chord conductance ( at 80 mv ) of the 5-ht3a(qda d293a ) receptor determined with solution e2 ( 29.8 0.5 , n = 7 ) was significantly less than that of the 5-ht3a(qda ) construct ( 40.8 ps ) . however , no difference in single channel conductance was observed between the 5-ht3a(qda ) and 5-ht3a(qda d293a ) constructs over a range of positive holding potentials ( 60 to 100 mv ) , indicating that the d293a mutation preferentially suppresses inwardly directed cation flux ( fig . figure 8.comparison of the relative ion permeability of wild - type , 5-ht3a(d293a ) , 5-ht3a(qda ) , and 5-ht3a(qda d293a ) receptor constructs with na and ca . a , the relationship between the reversal potential ( e5-ht ) of the macroscopic current response to pressure - applied 5-ht ( 10 m ) recorded from the 5-ht3a(qda d293a ) receptor construct and the activity of na in the extracellular medium ( ( na)o ) . of measurements performed on 34 outside - out membrane patches at a holding potential of 80 mv under the ionic conditions described in c. comparison of the relative ion permeability of wild - type , 5-ht3a(d293a ) , 5-ht3a(qda ) , and 5-ht3a(qda d293a ) receptor constructs with na and ca . a , the relationship between the reversal potential ( e5-ht ) of the macroscopic current response to pressure - applied 5-ht ( 10 m ) recorded from the 5-ht3a(qda d293a ) receptor construct and the activity of na in the extracellular medium ( ( na)o ) . notably , the 5-ht3a(qda ) construct retained perfect selectivity toward cations versus anions , a finding that accords with the results of a recent study in which deletion of the entire intracellular loop of the 5-ht3a receptor had no measurable influence upon pna / pcl ( 44 ) . the latter are suppressed by extracellular divalent cations by mechanisms that potentially include a reduction in agonist binding affinity ( 34 , 41 ) , an acceleration of the kinetics of deactivation and desensitization ( 34 ) , and depression of macroscopic and single channel current amplitudes ( 20 , 34 ) , the latter being inferred by fluctuation analysis ( 20 ) . a solution ( e3 ) , in which ca was the sole extracellular cation , supported outwardly rectifying single channel currents but with a chord conductance at negative potentials ( 5.7 ps ) , which was greatly reduced in comparison with the value of 40.8 ps obtained with solution e2 , in which na was the permeant species . moreover , the addition of ca to na - containing extracellular solutions caused a concentration - dependent inhibition of the inwardly directed single channel current amplitude , in a manner reminiscent of that observed for single channel currents mediated by the nicotinic ach receptor of skeletal muscle ( 45 ) . we considered the d293a residue of the human 5-ht3a receptor subunit , which forms the 20 negatively charged ring within the extracellular vestibule of the channel ( 2 , 28 ) , to be a strong candidate for the putative ca binding site that limits the rate of charge transfer through the channel ( fig . the potency of 5-ht to activate the mouse 5-ht3a(d298a ) construct is less than for the murine wild - type receptor ( 34 ) , a result concordant with our observation that the d293a mutation introduced into either the human wild - type or 5-ht3a(qda ) construct causes a dextral shift in the 5-ht concentration - response relationship . because of the very low conductance of the wild - type 5-ht3a receptor ( 20 , 21 ) and kinetic properties of the 5-ht3a(d293a ) receptor , which are unfavorable to fluctuation analysis ( 34),5 we could not estimate the effect of the d293a mutation upon single channel conductance in this construct . once more , such an effect can probably be attributed to a reduction in a local negative electrostatic potential that particularly favors the accumulation of ca at the entrance to the pore , as suggested by molecular dynamics simulations performed on models of the 7 and skeletal muscle nicotinic ach receptors ( 46 , 47 ) . a previous study of mouse 5-ht3a receptors in which cysteine residues engineered into the tm2 domain were successfully probed with a negatively charged sulfydryl - modifying agent ( methane - thiosulfonate ethylsulfonate ( mtses ) ) applied extracellularly also indicates that the extracellular ring does not totally select against anions gaining access to deeper regions of the pore ( 49 ) . heteromeric 5-ht3 receptors expressed in tsa-201 cells assemble with the stoichiometry ( 5-ht3a)2(5-ht3b)3 as a subunit rosette in the order b - b - a - b - a , yielding 2 a - b , 2 b - a , and 1 b - b subunit interfaces ( 16 ) . yet , the inclusion of the 5-ht3b subunit within heteromeric 5-ht3 receptors reduces permeability to ca ( pca / pcs = 0.50.6 ) in comparison with homomeric receptors ( pca / pcs = 1.11.4 ) ( ref . macroscopic current responses mediated by the mouse 5-ht3a(d298a ) receptor are insensitive to [ ca]o within the range of 0.1 to 10 mm ( 34 ) , but we observed that single channel currents recorded from the human 5-ht3a(qda d293a ) receptor were reduced in amplitude in the presence of extracellular ca .

the growing market of animal health products may account for the potential revenue source of companion animal care . the animal health care has resulted in more than one billion sales in 2007 for novartis , pfizer and heska ( kling 2007 ) . the opportunity to carry out preclinical trials based on spontaneous tumors has been considered suitable to develop new effective drugs that could be quickly translated into clinical practice ( de vico et al . ; vail and macewen 2000 ) . for instance , naturally occurring tumors in dogs share similarities with human cancer both in terms of histopathological features and biological behavior ( hahn et al . 1994 ) . in the second half of the 1800s , a substantial parallel between the animal and human oncopathology , evidence showed that spontaneous animal tumors were similar to their human counterpart and information retrieved from them might have clinical implications ( dobberstein 1937 ) . in the early 1970s , the first world health organization ( who ) international histological classification of tumors of domestic animals was published ( beveridge and sobin 1974 , 1976 ) . it was followed by the tnm classification of tumors in domestic animals based on the guidelines of the tnm classification of malignant tumors in man ( owen 1980 ) showing that spontaneous canine and feline tumors are relevant models to study human cancers . we summarized such models in table 1 according to the review from de vico et al . conversely , in vivo and in vitro models , based on rodents and cell lines , have displayed intrinsic limits , such as complexity of management ( schiffer 1997 ) and the experimental methods used to induce the pathology ( vail and thamm 2004 ) , which make them different from the human spontaneous diseases ( de vico et al . 2005 ; moore and siopes 2004).table 1summary of tumor features shared between animals and humans ( allen et al . 2002)modelkind of tumorfeaturescanine / felinenon - hodgkin s lymphomaequates to intermediate and high - grade non - hodgkin s lymphoma in humansimmune system alterations seem to be associated with a higher incidence of non - hodgkin s lymphomais very sensitive to chemotherapycurrently used as a model for testing new chemotherapeutics and new forms of immunotherapy , as well as for the study of multiple drug resistance ; it has been used as a relevant model for ( 1 ) developing hypoxic cell markers , ( 2 ) studying the effect of whole - body hyperthermia on the pharmacokinetics of systemic chemotherapy and ( 3 ) studying autologous bone transplantationcaninesoft tissue sarcomassimilar to pathological appearance , clinical presentation and behavior of human soft tissue sarcomasrespond to radiation therapy and chemotherapy in a manner similar to human pathologyhemangiosarcomas are extremely similar to human hemangiosarcomas with respect to histological pattern and metastatic behaviorcanineosteosarcomassimilar to human osteosarcomas with respect to the histology , metastatic behavior and clinical evolution of the diseasecanineoral malignant melanomasthey are chemoresistant and share many immunological targets with human oral malignant melanomasconsidered a relevant model for developing new immunotherapeutic approaches for both dogs and humansboth in dogs and humans , human tyrosinase dna vaccination is safe and potentially effectivecaninetransitional cell carcinomasshare histological appearance , biological behavior and response to therapy with invasive human transitional cell carcinomasconsidered a useful model for testing new photodynamic therapy technologies and chemotherapeutic combinationscaninemast cell tumorsmutation of exon 11 of c - kit occurs in 3050 % of advanced mast cell tumors and is similar to that usually occurring in 5090 % of human gastrointestinal stromal tumorscanine / felinemammary carcinomasvery similar to human breast cancers because of their hormonal dependence , spontaneous development in middle - aged to older animals , metastatic behavior toward regional lymph nodes and lungs , as well as adhesion molecules and neoangiogenesis patternsabnormalities in the nuclear dna content have been documented in both malignant and benign canine mammary tumors , but are more frequent in human mammary carcinomasmammary neoplasia in dogs may be a good molecular model for developing new antineoplastic strategies involving cyclin d1 and cyclin - dependent kinasesfeline mammary carcinoma shows age incidence , histopathology and pattern of metastasis similar to human breast cancerfeline mammary carcinoma shares common features with human inflammatory mammary carcinoma and human mammary carcinoma with osteoclast - like giant cellsepidermal growth factor receptor-2 ( her-2 ) and recepteur d'origine nantais ( ron ) overexpressions qualify feline mammary carcinoma as homologous to human breast carcinomas which have poor prognosiscanineseminomasmetastases occur only in a small percentage of cases , whereas human seminomas have a marked tendency to metastasizehave a histogenetic behavior similar to that of human seminomascaninetransmissible venereal tumoris considered a promising model to study human kaposi s sarcomapercutaneous inoculation and intraarterial transplantation of tumor fragments in the canine lung result in predictable patterns of tumor growth resembling the solitary pulmonary nodules and metastatic disease found in humans summary of tumor features shared between animals and humans ( allen et al . 2002 ) we searched pubmed / medline using the keywords spontaneous , animal , tumor , oncology and comparative alone or in combination and cross - referencing among published papers . selected papers from 1910 to 2013 were chosen on the basis of their content ( evidence - based quality and reliability ) . spontaneous canine tumors , such as prostate carcinoma , chondrosarcoma , which accounts for approximately 10 % of all primary bone tumors in dogs ( beveridge and sobin 1976 ) , axial osteosarcoma , which accounts for 80 % of all primary bone tumors in dogs ( thompson and pool 2002 ) and synovial cell sarcoma , have been extensively described representing valuable models to study carcinogenesis ( vail et al . 1994 ) . most canine prostate carcinomas affect both elderly sexually intact dogs and dogs which had undergone surgical castration after reaching sexual maturity . in 20052009 , a pilot study evaluated the incidence of spontaneous tumors in dogs living in the neighborhood of venice and vicenza ( vascellari et al . two thousand five hundred and nine out of 296,318 canine cases of neoplasia were diagnosed during the first three years . the estimated annual tumor incidence rate per 100,000 dogs was 282 , with an equal distribution of malignant and benign tumors between male and female dogs . moreover , in pure breeds , a twofold higher incidence of malignant tumors , with respect to mixed breeds , was observed . the incidence increased with age in both genders . due to the dissimilar methodologies and variable reference populations of both european and north american veterinary cancer registries , the occurrence of spontaneous tumors in pet animals has been underestimated ( bonnett et al . 1997 ; dobson et al . the animal tumor registry of genoa ( italy ) retrospectively showed that , from 1985 to 2002 , 70 % of all cancer cases in female dogs were located in the breast ( merlo et al . the overall incidence of cancer was 99.3 per 100,000 dogs per year in male dogs and 272.1 in female dogs . among domestic animal tumors , spontaneous squamous cell carcinomas provided additional information about the pathology of oral cancer ( gardner 1996 ) . as far as oral tumors affecting dogs are concerned , malignant melanoma accounts for the 4 % of all canine tumors ( macewen et al . canine malignant melanomas and human melanomas are treated similarly using surgery and/or fractionated radiation therapy and immunological therapies ( vail and thamm 2004 ; bergman et al . therefore , canine malignant melanomas are suitable models for new immunotherapeutic protocols in humans ( vail and thamm 2004 ) . dogs and cats also frequently develop squamous cell carcinomas ( dorn and priester 1976 ; strafuss et al . skin and subcutaneous tissue neoplasms are the most frequently recognized neoplastic disorders in domestic animals and can be caused by prolonged and continuous exposure to sunlight ( madewell 1981 ) . alimentary tract cancer in dogs has lower incidence , with ductal and acinar carcinomas occurring most frequently in females than in males while intranasal tumors account for 12 % of all canine neoplasms ( priester 1974 ) . primary bone cancer is the second most often detected in dogs , and its main risk factors are ionizing radiations , chemical carcinogens and viruses ( madewell 1981 ) . moreover , preexisting bone defects and skeletal abnormalities increase the risk factors from 60 up to 185 times for large and giant breeds with respect to small dogs ( tjalma 1966 ) . as to primary brain tumors , they account for approximately 2 % of all cancer in human adults ( mckinney 2004 ) and for 0.01 % in dogs ( lecouteur 1999 ) . specifically , gliomas are more common among brachycephalic breeds of dogs , especially boxers , english bulldogs and boston terriers ( hayes 1976 ) . high incidence rates were also detected for non - hodgkin s lymphoma in bitches and for non - hodgkin s lymphoma and skin cancer in male dogs ( merlo et al . 2008 ) . additionally , the incidence rate of cancer increased with age ranging between 23.7 and 763.2 in bitches and 16.5 and 237.6 in male dogs aged 3 and > 911 years . hemangiosarcoma , which can affect both cats and dogs , involves the musculoskeletal system , and mean survival time in dogs affected by this condition ranges from 267 ( ogilvie et al . the ovarian and epithelial tumors , although quite rare in domestic animals , have been reported mainly in cats , dogs and horses ( maclachlan 1987 ) . a single unique gonadoblastoma tumor affecting human males or females has been observed in the testicle of a dog ( turk et al . spontaneous testicular tumors , which are quite common in aged dogs , are mainly seminomas ( solitary , unilateral , more frequent in the right testicle and with a reduced possibility to develop metastases , if compared with humans ) ( kennedy et al . 1998 ) , sertoli cell tumors and leydig cell tumors , which can coexist together along with seminomas ( nielsen and kennedy 1990 ) and occur with the same frequency ( maiolino et al . 2004 ) . suggested a possible correspondence between human spermatocytic seminomas and most canine seminomas in order to justify their low metastatic behavior , showing a potential predictive model for the development of a treatment protocol ( maiolino et al . further , rare dog tumors are fibrosarcoma , rib , vertebral body and pelvis tumors ( both in dogs and in cats ) ( dernell et al . 1998 ; pirkey - ehrhart et al . 1992 ) and multiple myeloma ( rare in cats and uncommon in dogs ) , which accounts for 8 % of all canine hematopoietic tumors and affects older dogs with no breed or sex predilection ( matus et al . 1986 ) and intracranial central neurocytomas ( russell and burch 1959 ) . canine mast cell tumors , which account for 1621 % in this species ( thamm and vail 2001 ) , display a molecular alteration in the proto - oncogene c - kit , which is involved in mast cell differentiation , proliferation , survival and activation ( london et al . 1999 ) . specifically , the mutation of exon 11 of c - kit occurs in 3050 % of advanced mast cell tumors and is similar to that usually occurring in 5090 % of human gastrointestinal stromal tumors ( heinrich et al . 2002 ; london et al . this evidence emphasizes a parallel between human and canine mast cell tumors underlying the possibility of using the canine model to develop new beneficial therapeutics for both species ( pryer et al . non - hodgkin lymphoma , which accounts for 5 % of all malignant tumors and 83 % of all hematopoietic malignancies in dogs ( vail and thamm 2004 ) , affects mostly middle - aged , older dogs ( german shepherds , boxers , basset hounds and saint bernards ) ( macewen and young 1996 ) and both genders equally and corresponds to intermediate and high - grade non - hodgkin lymphoma in humans . it is generally associated with a poor prognosis , and in 7080 % of cases , it is b - cell mediated , whereas in 2030 % , it is t - cell mediated ( macewen 1995 ) . due to its sensitivity to chemotherapy , it is used as a model both for development of new chemotherapy drugs and multiple drug resistance studies ( vail and macewen 2000 ) . moreover , non - hodgkin lymphoma has been used to develop hypoxic cell markers , study the whole - body hyperthermia effects and evaluate autologous bone transplantation ( vail and thamm 2004 ) . spontaneous osteosarcomas account for more than 70 % of malignant bone tumors in cats ( thompson and pool 2002 ; brodey and riser 1969 ) . these tumors are relevant to human cancer biology , due to their relative resistance to chemotherapy ( he et al . injection - site sarcomas in felines , usually fibrosarcomas , are caused by an intense inflammatory reaction or vaccination adjuvants ( carwardine et al . they are highly locally invasive and metastasize in up to 23 % of the affected cases ( seguin 2002 ) . although aggressive surgical resection remains the gold standard therapy , chemotherapy and radiation can prolong the survival times ( hershey et al . 2000 ; kobayashi et al . 2002 ) . uterine tumors , rare in cats , account for 0.29 % of feline neoplasms ( miller et al . 2003 ) , and they include endometrial adenocarcinoma , mixed mullerian tumor , leiomyoma which occurs most frequently ( maclachlan and kennedy 2002 ) and myxoid leiomyosarcoma ( cooper et al . even if the etiology of nervous system tumors is not well understood , cats are commonly affected by meningiomas ( troxel et al . however , the biological behavior of such tumor , except for the anaplastic type , is generally considered benign in both humans ( louis et al . a pilot study evaluated the incidence of spontaneous tumors in cats living in the neighborhood of venice and vicenza ( vascellari et al . four hundred and ninety - four out of 214,683 feline cases of neoplasia were diagnosed during the first three years . for all tumors , the estimated annual incidence rate in cats per 100,000 was 77 . furthermore , a 4.6-fold higher incidence of malignant tumors was observed in cats , if compared to benign pathology . moreover , in pure breed cats , a twofold higher incidence of malignant tumors was observed and it increased with age in both malignant and benign pathologies , if compared with mixed breeds . in 2009 , wells introduced the potential role of pets in offering a therapeutic value to humans ( wells 2009b , 2012 ) . previous reports emphasized the role of dogs and cats as preventers of ill - health showing that pet owners are healthier than non - owners ( parslow et al . 2002 ; anderson et al . 1992 ; baun et al . 1984 ; dembicki and anderson 1996 ; friedmann et al . 1980 ; katcher 1981 ; katcher et al . 1983 ; larson et al . 2010 ; sebkova 1977 ; siegel 1993 ; vormbrock and grossberg 1988 ) . furthermore , the ability of pets to facilitate human recovery has been explored ( larson et al . 2010 ) . for instance , friedmann et al . reported that pet owners were significantly more likely to be alive 1 year after a heart attack than non - owners ( friedmann et al . 1980 ) and dogs were stronger facilitators to recovery from ill - health than cats ( friedmann and thomas 1995 ) . therefore , dogs may contribute indirectly to long - standing human physical health , which is of great importance , especially in immunocompromised patients ( hemsworth and pizer 2006 ) , possibly due to the increased physical activity which typically characterizes the ownership of a dog ( larson et al . dogs have also been used as warning systems for cancer ( williams and pembroke 1989 ) , epilepsy ( brown and strong 2001 ; dalziel et al . 2003 ; strong et al . 1999 , 2002 ) and diabetes ( chen et al . 2000 ; they have also been used as therapists in nursing homes ( crowley - robinson et al . 2002 ) , visitors in pediatric hospital wards ( moody et al . 2002 ) , assistants for the disabled ( davis et al . 2004 ; fishman 2003 ; sanders 2000 ) , enhancers of psychological welfare ( raina et al . 1999 ) and rehabilitators for prisoners ( merriam - arduini 2000 ; strimple 2003 ) . spontaneous canine tumors , such as prostate carcinoma , chondrosarcoma , which accounts for approximately 10 % of all primary bone tumors in dogs ( beveridge and sobin 1976 ) , axial osteosarcoma , which accounts for 80 % of all primary bone tumors in dogs ( thompson and pool 2002 ) and synovial cell sarcoma , have been extensively described representing valuable models to study carcinogenesis ( vail et al . 1994 ) . most canine prostate carcinomas affect both elderly sexually intact dogs and dogs which had undergone surgical castration after reaching sexual maturity . in 20052009 , a pilot study evaluated the incidence of spontaneous tumors in dogs living in the neighborhood of venice and vicenza ( vascellari et al . two thousand five hundred and nine out of 296,318 canine cases of neoplasia were diagnosed during the first three years . the estimated annual tumor incidence rate per 100,000 dogs was 282 , with an equal distribution of malignant and benign tumors between male and female dogs . moreover , in pure breeds , a twofold higher incidence of malignant tumors , with respect to mixed breeds , was observed . the incidence increased with age in both genders . due to the dissimilar methodologies and variable reference populations of both european and north american veterinary cancer registries , the occurrence of spontaneous tumors in pet animals has been underestimated ( bonnett et al . 1997 ; dobson et al . the animal tumor registry of genoa ( italy ) retrospectively showed that , from 1985 to 2002 , 70 % of all cancer cases in female dogs were located in the breast ( merlo et al . the overall incidence of cancer was 99.3 per 100,000 dogs per year in male dogs and 272.1 in female dogs . among domestic animal tumors , spontaneous squamous cell carcinomas provided additional information about the pathology of oral cancer ( gardner 1996 ) . as far as oral tumors affecting dogs are concerned , malignant melanoma accounts for the 4 % of all canine tumors ( macewen et al . canine malignant melanomas and human melanomas are treated similarly using surgery and/or fractionated radiation therapy and immunological therapies ( vail and thamm 2004 ; bergman et al . therefore , canine malignant melanomas are suitable models for new immunotherapeutic protocols in humans ( vail and thamm 2004 ) . dogs and cats also frequently develop squamous cell carcinomas ( dorn and priester 1976 ; strafuss et al . skin and subcutaneous tissue neoplasms are the most frequently recognized neoplastic disorders in domestic animals and can be caused by prolonged and continuous exposure to sunlight ( madewell 1981 ) . alimentary tract cancer in dogs has lower incidence , with ductal and acinar carcinomas occurring most frequently in females than in males while intranasal tumors account for 12 % of all canine neoplasms ( priester 1974 ) . primary bone cancer is the second most often detected in dogs , and its main risk factors are ionizing radiations , chemical carcinogens and viruses ( madewell 1981 ) . moreover , preexisting bone defects and skeletal abnormalities increase the risk factors from 60 up to 185 times for large and giant breeds with respect to small dogs ( tjalma 1966 ) . as to primary brain tumors , they account for approximately 2 % of all cancer in human adults ( mckinney 2004 ) and for 0.01 % in dogs ( lecouteur 1999 ) . specifically , gliomas are more common among brachycephalic breeds of dogs , especially boxers , english bulldogs and boston terriers ( hayes 1976 ) . high incidence rates were also detected for non - hodgkin s lymphoma in bitches and for non - hodgkin s lymphoma and skin cancer in male dogs ( merlo et al . 2008 ) . additionally , the incidence rate of cancer increased with age ranging between 23.7 and 763.2 in bitches and 16.5 and 237.6 in male dogs aged 3 and > 911 years . hemangiosarcoma , which can affect both cats and dogs , involves the musculoskeletal system , and mean survival time in dogs affected by this condition ranges from 267 ( ogilvie et al . although quite rare in domestic animals , have been reported mainly in cats , dogs and horses ( maclachlan 1987 ) . a single unique gonadoblastoma tumor affecting human males or females has been observed in the testicle of a dog ( turk et al . spontaneous testicular tumors , which are quite common in aged dogs , are mainly seminomas ( solitary , unilateral , more frequent in the right testicle and with a reduced possibility to develop metastases , if compared with humans ) ( kennedy et al . 1998 ) , sertoli cell tumors and leydig cell tumors , which can coexist together along with seminomas ( nielsen and kennedy 1990 ) and occur with the same frequency ( maiolino et al . suggested a possible correspondence between human spermatocytic seminomas and most canine seminomas in order to justify their low metastatic behavior , showing a potential predictive model for the development of a treatment protocol ( maiolino et al . further , rare dog tumors are fibrosarcoma , rib , vertebral body and pelvis tumors ( both in dogs and in cats ) ( dernell et al . 1998 ; pirkey - ehrhart et al . 1995 ; straw et al . 1992 ) and multiple myeloma ( rare in cats and uncommon in dogs ) , which accounts for 8 % of all canine hematopoietic tumors and affects older dogs with no breed or sex predilection ( matus et al . canine mast cell tumors , which account for 1621 % in this species ( thamm and vail 2001 ) , display a molecular alteration in the proto - oncogene c - kit , which is involved in mast cell differentiation , proliferation , survival and activation ( london et al . specifically , the mutation of exon 11 of c - kit occurs in 3050 % of advanced mast cell tumors and is similar to that usually occurring in 5090 % of human gastrointestinal stromal tumors ( heinrich et al . 2002 ; london et al . this evidence emphasizes a parallel between human and canine mast cell tumors underlying the possibility of using the canine model to develop new beneficial therapeutics for both species ( pryer et al . non - hodgkin lymphoma , which accounts for 5 % of all malignant tumors and 83 % of all hematopoietic malignancies in dogs ( vail and thamm 2004 ) , affects mostly middle - aged , older dogs ( german shepherds , boxers , basset hounds and saint bernards ) ( macewen and young 1996 ) and both genders equally and corresponds to intermediate and high - grade non - hodgkin lymphoma in humans . it is generally associated with a poor prognosis , and in 7080 % of cases , it is b - cell mediated , whereas in 2030 % , it is t - cell mediated ( macewen 1995 ) . due to its sensitivity to chemotherapy , it is used as a model both for development of new chemotherapy drugs and multiple drug resistance studies ( vail and macewen 2000 ) . moreover , non - hodgkin lymphoma has been used to develop hypoxic cell markers , study the whole - body hyperthermia effects and evaluate autologous bone transplantation ( vail and thamm 2004 ) . spontaneous osteosarcomas account for more than 70 % of malignant bone tumors in cats ( thompson and pool 2002 ; brodey and riser 1969 ) . these tumors are relevant to human cancer biology , due to their relative resistance to chemotherapy ( he et al . injection - site sarcomas in felines , usually fibrosarcomas , are caused by an intense inflammatory reaction or vaccination adjuvants ( carwardine et al . they are highly locally invasive and metastasize in up to 23 % of the affected cases ( seguin 2002 ) . although aggressive surgical resection remains the gold standard therapy , chemotherapy and radiation can prolong the survival times ( hershey et al . 2000 ; kobayashi et al . 2002 ) . uterine tumors , rare in cats , account for 0.29 % of feline neoplasms ( miller et al . 2003 ) , and they include endometrial adenocarcinoma , mixed mullerian tumor , leiomyoma which occurs most frequently ( maclachlan and kennedy 2002 ) and myxoid leiomyosarcoma ( cooper et al . 2006 ) . even if the etiology of nervous system tumors is not well understood , cats are commonly affected by meningiomas ( troxel et al . however , the biological behavior of such tumor , except for the anaplastic type , is generally considered benign in both humans ( louis et al . 2007 ) and common pets like dogs and cats ( motta et al . 2012 ) . a pilot study evaluated the incidence of spontaneous tumors in cats living in the neighborhood of venice and vicenza ( vascellari et al . four hundred and ninety - four out of 214,683 feline cases of neoplasia were diagnosed during the first three years . for all tumors , the estimated annual incidence rate in cats per 100,000 was 77 . furthermore , a 4.6-fold higher incidence of malignant tumors was observed in cats , if compared to benign pathology . moreover , in pure breed cats , a twofold higher incidence of malignant tumors was observed and it increased with age in both malignant and benign pathologies , if compared with mixed breeds . in 2009 , wells introduced the potential role of pets in offering a therapeutic value to humans ( wells 2009b , 2012 ) . previous reports emphasized the role of dogs and cats as preventers of ill - health showing that pet owners are healthier than non - owners ( parslow et al . 2002 ; anderson et al . 1992 ; baun et al . 1984 ; dembicki and anderson 1996 ; friedmann et al . 1980 ; katcher 1981 ; katcher et al . 1983 ; larson et al . 2010 ; sebkova 1977 ; siegel 1993 ; vormbrock and grossberg 1988 ) . furthermore , the ability of pets to facilitate human recovery has been explored ( larson et al . 2010 ) . for instance , friedmann et al . reported that pet owners were significantly more likely to be alive 1 year after a heart attack than non - owners ( friedmann et al . 1980 ) and dogs were stronger facilitators to recovery from ill - health than cats ( friedmann and thomas 1995 ) . therefore , dogs may contribute indirectly to long - standing human physical health , which is of great importance , especially in immunocompromised patients ( hemsworth and pizer 2006 ) , possibly due to the increased physical activity which typically characterizes the ownership of a dog ( larson et al . dogs have also been used as warning systems for cancer ( williams and pembroke 1989 ) , epilepsy ( brown and strong 2001 ; dalziel et al . 2003 ; strong et al . 1999 , 2002 ) and diabetes ( chen et al . they have also been used as therapists in nursing homes ( crowley - robinson et al . 2002 ) , visitors in pediatric hospital wards ( moody et al . 2002 ) , assistants for the disabled ( davis et al . 2004 ; fishman 2003 ; sanders 2000 ) , enhancers of psychological welfare ( raina et al . 1999 ) and rehabilitators for prisoners ( merriam - arduini 2000 ; strimple 2003 ) . comparative oncology research has extensively relied on domestic animals ( antinoff and hahn 2004 ; hansen and khanna 2004 ; hershey et al . 2005 ; nasir and reid 1999 ; seltenhammer et al . 2004 ; withrow and vail 2007 ) . the comparative oncology trials consortium program infrastructure , founded in 2009 to design and execute clinical trials involving dogs affected by cancer and in collaboration with the drug manufacturing industry and nongovernmental groups interested in cancer drug development , aimed to answer biological questions which could inform the development path of novel agents for future use in human cancer patients in a timely and integrated manner ( gordon et al . the recent identification of the canine genome has provided evidence of strong similarities with humans ( lindblad - toh et al . 2005 ; obrien and murphy 2003 ; ostrander et al . 2006 ) such as several cancer - associated gene families ( hoffman and birney 2007 ) , as well as the presence of the similar genetic cancer - associated molecular alterations ( haga et al . 2001 tumor initiation and progression are also influenced by age , nutrition , sex , reproductive status and environmental exposures in both human and canine cancers ( bukowski et al . 1998 ; hayes and fraumeni 1977 ; misdorp 1996 ; mukaratirwa 2005 ; olson 2007 ; patronek et al . 1997 ) . the lack of gold standards for the management of cancer in dogs and cats , as well as the high motivation of pet owners to seek out new options for its management , underlines the increasing need to evaluate novel therapeutics in these populations . in this ways , it is possible to assess the effectiveness of new agents when given alone or in combination with other therapies prior to the clinical testing of the drug . the enrollment of pet dogs in preclinical and clinical trials is now focusing on new anti - neoplastic drug research and development . although regulation of animal research possesses guidelines such as the three rs ( replacement , reduction and refinement ) ( russell and burch 1959 ) , legal implications regarding the use of animal models for research purposes are still debated ( griffin 1998 ; porrello et al . although the existence of spontaneous pet tumors could be a reliable model for human cancers , many open questions , related to the opportunities to select and recruit new types of animal models in oncology and to their legal and ethical implications , remain unanswered . unfortunately , the use of spontaneous animal models implicates several professional and ethical warnings with conflictory debates . in the recent years , researchers have also focused their exploration on a possible role of some dog species as early warning systems for cancer ( williams and pembroke 1989 ) , epilepsy ( brown and strong 2001 ; dalziel et al . 2003 ; strong et al . 1999 , 2002 ) and diabetes ( chen et al . 2000 ; lim et al . 1992 ; mcaulay et al . 2001 ) . while some dogs have showed innate ability to recognize with smell the human cancers in vivo , others have been trained to perform this skill ( willis et al . 2004 ) . from this overview , there is evidence that companion animal health care is impressively growing in terms of development of new therapies and diagnostic tools , nutrition and disease prevention ( kling 2007 ) . conclusively , we strongly encourage the animalists , veterinarians and clinical researchers to join us and support ethical observational studies on domestic and laboratory animal spontaneous cancer epidemiology , early prevention , diagnosis and treatment in order to detect and possibly eliminate alimentary and environmental factors which can also be cancerogenic for humans . in fact , the shorter life span of animals , compared with that of humans , can give a rapid overview of many clinical features which also characterize human cancer . furthermore , the pooling of anecdotal veterinary reports on drugs or multimodal animal cancer treatments , protocols and outcomes , including surgical pathology , oncoimmunology and molecular biology investigations , will give outstanding contribution to the interspecies translational information with undoubted mutual interspecies benefit . the pet psychological and physical support to the fitness and wellness of cancer patients should be more deeply investigated and largely extended in the palliation practice .

the spontaneous tumor biology has been investigated with the support of animalists using animals as a preclinical model allowing translation of results in clinical practice . this review provides an insight into the field of comparative oncology . evidence shows that companion animal health care is impressively growing in terms of development of new therapies and diagnostic tools , nutrition and disease prevention . however , even if most animal tumors might be a reliable model to study human carcinomas , many open questions , related to the opportunities to select and recruit new models in oncology , along with their legal and ethical implications , remain unanswered .

Introduction Searching criteria Epidemiology of cancer in dogs Epidemiology of cancer in cats Translation of experimental studies into the clinical setting Discussion Conclusions

the growing market of animal health products may account for the potential revenue source of companion animal care . the animal health care has resulted in more than one billion sales in 2007 for novartis , pfizer and heska ( kling 2007 ) . the opportunity to carry out preclinical trials based on spontaneous tumors has been considered suitable to develop new effective drugs that could be quickly translated into clinical practice ( de vico et al . ; vail and macewen 2000 ) . for instance , naturally occurring tumors in dogs share similarities with human cancer both in terms of histopathological features and biological behavior ( hahn et al . in the second half of the 1800s , a substantial parallel between the animal and human oncopathology , evidence showed that spontaneous animal tumors were similar to their human counterpart and information retrieved from them might have clinical implications ( dobberstein 1937 ) . in the early 1970s , the first world health organization ( who ) international histological classification of tumors of domestic animals was published ( beveridge and sobin 1974 , 1976 ) . it was followed by the tnm classification of tumors in domestic animals based on the guidelines of the tnm classification of malignant tumors in man ( owen 1980 ) showing that spontaneous canine and feline tumors are relevant models to study human cancers . we summarized such models in table 1 according to the review from de vico et al . 2002)modelkind of tumorfeaturescanine / felinenon - hodgkin s lymphomaequates to intermediate and high - grade non - hodgkin s lymphoma in humansimmune system alterations seem to be associated with a higher incidence of non - hodgkin s lymphomais very sensitive to chemotherapycurrently used as a model for testing new chemotherapeutics and new forms of immunotherapy , as well as for the study of multiple drug resistance ; it has been used as a relevant model for ( 1 ) developing hypoxic cell markers , ( 2 ) studying the effect of whole - body hyperthermia on the pharmacokinetics of systemic chemotherapy and ( 3 ) studying autologous bone transplantationcaninesoft tissue sarcomassimilar to pathological appearance , clinical presentation and behavior of human soft tissue sarcomasrespond to radiation therapy and chemotherapy in a manner similar to human pathologyhemangiosarcomas are extremely similar to human hemangiosarcomas with respect to histological pattern and metastatic behaviorcanineosteosarcomassimilar to human osteosarcomas with respect to the histology , metastatic behavior and clinical evolution of the diseasecanineoral malignant melanomasthey are chemoresistant and share many immunological targets with human oral malignant melanomasconsidered a relevant model for developing new immunotherapeutic approaches for both dogs and humansboth in dogs and humans , human tyrosinase dna vaccination is safe and potentially effectivecaninetransitional cell carcinomasshare histological appearance , biological behavior and response to therapy with invasive human transitional cell carcinomasconsidered a useful model for testing new photodynamic therapy technologies and chemotherapeutic combinationscaninemast cell tumorsmutation of exon 11 of c - kit occurs in 3050 % of advanced mast cell tumors and is similar to that usually occurring in 5090 % of human gastrointestinal stromal tumorscanine / felinemammary carcinomasvery similar to human breast cancers because of their hormonal dependence , spontaneous development in middle - aged to older animals , metastatic behavior toward regional lymph nodes and lungs , as well as adhesion molecules and neoangiogenesis patternsabnormalities in the nuclear dna content have been documented in both malignant and benign canine mammary tumors , but are more frequent in human mammary carcinomasmammary neoplasia in dogs may be a good molecular model for developing new antineoplastic strategies involving cyclin d1 and cyclin - dependent kinasesfeline mammary carcinoma shows age incidence , histopathology and pattern of metastasis similar to human breast cancerfeline mammary carcinoma shares common features with human inflammatory mammary carcinoma and human mammary carcinoma with osteoclast - like giant cellsepidermal growth factor receptor-2 ( her-2 ) and recepteur d'origine nantais ( ron ) overexpressions qualify feline mammary carcinoma as homologous to human breast carcinomas which have poor prognosiscanineseminomasmetastases occur only in a small percentage of cases , whereas human seminomas have a marked tendency to metastasizehave a histogenetic behavior similar to that of human seminomascaninetransmissible venereal tumoris considered a promising model to study human kaposi s sarcomapercutaneous inoculation and intraarterial transplantation of tumor fragments in the canine lung result in predictable patterns of tumor growth resembling the solitary pulmonary nodules and metastatic disease found in humans summary of tumor features shared between animals and humans ( allen et al . 2002 ) we searched pubmed / medline using the keywords spontaneous , animal , tumor , oncology and comparative alone or in combination and cross - referencing among published papers . selected papers from 1910 to 2013 were chosen on the basis of their content ( evidence - based quality and reliability ) . spontaneous canine tumors , such as prostate carcinoma , chondrosarcoma , which accounts for approximately 10 % of all primary bone tumors in dogs ( beveridge and sobin 1976 ) , axial osteosarcoma , which accounts for 80 % of all primary bone tumors in dogs ( thompson and pool 2002 ) and synovial cell sarcoma , have been extensively described representing valuable models to study carcinogenesis ( vail et al . most canine prostate carcinomas affect both elderly sexually intact dogs and dogs which had undergone surgical castration after reaching sexual maturity . due to the dissimilar methodologies and variable reference populations of both european and north american veterinary cancer registries , the occurrence of spontaneous tumors in pet animals has been underestimated ( bonnett et al . the overall incidence of cancer was 99.3 per 100,000 dogs per year in male dogs and 272.1 in female dogs . among domestic animal tumors , spontaneous squamous cell carcinomas provided additional information about the pathology of oral cancer ( gardner 1996 ) . canine malignant melanomas and human melanomas are treated similarly using surgery and/or fractionated radiation therapy and immunological therapies ( vail and thamm 2004 ; bergman et al . dogs and cats also frequently develop squamous cell carcinomas ( dorn and priester 1976 ; strafuss et al . skin and subcutaneous tissue neoplasms are the most frequently recognized neoplastic disorders in domestic animals and can be caused by prolonged and continuous exposure to sunlight ( madewell 1981 ) . specifically , gliomas are more common among brachycephalic breeds of dogs , especially boxers , english bulldogs and boston terriers ( hayes 1976 ) . high incidence rates were also detected for non - hodgkin s lymphoma in bitches and for non - hodgkin s lymphoma and skin cancer in male dogs ( merlo et al . hemangiosarcoma , which can affect both cats and dogs , involves the musculoskeletal system , and mean survival time in dogs affected by this condition ranges from 267 ( ogilvie et al . a single unique gonadoblastoma tumor affecting human males or females has been observed in the testicle of a dog ( turk et al . spontaneous testicular tumors , which are quite common in aged dogs , are mainly seminomas ( solitary , unilateral , more frequent in the right testicle and with a reduced possibility to develop metastases , if compared with humans ) ( kennedy et al . 1998 ) , sertoli cell tumors and leydig cell tumors , which can coexist together along with seminomas ( nielsen and kennedy 1990 ) and occur with the same frequency ( maiolino et al . suggested a possible correspondence between human spermatocytic seminomas and most canine seminomas in order to justify their low metastatic behavior , showing a potential predictive model for the development of a treatment protocol ( maiolino et al . this evidence emphasizes a parallel between human and canine mast cell tumors underlying the possibility of using the canine model to develop new beneficial therapeutics for both species ( pryer et al . due to its sensitivity to chemotherapy , it is used as a model both for development of new chemotherapy drugs and multiple drug resistance studies ( vail and macewen 2000 ) . moreover , non - hodgkin lymphoma has been used to develop hypoxic cell markers , study the whole - body hyperthermia effects and evaluate autologous bone transplantation ( vail and thamm 2004 ) . spontaneous osteosarcomas account for more than 70 % of malignant bone tumors in cats ( thompson and pool 2002 ; brodey and riser 1969 ) . these tumors are relevant to human cancer biology , due to their relative resistance to chemotherapy ( he et al . 2000 ; kobayashi et al . 2003 ) , and they include endometrial adenocarcinoma , mixed mullerian tumor , leiomyoma which occurs most frequently ( maclachlan and kennedy 2002 ) and myxoid leiomyosarcoma ( cooper et al . even if the etiology of nervous system tumors is not well understood , cats are commonly affected by meningiomas ( troxel et al . however , the biological behavior of such tumor , except for the anaplastic type , is generally considered benign in both humans ( louis et al . 2010 ; sebkova 1977 ; siegel 1993 ; vormbrock and grossberg 1988 ) . furthermore , the ability of pets to facilitate human recovery has been explored ( larson et al . therefore , dogs may contribute indirectly to long - standing human physical health , which is of great importance , especially in immunocompromised patients ( hemsworth and pizer 2006 ) , possibly due to the increased physical activity which typically characterizes the ownership of a dog ( larson et al . 2002 ) , assistants for the disabled ( davis et al . 2004 ; fishman 2003 ; sanders 2000 ) , enhancers of psychological welfare ( raina et al . 1999 ) and rehabilitators for prisoners ( merriam - arduini 2000 ; strimple 2003 ) . spontaneous canine tumors , such as prostate carcinoma , chondrosarcoma , which accounts for approximately 10 % of all primary bone tumors in dogs ( beveridge and sobin 1976 ) , axial osteosarcoma , which accounts for 80 % of all primary bone tumors in dogs ( thompson and pool 2002 ) and synovial cell sarcoma , have been extensively described representing valuable models to study carcinogenesis ( vail et al . 1994 ) . two thousand five hundred and nine out of 296,318 canine cases of neoplasia were diagnosed during the first three years . moreover , in pure breeds , a twofold higher incidence of malignant tumors , with respect to mixed breeds , was observed . due to the dissimilar methodologies and variable reference populations of both european and north american veterinary cancer registries , the occurrence of spontaneous tumors in pet animals has been underestimated ( bonnett et al . among domestic animal tumors , spontaneous squamous cell carcinomas provided additional information about the pathology of oral cancer ( gardner 1996 ) . as far as oral tumors affecting dogs are concerned , malignant melanoma accounts for the 4 % of all canine tumors ( macewen et al . canine malignant melanomas and human melanomas are treated similarly using surgery and/or fractionated radiation therapy and immunological therapies ( vail and thamm 2004 ; bergman et al . dogs and cats also frequently develop squamous cell carcinomas ( dorn and priester 1976 ; strafuss et al . although quite rare in domestic animals , have been reported mainly in cats , dogs and horses ( maclachlan 1987 ) . a single unique gonadoblastoma tumor affecting human males or females has been observed in the testicle of a dog ( turk et al . spontaneous testicular tumors , which are quite common in aged dogs , are mainly seminomas ( solitary , unilateral , more frequent in the right testicle and with a reduced possibility to develop metastases , if compared with humans ) ( kennedy et al . 1998 ) , sertoli cell tumors and leydig cell tumors , which can coexist together along with seminomas ( nielsen and kennedy 1990 ) and occur with the same frequency ( maiolino et al . suggested a possible correspondence between human spermatocytic seminomas and most canine seminomas in order to justify their low metastatic behavior , showing a potential predictive model for the development of a treatment protocol ( maiolino et al . 1995 ; straw et al . this evidence emphasizes a parallel between human and canine mast cell tumors underlying the possibility of using the canine model to develop new beneficial therapeutics for both species ( pryer et al . non - hodgkin lymphoma , which accounts for 5 % of all malignant tumors and 83 % of all hematopoietic malignancies in dogs ( vail and thamm 2004 ) , affects mostly middle - aged , older dogs ( german shepherds , boxers , basset hounds and saint bernards ) ( macewen and young 1996 ) and both genders equally and corresponds to intermediate and high - grade non - hodgkin lymphoma in humans . it is generally associated with a poor prognosis , and in 7080 % of cases , it is b - cell mediated , whereas in 2030 % , it is t - cell mediated ( macewen 1995 ) . due to its sensitivity to chemotherapy , it is used as a model both for development of new chemotherapy drugs and multiple drug resistance studies ( vail and macewen 2000 ) . moreover , non - hodgkin lymphoma has been used to develop hypoxic cell markers , study the whole - body hyperthermia effects and evaluate autologous bone transplantation ( vail and thamm 2004 ) . injection - site sarcomas in felines , usually fibrosarcomas , are caused by an intense inflammatory reaction or vaccination adjuvants ( carwardine et al . they are highly locally invasive and metastasize in up to 23 % of the affected cases ( seguin 2002 ) . although aggressive surgical resection remains the gold standard therapy , chemotherapy and radiation can prolong the survival times ( hershey et al . 2000 ; kobayashi et al . uterine tumors , rare in cats , account for 0.29 % of feline neoplasms ( miller et al . 2003 ) , and they include endometrial adenocarcinoma , mixed mullerian tumor , leiomyoma which occurs most frequently ( maclachlan and kennedy 2002 ) and myxoid leiomyosarcoma ( cooper et al . 2006 ) . even if the etiology of nervous system tumors is not well understood , cats are commonly affected by meningiomas ( troxel et al . however , the biological behavior of such tumor , except for the anaplastic type , is generally considered benign in both humans ( louis et al . 2012 ) . a pilot study evaluated the incidence of spontaneous tumors in cats living in the neighborhood of venice and vicenza ( vascellari et al . four hundred and ninety - four out of 214,683 feline cases of neoplasia were diagnosed during the first three years . for all tumors , the estimated annual incidence rate in cats per 100,000 was 77 . furthermore , a 4.6-fold higher incidence of malignant tumors was observed in cats , if compared to benign pathology . in 2009 , wells introduced the potential role of pets in offering a therapeutic value to humans ( wells 2009b , 2012 ) . previous reports emphasized the role of dogs and cats as preventers of ill - health showing that pet owners are healthier than non - owners ( parslow et al . 2002 ; anderson et al . 1980 ; katcher 1981 ; katcher et al . furthermore , the ability of pets to facilitate human recovery has been explored ( larson et al . 2010 ) . for instance , friedmann et al . 1980 ) and dogs were stronger facilitators to recovery from ill - health than cats ( friedmann and thomas 1995 ) . therefore , dogs may contribute indirectly to long - standing human physical health , which is of great importance , especially in immunocompromised patients ( hemsworth and pizer 2006 ) , possibly due to the increased physical activity which typically characterizes the ownership of a dog ( larson et al . dogs have also been used as warning systems for cancer ( williams and pembroke 1989 ) , epilepsy ( brown and strong 2001 ; dalziel et al . 1999 , 2002 ) and diabetes ( chen et al . 2002 ) , visitors in pediatric hospital wards ( moody et al . comparative oncology research has extensively relied on domestic animals ( antinoff and hahn 2004 ; hansen and khanna 2004 ; hershey et al . the comparative oncology trials consortium program infrastructure , founded in 2009 to design and execute clinical trials involving dogs affected by cancer and in collaboration with the drug manufacturing industry and nongovernmental groups interested in cancer drug development , aimed to answer biological questions which could inform the development path of novel agents for future use in human cancer patients in a timely and integrated manner ( gordon et al . 2005 ; obrien and murphy 2003 ; ostrander et al . 2001 tumor initiation and progression are also influenced by age , nutrition , sex , reproductive status and environmental exposures in both human and canine cancers ( bukowski et al . 1997 ) . in this ways , it is possible to assess the effectiveness of new agents when given alone or in combination with other therapies prior to the clinical testing of the drug . the enrollment of pet dogs in preclinical and clinical trials is now focusing on new anti - neoplastic drug research and development . although regulation of animal research possesses guidelines such as the three rs ( replacement , reduction and refinement ) ( russell and burch 1959 ) , legal implications regarding the use of animal models for research purposes are still debated ( griffin 1998 ; porrello et al . although the existence of spontaneous pet tumors could be a reliable model for human cancers , many open questions , related to the opportunities to select and recruit new types of animal models in oncology and to their legal and ethical implications , remain unanswered . unfortunately , the use of spontaneous animal models implicates several professional and ethical warnings with conflictory debates . in the recent years , researchers have also focused their exploration on a possible role of some dog species as early warning systems for cancer ( williams and pembroke 1989 ) , epilepsy ( brown and strong 2001 ; dalziel et al . 2003 ; strong et al . 1992 ; mcaulay et al . 2004 ) . from this overview , there is evidence that companion animal health care is impressively growing in terms of development of new therapies and diagnostic tools , nutrition and disease prevention ( kling 2007 ) . furthermore , the pooling of anecdotal veterinary reports on drugs or multimodal animal cancer treatments , protocols and outcomes , including surgical pathology , oncoimmunology and molecular biology investigations , will give outstanding contribution to the interspecies translational information with undoubted mutual interspecies benefit . the pet psychological and physical support to the fitness and wellness of cancer patients should be more deeply investigated and largely extended in the palliation practice .

for instance , naturally occurring tumors in dogs share similarities with human cancer both in terms of histopathological features and biological behavior ( hahn et al . in the second half of the 1800s , a substantial parallel between the animal and human oncopathology , evidence showed that spontaneous animal tumors were similar to their human counterpart and information retrieved from them might have clinical implications ( dobberstein 1937 ) . in the early 1970s , the first world health organization ( who ) international histological classification of tumors of domestic animals was published ( beveridge and sobin 1974 , 1976 ) . it was followed by the tnm classification of tumors in domestic animals based on the guidelines of the tnm classification of malignant tumors in man ( owen 1980 ) showing that spontaneous canine and feline tumors are relevant models to study human cancers . conversely , in vivo and in vitro models , based on rodents and cell lines , have displayed intrinsic limits , such as complexity of management ( schiffer 1997 ) and the experimental methods used to induce the pathology ( vail and thamm 2004 ) , which make them different from the human spontaneous diseases ( de vico et al . 2002)modelkind of tumorfeaturescanine / felinenon - hodgkin s lymphomaequates to intermediate and high - grade non - hodgkin s lymphoma in humansimmune system alterations seem to be associated with a higher incidence of non - hodgkin s lymphomais very sensitive to chemotherapycurrently used as a model for testing new chemotherapeutics and new forms of immunotherapy , as well as for the study of multiple drug resistance ; it has been used as a relevant model for ( 1 ) developing hypoxic cell markers , ( 2 ) studying the effect of whole - body hyperthermia on the pharmacokinetics of systemic chemotherapy and ( 3 ) studying autologous bone transplantationcaninesoft tissue sarcomassimilar to pathological appearance , clinical presentation and behavior of human soft tissue sarcomasrespond to radiation therapy and chemotherapy in a manner similar to human pathologyhemangiosarcomas are extremely similar to human hemangiosarcomas with respect to histological pattern and metastatic behaviorcanineosteosarcomassimilar to human osteosarcomas with respect to the histology , metastatic behavior and clinical evolution of the diseasecanineoral malignant melanomasthey are chemoresistant and share many immunological targets with human oral malignant melanomasconsidered a relevant model for developing new immunotherapeutic approaches for both dogs and humansboth in dogs and humans , human tyrosinase dna vaccination is safe and potentially effectivecaninetransitional cell carcinomasshare histological appearance , biological behavior and response to therapy with invasive human transitional cell carcinomasconsidered a useful model for testing new photodynamic therapy technologies and chemotherapeutic combinationscaninemast cell tumorsmutation of exon 11 of c - kit occurs in 3050 % of advanced mast cell tumors and is similar to that usually occurring in 5090 % of human gastrointestinal stromal tumorscanine / felinemammary carcinomasvery similar to human breast cancers because of their hormonal dependence , spontaneous development in middle - aged to older animals , metastatic behavior toward regional lymph nodes and lungs , as well as adhesion molecules and neoangiogenesis patternsabnormalities in the nuclear dna content have been documented in both malignant and benign canine mammary tumors , but are more frequent in human mammary carcinomasmammary neoplasia in dogs may be a good molecular model for developing new antineoplastic strategies involving cyclin d1 and cyclin - dependent kinasesfeline mammary carcinoma shows age incidence , histopathology and pattern of metastasis similar to human breast cancerfeline mammary carcinoma shares common features with human inflammatory mammary carcinoma and human mammary carcinoma with osteoclast - like giant cellsepidermal growth factor receptor-2 ( her-2 ) and recepteur d'origine nantais ( ron ) overexpressions qualify feline mammary carcinoma as homologous to human breast carcinomas which have poor prognosiscanineseminomasmetastases occur only in a small percentage of cases , whereas human seminomas have a marked tendency to metastasizehave a histogenetic behavior similar to that of human seminomascaninetransmissible venereal tumoris considered a promising model to study human kaposi s sarcomapercutaneous inoculation and intraarterial transplantation of tumor fragments in the canine lung result in predictable patterns of tumor growth resembling the solitary pulmonary nodules and metastatic disease found in humans summary of tumor features shared between animals and humans ( allen et al . 2002 ) we searched pubmed / medline using the keywords spontaneous , animal , tumor , oncology and comparative alone or in combination and cross - referencing among published papers . spontaneous canine tumors , such as prostate carcinoma , chondrosarcoma , which accounts for approximately 10 % of all primary bone tumors in dogs ( beveridge and sobin 1976 ) , axial osteosarcoma , which accounts for 80 % of all primary bone tumors in dogs ( thompson and pool 2002 ) and synovial cell sarcoma , have been extensively described representing valuable models to study carcinogenesis ( vail et al . in 20052009 , a pilot study evaluated the incidence of spontaneous tumors in dogs living in the neighborhood of venice and vicenza ( vascellari et al . moreover , in pure breeds , a twofold higher incidence of malignant tumors , with respect to mixed breeds , was observed . due to the dissimilar methodologies and variable reference populations of both european and north american veterinary cancer registries , the occurrence of spontaneous tumors in pet animals has been underestimated ( bonnett et al . the animal tumor registry of genoa ( italy ) retrospectively showed that , from 1985 to 2002 , 70 % of all cancer cases in female dogs were located in the breast ( merlo et al . the overall incidence of cancer was 99.3 per 100,000 dogs per year in male dogs and 272.1 in female dogs . therefore , canine malignant melanomas are suitable models for new immunotherapeutic protocols in humans ( vail and thamm 2004 ) . skin and subcutaneous tissue neoplasms are the most frequently recognized neoplastic disorders in domestic animals and can be caused by prolonged and continuous exposure to sunlight ( madewell 1981 ) . alimentary tract cancer in dogs has lower incidence , with ductal and acinar carcinomas occurring most frequently in females than in males while intranasal tumors account for 12 % of all canine neoplasms ( priester 1974 ) . primary bone cancer is the second most often detected in dogs , and its main risk factors are ionizing radiations , chemical carcinogens and viruses ( madewell 1981 ) . moreover , preexisting bone defects and skeletal abnormalities increase the risk factors from 60 up to 185 times for large and giant breeds with respect to small dogs ( tjalma 1966 ) . as to primary brain tumors , they account for approximately 2 % of all cancer in human adults ( mckinney 2004 ) and for 0.01 % in dogs ( lecouteur 1999 ) . high incidence rates were also detected for non - hodgkin s lymphoma in bitches and for non - hodgkin s lymphoma and skin cancer in male dogs ( merlo et al . additionally , the incidence rate of cancer increased with age ranging between 23.7 and 763.2 in bitches and 16.5 and 237.6 in male dogs aged 3 and > 911 years . hemangiosarcoma , which can affect both cats and dogs , involves the musculoskeletal system , and mean survival time in dogs affected by this condition ranges from 267 ( ogilvie et al . the ovarian and epithelial tumors , although quite rare in domestic animals , have been reported mainly in cats , dogs and horses ( maclachlan 1987 ) . spontaneous testicular tumors , which are quite common in aged dogs , are mainly seminomas ( solitary , unilateral , more frequent in the right testicle and with a reduced possibility to develop metastases , if compared with humans ) ( kennedy et al . suggested a possible correspondence between human spermatocytic seminomas and most canine seminomas in order to justify their low metastatic behavior , showing a potential predictive model for the development of a treatment protocol ( maiolino et al . 1992 ) and multiple myeloma ( rare in cats and uncommon in dogs ) , which accounts for 8 % of all canine hematopoietic tumors and affects older dogs with no breed or sex predilection ( matus et al . canine mast cell tumors , which account for 1621 % in this species ( thamm and vail 2001 ) , display a molecular alteration in the proto - oncogene c - kit , which is involved in mast cell differentiation , proliferation , survival and activation ( london et al . specifically , the mutation of exon 11 of c - kit occurs in 3050 % of advanced mast cell tumors and is similar to that usually occurring in 5090 % of human gastrointestinal stromal tumors ( heinrich et al . this evidence emphasizes a parallel between human and canine mast cell tumors underlying the possibility of using the canine model to develop new beneficial therapeutics for both species ( pryer et al . non - hodgkin lymphoma , which accounts for 5 % of all malignant tumors and 83 % of all hematopoietic malignancies in dogs ( vail and thamm 2004 ) , affects mostly middle - aged , older dogs ( german shepherds , boxers , basset hounds and saint bernards ) ( macewen and young 1996 ) and both genders equally and corresponds to intermediate and high - grade non - hodgkin lymphoma in humans . it is generally associated with a poor prognosis , and in 7080 % of cases , it is b - cell mediated , whereas in 2030 % , it is t - cell mediated ( macewen 1995 ) . due to its sensitivity to chemotherapy , it is used as a model both for development of new chemotherapy drugs and multiple drug resistance studies ( vail and macewen 2000 ) . moreover , non - hodgkin lymphoma has been used to develop hypoxic cell markers , study the whole - body hyperthermia effects and evaluate autologous bone transplantation ( vail and thamm 2004 ) . however , the biological behavior of such tumor , except for the anaplastic type , is generally considered benign in both humans ( louis et al . a pilot study evaluated the incidence of spontaneous tumors in cats living in the neighborhood of venice and vicenza ( vascellari et al . furthermore , a 4.6-fold higher incidence of malignant tumors was observed in cats , if compared to benign pathology . moreover , in pure breed cats , a twofold higher incidence of malignant tumors was observed and it increased with age in both malignant and benign pathologies , if compared with mixed breeds . previous reports emphasized the role of dogs and cats as preventers of ill - health showing that pet owners are healthier than non - owners ( parslow et al . therefore , dogs may contribute indirectly to long - standing human physical health , which is of great importance , especially in immunocompromised patients ( hemsworth and pizer 2006 ) , possibly due to the increased physical activity which typically characterizes the ownership of a dog ( larson et al . spontaneous canine tumors , such as prostate carcinoma , chondrosarcoma , which accounts for approximately 10 % of all primary bone tumors in dogs ( beveridge and sobin 1976 ) , axial osteosarcoma , which accounts for 80 % of all primary bone tumors in dogs ( thompson and pool 2002 ) and synovial cell sarcoma , have been extensively described representing valuable models to study carcinogenesis ( vail et al . in 20052009 , a pilot study evaluated the incidence of spontaneous tumors in dogs living in the neighborhood of venice and vicenza ( vascellari et al . moreover , in pure breeds , a twofold higher incidence of malignant tumors , with respect to mixed breeds , was observed . the animal tumor registry of genoa ( italy ) retrospectively showed that , from 1985 to 2002 , 70 % of all cancer cases in female dogs were located in the breast ( merlo et al . alimentary tract cancer in dogs has lower incidence , with ductal and acinar carcinomas occurring most frequently in females than in males while intranasal tumors account for 12 % of all canine neoplasms ( priester 1974 ) . primary bone cancer is the second most often detected in dogs , and its main risk factors are ionizing radiations , chemical carcinogens and viruses ( madewell 1981 ) . moreover , preexisting bone defects and skeletal abnormalities increase the risk factors from 60 up to 185 times for large and giant breeds with respect to small dogs ( tjalma 1966 ) . as to primary brain tumors , they account for approximately 2 % of all cancer in human adults ( mckinney 2004 ) and for 0.01 % in dogs ( lecouteur 1999 ) . high incidence rates were also detected for non - hodgkin s lymphoma in bitches and for non - hodgkin s lymphoma and skin cancer in male dogs ( merlo et al . additionally , the incidence rate of cancer increased with age ranging between 23.7 and 763.2 in bitches and 16.5 and 237.6 in male dogs aged 3 and > 911 years . hemangiosarcoma , which can affect both cats and dogs , involves the musculoskeletal system , and mean survival time in dogs affected by this condition ranges from 267 ( ogilvie et al . spontaneous testicular tumors , which are quite common in aged dogs , are mainly seminomas ( solitary , unilateral , more frequent in the right testicle and with a reduced possibility to develop metastases , if compared with humans ) ( kennedy et al . suggested a possible correspondence between human spermatocytic seminomas and most canine seminomas in order to justify their low metastatic behavior , showing a potential predictive model for the development of a treatment protocol ( maiolino et al . 1992 ) and multiple myeloma ( rare in cats and uncommon in dogs ) , which accounts for 8 % of all canine hematopoietic tumors and affects older dogs with no breed or sex predilection ( matus et al . canine mast cell tumors , which account for 1621 % in this species ( thamm and vail 2001 ) , display a molecular alteration in the proto - oncogene c - kit , which is involved in mast cell differentiation , proliferation , survival and activation ( london et al . specifically , the mutation of exon 11 of c - kit occurs in 3050 % of advanced mast cell tumors and is similar to that usually occurring in 5090 % of human gastrointestinal stromal tumors ( heinrich et al . this evidence emphasizes a parallel between human and canine mast cell tumors underlying the possibility of using the canine model to develop new beneficial therapeutics for both species ( pryer et al . non - hodgkin lymphoma , which accounts for 5 % of all malignant tumors and 83 % of all hematopoietic malignancies in dogs ( vail and thamm 2004 ) , affects mostly middle - aged , older dogs ( german shepherds , boxers , basset hounds and saint bernards ) ( macewen and young 1996 ) and both genders equally and corresponds to intermediate and high - grade non - hodgkin lymphoma in humans . it is generally associated with a poor prognosis , and in 7080 % of cases , it is b - cell mediated , whereas in 2030 % , it is t - cell mediated ( macewen 1995 ) . moreover , non - hodgkin lymphoma has been used to develop hypoxic cell markers , study the whole - body hyperthermia effects and evaluate autologous bone transplantation ( vail and thamm 2004 ) . a pilot study evaluated the incidence of spontaneous tumors in cats living in the neighborhood of venice and vicenza ( vascellari et al . moreover , in pure breed cats , a twofold higher incidence of malignant tumors was observed and it increased with age in both malignant and benign pathologies , if compared with mixed breeds . previous reports emphasized the role of dogs and cats as preventers of ill - health showing that pet owners are healthier than non - owners ( parslow et al . therefore , dogs may contribute indirectly to long - standing human physical health , which is of great importance , especially in immunocompromised patients ( hemsworth and pizer 2006 ) , possibly due to the increased physical activity which typically characterizes the ownership of a dog ( larson et al . the comparative oncology trials consortium program infrastructure , founded in 2009 to design and execute clinical trials involving dogs affected by cancer and in collaboration with the drug manufacturing industry and nongovernmental groups interested in cancer drug development , aimed to answer biological questions which could inform the development path of novel agents for future use in human cancer patients in a timely and integrated manner ( gordon et al . 2006 ) such as several cancer - associated gene families ( hoffman and birney 2007 ) , as well as the presence of the similar genetic cancer - associated molecular alterations ( haga et al . the lack of gold standards for the management of cancer in dogs and cats , as well as the high motivation of pet owners to seek out new options for its management , underlines the increasing need to evaluate novel therapeutics in these populations . in this ways , it is possible to assess the effectiveness of new agents when given alone or in combination with other therapies prior to the clinical testing of the drug . although regulation of animal research possesses guidelines such as the three rs ( replacement , reduction and refinement ) ( russell and burch 1959 ) , legal implications regarding the use of animal models for research purposes are still debated ( griffin 1998 ; porrello et al . although the existence of spontaneous pet tumors could be a reliable model for human cancers , many open questions , related to the opportunities to select and recruit new types of animal models in oncology and to their legal and ethical implications , remain unanswered . in the recent years , researchers have also focused their exploration on a possible role of some dog species as early warning systems for cancer ( williams and pembroke 1989 ) , epilepsy ( brown and strong 2001 ; dalziel et al . from this overview , there is evidence that companion animal health care is impressively growing in terms of development of new therapies and diagnostic tools , nutrition and disease prevention ( kling 2007 ) . conclusively , we strongly encourage the animalists , veterinarians and clinical researchers to join us and support ethical observational studies on domestic and laboratory animal spontaneous cancer epidemiology , early prevention , diagnosis and treatment in order to detect and possibly eliminate alimentary and environmental factors which can also be cancerogenic for humans . furthermore , the pooling of anecdotal veterinary reports on drugs or multimodal animal cancer treatments , protocols and outcomes , including surgical pathology , oncoimmunology and molecular biology investigations , will give outstanding contribution to the interspecies translational information with undoubted mutual interspecies benefit . the pet psychological and physical support to the fitness and wellness of cancer patients should be more deeply investigated and largely extended in the palliation practice .

health - related quality of life ( hrqol ) is a subjective assessment of the patient1 that is related to the perception of their health,14 well - being , and functioning,1 and also to their worries about the future.4 although the term hrqol is not yet precisely defined,5 it is a useful measure to assess the overall impact of a particular nosological entity in patient,2 and it complements the clinical information.3,6 there are several generic tools that can be used to assess hrqol ; they can be used not only in the healthy population but also in specific populations4 because they allow to assess a wide set of important dimensions that can be used in different diseases.4,68 wilson and cleary9 propose a conceptual model to explain the relationships between clinical variables and measures of hrqol in order to establish the connection between diagnosis and therapy and , thus , to increase patients hrqol . this classification is divided into five levels biological and physiological factors , symptoms , functioning , general health perceptions , and overall quality of life and proposes causal relationships between all of them . with regard to the relationship between hrqol , the presence of hypertension ( htn ) , and diabetes - related chronic complications , the literature is not consistent in pointing out that their presence decreases hrqol and , hence , the importance of evaluating the relationship between these variables and hrqol . moreover , as far as we know , the only two researches carried out in portugal5,10 that have assessed the relationship between the most frequent chronic complications of diabetes and hrqol did not make the adjustment of the hrqol scores for any demographic and/or clinical variables that can mediate some of these relationships . on the other hand , they evaluated simultaneously type 1 and type 2 diabetic patients and did not assess chronic complications according to diabetes type . lastly , the investigations that evaluated the relationship between the number of diabetes - related chronic complications and hrqol did not make stratified analyses according to sex that can bring important supplemental information to the one achieved for the whole sample . concerning microvascular complications of diabetes , while some studies1012 have found that the presence of diabetic retinopathy was related to worse hrqol , sakamaki et al13 found no relationship between the two variables . results of research on the relationship between diabetic nephropathy and hrqol have found , on one hand , that its presence was related to lower hrqol10,11 and , on the other hand , no relationship between both variables.13 concerning the relationship between diabetic neuropathy and hrqol , while sakamaki et al13 found no relationship between the two variables , some studies10,1417 have found that its presence was related to worse hrqol . as far as diabetes - related macrovascular complications are concerned , although there is evidence that the presence of cardiovascular disease ( cvd ) in persons with diabetes was related to lower hrqol,5,10,12,1416,1822 wasem et al17 found no relationship between the two variables . regarding the relationship between the presence of peripheral arterial disease ( pad ) and hrqol , silva et al,5 neves et al,10 javanbakht et al,11 and quah et al16 have shown that patients with this chronic complication had worse hrqol , whereas sakamaki et al13 and wasem et al17 found no relationship between the two variables , and venkataraman et al22 reported that its presence was related to better hrqol . regarding the relationship between htn and diabetes , while some studies5,10,23,24 have found that the presence of htn was related to lower hrqol , wexler et al20 and hill - briggs et al25 reported no relationship between the two variables . finally , some researchers , looking at the relationship between the number of diabetes - related chronic complications and hrqol , have found that an increased number of complications was related to worse hrqol.16,26 however , caldwell et al27 did not find a relationship between the two variables . the aim of this cross - sectional study was to assess the relationship between hrqol , the presence or absence of htn , and diabetes - related micro- and macrovascular complications in type 2 diabetes , as well as the association between hrqol and the number of chronic complications , for the whole sample and by sex . we focused on the assessment of htn and long - term complications on hrqol in type 2 diabetic patients for three reasons : first , because htn is a highly prevalent comorbidity in these individuals ; second , because it is highly associated with diabetes - related chronic complications ; and third , because it can have a negative association with hrqol . a consecutive sample of 100 type 2 diabetic patients from the outpatient department of endocrinology , diabetes and metabolism , centro hospitalar so joo , porto , portugal , was recruited . patients were included in the study if they had a diagnosis of type 2 diabetes , were at least 18 years old , and were not currently pregnant . the protocol for the study was approved by the ethics committee for health of centro hospitalar so joo , and informed consent was obtained from all participants before participating in the study . general hrqol was assessed by the portuguese version of the medical outcomes study 36-item short - form health survey ( sf-36),28,29 and it was administered by one of the researchers . we evaluated the presence of some clinical variables that are reported in the literature to be related to hrqol among type 2 diabetes;5,1012,1424,26 these factors included microvascular complications , macrovascular complications , and htn . type 2 diabetic patients were classified as on oral hypoglycemic agents or insulin therapy ( the latter including patients only on insulin therapy or on oral hypoglycemic agents plus insulin therapy ) . microvascular complications considered were diabetic retinopathy , diabetic nephropathy , and diabetic peripheral neuropathy ; macrovascular complications were cvd and pad . we considered that the patients had retinopathy if they had at least microaneurysms , nephropathy if they had at least albumin excretion rate higher than 30 mg / g creatinine , peripheral neuropathy if they had two or more insensate sites across two feet for the 10 g monofilament or one or more insensate sites for the 128 hz tuning fork . cvd was defined by the presence of at least one of the following conditions : angina pectoris or acute myocardial infarction or transient ischemic attack or stroke ; and pad was defined as an ankle - brachial index of 0.9 or a prior history of lower extremity revascularization . htn was defined by one of the following : systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or current use of antihypertensive agents or documented diagnosis of htn . the sf-36 healthy survey comprises 36 items covering functional health status and general health , currently and over the past 4 weeks , and measures eight dimensions : physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional , and mental health . raw dimension scores were transformed to scales ranging from 0 to 100 , in which higher scores indicate higher hrqol . statistical package for the social sciences version 20.0 for windows software ( ibm corporation , armonk , ny , usa ) was used for the analysis . descriptive statistical analysis included calculation of means and standard deviations for cardinal variables , and frequencies for ordinal and nominal variables . the degree of association between pairs of variables was measured by pearson s ( r ) or spearman s ( rs ) correlation coefficients . as there were age - discrepancies within the sample ( minimum age of 44 years old , and maximum age of 80 years old ) , we adjusted sf-36 results to the age of the participants ; therefore , all other variables were related to the residuals of simple linear regression models having each dimension as a dependent variable and age as the independent variable . a consecutive sample of 100 type 2 diabetic patients from the outpatient department of endocrinology , diabetes and metabolism , centro hospitalar so joo , porto , portugal , was recruited . patients were included in the study if they had a diagnosis of type 2 diabetes , were at least 18 years old , and were not currently pregnant . the protocol for the study was approved by the ethics committee for health of centro hospitalar so joo , and informed consent was obtained from all participants before participating in the study . general hrqol was assessed by the portuguese version of the medical outcomes study 36-item short - form health survey ( sf-36),28,29 and it was administered by one of the researchers . we evaluated the presence of some clinical variables that are reported in the literature to be related to hrqol among type 2 diabetes;5,1012,1424,26 these factors included microvascular complications , macrovascular complications , and htn . type 2 diabetic patients were classified as on oral hypoglycemic agents or insulin therapy ( the latter including patients only on insulin therapy or on oral hypoglycemic agents plus insulin therapy ) . microvascular complications considered were diabetic retinopathy , diabetic nephropathy , and diabetic peripheral neuropathy ; macrovascular complications were cvd and pad . we considered that the patients had retinopathy if they had at least microaneurysms , nephropathy if they had at least albumin excretion rate higher than 30 mg / g creatinine , peripheral neuropathy if they had two or more insensate sites across two feet for the 10 g monofilament or one or more insensate sites for the 128 hz tuning fork . cvd was defined by the presence of at least one of the following conditions : angina pectoris or acute myocardial infarction or transient ischemic attack or stroke ; and pad was defined as an ankle - brachial index of 0.9 or a prior history of lower extremity revascularization . htn was defined by one of the following : systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or current use of antihypertensive agents or documented diagnosis of htn . the sf-36 healthy survey comprises 36 items covering functional health status and general health , currently and over the past 4 weeks , and measures eight dimensions : physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional , and mental health . raw dimension scores were transformed to scales ranging from 0 to 100 , in which higher scores indicate higher hrqol . statistical package for the social sciences version 20.0 for windows software ( ibm corporation , armonk , ny , usa ) was used for the analysis . descriptive statistical analysis included calculation of means and standard deviations for cardinal variables , and frequencies for ordinal and nominal variables . the degree of association between pairs of variables was measured by pearson s ( r ) or spearman s ( rs ) correlation coefficients . as there were age - discrepancies within the sample ( minimum age of 44 years old , and maximum age of 80 years old ) , we adjusted sf-36 results to the age of the participants ; therefore , all other variables were related to the residuals of simple linear regression models having each dimension as a dependent variable and age as the independent variable . most patients were male , were receiving only oral hypoglycemic agents , and had the disease for < 10 years . only 21.0% of the patients did not have chronic complications related to diabetes . among the chronic complications considered , the most prevalent was diabetic retinopathy , followed by cvd . the average values obtained by patients in the eight dimensions of the sf-36 , as well as the association between these scores and age are shown in table 2 . there was no association between age and any of the eight dimensions of the sf-36 . the age - adjusted scores of hrqol by the presence or absence of each of the microvascular complications of diabetes are shown in table 3 , macrovascular complications of diabetes in table 4 , and htn in table 5 . there was an overall tendency for the presence of each of the chronic complications and htn to be related to worse hrqol in all dimensions of sf-36 , regardless of age ; the only exception was the dimension role - physical in diabetic nephropathy . the complications related to larger number of dimensions were peripheral neuropathy and cvd ( all dimensions except bodily pain ) . in type 2 diabetic patients with retinopathy , pad , and nephropathy , the hrqol only differed on four , three , and two dimensions , respectively retinopathy ( physical functioning , general health , vitality , and mental health ) , pad ( physical functioning , role - physical , and general health ) , and nephropathy ( general health and vitality ) . the most related dimensions to worse hrqol were general health and vitality ; the general health dimension was lower in patients with any of the long - term complications and htn , whereas the vitality dimension was lower in htn and all diabetes - related complications with the exception of pad . on the other hand , the bodily pain was the dimension least related to hrqol because it was not related to any of these variables . the association between hrqol and the number of chronic complications for the whole sample and by sex are shown in table 6 . an increased number of long - term complications tended to be associated with worse hrqol in all dimensions of sf-36 , regardless of age ; the only exceptions were the bodily pain dimension in males and the role - emotional dimension in females . this association reached statistical significance in all dimensions with the exception of bodily pain in the total sample , all except bodily pain and social functioning in men , and only in general health , vitality , and mental health dimensions in women . in men , the most associated dimension of hrqol with the number of chronic complications was physical functioning , followed by general health , and then by role - physical . in women , the most associated dimensions with the number of complications were general health , followed by vitality . the dimensions physical functioning , role - physical , and role - emotional were only associated with the number of complications in males . the presence of diabetes - related chronic complications and htn , as well as the number of complications were related to worse hrqol regardless of age , with the different conditions being related to different dimensions of hrqol . the adjustment of sf-36 scores for age may obscure relationships between diabetes - related complications , htn , and hrqol dimensions that strongly depend on a concomitant relationship with age . thereby , absolute scores on the sf-36 and their relationship with long - term complications and htn may have clinical usefulness , but to analyze their association with hrqol , which was our intention , it is recommended that values are adjusted for age if there are significant age - discrepancies within the sample . regarding the relationship between the presence of retinopathy and hrqol , we found that having retinopathy was related to lower hrqol in terms of physical functioning , general health , vitality , and mental health . our results partially overlap with the findings of neves et al10 for three reasons : first , these portuguese researchers have found that the diabetic patients with retinopathy had worse hrqol than those without retinopathy in all sf-36 dimensions ; second , because in that study the authors included in their analysis a quite heterogeneous sample ( ie , type 1 and type 2 diabetic patients ) , whereas we included a less heterogeneous one ; and third , because they did not make the adjustment of the hrqol scores for age . conversely , a cross - sectional study13 and a longitudinal study30 have found no relationship between the two variables . contrary to what we expected , which was a reduced hrqol in both physical and mental component scores of the sf-36 , we found that diabetic retinopathy appears to have a greater relationship with the physical component than the mental one . our results showed that the presence of nephropathy was related to worse hrqol in terms of general health and vitality . these results are partially in agreement with the findings of neves et al,10 because these authors reported that its presence was related to lower hrqol in all dimensions of sf-36 . however , as mentioned earlier , the study of neves et al10 has methodological limitations that might have compromised the results . in contrast , two studies13,30 reported no relationship between the presence of nephropathy and hrqol . surprisingly , we did not find differences between the two groups in the other dimensions of the sf-36 , particularly in terms of mental component . we found that the presence of peripheral neuropathy was related to worse hrqol in all dimensions of sf-36 with the exception of the bodily pain dimension . these results are different from those obtained in another study,13 which did not find a relationship between peripheral neuropathy and hrqol . our results are consistent with the findings of neves et al,10 given that in that study it was found that the presence of neuropathy was related to lower hrqol in all dimensions of sf-36 , and partially overlapping with the results of other investigations.15,16 in the first study it was reported that having neuropathy was related to worse hrqol in terms of physical functioning and bodily pain dimensions of sf-36 , while in the latter one it was found that the presence of peripheral neuropathy was related to lower hrqol in terms of physical and mental components of the aforementioned tool . concerning the relationship between the presence of cvd and hrqol , wasem et al17 reported no relationship between the two variables , whereas we found worse hrqol in patients with cvd in all dimensions of sf-36 with the exception of bodily pain dimension when comparing with those that did not have the disease . these findings are partially in agreement with the results of four cross - sectional studies5,10,15,22 because in those studies , the presence of cvd was related to lower hrqol in terms of physical functioning , general health,5,10,15,22 role - physical,5,10,22 bodily pain , vitality,5,10,15 social functioning,15 and physical component.15,22 on the other hand , our findings are also partially in accordance with the results of two longitudinal studies,30,31 which found that the presence of cvd was related to worse physical functioning , general health , vitality , mental component,30 and physical component.30,31 our results and those obtained in those studies suggest that cvd causes physical and emotional constraints in patients with diabetes . with regard to physical impairments , it appears that hrqol and life expectancy of these patients are more reduced , given that cvd is the major cause of death in both diabetic males and females.32 thereby , it is not surprising that these patients become physically more limited , and , consequently , end up becoming psychologically more vulnerable . regarding pad and its relationship with hrqol , sakamaki et al13 and wasem et al17 found no relation between the two variables , and surprisingly , venkataraman et al22 reported that patients with pad had better hrqol in terms of physical functioning , role - physical , and physical component than those without pad . conversely , we found that pad was related to worse hrqol in terms of physical functioning , role - physical , and general health . our results are partially in agreement with the findings of silva et al5 and neves et al,10 because it was found that its presence was related to worse hrqol in terms of physical functioning , bodily pain , vitality,5,10 role - physical , general health , social functioning , role - emotional , and mental health.10 however , it is worth noting that these studies5,10 have methodological limitations that might have jeopardized the results , namely , the inclusion of type 1 and type 2 diabetes , and the non - adjustment of the hrqol scores for age . as with diabetic retinopathy and nephropathy , although wexler et al20 and hill - briggs et al25 have found no relationship between the presence of htn and hrqol , in this study htn was related to worse hrqol in terms of general health and vitality . these findings are partially in agreement with the results of prior studies5,10,23,24 the presence of htn was related to lower hrqol in terms of physical functioning , role - physical , bodily pain,5,10 general health,5,10,23 vitality , social functioning , mental health,10,24 role - emotional,10 and mental component.24 these differences between the two groups may possibly be explained by its frequent association with cardiac , cerebrovascular , renal , and ocular complications . isolated htn ( except hypertensive crisis ) is usually asymptomatic , and it is through predisposition to associated diseases that eventually may help to explain the worse hrqol of the hypertensive patients . on the other hand , various items of the sf-36 are somewhat similar to some symptoms of hypotension and hypoglycemia and , thus , may have contributed to this worse hrqol in the hypertensive patients . with regard to the relationship between the number of diabetes - related chronic complications and hrqol , while caldwell et al27 reported no relationship between the two variables , we found that an increased number of complications was associated with worse hrqol in all dimensions of sf-36 with the exception of bodily pain dimension . these findings are partially in agreement with the results of two cross - sectional studies.16,26 in the first study , it was found that a higher number of chronic complications was related to worse hrqol in terms of physical and mental component , whereas in the latter investigation , an increased number of long - term complications was related to worse hrqol in terms of physical functioning , role - physical , role - emotional , social functioning , and mental health . on the other hand , our results are partially consistent with the findings of a longitudinal study33 that have found that an increased number of complications predicted lower hrqol in terms of physical and mental components . in the present study , the existence of higher number of diabetes - related complications suggests a set of physical , emotional , and social constraints , which result from the emergence of new symptoms , the intensification of the treatment regimen , and a feeling of frustration , anger , and failure after the worsening of diabetes . different hrqol dimensions were more strongly associated with the number of chronic complications in male and female patients . some differences in the results between this study and others may be explained by the different generic tools used to assess hrqol that do not measure exactly the same dimensions . this would also allow multivariate analysis , thus decreasing the confounder effect of the remaining variables . multiple testing also must be considered , as it increases the occurrence of statistically significant results ; however , the comparison of our results with prior studies enhances their adequate interpretation despite this limitation . furthermore , most of the patients interviewed were of low sociocultural status , which may have limited their understanding of the questions in the instrument used to assess hrqol . generalization of our findings may be limited because our sample was collected in a central public hospital and might not be representative of the population with type 2 diabetes in other care centers or in primary care . further studies are warranted to adjust the dimensions of hrqol to other variables ( eg , sex , body mass index , type of diabetes and treatment regimens , and duration of the disease ) to explore the relationships between hrqol and the presence of diabetes - related chronic complications and htn , which we think may explain some of these relationships . further investigations on stratifying the severity of diabetes - related chronic complications are required because they may bring important complementary information . more studies are needed to explore sex differences with regard to the association between the number of long - term complications and hrqol and considering different groups of complications . lastly , the use of qualitative methodology could be useful to obtain information complementary to that achieved by quantitative studies , allowing more accurate interpretation of some results . first , we included a more homogeneous sample ( ie , only type 2 diabetic patients ) in comparison to the two previous researches carried out in portugal5,10 which have included both type 1 and type 2 diabetic patients when assessing the relationship between diabetes - related chronic complications , htn and hrqol without making stratified analyses according to type of diabetes . second , the age - adjustment of the sf-36 values to evaluate the relationship between chronic complications , htn and hrqol , given the age - discrepancies within the sample , which was not made in the two mentioned portuguese investigations . third , we evaluated the association between hrqol and number of chronic complications by sex , which was not made previously,16,26,27 and we think may provide important supplemental information . in summary , our findings suggest that the presence and increased number of diabetes - related chronic complications , as well as the presence of htn were related to worse age - adjusted hrqol . it is worth noting that among diabetes - related long - term complications , peripheral neuropathy and cvd were more strongly related to patients age - adjusted hrqol . in males , there was a strong association between the number of chronic complications and hrqol than in their counterparts .

backgroundthe aim of this study was to assess the relationship between health - related quality of life ( hrqol ) and the presence or absence of hypertension and diabetes - related chronic complications in type 2 diabetes , and also the association between hrqol and the number of chronic complications.methodsone hundred patients with type 2 diabetes were interviewed . hrqol was evaluated using the age - adjusted short - form 36 dimensions ( physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional , and mental health).resultsthe mean age of the study population was 62.78.7 years ; 54.0% were male , and 51.0% were receiving only oral hypoglycemic agents . chronic complications were related to worse hrqol in different dimensions : peripheral neuropathy and cardiovascular disease ( all , except bodily pain ) , retinopathy ( physical functioning , general health , vitality , and mental health ) , peripheral arterial disease ( physical functioning , role - physical , and general health ) , and nephropathy ( general health and vitality ) . hypertension was related to worse general health and vitality . an increased number of chronic complications was associated with worse hrqol in all dimensions of short - form 36 except for the bodily pain dimension.conclusionthe presence and increased number of diabetes - related chronic complications , and the presence of hypertension were related to worse age - adjusted hrqol . peripheral neuropathy and cardiovascular disease were more strongly related to age - adjusted hrqol .

Introduction Subjects and methods Study design and participants Clinical outcomes HRQoL measurement tool Data analysis Results Discussion Conclusion

health - related quality of life ( hrqol ) is a subjective assessment of the patient1 that is related to the perception of their health,14 well - being , and functioning,1 and also to their worries about the future.4 although the term hrqol is not yet precisely defined,5 it is a useful measure to assess the overall impact of a particular nosological entity in patient,2 and it complements the clinical information.3,6 there are several generic tools that can be used to assess hrqol ; they can be used not only in the healthy population but also in specific populations4 because they allow to assess a wide set of important dimensions that can be used in different diseases.4,68 wilson and cleary9 propose a conceptual model to explain the relationships between clinical variables and measures of hrqol in order to establish the connection between diagnosis and therapy and , thus , to increase patients hrqol . this classification is divided into five levels biological and physiological factors , symptoms , functioning , general health perceptions , and overall quality of life and proposes causal relationships between all of them . with regard to the relationship between hrqol , the presence of hypertension ( htn ) , and diabetes - related chronic complications , the literature is not consistent in pointing out that their presence decreases hrqol and , hence , the importance of evaluating the relationship between these variables and hrqol . moreover , as far as we know , the only two researches carried out in portugal5,10 that have assessed the relationship between the most frequent chronic complications of diabetes and hrqol did not make the adjustment of the hrqol scores for any demographic and/or clinical variables that can mediate some of these relationships . lastly , the investigations that evaluated the relationship between the number of diabetes - related chronic complications and hrqol did not make stratified analyses according to sex that can bring important supplemental information to the one achieved for the whole sample . concerning microvascular complications of diabetes , while some studies1012 have found that the presence of diabetic retinopathy was related to worse hrqol , sakamaki et al13 found no relationship between the two variables . results of research on the relationship between diabetic nephropathy and hrqol have found , on one hand , that its presence was related to lower hrqol10,11 and , on the other hand , no relationship between both variables.13 concerning the relationship between diabetic neuropathy and hrqol , while sakamaki et al13 found no relationship between the two variables , some studies10,1417 have found that its presence was related to worse hrqol . as far as diabetes - related macrovascular complications are concerned , although there is evidence that the presence of cardiovascular disease ( cvd ) in persons with diabetes was related to lower hrqol,5,10,12,1416,1822 wasem et al17 found no relationship between the two variables . regarding the relationship between the presence of peripheral arterial disease ( pad ) and hrqol , silva et al,5 neves et al,10 javanbakht et al,11 and quah et al16 have shown that patients with this chronic complication had worse hrqol , whereas sakamaki et al13 and wasem et al17 found no relationship between the two variables , and venkataraman et al22 reported that its presence was related to better hrqol . regarding the relationship between htn and diabetes , while some studies5,10,23,24 have found that the presence of htn was related to lower hrqol , wexler et al20 and hill - briggs et al25 reported no relationship between the two variables . finally , some researchers , looking at the relationship between the number of diabetes - related chronic complications and hrqol , have found that an increased number of complications was related to worse hrqol.16,26 however , caldwell et al27 did not find a relationship between the two variables . the aim of this cross - sectional study was to assess the relationship between hrqol , the presence or absence of htn , and diabetes - related micro- and macrovascular complications in type 2 diabetes , as well as the association between hrqol and the number of chronic complications , for the whole sample and by sex . we focused on the assessment of htn and long - term complications on hrqol in type 2 diabetic patients for three reasons : first , because htn is a highly prevalent comorbidity in these individuals ; second , because it is highly associated with diabetes - related chronic complications ; and third , because it can have a negative association with hrqol . patients were included in the study if they had a diagnosis of type 2 diabetes , were at least 18 years old , and were not currently pregnant . the protocol for the study was approved by the ethics committee for health of centro hospitalar so joo , and informed consent was obtained from all participants before participating in the study . general hrqol was assessed by the portuguese version of the medical outcomes study 36-item short - form health survey ( sf-36),28,29 and it was administered by one of the researchers . we evaluated the presence of some clinical variables that are reported in the literature to be related to hrqol among type 2 diabetes;5,1012,1424,26 these factors included microvascular complications , macrovascular complications , and htn . the sf-36 healthy survey comprises 36 items covering functional health status and general health , currently and over the past 4 weeks , and measures eight dimensions : physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional , and mental health . as there were age - discrepancies within the sample ( minimum age of 44 years old , and maximum age of 80 years old ) , we adjusted sf-36 results to the age of the participants ; therefore , all other variables were related to the residuals of simple linear regression models having each dimension as a dependent variable and age as the independent variable . patients were included in the study if they had a diagnosis of type 2 diabetes , were at least 18 years old , and were not currently pregnant . the protocol for the study was approved by the ethics committee for health of centro hospitalar so joo , and informed consent was obtained from all participants before participating in the study . general hrqol was assessed by the portuguese version of the medical outcomes study 36-item short - form health survey ( sf-36),28,29 and it was administered by one of the researchers . we evaluated the presence of some clinical variables that are reported in the literature to be related to hrqol among type 2 diabetes;5,1012,1424,26 these factors included microvascular complications , macrovascular complications , and htn . we considered that the patients had retinopathy if they had at least microaneurysms , nephropathy if they had at least albumin excretion rate higher than 30 mg / g creatinine , peripheral neuropathy if they had two or more insensate sites across two feet for the 10 g monofilament or one or more insensate sites for the 128 hz tuning fork . cvd was defined by the presence of at least one of the following conditions : angina pectoris or acute myocardial infarction or transient ischemic attack or stroke ; and pad was defined as an ankle - brachial index of 0.9 or a prior history of lower extremity revascularization . the sf-36 healthy survey comprises 36 items covering functional health status and general health , currently and over the past 4 weeks , and measures eight dimensions : physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional , and mental health . as there were age - discrepancies within the sample ( minimum age of 44 years old , and maximum age of 80 years old ) , we adjusted sf-36 results to the age of the participants ; therefore , all other variables were related to the residuals of simple linear regression models having each dimension as a dependent variable and age as the independent variable . most patients were male , were receiving only oral hypoglycemic agents , and had the disease for < 10 years . only 21.0% of the patients did not have chronic complications related to diabetes . the average values obtained by patients in the eight dimensions of the sf-36 , as well as the association between these scores and age are shown in table 2 . there was no association between age and any of the eight dimensions of the sf-36 . the age - adjusted scores of hrqol by the presence or absence of each of the microvascular complications of diabetes are shown in table 3 , macrovascular complications of diabetes in table 4 , and htn in table 5 . there was an overall tendency for the presence of each of the chronic complications and htn to be related to worse hrqol in all dimensions of sf-36 , regardless of age ; the only exception was the dimension role - physical in diabetic nephropathy . the complications related to larger number of dimensions were peripheral neuropathy and cvd ( all dimensions except bodily pain ) . in type 2 diabetic patients with retinopathy , pad , and nephropathy , the hrqol only differed on four , three , and two dimensions , respectively retinopathy ( physical functioning , general health , vitality , and mental health ) , pad ( physical functioning , role - physical , and general health ) , and nephropathy ( general health and vitality ) . the most related dimensions to worse hrqol were general health and vitality ; the general health dimension was lower in patients with any of the long - term complications and htn , whereas the vitality dimension was lower in htn and all diabetes - related complications with the exception of pad . the association between hrqol and the number of chronic complications for the whole sample and by sex are shown in table 6 . an increased number of long - term complications tended to be associated with worse hrqol in all dimensions of sf-36 , regardless of age ; the only exceptions were the bodily pain dimension in males and the role - emotional dimension in females . this association reached statistical significance in all dimensions with the exception of bodily pain in the total sample , all except bodily pain and social functioning in men , and only in general health , vitality , and mental health dimensions in women . in men , the most associated dimension of hrqol with the number of chronic complications was physical functioning , followed by general health , and then by role - physical . in women , the most associated dimensions with the number of complications were general health , followed by vitality . the dimensions physical functioning , role - physical , and role - emotional were only associated with the number of complications in males . the presence of diabetes - related chronic complications and htn , as well as the number of complications were related to worse hrqol regardless of age , with the different conditions being related to different dimensions of hrqol . the adjustment of sf-36 scores for age may obscure relationships between diabetes - related complications , htn , and hrqol dimensions that strongly depend on a concomitant relationship with age . regarding the relationship between the presence of retinopathy and hrqol , we found that having retinopathy was related to lower hrqol in terms of physical functioning , general health , vitality , and mental health . our results partially overlap with the findings of neves et al10 for three reasons : first , these portuguese researchers have found that the diabetic patients with retinopathy had worse hrqol than those without retinopathy in all sf-36 dimensions ; second , because in that study the authors included in their analysis a quite heterogeneous sample ( ie , type 1 and type 2 diabetic patients ) , whereas we included a less heterogeneous one ; and third , because they did not make the adjustment of the hrqol scores for age . contrary to what we expected , which was a reduced hrqol in both physical and mental component scores of the sf-36 , we found that diabetic retinopathy appears to have a greater relationship with the physical component than the mental one . our results showed that the presence of nephropathy was related to worse hrqol in terms of general health and vitality . these results are partially in agreement with the findings of neves et al,10 because these authors reported that its presence was related to lower hrqol in all dimensions of sf-36 . we found that the presence of peripheral neuropathy was related to worse hrqol in all dimensions of sf-36 with the exception of the bodily pain dimension . these results are different from those obtained in another study,13 which did not find a relationship between peripheral neuropathy and hrqol . our results are consistent with the findings of neves et al,10 given that in that study it was found that the presence of neuropathy was related to lower hrqol in all dimensions of sf-36 , and partially overlapping with the results of other investigations.15,16 in the first study it was reported that having neuropathy was related to worse hrqol in terms of physical functioning and bodily pain dimensions of sf-36 , while in the latter one it was found that the presence of peripheral neuropathy was related to lower hrqol in terms of physical and mental components of the aforementioned tool . concerning the relationship between the presence of cvd and hrqol , wasem et al17 reported no relationship between the two variables , whereas we found worse hrqol in patients with cvd in all dimensions of sf-36 with the exception of bodily pain dimension when comparing with those that did not have the disease . these findings are partially in agreement with the results of four cross - sectional studies5,10,15,22 because in those studies , the presence of cvd was related to lower hrqol in terms of physical functioning , general health,5,10,15,22 role - physical,5,10,22 bodily pain , vitality,5,10,15 social functioning,15 and physical component.15,22 on the other hand , our findings are also partially in accordance with the results of two longitudinal studies,30,31 which found that the presence of cvd was related to worse physical functioning , general health , vitality , mental component,30 and physical component.30,31 our results and those obtained in those studies suggest that cvd causes physical and emotional constraints in patients with diabetes . regarding pad and its relationship with hrqol , sakamaki et al13 and wasem et al17 found no relation between the two variables , and surprisingly , venkataraman et al22 reported that patients with pad had better hrqol in terms of physical functioning , role - physical , and physical component than those without pad . conversely , we found that pad was related to worse hrqol in terms of physical functioning , role - physical , and general health . our results are partially in agreement with the findings of silva et al5 and neves et al,10 because it was found that its presence was related to worse hrqol in terms of physical functioning , bodily pain , vitality,5,10 role - physical , general health , social functioning , role - emotional , and mental health.10 however , it is worth noting that these studies5,10 have methodological limitations that might have jeopardized the results , namely , the inclusion of type 1 and type 2 diabetes , and the non - adjustment of the hrqol scores for age . as with diabetic retinopathy and nephropathy , although wexler et al20 and hill - briggs et al25 have found no relationship between the presence of htn and hrqol , in this study htn was related to worse hrqol in terms of general health and vitality . these findings are partially in agreement with the results of prior studies5,10,23,24 the presence of htn was related to lower hrqol in terms of physical functioning , role - physical , bodily pain,5,10 general health,5,10,23 vitality , social functioning , mental health,10,24 role - emotional,10 and mental component.24 these differences between the two groups may possibly be explained by its frequent association with cardiac , cerebrovascular , renal , and ocular complications . on the other hand , various items of the sf-36 are somewhat similar to some symptoms of hypotension and hypoglycemia and , thus , may have contributed to this worse hrqol in the hypertensive patients . with regard to the relationship between the number of diabetes - related chronic complications and hrqol , while caldwell et al27 reported no relationship between the two variables , we found that an increased number of complications was associated with worse hrqol in all dimensions of sf-36 with the exception of bodily pain dimension . these findings are partially in agreement with the results of two cross - sectional studies.16,26 in the first study , it was found that a higher number of chronic complications was related to worse hrqol in terms of physical and mental component , whereas in the latter investigation , an increased number of long - term complications was related to worse hrqol in terms of physical functioning , role - physical , role - emotional , social functioning , and mental health . on the other hand , our results are partially consistent with the findings of a longitudinal study33 that have found that an increased number of complications predicted lower hrqol in terms of physical and mental components . in the present study , the existence of higher number of diabetes - related complications suggests a set of physical , emotional , and social constraints , which result from the emergence of new symptoms , the intensification of the treatment regimen , and a feeling of frustration , anger , and failure after the worsening of diabetes . different hrqol dimensions were more strongly associated with the number of chronic complications in male and female patients . generalization of our findings may be limited because our sample was collected in a central public hospital and might not be representative of the population with type 2 diabetes in other care centers or in primary care . further studies are warranted to adjust the dimensions of hrqol to other variables ( eg , sex , body mass index , type of diabetes and treatment regimens , and duration of the disease ) to explore the relationships between hrqol and the presence of diabetes - related chronic complications and htn , which we think may explain some of these relationships . further investigations on stratifying the severity of diabetes - related chronic complications are required because they may bring important complementary information . more studies are needed to explore sex differences with regard to the association between the number of long - term complications and hrqol and considering different groups of complications . first , we included a more homogeneous sample ( ie , only type 2 diabetic patients ) in comparison to the two previous researches carried out in portugal5,10 which have included both type 1 and type 2 diabetic patients when assessing the relationship between diabetes - related chronic complications , htn and hrqol without making stratified analyses according to type of diabetes . second , the age - adjustment of the sf-36 values to evaluate the relationship between chronic complications , htn and hrqol , given the age - discrepancies within the sample , which was not made in the two mentioned portuguese investigations . third , we evaluated the association between hrqol and number of chronic complications by sex , which was not made previously,16,26,27 and we think may provide important supplemental information . in summary , our findings suggest that the presence and increased number of diabetes - related chronic complications , as well as the presence of htn were related to worse age - adjusted hrqol . it is worth noting that among diabetes - related long - term complications , peripheral neuropathy and cvd were more strongly related to patients age - adjusted hrqol . in males , there was a strong association between the number of chronic complications and hrqol than in their counterparts .

health - related quality of life ( hrqol ) is a subjective assessment of the patient1 that is related to the perception of their health,14 well - being , and functioning,1 and also to their worries about the future.4 although the term hrqol is not yet precisely defined,5 it is a useful measure to assess the overall impact of a particular nosological entity in patient,2 and it complements the clinical information.3,6 there are several generic tools that can be used to assess hrqol ; they can be used not only in the healthy population but also in specific populations4 because they allow to assess a wide set of important dimensions that can be used in different diseases.4,68 wilson and cleary9 propose a conceptual model to explain the relationships between clinical variables and measures of hrqol in order to establish the connection between diagnosis and therapy and , thus , to increase patients hrqol . with regard to the relationship between hrqol , the presence of hypertension ( htn ) , and diabetes - related chronic complications , the literature is not consistent in pointing out that their presence decreases hrqol and , hence , the importance of evaluating the relationship between these variables and hrqol . moreover , as far as we know , the only two researches carried out in portugal5,10 that have assessed the relationship between the most frequent chronic complications of diabetes and hrqol did not make the adjustment of the hrqol scores for any demographic and/or clinical variables that can mediate some of these relationships . on the other hand , they evaluated simultaneously type 1 and type 2 diabetic patients and did not assess chronic complications according to diabetes type . lastly , the investigations that evaluated the relationship between the number of diabetes - related chronic complications and hrqol did not make stratified analyses according to sex that can bring important supplemental information to the one achieved for the whole sample . concerning microvascular complications of diabetes , while some studies1012 have found that the presence of diabetic retinopathy was related to worse hrqol , sakamaki et al13 found no relationship between the two variables . results of research on the relationship between diabetic nephropathy and hrqol have found , on one hand , that its presence was related to lower hrqol10,11 and , on the other hand , no relationship between both variables.13 concerning the relationship between diabetic neuropathy and hrqol , while sakamaki et al13 found no relationship between the two variables , some studies10,1417 have found that its presence was related to worse hrqol . as far as diabetes - related macrovascular complications are concerned , although there is evidence that the presence of cardiovascular disease ( cvd ) in persons with diabetes was related to lower hrqol,5,10,12,1416,1822 wasem et al17 found no relationship between the two variables . regarding the relationship between the presence of peripheral arterial disease ( pad ) and hrqol , silva et al,5 neves et al,10 javanbakht et al,11 and quah et al16 have shown that patients with this chronic complication had worse hrqol , whereas sakamaki et al13 and wasem et al17 found no relationship between the two variables , and venkataraman et al22 reported that its presence was related to better hrqol . regarding the relationship between htn and diabetes , while some studies5,10,23,24 have found that the presence of htn was related to lower hrqol , wexler et al20 and hill - briggs et al25 reported no relationship between the two variables . finally , some researchers , looking at the relationship between the number of diabetes - related chronic complications and hrqol , have found that an increased number of complications was related to worse hrqol.16,26 however , caldwell et al27 did not find a relationship between the two variables . the aim of this cross - sectional study was to assess the relationship between hrqol , the presence or absence of htn , and diabetes - related micro- and macrovascular complications in type 2 diabetes , as well as the association between hrqol and the number of chronic complications , for the whole sample and by sex . we focused on the assessment of htn and long - term complications on hrqol in type 2 diabetic patients for three reasons : first , because htn is a highly prevalent comorbidity in these individuals ; second , because it is highly associated with diabetes - related chronic complications ; and third , because it can have a negative association with hrqol . a consecutive sample of 100 type 2 diabetic patients from the outpatient department of endocrinology , diabetes and metabolism , centro hospitalar so joo , porto , portugal , was recruited . general hrqol was assessed by the portuguese version of the medical outcomes study 36-item short - form health survey ( sf-36),28,29 and it was administered by one of the researchers . we evaluated the presence of some clinical variables that are reported in the literature to be related to hrqol among type 2 diabetes;5,1012,1424,26 these factors included microvascular complications , macrovascular complications , and htn . type 2 diabetic patients were classified as on oral hypoglycemic agents or insulin therapy ( the latter including patients only on insulin therapy or on oral hypoglycemic agents plus insulin therapy ) . we considered that the patients had retinopathy if they had at least microaneurysms , nephropathy if they had at least albumin excretion rate higher than 30 mg / g creatinine , peripheral neuropathy if they had two or more insensate sites across two feet for the 10 g monofilament or one or more insensate sites for the 128 hz tuning fork . cvd was defined by the presence of at least one of the following conditions : angina pectoris or acute myocardial infarction or transient ischemic attack or stroke ; and pad was defined as an ankle - brachial index of 0.9 or a prior history of lower extremity revascularization . the sf-36 healthy survey comprises 36 items covering functional health status and general health , currently and over the past 4 weeks , and measures eight dimensions : physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional , and mental health . as there were age - discrepancies within the sample ( minimum age of 44 years old , and maximum age of 80 years old ) , we adjusted sf-36 results to the age of the participants ; therefore , all other variables were related to the residuals of simple linear regression models having each dimension as a dependent variable and age as the independent variable . a consecutive sample of 100 type 2 diabetic patients from the outpatient department of endocrinology , diabetes and metabolism , centro hospitalar so joo , porto , portugal , was recruited . general hrqol was assessed by the portuguese version of the medical outcomes study 36-item short - form health survey ( sf-36),28,29 and it was administered by one of the researchers . we evaluated the presence of some clinical variables that are reported in the literature to be related to hrqol among type 2 diabetes;5,1012,1424,26 these factors included microvascular complications , macrovascular complications , and htn . type 2 diabetic patients were classified as on oral hypoglycemic agents or insulin therapy ( the latter including patients only on insulin therapy or on oral hypoglycemic agents plus insulin therapy ) . we considered that the patients had retinopathy if they had at least microaneurysms , nephropathy if they had at least albumin excretion rate higher than 30 mg / g creatinine , peripheral neuropathy if they had two or more insensate sites across two feet for the 10 g monofilament or one or more insensate sites for the 128 hz tuning fork . cvd was defined by the presence of at least one of the following conditions : angina pectoris or acute myocardial infarction or transient ischemic attack or stroke ; and pad was defined as an ankle - brachial index of 0.9 or a prior history of lower extremity revascularization . the sf-36 healthy survey comprises 36 items covering functional health status and general health , currently and over the past 4 weeks , and measures eight dimensions : physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional , and mental health . as there were age - discrepancies within the sample ( minimum age of 44 years old , and maximum age of 80 years old ) , we adjusted sf-36 results to the age of the participants ; therefore , all other variables were related to the residuals of simple linear regression models having each dimension as a dependent variable and age as the independent variable . the average values obtained by patients in the eight dimensions of the sf-36 , as well as the association between these scores and age are shown in table 2 . the age - adjusted scores of hrqol by the presence or absence of each of the microvascular complications of diabetes are shown in table 3 , macrovascular complications of diabetes in table 4 , and htn in table 5 . there was an overall tendency for the presence of each of the chronic complications and htn to be related to worse hrqol in all dimensions of sf-36 , regardless of age ; the only exception was the dimension role - physical in diabetic nephropathy . in type 2 diabetic patients with retinopathy , pad , and nephropathy , the hrqol only differed on four , three , and two dimensions , respectively retinopathy ( physical functioning , general health , vitality , and mental health ) , pad ( physical functioning , role - physical , and general health ) , and nephropathy ( general health and vitality ) . the most related dimensions to worse hrqol were general health and vitality ; the general health dimension was lower in patients with any of the long - term complications and htn , whereas the vitality dimension was lower in htn and all diabetes - related complications with the exception of pad . an increased number of long - term complications tended to be associated with worse hrqol in all dimensions of sf-36 , regardless of age ; the only exceptions were the bodily pain dimension in males and the role - emotional dimension in females . this association reached statistical significance in all dimensions with the exception of bodily pain in the total sample , all except bodily pain and social functioning in men , and only in general health , vitality , and mental health dimensions in women . in men , the most associated dimension of hrqol with the number of chronic complications was physical functioning , followed by general health , and then by role - physical . the dimensions physical functioning , role - physical , and role - emotional were only associated with the number of complications in males . the presence of diabetes - related chronic complications and htn , as well as the number of complications were related to worse hrqol regardless of age , with the different conditions being related to different dimensions of hrqol . the adjustment of sf-36 scores for age may obscure relationships between diabetes - related complications , htn , and hrqol dimensions that strongly depend on a concomitant relationship with age . thereby , absolute scores on the sf-36 and their relationship with long - term complications and htn may have clinical usefulness , but to analyze their association with hrqol , which was our intention , it is recommended that values are adjusted for age if there are significant age - discrepancies within the sample . regarding the relationship between the presence of retinopathy and hrqol , we found that having retinopathy was related to lower hrqol in terms of physical functioning , general health , vitality , and mental health . our results partially overlap with the findings of neves et al10 for three reasons : first , these portuguese researchers have found that the diabetic patients with retinopathy had worse hrqol than those without retinopathy in all sf-36 dimensions ; second , because in that study the authors included in their analysis a quite heterogeneous sample ( ie , type 1 and type 2 diabetic patients ) , whereas we included a less heterogeneous one ; and third , because they did not make the adjustment of the hrqol scores for age . contrary to what we expected , which was a reduced hrqol in both physical and mental component scores of the sf-36 , we found that diabetic retinopathy appears to have a greater relationship with the physical component than the mental one . our results showed that the presence of nephropathy was related to worse hrqol in terms of general health and vitality . surprisingly , we did not find differences between the two groups in the other dimensions of the sf-36 , particularly in terms of mental component . we found that the presence of peripheral neuropathy was related to worse hrqol in all dimensions of sf-36 with the exception of the bodily pain dimension . our results are consistent with the findings of neves et al,10 given that in that study it was found that the presence of neuropathy was related to lower hrqol in all dimensions of sf-36 , and partially overlapping with the results of other investigations.15,16 in the first study it was reported that having neuropathy was related to worse hrqol in terms of physical functioning and bodily pain dimensions of sf-36 , while in the latter one it was found that the presence of peripheral neuropathy was related to lower hrqol in terms of physical and mental components of the aforementioned tool . concerning the relationship between the presence of cvd and hrqol , wasem et al17 reported no relationship between the two variables , whereas we found worse hrqol in patients with cvd in all dimensions of sf-36 with the exception of bodily pain dimension when comparing with those that did not have the disease . these findings are partially in agreement with the results of four cross - sectional studies5,10,15,22 because in those studies , the presence of cvd was related to lower hrqol in terms of physical functioning , general health,5,10,15,22 role - physical,5,10,22 bodily pain , vitality,5,10,15 social functioning,15 and physical component.15,22 on the other hand , our findings are also partially in accordance with the results of two longitudinal studies,30,31 which found that the presence of cvd was related to worse physical functioning , general health , vitality , mental component,30 and physical component.30,31 our results and those obtained in those studies suggest that cvd causes physical and emotional constraints in patients with diabetes . with regard to physical impairments , it appears that hrqol and life expectancy of these patients are more reduced , given that cvd is the major cause of death in both diabetic males and females.32 thereby , it is not surprising that these patients become physically more limited , and , consequently , end up becoming psychologically more vulnerable . regarding pad and its relationship with hrqol , sakamaki et al13 and wasem et al17 found no relation between the two variables , and surprisingly , venkataraman et al22 reported that patients with pad had better hrqol in terms of physical functioning , role - physical , and physical component than those without pad . our results are partially in agreement with the findings of silva et al5 and neves et al,10 because it was found that its presence was related to worse hrqol in terms of physical functioning , bodily pain , vitality,5,10 role - physical , general health , social functioning , role - emotional , and mental health.10 however , it is worth noting that these studies5,10 have methodological limitations that might have jeopardized the results , namely , the inclusion of type 1 and type 2 diabetes , and the non - adjustment of the hrqol scores for age . as with diabetic retinopathy and nephropathy , although wexler et al20 and hill - briggs et al25 have found no relationship between the presence of htn and hrqol , in this study htn was related to worse hrqol in terms of general health and vitality . these findings are partially in agreement with the results of prior studies5,10,23,24 the presence of htn was related to lower hrqol in terms of physical functioning , role - physical , bodily pain,5,10 general health,5,10,23 vitality , social functioning , mental health,10,24 role - emotional,10 and mental component.24 these differences between the two groups may possibly be explained by its frequent association with cardiac , cerebrovascular , renal , and ocular complications . with regard to the relationship between the number of diabetes - related chronic complications and hrqol , while caldwell et al27 reported no relationship between the two variables , we found that an increased number of complications was associated with worse hrqol in all dimensions of sf-36 with the exception of bodily pain dimension . these findings are partially in agreement with the results of two cross - sectional studies.16,26 in the first study , it was found that a higher number of chronic complications was related to worse hrqol in terms of physical and mental component , whereas in the latter investigation , an increased number of long - term complications was related to worse hrqol in terms of physical functioning , role - physical , role - emotional , social functioning , and mental health . on the other hand , our results are partially consistent with the findings of a longitudinal study33 that have found that an increased number of complications predicted lower hrqol in terms of physical and mental components . in the present study , the existence of higher number of diabetes - related complications suggests a set of physical , emotional , and social constraints , which result from the emergence of new symptoms , the intensification of the treatment regimen , and a feeling of frustration , anger , and failure after the worsening of diabetes . furthermore , most of the patients interviewed were of low sociocultural status , which may have limited their understanding of the questions in the instrument used to assess hrqol . further studies are warranted to adjust the dimensions of hrqol to other variables ( eg , sex , body mass index , type of diabetes and treatment regimens , and duration of the disease ) to explore the relationships between hrqol and the presence of diabetes - related chronic complications and htn , which we think may explain some of these relationships . first , we included a more homogeneous sample ( ie , only type 2 diabetic patients ) in comparison to the two previous researches carried out in portugal5,10 which have included both type 1 and type 2 diabetic patients when assessing the relationship between diabetes - related chronic complications , htn and hrqol without making stratified analyses according to type of diabetes . second , the age - adjustment of the sf-36 values to evaluate the relationship between chronic complications , htn and hrqol , given the age - discrepancies within the sample , which was not made in the two mentioned portuguese investigations . third , we evaluated the association between hrqol and number of chronic complications by sex , which was not made previously,16,26,27 and we think may provide important supplemental information . in summary , our findings suggest that the presence and increased number of diabetes - related chronic complications , as well as the presence of htn were related to worse age - adjusted hrqol . it is worth noting that among diabetes - related long - term complications , peripheral neuropathy and cvd were more strongly related to patients age - adjusted hrqol .

osteoarthritis ( oa ) is a major cause of disability affecting millions of people worldwide . in the united states alone , to date there are no proven therapies for the prevention or treatment of oa . pain relief and visco - supplementation are prescribed to attenuate the symptoms of oa until disease progression significantly impairs joint function and joint replacements are required . the lack of disease modifying oa drugs ( dmoads ) may be a function of incongruence between in vitro models of oa and the pathogenesis in vivo , and between disease mechanisms in humans and model animals . to overcome these issues , there is increasing momentum to develop human cell - based organotypic models in vitro that functionally represent the osteochondral tissue directly affected by oa . the development of physiologically relevant models requires an understanding of the tissue architecture , physiology , and pathophysiological responses to biochemical ( or biophysical ) insults . this is especially the case for the osteochondral complex , where the main tissues , cartilage and bone , differ so substantially . cartilage is composed of a collagen type ii / aggrecan - rich , highly hydrated , viscoelastic , anisotropic matrix that encapsulates chondrocytes within biochemically distinct chondrons . in contrast , bone is composed of a collagen type i - rich , laminated or woven calcified structure that is much stiffer and encapsulates osteocytes , osteoblasts , and osteoclasts , blood vessels , and nerves . the cartilage and bone are intimately connected at the osteochondral junction ( ocj ) , a highly organized structure that represents a significant challenge to mimic in vitro by tissue engineering . it is composed of distinct , interacting layers that include ( epi - to - diaphyseally ) deep zone cartilage , a basophilic tidemark , calcified cartilage , the cement line , and the subchondral bone plate . interestingly , there is growing evidence of significant biochemical communication between cartilage and bone across the ocj . in the pathogenesis of oa , changes in the physical linkage between cartilage and bone at the ocj are critical components of disease progression . these include remodeling of the tidemark , microcracks , and fissures in both tissues and ingrowth from the underlying bone of blood vessels and nerves , all of which may enhance the cartilage - bone crosstalk allowing a better passage of growth factors , cytokines , and signaling molecules . these ocj changes accelerate cartilage degeneration and are associated with joint pain and disease morbidity , pointing to the need of a better understanding of the complex network of interactions between bone and cartilage in oa . cartilage and bone exist not only in a different matrix but also in very different biophysical environments . in vivo , there is a steep oxygen gradient from bone ( essentially normoxic ) to cartilage ( extremely hypoxic ) . these differences are reflected in the in vitro culture systems often used to maintain chondrocytes and osteoblasts . starved environment : low glucose , serum - free medium supplemented with pyruvate and abundant matrix precursors or -enhancing molecules ( proline and ascorbate ) in hypoxic conditions . however , osteoblasts are maintained in high glucose , serum - containing medium supplemented with -glycerol phosphate and vitamin d3 in normoxic conditions . with these fundamental environmental differences between chondrocytes and osteoblasts , it is not surprising that oa elicits specific responses from each tissue . oa disease progression is most frequently characterized by a net loss of cartilage matrix that results from an imbalance between cartilage matrix degradation and synthesis by chondrocytes in the cartilage . progressive chronic destruction of articular cartilage is the most obvious characteristic of oa , and the etiology of the disease is believed to be at the intersection of genetics and abnormal mechanical forces . therefore , the primary locus of the disease is traditionally presumed to be the cartilage , and as a result , most in vitro oa models focus exclusively on cartilage to study oa disease mechanisms and therapeutic intervention . however , there is increasing evidence from in vivo and clinical studies that subchondral bone lesions may precede cartilage degeneration , implying that oa is an osteochondral disease and possibly bone dependent . in addition , it has been often reported that the health of mature articular cartilage in vitro is positively impacted by the presence of subchondral bone . despite these observations , most in vitro oa research has not taken into account the effects of bone - cartilage interactions , focusing primarily on cartilage alone . this may account for the dearth of new therapeutics for the prevention and treatment of oa . we theorize that the development of a model system of osteochondral tissue using human cells in a physiologically relevant environment that can accurately replicate in vivo osteochondral tissue homeostasis and pathophysiology will lead to greater predictive power in the development of dmoads . the challenges in developing such a system include : ( 1 ) mimicking or inducing production of appropriate extracellular matrix critical to the function of cartilage and bone , ( 2 ) replicating the tissue architecture , ( 3 ) reconciling the different growth and maintenance conditions of bone and cartilage while promoting their interaction with each other , and ( 4 ) replicating the biomechanical environment known to be essential to cartilage and bone health . current in vitro models to investigate bone - cartilage interactions are mostly limited to cell co - culture systems in which bone and cartilage cells are both exposed to the same medium , arguably a very distant condition from the in vivo environment . here , we report the development of a bioreactor designed to accommodate the biphasic nature of an osteochondral plug by creating two separate compartments for the chondral and osseous microenvironments . these are separated only by the tissue itself and are supplied by a microfluidic system . the two microenvironments can be independently controlled and regulated via introductions of bioactive agents or candidate effecter cells , and the medium can be individually sampled for compositional assays . the central hypothesis of the study is that a gradient of tissue specific nutrients and conditions is required for the formation and maintenance of the osteochondral tissue . furthermore , we hypothesize that induction of an oa - like condition in the engineered osseous or the engineered chondral component alone will induce a corresponding oa - like response in the other component . to test these hypotheses , we have generated distinct chondral and osseous zones within the same construct by controlling the different media exposures within the bioreactor . then , we induced an oa - like response by exposing the osseous or chondral compartments to the pro - inflammatory cytokine ( il-1 ) and assayed the intervening changes in expression and secretion from both the engineered chondral and osseous components . all chemicals used in this study were purchased from sigma - aldrich ( st . louis , mo ) unless stated otherwise . the 3d structures of the bioreactor ( figure 1 ) were modeled using magics 14 ( materialise , belgium ) . the chamber and insert were fabricated using a stereolithography apparatus ( envisiontec , germany ) employing e - shell 300 as the resin . ( a ) an individual bioreactor composed of the removable insert ( dark gray ) within a chamber ( light gray ) of the microfluidic plate ( b ) and fixed in place with two o - rings . the osteochondral construct within the insert creates the final separation between the upper and lower medium conduits . opposing gradients of chondrogenic and osteogenic factors and stimulants will aid in forming an interface . ( b ) a single bioreactor formed by the inset and lid in the context of a 24-well plate . red circles indicate the o - rings that seal the joint space between lid / insert and chamber . hbmscs were isolated from the femoral heads of patients undergoing total joint arthroplasty with irb approval ( university of pittsburgh ) , cultured and expanded as previously described ( caterson , 2002 ; song , 2004 ) . briefly , bone marrow was flushed out from the trabecular bone of the femoral neck and head using an 18-gauge needle and resuspend in dulbecco s minimal essential medium ( dmem ) . the suspension was filtered through a 40 m strainer and the flow - through was centrifuged at 300 g for 5 min . after the supernatant was discarded , the pellets were suspended using growth medium ( gm , -mem containing 10% fetal bovine serum ( fbs , invitrogen ) , 1% antibiotics - antimycotic , and 1.5 ng / ml fgf-2 ( raybiotech , norcross , ga ) ) , and then plated into 150 cm tissue culture flasks at a density of 20,00040,000 nucleated cells / cm , and medium was changed every 3 to 4 days . the colony formation and trilineage mesenchymal differentiation capacity of hbmscs was validated before use ( data not shown ) . all experiments were performed with passage 3 ( p3 ) hbmscs from 3 patients ( 3 female patients 44 , 52 , and 72 years old ) , which were pooled for use in this study . the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate ( lap ) was synthesized as described by fairbanks et al . mgl was synthesized by reacting gelatin with methacrylic anhydride ( ma ) in water according to a procedure previously described . mha was prepared as previously reported using sodium hyaluronate powder ( research grade , mw 66 kda , lifecore ) . both mgl and mha were lyophilized and stored in a desiccator for future use . to test medium leakage between ( 1 ) the chamber wall and the insert and ( 2 ) the insert and scaffold material , the insert was filled with 10% mgl/0.15% lap in hbss and alexa fluor 488-conjugated soybean trypsin inhibitor ( ti488 , 21kd , molecular probes , ca ) and alexa fluor 555-conjugated albumin from bovine serum ( bsa ) ( bsa555 , 65kd , molecular probes ) were diluted in hbss individually at 10 g / ml and then perfused through the top and bottom of bioreactor , respectively , at 1 l / min . at different time points , effluent from the upper and lower medium conduits was collected and the fluorescence intensity at both wavelengths measured using a microplate reader ( synergy ht , biotek , winooski , vt ) . leaking between top and bottom conduits was estimated by the ratio of ti488 ( bottom)/ti488 ( top ) and bsa555(top)/bsa555 ( bottom ) . because of the permeable nature of gelatin scaffold used as the scaffold model , leaking was assayed for 24 h only . p3 hbmscs were pelleted and drained completely in order to prevent the unwanted dilution of polymers . chondrogenic cell suspension : hbmscs were resuspended in 10% mgl/1%mha/0.15% lap ( w / v ) hbss solution ( ph adjusted to 7.4 ) at a final density of 20 10/ml ( chondrogenic suspension ) . hbmscs were resuspended in the 10% mgl/1% hydroxyapatite/0.15% lap ( w / v ) hbss solution ( ph was adjusted to 7.4 ) at a final density of 20 10/ml . osteochondral construct preparation : first , the insert was placed within a hollow cylindrical well to prevent suspension leaking from the pores in the insert . second , 60 l of osteogenic suspension was pipetted into the insert and cross - linked using the uv light source . after 2 min of uv light exposure , the inset with photopolymerized osseous construct was removed from the chamber . third , 30 l of chondral suspension was added on the top of osseous construct within the same insert and cured for another 2 min . previous studies have shown cell viability within the scaffold > 90% after photo - cross - linking ( data not shown ) . the second round of cross - linking had the added benefit of bonding the osseous and chondral layers together as well , and the fabrication of the osteochondral construct within the insert created a tight seal . the inserts with nave osteochondral constructs were placed into the microfluidic plate as shown in figure 1 . chondrogenic medium ( cm ) was supplied through the upper conduit , while osteogenic medium ( om ) through the bottom conduit at a flow rate of 1 l / s . the following formulas were used for the differentiation media : om ( gm supplemented with 10 ng / ml bmp-2 ( peprotech , rocky hill , nj ) , 1% l - alanyl - l - glutamine ( glutamax ) , 10 nm dexamethasone ( dex ) , 0.1 mm l - ascorbic acid 2-phosphate ( asa2-p ) , and 10 mm beta - glycerophosphate ( -gp ) ; cm ( dmem supplemented with 10 ng / ml tgf-3 ( peprotech ) , 1% its , 50 m asa2-p , 55 m sodium pyruvate , and 23 m l - proline ) . the perfusion rate was 1 l / min , and used syringes were replaced with syringes and new medium every 3 days . after 4 weeks of differentiation , engineered osteochondral tissues were collected for validation using real - time pcr and histological analysis , or treated with il1 - 1. chondral and osseous constructs were collected separately . to avoid the potential contamination , total rna was extracted using trizol ( invitrogen ) following the standard protocol and purified with the rneasy plus mini kit ( qiagen , hilden , germany ) . superscript iii kit ( invitrogen ) was utilized with random hexamer primers to complete the reverse transcription . real - time rt - pcr was performed using the steponeplus thermocycler ( applied biosystems , foster city , ca ) and sybr green reaction mix ( applied biosystems ) . sox 9 , aggrecan ( acn ) , collagen type ii ( col2a1 ) , runx2 , osteocalcin ( ocn ) , and bone sialoprotein ( bsp ii ) expression were analyzed , and primer sequences are listed in table 1 . monolayers of hbmscs cultured in gm on 2d tissue culture plastic were used as negative controls . transcript level of 18s rrna was used as endogenous control , and gene expression folder changes were calculated using the comparative ct ( ct ) method . aggrecan ( acn ) , collagen type ii ( col2 ) , runt - related transcription factor 2 ( runx2 ) , osteocalcin ( ocn ) , bone sialoprotein ii ( bsp ii ) , and 18s rrna ( 18s ) . intact engineered osteochondral tissues were fixed in 10% neutral buffered formalin ( fisher scientific , pittsburgh , pa ) for 7 days , dehydrated , embedded in paraffin with 10 m sections cut from each sample . safranin o / fast green and alizarin red staining were used to detect the gag and calcium deposition , respectively . after 4 weeks of differentiation , engineered osteochondral constructs were treated with il-1 ( 10 ng / ml , r&d ) on the chondral or osseous sides only to investigate the cell / neo - tissue response to pro - inflammatory cytokines and possible communication through the osteochondral construct . the media used in this test were cm without tgf-3 ( chondral ) and om without bmp-2 ( osseous ) , both supplemented with 10 ng / ml il-1. there were three experimental groups : ( 1 ) cm / om , ( 2 ) cm + il1 - 1/om , and ( 3 ) cm / om + il1 - 1. the treatment lasted 7 days , with effluent medium collected and frozen at 1 and 7 days for elisa . after 7 days , the osteochondral constructs were bisected into the chondral and osseous halves and processed for gene expression analysis as described before . in addition to tissue specific gene expression , matrix metalloproteinase 1 , 3 , and 13 were also analyzed . media was collected separately from chondral and osseous constructs , cleared of cell debris via centrifugation ( 1000 g ) , and analyzed via il-1 ( abcam , cambridge , ma ) , mmp-1 ( r&d ) , mmp-3 ( abcam , cambridge , ma ) , and mmp-13 ( abcam ) elisas according to the manufacturers instructions . significant differences were determined with anova followed by a bonferroni post hoc analysis for multiple group comparisons using spss statistics 21 ( ibm , armonk , ny ) . significance was determined at p < 0.05 ( * ) and p < 0.01 ( * * ) . the robustness of the microbioreactor was tested by assessing the extent of leakage of two molecules perfused independently in the upper and lower medium conduits : ( 1 ) trypsin inhibitor ( 21 kda ) , with a molecular weight similar to the two commonly used osteoinductive ( bmp-2 , 26kd ) and chondroinductive ( tgf3 , 25kd ) factors , and ( 2 ) bsa ( 65kd ) , the most abundant protein in serum . as shown in figure 2a , after 24 h of perfusion , the extent of mixing between top and bottom was < 1% , indicating there was minimal medium exchange through the interfaces between the chamber wall and the inset and between the inset and scaffold - only construct . these results were further confirmed by elisa assay for il-1 in both medium conduits during the il-1 test ( figure 2b ) . ( a ) trypsin inhibitor-488 and bsa-555 were simultaneously perfused through the top and bottom space of bioreactor , respectively , and the percent leakage at different time was estimated based on the bottom / top ratio of 488 nm fluorescence readings ( bottom / top 488 ) and top / bottom 555 nm fluorescence readings ( top / bottom 555 ) . ( b ) il-1 was included in top or bottom stream and perfused for 24 h. its concentration in top or bottom medium was then measured . the naive hbmscs seeded within tissue - specific scaffolds in the freshly fabricated osteochondral construct were induced to differentiate using cm in the top stream and om in the bottom stream . we anticipated chondrogenesis in the upper , chondral half of the construct and osteogenesis in the bottom , osseous half . after 4 weeks of differentiation , biphasic osteochondral constructs were produced ( figure 4c ) . as shown in figure 3 , cells in the chondral half showed enhanced expression of chondrogenic genes , including sox 9 , aggrecan , and collagen type ii as compared to those in the osseous half , while the cells in the osseous half had higher expression of osteogenic genes , including runx2 , osteocalcin , and bsp ii . monolayers of hbmscs cultured in gm on 2d tissue culture plastic were used as negative controls . histological staining with alcian blue / alizarin red revealed high matrix gag content in the upper chondral half than the osseous half ( figure 4b ) ; although the amount of calcium deposition in the osseous half was not detectable . taken together , these results strongly indicate a spatially defined , biphasic differentiation of these engineered osteochondral constructs , with the chondral component undergoing more characteristic differentiation . in addition , h&e staining revealed a distinct , < 100 m wide basophilic band in the interface between the chondral and osseous halves , potentially indicative of a developing tidemark ( figure 4a ) . after 4 weeks of culture in the bioreactor , osteochondral constructs were separated into chondral ( cartilage ) and osseous ( bone ) components , and each were analyzed for expression of cartilage ( sox9 , col2 , and aggrecan ) or bone ( runx2 , osteocalcin , bspii ) markers . expression levels are normalized to 18s rrna and then to corresponding 2d control expression levels . expression of cartilage markers was found only in the chondral component , and bone markers in the osseous compartment . * p < 0.05 ; * * p < 0.01 ) . top , chondral component ( cc ) ; bottom , osseous component ( oc ) . ( a ) alizarin red staining ; ( b ) safranin o / fast green staining . dashed lines indicate the border between cc and oc . as described above , the microtissue bioreactor presented here , with its two separate medium flow systems and biphasic construct compartments , has the capability for targeted treatment of one ( or both ) tissue construct(s ) with soluble factors . osseous and chondral components were separately treated with the pro - inflammatory cytokine il-1 ( 10 ng / ml ) for 7 days ( control conditions consisted of untreated osteochondral constructs ) , and the responses of each of the two components were separately analyzed . media samples were collected from chondral and osseous components streams at days 1 and 7 , and after day 7 , the osteochondral constructs were separated into osseous and chondral components , and each was separately analyzed for gene expression of catabolic genes ( mmp-1 , mmp-3 , and mmp-13 ) and either cartilage markers ( sox9 , col2 , and aggrecan ) ( figure 5 ) or bone markers ( runx2 , osteocalcin , and bspii ) ( figure 6 ) . media samples from the chondral and osseous components stream collected at days 1 and 7 were analyzed via mmp-1 , mmp-3 , and mmp-13 elisas ( figure 7 ) . effects of il-1 treatment on cartilage gene expression in the engineered osteochondral microtissue . after treatment of either osseous ( bone ) or chondral ( cartilage ) component with 10 ng / ml il-1 for 7 days , cartilage components were analyzed for the expression of cartilage markers and mmps . expression levels were normalized to 18s rrna expression and then to corresponding gene expression under control conditions . * p < 0.05 ; * * p < 0.01 . effects of il-1 treatment on bone gene expression in the engineered osteochondral microtissue . after treatment of either osseous ( bone ) or chondral ( cartilage ) component with 10 ng / ml il-1 for 7 days , bone components were analyzed for expression of bone markers and mmps . expression levels were normalized to 18s rrna expression and then to corresponding gene expression under control conditions . effects of il-1 treatment on osseous and chondral mmp secretion in the engineered osteochondral microtissue . after treatment of either osseous ( bone ) or chondral ( cartilage ) component with 10 ng / ml il-1 for 1 or 7 days , medium samples collected from the bone or cartilage medium compartment were analyzed by elisa for the levels of secreted mmp-1 , mmp-3 , and mmp-13 . values were normalized to those measured under control conditions , which involved untreated osteochondral constructs . treatment of chondral constructs with il-1 caused decreases in expression of cartilage genes sox9 , col2 , and aggrecan , consistent with physiological outcomes of damaged or stressed cartilage ( figure 5 ) . chondral construct expression of these genes also decreased in response to il-1 treatment of osseous constructs , suggesting signaling between the osseous and chondral components . evidence of this osseous - to - chondral communication was even more apparent in results concerning expression of catabolic genes ; expression of mmp-1 , mmp-3 , and mmp-13 of the chondral constructs increased substantially in response to il-1 treatment of the osseous component . crosstalk between the two components was also detected in the case of chondral - to - osseous communication ( figure 6 ) . il-1 treatment of the chondral construct caused decreases in expression of the bone genes osteocalcin and bspii and increases in mmps production in the osseous construct , particularly mmp-13 , which is one of the most important mediators of oa cartilage degradation . elisa analysis of mmps secreted by the chondral and osseous components at different time points allowed for observations on the rate of signal propagation between the two components ( figure 7 ) . for example , the chondral construct responded to il-1 treatment of the osseous component with increases in mmp-1 , mmp-3 , and mmp-13 secretion . the chondral mmp-13 response occurred quickly , within 1 day , while the chondral mmp-1 response took 7 days . the chondral mmp-3 response time was intermediate between those of mmp-13 and mmp-1 . again , these results are interesting considering the central role mmp-13 plays in cartilage degeneration . the osseous construct response to treatment of chondral component with il-1 , however , was quick yet increased further over time , and by day 7 was overall stronger than the chondral responses to the osseous component treatment . it is worth noting that gene expression and protein levels of mmps should not be expected to be necessarily consistent . this stems from differences in the ways in which elisa and real - time pcr samples were collected and measured . elisa samples consisted of culture media conditioned by cells for 24 h and were collected at day 1 or day 7 for each experiment . the proteins contained in day 1 samples were secreted between days 0 and 1 , and day 7 samples contained proteins secreted between days 6 and 7 . pcr samples , however , were collected after 7 days of cultures and represent the expressional activities taking place at the moment of collection . in other words , the mrna levels analyzed by pcr at day 7 are not necessarily totally reflective of the protein levels analyzed by elisa in day 7 conditioned media samples . this disconnect between pcr and elisa measurements may be more pronounced in mmps , which need to be translated , secreted , and then diffuse out of the 3d construct before they are detected by elisa . furthermore , differences between pcr and elisa values also arise from differences in normalization . pcr results are normalized to 18s rrna expression , thereby taking into account cell number . elisa results are instead a representation of the entire culture and normalized to control conditions . thus , any experimental treatment that may affect cell number would have a larger impact on elisa results than pcr results . since chondrocytes are particularly sensitive to il-1 , this may explain why results concerning cells of the chondral component exhibit the greatest degree of inconsistency between pcr and elisa measurements when chondral constructs are directly stimulated by il-1. in this study , we have developed a novel bioreactor system for the engineering of osteochondral tissue . rt - pcr and histological analyses showed that hbmscs - derived nave constructs have been successfully differentiated into cartilage - like tissue on the top and bone - like tissue on the bottom , using separated culture medium for 4 weeks . a transition layer between 2 tissues is also observed . our results show that il-1 exposure decreases the ecm anabolic gene expression but greatly enhances the levels of mmp expression and secreted amount into the medium . interestingly , the il-1 insulted osseous construct induces a catabolic gene expression response into the untreated chondral component , which is not due to leakage of il-1 , suggesting active osseous chondral interaction and the likely importance of bone injury in oa development . in this study , a dual - chamber bioreactor has been developed to generate and maintain osteochondral constructs derived from human hbmscs . the design parameters included individual compartments to separate the chondral and osseous microenvironments that are individually accessible for the introduction of bioactive agents and/or candidate effector cells , tissue and medium sampling , and compositional assays , including noninvasive imaging techniques . furthermore , the total dimension and geometry of the bioreactor matches that of a multiwell culture plate chamber for the development of medium- to high - throughput analysis . validation of the system included successful , simultaneous differentiation of osseous and chondral constructs from hbmscs from the same source ( pooling of three donors ) and subsequent application of il-1 , a potent inflammatory mediator implicated in oa pathophysiology , to test the physiological response of the osteochondral construct . we have shown that in the course of 6 weeks , hbmscs undergo tissue - specific differentiation in response to the tissue specific growth media and hydrogel composition provided . the differentiating bbmscs expressed tissue - specific transcription factors and ecm molecules , as shown by rt - pcr and histological staining . most impressively , there was an indication of a basophilic , tidemark - like zone separating the chondral and osseous components . the recreation of the a tidemark - containing biphasic tissue is vital to drug testing using an osteochondral organotypic culture since changes in the ocj are mechanistically involved in oa progression and likely to be a target of toxicants and dmoads . we subsequently tested the response of the msc - based osteochondral tissue to il-1. the test served two purposes : ( 1 ) to validate the utility of the bioreactor in osteochondral studies and ( 2 ) to assess the physiological replication of the oc tissue by the mscs in this bioreactor . il-1 is almost ubiquitous in inflammatory diseases , is prominent in advanced oa in both the cartilage and synovial lining , and is frequently employed as a pathogenic initiator in in vitro models of oa . application of il-1 to both osseous and chondral components results in clear matrix degeneration and phenotypic changes in the resident cells , similar to what has been observed in monocultures of chondrocytes and osteoblasts . while chronic degeneration of the articular surface is most prevalent in oa , it is not clear whether alterations in the subchondral bone or articular cartilage is the primary trigger in oa . using the bioreactor in this study , we are able to study interactions between cartilage and bone that may contribute to oa progression clear osseous and chondral tissue interactions are observed when il-1 is applied to the osseous component , which results in low levels of anabolic gene expression ( sox9 , col2 , and aggrecan ) but robust expression of mmps in the cartilage component . conversely , application of il-1 to the cartilage induces in bone low levels of anabolic bone gene expression ( runx2 , opn , and bspii ) but robust expression of mmps . this apparently contradictory simultaneous induction of anabolic and catabolic processes within a tissue is entirely in keeping with the hypothesis that oa begins initially with a shift in the balance between anabolic and catabolic activities , followed by phenotypic changes in the cells in response to the modified environment . to some degree , inflammation can have beneficial effects on tissues , but at higher concentrations , inflammatory mediators induce tissue remodeling / destruction . focusing on the response of chondral component to il-1 treatment of the osseous component , it is interesting that direct application of il-1 to the chondral component has a less impressive catabolic response than indirect exposure via the osseous component . this result implies that the affected osteoblasts in the osseous component are producing bioactive factors , in addition to il-1 , that are causing greater catabolic responses than il-1 itself and vice versa . the formation of an ocj between regions of engineered cartilage and bone is often reported , but generally is not analyzed beyond histological identification . in our study , we have employed opposing chondrogenic and osteogenic nutrient gradients to stimulate ocj formation by nave , differentiating mscs . it is thus difficult to relate the potential tidemark development in our construct with frequently reported constructs that combine solid , porous polymeric sponges and hydrogels for osteochondral engineering in vitro or in vivo because of the great disparity in tissue architecture and scaffold biochemistry . tidemark development similar to what is seen here has been reported in studies employing microparticle - mediated spatially restricted growth factor release , microbead - encapsulated msc - derived chondrocytes and osteocytes , mscs encapsulated within scaffold material gradients , and mscs stimulated by growth factor gradients . in all cases , the basophilic tidemark is indistinct and broad , particularly in models without loading . we expect that appropriate mechanical loading and enhancement of cell differentiation , e.g. , with an oxygen gradient , may enhance the collection of metabolites to form the tidemark at the deep zone / calcified cartilage interface , particularly if differentiation is enhanced with an oxygen gradient and/or the addition of hydroxyapatite . while formation of ocj has been reported in in vivo implanted cell - seeded scaffold , there have been relatively few studies using a controlled bioreactor as reported here . the features of our bioreactor design , including separate compartments for the chondral and osseous microenvironments supplied by independent tissue - specific media that can be controlled and regulated via introductions of bioactive agents or candidate effecter cells , and capability of individual sampling of the different compartments , are thus of potential value in allowing more individual manipulations . specifically , we envision its application for the assessment of drug and environmental factor toxicity . we have postulated that catabolic insults to one tissue component comprising the osteochondral unit would influence the other in a manner reminiscent of tissue degeneration in oa . of particular interest is to investigate the communication of biomechanical signals / forces . to our knowledge , we are the first to provide evidence of communication between different compartments of an osteochondral construct in response to catabolic cues ( il-1 ) . the tissue responses reported here reflect a subset of pathophysiological conditions reported in in vitro and in vivo models of oa . il-1 is utilized in models of both rheumatoid and oa , with the effect of causing cartilage matrix breakdown and down regulation of cartilage matrix gene expression as shown here . in contrast , il-1 has been reported to induce increased bone matrix deposition , although it is a matrix of inferior quality , which may explain the response of the osseous component to direct exposure to il-1 in our model . the fact that catabolic gene expression in the osseous component is more enhanced by exposure of the overlying chondral component to il-1 suggests that the chondral construct is producing additional signals and catabolic factors that travel to and affect the osseous component below , possibly constituting a form of intercellular communication not previously reported . in summary , we have fabricated a new microfluidic - based , multichamber bioreactor for osteochondral differentiation and toxicity testing . we demonstrated clear biphasic tissue differentiation in response to opposing chondrogenic and osteogenic gradients produced by tissue - specific differentiation factors supplied by independent medium streams to the chondral and osseous components of the construct . finally we have shown that msc - based chondral and osseous tissues are capable of responding to il-1 in a relevant manner and that changes in one tissue compartment are communicated , and perhaps amplified , to the other along the osteochondral axis . this bioreactor / organotypic osteochondral culture combination will enable us to focus on the relationship of cartilage and bone in growth and degeneration and perhaps help to elucidate the roles of each tissue in oa . finally , with minimal modification and appropriate coupling , the bioreactor reported here can be adapted as a tissue - specific component of an interacting multitissue bioreactor platform to study systemic multitissue interactions .

osteoarthritis ( oa ) is a chronic degenerative disease of the articular joint that involves both bone and cartilage degenerative changes . an engineered osteochondral tissue within physiological conditions will be of significant utility in understanding the pathogenesis of oa and testing the efficacy of potential disease - modifying oa drugs ( dmoads ) . in this study , a multichamber bioreactor was fabricated and fitted into a microfluidic base . when the osteochondral construct is inserted , two chambers are formed on either side of the construct ( top , chondral ; bottom , osseous ) that is supplied by different medium streams . these medium conduits are critical to create tissue - specific microenvironments in which chondral and osseous tissues will develop and mature . human bone marrow stem cell ( hbmscs)-derived constructs were fabricated in situ and cultured within the bioreactor and induced to undergo spatially defined chondrogenic and osteogenic differentiation for 4 weeks in tissue - specific media . we observed tissue specific gene expression and matrix production as well as a basophilic interface suggesting a developing tidemark . introduction of interleukin-1 ( il-1 ) to either the chondral or osseous medium stream induced stronger degradative responses locally as well as in the opposing tissue type . for example , il-1 treatment of the osseous compartment resulted in a strong catabolic response in the chondral layer as indicated by increased matrix metalloproteinase ( mmp ) expression and activity , and tissue - specific gene expression . this induction was greater than that seen with il-1 application to the chondral component directly , indicative of active biochemical communication between the two tissue layers and supporting the osteochondral nature of oa . the microtissue culture system developed here offers novel capabilities for investigating the physiology of osteochondral tissue and pathogenic mechanisms of oa and serving as a high - throughput platform to test potential dmoads .

Introduction Experimental Section Results Discussion

osteoarthritis ( oa ) is a major cause of disability affecting millions of people worldwide . in the united states alone , to date there are no proven therapies for the prevention or treatment of oa . the lack of disease modifying oa drugs ( dmoads ) may be a function of incongruence between in vitro models of oa and the pathogenesis in vivo , and between disease mechanisms in humans and model animals . to overcome these issues , there is increasing momentum to develop human cell - based organotypic models in vitro that functionally represent the osteochondral tissue directly affected by oa . it is composed of distinct , interacting layers that include ( epi - to - diaphyseally ) deep zone cartilage , a basophilic tidemark , calcified cartilage , the cement line , and the subchondral bone plate . interestingly , there is growing evidence of significant biochemical communication between cartilage and bone across the ocj . in the pathogenesis of oa , changes in the physical linkage between cartilage and bone at the ocj are critical components of disease progression . these ocj changes accelerate cartilage degeneration and are associated with joint pain and disease morbidity , pointing to the need of a better understanding of the complex network of interactions between bone and cartilage in oa . progressive chronic destruction of articular cartilage is the most obvious characteristic of oa , and the etiology of the disease is believed to be at the intersection of genetics and abnormal mechanical forces . therefore , the primary locus of the disease is traditionally presumed to be the cartilage , and as a result , most in vitro oa models focus exclusively on cartilage to study oa disease mechanisms and therapeutic intervention . we theorize that the development of a model system of osteochondral tissue using human cells in a physiologically relevant environment that can accurately replicate in vivo osteochondral tissue homeostasis and pathophysiology will lead to greater predictive power in the development of dmoads . the challenges in developing such a system include : ( 1 ) mimicking or inducing production of appropriate extracellular matrix critical to the function of cartilage and bone , ( 2 ) replicating the tissue architecture , ( 3 ) reconciling the different growth and maintenance conditions of bone and cartilage while promoting their interaction with each other , and ( 4 ) replicating the biomechanical environment known to be essential to cartilage and bone health . current in vitro models to investigate bone - cartilage interactions are mostly limited to cell co - culture systems in which bone and cartilage cells are both exposed to the same medium , arguably a very distant condition from the in vivo environment . here , we report the development of a bioreactor designed to accommodate the biphasic nature of an osteochondral plug by creating two separate compartments for the chondral and osseous microenvironments . these are separated only by the tissue itself and are supplied by a microfluidic system . the central hypothesis of the study is that a gradient of tissue specific nutrients and conditions is required for the formation and maintenance of the osteochondral tissue . furthermore , we hypothesize that induction of an oa - like condition in the engineered osseous or the engineered chondral component alone will induce a corresponding oa - like response in the other component . to test these hypotheses , we have generated distinct chondral and osseous zones within the same construct by controlling the different media exposures within the bioreactor . then , we induced an oa - like response by exposing the osseous or chondral compartments to the pro - inflammatory cytokine ( il-1 ) and assayed the intervening changes in expression and secretion from both the engineered chondral and osseous components . the osteochondral construct within the insert creates the final separation between the upper and lower medium conduits . all experiments were performed with passage 3 ( p3 ) hbmscs from 3 patients ( 3 female patients 44 , 52 , and 72 years old ) , which were pooled for use in this study . because of the permeable nature of gelatin scaffold used as the scaffold model , leaking was assayed for 24 h only . osteochondral construct preparation : first , the insert was placed within a hollow cylindrical well to prevent suspension leaking from the pores in the insert . the second round of cross - linking had the added benefit of bonding the osseous and chondral layers together as well , and the fabrication of the osteochondral construct within the insert created a tight seal . after 4 weeks of differentiation , engineered osteochondral tissues were collected for validation using real - time pcr and histological analysis , or treated with il1 - 1. chondral and osseous constructs were collected separately . after 4 weeks of differentiation , engineered osteochondral constructs were treated with il-1 ( 10 ng / ml , r&d ) on the chondral or osseous sides only to investigate the cell / neo - tissue response to pro - inflammatory cytokines and possible communication through the osteochondral construct . the media used in this test were cm without tgf-3 ( chondral ) and om without bmp-2 ( osseous ) , both supplemented with 10 ng / ml il-1. after 7 days , the osteochondral constructs were bisected into the chondral and osseous halves and processed for gene expression analysis as described before . in addition to tissue specific gene expression , matrix metalloproteinase 1 , 3 , and 13 were also analyzed . media was collected separately from chondral and osseous constructs , cleared of cell debris via centrifugation ( 1000 g ) , and analyzed via il-1 ( abcam , cambridge , ma ) , mmp-1 ( r&d ) , mmp-3 ( abcam , cambridge , ma ) , and mmp-13 ( abcam ) elisas according to the manufacturers instructions . the robustness of the microbioreactor was tested by assessing the extent of leakage of two molecules perfused independently in the upper and lower medium conduits : ( 1 ) trypsin inhibitor ( 21 kda ) , with a molecular weight similar to the two commonly used osteoinductive ( bmp-2 , 26kd ) and chondroinductive ( tgf3 , 25kd ) factors , and ( 2 ) bsa ( 65kd ) , the most abundant protein in serum . the naive hbmscs seeded within tissue - specific scaffolds in the freshly fabricated osteochondral construct were induced to differentiate using cm in the top stream and om in the bottom stream . we anticipated chondrogenesis in the upper , chondral half of the construct and osteogenesis in the bottom , osseous half . after 4 weeks of differentiation , biphasic osteochondral constructs were produced ( figure 4c ) . as shown in figure 3 , cells in the chondral half showed enhanced expression of chondrogenic genes , including sox 9 , aggrecan , and collagen type ii as compared to those in the osseous half , while the cells in the osseous half had higher expression of osteogenic genes , including runx2 , osteocalcin , and bsp ii . taken together , these results strongly indicate a spatially defined , biphasic differentiation of these engineered osteochondral constructs , with the chondral component undergoing more characteristic differentiation . in addition , h&e staining revealed a distinct , < 100 m wide basophilic band in the interface between the chondral and osseous halves , potentially indicative of a developing tidemark ( figure 4a ) . after 4 weeks of culture in the bioreactor , osteochondral constructs were separated into chondral ( cartilage ) and osseous ( bone ) components , and each were analyzed for expression of cartilage ( sox9 , col2 , and aggrecan ) or bone ( runx2 , osteocalcin , bspii ) markers . expression of cartilage markers was found only in the chondral component , and bone markers in the osseous compartment . top , chondral component ( cc ) ; bottom , osseous component ( oc ) . as described above , the microtissue bioreactor presented here , with its two separate medium flow systems and biphasic construct compartments , has the capability for targeted treatment of one ( or both ) tissue construct(s ) with soluble factors . osseous and chondral components were separately treated with the pro - inflammatory cytokine il-1 ( 10 ng / ml ) for 7 days ( control conditions consisted of untreated osteochondral constructs ) , and the responses of each of the two components were separately analyzed . media samples were collected from chondral and osseous components streams at days 1 and 7 , and after day 7 , the osteochondral constructs were separated into osseous and chondral components , and each was separately analyzed for gene expression of catabolic genes ( mmp-1 , mmp-3 , and mmp-13 ) and either cartilage markers ( sox9 , col2 , and aggrecan ) ( figure 5 ) or bone markers ( runx2 , osteocalcin , and bspii ) ( figure 6 ) . media samples from the chondral and osseous components stream collected at days 1 and 7 were analyzed via mmp-1 , mmp-3 , and mmp-13 elisas ( figure 7 ) . effects of il-1 treatment on cartilage gene expression in the engineered osteochondral microtissue . effects of il-1 treatment on bone gene expression in the engineered osteochondral microtissue . effects of il-1 treatment on osseous and chondral mmp secretion in the engineered osteochondral microtissue . treatment of chondral constructs with il-1 caused decreases in expression of cartilage genes sox9 , col2 , and aggrecan , consistent with physiological outcomes of damaged or stressed cartilage ( figure 5 ) . chondral construct expression of these genes also decreased in response to il-1 treatment of osseous constructs , suggesting signaling between the osseous and chondral components . evidence of this osseous - to - chondral communication was even more apparent in results concerning expression of catabolic genes ; expression of mmp-1 , mmp-3 , and mmp-13 of the chondral constructs increased substantially in response to il-1 treatment of the osseous component . crosstalk between the two components was also detected in the case of chondral - to - osseous communication ( figure 6 ) . il-1 treatment of the chondral construct caused decreases in expression of the bone genes osteocalcin and bspii and increases in mmps production in the osseous construct , particularly mmp-13 , which is one of the most important mediators of oa cartilage degradation . elisa analysis of mmps secreted by the chondral and osseous components at different time points allowed for observations on the rate of signal propagation between the two components ( figure 7 ) . for example , the chondral construct responded to il-1 treatment of the osseous component with increases in mmp-1 , mmp-3 , and mmp-13 secretion . the osseous construct response to treatment of chondral component with il-1 , however , was quick yet increased further over time , and by day 7 was overall stronger than the chondral responses to the osseous component treatment . since chondrocytes are particularly sensitive to il-1 , this may explain why results concerning cells of the chondral component exhibit the greatest degree of inconsistency between pcr and elisa measurements when chondral constructs are directly stimulated by il-1. in this study , we have developed a novel bioreactor system for the engineering of osteochondral tissue . rt - pcr and histological analyses showed that hbmscs - derived nave constructs have been successfully differentiated into cartilage - like tissue on the top and bone - like tissue on the bottom , using separated culture medium for 4 weeks . in this study , a dual - chamber bioreactor has been developed to generate and maintain osteochondral constructs derived from human hbmscs . the design parameters included individual compartments to separate the chondral and osseous microenvironments that are individually accessible for the introduction of bioactive agents and/or candidate effector cells , tissue and medium sampling , and compositional assays , including noninvasive imaging techniques . furthermore , the total dimension and geometry of the bioreactor matches that of a multiwell culture plate chamber for the development of medium- to high - throughput analysis . validation of the system included successful , simultaneous differentiation of osseous and chondral constructs from hbmscs from the same source ( pooling of three donors ) and subsequent application of il-1 , a potent inflammatory mediator implicated in oa pathophysiology , to test the physiological response of the osteochondral construct . we have shown that in the course of 6 weeks , hbmscs undergo tissue - specific differentiation in response to the tissue specific growth media and hydrogel composition provided . most impressively , there was an indication of a basophilic , tidemark - like zone separating the chondral and osseous components . the test served two purposes : ( 1 ) to validate the utility of the bioreactor in osteochondral studies and ( 2 ) to assess the physiological replication of the oc tissue by the mscs in this bioreactor . il-1 is almost ubiquitous in inflammatory diseases , is prominent in advanced oa in both the cartilage and synovial lining , and is frequently employed as a pathogenic initiator in in vitro models of oa . while chronic degeneration of the articular surface is most prevalent in oa , it is not clear whether alterations in the subchondral bone or articular cartilage is the primary trigger in oa . using the bioreactor in this study , we are able to study interactions between cartilage and bone that may contribute to oa progression clear osseous and chondral tissue interactions are observed when il-1 is applied to the osseous component , which results in low levels of anabolic gene expression ( sox9 , col2 , and aggrecan ) but robust expression of mmps in the cartilage component . conversely , application of il-1 to the cartilage induces in bone low levels of anabolic bone gene expression ( runx2 , opn , and bspii ) but robust expression of mmps . this apparently contradictory simultaneous induction of anabolic and catabolic processes within a tissue is entirely in keeping with the hypothesis that oa begins initially with a shift in the balance between anabolic and catabolic activities , followed by phenotypic changes in the cells in response to the modified environment . focusing on the response of chondral component to il-1 treatment of the osseous component , it is interesting that direct application of il-1 to the chondral component has a less impressive catabolic response than indirect exposure via the osseous component . in our study , we have employed opposing chondrogenic and osteogenic nutrient gradients to stimulate ocj formation by nave , differentiating mscs . it is thus difficult to relate the potential tidemark development in our construct with frequently reported constructs that combine solid , porous polymeric sponges and hydrogels for osteochondral engineering in vitro or in vivo because of the great disparity in tissue architecture and scaffold biochemistry . the features of our bioreactor design , including separate compartments for the chondral and osseous microenvironments supplied by independent tissue - specific media that can be controlled and regulated via introductions of bioactive agents or candidate effecter cells , and capability of individual sampling of the different compartments , are thus of potential value in allowing more individual manipulations . we have postulated that catabolic insults to one tissue component comprising the osteochondral unit would influence the other in a manner reminiscent of tissue degeneration in oa . to our knowledge , we are the first to provide evidence of communication between different compartments of an osteochondral construct in response to catabolic cues ( il-1 ) . in contrast , il-1 has been reported to induce increased bone matrix deposition , although it is a matrix of inferior quality , which may explain the response of the osseous component to direct exposure to il-1 in our model . the fact that catabolic gene expression in the osseous component is more enhanced by exposure of the overlying chondral component to il-1 suggests that the chondral construct is producing additional signals and catabolic factors that travel to and affect the osseous component below , possibly constituting a form of intercellular communication not previously reported . we demonstrated clear biphasic tissue differentiation in response to opposing chondrogenic and osteogenic gradients produced by tissue - specific differentiation factors supplied by independent medium streams to the chondral and osseous components of the construct . finally we have shown that msc - based chondral and osseous tissues are capable of responding to il-1 in a relevant manner and that changes in one tissue compartment are communicated , and perhaps amplified , to the other along the osteochondral axis . finally , with minimal modification and appropriate coupling , the bioreactor reported here can be adapted as a tissue - specific component of an interacting multitissue bioreactor platform to study systemic multitissue interactions .

the lack of disease modifying oa drugs ( dmoads ) may be a function of incongruence between in vitro models of oa and the pathogenesis in vivo , and between disease mechanisms in humans and model animals . it is composed of distinct , interacting layers that include ( epi - to - diaphyseally ) deep zone cartilage , a basophilic tidemark , calcified cartilage , the cement line , and the subchondral bone plate . in the pathogenesis of oa , changes in the physical linkage between cartilage and bone at the ocj are critical components of disease progression . these include remodeling of the tidemark , microcracks , and fissures in both tissues and ingrowth from the underlying bone of blood vessels and nerves , all of which may enhance the cartilage - bone crosstalk allowing a better passage of growth factors , cytokines , and signaling molecules . these ocj changes accelerate cartilage degeneration and are associated with joint pain and disease morbidity , pointing to the need of a better understanding of the complex network of interactions between bone and cartilage in oa . progressive chronic destruction of articular cartilage is the most obvious characteristic of oa , and the etiology of the disease is believed to be at the intersection of genetics and abnormal mechanical forces . therefore , the primary locus of the disease is traditionally presumed to be the cartilage , and as a result , most in vitro oa models focus exclusively on cartilage to study oa disease mechanisms and therapeutic intervention . however , there is increasing evidence from in vivo and clinical studies that subchondral bone lesions may precede cartilage degeneration , implying that oa is an osteochondral disease and possibly bone dependent . we theorize that the development of a model system of osteochondral tissue using human cells in a physiologically relevant environment that can accurately replicate in vivo osteochondral tissue homeostasis and pathophysiology will lead to greater predictive power in the development of dmoads . the challenges in developing such a system include : ( 1 ) mimicking or inducing production of appropriate extracellular matrix critical to the function of cartilage and bone , ( 2 ) replicating the tissue architecture , ( 3 ) reconciling the different growth and maintenance conditions of bone and cartilage while promoting their interaction with each other , and ( 4 ) replicating the biomechanical environment known to be essential to cartilage and bone health . current in vitro models to investigate bone - cartilage interactions are mostly limited to cell co - culture systems in which bone and cartilage cells are both exposed to the same medium , arguably a very distant condition from the in vivo environment . here , we report the development of a bioreactor designed to accommodate the biphasic nature of an osteochondral plug by creating two separate compartments for the chondral and osseous microenvironments . furthermore , we hypothesize that induction of an oa - like condition in the engineered osseous or the engineered chondral component alone will induce a corresponding oa - like response in the other component . then , we induced an oa - like response by exposing the osseous or chondral compartments to the pro - inflammatory cytokine ( il-1 ) and assayed the intervening changes in expression and secretion from both the engineered chondral and osseous components . after the supernatant was discarded , the pellets were suspended using growth medium ( gm , -mem containing 10% fetal bovine serum ( fbs , invitrogen ) , 1% antibiotics - antimycotic , and 1.5 ng / ml fgf-2 ( raybiotech , norcross , ga ) ) , and then plated into 150 cm tissue culture flasks at a density of 20,00040,000 nucleated cells / cm , and medium was changed every 3 to 4 days . to test medium leakage between ( 1 ) the chamber wall and the insert and ( 2 ) the insert and scaffold material , the insert was filled with 10% mgl/0.15% lap in hbss and alexa fluor 488-conjugated soybean trypsin inhibitor ( ti488 , 21kd , molecular probes , ca ) and alexa fluor 555-conjugated albumin from bovine serum ( bsa ) ( bsa555 , 65kd , molecular probes ) were diluted in hbss individually at 10 g / ml and then perfused through the top and bottom of bioreactor , respectively , at 1 l / min . the second round of cross - linking had the added benefit of bonding the osseous and chondral layers together as well , and the fabrication of the osteochondral construct within the insert created a tight seal . the following formulas were used for the differentiation media : om ( gm supplemented with 10 ng / ml bmp-2 ( peprotech , rocky hill , nj ) , 1% l - alanyl - l - glutamine ( glutamax ) , 10 nm dexamethasone ( dex ) , 0.1 mm l - ascorbic acid 2-phosphate ( asa2-p ) , and 10 mm beta - glycerophosphate ( -gp ) ; cm ( dmem supplemented with 10 ng / ml tgf-3 ( peprotech ) , 1% its , 50 m asa2-p , 55 m sodium pyruvate , and 23 m l - proline ) . aggrecan ( acn ) , collagen type ii ( col2 ) , runt - related transcription factor 2 ( runx2 ) , osteocalcin ( ocn ) , bone sialoprotein ii ( bsp ii ) , and 18s rrna ( 18s ) . after 4 weeks of differentiation , engineered osteochondral constructs were treated with il-1 ( 10 ng / ml , r&d ) on the chondral or osseous sides only to investigate the cell / neo - tissue response to pro - inflammatory cytokines and possible communication through the osteochondral construct . there were three experimental groups : ( 1 ) cm / om , ( 2 ) cm + il1 - 1/om , and ( 3 ) cm / om + il1 - 1. the treatment lasted 7 days , with effluent medium collected and frozen at 1 and 7 days for elisa . media was collected separately from chondral and osseous constructs , cleared of cell debris via centrifugation ( 1000 g ) , and analyzed via il-1 ( abcam , cambridge , ma ) , mmp-1 ( r&d ) , mmp-3 ( abcam , cambridge , ma ) , and mmp-13 ( abcam ) elisas according to the manufacturers instructions . the robustness of the microbioreactor was tested by assessing the extent of leakage of two molecules perfused independently in the upper and lower medium conduits : ( 1 ) trypsin inhibitor ( 21 kda ) , with a molecular weight similar to the two commonly used osteoinductive ( bmp-2 , 26kd ) and chondroinductive ( tgf3 , 25kd ) factors , and ( 2 ) bsa ( 65kd ) , the most abundant protein in serum . as shown in figure 2a , after 24 h of perfusion , the extent of mixing between top and bottom was < 1% , indicating there was minimal medium exchange through the interfaces between the chamber wall and the inset and between the inset and scaffold - only construct . ( a ) trypsin inhibitor-488 and bsa-555 were simultaneously perfused through the top and bottom space of bioreactor , respectively , and the percent leakage at different time was estimated based on the bottom / top ratio of 488 nm fluorescence readings ( bottom / top 488 ) and top / bottom 555 nm fluorescence readings ( top / bottom 555 ) . as shown in figure 3 , cells in the chondral half showed enhanced expression of chondrogenic genes , including sox 9 , aggrecan , and collagen type ii as compared to those in the osseous half , while the cells in the osseous half had higher expression of osteogenic genes , including runx2 , osteocalcin , and bsp ii . taken together , these results strongly indicate a spatially defined , biphasic differentiation of these engineered osteochondral constructs , with the chondral component undergoing more characteristic differentiation . in addition , h&e staining revealed a distinct , < 100 m wide basophilic band in the interface between the chondral and osseous halves , potentially indicative of a developing tidemark ( figure 4a ) . after 4 weeks of culture in the bioreactor , osteochondral constructs were separated into chondral ( cartilage ) and osseous ( bone ) components , and each were analyzed for expression of cartilage ( sox9 , col2 , and aggrecan ) or bone ( runx2 , osteocalcin , bspii ) markers . osseous and chondral components were separately treated with the pro - inflammatory cytokine il-1 ( 10 ng / ml ) for 7 days ( control conditions consisted of untreated osteochondral constructs ) , and the responses of each of the two components were separately analyzed . media samples were collected from chondral and osseous components streams at days 1 and 7 , and after day 7 , the osteochondral constructs were separated into osseous and chondral components , and each was separately analyzed for gene expression of catabolic genes ( mmp-1 , mmp-3 , and mmp-13 ) and either cartilage markers ( sox9 , col2 , and aggrecan ) ( figure 5 ) or bone markers ( runx2 , osteocalcin , and bspii ) ( figure 6 ) . after treatment of either osseous ( bone ) or chondral ( cartilage ) component with 10 ng / ml il-1 for 1 or 7 days , medium samples collected from the bone or cartilage medium compartment were analyzed by elisa for the levels of secreted mmp-1 , mmp-3 , and mmp-13 . treatment of chondral constructs with il-1 caused decreases in expression of cartilage genes sox9 , col2 , and aggrecan , consistent with physiological outcomes of damaged or stressed cartilage ( figure 5 ) . evidence of this osseous - to - chondral communication was even more apparent in results concerning expression of catabolic genes ; expression of mmp-1 , mmp-3 , and mmp-13 of the chondral constructs increased substantially in response to il-1 treatment of the osseous component . il-1 treatment of the chondral construct caused decreases in expression of the bone genes osteocalcin and bspii and increases in mmps production in the osseous construct , particularly mmp-13 , which is one of the most important mediators of oa cartilage degradation . the osseous construct response to treatment of chondral component with il-1 , however , was quick yet increased further over time , and by day 7 was overall stronger than the chondral responses to the osseous component treatment . since chondrocytes are particularly sensitive to il-1 , this may explain why results concerning cells of the chondral component exhibit the greatest degree of inconsistency between pcr and elisa measurements when chondral constructs are directly stimulated by il-1. rt - pcr and histological analyses showed that hbmscs - derived nave constructs have been successfully differentiated into cartilage - like tissue on the top and bone - like tissue on the bottom , using separated culture medium for 4 weeks . interestingly , the il-1 insulted osseous construct induces a catabolic gene expression response into the untreated chondral component , which is not due to leakage of il-1 , suggesting active osseous chondral interaction and the likely importance of bone injury in oa development . the design parameters included individual compartments to separate the chondral and osseous microenvironments that are individually accessible for the introduction of bioactive agents and/or candidate effector cells , tissue and medium sampling , and compositional assays , including noninvasive imaging techniques . validation of the system included successful , simultaneous differentiation of osseous and chondral constructs from hbmscs from the same source ( pooling of three donors ) and subsequent application of il-1 , a potent inflammatory mediator implicated in oa pathophysiology , to test the physiological response of the osteochondral construct . we have shown that in the course of 6 weeks , hbmscs undergo tissue - specific differentiation in response to the tissue specific growth media and hydrogel composition provided . the recreation of the a tidemark - containing biphasic tissue is vital to drug testing using an osteochondral organotypic culture since changes in the ocj are mechanistically involved in oa progression and likely to be a target of toxicants and dmoads . the test served two purposes : ( 1 ) to validate the utility of the bioreactor in osteochondral studies and ( 2 ) to assess the physiological replication of the oc tissue by the mscs in this bioreactor . il-1 is almost ubiquitous in inflammatory diseases , is prominent in advanced oa in both the cartilage and synovial lining , and is frequently employed as a pathogenic initiator in in vitro models of oa . application of il-1 to both osseous and chondral components results in clear matrix degeneration and phenotypic changes in the resident cells , similar to what has been observed in monocultures of chondrocytes and osteoblasts . while chronic degeneration of the articular surface is most prevalent in oa , it is not clear whether alterations in the subchondral bone or articular cartilage is the primary trigger in oa . using the bioreactor in this study , we are able to study interactions between cartilage and bone that may contribute to oa progression clear osseous and chondral tissue interactions are observed when il-1 is applied to the osseous component , which results in low levels of anabolic gene expression ( sox9 , col2 , and aggrecan ) but robust expression of mmps in the cartilage component . conversely , application of il-1 to the cartilage induces in bone low levels of anabolic bone gene expression ( runx2 , opn , and bspii ) but robust expression of mmps . this apparently contradictory simultaneous induction of anabolic and catabolic processes within a tissue is entirely in keeping with the hypothesis that oa begins initially with a shift in the balance between anabolic and catabolic activities , followed by phenotypic changes in the cells in response to the modified environment . focusing on the response of chondral component to il-1 treatment of the osseous component , it is interesting that direct application of il-1 to the chondral component has a less impressive catabolic response than indirect exposure via the osseous component . this result implies that the affected osteoblasts in the osseous component are producing bioactive factors , in addition to il-1 , that are causing greater catabolic responses than il-1 itself and vice versa . it is thus difficult to relate the potential tidemark development in our construct with frequently reported constructs that combine solid , porous polymeric sponges and hydrogels for osteochondral engineering in vitro or in vivo because of the great disparity in tissue architecture and scaffold biochemistry . tidemark development similar to what is seen here has been reported in studies employing microparticle - mediated spatially restricted growth factor release , microbead - encapsulated msc - derived chondrocytes and osteocytes , mscs encapsulated within scaffold material gradients , and mscs stimulated by growth factor gradients . while formation of ocj has been reported in in vivo implanted cell - seeded scaffold , there have been relatively few studies using a controlled bioreactor as reported here . the features of our bioreactor design , including separate compartments for the chondral and osseous microenvironments supplied by independent tissue - specific media that can be controlled and regulated via introductions of bioactive agents or candidate effecter cells , and capability of individual sampling of the different compartments , are thus of potential value in allowing more individual manipulations . to our knowledge , we are the first to provide evidence of communication between different compartments of an osteochondral construct in response to catabolic cues ( il-1 ) . the fact that catabolic gene expression in the osseous component is more enhanced by exposure of the overlying chondral component to il-1 suggests that the chondral construct is producing additional signals and catabolic factors that travel to and affect the osseous component below , possibly constituting a form of intercellular communication not previously reported . finally we have shown that msc - based chondral and osseous tissues are capable of responding to il-1 in a relevant manner and that changes in one tissue compartment are communicated , and perhaps amplified , to the other along the osteochondral axis . this bioreactor / organotypic osteochondral culture combination will enable us to focus on the relationship of cartilage and bone in growth and degeneration and perhaps help to elucidate the roles of each tissue in oa .

enantioseparation ( es ) and enantiodiscrimination ( ed ) are , therefore , mandatory steps in the elucidation of enantiospecific biological functions on the molecular level . interestingly , ed with isotropic solution nmr was first demonstrated by pirkle,1 who also developed enantioseparating chromatographic phases.2 however , since these times , the two areas have evolved increasingly independently . although nmr studies are used to elucidate structural aspects of es,3a the role of enantioselective chromatography as a versatile tool in the understanding of ed obtained in nmr has been largely neglected . since the early report of pirkle and pochapsky in 1986,3b isotropic solutionnmr studies have been used to investigate structural aspects of chromatographic enantiorecognition processes.3c,3d for this purpose , chemical shifts and nuclear overhauser effects ( noes ) were largely used . one recent approach for ed in nmr is the use of chiral anisotropic media.4 in general , anisotropic achiral media permit the measurement of nmr parameters that require a partial alignment of the solute ( e.g. residual dipolar couplings or chemicalshift anisotropies ) . currently , a number of new alignment media that have been developed are becoming very important in the structural analysis of smalltolarge molecules.5 crosslinked acrylamidebased copolymers introduced in 20006a have been further developed into an alignment medium compatible with a wide range of solvents including dmso.6b recently , ed was reached by introducing a chiral , negatively charged monomer : ( r)aphes [ ( 2acrylamide)2phenylethanesulfonic acid , scheme s1 a].7 as expected from work with chiral ionexchange phases,8 ed was only reached for cationic ( protonated basic ) solutes ( erythromefloquine , menthylamine , and strychnine ) . no ed was observed for the neutral compound menthol.7 this approach had been inspired by previous work in the field of enantioselective ( chiral ) liquidphase chromatography . enantioselective chromatographic separation is based on the difference in the retention factor for a pair of enantiomers induced by a small difference in the phasetransfer equilibrium constants , owing to the involvement of diastereomeric associations ( between the chiral solute and the chiral selector ) in the phasetransfer process.8a a special class of stationary phases developed for enantioselective liquidphase chromatography ( chiral ionexchange phases ) employs positively and/or negatively charged immobilized chiral selectors , which can undergo the formation of diastereomeric associates ( ion pairs ) with protonated or deprotonated ( ionisable ) chiral solutes.8b , 8c it is now of interest to investigate whether or not the successful ed observed with a chiral alignment gel used for anisonmr ( nmr spectroscopy under anisotropic conditions ) can be further elucidated by using chromatographic studies employing the same alignment gel as a chiral separation medium in capillary electrochromatography ( cec ) ( figure 1 , and explanations given therein ) . for clarity , this approach should be clearly distinguished from techniques such as nmrce , capillary nmr , and chromatographic nmr spectroscopy.9 left side : schematic capillary / nmr tube cross section used for isotropic gelbased capillary electrochromatography ( cec ; upper part with isotropic mesh openings ) , and nmr under anisotropic conditions ( anisonmr ; lower part with anisotropic mesh openings ) . right side : electrochromatogram obtained by cec ( upper part ) , and twodimensional nmr spectrum with split signals attribtued to anisotropic alignment of the analyte ( lower part ) . ideally , for thermodynamic studies , the separation medium investigated through chromatographic studies should be identical to the alignment medium used for anisonmr . many alignment media ( e.g. bicelles , phages ) , however , are incompatible with cec instrumentation , whereas homogeneous gels are separation media in plate gel electrophoresis ( pge ) , in capillary gel electrophoresis ( cge ) , and in gelbased capillary electrochromatography ( gbcec).10 separation in pge and cge is based on differences in the migration velocity of the solute , induced by differences in the effective electrophoretic mobility of the solutes and the sieving effect . separation in gbcec ( in analogy to chromatography ) , however , is based on differences in the equilibrium constant characterizing the transfer of the solute from the electrokinetic transport zone ( etz , where the velocity of the solute is given by the electrophoretic velocity of this solute and the electroosmotic velocity ) into the interaction zone ( iz , where the velocity of the solute is zero ) . we describe this equilibrium as a distribution process , although the highly swollen gel employed in gbcec is a homogenous phase ( in contrast to the system of two interpenetrating continuous phases characteristic for cec with a monolith ) . gbcec was introduced by fujimoto,10a using charged amphiphilic polyacrylamide gels for the separation of uncharged lowmolecularweight compounds . gbcec with hydrophilic or amphiphilic gels with embedded or immobilized chiral selectors was also successfully applied to the separation of different enantiomers.10c10 g returning to previous results,7 we selected the synthetic antimalaria drug mefloquine ( figure 2 ) with two stereocenters as the test solute.11a in recent years , the correct absolute configurations of the enantiomers of the erythro form , which is administered as the racemate , and of the enantiomers of the threo form , have been subject to debate . they were not reliably established until 20122013.11b11f in contrast to these difficulties , es of the four stereoisomers had been successfully achieved much earlier by using capillary electrophoresis ( ce ) with cyclodextrin derivatives or other native cyclodextrins as chiral selectors,12 and by using highperformance liquid chromatography ( hplc ) either with amylose tris3,5dimethylphenyl carbamatecoated silica gel13 or with a quinidinebased zwitterionic chiral stationary phase ( chiral selector covalently anchored to the chemically modified silica gel).14 structural formulae of a ) ( + ) erythromefloquine [ ( + ) emq ] , b ) ( )erythromefloquine [ ( )emq ] , c ) ( + ) threomefloquine [ ( + ) tmq ] , and d ) ( )threomefloquine [ ( )tmq ] . against this background , in the present work , we study the chromatographic separation of the four stereoisomers of mefloquine [ ( )threomefloquine ( tmq ) and ( )erythromefloquine ( emq ) ] by using gbcec , employing the same polymeric gel as the chiral separation medium as that already successfully taken for the ed of emq through anisonmr . the retention data gained by using gbcec will permit the determination of the ratio of ( distribution ) equilibrium constants . with this ratio , we confirm those parameters that have been calculated in the interpretation of previously reported anisotropic nmr data.7 with all types of gels obtained by varying the solvent composition , a good diastereoselective and enantioselective separation of the four mefloquine stereoisomers was achieved ( figure 3 ) . although the peak of propranolol , taken as a marker of the holdup time ( see the supporting information ) , can be characterized to be symmetric , the peaks for the more retarded stereoisomers of mefloquine show a characteristic ( rightskewed ) triangular broadened shape , which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . separation of the four stereoisomers of mefloquine by gbcec with alignment gel as the separation medium with superimposed scaled trace for ( )propranolol . peak assignment : 1 ) ( )propranolol ; 2 , 3 ) ( )threomefloquine ; 4 ) ( )erythromefloquine ; 5 ) ( + ) erythromefloquine ; mobile phase = methanol / water ( 50:50 , v / v ) buffered with triethylamine / acetic acid , ph*=6.66 , electric conductivity=125 s cm , capillary dimensions=173 mm ( 102 mm)100 m , photometric ingel detection=283 nm , electrokinetic injection=2 kv2 s , separation voltage=2.0 kv , c ( analyte in sample)=0.25 g l ( analyte dissolved in mobile phase ) . we confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method , which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths ( see the supporting information ) . it should be noted that , in gbcec , the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . the depicted peak distortions ( figure 3 ) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase ( ionexchange cec).15 with analogously synthesized monolithic separation capillaries [ employing ( r)aphes as negatively charged comonomer ] synthesized for comparison purposes under phaseseparation conditions ( see the supporting information ) , leftskewed and rightskewed peaks can be obtained , dependent on the separation conditions ( figures s1 a and s1 b ) . under the conditions selected for recording the chromatogram depicted in figure 4 ( see legend ) , peak broadening by electromigration dispersion is absent and a higher peak efficiency is obtained . under these conditions , the method enabled baseline separation of the enantiomers of emq ( figure 4 ) . spiking the sample with ( + ) emq hcl revealed a higher retention factor for ( + ) emq than for ( )emq ( figure s2 ) . repeatability of the data was confirmed by sequential runs ( figure s3s5 ) . for five repeated runs , the relative standard deviations of the observed mobilities are 2.02.3 % ( table s1 ) . this corresponds to a relative confidence range of the mean of 2.52.9 % ( p=0.95 , twotailed test ) . separation of the enantiomers of erythromefloquine by gbcec with alignment gel as the separation medium . peak assignment : 1 ) ( )erythromefloquine ; 2 ) ( + ) erythromefloquine ; mobile phase = methanol buffered with 25 mm acetic acid and 2.66 mm triethylamine , ph*=4.46 , electric conductivity=55 s cm , capillary dimensions=234 mm ( 167 mm)100 m , photometric ingel detection=283 nm , electrokinetic injection=2.5 kv2 s , separation voltage=6.1 kv , c ( analyte in sample)=0.25 g l ( analyte dissolved in mobile phase ) . following two different approaches ( see the supporting information ) , we estimated the chromatographic separation factor ( = ratio of the two retention factors ) for tmq to be 1.08 and for emq to be 1.10 . these two factors correspond to the ratio of the two equilibrium constants related to the transfer from the electrokinetic transport zone to the interaction zone . hence , the energetic differences g for the involved ( distribution ) equilibria are 190 and 230 j mol for tmq and emq , respectively . taking propranolol as a nonretarded marker with an effective electrophoretic mobility similar to that of tmq and emq ( figure 3 and scheme s1 b ) , we obtained the following retention factors : 0.41 and 0.44 for the two enantiomers of tmq , 0.73 for ( )emq , and 0.80 for ( + ) emq . a larger difference in retention factors was obtained for the separation of tmq and emq , which corresponds to g=1.7 kj mol . under isotropic conditions , anisotropic parameters such as dipolar couplings between two nmrsensitive nuclei are averaged to zero.16a socalled alignment media lead to a low degree of orientation and reintroduce observable couplings . by using only a weak alignment , the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz ( residual dipolar couplings).16b with a set of experimentally determined rdcs , an alignment tensor ( a matrix with five independent elements ) can be calculated , which describes the degree and orientation of the alignment . in 2001 , the concept of the generalized degree of order ( gdo ) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor ( or the order tensor ) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments , as it is a quantity that is only dependent on the extent of dynamic averaging , arising from both overall alignment effects and internal motional effects ( the latter being neglected with rigid molecules).16a , 17 for ( )emq [ in the gel prepared with ( r)aphes ] gdo=5.1910 , which is smaller than for ( + ) emq [ gdo=5.8510 ; absolute difference : 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel ( being identical with a less stable diastereomeric association ) , the determined gdos correctly predict that ( )emq elutes first in a chromatographic separation . the significance of this result was confirmed by a duplicate determination of gdo for ( )emq.7 gdo1 = 5.2510 and gdo2 = 5.1310 , resulting in a difference of 0.1210 . the confidence range of the arithmetic mean ( p=0.95 , onetailed test ) is 0.3810 , which is smaller than the difference between the values for the two isomers . with the aim to identify fragments of a protein , where motion has an effect on the accuracy of structure determination , tolman et al.17 defined a fragmentspecific internal gdo as the ratio of the observed fragment gdo to the alignment tensor gdo . we propose here the enantioselectivity parameter ( = gdo ratio ) , referring to a nonracemic chiral alignment medium and a specific enantiomer pair . this parameter is quantified by determining the ratio of the ( higher ) gdo for enantiomer 1 to the ( smaller ) gdo for enantiomer 2 ( with 1 ) . in the present case , (emq)=gdo / gdo=1.13 . it is interesting to compare this value to the selectivity factor ( = k / k ) obtained for ( )emq by gbcec , which is 1.10 . both methods agree in terms of the sign [ ( + ) > ( ) ] and magnitude of the quantity describing ed / es , which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter ( here : rdc ) . based on the retention factors determined by gbcec , it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . this degree of transfer is quantified by the molar fraction x iz = n iz/(n it is directly accessible from chromatographic data through x iz = k/(1+k ) . for the different stereoisomers of mefloquine , it represents the fraction of those molecules being associated with the charged moieties of the crosslinked polymer acting as interaction sites . although n total ( = n etz+n iz ) is kept constant , the values for x iz are 0.29 and 0.31 for the two enantiomers of tmq , 0.42 for ( )emq , and 0.44 for ( + ) emq . with all types of gels obtained by varying the solvent composition , a good diastereoselective and enantioselective separation of the four mefloquine stereoisomers was achieved ( figure 3 ) . although the peak of propranolol , taken as a marker of the holdup time ( see the supporting information ) , can be characterized to be symmetric , the peaks for the more retarded stereoisomers of mefloquine show a characteristic ( rightskewed ) triangular broadened shape , which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . separation of the four stereoisomers of mefloquine by gbcec with alignment gel as the separation medium with superimposed scaled trace for ( )propranolol . peak assignment : 1 ) ( )propranolol ; 2 , 3 ) ( )threomefloquine ; 4 ) ( )erythromefloquine ; 5 ) ( + ) erythromefloquine ; mobile phase = methanol / water ( 50:50 , v / v ) buffered with triethylamine / acetic acid , ph*=6.66 , electric conductivity=125 s cm , capillary dimensions=173 mm ( 102 mm)100 m , photometric ingel detection=283 nm , electrokinetic injection=2 kv2 s , separation voltage=2.0 kv , c ( analyte in sample)=0.25 g l ( analyte dissolved in mobile phase ) . we confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method , which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths ( see the supporting information ) . it should be noted that , in gbcec , the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . the depicted peak distortions ( figure 3 ) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase ( ionexchange cec).15 with analogously synthesized monolithic separation capillaries [ employing ( r)aphes as negatively charged comonomer ] synthesized for comparison purposes under phaseseparation conditions ( see the supporting information ) , leftskewed and rightskewed peaks can be obtained , dependent on the separation conditions ( figures s1 a and s1 b ) . under the conditions selected for recording the chromatogram depicted in figure 4 ( see legend ) , peak broadening by electromigration dispersion is absent and a higher peak efficiency is obtained . under these conditions , the method enabled baseline separation of the enantiomers of emq ( figure 4 ) . spiking the sample with ( + ) emq hcl revealed a higher retention factor for ( + ) emq than for ( )emq ( figure s2 ) . repeatability of the data was confirmed by sequential runs ( figure s3s5 ) . for five repeated runs , the relative standard deviations of the observed mobilities are 2.02.3 % ( table s1 ) . this corresponds to a relative confidence range of the mean of 2.52.9 % ( p=0.95 , twotailed test ) . separation of the enantiomers of erythromefloquine by gbcec with alignment gel as the separation medium . peak assignment : 1 ) ( )erythromefloquine ; 2 ) ( + ) erythromefloquine ; mobile phase = methanol buffered with 25 mm acetic acid and 2.66 mm triethylamine , ph*=4.46 , electric conductivity=55 s cm , capillary dimensions=234 mm ( 167 mm)100 m , photometric ingel detection=283 nm , electrokinetic injection=2.5 kv2 s , separation voltage=6.1 kv , c ( analyte in sample)=0.25 g l ( analyte dissolved in mobile phase ) . following two different approaches ( see the supporting information ) , we estimated the chromatographic separation factor ( = ratio of the two retention factors ) for tmq to be 1.08 and for emq to be 1.10 . these two factors correspond to the ratio of the two equilibrium constants related to the transfer from the electrokinetic transport zone to the interaction zone . hence , the energetic differences g for the involved ( distribution ) equilibria are 190 and 230 j mol for tmq and emq , respectively . taking propranolol as a nonretarded marker with an effective electrophoretic mobility similar to that of tmq and emq ( figure 3 and scheme s1 b ) , we obtained the following retention factors : 0.41 and 0.44 for the two enantiomers of tmq , 0.73 for ( )emq , and 0.80 for ( + ) emq . a larger difference in retention factors was obtained for the separation of tmq and emq , which corresponds to g=1.7 kj mol . under isotropic conditions , anisotropic parameters such as dipolar couplings between two nmrsensitive nuclei are averaged to zero.16a socalled alignment media lead to a low degree of orientation and reintroduce observable couplings . by using only a weak alignment , the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz ( residual dipolar couplings).16b with a set of experimentally determined rdcs , an alignment tensor ( a matrix with five independent elements ) can be calculated , which describes the degree and orientation of the alignment . in 2001 , the concept of the generalized degree of order ( gdo ) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor ( or the order tensor ) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments , as it is a quantity that is only dependent on the extent of dynamic averaging , arising from both overall alignment effects and internal motional effects ( the latter being neglected with rigid molecules).16a , 17 for ( )emq [ in the gel prepared with ( r)aphes ] gdo=5.1910 , which is smaller than for ( + ) emq [ gdo=5.8510 ; absolute difference : 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel ( being identical with a less stable diastereomeric association ) , the determined gdos correctly predict that ( )emq elutes first in a chromatographic separation . the significance of this result was confirmed by a duplicate determination of gdo for ( )emq.7 gdo1 = 5.2510 and gdo2 = 5.1310 , resulting in a difference of 0.1210 . the confidence range of the arithmetic mean ( p=0.95 , onetailed test ) is 0.3810 , which is smaller than the difference between the values for the two isomers . with the aim to identify fragments of a protein , where motion has an effect on the accuracy of structure determination , tolman et al.17 defined a fragmentspecific internal gdo as the ratio of the observed fragment gdo to the alignment tensor gdo . we propose here the enantioselectivity parameter ( = gdo ratio ) , referring to a nonracemic chiral alignment medium and a specific enantiomer pair . this parameter is quantified by determining the ratio of the ( higher ) gdo for enantiomer 1 to the ( smaller ) gdo for enantiomer 2 ( with 1 ) . in the present case , (emq)=gdo / gdo=1.13 . it is interesting to compare this value to the selectivity factor ( = k / k ) obtained for ( )emq by gbcec , which is 1.10 . both methods agree in terms of the sign [ ( + ) > ( ) ] and magnitude of the quantity describing ed / es , which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter ( here : rdc ) . based on the retention factors determined by gbcec , it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . this degree of transfer is quantified by the molar fraction x iz = n iz/(n etz+n iz ) . it is directly accessible from chromatographic data through x iz = k/(1+k ) . for the different stereoisomers of mefloquine , it represents the fraction of those molecules being associated with the charged moieties of the crosslinked polymer acting as interaction sites . although n total ( = n etz+n iz ) is kept constant , the values for x iz are 0.29 and 0.31 for the two enantiomers of tmq , 0.42 for ( )emq , and 0.44 for ( + ) emq . taking a chiral alignment medium as the chiral separation medium , gbcec separates the four stereoisomers of mefloquine . this separation confirms our previous hypothesis that ed with this gel is based on the formation of localized diastereomeric associates between the charged solute and the oppositely charged moiety of the anisotropically deformed gel.7 consequently , ed is attributed to differences in the equilibrium constants related to associate formation . through gbcec , retention data are obtained that permit the calculation of the degrees of transfer of the solute from the electrokinetic transport zone into the interaction zone and the precise quantification of the gibbs energy difference regarding the two diastereomeric associates involved in the enantioselective separation process . hence , our approach provides physicochemical data that are important in the understanding and optimization of alignment processes.18 the capillary pretreatment procedure and the subsequent in situ synthesis of the polymeric gel are described in detail in the supporting information . briefly , the separation capillaries are treated with a 30 % ( v / v ) solution of 3(trimethoxysilyl ) propylmethacrylate ( bind silane ) in acetone to introduce vinylic anchoring groups to the inner wall of the fused silica capillary . these vinylic anchoring groups enable the covalent attachment of the synthesized gel to the inner capillary wall , which is a prerequisite to obtain homogeneously filled capillaries . subsequently , the chiral monomer ( 2acrylamide)2phenylethanesulfonic acid [ ( r)aphes ] , is copolymerized with n , ndimethylacrylamide ( dmaa ) and n , nmethylenebisacrylamide ( bis ) through a freesolution radical copolymerization ( 15 min at 70 c ) . the preparation procedure of the gel within the capillary is identical ( with respect to concentration of monomers , reaction time , reaction temperature ) to the one used for the measurement of anisonmr parameters with the exception of the absence or presence of a washing step ( removal of nonreacted monomers ) , an additional drying and swelling step ( in the case of anisonmr ) , and the solvent composition.7 although dmso was employed for anisonmr , the solvent is either methanol or methanol / water [ ( 50:50 , v / v ) or ( 25:75 , v / v ) ] in gbcec ( after equilibration ) . previous studies , however , with either methanol or dmso as the solvent demonstrated that the type of solvent has only a minor influence on the determined anisonmr parameters.19 in gbcec , the separation gel is buffered with triethylamine / acetic acid , ph*=4.56.7 , whereas in anisonmr all experiments were performed in unbuffered gels . the influence of the electrolyte concentration on the alignment properties of a very similar achiral polyacrylamide gel [ aphes replaced by 2(acrylamide)2methylpropanesulfonic acid ( amps ) ] was determined by trigomourio et al.20 according to these results , we can assume a negligible influence of the selected buffer concentration ( 2.7 mm ) on alignment properties and retention behavior . ( for details refer to the supporting information).21 a scheme of the apparatus allowing ingel detection at 283 nm is shown in scheme s2 . 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 reorganized for online delivery , but are not copyedited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .

abstractenantiodiscrimination and enantioseparation are two highly important processes in chemistry , often performed by using nmr spectroscopy and chromatography . for a better understanding of the mechanistic details , the same system should be studied by both methods . in addition , isotropic and anisotropic nmr parameters should be obtained , the latter using alignment media so that residual dipolar couplings and chemicalshift anisotropies can be measured . consequently , a chiral alignment medium was used for the first time in chiral gelbased capillary electrochromatography with the four stereoisomers of the antimalaria drug mefloquine as test compounds . chromatographic data verify that enantiodiscrimination obtained with this alignment gel is caused by differences in the equilibrium constants related to associate formation . hence , the chromatographic separation provides physicochemical data that form a basis for the understanding and optimization of alignment processes , and vice versa .

Introduction Results and Discussion Chromatographic Separation Selectivity Factor Generalized Degree of Order Conclusions Experimental Section Supporting information

although nmr studies are used to elucidate structural aspects of es,3a the role of enantioselective chromatography as a versatile tool in the understanding of ed obtained in nmr has been largely neglected . one recent approach for ed in nmr is the use of chiral anisotropic media.4 in general , anisotropic achiral media permit the measurement of nmr parameters that require a partial alignment of the solute ( e.g. residual dipolar couplings or chemicalshift anisotropies ) . currently , a number of new alignment media that have been developed are becoming very important in the structural analysis of smalltolarge molecules.5 crosslinked acrylamidebased copolymers introduced in 20006a have been further developed into an alignment medium compatible with a wide range of solvents including dmso.6b recently , ed was reached by introducing a chiral , negatively charged monomer : ( r)aphes [ ( 2acrylamide)2phenylethanesulfonic acid , scheme s1 a].7 as expected from work with chiral ionexchange phases,8 ed was only reached for cationic ( protonated basic ) solutes ( erythromefloquine , menthylamine , and strychnine ) . no ed was observed for the neutral compound menthol.7 this approach had been inspired by previous work in the field of enantioselective ( chiral ) liquidphase chromatography . enantioselective chromatographic separation is based on the difference in the retention factor for a pair of enantiomers induced by a small difference in the phasetransfer equilibrium constants , owing to the involvement of diastereomeric associations ( between the chiral solute and the chiral selector ) in the phasetransfer process.8a a special class of stationary phases developed for enantioselective liquidphase chromatography ( chiral ionexchange phases ) employs positively and/or negatively charged immobilized chiral selectors , which can undergo the formation of diastereomeric associates ( ion pairs ) with protonated or deprotonated ( ionisable ) chiral solutes.8b , 8c it is now of interest to investigate whether or not the successful ed observed with a chiral alignment gel used for anisonmr ( nmr spectroscopy under anisotropic conditions ) can be further elucidated by using chromatographic studies employing the same alignment gel as a chiral separation medium in capillary electrochromatography ( cec ) ( figure 1 , and explanations given therein ) . for clarity , this approach should be clearly distinguished from techniques such as nmrce , capillary nmr , and chromatographic nmr spectroscopy.9 left side : schematic capillary / nmr tube cross section used for isotropic gelbased capillary electrochromatography ( cec ; upper part with isotropic mesh openings ) , and nmr under anisotropic conditions ( anisonmr ; lower part with anisotropic mesh openings ) . ideally , for thermodynamic studies , the separation medium investigated through chromatographic studies should be identical to the alignment medium used for anisonmr . bicelles , phages ) , however , are incompatible with cec instrumentation , whereas homogeneous gels are separation media in plate gel electrophoresis ( pge ) , in capillary gel electrophoresis ( cge ) , and in gelbased capillary electrochromatography ( gbcec).10 separation in pge and cge is based on differences in the migration velocity of the solute , induced by differences in the effective electrophoretic mobility of the solutes and the sieving effect . separation in gbcec ( in analogy to chromatography ) , however , is based on differences in the equilibrium constant characterizing the transfer of the solute from the electrokinetic transport zone ( etz , where the velocity of the solute is given by the electrophoretic velocity of this solute and the electroosmotic velocity ) into the interaction zone ( iz , where the velocity of the solute is zero ) . gbcec with hydrophilic or amphiphilic gels with embedded or immobilized chiral selectors was also successfully applied to the separation of different enantiomers.10c10 g returning to previous results,7 we selected the synthetic antimalaria drug mefloquine ( figure 2 ) with two stereocenters as the test solute.11a in recent years , the correct absolute configurations of the enantiomers of the erythro form , which is administered as the racemate , and of the enantiomers of the threo form , have been subject to debate . they were not reliably established until 20122013.11b11f in contrast to these difficulties , es of the four stereoisomers had been successfully achieved much earlier by using capillary electrophoresis ( ce ) with cyclodextrin derivatives or other native cyclodextrins as chiral selectors,12 and by using highperformance liquid chromatography ( hplc ) either with amylose tris3,5dimethylphenyl carbamatecoated silica gel13 or with a quinidinebased zwitterionic chiral stationary phase ( chiral selector covalently anchored to the chemically modified silica gel).14 structural formulae of a ) ( + ) erythromefloquine [ ( + ) emq ] , b ) ( )erythromefloquine [ ( )emq ] , c ) ( + ) threomefloquine [ ( + ) tmq ] , and d ) ( )threomefloquine [ ( )tmq ] . against this background , in the present work , we study the chromatographic separation of the four stereoisomers of mefloquine [ ( )threomefloquine ( tmq ) and ( )erythromefloquine ( emq ) ] by using gbcec , employing the same polymeric gel as the chiral separation medium as that already successfully taken for the ed of emq through anisonmr . the retention data gained by using gbcec will permit the determination of the ratio of ( distribution ) equilibrium constants . with this ratio , we confirm those parameters that have been calculated in the interpretation of previously reported anisotropic nmr data.7 with all types of gels obtained by varying the solvent composition , a good diastereoselective and enantioselective separation of the four mefloquine stereoisomers was achieved ( figure 3 ) . although the peak of propranolol , taken as a marker of the holdup time ( see the supporting information ) , can be characterized to be symmetric , the peaks for the more retarded stereoisomers of mefloquine show a characteristic ( rightskewed ) triangular broadened shape , which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . separation of the four stereoisomers of mefloquine by gbcec with alignment gel as the separation medium with superimposed scaled trace for ( )propranolol . we confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method , which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths ( see the supporting information ) . it should be noted that , in gbcec , the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . the depicted peak distortions ( figure 3 ) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase ( ionexchange cec).15 with analogously synthesized monolithic separation capillaries [ employing ( r)aphes as negatively charged comonomer ] synthesized for comparison purposes under phaseseparation conditions ( see the supporting information ) , leftskewed and rightskewed peaks can be obtained , dependent on the separation conditions ( figures s1 a and s1 b ) . for five repeated runs , the relative standard deviations of the observed mobilities are 2.02.3 % ( table s1 ) . following two different approaches ( see the supporting information ) , we estimated the chromatographic separation factor ( = ratio of the two retention factors ) for tmq to be 1.08 and for emq to be 1.10 . these two factors correspond to the ratio of the two equilibrium constants related to the transfer from the electrokinetic transport zone to the interaction zone . hence , the energetic differences g for the involved ( distribution ) equilibria are 190 and 230 j mol for tmq and emq , respectively . by using only a weak alignment , the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz ( residual dipolar couplings).16b with a set of experimentally determined rdcs , an alignment tensor ( a matrix with five independent elements ) can be calculated , which describes the degree and orientation of the alignment . in 2001 , the concept of the generalized degree of order ( gdo ) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor ( or the order tensor ) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments , as it is a quantity that is only dependent on the extent of dynamic averaging , arising from both overall alignment effects and internal motional effects ( the latter being neglected with rigid molecules).16a , 17 for ( )emq [ in the gel prepared with ( r)aphes ] gdo=5.1910 , which is smaller than for ( + ) emq [ gdo=5.8510 ; absolute difference : 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel ( being identical with a less stable diastereomeric association ) , the determined gdos correctly predict that ( )emq elutes first in a chromatographic separation . the confidence range of the arithmetic mean ( p=0.95 , onetailed test ) is 0.3810 , which is smaller than the difference between the values for the two isomers . both methods agree in terms of the sign [ ( + ) > ( ) ] and magnitude of the quantity describing ed / es , which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter ( here : rdc ) . based on the retention factors determined by gbcec , it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . for the different stereoisomers of mefloquine , it represents the fraction of those molecules being associated with the charged moieties of the crosslinked polymer acting as interaction sites . although n total ( = n etz+n iz ) is kept constant , the values for x iz are 0.29 and 0.31 for the two enantiomers of tmq , 0.42 for ( )emq , and 0.44 for ( + ) emq . with all types of gels obtained by varying the solvent composition , a good diastereoselective and enantioselective separation of the four mefloquine stereoisomers was achieved ( figure 3 ) . although the peak of propranolol , taken as a marker of the holdup time ( see the supporting information ) , can be characterized to be symmetric , the peaks for the more retarded stereoisomers of mefloquine show a characteristic ( rightskewed ) triangular broadened shape , which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . separation of the four stereoisomers of mefloquine by gbcec with alignment gel as the separation medium with superimposed scaled trace for ( )propranolol . we confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method , which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths ( see the supporting information ) . it should be noted that , in gbcec , the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . the depicted peak distortions ( figure 3 ) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase ( ionexchange cec).15 with analogously synthesized monolithic separation capillaries [ employing ( r)aphes as negatively charged comonomer ] synthesized for comparison purposes under phaseseparation conditions ( see the supporting information ) , leftskewed and rightskewed peaks can be obtained , dependent on the separation conditions ( figures s1 a and s1 b ) . under these conditions , the method enabled baseline separation of the enantiomers of emq ( figure 4 ) . separation of the enantiomers of erythromefloquine by gbcec with alignment gel as the separation medium . following two different approaches ( see the supporting information ) , we estimated the chromatographic separation factor ( = ratio of the two retention factors ) for tmq to be 1.08 and for emq to be 1.10 . these two factors correspond to the ratio of the two equilibrium constants related to the transfer from the electrokinetic transport zone to the interaction zone . hence , the energetic differences g for the involved ( distribution ) equilibria are 190 and 230 j mol for tmq and emq , respectively . under isotropic conditions , anisotropic parameters such as dipolar couplings between two nmrsensitive nuclei are averaged to zero.16a socalled alignment media lead to a low degree of orientation and reintroduce observable couplings . by using only a weak alignment , the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz ( residual dipolar couplings).16b with a set of experimentally determined rdcs , an alignment tensor ( a matrix with five independent elements ) can be calculated , which describes the degree and orientation of the alignment . in 2001 , the concept of the generalized degree of order ( gdo ) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor ( or the order tensor ) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments , as it is a quantity that is only dependent on the extent of dynamic averaging , arising from both overall alignment effects and internal motional effects ( the latter being neglected with rigid molecules).16a , 17 for ( )emq [ in the gel prepared with ( r)aphes ] gdo=5.1910 , which is smaller than for ( + ) emq [ gdo=5.8510 ; absolute difference : 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel ( being identical with a less stable diastereomeric association ) , the determined gdos correctly predict that ( )emq elutes first in a chromatographic separation . the confidence range of the arithmetic mean ( p=0.95 , onetailed test ) is 0.3810 , which is smaller than the difference between the values for the two isomers . with the aim to identify fragments of a protein , where motion has an effect on the accuracy of structure determination , tolman et al.17 defined a fragmentspecific internal gdo as the ratio of the observed fragment gdo to the alignment tensor gdo . we propose here the enantioselectivity parameter ( = gdo ratio ) , referring to a nonracemic chiral alignment medium and a specific enantiomer pair . both methods agree in terms of the sign [ ( + ) > ( ) ] and magnitude of the quantity describing ed / es , which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter ( here : rdc ) . based on the retention factors determined by gbcec , it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . for the different stereoisomers of mefloquine , it represents the fraction of those molecules being associated with the charged moieties of the crosslinked polymer acting as interaction sites . although n total ( = n etz+n iz ) is kept constant , the values for x iz are 0.29 and 0.31 for the two enantiomers of tmq , 0.42 for ( )emq , and 0.44 for ( + ) emq . taking a chiral alignment medium as the chiral separation medium , gbcec separates the four stereoisomers of mefloquine . this separation confirms our previous hypothesis that ed with this gel is based on the formation of localized diastereomeric associates between the charged solute and the oppositely charged moiety of the anisotropically deformed gel.7 consequently , ed is attributed to differences in the equilibrium constants related to associate formation . hence , our approach provides physicochemical data that are important in the understanding and optimization of alignment processes.18 the capillary pretreatment procedure and the subsequent in situ synthesis of the polymeric gel are described in detail in the supporting information . the preparation procedure of the gel within the capillary is identical ( with respect to concentration of monomers , reaction time , reaction temperature ) to the one used for the measurement of anisonmr parameters with the exception of the absence or presence of a washing step ( removal of nonreacted monomers ) , an additional drying and swelling step ( in the case of anisonmr ) , and the solvent composition.7 although dmso was employed for anisonmr , the solvent is either methanol or methanol / water [ ( 50:50 , v / v ) or ( 25:75 , v / v ) ] in gbcec ( after equilibration ) . previous studies , however , with either methanol or dmso as the solvent demonstrated that the type of solvent has only a minor influence on the determined anisonmr parameters.19 in gbcec , the separation gel is buffered with triethylamine / acetic acid , ph*=4.56.7 , whereas in anisonmr all experiments were performed in unbuffered gels .

although nmr studies are used to elucidate structural aspects of es,3a the role of enantioselective chromatography as a versatile tool in the understanding of ed obtained in nmr has been largely neglected . since the early report of pirkle and pochapsky in 1986,3b isotropic solutionnmr studies have been used to investigate structural aspects of chromatographic enantiorecognition processes.3c,3d for this purpose , chemical shifts and nuclear overhauser effects ( noes ) were largely used . currently , a number of new alignment media that have been developed are becoming very important in the structural analysis of smalltolarge molecules.5 crosslinked acrylamidebased copolymers introduced in 20006a have been further developed into an alignment medium compatible with a wide range of solvents including dmso.6b recently , ed was reached by introducing a chiral , negatively charged monomer : ( r)aphes [ ( 2acrylamide)2phenylethanesulfonic acid , scheme s1 a].7 as expected from work with chiral ionexchange phases,8 ed was only reached for cationic ( protonated basic ) solutes ( erythromefloquine , menthylamine , and strychnine ) . no ed was observed for the neutral compound menthol.7 this approach had been inspired by previous work in the field of enantioselective ( chiral ) liquidphase chromatography . enantioselective chromatographic separation is based on the difference in the retention factor for a pair of enantiomers induced by a small difference in the phasetransfer equilibrium constants , owing to the involvement of diastereomeric associations ( between the chiral solute and the chiral selector ) in the phasetransfer process.8a a special class of stationary phases developed for enantioselective liquidphase chromatography ( chiral ionexchange phases ) employs positively and/or negatively charged immobilized chiral selectors , which can undergo the formation of diastereomeric associates ( ion pairs ) with protonated or deprotonated ( ionisable ) chiral solutes.8b , 8c it is now of interest to investigate whether or not the successful ed observed with a chiral alignment gel used for anisonmr ( nmr spectroscopy under anisotropic conditions ) can be further elucidated by using chromatographic studies employing the same alignment gel as a chiral separation medium in capillary electrochromatography ( cec ) ( figure 1 , and explanations given therein ) . for clarity , this approach should be clearly distinguished from techniques such as nmrce , capillary nmr , and chromatographic nmr spectroscopy.9 left side : schematic capillary / nmr tube cross section used for isotropic gelbased capillary electrochromatography ( cec ; upper part with isotropic mesh openings ) , and nmr under anisotropic conditions ( anisonmr ; lower part with anisotropic mesh openings ) . bicelles , phages ) , however , are incompatible with cec instrumentation , whereas homogeneous gels are separation media in plate gel electrophoresis ( pge ) , in capillary gel electrophoresis ( cge ) , and in gelbased capillary electrochromatography ( gbcec).10 separation in pge and cge is based on differences in the migration velocity of the solute , induced by differences in the effective electrophoretic mobility of the solutes and the sieving effect . separation in gbcec ( in analogy to chromatography ) , however , is based on differences in the equilibrium constant characterizing the transfer of the solute from the electrokinetic transport zone ( etz , where the velocity of the solute is given by the electrophoretic velocity of this solute and the electroosmotic velocity ) into the interaction zone ( iz , where the velocity of the solute is zero ) . we describe this equilibrium as a distribution process , although the highly swollen gel employed in gbcec is a homogenous phase ( in contrast to the system of two interpenetrating continuous phases characteristic for cec with a monolith ) . gbcec with hydrophilic or amphiphilic gels with embedded or immobilized chiral selectors was also successfully applied to the separation of different enantiomers.10c10 g returning to previous results,7 we selected the synthetic antimalaria drug mefloquine ( figure 2 ) with two stereocenters as the test solute.11a in recent years , the correct absolute configurations of the enantiomers of the erythro form , which is administered as the racemate , and of the enantiomers of the threo form , have been subject to debate . they were not reliably established until 20122013.11b11f in contrast to these difficulties , es of the four stereoisomers had been successfully achieved much earlier by using capillary electrophoresis ( ce ) with cyclodextrin derivatives or other native cyclodextrins as chiral selectors,12 and by using highperformance liquid chromatography ( hplc ) either with amylose tris3,5dimethylphenyl carbamatecoated silica gel13 or with a quinidinebased zwitterionic chiral stationary phase ( chiral selector covalently anchored to the chemically modified silica gel).14 structural formulae of a ) ( + ) erythromefloquine [ ( + ) emq ] , b ) ( )erythromefloquine [ ( )emq ] , c ) ( + ) threomefloquine [ ( + ) tmq ] , and d ) ( )threomefloquine [ ( )tmq ] . against this background , in the present work , we study the chromatographic separation of the four stereoisomers of mefloquine [ ( )threomefloquine ( tmq ) and ( )erythromefloquine ( emq ) ] by using gbcec , employing the same polymeric gel as the chiral separation medium as that already successfully taken for the ed of emq through anisonmr . with this ratio , we confirm those parameters that have been calculated in the interpretation of previously reported anisotropic nmr data.7 with all types of gels obtained by varying the solvent composition , a good diastereoselective and enantioselective separation of the four mefloquine stereoisomers was achieved ( figure 3 ) . although the peak of propranolol , taken as a marker of the holdup time ( see the supporting information ) , can be characterized to be symmetric , the peaks for the more retarded stereoisomers of mefloquine show a characteristic ( rightskewed ) triangular broadened shape , which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . peak assignment : 1 ) ( )propranolol ; 2 , 3 ) ( )threomefloquine ; 4 ) ( )erythromefloquine ; 5 ) ( + ) erythromefloquine ; mobile phase = methanol / water ( 50:50 , v / v ) buffered with triethylamine / acetic acid , ph*=6.66 , electric conductivity=125 s cm , capillary dimensions=173 mm ( 102 mm)100 m , photometric ingel detection=283 nm , electrokinetic injection=2 kv2 s , separation voltage=2.0 kv , c ( analyte in sample)=0.25 g l ( analyte dissolved in mobile phase ) . we confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method , which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths ( see the supporting information ) . it should be noted that , in gbcec , the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . the depicted peak distortions ( figure 3 ) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase ( ionexchange cec).15 with analogously synthesized monolithic separation capillaries [ employing ( r)aphes as negatively charged comonomer ] synthesized for comparison purposes under phaseseparation conditions ( see the supporting information ) , leftskewed and rightskewed peaks can be obtained , dependent on the separation conditions ( figures s1 a and s1 b ) . following two different approaches ( see the supporting information ) , we estimated the chromatographic separation factor ( = ratio of the two retention factors ) for tmq to be 1.08 and for emq to be 1.10 . taking propranolol as a nonretarded marker with an effective electrophoretic mobility similar to that of tmq and emq ( figure 3 and scheme s1 b ) , we obtained the following retention factors : 0.41 and 0.44 for the two enantiomers of tmq , 0.73 for ( )emq , and 0.80 for ( + ) emq . by using only a weak alignment , the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz ( residual dipolar couplings).16b with a set of experimentally determined rdcs , an alignment tensor ( a matrix with five independent elements ) can be calculated , which describes the degree and orientation of the alignment . in 2001 , the concept of the generalized degree of order ( gdo ) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor ( or the order tensor ) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments , as it is a quantity that is only dependent on the extent of dynamic averaging , arising from both overall alignment effects and internal motional effects ( the latter being neglected with rigid molecules).16a , 17 for ( )emq [ in the gel prepared with ( r)aphes ] gdo=5.1910 , which is smaller than for ( + ) emq [ gdo=5.8510 ; absolute difference : 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel ( being identical with a less stable diastereomeric association ) , the determined gdos correctly predict that ( )emq elutes first in a chromatographic separation . with the aim to identify fragments of a protein , where motion has an effect on the accuracy of structure determination , tolman et al.17 defined a fragmentspecific internal gdo as the ratio of the observed fragment gdo to the alignment tensor gdo . we propose here the enantioselectivity parameter ( = gdo ratio ) , referring to a nonracemic chiral alignment medium and a specific enantiomer pair . both methods agree in terms of the sign [ ( + ) > ( ) ] and magnitude of the quantity describing ed / es , which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter ( here : rdc ) . based on the retention factors determined by gbcec , it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . although n total ( = n etz+n iz ) is kept constant , the values for x iz are 0.29 and 0.31 for the two enantiomers of tmq , 0.42 for ( )emq , and 0.44 for ( + ) emq . although the peak of propranolol , taken as a marker of the holdup time ( see the supporting information ) , can be characterized to be symmetric , the peaks for the more retarded stereoisomers of mefloquine show a characteristic ( rightskewed ) triangular broadened shape , which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . we confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method , which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths ( see the supporting information ) . it should be noted that , in gbcec , the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . the depicted peak distortions ( figure 3 ) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase ( ionexchange cec).15 with analogously synthesized monolithic separation capillaries [ employing ( r)aphes as negatively charged comonomer ] synthesized for comparison purposes under phaseseparation conditions ( see the supporting information ) , leftskewed and rightskewed peaks can be obtained , dependent on the separation conditions ( figures s1 a and s1 b ) . following two different approaches ( see the supporting information ) , we estimated the chromatographic separation factor ( = ratio of the two retention factors ) for tmq to be 1.08 and for emq to be 1.10 . taking propranolol as a nonretarded marker with an effective electrophoretic mobility similar to that of tmq and emq ( figure 3 and scheme s1 b ) , we obtained the following retention factors : 0.41 and 0.44 for the two enantiomers of tmq , 0.73 for ( )emq , and 0.80 for ( + ) emq . by using only a weak alignment , the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz ( residual dipolar couplings).16b with a set of experimentally determined rdcs , an alignment tensor ( a matrix with five independent elements ) can be calculated , which describes the degree and orientation of the alignment . in 2001 , the concept of the generalized degree of order ( gdo ) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor ( or the order tensor ) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments , as it is a quantity that is only dependent on the extent of dynamic averaging , arising from both overall alignment effects and internal motional effects ( the latter being neglected with rigid molecules).16a , 17 for ( )emq [ in the gel prepared with ( r)aphes ] gdo=5.1910 , which is smaller than for ( + ) emq [ gdo=5.8510 ; absolute difference : 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel ( being identical with a less stable diastereomeric association ) , the determined gdos correctly predict that ( )emq elutes first in a chromatographic separation . with the aim to identify fragments of a protein , where motion has an effect on the accuracy of structure determination , tolman et al.17 defined a fragmentspecific internal gdo as the ratio of the observed fragment gdo to the alignment tensor gdo . we propose here the enantioselectivity parameter ( = gdo ratio ) , referring to a nonracemic chiral alignment medium and a specific enantiomer pair . both methods agree in terms of the sign [ ( + ) > ( ) ] and magnitude of the quantity describing ed / es , which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter ( here : rdc ) . based on the retention factors determined by gbcec , it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . this separation confirms our previous hypothesis that ed with this gel is based on the formation of localized diastereomeric associates between the charged solute and the oppositely charged moiety of the anisotropically deformed gel.7 consequently , ed is attributed to differences in the equilibrium constants related to associate formation . through gbcec , retention data are obtained that permit the calculation of the degrees of transfer of the solute from the electrokinetic transport zone into the interaction zone and the precise quantification of the gibbs energy difference regarding the two diastereomeric associates involved in the enantioselective separation process . hence , our approach provides physicochemical data that are important in the understanding and optimization of alignment processes.18 the capillary pretreatment procedure and the subsequent in situ synthesis of the polymeric gel are described in detail in the supporting information . the preparation procedure of the gel within the capillary is identical ( with respect to concentration of monomers , reaction time , reaction temperature ) to the one used for the measurement of anisonmr parameters with the exception of the absence or presence of a washing step ( removal of nonreacted monomers ) , an additional drying and swelling step ( in the case of anisonmr ) , and the solvent composition.7 although dmso was employed for anisonmr , the solvent is either methanol or methanol / water [ ( 50:50 , v / v ) or ( 25:75 , v / v ) ] in gbcec ( after equilibration ) . previous studies , however , with either methanol or dmso as the solvent demonstrated that the type of solvent has only a minor influence on the determined anisonmr parameters.19 in gbcec , the separation gel is buffered with triethylamine / acetic acid , ph*=4.56.7 , whereas in anisonmr all experiments were performed in unbuffered gels . the influence of the electrolyte concentration on the alignment properties of a very similar achiral polyacrylamide gel [ aphes replaced by 2(acrylamide)2methylpropanesulfonic acid ( amps ) ] was determined by trigomourio et al.20 according to these results , we can assume a negligible influence of the selected buffer concentration ( 2.7 mm ) on alignment properties and retention behavior .

peripheral nerves , especially those at the elbow region , such as the median nerve and ulnar nerve , are prone to entrapment neuropathies and injuries , such as cubital tunnel syndrome ( 1 , 2 ) , pronator teres syndrome ( 3 ) , and nerve laceration ( 4 ) . the traditional method used for evaluating these diseases with respect to preoperative diagnosis and postoperative rehabilitation is electrophysiological nerve conduction and electromyography ; however , accurate evaluations using these methods largely depend on the operator s experience , and false negative results are common ( 5 ) . a magnetic resonance ( mr ) image can display the soft tissue , including nerves , in t1 or t2 images , and the most commonly used method of mr for the diagnosis of peripheral nerve entrapment is to measure the cross sectional area ( csa ) and t2 signal intensity , which can not distinguish axonal injury from demyelination ( 6 ) . moreover , the continuity and integrity of a nerve can hardly be detected based on two - dimensional ( 2d ) images . therefore , obtaining a three - dimensional ( 3d ) display of a nerve that can be observed at any angle is essential for clinicians to make accurate assessments . currently , the methods and sequences of mr neurography are mostly represented through diffusion tensor imaging ( dti ) . dti with fiber tracking was the first method used to obtain a 3d view of the nerve , which was originally applied to image the central nervous system . diffusion is less restricted in the direction parallel to the nerve fiber than that perpendicular to the fiber ( possibly due to the layers of myelin ) . using special fiber - tracking software to reconstruct the dti data , it is possible to visualize the complete neural structure . in june 2004 , skorpil et al . ( 7 ) demonstrated that peripheral nerves ( the sciatic nerve in three healthy volunteers ) could be imaged in vivo by using dti with fiber tracking . this technique has now become the most widely used method for evaluating the status of the median nerves in carpal tunnel syndrome ( 8) , providing non - invasive imaging of peripheral nerve regeneration ( 9 - 11 ) , and characterizing the structure of the brachial plexus ( 12 ) . however , studies reporting the use of dti with fiber tracking post - processing at the elbow region are scarce . the following drawbacks of using dti for the elbow region , especially for the ulnar nerve , have been discussed : ( i ) it is superficial ; ( ii ) close to the bone surface ; and ( iii ) shows large angulations in the course of the nerve ( 13 ) . besides , these limitations of dti with respect to specific anatomical characteristics at the elbow region , the technique itself has many limitations . first , the quality of tractography depends on the parameters used in the acquisition step , such as the coils used , positioning of the patient , and other parameters including the b - value and the number of diffusion directions . ( 14 ) suggested using joint - specific coils at the joint region . when using this type of hard coil , the elbow , the subject must raise one hand above his / her head while in prone or supine position , which can be very uncomfortable . second , during the post - processing phase before fiber tracking , the operator must choose a series of regions of interest ( roi ) , which depends on the operator s anatomical knowledge . in addition to the dti fiber tracking technique , a novel post - processing method to reconstruct dti data was developed by skorpil et al . they used the maximum intensity projection ( mip ) technique to reconstruct sciatic nerves based on dti images in a single direction and in all directions . furthermore , based on preliminary experiments performed at the wrist region , we found that images of a single direction could also be reconstructed by using volume rendering ( vr ) and mip . these two post - processing methods ( vr and mip ) may offer a more convenient procedure for dti because they do not require prior choice of rois . we predicted that the peripheral nerve at the elbow region could be three - dimensionally displayed based on the mr dti data with multiple post - processing techniques . we aimed to determine whether the vr or mip reconstruction for single direction would be more convenient than fiber tracking post - processing while acquiring approximate qualities of nerve imaging , and the consistency of vr / mip and fiber tracking post - processing for the same sample . the study had prior approval by our institutional review board and ethics committee , and all subjects provided informed consent prior to the study . a total of 24 elbows of 12 healthy , young volunteers were studied ( six men , six women ; age range 22 - 32 years ) . nerves at the elbow region were analyzed , including the median nerve , ulnar nerve , and radial nerve . the exclusion criteria included general contraindications for mr imaging ( mri ) , a prior history of trauma or surgery of the elbow , presence of rheumatoid arthritis , and presence of space - occupying lesions at the elbow . the mr images were acquired with a siemens magnetom verio 3 t mri scanner ( siemens healthcare ; erlangen , germany ) using a 4-channal flex coil ( flex small 4 ; siemens ) . the mri system s maximum field gradient amplitude was 45 mt / m with a slew rate of 200 t m s. during the scan session , the soft coil was placed around the elbow , and then a polymer brace was placed on the whole upper limb in order to restrain movement . the subject s upper extremity with the coil was immobilized with cushions , sandbags , and bandages . the elbow was then positioned to the center of the magnet bore . for imaging of the elbow , dti was performed using an echo planar inversion recovery ( epir ) sequence ( tr = 15,300 ms ; te= 92 ms ; ti = 200 ms ; fa = 90 ; etl = 1 ; fov = 250 250 mm ; scan matrix = 128 128 ; average = 2 ) with 38 transversal slices of 3.0-mm thickness with no gap between the slices . diffusion weighting with a b value of 1200 s / mm was applied in 20 encoding diffusion directions . in addition to the diffusion - weighted images , a single reference image without diffusion weighting ( b = 0 s / mm ) was acquired . for anatomical reference , a t1-weighted image ( tr = 700 ms ; te = 11 ms ; fa = 150 ; etl = 3 ; thickness = 3 mm ; gap = 0 mm ; fov = 140 140 mm ; scan matrix = 320 240 ; average = 1 ) was acquired . the scanning times for dti and t1-weighted image were 11 min 29 s and 2 min 1 s , respectively , for a total scan time of approximately 15 min . the raw dti images were transferred automatically to the siemens mr workstation ( siemens syngo 3d ) . the x - axis represents the anterior posterior direction , the y - axis represents the left - right direction , and the z - axis represents the superior inferior direction . based on the gradient table of the dti scan , direction 1 with coordinates ( 1 , 0 , 0 ) represents the anterior posterior direction . ( 15 ) , the most suitable choice for single direction vr post - processing is direction 1 , which is perpendicular to the nerves . the original dti images usually contained a series of t2-weighted images ( b = 0 s / mm ) , which were generated as the first series in the result chart . the optimal choice ( direction 1 ) was the second series generated in the result chart . during vr / mip reconstruction of images of direction 1 , any high - signal noises of the veins or skin present in the reconstructed stereoscopic view were cut off in order to make the nerves as conspicuous as possible . one of the authors ( y.y ) was responsible for this part of the work . x - axis represents the anterior - posterior direction . y - axis represents the left - right direction . direction 1 with the coordinates ( 1 , 0 , 0 ) represents the anterior - posterior direction . the raw dti images were transferred to the siemens mr workstation ( siemens syngo neuro 3d ) . fiber tractography was then performed using a multiple seed roi technique , which employed fiber assignment by continuous tracking . seed rois were drawn freehand at three anatomical locations ( level to the supracondylar humerus , olecranon , and proximal radioulnar joint ) on the raw dti images and reference t1-weighted images . rois were confined precisely to the nerve border to avoid partial volume artefacts and to exclude any surrounding structure from the seed rois . in this study , the fractional anisotropy ( fa ) threshold value was 0.2 and the angulation tolerance was 30. the software s option step length was set to 0.47 mm . another author ( d .- s.j ) was responsible for this part of the work . in order to compare the imaging quality among vr / mip and fiber tractography , we created a custom evaluation scale to assess each nerve in vr / mip and fiber tractography images . we set the scale based on whether the target nerve could be shown and the length of the nerve was complete as follows : score of 2 , the target nerve ( the median , radial , or ulnar nerve ) at the elbow region was clearly shown and the entire length of the nerve was visible throughout the entire scanning section ; score of 1 , the target nerve at the elbow region was clearly shown , but its length was incomplete ; score of 0 , the target nerve was invisible . two authors ( w .- q.d and j .- h.g ) were responsible for independently evaluating the imaging qualities according to this scale . one author ( w .- q.d ) evaluated the imaging qualities again 4 weeks later . for each nerve , scores of vr / mip reconstruction of the single direction and fiber tracking were compared using friedman test . intraclass correlation coefficient ( icc ) analysis was used to compare the correlation of the imaging quality scores for each nerve among vr / mip and fiber tractography . kappa values of 0 - 0.20 were considered to indicate poor agreement ; 0.21 - 0.40 , fair agreement ; 0.41 - 0.60 , moderate agreement ; 0.61 - 0.80 , good agreement ; and 0.81 - 1.00 , excellent agreement . statistical analysis was performed by one author ( j .- h.g ) using spss statistical software , ver . the study had prior approval by our institutional review board and ethics committee , and all subjects provided informed consent prior to the study . a total of 24 elbows of 12 healthy , young volunteers were studied ( six men , six women ; age range 22 - 32 years ) . nerves at the elbow region were analyzed , including the median nerve , ulnar nerve , and radial nerve . the exclusion criteria included general contraindications for mr imaging ( mri ) , a prior history of trauma or surgery of the elbow , presence of rheumatoid arthritis , and presence of space - occupying lesions at the elbow . the mr images were acquired with a siemens magnetom verio 3 t mri scanner ( siemens healthcare ; erlangen , germany ) using a 4-channal flex coil ( flex small 4 ; siemens ) . the mri system s maximum field gradient amplitude was 45 mt / m with a slew rate of 200 t m s. during the scan session , the soft coil was placed around the elbow , and then a polymer brace was placed on the whole upper limb in order to restrain movement . the subject s upper extremity with the coil was immobilized with cushions , sandbags , and bandages . the elbow was then positioned to the center of the magnet bore . for imaging of the elbow , dti was performed using an echo planar inversion recovery ( epir ) sequence ( tr = 15,300 ms ; te= 92 ms ; ti = 200 ms ; fa = 90 ; etl = 1 ; fov = 250 250 mm ; scan matrix = 128 128 ; average = 2 ) with 38 transversal slices of 3.0-mm thickness with no gap between the slices . diffusion weighting with a b value of 1200 s / mm was applied in 20 encoding diffusion directions . in addition to the diffusion - weighted images , a single reference image without diffusion weighting ( b = 0 s / mm ) was acquired . for anatomical reference , a t1-weighted image ( tr = 700 ms ; te = 11 ms ; fa = 150 ; etl = 3 ; thickness = 3 mm ; gap = 0 mm ; fov = 140 140 mm ; scan matrix = 320 240 ; average = 1 ) was acquired . the scanning times for dti and t1-weighted image were 11 min 29 s and 2 min 1 s , respectively , for a total scan time of approximately 15 min . the raw dti images were transferred automatically to the siemens mr workstation ( siemens syngo 3d ) . the x - axis represents the anterior posterior direction , the y - axis represents the left - right direction , and the z - axis represents the superior inferior direction . based on the gradient table of the dti scan , direction 1 with coordinates ( 1 , 0 , 0 ) represents the anterior posterior direction . therefore , according to skorpil et al . ( 15 ) , the most suitable choice for single direction vr post - processing is direction 1 , which is perpendicular to the nerves . the original dti images usually contained a series of t2-weighted images ( b = 0 s / mm ) , which were generated as the first series in the result chart . the optimal choice ( direction 1 ) was the second series generated in the result chart . during vr / mip reconstruction of images of direction 1 , any high - signal noises of the veins or skin present in the reconstructed stereoscopic view were cut off in order to make the nerves as conspicuous as possible . one of the authors ( y.y ) was responsible for this part of the work . x - axis represents the anterior - posterior direction . y - axis represents the left - right direction . direction 1 with the coordinates ( 1 , 0 , 0 ) represents the anterior - posterior direction . the raw dti images were transferred to the siemens mr workstation ( siemens syngo neuro 3d ) . fiber tractography was then performed using a multiple seed roi technique , which employed fiber assignment by continuous tracking . seed rois were drawn freehand at three anatomical locations ( level to the supracondylar humerus , olecranon , and proximal radioulnar joint ) on the raw dti images and reference t1-weighted images . rois were confined precisely to the nerve border to avoid partial volume artefacts and to exclude any surrounding structure from the seed rois . in this study , the fractional anisotropy ( fa ) threshold value was 0.2 and the angulation tolerance was 30. the software s option step length was set to 0.47 mm . another author ( d .- s.j ) was responsible for this part of the work . in order to compare the imaging quality among vr / mip and fiber tractography , we created a custom evaluation scale to assess each nerve in vr / mip and fiber tractography images . we set the scale based on whether the target nerve could be shown and the length of the nerve was complete as follows : score of 2 , the target nerve ( the median , radial , or ulnar nerve ) at the elbow region was clearly shown and the entire length of the nerve was visible throughout the entire scanning section ; score of 1 , the target nerve at the elbow region was clearly shown , but its length was incomplete ; score of 0 , the target nerve was invisible . two authors ( w .- q.d and j .- h.g ) were responsible for independently evaluating the imaging qualities according to this scale . one author ( w .- q.d ) evaluated the imaging qualities again 4 weeks later . the imaging quality scores were expressed as medians with ranges and mean with sd . for each nerve , scores of vr / mip reconstruction of the single direction and fiber tracking were compared using friedman test . intraclass correlation coefficient ( icc ) analysis was used to compare the correlation of the imaging quality scores for each nerve among vr / mip and fiber tractography . kappa values of 0 - 0.20 were considered to indicate poor agreement ; 0.21 - 0.40 , fair agreement ; 0.41 - 0.60 , moderate agreement ; 0.61 - 0.80 , good agreement ; and 0.81 - 1.00 , excellent agreement . statistical analysis was performed by one author ( j .- h.g ) using spss statistical software , ver . all 24 elbows of the 12 young volunteers were tested , and stereoscopic displays of the nerves were obtained by using vr , mip , and fiber tractography post - processing methods ( figure 1 ) . for most elbows of volunteers , three main nerves could be seen clearly ( figure 1 ) , whereas only one nerve could be detected in other individual cases ( figure 2 ) . owing to the relatively simple and direct operations enabled without requiring rois selection , the vr and mip post - processing methods were easier to perform compared to fiber tractography . vr / mip reconstructions for single direction images and fiber tracking evaluation scores for each target nerve are listed in table 2 , which did not differ significantly ( p > 0.05 ) . the imaging quality scores of fiber tractography and vr / mip were significantly correlated based on intraclass correlation coefficient ( icc ) analysis ( icc ranged 0.709 - 0.901 ) , which suggested good consistency between the scores obtained based on the fiber tractography and vr / mip for the same sample . the values of inter- and intraobserver agreements ranged 0.674 - 0.909 , which showed good to excellent agreements . abbreviations : ft , fiber tractography ; icc , intraclass correlation coefficient ; 1 , interobserver value ; 2 , intraobserver value ; ob1 , observer 1 ; ob2 , observer 2 ; ob2 sec . , observer 2 second time . in this study , we found that some images belong to a single direction ( perpendicular to the nerve ) after dti scan could be used for vr or mip reconstruction to display the peripheral nerves clearly . this method is similar to unidirectional diffusion - weighted mr neurography ( dw - mrn ) ( 16 , 17 ) . the difference of applied diffusion directions between dti and dw - mrn scans is that dti scan can select many non - parallel diffusion directions , and the data can be used for fiber tracking and measuring the fa , apparent diffusion coefficient ( adc ) value , while dw - mrn can choose fewer number of diffusion directions , increase the excitation frequency so the scan time is shorter , and signal - to - noise ratio increased for better mip or vr display . ( 17 ) indicated when they applied diffusion direction which is perpendicular to the nerve axons , the water molecular diffusion in the nerve was restricted , so the nerve tissue could all be imaged and showed high signal . we speculated that in dti scan , the data collected from the perpendicular direction may play an important role in calculating fa value , which is the key parameter for fiber tracking . thus , vr and mip reconstruction of a single direction ( anterior - posterior direction , perpendicular to the nerve ) could predict the imaging quality of fiber tractography . the results of this study raise an important and essential question : under the same experimental conditions , why do the results show variation among different healthy volunteers ? for some elbows of volunteers , three main nerves could be seen clearly ( figure 1 ) , whereas only one nerve could be detected in other cases ( figure 2 ) . besides the reasons of anatomical characteristics at the elbow region and the limitations of the technique , one possible explanation for this variation is that slight movement of the limb is the key factor affecting imaging quality . in the image shown in figure 2 , the ulnar nerve is located outside of the outline of the elbow . the median nerve and radial nerve are covered by the outline of the elbow , which suggests that the signal values of the nerve fibers were calculated incorrectly , thereby affecting fiber tracking . vr and mip post - processing are more convenient to obtain compared to fiber tracking . the selection of rois is not required , which prevents subjective factors from influencing the results . the icc analysis result suggested that scores based on fiber tractography and vr / mip for the same samples were highly consistent . therefore , vr and mip reconstruction can be used as a simple preview tool before performing complicated fiber tracking . during the dti scanning process using siemens magenetom verio 3 t mri scanner , the dti original images are generated in the order of the gradient table ( table 1 ) from direction 1 to direction 20 , and from average 1 to average 2 . the images belonging to direction 1 of average 1 will be generated within the first few seconds , and then vr and mip post - processing can be immediately performed to observe whether or not the nerve is displayed . if the nerves display , then , a scan with more directions and more averages should be continued for fiber tracking . if the nerves do not display , the scan should be terminated , and more measures for restricting movement of the elbow should be adopted . using this method could help save a lot of time to the benefit of both the clinician and the patient . some potential applications of vr and mip reconstruction include evaluation of the continuity and integrity of the nerve as well as detection of abnormal morphology , whereas the application of fiber tracking is specific to assessing the status of axons . according to sunderland s nerve injury classification ( 18 ) , grade i , grade ii , and some grade iii injuries ( axons or endoneurial tube disruptions ) do not require surgical exploration , whereas grade iv - vi injuries ( perineurium or epineurium damages , defects , or mixed injuries ) do require surgical exploration as soon as possible . therefore , vr and mip reconstruction might be able to show more details of damaged nerves , which unlike fiber tracking , do not only focus on the axons . background tissue also can be reconstructed as reference landmark to show the damage site of nerve more clearly . cubital tunnel syndrome is the second - most common nerve compression syndrome after carpal tunnel syndrome . injury of the ulnar nerve at the elbow region is also common , because it is superficial and close to the bone surface . stereoscopic display of the nerves at the elbow region can help the clinician understand the morphology of the target nerve . for example , in nerve entrapment patients , nerves can be detected as the features of increasing csa and flatten ratio ( fr ) , which can be observed more easily in stereoscopic displays compared with conventional 2d images . by using these methods with the addition of fa and adc value measurements , dti appears to be a promising tool for future evaluations of the severity and rehabilitation of ulnar neuropathy . first , due to the limited choice of equipment and coil , the parameters and coil of the dti scan at the elbow region did not reach adequate optimization . second , the measures adopted for restricting slight movement of the limb were not highly efficient . stereoscopic displays of the peripheral nerve at the elbow region were achieved by using vr , mip , and fiber tracking post - processing methods based on raw dti images . the scores based on vr / mip and fiber tracking post - processing had good consistency for the same samples indicating that vr and mip reconstruction for single direction could be used as preview tools before fiber tracking to determine whether the raw images are satisfactory .

background : peripheral nerves at the elbow region are prone to entrapment neuropathies and injuries . to make accurate assessment , clinicians need stereoscopic display of the nerves to observe them at all angles.objectives:to obtain a stereoscopic display of the peripheral nerves at the elbow region based on magnetic resonance ( mr ) diffusion tensor imaging ( dti ) data using three post - processing methods of volume rendering ( vr ) , maximum intensity projection ( mip ) , and fiber tractography , and to evaluate the difference and correlation between them.subjects and methods : twenty - four elbows of 12 healthy young volunteers were assessed by 20 encoding diffusion direction mr dti scans . images belonging to a single direction ( anterior - posterior direction , perpendicular to the nerve ) were subjected to vr and mip reconstruction . all raw dti data were transferred to the siemens mr workstation for fiber tractography post - processing . imaging qualities of fiber tractography and vr / mip were evaluated by two observers independently based on a custom evaluation scale.results:stereoscopic displays of the nerves were obtained in all 24 elbows by vr , mip , and fiber tractography post - processing methods . the vr / mip post - processing methods were easier to perform compared to fiber tractography . there was no significant difference among the scores of fiber tracking and vr / mip reconstruction for single direction . the imaging quality scores of fiber tractography and vr / mip were significantly correlated based on intraclass correlation coefficient ( icc ) analysis ( icc ranged 0.709 - 0.901 ) , which suggested that the scores based on fiber tractography and vr / mip for the same sample were consistent . inter- and intraobserver agreements were good to excellent.conclusion:stereoscopic displays of the peripheral nerves at the elbow region can be achieved by using vr , mip , and fiber tracking post - processing methods based on raw dti images . vr and mip reconstruction could be used as preview tools before fiber tracking to determine whether the raw images are satisfactory .

1. Background 2. Objectives 3. Subjects and Methods 3.1. Study Subjects 3.2. MR Imaging Protocol 3.3. VR and MIP Post-Processing 3.4. Fiber Tractography 3.5. Imaging Quality Evaluation 3.6. Statistical Analysis 4. Results 5. Discussion

peripheral nerves , especially those at the elbow region , such as the median nerve and ulnar nerve , are prone to entrapment neuropathies and injuries , such as cubital tunnel syndrome ( 1 , 2 ) , pronator teres syndrome ( 3 ) , and nerve laceration ( 4 ) . a magnetic resonance ( mr ) image can display the soft tissue , including nerves , in t1 or t2 images , and the most commonly used method of mr for the diagnosis of peripheral nerve entrapment is to measure the cross sectional area ( csa ) and t2 signal intensity , which can not distinguish axonal injury from demyelination ( 6 ) . therefore , obtaining a three - dimensional ( 3d ) display of a nerve that can be observed at any angle is essential for clinicians to make accurate assessments . currently , the methods and sequences of mr neurography are mostly represented through diffusion tensor imaging ( dti ) . dti with fiber tracking was the first method used to obtain a 3d view of the nerve , which was originally applied to image the central nervous system . diffusion is less restricted in the direction parallel to the nerve fiber than that perpendicular to the fiber ( possibly due to the layers of myelin ) . ( 7 ) demonstrated that peripheral nerves ( the sciatic nerve in three healthy volunteers ) could be imaged in vivo by using dti with fiber tracking . this technique has now become the most widely used method for evaluating the status of the median nerves in carpal tunnel syndrome ( 8) , providing non - invasive imaging of peripheral nerve regeneration ( 9 - 11 ) , and characterizing the structure of the brachial plexus ( 12 ) . however , studies reporting the use of dti with fiber tracking post - processing at the elbow region are scarce . the following drawbacks of using dti for the elbow region , especially for the ulnar nerve , have been discussed : ( i ) it is superficial ; ( ii ) close to the bone surface ; and ( iii ) shows large angulations in the course of the nerve ( 13 ) . besides , these limitations of dti with respect to specific anatomical characteristics at the elbow region , the technique itself has many limitations . first , the quality of tractography depends on the parameters used in the acquisition step , such as the coils used , positioning of the patient , and other parameters including the b - value and the number of diffusion directions . ( 14 ) suggested using joint - specific coils at the joint region . when using this type of hard coil , the elbow , the subject must raise one hand above his / her head while in prone or supine position , which can be very uncomfortable . second , during the post - processing phase before fiber tracking , the operator must choose a series of regions of interest ( roi ) , which depends on the operator s anatomical knowledge . in addition to the dti fiber tracking technique , a novel post - processing method to reconstruct dti data was developed by skorpil et al . they used the maximum intensity projection ( mip ) technique to reconstruct sciatic nerves based on dti images in a single direction and in all directions . furthermore , based on preliminary experiments performed at the wrist region , we found that images of a single direction could also be reconstructed by using volume rendering ( vr ) and mip . these two post - processing methods ( vr and mip ) may offer a more convenient procedure for dti because they do not require prior choice of rois . we predicted that the peripheral nerve at the elbow region could be three - dimensionally displayed based on the mr dti data with multiple post - processing techniques . we aimed to determine whether the vr or mip reconstruction for single direction would be more convenient than fiber tracking post - processing while acquiring approximate qualities of nerve imaging , and the consistency of vr / mip and fiber tracking post - processing for the same sample . the study had prior approval by our institutional review board and ethics committee , and all subjects provided informed consent prior to the study . a total of 24 elbows of 12 healthy , young volunteers were studied ( six men , six women ; age range 22 - 32 years ) . nerves at the elbow region were analyzed , including the median nerve , ulnar nerve , and radial nerve . the exclusion criteria included general contraindications for mr imaging ( mri ) , a prior history of trauma or surgery of the elbow , presence of rheumatoid arthritis , and presence of space - occupying lesions at the elbow . the mri system s maximum field gradient amplitude was 45 mt / m with a slew rate of 200 t m s. during the scan session , the soft coil was placed around the elbow , and then a polymer brace was placed on the whole upper limb in order to restrain movement . the elbow was then positioned to the center of the magnet bore . for imaging of the elbow , dti was performed using an echo planar inversion recovery ( epir ) sequence ( tr = 15,300 ms ; te= 92 ms ; ti = 200 ms ; fa = 90 ; etl = 1 ; fov = 250 250 mm ; scan matrix = 128 128 ; average = 2 ) with 38 transversal slices of 3.0-mm thickness with no gap between the slices . diffusion weighting with a b value of 1200 s / mm was applied in 20 encoding diffusion directions . in addition to the diffusion - weighted images , a single reference image without diffusion weighting ( b = 0 s / mm ) was acquired . the raw dti images were transferred automatically to the siemens mr workstation ( siemens syngo 3d ) . the x - axis represents the anterior posterior direction , the y - axis represents the left - right direction , and the z - axis represents the superior inferior direction . based on the gradient table of the dti scan , direction 1 with coordinates ( 1 , 0 , 0 ) represents the anterior posterior direction . ( 15 ) , the most suitable choice for single direction vr post - processing is direction 1 , which is perpendicular to the nerves . the original dti images usually contained a series of t2-weighted images ( b = 0 s / mm ) , which were generated as the first series in the result chart . during vr / mip reconstruction of images of direction 1 , any high - signal noises of the veins or skin present in the reconstructed stereoscopic view were cut off in order to make the nerves as conspicuous as possible . x - axis represents the anterior - posterior direction . direction 1 with the coordinates ( 1 , 0 , 0 ) represents the anterior - posterior direction . the raw dti images were transferred to the siemens mr workstation ( siemens syngo neuro 3d ) . fiber tractography was then performed using a multiple seed roi technique , which employed fiber assignment by continuous tracking . seed rois were drawn freehand at three anatomical locations ( level to the supracondylar humerus , olecranon , and proximal radioulnar joint ) on the raw dti images and reference t1-weighted images . rois were confined precisely to the nerve border to avoid partial volume artefacts and to exclude any surrounding structure from the seed rois . another author ( d .- s.j ) was responsible for this part of the work . in order to compare the imaging quality among vr / mip and fiber tractography , we created a custom evaluation scale to assess each nerve in vr / mip and fiber tractography images . we set the scale based on whether the target nerve could be shown and the length of the nerve was complete as follows : score of 2 , the target nerve ( the median , radial , or ulnar nerve ) at the elbow region was clearly shown and the entire length of the nerve was visible throughout the entire scanning section ; score of 1 , the target nerve at the elbow region was clearly shown , but its length was incomplete ; score of 0 , the target nerve was invisible . two authors ( w .- q.d and j .- h.g ) were responsible for independently evaluating the imaging qualities according to this scale . one author ( w .- q.d ) evaluated the imaging qualities again 4 weeks later . for each nerve , scores of vr / mip reconstruction of the single direction and fiber tracking were compared using friedman test . intraclass correlation coefficient ( icc ) analysis was used to compare the correlation of the imaging quality scores for each nerve among vr / mip and fiber tractography . the study had prior approval by our institutional review board and ethics committee , and all subjects provided informed consent prior to the study . a total of 24 elbows of 12 healthy , young volunteers were studied ( six men , six women ; age range 22 - 32 years ) . nerves at the elbow region were analyzed , including the median nerve , ulnar nerve , and radial nerve . the exclusion criteria included general contraindications for mr imaging ( mri ) , a prior history of trauma or surgery of the elbow , presence of rheumatoid arthritis , and presence of space - occupying lesions at the elbow . the mri system s maximum field gradient amplitude was 45 mt / m with a slew rate of 200 t m s. during the scan session , the soft coil was placed around the elbow , and then a polymer brace was placed on the whole upper limb in order to restrain movement . the subject s upper extremity with the coil was immobilized with cushions , sandbags , and bandages . the elbow was then positioned to the center of the magnet bore . for imaging of the elbow , dti was performed using an echo planar inversion recovery ( epir ) sequence ( tr = 15,300 ms ; te= 92 ms ; ti = 200 ms ; fa = 90 ; etl = 1 ; fov = 250 250 mm ; scan matrix = 128 128 ; average = 2 ) with 38 transversal slices of 3.0-mm thickness with no gap between the slices . diffusion weighting with a b value of 1200 s / mm was applied in 20 encoding diffusion directions . in addition to the diffusion - weighted images , a single reference image without diffusion weighting ( b = 0 s / mm ) was acquired . the raw dti images were transferred automatically to the siemens mr workstation ( siemens syngo 3d ) . the x - axis represents the anterior posterior direction , the y - axis represents the left - right direction , and the z - axis represents the superior inferior direction . based on the gradient table of the dti scan , direction 1 with coordinates ( 1 , 0 , 0 ) represents the anterior posterior direction . ( 15 ) , the most suitable choice for single direction vr post - processing is direction 1 , which is perpendicular to the nerves . the original dti images usually contained a series of t2-weighted images ( b = 0 s / mm ) , which were generated as the first series in the result chart . during vr / mip reconstruction of images of direction 1 , any high - signal noises of the veins or skin present in the reconstructed stereoscopic view were cut off in order to make the nerves as conspicuous as possible . x - axis represents the anterior - posterior direction . direction 1 with the coordinates ( 1 , 0 , 0 ) represents the anterior - posterior direction . the raw dti images were transferred to the siemens mr workstation ( siemens syngo neuro 3d ) . fiber tractography was then performed using a multiple seed roi technique , which employed fiber assignment by continuous tracking . seed rois were drawn freehand at three anatomical locations ( level to the supracondylar humerus , olecranon , and proximal radioulnar joint ) on the raw dti images and reference t1-weighted images . rois were confined precisely to the nerve border to avoid partial volume artefacts and to exclude any surrounding structure from the seed rois . another author ( d .- s.j ) was responsible for this part of the work . in order to compare the imaging quality among vr / mip and fiber tractography , we created a custom evaluation scale to assess each nerve in vr / mip and fiber tractography images . we set the scale based on whether the target nerve could be shown and the length of the nerve was complete as follows : score of 2 , the target nerve ( the median , radial , or ulnar nerve ) at the elbow region was clearly shown and the entire length of the nerve was visible throughout the entire scanning section ; score of 1 , the target nerve at the elbow region was clearly shown , but its length was incomplete ; score of 0 , the target nerve was invisible . two authors ( w .- q.d and j .- h.g ) were responsible for independently evaluating the imaging qualities according to this scale . one author ( w .- q.d ) evaluated the imaging qualities again 4 weeks later . the imaging quality scores were expressed as medians with ranges and mean with sd . for each nerve , scores of vr / mip reconstruction of the single direction and fiber tracking were compared using friedman test . intraclass correlation coefficient ( icc ) analysis was used to compare the correlation of the imaging quality scores for each nerve among vr / mip and fiber tractography . kappa values of 0 - 0.20 were considered to indicate poor agreement ; 0.21 - 0.40 , fair agreement ; 0.41 - 0.60 , moderate agreement ; 0.61 - 0.80 , good agreement ; and 0.81 - 1.00 , excellent agreement . all 24 elbows of the 12 young volunteers were tested , and stereoscopic displays of the nerves were obtained by using vr , mip , and fiber tractography post - processing methods ( figure 1 ) . for most elbows of volunteers , three main nerves could be seen clearly ( figure 1 ) , whereas only one nerve could be detected in other individual cases ( figure 2 ) . owing to the relatively simple and direct operations enabled without requiring rois selection , the vr and mip post - processing methods were easier to perform compared to fiber tractography . vr / mip reconstructions for single direction images and fiber tracking evaluation scores for each target nerve are listed in table 2 , which did not differ significantly ( p > 0.05 ) . the imaging quality scores of fiber tractography and vr / mip were significantly correlated based on intraclass correlation coefficient ( icc ) analysis ( icc ranged 0.709 - 0.901 ) , which suggested good consistency between the scores obtained based on the fiber tractography and vr / mip for the same sample . the values of inter- and intraobserver agreements ranged 0.674 - 0.909 , which showed good to excellent agreements . abbreviations : ft , fiber tractography ; icc , intraclass correlation coefficient ; 1 , interobserver value ; 2 , intraobserver value ; ob1 , observer 1 ; ob2 , observer 2 ; ob2 sec . in this study , we found that some images belong to a single direction ( perpendicular to the nerve ) after dti scan could be used for vr or mip reconstruction to display the peripheral nerves clearly . the difference of applied diffusion directions between dti and dw - mrn scans is that dti scan can select many non - parallel diffusion directions , and the data can be used for fiber tracking and measuring the fa , apparent diffusion coefficient ( adc ) value , while dw - mrn can choose fewer number of diffusion directions , increase the excitation frequency so the scan time is shorter , and signal - to - noise ratio increased for better mip or vr display . ( 17 ) indicated when they applied diffusion direction which is perpendicular to the nerve axons , the water molecular diffusion in the nerve was restricted , so the nerve tissue could all be imaged and showed high signal . we speculated that in dti scan , the data collected from the perpendicular direction may play an important role in calculating fa value , which is the key parameter for fiber tracking . thus , vr and mip reconstruction of a single direction ( anterior - posterior direction , perpendicular to the nerve ) could predict the imaging quality of fiber tractography . for some elbows of volunteers , three main nerves could be seen clearly ( figure 1 ) , whereas only one nerve could be detected in other cases ( figure 2 ) . besides the reasons of anatomical characteristics at the elbow region and the limitations of the technique , one possible explanation for this variation is that slight movement of the limb is the key factor affecting imaging quality . in the image shown in figure 2 , the ulnar nerve is located outside of the outline of the elbow . the median nerve and radial nerve are covered by the outline of the elbow , which suggests that the signal values of the nerve fibers were calculated incorrectly , thereby affecting fiber tracking . vr and mip post - processing are more convenient to obtain compared to fiber tracking . the icc analysis result suggested that scores based on fiber tractography and vr / mip for the same samples were highly consistent . therefore , vr and mip reconstruction can be used as a simple preview tool before performing complicated fiber tracking . during the dti scanning process using siemens magenetom verio 3 t mri scanner , the dti original images are generated in the order of the gradient table ( table 1 ) from direction 1 to direction 20 , and from average 1 to average 2 . the images belonging to direction 1 of average 1 will be generated within the first few seconds , and then vr and mip post - processing can be immediately performed to observe whether or not the nerve is displayed . if the nerves display , then , a scan with more directions and more averages should be continued for fiber tracking . if the nerves do not display , the scan should be terminated , and more measures for restricting movement of the elbow should be adopted . some potential applications of vr and mip reconstruction include evaluation of the continuity and integrity of the nerve as well as detection of abnormal morphology , whereas the application of fiber tracking is specific to assessing the status of axons . according to sunderland s nerve injury classification ( 18 ) , grade i , grade ii , and some grade iii injuries ( axons or endoneurial tube disruptions ) do not require surgical exploration , whereas grade iv - vi injuries ( perineurium or epineurium damages , defects , or mixed injuries ) do require surgical exploration as soon as possible . therefore , vr and mip reconstruction might be able to show more details of damaged nerves , which unlike fiber tracking , do not only focus on the axons . injury of the ulnar nerve at the elbow region is also common , because it is superficial and close to the bone surface . stereoscopic display of the nerves at the elbow region can help the clinician understand the morphology of the target nerve . for example , in nerve entrapment patients , nerves can be detected as the features of increasing csa and flatten ratio ( fr ) , which can be observed more easily in stereoscopic displays compared with conventional 2d images . by using these methods with the addition of fa and adc value measurements , dti appears to be a promising tool for future evaluations of the severity and rehabilitation of ulnar neuropathy . first , due to the limited choice of equipment and coil , the parameters and coil of the dti scan at the elbow region did not reach adequate optimization . second , the measures adopted for restricting slight movement of the limb were not highly efficient . stereoscopic displays of the peripheral nerve at the elbow region were achieved by using vr , mip , and fiber tracking post - processing methods based on raw dti images . the scores based on vr / mip and fiber tracking post - processing had good consistency for the same samples indicating that vr and mip reconstruction for single direction could be used as preview tools before fiber tracking to determine whether the raw images are satisfactory .

peripheral nerves , especially those at the elbow region , such as the median nerve and ulnar nerve , are prone to entrapment neuropathies and injuries , such as cubital tunnel syndrome ( 1 , 2 ) , pronator teres syndrome ( 3 ) , and nerve laceration ( 4 ) . the traditional method used for evaluating these diseases with respect to preoperative diagnosis and postoperative rehabilitation is electrophysiological nerve conduction and electromyography ; however , accurate evaluations using these methods largely depend on the operator s experience , and false negative results are common ( 5 ) . a magnetic resonance ( mr ) image can display the soft tissue , including nerves , in t1 or t2 images , and the most commonly used method of mr for the diagnosis of peripheral nerve entrapment is to measure the cross sectional area ( csa ) and t2 signal intensity , which can not distinguish axonal injury from demyelination ( 6 ) . moreover , the continuity and integrity of a nerve can hardly be detected based on two - dimensional ( 2d ) images . therefore , obtaining a three - dimensional ( 3d ) display of a nerve that can be observed at any angle is essential for clinicians to make accurate assessments . currently , the methods and sequences of mr neurography are mostly represented through diffusion tensor imaging ( dti ) . dti with fiber tracking was the first method used to obtain a 3d view of the nerve , which was originally applied to image the central nervous system . diffusion is less restricted in the direction parallel to the nerve fiber than that perpendicular to the fiber ( possibly due to the layers of myelin ) . using special fiber - tracking software to reconstruct the dti data , it is possible to visualize the complete neural structure . ( 7 ) demonstrated that peripheral nerves ( the sciatic nerve in three healthy volunteers ) could be imaged in vivo by using dti with fiber tracking . this technique has now become the most widely used method for evaluating the status of the median nerves in carpal tunnel syndrome ( 8) , providing non - invasive imaging of peripheral nerve regeneration ( 9 - 11 ) , and characterizing the structure of the brachial plexus ( 12 ) . however , studies reporting the use of dti with fiber tracking post - processing at the elbow region are scarce . the following drawbacks of using dti for the elbow region , especially for the ulnar nerve , have been discussed : ( i ) it is superficial ; ( ii ) close to the bone surface ; and ( iii ) shows large angulations in the course of the nerve ( 13 ) . besides , these limitations of dti with respect to specific anatomical characteristics at the elbow region , the technique itself has many limitations . first , the quality of tractography depends on the parameters used in the acquisition step , such as the coils used , positioning of the patient , and other parameters including the b - value and the number of diffusion directions . when using this type of hard coil , the elbow , the subject must raise one hand above his / her head while in prone or supine position , which can be very uncomfortable . second , during the post - processing phase before fiber tracking , the operator must choose a series of regions of interest ( roi ) , which depends on the operator s anatomical knowledge . in addition to the dti fiber tracking technique , a novel post - processing method to reconstruct dti data was developed by skorpil et al . they used the maximum intensity projection ( mip ) technique to reconstruct sciatic nerves based on dti images in a single direction and in all directions . furthermore , based on preliminary experiments performed at the wrist region , we found that images of a single direction could also be reconstructed by using volume rendering ( vr ) and mip . these two post - processing methods ( vr and mip ) may offer a more convenient procedure for dti because they do not require prior choice of rois . we predicted that the peripheral nerve at the elbow region could be three - dimensionally displayed based on the mr dti data with multiple post - processing techniques . we aimed to determine whether the vr or mip reconstruction for single direction would be more convenient than fiber tracking post - processing while acquiring approximate qualities of nerve imaging , and the consistency of vr / mip and fiber tracking post - processing for the same sample . a total of 24 elbows of 12 healthy , young volunteers were studied ( six men , six women ; age range 22 - 32 years ) . nerves at the elbow region were analyzed , including the median nerve , ulnar nerve , and radial nerve . the exclusion criteria included general contraindications for mr imaging ( mri ) , a prior history of trauma or surgery of the elbow , presence of rheumatoid arthritis , and presence of space - occupying lesions at the elbow . the mri system s maximum field gradient amplitude was 45 mt / m with a slew rate of 200 t m s. during the scan session , the soft coil was placed around the elbow , and then a polymer brace was placed on the whole upper limb in order to restrain movement . the subject s upper extremity with the coil was immobilized with cushions , sandbags , and bandages . for imaging of the elbow , dti was performed using an echo planar inversion recovery ( epir ) sequence ( tr = 15,300 ms ; te= 92 ms ; ti = 200 ms ; fa = 90 ; etl = 1 ; fov = 250 250 mm ; scan matrix = 128 128 ; average = 2 ) with 38 transversal slices of 3.0-mm thickness with no gap between the slices . in addition to the diffusion - weighted images , a single reference image without diffusion weighting ( b = 0 s / mm ) was acquired . for anatomical reference , a t1-weighted image ( tr = 700 ms ; te = 11 ms ; fa = 150 ; etl = 3 ; thickness = 3 mm ; gap = 0 mm ; fov = 140 140 mm ; scan matrix = 320 240 ; average = 1 ) was acquired . the scanning times for dti and t1-weighted image were 11 min 29 s and 2 min 1 s , respectively , for a total scan time of approximately 15 min . the x - axis represents the anterior posterior direction , the y - axis represents the left - right direction , and the z - axis represents the superior inferior direction . based on the gradient table of the dti scan , direction 1 with coordinates ( 1 , 0 , 0 ) represents the anterior posterior direction . ( 15 ) , the most suitable choice for single direction vr post - processing is direction 1 , which is perpendicular to the nerves . the original dti images usually contained a series of t2-weighted images ( b = 0 s / mm ) , which were generated as the first series in the result chart . the optimal choice ( direction 1 ) was the second series generated in the result chart . during vr / mip reconstruction of images of direction 1 , any high - signal noises of the veins or skin present in the reconstructed stereoscopic view were cut off in order to make the nerves as conspicuous as possible . one of the authors ( y.y ) was responsible for this part of the work . direction 1 with the coordinates ( 1 , 0 , 0 ) represents the anterior - posterior direction . seed rois were drawn freehand at three anatomical locations ( level to the supracondylar humerus , olecranon , and proximal radioulnar joint ) on the raw dti images and reference t1-weighted images . rois were confined precisely to the nerve border to avoid partial volume artefacts and to exclude any surrounding structure from the seed rois . in this study , the fractional anisotropy ( fa ) threshold value was 0.2 and the angulation tolerance was 30. the software s option step length was set to 0.47 mm . another author ( d .- s.j ) was responsible for this part of the work . in order to compare the imaging quality among vr / mip and fiber tractography , we created a custom evaluation scale to assess each nerve in vr / mip and fiber tractography images . we set the scale based on whether the target nerve could be shown and the length of the nerve was complete as follows : score of 2 , the target nerve ( the median , radial , or ulnar nerve ) at the elbow region was clearly shown and the entire length of the nerve was visible throughout the entire scanning section ; score of 1 , the target nerve at the elbow region was clearly shown , but its length was incomplete ; score of 0 , the target nerve was invisible . two authors ( w .- q.d and j .- h.g ) were responsible for independently evaluating the imaging qualities according to this scale . one author ( w .- q.d ) evaluated the imaging qualities again 4 weeks later . for each nerve , scores of vr / mip reconstruction of the single direction and fiber tracking were compared using friedman test . intraclass correlation coefficient ( icc ) analysis was used to compare the correlation of the imaging quality scores for each nerve among vr / mip and fiber tractography . kappa values of 0 - 0.20 were considered to indicate poor agreement ; 0.21 - 0.40 , fair agreement ; 0.41 - 0.60 , moderate agreement ; 0.61 - 0.80 , good agreement ; and 0.81 - 1.00 , excellent agreement . a total of 24 elbows of 12 healthy , young volunteers were studied ( six men , six women ; age range 22 - 32 years ) . nerves at the elbow region were analyzed , including the median nerve , ulnar nerve , and radial nerve . the exclusion criteria included general contraindications for mr imaging ( mri ) , a prior history of trauma or surgery of the elbow , presence of rheumatoid arthritis , and presence of space - occupying lesions at the elbow . the mri system s maximum field gradient amplitude was 45 mt / m with a slew rate of 200 t m s. during the scan session , the soft coil was placed around the elbow , and then a polymer brace was placed on the whole upper limb in order to restrain movement . the subject s upper extremity with the coil was immobilized with cushions , sandbags , and bandages . for imaging of the elbow , dti was performed using an echo planar inversion recovery ( epir ) sequence ( tr = 15,300 ms ; te= 92 ms ; ti = 200 ms ; fa = 90 ; etl = 1 ; fov = 250 250 mm ; scan matrix = 128 128 ; average = 2 ) with 38 transversal slices of 3.0-mm thickness with no gap between the slices . in addition to the diffusion - weighted images , a single reference image without diffusion weighting ( b = 0 s / mm ) was acquired . for anatomical reference , a t1-weighted image ( tr = 700 ms ; te = 11 ms ; fa = 150 ; etl = 3 ; thickness = 3 mm ; gap = 0 mm ; fov = 140 140 mm ; scan matrix = 320 240 ; average = 1 ) was acquired . the x - axis represents the anterior posterior direction , the y - axis represents the left - right direction , and the z - axis represents the superior inferior direction . based on the gradient table of the dti scan , direction 1 with coordinates ( 1 , 0 , 0 ) represents the anterior posterior direction . ( 15 ) , the most suitable choice for single direction vr post - processing is direction 1 , which is perpendicular to the nerves . the original dti images usually contained a series of t2-weighted images ( b = 0 s / mm ) , which were generated as the first series in the result chart . during vr / mip reconstruction of images of direction 1 , any high - signal noises of the veins or skin present in the reconstructed stereoscopic view were cut off in order to make the nerves as conspicuous as possible . one of the authors ( y.y ) was responsible for this part of the work . direction 1 with the coordinates ( 1 , 0 , 0 ) represents the anterior - posterior direction . seed rois were drawn freehand at three anatomical locations ( level to the supracondylar humerus , olecranon , and proximal radioulnar joint ) on the raw dti images and reference t1-weighted images . rois were confined precisely to the nerve border to avoid partial volume artefacts and to exclude any surrounding structure from the seed rois . in this study , the fractional anisotropy ( fa ) threshold value was 0.2 and the angulation tolerance was 30. the software s option step length was set to 0.47 mm . another author ( d .- s.j ) was responsible for this part of the work . in order to compare the imaging quality among vr / mip and fiber tractography , we created a custom evaluation scale to assess each nerve in vr / mip and fiber tractography images . we set the scale based on whether the target nerve could be shown and the length of the nerve was complete as follows : score of 2 , the target nerve ( the median , radial , or ulnar nerve ) at the elbow region was clearly shown and the entire length of the nerve was visible throughout the entire scanning section ; score of 1 , the target nerve at the elbow region was clearly shown , but its length was incomplete ; score of 0 , the target nerve was invisible . two authors ( w .- q.d and j .- h.g ) were responsible for independently evaluating the imaging qualities according to this scale . one author ( w .- q.d ) evaluated the imaging qualities again 4 weeks later . for each nerve , scores of vr / mip reconstruction of the single direction and fiber tracking were compared using friedman test . intraclass correlation coefficient ( icc ) analysis was used to compare the correlation of the imaging quality scores for each nerve among vr / mip and fiber tractography . kappa values of 0 - 0.20 were considered to indicate poor agreement ; 0.21 - 0.40 , fair agreement ; 0.41 - 0.60 , moderate agreement ; 0.61 - 0.80 , good agreement ; and 0.81 - 1.00 , excellent agreement . all 24 elbows of the 12 young volunteers were tested , and stereoscopic displays of the nerves were obtained by using vr , mip , and fiber tractography post - processing methods ( figure 1 ) . for most elbows of volunteers , three main nerves could be seen clearly ( figure 1 ) , whereas only one nerve could be detected in other individual cases ( figure 2 ) . owing to the relatively simple and direct operations enabled without requiring rois selection , the vr and mip post - processing methods were easier to perform compared to fiber tractography . vr / mip reconstructions for single direction images and fiber tracking evaluation scores for each target nerve are listed in table 2 , which did not differ significantly ( p > 0.05 ) . the imaging quality scores of fiber tractography and vr / mip were significantly correlated based on intraclass correlation coefficient ( icc ) analysis ( icc ranged 0.709 - 0.901 ) , which suggested good consistency between the scores obtained based on the fiber tractography and vr / mip for the same sample . the values of inter- and intraobserver agreements ranged 0.674 - 0.909 , which showed good to excellent agreements . in this study , we found that some images belong to a single direction ( perpendicular to the nerve ) after dti scan could be used for vr or mip reconstruction to display the peripheral nerves clearly . the difference of applied diffusion directions between dti and dw - mrn scans is that dti scan can select many non - parallel diffusion directions , and the data can be used for fiber tracking and measuring the fa , apparent diffusion coefficient ( adc ) value , while dw - mrn can choose fewer number of diffusion directions , increase the excitation frequency so the scan time is shorter , and signal - to - noise ratio increased for better mip or vr display . ( 17 ) indicated when they applied diffusion direction which is perpendicular to the nerve axons , the water molecular diffusion in the nerve was restricted , so the nerve tissue could all be imaged and showed high signal . we speculated that in dti scan , the data collected from the perpendicular direction may play an important role in calculating fa value , which is the key parameter for fiber tracking . thus , vr and mip reconstruction of a single direction ( anterior - posterior direction , perpendicular to the nerve ) could predict the imaging quality of fiber tractography . the results of this study raise an important and essential question : under the same experimental conditions , why do the results show variation among different healthy volunteers ? besides the reasons of anatomical characteristics at the elbow region and the limitations of the technique , one possible explanation for this variation is that slight movement of the limb is the key factor affecting imaging quality . in the image shown in figure 2 , the ulnar nerve is located outside of the outline of the elbow . the median nerve and radial nerve are covered by the outline of the elbow , which suggests that the signal values of the nerve fibers were calculated incorrectly , thereby affecting fiber tracking . the icc analysis result suggested that scores based on fiber tractography and vr / mip for the same samples were highly consistent . during the dti scanning process using siemens magenetom verio 3 t mri scanner , the dti original images are generated in the order of the gradient table ( table 1 ) from direction 1 to direction 20 , and from average 1 to average 2 . the images belonging to direction 1 of average 1 will be generated within the first few seconds , and then vr and mip post - processing can be immediately performed to observe whether or not the nerve is displayed . some potential applications of vr and mip reconstruction include evaluation of the continuity and integrity of the nerve as well as detection of abnormal morphology , whereas the application of fiber tracking is specific to assessing the status of axons . according to sunderland s nerve injury classification ( 18 ) , grade i , grade ii , and some grade iii injuries ( axons or endoneurial tube disruptions ) do not require surgical exploration , whereas grade iv - vi injuries ( perineurium or epineurium damages , defects , or mixed injuries ) do require surgical exploration as soon as possible . therefore , vr and mip reconstruction might be able to show more details of damaged nerves , which unlike fiber tracking , do not only focus on the axons . cubital tunnel syndrome is the second - most common nerve compression syndrome after carpal tunnel syndrome . stereoscopic display of the nerves at the elbow region can help the clinician understand the morphology of the target nerve . for example , in nerve entrapment patients , nerves can be detected as the features of increasing csa and flatten ratio ( fr ) , which can be observed more easily in stereoscopic displays compared with conventional 2d images . by using these methods with the addition of fa and adc value measurements , dti appears to be a promising tool for future evaluations of the severity and rehabilitation of ulnar neuropathy . first , due to the limited choice of equipment and coil , the parameters and coil of the dti scan at the elbow region did not reach adequate optimization . stereoscopic displays of the peripheral nerve at the elbow region were achieved by using vr , mip , and fiber tracking post - processing methods based on raw dti images . the scores based on vr / mip and fiber tracking post - processing had good consistency for the same samples indicating that vr and mip reconstruction for single direction could be used as preview tools before fiber tracking to determine whether the raw images are satisfactory .

since the initial studies by murugan et al . in 2007 , li7la3zr2o12 ( llzo ) has received much scientific attention as a solid electrolyte for beyond li ion battery llzo provides a high li - ion conductivity ( 10 to 10 s cm at ambient temperature ) and a li transference number approaching 1 , superior chemical stability against high voltage cathodes , and electrochemical inertness in a wide potential window of up to 6 v. in particular , its stability against li - metal as well as its thermal and mechanical robustness makes llzo garnet exceptionally well - suited to be used as a protecting layer for li - metal - based batteries . for pure llzo , two structural polymorphs are described in the literature . a low - temperature tetragonal modification with a completely ordered arrangement of li crystallizes in space group ( sg ) i41/acd ( no . 142 ) . this structure type is described as garnet - related framework with two types of dodecahedral lao8 polyhedra ( 8b and 16e ) and zro6 octahedra ( 16c ) . li occupies three different sites : the tetrahedral 8a site as well as distorted octahedral 16f and 32 g sites . in contrast to the tetragonal modification , the cubic high - temperature modification exhibits a disorder in the li distribution . this cubic modification shows sg ia-3d ( no . 230 ) and has the garnet structure composed of a framework of 8-fold coordinated lao8 dodecahedra ( 24c ) and 6-fold coordinated zro6 octahedra ( 16a ) . li is located at interstitial sites , showing tetrahedral ( 24d ) , octahedral ( 48 g ) , and distorted 4-fold ( 96h ) coordination . for use as li - ion conductors , the cubic modification is much more desirable as its li - ion conductivity is 2 orders of magnitude higher than for the tetragonal modification . the cubic modification of pure llzo is not stable at room temperature ( rt ) . however , it can be stabilized at rt by the introduction of supervalent cations , which cause a reduction of the li content that leads to the introduction of additional vacancies at the li sites . the stabilization of the cubic modification was originally achieved by the incorporation of al . further research led to the discovery of other cations that are capable to stabilize the cubic modification . among these , ga turned out to be a promising candidate which has been studied extensively . ga is incorporated at the li sites according to 3 li ga + 2 li , i.e. , creating two vacant sites , li , in the li - sublattice . ga - stabilized llzo is characterized by a li conductivity of 1.3 ms cm at rt , which is twice as high as for llzo stabilized with al . the reason for the higher li - ion conductivity of ga - stabilized llzo is , however , not yet fully understood . because of their blocking effect , some of these ions are suspected to hinder the long - range li - ion transport and , therefore , to reduce li - ion conductivity . ( llzto ) with those of li6.15la3zr1.75ta0.25m0.2o12 ( m = al , ga ) ; according to their study pure llzto shows the highest li - ion conductivity , followed by llzto : ga and llzto : al . on the basis of the crystal chemical considerations , they attributed this behavior to the different site preference of al and ga . in particular , the larger ga prefers the 96h site , which seems to be less hindering for long - range li - ion transport , compared to al , located at the 24d site , which is a junction for li - ion diffusion . however , the site preference of ga is still under discussion as ga nuclear magnetic resonance ( nmr ) spectroscopy studies revealed different results . ( 2015 ) , using very high magnetic fields ( 21.1 t ) , showed that the site preference of ga and al to occupy 24d and 96h voids in samples crystallizing with ia-3d is practically the same . thus , despite all efforts , there is , so far , no satisfying explanation why some of the ga - stabilized llzo samples presented in literature show higher ionic conductivities . investigations on ga - stabilized llzo single crystals might help us to shed light on this question . up to now , very little research has been performed on llzo single crystals . the synthesis of applicable llzo single crystals is delicate . to the best of our knowledge , no single - crystal x - ray diffraction ( sc - xrd ) study on ga - stabilized llzo has been published yet . in the present study , another cubic modification of llzo , showing the acentric sg i-43d , has been observed for ga - stabilized llzo for the first time . coarse - grained ga - stabilized llzo samples were synthesized via solid - state reaction and characterized by a rich portfolio of techniques including x - ray powder diffraction ( xrpd ) , single - crystal x - ray diffraction ( sc - xrd ) , neutron powder diffraction ( npd ) , scanning electron microscopy ( sem)/ backscattered electron ( bse ) imaging , energy - dispersive x - ray spectroscopy ( edx ) , and li nmr spectroscopy . a series of li73xgaxla3zr2o12 with intended ga contents xga = 0.10 , 0.15 , 0.20 , 0.30 , 0.40 , 0.50 , 0.60 pfu was synthesized by high - temperature sintering in air . for reason of comparison , also one sample of al - stabilized llzo with an intended al content xal of 0.20 al pfu ( li6.4al0.2la3zr2o12 ) was prepared under the same conditions . results obtained by cheng et al . concerning the densification and coarsening of llzo samples were taken into consideration for this synthesis . li2co3 ( 99% , merck ) , ga2o3 ( 99.0% , aldrich ) , al2o3 ( 99.5% , aldrich ) , la2o3 ( 99.99% , roth ) , and zro2 ( 99.0% , roth ) were used as reagents . the starting materials were weighed out in their intended stoichiometric proportions with an excess of 10 wt % li2co3 to compensate the loss of li2o during sintering . the reagents were mixed in an agate mortar under addition of isopropyl alcohol and then cold - pressed into pellets with a uniaxial press . the resulting pellets were put in an alumina crucible . to avoid undesired contamination with al from the crucible , the samples were placed on a pellet of pure llzo . the samples were heated to 850 c with a rate of 5 c / min and calcinated for 4 h. the resulting pellets were then removed from the furnace , ground in an agate mortar , and ball - milled for 1 h under isopropyl alcohol ( fritsch pulverisette 7 , 800 rpm , 2 mm zro2 balls ) . after drying under air , to avoid incorporation of al from the crucible , the samples were again placed on a pellet of pure llzo . to suppress formation of extra phases due to li loss during sintering , the sample pellets were covered with a pellet of pure llzo . for the final sintering , the pellets were heated in a muffle furnace in air with a rate of 5 c / min to 1230 c and sintered for 6 h. for xrpd investigations , small fragments of the sintered pellets were ground using an agate mortar . pellets used for npd were stored in a glovebox under ar atmosphere to avoid reaction with co2 and h2o ; also the whole processing after sintering , including grinding using an agate mortar and filling of the sample container has been performed under ar atmosphere . for sem analysis , polycrystalline chips from the pellets were embedded in an epoxy holder . the surface was ground and then polished using diamond paste . in particular , we put emphasis on the investigation of the grain size , morphology , phase composition , and the chemical homogeneity , i.e. , the distribution , of ga , la , zr , using a backscattered electrons detector ( bse ) and energy - dispersive x - ray spectroscopy ( edx ) measurements , respectively . patterns were recorded using a bruker d8 advance davinci design diffractometer with a lynxeye solid - state detector using cu k radiation . the synthesis products were characterized regarding the presence of extra phases as well as to determine the symmetry and unit - cell dimension of the samples . data were collected between 10 and 80 2. evaluation of xrpd data was performed by rietveld refinement using the program bruker diffrac topas ( version 4.2 ) . single - crystal x - ray diffraction data were collected on a bruker smart apex ccd - diffractometer . samples for single - crystal studies were selected readily after removing of samples from the furnace , and data collection was done within 24 h after synthesis . so we exclude that the change in sg symmetry is due to the incorporation of h in the structure as it was suggested in literature . single crystals were carefully hand - picked under the binocular loupe from the gently crushed pellets . for each composition , more than 10 crystals were selected on the basis of their optical properties ( regular shape , clearness , and homogeneity in color ) . selected crystals were glued on top of glass capillaries ( 0.1 mm ) and tested on the diffractometer . a full set of intensity data was collected on 24 crystals for each composition to obtain good statistics and data sets for crystal chemical interpretation , resulting in a total of 21 data sets . intensity data were collected with graphite - monochromatized mo k x - radiation ( 50 kv , 30 ma ) ; the crystal - to - detector distance was 30 mm , and the detector was positioned at 30 and 50 2 using an -scan mode strategy at four different positions ( 0 , 90 , 180 , and 270 ) for each 2 position . in total , 630 frames with = 0.3 were acquired for each run . with this strategy , data in a large q - range up to minimum d - values d = 0.53 could be acquired . this is necessary for accurate determination of anisotropic displacement parameters and to reduce correlation effects between atomic displacement parameters and site occupation numbers . three - dimensional data were integrated and corrected for lorentz- , polarization , and background effects using the apex2 software . structure solution ( using direct methods ) and subsequent weighted full - matrix least - squares refinements on f were done with shelx-2012 as implemented in the program suite wingx 2014.1 . neutron powder diffraction data for a sample with a nominal ga content xga = 0.20 pfu were collected at the maier - leibnitz zentrum ( mlz ) , frm - ii , munich , germany . powder diffraction data were acquired at 298 k in constant wavelength mode using the high - resolution powder diffractometer spodi with ge551 monochromatized neutron radiation ( = 1.5482 ) . experiments were performed in a 2 range of 3 2 154 , step width of 0.045. data treatment of powder diffraction data sets as well as a combined refinement of neutron powder diffraction data and single - crystal x - ray diffraction data ( with special emphasis of li - cationic distribution ) was performed using the fullprof - suite of programs . variable - temperature li nmr spectra and spin lattice relaxation rates were recorded with a bruker avance iii solid - state spectrometer connected to a 7-t magnet ( bruker ) . the resonance frequency was 116 mhz ; 90 pulse lengths ranged from 2 to 3 s depending on temperature . we used single - pulse and solid - echo experiments to record nmr line shapes . lattice relaxation rates in the laboratory frame were measured by means of the saturation recovery sequence ; the rates in the rotating frame were acquired by using the spin - lock technique , see epp et al . for details . to protect the samples permanently from humidity during the nmr measurements small pieces of the sintered garnets were put into glass ampules that were sealed under vacuum . polished samples and pellet fragments were examined by sem - bse . for all compositions , grain sizes of > minor amounts of extra phases were documented by sem - bse due to a contrast in brightness and a different morphology . as already observed visually , extra phases are commonly found in the peripheral part of the pellets . single grains are commonly separated by gaps , and small amounts of extra phases are located in these gaps . only small open voids with a size of < 10 m were found as inclusions within llzo grains . a representative sem - bse image of a pellet fragment is shown in figure 1 . standard - free sem - edx analysis of llzo grains showed the presence of la , zr , and ga . the la / zr ratio of llzo grains was slightly below the theoretical values of 3:2 , which is in accordance with site occupation refinements from sc - xrd data and microprobe measurements of ga - stabilized llzo by rettenwander et al . ga contents of llzo grains were partially lower than the target stoichiometry ; additionally , a slight variation of the ga content within single samples was noted . edx mapping of llzo grains confirmed their homogeneity with regards to the distribution of la and zr within single grains . extra phases observed by sem - bse can be divided due to their chemical composition seen by edx into three different phases . an extra phase with a la / zr ratio of 1:1 and another extra phase showing a la signal only were mainly found in the peripheral part of the pellets . for samples with nominal xga = ( 0.50 , 0.60 ) per formula unit ( pfu ) , another extra phase that only showed a ga signal in edx was found . the xrpd patterns of li73xgaxla3zr2o12 ( gax ) with nominal ga concentration xga = 0.10 , 0.15 , 0.20 , 0.30 , 0.40 , 0.50 , 0.60 pfu are shown in figure 1 . all garnet compositions exhibit reflections indicating cubic symmetry . for sample with xga = 0.10 , tetragonal llzo was still present with a share of 18 wt % as determined by quantitative phase analysis using the rietveld method . for samples with xga 0.15 , no tetragonal llzo phase was observed . la2zr2o7 and la(oh)3 were identified as extra phases and confirmed results of sem - edx investigations . considering the sem - bse results , these extra phases occur predominantly in the rim of the pellets and formed due to evaporation of li during high temperature sintering . the presence of extra phases was accepted as they facilitate the extraction of single crystals . for sample with xga = 0.60 , it has to be noted that for most samples , an additional weak reflection at 2 = 21.65 ( equal to d = 4.10 ) was observed . this peak can not be explained with llzo showing sg ia-3d or any extra phase . comparison of xrpd patterns for samples ga10 ( lowermost pattern ) , ga15 , ga20 , ga30 , ga40 , ga50 , ga60 ( uppermost pattern ) . pattern contributions from extra phases are marked with colors : la2zr2o7 ( blue ) , la(oh)3 ( green ) , ligao2 ( violet ) . the peak at 2 = 21.65 ( red ) could neither be attributed to cubic llzo with sg ia-3d nor to any extra phase . the inset shows a representative sem - bse image of a pellet fragment of sample ga40 to demonstrate the grain size and morphology . the experimental data and results of structure refinement for selected samples are reported in table 1 and table 2 , while the fractional atomic coordinates , occupation numbers and equivalent isotropic , and anisotropic atomic displacement parameters are given in table s1 . crystallographic information files ( cif ) with full structural information are deposited as supporting information . xal value instead of xga_ref ; fixed to the data obtained from edx analysis for sample al20_7 . single - crystal x - ray intensity data processing gave strong evidence for the cubic crystal system for all data collected . for the data of sample ga10 , with nominal ga concentration xga = 0.10 pfu , sg determination yields ia-3d symmetry as is widely accepted for llzo garnets . however , for all samples with nominal ga concentrations xga 0.15 pfu , intensity statistics and systematic extinctions did not result in sg ia-3d but yielded an acentric sg i-43d ( no . 220 ) . this sg was consistently obtained using all the different sg determination tools as implemented in wingx . subsequent structure solution tests for samples with xga 0.15 pfu using commonly occurring sg ia-3d or tetragonal sg i41/acd and sg i41/a failed . simulated precession images of the 0kl layer of samples ga10_1 and ga40_2 , displayed in figure 2 , obviously show the presence of bragg peaks with k = odd and l = odd in sample ga40 . these peaks are forbidden in sg ia-3d ( as is also the case for sample ga10_1 ) but allowed in sg i-43d . the presence of bragg peaks with k = odd and l = odd in ( b ) is evident . structure solution with direct methods using i-43d symmetry resulted in a structure model which could be refined down to wr2 values for all data < 4% . the li - positions were located from difference fourier map calculations , in the final structure all atoms could be treated with anisotropic atomic displacement parameters . our structure model has been found consistent with the one given by lager et al . and galven et al . li leaching and replacement by h and up to now , no doubt on the violation of ia-3d symmetry for ga - stabilized llzo was reported . samples of al - stabilized llzo , which are produced with the very same synthesis strategies , show ia-3d symmetry up to nominal compositions of xal = 0.30 pfu in single - crystal structure refinements . different to the ia-3d structure of sample ga10 with a nominal ga content xga = 0.10 pfu , which exhibits 5 different atomic positions in the asymmetric unit , the crystal structure of ga - stabilized llzo with a nominal ga content xga 0.15 pfu , showing sg i-43d , exhibits seven different atomic positions : la occupies the 8-fold coordinated 24d position ( site symmetry 2 .. ) , and zr is located at the octahedrally coordinated 16c position ( site symmetry .3 . ) . li is found distributed over three different positions in i-43d : two of them correspond to the regular tetrahedral coordinated site of the silicate garnets ( 24d in ia-3d ) , nevertheless , they split into two positions , namely li1 ( 12a ) and li2 ( 12b ) , both with site symmetry 4 .. , and differ in both bond lengths and polyhedral distortion . the third li position , namely li3 , has been found located on general position 48e , site symmetry 1 , which is only partly occupied as is also common in llzo compounds . in contrast to common silicate garnets , the acentric structure exhibits two independent o positions , both in general position 48e . table 1 shows a summary of the sc - xrd results of selected samples . more detailed information is given in table 2 , while a comparison of the two structural models ia-3d and i-43d is given in table s1 . a graphical representation of the ia-3d and i-43d structures , including the li - network , is displayed in figure 3 . ( a ) crystal structure of ga - stabilized llzo with xga = 0.10 and sg ia-3d . blue dodecahedra represent 8-fold coordinated la ( at the wyckoff position 24c ) ; green octahedra 6-fold coordinated zr ( 16a ) . the red spheres correspond to tetrahedrally coordinated li at the 24d ( li1 ) site , yellow spheres correspond to distorted 4-fold coordinated li at wyckoff position 96h ( li2 ) . ( b ) crystal structure of ga - stabilized llzo with xga = 0.30 and sg i-43d . blue dodecahedra represent 8-fold coordinated la ( at the wyckoff position 24d ) ; green octahedra 6-fold coordinated zr ( 16c ) . the red spheres correspond to tetrahedrally coordinated li at the 12a site ( li1 ) , orange spheres represent tetrahedrally coordinated li at the 12b site ( li2 ) ; yellow spheres correspond to distorted 6-fold coordinated li at wyckoff position 48e ( li3 ) . ( c ) projection of li - network for sg ia-3d ( left ) and sg i-43d ( right ) . both , for the ia-3d and the i-43d structure , site occupation refinements yielded the octahedral 16c sites to be fully occupied by zr , no evidence was found for a ga substitution onto this site , so during refinements , the occupation of zr was fixed to the ideal value . for both structures , however , there is a small deficiency of la , and thus , the site occupation factor was allowed to vary freely during the refinements . the finding of a small la deficit is consistent with results from edx analysis . the amount of vacancies varies between 0.08(2 ) in ga - poor samples to 0.04(2 ) in ga - rich samples , and there are some evidence that the amount of vacancies at the la site steadily decreases upon increasing of ga content , see figure 4a . ( b ) ga is almost exclusively located at the smaller li1 site ( 12a ) , only for samples with high ga contents , a small amount of ga is located at the li2 site ( 12b ) . ( c ) as a function of the decreased total li content for samples with higher ga contents , the occupation of the li3 site decreases . for the ia-3d structure for the i-43d structure , the refinement strategy was modified slightly . in a first refinement cycle , the site occupation numbers of the li1 , li2 , and li3 sites were allowed to refine freely without constraints , assuming only the scattering power of li . these preliminary refinements showed a surplus of electron density at the li1 site ( occupation of li larger than 1.00 , the value of full occupation ) , and thus , a larger scatterer in this case ga must be present , whereas at the li2 site , the refined occupation was close to 1.00 or even lower , indicating some vacancies at this site . li2 site values > 1.00 were observed only for higher overall ga contents . for occupation numbers > 1.00 , a mixed occupation with li and ga was refined , assuming full occupation of these sites . with the model of site occupation refinement , we have strong evidence that ga shows a strong preference for the li1 site , only very small amounts of ga are found at the li2 site ( figure 4b ) . it should be noted that the li2 site , thereby , is fully occupied in most cases and the li - content is 1.441.48 pfu . we are aware that this model of full occupation of li1 and li2 sites may suffer small deficits . however , it yields total ga contents which are very close to or even higher than those determined from edx analysis of the samples studied by sc - xrd . assuming vacancy concentrations of 20% on both sites , this does not change the site preference of ga for the li1 site but only increases the overall ga content by 15% . additionally , there is good evidence from neutron diffraction data that the amount of vacancies indeed is below 20% in ga - stabilized llzo samples ( see below ) . the occupation of the li3 site generally was refined freely and gave a partial occupation of this site . with increasing ga substitution , the occupation of the li3 site decreases ; that is , the trivalent cationic substitution reduces the amount of interstitial li ( see figure 4c ) . the decrease of li with increasing ga is even more evident and with smaller scatter in the data when the total li content of the samples is taken into account . therefore , with our high - quality , high - resolution x - ray data , we are able to deduce smooth trends in li - occupation in the llzo : ga series . for one composition ( xga = 0.20 ) , the derived structural model as well as the cationic distribution were simultaneously refined against high - resolution neutron powder diffraction ( npd ) and sc - xrd data . the best fit to the neutron data was indeed obtained with the i-43d model of this study , and the cationic distribution of x - ray data was proven . also , in this combined simultaneous refinements , the la site shows the presence of a small amount of vacancies . again , there is no clear evidence for ga on the li2 position , while it is enriched on li1 ; in the combined refinement , the ga and li occupation was refined freely without constraints : this strategy gave evidence for a rather low concentration of vacancies , both on li1 ( 14% ) and li2 ( 12% ) sites . the occupation of the li3 site is somewhat higher than in the single - crystal study but still comparable within estimated standard deviations . the substitution of ga into the structure of pure llzo stabilizes the cubic structure . the lattice parameters thereby are reduced from 12.985 to 12.965 , and the decrease is almost linear , see figure 5 . the phase transition from ia-3d to i-43d does not cause a significant change in the lattice parameter . for small ga concentrations , we find as mentioned the ia-3d symmetry , while for refined ga contents xga_ref > 0.07 , a reduction in symmetry to sg i-43d takes place . the reduction in symmetry most probably is induced by the strong ordering of ga onto one of the two possible tetrahedrally coordinated li sites , namely , onto li1 . the li1 site in i-43d is smaller as compared to the corresponding li1 site in ia-3d , and it is also slightly smaller as compared to the li2 site in i-43d , expressed by the smaller li o bond lengths and smaller volume . with increasing ga substitution , the li1o bond lengths successively decrease due to the smaller cationic size of ga as compared to li in tetrahedral coordination ( 0.47 and 0.59 , respectively ) . the li2o bond lengths remain constant , or increase slightly with increasing ga content ( see figure s2a , supporting information ) . this is seen as a further evidence that ga preferentially eenters the li1 site ( 12a ) . obviously , it is the smaller and more distorted character of the li1 tetrahedron , which favors the ga substitution . the li3 site is a large cavity which has four li o bonds within 1.9 and 2.25 and two more distant li o bonds at 2.65 and 2.75 , and thus , one may consider the coordination polyhedron of the li3 site as a strongly distorted octahedron . each li3 site shares two of its triangular faces with neighboring li1 and li2 sites , while both the li1 and the li2 site share all of their four triangular faces with li3 sites . thus , a three - dimensional network of face sharing li - sites is present , which forms a diffusion pathway for li ions . li distances within the li - network : li1li3 is the shorter one with distances of 1.6 , whereas the li2li3 distance is around 2.3 . further crystal chemical considerations are given as supporting information . in figure 6 , the li nmr line shapes and line widths of the central transition of ga20 and al20 are shown . considering the li nmr central lines shown in figure 6b it is seen that the lines are stepwise narrowed with increasing temperature . as an example , at 213 k ( 60 c ) the nmr signal of ga20 with xga = 0.20 is composed of two components viz . a broad gaussian shaped line and a motionally narrowed lorentzian one . those ions contributing to the latter are already exposed to sufficiently fast exchange processes with rates exceeding the rigid - lattice line , the latter is estimated to be in the order of 8 to 9 khz . at even higher temperature , see the spectrum recorded at 273 k ( 0 c ) , all of the li ions participate in sufficiently fast li diffusivity to average dipole dipole couplings . above 273 k , the line widths ( see figure 6a ) reached the so - called extreme narrowing regime . ( a ) motional narrowing of the li nmr central line of ga20 and al20 ; the inset shows a magnification of the quadrupole intensities of the two nmr lines recorded at 223 k ( 50 c ) by means of a nonselective solid - echo experiment . ( b ) li nmr lines recorded at 116 mhz via single - pulse excitation . essentially the same behavior is found for the sample al20 stabilized with 0.20 al pfu . the corresponding motional narrowing curve , however , is slightly shifted toward higher temperatures indicating somewhat lower diffusivity in the sample containing al . looking at the quadrupole intensities , visualized by the solid - echo technique ( see the inset of figure 6a ) , the contribution to the nmr line of ga20 is slightly reduced . the satellite transitions reflect the interaction of the spin-3/2 nucleus with the electric field gradients at the li sites in garnet - type llzo . a reduction in intensity ( note that the spectra shown are scaled such that they have the same height ) might be explained by faster li exchange processes in ga - bearing llzo . at very high temperatures , full averaging of the satellite intensities is seen for the two samples . in order to quantify ionic motion , we recorded li nmr laboratory - frame ( 1/t1 ) and rotating - frame ( 1/t1 ) spin lattice relaxation rates . the rates obtained are shown as a function of the inverse temperature in a semilogarithmic plot in figure 7a ; in figure 7b , selected magnetization transients of the experiments in the rotating frame are displayed . solid lines represent fits with stretched exponentials to extract the rates 1/t1. the inset in figure 7a shows the temperature dependence of the stretching exponent . as temperature decreases , the transients become more stretched . 200 k. as has been observed for other li - ion conductors this feature might correspond to a ( local ) maximum in the 1/t1(1/t ) plot . indeed , this behavior is seen in figure 7b , see the vertically drawn arrow . it could be interpreted as an additional li ion diffusion process that is absent for the sample stabilized by al instead of ga . for ga20 , an activation energy of 0.39 ev can be roughly estimated . ( a ) 1/t1 magnetization transients , ranging from 233 to 373 k in steps of 20 k , which were analyzed by stretched exponentials ( solid lines ) . inset : variation of the stretching exponent as a function of the inverse temperature . the arrow points to a local minimum in the case of the ga20 sample . besides this slight difference observed by rotating - frame spin - lock nmr , the two samples show two marked similarities that were also observed in earlier studies focusing on garnets stabilized by m cations : ( i ) 1/t1 passes through an extremely broad rate peak from which the high - t flank , characterized by a mean activation energy of 0.41 ev , is only partly accessible . on the other hand , ( ii ) up to ca . 400 k , the rates 1/t1(1/t ) follow linear behavior in the arrhenius plot pointing to activation energies of 0.13 to 0.14 ev . these values characterize local li ion jumps in the garnet structure , whereas those deduced from 1/t1 might already be influenced by long - range ion transport . it is generally accepted that the garnets and garnet - related ( synthetic ) materials can adopt both cubic and tetragonal symmetries . the inorganic crystal structure database reports almost 500 entries for garnets , with the vast majority of 95% showing the cubic space group ia-3d . few natural garnets like henritermierite ca3(mn , al)2(sio4)2(oh)4 and synthetic materials such as llzo are known to show tetragonal i41/acd symmetry . it is accepted that symmetry breaking from cubic ia-3d to lower symmetry takes place among others as a consequence of jahn teller distortion for mn bearing garnets , strain , and growth effects , cation ordering and incorporation of hydrogen atoms . in pure llzo , the symmetry breaking is explained by the complete ordering of li onto tetrahedral site . in a recent paper , report on the symmetry change from ia-3d to i213 during li / h exchange in the li7xhxla3sn2o12 and li7xhxla3nb2o12 systems . for li6-xhxcala2nb2o12 , a change from ia-3d to the acentric space group i-43d is described as a consequence of li / h exchange upon leaching in acetic acid for 4 days by galven et al . were not the first to report onto the unusual acentric cubic space group : the structure was first reported and solved by lager et al . in the unusual mineral compound katoite ca3al2(o4h4)3 . using single - crystal x - ray diffraction , the authors describe a phase transition from ia-3d to i-43d occurring at high pressures ( above 5 gpa ) . in this study , we observed the acentric sg i-43d for ga - stabilized llzo samples with nominal ga contents xga 0.15 pfu . this deviates from al stabilized llzo prepared under the same conditions , which still shows sg ia-3d . the present study indicates that for li - oxide garnets , sg i-43d seems to be more common than expected . ga - stabilized llzo gets ordered in this acentric sg even without explicit aging or protonation . previous studies reporting sg i-43d for li - oxide garnets correlated this phase transformation with a protonation process . as our samples were characterized immediately after the final calcination step , we exclude a phase transformation due to hydration caused by humidity from exposure to air as supposed by larraz et al . for pure llzo from i41/acd to ia-3d . recently , larraz et al . documented some additional weak reflections for pure llzo that was stored for three years , washed , and then heated to 300 c that can not be explained using sg ia-3d . in contrast to this , ma et al . did not observe any phase transformation for cubic llzo exposed to aqueous solutions and suggested that llzo with sg ia-3d is relatively stable , even at very high li / h exchange rates of 63.6% , which affected almost solely 4-fold - coordinated li at the 96h position . a particular focus of their study was on the presence of any other space groups such as i213 or i-43d , which was clearly denied by this paper . as the phase transition seems to be triggered by a splitting of the 24d position of sg ia-3d , which is not affected by the li / h exchange , it is hardly imaginable that a phase transition can be caused by a li / h exchange at the 96h position only ; so possibly other processes such as the heating to 300 c might have caused the phase transition to i-43d mentioned by larraz et al . the additional reflections observed by larraz et al . at 2 = 21.5 , 40.3 , and 53.4 , respectively , can indeed be attributed to the ( 310 ) , ( 530 ) , and ( 710 ) reflections of sg i-43d , as these reflections are forbidden for sg ia-3d . in this study , the reflection at 2 = 21.65 ( d = 4.101 ) can be attributed to the ( 310 ) reflection of sg i-43d , which is expected to show a relative intensity of 1.2% compared to the strongest reflection at 2 = 30.81 ( d = 2.90 ) . the other additional reflections show an even lower relative intensity of 0.4% and 0.3% , respectively . however , due to their low intensity , these peaks might have been overlooked by previous studies on ga - stabilized llzo or incorrectly attributed to ligao2 , which is a common extra phase and shows a reflection at 2 = 21.52 ( d = 4.126 ) . for the detection of ligao2 , the use of most intense reflection at 2 = 22.55 ( d = 3.94 ) is more advantageous . for a definite determination of the sg of ga - stabilized llzo , much experimental as well as theoretical effort has been undertaken to collect information on the site preference of ga and its influence on li - ion dynamics and li - ion conductivity . the position of these cations might influence the mobility of li ions due to a possible blockage of the diffusion path . several studies using ga nmr spectroscopy lead to different interpretations , indicating either one or two ga positions . in this study , the refinement of sc - xrd and npd data suggests that ga is preferentially located at a single position , namely the tetrahedral 12a position , and only minor amounts of ga occupy the 12b position . as the coarse - grained llzo samples in this study were prepared at high temperatures , we can not assess whether ga - stabilized llzo prepared under different conditions ( e.g. , at lower temperatures ) still orders in sg i-43d . further research will be needed to clarify the influence of the preparation condition on the crystal structure of ga - stabilized llzo . despite that ga - stabilized llzo shows a different sg than al - stabilized llzo , the li - ion mobility is still comparable or even better than for al - stabilized llzo with sg ia-3d . it stands to reason that the structural differences will have a significant impact on the li - ion dynamics . however , investigations on the influence of the new cubic modification on the li - ion mobility are beyond the scope of this study and will therefore be reported in a subsequent paper . the present study reveals that ga - stabilized llzo shows the acentric cubic sg i-43d ( no . 220 ) , which is different from other members of the li - oxide garnet group that show sg ia-3d ( no . 230 ) . in contrast to other studies which observed this sg due to li / h exchange , the structural changes might be caused by the site preference of ga . li nmr relaxometry and line shape studies support the findings by impedance spectroscopy revealing enhanced ion dynamics in ga - stabilized llzo as compared to llzo stabilized by al . this study stimulates further research for an understanding of the structureproperty relationship as a basis to improve the electrochemical capabilities of these electrolyte materials .

li - oxide garnets such as li7la3zr2o12 ( llzo ) are among the most promising candidates for solid - state electrolytes to be used in next - generation li - ion batteries . the garnet - structured cubic modification of llzo , showing space group ia-3d , has to be stabilized with supervalent cations . llzo stabilized with ga3 + shows superior properties compared to llzo stabilized with similar cations ; however , the reason for this behavior is still unknown . in this study , a comprehensive structural characterization of ga - stabilized llzo is performed by means of single - crystal x - ray diffraction . coarse - grained samples with crystal sizes of several hundred micrometers are obtained by solid - state reaction . single - crystal x - ray diffraction results show that li73xgaxla3zr2o12 with x > 0.07 crystallizes in the acentric cubic space group i-43d . this is the first definite record of this cubic modification for llzo materials and might explain the superior electrochemical performance of ga - stabilized llzo compared to its al - stabilized counterpart . the phase transition seems to be caused by the site preference of ga3 + . 7li nmr spectroscopy indicates an additional li - ion diffusion process for llzo with space group i-43d compared to space group ia-3d . despite all efforts undertaken to reveal structure property relationships for this class of materials , this study highlights the potential for new discoveries .

Introduction Experimental Section Results Discussion Conclusions

in 2007 , li7la3zr2o12 ( llzo ) has received much scientific attention as a solid electrolyte for beyond li ion battery llzo provides a high li - ion conductivity ( 10 to 10 s cm at ambient temperature ) and a li transference number approaching 1 , superior chemical stability against high voltage cathodes , and electrochemical inertness in a wide potential window of up to 6 v. in particular , its stability against li - metal as well as its thermal and mechanical robustness makes llzo garnet exceptionally well - suited to be used as a protecting layer for li - metal - based batteries . for pure llzo , two structural polymorphs are described in the literature . a low - temperature tetragonal modification with a completely ordered arrangement of li crystallizes in space group ( sg ) i41/acd ( no . in contrast to the tetragonal modification , the cubic high - temperature modification exhibits a disorder in the li distribution . this cubic modification shows sg ia-3d ( no . for use as li - ion conductors , the cubic modification is much more desirable as its li - ion conductivity is 2 orders of magnitude higher than for the tetragonal modification . the cubic modification of pure llzo is not stable at room temperature ( rt ) . however , it can be stabilized at rt by the introduction of supervalent cations , which cause a reduction of the li content that leads to the introduction of additional vacancies at the li sites . the stabilization of the cubic modification was originally achieved by the incorporation of al . , creating two vacant sites , li , in the li - sublattice . ga - stabilized llzo is characterized by a li conductivity of 1.3 ms cm at rt , which is twice as high as for llzo stabilized with al . the reason for the higher li - ion conductivity of ga - stabilized llzo is , however , not yet fully understood . because of their blocking effect , some of these ions are suspected to hinder the long - range li - ion transport and , therefore , to reduce li - ion conductivity . ( llzto ) with those of li6.15la3zr1.75ta0.25m0.2o12 ( m = al , ga ) ; according to their study pure llzto shows the highest li - ion conductivity , followed by llzto : ga and llzto : al . on the basis of the crystal chemical considerations , they attributed this behavior to the different site preference of al and ga . in particular , the larger ga prefers the 96h site , which seems to be less hindering for long - range li - ion transport , compared to al , located at the 24d site , which is a junction for li - ion diffusion . however , the site preference of ga is still under discussion as ga nuclear magnetic resonance ( nmr ) spectroscopy studies revealed different results . ( 2015 ) , using very high magnetic fields ( 21.1 t ) , showed that the site preference of ga and al to occupy 24d and 96h voids in samples crystallizing with ia-3d is practically the same . thus , despite all efforts , there is , so far , no satisfying explanation why some of the ga - stabilized llzo samples presented in literature show higher ionic conductivities . investigations on ga - stabilized llzo single crystals might help us to shed light on this question . to the best of our knowledge , no single - crystal x - ray diffraction ( sc - xrd ) study on ga - stabilized llzo has been published yet . in the present study , another cubic modification of llzo , showing the acentric sg i-43d , has been observed for ga - stabilized llzo for the first time . coarse - grained ga - stabilized llzo samples were synthesized via solid - state reaction and characterized by a rich portfolio of techniques including x - ray powder diffraction ( xrpd ) , single - crystal x - ray diffraction ( sc - xrd ) , neutron powder diffraction ( npd ) , scanning electron microscopy ( sem)/ backscattered electron ( bse ) imaging , energy - dispersive x - ray spectroscopy ( edx ) , and li nmr spectroscopy . for reason of comparison , also one sample of al - stabilized llzo with an intended al content xal of 0.20 al pfu ( li6.4al0.2la3zr2o12 ) was prepared under the same conditions . , the distribution , of ga , la , zr , using a backscattered electrons detector ( bse ) and energy - dispersive x - ray spectroscopy ( edx ) measurements , respectively . patterns were recorded using a bruker d8 advance davinci design diffractometer with a lynxeye solid - state detector using cu k radiation . single - crystal x - ray diffraction data were collected on a bruker smart apex ccd - diffractometer . experiments were performed in a 2 range of 3 2 154 , step width of 0.045. data treatment of powder diffraction data sets as well as a combined refinement of neutron powder diffraction data and single - crystal x - ray diffraction data ( with special emphasis of li - cationic distribution ) was performed using the fullprof - suite of programs . variable - temperature li nmr spectra and spin lattice relaxation rates were recorded with a bruker avance iii solid - state spectrometer connected to a 7-t magnet ( bruker ) . we used single - pulse and solid - echo experiments to record nmr line shapes . lattice relaxation rates in the laboratory frame were measured by means of the saturation recovery sequence ; the rates in the rotating frame were acquired by using the spin - lock technique , see epp et al . the la / zr ratio of llzo grains was slightly below the theoretical values of 3:2 , which is in accordance with site occupation refinements from sc - xrd data and microprobe measurements of ga - stabilized llzo by rettenwander et al . ga contents of llzo grains were partially lower than the target stoichiometry ; additionally , a slight variation of the ga content within single samples was noted . for sample with xga = 0.60 , it has to be noted that for most samples , an additional weak reflection at 2 = 21.65 ( equal to d = 4.10 ) was observed . single - crystal x - ray intensity data processing gave strong evidence for the cubic crystal system for all data collected . however , for all samples with nominal ga concentrations xga 0.15 pfu , intensity statistics and systematic extinctions did not result in sg ia-3d but yielded an acentric sg i-43d ( no . li leaching and replacement by h and up to now , no doubt on the violation of ia-3d symmetry for ga - stabilized llzo was reported . samples of al - stabilized llzo , which are produced with the very same synthesis strategies , show ia-3d symmetry up to nominal compositions of xal = 0.30 pfu in single - crystal structure refinements . different to the ia-3d structure of sample ga10 with a nominal ga content xga = 0.10 pfu , which exhibits 5 different atomic positions in the asymmetric unit , the crystal structure of ga - stabilized llzo with a nominal ga content xga 0.15 pfu , showing sg i-43d , exhibits seven different atomic positions : la occupies the 8-fold coordinated 24d position ( site symmetry 2 .. ) , and zr is located at the octahedrally coordinated 16c position ( site symmetry .3 . ) in contrast to common silicate garnets , the acentric structure exhibits two independent o positions , both in general position 48e . ( a ) crystal structure of ga - stabilized llzo with xga = 0.10 and sg ia-3d . ( b ) crystal structure of ga - stabilized llzo with xga = 0.30 and sg i-43d . both , for the ia-3d and the i-43d structure , site occupation refinements yielded the octahedral 16c sites to be fully occupied by zr , no evidence was found for a ga substitution onto this site , so during refinements , the occupation of zr was fixed to the ideal value . for both structures , however , there is a small deficiency of la , and thus , the site occupation factor was allowed to vary freely during the refinements . the amount of vacancies varies between 0.08(2 ) in ga - poor samples to 0.04(2 ) in ga - rich samples , and there are some evidence that the amount of vacancies at the la site steadily decreases upon increasing of ga content , see figure 4a . ( b ) ga is almost exclusively located at the smaller li1 site ( 12a ) , only for samples with high ga contents , a small amount of ga is located at the li2 site ( 12b ) . ( c ) as a function of the decreased total li content for samples with higher ga contents , the occupation of the li3 site decreases . in a first refinement cycle , the site occupation numbers of the li1 , li2 , and li3 sites were allowed to refine freely without constraints , assuming only the scattering power of li . these preliminary refinements showed a surplus of electron density at the li1 site ( occupation of li larger than 1.00 , the value of full occupation ) , and thus , a larger scatterer in this case ga must be present , whereas at the li2 site , the refined occupation was close to 1.00 or even lower , indicating some vacancies at this site . assuming vacancy concentrations of 20% on both sites , this does not change the site preference of ga for the li1 site but only increases the overall ga content by 15% . additionally , there is good evidence from neutron diffraction data that the amount of vacancies indeed is below 20% in ga - stabilized llzo samples ( see below ) . therefore , with our high - quality , high - resolution x - ray data , we are able to deduce smooth trends in li - occupation in the llzo : ga series . the best fit to the neutron data was indeed obtained with the i-43d model of this study , and the cationic distribution of x - ray data was proven . also , in this combined simultaneous refinements , the la site shows the presence of a small amount of vacancies . again , there is no clear evidence for ga on the li2 position , while it is enriched on li1 ; in the combined refinement , the ga and li occupation was refined freely without constraints : this strategy gave evidence for a rather low concentration of vacancies , both on li1 ( 14% ) and li2 ( 12% ) sites . the occupation of the li3 site is somewhat higher than in the single - crystal study but still comparable within estimated standard deviations . the phase transition from ia-3d to i-43d does not cause a significant change in the lattice parameter . for small ga concentrations , we find as mentioned the ia-3d symmetry , while for refined ga contents xga_ref > 0.07 , a reduction in symmetry to sg i-43d takes place . the reduction in symmetry most probably is induced by the strong ordering of ga onto one of the two possible tetrahedrally coordinated li sites , namely , onto li1 . the li1 site in i-43d is smaller as compared to the corresponding li1 site in ia-3d , and it is also slightly smaller as compared to the li2 site in i-43d , expressed by the smaller li o bond lengths and smaller volume . with increasing ga substitution , the li1o bond lengths successively decrease due to the smaller cationic size of ga as compared to li in tetrahedral coordination ( 0.47 and 0.59 , respectively ) . thus , a three - dimensional network of face sharing li - sites is present , which forms a diffusion pathway for li ions . those ions contributing to the latter are already exposed to sufficiently fast exchange processes with rates exceeding the rigid - lattice line , the latter is estimated to be in the order of 8 to 9 khz . ( a ) motional narrowing of the li nmr central line of ga20 and al20 ; the inset shows a magnification of the quadrupole intensities of the two nmr lines recorded at 223 k ( 50 c ) by means of a nonselective solid - echo experiment . essentially the same behavior is found for the sample al20 stabilized with 0.20 al pfu . looking at the quadrupole intensities , visualized by the solid - echo technique ( see the inset of figure 6a ) , the contribution to the nmr line of ga20 is slightly reduced . 200 k. as has been observed for other li - ion conductors this feature might correspond to a ( local ) maximum in the 1/t1(1/t ) plot . indeed , this behavior is seen in figure 7b , see the vertically drawn arrow . it could be interpreted as an additional li ion diffusion process that is absent for the sample stabilized by al instead of ga . 400 k , the rates 1/t1(1/t ) follow linear behavior in the arrhenius plot pointing to activation energies of 0.13 to 0.14 ev . these values characterize local li ion jumps in the garnet structure , whereas those deduced from 1/t1 might already be influenced by long - range ion transport . the inorganic crystal structure database reports almost 500 entries for garnets , with the vast majority of 95% showing the cubic space group ia-3d . in pure llzo , the symmetry breaking is explained by the complete ordering of li onto tetrahedral site . for li6-xhxcala2nb2o12 , a change from ia-3d to the acentric space group i-43d is described as a consequence of li / h exchange upon leaching in acetic acid for 4 days by galven et al . were not the first to report onto the unusual acentric cubic space group : the structure was first reported and solved by lager et al . using single - crystal x - ray diffraction , the authors describe a phase transition from ia-3d to i-43d occurring at high pressures ( above 5 gpa ) . in this study , we observed the acentric sg i-43d for ga - stabilized llzo samples with nominal ga contents xga 0.15 pfu . the present study indicates that for li - oxide garnets , sg i-43d seems to be more common than expected . ga - stabilized llzo gets ordered in this acentric sg even without explicit aging or protonation . previous studies reporting sg i-43d for li - oxide garnets correlated this phase transformation with a protonation process . as the phase transition seems to be triggered by a splitting of the 24d position of sg ia-3d , which is not affected by the li / h exchange , it is hardly imaginable that a phase transition can be caused by a li / h exchange at the 96h position only ; so possibly other processes such as the heating to 300 c might have caused the phase transition to i-43d mentioned by larraz et al . in this study , the reflection at 2 = 21.65 ( d = 4.101 ) can be attributed to the ( 310 ) reflection of sg i-43d , which is expected to show a relative intensity of 1.2% compared to the strongest reflection at 2 = 30.81 ( d = 2.90 ) . however , due to their low intensity , these peaks might have been overlooked by previous studies on ga - stabilized llzo or incorrectly attributed to ligao2 , which is a common extra phase and shows a reflection at 2 = 21.52 ( d = 4.126 ) . for a definite determination of the sg of ga - stabilized llzo , much experimental as well as theoretical effort has been undertaken to collect information on the site preference of ga and its influence on li - ion dynamics and li - ion conductivity . in this study , the refinement of sc - xrd and npd data suggests that ga is preferentially located at a single position , namely the tetrahedral 12a position , and only minor amounts of ga occupy the 12b position . as the coarse - grained llzo samples in this study were prepared at high temperatures , we can not assess whether ga - stabilized llzo prepared under different conditions ( e.g. further research will be needed to clarify the influence of the preparation condition on the crystal structure of ga - stabilized llzo . despite that ga - stabilized llzo shows a different sg than al - stabilized llzo , the li - ion mobility is still comparable or even better than for al - stabilized llzo with sg ia-3d . it stands to reason that the structural differences will have a significant impact on the li - ion dynamics . however , investigations on the influence of the new cubic modification on the li - ion mobility are beyond the scope of this study and will therefore be reported in a subsequent paper . the present study reveals that ga - stabilized llzo shows the acentric cubic sg i-43d ( no . 220 ) , which is different from other members of the li - oxide garnet group that show sg ia-3d ( no . in contrast to other studies which observed this sg due to li / h exchange , the structural changes might be caused by the site preference of ga . li nmr relaxometry and line shape studies support the findings by impedance spectroscopy revealing enhanced ion dynamics in ga - stabilized llzo as compared to llzo stabilized by al .

in 2007 , li7la3zr2o12 ( llzo ) has received much scientific attention as a solid electrolyte for beyond li ion battery llzo provides a high li - ion conductivity ( 10 to 10 s cm at ambient temperature ) and a li transference number approaching 1 , superior chemical stability against high voltage cathodes , and electrochemical inertness in a wide potential window of up to 6 v. in particular , its stability against li - metal as well as its thermal and mechanical robustness makes llzo garnet exceptionally well - suited to be used as a protecting layer for li - metal - based batteries . this structure type is described as garnet - related framework with two types of dodecahedral lao8 polyhedra ( 8b and 16e ) and zro6 octahedra ( 16c ) . in contrast to the tetragonal modification , the cubic high - temperature modification exhibits a disorder in the li distribution . 230 ) and has the garnet structure composed of a framework of 8-fold coordinated lao8 dodecahedra ( 24c ) and 6-fold coordinated zro6 octahedra ( 16a ) . for use as li - ion conductors , the cubic modification is much more desirable as its li - ion conductivity is 2 orders of magnitude higher than for the tetragonal modification . however , it can be stabilized at rt by the introduction of supervalent cations , which cause a reduction of the li content that leads to the introduction of additional vacancies at the li sites . the reason for the higher li - ion conductivity of ga - stabilized llzo is , however , not yet fully understood . because of their blocking effect , some of these ions are suspected to hinder the long - range li - ion transport and , therefore , to reduce li - ion conductivity . ( llzto ) with those of li6.15la3zr1.75ta0.25m0.2o12 ( m = al , ga ) ; according to their study pure llzto shows the highest li - ion conductivity , followed by llzto : ga and llzto : al . on the basis of the crystal chemical considerations , they attributed this behavior to the different site preference of al and ga . in particular , the larger ga prefers the 96h site , which seems to be less hindering for long - range li - ion transport , compared to al , located at the 24d site , which is a junction for li - ion diffusion . however , the site preference of ga is still under discussion as ga nuclear magnetic resonance ( nmr ) spectroscopy studies revealed different results . ( 2015 ) , using very high magnetic fields ( 21.1 t ) , showed that the site preference of ga and al to occupy 24d and 96h voids in samples crystallizing with ia-3d is practically the same . thus , despite all efforts , there is , so far , no satisfying explanation why some of the ga - stabilized llzo samples presented in literature show higher ionic conductivities . to the best of our knowledge , no single - crystal x - ray diffraction ( sc - xrd ) study on ga - stabilized llzo has been published yet . in the present study , another cubic modification of llzo , showing the acentric sg i-43d , has been observed for ga - stabilized llzo for the first time . coarse - grained ga - stabilized llzo samples were synthesized via solid - state reaction and characterized by a rich portfolio of techniques including x - ray powder diffraction ( xrpd ) , single - crystal x - ray diffraction ( sc - xrd ) , neutron powder diffraction ( npd ) , scanning electron microscopy ( sem)/ backscattered electron ( bse ) imaging , energy - dispersive x - ray spectroscopy ( edx ) , and li nmr spectroscopy . a series of li73xgaxla3zr2o12 with intended ga contents xga = 0.10 , 0.15 , 0.20 , 0.30 , 0.40 , 0.50 , 0.60 pfu was synthesized by high - temperature sintering in air . the samples were heated to 850 c with a rate of 5 c / min and calcinated for 4 h. the resulting pellets were then removed from the furnace , ground in an agate mortar , and ball - milled for 1 h under isopropyl alcohol ( fritsch pulverisette 7 , 800 rpm , 2 mm zro2 balls ) . for the final sintering , the pellets were heated in a muffle furnace in air with a rate of 5 c / min to 1230 c and sintered for 6 h. for xrpd investigations , small fragments of the sintered pellets were ground using an agate mortar . in particular , we put emphasis on the investigation of the grain size , morphology , phase composition , and the chemical homogeneity , i.e. , the distribution , of ga , la , zr , using a backscattered electrons detector ( bse ) and energy - dispersive x - ray spectroscopy ( edx ) measurements , respectively . the synthesis products were characterized regarding the presence of extra phases as well as to determine the symmetry and unit - cell dimension of the samples . so we exclude that the change in sg symmetry is due to the incorporation of h in the structure as it was suggested in literature . intensity data were collected with graphite - monochromatized mo k x - radiation ( 50 kv , 30 ma ) ; the crystal - to - detector distance was 30 mm , and the detector was positioned at 30 and 50 2 using an -scan mode strategy at four different positions ( 0 , 90 , 180 , and 270 ) for each 2 position . experiments were performed in a 2 range of 3 2 154 , step width of 0.045. data treatment of powder diffraction data sets as well as a combined refinement of neutron powder diffraction data and single - crystal x - ray diffraction data ( with special emphasis of li - cationic distribution ) was performed using the fullprof - suite of programs . lattice relaxation rates in the laboratory frame were measured by means of the saturation recovery sequence ; the rates in the rotating frame were acquired by using the spin - lock technique , see epp et al . the la / zr ratio of llzo grains was slightly below the theoretical values of 3:2 , which is in accordance with site occupation refinements from sc - xrd data and microprobe measurements of ga - stabilized llzo by rettenwander et al . the xrpd patterns of li73xgaxla3zr2o12 ( gax ) with nominal ga concentration xga = 0.10 , 0.15 , 0.20 , 0.30 , 0.40 , 0.50 , 0.60 pfu are shown in figure 1 . for sample with xga = 0.10 , tetragonal llzo was still present with a share of 18 wt % as determined by quantitative phase analysis using the rietveld method . considering the sem - bse results , these extra phases occur predominantly in the rim of the pellets and formed due to evaporation of li during high temperature sintering . comparison of xrpd patterns for samples ga10 ( lowermost pattern ) , ga15 , ga20 , ga30 , ga40 , ga50 , ga60 ( uppermost pattern ) . the experimental data and results of structure refinement for selected samples are reported in table 1 and table 2 , while the fractional atomic coordinates , occupation numbers and equivalent isotropic , and anisotropic atomic displacement parameters are given in table s1 . simulated precession images of the 0kl layer of samples ga10_1 and ga40_2 , displayed in figure 2 , obviously show the presence of bragg peaks with k = odd and l = odd in sample ga40 . samples of al - stabilized llzo , which are produced with the very same synthesis strategies , show ia-3d symmetry up to nominal compositions of xal = 0.30 pfu in single - crystal structure refinements . different to the ia-3d structure of sample ga10 with a nominal ga content xga = 0.10 pfu , which exhibits 5 different atomic positions in the asymmetric unit , the crystal structure of ga - stabilized llzo with a nominal ga content xga 0.15 pfu , showing sg i-43d , exhibits seven different atomic positions : la occupies the 8-fold coordinated 24d position ( site symmetry 2 .. ) , and zr is located at the octahedrally coordinated 16c position ( site symmetry .3 . ) li is found distributed over three different positions in i-43d : two of them correspond to the regular tetrahedral coordinated site of the silicate garnets ( 24d in ia-3d ) , nevertheless , they split into two positions , namely li1 ( 12a ) and li2 ( 12b ) , both with site symmetry 4 .. , and differ in both bond lengths and polyhedral distortion . both , for the ia-3d and the i-43d structure , site occupation refinements yielded the octahedral 16c sites to be fully occupied by zr , no evidence was found for a ga substitution onto this site , so during refinements , the occupation of zr was fixed to the ideal value . for both structures , however , there is a small deficiency of la , and thus , the site occupation factor was allowed to vary freely during the refinements . the amount of vacancies varies between 0.08(2 ) in ga - poor samples to 0.04(2 ) in ga - rich samples , and there are some evidence that the amount of vacancies at the la site steadily decreases upon increasing of ga content , see figure 4a . ( b ) ga is almost exclusively located at the smaller li1 site ( 12a ) , only for samples with high ga contents , a small amount of ga is located at the li2 site ( 12b ) . ( c ) as a function of the decreased total li content for samples with higher ga contents , the occupation of the li3 site decreases . in a first refinement cycle , the site occupation numbers of the li1 , li2 , and li3 sites were allowed to refine freely without constraints , assuming only the scattering power of li . these preliminary refinements showed a surplus of electron density at the li1 site ( occupation of li larger than 1.00 , the value of full occupation ) , and thus , a larger scatterer in this case ga must be present , whereas at the li2 site , the refined occupation was close to 1.00 or even lower , indicating some vacancies at this site . with the model of site occupation refinement , we have strong evidence that ga shows a strong preference for the li1 site , only very small amounts of ga are found at the li2 site ( figure 4b ) . assuming vacancy concentrations of 20% on both sites , this does not change the site preference of ga for the li1 site but only increases the overall ga content by 15% . additionally , there is good evidence from neutron diffraction data that the amount of vacancies indeed is below 20% in ga - stabilized llzo samples ( see below ) . with increasing ga substitution , the occupation of the li3 site decreases ; that is , the trivalent cationic substitution reduces the amount of interstitial li ( see figure 4c ) . therefore , with our high - quality , high - resolution x - ray data , we are able to deduce smooth trends in li - occupation in the llzo : ga series . for one composition ( xga = 0.20 ) , the derived structural model as well as the cationic distribution were simultaneously refined against high - resolution neutron powder diffraction ( npd ) and sc - xrd data . the best fit to the neutron data was indeed obtained with the i-43d model of this study , and the cationic distribution of x - ray data was proven . again , there is no clear evidence for ga on the li2 position , while it is enriched on li1 ; in the combined refinement , the ga and li occupation was refined freely without constraints : this strategy gave evidence for a rather low concentration of vacancies , both on li1 ( 14% ) and li2 ( 12% ) sites . the occupation of the li3 site is somewhat higher than in the single - crystal study but still comparable within estimated standard deviations . for small ga concentrations , we find as mentioned the ia-3d symmetry , while for refined ga contents xga_ref > 0.07 , a reduction in symmetry to sg i-43d takes place . the li1 site in i-43d is smaller as compared to the corresponding li1 site in ia-3d , and it is also slightly smaller as compared to the li2 site in i-43d , expressed by the smaller li o bond lengths and smaller volume . with increasing ga substitution , the li1o bond lengths successively decrease due to the smaller cationic size of ga as compared to li in tetrahedral coordination ( 0.47 and 0.59 , respectively ) . the li3 site is a large cavity which has four li o bonds within 1.9 and 2.25 and two more distant li o bonds at 2.65 and 2.75 , and thus , one may consider the coordination polyhedron of the li3 site as a strongly distorted octahedron . li distances within the li - network : li1li3 is the shorter one with distances of 1.6 , whereas the li2li3 distance is around 2.3 . ( a ) motional narrowing of the li nmr central line of ga20 and al20 ; the inset shows a magnification of the quadrupole intensities of the two nmr lines recorded at 223 k ( 50 c ) by means of a nonselective solid - echo experiment . the rates obtained are shown as a function of the inverse temperature in a semilogarithmic plot in figure 7a ; in figure 7b , selected magnetization transients of the experiments in the rotating frame are displayed . besides this slight difference observed by rotating - frame spin - lock nmr , the two samples show two marked similarities that were also observed in earlier studies focusing on garnets stabilized by m cations : ( i ) 1/t1 passes through an extremely broad rate peak from which the high - t flank , characterized by a mean activation energy of 0.41 ev , is only partly accessible . in a recent paper , report on the symmetry change from ia-3d to i213 during li / h exchange in the li7xhxla3sn2o12 and li7xhxla3nb2o12 systems . using single - crystal x - ray diffraction , the authors describe a phase transition from ia-3d to i-43d occurring at high pressures ( above 5 gpa ) . in this study , we observed the acentric sg i-43d for ga - stabilized llzo samples with nominal ga contents xga 0.15 pfu . did not observe any phase transformation for cubic llzo exposed to aqueous solutions and suggested that llzo with sg ia-3d is relatively stable , even at very high li / h exchange rates of 63.6% , which affected almost solely 4-fold - coordinated li at the 96h position . as the phase transition seems to be triggered by a splitting of the 24d position of sg ia-3d , which is not affected by the li / h exchange , it is hardly imaginable that a phase transition can be caused by a li / h exchange at the 96h position only ; so possibly other processes such as the heating to 300 c might have caused the phase transition to i-43d mentioned by larraz et al . at 2 = 21.5 , 40.3 , and 53.4 , respectively , can indeed be attributed to the ( 310 ) , ( 530 ) , and ( 710 ) reflections of sg i-43d , as these reflections are forbidden for sg ia-3d . in this study , the reflection at 2 = 21.65 ( d = 4.101 ) can be attributed to the ( 310 ) reflection of sg i-43d , which is expected to show a relative intensity of 1.2% compared to the strongest reflection at 2 = 30.81 ( d = 2.90 ) . for a definite determination of the sg of ga - stabilized llzo , much experimental as well as theoretical effort has been undertaken to collect information on the site preference of ga and its influence on li - ion dynamics and li - ion conductivity . in this study , the refinement of sc - xrd and npd data suggests that ga is preferentially located at a single position , namely the tetrahedral 12a position , and only minor amounts of ga occupy the 12b position . as the coarse - grained llzo samples in this study were prepared at high temperatures , we can not assess whether ga - stabilized llzo prepared under different conditions ( e.g. despite that ga - stabilized llzo shows a different sg than al - stabilized llzo , the li - ion mobility is still comparable or even better than for al - stabilized llzo with sg ia-3d . however , investigations on the influence of the new cubic modification on the li - ion mobility are beyond the scope of this study and will therefore be reported in a subsequent paper .

dynamic morphological changes are mediated by continual , highly- regulated fusion and fission events , collectively termed mitochondrial dynamics . for example , the evolutionarily conserved gtpase , dynamin related protein1 ( drp1 ) , is known to participate in the process of mitochondrial fission . when mitochondria undergo fission , cytosolic drp1 translocates to the outer mitochondrial membrane , oligomerizes around mitochondrial tubules , and in conjunction with fis1 promotes division or fission . the fusion machinery consists mainly of three additional large dynamin - like gtpases : both mitofusin1/2 ( mfn1/2 ) and optic atrophy protein 1 ( opa1 ) . the strict regulation of the equilibrium between fusion and fission events governs mitochondrial dynamics in a healthy cell and is crucial to maintaining mitochondrial function affecting energy supply , response to apoptotic stimuli , mitochondrial inheritance via admixture of mitochondrial - dna ( mtdna ) , and translocation of mitochondria to regions of high - energy demand . although mitochondrial dynamics are crucial in all cell types , neurons are thought to be especially dependent on a dynamic mitochondrial network . with their often long processes , neurons require proper distribution of mitochondria across long distances to satisfy the energy requirements of electrical excitability and synaptic transmission ( fig . , healthy neurons precisely control mitochondrial dynamics in order to efficiently transport mitochondria to distal locations , including dendrites , axons , and synapses . thus , mitochondrial function is essential to many neuronal functions , including energy production , ca buffering , axonal and dendritic transport , and release and re - uptake of neurotransmitter . in order to maintain a proper distribution of functional mitochondria , basal activity of the fission machinery is required to divide the mitochondrial network into transportable units which can then be efficiently distributed to synapses . fusion is necessary to maintain bioenergetic integrity of mitochondria . if the balance of fission and fusion becomes biased ( e.g. , with increased fission or impaired fusion ) excessive mitochondrial fragmentation is observed , resulting in impaired mitochondrial translocation and energy production at synaptic sites ( fig . 1 ) . the synaptic dysfunction thus induced is typically followed by dendritic and axonal degeneration , leading to neurodegeneration . indeed , excessive mitochondrial fragmentation with damaged cristae and resulting impaired bioenergetics are often associated with many neurological conditions , including stroke , alzheimer s disease ( ad ) , parkinson s disease ( pd ) , and huntington s disease ( hd ) , as well as several metabolic diseases . pathological disturbance of mitochondrial dynamics can result from either : 1 ) rare genetic mutations , such as those described for mfn1/2 , opa1 and drp1 , which underlie charcot - marie - tooth neuropathy type 2a ( cmt2a ) , autosomal dominant optic atrophy ( adoa ) , and a rare disorder of brain development , respectively ; or 2 ) altered posttranslational modifications ( ptms ) of the proteins encoded by these genes that are responsible for the fission / fusion machinery . in this review , we discuss recent findings concerning aberrant ptms which regulate the mitochondrial fusion and fission machinery and thus contribute to the pathophysiology of neurodegenerative diseases . in particular , we focus on involvement of nitrosative stress - induced drp1 activation , which results in excessive mitochondrial fragmentation , impaired bioenergetics , and consequent synaptic damage in neurodegenerative disorders . as mentioned above , tight regulation of the mitochondrial fission / fusion machinery is essential for maintaining healthy neuronal physiology . however , dysfunctional mitochondrial dynamics caused by impaired mitochondrial fission or fusion is a hallmark of many neurodegenerative diseases and contributes to their pathophysiology . mitochondrial fusion is the process by which the inner and outer mitochondrial membranes are separately fused by opa1 and mfn1/2 , respectively . for example , loss of function mutations in the mfn2 gene disrupts mitochondrial fusion , resulting in distal muscle weakness and sensory loss in cmt2a . to date , more than 40 mutations have been identified , most of which are located in the conserved gtpase domain of mfn2 . in mouse models , deletion in the mouse mfn2 gene causes severe mtdna loss and the accumulation of mtdna mutations . in addition , mfn2 deficiency results in decreased axonal transport of mitochondria , causing a lack of functional mitochondria at pre- and postsynaptic sites . collectively , these findings suggest that mfn2 mutations in cmt2a patients may trigger defects both in mtdna integrity and mitochondrial transport , contributing to disease pathogenesis . another neurologic disorder caused by disruption in the fusion machinery is adoa , representing an hereditary optic nerve atrophy characterized by degeneration of retinal ganglion cells and progressive loss of vision . mutations in the opa1 gene , and to a lesser extent in opa3 , underlie adoa . similar to mfn2 mutations , over 100 reported mutations in opa1 target predominantly the gtpase domain . opa1 is located at the inner mitochondrial membrane , regulating fusion and modeling of the cristae structures . interestingly , most opa1 mutations identified in the gtpase domain cause adoa via a dominant - negative effect . furthermore , heterozygous opa1 knockout mice manifest severe degeneration of the optic nerves , similar to human adoa . due to decreased mitochondrial fusion activity , disruption of opa1 gtpase activity results in increased mitochondrial fragmentation , possibly making the retinal ganglion cells more susceptible to apoptosis . however , further analyses are needed to determine whether mitochondrially - mediated apoptotic pathways are implicated in the pathogenesis of adoa . increased mitochondrial fragmentation due to excessive fission is found in many neurodegenerative disorders , including ad , pd and hd , as well as in ischemic brain damage . drp1 consists of an n - terminal gtpase domain , followed by a helical domain ( also termed dynamin - like middle domain ) , an insert b domain , and a c - terminal gtpase effector domain ( ged ) ( fig . 2 ) . the helical domain allows drp1 to form ring - like oligomeric structures at the outer membrane of mitochondria to induce mitochondrial fission upon hydrolysis of gtp . a recent case study reported a rare dominant negative mutation in the human drp1 gene . this mutation inhibits drp1 oligomerization and thus results in the appearance of elongated mitochondrial morphologies . these abnormal mitochondria lose their respiratory function and can no longer be effectively distributed to regions of high - energy demand , such as neuronal synapses . the dominant negative drp1 that results from this mutation caused impaired brain development , microcephaly , optic atrophy , lactic academia , and eventual death by 37 days of age . additionally , drp1 activity can be tightly regulated by a series of ptms including s - nitrosylation , phosphorylation , sumoylation and ubiquitination , as depicted in figure 2 . these ptms generally do not target the drp1 gtpase domain but instead are located within or in close proximity to the ged domain , thereby allosterically influencing gtpase activity . recently , we discovered that drp1 is a target of s - nitrosylation , a covalent modification of free thiol by a nitric oxide ( no ) group , forming an s- nitrosothiol ( r - sno ) . specifically , we found that s - nitrosylation of cys644 within the ged domain of drp1 enhances its gtpase activity and results in excessive mitochondrial fragmentation . s - nitrosylation of drp1 and its involvement in neurologic disorders will be discussed in detail in later sections . in addition to s - nitrosylation , drp1 undergoes multiple other ptms including phosphorylation , sumoylation , and ubiquitination . among these drp1 , it is known that phosphorylation of drp1 at ser616 by cdk1/cyclin b can increase its gtpase activity . in addition , cyclic amp - dependent protein kinase ( pka ) phosphorylates drp1 at ser637 and decreases drp1 activity by inhibiting molecular interactions . pka - mediated phosphorylation can be counteracted by calcineurin - induced dephosphorylation of ser637 , resulting in translocation of drp1 to the mitochondrial membrane . in an apparently paradoxical manner , however , calcium / calmodulin - dependent pka 1 ( camki ) can phosphorylate the same cysteine ( ser637 ) as pka but rather induces recruitment of drp1 to the mitochondrial membrane to form a complex with fis1 , resulting in increased mitochondrial fission . therefore , additional studies are needed to clarify the basis for the differential effects of pka and camki. drp1 activity is also be affected by sumoylation , which occurs within the insert b domain ( fig . the attachment of a small ubiquitin - related modifier 1 ( sumo1 ) to the lysine residues of drp1 stabilizes the protein and thus increases mitochondrial fragmentation . several proteins , including sumo1 , ubc9 , and mapl , have been shown to mediate drp1 sumoylation . this stabilizing modification can be reversed by sentrin / sumo - specific protease ( senp5 ) . parkin and mitochondrial e3 ligase protein march5 can ubiquitinate drp1 on lysine residues located in the middle domain . initial studies reported that march5 activity promoted mitochondrial fusion and elongation , suggesting that march - dependent ubiquitination of drp1 inhibited its activity . although one could speculate that different expression levels of march5 might cause these opposite effects , the physiological action of march5-mediated ubiquitination on drp1 certainly warrants further investigation . parkin is a ring - type ubiquitin e3 ligase expressed in many human tissues , including brain , heart , testis , and skeletal muscle . in the human brain , parkin is primarily expressed in neurons in various regions , including the substantia nigra in the midbrain as well as in the ca1/3 region of the hippocampus . since mutations in the parkin gene cause a familial form of pd , it is tempting to speculate that abnormal regulation of the parkin - drp1 pathway , which would lead to impaired mitochondrial dynamics , might contribute to the pathogenesis of pd . mitochondrial fusion is the process by which the inner and outer mitochondrial membranes are separately fused by opa1 and mfn1/2 , respectively . for example , loss of function mutations in the mfn2 gene disrupts mitochondrial fusion , resulting in distal muscle weakness and sensory loss in cmt2a . to date , more than 40 mutations have been identified , most of which are located in the conserved gtpase domain of mfn2 . in mouse models , deletion in the mouse mfn2 gene causes severe mtdna loss and the accumulation of mtdna mutations . in addition , mfn2 deficiency results in decreased axonal transport of mitochondria , causing a lack of functional mitochondria at pre- and postsynaptic sites . collectively , these findings suggest that mfn2 mutations in cmt2a patients may trigger defects both in mtdna integrity and mitochondrial transport , contributing to disease pathogenesis . another neurologic disorder caused by disruption in the fusion machinery is adoa , representing an hereditary optic nerve atrophy characterized by degeneration of retinal ganglion cells and progressive loss of vision . mutations in the opa1 gene , and to a lesser extent in opa3 , underlie adoa . similar to mfn2 mutations , over 100 reported mutations in opa1 target predominantly the gtpase domain . opa1 is located at the inner mitochondrial membrane , regulating fusion and modeling of the cristae structures . interestingly , most opa1 mutations identified in the gtpase domain cause adoa via a dominant - negative effect . furthermore , heterozygous opa1 knockout mice manifest severe degeneration of the optic nerves , similar to human adoa . due to decreased mitochondrial fusion activity , disruption of opa1 gtpase activity results in increased mitochondrial fragmentation , possibly making the retinal ganglion cells more susceptible to apoptosis . however , further analyses are needed to determine whether mitochondrially - mediated apoptotic pathways are implicated in the pathogenesis of adoa . increased mitochondrial fragmentation due to excessive fission is found in many neurodegenerative disorders , including ad , pd and hd , as well as in ischemic brain damage . drp1 consists of an n - terminal gtpase domain , followed by a helical domain ( also termed dynamin - like middle domain ) , an insert b domain , and a c - terminal gtpase effector domain ( ged ) ( fig . 2 ) . the helical domain allows drp1 to form ring - like oligomeric structures at the outer membrane of mitochondria to induce mitochondrial fission upon hydrolysis of gtp . a recent case study reported a rare dominant negative mutation in the human drp1 gene . this mutation inhibits drp1 oligomerization and thus results in the appearance of elongated mitochondrial morphologies . these abnormal mitochondria lose their respiratory function and can no longer be effectively distributed to regions of high - energy demand , such as neuronal synapses . the dominant negative drp1 that results from this mutation caused impaired brain development , microcephaly , optic atrophy , lactic academia , and eventual death by 37 days of age . additionally , drp1 activity can be tightly regulated by a series of ptms including s - nitrosylation , phosphorylation , sumoylation and ubiquitination , as depicted in figure 2 . these ptms generally do not target the drp1 gtpase domain but instead are located within or in close proximity to the ged domain , thereby allosterically influencing gtpase activity . recently , we discovered that drp1 is a target of s - nitrosylation , a covalent modification of free thiol by a nitric oxide ( no ) group , forming an s- nitrosothiol ( r - sno ) . specifically , we found that s - nitrosylation of cys644 within the ged domain of drp1 enhances its gtpase activity and results in excessive mitochondrial fragmentation . s - nitrosylation of drp1 and its involvement in neurologic disorders will be discussed in detail in later sections . in addition to s - nitrosylation , drp1 undergoes multiple other ptms including phosphorylation , sumoylation , and ubiquitination . among these drp1 for example , it is known that phosphorylation of drp1 at ser616 by cdk1/cyclin b can increase its gtpase activity . in addition , cyclic amp - dependent protein kinase ( pka ) phosphorylates drp1 at ser637 and decreases drp1 activity by inhibiting molecular interactions . pka - mediated phosphorylation can be counteracted by calcineurin - induced dephosphorylation of ser637 , resulting in translocation of drp1 to the mitochondrial membrane . in an apparently paradoxical manner , however , calcium / calmodulin - dependent pka 1 ( camki ) can phosphorylate the same cysteine ( ser637 ) as pka but rather induces recruitment of drp1 to the mitochondrial membrane to form a complex with fis1 , resulting in increased mitochondrial fission . therefore , additional studies are needed to clarify the basis for the differential effects of pka and camki. drp1 activity is also be affected by sumoylation , which occurs within the insert b domain ( fig . 2 ) . the attachment of a small ubiquitin - related modifier 1 ( sumo1 ) to the lysine residues of drp1 stabilizes the protein and thus increases mitochondrial fragmentation . several proteins , including sumo1 , ubc9 , and mapl , have been shown to mediate drp1 sumoylation . this stabilizing modification can be reversed by sentrin / sumo - specific protease ( senp5 ) . parkin and mitochondrial e3 ligase protein march5 can ubiquitinate drp1 on lysine residues located in the middle domain . initial studies reported that march5 activity promoted mitochondrial fusion and elongation , suggesting that march - dependent ubiquitination of drp1 inhibited its activity . however , a subsequent report by karbowski et al . found that march5 activity is required for mitochondrial fission . although one could speculate that different expression levels of march5 might cause these opposite effects , the physiological action of march5-mediated ubiquitination on drp1 certainly warrants further investigation . parkin is a ring - type ubiquitin e3 ligase expressed in many human tissues , including brain , heart , testis , and skeletal muscle . in the human brain , parkin is primarily expressed in neurons in various regions , including the substantia nigra in the midbrain as well as in the ca1/3 region of the hippocampus . since mutations in the parkin gene cause a familial form of pd , it is tempting to speculate that abnormal regulation of the parkin - drp1 pathway , which would lead to impaired mitochondrial dynamics , might contribute to the pathogenesis of pd . over the past twenty years , numerous reports identified no as a crucial protein - modifying molecule in s - nitrosylation . depending on the target protein , the effects of s - nitrosylation can be quite diverse . no is typically generated from no synthase ( nos ) during the conversion of l - arginine to l - citrulline . no production can be controlled by transcriptional activation of inducible nos ( inos ) , or by ca / calmodulin dependent activation of neuronal or endothelial nos ( nnos or enos ) . a well - characterized pathway for no production in the nervous system occurs via activation of the n - methyl - d - aspartate - type glutamate receptor ( nmdar ) . nmdar stimulation results in the influx of ca , which , together with calmodulin , stimulates nnos to generate no ( fig . 3 ) . prior to the discovery of s - nitrosylation , initial studies of no signaling concentrated on its physiological and neuroprotective functions mediated by activation of soluble guanylate cyclase ( sgc ) and subsequent production of cgmp ( fig . subsequently , accumulating evidence has suggested that in a variety of diseases , including ad , hd and pd , nitrosative and oxidative stress appear to trigger aberrant s - nitrosylation events that contribute to neurodegeneration . for example , pathological s - nitrosylation reactions include no - mediated modifications to parkin and protein disulfide isomerase ( pdi ) ( fig . 3 ) . additionally , we recently discovered that aberrant s - nitrosylation of drp1 hyperactivates its mitochondrial fission activity . in this case , no triggers excessive mitochondrial fission accompanied by increased autophagy ( mitophagy ) and decreased atp production , which contributes to synaptic damage conditions such as ad . in addition to sno signaling pathways , no can react with superoxide anion , generated from mitochondrial as well as non- mitochondrial sources ( e.g. , nadph oxidase ) , to form peroxynitrite ( onoo- ) ( fig . peroxynitrite can result in lipid peroxidation and tyrosine nitration , another no - mediated ptm that causes pathological alteration in target protein activity , as reviewed elsewhere . ad is the most common neurodegenerative disorder with increasing prevalence because of the aging demographic of our society . ad is clinically characterized by loss of cognitive functions accompanied by intracellular neurofibrillary tangles , composed of hyperphosphorylated tau , and extracellular amyloid plaques , comprised of amyloid- ( a ) peptide . soluble oligomers of a arise from the proteolytic cleavage of amyloid precursor protein ( app ) and are thought to contribute to the pathogenesis of ad . additional evidence accumulated over the past decade has suggested that altered mitochondrial function associated with dysregulated mitochondrial dynamics is another key feature of ad . recently , the neurotoxic a oligomers were reported to cause excessive mitochondrial fragmentation in cell - based models of ad . specifically , a oligomer - induced generation of no resulted in s - nitrosylation of drp1 ( forming sno - drp1 ) , which was found to hyperactivate its gtpase activity and induce excessive mitochondrial fragmentation . the resulting disruption of mitochondrial bioenergetics caused a drop in atp levels and consequent synaptic damage . drp1 as shown to be s - nitrosylated at cysteine residue 644 , located in the ged domain . importantly , mutation of drp1 cys644 to an alanine abrogated a/no - induced mitochondrial fission , consistent with the notion that the mitochondrial fragmentation was at least in part mediated by formation of sno - drp1 . in ad , the only neuropathological correlation with cognitive decline is the degree of synaptic loss . interestingly , drp1-c644a ( representing a nitrosylation - resistant mutant ) prevented a-induced synaptic damage , suggesting that s- nitrosylation of drp1 at cys644 contributes to a toxicity in ad . moreover , the non - nitrosylatable drp1 mutant prevented nitrosative stress - induced neuronal cell death , lending support to the role of sno - drp1 in no - mediated neuronal damage . although this pathway of aberrant sno - drp1 formation was initially discovered in human ad brains and animal models of ad , many other neurodegenerative conditions , including pd and hd , are characterized by increased oxidative and nitrosative stress . consistent with this notion , sno - drp1 is present at very early stages in the substantia nigra of pd animal models induced by pesticide exposure . additionally , sno - drp1 appears to contribute to mutant huntingtin ( mthtt)-induced neurotoxicity in cell - based and animal models of hd . hd is an adult onset , genetic neurodegenerative disorder caused by an aberrant expansion of a trinucleotide cag repeat in the htt gene . the cag expansion , translated into an expanded polyglutamine ( polyq ) repeat in the mthtt protein , accelerates toxic misfolding of mthtt , leading to interference with several critical molecular cascades in the cell , and resultant degeneration of synapses and neurons in the affected brain area ( i.e. , the striatum and cortex ) . similar to the effects of a oligomers on sno - drp1 , expression of mthtt significantly increased no production and subsequently up - regulated the levels of sno - drp1 in primary cultures of cortical neurons . further along these lines , sno - drp1 formation was considerably elevated in the striatum of a transgenic mouse model of hd as well as in human postmortem brains from hd patients . in a cell culture model of hd , transfection of a non - nitrosylatable mutant form of drp1 abrogated the neurotoxic effects of mthtt on mitochondrial fragmentation and dendritic spine damage , suggesting that sno - drp1 is a key mediator of this form of mthtt toxicity . taken together , these findings imply that s - nitrosylation of drp1 may occur in other neurodegenerative conditions , contributing to aberrant mitochondrial dynamics and downstream neuronal damage . over the past twenty years , numerous reports identified no as a crucial protein - modifying molecule in s - nitrosylation . depending on the target protein , the effects of s - nitrosylation can be quite diverse . no is typically generated from no synthase ( nos ) during the conversion of l - arginine to l - citrulline . no production can be controlled by transcriptional activation of inducible nos ( inos ) , or by ca / calmodulin dependent activation of neuronal or endothelial nos ( nnos or enos ) . a well - characterized pathway for no production in the nervous system occurs via activation of the n - methyl - d - aspartate - type glutamate receptor ( nmdar ) . nmdar stimulation results in the influx of ca , which , together with calmodulin , stimulates nnos to generate no ( fig . 3 ) . prior to the discovery of s - nitrosylation , initial studies of no signaling concentrated on its physiological and neuroprotective functions mediated by activation of soluble guanylate cyclase ( sgc ) and subsequent production of cgmp ( fig . subsequently , accumulating evidence has suggested that in a variety of diseases , including ad , hd and pd , nitrosative and oxidative stress appear to trigger aberrant s - nitrosylation events that contribute to neurodegeneration . for example , pathological s - nitrosylation reactions include no - mediated modifications to parkin and protein disulfide isomerase ( pdi ) ( fig . 3 ) . additionally , we recently discovered that aberrant s - nitrosylation of drp1 hyperactivates its mitochondrial fission activity . in this case , no triggers excessive mitochondrial fission accompanied by increased autophagy ( mitophagy ) and decreased atp production , which contributes to synaptic damage conditions such as ad . in addition to sno signaling pathways , no can react with superoxide anion , generated from mitochondrial as well as non- mitochondrial sources ( e.g. , nadph oxidase ) , to form peroxynitrite ( onoo- ) ( fig . peroxynitrite can result in lipid peroxidation and tyrosine nitration , another no - mediated ptm that causes pathological alteration in target protein activity , as reviewed elsewhere . ad is the most common neurodegenerative disorder with increasing prevalence because of the aging demographic of our society . ad is clinically characterized by loss of cognitive functions accompanied by intracellular neurofibrillary tangles , composed of hyperphosphorylated tau , and extracellular amyloid plaques , comprised of amyloid- ( a ) peptide . soluble oligomers of a arise from the proteolytic cleavage of amyloid precursor protein ( app ) and are thought to contribute to the pathogenesis of ad . additional evidence accumulated over the past decade has suggested that altered mitochondrial function associated with dysregulated mitochondrial dynamics is another key feature of ad . recently , the neurotoxic a oligomers were reported to cause excessive mitochondrial fragmentation in cell - based models of ad . specifically , a oligomer - induced generation of no resulted in s - nitrosylation of drp1 ( forming sno - drp1 ) , which was found to hyperactivate its gtpase activity and induce excessive mitochondrial fragmentation . the resulting disruption of mitochondrial bioenergetics caused a drop in atp levels and consequent synaptic damage . drp1 as shown to be s - nitrosylated at cysteine residue 644 , located in the ged domain . importantly , mutation of drp1 cys644 to an alanine abrogated a/no - induced mitochondrial fission , consistent with the notion that the mitochondrial fragmentation was at least in part mediated by formation of sno - drp1 . in ad , interestingly , drp1-c644a ( representing a nitrosylation - resistant mutant ) prevented a-induced synaptic damage , suggesting that s- nitrosylation of drp1 at cys644 contributes to a toxicity in ad . moreover , the non - nitrosylatable drp1 mutant prevented nitrosative stress - induced neuronal cell death , lending support to the role of sno - drp1 in no - mediated neuronal damage . although this pathway of aberrant sno - drp1 formation was initially discovered in human ad brains and animal models of ad , many other neurodegenerative conditions , including pd and hd , are characterized by increased oxidative and nitrosative stress . consistent with this notion , sno - drp1 is present at very early stages in the substantia nigra of pd animal models induced by pesticide exposure . additionally , sno - drp1 appears to contribute to mutant huntingtin ( mthtt)-induced neurotoxicity in cell - based and animal models of hd . hd is an adult onset , genetic neurodegenerative disorder caused by an aberrant expansion of a trinucleotide cag repeat in the htt gene . the cag expansion , translated into an expanded polyglutamine ( polyq ) repeat in the mthtt protein , accelerates toxic misfolding of mthtt , leading to interference with several critical molecular cascades in the cell , and resultant degeneration of synapses and neurons in the affected brain area ( i.e. , the striatum and cortex ) . similar to the effects of a oligomers on sno - drp1 , expression of mthtt significantly increased no production and subsequently up - regulated the levels of sno - drp1 in primary cultures of cortical neurons . further along these lines , sno - drp1 formation was considerably elevated in the striatum of a transgenic mouse model of hd as well as in human postmortem brains from hd patients . in a cell culture model of hd , transfection of a non - nitrosylatable mutant form of drp1 abrogated the neurotoxic effects of mthtt on mitochondrial fragmentation and dendritic spine damage , suggesting that sno - drp1 is a key mediator of this form of mthtt toxicity . taken together , these findings imply that s - nitrosylation of drp1 may occur in other neurodegenerative conditions , contributing to aberrant mitochondrial dynamics and downstream neuronal damage . in the present study , we review how ptms of drp1 , particularly s - nitrosylation , may contribute to excessive mitochondrial fragmentation in neurodegenerative diseases . these findings strengthen the hypothesis that aberrant protein s - nitrosylation may play a key role in the contribution of mitochondrial pathology to many neurodegenerative diseases . importantly , the demonstration that preventing formation of sno - drp1 can ameliorate a- or mthtt - induced synaptic damage in models of ad and hd , respectively , suggests a new therapeutic approach based on developing drugs to affect disease - related protein s - nitrosylation . additionally , we anticipate that additional examples of aberrant s - nitrosylation as well as other ptm events will be discovered which influence mitochondrial dynamics and function in a number of other neurodegenerative disorders . another important area for future research into the effect of sno - drp1 concerns the discovery of endogenous compounds , including nitrosylase and denitrosylase enzymes which regulate its formation and destruction . related to this question , we recently discovered that sno - cdk5 can act as an endogenous nitrosylase , enhancing sno - drp1 levels via transnitrosylation of drp1 ( i.e. , sno - cdk5 donates an no group to drp1 to form sno - drp1 ) . further efforts to identify additional molecular controls of aberrant s - nitrosylation of drp1 will be critical in determining not only the downstream signaling consequences but also for formulating new therapies for several neurological disorders .

mitochondrial dysfunction occurs in neurodegenerative diseases , however molecular mechanisms underlying this process remain elusive . emerging evidence suggests that nitrosative stress , mediated by reactive nitrogen species ( rns ) , may play a role in mitochondrial pathology . here , we review findings that highlight the abnormal mitochondrial morphology observed in many neurode - generative disorders including alzheimer s , parkinson s , and huntington s diseases . one mechanism whereby rns can affect mitochondrial function and thus neuronal survival occurs via protein s - nitrosylation , representing chemical reaction of a nitric oxide ( no ) group with a critical cysteine thiol . in this review , we focus on the signaling pathway whereby s - nitrosylation of the mitochondrial fission protein drp1 ( dynamin - related protein 1 ; forming s - nitrosothiol ( sno)-drp1 ) precipitates excessive mitochondrial fission or fragmentation and consequent bioenergetic compromise . subsequently , the formation of sno - drp1 leads to synaptic damage and neuronal death . thus , intervention in the sno - drp1 pathway may provide therapeutic benefit in neurodegenerative diseases .

Introduction Mitochondrial Dynamics in Neurodegeneration Impaired mitochondrial fission and fusion in neurodegenerative diseases PTM regulation of Drp1 activity and mitochondrial dynamics S-Nitrosylation, Enhanced Mitochondrial Fission, and Neurodegeneration NO/reactive nitrogen species and neurodegeneration S-Nitrosylation of Drp1 and impaired mitochondrial dynamics in AD and HD Conclusions and Future Directions

for example , the evolutionarily conserved gtpase , dynamin related protein1 ( drp1 ) , is known to participate in the process of mitochondrial fission . the fusion machinery consists mainly of three additional large dynamin - like gtpases : both mitofusin1/2 ( mfn1/2 ) and optic atrophy protein 1 ( opa1 ) . the strict regulation of the equilibrium between fusion and fission events governs mitochondrial dynamics in a healthy cell and is crucial to maintaining mitochondrial function affecting energy supply , response to apoptotic stimuli , mitochondrial inheritance via admixture of mitochondrial - dna ( mtdna ) , and translocation of mitochondria to regions of high - energy demand . thus , mitochondrial function is essential to many neuronal functions , including energy production , ca buffering , axonal and dendritic transport , and release and re - uptake of neurotransmitter . in order to maintain a proper distribution of functional mitochondria , basal activity of the fission machinery is required to divide the mitochondrial network into transportable units which can then be efficiently distributed to synapses . , with increased fission or impaired fusion ) excessive mitochondrial fragmentation is observed , resulting in impaired mitochondrial translocation and energy production at synaptic sites ( fig . indeed , excessive mitochondrial fragmentation with damaged cristae and resulting impaired bioenergetics are often associated with many neurological conditions , including stroke , alzheimer s disease ( ad ) , parkinson s disease ( pd ) , and huntington s disease ( hd ) , as well as several metabolic diseases . pathological disturbance of mitochondrial dynamics can result from either : 1 ) rare genetic mutations , such as those described for mfn1/2 , opa1 and drp1 , which underlie charcot - marie - tooth neuropathy type 2a ( cmt2a ) , autosomal dominant optic atrophy ( adoa ) , and a rare disorder of brain development , respectively ; or 2 ) altered posttranslational modifications ( ptms ) of the proteins encoded by these genes that are responsible for the fission / fusion machinery . in this review , we discuss recent findings concerning aberrant ptms which regulate the mitochondrial fusion and fission machinery and thus contribute to the pathophysiology of neurodegenerative diseases . in particular , we focus on involvement of nitrosative stress - induced drp1 activation , which results in excessive mitochondrial fragmentation , impaired bioenergetics , and consequent synaptic damage in neurodegenerative disorders . as mentioned above , tight regulation of the mitochondrial fission / fusion machinery is essential for maintaining healthy neuronal physiology . however , dysfunctional mitochondrial dynamics caused by impaired mitochondrial fission or fusion is a hallmark of many neurodegenerative diseases and contributes to their pathophysiology . in mouse models , deletion in the mouse mfn2 gene causes severe mtdna loss and the accumulation of mtdna mutations . another neurologic disorder caused by disruption in the fusion machinery is adoa , representing an hereditary optic nerve atrophy characterized by degeneration of retinal ganglion cells and progressive loss of vision . mutations in the opa1 gene , and to a lesser extent in opa3 , underlie adoa . drp1 consists of an n - terminal gtpase domain , followed by a helical domain ( also termed dynamin - like middle domain ) , an insert b domain , and a c - terminal gtpase effector domain ( ged ) ( fig . this mutation inhibits drp1 oligomerization and thus results in the appearance of elongated mitochondrial morphologies . additionally , drp1 activity can be tightly regulated by a series of ptms including s - nitrosylation , phosphorylation , sumoylation and ubiquitination , as depicted in figure 2 . recently , we discovered that drp1 is a target of s - nitrosylation , a covalent modification of free thiol by a nitric oxide ( no ) group , forming an s- nitrosothiol ( r - sno ) . specifically , we found that s - nitrosylation of cys644 within the ged domain of drp1 enhances its gtpase activity and results in excessive mitochondrial fragmentation . s - nitrosylation of drp1 and its involvement in neurologic disorders will be discussed in detail in later sections . in addition to s - nitrosylation , drp1 undergoes multiple other ptms including phosphorylation , sumoylation , and ubiquitination . in an apparently paradoxical manner , however , calcium / calmodulin - dependent pka 1 ( camki ) can phosphorylate the same cysteine ( ser637 ) as pka but rather induces recruitment of drp1 to the mitochondrial membrane to form a complex with fis1 , resulting in increased mitochondrial fission . the attachment of a small ubiquitin - related modifier 1 ( sumo1 ) to the lysine residues of drp1 stabilizes the protein and thus increases mitochondrial fragmentation . parkin is a ring - type ubiquitin e3 ligase expressed in many human tissues , including brain , heart , testis , and skeletal muscle . in the human brain , parkin is primarily expressed in neurons in various regions , including the substantia nigra in the midbrain as well as in the ca1/3 region of the hippocampus . since mutations in the parkin gene cause a familial form of pd , it is tempting to speculate that abnormal regulation of the parkin - drp1 pathway , which would lead to impaired mitochondrial dynamics , might contribute to the pathogenesis of pd . another neurologic disorder caused by disruption in the fusion machinery is adoa , representing an hereditary optic nerve atrophy characterized by degeneration of retinal ganglion cells and progressive loss of vision . mutations in the opa1 gene , and to a lesser extent in opa3 , underlie adoa . drp1 consists of an n - terminal gtpase domain , followed by a helical domain ( also termed dynamin - like middle domain ) , an insert b domain , and a c - terminal gtpase effector domain ( ged ) ( fig . this mutation inhibits drp1 oligomerization and thus results in the appearance of elongated mitochondrial morphologies . additionally , drp1 activity can be tightly regulated by a series of ptms including s - nitrosylation , phosphorylation , sumoylation and ubiquitination , as depicted in figure 2 . recently , we discovered that drp1 is a target of s - nitrosylation , a covalent modification of free thiol by a nitric oxide ( no ) group , forming an s- nitrosothiol ( r - sno ) . specifically , we found that s - nitrosylation of cys644 within the ged domain of drp1 enhances its gtpase activity and results in excessive mitochondrial fragmentation . s - nitrosylation of drp1 and its involvement in neurologic disorders will be discussed in detail in later sections . in addition to s - nitrosylation , drp1 undergoes multiple other ptms including phosphorylation , sumoylation , and ubiquitination . in an apparently paradoxical manner , however , calcium / calmodulin - dependent pka 1 ( camki ) can phosphorylate the same cysteine ( ser637 ) as pka but rather induces recruitment of drp1 to the mitochondrial membrane to form a complex with fis1 , resulting in increased mitochondrial fission . the attachment of a small ubiquitin - related modifier 1 ( sumo1 ) to the lysine residues of drp1 stabilizes the protein and thus increases mitochondrial fragmentation . parkin is a ring - type ubiquitin e3 ligase expressed in many human tissues , including brain , heart , testis , and skeletal muscle . in the human brain , parkin is primarily expressed in neurons in various regions , including the substantia nigra in the midbrain as well as in the ca1/3 region of the hippocampus . since mutations in the parkin gene cause a familial form of pd , it is tempting to speculate that abnormal regulation of the parkin - drp1 pathway , which would lead to impaired mitochondrial dynamics , might contribute to the pathogenesis of pd . over the past twenty years , numerous reports identified no as a crucial protein - modifying molecule in s - nitrosylation . depending on the target protein , the effects of s - nitrosylation can be quite diverse . a well - characterized pathway for no production in the nervous system occurs via activation of the n - methyl - d - aspartate - type glutamate receptor ( nmdar ) . prior to the discovery of s - nitrosylation , initial studies of no signaling concentrated on its physiological and neuroprotective functions mediated by activation of soluble guanylate cyclase ( sgc ) and subsequent production of cgmp ( fig . subsequently , accumulating evidence has suggested that in a variety of diseases , including ad , hd and pd , nitrosative and oxidative stress appear to trigger aberrant s - nitrosylation events that contribute to neurodegeneration . for example , pathological s - nitrosylation reactions include no - mediated modifications to parkin and protein disulfide isomerase ( pdi ) ( fig . additionally , we recently discovered that aberrant s - nitrosylation of drp1 hyperactivates its mitochondrial fission activity . in this case , no triggers excessive mitochondrial fission accompanied by increased autophagy ( mitophagy ) and decreased atp production , which contributes to synaptic damage conditions such as ad . recently , the neurotoxic a oligomers were reported to cause excessive mitochondrial fragmentation in cell - based models of ad . specifically , a oligomer - induced generation of no resulted in s - nitrosylation of drp1 ( forming sno - drp1 ) , which was found to hyperactivate its gtpase activity and induce excessive mitochondrial fragmentation . the resulting disruption of mitochondrial bioenergetics caused a drop in atp levels and consequent synaptic damage . drp1 as shown to be s - nitrosylated at cysteine residue 644 , located in the ged domain . importantly , mutation of drp1 cys644 to an alanine abrogated a/no - induced mitochondrial fission , consistent with the notion that the mitochondrial fragmentation was at least in part mediated by formation of sno - drp1 . interestingly , drp1-c644a ( representing a nitrosylation - resistant mutant ) prevented a-induced synaptic damage , suggesting that s- nitrosylation of drp1 at cys644 contributes to a toxicity in ad . moreover , the non - nitrosylatable drp1 mutant prevented nitrosative stress - induced neuronal cell death , lending support to the role of sno - drp1 in no - mediated neuronal damage . although this pathway of aberrant sno - drp1 formation was initially discovered in human ad brains and animal models of ad , many other neurodegenerative conditions , including pd and hd , are characterized by increased oxidative and nitrosative stress . consistent with this notion , sno - drp1 is present at very early stages in the substantia nigra of pd animal models induced by pesticide exposure . additionally , sno - drp1 appears to contribute to mutant huntingtin ( mthtt)-induced neurotoxicity in cell - based and animal models of hd . hd is an adult onset , genetic neurodegenerative disorder caused by an aberrant expansion of a trinucleotide cag repeat in the htt gene . the cag expansion , translated into an expanded polyglutamine ( polyq ) repeat in the mthtt protein , accelerates toxic misfolding of mthtt , leading to interference with several critical molecular cascades in the cell , and resultant degeneration of synapses and neurons in the affected brain area ( i.e. similar to the effects of a oligomers on sno - drp1 , expression of mthtt significantly increased no production and subsequently up - regulated the levels of sno - drp1 in primary cultures of cortical neurons . further along these lines , sno - drp1 formation was considerably elevated in the striatum of a transgenic mouse model of hd as well as in human postmortem brains from hd patients . in a cell culture model of hd , transfection of a non - nitrosylatable mutant form of drp1 abrogated the neurotoxic effects of mthtt on mitochondrial fragmentation and dendritic spine damage , suggesting that sno - drp1 is a key mediator of this form of mthtt toxicity . taken together , these findings imply that s - nitrosylation of drp1 may occur in other neurodegenerative conditions , contributing to aberrant mitochondrial dynamics and downstream neuronal damage . over the past twenty years , numerous reports identified no as a crucial protein - modifying molecule in s - nitrosylation . depending on the target protein , the effects of s - nitrosylation can be quite diverse . a well - characterized pathway for no production in the nervous system occurs via activation of the n - methyl - d - aspartate - type glutamate receptor ( nmdar ) . nmdar stimulation results in the influx of ca , which , together with calmodulin , stimulates nnos to generate no ( fig . prior to the discovery of s - nitrosylation , initial studies of no signaling concentrated on its physiological and neuroprotective functions mediated by activation of soluble guanylate cyclase ( sgc ) and subsequent production of cgmp ( fig . subsequently , accumulating evidence has suggested that in a variety of diseases , including ad , hd and pd , nitrosative and oxidative stress appear to trigger aberrant s - nitrosylation events that contribute to neurodegeneration . for example , pathological s - nitrosylation reactions include no - mediated modifications to parkin and protein disulfide isomerase ( pdi ) ( fig . additionally , we recently discovered that aberrant s - nitrosylation of drp1 hyperactivates its mitochondrial fission activity . in this case , no triggers excessive mitochondrial fission accompanied by increased autophagy ( mitophagy ) and decreased atp production , which contributes to synaptic damage conditions such as ad . ad is clinically characterized by loss of cognitive functions accompanied by intracellular neurofibrillary tangles , composed of hyperphosphorylated tau , and extracellular amyloid plaques , comprised of amyloid- ( a ) peptide . soluble oligomers of a arise from the proteolytic cleavage of amyloid precursor protein ( app ) and are thought to contribute to the pathogenesis of ad . recently , the neurotoxic a oligomers were reported to cause excessive mitochondrial fragmentation in cell - based models of ad . specifically , a oligomer - induced generation of no resulted in s - nitrosylation of drp1 ( forming sno - drp1 ) , which was found to hyperactivate its gtpase activity and induce excessive mitochondrial fragmentation . the resulting disruption of mitochondrial bioenergetics caused a drop in atp levels and consequent synaptic damage . drp1 as shown to be s - nitrosylated at cysteine residue 644 , located in the ged domain . importantly , mutation of drp1 cys644 to an alanine abrogated a/no - induced mitochondrial fission , consistent with the notion that the mitochondrial fragmentation was at least in part mediated by formation of sno - drp1 . in ad , interestingly , drp1-c644a ( representing a nitrosylation - resistant mutant ) prevented a-induced synaptic damage , suggesting that s- nitrosylation of drp1 at cys644 contributes to a toxicity in ad . moreover , the non - nitrosylatable drp1 mutant prevented nitrosative stress - induced neuronal cell death , lending support to the role of sno - drp1 in no - mediated neuronal damage . although this pathway of aberrant sno - drp1 formation was initially discovered in human ad brains and animal models of ad , many other neurodegenerative conditions , including pd and hd , are characterized by increased oxidative and nitrosative stress . consistent with this notion , sno - drp1 is present at very early stages in the substantia nigra of pd animal models induced by pesticide exposure . additionally , sno - drp1 appears to contribute to mutant huntingtin ( mthtt)-induced neurotoxicity in cell - based and animal models of hd . hd is an adult onset , genetic neurodegenerative disorder caused by an aberrant expansion of a trinucleotide cag repeat in the htt gene . the cag expansion , translated into an expanded polyglutamine ( polyq ) repeat in the mthtt protein , accelerates toxic misfolding of mthtt , leading to interference with several critical molecular cascades in the cell , and resultant degeneration of synapses and neurons in the affected brain area ( i.e. similar to the effects of a oligomers on sno - drp1 , expression of mthtt significantly increased no production and subsequently up - regulated the levels of sno - drp1 in primary cultures of cortical neurons . further along these lines , sno - drp1 formation was considerably elevated in the striatum of a transgenic mouse model of hd as well as in human postmortem brains from hd patients . in a cell culture model of hd , transfection of a non - nitrosylatable mutant form of drp1 abrogated the neurotoxic effects of mthtt on mitochondrial fragmentation and dendritic spine damage , suggesting that sno - drp1 is a key mediator of this form of mthtt toxicity . taken together , these findings imply that s - nitrosylation of drp1 may occur in other neurodegenerative conditions , contributing to aberrant mitochondrial dynamics and downstream neuronal damage . in the present study , we review how ptms of drp1 , particularly s - nitrosylation , may contribute to excessive mitochondrial fragmentation in neurodegenerative diseases . these findings strengthen the hypothesis that aberrant protein s - nitrosylation may play a key role in the contribution of mitochondrial pathology to many neurodegenerative diseases . importantly , the demonstration that preventing formation of sno - drp1 can ameliorate a- or mthtt - induced synaptic damage in models of ad and hd , respectively , suggests a new therapeutic approach based on developing drugs to affect disease - related protein s - nitrosylation . additionally , we anticipate that additional examples of aberrant s - nitrosylation as well as other ptm events will be discovered which influence mitochondrial dynamics and function in a number of other neurodegenerative disorders . another important area for future research into the effect of sno - drp1 concerns the discovery of endogenous compounds , including nitrosylase and denitrosylase enzymes which regulate its formation and destruction . related to this question , we recently discovered that sno - cdk5 can act as an endogenous nitrosylase , enhancing sno - drp1 levels via transnitrosylation of drp1 ( i.e. , sno - cdk5 donates an no group to drp1 to form sno - drp1 ) . further efforts to identify additional molecular controls of aberrant s - nitrosylation of drp1 will be critical in determining not only the downstream signaling consequences but also for formulating new therapies for several neurological disorders .

for example , the evolutionarily conserved gtpase , dynamin related protein1 ( drp1 ) , is known to participate in the process of mitochondrial fission . the fusion machinery consists mainly of three additional large dynamin - like gtpases : both mitofusin1/2 ( mfn1/2 ) and optic atrophy protein 1 ( opa1 ) . the strict regulation of the equilibrium between fusion and fission events governs mitochondrial dynamics in a healthy cell and is crucial to maintaining mitochondrial function affecting energy supply , response to apoptotic stimuli , mitochondrial inheritance via admixture of mitochondrial - dna ( mtdna ) , and translocation of mitochondria to regions of high - energy demand . although mitochondrial dynamics are crucial in all cell types , neurons are thought to be especially dependent on a dynamic mitochondrial network . , healthy neurons precisely control mitochondrial dynamics in order to efficiently transport mitochondria to distal locations , including dendrites , axons , and synapses . thus , mitochondrial function is essential to many neuronal functions , including energy production , ca buffering , axonal and dendritic transport , and release and re - uptake of neurotransmitter . in order to maintain a proper distribution of functional mitochondria , basal activity of the fission machinery is required to divide the mitochondrial network into transportable units which can then be efficiently distributed to synapses . , with increased fission or impaired fusion ) excessive mitochondrial fragmentation is observed , resulting in impaired mitochondrial translocation and energy production at synaptic sites ( fig . indeed , excessive mitochondrial fragmentation with damaged cristae and resulting impaired bioenergetics are often associated with many neurological conditions , including stroke , alzheimer s disease ( ad ) , parkinson s disease ( pd ) , and huntington s disease ( hd ) , as well as several metabolic diseases . pathological disturbance of mitochondrial dynamics can result from either : 1 ) rare genetic mutations , such as those described for mfn1/2 , opa1 and drp1 , which underlie charcot - marie - tooth neuropathy type 2a ( cmt2a ) , autosomal dominant optic atrophy ( adoa ) , and a rare disorder of brain development , respectively ; or 2 ) altered posttranslational modifications ( ptms ) of the proteins encoded by these genes that are responsible for the fission / fusion machinery . in this review , we discuss recent findings concerning aberrant ptms which regulate the mitochondrial fusion and fission machinery and thus contribute to the pathophysiology of neurodegenerative diseases . in particular , we focus on involvement of nitrosative stress - induced drp1 activation , which results in excessive mitochondrial fragmentation , impaired bioenergetics , and consequent synaptic damage in neurodegenerative disorders . as mentioned above , tight regulation of the mitochondrial fission / fusion machinery is essential for maintaining healthy neuronal physiology . however , dysfunctional mitochondrial dynamics caused by impaired mitochondrial fission or fusion is a hallmark of many neurodegenerative diseases and contributes to their pathophysiology . mitochondrial fusion is the process by which the inner and outer mitochondrial membranes are separately fused by opa1 and mfn1/2 , respectively . for example , loss of function mutations in the mfn2 gene disrupts mitochondrial fusion , resulting in distal muscle weakness and sensory loss in cmt2a . to date , more than 40 mutations have been identified , most of which are located in the conserved gtpase domain of mfn2 . in mouse models , deletion in the mouse mfn2 gene causes severe mtdna loss and the accumulation of mtdna mutations . collectively , these findings suggest that mfn2 mutations in cmt2a patients may trigger defects both in mtdna integrity and mitochondrial transport , contributing to disease pathogenesis . another neurologic disorder caused by disruption in the fusion machinery is adoa , representing an hereditary optic nerve atrophy characterized by degeneration of retinal ganglion cells and progressive loss of vision . mutations in the opa1 gene , and to a lesser extent in opa3 , underlie adoa . interestingly , most opa1 mutations identified in the gtpase domain cause adoa via a dominant - negative effect . due to decreased mitochondrial fusion activity , disruption of opa1 gtpase activity results in increased mitochondrial fragmentation , possibly making the retinal ganglion cells more susceptible to apoptosis . however , further analyses are needed to determine whether mitochondrially - mediated apoptotic pathways are implicated in the pathogenesis of adoa . increased mitochondrial fragmentation due to excessive fission is found in many neurodegenerative disorders , including ad , pd and hd , as well as in ischemic brain damage . drp1 consists of an n - terminal gtpase domain , followed by a helical domain ( also termed dynamin - like middle domain ) , an insert b domain , and a c - terminal gtpase effector domain ( ged ) ( fig . the helical domain allows drp1 to form ring - like oligomeric structures at the outer membrane of mitochondria to induce mitochondrial fission upon hydrolysis of gtp . the dominant negative drp1 that results from this mutation caused impaired brain development , microcephaly , optic atrophy , lactic academia , and eventual death by 37 days of age . additionally , drp1 activity can be tightly regulated by a series of ptms including s - nitrosylation , phosphorylation , sumoylation and ubiquitination , as depicted in figure 2 . recently , we discovered that drp1 is a target of s - nitrosylation , a covalent modification of free thiol by a nitric oxide ( no ) group , forming an s- nitrosothiol ( r - sno ) . specifically , we found that s - nitrosylation of cys644 within the ged domain of drp1 enhances its gtpase activity and results in excessive mitochondrial fragmentation . pka - mediated phosphorylation can be counteracted by calcineurin - induced dephosphorylation of ser637 , resulting in translocation of drp1 to the mitochondrial membrane . in an apparently paradoxical manner , however , calcium / calmodulin - dependent pka 1 ( camki ) can phosphorylate the same cysteine ( ser637 ) as pka but rather induces recruitment of drp1 to the mitochondrial membrane to form a complex with fis1 , resulting in increased mitochondrial fission . the attachment of a small ubiquitin - related modifier 1 ( sumo1 ) to the lysine residues of drp1 stabilizes the protein and thus increases mitochondrial fragmentation . initial studies reported that march5 activity promoted mitochondrial fusion and elongation , suggesting that march - dependent ubiquitination of drp1 inhibited its activity . in the human brain , parkin is primarily expressed in neurons in various regions , including the substantia nigra in the midbrain as well as in the ca1/3 region of the hippocampus . since mutations in the parkin gene cause a familial form of pd , it is tempting to speculate that abnormal regulation of the parkin - drp1 pathway , which would lead to impaired mitochondrial dynamics , might contribute to the pathogenesis of pd . mitochondrial fusion is the process by which the inner and outer mitochondrial membranes are separately fused by opa1 and mfn1/2 , respectively . for example , loss of function mutations in the mfn2 gene disrupts mitochondrial fusion , resulting in distal muscle weakness and sensory loss in cmt2a . to date , more than 40 mutations have been identified , most of which are located in the conserved gtpase domain of mfn2 . in mouse models , deletion in the mouse mfn2 gene causes severe mtdna loss and the accumulation of mtdna mutations . collectively , these findings suggest that mfn2 mutations in cmt2a patients may trigger defects both in mtdna integrity and mitochondrial transport , contributing to disease pathogenesis . another neurologic disorder caused by disruption in the fusion machinery is adoa , representing an hereditary optic nerve atrophy characterized by degeneration of retinal ganglion cells and progressive loss of vision . interestingly , most opa1 mutations identified in the gtpase domain cause adoa via a dominant - negative effect . due to decreased mitochondrial fusion activity , disruption of opa1 gtpase activity results in increased mitochondrial fragmentation , possibly making the retinal ganglion cells more susceptible to apoptosis . however , further analyses are needed to determine whether mitochondrially - mediated apoptotic pathways are implicated in the pathogenesis of adoa . increased mitochondrial fragmentation due to excessive fission is found in many neurodegenerative disorders , including ad , pd and hd , as well as in ischemic brain damage . drp1 consists of an n - terminal gtpase domain , followed by a helical domain ( also termed dynamin - like middle domain ) , an insert b domain , and a c - terminal gtpase effector domain ( ged ) ( fig . the dominant negative drp1 that results from this mutation caused impaired brain development , microcephaly , optic atrophy , lactic academia , and eventual death by 37 days of age . recently , we discovered that drp1 is a target of s - nitrosylation , a covalent modification of free thiol by a nitric oxide ( no ) group , forming an s- nitrosothiol ( r - sno ) . specifically , we found that s - nitrosylation of cys644 within the ged domain of drp1 enhances its gtpase activity and results in excessive mitochondrial fragmentation . in an apparently paradoxical manner , however , calcium / calmodulin - dependent pka 1 ( camki ) can phosphorylate the same cysteine ( ser637 ) as pka but rather induces recruitment of drp1 to the mitochondrial membrane to form a complex with fis1 , resulting in increased mitochondrial fission . the attachment of a small ubiquitin - related modifier 1 ( sumo1 ) to the lysine residues of drp1 stabilizes the protein and thus increases mitochondrial fragmentation . initial studies reported that march5 activity promoted mitochondrial fusion and elongation , suggesting that march - dependent ubiquitination of drp1 inhibited its activity . in the human brain , parkin is primarily expressed in neurons in various regions , including the substantia nigra in the midbrain as well as in the ca1/3 region of the hippocampus . since mutations in the parkin gene cause a familial form of pd , it is tempting to speculate that abnormal regulation of the parkin - drp1 pathway , which would lead to impaired mitochondrial dynamics , might contribute to the pathogenesis of pd . over the past twenty years , numerous reports identified no as a crucial protein - modifying molecule in s - nitrosylation . no production can be controlled by transcriptional activation of inducible nos ( inos ) , or by ca / calmodulin dependent activation of neuronal or endothelial nos ( nnos or enos ) . a well - characterized pathway for no production in the nervous system occurs via activation of the n - methyl - d - aspartate - type glutamate receptor ( nmdar ) . prior to the discovery of s - nitrosylation , initial studies of no signaling concentrated on its physiological and neuroprotective functions mediated by activation of soluble guanylate cyclase ( sgc ) and subsequent production of cgmp ( fig . subsequently , accumulating evidence has suggested that in a variety of diseases , including ad , hd and pd , nitrosative and oxidative stress appear to trigger aberrant s - nitrosylation events that contribute to neurodegeneration . soluble oligomers of a arise from the proteolytic cleavage of amyloid precursor protein ( app ) and are thought to contribute to the pathogenesis of ad . additional evidence accumulated over the past decade has suggested that altered mitochondrial function associated with dysregulated mitochondrial dynamics is another key feature of ad . specifically , a oligomer - induced generation of no resulted in s - nitrosylation of drp1 ( forming sno - drp1 ) , which was found to hyperactivate its gtpase activity and induce excessive mitochondrial fragmentation . importantly , mutation of drp1 cys644 to an alanine abrogated a/no - induced mitochondrial fission , consistent with the notion that the mitochondrial fragmentation was at least in part mediated by formation of sno - drp1 . interestingly , drp1-c644a ( representing a nitrosylation - resistant mutant ) prevented a-induced synaptic damage , suggesting that s- nitrosylation of drp1 at cys644 contributes to a toxicity in ad . moreover , the non - nitrosylatable drp1 mutant prevented nitrosative stress - induced neuronal cell death , lending support to the role of sno - drp1 in no - mediated neuronal damage . although this pathway of aberrant sno - drp1 formation was initially discovered in human ad brains and animal models of ad , many other neurodegenerative conditions , including pd and hd , are characterized by increased oxidative and nitrosative stress . consistent with this notion , sno - drp1 is present at very early stages in the substantia nigra of pd animal models induced by pesticide exposure . additionally , sno - drp1 appears to contribute to mutant huntingtin ( mthtt)-induced neurotoxicity in cell - based and animal models of hd . the cag expansion , translated into an expanded polyglutamine ( polyq ) repeat in the mthtt protein , accelerates toxic misfolding of mthtt , leading to interference with several critical molecular cascades in the cell , and resultant degeneration of synapses and neurons in the affected brain area ( i.e. similar to the effects of a oligomers on sno - drp1 , expression of mthtt significantly increased no production and subsequently up - regulated the levels of sno - drp1 in primary cultures of cortical neurons . further along these lines , sno - drp1 formation was considerably elevated in the striatum of a transgenic mouse model of hd as well as in human postmortem brains from hd patients . in a cell culture model of hd , transfection of a non - nitrosylatable mutant form of drp1 abrogated the neurotoxic effects of mthtt on mitochondrial fragmentation and dendritic spine damage , suggesting that sno - drp1 is a key mediator of this form of mthtt toxicity . taken together , these findings imply that s - nitrosylation of drp1 may occur in other neurodegenerative conditions , contributing to aberrant mitochondrial dynamics and downstream neuronal damage . a well - characterized pathway for no production in the nervous system occurs via activation of the n - methyl - d - aspartate - type glutamate receptor ( nmdar ) . prior to the discovery of s - nitrosylation , initial studies of no signaling concentrated on its physiological and neuroprotective functions mediated by activation of soluble guanylate cyclase ( sgc ) and subsequent production of cgmp ( fig . subsequently , accumulating evidence has suggested that in a variety of diseases , including ad , hd and pd , nitrosative and oxidative stress appear to trigger aberrant s - nitrosylation events that contribute to neurodegeneration . additional evidence accumulated over the past decade has suggested that altered mitochondrial function associated with dysregulated mitochondrial dynamics is another key feature of ad . specifically , a oligomer - induced generation of no resulted in s - nitrosylation of drp1 ( forming sno - drp1 ) , which was found to hyperactivate its gtpase activity and induce excessive mitochondrial fragmentation . importantly , mutation of drp1 cys644 to an alanine abrogated a/no - induced mitochondrial fission , consistent with the notion that the mitochondrial fragmentation was at least in part mediated by formation of sno - drp1 . in ad , interestingly , drp1-c644a ( representing a nitrosylation - resistant mutant ) prevented a-induced synaptic damage , suggesting that s- nitrosylation of drp1 at cys644 contributes to a toxicity in ad . moreover , the non - nitrosylatable drp1 mutant prevented nitrosative stress - induced neuronal cell death , lending support to the role of sno - drp1 in no - mediated neuronal damage . although this pathway of aberrant sno - drp1 formation was initially discovered in human ad brains and animal models of ad , many other neurodegenerative conditions , including pd and hd , are characterized by increased oxidative and nitrosative stress . consistent with this notion , sno - drp1 is present at very early stages in the substantia nigra of pd animal models induced by pesticide exposure . the cag expansion , translated into an expanded polyglutamine ( polyq ) repeat in the mthtt protein , accelerates toxic misfolding of mthtt , leading to interference with several critical molecular cascades in the cell , and resultant degeneration of synapses and neurons in the affected brain area ( i.e. similar to the effects of a oligomers on sno - drp1 , expression of mthtt significantly increased no production and subsequently up - regulated the levels of sno - drp1 in primary cultures of cortical neurons . further along these lines , sno - drp1 formation was considerably elevated in the striatum of a transgenic mouse model of hd as well as in human postmortem brains from hd patients . in a cell culture model of hd , transfection of a non - nitrosylatable mutant form of drp1 abrogated the neurotoxic effects of mthtt on mitochondrial fragmentation and dendritic spine damage , suggesting that sno - drp1 is a key mediator of this form of mthtt toxicity . taken together , these findings imply that s - nitrosylation of drp1 may occur in other neurodegenerative conditions , contributing to aberrant mitochondrial dynamics and downstream neuronal damage . in the present study , we review how ptms of drp1 , particularly s - nitrosylation , may contribute to excessive mitochondrial fragmentation in neurodegenerative diseases . these findings strengthen the hypothesis that aberrant protein s - nitrosylation may play a key role in the contribution of mitochondrial pathology to many neurodegenerative diseases . importantly , the demonstration that preventing formation of sno - drp1 can ameliorate a- or mthtt - induced synaptic damage in models of ad and hd , respectively , suggests a new therapeutic approach based on developing drugs to affect disease - related protein s - nitrosylation . additionally , we anticipate that additional examples of aberrant s - nitrosylation as well as other ptm events will be discovered which influence mitochondrial dynamics and function in a number of other neurodegenerative disorders . another important area for future research into the effect of sno - drp1 concerns the discovery of endogenous compounds , including nitrosylase and denitrosylase enzymes which regulate its formation and destruction . related to this question , we recently discovered that sno - cdk5 can act as an endogenous nitrosylase , enhancing sno - drp1 levels via transnitrosylation of drp1 ( i.e. further efforts to identify additional molecular controls of aberrant s - nitrosylation of drp1 will be critical in determining not only the downstream signaling consequences but also for formulating new therapies for several neurological disorders .

fabry disease ( fd ) is an x - linked recessive lysosomal storage disorder caused by a defect in the gene that encodes lysosomal -galactosidase a and is known to affect various tissues including the kidney and heart . patients with fd manifest a variety of clinical symptoms , such as acroparesthesias , angiokeratoma , hypohidrosis , corneal opacities , stroke , renal disorder and cardiac abnormalities . the manifestations are thought to be due to progressive accumulation of globotriaosylceramide ( gl-3 ) and other glycosphingolipids within various cells such as vascular endothelium , cardiomyocytes , glomerular cells and renal tubular cells . the precise pathophysiological mechanisms of fd remain unclear , and coronary microvascular dysfunction appears to be one of the causes of cardiac complications in fd [ 14 ] . in several studies , gl-3 accumulation within vascular endothelial cells in the kidney and heart many studies have demonstrated that nitric oxide ( no ) plays an important role in the progression of endothelial dysfunction , atherosclerosis and chronic kidney disease ( ckd ) [ 811 ] . no is synthesized by endothelial , neuronal and macrophage isoforms of the enzyme no synthase ( nos ) . asymmetric dimethylarginine ( adma ) is an endogenous competitive inhibitor of nos , and serum adma levels have been suggested to be a surrogate marker of endothelial dysfunction and/or angiosclerosis . recently , we revealed a significant relationship between adma , glomerular filtration rate and endothelial dysfunction of the coronary and peripheral arteries [ 12 , 13 ] . from the results of not only our studies but also various earlier studies , we thought that adma may be involved in the underlying mechanism connecting the two pathological conditions between the kidney and the heart and may be a useful biochemical marker even in fd . the aim of our study was to examine the effect of ert on cardiac abnormalities and to determine the relationship between adma , coronary microvascular function and left ventricular hypertrophy ( lvh ) in fd patients . for these patients , -galactosidase a ( agalsidase , fabrazyme ; genzyme corp . , cambridge , ma , or agalsidase , replagal ; shire plc , dublin , ireland ) was administered intravenously every 2 weeks for a period of 12 months ( at a dosage of 1 or 0.2 mg / kg body weight , respectively ) . ten hypertensive patients without diabetes mellitus , inflammatory disease , infection , ckd or any cardiovascular complications were also evaluated as controls . in addition , 20 healthy volunteers ( mean age : 29 4 years ) were also evaluated to determine serum adma levels . patients were excluded only if they did not agree to participate in the study . for the fd patients , we prospectively performed blood examination , echocardiography , exercise stress myocardial scintigraphy and coronary flow reserve ( cfr ) measurements before starting ert and at 3 , 6 and 12 months . the experimental protocols were approved by the appropriate institutional review committee and performed in accordance with the helsinki declaration of 1975 , as revised in 2008 . transthoracic doppler echocardiography , which has been described previously , was used to measure cfr . the ultrasound beam was transmitted toward the heart to visualize coronary blood flow in the distal portion of the left anterior descending ( lad ) coronary artery by color doppler flow mapping . first , the left ventricle was imaged in cross - section along the longitudinal axis , and then , the ultrasound beam was inclined laterally . next , coronary blood flow in the distal lad was examined under the guidance of color doppler flow mapping . after positioning a sample volume on the color signal in the distal lad , doppler spectral tracings of flow velocity were recorded by fast fourier transformation analysis . adenosine-5-triphosphate was administered ( 140 g / kg / min , i.v . ) for 3 min to record spectral doppler signals during hyperemic conditions . all of the patients underwent continuous heart rate and blood pressure monitoring throughout the study period . an ultrasound system computer was used to trace the contour of the spectral doppler signal for off - line analysis of coronary flow velocity . peak diastolic velocity ( pdv ) was measured at the baseline and peak hyperemic conditions . we adopted a cfr of < 2.0 as the cut - off value for the presence of significant coronary microvascular disease , as used previously . two - dimensional guided m - mode echocardiography was performed to measure left ventricular wall mass . left ventricular diastolic diameter ( lvdd ) , left ventricular systolic diameter , diastolic thickness of the left ventricular posterior wall ( lvpwt ) and interventricular septum ( ivst ) were assessed in m - mode images in the parasternal longitudinal axis view . the m - mode analysis was performed according to the guidelines of the american society of echocardiography . the following formula was used to calculate the left ventricular mass index ( lvmi ) ( g / m ) : on the same day as the cfr measurement , venous blood was collected from the patients after a 20-min period of supine rest in the morning following overnight fasting . in two dialysis patients , according to japanese society of dialysis therapy ( jsdt ) guidelines , blood samples were collected before each dialysis session at 2-day intervals . serum was separated by centrifugation at 2500 g for 10 min at 4c and stored at 20c until analysis . the ckd epidemiology collaboration ( ckd - epi ) equation was used to estimate the glomerular filtration rate for evaluation of renal function . we used the computer software application statview 5.0 ( sas institute , cary , nc ) for all statistical analyses . whitney u - test was used to analyze the significance of differences between two groups . pearson 's correlation coefficient was used to analyze relationships between variables . results with p - values for these patients , -galactosidase a ( agalsidase , fabrazyme ; genzyme corp . , cambridge , ma , or agalsidase , replagal ; shire plc , dublin , ireland ) was administered intravenously every 2 weeks for a period of 12 months ( at a dosage of 1 or 0.2 mg / kg body weight , respectively ) . ten hypertensive patients without diabetes mellitus , inflammatory disease , infection , ckd or any cardiovascular complications were also evaluated as controls . in addition , 20 healthy volunteers ( mean age : 29 4 years ) were also evaluated to determine serum adma levels . patients were excluded only if they did not agree to participate in the study . for the fd patients , we prospectively performed blood examination , echocardiography , exercise stress myocardial scintigraphy and coronary flow reserve ( cfr ) measurements before starting ert and at 3 , 6 and 12 months . the experimental protocols were approved by the appropriate institutional review committee and performed in accordance with the helsinki declaration of 1975 , as revised in 2008 . transthoracic doppler echocardiography , which has been described previously , was used to measure cfr . the ultrasound beam was transmitted toward the heart to visualize coronary blood flow in the distal portion of the left anterior descending ( lad ) coronary artery by color doppler flow mapping . first , the left ventricle was imaged in cross - section along the longitudinal axis , and then , the ultrasound beam was inclined laterally . next , coronary blood flow in the distal lad was examined under the guidance of color doppler flow mapping . after positioning a sample volume on the color signal in the distal lad , adenosine-5-triphosphate was administered ( 140 g / kg / min , i.v . ) for 3 min to record spectral doppler signals during hyperemic conditions . all of the patients underwent continuous heart rate and blood pressure monitoring throughout the study period . an ultrasound system computer was used to trace the contour of the spectral doppler signal for off - line analysis of coronary flow velocity . peak diastolic velocity ( pdv ) was measured at the baseline and peak hyperemic conditions . we adopted a cfr of < 2.0 as the cut - off value for the presence of significant coronary microvascular disease , as used previously . two - dimensional guided m - mode echocardiography was performed to measure left ventricular wall mass . left ventricular diastolic diameter ( lvdd ) , left ventricular systolic diameter , diastolic thickness of the left ventricular posterior wall ( lvpwt ) and interventricular septum ( ivst ) were assessed in m - mode images in the parasternal longitudinal axis view . the m - mode analysis was performed according to the guidelines of the american society of echocardiography . the following formula was used to calculate the left ventricular mass index ( lvmi ) ( g / m ) : on the same day as the cfr measurement , venous blood was collected from the patients after a 20-min period of supine rest in the morning following overnight fasting . in two dialysis patients , according to japanese society of dialysis therapy ( jsdt ) guidelines , blood samples were collected before each dialysis session at 2-day intervals . serum was separated by centrifugation at 2500 g for 10 min at 4c and stored at 20c until analysis . the ckd epidemiology collaboration ( ckd - epi ) equation was used to estimate the glomerular filtration rate for evaluation of renal function . we used the computer software application statview 5.0 ( sas institute , cary , nc ) for all statistical analyses . whitney u - test was used to analyze the significance of differences between two groups . the main characteristics of the subjects are shown in table 1 . among the study patients , three had a history of stroke ( cases 1 , 3 and 4 ) and two had received hemodialysis therapy ( cases 1 and 4 ) . at the baseline , -galactosidase activity was low in all patients , as determined by the measurement of the dried blood spots in a fluorescence assay using 4-methylumbelliferyl . in contrast , -galactosidase activity measured in leukocytes was low in only one patient ( case 4 ) . serum adma levels were significantly higher in the fd group than in the hypertensive control and healthy control groups ( figure 1a ) . as shown in table 2 , the lvmi increased in three patients ( cases 1 , 3 and 4 ) , and systolic function decreased in two ( cases 1 and 4 ) . although findings of ischemia were not observed using exercise stress myocardial scintigraphy in all the study patients , cfr decreased in three patients ( cases 1 , 3 and 4 ) and two had angina - like chest pain ( cases 1 and 3 ) . needless to say , cfr was significantly lower in the fd group than in the hypertensive control group ( figure 1b ) . table 1.patient characteristicscase 1case 2case 3case 4normal valuesage ( years old)45197151sexfmfmhd duration ( years)1223hypertension(+)()(+)()diabetes mellitus(+)(+)()()hyperlipidemia()()()()stroke(+)()(+)(+)acroparesthesias()()(+)()hypohidrosis()()()()angiokeratoma()()()()corneal opacities(+)()()()chest pain(+)()(+)()cr ( mol / l)872.451.165.6976.1egfr ( ml / min/1.73 m)13968rbc ( 10/l)4.105.533.054.20hemoglobin ( g / l)114161103121tp ( g / l)686968796482alb ( g / l)394539363552t - chol ( mmol / l)3.236.214.864.093.366.21tg ( mmol / l)2.019.043.921.260.342.03hdl ( mmol / l)0.980.961.140.961.03hba1c ( % ) 6.112.65.04.64.35.8ca ( mmol / l)2.222.422.222.592.122.55p ( mmol / l)1.681.190.872.160.771.39bnp ( ng / l)321.52.459.6272.218.4hscrp ( nmol / l)28.645.712.421.9<28.6adma ( ng / l)0.850.480.550.70-galactosidase activitydried blood spot ( agal u)16.911.215.115.220leukocyte ( nmol / mg protein / h)61.252.375.532.149.8116.4hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . table 2.echocardiographic parameters at baselinecase 1case 2case 3case 4lad ( mm)41394144lvdd ( mm)49454847lvds ( mm)37313333ivst ( mm)16101212pwt ( mm)13101112fs ( % ) 24303129ef ( % ) 58606657lvmi ( g / m)203.293.8158.2151.9e / a ratio0.772.260.620.65dct ( ms)346146200212ivcd ( mm)168.95.713cfr1.773.412.311.45lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . fig . ( a ) serum adma levels in the healthy subjects , hypertensive patients and fd patients . ( b ) cfr in the hypertensive patients and fd patients . * p < 0.05 patient characteristics hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . echocardiographic parameters at baseline lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . serum adma levels and cfr in the control patients and fd patients . ( a ) serum adma levels in the healthy subjects , hypertensive patients and fd patients . p < 0.05 we evaluated the changes in serum adma levels , cfr and lvmi after starting ert , and these parameters mostly improved 12 months after starting ert compared with the values at baseline ( figure 2a c ) . two study patients had angina - like chest pain before the treatment ; their symptoms disappeared following ert . unfortunately , because of a worldwide shortage of available recombinant -galactosidase , cases 2 and 4 did not receive sufficient ert for the last 2 months of treatment . table 3.laboratory data during ertcase 1case 2case 3case 4months03612036120361203612cr ( mol / l)872.4879.3854.1903.740.438.141.243.565.674.773.254.9976.1995.91060.0986.0egfr ( ml / min/1.73 m)15315715214968586085rbc ( 10/l)4.103.393.614.055.535.675.715.643.053.143.323.404.203.803.783.75hemoglobin ( g / l)11492103116161163167167103102105110121108114114tp ( g / l)68697071696968706863646979727373alb ( g / l)39414242454545483934363736333232t - chol ( mmol / l)3.233.473.133.706.214.975.904.424.864.504.035.174.092.772.842.64tg ( mmol / l)2.011.680.821.559.403.425.013.133.921.102.673.411.261.461.240.69hdl ( mmol / l)0.981.241.321.320.960.960.960.911.141.531.241.810.960.800.720.88hba1c ( % ) 6.15.95.16.312.610.611.811.25.04.6ca ( mmol / l)2.222.252.102.302.422.322.322.472.222.322.402.252.592.152.252.17p ( mmol / l)1.681.941.522.231.191.191.231.130.871.001.190.902.161.781.841.84bnp ( ng / l)321.5169.268.353.32.43.12.26.459.669.127.677.7272.2173.2183.1144.4cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide . fig . laboratory data during ert cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide . change of cfr , lvmi and adma during ert . ( a ) serum adma levels in individual patients . we examined the correlation of serum adma levels with cfr and lvmi and found that serum adma levels were significantly correlated with both cfr ( r = 0.576 , p < 0.05 ) and lvmi ( r = 0.874 , p < 0.0001 ) ( figure 3a and b ) . there was also a significant relationship between cfr and lvmi ( r = 0.545 , p < 0.05 ) ( figure 3c ) . in addition , serum adma levels were significantly correlated with plasma brain natriuretic peptide ( bnp ) levels ( r = 0.770 , p = 0.0005 ) . the main characteristics of the subjects are shown in table 1 . among the study patients , three had a history of stroke ( cases 1 , 3 and 4 ) and two had received hemodialysis therapy ( cases 1 and 4 ) . at the baseline , -galactosidase activity was low in all patients , as determined by the measurement of the dried blood spots in a fluorescence assay using 4-methylumbelliferyl . in contrast , -galactosidase activity measured in leukocytes was low in only one patient ( case 4 ) . serum adma levels were significantly higher in the fd group than in the hypertensive control and healthy control groups ( figure 1a ) . as shown in table 2 , the lvmi increased in three patients ( cases 1 , 3 and 4 ) , and systolic function decreased in two ( cases 1 and 4 ) . although findings of ischemia were not observed using exercise stress myocardial scintigraphy in all the study patients , cfr decreased in three patients ( cases 1 , 3 and 4 ) and two had angina - like chest pain ( cases 1 and 3 ) . needless to say , cfr was significantly lower in the fd group than in the hypertensive control group ( figure 1b ) . table 1.patient characteristicscase 1case 2case 3case 4normal valuesage ( years old)45197151sexfmfmhd duration ( years)1223hypertension(+)()(+)()diabetes mellitus(+)(+)()()hyperlipidemia()()()()stroke(+)()(+)(+)acroparesthesias()()(+)()hypohidrosis()()()()angiokeratoma()()()()corneal opacities(+)()()()chest pain(+)()(+)()cr ( mol / l)872.451.165.6976.1egfr ( ml / min/1.73 m)13968rbc ( 10/l)4.105.533.054.20hemoglobin ( g / l)114161103121tp ( g / l)686968796482alb ( g / l)394539363552t - chol ( mmol / l)3.236.214.864.093.366.21tg ( mmol / l)2.019.043.921.260.342.03hdl ( mmol / l)0.980.961.140.961.03hba1c ( % ) 6.112.65.04.64.35.8ca ( mmol / l)2.222.422.222.592.122.55p ( mmol / l)1.681.190.872.160.771.39bnp ( ng / l)321.52.459.6272.218.4hscrp ( nmol / l)28.645.712.421.9<28.6adma ( ng / l)0.850.480.550.70-galactosidase activitydried blood spot ( agal u)16.911.215.115.220leukocyte ( nmol / mg protein / h)61.252.375.532.149.8116.4hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . table 2.echocardiographic parameters at baselinecase 1case 2case 3case 4lad ( mm)41394144lvdd ( mm)49454847lvds ( mm)37313333ivst ( mm)16101212pwt ( mm)13101112fs ( % ) 24303129ef ( % ) 58606657lvmi ( g / m)203.293.8158.2151.9e / a ratio0.772.260.620.65dct ( ms)346146200212ivcd ( mm)168.95.713cfr1.773.412.311.45lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . fig . ( a ) serum adma levels in the healthy subjects , hypertensive patients and fd patients . ( b ) cfr in the hypertensive patients and fd patients . * p < 0.05 patient characteristics hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . echocardiographic parameters at baseline lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . serum adma levels and cfr in the control patients and fd patients . ( a ) serum adma levels in the healthy subjects , hypertensive patients and fd patients . we evaluated the changes in serum adma levels , cfr and lvmi after starting ert , and these parameters mostly improved 12 months after starting ert compared with the values at baseline ( figure 2a c ) . two study patients had angina - like chest pain before the treatment ; their symptoms disappeared following ert . unfortunately , because of a worldwide shortage of available recombinant -galactosidase , cases 2 and 4 did not receive sufficient ert for the last 2 months of treatment . table 3.laboratory data during ertcase 1case 2case 3case 4months03612036120361203612cr ( mol / l)872.4879.3854.1903.740.438.141.243.565.674.773.254.9976.1995.91060.0986.0egfr ( ml / min/1.73 m)15315715214968586085rbc ( 10/l)4.103.393.614.055.535.675.715.643.053.143.323.404.203.803.783.75hemoglobin ( g / l)11492103116161163167167103102105110121108114114tp ( g / l)68697071696968706863646979727373alb ( g / l)39414242454545483934363736333232t - chol ( mmol / l)3.233.473.133.706.214.975.904.424.864.504.035.174.092.772.842.64tg ( mmol / l)2.011.680.821.559.403.425.013.133.921.102.673.411.261.461.240.69hdl ( mmol / l)0.981.241.321.320.960.960.960.911.141.531.241.810.960.800.720.88hba1c ( % ) 6.15.95.16.312.610.611.811.25.04.6ca ( mmol / l)2.222.252.102.302.422.322.322.472.222.322.402.252.592.152.252.17p ( mmol / l)1.681.941.522.231.191.191.231.130.871.001.190.902.161.781.841.84bnp ( ng / l)321.5169.268.353.32.43.12.26.459.669.127.677.7272.2173.2183.1144.4cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide . fig . laboratory data during ert cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide . change of cfr , lvmi and adma during ert . we examined the correlation of serum adma levels with cfr and lvmi and found that serum adma levels were significantly correlated with both cfr ( r = 0.576 , p < 0.05 ) and lvmi ( r = 0.874 , p < 0.0001 ) ( figure 3a and b ) . there was also a significant relationship between cfr and lvmi ( r = 0.545 , p < 0.05 ) ( figure 3c ) . in addition , serum adma levels were significantly correlated with plasma brain natriuretic peptide ( bnp ) levels ( r = 0.770 , p = 0.0005 ) . our study demonstrated that ( i ) cfr was significantly lower and serum adma levels and lvmi were significantly higher in the fd group than in the control group ; ( ii ) ert increased the cfr , decreased the elevated serum adma levels and increased lvmi ; and ( iii ) serum adma levels showed a significant relationship with cfr and lvmi in the fd patients . it has been thought that fd patients show changes not only in the myocardium but also in coronary vascular function . in fact , some studies have demonstrated that cfr is markedly decreased in fd [ 4 , 1618 ] . previous studies have also reported that 50% of fd patients complain of angina - like chest pain [ 19 , 20 ] . in our study , two patients had angina - like chest pain and three had markedly impaired cfr , despite having no manifest coronary artery disease . thus , coronary microvascular dysfunction may play a key role in the development of these symptoms . in many cases with cardiac symptoms , gl-3 accumulates in the endothelial , myocardial and smooth muscle cells , which causes microvascular dysfunction in coronary arteries and lvh . it has been shown that ert reverses the accumulation of microvascular endothelial deposits of gl-3 in several organs , including the heart [ 13 , 7 ] . furthermore , it is thought that the increased oxygen demand of the hypertrophied myocardium is related to myocardial ischemia in fd . there are also some studies that have reported that ert was effective in decreasing lvh and improving regional myocardial function [ 5 , 21 , 22 ] . therefore , we speculated that ert could ameliorate coronary microvascular dysfunction in fd through these two mechanisms . on the other hand , two previous studies reported that no improvements in hyperemic myocardial blood flow , cfr and lvmi were observed by ert [ 23 , 24 ] . however , the result of one study showed that the study patients felt subjectively better and suffered less pain after starting ert . in addition , plasma gl-3 levels decreased significantly after ert in both studies . from the results of these studies , ert could prevent the progression of impaired cfr and lvh but could not improve cfr and lvh in their study patients . therefore , it also has been speculated that ert is less effective in patients with severe lvh . in the present study , ert improved not only cfr but also lvmi in most patients . we believe that the reason is that our study patients did not have such severe lvh . several studies have demonstrated that serum adma levels increased in ckd patients and correlated with the severity of atherosclerotic lesions [ 911 ] . thus , adma is thought to be an available biochemical marker of endothelial dysfunction and/or vascular lesions in patients with ckd . our previous studies demonstrated that adma was significantly associated with cfr , endothelium - mediated vasodilation of the brachial artery and renal function [ 12 , 13 ] . adma is also known to be related to lvh in patients with ckd [ 26 , 27 ] . in the present study , to ascertain a possible role of adma in fd and determine whether adma is a useful marker of cardiac manifestation in fd , we assessed the relationship between serum adma levels , cfr and lvmi . we found that serum adma levels were significantly correlated with cfr and lvmi in patients with fd as well as in non - fd patients . furthermore , the levels tended to decrease with improving cfr and lvmi after starting ert . in the present study , serum bnp levels were also significantly associated with cfr and lvmi . however , because many papers demonstrated that adma induces cardiovascular dysfunction , it is reasonable to suppose that adma plays a key role in kidney and heart disease . moreover , it has been reported that adma is more sensitive than n - terminal pro - bnp to diagnose heart failure in patients with congenital heart disease . considering these findings , there is a possibility that serum adma levels may reflect the degree of cardiac damage even in fd . acroparesthesia is one of the major symptoms in fd and it is known that ert could reduce this symptom . as mentioned above , our results show that serum adma levels increased in fd . taken together , we speculated that adma might be involved in the acroparesthesia of fd though we do not have objective data on polyneuropathy . the main limitation of our study was the small number of study patients and heterogeneous patient group . we believe this is important because there are a few prospective and longitudinal studies that evaluated cardiac parameters on this topic . therefore , we consider the results of the present study to be valuable for the management of patients with fd . another limitation of the present study was that two study patients did not get adequate therapy for the last 2 months of the study period because of a worldwide shortage of recombinant -galactosidase . there is a possibility that the insufficient therapy might have affected these patients ' conditions and clinical data . clearly , if we were to evaluate a larger number of appropriately treated patients , the efficacy of ert and the usefulness of serum adma measurement would be more evident . our data suggest that ert prevents progression of cardiac abnormalities , possibly by improving coronary microvascular dysfunction . in addition , we believe that adma may be a useful surrogate marker for cardiac lesions in fd . further study is required to elucidate the precise mechanisms underlying ert and the change in adma among patients with fd and to establish a useful strategy for the prevention of organ damage . this study was presented in part at the annual meeting of the american society of nephrology , 2011 .

backgroundfabry disease ( fd ) is a rare disorder and one of the causes of progressive renal and cardiac dysfunction . fd results from an x - linked recessive lysosomal storage disorder caused by a defect in the gene encoding lysosomal -galactosidase a. although accumulation of globotriaosylceramide leads to renal and cardiac manifestations , the precise mechanisms remain unclear . coronary microvascular dysfunction may be one of the causes of cardiac complications in fd . we aimed to assess coronary flow reserve ( cfr ) and the effect of enzyme replacement therapy ( ert ) on coronary microvascular dysfunction.methodsfour fd patients who had never received ert were included . the serum asymmetric dimethylarginine ( adma ) level , as a marker of endothelial dysfunction , was measured . two - dimensional guided m - mode echocardiography was performed to measure left ventricular wall mass . adenosine - triphosphate stress transthoracic doppler echocardiography was used to measure cfr before starting ert and at 3 , 6 and 12 months.resultsall the patients tolerated ert without any side effects . at the baseline , two patients had impaired cfr , increased left ventricular mass index ( lvmi ) and elevated serum adma levels . twelve months after starting ert , cfr was increased in all patients , and lvmi and serum adma levels decreased in two patients . furthermore , serum adma levels significantly correlated with cfr ( r = 0.576 , p < 0.05 ) and lvmi ( r = 0.874 , p < 0.0001).conclusionsthe results suggest that ert prevented the progression of cardiac abnormalities , possibly by improving coronary microvascular dysfunction . adma may be a useful surrogate marker in fd .

Introduction Materials and methods Study population Measurement of CFR Echocardiographic study Measurement of serum ADMA levels and other laboratory determinations Statistical analysis Results Patient characteristics Change of ADMA levels, CFR and LVMI during ERT Correlation between ADMA, CFR and LVMI Discussion Conclusions Transparency declaration Conflict of interest statement

fabry disease ( fd ) is an x - linked recessive lysosomal storage disorder caused by a defect in the gene that encodes lysosomal -galactosidase a and is known to affect various tissues including the kidney and heart . patients with fd manifest a variety of clinical symptoms , such as acroparesthesias , angiokeratoma , hypohidrosis , corneal opacities , stroke , renal disorder and cardiac abnormalities . the manifestations are thought to be due to progressive accumulation of globotriaosylceramide ( gl-3 ) and other glycosphingolipids within various cells such as vascular endothelium , cardiomyocytes , glomerular cells and renal tubular cells . the precise pathophysiological mechanisms of fd remain unclear , and coronary microvascular dysfunction appears to be one of the causes of cardiac complications in fd [ 14 ] . in several studies , gl-3 accumulation within vascular endothelial cells in the kidney and heart many studies have demonstrated that nitric oxide ( no ) plays an important role in the progression of endothelial dysfunction , atherosclerosis and chronic kidney disease ( ckd ) [ 811 ] . asymmetric dimethylarginine ( adma ) is an endogenous competitive inhibitor of nos , and serum adma levels have been suggested to be a surrogate marker of endothelial dysfunction and/or angiosclerosis . recently , we revealed a significant relationship between adma , glomerular filtration rate and endothelial dysfunction of the coronary and peripheral arteries [ 12 , 13 ] . from the results of not only our studies but also various earlier studies , we thought that adma may be involved in the underlying mechanism connecting the two pathological conditions between the kidney and the heart and may be a useful biochemical marker even in fd . the aim of our study was to examine the effect of ert on cardiac abnormalities and to determine the relationship between adma , coronary microvascular function and left ventricular hypertrophy ( lvh ) in fd patients . for these patients , -galactosidase a ( agalsidase , fabrazyme ; genzyme corp . in addition , 20 healthy volunteers ( mean age : 29 4 years ) were also evaluated to determine serum adma levels . for the fd patients , we prospectively performed blood examination , echocardiography , exercise stress myocardial scintigraphy and coronary flow reserve ( cfr ) measurements before starting ert and at 3 , 6 and 12 months . transthoracic doppler echocardiography , which has been described previously , was used to measure cfr . the ultrasound beam was transmitted toward the heart to visualize coronary blood flow in the distal portion of the left anterior descending ( lad ) coronary artery by color doppler flow mapping . an ultrasound system computer was used to trace the contour of the spectral doppler signal for off - line analysis of coronary flow velocity . peak diastolic velocity ( pdv ) was measured at the baseline and peak hyperemic conditions . two - dimensional guided m - mode echocardiography was performed to measure left ventricular wall mass . left ventricular diastolic diameter ( lvdd ) , left ventricular systolic diameter , diastolic thickness of the left ventricular posterior wall ( lvpwt ) and interventricular septum ( ivst ) were assessed in m - mode images in the parasternal longitudinal axis view . the m - mode analysis was performed according to the guidelines of the american society of echocardiography . the following formula was used to calculate the left ventricular mass index ( lvmi ) ( g / m ) : on the same day as the cfr measurement , venous blood was collected from the patients after a 20-min period of supine rest in the morning following overnight fasting . in two dialysis patients , according to japanese society of dialysis therapy ( jsdt ) guidelines , blood samples were collected before each dialysis session at 2-day intervals . the ckd epidemiology collaboration ( ckd - epi ) equation was used to estimate the glomerular filtration rate for evaluation of renal function . pearson 's correlation coefficient was used to analyze relationships between variables . in addition , 20 healthy volunteers ( mean age : 29 4 years ) were also evaluated to determine serum adma levels . for the fd patients , we prospectively performed blood examination , echocardiography , exercise stress myocardial scintigraphy and coronary flow reserve ( cfr ) measurements before starting ert and at 3 , 6 and 12 months . transthoracic doppler echocardiography , which has been described previously , was used to measure cfr . first , the left ventricle was imaged in cross - section along the longitudinal axis , and then , the ultrasound beam was inclined laterally . all of the patients underwent continuous heart rate and blood pressure monitoring throughout the study period . an ultrasound system computer was used to trace the contour of the spectral doppler signal for off - line analysis of coronary flow velocity . peak diastolic velocity ( pdv ) was measured at the baseline and peak hyperemic conditions . we adopted a cfr of < 2.0 as the cut - off value for the presence of significant coronary microvascular disease , as used previously . two - dimensional guided m - mode echocardiography was performed to measure left ventricular wall mass . left ventricular diastolic diameter ( lvdd ) , left ventricular systolic diameter , diastolic thickness of the left ventricular posterior wall ( lvpwt ) and interventricular septum ( ivst ) were assessed in m - mode images in the parasternal longitudinal axis view . the m - mode analysis was performed according to the guidelines of the american society of echocardiography . the following formula was used to calculate the left ventricular mass index ( lvmi ) ( g / m ) : on the same day as the cfr measurement , venous blood was collected from the patients after a 20-min period of supine rest in the morning following overnight fasting . in two dialysis patients , according to japanese society of dialysis therapy ( jsdt ) guidelines , blood samples were collected before each dialysis session at 2-day intervals . the ckd epidemiology collaboration ( ckd - epi ) equation was used to estimate the glomerular filtration rate for evaluation of renal function . whitney u - test was used to analyze the significance of differences between two groups . among the study patients , three had a history of stroke ( cases 1 , 3 and 4 ) and two had received hemodialysis therapy ( cases 1 and 4 ) . at the baseline , -galactosidase activity was low in all patients , as determined by the measurement of the dried blood spots in a fluorescence assay using 4-methylumbelliferyl . serum adma levels were significantly higher in the fd group than in the hypertensive control and healthy control groups ( figure 1a ) . as shown in table 2 , the lvmi increased in three patients ( cases 1 , 3 and 4 ) , and systolic function decreased in two ( cases 1 and 4 ) . although findings of ischemia were not observed using exercise stress myocardial scintigraphy in all the study patients , cfr decreased in three patients ( cases 1 , 3 and 4 ) and two had angina - like chest pain ( cases 1 and 3 ) . needless to say , cfr was significantly lower in the fd group than in the hypertensive control group ( figure 1b ) . table 2.echocardiographic parameters at baselinecase 1case 2case 3case 4lad ( mm)41394144lvdd ( mm)49454847lvds ( mm)37313333ivst ( mm)16101212pwt ( mm)13101112fs ( % ) 24303129ef ( % ) 58606657lvmi ( g / m)203.293.8158.2151.9e / a ratio0.772.260.620.65dct ( ms)346146200212ivcd ( mm)168.95.713cfr1.773.412.311.45lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . ( a ) serum adma levels in the healthy subjects , hypertensive patients and fd patients . ( b ) cfr in the hypertensive patients and fd patients . * p < 0.05 patient characteristics hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . echocardiographic parameters at baseline lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . serum adma levels and cfr in the control patients and fd patients . ( a ) serum adma levels in the healthy subjects , hypertensive patients and fd patients . p < 0.05 we evaluated the changes in serum adma levels , cfr and lvmi after starting ert , and these parameters mostly improved 12 months after starting ert compared with the values at baseline ( figure 2a c ) . change of cfr , lvmi and adma during ert . ( a ) serum adma levels in individual patients . we examined the correlation of serum adma levels with cfr and lvmi and found that serum adma levels were significantly correlated with both cfr ( r = 0.576 , p < 0.05 ) and lvmi ( r = 0.874 , p < 0.0001 ) ( figure 3a and b ) . there was also a significant relationship between cfr and lvmi ( r = 0.545 , p < 0.05 ) ( figure 3c ) . in addition , serum adma levels were significantly correlated with plasma brain natriuretic peptide ( bnp ) levels ( r = 0.770 , p = 0.0005 ) . among the study patients , three had a history of stroke ( cases 1 , 3 and 4 ) and two had received hemodialysis therapy ( cases 1 and 4 ) . at the baseline , -galactosidase activity was low in all patients , as determined by the measurement of the dried blood spots in a fluorescence assay using 4-methylumbelliferyl . serum adma levels were significantly higher in the fd group than in the hypertensive control and healthy control groups ( figure 1a ) . as shown in table 2 , the lvmi increased in three patients ( cases 1 , 3 and 4 ) , and systolic function decreased in two ( cases 1 and 4 ) . although findings of ischemia were not observed using exercise stress myocardial scintigraphy in all the study patients , cfr decreased in three patients ( cases 1 , 3 and 4 ) and two had angina - like chest pain ( cases 1 and 3 ) . needless to say , cfr was significantly lower in the fd group than in the hypertensive control group ( figure 1b ) . table 2.echocardiographic parameters at baselinecase 1case 2case 3case 4lad ( mm)41394144lvdd ( mm)49454847lvds ( mm)37313333ivst ( mm)16101212pwt ( mm)13101112fs ( % ) 24303129ef ( % ) 58606657lvmi ( g / m)203.293.8158.2151.9e / a ratio0.772.260.620.65dct ( ms)346146200212ivcd ( mm)168.95.713cfr1.773.412.311.45lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . ( a ) serum adma levels in the healthy subjects , hypertensive patients and fd patients . ( b ) cfr in the hypertensive patients and fd patients . * p < 0.05 patient characteristics hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . echocardiographic parameters at baseline lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . serum adma levels and cfr in the control patients and fd patients . ( a ) serum adma levels in the healthy subjects , hypertensive patients and fd patients . we evaluated the changes in serum adma levels , cfr and lvmi after starting ert , and these parameters mostly improved 12 months after starting ert compared with the values at baseline ( figure 2a c ) . change of cfr , lvmi and adma during ert . we examined the correlation of serum adma levels with cfr and lvmi and found that serum adma levels were significantly correlated with both cfr ( r = 0.576 , p < 0.05 ) and lvmi ( r = 0.874 , p < 0.0001 ) ( figure 3a and b ) . there was also a significant relationship between cfr and lvmi ( r = 0.545 , p < 0.05 ) ( figure 3c ) . in addition , serum adma levels were significantly correlated with plasma brain natriuretic peptide ( bnp ) levels ( r = 0.770 , p = 0.0005 ) . our study demonstrated that ( i ) cfr was significantly lower and serum adma levels and lvmi were significantly higher in the fd group than in the control group ; ( ii ) ert increased the cfr , decreased the elevated serum adma levels and increased lvmi ; and ( iii ) serum adma levels showed a significant relationship with cfr and lvmi in the fd patients . it has been thought that fd patients show changes not only in the myocardium but also in coronary vascular function . in our study , two patients had angina - like chest pain and three had markedly impaired cfr , despite having no manifest coronary artery disease . thus , coronary microvascular dysfunction may play a key role in the development of these symptoms . it has been shown that ert reverses the accumulation of microvascular endothelial deposits of gl-3 in several organs , including the heart [ 13 , 7 ] . furthermore , it is thought that the increased oxygen demand of the hypertrophied myocardium is related to myocardial ischemia in fd . therefore , we speculated that ert could ameliorate coronary microvascular dysfunction in fd through these two mechanisms . on the other hand , two previous studies reported that no improvements in hyperemic myocardial blood flow , cfr and lvmi were observed by ert [ 23 , 24 ] . however , the result of one study showed that the study patients felt subjectively better and suffered less pain after starting ert . from the results of these studies , ert could prevent the progression of impaired cfr and lvh but could not improve cfr and lvh in their study patients . several studies have demonstrated that serum adma levels increased in ckd patients and correlated with the severity of atherosclerotic lesions [ 911 ] . thus , adma is thought to be an available biochemical marker of endothelial dysfunction and/or vascular lesions in patients with ckd . our previous studies demonstrated that adma was significantly associated with cfr , endothelium - mediated vasodilation of the brachial artery and renal function [ 12 , 13 ] . in the present study , to ascertain a possible role of adma in fd and determine whether adma is a useful marker of cardiac manifestation in fd , we assessed the relationship between serum adma levels , cfr and lvmi . we found that serum adma levels were significantly correlated with cfr and lvmi in patients with fd as well as in non - fd patients . furthermore , the levels tended to decrease with improving cfr and lvmi after starting ert . in the present study , serum bnp levels were also significantly associated with cfr and lvmi . considering these findings , there is a possibility that serum adma levels may reflect the degree of cardiac damage even in fd . acroparesthesia is one of the major symptoms in fd and it is known that ert could reduce this symptom . as mentioned above , our results show that serum adma levels increased in fd . clearly , if we were to evaluate a larger number of appropriately treated patients , the efficacy of ert and the usefulness of serum adma measurement would be more evident . our data suggest that ert prevents progression of cardiac abnormalities , possibly by improving coronary microvascular dysfunction . in addition , we believe that adma may be a useful surrogate marker for cardiac lesions in fd . further study is required to elucidate the precise mechanisms underlying ert and the change in adma among patients with fd and to establish a useful strategy for the prevention of organ damage . this study was presented in part at the annual meeting of the american society of nephrology , 2011 .

fabry disease ( fd ) is an x - linked recessive lysosomal storage disorder caused by a defect in the gene that encodes lysosomal -galactosidase a and is known to affect various tissues including the kidney and heart . patients with fd manifest a variety of clinical symptoms , such as acroparesthesias , angiokeratoma , hypohidrosis , corneal opacities , stroke , renal disorder and cardiac abnormalities . the manifestations are thought to be due to progressive accumulation of globotriaosylceramide ( gl-3 ) and other glycosphingolipids within various cells such as vascular endothelium , cardiomyocytes , glomerular cells and renal tubular cells . the precise pathophysiological mechanisms of fd remain unclear , and coronary microvascular dysfunction appears to be one of the causes of cardiac complications in fd [ 14 ] . in several studies , gl-3 accumulation within vascular endothelial cells in the kidney and heart many studies have demonstrated that nitric oxide ( no ) plays an important role in the progression of endothelial dysfunction , atherosclerosis and chronic kidney disease ( ckd ) [ 811 ] . asymmetric dimethylarginine ( adma ) is an endogenous competitive inhibitor of nos , and serum adma levels have been suggested to be a surrogate marker of endothelial dysfunction and/or angiosclerosis . recently , we revealed a significant relationship between adma , glomerular filtration rate and endothelial dysfunction of the coronary and peripheral arteries [ 12 , 13 ] . from the results of not only our studies but also various earlier studies , we thought that adma may be involved in the underlying mechanism connecting the two pathological conditions between the kidney and the heart and may be a useful biochemical marker even in fd . the aim of our study was to examine the effect of ert on cardiac abnormalities and to determine the relationship between adma , coronary microvascular function and left ventricular hypertrophy ( lvh ) in fd patients . for the fd patients , we prospectively performed blood examination , echocardiography , exercise stress myocardial scintigraphy and coronary flow reserve ( cfr ) measurements before starting ert and at 3 , 6 and 12 months . the ultrasound beam was transmitted toward the heart to visualize coronary blood flow in the distal portion of the left anterior descending ( lad ) coronary artery by color doppler flow mapping . first , the left ventricle was imaged in cross - section along the longitudinal axis , and then , the ultrasound beam was inclined laterally . next , coronary blood flow in the distal lad was examined under the guidance of color doppler flow mapping . an ultrasound system computer was used to trace the contour of the spectral doppler signal for off - line analysis of coronary flow velocity . we adopted a cfr of < 2.0 as the cut - off value for the presence of significant coronary microvascular disease , as used previously . left ventricular diastolic diameter ( lvdd ) , left ventricular systolic diameter , diastolic thickness of the left ventricular posterior wall ( lvpwt ) and interventricular septum ( ivst ) were assessed in m - mode images in the parasternal longitudinal axis view . the following formula was used to calculate the left ventricular mass index ( lvmi ) ( g / m ) : on the same day as the cfr measurement , venous blood was collected from the patients after a 20-min period of supine rest in the morning following overnight fasting . the ckd epidemiology collaboration ( ckd - epi ) equation was used to estimate the glomerular filtration rate for evaluation of renal function . for the fd patients , we prospectively performed blood examination , echocardiography , exercise stress myocardial scintigraphy and coronary flow reserve ( cfr ) measurements before starting ert and at 3 , 6 and 12 months . the ultrasound beam was transmitted toward the heart to visualize coronary blood flow in the distal portion of the left anterior descending ( lad ) coronary artery by color doppler flow mapping . first , the left ventricle was imaged in cross - section along the longitudinal axis , and then , the ultrasound beam was inclined laterally . next , coronary blood flow in the distal lad was examined under the guidance of color doppler flow mapping . an ultrasound system computer was used to trace the contour of the spectral doppler signal for off - line analysis of coronary flow velocity . we adopted a cfr of < 2.0 as the cut - off value for the presence of significant coronary microvascular disease , as used previously . left ventricular diastolic diameter ( lvdd ) , left ventricular systolic diameter , diastolic thickness of the left ventricular posterior wall ( lvpwt ) and interventricular septum ( ivst ) were assessed in m - mode images in the parasternal longitudinal axis view . the following formula was used to calculate the left ventricular mass index ( lvmi ) ( g / m ) : on the same day as the cfr measurement , venous blood was collected from the patients after a 20-min period of supine rest in the morning following overnight fasting . among the study patients , three had a history of stroke ( cases 1 , 3 and 4 ) and two had received hemodialysis therapy ( cases 1 and 4 ) . at the baseline , -galactosidase activity was low in all patients , as determined by the measurement of the dried blood spots in a fluorescence assay using 4-methylumbelliferyl . serum adma levels were significantly higher in the fd group than in the hypertensive control and healthy control groups ( figure 1a ) . as shown in table 2 , the lvmi increased in three patients ( cases 1 , 3 and 4 ) , and systolic function decreased in two ( cases 1 and 4 ) . although findings of ischemia were not observed using exercise stress myocardial scintigraphy in all the study patients , cfr decreased in three patients ( cases 1 , 3 and 4 ) and two had angina - like chest pain ( cases 1 and 3 ) . needless to say , cfr was significantly lower in the fd group than in the hypertensive control group ( figure 1b ) . table 1.patient characteristicscase 1case 2case 3case 4normal valuesage ( years old)45197151sexfmfmhd duration ( years)1223hypertension(+)()(+)()diabetes mellitus(+)(+)()()hyperlipidemia()()()()stroke(+)()(+)(+)acroparesthesias()()(+)()hypohidrosis()()()()angiokeratoma()()()()corneal opacities(+)()()()chest pain(+)()(+)()cr ( mol / l)872.451.165.6976.1egfr ( ml / min/1.73 m)13968rbc ( 10/l)4.105.533.054.20hemoglobin ( g / l)114161103121tp ( g / l)686968796482alb ( g / l)394539363552t - chol ( mmol / l)3.236.214.864.093.366.21tg ( mmol / l)2.019.043.921.260.342.03hdl ( mmol / l)0.980.961.140.961.03hba1c ( % ) 6.112.65.04.64.35.8ca ( mmol / l)2.222.422.222.592.122.55p ( mmol / l)1.681.190.872.160.771.39bnp ( ng / l)321.52.459.6272.218.4hscrp ( nmol / l)28.645.712.421.9<28.6adma ( ng / l)0.850.480.550.70-galactosidase activitydried blood spot ( agal u)16.911.215.115.220leukocyte ( nmol / mg protein / h)61.252.375.532.149.8116.4hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . table 2.echocardiographic parameters at baselinecase 1case 2case 3case 4lad ( mm)41394144lvdd ( mm)49454847lvds ( mm)37313333ivst ( mm)16101212pwt ( mm)13101112fs ( % ) 24303129ef ( % ) 58606657lvmi ( g / m)203.293.8158.2151.9e / a ratio0.772.260.620.65dct ( ms)346146200212ivcd ( mm)168.95.713cfr1.773.412.311.45lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . * p < 0.05 patient characteristics hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . echocardiographic parameters at baseline lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . p < 0.05 we evaluated the changes in serum adma levels , cfr and lvmi after starting ert , and these parameters mostly improved 12 months after starting ert compared with the values at baseline ( figure 2a c ) . laboratory data during ert cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide . we examined the correlation of serum adma levels with cfr and lvmi and found that serum adma levels were significantly correlated with both cfr ( r = 0.576 , p < 0.05 ) and lvmi ( r = 0.874 , p < 0.0001 ) ( figure 3a and b ) . there was also a significant relationship between cfr and lvmi ( r = 0.545 , p < 0.05 ) ( figure 3c ) . in addition , serum adma levels were significantly correlated with plasma brain natriuretic peptide ( bnp ) levels ( r = 0.770 , p = 0.0005 ) . among the study patients , three had a history of stroke ( cases 1 , 3 and 4 ) and two had received hemodialysis therapy ( cases 1 and 4 ) . at the baseline , -galactosidase activity was low in all patients , as determined by the measurement of the dried blood spots in a fluorescence assay using 4-methylumbelliferyl . serum adma levels were significantly higher in the fd group than in the hypertensive control and healthy control groups ( figure 1a ) . as shown in table 2 , the lvmi increased in three patients ( cases 1 , 3 and 4 ) , and systolic function decreased in two ( cases 1 and 4 ) . although findings of ischemia were not observed using exercise stress myocardial scintigraphy in all the study patients , cfr decreased in three patients ( cases 1 , 3 and 4 ) and two had angina - like chest pain ( cases 1 and 3 ) . table 1.patient characteristicscase 1case 2case 3case 4normal valuesage ( years old)45197151sexfmfmhd duration ( years)1223hypertension(+)()(+)()diabetes mellitus(+)(+)()()hyperlipidemia()()()()stroke(+)()(+)(+)acroparesthesias()()(+)()hypohidrosis()()()()angiokeratoma()()()()corneal opacities(+)()()()chest pain(+)()(+)()cr ( mol / l)872.451.165.6976.1egfr ( ml / min/1.73 m)13968rbc ( 10/l)4.105.533.054.20hemoglobin ( g / l)114161103121tp ( g / l)686968796482alb ( g / l)394539363552t - chol ( mmol / l)3.236.214.864.093.366.21tg ( mmol / l)2.019.043.921.260.342.03hdl ( mmol / l)0.980.961.140.961.03hba1c ( % ) 6.112.65.04.64.35.8ca ( mmol / l)2.222.422.222.592.122.55p ( mmol / l)1.681.190.872.160.771.39bnp ( ng / l)321.52.459.6272.218.4hscrp ( nmol / l)28.645.712.421.9<28.6adma ( ng / l)0.850.480.550.70-galactosidase activitydried blood spot ( agal u)16.911.215.115.220leukocyte ( nmol / mg protein / h)61.252.375.532.149.8116.4hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . table 2.echocardiographic parameters at baselinecase 1case 2case 3case 4lad ( mm)41394144lvdd ( mm)49454847lvds ( mm)37313333ivst ( mm)16101212pwt ( mm)13101112fs ( % ) 24303129ef ( % ) 58606657lvmi ( g / m)203.293.8158.2151.9e / a ratio0.772.260.620.65dct ( ms)346146200212ivcd ( mm)168.95.713cfr1.773.412.311.45lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . * p < 0.05 patient characteristics hd , hemodialysis ; cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide ; hscrp , high - sensitivity c - reactive protein ; adma , asymmetric dimethylarginine . echocardiographic parameters at baseline lad , left atrial dimension ; lvdd , left ventricular end - diastolic dimension ; lvds , left ventricular end - systolic dimension ; ivst , interventricular septum thickness ; pwt , posterior wall thickness ; fs , fractional shortening ; ef , ejection fraction ; lvmi , left ventricular mass index ; dct , deceleration time ; ivcd , inferior vena cava diameter ; cfr , coronary flow reserve . we evaluated the changes in serum adma levels , cfr and lvmi after starting ert , and these parameters mostly improved 12 months after starting ert compared with the values at baseline ( figure 2a c ) . laboratory data during ert cr , creatinine ; egfr , estimated glomerular filtration rate ; rbc , red blood cell count ; tp , total protein ; alb , albumin ; t - chol , total cholesterol ; tg , triglyceride ; hdl , high - density lipoprotein cholesterol ; hba1c , hemoglobin a1c ; bnp , brain natriuretic peptide . we examined the correlation of serum adma levels with cfr and lvmi and found that serum adma levels were significantly correlated with both cfr ( r = 0.576 , p < 0.05 ) and lvmi ( r = 0.874 , p < 0.0001 ) ( figure 3a and b ) . in addition , serum adma levels were significantly correlated with plasma brain natriuretic peptide ( bnp ) levels ( r = 0.770 , p = 0.0005 ) . our study demonstrated that ( i ) cfr was significantly lower and serum adma levels and lvmi were significantly higher in the fd group than in the control group ; ( ii ) ert increased the cfr , decreased the elevated serum adma levels and increased lvmi ; and ( iii ) serum adma levels showed a significant relationship with cfr and lvmi in the fd patients . several studies have demonstrated that serum adma levels increased in ckd patients and correlated with the severity of atherosclerotic lesions [ 911 ] . our previous studies demonstrated that adma was significantly associated with cfr , endothelium - mediated vasodilation of the brachial artery and renal function [ 12 , 13 ] . in the present study , to ascertain a possible role of adma in fd and determine whether adma is a useful marker of cardiac manifestation in fd , we assessed the relationship between serum adma levels , cfr and lvmi . we found that serum adma levels were significantly correlated with cfr and lvmi in patients with fd as well as in non - fd patients . taken together , we speculated that adma might be involved in the acroparesthesia of fd though we do not have objective data on polyneuropathy . therefore , we consider the results of the present study to be valuable for the management of patients with fd . another limitation of the present study was that two study patients did not get adequate therapy for the last 2 months of the study period because of a worldwide shortage of recombinant -galactosidase . clearly , if we were to evaluate a larger number of appropriately treated patients , the efficacy of ert and the usefulness of serum adma measurement would be more evident . further study is required to elucidate the precise mechanisms underlying ert and the change in adma among patients with fd and to establish a useful strategy for the prevention of organ damage .

skeletal muscle exhibits a highly adaptable protein organization whose properties can be easily changed by aging , temperature , exercise , nutrition , and incretion , to name a few . skeletal muscle adaptability results in a hypertrophied or atrophied state ; these states are primarily associated with slow or fast fibers . slow or fast fiber characteristics are attributed to changes in the population of intracellular proteins of the muscle cells . one study concerning the content of b - crystallin and tubulin in 10 days of atrophied soleus muscle was previously reported . b - crystallin and tubulin expression is dependent on muscle fiber type ; however , no study has investigated the response of b - crystallin and -tubulin with transition of myosin heavy chain ( mhc ) isoforms in plantar flexor muscles followed by hindlimb unloading . in this study , we focused on b - crystallin and tubulin because these proteins exhibit functional properties associated with the adaptability of skeletal muscles . b - crystallin is localized in the z - line of the sarcomere in cardiac and slow - twitch muscle . it acts as a molecular chaperone during the folding process of other specific proteins to prevent protein degradation . b - crystallin is activated in a phosphorylation - dependent manner and also acts to stabilize intermediate filament and cytoskeletal proteins in vitro . another study showed that disorganized cytoskeletal components trigger the phosphorylation of b - crystallin through activating the p38 mitogen - activated protein kinase ( mapk ) pathway . b - crystallin is also known to be upregulated in slow twitch muscles , which have high oxidative capability and higher protein turnover rates compared to fast twitch muscles , which are characterized by their high glycolytic capability . microtubules function as an intracellular frame to shape the cell , maintain cellular morphology , and transport proteins throughout the cell [ 11 - 13 ] . microtubules exhibit a morphological , structural , and dynamic property called dynamic instability , which is regulated by the concentration of free- tubulin dimers . microtubules are directly associated with myosin , and the localization of myosin filaments during sarcomere formation and myogenic cell morphology is partly dependent on the microtubule networking organization . it can be inferred that different muscle types are due to the different myogenic cell morphologies and their localization is related to differences in the formation and content of microtubules . the mhc is a major component of muscle and can be transformed into various isoforms in muscle cells in response to various stimuli . the increase in directional shifts from the mhc i to the mhc iia , mhc iid / x , and mhc iib isoforms , augments the velocity of the shortening and power generation capacity of the mhc , but decreases its atpase activity . reported that an atrophy response due to unloading induces the shift of mhc isoforms from slow - twitch to fast - twitch muscle types . it is thought that b - crystallin , tubulin , and myosin work closely with one another during the processes of myocyte formation . b - crystallin plays a critical role in chaeperoning for tubulin , which functions to maintain muscle cell morphology and transport organelles in the cell . b - crystallin also indirectly supports power transmission since it acts as a chaperone protein for myosin , one of the main components of muscle . microtubules , which are composed of tubulin , also directly associate with myosin to ensure its proper localization during sarcomere formation . these data suggest that the functional relationship between b - crystallin , tubulin , and myosin are important for muscular function and adaptability . we hypothesized that hindlimb unloading induces muscular atrophy with mhc isoform transformation toward the mhc imhc furthermore , we evaluated whether b - crystallin and -tubulin would show different expression patterns in different types of atrophied muscles as atrophied slow muscle shows a more sensitive response to both proteins compared to atrophied fast muscle . we examined the triceps surae in the hindlimbs ( soleus , plantaris , and gastrocnemius muscles ) because these muscles might exhibit similar functions and endure similar mechanical loads compared to other muscles in the hindlimbs , such as the tibialis anterior . we employed the unloading model to induce muscle atrophy and to quantitatively determine the relationship between the proteins ( b - crystallin and tubulin ) with the transition of mhc isoforms , and its significance to the adaptability of skeletal muscles . in this study , we utilized 12 seven - week - old adult male wistar rats that weighed 238.2 9.1 g. after 2 - 3 days of adaptation , the rats were randomly divided , with half placed in a control group and the half placed in an unloading group . the unloading group was tested using the hindlimb unloading model based on modified methods described by morey and atomi . all animals were provided standard rat chow and water ad libitum , and were housed at 22 - 24 c with a 12:12 h light - dark cycle . after 15 days , all rats were euthanized by cervical dislocation under anesthesia induced by diethyl ether for relieving pain ( 50 ml / kg body mass ) . the right sides of the whole soleus muscle , plantaris muscle , and gastrocnemius muscle were isolated , and the muscles were immediately frozen in liquid nitrogen and stored at -80 c until use . animal use and maintenance was approved by the university of tokyo animal care and use committee . the three isolated whole muscles used in this study were crushed in liquid nitrogen and solubilized in a low - salt buffer containing 20 mm potassium chloride ( kcl ) , 2 mm sodium phosphate ( ph 6.8 ) , 2 mm ethylene glycol tetraacetic acid ( egta ) , 5 mm ethylene diamine tetraacetic acid ( edta ) , 20 mm sodium fluoride , 1 mm sodium orthovanadate , a protease inhibitor containing 1 mm phenylmethylsulfonyl fluoride , 2 g / ml leupeptin , 2 g / ml aprotinin , 10 g / ml soybean trypsin inhibitor , and a phosphatase inhibitor containing 100 nm okadaic acid and 10 mm sodium -glycerophosphate . the homogenate was solubilized with an equal volume of 2 sodium dodecyl sulfate ( sds ) sample buffer [ 2 mm sodium phosphate ( ph 6.8 ) , 2% sds , and 16% 2-mercaptoethanol ] and boiled for 2 min . the supernatant was collected and the protein concentration was determined using a protein determination kit ( bio - rad ; richmond , va , usa ) . polyacrylamide gels ( 8.5% to 12% ) were used as running gels to detect b - crystallin , tubulin , and myosin proteins . as stacking gels 1.6 g of sample was electrophoresed over a 24-h period at a constant voltage of 150 v and 4 c . gels were stained with coomasie brilliant blue r-250 dye ( sigma - aldrich co. ; st . the relative percentage of mhc isoform content was calculated using imagej ( national institutes of health , rockville pike bethesda , md , usa ) . the extracted plantaris muscle was used as a reference marker for analyzing the extracted soleus muscle because not all mhc isoforms in the plantaris muscle overlap with the mhc isoforms in the soleus muscle , and vice versa . the markers used for the gastrocnemius muscle were a mixture of extracted soleus and plantaris muscles . the following primary antibodies were used for immunoblotting : anti--tubulin ( 1:1000 ; sigma - aldrich co. ; st . louis , mo , usa ) and anti-b - crystallin ( 1:5000 , raised in rabbit against the c - terminal 10 peptides [ ( sh ) kpavtaapkk ] of human b - crystallin ) . after incubation with secondary antibodies , the membrane was incubated with an enhanced chemiluminescence kit ( amersham biosciences corp . ; buckinghamshire , uk ) . b - crystallin was expressed in escherichia coli and tubulin ( phosphocellulose column - purified tubulin , pct ) was purified from the porcine brain . to estimate the exact quantity of each protein in the extracted samples , we used the purified proteins ( b - crystallin , 5 , 10 , 20 , 30 , and 40 ng ; -tubulin , 1 , 3 , 5 , 7 , 9 , and 11 ng ) that were used as standards for each protein because there was a linear relationship between the gradual contents of each protein and the band intensity ( fig . we subsequently loaded different quantities of proteins extracted from the soleus ( 0.59 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles and their corresponding protein standards onto the same gel . the contents of b - crystallin and -tubulin estimated from the extracted soleus ( 0.5 - 9 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles are presented as ng/g . all data are presented as the mean standard deviation ( sd ) and the differences between control and experiment groups were analyzed using independent t - tests ( spss ver . 1 ) whole values were considered statistically significant when p < 0.05 or p < 0.01 . in this study , we utilized 12 seven - week - old adult male wistar rats that weighed 238.2 9.1 g. after 2 - 3 days of adaptation , the rats were randomly divided , with half placed in a control group and the half placed in an unloading group . the unloading group was tested using the hindlimb unloading model based on modified methods described by morey and atomi . all animals were provided standard rat chow and water ad libitum , and were housed at 22 - 24 c with a 12:12 h light - dark cycle . after 15 days , all rats were euthanized by cervical dislocation under anesthesia induced by diethyl ether for relieving pain ( 50 ml / kg body mass ) . the right sides of the whole soleus muscle , plantaris muscle , and gastrocnemius muscle were isolated , and the muscles were immediately frozen in liquid nitrogen and stored at -80 c until use . animal use and maintenance was approved by the university of tokyo animal care and use committee . the three isolated whole muscles used in this study were crushed in liquid nitrogen and solubilized in a low - salt buffer containing 20 mm potassium chloride ( kcl ) , 2 mm sodium phosphate ( ph 6.8 ) , 2 mm ethylene glycol tetraacetic acid ( egta ) , 5 mm ethylene diamine tetraacetic acid ( edta ) , 20 mm sodium fluoride , 1 mm sodium orthovanadate , a protease inhibitor containing 1 mm phenylmethylsulfonyl fluoride , 2 g / ml leupeptin , 2 g / ml aprotinin , 10 g / ml soybean trypsin inhibitor , and a phosphatase inhibitor containing 100 nm okadaic acid and 10 mm sodium -glycerophosphate . the homogenate was solubilized with an equal volume of 2 sodium dodecyl sulfate ( sds ) sample buffer [ 2 mm sodium phosphate ( ph 6.8 ) , 2% sds , and 16% 2-mercaptoethanol ] and boiled for 2 min . the supernatant was collected and the protein concentration was determined using a protein determination kit ( bio - rad ; richmond , va , usa ) . polyacrylamide gels ( 8.5% to 12% ) were used as running gels to detect b - crystallin , tubulin , and myosin proteins . as stacking gels 1.6 g of sample was electrophoresed over a 24-h period at a constant voltage of 150 v and 4 c . gels were stained with coomasie brilliant blue r-250 dye ( sigma - aldrich co. ; st . the relative percentage of mhc isoform content was calculated using imagej ( national institutes of health , rockville pike bethesda , md , usa ) . the extracted plantaris muscle was used as a reference marker for analyzing the extracted soleus muscle because not all mhc isoforms in the plantaris muscle overlap with the mhc isoforms in the soleus muscle , and vice versa . the markers used for the gastrocnemius muscle were a mixture of extracted soleus and plantaris muscles . the following primary antibodies were used for immunoblotting : anti--tubulin ( 1:1000 ; sigma - aldrich co. ; st . louis , mo , usa ) and anti-b - crystallin ( 1:5000 , raised in rabbit against the c - terminal 10 peptides [ ( sh ) kpavtaapkk ] of human b - crystallin ) . after incubation with secondary antibodies , the membrane was incubated with an enhanced chemiluminescence kit ( amersham biosciences corp . ; buckinghamshire , uk ) . b - crystallin was expressed in escherichia coli and tubulin ( phosphocellulose column - purified tubulin , pct ) was purified from the porcine brain . to estimate the exact quantity of each protein in the extracted samples , we used the purified proteins ( b - crystallin , 5 , 10 , 20 , 30 , and 40 ng ; -tubulin , 1 , 3 , 5 , 7 , 9 , and 11 ng ) that were used as standards for each protein because there was a linear relationship between the gradual contents of each protein and the band intensity ( fig . we subsequently loaded different quantities of proteins extracted from the soleus ( 0.59 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles and their corresponding protein standards onto the same gel . the contents of b - crystallin and -tubulin estimated from the extracted soleus ( 0.5 - 9 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles are presented as ng/g . all data are presented as the mean standard deviation ( sd ) and the differences between control and experiment groups were analyzed using independent t - tests ( spss ver . 1 ) whole values were considered statistically significant when p < 0.05 or p < 0.01 . the body and tissue weight of the control and unloading groups were measured after 15 days of hindlimb unloading ( table 1 ) . there was a significant reduction in body weight in the unloading group compared to the control group ( p < 0.05 ) . in addition , the weights of the soleus , plantaris , and gastrocnemius muscles of the unloading groups were each significantly reduced compared to the corresponding muscle in the control group , such as 121.6 18.0 ( mg ) to 54.9 4.2 ( mg ) , 299.3 37.5 ( mg ) to 218.5 22.9 ( mg ) , and 1454.8 140.3 ( mg ) to 988.3 57.0 ( mg ) , respectively ( p < 0.01 ) . based on the whole muscle to body weight ratios , the unloading soleus ( p < 0.01 ) , plantaris ( p < 0.05 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly atrophied . b - crystallin levels in the soleus ( p < 0.01 ) , plantaris ( p < 0.01 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly changed in the unloading group compared to the corresponding muscles of the control group ( fig . b - crystallin levels in the unloading group were approximately 28% lower than those in the control group . the b - crystallin content in the unloading plantaris and gastrocnemius muscles was reduced by more than 37% and 42% compared to the control group , respectively . the soleus muscle contained approximately 7-fold as much b - crystallin as the plantaris or gastrocnemius muscles did . there was a significant decrease of -tubulin in the unloading soleus muscle compared to the control muscle ( p < 0.01 ) ( fig . 2b ) ; however , we did not observe significant differences in -tubulin levels in the plantaris and gastrocnemius muscles between the control group and unloading group . the mhc isoforms in the soleus , plantaris , and gastrocnemius muscles were quantified as comparative values ( fig . each muscle expressed a different primary mhc isoform as shown in figures 2c , 3c , and 4c . mhc i in the soleus muscle was significantly reduced in the unloading group compared to the control group ( p < 0.05 ) . however , mhc ii was significantly increased in the unloading soleus muscle compared to the control soleus muscle ( fig . high levels of mhc iid / x and iib expression were found in the plantaris muscle . mhc iid / x was significantly reduced in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iia and iib were significantly increased in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iib was predominantly expressed in the gastrocnemius muscle and was significantly increased after hindlimb unloading , whereas mhc i significantly decreased after hindlimb unloading ( fig . the body and tissue weight of the control and unloading groups were measured after 15 days of hindlimb unloading ( table 1 ) . there was a significant reduction in body weight in the unloading group compared to the control group ( p < 0.05 ) . in addition , the weights of the soleus , plantaris , and gastrocnemius muscles of the unloading groups were each significantly reduced compared to the corresponding muscle in the control group , such as 121.6 18.0 ( mg ) to 54.9 4.2 ( mg ) , 299.3 37.5 ( mg ) to 218.5 22.9 ( mg ) , and 1454.8 140.3 ( mg ) to 988.3 57.0 ( mg ) , respectively ( p < 0.01 ) . based on the whole muscle to body weight ratios , the unloading soleus ( p < 0.01 ) , plantaris ( p < 0.05 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly atrophied . b - crystallin levels in the soleus ( p < 0.01 ) , plantaris ( p < 0.01 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly changed in the unloading group compared to the corresponding muscles of the control group ( fig . b - crystallin levels in the unloading group were approximately 28% lower than those in the control group . the b - crystallin content in the unloading plantaris and gastrocnemius muscles was reduced by more than 37% and 42% compared to the control group , respectively . the soleus muscle contained approximately 7-fold as much b - crystallin as the plantaris or gastrocnemius muscles did . there was a significant decrease of -tubulin in the unloading soleus muscle compared to the control muscle ( p < 0.01 ) ( fig . 2b ) ; however , we did not observe significant differences in -tubulin levels in the plantaris and gastrocnemius muscles between the control group and unloading group . the mhc isoforms in the soleus , plantaris , and gastrocnemius muscles were quantified as comparative values ( fig . 2c , 3c , and 4c ) . each muscle expressed a different primary mhc isoform as shown in figures 2c , 3c , and 4c . mhc i in the soleus muscle was significantly reduced in the unloading group compared to the control group ( p < 0.05 ) . however , mhc ii was significantly increased in the unloading soleus muscle compared to the control soleus muscle ( fig . high levels of mhc iid / x and iib expression were found in the plantaris muscle . mhc iid / x was significantly reduced in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iia and iib were significantly increased in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iib was predominantly expressed in the gastrocnemius muscle and was significantly increased after hindlimb unloading , whereas mhc i significantly decreased after hindlimb unloading ( fig . there were obvious correlations between the changes observed in b - crystallin , -tubulin , and dominant mhc isoforms in the muscles . muscle atrophy induced a reduced expression pattern of b - crystallin and -tubulin in plantar flexor muscles when the muscles shifted to the fast muscle fiber in the unloading group . both proteins , b - crystallin and -tubulin , were downregulated in slow muscle ( p < 0.01 ) . b - crystallin was significantly decreased in the fast muscles of the unloading group compared to the control group ( p < 0.01 ) ; however , -tubulin was not reduced . hindlimb unloading leads to proteolytic processes related muscle atrophy . the proteolytic process induced by hindlimb unloading for example , chaperone - mediated autophagy ( cma ) is believed to be related to muscle atrophy . hindlimb unloading leads to the loss of muscle activity in the hindlimbs against the gravitational force and induces muscular atrophy . a reduced gravitational stimulus due to suspending hindlimbs was designed to investigate the transition and degradation of proteins that contribute to hindlimb muscle atrophy . we show that hindlimb unloading results in reduced -tubulin expression , followed by reduced ab - crystallin expression in atrophied triceps surae muscles . furthermore , our results show the expression pattern of these proteins in fast triceps surae muscles , plantaris , and gastrocnemius muscles , in conjunction with alterations of mhc isoforms by hindlimb unloading . skeletal muscles are categorized as slow - twitch or fast - twitch muscles based on properties related to adaptable characteristics such as , tension , contraction velocity , force , and glycolytic and oxidative metabolism . among the triceps surae muscles , we selected the soleus muscle as representative of slow - twitch muscle and the plantaris and gastrocnemius muscles as representatives of fast - twitch muscles . we investigated the responses of b - crystallin and -tubulin in these muscles after hindlimb unloading . it is thought that significant changes in the protein population contribute to muscular function , generation of force , or sensitivity to gravity stimuli . the different expression levels of proteins in these muscles are caused by different recruitment and loading patterns in response to mechanical unloading . this suggests that the different tensile contractions of a muscle that causes different loading force also affects the quantity of tubulin . chaperone proteins are also involved in the passive and active tensile stress to protect its substrates . in this study , we showed that b - crystallin and -tubulin respond to unloading in mhc i - dominant slow muscle but not in mhc ii - dominant muscles . in our study , the -tubulin and b - crystallin levels exhibited similar changes between control and unloading soleus muscle ( fig . however , we did not observe significant changes in -tubulin levels between the control and unloading group in the plantaris and gastrocnemius muscles ( fig . sakurai et al . reported that the -tubulin transcript level did not change after 10 d of tail suspension even though the b - crystallin transcript level was attenuated when compared to controls . based on a previous report and our results , tubulin appears to have a less sensitive response than does b - crystallin . in atrophied muscles tubulin is a scaffold protein , and is a net component of the cytoskeleton that maintains the cellular structure . the hypothesis that the degradation of tubulin accelerates muscular atrophy is supported by the fact that the most significant decrease in tubulin levels ( p < 0.01 ) was observed in the atrophied soleus muscle , which also showed the greatest extent of atrophy among the three skeletal muscles ( 47% reduction ) . by contrast , relatively less prominent atrophy was observed in the gastrocnemius ( 21% atrophied ) and plantaris ( 16% atrophied ) muscles , in which tubulin was not significantly altered when compared to the controls . mhc is also associated with b - crystallin . b - crystallin assists in preventing the aggregation of myosin and maintains atpase activation . it is thought that hindlimb unloading directly or indirectly affects the activation of quiescent satellite cells in the disruption of sarcolemma . these stimulated satellite cells activate hepatocyte growth factor ( hgf ) and the activated hgf initiates a signaling cascade , such as the 38p mapk pathway . this signaling pathway is likely related to the expression of ab - crystallin and the mhc isoforms in this study . we used 4 mhc isoforms to reliably identify the dominant mhc isoform in slow - twitch and fast - twitch muscles . we utilized these isoforms because the dominant mhc isoform is partly related to the morphological , contractile , metabolic , and fatigue - resistant properties of muscles [ 34 - 36 ] . we analyzed whole plantar flexor muscles because a whole muscle unit , such as the soleus , plantaris , and gastrocnemius muscles , can be linked to its explicit function and adaptability , which were the factors of interest for this study , and because at the molecular level , the border of the medial or lateral portion of the muscle is quite ambiguous , which could lead to inconclusive or erroneous results . our results indicate that the atrophied plantar flexor muscles began to shift and resemble a fast twitch muscle . this is consistent with other studies that demonstrated a fiber type transition caused by atrophy mainly due to a reduction in the fiber size of the muscle . these results may be related to b - crystallin and tubulin expressions since the expressions of these proteins are dependent on fiber type . we could not clearly detect changes of tubulin because we used whole fast muscles in this study ; therefore , in future studies , we will analyze changes of tubulin in muscle fibers . in addition , future studies will also focus on identifying changes in protein levels in hypertrophied slow and fast muscles . hindlimb unloading leads to proteolytic processes related muscle atrophy . the proteolytic process induced by hindlimb unloading for example , chaperone - mediated autophagy ( cma ) is believed to be related to muscle atrophy . hindlimb unloading leads to the loss of muscle activity in the hindlimbs against the gravitational force and induces muscular atrophy . a reduced gravitational stimulus due to suspending hindlimbs was designed to investigate the transition and degradation of proteins that contribute to hindlimb muscle atrophy . we show that hindlimb unloading results in reduced -tubulin expression , followed by reduced ab - crystallin expression in atrophied triceps surae muscles . furthermore , our results show the expression pattern of these proteins in fast triceps surae muscles , plantaris , and gastrocnemius muscles , in conjunction with alterations of mhc isoforms by hindlimb unloading . skeletal muscles are categorized as slow - twitch or fast - twitch muscles based on properties related to adaptable characteristics such as , tension , contraction velocity , force , and glycolytic and oxidative metabolism . among the triceps surae muscles , we selected the soleus muscle as representative of slow - twitch muscle and the plantaris and gastrocnemius muscles as representatives of fast - twitch muscles . we investigated the responses of b - crystallin and -tubulin in these muscles after hindlimb unloading . it is thought that significant changes in the protein population contribute to muscular function , generation of force , or sensitivity to gravity stimuli . the different expression levels of proteins in these muscles are caused by different recruitment and loading patterns in response to mechanical unloading . this suggests that the different tensile contractions of a muscle that causes different loading force also affects the quantity of tubulin . chaperone proteins are also involved in the passive and active tensile stress to protect its substrates . in this study , we showed that b - crystallin and -tubulin respond to unloading in mhc i - dominant slow muscle but not in mhc ii - dominant muscles . in our study , the -tubulin and b - crystallin levels exhibited similar changes between control and unloading soleus muscle ( fig . however , we did not observe significant changes in -tubulin levels between the control and unloading group in the plantaris and gastrocnemius muscles ( fig . sakurai et al . reported that the -tubulin transcript level did not change after 10 d of tail suspension even though the b - crystallin transcript level was attenuated when compared to controls . based on a previous report and our results , tubulin appears to have a less sensitive response than does b - crystallin . in atrophied muscles , tubulin begins to degrade after b - crystallin has degraded . tubulin is a scaffold protein , and is a net component of the cytoskeleton that maintains the cellular structure . the hypothesis that the degradation of tubulin accelerates muscular atrophy is supported by the fact that the most significant decrease in tubulin levels ( p < 0.01 ) was observed in the atrophied soleus muscle , which also showed the greatest extent of atrophy among the three skeletal muscles ( 47% reduction ) . by contrast , relatively less prominent atrophy was observed in the gastrocnemius ( 21% atrophied ) and plantaris ( 16% atrophied ) muscles , in which tubulin was not significantly altered when compared to the controls . mhc is also associated with b - crystallin . b - crystallin assists in preventing the aggregation of myosin and maintains atpase activation . it is thought that hindlimb unloading directly or indirectly affects the activation of quiescent satellite cells in the disruption of sarcolemma . these stimulated satellite cells activate hepatocyte growth factor ( hgf ) and the activated hgf initiates a signaling cascade , such as the 38p mapk pathway . this signaling pathway is likely related to the expression of ab - crystallin and the mhc isoforms in this study . we used 4 mhc isoforms to reliably identify the dominant mhc isoform in slow - twitch and fast - twitch muscles . we utilized these isoforms because the dominant mhc isoform is partly related to the morphological , contractile , metabolic , and fatigue - resistant properties of muscles [ 34 - 36 ] . we analyzed whole plantar flexor muscles because a whole muscle unit , such as the soleus , plantaris , and gastrocnemius muscles , can be linked to its explicit function and adaptability , which were the factors of interest for this study , and because at the molecular level , the border of the medial or lateral portion of the muscle is quite ambiguous , which could lead to inconclusive or erroneous results . our results indicate that the atrophied plantar flexor muscles began to shift and resemble a fast twitch muscle . this is consistent with other studies that demonstrated a fiber type transition caused by atrophy mainly due to a reduction in the fiber size of the muscle . these results may be related to b - crystallin and tubulin expressions since the expressions of these proteins are dependent on fiber type . we could not clearly detect changes of tubulin because we used whole fast muscles in this study ; therefore , in future studies , we will analyze changes of tubulin in muscle fibers . in addition , future studies will also focus on identifying changes in protein levels in hypertrophied slow and fast muscles . in conclusion , we found that b - crystallin and tubulin related to the transition of mhc isoforms were downregulated more explicitly in the atrophied soleus muscle rather than in the atrophied plantaris and gastrocnemius muscles . this finding may explain the phenomenon that the target proteins in the fast muscles reacted less sensitive to microgravity than those in the slow muscle . further studies are needed to provide more clear mechanism regarding the adaptability of skeletal muscles from disuse or immobilization .

[ purpose]b - crystallin is a small heat shock protein that acts as a molecular chaperone under various stress conditions . microtubules , which consist of tubulin , are related to maintain the intracellular organelles and cellular morphology . these two proteins have been shown to be related to the properties of different types of myofibers based on their contractile properties . the response of these proteins during muscular atrophy , which induces a myofibril component change , is not clearly understood.[methods]we performed 15 days of hindlimb unloading on rats to investigate the transitions of these proteins by analyzing their absolute quantities . protein contents were analyzed in the soleus , plantaris , and gastrocnemius muscles of the unloading and control groups ( n = 6).[results]all three muscles were significantly atrophied by hindlimb unloading ( p < 0.01 ) : soleus ( 47.5% ) , plantaris ( 16.3% ) , and gastrocnemius ( 21.3% ) compared to each control group . b - crystallin was significantly reduced in all three examined unloaded hindlimb muscles compared to controls ( p < 0.01 ) during the transition of the myosin heavy chain to fast twitch muscles . -tubulin responded only in the unloaded soleus muscle . muscle atrophy induced the reduction of b - crystallin and -tubulin expressions in plantar flexor muscles with a shift to the fast muscle fiber compared to the control.[conclusion]the novel finding of this study is that both proteins , b - crystallin and -tubulin , were downregulated in slow muscles ( p < 0.01 ) ; however , -tubulin was not significantly reduced compared to the control in fast muscles ( p < 0.01 ) .

INTRODUCTION METHODS Animals and unloading procedure Preparation of muscle samples SDS polyacrylamide gel electrophoresis (PAGE) and immunoblotting Statistical analysis RESULTS Body and muscle weight B-crystallin content in the soleus, plantaris, and gastrocnemius muscles -tubulin content in the soleus, plantaris, and gastrocnemius muscles Percentages of the four MHC isoforms in the soleus, plantaris, and gastrocnemius muscles DISCUSSION Hindlimb unloading Protein transition in atrophied muscles Role of MHC in atrophied muscles CONCLUSION

one study concerning the content of b - crystallin and tubulin in 10 days of atrophied soleus muscle was previously reported . b - crystallin and tubulin expression is dependent on muscle fiber type ; however , no study has investigated the response of b - crystallin and -tubulin with transition of myosin heavy chain ( mhc ) isoforms in plantar flexor muscles followed by hindlimb unloading . in this study , we focused on b - crystallin and tubulin because these proteins exhibit functional properties associated with the adaptability of skeletal muscles . b - crystallin is localized in the z - line of the sarcomere in cardiac and slow - twitch muscle . it acts as a molecular chaperone during the folding process of other specific proteins to prevent protein degradation . b - crystallin is also known to be upregulated in slow twitch muscles , which have high oxidative capability and higher protein turnover rates compared to fast twitch muscles , which are characterized by their high glycolytic capability . it is thought that b - crystallin , tubulin , and myosin work closely with one another during the processes of myocyte formation . b - crystallin plays a critical role in chaeperoning for tubulin , which functions to maintain muscle cell morphology and transport organelles in the cell . b - crystallin also indirectly supports power transmission since it acts as a chaperone protein for myosin , one of the main components of muscle . microtubules , which are composed of tubulin , also directly associate with myosin to ensure its proper localization during sarcomere formation . these data suggest that the functional relationship between b - crystallin , tubulin , and myosin are important for muscular function and adaptability . we hypothesized that hindlimb unloading induces muscular atrophy with mhc isoform transformation toward the mhc imhc furthermore , we evaluated whether b - crystallin and -tubulin would show different expression patterns in different types of atrophied muscles as atrophied slow muscle shows a more sensitive response to both proteins compared to atrophied fast muscle . we examined the triceps surae in the hindlimbs ( soleus , plantaris , and gastrocnemius muscles ) because these muscles might exhibit similar functions and endure similar mechanical loads compared to other muscles in the hindlimbs , such as the tibialis anterior . we employed the unloading model to induce muscle atrophy and to quantitatively determine the relationship between the proteins ( b - crystallin and tubulin ) with the transition of mhc isoforms , and its significance to the adaptability of skeletal muscles . the right sides of the whole soleus muscle , plantaris muscle , and gastrocnemius muscle were isolated , and the muscles were immediately frozen in liquid nitrogen and stored at -80 c until use . the three isolated whole muscles used in this study were crushed in liquid nitrogen and solubilized in a low - salt buffer containing 20 mm potassium chloride ( kcl ) , 2 mm sodium phosphate ( ph 6.8 ) , 2 mm ethylene glycol tetraacetic acid ( egta ) , 5 mm ethylene diamine tetraacetic acid ( edta ) , 20 mm sodium fluoride , 1 mm sodium orthovanadate , a protease inhibitor containing 1 mm phenylmethylsulfonyl fluoride , 2 g / ml leupeptin , 2 g / ml aprotinin , 10 g / ml soybean trypsin inhibitor , and a phosphatase inhibitor containing 100 nm okadaic acid and 10 mm sodium -glycerophosphate . polyacrylamide gels ( 8.5% to 12% ) were used as running gels to detect b - crystallin , tubulin , and myosin proteins . the extracted plantaris muscle was used as a reference marker for analyzing the extracted soleus muscle because not all mhc isoforms in the plantaris muscle overlap with the mhc isoforms in the soleus muscle , and vice versa . b - crystallin was expressed in escherichia coli and tubulin ( phosphocellulose column - purified tubulin , pct ) was purified from the porcine brain . to estimate the exact quantity of each protein in the extracted samples , we used the purified proteins ( b - crystallin , 5 , 10 , 20 , 30 , and 40 ng ; -tubulin , 1 , 3 , 5 , 7 , 9 , and 11 ng ) that were used as standards for each protein because there was a linear relationship between the gradual contents of each protein and the band intensity ( fig . we subsequently loaded different quantities of proteins extracted from the soleus ( 0.59 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles and their corresponding protein standards onto the same gel . the contents of b - crystallin and -tubulin estimated from the extracted soleus ( 0.5 - 9 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles are presented as ng/g . the right sides of the whole soleus muscle , plantaris muscle , and gastrocnemius muscle were isolated , and the muscles were immediately frozen in liquid nitrogen and stored at -80 c until use . polyacrylamide gels ( 8.5% to 12% ) were used as running gels to detect b - crystallin , tubulin , and myosin proteins . the extracted plantaris muscle was used as a reference marker for analyzing the extracted soleus muscle because not all mhc isoforms in the plantaris muscle overlap with the mhc isoforms in the soleus muscle , and vice versa . b - crystallin was expressed in escherichia coli and tubulin ( phosphocellulose column - purified tubulin , pct ) was purified from the porcine brain . to estimate the exact quantity of each protein in the extracted samples , we used the purified proteins ( b - crystallin , 5 , 10 , 20 , 30 , and 40 ng ; -tubulin , 1 , 3 , 5 , 7 , 9 , and 11 ng ) that were used as standards for each protein because there was a linear relationship between the gradual contents of each protein and the band intensity ( fig . we subsequently loaded different quantities of proteins extracted from the soleus ( 0.59 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles and their corresponding protein standards onto the same gel . the contents of b - crystallin and -tubulin estimated from the extracted soleus ( 0.5 - 9 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles are presented as ng/g . the body and tissue weight of the control and unloading groups were measured after 15 days of hindlimb unloading ( table 1 ) . there was a significant reduction in body weight in the unloading group compared to the control group ( p < 0.05 ) . in addition , the weights of the soleus , plantaris , and gastrocnemius muscles of the unloading groups were each significantly reduced compared to the corresponding muscle in the control group , such as 121.6 18.0 ( mg ) to 54.9 4.2 ( mg ) , 299.3 37.5 ( mg ) to 218.5 22.9 ( mg ) , and 1454.8 140.3 ( mg ) to 988.3 57.0 ( mg ) , respectively ( p < 0.01 ) . based on the whole muscle to body weight ratios , the unloading soleus ( p < 0.01 ) , plantaris ( p < 0.05 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly atrophied . b - crystallin levels in the soleus ( p < 0.01 ) , plantaris ( p < 0.01 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly changed in the unloading group compared to the corresponding muscles of the control group ( fig . b - crystallin levels in the unloading group were approximately 28% lower than those in the control group . the b - crystallin content in the unloading plantaris and gastrocnemius muscles was reduced by more than 37% and 42% compared to the control group , respectively . the soleus muscle contained approximately 7-fold as much b - crystallin as the plantaris or gastrocnemius muscles did . there was a significant decrease of -tubulin in the unloading soleus muscle compared to the control muscle ( p < 0.01 ) ( fig . 2b ) ; however , we did not observe significant differences in -tubulin levels in the plantaris and gastrocnemius muscles between the control group and unloading group . the mhc isoforms in the soleus , plantaris , and gastrocnemius muscles were quantified as comparative values ( fig . mhc i in the soleus muscle was significantly reduced in the unloading group compared to the control group ( p < 0.05 ) . however , mhc ii was significantly increased in the unloading soleus muscle compared to the control soleus muscle ( fig . mhc iid / x was significantly reduced in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iia and iib were significantly increased in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iib was predominantly expressed in the gastrocnemius muscle and was significantly increased after hindlimb unloading , whereas mhc i significantly decreased after hindlimb unloading ( fig . the body and tissue weight of the control and unloading groups were measured after 15 days of hindlimb unloading ( table 1 ) . there was a significant reduction in body weight in the unloading group compared to the control group ( p < 0.05 ) . in addition , the weights of the soleus , plantaris , and gastrocnemius muscles of the unloading groups were each significantly reduced compared to the corresponding muscle in the control group , such as 121.6 18.0 ( mg ) to 54.9 4.2 ( mg ) , 299.3 37.5 ( mg ) to 218.5 22.9 ( mg ) , and 1454.8 140.3 ( mg ) to 988.3 57.0 ( mg ) , respectively ( p < 0.01 ) . based on the whole muscle to body weight ratios , the unloading soleus ( p < 0.01 ) , plantaris ( p < 0.05 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly atrophied . b - crystallin levels in the soleus ( p < 0.01 ) , plantaris ( p < 0.01 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly changed in the unloading group compared to the corresponding muscles of the control group ( fig . b - crystallin levels in the unloading group were approximately 28% lower than those in the control group . the b - crystallin content in the unloading plantaris and gastrocnemius muscles was reduced by more than 37% and 42% compared to the control group , respectively . the soleus muscle contained approximately 7-fold as much b - crystallin as the plantaris or gastrocnemius muscles did . there was a significant decrease of -tubulin in the unloading soleus muscle compared to the control muscle ( p < 0.01 ) ( fig . 2b ) ; however , we did not observe significant differences in -tubulin levels in the plantaris and gastrocnemius muscles between the control group and unloading group . the mhc isoforms in the soleus , plantaris , and gastrocnemius muscles were quantified as comparative values ( fig . mhc i in the soleus muscle was significantly reduced in the unloading group compared to the control group ( p < 0.05 ) . however , mhc ii was significantly increased in the unloading soleus muscle compared to the control soleus muscle ( fig . mhc iid / x was significantly reduced in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iia and iib were significantly increased in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iib was predominantly expressed in the gastrocnemius muscle and was significantly increased after hindlimb unloading , whereas mhc i significantly decreased after hindlimb unloading ( fig . there were obvious correlations between the changes observed in b - crystallin , -tubulin , and dominant mhc isoforms in the muscles . muscle atrophy induced a reduced expression pattern of b - crystallin and -tubulin in plantar flexor muscles when the muscles shifted to the fast muscle fiber in the unloading group . both proteins , b - crystallin and -tubulin , were downregulated in slow muscle ( p < 0.01 ) . b - crystallin was significantly decreased in the fast muscles of the unloading group compared to the control group ( p < 0.01 ) ; however , -tubulin was not reduced . the proteolytic process induced by hindlimb unloading for example , chaperone - mediated autophagy ( cma ) is believed to be related to muscle atrophy . hindlimb unloading leads to the loss of muscle activity in the hindlimbs against the gravitational force and induces muscular atrophy . a reduced gravitational stimulus due to suspending hindlimbs was designed to investigate the transition and degradation of proteins that contribute to hindlimb muscle atrophy . furthermore , our results show the expression pattern of these proteins in fast triceps surae muscles , plantaris , and gastrocnemius muscles , in conjunction with alterations of mhc isoforms by hindlimb unloading . skeletal muscles are categorized as slow - twitch or fast - twitch muscles based on properties related to adaptable characteristics such as , tension , contraction velocity , force , and glycolytic and oxidative metabolism . among the triceps surae muscles , we selected the soleus muscle as representative of slow - twitch muscle and the plantaris and gastrocnemius muscles as representatives of fast - twitch muscles . we investigated the responses of b - crystallin and -tubulin in these muscles after hindlimb unloading . in this study , we showed that b - crystallin and -tubulin respond to unloading in mhc i - dominant slow muscle but not in mhc ii - dominant muscles . however , we did not observe significant changes in -tubulin levels between the control and unloading group in the plantaris and gastrocnemius muscles ( fig . reported that the -tubulin transcript level did not change after 10 d of tail suspension even though the b - crystallin transcript level was attenuated when compared to controls . the hypothesis that the degradation of tubulin accelerates muscular atrophy is supported by the fact that the most significant decrease in tubulin levels ( p < 0.01 ) was observed in the atrophied soleus muscle , which also showed the greatest extent of atrophy among the three skeletal muscles ( 47% reduction ) . by contrast , relatively less prominent atrophy was observed in the gastrocnemius ( 21% atrophied ) and plantaris ( 16% atrophied ) muscles , in which tubulin was not significantly altered when compared to the controls . this signaling pathway is likely related to the expression of ab - crystallin and the mhc isoforms in this study . we utilized these isoforms because the dominant mhc isoform is partly related to the morphological , contractile , metabolic , and fatigue - resistant properties of muscles [ 34 - 36 ] . we analyzed whole plantar flexor muscles because a whole muscle unit , such as the soleus , plantaris , and gastrocnemius muscles , can be linked to its explicit function and adaptability , which were the factors of interest for this study , and because at the molecular level , the border of the medial or lateral portion of the muscle is quite ambiguous , which could lead to inconclusive or erroneous results . these results may be related to b - crystallin and tubulin expressions since the expressions of these proteins are dependent on fiber type . we could not clearly detect changes of tubulin because we used whole fast muscles in this study ; therefore , in future studies , we will analyze changes of tubulin in muscle fibers . the proteolytic process induced by hindlimb unloading for example , chaperone - mediated autophagy ( cma ) is believed to be related to muscle atrophy . hindlimb unloading leads to the loss of muscle activity in the hindlimbs against the gravitational force and induces muscular atrophy . a reduced gravitational stimulus due to suspending hindlimbs was designed to investigate the transition and degradation of proteins that contribute to hindlimb muscle atrophy . furthermore , our results show the expression pattern of these proteins in fast triceps surae muscles , plantaris , and gastrocnemius muscles , in conjunction with alterations of mhc isoforms by hindlimb unloading . skeletal muscles are categorized as slow - twitch or fast - twitch muscles based on properties related to adaptable characteristics such as , tension , contraction velocity , force , and glycolytic and oxidative metabolism . among the triceps surae muscles , we selected the soleus muscle as representative of slow - twitch muscle and the plantaris and gastrocnemius muscles as representatives of fast - twitch muscles . we investigated the responses of b - crystallin and -tubulin in these muscles after hindlimb unloading . in this study , we showed that b - crystallin and -tubulin respond to unloading in mhc i - dominant slow muscle but not in mhc ii - dominant muscles . however , we did not observe significant changes in -tubulin levels between the control and unloading group in the plantaris and gastrocnemius muscles ( fig . reported that the -tubulin transcript level did not change after 10 d of tail suspension even though the b - crystallin transcript level was attenuated when compared to controls . the hypothesis that the degradation of tubulin accelerates muscular atrophy is supported by the fact that the most significant decrease in tubulin levels ( p < 0.01 ) was observed in the atrophied soleus muscle , which also showed the greatest extent of atrophy among the three skeletal muscles ( 47% reduction ) . by contrast , relatively less prominent atrophy was observed in the gastrocnemius ( 21% atrophied ) and plantaris ( 16% atrophied ) muscles , in which tubulin was not significantly altered when compared to the controls . this signaling pathway is likely related to the expression of ab - crystallin and the mhc isoforms in this study . we utilized these isoforms because the dominant mhc isoform is partly related to the morphological , contractile , metabolic , and fatigue - resistant properties of muscles [ 34 - 36 ] . we analyzed whole plantar flexor muscles because a whole muscle unit , such as the soleus , plantaris , and gastrocnemius muscles , can be linked to its explicit function and adaptability , which were the factors of interest for this study , and because at the molecular level , the border of the medial or lateral portion of the muscle is quite ambiguous , which could lead to inconclusive or erroneous results . these results may be related to b - crystallin and tubulin expressions since the expressions of these proteins are dependent on fiber type . we could not clearly detect changes of tubulin because we used whole fast muscles in this study ; therefore , in future studies , we will analyze changes of tubulin in muscle fibers . in conclusion , we found that b - crystallin and tubulin related to the transition of mhc isoforms were downregulated more explicitly in the atrophied soleus muscle rather than in the atrophied plantaris and gastrocnemius muscles .

one study concerning the content of b - crystallin and tubulin in 10 days of atrophied soleus muscle was previously reported . b - crystallin and tubulin expression is dependent on muscle fiber type ; however , no study has investigated the response of b - crystallin and -tubulin with transition of myosin heavy chain ( mhc ) isoforms in plantar flexor muscles followed by hindlimb unloading . in this study , we focused on b - crystallin and tubulin because these proteins exhibit functional properties associated with the adaptability of skeletal muscles . b - crystallin is localized in the z - line of the sarcomere in cardiac and slow - twitch muscle . b - crystallin is activated in a phosphorylation - dependent manner and also acts to stabilize intermediate filament and cytoskeletal proteins in vitro . another study showed that disorganized cytoskeletal components trigger the phosphorylation of b - crystallin through activating the p38 mitogen - activated protein kinase ( mapk ) pathway . b - crystallin is also known to be upregulated in slow twitch muscles , which have high oxidative capability and higher protein turnover rates compared to fast twitch muscles , which are characterized by their high glycolytic capability . microtubules are directly associated with myosin , and the localization of myosin filaments during sarcomere formation and myogenic cell morphology is partly dependent on the microtubule networking organization . it can be inferred that different muscle types are due to the different myogenic cell morphologies and their localization is related to differences in the formation and content of microtubules . the increase in directional shifts from the mhc i to the mhc iia , mhc iid / x , and mhc iib isoforms , augments the velocity of the shortening and power generation capacity of the mhc , but decreases its atpase activity . b - crystallin plays a critical role in chaeperoning for tubulin , which functions to maintain muscle cell morphology and transport organelles in the cell . these data suggest that the functional relationship between b - crystallin , tubulin , and myosin are important for muscular function and adaptability . we hypothesized that hindlimb unloading induces muscular atrophy with mhc isoform transformation toward the mhc imhc furthermore , we evaluated whether b - crystallin and -tubulin would show different expression patterns in different types of atrophied muscles as atrophied slow muscle shows a more sensitive response to both proteins compared to atrophied fast muscle . we examined the triceps surae in the hindlimbs ( soleus , plantaris , and gastrocnemius muscles ) because these muscles might exhibit similar functions and endure similar mechanical loads compared to other muscles in the hindlimbs , such as the tibialis anterior . we employed the unloading model to induce muscle atrophy and to quantitatively determine the relationship between the proteins ( b - crystallin and tubulin ) with the transition of mhc isoforms , and its significance to the adaptability of skeletal muscles . in this study , we utilized 12 seven - week - old adult male wistar rats that weighed 238.2 9.1 g. after 2 - 3 days of adaptation , the rats were randomly divided , with half placed in a control group and the half placed in an unloading group . the right sides of the whole soleus muscle , plantaris muscle , and gastrocnemius muscle were isolated , and the muscles were immediately frozen in liquid nitrogen and stored at -80 c until use . the three isolated whole muscles used in this study were crushed in liquid nitrogen and solubilized in a low - salt buffer containing 20 mm potassium chloride ( kcl ) , 2 mm sodium phosphate ( ph 6.8 ) , 2 mm ethylene glycol tetraacetic acid ( egta ) , 5 mm ethylene diamine tetraacetic acid ( edta ) , 20 mm sodium fluoride , 1 mm sodium orthovanadate , a protease inhibitor containing 1 mm phenylmethylsulfonyl fluoride , 2 g / ml leupeptin , 2 g / ml aprotinin , 10 g / ml soybean trypsin inhibitor , and a phosphatase inhibitor containing 100 nm okadaic acid and 10 mm sodium -glycerophosphate . the extracted plantaris muscle was used as a reference marker for analyzing the extracted soleus muscle because not all mhc isoforms in the plantaris muscle overlap with the mhc isoforms in the soleus muscle , and vice versa . louis , mo , usa ) and anti-b - crystallin ( 1:5000 , raised in rabbit against the c - terminal 10 peptides [ ( sh ) kpavtaapkk ] of human b - crystallin ) . to estimate the exact quantity of each protein in the extracted samples , we used the purified proteins ( b - crystallin , 5 , 10 , 20 , 30 , and 40 ng ; -tubulin , 1 , 3 , 5 , 7 , 9 , and 11 ng ) that were used as standards for each protein because there was a linear relationship between the gradual contents of each protein and the band intensity ( fig . we subsequently loaded different quantities of proteins extracted from the soleus ( 0.59 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles and their corresponding protein standards onto the same gel . the contents of b - crystallin and -tubulin estimated from the extracted soleus ( 0.5 - 9 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles are presented as ng/g . in this study , we utilized 12 seven - week - old adult male wistar rats that weighed 238.2 9.1 g. after 2 - 3 days of adaptation , the rats were randomly divided , with half placed in a control group and the half placed in an unloading group . the right sides of the whole soleus muscle , plantaris muscle , and gastrocnemius muscle were isolated , and the muscles were immediately frozen in liquid nitrogen and stored at -80 c until use . the three isolated whole muscles used in this study were crushed in liquid nitrogen and solubilized in a low - salt buffer containing 20 mm potassium chloride ( kcl ) , 2 mm sodium phosphate ( ph 6.8 ) , 2 mm ethylene glycol tetraacetic acid ( egta ) , 5 mm ethylene diamine tetraacetic acid ( edta ) , 20 mm sodium fluoride , 1 mm sodium orthovanadate , a protease inhibitor containing 1 mm phenylmethylsulfonyl fluoride , 2 g / ml leupeptin , 2 g / ml aprotinin , 10 g / ml soybean trypsin inhibitor , and a phosphatase inhibitor containing 100 nm okadaic acid and 10 mm sodium -glycerophosphate . the extracted plantaris muscle was used as a reference marker for analyzing the extracted soleus muscle because not all mhc isoforms in the plantaris muscle overlap with the mhc isoforms in the soleus muscle , and vice versa . louis , mo , usa ) and anti-b - crystallin ( 1:5000 , raised in rabbit against the c - terminal 10 peptides [ ( sh ) kpavtaapkk ] of human b - crystallin ) . to estimate the exact quantity of each protein in the extracted samples , we used the purified proteins ( b - crystallin , 5 , 10 , 20 , 30 , and 40 ng ; -tubulin , 1 , 3 , 5 , 7 , 9 , and 11 ng ) that were used as standards for each protein because there was a linear relationship between the gradual contents of each protein and the band intensity ( fig . we subsequently loaded different quantities of proteins extracted from the soleus ( 0.59 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles and their corresponding protein standards onto the same gel . the contents of b - crystallin and -tubulin estimated from the extracted soleus ( 0.5 - 9 g ) , plantaris ( 5 - 13 g ) , or gastrocnemius ( 2 - 35 g ) muscles are presented as ng/g . the body and tissue weight of the control and unloading groups were measured after 15 days of hindlimb unloading ( table 1 ) . there was a significant reduction in body weight in the unloading group compared to the control group ( p < 0.05 ) . in addition , the weights of the soleus , plantaris , and gastrocnemius muscles of the unloading groups were each significantly reduced compared to the corresponding muscle in the control group , such as 121.6 18.0 ( mg ) to 54.9 4.2 ( mg ) , 299.3 37.5 ( mg ) to 218.5 22.9 ( mg ) , and 1454.8 140.3 ( mg ) to 988.3 57.0 ( mg ) , respectively ( p < 0.01 ) . based on the whole muscle to body weight ratios , the unloading soleus ( p < 0.01 ) , plantaris ( p < 0.05 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly atrophied . b - crystallin levels in the soleus ( p < 0.01 ) , plantaris ( p < 0.01 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly changed in the unloading group compared to the corresponding muscles of the control group ( fig . b - crystallin levels in the unloading group were approximately 28% lower than those in the control group . the b - crystallin content in the unloading plantaris and gastrocnemius muscles was reduced by more than 37% and 42% compared to the control group , respectively . there was a significant decrease of -tubulin in the unloading soleus muscle compared to the control muscle ( p < 0.01 ) ( fig . 2b ) ; however , we did not observe significant differences in -tubulin levels in the plantaris and gastrocnemius muscles between the control group and unloading group . mhc i in the soleus muscle was significantly reduced in the unloading group compared to the control group ( p < 0.05 ) . however , mhc ii was significantly increased in the unloading soleus muscle compared to the control soleus muscle ( fig . mhc iid / x was significantly reduced in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iia and iib were significantly increased in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iib was predominantly expressed in the gastrocnemius muscle and was significantly increased after hindlimb unloading , whereas mhc i significantly decreased after hindlimb unloading ( fig . there was a significant reduction in body weight in the unloading group compared to the control group ( p < 0.05 ) . in addition , the weights of the soleus , plantaris , and gastrocnemius muscles of the unloading groups were each significantly reduced compared to the corresponding muscle in the control group , such as 121.6 18.0 ( mg ) to 54.9 4.2 ( mg ) , 299.3 37.5 ( mg ) to 218.5 22.9 ( mg ) , and 1454.8 140.3 ( mg ) to 988.3 57.0 ( mg ) , respectively ( p < 0.01 ) . based on the whole muscle to body weight ratios , the unloading soleus ( p < 0.01 ) , plantaris ( p < 0.05 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly atrophied . b - crystallin levels in the soleus ( p < 0.01 ) , plantaris ( p < 0.01 ) , and gastrocnemius ( p < 0.01 ) muscles were significantly changed in the unloading group compared to the corresponding muscles of the control group ( fig . b - crystallin levels in the unloading group were approximately 28% lower than those in the control group . the b - crystallin content in the unloading plantaris and gastrocnemius muscles was reduced by more than 37% and 42% compared to the control group , respectively . there was a significant decrease of -tubulin in the unloading soleus muscle compared to the control muscle ( p < 0.01 ) ( fig . 2b ) ; however , we did not observe significant differences in -tubulin levels in the plantaris and gastrocnemius muscles between the control group and unloading group . mhc i in the soleus muscle was significantly reduced in the unloading group compared to the control group ( p < 0.05 ) . however , mhc ii was significantly increased in the unloading soleus muscle compared to the control soleus muscle ( fig . mhc iid / x was significantly reduced in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iia and iib were significantly increased in the unloading plantaris muscle compared to the control plantaris muscle ( p < 0.01 ) . mhc iib was predominantly expressed in the gastrocnemius muscle and was significantly increased after hindlimb unloading , whereas mhc i significantly decreased after hindlimb unloading ( fig . there were obvious correlations between the changes observed in b - crystallin , -tubulin , and dominant mhc isoforms in the muscles . muscle atrophy induced a reduced expression pattern of b - crystallin and -tubulin in plantar flexor muscles when the muscles shifted to the fast muscle fiber in the unloading group . b - crystallin was significantly decreased in the fast muscles of the unloading group compared to the control group ( p < 0.01 ) ; however , -tubulin was not reduced . we show that hindlimb unloading results in reduced -tubulin expression , followed by reduced ab - crystallin expression in atrophied triceps surae muscles . furthermore , our results show the expression pattern of these proteins in fast triceps surae muscles , plantaris , and gastrocnemius muscles , in conjunction with alterations of mhc isoforms by hindlimb unloading . among the triceps surae muscles , we selected the soleus muscle as representative of slow - twitch muscle and the plantaris and gastrocnemius muscles as representatives of fast - twitch muscles . we investigated the responses of b - crystallin and -tubulin in these muscles after hindlimb unloading . in this study , we showed that b - crystallin and -tubulin respond to unloading in mhc i - dominant slow muscle but not in mhc ii - dominant muscles . in our study , the -tubulin and b - crystallin levels exhibited similar changes between control and unloading soleus muscle ( fig . however , we did not observe significant changes in -tubulin levels between the control and unloading group in the plantaris and gastrocnemius muscles ( fig . reported that the -tubulin transcript level did not change after 10 d of tail suspension even though the b - crystallin transcript level was attenuated when compared to controls . the hypothesis that the degradation of tubulin accelerates muscular atrophy is supported by the fact that the most significant decrease in tubulin levels ( p < 0.01 ) was observed in the atrophied soleus muscle , which also showed the greatest extent of atrophy among the three skeletal muscles ( 47% reduction ) . by contrast , relatively less prominent atrophy was observed in the gastrocnemius ( 21% atrophied ) and plantaris ( 16% atrophied ) muscles , in which tubulin was not significantly altered when compared to the controls . this signaling pathway is likely related to the expression of ab - crystallin and the mhc isoforms in this study . we utilized these isoforms because the dominant mhc isoform is partly related to the morphological , contractile , metabolic , and fatigue - resistant properties of muscles [ 34 - 36 ] . we analyzed whole plantar flexor muscles because a whole muscle unit , such as the soleus , plantaris , and gastrocnemius muscles , can be linked to its explicit function and adaptability , which were the factors of interest for this study , and because at the molecular level , the border of the medial or lateral portion of the muscle is quite ambiguous , which could lead to inconclusive or erroneous results . we could not clearly detect changes of tubulin because we used whole fast muscles in this study ; therefore , in future studies , we will analyze changes of tubulin in muscle fibers . we show that hindlimb unloading results in reduced -tubulin expression , followed by reduced ab - crystallin expression in atrophied triceps surae muscles . furthermore , our results show the expression pattern of these proteins in fast triceps surae muscles , plantaris , and gastrocnemius muscles , in conjunction with alterations of mhc isoforms by hindlimb unloading . among the triceps surae muscles , we selected the soleus muscle as representative of slow - twitch muscle and the plantaris and gastrocnemius muscles as representatives of fast - twitch muscles . we investigated the responses of b - crystallin and -tubulin in these muscles after hindlimb unloading . in this study , we showed that b - crystallin and -tubulin respond to unloading in mhc i - dominant slow muscle but not in mhc ii - dominant muscles . in our study , the -tubulin and b - crystallin levels exhibited similar changes between control and unloading soleus muscle ( fig . however , we did not observe significant changes in -tubulin levels between the control and unloading group in the plantaris and gastrocnemius muscles ( fig . the hypothesis that the degradation of tubulin accelerates muscular atrophy is supported by the fact that the most significant decrease in tubulin levels ( p < 0.01 ) was observed in the atrophied soleus muscle , which also showed the greatest extent of atrophy among the three skeletal muscles ( 47% reduction ) . by contrast , relatively less prominent atrophy was observed in the gastrocnemius ( 21% atrophied ) and plantaris ( 16% atrophied ) muscles , in which tubulin was not significantly altered when compared to the controls . this signaling pathway is likely related to the expression of ab - crystallin and the mhc isoforms in this study . we analyzed whole plantar flexor muscles because a whole muscle unit , such as the soleus , plantaris , and gastrocnemius muscles , can be linked to its explicit function and adaptability , which were the factors of interest for this study , and because at the molecular level , the border of the medial or lateral portion of the muscle is quite ambiguous , which could lead to inconclusive or erroneous results . we could not clearly detect changes of tubulin because we used whole fast muscles in this study ; therefore , in future studies , we will analyze changes of tubulin in muscle fibers . in conclusion , we found that b - crystallin and tubulin related to the transition of mhc isoforms were downregulated more explicitly in the atrophied soleus muscle rather than in the atrophied plantaris and gastrocnemius muscles .

sigmund freud is rarely mentioned in scientific discourse without also belittling the lack of quantitative statistical evidence for his elaborate models . at the same time , his qualitative case reports and the conclusions he drew from them by far belong to the most well - known research in psychosomatic medicine . despite all valid critique , one reason , we argue , may very well be the superiority of the single case study in first observing , describing , capturing , evaluating , and creatively reflecting on an infinite set of parameters surrounding any chosen topic . out of this primary assessment , novel hypotheses and it is our objective to reapply such primary assessment to the case of adolescent brittle diabetes ( or more generally speaking , the psychosomatic underpinnings of diabetes type 1 in minors and young adults ) , while also trying to answer calls for more quantitative and statistically reliable approaches to doing so . this in mind , we have first selected a highly quantitative case study on family dynamics and brittle diabetes and reviewed and reanalyzed its raw data through implementation of a new statistical procedure increasing the coefficient of determination in the new model by factor ten ( while also presenting new and clearer findings ) , in order to then , in a second step , discuss and compare our results to possibly the historically most well - known set of qualitative case studies on the topic . we will start by briefly revisiting the literature on the psychosomatics of adolescent instable diabetes type 1 , present a case vignette and basic data collection method of the original case study we reexamine ( which may be skipped by those familiar with the work published by ) , followed by a detailed description of our new statistical approach and its results , concluding with a clear clinically oriented graphical presentation of our findings and their discussion in light of minuchin et al . one out of 600 us or european school - age children suffers from insulin dependent diabetes mellitus [ 3 , 4 ] . just about 33 percent of diabetics between 13 and 19 years of age manage to maintain tolerable glycemic control and a hba1c below 8 ; 6.3 percent suffered at least one episode of major hypoglycemia within the last three months [ 5 , 6 ] . the devastating immediate and long - term effects of poor diabetic control are widely known and feared . 44 percent of the variance in blood glucose control can be statistically explained by psychological variables in these patients and their parents . a randomized controlled study further demonstrates how an intensive inpatient treatment program including psychoanalytic psychotherapy could effectively improve diabetic control in children . these cases of glycemic instability with no somatic explanation have been termed brittle diabetes by some authors and there is no doubt concerning the importance of further exploration of the causes and remedies surrounding this truly psychosomatic disease . while various aspects of brittle diabetes have been explored in recent years , including its exact definition , there seems to be a gap in the literature in exploring how emotional variables of all individuals within the family system may interact to affect glycemic control of the diabetic adolescent , the index patient of a dysfunctional family system . the little research which has sought to fill this gap ( i.e. , [ 2 , 10 , 11 ] ) is primarily qualitative in nature and must face similar critique as all such work , as will be discussed in the last section of this study . the case and its psychosomatic background ( adopted and revised from ) . the adolescent index patient of this case study was diagnosed with diabetes type 1 at age of four ( clinical clues were polyuria , polydipsia , loss of appetite , a fungal infection , hba1c of 9.1 per cent , antibodies against islet cells , and gad65 ) . family dynamics surrounding this classic family of three ( biological parents , single child ) appeared unsuspicious notwithstanding the girl 's history of poorly controlled bronchial asthma and allergic diseases . yet at age of six , nocturnal hypoglycemia with loss of consciousness led to readmission to the hospital , during which another episode of profound hypoglycemia , this time in conjunction with a tonic - clonic seizure , occurred , thus further consolidating her parents ' distress concerning hypoglycemia and hospital treatment . once all educational efforts concerning the diabetic management were exhausted ( including individual and family - based counseling , detailed and repetitive disease - specific education , and information about glycemic control mechanisms including the influence of nutrition , sport , and other aspects of blood sugar regulation ) , but a hba1c below 7 percent was never achieved , the family finally sought for psychosomatic family treatment . psychodynamically based therapeutic analysis of the family dynamic suggested a conflict between the adolescent and her mother about who had control of the blood sugar levels . the mother 's dominance seemed to have negative effects on her daughter 's glycemic control . fears of hypoglycemia were somewhat irrational with all three family members , including the father , who , at first sight , seemed rather more distant to the matter ( literature proposes parental hypoglycemia avoidance behaviours to adversely affect glycemic control ) . the family 's shock in relation to the diagnosis and mistrust of hospital personnel was discussed . finally , a therapeutic intervention confronted them with their specific type of collusion concerning ( in-)dependence , in which both parents , in their manifest statements , advocated for more self - confidence and extended duties on the side of the daughter , but on a more latent level , gave hints to their beloved little girl not being ready to take control over the blood sugar monitoring by herself . this mostly unconscious conflict had culminated in cloudy paths of communication concerning glycemic control , in nebulous distributions of duties within the family members , and , as a result of the arrangement , in deep dissatisfaction over the failure of proper diabetic control . while traditional case studies would focus on the qualitative data outlined above , we sought to amend such observations by a highly quantitative approach in order to produce more evidence based and reproducible results . therefore , we aimed to statistically explore how specific basic affect states of all three individual family members may impact each other and the success of the diabetic management over a period of 120 days . to operationalize this quest , we drew on the standardized self - assessment manikin ( sam ) , as developed by bradley and lang ( for details see [ 13 , 14 ] ) , asking all three family members to individually record on a daily basis their valence ( mood ) , arousal ( high versus low ) , and dominance ( a sense of presence in the current environment ) . in addition the index patient was asked to obtain at least three daily blood glucose measurements ( or more if required by the disease ) over the same period utilizing a common standardized technique . this form of diary based data collection is also referred to as ecological momentary assessment with many benefits in terms of accuracy and validity of measurements . standard deviations of the daily blood glucose measurements served as an indicator for glycemic variability , a measure which recent research has identified as the most precise predictor of diabetic control , followed by the hba1c - value in second place [ 1619 ] , due to it being the best known predictor for diabetic complications and microvascular derailments in particular . resulting from this data collection and primary analysis are ten time series : three time series for each of the three family members from the sam , affective valence ( happy , sad ) , arousal ( excited , calm ) , and dominance ( a sense of presence , distance to the current environment ) , as well as one time series recording glycemic variability ( daily standard deviations of measurements ) . , these ten time series were further analyzed by a completely new statistical approach to vector autoregressive ( var ) modeling . while past analysis of this same set of data ( see ) has also relied on basic var analysis , there had been some common shortcomings to the validity and scope of results , which we were able to remedy here , thus solving statistical shortcomings while also presenting completely new results in a clearer more clinically oriented fashion . how we were able to achieve this , the presentation of a newly developed optimized multivariate lag selection process in var analysis , and a comprehensive review of the principles of vector autoregression will be presented next . the use of vector autoregressive models ( var ) for the analysis of time series data in psychosomatic medicine ( also widely used in neuroscience ) allows treating a set of variables as jointly driven by the lagged values of all variables in the system with no a priori assignment of dependent and independent status being necessary . this technique seems particularly apt for research in psychosomatic medicine , where , among others , has long called for a more integrated ( monistic ) view on the complexity of dynamic dependencies and intertemporal reciprocal cause and effect relationships among different psychic as well as somatic variables . any var model requires the user to select a maximum number of lags , which , in more practical terms , refers to how far back in time the user wants to go in the search for past recordings of all variables to predict the present value of one variable . the farther back in time the user decides to go , the more explanatory variables ( lags ) need to be included in the model because it used to be improper to exclude past recordings of explanatory variables , which lay in - between the present value and the most historic one [ 22 , 23 ] . unfortunately including more explanatory variables ( going back further in time ) is a double edged sword , since this would provide a var model more representative of reality ( goodness of fit ) , but would also endorse one with less explanatory power ( lower adjusted r ) . the latter is due to the tremendous penalty inflicted by the large number of explanatory variables ( lags ) in the model resulting in high estimation variance [ 22 , 23 ] . this substantial drawback weakened the substance of empirical findings derived from var models , because researchers would either present results through models with teeth chattering low r values ( see previously published results from the same raw data as one example ) or adopt models only incorporating the effects of events preceding the predicted value of a variable by one day / one unit of time in the var ( e.g. , see ) . in order to alleviate this shortcoming of low adjusted r values in the standard vector autoregressive modeling approach , we developed a computer code implementing a statistical procedure recently published in parts in savin and winker and winker [ 26 , 27 ] , referred to as the optimized multivariate lag selection process , which allows ( contrary to previous practice ) excluding such explanatory variables ( lags ) from the var model which add little to its goodness of fit ( estimated representativeness of reality ) while nonetheless reducing its explanatory power ( adjusted r ) . this admittance of holes to the lag structure ( equations organizing the explanatory variables ) allows us to now present an entirely new model exhibiting more detailed dynamics with a smaller number of parameters , for the data in this case resulting in about tenfold increase of the adjusted r value . mathematical details of applying the optimized multivariate lag selection process to this var analysis of the ten time series of the data set at hand will be presented next ( and may be skipped by the more clinically focused researcher ) . a standard vector autoregressive ( var ) model was constructed , using eviews 7.1 ( qms , quantitative micro software , irvine ca ) , based on the ten time series we mentioned above . in order to focus on the innovative aspects of our methodology we will not delve into the details of var model construction , which have been described at length in preceding publications ( i.e. , [ 1 , 24 ] ) . given the large number of explanatory variables ( the more lags , the more variables ) and the limited number of observations , only a very limited number of lags ( past days ) could be considered while adjusted r would still be low , if we were to follow the standard modeling approach [ 22 , 23 ] . the novel contribution is to maximize the informational content of the model by minimizing an information criterion [ 2527 ] . in more concrete terms , if we assume that any one value within the ten time series may have effects on any of the other values of all - time series with a delay of up to one week , a total of 710 parameters would have to be estimated . given 120 observations in each time series , this results in tremendous estimation variance ( very low r ) . model selection criteria suggest using only one lag ( assuming effects will take place within a day instead of within a week , which seems highly unrealistic but is a common approach adopted by other researchers in the field , including wild et al . , 2010 ) resulting in a total of only 110 parameters to be estimated with a still low r value of 0.02 for the model explaining glycemic variance . to resolve this dilemma , we drew on winker [ 26 , 27 ] and savin and winker engaging in optimized multivariate lag structure analysis . given the huge discrete search space of all possible lag structures , for example , for a maximum lag length of seven , heuristic optimization algorithms are used to this end . for this process , a computer code was developed using matlab r2011b with an interface to eviews 7.1 , which implements a genetic algorithm for the search of an optimized lag structure making use of information criteria ( bic ) as in the standard selection procedure ( see for more details ) . by providing an approximation to the minimum of the information criterion , the resulting model exhibits an optimized tradeoff between a good fit to the multivariate dynamics of the data and model parsimony . as a result , we obtained a model with only 70 parameters , but still cover effect delays up to one week . since the maintained lags are selected based on their joint informational content ( as measured by the information criteria ) , the procedure results in a model with much higher explanatory power ( for predicting glycemic variability adjusted r value of 0.20 as opposed to 0.02 for the standard model with only one lag ) and a richer dynamic . given the rich dynamics between all variables of the model , besides considering single equations , the calculation of impulse response functions as in would be of interest . however , the zero constraints of the var model with holes preclude the application of standard methods for the calculation of confidence bands . similarly , poor glycemic control ( high glycemic variability ) will correlate with low glycemic variability four days earlier , a calm mother three days earlier , an excited mother seven days earlier , a dominating mother four days earlier , a nondominating mother seven days earlier ( although statistically insignificant ) , a sad father both five and six days earlier , a calm father both three and seven days earlier , and a dominating father both two and five days earlier . high glycemic variability will also correlate with a sad child six days later , an excited mother three days later , and a dominating father one day later . for a graphical representation see figure 2 . the optimized multivariate lag structure selection process provides one equation of seemingly unrelated multiple regression for each of the ten time series to be presented next . three of them directly involve glycemic variability in addition to the one for glycemic variability itself , which shall be presented last ( lags in parentheses ) : affective valence of the adolescent = 1 glycemic variability ( 6 ) + 2 valence adolescent ( 1 ) ( r = 0.25 , adj . r = 0.24 ) ; affective valence of the mother = 3 dominance adolescent ( 7 ) + 4 valence mother ( 5 ) + 5 arousal mother ( 6 ) + 6 arousal father ( 4 ) + 7 arousal father ( 6 ) ( r = 0.21 , adj . r = 0.18 ) ; affective valence of the father = 8 valence adolescent ( 3 ) + 9 valence adolescent ( 5 ) + 10 arousal mother ( 5 ) + 11 dominance father ( 3 ) ( r = 0.21 , adj . r = 0.18 ) ; arousal of the adolescent = 12 arousal adolescent ( 1 ) + 13 arousal adolescent ( 3 ) + 14 arousal adolescent ( 7 ) + 15 valence mother ( 4 ) + 16 arousal mother ( 3 ) + 17 valence father ( 2 ) + 18 valence father ( 6 ) ( r = 0.30 , adj . r = 0.25 ) ; arousal of the mother = 19 glycemic variability ( 3 ) + 20 arousal adolescent ( 7 ) + 21 dominance adolescent ( 5 ) + 22 arousal mother ( 5 ) + 23 arousal mother ( 7 ) + 24 dominance mother ( 1 ) + 25 dominance father ( 6 ) ( r = 0.29 , adj . r = 0.24 ) ; arousal of the father = 26 valence mother ( 4 ) + 27 dominance mother ( 6 ) + 28 arousal father ( 1 ) + 29 arousal father ( 2 ) + 30 arousal father ( 6 ) + 31 dominance father ( 1 ) ( r = 0.19 , adj . r = 0.15 ) ; dominance of the adolescent = 32 valence adolescent ( 1 ) + 33 arousal adolescent ( 5 ) + 34 arousal father ( 1 ) + 35 dominance father ( 1 ) ( r = 0.25 , adj . r = 0.22 ) ; dominance of the mother = 36 valence mother ( 7 ) + 37 dominance mother ( 1 ) + 38 dominance mother ( 3 ) + 39 dominance father ( 5 ) ( r = 0.65 , adj . r = 0.64 ) ; dominance of the father = 40 glycemic variability ( 1 ) + 41 dominance child ( 6 ) + 42 valence mother ( 5 ) + 43 valence mother ( 7 ) + 44 dominance mother ( 4 ) + 45 dominance mother ( 6 ) + 46 valence father ( 1 ) + 47 valence father ( 3 ) + 48 arousal father ( 3 ) + 49 dominance father ( 2 ) ( r = 0.34 , adj . r = 0.27 ) ; glycemic variability = 1 glycemic variability ( 4 ) + 2 arousal mother ( 3 ) + 3 arousal mother ( 7 ) + 4 dominance mother ( 4 ) + 5 dominance mother ( 7 ) + 6 valence father ( 5 ) + 7 valence father ( 6 ) + 8 arousal father ( 3 ) + 9 arousal father ( 7 ) + 10 dominance father ( 2 ) + 11 dominance father ( 5 ) ( r = 0.28 , adj . affective valence of the adolescent = 1 glycemic variability ( 6 ) + 2 valence adolescent ( 1 ) ( r = 0.25 , adj . r = 0.24 ) ; affective valence of the mother = 3 dominance adolescent ( 7 ) + 4 valence mother ( 5 ) + 5 arousal mother ( 6 ) + 6 arousal father ( 4 ) + 7 arousal father ( 6 ) ( r = 0.21 , adj . r = 0.18 ) ; affective valence of the father = 8 valence adolescent ( 3 ) + 9 valence adolescent ( 5 ) + 10 arousal mother ( 5 ) + 11 dominance father ( 3 ) ( r = 0.21 , adj . r = 0.18 ) ; arousal of the adolescent = 12 arousal adolescent ( 1 ) + 13 arousal adolescent ( 3 ) + 14 arousal adolescent ( 7 ) + 15 valence mother ( 4 ) + 16 arousal mother ( 3 ) + 17 valence father ( 2 ) + 18 valence father ( 6 ) ( r = 0.30 , adj . r = 0.25 ) ; arousal of the mother = 19 glycemic variability ( 3 ) + 20 arousal adolescent ( 7 ) + 21 dominance adolescent ( 5 ) + 22 arousal mother ( 5 ) + 23 arousal mother ( 7 ) + 24 dominance mother ( 1 ) + 25 dominance father ( 6 ) ( r = 0.29 , adj . r = 0.24 ) ; arousal of the father = 26 valence mother ( 4 ) + 27 dominance mother ( 6 ) + 28 arousal father ( 1 ) + 29 arousal father ( 2 ) + 30 arousal father ( 6 ) + 31 dominance father ( 1 ) ( r = 0.19 , adj . r = 0.15 ) ; dominance of the adolescent = 32 valence adolescent ( 1 ) + 33 arousal adolescent ( 5 ) + 34 arousal father ( 1 ) + 35 dominance father ( 1 ) ( r = 0.25 , adj . r = 0.22 ) ; dominance of the mother = 36 valence mother ( 7 ) + 37 dominance mother ( 1 ) + 38 dominance mother ( 3 ) + 39 dominance father ( 5 ) ( r = 0.65 , adj . r = 0.64 ) ; dominance of the father = 40 glycemic variability ( 1 ) + 41 dominance child ( 6 ) + 42 valence mother ( 5 ) + 43 valence mother ( 7 ) + 44 dominance mother ( 4 ) + 45 dominance mother ( 6 ) + 46 valence father ( 1 ) + 47 valence father ( 3 ) + 48 arousal father ( 3 ) + 49 dominance father ( 2 ) ( r = 0.34 , adj . r = 0.27 ) ; glycemic variability = 1 glycemic variability ( 4 ) + 2 arousal mother ( 3 ) + 3 arousal mother ( 7 ) + 4 dominance mother ( 4 ) + 5 dominance mother ( 7 ) + 6 valence father ( 5 ) + 7 valence father ( 6 ) + 8 arousal father ( 3 ) + 9 arousal father ( 7 ) + 10 dominance father ( 2 ) + 11 dominance father ( 5 ) ( r = 0.28 , adj . the coefficients , their standard error , t - statistic , and probability referred to above , can be reviewed in table 1 . the development of a novel statistical methodology allowed us to disentangle the data and generate statistically reliable results in the form of ten equations . the dynamic of the results pertaining to glycemic variability , ( thereby , it has to be taken into account that additional dynamic interactions arise due to spillover between equations , which are not considered here ) , taking into account the direction of coefficients , can be summarized in the following words and graphical representations . low glycemic variability and , therefore , good diabetic control will correlate with the following : high glycemic variability four days earlier , an excited mother three days earlier , a calm mother seven days earlier , a non - dominating mother four days earlier , a dominating mother seven days earlier ( although statistically insignificant ) , a happy father both five and six days earlier , an excited father both three and seven days earlier , and a non - dominating father both two and five days earlier . low glycemic variability will also correlate with a happy child six days later , a calm mother three days later , and a non - dominating father one day later . for a graphical representation of this paragraph refer to figure 1 . similarly , poor glycemic control ( high glycemic variability ) will correlate with low glycemic variability four days earlier , a calm mother three days earlier , an excited mother seven days earlier , a dominating mother four days earlier , a non - dominating mother seven days earlier ( although statistically insignificant ) , a sad father both five and six days earlier , a calm father both three and seven days earlier , and a dominating father both two and five days earlier . high glycemic variability will also correlate with a sad child six days later , an excited mother three days later , and a dominating father one day later . a graphical representation of this paragraph is presented in figure 2 in clinical terms , this means , good diabetic control was preceded by attentive and alert ( high arousal , excited ) parents with a positive attitude ( happy father ) , at the same time refraining from too much overwhelming presence ( low dominance ) . likewise , phases of good diabetic management were followed by a continuously distant father ( low dominance ) , unfortunately a less alert mother ( low arousal ) , and a content ( happy ) adolescent index patient . similarly , mostly self - explanatory , graphical representations were constructed for the effects surrounding the affective valence of all three family members ( see figures 3 , 4 , and 5 ) . we picked these three timelines for more detailed examination , because the appropriate measurement of depressive symptoms ( which at least at a distance somewhat relates to affective valence ) in diabetics in general , remains to be a topic of current debate in the literature . in comparison to the results derived from the same set of raw data with a different statistical approach in an earlier publication , there are several improvements we were able to achieve:(i)increasing the coefficient of determination r for the model prediction of glycemic variability by factor ten ( adjusted r value of 0.20 as opposed to 0.02 ) while incorporating significant effects of explanatory variables ( lags ) stemming from a longer period of time preceding the predicted event;(ii)presenting a more precise timeline of effects of various variables on each other , including glycemic variability and vice versa ( e.g. , a nondominating mother four days prior to a set day will increase glycemic control instead of a nondominating mother somewhere up to four days prior to a set day will increase glycemic control);(iii)isolating additional relationships between variables which did not reach statistical significance earlier or took more time to take effect than the time frame of the earlier models allowed for . increasing the coefficient of determination r for the model prediction of glycemic variability by factor ten ( adjusted r value of 0.20 as opposed to 0.02 ) while incorporating significant effects of explanatory variables ( lags ) stemming from a longer period of time preceding the predicted event ; presenting a more precise timeline of effects of various variables on each other , including glycemic variability and vice versa ( e.g. , a nondominating mother four days prior to a set day will increase glycemic control instead of a nondominating mother somewhere up to four days prior to a set day will increase glycemic control ) ; isolating additional relationships between variables which did not reach statistical significance earlier or took more time to take effect than the time frame of the earlier models allowed for . a more substantial contribution of this paper is the demonstration and practical application of the multivariate lag selection process to var analysis , resolving an essential shortcoming in var analysis of ( relatively ) small samples . hence , this contribution to literature will have relevance beyond the case study approach but also to var - based studies of larger cohorts of patients ( as e.g. , ) , significantly increasing either the number of effects analyzed ( as in ) or the statistical reliability ( i.e. , the adjusted r ) with which results are presented . all in all , however , mathematically refined quantitative methodological approaches relying on modern computational technology can generate more specific , reproducible , and thus trustworthy results than purely qualitative ( narrative ) accounts , while still honoring the benefits of the case study approach aiming to explore previously unforeseen avenues fit for further vested inquiry ( often costly to perform ) . yet , we have to ask ourselves critically if the added mathematical complexity honors the overall value of the results a case study approach can provide . revisiting the opening comments of this report in the context of brittle diabetes , it seems interesting to note that particularly the most highly acclaimed and clinically widely trusted research on brittle diabetes has also been the most severely and broadly criticized . so , for instance , more than ten years after the initial publication of the pioneering work of minuchin et al . in 1978 ( on what they called psychosomatic diabetes ) entitled psychosomatic families , critics commented as follows : as we conducted research and therapy with the families of diabetic children , we were impressed with both the limit of the formulation of the family 's role in diabetes offered in psychosomatic families ' and the uncritical acceptance that the book continued to enjoy . in their rather pointed article entitled the psychosomatic family ' reconsidered ii : recalling a defective model and looking ahead coyne and anderson criticize minuchin et al . primarily for their bold , yet statistically ( allegedly ) poorly supported , statements on the typical psychosomatic family psychosomatic family as featuring enmeshment , rigidity , overprotectiveness , and lack of conflict resolution and the children affected by brittle diabetes as having difficulty in handling stress , showing a tendency to internalize anger and being somewhat immature in their ability to cope with challenging situations ) and their overgeneralizations of these overall weak findings on familial situations in one psychosomatic illness to various psychosomatic illnesses . more specifically , small sample sizes and poor documentation of methodology ( or lack thereof ) are being highlighted . reflecting on such valid criticism in light of our own extensive research both on the subject of brittle diabetes in adolescents and on the various shortcomings of contemporary statistical approaches to time series data in psychosomatic medicine , we believe there is a case for both sides . on the one hand , we must vigorously support critics ( i.e. , ) in their call for much more detailed and sophisticated reports on and publication of statistical methodology in such complex and intricate research situations as are present in multivariate time series analysis . the reason lies in the fact that there is vast room for pitfalls and error with this type of research , if left in the hands of the mathematically inexperienced . on the other hand , however , we found for fact , that with the change of statistical approach , the results drawn from a given set of data may change somewhat , despite both methodologies being perfectly valid and academically accepted . so one wonders how this ( agreeably small ) imprecision of highly quantitative research is any different from the ( possibly but not necessarily larger ) inaccuracy of qualitative research due to subjectivity . [ 2 , 11 ] , the one finding which we were able to observe clinically before conducting any statistical testing at all , namely , that of a dominating mother having a negative effect on glycemic control of her child , was also a finding that both of our methodologies were able to report at a high level of significance . described as overprotectiveness in families with brittle diabetes is very similar , if not the same , to what we were able to pinpoint in terms of exaggerated control of a mother over her glycemically out of control child . ) additionally , we also fear that critics of primarily qualitative case research ( i.e. , ) may not have realized the vastness of data inherent even in a small sample in time series analysis , an apprehension possibly supported by the fait accompli of not too many critics providing any statistically evidenced findings on the subject of brittle diabetes themselves ( i.e. , ) . so in conclusion , we believe the careful observation of the clinically experienced therapist to be almost as valuable as the most substantiated and savvy statistical approach .

background . in questing for a more refined quantitative research approach , we revisited vector autoregressive ( var ) modeling for the analysis of time series data in the context of the so far poorly explored concept of family dynamics surrounding instable diabetes type 1 ( or brittle diabetes ) . method . we adopted a new approach to var analysis from econometrics referred to as the optimized multivariate lag selection process and applied it to a set of raw data previously analyzed through standard approaches . results . we illustrated recurring psychosomatic circles of cause and effect relationships between emotional and somatic parameters surrounding glycemic control of the child 's diabetes and the affective states of all family members . conclusion . the optimized multivariate lag selection process allowed for more specific , dynamic , and statistically reliable results ( increasing r2 tenfold in explaining glycemic variability ) , which were derived from a larger window of past explanatory variables ( lags ) . such highly quantitative versus historic more qualitative approaches to case study analysis of psychosomatics surrounding diabetes in adolescents were reflected critically .

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

despite all valid critique , one reason , we argue , may very well be the superiority of the single case study in first observing , describing , capturing , evaluating , and creatively reflecting on an infinite set of parameters surrounding any chosen topic . out of this primary assessment , novel hypotheses and it is our objective to reapply such primary assessment to the case of adolescent brittle diabetes ( or more generally speaking , the psychosomatic underpinnings of diabetes type 1 in minors and young adults ) , while also trying to answer calls for more quantitative and statistically reliable approaches to doing so . this in mind , we have first selected a highly quantitative case study on family dynamics and brittle diabetes and reviewed and reanalyzed its raw data through implementation of a new statistical procedure increasing the coefficient of determination in the new model by factor ten ( while also presenting new and clearer findings ) , in order to then , in a second step , discuss and compare our results to possibly the historically most well - known set of qualitative case studies on the topic . we will start by briefly revisiting the literature on the psychosomatics of adolescent instable diabetes type 1 , present a case vignette and basic data collection method of the original case study we reexamine ( which may be skipped by those familiar with the work published by ) , followed by a detailed description of our new statistical approach and its results , concluding with a clear clinically oriented graphical presentation of our findings and their discussion in light of minuchin et al . these cases of glycemic instability with no somatic explanation have been termed brittle diabetes by some authors and there is no doubt concerning the importance of further exploration of the causes and remedies surrounding this truly psychosomatic disease . while various aspects of brittle diabetes have been explored in recent years , including its exact definition , there seems to be a gap in the literature in exploring how emotional variables of all individuals within the family system may interact to affect glycemic control of the diabetic adolescent , the index patient of a dysfunctional family system . the adolescent index patient of this case study was diagnosed with diabetes type 1 at age of four ( clinical clues were polyuria , polydipsia , loss of appetite , a fungal infection , hba1c of 9.1 per cent , antibodies against islet cells , and gad65 ) . once all educational efforts concerning the diabetic management were exhausted ( including individual and family - based counseling , detailed and repetitive disease - specific education , and information about glycemic control mechanisms including the influence of nutrition , sport , and other aspects of blood sugar regulation ) , but a hba1c below 7 percent was never achieved , the family finally sought for psychosomatic family treatment . psychodynamically based therapeutic analysis of the family dynamic suggested a conflict between the adolescent and her mother about who had control of the blood sugar levels . fears of hypoglycemia were somewhat irrational with all three family members , including the father , who , at first sight , seemed rather more distant to the matter ( literature proposes parental hypoglycemia avoidance behaviours to adversely affect glycemic control ) . finally , a therapeutic intervention confronted them with their specific type of collusion concerning ( in-)dependence , in which both parents , in their manifest statements , advocated for more self - confidence and extended duties on the side of the daughter , but on a more latent level , gave hints to their beloved little girl not being ready to take control over the blood sugar monitoring by herself . this mostly unconscious conflict had culminated in cloudy paths of communication concerning glycemic control , in nebulous distributions of duties within the family members , and , as a result of the arrangement , in deep dissatisfaction over the failure of proper diabetic control . therefore , we aimed to statistically explore how specific basic affect states of all three individual family members may impact each other and the success of the diabetic management over a period of 120 days . to operationalize this quest , we drew on the standardized self - assessment manikin ( sam ) , as developed by bradley and lang ( for details see [ 13 , 14 ] ) , asking all three family members to individually record on a daily basis their valence ( mood ) , arousal ( high versus low ) , and dominance ( a sense of presence in the current environment ) . this form of diary based data collection is also referred to as ecological momentary assessment with many benefits in terms of accuracy and validity of measurements . standard deviations of the daily blood glucose measurements served as an indicator for glycemic variability , a measure which recent research has identified as the most precise predictor of diabetic control , followed by the hba1c - value in second place [ 1619 ] , due to it being the best known predictor for diabetic complications and microvascular derailments in particular . resulting from this data collection and primary analysis are ten time series : three time series for each of the three family members from the sam , affective valence ( happy , sad ) , arousal ( excited , calm ) , and dominance ( a sense of presence , distance to the current environment ) , as well as one time series recording glycemic variability ( daily standard deviations of measurements ) . , these ten time series were further analyzed by a completely new statistical approach to vector autoregressive ( var ) modeling . while past analysis of this same set of data ( see ) has also relied on basic var analysis , there had been some common shortcomings to the validity and scope of results , which we were able to remedy here , thus solving statistical shortcomings while also presenting completely new results in a clearer more clinically oriented fashion . how we were able to achieve this , the presentation of a newly developed optimized multivariate lag selection process in var analysis , and a comprehensive review of the principles of vector autoregression will be presented next . the use of vector autoregressive models ( var ) for the analysis of time series data in psychosomatic medicine ( also widely used in neuroscience ) allows treating a set of variables as jointly driven by the lagged values of all variables in the system with no a priori assignment of dependent and independent status being necessary . this technique seems particularly apt for research in psychosomatic medicine , where , among others , has long called for a more integrated ( monistic ) view on the complexity of dynamic dependencies and intertemporal reciprocal cause and effect relationships among different psychic as well as somatic variables . any var model requires the user to select a maximum number of lags , which , in more practical terms , refers to how far back in time the user wants to go in the search for past recordings of all variables to predict the present value of one variable . the farther back in time the user decides to go , the more explanatory variables ( lags ) need to be included in the model because it used to be improper to exclude past recordings of explanatory variables , which lay in - between the present value and the most historic one [ 22 , 23 ] . unfortunately including more explanatory variables ( going back further in time ) is a double edged sword , since this would provide a var model more representative of reality ( goodness of fit ) , but would also endorse one with less explanatory power ( lower adjusted r ) . the latter is due to the tremendous penalty inflicted by the large number of explanatory variables ( lags ) in the model resulting in high estimation variance [ 22 , 23 ] . this substantial drawback weakened the substance of empirical findings derived from var models , because researchers would either present results through models with teeth chattering low r values ( see previously published results from the same raw data as one example ) or adopt models only incorporating the effects of events preceding the predicted value of a variable by one day / one unit of time in the var ( e.g. in order to alleviate this shortcoming of low adjusted r values in the standard vector autoregressive modeling approach , we developed a computer code implementing a statistical procedure recently published in parts in savin and winker and winker [ 26 , 27 ] , referred to as the optimized multivariate lag selection process , which allows ( contrary to previous practice ) excluding such explanatory variables ( lags ) from the var model which add little to its goodness of fit ( estimated representativeness of reality ) while nonetheless reducing its explanatory power ( adjusted r ) . this admittance of holes to the lag structure ( equations organizing the explanatory variables ) allows us to now present an entirely new model exhibiting more detailed dynamics with a smaller number of parameters , for the data in this case resulting in about tenfold increase of the adjusted r value . mathematical details of applying the optimized multivariate lag selection process to this var analysis of the ten time series of the data set at hand will be presented next ( and may be skipped by the more clinically focused researcher ) . a standard vector autoregressive ( var ) model was constructed , using eviews 7.1 ( qms , quantitative micro software , irvine ca ) , based on the ten time series we mentioned above . given the large number of explanatory variables ( the more lags , the more variables ) and the limited number of observations , only a very limited number of lags ( past days ) could be considered while adjusted r would still be low , if we were to follow the standard modeling approach [ 22 , 23 ] . in more concrete terms , if we assume that any one value within the ten time series may have effects on any of the other values of all - time series with a delay of up to one week , a total of 710 parameters would have to be estimated . to resolve this dilemma , we drew on winker [ 26 , 27 ] and savin and winker engaging in optimized multivariate lag structure analysis . for this process , a computer code was developed using matlab r2011b with an interface to eviews 7.1 , which implements a genetic algorithm for the search of an optimized lag structure making use of information criteria ( bic ) as in the standard selection procedure ( see for more details ) . since the maintained lags are selected based on their joint informational content ( as measured by the information criteria ) , the procedure results in a model with much higher explanatory power ( for predicting glycemic variability adjusted r value of 0.20 as opposed to 0.02 for the standard model with only one lag ) and a richer dynamic . however , the zero constraints of the var model with holes preclude the application of standard methods for the calculation of confidence bands . similarly , poor glycemic control ( high glycemic variability ) will correlate with low glycemic variability four days earlier , a calm mother three days earlier , an excited mother seven days earlier , a dominating mother four days earlier , a nondominating mother seven days earlier ( although statistically insignificant ) , a sad father both five and six days earlier , a calm father both three and seven days earlier , and a dominating father both two and five days earlier . high glycemic variability will also correlate with a sad child six days later , an excited mother three days later , and a dominating father one day later . the optimized multivariate lag structure selection process provides one equation of seemingly unrelated multiple regression for each of the ten time series to be presented next . three of them directly involve glycemic variability in addition to the one for glycemic variability itself , which shall be presented last ( lags in parentheses ) : affective valence of the adolescent = 1 glycemic variability ( 6 ) + 2 valence adolescent ( 1 ) ( r = 0.25 , adj . r = 0.25 ) ; arousal of the mother = 19 glycemic variability ( 3 ) + 20 arousal adolescent ( 7 ) + 21 dominance adolescent ( 5 ) + 22 arousal mother ( 5 ) + 23 arousal mother ( 7 ) + 24 dominance mother ( 1 ) + 25 dominance father ( 6 ) ( r = 0.29 , adj . r = 0.64 ) ; dominance of the father = 40 glycemic variability ( 1 ) + 41 dominance child ( 6 ) + 42 valence mother ( 5 ) + 43 valence mother ( 7 ) + 44 dominance mother ( 4 ) + 45 dominance mother ( 6 ) + 46 valence father ( 1 ) + 47 valence father ( 3 ) + 48 arousal father ( 3 ) + 49 dominance father ( 2 ) ( r = 0.34 , adj . r = 0.25 ) ; arousal of the mother = 19 glycemic variability ( 3 ) + 20 arousal adolescent ( 7 ) + 21 dominance adolescent ( 5 ) + 22 arousal mother ( 5 ) + 23 arousal mother ( 7 ) + 24 dominance mother ( 1 ) + 25 dominance father ( 6 ) ( r = 0.29 , adj . r = 0.64 ) ; dominance of the father = 40 glycemic variability ( 1 ) + 41 dominance child ( 6 ) + 42 valence mother ( 5 ) + 43 valence mother ( 7 ) + 44 dominance mother ( 4 ) + 45 dominance mother ( 6 ) + 46 valence father ( 1 ) + 47 valence father ( 3 ) + 48 arousal father ( 3 ) + 49 dominance father ( 2 ) ( r = 0.34 , adj . the coefficients , their standard error , t - statistic , and probability referred to above , can be reviewed in table 1 . the development of a novel statistical methodology allowed us to disentangle the data and generate statistically reliable results in the form of ten equations . the dynamic of the results pertaining to glycemic variability , ( thereby , it has to be taken into account that additional dynamic interactions arise due to spillover between equations , which are not considered here ) , taking into account the direction of coefficients , can be summarized in the following words and graphical representations . low glycemic variability and , therefore , good diabetic control will correlate with the following : high glycemic variability four days earlier , an excited mother three days earlier , a calm mother seven days earlier , a non - dominating mother four days earlier , a dominating mother seven days earlier ( although statistically insignificant ) , a happy father both five and six days earlier , an excited father both three and seven days earlier , and a non - dominating father both two and five days earlier . low glycemic variability will also correlate with a happy child six days later , a calm mother three days later , and a non - dominating father one day later . similarly , poor glycemic control ( high glycemic variability ) will correlate with low glycemic variability four days earlier , a calm mother three days earlier , an excited mother seven days earlier , a dominating mother four days earlier , a non - dominating mother seven days earlier ( although statistically insignificant ) , a sad father both five and six days earlier , a calm father both three and seven days earlier , and a dominating father both two and five days earlier . likewise , phases of good diabetic management were followed by a continuously distant father ( low dominance ) , unfortunately a less alert mother ( low arousal ) , and a content ( happy ) adolescent index patient . similarly , mostly self - explanatory , graphical representations were constructed for the effects surrounding the affective valence of all three family members ( see figures 3 , 4 , and 5 ) . we picked these three timelines for more detailed examination , because the appropriate measurement of depressive symptoms ( which at least at a distance somewhat relates to affective valence ) in diabetics in general , remains to be a topic of current debate in the literature . in comparison to the results derived from the same set of raw data with a different statistical approach in an earlier publication , there are several improvements we were able to achieve:(i)increasing the coefficient of determination r for the model prediction of glycemic variability by factor ten ( adjusted r value of 0.20 as opposed to 0.02 ) while incorporating significant effects of explanatory variables ( lags ) stemming from a longer period of time preceding the predicted event;(ii)presenting a more precise timeline of effects of various variables on each other , including glycemic variability and vice versa ( e.g. , a nondominating mother four days prior to a set day will increase glycemic control instead of a nondominating mother somewhere up to four days prior to a set day will increase glycemic control);(iii)isolating additional relationships between variables which did not reach statistical significance earlier or took more time to take effect than the time frame of the earlier models allowed for . increasing the coefficient of determination r for the model prediction of glycemic variability by factor ten ( adjusted r value of 0.20 as opposed to 0.02 ) while incorporating significant effects of explanatory variables ( lags ) stemming from a longer period of time preceding the predicted event ; presenting a more precise timeline of effects of various variables on each other , including glycemic variability and vice versa ( e.g. , a nondominating mother four days prior to a set day will increase glycemic control instead of a nondominating mother somewhere up to four days prior to a set day will increase glycemic control ) ; isolating additional relationships between variables which did not reach statistical significance earlier or took more time to take effect than the time frame of the earlier models allowed for . a more substantial contribution of this paper is the demonstration and practical application of the multivariate lag selection process to var analysis , resolving an essential shortcoming in var analysis of ( relatively ) small samples . hence , this contribution to literature will have relevance beyond the case study approach but also to var - based studies of larger cohorts of patients ( as e.g. all in all , however , mathematically refined quantitative methodological approaches relying on modern computational technology can generate more specific , reproducible , and thus trustworthy results than purely qualitative ( narrative ) accounts , while still honoring the benefits of the case study approach aiming to explore previously unforeseen avenues fit for further vested inquiry ( often costly to perform ) . yet , we have to ask ourselves critically if the added mathematical complexity honors the overall value of the results a case study approach can provide . revisiting the opening comments of this report in the context of brittle diabetes , it seems interesting to note that particularly the most highly acclaimed and clinically widely trusted research on brittle diabetes has also been the most severely and broadly criticized . in 1978 ( on what they called psychosomatic diabetes ) entitled psychosomatic families , critics commented as follows : as we conducted research and therapy with the families of diabetic children , we were impressed with both the limit of the formulation of the family 's role in diabetes offered in psychosomatic families ' and the uncritical acceptance that the book continued to enjoy . primarily for their bold , yet statistically ( allegedly ) poorly supported , statements on the typical psychosomatic family psychosomatic family as featuring enmeshment , rigidity , overprotectiveness , and lack of conflict resolution and the children affected by brittle diabetes as having difficulty in handling stress , showing a tendency to internalize anger and being somewhat immature in their ability to cope with challenging situations ) and their overgeneralizations of these overall weak findings on familial situations in one psychosomatic illness to various psychosomatic illnesses . reflecting on such valid criticism in light of our own extensive research both on the subject of brittle diabetes in adolescents and on the various shortcomings of contemporary statistical approaches to time series data in psychosomatic medicine , we believe there is a case for both sides . on the other hand , however , we found for fact , that with the change of statistical approach , the results drawn from a given set of data may change somewhat , despite both methodologies being perfectly valid and academically accepted . [ 2 , 11 ] , the one finding which we were able to observe clinically before conducting any statistical testing at all , namely , that of a dominating mother having a negative effect on glycemic control of her child , was also a finding that both of our methodologies were able to report at a high level of significance . described as overprotectiveness in families with brittle diabetes is very similar , if not the same , to what we were able to pinpoint in terms of exaggerated control of a mother over her glycemically out of control child . ) so in conclusion , we believe the careful observation of the clinically experienced therapist to be almost as valuable as the most substantiated and savvy statistical approach .

despite all valid critique , one reason , we argue , may very well be the superiority of the single case study in first observing , describing , capturing , evaluating , and creatively reflecting on an infinite set of parameters surrounding any chosen topic . out of this primary assessment , novel hypotheses and it is our objective to reapply such primary assessment to the case of adolescent brittle diabetes ( or more generally speaking , the psychosomatic underpinnings of diabetes type 1 in minors and young adults ) , while also trying to answer calls for more quantitative and statistically reliable approaches to doing so . this in mind , we have first selected a highly quantitative case study on family dynamics and brittle diabetes and reviewed and reanalyzed its raw data through implementation of a new statistical procedure increasing the coefficient of determination in the new model by factor ten ( while also presenting new and clearer findings ) , in order to then , in a second step , discuss and compare our results to possibly the historically most well - known set of qualitative case studies on the topic . we will start by briefly revisiting the literature on the psychosomatics of adolescent instable diabetes type 1 , present a case vignette and basic data collection method of the original case study we reexamine ( which may be skipped by those familiar with the work published by ) , followed by a detailed description of our new statistical approach and its results , concluding with a clear clinically oriented graphical presentation of our findings and their discussion in light of minuchin et al . just about 33 percent of diabetics between 13 and 19 years of age manage to maintain tolerable glycemic control and a hba1c below 8 ; 6.3 percent suffered at least one episode of major hypoglycemia within the last three months [ 5 , 6 ] . these cases of glycemic instability with no somatic explanation have been termed brittle diabetes by some authors and there is no doubt concerning the importance of further exploration of the causes and remedies surrounding this truly psychosomatic disease . while various aspects of brittle diabetes have been explored in recent years , including its exact definition , there seems to be a gap in the literature in exploring how emotional variables of all individuals within the family system may interact to affect glycemic control of the diabetic adolescent , the index patient of a dysfunctional family system . the adolescent index patient of this case study was diagnosed with diabetes type 1 at age of four ( clinical clues were polyuria , polydipsia , loss of appetite , a fungal infection , hba1c of 9.1 per cent , antibodies against islet cells , and gad65 ) . family dynamics surrounding this classic family of three ( biological parents , single child ) appeared unsuspicious notwithstanding the girl 's history of poorly controlled bronchial asthma and allergic diseases . yet at age of six , nocturnal hypoglycemia with loss of consciousness led to readmission to the hospital , during which another episode of profound hypoglycemia , this time in conjunction with a tonic - clonic seizure , occurred , thus further consolidating her parents ' distress concerning hypoglycemia and hospital treatment . once all educational efforts concerning the diabetic management were exhausted ( including individual and family - based counseling , detailed and repetitive disease - specific education , and information about glycemic control mechanisms including the influence of nutrition , sport , and other aspects of blood sugar regulation ) , but a hba1c below 7 percent was never achieved , the family finally sought for psychosomatic family treatment . psychodynamically based therapeutic analysis of the family dynamic suggested a conflict between the adolescent and her mother about who had control of the blood sugar levels . fears of hypoglycemia were somewhat irrational with all three family members , including the father , who , at first sight , seemed rather more distant to the matter ( literature proposes parental hypoglycemia avoidance behaviours to adversely affect glycemic control ) . finally , a therapeutic intervention confronted them with their specific type of collusion concerning ( in-)dependence , in which both parents , in their manifest statements , advocated for more self - confidence and extended duties on the side of the daughter , but on a more latent level , gave hints to their beloved little girl not being ready to take control over the blood sugar monitoring by herself . this mostly unconscious conflict had culminated in cloudy paths of communication concerning glycemic control , in nebulous distributions of duties within the family members , and , as a result of the arrangement , in deep dissatisfaction over the failure of proper diabetic control . while traditional case studies would focus on the qualitative data outlined above , we sought to amend such observations by a highly quantitative approach in order to produce more evidence based and reproducible results . therefore , we aimed to statistically explore how specific basic affect states of all three individual family members may impact each other and the success of the diabetic management over a period of 120 days . to operationalize this quest , we drew on the standardized self - assessment manikin ( sam ) , as developed by bradley and lang ( for details see [ 13 , 14 ] ) , asking all three family members to individually record on a daily basis their valence ( mood ) , arousal ( high versus low ) , and dominance ( a sense of presence in the current environment ) . standard deviations of the daily blood glucose measurements served as an indicator for glycemic variability , a measure which recent research has identified as the most precise predictor of diabetic control , followed by the hba1c - value in second place [ 1619 ] , due to it being the best known predictor for diabetic complications and microvascular derailments in particular . resulting from this data collection and primary analysis are ten time series : three time series for each of the three family members from the sam , affective valence ( happy , sad ) , arousal ( excited , calm ) , and dominance ( a sense of presence , distance to the current environment ) , as well as one time series recording glycemic variability ( daily standard deviations of measurements ) . while past analysis of this same set of data ( see ) has also relied on basic var analysis , there had been some common shortcomings to the validity and scope of results , which we were able to remedy here , thus solving statistical shortcomings while also presenting completely new results in a clearer more clinically oriented fashion . how we were able to achieve this , the presentation of a newly developed optimized multivariate lag selection process in var analysis , and a comprehensive review of the principles of vector autoregression will be presented next . the use of vector autoregressive models ( var ) for the analysis of time series data in psychosomatic medicine ( also widely used in neuroscience ) allows treating a set of variables as jointly driven by the lagged values of all variables in the system with no a priori assignment of dependent and independent status being necessary . this technique seems particularly apt for research in psychosomatic medicine , where , among others , has long called for a more integrated ( monistic ) view on the complexity of dynamic dependencies and intertemporal reciprocal cause and effect relationships among different psychic as well as somatic variables . any var model requires the user to select a maximum number of lags , which , in more practical terms , refers to how far back in time the user wants to go in the search for past recordings of all variables to predict the present value of one variable . the farther back in time the user decides to go , the more explanatory variables ( lags ) need to be included in the model because it used to be improper to exclude past recordings of explanatory variables , which lay in - between the present value and the most historic one [ 22 , 23 ] . this substantial drawback weakened the substance of empirical findings derived from var models , because researchers would either present results through models with teeth chattering low r values ( see previously published results from the same raw data as one example ) or adopt models only incorporating the effects of events preceding the predicted value of a variable by one day / one unit of time in the var ( e.g. in order to alleviate this shortcoming of low adjusted r values in the standard vector autoregressive modeling approach , we developed a computer code implementing a statistical procedure recently published in parts in savin and winker and winker [ 26 , 27 ] , referred to as the optimized multivariate lag selection process , which allows ( contrary to previous practice ) excluding such explanatory variables ( lags ) from the var model which add little to its goodness of fit ( estimated representativeness of reality ) while nonetheless reducing its explanatory power ( adjusted r ) . this admittance of holes to the lag structure ( equations organizing the explanatory variables ) allows us to now present an entirely new model exhibiting more detailed dynamics with a smaller number of parameters , for the data in this case resulting in about tenfold increase of the adjusted r value . mathematical details of applying the optimized multivariate lag selection process to this var analysis of the ten time series of the data set at hand will be presented next ( and may be skipped by the more clinically focused researcher ) . in order to focus on the innovative aspects of our methodology we will not delve into the details of var model construction , which have been described at length in preceding publications ( i.e. given the large number of explanatory variables ( the more lags , the more variables ) and the limited number of observations , only a very limited number of lags ( past days ) could be considered while adjusted r would still be low , if we were to follow the standard modeling approach [ 22 , 23 ] . in more concrete terms , if we assume that any one value within the ten time series may have effects on any of the other values of all - time series with a delay of up to one week , a total of 710 parameters would have to be estimated . for this process , a computer code was developed using matlab r2011b with an interface to eviews 7.1 , which implements a genetic algorithm for the search of an optimized lag structure making use of information criteria ( bic ) as in the standard selection procedure ( see for more details ) . since the maintained lags are selected based on their joint informational content ( as measured by the information criteria ) , the procedure results in a model with much higher explanatory power ( for predicting glycemic variability adjusted r value of 0.20 as opposed to 0.02 for the standard model with only one lag ) and a richer dynamic . given the rich dynamics between all variables of the model , besides considering single equations , the calculation of impulse response functions as in would be of interest . similarly , poor glycemic control ( high glycemic variability ) will correlate with low glycemic variability four days earlier , a calm mother three days earlier , an excited mother seven days earlier , a dominating mother four days earlier , a nondominating mother seven days earlier ( although statistically insignificant ) , a sad father both five and six days earlier , a calm father both three and seven days earlier , and a dominating father both two and five days earlier . three of them directly involve glycemic variability in addition to the one for glycemic variability itself , which shall be presented last ( lags in parentheses ) : affective valence of the adolescent = 1 glycemic variability ( 6 ) + 2 valence adolescent ( 1 ) ( r = 0.25 , adj . r = 0.18 ) ; arousal of the adolescent = 12 arousal adolescent ( 1 ) + 13 arousal adolescent ( 3 ) + 14 arousal adolescent ( 7 ) + 15 valence mother ( 4 ) + 16 arousal mother ( 3 ) + 17 valence father ( 2 ) + 18 valence father ( 6 ) ( r = 0.30 , adj . r = 0.25 ) ; arousal of the mother = 19 glycemic variability ( 3 ) + 20 arousal adolescent ( 7 ) + 21 dominance adolescent ( 5 ) + 22 arousal mother ( 5 ) + 23 arousal mother ( 7 ) + 24 dominance mother ( 1 ) + 25 dominance father ( 6 ) ( r = 0.29 , adj . r = 0.64 ) ; dominance of the father = 40 glycemic variability ( 1 ) + 41 dominance child ( 6 ) + 42 valence mother ( 5 ) + 43 valence mother ( 7 ) + 44 dominance mother ( 4 ) + 45 dominance mother ( 6 ) + 46 valence father ( 1 ) + 47 valence father ( 3 ) + 48 arousal father ( 3 ) + 49 dominance father ( 2 ) ( r = 0.34 , adj . r = 0.27 ) ; glycemic variability = 1 glycemic variability ( 4 ) + 2 arousal mother ( 3 ) + 3 arousal mother ( 7 ) + 4 dominance mother ( 4 ) + 5 dominance mother ( 7 ) + 6 valence father ( 5 ) + 7 valence father ( 6 ) + 8 arousal father ( 3 ) + 9 arousal father ( 7 ) + 10 dominance father ( 2 ) + 11 dominance father ( 5 ) ( r = 0.28 , adj . r = 0.18 ) ; arousal of the adolescent = 12 arousal adolescent ( 1 ) + 13 arousal adolescent ( 3 ) + 14 arousal adolescent ( 7 ) + 15 valence mother ( 4 ) + 16 arousal mother ( 3 ) + 17 valence father ( 2 ) + 18 valence father ( 6 ) ( r = 0.30 , adj . r = 0.25 ) ; arousal of the mother = 19 glycemic variability ( 3 ) + 20 arousal adolescent ( 7 ) + 21 dominance adolescent ( 5 ) + 22 arousal mother ( 5 ) + 23 arousal mother ( 7 ) + 24 dominance mother ( 1 ) + 25 dominance father ( 6 ) ( r = 0.29 , adj . r = 0.64 ) ; dominance of the father = 40 glycemic variability ( 1 ) + 41 dominance child ( 6 ) + 42 valence mother ( 5 ) + 43 valence mother ( 7 ) + 44 dominance mother ( 4 ) + 45 dominance mother ( 6 ) + 46 valence father ( 1 ) + 47 valence father ( 3 ) + 48 arousal father ( 3 ) + 49 dominance father ( 2 ) ( r = 0.34 , adj . r = 0.27 ) ; glycemic variability = 1 glycemic variability ( 4 ) + 2 arousal mother ( 3 ) + 3 arousal mother ( 7 ) + 4 dominance mother ( 4 ) + 5 dominance mother ( 7 ) + 6 valence father ( 5 ) + 7 valence father ( 6 ) + 8 arousal father ( 3 ) + 9 arousal father ( 7 ) + 10 dominance father ( 2 ) + 11 dominance father ( 5 ) ( r = 0.28 , adj . the dynamic of the results pertaining to glycemic variability , ( thereby , it has to be taken into account that additional dynamic interactions arise due to spillover between equations , which are not considered here ) , taking into account the direction of coefficients , can be summarized in the following words and graphical representations . low glycemic variability and , therefore , good diabetic control will correlate with the following : high glycemic variability four days earlier , an excited mother three days earlier , a calm mother seven days earlier , a non - dominating mother four days earlier , a dominating mother seven days earlier ( although statistically insignificant ) , a happy father both five and six days earlier , an excited father both three and seven days earlier , and a non - dominating father both two and five days earlier . similarly , poor glycemic control ( high glycemic variability ) will correlate with low glycemic variability four days earlier , a calm mother three days earlier , an excited mother seven days earlier , a dominating mother four days earlier , a non - dominating mother seven days earlier ( although statistically insignificant ) , a sad father both five and six days earlier , a calm father both three and seven days earlier , and a dominating father both two and five days earlier . a graphical representation of this paragraph is presented in figure 2 in clinical terms , this means , good diabetic control was preceded by attentive and alert ( high arousal , excited ) parents with a positive attitude ( happy father ) , at the same time refraining from too much overwhelming presence ( low dominance ) . in comparison to the results derived from the same set of raw data with a different statistical approach in an earlier publication , there are several improvements we were able to achieve:(i)increasing the coefficient of determination r for the model prediction of glycemic variability by factor ten ( adjusted r value of 0.20 as opposed to 0.02 ) while incorporating significant effects of explanatory variables ( lags ) stemming from a longer period of time preceding the predicted event;(ii)presenting a more precise timeline of effects of various variables on each other , including glycemic variability and vice versa ( e.g. , a nondominating mother four days prior to a set day will increase glycemic control instead of a nondominating mother somewhere up to four days prior to a set day will increase glycemic control);(iii)isolating additional relationships between variables which did not reach statistical significance earlier or took more time to take effect than the time frame of the earlier models allowed for . increasing the coefficient of determination r for the model prediction of glycemic variability by factor ten ( adjusted r value of 0.20 as opposed to 0.02 ) while incorporating significant effects of explanatory variables ( lags ) stemming from a longer period of time preceding the predicted event ; presenting a more precise timeline of effects of various variables on each other , including glycemic variability and vice versa ( e.g. a more substantial contribution of this paper is the demonstration and practical application of the multivariate lag selection process to var analysis , resolving an essential shortcoming in var analysis of ( relatively ) small samples . all in all , however , mathematically refined quantitative methodological approaches relying on modern computational technology can generate more specific , reproducible , and thus trustworthy results than purely qualitative ( narrative ) accounts , while still honoring the benefits of the case study approach aiming to explore previously unforeseen avenues fit for further vested inquiry ( often costly to perform ) . revisiting the opening comments of this report in the context of brittle diabetes , it seems interesting to note that particularly the most highly acclaimed and clinically widely trusted research on brittle diabetes has also been the most severely and broadly criticized . in 1978 ( on what they called psychosomatic diabetes ) entitled psychosomatic families , critics commented as follows : as we conducted research and therapy with the families of diabetic children , we were impressed with both the limit of the formulation of the family 's role in diabetes offered in psychosomatic families ' and the uncritical acceptance that the book continued to enjoy . primarily for their bold , yet statistically ( allegedly ) poorly supported , statements on the typical psychosomatic family psychosomatic family as featuring enmeshment , rigidity , overprotectiveness , and lack of conflict resolution and the children affected by brittle diabetes as having difficulty in handling stress , showing a tendency to internalize anger and being somewhat immature in their ability to cope with challenging situations ) and their overgeneralizations of these overall weak findings on familial situations in one psychosomatic illness to various psychosomatic illnesses . reflecting on such valid criticism in light of our own extensive research both on the subject of brittle diabetes in adolescents and on the various shortcomings of contemporary statistical approaches to time series data in psychosomatic medicine , we believe there is a case for both sides . on the other hand , however , we found for fact , that with the change of statistical approach , the results drawn from a given set of data may change somewhat , despite both methodologies being perfectly valid and academically accepted . [ 2 , 11 ] , the one finding which we were able to observe clinically before conducting any statistical testing at all , namely , that of a dominating mother having a negative effect on glycemic control of her child , was also a finding that both of our methodologies were able to report at a high level of significance . described as overprotectiveness in families with brittle diabetes is very similar , if not the same , to what we were able to pinpoint in terms of exaggerated control of a mother over her glycemically out of control child . )

sepsis affects approximately 700,000 people annually and accounts for about 210,000 deaths per year in the us . its incidence is rising at rates between 1.5% and 8% per year , despite continuous progress in the development of antimicrobial therapeutics and supportive cares . most of the sepsis is caused by bacteria and bacteria of abdominal origin contribute to the second major reason for sepsis . the septic peritonitis is the host 's systemic inflammatory response to the bacteria , initiated by the pathogen associated molecule patterns ( pamps ) including lipopolysaccharide and lipid a from gram - negative bacteria and lipoteichoic acid and peptidoglycan from gram - positive bacteria . the inflammatory initiation leads to the release of chemokines , such as interleukin-8 ( il-8 ) , and proinflammatory cytokines , such as tumor necrosis factor- ( tnf- ) , il-1 , il-6 , and il-12 , in a massive amount . the cytokines , if not appropriately controlled , may severely impair the functions of vital organs or systems , resulting in death . data from the ciaow study ( complicated intra - abdominal infections worldwide observational study ) showed that the overall mortality rate was 10.5% in the patients with septic peritonitis in a worldwide context . septic peritonitis is characterized by a massive infiltration of neutrophils into the peritoneum in response to bacterial invasion , where they are activated and act as a first line of defense against the bacteria . morphologically , the mature neutrophils are unique among the other white blood cells by their lobulated nucleus , which inspired the renaming of them as polymorphonuclear neutrophils ( pmns ) . based on cytokine production , macrophage activation , expression of toll - like receptor ( tlr ) , and surface antigen expression , murine pmns have been classified into three types : normal pmn ( pmn - n ) , pmn - i , and pmn - ii . pmn - i produces il-12/ccl3 , activates macrophages in a classical way , expresses tlr2/tlr4/tlr5/tlr8 , and has a cd49dcd11b phenotype . pmn - ii produces il-10/ccl2 , activates macrophages in an alternative manner , expresses tlr2/tlr4/tlr7/tlr9 , and possesses a cd49dcd11b phenotype . pmn - n rarely produces cytokine / chemokine , displays no effect on macrophage activation , and expresses tlr2/tlr4/tlr9 , with cd49dcd11b phenotypes . the pmn - n may convert to pmn - i or pmn - ii in response to bacterial infections . recently , macrophages have been reported to cooperate with neutrophils by promoting extravasation and activation of pmns , even their clearance of bacteria . accumulated evidence revealed that toll - like receptors ( tlrs ) could play a fundamental role in induction of hyperinflammation and tissue damage in sepsis . tlrs are pattern recognition receptors ( prrs ) that sense microbial invasion and initiate innate immune responses . positioned at the cell surface , tlr4 is essential for sensing lipopolysaccharide of gram - negative bacteria and tlr2 is involved in the recognition of a large panel of microbial ligands , while tlr5 recognizes flagellin . endosomal tlr3 , tlr7 , tlr8 , and tlr9 are specialized in the sensing of nucleic acids produced notably during viral infections or during bacterial infections . conventionally , tlr9 is thought to sense microbial dna in endolysosomes and not at the cell surface . recently , it has been found that tlr9 is expressed on the surface of human and mouse pmns , that the surface tlr9 ( stlr9 ) senses dna in pmns , and that stlr9 expressing pmns ( stlr9 pmns ) have roles in inducing rapid inflammation . human and mouse pmns spontaneously express stlr9 , and the stlr9 expression is upregulated in response to pmn stimulation . in front of tlr9 ligands , tlr9 mice exhibited lower serum inflammatory cytokine levels , higher bacterial clearance , and greater survival after experimental peritonitis induced by cecal ligation and puncture ( clp ) . a single injection of tlr9 antagonist protected the wide type mice , even when administered as late as 12 hours after clp . it has been proposed that , during infection , stlr9 pmns could be involved in the detrimental effect by releasing massive proinflammatory cytokines , including il-6 and tnf- , and possibly other cytokines in response to pamps [ 12 , 13 ] . interleukin-17 ( il-17 ) is a cytokine family that signatures t helper 17 ( th17 ) cell subset and contains six members ( il-17a to il-17f ) , among which il-17a is considered as one of the major proinflammatory cytokines mediating the innate and adaptive immune responses against bacterial infections . in addition to th17 cells , t cells , innate lymphoid cells ( ilcs ) , mast cells , pmns , and macrophages are also il-17 producing cells . notably , innate immune cell - derived il-17 constitutes a major element in the immune response against infectious agents by recruiting pmns to the sites of infections and by inducing the production of antimicrobial peptides , cxc chemokines , and granulocyte colony stimulating factor ( g - csf ) [ 15 , 16 ] . in bacterial infection , pmns act to produce increased il-17 , which in turn recruits more pmns to join the fighting against invaded bacteria , resulting in increased production of innate immune cell - derived il-17 [ 16 , 17 ] . il-17 was found to work in an il-23-il-17 axis which was critical for the survival of the host infected with bacteria . in the axis , tissue - infiltrating pmns could be the main source of il-23 which induces production of il-17 from macrophages . in the present study , we kinetically observed the infiltration of pmns and expression of stlr9 and il-17 on / in the pmns in the peritoneal lavage cells ( plcs ) of mice intraperitoneally infected with various doses of escherichia coli ( e. coli ) , aiming to find the correlation of the expression of stlr9 and il-17 on / in the pmns during the development of bacterial septic peritonitis . the achieved results could provide a basis for further investigation on the roles of stlr9 expressing pmns and il-17 producing pmns in bacterium caused septic peritonitis . the monoclonal antibodies of apc - conjugated rat anti - mouse cd45 antibody ( 561689 ) , pe - conjugated rat anti - mouse cd14 antibody ( 561711 ) , pe - conjugated rat anti - mouse cd11b antibody ( 561689 ) , apc - cy 7-cojugated rat anti - mouse cd3 antibody ( 560590 ) , and apc - conjugated rat anti - mouse ly-6 g antibody ( 560599 ) were purchased from bd bioscience . the fitc - conjugated active anti - tlr9 monoclonal antibody ( 26c593.2 ) was from abcam ; fitc - conjugated anti - mouse il-17a antibody ( 506908 ) and percp - cy 5.5-conjugated rat anti - mouse il-17a ( 3354955 ) antibody were from biolegend . cdna synthesis kit ( transgene biotech , i21021 ) and two - step sybr green qpcr assays ( transgene biotech , g31227 ) were from biotech . e. coli strain of jm109 was recovered from lyophilized powder by being suspended into lb medium and then cultured on lb agar plate at 37c overnight . the single colonies of e. coli on the plate were used as a group of original seed e. coli . female icr mice were purchased from the experimental animal center , medical college of norman bethune , jilin university , and maintained in microisolator cages under specific pathogen - free conditions . the experimental manipulation of mice was undertaken in accordance with the national institute of health guide for the care and use of laboratory animals , with the approval of the scientific investigation board of science & technology of jilin province . to prepare e. coli culture , each single colony of e. coli was picked up and cultured in 5 ml lb medium at 37c with shaking at 200 rpm . when the od value ( a600 ) of the culture reached 1.4 , e. coli was harvested from the culture by centrifugation and mixed with 20% glycerol solution . the working seed e. coli were seeded into lb medium and cultured at 37c till the od value reached 0.7 followed by harvesting the e. coli pellet after centrifugation at 4000 g for 5 min . the e. coli pellet was diluted in a serial tenfold , plated on an eosin methylene blue agar plate , and then cultured at 37c for 14 hours . the colony - forming units ( cfus ) of the e. coli culture were calculated and determined as approximately 0.8 10 cfus / ml . for animal experiment use , the e. coli pellet was washed twice using sterile 0.9% nacl ( saline ) and then resuspended in 1.0 ml saline containing 0.4 , 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , and was ready to use for infecting mice . for inducing bacterial septic peritonitis , mice were injected intraperitoneally ( i.p . ) with 1.0 ml of e. coli preparations containing 0.4 , 0.8 , 1.6 , and 2.4 10 cfus / ml in sterile saline , respectively . the survival of the mice was monitored every 4 hours for 4 days . to harvest the peritoneal lavage cells ( plcs ) from mice either infected with e. coli or injected with saline , the mice ( n 3 per group ) were euthanized at defined time points with 50 mg / kg pentobarbital sodium followed by washing peritoneal cavities using 6 ml per mouse of ice - cold phosphate - buffered saline ( pbs ) . the peritoneal lavage fluid was centrifuged at 750 g for 5 minutes at 4c for harvesting the plcs . plcs were harvested from the nave mice and maintained in rpmi 1640 supplemented with 10% ( v / v ) fetal bovine serum ( fbs ) ( gibco ) and antibiotics ( 100 iu of penicillin / ml and 100 iu of streptomycin / ml ) . in experiments using viable bacteria , antibiotics were not added to the culture medium during the isolation , washing , or subsequent culturing period . no bacterial contamination was observed in plcs cultured in the absence of antibiotics . cells were counted and then plated in 24-well cell culture plates ( costar , cambridge , ma ) at an approximate density of 1 10 cells / well . the plcs were cocultured with e. coli at 1 10 or 1 10 cfus / well or saline as a vehicle control for 14 hours , and then they were cultured with bfa for another 4 hours , in a 5% co2 humidified incubator at 37c . the plcs were collected and stained with fitc - labeled anti - il-17 mab , followed by flow cytometry analysis to detect the expression of il-17 . to count the numbers of cells in each peritoneal lavage sample the cell pellets were smeared on slides and then stained using hematoxylin - eosin ( he ) followed by counting the cell numbers on hemocytometer ( beckman coulter , fullerton , ca ) and taking photos of the cells . for surface staining , the plcs were stained with fluorescence - conjugated mabs against cd45 , cd11b , and stlr9 , respectively , for 30 minutes at room temperature in the dark followed by washing twice with pbs . for il-17 intracellular staining , the plcs were surface stained as described above and then fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin followed by staining with fitc - conjugated anti - il17a monoclonal antibody . all stained cells were analyzed by flow cytometer facscalibur ( bd ) and cytoflex ( beckman coulter ) . the plcs were harvested from mice ( n 6 per group ) either infected with e. coli or injected with saline . the plcs were stained with fluorescence - conjugated mabs against cd14 , cd11b , and cd3 for 30 minutes at room temperature in the dark , followed by washing twice with pbs . the stained cells were sorted using bd facs aria ii by the methods described in figure 5(g ) . the neutrophils were sorted by selection for cd3cd14cd11b cells , and t cells were sorted by selection for cd3 cells , according to the manufacturer 's recommendations . t cells ( defined as cd3 cells with a purity > 83% of living cells ) and pmns ( defined as cd3cd14cd11b cells with a purity > 96.7% of living cells ) were obtained . total rna was isolated from the plcs with trizol reagent and reverse - transcribed using cdna synthesis kit . quantitative real - time pcr ( qrt - pcr ) was performed using two - step sybr green qpcr assays and the target mrnas were identified by the specific primers as follows : il-17a , forward : 5-aaggcagcagcgatcatccct ; reverse : 3-tcttcattgcggtggagagtcc ; gapdh , forward : 5-atcaccatcttccaggagcga ; reverse : 3-tctcgtggttcacacccatca . the data were acquired using the step one real - time pcr system ( applied biosystems ) . the procedure of the target mrna amplification was as follows : 1 cycle at 95c ( 30 seconds ) followed by 40 cycles at 95c ( 5 seconds ) and 64c ( 31 seconds ) . the relative amount of gene expression was calculated according to the formula 2 , in which ct = [ ct(gene ) ct(gapdh ) ] and ct is the crossing threshold value returned by the pcr instrument for every gene amplification . survival curves of mice were estimated using the kaplan - meier method and compared using the log - rank test . we considered the resulting p values of less than 0.05 ( 95% ci ) to be statistically significant . statistics were analyzed using graphpad prism 5.0 for windows ( san diego , ca ) . to understand how neutrophils contribute to the development of sepsis , we first established a murine model of septic peritonitis induced by escherichia coli ( e. coli ) . the female icr mice , 10 in each group , were intraperitoneally injected with 2.4 10 , 1.6 10 , 0.8 10 , and 0.4 10 colony forming units ( cfus ) of e. coli , respectively , and then monitored for their physical conditions and survival . around 6 hours after the infection , all of the 10 mice received 2.4 10 cfus of e. coli , 8 out of the 10 mice received 1.6 10 cfus of e. coli , and 5 out of the 10 mice that received 0.8 10 cfus of e. coli began to exhibit multiple neurological symptoms , including dispirited behavior , staggered gait , and trembling , but these phenomena were not present in all of the 10 mice that received 0.4 10 cfus of e. coli or saline . around 18 hours after the infection , the infected mice began to die . around 48 hours , the infected mice underwent a massive death , especially in those that received high infectious dose of e. coli . by 72 hours after the infection , 80% , 60% , and 40% of the mice infected with 2.4 , 1.6 , and 0.8 10 cfus of e. coli , respectively , died and yet 100% of the mice which were infected with 0.4 10 cfus of e. coli or received saline survived ( figure 1 ) . the results indicated that the infection with e. coli at 1.6 10 cfus and 2.4 10 cfus could induce septic peritonitis in icr mice . since the polymorphonuclear cells ( pmns ) are documented as the first innate immune cells recruited at inflammation sites and play a central role in host defense , we next collected the peritoneal lavage cells ( plcs ) of the mice at 18 , 48 , and 72 hours after infection , respectively , for observing the infiltration and morphology of the pmns in the plcs . the collected plcs were fixed on glass slides , stained with h&e dye , and photographed . a massive infiltration of pmns was observed in the plcs from the infected mice . at 18 hours after infection , in the plcs of the mice infected with 1.6 and 2.4 10 cfus of e. coli , the majority of the cells with the size of 0.2~0.4 micrometer were pmns with a typical lobulated nucleus . interestingly , there occurred a portion of giant cells , with the size of nearly 1.0~1.6 micrometer , characterized by lobulated nucleus that was squeezed to the marginal zone of the inner cell membrane . however , at 48 or 72 hours , the giant cells disappeared in the plcs of the mice infected with 1.6 and 2.4 10 cfus of e. coli ( figure 2(a ) ) . morphologically , there are four types of cells in the plcs , including pmns , macrophages , lymphocytes , and the giant cells ( figure 2(b ) ) . when counting the cells , we found that at 18 , 48 , and 72 hours , pmns constituted 50%80% and macrophages constituted 20%40% of the plcs from the mice infected with 4 doses of e. coli , respectively , compared with nearly 30% for pmns and 60% for macrophages in the plcs from the mice that received saline . at 18 hours , interestingly , there were about 20% of the giant cells in the plcs of the mice infected with 1.6 and 2.4 10 cfus of e. coli ( figure 2(b ) , upper right ) . noticeably , when the mice died of the infection , we collected the plcs immediately and observed and found that plenty of bacteria coexisted with the pmns in the plcs of the mouse infected with 1.6 10 cfus of e. coli for 31 hours ( figure 2(c ) ) . these results indicate that neutrophils can move to infected peritoneal cavity by infiltration and change their morphologies after engulfing bacteria possibly . during septic peritonitis , a large number of pmns were recruited to the peritoneal cavity of the infected mice . to find the correlation of the numbers of pmns at various time points with the severity of the disease in mice infected with e. coli , the plcs of the mice were collected at 18 , 48 , and 72 hours after infection , counted by hemocytometer or flow cytometry after staining with fluorescence - labeled mab of anti - cd45 and anti - cd11b . at 18 hours , the numbers of plcs were nearly 1 10 cells / ml of peritoneal lavage fluid ( plf ) in the mice received saline ( control mice ) , significantly increased in the mice infected with 3 doses of e. coli in a dose dependent manner , and even reached nearly 3 10 cells / ml of plf in the mice infected with 2.4 10 cfus of e. coli ( p < 0.05 ) . at 48 hours , the numbers of plcs were less than 1 10 cells / ml of plf in control mice and significantly increased to over 3 10 cells / ml of plf in the mice infected with three doses of e. coli and even to about 4 10 cells / ml of plf in the mice infected with 1.6 and 2.4 10 cfus of e. coli ( p < 0.05 ) . at 72 hours , the numbers of plcs were decreased to 0.5 10 cells / ml of plf in control mice and decreased to nearly 0.5 10 cells / ml of plf in the mice infected with three doses of e. coli ( figure 3(a ) ) . in the plcs from the infected mice , most of the cells were cd45 nucleated leukocytes , representing pmns , macrophages , and lymphocytes , respectively . at 18 hours , the pmns , macrophages , and lymphocytes were displayed on the left , middle , and right of the histogram , respectively , sequentially based on their nuclear structure and fluorescence intensity . at 48 and 72 hours , the pmns were found to be shifted to the right upper quadrant , indicating that the pmns were with more ovulated nuclei and expressed elevated cd45 . as shown in figure 3(b ) , at 18 hours after infection , the cd45 pmns constituted 61.5% , 49.6% , and 56.4% of the plcs in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , compared with 32.5% in the control mice . at 48 hours , the cd45 pmns were increased to 59.5% , 63.7% , and 54.7% in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , compared with 41.2% from the control mice . at 72 hours , the cd45 pmns constituted 62.2% , 62.8% , and 63.5% of the plcs in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , compared with 39.5% from the control mice . the pmns were further detected as cd11bcd45 pmns and cd11bcd45 pmns , and their ratios in the pmns were calculated . as shown in figure 3(c ) , the cd11bcd45 pmns constituted 49% , 36% , and 13% of the pmns in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli for 18 hours , respectively . at 48 hours after the infection , the cd11bcd45 pmns constituted 91% , 88% , and 86% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively . at 72 hours after the infection , the cd11b pmns reached around 96% in all infected mice . oppositely , at 18 hours , the cd11b pmns constituted 51% , 64% , and 87% of the pmns in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively . at 48 hours after the infection , the cd11b pmns constituted 9% , 12% , and 14% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively . at 72 hours after the infection , the cd11b pmns only accounted for around 4% in all infected mice ( figure 3(c ) ) . the results indicated that the peritoneal infection of e. coli induced massive infiltration of cd45 pmns . among the cd45 pmns , cd11b pmns were increased with the progression of infection . in parallel , the cd11b pmns were decreased . at 18 and 48 hours after infection , the ratios of cd11b pmns or cd11b pmns were correlated with the doses of e. coli negatively or positively . since primary human and mouse blood neutrophils were reported to be able to express a functional stlr9 , we tried to detect whether the expression of stlr9 was different on cd11b pmns and cd11b pmns and correlates with development of septic peritonitis in mice . the plcs collected from the mice which were infected with e. coli or received saline were gated for detecting the expression of stlr9 on both cd11b and cd11b pmns ( figure 4(a ) ) . results showed that , at 18 hours after infection , the ratios of stlr9cd11b pmns were increased to 57% , 51% , and 70% of the cd11b pmns in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively ( p < 0.05 ) , compared to 12% in the mice that received saline control ( control mice ) . at 48 hours , the ratios of stlr9cd11b pmns were decreased to 2.3% , 2.3% , ( p < 0.05 ) and 8% in the infected mice , respectively , and less than 13% as in control mice . at 72 hours , the ratios of stlr9cd11b pmns were 7% , 5.5% , and 9% in the infected mice , respectively , compared with 18% in control mice ( figure 4(b ) ) . the ratios of stlr9cd11b pmns constituted 52% , 53% , and 68.5% of the cd11b pmns in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours ( p < 0.05 ) , compared with 3.5% in control mice , sharply decreased to 9% , 6% , and 9.5% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 8% in control mice , and reached 10% , 6% , and 10% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours , compared with 8% in control mice ( figure 4(c ) ) . these results indicated that e. coli could significantly induce the occurrence of both of the stlr9cd11b pmns and stlr9cd11b pmns in the peritoneal cavity at early stage of infection . considering the fact that macrophages are the inflammatory cells which overlapped with the pmns in the inflammatory sites , we also observed stlr9 expression on macrophages in the plcs . the results showed that the ratios of stlr9 macrophages constituted 5% , 6% , and 6.7% ( p < 0.05 ) of the macrophages in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours , compared with 3% in control mice , decreased to 0.9% ( p < 0.05 ) , 0.45% ( p < 0.05 ) , and 3% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 5% in control mice , and reached 4.8% , 2% ( p < 0.05 ) , and 2.8% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours , compared with 5.5% in control mice ( figure 4(d ) ) . these results indicated that , unlike pmns , the macrophages barely express increased stlr9 during septic peritonitis . in recent years , primary human and mouse neutrophils have been found to be able to display autocrine il-17 activity that probably contributes to the etiology of microbial and inflammatory diseases . to find whether the different subtype neutrophil derived il-17 was involved in the development of septic peritonitis , we detected il-17 expression in the cd11b pmns and cd11b pmns ( figure 5(a ) ) of the plcs collected from the mice which were infected with e. coli or received saline ( control mice ) . it was found that the ratios of il-17cd11b pmns constituted 7% , 8.5% , and 10% of the cd11b pmns in plcs of the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours ( p < 0.05 ) , compared with 25% in control mice , was 2% ( p < 0.05 ) , 7.5% ( p < 0.05 ) , and 25.5% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 25% in control mice , and reached 6% , 10% , and 8% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours ( p < 0.05 ) , compared with 27% in control mice ( figure 5(b ) ) . noticeably , the saline injection seemed to stimulate neutrophils to express il-17 . to confirm this , the plcs were collected from the mice injected with saline at 18 , 48 , or 72 hours after the injection , respectively , and stained with fluorescence - labeled mab against cd45 , cd11b , and il-17 , followed by detection using flow cytometry . we found that saline stimulation tended to induce il-17 expression ( not statistically significant ) with a big range of variation individually ( data not shown ) . the ratios of the il-17cd11b pmns were 5% , 4% , and 3% of the cd11b pmns in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours , compared with 7% in control mice ; 3% , 11% , and 21.5% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 7% in control mice ; and 7% , 8% , and 13% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours , compared with 9% in control mice ( figure 5(c ) ) . the results indicated that both of the cd11b pmns and the cd11b pmns could not increase il-17 expression during the development of septic peritonitis in mice induced by e. coli . also , as shown in figure 5(d ) , macrophages could not increase the il-17 expression in response to e. coli . obviously , the results were in disagreement with the reports showing that pmns could increase the production of il-17 during bacterial infections . to validate the in vivo expression of il17 in the plcs , the plcs pooled from the nave mice were cocultured with e. coli at 1 10 cfus , 1 10 cfus or saline for 14 hours , and then with bfa for another 4 hours . the cultured cells were harvested , stained with fitc - labeled anti - il-17 mab , and detected by flow cytometry . as shown in figure 5(e ) , e. coli could significantly increase il-17 plcs of the nave mice , indicating that e. coli could stimulate the plcs to express il-17 . to clarify this , we set up an experiment to detect il-17 mrna expression in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli for 18 hours , respectively , or from the control mice and found that the il-17 mrna levels in the mice infected with 3 doses of e. coli were significantly elevated in a dose dependent manner ( figure 5(f ) ) . next we sorted cd3 cells and cd3cd14cd11b cells from the plcs of the mice which were infected with e. coli or received saline for 18 hours , detected their il-17 mrna expression by qrt - pcr , and found that il-17 mrna levels were significantly increased in both of the cd3 t cells ( p = 0.0006 ) and the cd3cd14cd11b cells ( p = 0.0434 ) in the plcs from the e. coli infected mice . comparatively , the il-17 mrna levels in the cd3 t cells were much higher than those in the cd3cd14cd11b neutrophils . the result indicated that both of t cells and pmns were the il-17 producers in the peritoneal cavity during the development of septic peritonitis ( figure 5(g ) ) . the result suggests that the plcs from the infected mice did express il-17 and il-17 in the pmns might be secreted out during the development of septic peritonitis . to find the possible correlation between the production of il-17 and the expression of stlr9 in / on the pmns and macrophages in peritoneal cavity of the mice during septic peritonitis , we harvested the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , at early stage of 8 hours after infection and detected their expression of il-17 and stlr9 . the expression was represented by mean fluorescence intensity ( mfi ) to emphasize the increased expression of il-17 and stlr9 per cell . as shown in figure 6 , il-17 expression was significantly upregulated in the cd11b pmns from the infected mice and their levels were positively correlated with the doses of e. coli ( figure 6(a ) ) , while the upregulated il-17 expression was only observed in the cd11b pmns or macrophages from the mice infected with 0.8 10 cfus and 1.6 10 cfus of e. coli . the highest dose of e. coli could not induce the highest levels of il-17 in the cd11b pmns or macrophages after infection for 8 hours ( figures 6(b ) and 6(c ) ) . this result reveals that il-17 may be mainly derived from cd11b pmns and secrete out earlier during bacterial infection in mice infected with high dose of e. coli . in parallel , we also tested the expression of stlr9 on pmns and macrophages and found that stlr9 expression was significantly downregulated in the cd11b pmns from the infected mice and the lowest downregulation happened in the cd11b pmns from the mice infected with 2.4 10 cfus of e. coli ( figure 6(d ) ) . in contrast , stlr9 expression was significantly upregulated in the cd11b pmns from the infected mice ( figure 6(e ) ) . interestingly , the significantly upregulated expression of stlr9 was observed also in the macrophages from the mice infected with 2.4 10 cfus of e. coli ( figure 6(f ) ) . to validate whether other types of cells in the plcs also expressed il-17 and stlr9 , the plcs from mice infected with e. coli for 8 hours were stained with fluorescence - labeled mabs against ly6 g , cd11b , il-17 , or tlr9 and then analyzed by flow cytometry . the results showed that e. coli infection significantly increased il-17 expressing cd11bly6 g neutrophils ( figure 6(h ) ) as well as stlr9 expressing cd11bly6 g neutrophils ( figure 6(g ) ) in a dose dependent manner but could not increase il-17 expression in the stlr9cd11bly6 g neutrophils ( figure 6(i ) ) at the time point . in parallel , the plcs were stained with fluorescence - labeled mabs against cd3 , cd11b , and il-17 and analyzed . it was found that both cd3 t cells ( figure 6(j ) ) in the plcs and cd3cd11b pmns ( figure 6(k ) ) increased their il-17 expression in response to the infection with e. coli for 8 hours . this result indicates that , at early stage , stlr9 may be mainly expressed on cd11b pmns in plcs of the mice infected with e. coli , and with the infection progression , macrophages may become successors to replace the cd11b pmns as the major stlr9 cells . in this study , we tried to investigate the correlation of tlr9 expression with il-17 production in pmns during septic peritonitis and found that both stlr9 and il-7 could be expressed in the pmns infiltrated into the peritoneal cavity of the mice infected with e. coli . at early stage of the infection , stlr9 was increasingly expressed in the infiltrated cd11b pmns , and il-17 was increasingly expressed in both of the cd11b pmns and cd11b pmns . il-17 expression in cd11b pmns was positively correlated with the doses of e. coli . when infected with the highest dose of e. coli ( 2.4 10 cfus ) , il-17 was increasingly expressed and stlr9 was decreasingly expressed in / on the cd11b pmn . when infected with the lowest dose of e. coli ( 0.8 10 cfus ) , both il-17 and stlr9 furthermore , we stained the plcs with both cd11b mab and ly6 g mab and confirmed that the cd11bly6 g pmns in the plcs could increase their expression of both il-17 and stlr9 in response to e. coli infection . taken together , with the fact that highest dose of e. coli ( 2.4 10 cfus ) was the most deadly and the lowest dose of e. coli was the least deadly to the mice with septic peritonitis , we may deduce that increased expression of both stlr9 and il-17 in cd11b pmns might benefit the survival of the mice with septic peritonitis and that the decreased expression of stlr9 and increased expression of il-17 in cd11b pmns may be detrimental to the mice . in recent years , pmns have been found to be able to express stlr9 which engage pamps , such as bacterial dna generated during infection , and damage - associated molecular patterns ( damps ) , such as mitochondrial dna released from necrotic cells during sterile inflammation . the engagement , if intense , can prime pmns to release massively produced cytokines , leading to sepsis or systemic inflammatory response syndrome ( sirs ) . in the present work , we found that the ratios of the stlr9cd11b pmns and stlr9cd11b pmns were significantly increased in the infiltrated cd11b pmns and cd11b pmns of the mice infected with e. coli for 18 hours , respectively . at this stage , we observed that a plenty of e. coli coexisted with the pmns . when observed at 48 hours and 72 hours after infection , the ratios of both of the stlr9cd11b pmns and stlr9cd11b pmns were sharply decreased to the levels as those in saline control . possibly , the increased tlr9cd11b pmns and stlr9cd11b pmns present a rapid innate immune response of the pmns to bacterial invasion at the early stage of infection . pmns armed with stlr9 were demonstrated to be able to sense extracellular ligands and consequently initiate tlr9 mediated signaling in an intracellular tlr9 independent way . the response could offer a rescue mechanism for pmn activation when pathogen derived tlr9 ligands can not reach the endosome in the early stage of infection . practically , the increased expression of stlr9 on pmns may be a proinflammatory activation marker and stlr9 pmn should benefit antibacterial defense during infection , evidenced by the following : ( 1 ) tlr9 agonists , if they exist , could activate stlr9 pmn , not tlr9-deficient pmns , to upregulate cd11b and secrete mip-2 and il-8 ( cxcl8 ) ; ( 2 ) stlr9 pmns are involved in inducing the rapid inflammation which is needed in the initial phase of bacterial infection by secreting antimicrobial peptides and elastases ; ( 3 ) encountering microbial dna , stlr9 signaling is able to activate pmns , and the activated pmn increases stlr9 expression . however , it is worthy to note that stlr9 pmns could be culprit for causing more severe pathological consequences . for instance , bacterial dna or formulated peptides released following sepsis were reported to activate p38 map kinase through binding stlr9 on pmns , leading to acute lung injury which is characterized by protein - rich pulmonary oedema ( swelling ) and accumulation of large numbers of pmns in the lungs . it is well established that pmns , as the firstly recruited inflammatory cells in infection sites , are il-17 producing cells . il-17 secreted from pmns induces the release of proinflammatory factors from mesenchymal and myeloid cells , recruiting additional pmns . in addition to pmns , macrophages located in the epithelial barriers are also important sources of il-17 . in this study , we initially found that the average ratios of the il17cd11b pmns and il17 macrophages were significantly decreased in the plcs at 18 , 48 , and 72 hours in the mice infected with 3 doses of e. coli . seemingly , the cd11b pmns and macrophages , in response to the peritoneal infection , could not produce increased il-17 . however , when checking mrna expression , we found significantly upregulated il-17 mrna in the plcs from the mice infected with e. coli for 18 hours ( figure 5(e ) ) . the data imply that the infiltrated cells in peritoneal cavity of the mice infected with e. coli could produce il-17 and the produced il-17 may promptly secrete out the cells , therefore resulting in the decreased ratios of il17cd11b pmns and il17 macrophages . to clarify this , we harvested the plcs from the mice infected with e. coli at early stage of 8 hours after infection and detected their il-17 levels which were expressed as mean fluorescence intensity ( mfi ) . by doing this , we found that both of the cd11b pmns and cd11b pmns obviously produced il-17 in the early stage of the peritoneal infection caused by e. coli . to confirm this , we set up another experiment in which the plcs were harvested at 12 and 16 hours after the infection , cultured with bfa , a protein transport inhibitor capable of retaining the produced cytokines inside the cells , for additional 4 hours , and then detected their il-17 expression . we found that the bfa in vitro treatment significantly increased the average ratios of the il17cd11b pmns in cd11b pmns and il17 macrophages in macrophages ( data not shown ) . the results support the assumption that produced il-17 in cd11b pmns and macrophages could be secreted out promptly in response to e. coli at the early stage of septic peritonitis in mice . theoretically , deducing based on the published data , il-17 derived from cd11b pmns and macrophages may initiate a response to induce chemokines which in turn recruit more pmns [ 23 , 24 ] to the site of infection [ 18 , 25 ] . the new comer pmns join the fighting against invaded bacteria , resulting in more il-17 [ 16 , 17 ] . in addition to the pmns , macrophages could be recruited by il-17 [ 20 , 26 ] , specifically by cd11b pmns derived il-17 . the increased macrophages could produce more il-17 to join the cd11b pmns produced il-17 , playing a defense role in fighting bacterial infection and reducing mortality of mice with septic peritonitis . in addition to the pmns and macrophages , t cells were found as a major producer of il-17 in the mice with experimental sepsis induced by clp ; t cell - derived il-17 could promote production of proinflammatory mediators , resulting in enhanced lethality . compatibly , t cells , in this study , were confirmed to be involved in the development of septic peritonitis in mice by the evidence that ( 1 ) the il-17 expression was increased in the cd3 cells of the plcs from the mice infected with e. coli and ( 2 ) il-17 mrna levels were significantly increased in the sorted cd3 cells in the plcs from the mice infected with e. coli . furthermore , il-17 expression was obviously increased in the sorted cd3cd11b pmns in the plcs from the mice infected with e. coli . together , these data suggest that the pmns , like t cells , do express il-17 as a response to bacterial infection during septic peritonitis . although cd3 cells were one of the major producers of il-17 during the development of septic peritonitis , probably , neutrophils might produce more il-17 compared to cd3 cells because the neutrophils , as we found in this study , could constitute up to 70% of the plcs ( figure 3(b ) ) , whereas cd3 cells only constituted 1015% of the plcs ( data not shown ) . interestingly , stlr9cd11bly6 g pmns , not stlr9cd11bly6 g pmns , were found to be able to increase their il-17 expression during septic peritonitis . interestingly , we found a type of giant cells with increasing numbers in the plcs from the mice infected with e. coli up to 1.6 10 cfus . morphologically , the giant cells ( figure 2(a ) ) could be the pmns with a changed lobulated nucleus located near to cell membrane , being at least 4 times larger than the regular pmns . obviously , the giant cells are the newly described cells and have several facets worthy of note . ( 1 ) kinetically , the giant cells only appeared at the early stage of the infection and were not observed in the plcs of the mice infected for 48 and 72 hours . ( 2 ) the giant cells only occurred in the plcs of the severely infected mice , not in the plcs of the mice infected with less amount ( 0.40.8 10 cfus ) of e. coli . possibly , the giant cells were developed from the regular pmns after engulfing a large amount of bacterial pathogens . biologically , the appearance of the giant cells could be used as a cell marker for signifying a real danger because the giant cells were intimately correlated with the severity of the infection and the death of the mice .

il-17 is a proinflammatory cytokine produced by various immune cells . polymorphonuclear neutrophils ( pmns ) are the first line of defense in bacterial infection and express surface toll - like receptor 9 ( stlr9 ) . to study the relationship of stlr9 and il-17 in pmns during bacterial infection , we infected mice with e. coli intraperitoneally to establish a septic peritonitis model for studying the pmns response in peritoneal cavity . we found that pmns and some of giant cells were massively accumulated in the peritoneal cavity of mice with fatal septic peritonitis induced by e. coli . kinetically , the cd11b+ pmns were increased from 2040% at 18 hours to > 80% at 72 hours after infection . after e. coli infection , stlr9 expression on cd11b+ and cd11b pmns and macrophages in the plcs were increased at early stage and deceased at late stage ; il-17 expression was also increased in cd11b+ pmns , cd11b pmns , macrophages , and cd3 + t cells . using experiments of in vitro blockage , qrt - pcr and cell sorting , we confirmed that pmns in the plcs did increase their il-17 expression during e. coli infection . interestingly , stlr9cd11b+ly6g+ pmns , not stlr9+cd11b+ly6g+ pmns , were found to be able to increase their il-17 expression . together , the data may help understand novel roles of pmns in septic peritonitis .

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

recently , it has been found that tlr9 is expressed on the surface of human and mouse pmns , that the surface tlr9 ( stlr9 ) senses dna in pmns , and that stlr9 expressing pmns ( stlr9 pmns ) have roles in inducing rapid inflammation . in the present study , we kinetically observed the infiltration of pmns and expression of stlr9 and il-17 on / in the pmns in the peritoneal lavage cells ( plcs ) of mice intraperitoneally infected with various doses of escherichia coli ( e. coli ) , aiming to find the correlation of the expression of stlr9 and il-17 on / in the pmns during the development of bacterial septic peritonitis . since the polymorphonuclear cells ( pmns ) are documented as the first innate immune cells recruited at inflammation sites and play a central role in host defense , we next collected the peritoneal lavage cells ( plcs ) of the mice at 18 , 48 , and 72 hours after infection , respectively , for observing the infiltration and morphology of the pmns in the plcs . at 18 hours after infection , in the plcs of the mice infected with 1.6 and 2.4 10 cfus of e. coli , the majority of the cells with the size of 0.2~0.4 micrometer were pmns with a typical lobulated nucleus . when counting the cells , we found that at 18 , 48 , and 72 hours , pmns constituted 50%80% and macrophages constituted 20%40% of the plcs from the mice infected with 4 doses of e. coli , respectively , compared with nearly 30% for pmns and 60% for macrophages in the plcs from the mice that received saline . at 18 hours , interestingly , there were about 20% of the giant cells in the plcs of the mice infected with 1.6 and 2.4 10 cfus of e. coli ( figure 2(b ) , upper right ) . noticeably , when the mice died of the infection , we collected the plcs immediately and observed and found that plenty of bacteria coexisted with the pmns in the plcs of the mouse infected with 1.6 10 cfus of e. coli for 31 hours ( figure 2(c ) ) . to find the correlation of the numbers of pmns at various time points with the severity of the disease in mice infected with e. coli , the plcs of the mice were collected at 18 , 48 , and 72 hours after infection , counted by hemocytometer or flow cytometry after staining with fluorescence - labeled mab of anti - cd45 and anti - cd11b . at 18 hours , the numbers of plcs were nearly 1 10 cells / ml of peritoneal lavage fluid ( plf ) in the mice received saline ( control mice ) , significantly increased in the mice infected with 3 doses of e. coli in a dose dependent manner , and even reached nearly 3 10 cells / ml of plf in the mice infected with 2.4 10 cfus of e. coli ( p < 0.05 ) . in the plcs from the infected mice , most of the cells were cd45 nucleated leukocytes , representing pmns , macrophages , and lymphocytes , respectively . at 18 hours , the pmns , macrophages , and lymphocytes were displayed on the left , middle , and right of the histogram , respectively , sequentially based on their nuclear structure and fluorescence intensity . as shown in figure 3(b ) , at 18 hours after infection , the cd45 pmns constituted 61.5% , 49.6% , and 56.4% of the plcs in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , compared with 32.5% in the control mice . at 48 hours , the cd45 pmns were increased to 59.5% , 63.7% , and 54.7% in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , compared with 41.2% from the control mice . at 72 hours , the cd45 pmns constituted 62.2% , 62.8% , and 63.5% of the plcs in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , compared with 39.5% from the control mice . as shown in figure 3(c ) , the cd11bcd45 pmns constituted 49% , 36% , and 13% of the pmns in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli for 18 hours , respectively . at 72 hours after the infection , the cd11b pmns reached around 96% in all infected mice . oppositely , at 18 hours , the cd11b pmns constituted 51% , 64% , and 87% of the pmns in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively . at 48 hours after the infection , the cd11b pmns constituted 9% , 12% , and 14% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively . at 72 hours after the infection , the cd11b pmns only accounted for around 4% in all infected mice ( figure 3(c ) ) . at 18 and 48 hours after infection , the ratios of cd11b pmns or cd11b pmns were correlated with the doses of e. coli negatively or positively . since primary human and mouse blood neutrophils were reported to be able to express a functional stlr9 , we tried to detect whether the expression of stlr9 was different on cd11b pmns and cd11b pmns and correlates with development of septic peritonitis in mice . results showed that , at 18 hours after infection , the ratios of stlr9cd11b pmns were increased to 57% , 51% , and 70% of the cd11b pmns in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively ( p < 0.05 ) , compared to 12% in the mice that received saline control ( control mice ) . at 72 hours , the ratios of stlr9cd11b pmns were 7% , 5.5% , and 9% in the infected mice , respectively , compared with 18% in control mice ( figure 4(b ) ) . the ratios of stlr9cd11b pmns constituted 52% , 53% , and 68.5% of the cd11b pmns in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours ( p < 0.05 ) , compared with 3.5% in control mice , sharply decreased to 9% , 6% , and 9.5% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 8% in control mice , and reached 10% , 6% , and 10% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours , compared with 8% in control mice ( figure 4(c ) ) . these results indicated that e. coli could significantly induce the occurrence of both of the stlr9cd11b pmns and stlr9cd11b pmns in the peritoneal cavity at early stage of infection . considering the fact that macrophages are the inflammatory cells which overlapped with the pmns in the inflammatory sites , we also observed stlr9 expression on macrophages in the plcs . the results showed that the ratios of stlr9 macrophages constituted 5% , 6% , and 6.7% ( p < 0.05 ) of the macrophages in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours , compared with 3% in control mice , decreased to 0.9% ( p < 0.05 ) , 0.45% ( p < 0.05 ) , and 3% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 5% in control mice , and reached 4.8% , 2% ( p < 0.05 ) , and 2.8% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours , compared with 5.5% in control mice ( figure 4(d ) ) . to find whether the different subtype neutrophil derived il-17 was involved in the development of septic peritonitis , we detected il-17 expression in the cd11b pmns and cd11b pmns ( figure 5(a ) ) of the plcs collected from the mice which were infected with e. coli or received saline ( control mice ) . it was found that the ratios of il-17cd11b pmns constituted 7% , 8.5% , and 10% of the cd11b pmns in plcs of the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours ( p < 0.05 ) , compared with 25% in control mice , was 2% ( p < 0.05 ) , 7.5% ( p < 0.05 ) , and 25.5% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 25% in control mice , and reached 6% , 10% , and 8% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours ( p < 0.05 ) , compared with 27% in control mice ( figure 5(b ) ) . to confirm this , the plcs were collected from the mice injected with saline at 18 , 48 , or 72 hours after the injection , respectively , and stained with fluorescence - labeled mab against cd45 , cd11b , and il-17 , followed by detection using flow cytometry . the ratios of the il-17cd11b pmns were 5% , 4% , and 3% of the cd11b pmns in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours , compared with 7% in control mice ; 3% , 11% , and 21.5% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 7% in control mice ; and 7% , 8% , and 13% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours , compared with 9% in control mice ( figure 5(c ) ) . the results indicated that both of the cd11b pmns and the cd11b pmns could not increase il-17 expression during the development of septic peritonitis in mice induced by e. coli . next we sorted cd3 cells and cd3cd14cd11b cells from the plcs of the mice which were infected with e. coli or received saline for 18 hours , detected their il-17 mrna expression by qrt - pcr , and found that il-17 mrna levels were significantly increased in both of the cd3 t cells ( p = 0.0006 ) and the cd3cd14cd11b cells ( p = 0.0434 ) in the plcs from the e. coli infected mice . to find the possible correlation between the production of il-17 and the expression of stlr9 in / on the pmns and macrophages in peritoneal cavity of the mice during septic peritonitis , we harvested the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , at early stage of 8 hours after infection and detected their expression of il-17 and stlr9 . in parallel , we also tested the expression of stlr9 on pmns and macrophages and found that stlr9 expression was significantly downregulated in the cd11b pmns from the infected mice and the lowest downregulation happened in the cd11b pmns from the mice infected with 2.4 10 cfus of e. coli ( figure 6(d ) ) . it was found that both cd3 t cells ( figure 6(j ) ) in the plcs and cd3cd11b pmns ( figure 6(k ) ) increased their il-17 expression in response to the infection with e. coli for 8 hours . this result indicates that , at early stage , stlr9 may be mainly expressed on cd11b pmns in plcs of the mice infected with e. coli , and with the infection progression , macrophages may become successors to replace the cd11b pmns as the major stlr9 cells . in this study , we tried to investigate the correlation of tlr9 expression with il-17 production in pmns during septic peritonitis and found that both stlr9 and il-7 could be expressed in the pmns infiltrated into the peritoneal cavity of the mice infected with e. coli . at early stage of the infection , stlr9 was increasingly expressed in the infiltrated cd11b pmns , and il-17 was increasingly expressed in both of the cd11b pmns and cd11b pmns . when infected with the lowest dose of e. coli ( 0.8 10 cfus ) , both il-17 and stlr9 furthermore , we stained the plcs with both cd11b mab and ly6 g mab and confirmed that the cd11bly6 g pmns in the plcs could increase their expression of both il-17 and stlr9 in response to e. coli infection . taken together , with the fact that highest dose of e. coli ( 2.4 10 cfus ) was the most deadly and the lowest dose of e. coli was the least deadly to the mice with septic peritonitis , we may deduce that increased expression of both stlr9 and il-17 in cd11b pmns might benefit the survival of the mice with septic peritonitis and that the decreased expression of stlr9 and increased expression of il-17 in cd11b pmns may be detrimental to the mice . in the present work , we found that the ratios of the stlr9cd11b pmns and stlr9cd11b pmns were significantly increased in the infiltrated cd11b pmns and cd11b pmns of the mice infected with e. coli for 18 hours , respectively . practically , the increased expression of stlr9 on pmns may be a proinflammatory activation marker and stlr9 pmn should benefit antibacterial defense during infection , evidenced by the following : ( 1 ) tlr9 agonists , if they exist , could activate stlr9 pmn , not tlr9-deficient pmns , to upregulate cd11b and secrete mip-2 and il-8 ( cxcl8 ) ; ( 2 ) stlr9 pmns are involved in inducing the rapid inflammation which is needed in the initial phase of bacterial infection by secreting antimicrobial peptides and elastases ; ( 3 ) encountering microbial dna , stlr9 signaling is able to activate pmns , and the activated pmn increases stlr9 expression . in this study , we initially found that the average ratios of the il17cd11b pmns and il17 macrophages were significantly decreased in the plcs at 18 , 48 , and 72 hours in the mice infected with 3 doses of e. coli . the data imply that the infiltrated cells in peritoneal cavity of the mice infected with e. coli could produce il-17 and the produced il-17 may promptly secrete out the cells , therefore resulting in the decreased ratios of il17cd11b pmns and il17 macrophages . to clarify this , we harvested the plcs from the mice infected with e. coli at early stage of 8 hours after infection and detected their il-17 levels which were expressed as mean fluorescence intensity ( mfi ) . by doing this , we found that both of the cd11b pmns and cd11b pmns obviously produced il-17 in the early stage of the peritoneal infection caused by e. coli . to confirm this , we set up another experiment in which the plcs were harvested at 12 and 16 hours after the infection , cultured with bfa , a protein transport inhibitor capable of retaining the produced cytokines inside the cells , for additional 4 hours , and then detected their il-17 expression . we found that the bfa in vitro treatment significantly increased the average ratios of the il17cd11b pmns in cd11b pmns and il17 macrophages in macrophages ( data not shown ) . the results support the assumption that produced il-17 in cd11b pmns and macrophages could be secreted out promptly in response to e. coli at the early stage of septic peritonitis in mice . in addition to the pmns and macrophages , t cells were found as a major producer of il-17 in the mice with experimental sepsis induced by clp ; t cell - derived il-17 could promote production of proinflammatory mediators , resulting in enhanced lethality . compatibly , t cells , in this study , were confirmed to be involved in the development of septic peritonitis in mice by the evidence that ( 1 ) the il-17 expression was increased in the cd3 cells of the plcs from the mice infected with e. coli and ( 2 ) il-17 mrna levels were significantly increased in the sorted cd3 cells in the plcs from the mice infected with e. coli . furthermore , il-17 expression was obviously increased in the sorted cd3cd11b pmns in the plcs from the mice infected with e. coli . interestingly , stlr9cd11bly6 g pmns , not stlr9cd11bly6 g pmns , were found to be able to increase their il-17 expression during septic peritonitis . interestingly , we found a type of giant cells with increasing numbers in the plcs from the mice infected with e. coli up to 1.6 10 cfus . ( 1 ) kinetically , the giant cells only appeared at the early stage of the infection and were not observed in the plcs of the mice infected for 48 and 72 hours .

recently , it has been found that tlr9 is expressed on the surface of human and mouse pmns , that the surface tlr9 ( stlr9 ) senses dna in pmns , and that stlr9 expressing pmns ( stlr9 pmns ) have roles in inducing rapid inflammation . notably , innate immune cell - derived il-17 constitutes a major element in the immune response against infectious agents by recruiting pmns to the sites of infections and by inducing the production of antimicrobial peptides , cxc chemokines , and granulocyte colony stimulating factor ( g - csf ) [ 15 , 16 ] . in the present study , we kinetically observed the infiltration of pmns and expression of stlr9 and il-17 on / in the pmns in the peritoneal lavage cells ( plcs ) of mice intraperitoneally infected with various doses of escherichia coli ( e. coli ) , aiming to find the correlation of the expression of stlr9 and il-17 on / in the pmns during the development of bacterial septic peritonitis . quantitative real - time pcr ( qrt - pcr ) was performed using two - step sybr green qpcr assays and the target mrnas were identified by the specific primers as follows : il-17a , forward : 5-aaggcagcagcgatcatccct ; reverse : 3-tcttcattgcggtggagagtcc ; gapdh , forward : 5-atcaccatcttccaggagcga ; reverse : 3-tctcgtggttcacacccatca . around 6 hours after the infection , all of the 10 mice received 2.4 10 cfus of e. coli , 8 out of the 10 mice received 1.6 10 cfus of e. coli , and 5 out of the 10 mice that received 0.8 10 cfus of e. coli began to exhibit multiple neurological symptoms , including dispirited behavior , staggered gait , and trembling , but these phenomena were not present in all of the 10 mice that received 0.4 10 cfus of e. coli or saline . by 72 hours after the infection , 80% , 60% , and 40% of the mice infected with 2.4 , 1.6 , and 0.8 10 cfus of e. coli , respectively , died and yet 100% of the mice which were infected with 0.4 10 cfus of e. coli or received saline survived ( figure 1 ) . since the polymorphonuclear cells ( pmns ) are documented as the first innate immune cells recruited at inflammation sites and play a central role in host defense , we next collected the peritoneal lavage cells ( plcs ) of the mice at 18 , 48 , and 72 hours after infection , respectively , for observing the infiltration and morphology of the pmns in the plcs . at 18 hours after infection , in the plcs of the mice infected with 1.6 and 2.4 10 cfus of e. coli , the majority of the cells with the size of 0.2~0.4 micrometer were pmns with a typical lobulated nucleus . when counting the cells , we found that at 18 , 48 , and 72 hours , pmns constituted 50%80% and macrophages constituted 20%40% of the plcs from the mice infected with 4 doses of e. coli , respectively , compared with nearly 30% for pmns and 60% for macrophages in the plcs from the mice that received saline . noticeably , when the mice died of the infection , we collected the plcs immediately and observed and found that plenty of bacteria coexisted with the pmns in the plcs of the mouse infected with 1.6 10 cfus of e. coli for 31 hours ( figure 2(c ) ) . to find the correlation of the numbers of pmns at various time points with the severity of the disease in mice infected with e. coli , the plcs of the mice were collected at 18 , 48 , and 72 hours after infection , counted by hemocytometer or flow cytometry after staining with fluorescence - labeled mab of anti - cd45 and anti - cd11b . at 18 hours , the numbers of plcs were nearly 1 10 cells / ml of peritoneal lavage fluid ( plf ) in the mice received saline ( control mice ) , significantly increased in the mice infected with 3 doses of e. coli in a dose dependent manner , and even reached nearly 3 10 cells / ml of plf in the mice infected with 2.4 10 cfus of e. coli ( p < 0.05 ) . at 48 hours , the numbers of plcs were less than 1 10 cells / ml of plf in control mice and significantly increased to over 3 10 cells / ml of plf in the mice infected with three doses of e. coli and even to about 4 10 cells / ml of plf in the mice infected with 1.6 and 2.4 10 cfus of e. coli ( p < 0.05 ) . at 72 hours , the numbers of plcs were decreased to 0.5 10 cells / ml of plf in control mice and decreased to nearly 0.5 10 cells / ml of plf in the mice infected with three doses of e. coli ( figure 3(a ) ) . as shown in figure 3(b ) , at 18 hours after infection , the cd45 pmns constituted 61.5% , 49.6% , and 56.4% of the plcs in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , compared with 32.5% in the control mice . at 48 hours , the cd45 pmns were increased to 59.5% , 63.7% , and 54.7% in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , compared with 41.2% from the control mice . at 72 hours , the cd45 pmns constituted 62.2% , 62.8% , and 63.5% of the plcs in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , compared with 39.5% from the control mice . as shown in figure 3(c ) , the cd11bcd45 pmns constituted 49% , 36% , and 13% of the pmns in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli for 18 hours , respectively . oppositely , at 18 hours , the cd11b pmns constituted 51% , 64% , and 87% of the pmns in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively . results showed that , at 18 hours after infection , the ratios of stlr9cd11b pmns were increased to 57% , 51% , and 70% of the cd11b pmns in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively ( p < 0.05 ) , compared to 12% in the mice that received saline control ( control mice ) . the ratios of stlr9cd11b pmns constituted 52% , 53% , and 68.5% of the cd11b pmns in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours ( p < 0.05 ) , compared with 3.5% in control mice , sharply decreased to 9% , 6% , and 9.5% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 8% in control mice , and reached 10% , 6% , and 10% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours , compared with 8% in control mice ( figure 4(c ) ) . the results showed that the ratios of stlr9 macrophages constituted 5% , 6% , and 6.7% ( p < 0.05 ) of the macrophages in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours , compared with 3% in control mice , decreased to 0.9% ( p < 0.05 ) , 0.45% ( p < 0.05 ) , and 3% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 5% in control mice , and reached 4.8% , 2% ( p < 0.05 ) , and 2.8% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours , compared with 5.5% in control mice ( figure 4(d ) ) . to find whether the different subtype neutrophil derived il-17 was involved in the development of septic peritonitis , we detected il-17 expression in the cd11b pmns and cd11b pmns ( figure 5(a ) ) of the plcs collected from the mice which were infected with e. coli or received saline ( control mice ) . it was found that the ratios of il-17cd11b pmns constituted 7% , 8.5% , and 10% of the cd11b pmns in plcs of the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours ( p < 0.05 ) , compared with 25% in control mice , was 2% ( p < 0.05 ) , 7.5% ( p < 0.05 ) , and 25.5% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 25% in control mice , and reached 6% , 10% , and 8% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours ( p < 0.05 ) , compared with 27% in control mice ( figure 5(b ) ) . the ratios of the il-17cd11b pmns were 5% , 4% , and 3% of the cd11b pmns in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 18 hours , compared with 7% in control mice ; 3% , 11% , and 21.5% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 48 hours , compared with 7% in control mice ; and 7% , 8% , and 13% in the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , for 72 hours , compared with 9% in control mice ( figure 5(c ) ) . to validate the in vivo expression of il17 in the plcs , the plcs pooled from the nave mice were cocultured with e. coli at 1 10 cfus , 1 10 cfus or saline for 14 hours , and then with bfa for another 4 hours . to clarify this , we set up an experiment to detect il-17 mrna expression in the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli for 18 hours , respectively , or from the control mice and found that the il-17 mrna levels in the mice infected with 3 doses of e. coli were significantly elevated in a dose dependent manner ( figure 5(f ) ) . next we sorted cd3 cells and cd3cd14cd11b cells from the plcs of the mice which were infected with e. coli or received saline for 18 hours , detected their il-17 mrna expression by qrt - pcr , and found that il-17 mrna levels were significantly increased in both of the cd3 t cells ( p = 0.0006 ) and the cd3cd14cd11b cells ( p = 0.0434 ) in the plcs from the e. coli infected mice . to find the possible correlation between the production of il-17 and the expression of stlr9 in / on the pmns and macrophages in peritoneal cavity of the mice during septic peritonitis , we harvested the plcs from the mice infected with 0.8 , 1.6 , and 2.4 10 cfus of e. coli , respectively , at early stage of 8 hours after infection and detected their expression of il-17 and stlr9 . as shown in figure 6 , il-17 expression was significantly upregulated in the cd11b pmns from the infected mice and their levels were positively correlated with the doses of e. coli ( figure 6(a ) ) , while the upregulated il-17 expression was only observed in the cd11b pmns or macrophages from the mice infected with 0.8 10 cfus and 1.6 10 cfus of e. coli . in parallel , we also tested the expression of stlr9 on pmns and macrophages and found that stlr9 expression was significantly downregulated in the cd11b pmns from the infected mice and the lowest downregulation happened in the cd11b pmns from the mice infected with 2.4 10 cfus of e. coli ( figure 6(d ) ) . to validate whether other types of cells in the plcs also expressed il-17 and stlr9 , the plcs from mice infected with e. coli for 8 hours were stained with fluorescence - labeled mabs against ly6 g , cd11b , il-17 , or tlr9 and then analyzed by flow cytometry . the results showed that e. coli infection significantly increased il-17 expressing cd11bly6 g neutrophils ( figure 6(h ) ) as well as stlr9 expressing cd11bly6 g neutrophils ( figure 6(g ) ) in a dose dependent manner but could not increase il-17 expression in the stlr9cd11bly6 g neutrophils ( figure 6(i ) ) at the time point . this result indicates that , at early stage , stlr9 may be mainly expressed on cd11b pmns in plcs of the mice infected with e. coli , and with the infection progression , macrophages may become successors to replace the cd11b pmns as the major stlr9 cells . in this study , we tried to investigate the correlation of tlr9 expression with il-17 production in pmns during septic peritonitis and found that both stlr9 and il-7 could be expressed in the pmns infiltrated into the peritoneal cavity of the mice infected with e. coli . when infected with the lowest dose of e. coli ( 0.8 10 cfus ) , both il-17 and stlr9 furthermore , we stained the plcs with both cd11b mab and ly6 g mab and confirmed that the cd11bly6 g pmns in the plcs could increase their expression of both il-17 and stlr9 in response to e. coli infection . taken together , with the fact that highest dose of e. coli ( 2.4 10 cfus ) was the most deadly and the lowest dose of e. coli was the least deadly to the mice with septic peritonitis , we may deduce that increased expression of both stlr9 and il-17 in cd11b pmns might benefit the survival of the mice with septic peritonitis and that the decreased expression of stlr9 and increased expression of il-17 in cd11b pmns may be detrimental to the mice . in the present work , we found that the ratios of the stlr9cd11b pmns and stlr9cd11b pmns were significantly increased in the infiltrated cd11b pmns and cd11b pmns of the mice infected with e. coli for 18 hours , respectively . practically , the increased expression of stlr9 on pmns may be a proinflammatory activation marker and stlr9 pmn should benefit antibacterial defense during infection , evidenced by the following : ( 1 ) tlr9 agonists , if they exist , could activate stlr9 pmn , not tlr9-deficient pmns , to upregulate cd11b and secrete mip-2 and il-8 ( cxcl8 ) ; ( 2 ) stlr9 pmns are involved in inducing the rapid inflammation which is needed in the initial phase of bacterial infection by secreting antimicrobial peptides and elastases ; ( 3 ) encountering microbial dna , stlr9 signaling is able to activate pmns , and the activated pmn increases stlr9 expression . the data imply that the infiltrated cells in peritoneal cavity of the mice infected with e. coli could produce il-17 and the produced il-17 may promptly secrete out the cells , therefore resulting in the decreased ratios of il17cd11b pmns and il17 macrophages . to confirm this , we set up another experiment in which the plcs were harvested at 12 and 16 hours after the infection , cultured with bfa , a protein transport inhibitor capable of retaining the produced cytokines inside the cells , for additional 4 hours , and then detected their il-17 expression . in addition to the pmns and macrophages , t cells were found as a major producer of il-17 in the mice with experimental sepsis induced by clp ; t cell - derived il-17 could promote production of proinflammatory mediators , resulting in enhanced lethality . compatibly , t cells , in this study , were confirmed to be involved in the development of septic peritonitis in mice by the evidence that ( 1 ) the il-17 expression was increased in the cd3 cells of the plcs from the mice infected with e. coli and ( 2 ) il-17 mrna levels were significantly increased in the sorted cd3 cells in the plcs from the mice infected with e. coli . although cd3 cells were one of the major producers of il-17 during the development of septic peritonitis , probably , neutrophils might produce more il-17 compared to cd3 cells because the neutrophils , as we found in this study , could constitute up to 70% of the plcs ( figure 3(b ) ) , whereas cd3 cells only constituted 1015% of the plcs ( data not shown ) . ( 2 ) the giant cells only occurred in the plcs of the severely infected mice , not in the plcs of the mice infected with less amount ( 0.40.8 10 cfus ) of e. coli .

the study base was the participants of the ongoing tsuruoka metabolomic cohort study , initiated in april 2012 ( yamagata prefecture , japan ) , comprising individuals aged 35 to 74 years who were recruited among attendees of annual municipal or worksite health check - up programs in the city . a total of 1,422 women consented to participate during the first few months of the baseline period , and plasma metabolomic profiling in these women was completed by the end of 2014 . only postmenopausal female participants were ultimately included in the analysis due to the small number of premenopausal women . because there are known metabolic differences compared with natural menopause , participants with surgical or medical menopause were excluded . in addition , women who had used hormone therapy within 1 year or had undergone oophorectomy after menopause were excluded to eliminate the possible effect on the metabolic profile of administered estrogen and hormones excreted from postmenopausal ovaries . the study was approved by the medical ethics committee of the school of medicine , keio university , tokyo , japan ( approval no . all data and samples were obtained in the baseline study , including anthropometrics , clinical biochemistry , and blood specimens for metabolomic profiling . detailed information was collected through a standardized self - administered questionnaire on medical history , including treatment of hypertension , dyslipidemia , and diabetes ; gynecological and reproductive history , including menopause status ( pre / peri / post ) , ages at menarche and menopause , reason for menopause ( natural / medical / others ) , number of pregnancies and deliveries , age at first delivery , use of hormone therapy ( if yes , number of years used and when it was used last ) , and whether oophorectomy was ever performed ; and lifestyle parameters such as smoking habits , alcohol intake , diet , stress , and physical activity . wc was measured to the nearest 0.1 cm at the umbilicus at the end of a normal breath . if the umbilicus drooped down , the measurement was made midway between the inferior margin of the last rib and the top of the iliac crest in a horizontal plane . bp was measured twice on one occasion in the sitting position using an automated sphygmomanometer ( omron hbp - t105s - n ) , and the mean of the two measurements was used for analysis . blood samples were collected in the morning between 8:30 and 10:30 after overnight fasting to avoid variation due to fasting and circadian rhythm . plasma samples were collected with ethylenediaminetetraacetic acid-2na as an anticoagulant and kept at 4c immediately after collection . the samples were centrifuged for 15 minutes ( 1,500 g at 4c ) within 1 hour of collection , divided into aliquots , and kept for a maximum of 6 hours at 4c until extraction of metabolites . serum samples were collected with serum - separating medium and kept at room temperature after collection . serum levels of total cholesterol , tg , and fasting plasma glucose were analyzed using enzymatic methods , and glycated hemoglobin ( hba1c ) was determined by immunoassay . mets was defined using the modified national cholesterol education program adult treatment panel iii definition with 100 mg / dl as the cutoff for the glucose level . because lower overall adiposity has been associated with an increased risk of medical conditions such as type 2 diabetes in asian countries , the threshold for wc was set according to the recommendation for asians established by several organizations , including the national cholesterol education program . specifically , central obesity was defined as wc at least 80 cm , high bp as mean systolic / diastolic bp at least 130/85 mm hg or currently on antihypertensive therapy , high serum tg as at least 1.7 mmol / l ( 150 mg / dl ) , low hdl - c as less than 1.3 mmol / l ( 50 mg / dl ) , and high fasting plasma glucose as at least 5.5 mmol / l ( 100 mg / dl ) or current use of antidiabetic medication . nontargeted mass spectrometry - based metabolomic profiling was performed with fasting plasma samples via capillary electrophoresis time - of - flight mass spectrometry . metabolite extraction from plasma was completed within 6 hours after collection to minimize the effect of metabolic changes in plasma . capillary electrophoresis time - of - flight mass spectrometry analysis of cationic and anionic metabolites was performed as described previously . raw data were processed using our proprietary software ( masterhands ) . as a preliminary study , we identified 290 metabolite peaks ( 131 cations and 159 anions ) in plasma : 154 known with standard compounds and 136 unknown . we decided a priori to measure absolute concentrations of 115 metabolites ( 63 cations and 52 anions ) that were expected to be observed stably in most human plasma samples and matched with standard compounds . to monitor the stability of metabolome analysis , quality control samples were injected every 10 samples and assessed at the start of the analytical run and at intervals throughout the analysis . the technical variability and between - subject variability for each metabolite assayed is shown in supplementary table 1 . participants were randomly allocated into two groups ( 2:1 ) to create two independent original and replication populations . analyses were first performed in the original population , and then the same analyses were performed in the replication population to confirm the robustness of the study outcome . the two groups were stratified for age and batch number to eliminate any confounding effects of age and analytical variability between multiple batches . out of 115 metabolites , 37 had plasma levels below the assay limits of detection ( lod ) in more than 90% of the participants , and therefore were excluded from further analyses . the remaining 78 metabolites were analyzed . for samples with levels below lod , which comprised less than 1% of all samples , the distribution of each metabolite concentration was tested for normality using quantile - quantile plots . on the basis of the shape of the distribution , most of the metabolite concentrations were log - transformed . the characteristics of participants with and without mets were compared by t test and test for continuous and categorical variables , respectively . age - adjusted spearman correlation coefficients were calculated for each metabolite pair in the original population . linear regression analysis was performed between the mets and non - mets groups with each metabolite concentration used as the outcome to examine the association between plasma metabolite concentrations and mets . analysis of covariance was performed to adjust for possible confounders using age , ldl - c levels , current smoking status , current alcohol consumption , physical activity , and dietary intake level . we calculated p values using the benjamini and hochberg false discovery rate method ( = 0.05 ) for analysis of the original population to adjust for independent multiple comparisons . sensitivity analyses were performed by excluding ( 1 ) women with bmi at least 25 kg / m and ( 2 ) those taking medications for hypertension , dyslipidemia , and diabetes . both sensitivity and replication analyses were conducted using the metabolites shown to be significantly associated with mets in the original population ( unadjusted false discovery rate p < 0.05 ) . sas 9.3 ( sas institute inc , cary , nc ) was used for all analyses . the study base was the participants of the ongoing tsuruoka metabolomic cohort study , initiated in april 2012 ( yamagata prefecture , japan ) , comprising individuals aged 35 to 74 years who were recruited among attendees of annual municipal or worksite health check - up programs in the city . a total of 1,422 women consented to participate during the first few months of the baseline period , and plasma metabolomic profiling in these women was completed by the end of 2014 . only postmenopausal female participants were ultimately included in the analysis due to the small number of premenopausal women . because there are known metabolic differences compared with natural menopause , participants with surgical or medical menopause were excluded . in addition , women who had used hormone therapy within 1 year or had undergone oophorectomy after menopause were excluded to eliminate the possible effect on the metabolic profile of administered estrogen and hormones excreted from postmenopausal ovaries . the study was approved by the medical ethics committee of the school of medicine , keio university , tokyo , japan ( approval no . all data and samples were obtained in the baseline study , including anthropometrics , clinical biochemistry , and blood specimens for metabolomic profiling . detailed information was collected through a standardized self - administered questionnaire on medical history , including treatment of hypertension , dyslipidemia , and diabetes ; gynecological and reproductive history , including menopause status ( pre / peri / post ) , ages at menarche and menopause , reason for menopause ( natural / medical / others ) , number of pregnancies and deliveries , age at first delivery , use of hormone therapy ( if yes , number of years used and when it was used last ) , and whether oophorectomy was ever performed ; and lifestyle parameters such as smoking habits , alcohol intake , diet , stress , and physical activity . wc was measured to the nearest 0.1 cm at the umbilicus at the end of a normal breath . if the umbilicus drooped down , the measurement was made midway between the inferior margin of the last rib and the top of the iliac crest in a horizontal plane . bp was measured twice on one occasion in the sitting position using an automated sphygmomanometer ( omron hbp - t105s - n ) , and the mean of the two measurements was used for analysis . blood samples were collected in the morning between 8:30 and 10:30 after overnight fasting to avoid variation due to fasting and circadian rhythm . plasma samples were collected with ethylenediaminetetraacetic acid-2na as an anticoagulant and kept at 4c immediately after collection . the samples were centrifuged for 15 minutes ( 1,500 g at 4c ) within 1 hour of collection , divided into aliquots , and kept for a maximum of 6 hours at 4c until extraction of metabolites . serum samples were collected with serum - separating medium and kept at room temperature after collection . serum levels of total cholesterol , tg , and fasting plasma glucose were analyzed using enzymatic methods , and glycated hemoglobin ( hba1c ) was determined by immunoassay . mets was defined using the modified national cholesterol education program adult treatment panel iii definition with 100 mg / dl as the cutoff for the glucose level . because lower overall adiposity has been associated with an increased risk of medical conditions such as type 2 diabetes in asian countries , the threshold for wc was set according to the recommendation for asians established by several organizations , including the national cholesterol education program . specifically , central obesity was defined as wc at least 80 cm , high bp as mean systolic / diastolic bp at least 130/85 mm hg or currently on antihypertensive therapy , high serum tg as at least 1.7 mmol / l ( 150 mg / dl ) , low hdl - c as less than 1.3 mmol / l ( 50 mg / dl ) , and high fasting plasma glucose as at least 5.5 mmol / l ( 100 mg / dl ) or current use of antidiabetic medication . nontargeted mass spectrometry - based metabolomic profiling was performed with fasting plasma samples via capillary electrophoresis time - of - flight mass spectrometry . metabolite extraction from plasma was completed within 6 hours after collection to minimize the effect of metabolic changes in plasma . capillary electrophoresis time - of - flight mass spectrometry analysis of cationic and anionic metabolites was performed as described previously . raw data were processed using our proprietary software ( masterhands ) . as a preliminary study , we identified 290 metabolite peaks ( 131 cations and 159 anions ) in plasma : 154 known with standard compounds and 136 unknown . we decided a priori to measure absolute concentrations of 115 metabolites ( 63 cations and 52 anions ) that were expected to be observed stably in most human plasma samples and matched with standard compounds . to monitor the stability of metabolome analysis , quality control samples were injected every 10 samples and assessed at the start of the analytical run and at intervals throughout the analysis . the technical variability and between - subject variability for each metabolite assayed is shown in supplementary table 1 . participants were randomly allocated into two groups ( 2:1 ) to create two independent original and replication populations . analyses were first performed in the original population , and then the same analyses were performed in the replication population to confirm the robustness of the study outcome . the two groups were stratified for age and batch number to eliminate any confounding effects of age and analytical variability between multiple batches . out of 115 metabolites , 37 had plasma levels below the assay limits of detection ( lod ) in more than 90% of the participants , and therefore were excluded from further analyses . the remaining 78 metabolites were analyzed . for samples with levels below lod , which comprised less than 1% of all samples , the distribution of each metabolite concentration was tested for normality using quantile - quantile plots . on the basis of the shape of the distribution , most of the metabolite concentrations were log - transformed . the characteristics of participants with and without mets were compared by t test and test for continuous and categorical variables , respectively . age - adjusted spearman correlation coefficients were calculated for each metabolite pair in the original population . linear regression analysis was performed between the mets and non - mets groups with each metabolite concentration used as the outcome to examine the association between plasma metabolite concentrations and mets . analysis of covariance was performed to adjust for possible confounders using age , ldl - c levels , current smoking status , current alcohol consumption , physical activity , and dietary intake level . we calculated p values using the benjamini and hochberg false discovery rate method ( = 0.05 ) for analysis of the original population to adjust for independent multiple comparisons . sensitivity analyses were performed by excluding ( 1 ) women with bmi at least 25 kg / m and ( 2 ) those taking medications for hypertension , dyslipidemia , and diabetes . both sensitivity and replication analyses were conducted using the metabolites shown to be significantly associated with mets in the original population ( unadjusted false discovery rate p < 0.05 ) . sas 9.3 ( sas institute inc , cary , nc ) was used for all analyses . the final data set included 877 participants , with 594 women in the original population and 283 in the replication population . in the original population , 149 women were diagnosed with mets and 445 women did not meet the criteria ( non - mets ) . in the replication population , 72 women were diagnosed with mets and 211 were free of mets . the ratio of mets to non - mets participants was similar in the two populations ( 1:3 ) . women with mets were older than those without mets ( 65.5 vs 64.1 y , p = 0.004 ) , and had higher tg ( 111.0 vs 76.0 mg / dl ) and lower hdl - c ( 64.8 vs 75.3 mg / dl ) levels . there was no significant difference in serum ldl - c levels ( 126.1 vs 124.9 mg / dl ) or in lifestyle characteristics ( current smoking , current alcohol consumption , calorie intake , and physical activity ) between the two groups . most characteristics of the replication population were similar to those of the original population ( data not shown ) . the only discrepancy between the two populations was for alcohol intake , with significantly fewer women with mets having current alcohol intake in the replication population ( 7.0% vs 51.0% , p = 0.01 ) . high correlations within groups of related metabolites were observed for amino acids and their metabolites , and for intermediates of the tricarboxylic acid ( tca ) cycle , suggesting the importance of these metabolites in revealing the network of biochemical reactions in humans ( fig . 2 ) . we discovered a total of 19 metabolites with plasma concentrations that differed significantly between mets and non - mets in the original population , of which 11 remained significant after adjusting for possible confounders ( table 2 ) . plasma levels of branched - chain amino acids ( bcaas ) and their derivatives were 7% to 10% higher in mets . levels of phenylalanine and tyrosine tended to be slightly higher in mets , but significance disappeared after adjustments . other metabolites that showed significantly higher concentrations in mets included alanine , glutamate , alpha - aminoadipate , cystine , and proline . a suggestive positive trend could also be seen for lactate and pyruvate ( p = 0.06 and 0.07 , respectively ) . 3-hydroxybutyrate levels were 18% lower in the mets group compared with the non - mets group . correlation matrix for plasma metabolite concentrations in the original population ( n = 594 ) . age - adjusted spearman coefficients were calculated for each pair of metabolite levels in the original population . out of the 19 metabolites , 16 ( 84.2% ) could be replicated ( table 2 ) , including those related to bcaa metabolism , aromatic amino acid metabolism , alanine , aspartate and glutamate metabolism , glucose - alanine cycle , lysine metabolism , proline and arginine metabolism , and the tca cycle . among these comparisons of the adjusted means of the 13 metabolites between non - mets and mets in the original population are shown in figure 3 . sensitivity analyses excluding ( 1 ) participants with bmi at least 25 kg / m and ( 2 ) those taking medications gave similar results ( data not shown ) . the adjusted mean concentration of each metabolite was calculated using the fully adjusted model of the original population . the final data set included 877 participants , with 594 women in the original population and 283 in the replication population . in the original population , 149 women were diagnosed with mets and 445 women did not meet the criteria ( non - mets ) . in the replication population , 72 women were diagnosed with mets and 211 were free of mets . the ratio of mets to non - mets participants was similar in the two populations ( 1:3 ) . women with mets were older than those without mets ( 65.5 vs 64.1 y , p = 0.004 ) , and had higher tg ( 111.0 vs 76.0 mg / dl ) and lower hdl - c ( 64.8 vs 75.3 mg / dl ) levels . there was no significant difference in serum ldl - c levels ( 126.1 vs 124.9 mg / dl ) or in lifestyle characteristics ( current smoking , current alcohol consumption , calorie intake , and physical activity ) between the two groups . most characteristics of the replication population were similar to those of the original population ( data not shown ) . the only discrepancy between the two populations was for alcohol intake , with significantly fewer women with mets having current alcohol intake in the replication population ( 7.0% vs 51.0% , p = 0.01 ) . high correlations within groups of related metabolites were observed for amino acids and their metabolites , and for intermediates of the tricarboxylic acid ( tca ) cycle , suggesting the importance of these metabolites in revealing the network of biochemical reactions in humans ( fig . 2 ) . we discovered a total of 19 metabolites with plasma concentrations that differed significantly between mets and non - mets in the original population , of which 11 remained significant after adjusting for possible confounders ( table 2 ) . plasma levels of branched - chain amino acids ( bcaas ) and their derivatives were 7% to 10% higher in mets . levels of phenylalanine and tyrosine tended to be slightly higher in mets , but significance disappeared after adjustments . other metabolites that showed significantly higher concentrations in mets included alanine , glutamate , alpha - aminoadipate , cystine , and proline . a suggestive positive trend could also be seen for lactate and pyruvate ( p = 0.06 and 0.07 , respectively ) . 3-hydroxybutyrate levels were 18% lower in the mets group compared with the non - mets group . correlation matrix for plasma metabolite concentrations in the original population ( n = 594 ) . age - adjusted spearman coefficients were calculated for each pair of metabolite levels in the original population . bcaa , branched - chain amino acid ; tca , tricarboxylic acid . out of the 19 metabolites , 16 ( 84.2% ) could be replicated ( table 2 ) , including those related to bcaa metabolism , aromatic amino acid metabolism , alanine , aspartate and glutamate metabolism , glucose - alanine cycle , lysine metabolism , proline and arginine metabolism , and the tca cycle . among these comparisons of the adjusted means of the 13 metabolites between non - mets and mets in the original population are shown in figure 3 . sensitivity analyses excluding ( 1 ) participants with bmi at least 25 kg / m and ( 2 ) those taking medications gave similar results ( data not shown ) . the adjusted mean concentration of each metabolite was calculated using the fully adjusted model of the original population . to the best of our knowledge , this is the first population - based cross - sectional study to report associations between polar metabolites and mets in postmenopausal women in a lean asian population ( average bmi 23 kg / m ) with average hdl - c levels of 72.1 mg / dl . our data indicate that alterations in amino acid metabolism are present in both obese individuals and in women with normal bmi with mets after menopause . 4-methyl-2-oxopentaonate and 2-oxoisopentanoate , which are degradation products of bcaas , were significantly elevated in mets , in addition to bcaas themselves . the significant increase of bcaas in plasma in mets is consistent with many prior experimental and physiological studies . in a study of lean and obese individuals , bcaas accounted for the largest amount of variance in principal components analysis of metabolite data , along with derivatives from oxidation products of bcaas . this increase in levels of downstream metabolites indicates that the associations with mets are related to an alteration in the flux of bcaas through this catabolic pathway . a leading theory of the mechanism behind this physiological change is suppression of bcaa catabolism under insulin resistance . diabetic rats and humans have significantly lower levels of branched - chain keto acid dehydrogenase ( bckdh ) enzyme activity , which is the rate - limiting step in overall catabolism of bcaas . several theories have been proposed for the mechanisms underlying suppressed bckdh activity , including increased activity of bckdh kinase that inactivates bckdh , and reduction of adiponectin signaling via an adenosine monophosphate - activated protein kinase 2-dependent pathway . the increase of bcaas has also been explained based on amino acid breakdown products being released into blood because insulin resistance causes excess protein breakdown in skeletal muscle . a causal role for bcaas in metabolic disease via the mammalian target of rapamycin pathway or inhibition of glucose transport / phosphorylation has also been suggested . whether the increase in bcaas reflects the consequence of diabetes pathogenesis or has a causal role in disease development has yet to be proven because many large epidemiological studies have had a cross - sectional or case - control design . further work is needed to determine the mechanisms through which increased amino acid levels might contribute to metabolic diseases . in our study , alpha - aminoadipate levels were 6.4% to 17.6% higher in plasma of women with mets compared with those without mets . aminoadipate was found to be the strongest biomarker of diabetes risk out of 70 metabolites screened in the framingham heart study , in which participants with the highest quartile of plasma aminoadipate had fourfold higher odds of developing diabetes over a 12-year follow - up period compared with those in the lowest quartile . there were no correlations between aminoadipate and bcaas . because aminoadipate is generated by lysine degradation and may also serve as a substrate for enzymes downstream of tryptophan metabolism , the current and previous findings collectively suggest that the mechanism behind mets and insulin resistance involves alterations in these metabolic pathways , distinct from pathways of bcaas . 3-hydroxybutyrate , a ketone body , was significantly lower in mets compared with non - mets participants ( 5.7%-28.4% ) . this is in contrast to previous studies showing that alpha - hydroxybutyrate is a positive predictor of type 2 diabetes . alpha - hydroxybutyrate is generated in the liver under increased oxidative stress ; therefore , the different results and the suggestive trends in lactate and pyruvate concentrations may be indicative of down- and up - regulation of ketogenesis and its relationship to the tca cycle . another hypothesis is that the phenotype of mets in our population differs from those in previous studies , especially with respect to the very high levels of hdl - c . a recent review article on bcaas in insulin resistance described two types of obesity in rodents with regard to impairment of bcaa metabolism . similarly , the alpha - hydroxybutyrate levels might be regulated in a more complex manner . one of the strengths of our study is the characteristics of the participants . to our knowledge , metabolomic profiling of mets on a population of lean , older females has not been performed previously . the only study in women with normal bmi ( average 24.4 kg / m ) was reported by wrtz et al ; however , the average age of the participants was 32.1 years . metabolism changes significantly with age and data should be carefully interpreted , even with adjustment of statistical models for age . in addition , the average hdl - c level of our participants was much higher than those in other populations . hdl - c levels are indeed high among japanese people in general , and have increased in the past few decades . as one of the components of mets , decreased hdl - c is an independent risk factor for cvd , and the tg / hdl - c ratio has a positive correlation with the insulin resistance index . our data suggest that alterations in plasma amino acids may precede the reduction of hdl - c levels in the development of insulin resistance . the study has several limitations . when interpreting metabolic profiling data in a cross - sectional study , the temporality of cause - effect relationships is not assured and possible confounding of unmeasured factors may exist , even after adjustments . equally of concern is that our data were derived from a single site , lacking test marker performance in an independent cohort . therefore , follow - up and intervention studies along with those at different facilities are needed . comparisons between pre- and postmenopausal women could not be performed because of the few premenopausal women enrolled in the study . completion of a separate ongoing cohort with a working age population is awaited to investigate the impact of menopause on these metabolic changes . other common sites for wc measurements include the midpoint between the lowest rib and the iliac crest ; narrowest or widest wc ; just below the lowest rib ; and just above the iliac crest . however , there is no consensus on the optimal protocol for measurement of wc . an effort was made to eliminate conditions that could affect the metabolic profile , such as history of cancer and nonfasting status , but multiple drugs for treated participants may have acted as confounders . various factors might also have influenced measurement variability in the metabolomics analysis , potentially causing nondifferential misclassification of metabolomics data . to minimize these variabilities , we set a uniform fasting condition for participants and standardized the quality control procedures for metabolomics analysis . this study shows that japanese postmenopausal women who develop mets may have elevated concentrations of multiple amino acids , including bcaas , alanine , glutamate , lysine , proline , and other polar metabolites such as alpha - aminoadipate . these findings may not be translatable to populations of non - japanese women without very high hdl - c levels , but identifying these metabolic changes may be useful for detection of high - risk individuals , including those with normal bmi and relatively high hdl - c levels . a follow - up survey is needed to examine whether these candidate metabolites provide better prediction than standard measures for future development of mets .

abstractobjective : the aim of the study was to investigate the associations of amino acids and other polar metabolites with metabolic syndrome ( mets ) in postmenopausal women in a lean asian population.methods:the participants were 1,422 female residents enrolled in a cohort study from april to august 2012 . mets was defined according to the national cholesterol education program adult treatment panel iii modified for japanese women . associations were examined between mets and 78 metabolites assayed in fasting plasma samples using capillary electrophoresis - mass spectrometry . replication analysis was performed to confirm the robustness of the results in a separate population created by random allocation.results:analysis was performed for 877 naturally postmenopausal women , including 594 in the original population and 283 in the replication population . the average age , body mass index , and levels of high- and low - density lipoprotein cholesterol of the entire population were 64.6 years , 23.0 kg / m2 , 72.1 mg / dl , and 126.1 mg / dl , respectively . there was no significant difference in low - density lipoprotein cholesterol levels between women with and without mets . thirteen metabolites were significantly related to mets : multiple plasma amino acids were elevated in women with mets , including branched - chain amino acids , alanine , glutamate , and proline ; and alpha - aminoadipate , which is generated by lysine degradation , was also significantly increased.conclusions:our large - scale metabolomic profiling indicates that japanese postmenopausal women with mets have abnormal polar metabolites , suggesting altered catabolic pathways . these results may help to understand metabolic disturbance , including in persons with normal body mass index and relatively high levels of high - density lipoprotein cholesterol , and may have clinical utility based on further studies .

METHODS Study population Data collection, anthropometric measurements, and biochemical examinations Definition of MetS Metabolomics measurement Statistical analysis RESULTS Population characteristics Potential biomarkers of MetS in plasma metabolites DISCUSSION CONCLUSIONS Supplementary Material Supplementary Material

the study base was the participants of the ongoing tsuruoka metabolomic cohort study , initiated in april 2012 ( yamagata prefecture , japan ) , comprising individuals aged 35 to 74 years who were recruited among attendees of annual municipal or worksite health check - up programs in the city . a total of 1,422 women consented to participate during the first few months of the baseline period , and plasma metabolomic profiling in these women was completed by the end of 2014 . only postmenopausal female participants were ultimately included in the analysis due to the small number of premenopausal women . the study was approved by the medical ethics committee of the school of medicine , keio university , tokyo , japan ( approval no . all data and samples were obtained in the baseline study , including anthropometrics , clinical biochemistry , and blood specimens for metabolomic profiling . detailed information was collected through a standardized self - administered questionnaire on medical history , including treatment of hypertension , dyslipidemia , and diabetes ; gynecological and reproductive history , including menopause status ( pre / peri / post ) , ages at menarche and menopause , reason for menopause ( natural / medical / others ) , number of pregnancies and deliveries , age at first delivery , use of hormone therapy ( if yes , number of years used and when it was used last ) , and whether oophorectomy was ever performed ; and lifestyle parameters such as smoking habits , alcohol intake , diet , stress , and physical activity . if the umbilicus drooped down , the measurement was made midway between the inferior margin of the last rib and the top of the iliac crest in a horizontal plane . bp was measured twice on one occasion in the sitting position using an automated sphygmomanometer ( omron hbp - t105s - n ) , and the mean of the two measurements was used for analysis . serum levels of total cholesterol , tg , and fasting plasma glucose were analyzed using enzymatic methods , and glycated hemoglobin ( hba1c ) was determined by immunoassay . mets was defined using the modified national cholesterol education program adult treatment panel iii definition with 100 mg / dl as the cutoff for the glucose level . because lower overall adiposity has been associated with an increased risk of medical conditions such as type 2 diabetes in asian countries , the threshold for wc was set according to the recommendation for asians established by several organizations , including the national cholesterol education program . specifically , central obesity was defined as wc at least 80 cm , high bp as mean systolic / diastolic bp at least 130/85 mm hg or currently on antihypertensive therapy , high serum tg as at least 1.7 mmol / l ( 150 mg / dl ) , low hdl - c as less than 1.3 mmol / l ( 50 mg / dl ) , and high fasting plasma glucose as at least 5.5 mmol / l ( 100 mg / dl ) or current use of antidiabetic medication . nontargeted mass spectrometry - based metabolomic profiling was performed with fasting plasma samples via capillary electrophoresis time - of - flight mass spectrometry . capillary electrophoresis time - of - flight mass spectrometry analysis of cationic and anionic metabolites was performed as described previously . to monitor the stability of metabolome analysis , quality control samples were injected every 10 samples and assessed at the start of the analytical run and at intervals throughout the analysis . analyses were first performed in the original population , and then the same analyses were performed in the replication population to confirm the robustness of the study outcome . out of 115 metabolites , 37 had plasma levels below the assay limits of detection ( lod ) in more than 90% of the participants , and therefore were excluded from further analyses . the remaining 78 metabolites were analyzed . the characteristics of participants with and without mets were compared by t test and test for continuous and categorical variables , respectively . age - adjusted spearman correlation coefficients were calculated for each metabolite pair in the original population . linear regression analysis was performed between the mets and non - mets groups with each metabolite concentration used as the outcome to examine the association between plasma metabolite concentrations and mets . analysis of covariance was performed to adjust for possible confounders using age , ldl - c levels , current smoking status , current alcohol consumption , physical activity , and dietary intake level . we calculated p values using the benjamini and hochberg false discovery rate method ( = 0.05 ) for analysis of the original population to adjust for independent multiple comparisons . sensitivity analyses were performed by excluding ( 1 ) women with bmi at least 25 kg / m and ( 2 ) those taking medications for hypertension , dyslipidemia , and diabetes . both sensitivity and replication analyses were conducted using the metabolites shown to be significantly associated with mets in the original population ( unadjusted false discovery rate p < 0.05 ) . the study base was the participants of the ongoing tsuruoka metabolomic cohort study , initiated in april 2012 ( yamagata prefecture , japan ) , comprising individuals aged 35 to 74 years who were recruited among attendees of annual municipal or worksite health check - up programs in the city . a total of 1,422 women consented to participate during the first few months of the baseline period , and plasma metabolomic profiling in these women was completed by the end of 2014 . only postmenopausal female participants were ultimately included in the analysis due to the small number of premenopausal women . the study was approved by the medical ethics committee of the school of medicine , keio university , tokyo , japan ( approval no . all data and samples were obtained in the baseline study , including anthropometrics , clinical biochemistry , and blood specimens for metabolomic profiling . detailed information was collected through a standardized self - administered questionnaire on medical history , including treatment of hypertension , dyslipidemia , and diabetes ; gynecological and reproductive history , including menopause status ( pre / peri / post ) , ages at menarche and menopause , reason for menopause ( natural / medical / others ) , number of pregnancies and deliveries , age at first delivery , use of hormone therapy ( if yes , number of years used and when it was used last ) , and whether oophorectomy was ever performed ; and lifestyle parameters such as smoking habits , alcohol intake , diet , stress , and physical activity . if the umbilicus drooped down , the measurement was made midway between the inferior margin of the last rib and the top of the iliac crest in a horizontal plane . bp was measured twice on one occasion in the sitting position using an automated sphygmomanometer ( omron hbp - t105s - n ) , and the mean of the two measurements was used for analysis . serum levels of total cholesterol , tg , and fasting plasma glucose were analyzed using enzymatic methods , and glycated hemoglobin ( hba1c ) was determined by immunoassay . mets was defined using the modified national cholesterol education program adult treatment panel iii definition with 100 mg / dl as the cutoff for the glucose level . because lower overall adiposity has been associated with an increased risk of medical conditions such as type 2 diabetes in asian countries , the threshold for wc was set according to the recommendation for asians established by several organizations , including the national cholesterol education program . specifically , central obesity was defined as wc at least 80 cm , high bp as mean systolic / diastolic bp at least 130/85 mm hg or currently on antihypertensive therapy , high serum tg as at least 1.7 mmol / l ( 150 mg / dl ) , low hdl - c as less than 1.3 mmol / l ( 50 mg / dl ) , and high fasting plasma glucose as at least 5.5 mmol / l ( 100 mg / dl ) or current use of antidiabetic medication . nontargeted mass spectrometry - based metabolomic profiling was performed with fasting plasma samples via capillary electrophoresis time - of - flight mass spectrometry . capillary electrophoresis time - of - flight mass spectrometry analysis of cationic and anionic metabolites was performed as described previously . analyses were first performed in the original population , and then the same analyses were performed in the replication population to confirm the robustness of the study outcome . out of 115 metabolites , 37 had plasma levels below the assay limits of detection ( lod ) in more than 90% of the participants , and therefore were excluded from further analyses . the remaining 78 metabolites were analyzed . the characteristics of participants with and without mets were compared by t test and test for continuous and categorical variables , respectively . age - adjusted spearman correlation coefficients were calculated for each metabolite pair in the original population . linear regression analysis was performed between the mets and non - mets groups with each metabolite concentration used as the outcome to examine the association between plasma metabolite concentrations and mets . analysis of covariance was performed to adjust for possible confounders using age , ldl - c levels , current smoking status , current alcohol consumption , physical activity , and dietary intake level . we calculated p values using the benjamini and hochberg false discovery rate method ( = 0.05 ) for analysis of the original population to adjust for independent multiple comparisons . sensitivity analyses were performed by excluding ( 1 ) women with bmi at least 25 kg / m and ( 2 ) those taking medications for hypertension , dyslipidemia , and diabetes . both sensitivity and replication analyses were conducted using the metabolites shown to be significantly associated with mets in the original population ( unadjusted false discovery rate p < 0.05 ) . the final data set included 877 participants , with 594 women in the original population and 283 in the replication population . in the original population , 149 women were diagnosed with mets and 445 women did not meet the criteria ( non - mets ) . in the replication population , 72 women were diagnosed with mets and 211 were free of mets . women with mets were older than those without mets ( 65.5 vs 64.1 y , p = 0.004 ) , and had higher tg ( 111.0 vs 76.0 mg / dl ) and lower hdl - c ( 64.8 vs 75.3 mg / dl ) levels . there was no significant difference in serum ldl - c levels ( 126.1 vs 124.9 mg / dl ) or in lifestyle characteristics ( current smoking , current alcohol consumption , calorie intake , and physical activity ) between the two groups . most characteristics of the replication population were similar to those of the original population ( data not shown ) . the only discrepancy between the two populations was for alcohol intake , with significantly fewer women with mets having current alcohol intake in the replication population ( 7.0% vs 51.0% , p = 0.01 ) . high correlations within groups of related metabolites were observed for amino acids and their metabolites , and for intermediates of the tricarboxylic acid ( tca ) cycle , suggesting the importance of these metabolites in revealing the network of biochemical reactions in humans ( fig . we discovered a total of 19 metabolites with plasma concentrations that differed significantly between mets and non - mets in the original population , of which 11 remained significant after adjusting for possible confounders ( table 2 ) . plasma levels of branched - chain amino acids ( bcaas ) and their derivatives were 7% to 10% higher in mets . levels of phenylalanine and tyrosine tended to be slightly higher in mets , but significance disappeared after adjustments . other metabolites that showed significantly higher concentrations in mets included alanine , glutamate , alpha - aminoadipate , cystine , and proline . correlation matrix for plasma metabolite concentrations in the original population ( n = 594 ) . age - adjusted spearman coefficients were calculated for each pair of metabolite levels in the original population . out of the 19 metabolites , 16 ( 84.2% ) could be replicated ( table 2 ) , including those related to bcaa metabolism , aromatic amino acid metabolism , alanine , aspartate and glutamate metabolism , glucose - alanine cycle , lysine metabolism , proline and arginine metabolism , and the tca cycle . among these comparisons of the adjusted means of the 13 metabolites between non - mets and mets in the original population are shown in figure 3 . the adjusted mean concentration of each metabolite was calculated using the fully adjusted model of the original population . the final data set included 877 participants , with 594 women in the original population and 283 in the replication population . in the original population , 149 women were diagnosed with mets and 445 women did not meet the criteria ( non - mets ) . in the replication population , 72 women were diagnosed with mets and 211 were free of mets . women with mets were older than those without mets ( 65.5 vs 64.1 y , p = 0.004 ) , and had higher tg ( 111.0 vs 76.0 mg / dl ) and lower hdl - c ( 64.8 vs 75.3 mg / dl ) levels . there was no significant difference in serum ldl - c levels ( 126.1 vs 124.9 mg / dl ) or in lifestyle characteristics ( current smoking , current alcohol consumption , calorie intake , and physical activity ) between the two groups . most characteristics of the replication population were similar to those of the original population ( data not shown ) . the only discrepancy between the two populations was for alcohol intake , with significantly fewer women with mets having current alcohol intake in the replication population ( 7.0% vs 51.0% , p = 0.01 ) . high correlations within groups of related metabolites were observed for amino acids and their metabolites , and for intermediates of the tricarboxylic acid ( tca ) cycle , suggesting the importance of these metabolites in revealing the network of biochemical reactions in humans ( fig . we discovered a total of 19 metabolites with plasma concentrations that differed significantly between mets and non - mets in the original population , of which 11 remained significant after adjusting for possible confounders ( table 2 ) . plasma levels of branched - chain amino acids ( bcaas ) and their derivatives were 7% to 10% higher in mets . levels of phenylalanine and tyrosine tended to be slightly higher in mets , but significance disappeared after adjustments . other metabolites that showed significantly higher concentrations in mets included alanine , glutamate , alpha - aminoadipate , cystine , and proline . correlation matrix for plasma metabolite concentrations in the original population ( n = 594 ) . age - adjusted spearman coefficients were calculated for each pair of metabolite levels in the original population . bcaa , branched - chain amino acid ; tca , tricarboxylic acid . out of the 19 metabolites , 16 ( 84.2% ) could be replicated ( table 2 ) , including those related to bcaa metabolism , aromatic amino acid metabolism , alanine , aspartate and glutamate metabolism , glucose - alanine cycle , lysine metabolism , proline and arginine metabolism , and the tca cycle . among these comparisons of the adjusted means of the 13 metabolites between non - mets and mets in the original population are shown in figure 3 . the adjusted mean concentration of each metabolite was calculated using the fully adjusted model of the original population . to the best of our knowledge , this is the first population - based cross - sectional study to report associations between polar metabolites and mets in postmenopausal women in a lean asian population ( average bmi 23 kg / m ) with average hdl - c levels of 72.1 mg / dl . our data indicate that alterations in amino acid metabolism are present in both obese individuals and in women with normal bmi with mets after menopause . 4-methyl-2-oxopentaonate and 2-oxoisopentanoate , which are degradation products of bcaas , were significantly elevated in mets , in addition to bcaas themselves . this increase in levels of downstream metabolites indicates that the associations with mets are related to an alteration in the flux of bcaas through this catabolic pathway . diabetic rats and humans have significantly lower levels of branched - chain keto acid dehydrogenase ( bckdh ) enzyme activity , which is the rate - limiting step in overall catabolism of bcaas . several theories have been proposed for the mechanisms underlying suppressed bckdh activity , including increased activity of bckdh kinase that inactivates bckdh , and reduction of adiponectin signaling via an adenosine monophosphate - activated protein kinase 2-dependent pathway . in our study , alpha - aminoadipate levels were 6.4% to 17.6% higher in plasma of women with mets compared with those without mets . because aminoadipate is generated by lysine degradation and may also serve as a substrate for enzymes downstream of tryptophan metabolism , the current and previous findings collectively suggest that the mechanism behind mets and insulin resistance involves alterations in these metabolic pathways , distinct from pathways of bcaas . alpha - hydroxybutyrate is generated in the liver under increased oxidative stress ; therefore , the different results and the suggestive trends in lactate and pyruvate concentrations may be indicative of down- and up - regulation of ketogenesis and its relationship to the tca cycle . another hypothesis is that the phenotype of mets in our population differs from those in previous studies , especially with respect to the very high levels of hdl - c . similarly , the alpha - hydroxybutyrate levels might be regulated in a more complex manner . the only study in women with normal bmi ( average 24.4 kg / m ) was reported by wrtz et al ; however , the average age of the participants was 32.1 years . hdl - c levels are indeed high among japanese people in general , and have increased in the past few decades . as one of the components of mets , decreased hdl - c is an independent risk factor for cvd , and the tg / hdl - c ratio has a positive correlation with the insulin resistance index . our data suggest that alterations in plasma amino acids may precede the reduction of hdl - c levels in the development of insulin resistance . comparisons between pre- and postmenopausal women could not be performed because of the few premenopausal women enrolled in the study . completion of a separate ongoing cohort with a working age population is awaited to investigate the impact of menopause on these metabolic changes . this study shows that japanese postmenopausal women who develop mets may have elevated concentrations of multiple amino acids , including bcaas , alanine , glutamate , lysine , proline , and other polar metabolites such as alpha - aminoadipate . these findings may not be translatable to populations of non - japanese women without very high hdl - c levels , but identifying these metabolic changes may be useful for detection of high - risk individuals , including those with normal bmi and relatively high hdl - c levels .

the study base was the participants of the ongoing tsuruoka metabolomic cohort study , initiated in april 2012 ( yamagata prefecture , japan ) , comprising individuals aged 35 to 74 years who were recruited among attendees of annual municipal or worksite health check - up programs in the city . a total of 1,422 women consented to participate during the first few months of the baseline period , and plasma metabolomic profiling in these women was completed by the end of 2014 . in addition , women who had used hormone therapy within 1 year or had undergone oophorectomy after menopause were excluded to eliminate the possible effect on the metabolic profile of administered estrogen and hormones excreted from postmenopausal ovaries . all data and samples were obtained in the baseline study , including anthropometrics , clinical biochemistry , and blood specimens for metabolomic profiling . detailed information was collected through a standardized self - administered questionnaire on medical history , including treatment of hypertension , dyslipidemia , and diabetes ; gynecological and reproductive history , including menopause status ( pre / peri / post ) , ages at menarche and menopause , reason for menopause ( natural / medical / others ) , number of pregnancies and deliveries , age at first delivery , use of hormone therapy ( if yes , number of years used and when it was used last ) , and whether oophorectomy was ever performed ; and lifestyle parameters such as smoking habits , alcohol intake , diet , stress , and physical activity . if the umbilicus drooped down , the measurement was made midway between the inferior margin of the last rib and the top of the iliac crest in a horizontal plane . bp was measured twice on one occasion in the sitting position using an automated sphygmomanometer ( omron hbp - t105s - n ) , and the mean of the two measurements was used for analysis . serum levels of total cholesterol , tg , and fasting plasma glucose were analyzed using enzymatic methods , and glycated hemoglobin ( hba1c ) was determined by immunoassay . mets was defined using the modified national cholesterol education program adult treatment panel iii definition with 100 mg / dl as the cutoff for the glucose level . because lower overall adiposity has been associated with an increased risk of medical conditions such as type 2 diabetes in asian countries , the threshold for wc was set according to the recommendation for asians established by several organizations , including the national cholesterol education program . specifically , central obesity was defined as wc at least 80 cm , high bp as mean systolic / diastolic bp at least 130/85 mm hg or currently on antihypertensive therapy , high serum tg as at least 1.7 mmol / l ( 150 mg / dl ) , low hdl - c as less than 1.3 mmol / l ( 50 mg / dl ) , and high fasting plasma glucose as at least 5.5 mmol / l ( 100 mg / dl ) or current use of antidiabetic medication . nontargeted mass spectrometry - based metabolomic profiling was performed with fasting plasma samples via capillary electrophoresis time - of - flight mass spectrometry . capillary electrophoresis time - of - flight mass spectrometry analysis of cationic and anionic metabolites was performed as described previously . as a preliminary study , we identified 290 metabolite peaks ( 131 cations and 159 anions ) in plasma : 154 known with standard compounds and 136 unknown . we decided a priori to measure absolute concentrations of 115 metabolites ( 63 cations and 52 anions ) that were expected to be observed stably in most human plasma samples and matched with standard compounds . to monitor the stability of metabolome analysis , quality control samples were injected every 10 samples and assessed at the start of the analytical run and at intervals throughout the analysis . analyses were first performed in the original population , and then the same analyses were performed in the replication population to confirm the robustness of the study outcome . out of 115 metabolites , 37 had plasma levels below the assay limits of detection ( lod ) in more than 90% of the participants , and therefore were excluded from further analyses . for samples with levels below lod , which comprised less than 1% of all samples , the distribution of each metabolite concentration was tested for normality using quantile - quantile plots . on the basis of the shape of the distribution , most of the metabolite concentrations were log - transformed . linear regression analysis was performed between the mets and non - mets groups with each metabolite concentration used as the outcome to examine the association between plasma metabolite concentrations and mets . analysis of covariance was performed to adjust for possible confounders using age , ldl - c levels , current smoking status , current alcohol consumption , physical activity , and dietary intake level . both sensitivity and replication analyses were conducted using the metabolites shown to be significantly associated with mets in the original population ( unadjusted false discovery rate p < 0.05 ) . the study base was the participants of the ongoing tsuruoka metabolomic cohort study , initiated in april 2012 ( yamagata prefecture , japan ) , comprising individuals aged 35 to 74 years who were recruited among attendees of annual municipal or worksite health check - up programs in the city . a total of 1,422 women consented to participate during the first few months of the baseline period , and plasma metabolomic profiling in these women was completed by the end of 2014 . in addition , women who had used hormone therapy within 1 year or had undergone oophorectomy after menopause were excluded to eliminate the possible effect on the metabolic profile of administered estrogen and hormones excreted from postmenopausal ovaries . all data and samples were obtained in the baseline study , including anthropometrics , clinical biochemistry , and blood specimens for metabolomic profiling . detailed information was collected through a standardized self - administered questionnaire on medical history , including treatment of hypertension , dyslipidemia , and diabetes ; gynecological and reproductive history , including menopause status ( pre / peri / post ) , ages at menarche and menopause , reason for menopause ( natural / medical / others ) , number of pregnancies and deliveries , age at first delivery , use of hormone therapy ( if yes , number of years used and when it was used last ) , and whether oophorectomy was ever performed ; and lifestyle parameters such as smoking habits , alcohol intake , diet , stress , and physical activity . if the umbilicus drooped down , the measurement was made midway between the inferior margin of the last rib and the top of the iliac crest in a horizontal plane . bp was measured twice on one occasion in the sitting position using an automated sphygmomanometer ( omron hbp - t105s - n ) , and the mean of the two measurements was used for analysis . serum levels of total cholesterol , tg , and fasting plasma glucose were analyzed using enzymatic methods , and glycated hemoglobin ( hba1c ) was determined by immunoassay . because lower overall adiposity has been associated with an increased risk of medical conditions such as type 2 diabetes in asian countries , the threshold for wc was set according to the recommendation for asians established by several organizations , including the national cholesterol education program . specifically , central obesity was defined as wc at least 80 cm , high bp as mean systolic / diastolic bp at least 130/85 mm hg or currently on antihypertensive therapy , high serum tg as at least 1.7 mmol / l ( 150 mg / dl ) , low hdl - c as less than 1.3 mmol / l ( 50 mg / dl ) , and high fasting plasma glucose as at least 5.5 mmol / l ( 100 mg / dl ) or current use of antidiabetic medication . nontargeted mass spectrometry - based metabolomic profiling was performed with fasting plasma samples via capillary electrophoresis time - of - flight mass spectrometry . as a preliminary study , we identified 290 metabolite peaks ( 131 cations and 159 anions ) in plasma : 154 known with standard compounds and 136 unknown . we decided a priori to measure absolute concentrations of 115 metabolites ( 63 cations and 52 anions ) that were expected to be observed stably in most human plasma samples and matched with standard compounds . to monitor the stability of metabolome analysis , quality control samples were injected every 10 samples and assessed at the start of the analytical run and at intervals throughout the analysis . analyses were first performed in the original population , and then the same analyses were performed in the replication population to confirm the robustness of the study outcome . out of 115 metabolites , 37 had plasma levels below the assay limits of detection ( lod ) in more than 90% of the participants , and therefore were excluded from further analyses . for samples with levels below lod , which comprised less than 1% of all samples , the distribution of each metabolite concentration was tested for normality using quantile - quantile plots . on the basis of the shape of the distribution , most of the metabolite concentrations were log - transformed . linear regression analysis was performed between the mets and non - mets groups with each metabolite concentration used as the outcome to examine the association between plasma metabolite concentrations and mets . analysis of covariance was performed to adjust for possible confounders using age , ldl - c levels , current smoking status , current alcohol consumption , physical activity , and dietary intake level . both sensitivity and replication analyses were conducted using the metabolites shown to be significantly associated with mets in the original population ( unadjusted false discovery rate p < 0.05 ) . in the original population , 149 women were diagnosed with mets and 445 women did not meet the criteria ( non - mets ) . the ratio of mets to non - mets participants was similar in the two populations ( 1:3 ) . women with mets were older than those without mets ( 65.5 vs 64.1 y , p = 0.004 ) , and had higher tg ( 111.0 vs 76.0 mg / dl ) and lower hdl - c ( 64.8 vs 75.3 mg / dl ) levels . there was no significant difference in serum ldl - c levels ( 126.1 vs 124.9 mg / dl ) or in lifestyle characteristics ( current smoking , current alcohol consumption , calorie intake , and physical activity ) between the two groups . the only discrepancy between the two populations was for alcohol intake , with significantly fewer women with mets having current alcohol intake in the replication population ( 7.0% vs 51.0% , p = 0.01 ) . high correlations within groups of related metabolites were observed for amino acids and their metabolites , and for intermediates of the tricarboxylic acid ( tca ) cycle , suggesting the importance of these metabolites in revealing the network of biochemical reactions in humans ( fig . we discovered a total of 19 metabolites with plasma concentrations that differed significantly between mets and non - mets in the original population , of which 11 remained significant after adjusting for possible confounders ( table 2 ) . plasma levels of branched - chain amino acids ( bcaas ) and their derivatives were 7% to 10% higher in mets . other metabolites that showed significantly higher concentrations in mets included alanine , glutamate , alpha - aminoadipate , cystine , and proline . 3-hydroxybutyrate levels were 18% lower in the mets group compared with the non - mets group . out of the 19 metabolites , 16 ( 84.2% ) could be replicated ( table 2 ) , including those related to bcaa metabolism , aromatic amino acid metabolism , alanine , aspartate and glutamate metabolism , glucose - alanine cycle , lysine metabolism , proline and arginine metabolism , and the tca cycle . among these comparisons of the adjusted means of the 13 metabolites between non - mets and mets in the original population are shown in figure 3 . in the original population , 149 women were diagnosed with mets and 445 women did not meet the criteria ( non - mets ) . women with mets were older than those without mets ( 65.5 vs 64.1 y , p = 0.004 ) , and had higher tg ( 111.0 vs 76.0 mg / dl ) and lower hdl - c ( 64.8 vs 75.3 mg / dl ) levels . there was no significant difference in serum ldl - c levels ( 126.1 vs 124.9 mg / dl ) or in lifestyle characteristics ( current smoking , current alcohol consumption , calorie intake , and physical activity ) between the two groups . the only discrepancy between the two populations was for alcohol intake , with significantly fewer women with mets having current alcohol intake in the replication population ( 7.0% vs 51.0% , p = 0.01 ) . high correlations within groups of related metabolites were observed for amino acids and their metabolites , and for intermediates of the tricarboxylic acid ( tca ) cycle , suggesting the importance of these metabolites in revealing the network of biochemical reactions in humans ( fig . we discovered a total of 19 metabolites with plasma concentrations that differed significantly between mets and non - mets in the original population , of which 11 remained significant after adjusting for possible confounders ( table 2 ) . plasma levels of branched - chain amino acids ( bcaas ) and their derivatives were 7% to 10% higher in mets . other metabolites that showed significantly higher concentrations in mets included alanine , glutamate , alpha - aminoadipate , cystine , and proline . 3-hydroxybutyrate levels were 18% lower in the mets group compared with the non - mets group . out of the 19 metabolites , 16 ( 84.2% ) could be replicated ( table 2 ) , including those related to bcaa metabolism , aromatic amino acid metabolism , alanine , aspartate and glutamate metabolism , glucose - alanine cycle , lysine metabolism , proline and arginine metabolism , and the tca cycle . among these comparisons of the adjusted means of the 13 metabolites between non - mets and mets in the original population are shown in figure 3 . to the best of our knowledge , this is the first population - based cross - sectional study to report associations between polar metabolites and mets in postmenopausal women in a lean asian population ( average bmi 23 kg / m ) with average hdl - c levels of 72.1 mg / dl . our data indicate that alterations in amino acid metabolism are present in both obese individuals and in women with normal bmi with mets after menopause . 4-methyl-2-oxopentaonate and 2-oxoisopentanoate , which are degradation products of bcaas , were significantly elevated in mets , in addition to bcaas themselves . in a study of lean and obese individuals , bcaas accounted for the largest amount of variance in principal components analysis of metabolite data , along with derivatives from oxidation products of bcaas . this increase in levels of downstream metabolites indicates that the associations with mets are related to an alteration in the flux of bcaas through this catabolic pathway . diabetic rats and humans have significantly lower levels of branched - chain keto acid dehydrogenase ( bckdh ) enzyme activity , which is the rate - limiting step in overall catabolism of bcaas . several theories have been proposed for the mechanisms underlying suppressed bckdh activity , including increased activity of bckdh kinase that inactivates bckdh , and reduction of adiponectin signaling via an adenosine monophosphate - activated protein kinase 2-dependent pathway . a causal role for bcaas in metabolic disease via the mammalian target of rapamycin pathway or inhibition of glucose transport / phosphorylation has also been suggested . whether the increase in bcaas reflects the consequence of diabetes pathogenesis or has a causal role in disease development has yet to be proven because many large epidemiological studies have had a cross - sectional or case - control design . in our study , alpha - aminoadipate levels were 6.4% to 17.6% higher in plasma of women with mets compared with those without mets . aminoadipate was found to be the strongest biomarker of diabetes risk out of 70 metabolites screened in the framingham heart study , in which participants with the highest quartile of plasma aminoadipate had fourfold higher odds of developing diabetes over a 12-year follow - up period compared with those in the lowest quartile . because aminoadipate is generated by lysine degradation and may also serve as a substrate for enzymes downstream of tryptophan metabolism , the current and previous findings collectively suggest that the mechanism behind mets and insulin resistance involves alterations in these metabolic pathways , distinct from pathways of bcaas . 3-hydroxybutyrate , a ketone body , was significantly lower in mets compared with non - mets participants ( 5.7%-28.4% ) . alpha - hydroxybutyrate is generated in the liver under increased oxidative stress ; therefore , the different results and the suggestive trends in lactate and pyruvate concentrations may be indicative of down- and up - regulation of ketogenesis and its relationship to the tca cycle . another hypothesis is that the phenotype of mets in our population differs from those in previous studies , especially with respect to the very high levels of hdl - c . a recent review article on bcaas in insulin resistance described two types of obesity in rodents with regard to impairment of bcaa metabolism . to our knowledge , metabolomic profiling of mets on a population of lean , older females has not been performed previously . the only study in women with normal bmi ( average 24.4 kg / m ) was reported by wrtz et al ; however , the average age of the participants was 32.1 years . as one of the components of mets , decreased hdl - c is an independent risk factor for cvd , and the tg / hdl - c ratio has a positive correlation with the insulin resistance index . our data suggest that alterations in plasma amino acids may precede the reduction of hdl - c levels in the development of insulin resistance . when interpreting metabolic profiling data in a cross - sectional study , the temporality of cause - effect relationships is not assured and possible confounding of unmeasured factors may exist , even after adjustments . completion of a separate ongoing cohort with a working age population is awaited to investigate the impact of menopause on these metabolic changes . this study shows that japanese postmenopausal women who develop mets may have elevated concentrations of multiple amino acids , including bcaas , alanine , glutamate , lysine , proline , and other polar metabolites such as alpha - aminoadipate . these findings may not be translatable to populations of non - japanese women without very high hdl - c levels , but identifying these metabolic changes may be useful for detection of high - risk individuals , including those with normal bmi and relatively high hdl - c levels . a follow - up survey is needed to examine whether these candidate metabolites provide better prediction than standard measures for future development of mets .

although conservative treatment has its indications , surgical treatment for acl injuries is the ideal choice for young and active patients because it allows them to return to sports but prevents the onset of additional knee injuries.1 surgical techniques have evolved over recent decades , and the current gold standard treatment is arthroscopic intra - articular acl reconstruction with autogenous grafts . the main aspects of the surgical technique required to achieve favorable clinical results include graft quality , tunnel positioning and efficient graft fixation , with sufficient tension to provide joint stability and a physiological range of motion with normal flexion and extension.2 , 3 along with the increasing number of indications for surgery , increases in the frequency of revision surgery have also been reported . revision surgery is now performed in 1040% of cases.4 the main cause of surgical failure and acl revision is inadequate tunnel positioning.5 , 6 anatomical studies have reported that the anatomical acl origin and insertion is the most isometric graft position710 . however , it is known that even senior surgeons using conventional guides frequently fail to place the tunnels in the desired positions.11 , 12 a recent resource for improving orthopedic surgery precision is the computer assisted orthopedic surgery ( caos ) system.13 navigation systems for acl reconstruction have been developed,1418 but no comparative clinical studies have unequivocally demonstrated their superiority in relation to conventional guides . in addition to enhancement of precision , such navigation systems can also facilitate choosing the location for bony tunnels based on the new isometry criterion . before the advent of such systems , these data could not be supplied to surgeons intraoperatively . the purpose of the present study was to compare isometry and tunnel positioning using the caos system and using conventional guides . the inclusion criteria for the knee specimens used were that the cause of death was not traumatic or due to infectious disease and that there were no scars , hematomas or deformities in the lower limbs . the exclusion criteria diagnosed during arthroscopy were the presence of any ligament lesions , meniscal lesions or degenerative joint disease . thirty - six freshly frozen , undamaged human knees from cadavers ( 18 pairs from 18 cadavers ) were used in this study . four of the cadavers were female and 14 were male , ranging in age from 38 to 76 years ( mean , 51.9 ; standard deviation ( sd ) , 11.9 ) . the femur was cut 20 cm and the tibia 30 cm from the joint line . the iliotibial tract up to mid - thigh , the popliteus musculotendinous unit and the joint capsule were left intact . the knees were stored at 20 c and thawed for 12 hours at room temperature before testing . prior to the procedures , the knees were subjected to an initial arthroscopic inspection to rule out any previous intra - articular lesions . the specimens were divided in two groups , each comprising one knee of each pair ( randomized side distribution by means of flipping a coin ) . these groups were named group 1 , which consisted of 18 knees that underwent acl reconstruction using conventional guides , and group 2 , which consisted of the 18 opposite - side knees that underwent acl reconstruction assisted by the caos system and navigation instruments . all procedures were performed by the same surgeon , who had prior experience in acl reconstruction and navigation . in group 1 , an acufex tibial guide was used ( director model ; smith & nephew , inc . , u.s.a . ) with 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia , halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . in this position , a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . a polyester suture wire ( ethibond 5 ) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint , exiting from the central point of the tibial tunnel , which was not drilled ( figure 1 ) . for the femoral tunnel , a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch , with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . the end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur ( figure 2 ) . in this way , the suture wire was positioned at the central point of the femoral tunnel , which was also not drilled . orthopilot ( aesculap , tuttlingen , germany ) is a computer system that provides three - dimensional ( 3d ) real - time tracking of specific surgical instruments in relation to anatomical reference points that are acquired during surgery . the 3d tracking is performed without the need for additional preoperative planning or imaging such as tomography . the anterior cruciate ligament replacement software , version 1.1 ( 2002 ) was used together with its specific surgical instruments . to provide tracking , two passive rigid bodies with four reflective spheres each the hardwired cameras detected the signals reflected by the spheres and determined their spatial position . a third passive rigid body was connected to the specific instruments for each step of the surgery . in the orthopilot setup , the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software , thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . the anatomical reference points input to the system were obtained using a pointer connected to the mobile and passive rigid bodies ( figure 3 ) . superficial anatomical landmarks were registered in sequence , starting with the anterior tibial tuberosity and extending to the anterior , medial and lateral tibial plateau borders . the following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal : posterior cruciate ligament ( pcl ) , anterior tibial insertion , medial intercondylar tubercle , posteromedial margin of the anterior horn of the lateral meniscus , at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions , eleven and twelve oclock for the right knee and twelve and one oclock for the left knee , was carried out with a navigation - linked pointer . the first step in the navigation was the positioning of the tibial tunnel ; a specific tibial guide connected to the mobile and passive rigid bodies was tracked , and its position was monitored on the computer screen ( figure 4 ) . as soon as the desired position of the tibial guide was achieved , the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . a polyester suture wire ( ethibond 5 ) was pulled through the four - hole wire guide along the drilled path . the wire remained free inside the joint at the center of what would be the tibial tunnel , which was not drilled as in group 1 . the data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . a navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system , allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . in addition , it permitted the calculation of the following parameters : coronal angulation , distance from the femur posterior wall and graft isometry . the point selected for the center of the femoral tunnel respected the previously defined parameters and provided the best isometry . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . the same procedures as followed for group 1 were performed in relation to the suture wire , and the femoral tunnel was not drilled . thus , no tunnel was drilled , but the suture wire was positioned inside the knees , through the centers of what would be the femoral and tibial tunnels . a knot was made at the end of the suture wire , proximally to the femur . this knot had a diameter larger than 2.5 mm , and therefore , when the opposite end emerging from the tibia was pulled , it could not migrate beyond the femoral cortex . another knot was made at the extra - articular tibial end of the wire at an arbitrary distance from its tibial exit . the wire length between the tibial exit and the distal knot , with the knee at 90 of flexion and total extension ( figure 5 ) , was measured using a manual pachymeter . the difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . the graft was considered isometric if the length variation was zero . through arthrotomy , the distances to the pcl , medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface were collected using a manual pachymeter . a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . a line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch ( twelve oclock ) . the intersection of this straight line with the image of the cranial edge of the tibia determined the point that was considered the angular vertex . from this point the angle formed by these two straight lines was measured by the software ( figure 6 ) . thirty - six freshly frozen , undamaged human knees from cadavers ( 18 pairs from 18 cadavers ) were used in this study . four of the cadavers were female and 14 were male , ranging in age from 38 to 76 years ( mean , 51.9 ; standard deviation ( sd ) , 11.9 ) . the femur was cut 20 cm and the tibia 30 cm from the joint line . the iliotibial tract up to mid - thigh , the popliteus musculotendinous unit and the joint capsule were left intact . the knees were stored at 20 c and thawed for 12 hours at room temperature before testing . prior to the procedures , the knees were subjected to an initial arthroscopic inspection to rule out any previous intra - articular lesions . the specimens were divided in two groups , each comprising one knee of each pair ( randomized side distribution by means of flipping a coin ) . these groups were named group 1 , which consisted of 18 knees that underwent acl reconstruction using conventional guides , and group 2 , which consisted of the 18 opposite - side knees that underwent acl reconstruction assisted by the caos system and navigation instruments . all procedures were performed by the same surgeon , who had prior experience in acl reconstruction and navigation . in group 1 , guide wire positioning was performed using conventional surgical instruments . for the tibial tunnel , an acufex tibial guide was used ( director model ; smith & nephew , inc . , u.s.a . ) with 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia , halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . in this position , a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . a polyester suture wire ( ethibond 5 ) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint , exiting from the central point of the tibial tunnel , which was not drilled ( figure 1 ) . for the femoral tunnel , a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch , with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . the end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur ( figure 2 ) . in this way , the suture wire was positioned at the central point of the femoral tunnel , which was also not drilled . orthopilot ( aesculap , tuttlingen , germany ) is a computer system that provides three - dimensional ( 3d ) real - time tracking of specific surgical instruments in relation to anatomical reference points that are acquired during surgery . the 3d tracking is performed without the need for additional preoperative planning or imaging such as tomography . the anterior cruciate ligament replacement software , version 1.1 ( 2002 ) was used together with its specific surgical instruments . to provide tracking , two passive rigid bodies with four reflective spheres each the hardwired cameras detected the signals reflected by the spheres and determined their spatial position . a third passive rigid body was connected to the specific instruments for each step of the surgery . in the orthopilot setup , the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software , thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . the anatomical reference points input to the system were obtained using a pointer connected to the mobile and passive rigid bodies ( figure 3 ) . superficial anatomical landmarks were registered in sequence , starting with the anterior tibial tuberosity and extending to the anterior , medial and lateral tibial plateau borders . the following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal : posterior cruciate ligament ( pcl ) , anterior tibial insertion , medial intercondylar tubercle , posteromedial margin of the anterior horn of the lateral meniscus , at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions , eleven and twelve oclock for the right knee and twelve and one oclock for the left knee , was carried out with a navigation - linked pointer . the first step in the navigation was the positioning of the tibial tunnel ; a specific tibial guide connected to the mobile and passive rigid bodies was tracked , and its position was monitored on the computer screen ( figure 4 ) . as soon as the desired position of the tibial guide was achieved , the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . a polyester suture wire ( ethibond 5 ) was pulled through the four - hole wire guide along the drilled path . the wire remained free inside the joint at the center of what would be the tibial tunnel , which was not drilled as in group 1 . the data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . a navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system , allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . in addition , it permitted the calculation of the following parameters : coronal angulation , distance from the femur posterior wall and graft isometry . the point selected for the center of the femoral tunnel respected the previously defined parameters and the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . the same procedures as followed for group 1 were performed in relation to the suture wire , and the femoral tunnel was not drilled . thus , no tunnel was drilled , but the suture wire was positioned inside the knees , through the centers of what would be the femoral and tibial tunnels . a knot was made at the end of the suture wire , proximally to the femur . this knot had a diameter larger than 2.5 mm , and therefore , when the opposite end emerging from the tibia was pulled , it could not migrate beyond the femoral cortex . another knot was made at the extra - articular tibial end of the wire at an arbitrary distance from its tibial exit . the wire length between the tibial exit and the distal knot , with the knee at 90 of flexion and total extension ( figure 5 ) , was measured using a manual pachymeter . the difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . the graft was considered isometric if the length variation was zero . through arthrotomy , the distances to the pcl , medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . a line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch ( twelve oclock ) . the intersection of this straight line with the image of the cranial edge of the tibia determined the point that was considered the angular vertex . from this point the angle formed by these two straight lines was measured by the software ( figure 6 ) . in group 1 , guide wire positioning was performed using conventional surgical instruments . for the tibial tunnel , an acufex tibial guide was used ( director model ; smith & nephew , inc . , u.s.a . ) with 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia , halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . in this position , a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . a polyester suture wire ( ethibond 5 ) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint , exiting from the central point of the tibial tunnel , which was not drilled ( figure 1 ) . for the femoral tunnel , a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch , with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . the end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur ( figure 2 ) . in this way , the suture wire was positioned at the central point of the femoral tunnel , which was also not drilled . orthopilot ( aesculap , tuttlingen , germany ) is a computer system that provides three - dimensional ( 3d ) real - time tracking of specific surgical instruments in relation to anatomical reference points that are acquired during surgery . the 3d tracking is performed without the need for additional preoperative planning or imaging such as tomography . the anterior cruciate ligament replacement software , version 1.1 ( 2002 ) was used together with its specific surgical instruments . to provide tracking , two passive rigid bodies with four reflective spheres each the hardwired cameras detected the signals reflected by the spheres and determined their spatial position . a third passive rigid body was connected to the specific instruments for each step of the surgery . in the orthopilot setup , the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software , thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . the anatomical reference points input to the system were obtained using a pointer connected to the mobile and passive rigid bodies ( figure 3 ) . superficial anatomical landmarks were registered in sequence , starting with the anterior tibial tuberosity and extending to the anterior , medial and lateral tibial plateau borders . the following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal : posterior cruciate ligament ( pcl ) , anterior tibial insertion , medial intercondylar tubercle , posteromedial margin of the anterior horn of the lateral meniscus , at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions , eleven and twelve oclock for the right knee and twelve and one oclock for the left knee , was carried out with a navigation - linked pointer . the first step in the navigation was the positioning of the tibial tunnel ; a specific tibial guide connected to the mobile and passive rigid bodies was tracked , and its position was monitored on the computer screen ( figure 4 ) . as soon as the desired position of the tibial guide was achieved , the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . a polyester suture wire ( ethibond 5 ) was pulled through the four - hole wire guide along the drilled path . the wire remained free inside the joint at the center of what would be the tibial tunnel , which was not drilled as in group 1 . the data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . a navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system , allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . in addition , it permitted the calculation of the following parameters : coronal angulation , distance from the femur posterior wall and graft isometry . the point selected for the center of the femoral tunnel respected the previously defined parameters and provided the best isometry . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . the same procedures as followed for group 1 were performed in relation to the suture wire , and the femoral tunnel was not drilled . thus , no tunnel was drilled , but the suture wire was positioned inside the knees , through the centers of what would be the femoral and tibial tunnels . a knot was made at the end of the suture wire , proximally to the femur . this knot had a diameter larger than 2.5 mm , and therefore , when the opposite end emerging from the tibia was pulled , it could not migrate beyond the femoral cortex . another knot was made at the extra - articular tibial end of the wire at an arbitrary distance from its tibial exit . the wire length between the tibial exit and the distal knot , with the knee at 90 of flexion and total extension ( figure 5 ) , was measured using a manual pachymeter . the difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . the graft was considered isometric if the length variation was zero . through arthrotomy , the distances to the pcl , medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . a line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch ( twelve oclock ) . the intersection of this straight line with the image of the cranial edge of the tibia determined the point that was considered the angular vertex . from this point the angle formed by these two straight lines was measured by the software ( figure 6 ) . the variation of the distance from the femoral tunnel to the tibial tunnel between flexion and extension was smaller in the orthopilot group [ better isometry ; group 1 , mean = 4.2 mm ( range , 1.19.4 mm ) ; group 2 , mean = 2.8 mm ( range , 0.36.7 mm ) ; p < 0.05 ] . none of the other parameters showed any significant difference between the groups , and all of the tunnels were considered to be in satisfactory positions ( table 1 ) . the design of this study aimed to make simple comparisons using direct measurements between the positions of the acl reconstruction tunnels achieved using the orthopilot navigation system and using conventional guides . furthermore , and perhaps more importantly , this study aimed to determine which of these techniques provided better isometry . the knees used in this study did not present any acl injury , and the acls were arthroscopically removed . the resection invariably leaves clear signs of the anatomical origin and insertion of the acl , and this may have assisted in positioning the guides , thereby potentially improving the results in both groups . direct measurements using a manual pachymeter have the advantage of being simple and reproducible , particularly when performed by a single surgeon . the measurements were based on absolute points that were covered by radiolucent structures , including cartilage and other tissues . thus , our data can not be compared with radiographic data.19 , 20 the measurements of the distance from the tibial tunnel to the pcl showed averages close to 10.2 mm in both groups . this result was higher than the expected result of 7 mm , potentially indicating that this was not a reliable reference point for tunnel positioning . measurement ( in degrees ) of the coronal angle of the femoral tunnel using digital photography has not been reported previously in the literature . we chose to use this measurement because of its reproducibility and because it is easy to perform . therefore , it was surprising that the two groups , which demonstrated no significant differences in any anatomical parameters , had different isometric behaviors . the orthopilot group not only had a lower average but also a lower interval between the minimum and maximum values , and this result was very consistent . our interpretation of this finding is that there is a relatively broad acceptable area for positioning the centers of the tibial and femoral tunnels . positioning the center of the tunnel at any of the infinite number of points inside these areas would be appropriate . therefore , an infinite number of possible combinations of appropriate tibial tunnels with appropriate femoral tunnels for each knee are possible . the navigation system seemed to provide us with a femoral tunnel location ( chosen after positioning the tibial tunnel ) in an acceptable area of the femur in relation to the selected tibial position , thereby providing decreased variation of the graft length or better isometry . however , the navigation system presents some potential inconveniences , such as the longer time required for the procedure21 and morbidity related to the fixation of rigid bodies to bones . the results of this study suggested that there was no difference in the anatomical tunnel position between the groups . however , further studies with a larger number of cases are needed to confirm these results . finally , the results of our study encourage us to continue evaluating the use of navigation for the improvement of knee surgery . better isometry was achieved using the navigation technique ( group 2 ) compared to the conventional technique ( group 1 ) .

objectives : to compare the accuracy of tunnel placement and graft isometry for anterior cruciate ligament reconstruction performed using a computer - assisted navigation system ( orthopilot ) and using traditional instruments.methods:the anterior cruciate ligament was removed intact from 36 pairs of human cadaver knees . from each pair , one knee was randomized to group 1 ( conventional ) and the other to group 2 ( orthopilot ) . an inelastic suture was then passed through the central points of the tibial and femoral tunnels . neither of the tunnels was drilled . all knees were then dissected , and six parameters were obtained : distances from the tibial tunnel center to the 1 ) posterior cruciate ligament , 2 ) anterior horn of the lateral meniscus and 3 ) medial tibial spine ; 4 ) distance from the femoral tunnel center to the posterior femoral cortex ; 5 ) femoral tunnel coronal angle ; and 6 ) variation of the distance from the femoral to the tibial tunnel with the knee extended and at 90 degrees of flexion.results:the variation of the distance from the femoral to the tibial tunnel during flexion and extension was smaller in the orthopilot group ( better isometry ) compared to the conventional group . there were no statistical differences in any other parameters between the groups , and all tunnels were considered to be in satisfactory positions.discussion:the results obtained for anterior cruciate ligament reconstruction depend on precise isometric point positioning , and a navigation system is a precision tool that can assist surgeons in tunnel positioning.conclusion:no differences in tunnel position were observed between the groups . nonetheless , better isometry was achieved in the orthopilot group than with conventional instruments .

INTRODUCTION MATERIALS AND METHODS Specimen Preparation Groups Surgical technique Group 1: Conventional guide technique Group 2 Navigation technique Statistics RESULTS DISCUSSION CONCLUSION

the main aspects of the surgical technique required to achieve favorable clinical results include graft quality , tunnel positioning and efficient graft fixation , with sufficient tension to provide joint stability and a physiological range of motion with normal flexion and extension.2 , 3 along with the increasing number of indications for surgery , increases in the frequency of revision surgery have also been reported . the purpose of the present study was to compare isometry and tunnel positioning using the caos system and using conventional guides . the inclusion criteria for the knee specimens used were that the cause of death was not traumatic or due to infectious disease and that there were no scars , hematomas or deformities in the lower limbs . the specimens were divided in two groups , each comprising one knee of each pair ( randomized side distribution by means of flipping a coin ) . these groups were named group 1 , which consisted of 18 knees that underwent acl reconstruction using conventional guides , and group 2 , which consisted of the 18 opposite - side knees that underwent acl reconstruction assisted by the caos system and navigation instruments . with 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia , halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . in this position , a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . a polyester suture wire ( ethibond 5 ) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint , exiting from the central point of the tibial tunnel , which was not drilled ( figure 1 ) . for the femoral tunnel , a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch , with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . the end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur ( figure 2 ) . in this way , the suture wire was positioned at the central point of the femoral tunnel , which was also not drilled . the anterior cruciate ligament replacement software , version 1.1 ( 2002 ) was used together with its specific surgical instruments . in the orthopilot setup , the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software , thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . the anatomical reference points input to the system were obtained using a pointer connected to the mobile and passive rigid bodies ( figure 3 ) . the following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal : posterior cruciate ligament ( pcl ) , anterior tibial insertion , medial intercondylar tubercle , posteromedial margin of the anterior horn of the lateral meniscus , at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions , eleven and twelve oclock for the right knee and twelve and one oclock for the left knee , was carried out with a navigation - linked pointer . the first step in the navigation was the positioning of the tibial tunnel ; a specific tibial guide connected to the mobile and passive rigid bodies was tracked , and its position was monitored on the computer screen ( figure 4 ) . as soon as the desired position of the tibial guide was achieved , the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . the wire remained free inside the joint at the center of what would be the tibial tunnel , which was not drilled as in group 1 . the data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . a navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system , allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . in addition , it permitted the calculation of the following parameters : coronal angulation , distance from the femur posterior wall and graft isometry . the point selected for the center of the femoral tunnel respected the previously defined parameters and provided the best isometry . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . the same procedures as followed for group 1 were performed in relation to the suture wire , and the femoral tunnel was not drilled . thus , no tunnel was drilled , but the suture wire was positioned inside the knees , through the centers of what would be the femoral and tibial tunnels . this knot had a diameter larger than 2.5 mm , and therefore , when the opposite end emerging from the tibia was pulled , it could not migrate beyond the femoral cortex . the wire length between the tibial exit and the distal knot , with the knee at 90 of flexion and total extension ( figure 5 ) , was measured using a manual pachymeter . the difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . through arthrotomy , the distances to the pcl , medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface were collected using a manual pachymeter . a line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch ( twelve oclock ) . the femur was cut 20 cm and the tibia 30 cm from the joint line . the specimens were divided in two groups , each comprising one knee of each pair ( randomized side distribution by means of flipping a coin ) . these groups were named group 1 , which consisted of 18 knees that underwent acl reconstruction using conventional guides , and group 2 , which consisted of the 18 opposite - side knees that underwent acl reconstruction assisted by the caos system and navigation instruments . with 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia , halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . in this position , a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . a polyester suture wire ( ethibond 5 ) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint , exiting from the central point of the tibial tunnel , which was not drilled ( figure 1 ) . for the femoral tunnel , a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch , with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . the end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur ( figure 2 ) . in this way , the suture wire was positioned at the central point of the femoral tunnel , which was also not drilled . the anterior cruciate ligament replacement software , version 1.1 ( 2002 ) was used together with its specific surgical instruments . in the orthopilot setup , the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software , thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . the anatomical reference points input to the system were obtained using a pointer connected to the mobile and passive rigid bodies ( figure 3 ) . the following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal : posterior cruciate ligament ( pcl ) , anterior tibial insertion , medial intercondylar tubercle , posteromedial margin of the anterior horn of the lateral meniscus , at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions , eleven and twelve oclock for the right knee and twelve and one oclock for the left knee , was carried out with a navigation - linked pointer . the first step in the navigation was the positioning of the tibial tunnel ; a specific tibial guide connected to the mobile and passive rigid bodies was tracked , and its position was monitored on the computer screen ( figure 4 ) . as soon as the desired position of the tibial guide was achieved , the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . the wire remained free inside the joint at the center of what would be the tibial tunnel , which was not drilled as in group 1 . the data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . a navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system , allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . in addition , it permitted the calculation of the following parameters : coronal angulation , distance from the femur posterior wall and graft isometry . the point selected for the center of the femoral tunnel respected the previously defined parameters and the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . the same procedures as followed for group 1 were performed in relation to the suture wire , and the femoral tunnel was not drilled . thus , no tunnel was drilled , but the suture wire was positioned inside the knees , through the centers of what would be the femoral and tibial tunnels . this knot had a diameter larger than 2.5 mm , and therefore , when the opposite end emerging from the tibia was pulled , it could not migrate beyond the femoral cortex . the wire length between the tibial exit and the distal knot , with the knee at 90 of flexion and total extension ( figure 5 ) , was measured using a manual pachymeter . the difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . through arthrotomy , the distances to the pcl , medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . a line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch ( twelve oclock ) . with 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia , halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . in this position , a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . a polyester suture wire ( ethibond 5 ) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint , exiting from the central point of the tibial tunnel , which was not drilled ( figure 1 ) . for the femoral tunnel , a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch , with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . the end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur ( figure 2 ) . in this way , the suture wire was positioned at the central point of the femoral tunnel , which was also not drilled . the anterior cruciate ligament replacement software , version 1.1 ( 2002 ) was used together with its specific surgical instruments . in the orthopilot setup , the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software , thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . the anatomical reference points input to the system were obtained using a pointer connected to the mobile and passive rigid bodies ( figure 3 ) . the following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal : posterior cruciate ligament ( pcl ) , anterior tibial insertion , medial intercondylar tubercle , posteromedial margin of the anterior horn of the lateral meniscus , at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions , eleven and twelve oclock for the right knee and twelve and one oclock for the left knee , was carried out with a navigation - linked pointer . the first step in the navigation was the positioning of the tibial tunnel ; a specific tibial guide connected to the mobile and passive rigid bodies was tracked , and its position was monitored on the computer screen ( figure 4 ) . as soon as the desired position of the tibial guide was achieved , the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . the wire remained free inside the joint at the center of what would be the tibial tunnel , which was not drilled as in group 1 . the data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . a navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system , allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . in addition , it permitted the calculation of the following parameters : coronal angulation , distance from the femur posterior wall and graft isometry . the point selected for the center of the femoral tunnel respected the previously defined parameters and provided the best isometry . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . the same procedures as followed for group 1 were performed in relation to the suture wire , and the femoral tunnel was not drilled . thus , no tunnel was drilled , but the suture wire was positioned inside the knees , through the centers of what would be the femoral and tibial tunnels . this knot had a diameter larger than 2.5 mm , and therefore , when the opposite end emerging from the tibia was pulled , it could not migrate beyond the femoral cortex . the wire length between the tibial exit and the distal knot , with the knee at 90 of flexion and total extension ( figure 5 ) , was measured using a manual pachymeter . the difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . through arthrotomy , the distances to the pcl , medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . a line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch ( twelve oclock ) . the variation of the distance from the femoral tunnel to the tibial tunnel between flexion and extension was smaller in the orthopilot group [ better isometry ; group 1 , mean = 4.2 mm ( range , 1.19.4 mm ) ; group 2 , mean = 2.8 mm ( range , 0.36.7 mm ) ; p < 0.05 ] . none of the other parameters showed any significant difference between the groups , and all of the tunnels were considered to be in satisfactory positions ( table 1 ) . the design of this study aimed to make simple comparisons using direct measurements between the positions of the acl reconstruction tunnels achieved using the orthopilot navigation system and using conventional guides . thus , our data can not be compared with radiographic data.19 , 20 the measurements of the distance from the tibial tunnel to the pcl showed averages close to 10.2 mm in both groups . measurement ( in degrees ) of the coronal angle of the femoral tunnel using digital photography has not been reported previously in the literature . therefore , it was surprising that the two groups , which demonstrated no significant differences in any anatomical parameters , had different isometric behaviors . the orthopilot group not only had a lower average but also a lower interval between the minimum and maximum values , and this result was very consistent . our interpretation of this finding is that there is a relatively broad acceptable area for positioning the centers of the tibial and femoral tunnels . the navigation system seemed to provide us with a femoral tunnel location ( chosen after positioning the tibial tunnel ) in an acceptable area of the femur in relation to the selected tibial position , thereby providing decreased variation of the graft length or better isometry . the results of this study suggested that there was no difference in the anatomical tunnel position between the groups . better isometry was achieved using the navigation technique ( group 2 ) compared to the conventional technique ( group 1 ) .

although conservative treatment has its indications , surgical treatment for acl injuries is the ideal choice for young and active patients because it allows them to return to sports but prevents the onset of additional knee injuries.1 surgical techniques have evolved over recent decades , and the current gold standard treatment is arthroscopic intra - articular acl reconstruction with autogenous grafts . the main aspects of the surgical technique required to achieve favorable clinical results include graft quality , tunnel positioning and efficient graft fixation , with sufficient tension to provide joint stability and a physiological range of motion with normal flexion and extension.2 , 3 along with the increasing number of indications for surgery , increases in the frequency of revision surgery have also been reported . revision surgery is now performed in 1040% of cases.4 the main cause of surgical failure and acl revision is inadequate tunnel positioning.5 , 6 anatomical studies have reported that the anatomical acl origin and insertion is the most isometric graft position710 . however , it is known that even senior surgeons using conventional guides frequently fail to place the tunnels in the desired positions.11 , 12 a recent resource for improving orthopedic surgery precision is the computer assisted orthopedic surgery ( caos ) system.13 navigation systems for acl reconstruction have been developed,1418 but no comparative clinical studies have unequivocally demonstrated their superiority in relation to conventional guides . in addition to enhancement of precision , such navigation systems can also facilitate choosing the location for bony tunnels based on the new isometry criterion . the purpose of the present study was to compare isometry and tunnel positioning using the caos system and using conventional guides . the inclusion criteria for the knee specimens used were that the cause of death was not traumatic or due to infectious disease and that there were no scars , hematomas or deformities in the lower limbs . four of the cadavers were female and 14 were male , ranging in age from 38 to 76 years ( mean , 51.9 ; standard deviation ( sd ) , 11.9 ) . the iliotibial tract up to mid - thigh , the popliteus musculotendinous unit and the joint capsule were left intact . these groups were named group 1 , which consisted of 18 knees that underwent acl reconstruction using conventional guides , and group 2 , which consisted of the 18 opposite - side knees that underwent acl reconstruction assisted by the caos system and navigation instruments . with 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia , halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . in this position , a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . a polyester suture wire ( ethibond 5 ) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint , exiting from the central point of the tibial tunnel , which was not drilled ( figure 1 ) . for the femoral tunnel , a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch , with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . the end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur ( figure 2 ) . in this way , the suture wire was positioned at the central point of the femoral tunnel , which was also not drilled . orthopilot ( aesculap , tuttlingen , germany ) is a computer system that provides three - dimensional ( 3d ) real - time tracking of specific surgical instruments in relation to anatomical reference points that are acquired during surgery . in the orthopilot setup , the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software , thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . superficial anatomical landmarks were registered in sequence , starting with the anterior tibial tuberosity and extending to the anterior , medial and lateral tibial plateau borders . the following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal : posterior cruciate ligament ( pcl ) , anterior tibial insertion , medial intercondylar tubercle , posteromedial margin of the anterior horn of the lateral meniscus , at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions , eleven and twelve oclock for the right knee and twelve and one oclock for the left knee , was carried out with a navigation - linked pointer . the first step in the navigation was the positioning of the tibial tunnel ; a specific tibial guide connected to the mobile and passive rigid bodies was tracked , and its position was monitored on the computer screen ( figure 4 ) . as soon as the desired position of the tibial guide was achieved , the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . the wire remained free inside the joint at the center of what would be the tibial tunnel , which was not drilled as in group 1 . the data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . a navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system , allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . the point selected for the center of the femoral tunnel respected the previously defined parameters and provided the best isometry . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . the same procedures as followed for group 1 were performed in relation to the suture wire , and the femoral tunnel was not drilled . thus , no tunnel was drilled , but the suture wire was positioned inside the knees , through the centers of what would be the femoral and tibial tunnels . another knot was made at the extra - articular tibial end of the wire at an arbitrary distance from its tibial exit . the wire length between the tibial exit and the distal knot , with the knee at 90 of flexion and total extension ( figure 5 ) , was measured using a manual pachymeter . the difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . through arthrotomy , the distances to the pcl , medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface were collected using a manual pachymeter . a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . a line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch ( twelve oclock ) . the intersection of this straight line with the image of the cranial edge of the tibia determined the point that was considered the angular vertex . four of the cadavers were female and 14 were male , ranging in age from 38 to 76 years ( mean , 51.9 ; standard deviation ( sd ) , 11.9 ) . these groups were named group 1 , which consisted of 18 knees that underwent acl reconstruction using conventional guides , and group 2 , which consisted of the 18 opposite - side knees that underwent acl reconstruction assisted by the caos system and navigation instruments . with 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia , halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . in this position , a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . a polyester suture wire ( ethibond 5 ) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint , exiting from the central point of the tibial tunnel , which was not drilled ( figure 1 ) . for the femoral tunnel , a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch , with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . the end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur ( figure 2 ) . orthopilot ( aesculap , tuttlingen , germany ) is a computer system that provides three - dimensional ( 3d ) real - time tracking of specific surgical instruments in relation to anatomical reference points that are acquired during surgery . in the orthopilot setup , the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software , thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . superficial anatomical landmarks were registered in sequence , starting with the anterior tibial tuberosity and extending to the anterior , medial and lateral tibial plateau borders . the following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal : posterior cruciate ligament ( pcl ) , anterior tibial insertion , medial intercondylar tubercle , posteromedial margin of the anterior horn of the lateral meniscus , at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions , eleven and twelve oclock for the right knee and twelve and one oclock for the left knee , was carried out with a navigation - linked pointer . the first step in the navigation was the positioning of the tibial tunnel ; a specific tibial guide connected to the mobile and passive rigid bodies was tracked , and its position was monitored on the computer screen ( figure 4 ) . as soon as the desired position of the tibial guide was achieved , the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . the wire remained free inside the joint at the center of what would be the tibial tunnel , which was not drilled as in group 1 . the data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . a navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system , allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . the point selected for the center of the femoral tunnel respected the previously defined parameters and the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . thus , no tunnel was drilled , but the suture wire was positioned inside the knees , through the centers of what would be the femoral and tibial tunnels . the wire length between the tibial exit and the distal knot , with the knee at 90 of flexion and total extension ( figure 5 ) , was measured using a manual pachymeter . the difference between these measurements corresponded to the difference between the distances from the femoral tunnel to the tibial tunnel at these positions . through arthrotomy , the distances to the pcl , medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . a line perpendicular to the tibial joint surface was drawn through the center of the intercondylar notch ( twelve oclock ) . the intersection of this straight line with the image of the cranial edge of the tibia determined the point that was considered the angular vertex . with 55 sagittal angulation and 15 lateral inclination and with the tip of the guide directed toward the intercondylar eminence of the tibia , halfway between the anterior horn of the lateral meniscus and the medial tibial intercondylar tubercle . in this position , a four - hole wire guide was drilled from the anteromedial surface of the tibia to the tip of the guide inside the joint . a polyester suture wire ( ethibond 5 ) was then pulled by the metal guide wire from the extra - articular anteromedial cortical surface of the tibia to the inside of the joint , exiting from the central point of the tibial tunnel , which was not drilled ( figure 1 ) . for the femoral tunnel , a 7-mm offset acufex femoral guide was introduced through the anteromedial portal and positioned at the femoral intercondylar notch , with the tip of the guide on the posterior wall at eleven oclock for the right knee and one oclock for the left knee . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femoral condyle to the anterolateral surface of the thigh of the specimen . the end of the suture wire that had been left inside the joint was then pulled through the anteromedial portal and was led by the four - hole wire guide through the femur ( figure 2 ) . orthopilot ( aesculap , tuttlingen , germany ) is a computer system that provides three - dimensional ( 3d ) real - time tracking of specific surgical instruments in relation to anatomical reference points that are acquired during surgery . in the orthopilot setup , the anatomical reference points and kinematics of the knee were entered and registered by the orthopilot software , thus allowing it to calculate the relative positions between the fixed sensors and anatomical landmarks . the following intra - articular structures were viewed by means of arthroscopy and registered with the pointer through the anteromedial portal : posterior cruciate ligament ( pcl ) , anterior tibial insertion , medial intercondylar tubercle , posteromedial margin of the anterior horn of the lateral meniscus , at least five points of the anterior border of the femoral intercondylar notch at its transition with the joint cartilage and at least five points on the medial surface of the femoral lateral condyle . subsequent palpation of the posterior wall of the femoral intercondylar notch in two positions , eleven and twelve oclock for the right knee and twelve and one oclock for the left knee , was carried out with a navigation - linked pointer . the first step in the navigation was the positioning of the tibial tunnel ; a specific tibial guide connected to the mobile and passive rigid bodies was tracked , and its position was monitored on the computer screen ( figure 4 ) . as soon as the desired position of the tibial guide was achieved , the guide wire was drilled through the guide from the tibial anteromedial surface to the guide tip inside the joint . the wire remained free inside the joint at the center of what would be the tibial tunnel , which was not drilled as in group 1 . the data for the tibial tunnel were stored in the computer and used to calculate the optimum entry point for the femoral tunnel in relation to that specific tibial tunnel . a navigated femoral guide was introduced through the anteromedial portal and was tracked by the navigation system , allowing the guide position on the medial wall of the lateral femoral condyle to be followed on the computer screen . in this position , the four - hole wire guide was drilled from the medial surface of the lateral femur condyle to the anterolateral surface of the thigh of the specimen . thus , no tunnel was drilled , but the suture wire was positioned inside the knees , through the centers of what would be the femoral and tibial tunnels . the wire length between the tibial exit and the distal knot , with the knee at 90 of flexion and total extension ( figure 5 ) , was measured using a manual pachymeter . through arthrotomy , the distances to the pcl , medial intercondylar tubercle and margin of the anterior horn of the lateral meniscus relative to the guide wire exit on the tibial joint surface a digital photograph of the frontal view of the femoral intercondylar notch was taken with 90 knee flexion . the intersection of this straight line with the image of the cranial edge of the tibia determined the point that was considered the angular vertex . the variation of the distance from the femoral tunnel to the tibial tunnel between flexion and extension was smaller in the orthopilot group [ better isometry ; group 1 , mean = 4.2 mm ( range , 1.19.4 mm ) ; group 2 , mean = 2.8 mm ( range , 0.36.7 mm ) ; p < 0.05 ] . none of the other parameters showed any significant difference between the groups , and all of the tunnels were considered to be in satisfactory positions ( table 1 ) . the design of this study aimed to make simple comparisons using direct measurements between the positions of the acl reconstruction tunnels achieved using the orthopilot navigation system and using conventional guides . the resection invariably leaves clear signs of the anatomical origin and insertion of the acl , and this may have assisted in positioning the guides , thereby potentially improving the results in both groups . thus , our data can not be compared with radiographic data.19 , 20 the measurements of the distance from the tibial tunnel to the pcl showed averages close to 10.2 mm in both groups . the navigation system seemed to provide us with a femoral tunnel location ( chosen after positioning the tibial tunnel ) in an acceptable area of the femur in relation to the selected tibial position , thereby providing decreased variation of the graft length or better isometry . however , the navigation system presents some potential inconveniences , such as the longer time required for the procedure21 and morbidity related to the fixation of rigid bodies to bones .

methyleugenol ( chart 1a , cas # 93 - 15 - 2 ) is the methyl ether of eugenol ( chart1b , cas # 97 - 53 - 0 ) , which is itself a major constituent of spices such as clove , allspice , and bay leaves and is known to induce contact allergies . methyleugenol ( meu ) and eugenol ( eug ) are allylalkoxybenzene derivatives that are present as flavoring constituents in foodstuffs or are added as flavor extenders . their chemical structures resemble those of safrole , a known carcinogen , and estragole . a safety assessment of allylalkoxybenzene derivatives was prepared by the expert panel of the flavor and extract manufacturers association , which summarized previous findings about the safety of meu . they conclude that the present human exposure to meu does not pose a significant cancer risk . by contrast , the twelfth report on carcinogens of the national toxicology program concludes that meu is reasonably anticipated to be a human carcinogen . this finding is based on the observation of increased incidence of malignant tumors in the liver , bile duct , glandular stomach , kidney , mesothelium , mammary gland , and skin in experimental animals . the mechanism of hepatic carcinogenesis appears to result from the bioactivation of meu to dna - reactive intermediates via hydroxylation of the 1 position of the allylic side chain and subsequent sulfation to generate 1-sulfoxymethyleugenol , conversions that can occur in the liver and that have also been demonstrated to occur in the human liver.in vitro studies have identified the human liver cytochrome p450 1a2 as the enzyme that is the likely bioactivator of meu . a 5-fold difference in activities occurred among 15 human microsome samples in a correlation study , which suggested that individual differences , arising from lifestyle factors such as smoking or drug use , can influence the likelihood that meu exposure will lead to harmful effects . additionally , the european commission s scientific committee on food formulated an opinion on meu , based largely on the rodent studies , concluding it to be a multisite , multispecies genotoxin and carcinogen for which no threshold could be assumed and for which no safe exposure limit could be established . the use of pure meu has been banned in the european union but not in the u.s.a . the biological and biochemical mechanisms by which alkenylbenzenes cause mutagenesis and carcinogenesis are areas of active research . bioactivation of these molecules to their ultimate carcinogens takes place through cytochromes p450 and sulfotransferases . kinetic studies in vitro of [ c]meu and [ c]eug in human , mouse , and rat liver and lung tissue fractions showed that the compounds are metabolized differently : meu is converted to significant amounts of the 1-hydroxy proximate carcinogen , but eug is glucuronidated , thus avoiding the formation of 1-hydroxyeugenol . in a study of meu and its oxidative metabolites in chinese hamster lung fibroblasts , marginal cytotoxic effects were not observed , but meu and 1-hydroxymethyleugenol at concentrations of 10 m caused dna strand breaks . meu bioactivation and detoxification were studied by a physiologically based kinetic approach comparing human and rat models , and the results obtained revealed that there were no substantial species differences in the formation of the reactive ultimate carcinogenic l-sulfoxymethyleugenol metabolite . meu is a ubiquitous environmental component with annual use in excess of 30,000 kg in the united states food , perfume , and pesticide industries . it is present in soaps and perfumes as a fragrance , in commercial food products as a flavorant , and in pesticides as an insect attractant . high resolution mass spectrometric analyses of serum samples from 213 nonfasting adult participants in the third national health and nutrition examination survey detected methyleugenol in 98% of samples at a mean concentration of 24 pg / g ( range < 3.1390 pg / g ) . fasted human volunteers who consumed controlled amounts of meu had prefeeding serum concentrations averaging 16.2 pg / g meu . mean serum levels increased following meu consumption and then decreased over time with metabolism : 53.9 pg / g ( 15 min ) ; 42.9 ( 30 min ) ; 37.0 ( 60 min ) ; and 25.2 ( 120 min ) with an elimination half - life of ca . the total human oral intake of meu has been estimated as 0.77 g / kg - bw / d , of which 14% is added meu . since meu is carcinogenic in extrahepatic tissues which have lower levels of cytochrome p450s and sulfotransferases , we decided to investigate the oxidation of meu and the related eug by peroxidases , which are widely distributed in extrahepatic tissues . although phenoxyl radicals of eug have been investigated by electron paramagnetic resonance spectroscopy ( epr ) , no reports of meu radicals have appeared . okada , et al . , satoh et al . , and fujisawa et al . observed phenoxyl radicals of eug in alkaline solution but do not report any epr hyperfine analysis . nakagawa et al . reported resolved and assigned epr spectra for sesmolyl and related phenoxyl radicals that were generated by continuous uv - irradiation of benzene solutions of the corresponding phenols . oxidized eug to its phenoxyl radical using hrp in a fast - flow system and report epr parameters similar to those for related methoxy - substituted phenoxyl radicals . although there have been no previous reports of epr observations of meu radical cations , there have been literature reports of chemical or electrochemical production of methoxybenzene radical cations . zweig and co - workers used controlled potential electrolysis to generate methoxy - substituted benzene cation radicals in situ for epr studies . siero and co - workers observed highly resolved epr spectra of radical cations generated by phenyl iodine(iii)bis(trifluoroacetate ) [ pifa ] as the electron acceptor in a charge transfer complex formed by methoxybenzenes and pifa in hexafluoropropanol solution . also kersten and co - workers reported the biochemical generation of methoxybenzene radical cations of 10 of the 12 possible methoxybenzene congeners , including cis- and trans-1,4-dimethoxybenzene , 1,2,3,4-tetramethoxybenzene , and 1,2,4,5-tetramethoxybenzene by ligninase peroxidase and hydrogen peroxide . by contrast , hrp was only able to oxidize the four congeners with the lowest half - wave oxidation potentials . biochemical oxidation of these molecules produced methoxy - substituted benzoquinones and methanol and demonstrated that the quinone oxygen atoms were derived from the solvent water . in this study , we report the first observation of radical cations of meu produced by hrp metabolism . observation of radical cations of several related compounds is also reported , and the epr spectral assignments for this series of radicals form a consistent pattern . free radical metabolites have been widely implicated as contributing to disease states and may be involved in meu carcinogenicity in extrahepatic tissues . methyleugenol ( 4-allyl-1,2-dimethoxybenzene ) ; eugenol ( 4-allyl-2-methoxyphenol ) ; 3,4-dimethoxytoluene ; 2-methoxy-4-methylphenol ; 3,4-dimethoxyphenol ; cerium(iv ) sulfate ; sulfuric acid ; dtpa ; hemoglobin ; and hrp ( type vi ) were purchased from sigma - aldrich ( st . louis , mo ) . the buffer for those reactions using it was 50 mm , ph 7.4 , phosphate buffer prepared by diluting a 500 mm solution treated with chelex 100 resin ( bio - rad , hercules , ca ) to remove adventitious transition metal ions . these solutions also contained either 1.0 mm or 0.1 mm dtpa to further hinder trace metal catalysis . the h2o2 concentration was confirmed by absorbance measurements at 240 nm ( 240 = 39.4 m cm ) . in some experiments , the commercial preparation of hrp was loaded onto a pd-10 gel filtration column and eluted with 50 mm phosphate buffer , ph 7.4 , treated with chelex before use . the concentration of hrp was determined from the soret maximum at 412 nm ( native hrp = 102 mm cm ) . unstable intermediates were detected by means of fast - flow epr experiments performed in a manner similar to that in our previous reports . reagents placed in solution reservoirs were pumped though plastic hoses by means of a masterflex l / s microprocessor - controlled peristaltic pump ( cole - parmer , vernon hills , il ) to a quartz fast - flow mixing chamber flat cell ( type wg-804 , 10 mm width , wilmad glass co. , buena , nj ) . flow rates for each solution could be selected from 10 ml / min to greater than 100 ml / min . epr measurements were made using an elexsys epr spectrometer operated at a frequency near 9.7 ghz and a magnetic field near 348 mt ( bruker biospin , billerica , ma ) . the magnetic field at the er4122 super hi - q microwave cavity was modulated at 100 khz , affording the first - derivative epr spectra that were recorded as computer files using bruker s software . subsequent analysis , interpretation , and simulation of the spectra used locally produced software . absorption spectra were recorded on a cary 100 uv visible spectrometer ( varian , victoria , australia ) using 1.0 cm - path length quartz cuvettes . hydroperoxide tests used quantofix peroxide test strips ( sigma - aldrich , inc . , milwaukee , wi ) that have a sensitivity reported to detect a lower limit of 1 mg / l peroxide . northampton , ma ) or coreldraw ( corel corporation , ottawa , canada ) software packages . meu oxidized by hrp activated by hydrogen peroxide in a fast - flow system produced a well - resolved epr spectrum of the corresponding radical cation ( figure 1a ) . essentially the same spectrum resulted when meu was oxidized chemically by ce(iv ) in sulfuric acid solution ( data not shown ) . the same spectrum was also produced when meu was oxidized by hemoglobin and hydrogen peroxide in a fast - flow system ( data not shown ) . when either meu ( figure 2b ) or hrp ( figure 2c ) was omitted , no epr spectrum was observed , as expected . however , when hydrogen peroxide was omitted , a strong epr signal was still observed ( figure 2d ) , and the same spectrum was observed in the presence of catalase at a concentration 70 times that of the peroxidase ( figure 2e ) . epr fast - flow spectra of the meu radical produced in a system of meu , h2o2 , and hrp . the concentrations of meu , h2o2 , and hrp in the flat cell were 1.75 mm , 13 mm , and 0.46 m , respectively . oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . equal volumes of a solution of meu / h2o2 and a solution of hrp in 100 mm phosphate buffer , ph 7.4 , were mixed milliseconds before they entered the flat cell at a total flow rate of 40 ml / min . the final solution also contained 10% v / v ethanol and 0.10 mm dtpa . ( b ) simulated epr spectrum with the coupling constants given in table 1 . ( c ) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw microwave power , 0.10 mt field modulation , 5.0 mt field sweep width , 164 ms time constant , 82 ms conversion time , and 33 scans of 1024 data points . epr fast - flow spectra of the meu radical produced in a system of meu , h2o2 , and hrp . the concentrations of meu , h2o2 , and hrp in the flat cell were 1.75 mm , 13 mm , and 0.46 m , respectively . oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . equal volumes of a solution of meu / h2o2 and a solution of hrp in 100 mm phosphate buffer , ph 7.4 , were mixed milliseconds before they entered the flat cell at a total flow rate of 40 ml / min . the final solution also contained 10% v / v ethanol and 0.10 mm dtpa . ( b ) as in panel a but with no meu . ( c ) as in panel a but with no hrp . ( e ) as in panel a but with no h2o2 and 32 m catalase added . numbers in parentheses are numbers of equivalent hydrogen atoms contributing to the stated hyperfine coupling . coupling constants were not resolved and are less than the linewidths of epr lines , ca . coupling constants in square brackets are literature values reported by ref ( 26 ) . alkenylbenzenes , such as meu , with allylic hydrogen atoms are known to be susceptible to autoxidation reactions that produce organic hydroperoxides . the quantofix test strip assay of stock meu indicated the presence of hydroperoxides in the range of 10 to 30 mg / l . the presence of endogenous hydroperoxides in stock meu and the ability of hydroperoxide to activate hrp and sustain the oxidation of meu to its radical were established by uv the spectrum obtained for native hrp before the addition of h2o2 and/or meu exhibits two characteristic bands at 502 and 642 nm and the soret band at 402 nm ( figure 3a ) . the addition of h2o2 resulted in the immediate decrease in absorbance of the soret band and the appearance of peaks with maxima at approximately 565 and 650 nm , which are associated with the formation of compound i. the addition of 1.75 mm meu 2 min after the addition of h2o2 caused a shift of the soret peak from 402 to 419 nm and the appearance of two peaks at 528 and 557 nm that are characteristic of compound ii ( figure 3b ) . during the first 4 min following the addition of meu , the soret intensity increased quickly , after which it remained at a constant level . visible detection of enzymatic intermediates produced in a system of meu , h2o2 , and hrp . the reaction mixtures consisted of hrp ( 5.3 m ) in 50 mm phosphate buffer , ph 7.4 , at 25 c . the dotted lines are for native hrp before the addition of h2o2 and/or meu . absorption spectra were recorded every 2 min after the initiation of the reaction . ( a ) spectral changes from native hrp to compound i. the reaction was initiated by the addition of 10 m h2o2 . compound i was prepared as described in a , and 1.75 mm meu was added 2 min after the addition of peroxide . ( c ) formation and spontaneous decomposition of compound ii in the absence of exogenously added h2o2 . in this case , the reaction was initiated by the addition of 2.5 mm meu , and scans were recorded every 2 min . the presence of endogenous hydroperoxide in the stock of meu was investigated by incubating hrp with meu in the absence of exogenously added h2o2 . the spectral variation observed between 2 and 10 min after the addition of meu is presented in figure 3c . the first scan following the addition of meu to the native enzyme showed the formation of species similar to compound ii , with absorption maxima at 419 , 528 , and 557 nm . the subsequent spectra showed that compound ii slowly reverted to the native form . none of the spectra observed showed the dramatic decrease in absorbance of the soret band and the absorption peaks at 565 and 650 nm that are characteristic of compound i , in accordance with the instability of this intermediate in the presence of one - electron reductants . that an endogenous hydroperoxide was effectively responsible for the accumulation of compound ii in the reaction system has been tested by the addition of catalase . similar changes in spectra were obtained when catalase was added immediately before the injection of meu in the above reaction mixture ( not shown ) . however , in the presence of the h2o2 scavenger , slightly less compound ii was generated , but the effect was small , indicating that the endogenous hydroperoxide did not react well with catalase ( data not shown ) . the epr spectrum of the 3,4-dimethoxytoluene radical cation ( figure 4 ) was recorded and interpreted to assist the assignment of epr hyperfine coupling constants in the meu radical cation with ce(iv ) in 0.23 m sulfuric acid solution serving as the oxidizing agent . oxidation of 3,4-dimethoxytoluene by hrp and hydrogen peroxide produced only a very weak epr signal under our fast flow conditions ( data not shown ) . epr fast - flow spectra of the 3,4-dimethoxytoluene radical cation produced in a system of 3,4-dimethoxytoluene , cerium(iv ) sulfate , and h2so4 at concentrations in the flat cell of 1.0 mm , 0.9 mm , and 0.225 m , respectively . oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . equal volumes of 3,4-dimethoxytoluene and ce(iv ) solutions were mixed milliseconds before entering the flat cell at a total flow rate of 20 ml / min . ( b ) simulated epr spectrum with the coupling constants given in table 1 . ( c ) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw microwave power , 0.025 mt field modulation , 6.0 mt field sweep width , 1.3 s time constant , 655 ms conversion time , and 36 scans of 1024 data points . a well - resolved epr spectrum of eugenol phenoxyl radical , reported in figure 5 , was observed when eugenol was oxidized by reaction with hrp and hydrogen peroxide in a fast flow system . this spectrum was recorded with somewhat better signal - to - noise and resolution than that reported previously . essentially the same spectrum was observed when eugenol was oxidized chemically by ce(iv ) in 0.23 m sulfuric acid solution ( data not shown ) . epr fast - flow spectra of the eugenol phenoxyl radical produced in a system of eugenol , h2o2 , and hrp at concentrations in the flat cell of 1.0 mm , 13 mm , and 0.23 m , respectively . oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . equal volumes of a solution of eugenol / h2o2 and a solution of hrp in 100 mm , ph 7.4 , phosphate buffer were mixed milliseconds before entering the flat cell at a total flow rate of 60 ml / min . the final solution also contained 10% v / v ethanol and 0.10 mm dtpa . ( a ) complete system with eugenol , h2o2 , and hrp . ( b ) simulated epr spectrum with the coupling constants given in table 1 . ( c ) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw of microwave power , 0.025 mt of field modulation , 5.0 mt of field sweep width , 164 ms time constant , 82 ms conversion time , and 12 scans of 1024 data points . the epr spectrum of 2-methoxy-4-methylphenol phenoxyl radical ( figure 6 ) was recorded and interpreted to assist the assignment of epr hyperfine coupling constants in the eugenol phenoxyl radical . epr fast - flow spectra of the 2-methoxy-4-methylphenol radical cation produced in a system of 2-methoxy-4-methylphenol , cerium(iv ) sulfate , and h2so4 at concentrations in the flat cell of 1.0 mm , 0.9 mm , and 0.225 m , respectively . oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . equal volumes of 2-methoxy-4-methylphenol and ce(iv ) solutions were mixed milliseconds before they entered the flat cell at a total flow rate of 40 ml / min . ( b ) simulated epr spectrum with the coupling constants given in table 1 . ( c ) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw of microwave power , 0.050 mt of field modulation , 6.0 mt of field sweep width , 164 ms time constant , 82 ms conversion time , and 33 scans of 1024 points . kersten et al . were unable to oxidize any of the dimethoxybenzene compounds ( or any related compounds with oxidation potentials greater than about 1.36 v ) with the hrp - hydrogen peroxide system . ( all oxidation potentials have been corrected to volts vs the standard hydrogen electrode . ) the oxidation potential of 1,2-dimethoxybenzene is reported as 1.69 v , while that for allylbenzene is about 2.6 v. the structurally similar compound ( 3,4-dimethoxy)methylcinnamate has an oxidation potential of 1.7 v. the oxidation potential of hrp compounds i and ii is about 0.9 v near ph 7.4 depending on the isozyme , which suggests that meu might be oxidized successfully by hrp . the meu radical cation has a well resolved epr spectrum ( figures 1a and 2a ) and is accurately simulated ( figure 1b ) by the hyperfine coupling constants reported in table 1 . subtraction of the simulation from the experimental spectrum results in a residual pattern that is essentially noise ( figure 1c ) . when either meu ( figure 2b ) or hrp ( figure 2c ) is omitted from the incubation , no epr spectrum is recorded . however , even when h2o2 is omitted from the incubation , a strong meu epr signal is seen ( figure 2d ) , and the same spectrum is observed when the incubation lacks h2o2 but contains 32 m catalase ( figure 2e ) . these observations are consistent with the presence of an organic hydroperoxide in meu ; this was confirmed by peroxide test strips that provided a semiquantitative estimate of 1050 mg / l peroxide concentration . treating meu with alumina to remove data presented in figure 3a and b are the first clear spectral evidence that the oxidation of meu proceeds by a cycle typical of peroxidase involving compounds i and ii . hrp compound ii was the predominant form of peroxidase observed in the presence of a high concentration of meu , and this intermediate was detected even in the absence of exogenously added h2o2 . the intensity of the characteristic absorption peaks of hrp compound ii decreases over time as the reaction proceeds , and the resting state of hrp is regenerated ( figure 3c ) . a similar experiment with catalase added to the incubation demonstrated that the hydroperoxide is not affected by the presence of catalase , a result that is consistent with the epr observation ( figure 2e ) . this indicates that the hydroperoxide formed by autoxidation of meu is not a good substrate for catalase and explains why inclusion of catalase in the epr incubation of meu had no effect ( figure 2e ) . presumably glutathione peroxidase , which is nonspecific , will reduce this unknown hydroperoxide and thereby inhibit oxidation of meu . the meu radical epr spectrum hyperfine pattern arises from large hyperfine couplings of the two hydrogens of the methylene group attached at the 4 position and one hydrogen atom attached at the 5 position , as would be predicted from the simple model of benzene molecular orbital ordering that results from two ortho - methoxy electron - releasing substituents . that the methylene hyperfine couplings are large is also evident from the epr spectrum of the 3,4-dimethoxytoluene radical cation ( see figure 4a ) . this epr spectrum is simulated successfully ( figure 4b ) with hyperfine parameters quite close to those for meu ( see table 1 ) , and the residual pattern that results from subtracting the simulated from experimental spectrum is essentially noise ( figure 4c ) . successful simulation of the spectra of both radicals also requires the inclusion of hyperfine coupling from two sets of methoxy hydrogen atoms , which indicates that we are seeing the initial free radical species before any demethoxylation reaction can occur . the epr spectrum of the eug phenoxyl radical ( figure 5a ) has been reported previously but recorded at somewhat lower resolution and poorer s / n . hyperfine coupling constants for this radical and for the 2-methoxy-4-methylphenol phenoxyl radical ( figure 6a ) are reported in table 1 and are consistent with the related radicals . they are characterized by large hyperfine couplings to two and three hydrogen atoms , respectively , substituted at the number 4 carbon of the aromatic ring . both spectra are accurately simulated by the assigned hyperfine parameters ( figures 5b and 6b ) and have residuals that are essentially noise ( figures 5c and 6c ) . methyleugenol undergoes autoxidation such that the commercial product contains 1030 mg / l hydroperoxide and is capable of activating peroxidases without the presence of added hydrogen peroxide . our spectroscopic studies show that the hydroperoxide is not a good substrate for catalase , which suggests that glutathione peroxidase may be important in the inhibition of this pathway in vivo . thus , we suggest that peroxidase metabolism may contribute to the observed carcinogenicity of meu in extrahepatic tissues . whether the peroxidase metabolism may contribute to the observed carcinogenicity of meu in extrahepatic tissues remains to be investigated .

methyleugenol , the methyl ether of eugenol , both of which are flavorant constituents of spices , has been listed by the national toxicology program s report on carcinogens as reasonably anticipated to be a human carcinogen . this finding is based on the observation of increased incidence of malignant tumors at multiple tissue sites in experimental animals of different species . by contrast , eugenol is not listed . in this study , we show that both methyleugenol and eugenol readily undergo peroxidative metabolism in vitro to form free radicals with large hyperfine interactions of the methylene allylic hydrogen atoms . these large hyperfine splittings indicate large electron densities adjacent to those hydrogen atoms . methyleugenol undergoes autoxidation such that the commercial product contains 1030 mg / l hydroperoxide and is capable of activating peroxidases without the presence of added hydrogen peroxide . additionally , the hydroperoxide is not a good substrate for catalase , which demonstrates that these antioxidant defenses will not be effective in protecting against methyleugenol exposure .

Introduction Materials and Methods Results Discussion

methyleugenol ( chart 1a , cas # 93 - 15 - 2 ) is the methyl ether of eugenol ( chart1b , cas # 97 - 53 - 0 ) , which is itself a major constituent of spices such as clove , allspice , and bay leaves and is known to induce contact allergies . methyleugenol ( meu ) and eugenol ( eug ) are allylalkoxybenzene derivatives that are present as flavoring constituents in foodstuffs or are added as flavor extenders . a safety assessment of allylalkoxybenzene derivatives was prepared by the expert panel of the flavor and extract manufacturers association , which summarized previous findings about the safety of meu . they conclude that the present human exposure to meu does not pose a significant cancer risk . by contrast , the twelfth report on carcinogens of the national toxicology program concludes that meu is reasonably anticipated to be a human carcinogen . this finding is based on the observation of increased incidence of malignant tumors in the liver , bile duct , glandular stomach , kidney , mesothelium , mammary gland , and skin in experimental animals . the mechanism of hepatic carcinogenesis appears to result from the bioactivation of meu to dna - reactive intermediates via hydroxylation of the 1 position of the allylic side chain and subsequent sulfation to generate 1-sulfoxymethyleugenol , conversions that can occur in the liver and that have also been demonstrated to occur in the human liver.in vitro studies have identified the human liver cytochrome p450 1a2 as the enzyme that is the likely bioactivator of meu . a 5-fold difference in activities occurred among 15 human microsome samples in a correlation study , which suggested that individual differences , arising from lifestyle factors such as smoking or drug use , can influence the likelihood that meu exposure will lead to harmful effects . additionally , the european commission s scientific committee on food formulated an opinion on meu , based largely on the rodent studies , concluding it to be a multisite , multispecies genotoxin and carcinogen for which no threshold could be assumed and for which no safe exposure limit could be established . the use of pure meu has been banned in the european union but not in the u.s.a . bioactivation of these molecules to their ultimate carcinogens takes place through cytochromes p450 and sulfotransferases . kinetic studies in vitro of [ c]meu and [ c]eug in human , mouse , and rat liver and lung tissue fractions showed that the compounds are metabolized differently : meu is converted to significant amounts of the 1-hydroxy proximate carcinogen , but eug is glucuronidated , thus avoiding the formation of 1-hydroxyeugenol . in a study of meu and its oxidative metabolites in chinese hamster lung fibroblasts , marginal cytotoxic effects were not observed , but meu and 1-hydroxymethyleugenol at concentrations of 10 m caused dna strand breaks . meu bioactivation and detoxification were studied by a physiologically based kinetic approach comparing human and rat models , and the results obtained revealed that there were no substantial species differences in the formation of the reactive ultimate carcinogenic l-sulfoxymethyleugenol metabolite . the total human oral intake of meu has been estimated as 0.77 g / kg - bw / d , of which 14% is added meu . since meu is carcinogenic in extrahepatic tissues which have lower levels of cytochrome p450s and sulfotransferases , we decided to investigate the oxidation of meu and the related eug by peroxidases , which are widely distributed in extrahepatic tissues . , satoh et al . nakagawa et al . reported resolved and assigned epr spectra for sesmolyl and related phenoxyl radicals that were generated by continuous uv - irradiation of benzene solutions of the corresponding phenols . oxidized eug to its phenoxyl radical using hrp in a fast - flow system and report epr parameters similar to those for related methoxy - substituted phenoxyl radicals . although there have been no previous reports of epr observations of meu radical cations , there have been literature reports of chemical or electrochemical production of methoxybenzene radical cations . also kersten and co - workers reported the biochemical generation of methoxybenzene radical cations of 10 of the 12 possible methoxybenzene congeners , including cis- and trans-1,4-dimethoxybenzene , 1,2,3,4-tetramethoxybenzene , and 1,2,4,5-tetramethoxybenzene by ligninase peroxidase and hydrogen peroxide . by contrast , hrp was only able to oxidize the four congeners with the lowest half - wave oxidation potentials . biochemical oxidation of these molecules produced methoxy - substituted benzoquinones and methanol and demonstrated that the quinone oxygen atoms were derived from the solvent water . in this study , we report the first observation of radical cations of meu produced by hrp metabolism . observation of radical cations of several related compounds is also reported , and the epr spectral assignments for this series of radicals form a consistent pattern . free radical metabolites have been widely implicated as contributing to disease states and may be involved in meu carcinogenicity in extrahepatic tissues . the h2o2 concentration was confirmed by absorbance measurements at 240 nm ( 240 = 39.4 m cm ) . in some experiments , the commercial preparation of hrp was loaded onto a pd-10 gel filtration column and eluted with 50 mm phosphate buffer , ph 7.4 , treated with chelex before use . the magnetic field at the er4122 super hi - q microwave cavity was modulated at 100 khz , affording the first - derivative epr spectra that were recorded as computer files using bruker s software . subsequent analysis , interpretation , and simulation of the spectra used locally produced software . , milwaukee , wi ) that have a sensitivity reported to detect a lower limit of 1 mg / l peroxide . meu oxidized by hrp activated by hydrogen peroxide in a fast - flow system produced a well - resolved epr spectrum of the corresponding radical cation ( figure 1a ) . the same spectrum was also produced when meu was oxidized by hemoglobin and hydrogen peroxide in a fast - flow system ( data not shown ) . however , when hydrogen peroxide was omitted , a strong epr signal was still observed ( figure 2d ) , and the same spectrum was observed in the presence of catalase at a concentration 70 times that of the peroxidase ( figure 2e ) . epr fast - flow spectra of the meu radical produced in a system of meu , h2o2 , and hrp . the concentrations of meu , h2o2 , and hrp in the flat cell were 1.75 mm , 13 mm , and 0.46 m , respectively . oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . the final solution also contained 10% v / v ethanol and 0.10 mm dtpa . epr fast - flow spectra of the meu radical produced in a system of meu , h2o2 , and hrp . numbers in parentheses are numbers of equivalent hydrogen atoms contributing to the stated hyperfine coupling . alkenylbenzenes , such as meu , with allylic hydrogen atoms are known to be susceptible to autoxidation reactions that produce organic hydroperoxides . the quantofix test strip assay of stock meu indicated the presence of hydroperoxides in the range of 10 to 30 mg / l . the presence of endogenous hydroperoxides in stock meu and the ability of hydroperoxide to activate hrp and sustain the oxidation of meu to its radical were established by uv the spectrum obtained for native hrp before the addition of h2o2 and/or meu exhibits two characteristic bands at 502 and 642 nm and the soret band at 402 nm ( figure 3a ) . the addition of h2o2 resulted in the immediate decrease in absorbance of the soret band and the appearance of peaks with maxima at approximately 565 and 650 nm , which are associated with the formation of compound i. the addition of 1.75 mm meu 2 min after the addition of h2o2 caused a shift of the soret peak from 402 to 419 nm and the appearance of two peaks at 528 and 557 nm that are characteristic of compound ii ( figure 3b ) . during the first 4 min following the addition of meu , the soret intensity increased quickly , after which it remained at a constant level . absorption spectra were recorded every 2 min after the initiation of the reaction . ( a ) spectral changes from native hrp to compound i. the reaction was initiated by the addition of 10 m h2o2 . compound i was prepared as described in a , and 1.75 mm meu was added 2 min after the addition of peroxide . ( c ) formation and spontaneous decomposition of compound ii in the absence of exogenously added h2o2 . in this case , the reaction was initiated by the addition of 2.5 mm meu , and scans were recorded every 2 min . the presence of endogenous hydroperoxide in the stock of meu was investigated by incubating hrp with meu in the absence of exogenously added h2o2 . the subsequent spectra showed that compound ii slowly reverted to the native form . none of the spectra observed showed the dramatic decrease in absorbance of the soret band and the absorption peaks at 565 and 650 nm that are characteristic of compound i , in accordance with the instability of this intermediate in the presence of one - electron reductants . that an endogenous hydroperoxide was effectively responsible for the accumulation of compound ii in the reaction system has been tested by the addition of catalase . however , in the presence of the h2o2 scavenger , slightly less compound ii was generated , but the effect was small , indicating that the endogenous hydroperoxide did not react well with catalase ( data not shown ) . the epr spectrum of the 3,4-dimethoxytoluene radical cation ( figure 4 ) was recorded and interpreted to assist the assignment of epr hyperfine coupling constants in the meu radical cation with ce(iv ) in 0.23 m sulfuric acid solution serving as the oxidizing agent . oxidation of 3,4-dimethoxytoluene by hrp and hydrogen peroxide produced only a very weak epr signal under our fast flow conditions ( data not shown ) . epr fast - flow spectra of the 3,4-dimethoxytoluene radical cation produced in a system of 3,4-dimethoxytoluene , cerium(iv ) sulfate , and h2so4 at concentrations in the flat cell of 1.0 mm , 0.9 mm , and 0.225 m , respectively . equal volumes of 3,4-dimethoxytoluene and ce(iv ) solutions were mixed milliseconds before entering the flat cell at a total flow rate of 20 ml / min . ( b ) simulated epr spectrum with the coupling constants given in table 1 . a well - resolved epr spectrum of eugenol phenoxyl radical , reported in figure 5 , was observed when eugenol was oxidized by reaction with hrp and hydrogen peroxide in a fast flow system . epr fast - flow spectra of the eugenol phenoxyl radical produced in a system of eugenol , h2o2 , and hrp at concentrations in the flat cell of 1.0 mm , 13 mm , and 0.23 m , respectively . oxygen was removed from the reagent solutions by bubbling 99.9999% pure nitrogen gas through them . equal volumes of a solution of eugenol / h2o2 and a solution of hrp in 100 mm , ph 7.4 , phosphate buffer were mixed milliseconds before entering the flat cell at a total flow rate of 60 ml / min . ( a ) complete system with eugenol , h2o2 , and hrp . epr fast - flow spectra of the 2-methoxy-4-methylphenol radical cation produced in a system of 2-methoxy-4-methylphenol , cerium(iv ) sulfate , and h2so4 at concentrations in the flat cell of 1.0 mm , 0.9 mm , and 0.225 m , respectively . were unable to oxidize any of the dimethoxybenzene compounds ( or any related compounds with oxidation potentials greater than about 1.36 v ) with the hrp - hydrogen peroxide system . the oxidation potential of 1,2-dimethoxybenzene is reported as 1.69 v , while that for allylbenzene is about 2.6 v. the structurally similar compound ( 3,4-dimethoxy)methylcinnamate has an oxidation potential of 1.7 v. the oxidation potential of hrp compounds i and ii is about 0.9 v near ph 7.4 depending on the isozyme , which suggests that meu might be oxidized successfully by hrp . the meu radical cation has a well resolved epr spectrum ( figures 1a and 2a ) and is accurately simulated ( figure 1b ) by the hyperfine coupling constants reported in table 1 . subtraction of the simulation from the experimental spectrum results in a residual pattern that is essentially noise ( figure 1c ) . these observations are consistent with the presence of an organic hydroperoxide in meu ; this was confirmed by peroxide test strips that provided a semiquantitative estimate of 1050 mg / l peroxide concentration . treating meu with alumina to remove data presented in figure 3a and b are the first clear spectral evidence that the oxidation of meu proceeds by a cycle typical of peroxidase involving compounds i and ii . hrp compound ii was the predominant form of peroxidase observed in the presence of a high concentration of meu , and this intermediate was detected even in the absence of exogenously added h2o2 . the intensity of the characteristic absorption peaks of hrp compound ii decreases over time as the reaction proceeds , and the resting state of hrp is regenerated ( figure 3c ) . a similar experiment with catalase added to the incubation demonstrated that the hydroperoxide is not affected by the presence of catalase , a result that is consistent with the epr observation ( figure 2e ) . this indicates that the hydroperoxide formed by autoxidation of meu is not a good substrate for catalase and explains why inclusion of catalase in the epr incubation of meu had no effect ( figure 2e ) . presumably glutathione peroxidase , which is nonspecific , will reduce this unknown hydroperoxide and thereby inhibit oxidation of meu . the meu radical epr spectrum hyperfine pattern arises from large hyperfine couplings of the two hydrogens of the methylene group attached at the 4 position and one hydrogen atom attached at the 5 position , as would be predicted from the simple model of benzene molecular orbital ordering that results from two ortho - methoxy electron - releasing substituents . that the methylene hyperfine couplings are large is also evident from the epr spectrum of the 3,4-dimethoxytoluene radical cation ( see figure 4a ) . this epr spectrum is simulated successfully ( figure 4b ) with hyperfine parameters quite close to those for meu ( see table 1 ) , and the residual pattern that results from subtracting the simulated from experimental spectrum is essentially noise ( figure 4c ) . successful simulation of the spectra of both radicals also requires the inclusion of hyperfine coupling from two sets of methoxy hydrogen atoms , which indicates that we are seeing the initial free radical species before any demethoxylation reaction can occur . the epr spectrum of the eug phenoxyl radical ( figure 5a ) has been reported previously but recorded at somewhat lower resolution and poorer s / n . they are characterized by large hyperfine couplings to two and three hydrogen atoms , respectively , substituted at the number 4 carbon of the aromatic ring . both spectra are accurately simulated by the assigned hyperfine parameters ( figures 5b and 6b ) and have residuals that are essentially noise ( figures 5c and 6c ) . methyleugenol undergoes autoxidation such that the commercial product contains 1030 mg / l hydroperoxide and is capable of activating peroxidases without the presence of added hydrogen peroxide . our spectroscopic studies show that the hydroperoxide is not a good substrate for catalase , which suggests that glutathione peroxidase may be important in the inhibition of this pathway in vivo . thus , we suggest that peroxidase metabolism may contribute to the observed carcinogenicity of meu in extrahepatic tissues . whether the peroxidase metabolism may contribute to the observed carcinogenicity of meu in extrahepatic tissues remains to be investigated .

methyleugenol ( chart 1a , cas # 93 - 15 - 2 ) is the methyl ether of eugenol ( chart1b , cas # 97 - 53 - 0 ) , which is itself a major constituent of spices such as clove , allspice , and bay leaves and is known to induce contact allergies . a safety assessment of allylalkoxybenzene derivatives was prepared by the expert panel of the flavor and extract manufacturers association , which summarized previous findings about the safety of meu . they conclude that the present human exposure to meu does not pose a significant cancer risk . by contrast , the twelfth report on carcinogens of the national toxicology program concludes that meu is reasonably anticipated to be a human carcinogen . this finding is based on the observation of increased incidence of malignant tumors in the liver , bile duct , glandular stomach , kidney , mesothelium , mammary gland , and skin in experimental animals . the mechanism of hepatic carcinogenesis appears to result from the bioactivation of meu to dna - reactive intermediates via hydroxylation of the 1 position of the allylic side chain and subsequent sulfation to generate 1-sulfoxymethyleugenol , conversions that can occur in the liver and that have also been demonstrated to occur in the human liver.in vitro studies have identified the human liver cytochrome p450 1a2 as the enzyme that is the likely bioactivator of meu . a 5-fold difference in activities occurred among 15 human microsome samples in a correlation study , which suggested that individual differences , arising from lifestyle factors such as smoking or drug use , can influence the likelihood that meu exposure will lead to harmful effects . additionally , the european commission s scientific committee on food formulated an opinion on meu , based largely on the rodent studies , concluding it to be a multisite , multispecies genotoxin and carcinogen for which no threshold could be assumed and for which no safe exposure limit could be established . kinetic studies in vitro of [ c]meu and [ c]eug in human , mouse , and rat liver and lung tissue fractions showed that the compounds are metabolized differently : meu is converted to significant amounts of the 1-hydroxy proximate carcinogen , but eug is glucuronidated , thus avoiding the formation of 1-hydroxyeugenol . in a study of meu and its oxidative metabolites in chinese hamster lung fibroblasts , marginal cytotoxic effects were not observed , but meu and 1-hydroxymethyleugenol at concentrations of 10 m caused dna strand breaks . meu bioactivation and detoxification were studied by a physiologically based kinetic approach comparing human and rat models , and the results obtained revealed that there were no substantial species differences in the formation of the reactive ultimate carcinogenic l-sulfoxymethyleugenol metabolite . meu is a ubiquitous environmental component with annual use in excess of 30,000 kg in the united states food , perfume , and pesticide industries . it is present in soaps and perfumes as a fragrance , in commercial food products as a flavorant , and in pesticides as an insect attractant . high resolution mass spectrometric analyses of serum samples from 213 nonfasting adult participants in the third national health and nutrition examination survey detected methyleugenol in 98% of samples at a mean concentration of 24 pg / g ( range < 3.1390 pg / g ) . mean serum levels increased following meu consumption and then decreased over time with metabolism : 53.9 pg / g ( 15 min ) ; 42.9 ( 30 min ) ; 37.0 ( 60 min ) ; and 25.2 ( 120 min ) with an elimination half - life of ca . the total human oral intake of meu has been estimated as 0.77 g / kg - bw / d , of which 14% is added meu . since meu is carcinogenic in extrahepatic tissues which have lower levels of cytochrome p450s and sulfotransferases , we decided to investigate the oxidation of meu and the related eug by peroxidases , which are widely distributed in extrahepatic tissues . although phenoxyl radicals of eug have been investigated by electron paramagnetic resonance spectroscopy ( epr ) , no reports of meu radicals have appeared . reported resolved and assigned epr spectra for sesmolyl and related phenoxyl radicals that were generated by continuous uv - irradiation of benzene solutions of the corresponding phenols . oxidized eug to its phenoxyl radical using hrp in a fast - flow system and report epr parameters similar to those for related methoxy - substituted phenoxyl radicals . although there have been no previous reports of epr observations of meu radical cations , there have been literature reports of chemical or electrochemical production of methoxybenzene radical cations . siero and co - workers observed highly resolved epr spectra of radical cations generated by phenyl iodine(iii)bis(trifluoroacetate ) [ pifa ] as the electron acceptor in a charge transfer complex formed by methoxybenzenes and pifa in hexafluoropropanol solution . also kersten and co - workers reported the biochemical generation of methoxybenzene radical cations of 10 of the 12 possible methoxybenzene congeners , including cis- and trans-1,4-dimethoxybenzene , 1,2,3,4-tetramethoxybenzene , and 1,2,4,5-tetramethoxybenzene by ligninase peroxidase and hydrogen peroxide . by contrast , hrp was only able to oxidize the four congeners with the lowest half - wave oxidation potentials . biochemical oxidation of these molecules produced methoxy - substituted benzoquinones and methanol and demonstrated that the quinone oxygen atoms were derived from the solvent water . in this study , we report the first observation of radical cations of meu produced by hrp metabolism . observation of radical cations of several related compounds is also reported , and the epr spectral assignments for this series of radicals form a consistent pattern . methyleugenol ( 4-allyl-1,2-dimethoxybenzene ) ; eugenol ( 4-allyl-2-methoxyphenol ) ; 3,4-dimethoxytoluene ; 2-methoxy-4-methylphenol ; 3,4-dimethoxyphenol ; cerium(iv ) sulfate ; sulfuric acid ; dtpa ; hemoglobin ; and hrp ( type vi ) were purchased from sigma - aldrich ( st . reagents placed in solution reservoirs were pumped though plastic hoses by means of a masterflex l / s microprocessor - controlled peristaltic pump ( cole - parmer , vernon hills , il ) to a quartz fast - flow mixing chamber flat cell ( type wg-804 , 10 mm width , wilmad glass co. , buena , nj ) . the magnetic field at the er4122 super hi - q microwave cavity was modulated at 100 khz , affording the first - derivative epr spectra that were recorded as computer files using bruker s software . meu oxidized by hrp activated by hydrogen peroxide in a fast - flow system produced a well - resolved epr spectrum of the corresponding radical cation ( figure 1a ) . however , when hydrogen peroxide was omitted , a strong epr signal was still observed ( figure 2d ) , and the same spectrum was observed in the presence of catalase at a concentration 70 times that of the peroxidase ( figure 2e ) . epr fast - flow spectra of the meu radical produced in a system of meu , h2o2 , and hrp . the concentrations of meu , h2o2 , and hrp in the flat cell were 1.75 mm , 13 mm , and 0.46 m , respectively . equal volumes of a solution of meu / h2o2 and a solution of hrp in 100 mm phosphate buffer , ph 7.4 , were mixed milliseconds before they entered the flat cell at a total flow rate of 40 ml / min . ( c ) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw microwave power , 0.10 mt field modulation , 5.0 mt field sweep width , 164 ms time constant , 82 ms conversion time , and 33 scans of 1024 data points . epr fast - flow spectra of the meu radical produced in a system of meu , h2o2 , and hrp . the concentrations of meu , h2o2 , and hrp in the flat cell were 1.75 mm , 13 mm , and 0.46 m , respectively . equal volumes of a solution of meu / h2o2 and a solution of hrp in 100 mm phosphate buffer , ph 7.4 , were mixed milliseconds before they entered the flat cell at a total flow rate of 40 ml / min . the presence of endogenous hydroperoxides in stock meu and the ability of hydroperoxide to activate hrp and sustain the oxidation of meu to its radical were established by uv the spectrum obtained for native hrp before the addition of h2o2 and/or meu exhibits two characteristic bands at 502 and 642 nm and the soret band at 402 nm ( figure 3a ) . the addition of h2o2 resulted in the immediate decrease in absorbance of the soret band and the appearance of peaks with maxima at approximately 565 and 650 nm , which are associated with the formation of compound i. the addition of 1.75 mm meu 2 min after the addition of h2o2 caused a shift of the soret peak from 402 to 419 nm and the appearance of two peaks at 528 and 557 nm that are characteristic of compound ii ( figure 3b ) . during the first 4 min following the addition of meu , the soret intensity increased quickly , after which it remained at a constant level . ( a ) spectral changes from native hrp to compound i. the reaction was initiated by the addition of 10 m h2o2 . the presence of endogenous hydroperoxide in the stock of meu was investigated by incubating hrp with meu in the absence of exogenously added h2o2 . the first scan following the addition of meu to the native enzyme showed the formation of species similar to compound ii , with absorption maxima at 419 , 528 , and 557 nm . none of the spectra observed showed the dramatic decrease in absorbance of the soret band and the absorption peaks at 565 and 650 nm that are characteristic of compound i , in accordance with the instability of this intermediate in the presence of one - electron reductants . that an endogenous hydroperoxide was effectively responsible for the accumulation of compound ii in the reaction system has been tested by the addition of catalase . however , in the presence of the h2o2 scavenger , slightly less compound ii was generated , but the effect was small , indicating that the endogenous hydroperoxide did not react well with catalase ( data not shown ) . the epr spectrum of the 3,4-dimethoxytoluene radical cation ( figure 4 ) was recorded and interpreted to assist the assignment of epr hyperfine coupling constants in the meu radical cation with ce(iv ) in 0.23 m sulfuric acid solution serving as the oxidizing agent . epr fast - flow spectra of the 3,4-dimethoxytoluene radical cation produced in a system of 3,4-dimethoxytoluene , cerium(iv ) sulfate , and h2so4 at concentrations in the flat cell of 1.0 mm , 0.9 mm , and 0.225 m , respectively . ( c ) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw microwave power , 0.025 mt field modulation , 6.0 mt field sweep width , 1.3 s time constant , 655 ms conversion time , and 36 scans of 1024 data points . a well - resolved epr spectrum of eugenol phenoxyl radical , reported in figure 5 , was observed when eugenol was oxidized by reaction with hrp and hydrogen peroxide in a fast flow system . epr fast - flow spectra of the eugenol phenoxyl radical produced in a system of eugenol , h2o2 , and hrp at concentrations in the flat cell of 1.0 mm , 13 mm , and 0.23 m , respectively . equal volumes of a solution of eugenol / h2o2 and a solution of hrp in 100 mm , ph 7.4 , phosphate buffer were mixed milliseconds before entering the flat cell at a total flow rate of 60 ml / min . ( c ) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw of microwave power , 0.025 mt of field modulation , 5.0 mt of field sweep width , 164 ms time constant , 82 ms conversion time , and 12 scans of 1024 data points . the epr spectrum of 2-methoxy-4-methylphenol phenoxyl radical ( figure 6 ) was recorded and interpreted to assist the assignment of epr hyperfine coupling constants in the eugenol phenoxyl radical . epr fast - flow spectra of the 2-methoxy-4-methylphenol radical cation produced in a system of 2-methoxy-4-methylphenol , cerium(iv ) sulfate , and h2so4 at concentrations in the flat cell of 1.0 mm , 0.9 mm , and 0.225 m , respectively . ( c ) residual plot produced when simulated spectrum b is subtracted from experimental spectrum a. epr spectra were recorded at 20 mw of microwave power , 0.050 mt of field modulation , 6.0 mt of field sweep width , 164 ms time constant , 82 ms conversion time , and 33 scans of 1024 points . the oxidation potential of 1,2-dimethoxybenzene is reported as 1.69 v , while that for allylbenzene is about 2.6 v. the structurally similar compound ( 3,4-dimethoxy)methylcinnamate has an oxidation potential of 1.7 v. the oxidation potential of hrp compounds i and ii is about 0.9 v near ph 7.4 depending on the isozyme , which suggests that meu might be oxidized successfully by hrp . however , even when h2o2 is omitted from the incubation , a strong meu epr signal is seen ( figure 2d ) , and the same spectrum is observed when the incubation lacks h2o2 but contains 32 m catalase ( figure 2e ) . treating meu with alumina to remove data presented in figure 3a and b are the first clear spectral evidence that the oxidation of meu proceeds by a cycle typical of peroxidase involving compounds i and ii . hrp compound ii was the predominant form of peroxidase observed in the presence of a high concentration of meu , and this intermediate was detected even in the absence of exogenously added h2o2 . the intensity of the characteristic absorption peaks of hrp compound ii decreases over time as the reaction proceeds , and the resting state of hrp is regenerated ( figure 3c ) . a similar experiment with catalase added to the incubation demonstrated that the hydroperoxide is not affected by the presence of catalase , a result that is consistent with the epr observation ( figure 2e ) . this indicates that the hydroperoxide formed by autoxidation of meu is not a good substrate for catalase and explains why inclusion of catalase in the epr incubation of meu had no effect ( figure 2e ) . the meu radical epr spectrum hyperfine pattern arises from large hyperfine couplings of the two hydrogens of the methylene group attached at the 4 position and one hydrogen atom attached at the 5 position , as would be predicted from the simple model of benzene molecular orbital ordering that results from two ortho - methoxy electron - releasing substituents . this epr spectrum is simulated successfully ( figure 4b ) with hyperfine parameters quite close to those for meu ( see table 1 ) , and the residual pattern that results from subtracting the simulated from experimental spectrum is essentially noise ( figure 4c ) . successful simulation of the spectra of both radicals also requires the inclusion of hyperfine coupling from two sets of methoxy hydrogen atoms , which indicates that we are seeing the initial free radical species before any demethoxylation reaction can occur . hyperfine coupling constants for this radical and for the 2-methoxy-4-methylphenol phenoxyl radical ( figure 6a ) are reported in table 1 and are consistent with the related radicals . our spectroscopic studies show that the hydroperoxide is not a good substrate for catalase , which suggests that glutathione peroxidase may be important in the inhibition of this pathway in vivo . thus , we suggest that peroxidase metabolism may contribute to the observed carcinogenicity of meu in extrahepatic tissues .

in terms of stability and retention , attachment system provides superior effectiveness for complete dentures . biological and technical ( mechanical ) are the two types of complications encountered in implant therapy . prosthetic maintenance ( the capability to adapt or repair ) is of daily clinical interest . technical aspects are the parameters to be considered now . durable retention remains the subject to debate . technical complications include mechanical damage to the implant and prosthetic components . retention loss or adjustments are the most encountered problems ( 30% of the reported maintenance ) . the following keywords directed the search : complications , retention , wear , attachment biomaterial , overdenture attachments , attachment systems , implant - retained overdentures , implant - supported overdentures , and locator . peer - reviewed articles reporting on investigations of retention , wear , or complications of overdenture attachment systems used specifically for mandibular two - implant overdentures were identified . the search included articles published in english up to july 2014 which contained all or part of the key words in their headings . divers attachment systems are released often without evidence - based support for long - term maintenance or repair . industry proposes to replace the well - documented ball and bars attachments by new connector type introduced in 2001 , the locator attachment ( zest anchors , escondido , ca , usa ) combined the best features of a ball attachment , an era attachment ( sterngold ) , and a cap attachment , with its appreciated dual retention and different retention values . it is classified as a resilient universal hinge , and is indicated for limited inter - arch distance . limited in vitro reports on the locator retentive force are published . the dual retention ( inner and outer ) enhances its cross - section strength by mechanical and also frictional mode . the slightly oversized nylon male insert and the smaller diameter inner ring of the female abutment while the central stud of the nylon male insert of the locator attachment press fits within the inner metal ring of the female abutment , the outer margin engages the shallow undercut area present at the outer margin of the abutment simultaneously and completely . the color of the patrix ( replaceable nylon insert ) indicates the retention value . to correct implant angulation , implant manufacturers provide only little data about the retentive strength and wear of attachments . for a single individual unsplinted attachment , 4 n is mentioned as the minimum retentive force , whereas this retention may vary from as low as 1 n to as high as 85 n when mandibular overdentures are retained by two or more implants . when a rough estimate of 20 n of adequate retentive force has been proposed for a two - implant mandibular overdenture , pigozzo et al . mechanical and frictional contacts , as well as magnetic forces of attraction between the patrix and matrix can be the basis of retentive force . studies have classified the attachment systems into high ( era gray ) , medium ( locator lr white , spheroflex ball , hader bar and metal clip , era white ) , low ( locator lr pink ) , and very low ( shiner magnet , maxi magnet , magnedisc magnet ) retention groups . significantly higher retention and stability are provided by locator connectors and sterngold era , compared to nobel biocare ball connectors . pointed that the locator white , pink , and blue connectors demonstrated higher retentive forces than either a 7.9-mm prototype ball attachment design or the standard 2.25-mm ball attachment . the greatest reported value for the peak load was for the zest anchor advanced generation ( zaag ) attachment , compared to the nobel biocare ball , the zest anchor , and the sterngold era . the peak load is defined as the maximum forces developed before complete separation of the attachment components from the implant abutments . the zaag attachment exhibited significantly the highest retentive vertical and oblique forces under dislodging tensile forces applied to the housings in two directions simulating function : vertical and oblique . the lowest vertical force corresponds to the zest anchor and the lowest oblique retentive force to the nobel biocare standard . furthermore , according to lehmann , an attachment system must be able to maintain its retentive force during a 10-year shelf life . however , evidence from past and current studies demonstrates that the wear - induced structural changes undergone by an attachment system inevitably lead to a reduction or total loss of retention . a mechanical action alone or a combination of chemical and mechanical actions will induce loss of material from the surface . components wear decrease ball attachment 's retention . deterioration and deformation , along with work hardening , can also lead to attachment fracture . the variations in the wear patterns seen with different attachment systems still need better understanding . study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . this would represent 6 months to 9 years of clinical function , on the basis of three daily overdenture removals and insertions for hygienic purposes . a common trend toward reduction or total loss in retentive force this loss can be abrupt after approximately 500 cycles and may reach 80% of the initial value after 2000 cycles . to evaluate the effects of wear on overdenture resilient attachments , rutkunas et al . determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy ( sem ) of era orange and white ( eo and ew ) , locator pink , white , and blue ( lrp , lrw and lrb ) , and op anchor ( op ) , respectively , after simulated 15,000 insertion removal cycles . the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . the dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . a literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . a dramatic loss of retention for era attachments was observed at the conclusion of wear simulation test . upon microscopic measurement , gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices , with the metallic matrices remaining unchanged . studies reported similar observations with four ball attachment systems , whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . this may indicate that severe mechanical wear on both surfaces may occur after long periods of use . while some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force , others reported the highest wear on the ball attachments for implant - retained overdentures . moreover , even among samples of the same attachment systems , differences in the retentive forces were evident . to increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . when tested under identical conditions , less physical deterioration was found , despite microscopic corrosion signs observed within the stainless steel magnet case . in contrast , under long - term simulated function , in the case of telescopic attachments made of different alloys ( titanium , gold , and cobalt - chromium ) , a steady increase in the retentive force has been observed . the authors explained their results by the increased mechanical adaptation of the attachment components under cyclic loading . mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles , even though it was of little valuewhen compared to the initial retentive forces . eight hundred cycles are needed to attain relatively stable retention of overdenture attachments , especially for the most retentive systems . the clinician has to place and remove the overdenture multiple times before delivery , although this reduction might not be noticeable to the patient but only to the examiner . nevertheless , cakarer did not find any advantage of ball and bar designs over the locator in terms of retention . also , the locator root pink remained the most retentive after fatigue , compared to the era orange and white . the wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion removal cycles . when occlusal loads are applied , the mucosa is displaced under the denture base resulting in denture rotation around the attachments . the amount of occlusal load transmitted to the attachments is a factor of their resiliency . an optimal stress distribution reduces the denture movement and , thereby , the forces on the implants . simulated mastication reduced the locator retention to 40% of the baseline values with a non - linear descending curve . kleis declared that after 12 months of overdentures delivery , the male parts of the locator have to be changed , as 75.5% loss of retention has been noticed . however , the reduction in peak load - to - dislodgement for these attachments is more apparent in case of non - parallel implants . length , number , implant 's angulation , opposing dentition , and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . excessive masticatory forces , as off - axis centric contacts , excursive contacts , and cantilevered loading , may be generated in severely resorbed mandible . correct implant placement diminishes attachment systems maintenance . to overcome the inevitable continuous resorption of the underlying residual ridge , frequent rebasing of implant - retained overdentures ( ovds ) may restore the proper occlusion and reduce possible rotation of the denture around the retentive components . no major differences in prosthetic complications have been observed for bar or ball attachments , thus both are considered as reliable connectors . most of them reported more prosthetic maintenance for separated attachments , whereas others found found more technical complications / repairs per patient around bar than ball attachments . most of the separated attachments need more prosthetic maintenance ; but for others , the frequency of technical complications / repairs per patient was higher around bar than ball attachments , with an increased failure rate for the cantilevered extended bars . meanwhile , no difference was seen in the implant survival rate among splinted and unsplinted schemes . recent studies sought to compare the incidence of mechanical complications of the locator attachments with that of the commonly used overdenture attachment systems . a recent survey showed that british general dental practitioners are not familiar with the locator attachment system and are reluctant to do implant - retained ovds maintenance . general dentist practitioner general practice dentists ( gdps ) would like further training in this area . observed less prosthodontic complications and maintenance of the oral function for the locator system than for ball and bar attachments . 's findings were in agreement with this when they compared locator to southern plastic and straumann gold over a 3-year period . in contrast , kleis et al . argued that the creep response of the matrices and the hardness of the patrices provoked extensive deformation and deterioration of the locator nylon parts , with a higher substantial need for maintenance , compared to ball attachments . differences in the dimensions or material composition , and a large variation in retentive forces have been found between different batches of the same attachment system due to the poor manufacturing quality control . researchers concluded that implant parallelism has more impact on the complications that occur than the choice of type of attachments . any installation load greater than 0 n is recommended for the connection of ball , locator , or magnetic attachments to a denture base . by increasing this installation load , the resultant force acting on the implant may be decreased . however , when this load surpasses 100 n , the harmful denture movement may be increased . implant manufacturers provide only little data about the retentive strength and wear of attachments . for a single individual unsplinted attachment , 4 n is mentioned as the minimum retentive force , whereas this retention may vary from as low as 1 n to as high as 85 n when mandibular overdentures are retained by two or more implants . when a rough estimate of 20 n of adequate retentive force has been proposed for a two - implant mandibular overdenture , pigozzo et al . mechanical and frictional contacts , as well as magnetic forces of attraction between the patrix and matrix can be the basis of retentive force . studies have classified the attachment systems into high ( era gray ) , medium ( locator lr white , spheroflex ball , hader bar and metal clip , era white ) , low ( locator lr pink ) , and very low ( shiner magnet , maxi magnet , magnedisc magnet ) retention groups . significantly higher retention and stability are provided by locator connectors and sterngold era , compared to nobel biocare ball connectors . pointed that the locator white , pink , and blue connectors demonstrated higher retentive forces than either a 7.9-mm prototype ball attachment design or the standard 2.25-mm ball attachment . the greatest reported value for the peak load was for the zest anchor advanced generation ( zaag ) attachment , compared to the nobel biocare ball , the zest anchor , and the sterngold era . the peak load is defined as the maximum forces developed before complete separation of the attachment components from the implant abutments . the zaag attachment exhibited significantly the highest retentive vertical and oblique forces under dislodging tensile forces applied to the housings in two directions simulating function : vertical and oblique . the lowest vertical force corresponds to the zest anchor and the lowest oblique retentive force to the nobel biocare standard . furthermore , according to lehmann , an attachment system must be able to maintain its retentive force during a 10-year shelf life . however , evidence from past and current studies demonstrates that the wear - induced structural changes undergone by an attachment system inevitably lead to a reduction or total loss of retention . a mechanical action alone or a combination of chemical and mechanical actions will induce loss of material from the surface . components wear decrease ball attachment 's retention . deterioration and deformation , along with work hardening , can also lead to attachment fracture . the variations in the wear patterns seen with different attachment systems still need better understanding . study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . this would represent 6 months to 9 years of clinical function , on the basis of three daily overdenture removals and insertions for hygienic purposes . a common trend toward reduction or total loss in retentive force . this loss can be abrupt after approximately 500 cycles and may reach 80% of the initial value after 2000 cycles . to evaluate the effects of wear on overdenture resilient attachments , rutkunas et al . determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy ( sem ) of era orange and white ( eo and ew ) , locator pink , white , and blue ( lrp , lrw and lrb ) , and op anchor ( op ) , respectively , after simulated 15,000 insertion removal cycles . the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . the dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . a literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . a dramatic loss of retention for era attachments was observed at the conclusion of wear simulation test . upon microscopic measurement , gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices , with the metallic matrices remaining unchanged . studies reported similar observations with four ball attachment systems , whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . this may indicate that severe mechanical wear on both surfaces may occur after long periods of use . while some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force , others reported the highest wear on the ball attachments for implant - retained overdentures . when antagonist to titanium matrix . moreover , even among samples of the same attachment systems , differences in the retentive forces were evident . to increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . when tested under identical conditions , less physical deterioration was found , despite microscopic corrosion signs observed within the stainless steel magnet case . in contrast , under long - term simulated function , in the case of telescopic attachments made of different alloys ( titanium , gold , and cobalt - chromium ) , a steady increase in the retentive force has been observed . the authors explained their results by the increased mechanical adaptation of the attachment components under cyclic loading . mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles , even though it was of little valuewhen compared to the initial retentive forces . eight hundred cycles are needed to attain relatively stable retention of overdenture attachments , especially for the most retentive systems . the clinician has to place and remove the overdenture multiple times before delivery , although this reduction might not be noticeable to the patient but only to the examiner . nevertheless , cakarer did not find any advantage of ball and bar designs over the locator in terms of retention . also , the locator root pink remained the most retentive after fatigue , compared to the era orange and white . the wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion when occlusal loads are applied , the mucosa is displaced under the denture base resulting in denture rotation around the attachments . the amount of occlusal load transmitted to the attachments is a factor of their resiliency . an optimal stress distribution reduces the denture movement and , thereby , the forces on the implants . simulated mastication reduced the locator retention to 40% of the baseline values with a non - linear descending curve . kleis declared that after 12 months of overdentures delivery , the male parts of the locator have to be changed , as 75.5% loss of retention has been noticed . however , the reduction in peak load - to - dislodgement for these attachments is more apparent in case of non - parallel implants . length , number , implant 's angulation , opposing dentition , and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . excessive masticatory forces , as off - axis centric contacts , excursive contacts , and cantilevered loading , may be generated in severely resorbed mandible . correct implant placement diminishes attachment systems maintenance . to overcome the inevitable continuous resorption of the underlying residual ridge , frequent rebasing of implant - retained overdentures ( ovds ) may restore the proper occlusion and reduce possible rotation of the denture around the retentive components . study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . this would represent 6 months to 9 years of clinical function , on the basis of three daily overdenture removals and insertions for hygienic purposes . a common trend toward reduction or total loss in retentive force . this loss can be abrupt after approximately 500 cycles and may reach 80% of the initial value after 2000 cycles . to evaluate the effects of wear on overdenture resilient attachments , rutkunas et al . determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy ( sem ) of era orange and white ( eo and ew ) , locator pink , white , and blue ( lrp , lrw and lrb ) , and op anchor ( op ) , respectively , after simulated 15,000 insertion the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . the dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . a literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . a dramatic loss of retention for era attachments was observed at the conclusion of wear simulation test . upon microscopic measurement , gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices , with the metallic matrices remaining unchanged . studies reported similar observations with four ball attachment systems , whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . this may indicate that severe mechanical wear on both surfaces may occur after long periods of use . while some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force , others reported the highest wear on the ball attachments for implant - retained overdentures . when antagonist to titanium matrix . moreover , even among samples of the same attachment systems , differences in the retentive forces were evident . to increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . when tested under identical conditions , less physical deterioration was found , despite microscopic corrosion signs observed within the stainless steel magnet case . in contrast , under long - term simulated function , in the case of telescopic attachments made of different alloys ( titanium , gold , and cobalt - chromium ) , a steady increase in the retentive force has been observed . the authors explained their results by the increased mechanical adaptation of the attachment components under cyclic loading . mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles , even though it was of little valuewhen compared to the initial retentive forces . eight hundred cycles are needed to attain relatively stable retention of overdenture attachments , especially for the most retentive systems . the clinician has to place and remove the overdenture multiple times before delivery , although this reduction might not be noticeable to the patient but only to the examiner . nevertheless , cakarer did not find any advantage of ball and bar designs over the locator in terms of retention . also , the locator root pink remained the most retentive after fatigue , compared to the era orange and white . the wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion when occlusal loads are applied , the mucosa is displaced under the denture base resulting in denture rotation around the attachments . the amount of occlusal load transmitted to the attachments is a factor of their resiliency . an optimal stress distribution reduces the denture movement and , thereby , the forces on the implants . simulated mastication reduced the locator retention to 40% of the baseline values with a non - linear descending curve . kleis declared that after 12 months of overdentures delivery , the male parts of the locator have to be changed , as 75.5% loss of retention has been noticed . however , the reduction in peak load - to - dislodgement for these attachments is more apparent in case of non - parallel implants . length , number , implant 's angulation , opposing dentition , and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . excessive masticatory forces , as off - axis centric contacts , excursive contacts , and cantilevered loading , may be generated in severely resorbed mandible . correct implant placement diminishes attachment systems maintenance . to overcome the inevitable continuous resorption of the underlying residual ridge , frequent rebasing of implant - retained overdentures ( ovds ) may restore the proper occlusion and reduce possible rotation of the denture around the retentive components . no major differences in prosthetic complications have been observed for bar or ball attachments , thus both are considered as reliable connectors . most of them reported more prosthetic maintenance for separated attachments , whereas others found found more technical complications / repairs per patient around bar than ball attachments . most of the separated attachments need more prosthetic maintenance ; but for others , the frequency of technical complications / repairs per patient was higher around bar than ball attachments , with an increased failure rate for the cantilevered extended bars . meanwhile , no difference was seen in the implant survival rate among splinted and unsplinted schemes . recent studies sought to compare the incidence of mechanical complications of the locator attachments with that of the commonly used overdenture attachment systems . a recent survey showed that british general dental practitioners are not familiar with the locator attachment system and are reluctant to do implant - retained ovds maintenance . general dentist practitioner general practice dentists ( gdps ) would like further training in this area . observed less prosthodontic complications and maintenance of the oral function for the locator system than for ball and bar attachments . 's findings were in agreement with this when they compared locator to southern plastic and straumann gold over a 3-year period . in contrast , kleis et al . argued that the creep response of the matrices and the hardness of the patrices provoked extensive deformation and deterioration of the locator nylon parts , with a higher substantial need for maintenance , compared to ball attachments . differences in the dimensions or material composition , and a large variation in retentive forces have been found between different batches of the same attachment system due to the poor manufacturing quality control . researchers concluded that implant parallelism has more impact on the complications that occur than the choice of type of attachments . any installation load greater than 0 n is recommended for the connection of ball , locator , or magnetic attachments to a denture base . by increasing this installation load , the resultant force acting on the implant may be decreased . however , when this load surpasses 100 n , the harmful denture movement may be increased . however patient satisfaction is somehow , independent of the attachment system ( 3 ) . in - depth studies following standardized criteria to compare different options for mandibular implant overdentures ( iovd ) treatment remain scarce . in particular , similar clinical protocols are still insufficient to allow the calculation of an overall complication incidence for implant overdentures iovds . a limitation of this literature review is that it includes studies with non - declared sample size , or measurement methods . other studies are in vitro experiments , whereas non - randomized controlled studies are lacking . well - structured clinical prospective studies remain essential , in addition to well - designed in vitro studies . the accuracy of parameters applied to the model , including geometry , constraints , and mechanical properties , determines the value of the finite element analysis experiment . masticatory loading submits the prosthesis to a hardly reproducible scheme of three - dimensional movements . the clinical wear can be influenced by saliva , denture cleansers , or food particles . as a result factors should be investigated separately under well - controlled conditions , in order to limit the influence of confounding variables . evidence - based studies do not allow us to select the most effective implant ovd connection . equal atraumatic distribution of forces between mechanical and biological supporting structures , and minimal complications are the ultimate goals when placing any connector . the amount of retention desired and the specific clinical situation guide the clinician in selecting an appropriate attachment . the retention measurement values provided by the manufacturer at treatment beginning and after function would help to respond to individual needs of patients . scientific evidence related to the material 's clinical performance , objective oral function , and patients appreciation of the treatment should guide the clinician in making the ultimate choice of a specified attachment . adequate aftercare may be difficult or impossible when treating aging patients . in the author 's opinion and based on the presented data , the locator attachment system is an easy - to - use connector , with less post - insertion complications . the choice of implant location and retentive attachments in implant - retained overdentures is based on clinician 's preference , expert opinion , and empirical information . careful post - insertion maintenance of the prosthesis , attachment system , and mucosa is mandatory . out - of - pocket treatment and post - treatment expenses should be less and the treatment should not be time consuming .

background : new attachment systems are released for mandibular two - implant overdentures often without evidence - based support . biomaterial aspects are now the parameters considered when choosing the appropriate attachment . studies regarding their properties remain scarce.purpose:the purpose of this review was to help the clinician in selrcting the most adapted stud attachments according evidence - based dentistry.materials and methods : an electronic search was conducted using specific databases ( pubmed , medline , and elsevier libraries ) . peer - reviewed articles published in english up to july 2014 were identified . emphasis was given on the biomaterial aspects and technical complications . no hand search was added.results:the electronic search generated 115 full - text papers , of which 84 papers were included in the review . the majority were clinical and in vitro studies . some review articles were also considered . papers reported survival and failures of overdenture connection systems . emphasis was laid on attachment deformation.conclusion:implant overdentures long - term follow - up studies may provide useful guidelines for the clinician in selecting the type of attachment system and overdenture design . locator attachments are more and more used , with lesser complications reported .

INTRODUCTION MATERIALS AND METHODS Adequate retentive force Connector type and retention Change of retentive values over time Wear simulation tests Related to reproduced oral environment Related to material components Related to insertionremoval cycles Related to mastication Type of attachment and incidence of mechanical complications DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

in terms of stability and retention , attachment system provides superior effectiveness for complete dentures . biological and technical ( mechanical ) are the two types of complications encountered in implant therapy . technical aspects are the parameters to be considered now . technical complications include mechanical damage to the implant and prosthetic components . retention loss or adjustments are the most encountered problems ( 30% of the reported maintenance ) . the following keywords directed the search : complications , retention , wear , attachment biomaterial , overdenture attachments , attachment systems , implant - retained overdentures , implant - supported overdentures , and locator . peer - reviewed articles reporting on investigations of retention , wear , or complications of overdenture attachment systems used specifically for mandibular two - implant overdentures were identified . the search included articles published in english up to july 2014 which contained all or part of the key words in their headings . divers attachment systems are released often without evidence - based support for long - term maintenance or repair . industry proposes to replace the well - documented ball and bars attachments by new connector type introduced in 2001 , the locator attachment ( zest anchors , escondido , ca , usa ) combined the best features of a ball attachment , an era attachment ( sterngold ) , and a cap attachment , with its appreciated dual retention and different retention values . it is classified as a resilient universal hinge , and is indicated for limited inter - arch distance . limited in vitro reports on the locator retentive force are published . when a rough estimate of 20 n of adequate retentive force has been proposed for a two - implant mandibular overdenture , pigozzo et al . studies have classified the attachment systems into high ( era gray ) , medium ( locator lr white , spheroflex ball , hader bar and metal clip , era white ) , low ( locator lr pink ) , and very low ( shiner magnet , maxi magnet , magnedisc magnet ) retention groups . pointed that the locator white , pink , and blue connectors demonstrated higher retentive forces than either a 7.9-mm prototype ball attachment design or the standard 2.25-mm ball attachment . the greatest reported value for the peak load was for the zest anchor advanced generation ( zaag ) attachment , compared to the nobel biocare ball , the zest anchor , and the sterngold era . furthermore , according to lehmann , an attachment system must be able to maintain its retentive force during a 10-year shelf life . however , evidence from past and current studies demonstrates that the wear - induced structural changes undergone by an attachment system inevitably lead to a reduction or total loss of retention . the variations in the wear patterns seen with different attachment systems still need better understanding . study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . this would represent 6 months to 9 years of clinical function , on the basis of three daily overdenture removals and insertions for hygienic purposes . determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy ( sem ) of era orange and white ( eo and ew ) , locator pink , white , and blue ( lrp , lrw and lrb ) , and op anchor ( op ) , respectively , after simulated 15,000 insertion removal cycles . the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . the dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . a literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . upon microscopic measurement , gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices , with the metallic matrices remaining unchanged . studies reported similar observations with four ball attachment systems , whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . this may indicate that severe mechanical wear on both surfaces may occur after long periods of use . while some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force , others reported the highest wear on the ball attachments for implant - retained overdentures . moreover , even among samples of the same attachment systems , differences in the retentive forces were evident . to increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . in contrast , under long - term simulated function , in the case of telescopic attachments made of different alloys ( titanium , gold , and cobalt - chromium ) , a steady increase in the retentive force has been observed . mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles , even though it was of little valuewhen compared to the initial retentive forces . eight hundred cycles are needed to attain relatively stable retention of overdenture attachments , especially for the most retentive systems . the clinician has to place and remove the overdenture multiple times before delivery , although this reduction might not be noticeable to the patient but only to the examiner . also , the locator root pink remained the most retentive after fatigue , compared to the era orange and white . the wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion removal cycles . an optimal stress distribution reduces the denture movement and , thereby , the forces on the implants . length , number , implant 's angulation , opposing dentition , and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . excessive masticatory forces , as off - axis centric contacts , excursive contacts , and cantilevered loading , may be generated in severely resorbed mandible . correct implant placement diminishes attachment systems maintenance . most of them reported more prosthetic maintenance for separated attachments , whereas others found found more technical complications / repairs per patient around bar than ball attachments . most of the separated attachments need more prosthetic maintenance ; but for others , the frequency of technical complications / repairs per patient was higher around bar than ball attachments , with an increased failure rate for the cantilevered extended bars . meanwhile , no difference was seen in the implant survival rate among splinted and unsplinted schemes . recent studies sought to compare the incidence of mechanical complications of the locator attachments with that of the commonly used overdenture attachment systems . a recent survey showed that british general dental practitioners are not familiar with the locator attachment system and are reluctant to do implant - retained ovds maintenance . observed less prosthodontic complications and maintenance of the oral function for the locator system than for ball and bar attachments . argued that the creep response of the matrices and the hardness of the patrices provoked extensive deformation and deterioration of the locator nylon parts , with a higher substantial need for maintenance , compared to ball attachments . differences in the dimensions or material composition , and a large variation in retentive forces have been found between different batches of the same attachment system due to the poor manufacturing quality control . researchers concluded that implant parallelism has more impact on the complications that occur than the choice of type of attachments . any installation load greater than 0 n is recommended for the connection of ball , locator , or magnetic attachments to a denture base . by increasing this installation load , the resultant force acting on the implant may be decreased . for a single individual unsplinted attachment , 4 n is mentioned as the minimum retentive force , whereas this retention may vary from as low as 1 n to as high as 85 n when mandibular overdentures are retained by two or more implants . when a rough estimate of 20 n of adequate retentive force has been proposed for a two - implant mandibular overdenture , pigozzo et al . studies have classified the attachment systems into high ( era gray ) , medium ( locator lr white , spheroflex ball , hader bar and metal clip , era white ) , low ( locator lr pink ) , and very low ( shiner magnet , maxi magnet , magnedisc magnet ) retention groups . pointed that the locator white , pink , and blue connectors demonstrated higher retentive forces than either a 7.9-mm prototype ball attachment design or the standard 2.25-mm ball attachment . the greatest reported value for the peak load was for the zest anchor advanced generation ( zaag ) attachment , compared to the nobel biocare ball , the zest anchor , and the sterngold era . furthermore , according to lehmann , an attachment system must be able to maintain its retentive force during a 10-year shelf life . however , evidence from past and current studies demonstrates that the wear - induced structural changes undergone by an attachment system inevitably lead to a reduction or total loss of retention . the variations in the wear patterns seen with different attachment systems still need better understanding . study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . this would represent 6 months to 9 years of clinical function , on the basis of three daily overdenture removals and insertions for hygienic purposes . determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy ( sem ) of era orange and white ( eo and ew ) , locator pink , white , and blue ( lrp , lrw and lrb ) , and op anchor ( op ) , respectively , after simulated 15,000 insertion removal cycles . the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . the dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . a literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . upon microscopic measurement , gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices , with the metallic matrices remaining unchanged . studies reported similar observations with four ball attachment systems , whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . while some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force , others reported the highest wear on the ball attachments for implant - retained overdentures . moreover , even among samples of the same attachment systems , differences in the retentive forces were evident . to increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . in contrast , under long - term simulated function , in the case of telescopic attachments made of different alloys ( titanium , gold , and cobalt - chromium ) , a steady increase in the retentive force has been observed . mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles , even though it was of little valuewhen compared to the initial retentive forces . eight hundred cycles are needed to attain relatively stable retention of overdenture attachments , especially for the most retentive systems . the clinician has to place and remove the overdenture multiple times before delivery , although this reduction might not be noticeable to the patient but only to the examiner . also , the locator root pink remained the most retentive after fatigue , compared to the era orange and white . the wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion when occlusal loads are applied , the mucosa is displaced under the denture base resulting in denture rotation around the attachments . an optimal stress distribution reduces the denture movement and , thereby , the forces on the implants . length , number , implant 's angulation , opposing dentition , and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . excessive masticatory forces , as off - axis centric contacts , excursive contacts , and cantilevered loading , may be generated in severely resorbed mandible . correct implant placement diminishes attachment systems maintenance . study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . this would represent 6 months to 9 years of clinical function , on the basis of three daily overdenture removals and insertions for hygienic purposes . determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy ( sem ) of era orange and white ( eo and ew ) , locator pink , white , and blue ( lrp , lrw and lrb ) , and op anchor ( op ) , respectively , after simulated 15,000 insertion the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . the dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . a literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . upon microscopic measurement , gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices , with the metallic matrices remaining unchanged . studies reported similar observations with four ball attachment systems , whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . while some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force , others reported the highest wear on the ball attachments for implant - retained overdentures . moreover , even among samples of the same attachment systems , differences in the retentive forces were evident . to increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . in contrast , under long - term simulated function , in the case of telescopic attachments made of different alloys ( titanium , gold , and cobalt - chromium ) , a steady increase in the retentive force has been observed . mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles , even though it was of little valuewhen compared to the initial retentive forces . eight hundred cycles are needed to attain relatively stable retention of overdenture attachments , especially for the most retentive systems . the clinician has to place and remove the overdenture multiple times before delivery , although this reduction might not be noticeable to the patient but only to the examiner . also , the locator root pink remained the most retentive after fatigue , compared to the era orange and white . the wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion when occlusal loads are applied , the mucosa is displaced under the denture base resulting in denture rotation around the attachments . an optimal stress distribution reduces the denture movement and , thereby , the forces on the implants . length , number , implant 's angulation , opposing dentition , and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . excessive masticatory forces , as off - axis centric contacts , excursive contacts , and cantilevered loading , may be generated in severely resorbed mandible . correct implant placement diminishes attachment systems maintenance . most of them reported more prosthetic maintenance for separated attachments , whereas others found found more technical complications / repairs per patient around bar than ball attachments . most of the separated attachments need more prosthetic maintenance ; but for others , the frequency of technical complications / repairs per patient was higher around bar than ball attachments , with an increased failure rate for the cantilevered extended bars . meanwhile , no difference was seen in the implant survival rate among splinted and unsplinted schemes . recent studies sought to compare the incidence of mechanical complications of the locator attachments with that of the commonly used overdenture attachment systems . a recent survey showed that british general dental practitioners are not familiar with the locator attachment system and are reluctant to do implant - retained ovds maintenance . observed less prosthodontic complications and maintenance of the oral function for the locator system than for ball and bar attachments . argued that the creep response of the matrices and the hardness of the patrices provoked extensive deformation and deterioration of the locator nylon parts , with a higher substantial need for maintenance , compared to ball attachments . differences in the dimensions or material composition , and a large variation in retentive forces have been found between different batches of the same attachment system due to the poor manufacturing quality control . researchers concluded that implant parallelism has more impact on the complications that occur than the choice of type of attachments . any installation load greater than 0 n is recommended for the connection of ball , locator , or magnetic attachments to a denture base . by increasing this installation load , the resultant force acting on the implant may be decreased . however patient satisfaction is somehow , independent of the attachment system ( 3 ) . in - depth studies following standardized criteria to compare different options for mandibular implant overdentures ( iovd ) treatment remain scarce . in particular , similar clinical protocols are still insufficient to allow the calculation of an overall complication incidence for implant overdentures iovds . a limitation of this literature review is that it includes studies with non - declared sample size , or measurement methods . other studies are in vitro experiments , whereas non - randomized controlled studies are lacking . well - structured clinical prospective studies remain essential , in addition to well - designed in vitro studies . the accuracy of parameters applied to the model , including geometry , constraints , and mechanical properties , determines the value of the finite element analysis experiment . evidence - based studies do not allow us to select the most effective implant ovd connection . equal atraumatic distribution of forces between mechanical and biological supporting structures , and minimal complications are the ultimate goals when placing any connector . the amount of retention desired and the specific clinical situation guide the clinician in selecting an appropriate attachment . scientific evidence related to the material 's clinical performance , objective oral function , and patients appreciation of the treatment should guide the clinician in making the ultimate choice of a specified attachment . in the author 's opinion and based on the presented data , the locator attachment system is an easy - to - use connector , with less post - insertion complications . the choice of implant location and retentive attachments in implant - retained overdentures is based on clinician 's preference , expert opinion , and empirical information . careful post - insertion maintenance of the prosthesis , attachment system , and mucosa is mandatory .

in terms of stability and retention , attachment system provides superior effectiveness for complete dentures . the following keywords directed the search : complications , retention , wear , attachment biomaterial , overdenture attachments , attachment systems , implant - retained overdentures , implant - supported overdentures , and locator . peer - reviewed articles reporting on investigations of retention , wear , or complications of overdenture attachment systems used specifically for mandibular two - implant overdentures were identified . the search included articles published in english up to july 2014 which contained all or part of the key words in their headings . divers attachment systems are released often without evidence - based support for long - term maintenance or repair . industry proposes to replace the well - documented ball and bars attachments by new connector type introduced in 2001 , the locator attachment ( zest anchors , escondido , ca , usa ) combined the best features of a ball attachment , an era attachment ( sterngold ) , and a cap attachment , with its appreciated dual retention and different retention values . it is classified as a resilient universal hinge , and is indicated for limited inter - arch distance . the slightly oversized nylon male insert and the smaller diameter inner ring of the female abutment while the central stud of the nylon male insert of the locator attachment press fits within the inner metal ring of the female abutment , the outer margin engages the shallow undercut area present at the outer margin of the abutment simultaneously and completely . to correct implant angulation , implant manufacturers provide only little data about the retentive strength and wear of attachments . for a single individual unsplinted attachment , 4 n is mentioned as the minimum retentive force , whereas this retention may vary from as low as 1 n to as high as 85 n when mandibular overdentures are retained by two or more implants . when a rough estimate of 20 n of adequate retentive force has been proposed for a two - implant mandibular overdenture , pigozzo et al . mechanical and frictional contacts , as well as magnetic forces of attraction between the patrix and matrix can be the basis of retentive force . studies have classified the attachment systems into high ( era gray ) , medium ( locator lr white , spheroflex ball , hader bar and metal clip , era white ) , low ( locator lr pink ) , and very low ( shiner magnet , maxi magnet , magnedisc magnet ) retention groups . the greatest reported value for the peak load was for the zest anchor advanced generation ( zaag ) attachment , compared to the nobel biocare ball , the zest anchor , and the sterngold era . the peak load is defined as the maximum forces developed before complete separation of the attachment components from the implant abutments . the zaag attachment exhibited significantly the highest retentive vertical and oblique forces under dislodging tensile forces applied to the housings in two directions simulating function : vertical and oblique . the lowest vertical force corresponds to the zest anchor and the lowest oblique retentive force to the nobel biocare standard . however , evidence from past and current studies demonstrates that the wear - induced structural changes undergone by an attachment system inevitably lead to a reduction or total loss of retention . a mechanical action alone or a combination of chemical and mechanical actions will induce loss of material from the surface . the variations in the wear patterns seen with different attachment systems still need better understanding . study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . attachment systems were then submitted to cyclic loading under either axial or paraxial forces in the range of 54010,000 cycles of repeated insertion and removal . this would represent 6 months to 9 years of clinical function , on the basis of three daily overdenture removals and insertions for hygienic purposes . a common trend toward reduction or total loss in retentive force this loss can be abrupt after approximately 500 cycles and may reach 80% of the initial value after 2000 cycles . to evaluate the effects of wear on overdenture resilient attachments , rutkunas et al . determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy ( sem ) of era orange and white ( eo and ew ) , locator pink , white , and blue ( lrp , lrw and lrb ) , and op anchor ( op ) , respectively , after simulated 15,000 insertion removal cycles . the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . the dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . a literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . a dramatic loss of retention for era attachments was observed at the conclusion of wear simulation test . upon microscopic measurement , gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices , with the metallic matrices remaining unchanged . studies reported similar observations with four ball attachment systems , whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . while some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force , others reported the highest wear on the ball attachments for implant - retained overdentures . moreover , even among samples of the same attachment systems , differences in the retentive forces were evident . to increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . in contrast , under long - term simulated function , in the case of telescopic attachments made of different alloys ( titanium , gold , and cobalt - chromium ) , a steady increase in the retentive force has been observed . the authors explained their results by the increased mechanical adaptation of the attachment components under cyclic loading . mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles , even though it was of little valuewhen compared to the initial retentive forces . the clinician has to place and remove the overdenture multiple times before delivery , although this reduction might not be noticeable to the patient but only to the examiner . also , the locator root pink remained the most retentive after fatigue , compared to the era orange and white . the wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion removal cycles . when occlusal loads are applied , the mucosa is displaced under the denture base resulting in denture rotation around the attachments . an optimal stress distribution reduces the denture movement and , thereby , the forces on the implants . simulated mastication reduced the locator retention to 40% of the baseline values with a non - linear descending curve . kleis declared that after 12 months of overdentures delivery , the male parts of the locator have to be changed , as 75.5% loss of retention has been noticed . however , the reduction in peak load - to - dislodgement for these attachments is more apparent in case of non - parallel implants . length , number , implant 's angulation , opposing dentition , and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . excessive masticatory forces , as off - axis centric contacts , excursive contacts , and cantilevered loading , may be generated in severely resorbed mandible . to overcome the inevitable continuous resorption of the underlying residual ridge , frequent rebasing of implant - retained overdentures ( ovds ) may restore the proper occlusion and reduce possible rotation of the denture around the retentive components . most of the separated attachments need more prosthetic maintenance ; but for others , the frequency of technical complications / repairs per patient was higher around bar than ball attachments , with an increased failure rate for the cantilevered extended bars . meanwhile , no difference was seen in the implant survival rate among splinted and unsplinted schemes . recent studies sought to compare the incidence of mechanical complications of the locator attachments with that of the commonly used overdenture attachment systems . a recent survey showed that british general dental practitioners are not familiar with the locator attachment system and are reluctant to do implant - retained ovds maintenance . observed less prosthodontic complications and maintenance of the oral function for the locator system than for ball and bar attachments . argued that the creep response of the matrices and the hardness of the patrices provoked extensive deformation and deterioration of the locator nylon parts , with a higher substantial need for maintenance , compared to ball attachments . differences in the dimensions or material composition , and a large variation in retentive forces have been found between different batches of the same attachment system due to the poor manufacturing quality control . researchers concluded that implant parallelism has more impact on the complications that occur than the choice of type of attachments . any installation load greater than 0 n is recommended for the connection of ball , locator , or magnetic attachments to a denture base . for a single individual unsplinted attachment , 4 n is mentioned as the minimum retentive force , whereas this retention may vary from as low as 1 n to as high as 85 n when mandibular overdentures are retained by two or more implants . when a rough estimate of 20 n of adequate retentive force has been proposed for a two - implant mandibular overdenture , pigozzo et al . mechanical and frictional contacts , as well as magnetic forces of attraction between the patrix and matrix can be the basis of retentive force . studies have classified the attachment systems into high ( era gray ) , medium ( locator lr white , spheroflex ball , hader bar and metal clip , era white ) , low ( locator lr pink ) , and very low ( shiner magnet , maxi magnet , magnedisc magnet ) retention groups . the greatest reported value for the peak load was for the zest anchor advanced generation ( zaag ) attachment , compared to the nobel biocare ball , the zest anchor , and the sterngold era . the peak load is defined as the maximum forces developed before complete separation of the attachment components from the implant abutments . the zaag attachment exhibited significantly the highest retentive vertical and oblique forces under dislodging tensile forces applied to the housings in two directions simulating function : vertical and oblique . the lowest vertical force corresponds to the zest anchor and the lowest oblique retentive force to the nobel biocare standard . however , evidence from past and current studies demonstrates that the wear - induced structural changes undergone by an attachment system inevitably lead to a reduction or total loss of retention . a mechanical action alone or a combination of chemical and mechanical actions will induce loss of material from the surface . the variations in the wear patterns seen with different attachment systems still need better understanding . study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . this would represent 6 months to 9 years of clinical function , on the basis of three daily overdenture removals and insertions for hygienic purposes . to evaluate the effects of wear on overdenture resilient attachments , rutkunas et al . determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy ( sem ) of era orange and white ( eo and ew ) , locator pink , white , and blue ( lrp , lrw and lrb ) , and op anchor ( op ) , respectively , after simulated 15,000 insertion removal cycles . the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . the dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . a literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . a dramatic loss of retention for era attachments was observed at the conclusion of wear simulation test . upon microscopic measurement , gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices , with the metallic matrices remaining unchanged . studies reported similar observations with four ball attachment systems , whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . while some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force , others reported the highest wear on the ball attachments for implant - retained overdentures . moreover , even among samples of the same attachment systems , differences in the retentive forces were evident . to increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . in contrast , under long - term simulated function , in the case of telescopic attachments made of different alloys ( titanium , gold , and cobalt - chromium ) , a steady increase in the retentive force has been observed . mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles , even though it was of little valuewhen compared to the initial retentive forces . also , the locator root pink remained the most retentive after fatigue , compared to the era orange and white . the wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion when occlusal loads are applied , the mucosa is displaced under the denture base resulting in denture rotation around the attachments . simulated mastication reduced the locator retention to 40% of the baseline values with a non - linear descending curve . kleis declared that after 12 months of overdentures delivery , the male parts of the locator have to be changed , as 75.5% loss of retention has been noticed . however , the reduction in peak load - to - dislodgement for these attachments is more apparent in case of non - parallel implants . length , number , implant 's angulation , opposing dentition , and parafunctional habits are among the factors that may increase susceptibility to mechanical complications . to overcome the inevitable continuous resorption of the underlying residual ridge , frequent rebasing of implant - retained overdentures ( ovds ) may restore the proper occlusion and reduce possible rotation of the denture around the retentive components . study designs that attempted to emulate the actual oral environment tried to investigate the effect of short- and long - term simulated function on the attachment systems retentive force . this would represent 6 months to 9 years of clinical function , on the basis of three daily overdenture removals and insertions for hygienic purposes . determined the dimensional changes and surface characteristics using light microscopy and scanning electron microscopy ( sem ) of era orange and white ( eo and ew ) , locator pink , white , and blue ( lrp , lrw and lrb ) , and op anchor ( op ) , respectively , after simulated 15,000 insertion the sudden decrease of retentive force of era attachments was opposed to the retentive force fluctuation of locator attachments throughout the wear simulation period . the dimensional changes and surface wear on the plastic rings of attachment males were less expressed than on plastic cores . a literature review confirmed the reduction of the retentive force of the majority of attachment systems under in vitro conditions . upon microscopic measurement , gamborena noticed distinct wear patterns that arise due to distortion of the plastic patrices , with the metallic matrices remaining unchanged . studies reported similar observations with four ball attachment systems , whereas some studies reported that significant and maximal amount of wear of the diameter of ball abutment was reached after 3 years of clinical use . while some studies sustained that attachment systems which possess a male and female component of different material composition exhibit smaller changes in the retention force , others reported the highest wear on the ball attachments for implant - retained overdentures . moreover , even among samples of the same attachment systems , differences in the retentive forces were evident . to increase the wear compensation and retention force it is noteworthy that a minimal reduction in retentive force was achieved by the magnetic attachments compared to the gradual decrease of the stud attachments retention . in contrast , under long - term simulated function , in the case of telescopic attachments made of different alloys ( titanium , gold , and cobalt - chromium ) , a steady increase in the retentive force has been observed . mechanical fatigue also induced some retention loss of the attachment systems during the experiment after 15,000 fatigue cycles , even though it was of little valuewhen compared to the initial retentive forces . also , the locator root pink remained the most retentive after fatigue , compared to the era orange and white . the wear patterns and their related attachment deformation generated by the mastication are different from those induced by insertion when occlusal loads are applied , the mucosa is displaced under the denture base resulting in denture rotation around the attachments . simulated mastication reduced the locator retention to 40% of the baseline values with a non - linear descending curve . kleis declared that after 12 months of overdentures delivery , the male parts of the locator have to be changed , as 75.5% loss of retention has been noticed . however , the reduction in peak load - to - dislodgement for these attachments is more apparent in case of non - parallel implants . to overcome the inevitable continuous resorption of the underlying residual ridge , frequent rebasing of implant - retained overdentures ( ovds ) may restore the proper occlusion and reduce possible rotation of the denture around the retentive components . most of the separated attachments need more prosthetic maintenance ; but for others , the frequency of technical complications / repairs per patient was higher around bar than ball attachments , with an increased failure rate for the cantilevered extended bars . recent studies sought to compare the incidence of mechanical complications of the locator attachments with that of the commonly used overdenture attachment systems . observed less prosthodontic complications and maintenance of the oral function for the locator system than for ball and bar attachments . argued that the creep response of the matrices and the hardness of the patrices provoked extensive deformation and deterioration of the locator nylon parts , with a higher substantial need for maintenance , compared to ball attachments . differences in the dimensions or material composition , and a large variation in retentive forces have been found between different batches of the same attachment system due to the poor manufacturing quality control . in - depth studies following standardized criteria to compare different options for mandibular implant overdentures ( iovd ) treatment remain scarce . the accuracy of parameters applied to the model , including geometry , constraints , and mechanical properties , determines the value of the finite element analysis experiment . scientific evidence related to the material 's clinical performance , objective oral function , and patients appreciation of the treatment should guide the clinician in making the ultimate choice of a specified attachment . in the author 's opinion and based on the presented data , the locator attachment system is an easy - to - use connector , with less post - insertion complications .

single - walled carbon nanotubes ( swcnts ) dispersed in aqueous media hold great promise for exciting applications in chemistry , biology , and nanomedicine . central to these applications is the stability of these nanoscale dispersions in aqueous media . various reagents or perturbations have been applied to control the aggregation status of swcnts , such as solution ph , light , oxidation and reduction of dispersants , temperature , and addition of salt . however , the molecular mechanisms that underlie swcnt aggregation have not been explored systematically , and there have been inconsistent views on this very important issue . the fundamental question that we concern is what the molecular interactions that drive this aggregation process are in aqueous medium . although the surface of swcnts is hydrophobic in nature , which can in principle promote their self - association and aggregation through hydrophobic and van der waals interactions , this is not true for swcnts that have been dispersed into aqueous media assisted by various charged dispersants . for these dispersed swcnts , the charge status of these dispersant molecules on swcnt surface will have profound impact on the physical properties of swcnts in water . as demonstrated by wang and chen , swcnts dispersed by poly - l - lysine can undergo reversible aggregation that is dependent on the solution ph . when solution ph is close to the isoelectric point of lysine , dispersed swcnts become aggregated , suggesting that electrostatic interactions may play a significant role in this aggregation and redispersion process . however , how electrostatic interactions may tune the interactions among individual swcnts remains largely unexplored . in several aspects , individual swcnts coated with charged dispersants on their surface share similarity with polyelectrolytes such as double - stranded dna in solution . the sp2 carbons of swcnts form the hydrophobic backbones , and resemble the aromatic base groups located at the center of double - helical dna . the charged dispersant molecules are coated on this hydrophobic surface , very similar to the phosphate groups that lie on the exterior of double - stranded dna . therefore , mechanisms that operate in dna condensation might work similarly in dispersed swcnts . for swcnts dispersed with charged molecules , we hypothesize that their aggregation in aqueous medium is mediated by electrostatic instead of van der waals interactions , and modulation of these electrostatic interactions can lead to reversion of swcnt aggregates . herein we test this hypothesis by systematically investigating swcnt aggregation in aqueous medium mediated by a variety of dispersant molecules . we measure the surface charge status of swcnts during this aggregation process through measurement of the zeta potential , and correlate the extent of swcnt aggregation with zeta potential measurement . these results allow us to clarify the mechanisms of swcnt aggregation in aqueous medium . by further exploring these mechanisms , we demonstrate various strategies that can be used to reverse the aggregation of swcnts in solution . these mechanisms that we identified also apply to typical cell culture conditions and are thus relevant to the potential biological applications of swcnts in vivo . one milligram of arc - discharge ( ad ) swcnts ( helix materials solution , tx ) were dispersed in 1 ml of distilled and deionized water ( ddh2o , synergy uv , millipore corporation , billerica , ma ) with 1 h sonication in the presence of various dispersing reagents in a tip sonicator ( ultrasonic processor s-4000 , misonix , farmingdale , ny ) as described previously to obtain singly dispersed tubes . this condition does not introduce defects to swcnt sidewalls , as indicated by the intensity ratio between d and g bands from raman spectrum measurements . the concentrations of dispersing reagents are 1 mg / ml for dna oligo ( dt)30 , fluorescein isothiocyanate ( fitc ) , rhodamine b ( rb ) , crystal violet ( cv ) and poly - l - lysine ( pll ) , and 40 mg / ml for pluronic f 108 . ice was constantly added to the bath to prevent heating of the sample during sonication . p3-swnts ( swcnt cooh , carbon solutions , riverside , ca ) were dispersed in ddh2o with the same procedure described above . we adjusted concentration of dispersed swcnts in aqueous medium as 0.02 g/l for consistency throughout the experiments . this concentration was determined based on the absorbance at 1023 nm , using an extinction coefficient of 11.9 ( mg / ml)cm for ad swcnts that we estimated previously . all other reagents were purchased from sigma - aldrich ( st . louis , mo ) unless specified otherwise . seven hundred microliters of individually dispersed swcnts was agitated in an orbital shaker ( excella e-24r incubator shakers , new brunswick scientific co , edison , nj ) in the presence of different concentrations of various electrolytes at 20 c for 30 min at 200 rpm . after agitation , 600 l samples were used immediately for measurement of zeta potential in zetasizer zs90 ( malvern , uk ) . the rest of the samples were centrifuged for 30 min at 17 000 g , and supernatants were collected for ultraviolet visible near - infrared ( uv vis nir ) absorbance measurement ( shimadzu uv-1800 , kyoto , japan ) to determine the fraction of swcnts that remained in solution relative to the original quantity of dispersed swcnts . aggregated swcnts were not redispersed by themselves for greater than 2 weeks . throughout this paper , aggregation and zeta potential profiles of ad swcnts all the starting materials for aggregation experiments are supernatants of swcnts after centrifugation to remove nondispersed swcnts . for ethylenediaminetetraacetic acid ( edta)- , dithiothreitol ( dtt)- , 2-mercaptoethanol- , or nacl - mediated redispersion of various swcnt aggregates , we first induced aggregation of swcnts with various electrolytes at designated concentrations by agitation at a speed of 200 rpm for 30 min at room temperature ( 22 c ) . we then titrated in edta , dtt , 2-mercaptoethanol , or nacl , and agitated the sample at 20 c for 30 min at 200 rpm . after agitation , 600 l samples were used immediately for measurement of zeta potential . the rest of the samples were centrifuged for 30 min at 17 000 g , and supernatants were collected for uv vis nir absorbance measurement to determine the fraction of swcnts that were redispersed back to solution relative to the original quantity of dispersed swcnts . to determine the visible - nir absorbance spectra before and after inducing aggregation and redispersion , ad swcnts , chemical vapor deposition ( cvd ) swcnts ( ses research , richardson , tx ) and high pressure carbon monoxide ( hipco ) swcnts ( super purified grade , unidym , sunnyvale , ca ) were dispersed in the presence of ( dt)30 and agitated for 30 min with 5 mm of cacl2 in order to induce aggregation . swcnt samples were then agitated in the presence of 20 mm edta for redispersion . for side - by - side comparison , each untreated ad swcnt/(dt)30 , cvd swcnt/(dt)30 , and hipco swcnt/(dt)30 sample also went through the same procedures as described above , except that ddh2o was used in lieu of either cacl2 or edta . nir absorbance spectra were recorded and plotted in parallel for side - by - side comparison . we first induced aggregation of negatively charged swcnts with 1 mm ka8k and positively charged swcnts with 5 mm ea8e ( custom synthesized polypeptides , 95% purity , pierce protein , il . ) . we then titrated in either protease ( 0.1% of trypsin and 1 mg / ml proteinase k ) , and agitated the sample at 20 c for 30 min at 200 rpm . after agitation , the samples were centrifuged for 30 min at 17 000 g , and supernatants were collected for uv vis nir absorbance measurement to determine the fraction of swcnts that were redispersed back to solution relative to the original quantity of dispersed swcnts . for inhibition of protease activity as a control experiment , swcnt aggregates induced by polypeptides ka8k or ea8e were treated with either 0.2 mg / ml trypsin inhibitor ( from soybean ) or 10 mm pmsf , respectively . trypsin and proteinase k were then added to swcnt aggregates to monitor the redispersion of swcnts as described above . one microgram of dispersed swcnts was agitated with various fractions of complete cell culture media { 90% atcc dulbecco s modified eagle medium ( dmem ) + 10% fetal bovine serum ( fbs ) } in a total volume of 100 l . after agitation at 200 rpm for various durations at various temperatures as indicated , samples were centrifuged at 17 000 g for 30 min ( legend pro 17 , thermo fisher scientific , ma ) . supernatants were collected after centrifugation , and the fraction of individual swcnts that remained in solution was measured with uv vis previous studies on swcnts dispersed in h2o using sodium dodecyl sulfate ( sds ) showed that swcnts could undergo selective aggregation upon addition of salt . this phenomenon has been interpreted as aggregation mediated by van der waals attractions . when the concentration of counterions was increased , the surface of individually dispersed swcnts could be completely neutralized , at which point the adjacent swcnts would come into contact due to van der waals attractions . this leads to aggregation of swcnts upon addition of salt . to further explore this phenomenon in similar systems , we dispersed swcnts in h2o using dna oligos ( dt)30 , which carried negative charges under current experimental conditions . we then titrated swcnt/(dt)30 with various electrolytes , and monitored the surface charge status of swcnts by measuring the zeta potential of the swcnts after addition of the electrolytes at various concentrations . we then monitored the concentration of individually - dispersed swcnt/(dt)30 left in the aqueous medium after sedimentation . as shown in figure 1a , top panel , the fraction of dispersed swcnts that remained in solution did not change significantly until the concentration of the added electrolyte reached a certain threshold , which resulted in very sharp transitions in these curves as we progressively increased the concentrations of the electrolytes . this threshold behavior depends on the type of the electrolytes added , so that the higher the valence for the counterion , the lower the threshold concentration , which follows the order of fe < mg ca < na . in contrast to this threshold behavior , the zeta potential of the swcnts displayed a gradual instead of a sharp transition for all cases ( figure 1a , bottom panel ) . upon addition of various electrolytes , the surface charge of swcnts diminished monotonically , suggesting either the binding of counterions to swcnts that lead to charge neutralization or perhaps , dissociation of ( dt)30 from swcnt surface . notably , this charge decrease occurred gradually and never reached 100% even when all the swcnts in solution were aggregated . comparison between figure 1a top and bottom panels suggests that , upon addition of electrolytes , aggregation of the swcnts did not occur until the overall surface charge was reduced below a certain threshold . similar results were also observed for swcnts dispersed by fitc , another negatively charged dispersant for swcnts under these conditions that we described recently ( figure 1b ) . fraction of individual ( a ) swcnt/(dt)30 , ( b ) swcnt / fitc , ( c ) swcnt cooh , and ( d ) swcnt / pluronic that remained in solution upon titration with various electrolytes ( upper panel ) and the zeta potential changes on swcnts associated with this process ( lower panel ) . error bars represent standard deviation from three independent repeats of the same experiments . throughout this paper , aggregation and zeta potential profiles of ad swcnts the explanation offered for the aggregation of swcnts dispersed by sds upon salt addition is that salt reduces the solubility of sds in water , and the loss of sds from swcnt surface leads to charge depletion and aggregation . to test this type of mechanism , we repeated the above experiments using swcnts that carried covalent carboxylic functional moieties ( swcnt cooh ) . the presence of cooh groups renders these swcnts readily dispersed in h2o without addition of any external dispersant molecules . as shown in figure 1c , the fraction of dispersed swcnts that remained in solution decreased sharply when the concentration of the added electrolyte reached a certain threshold . cooh displayed a gradual instead of a sharp transition for all cases ( figure 1c , bottom panel ) . this apparent charge decrease never reached 100% even when all the swcnts in solution were aggregated . because cooh groups were covalently attached to swcnts , this result indicates that it is the binding of counterions to swcnt surfaces that partially neutralizes surface charge , which leads to aggregation of swcnts when the surface charge is neutralized beyond a threshold . this threshold pattern follows the same order of fe < mg ca < na , as in figure 1a , b , although the threshold concentrations of the electrolytes were different . for swcnt cooh , only 0.01 mm fecl3 is needed to aggregate more than 90% of swcnts , in contrast to 0.5 mm fecl3 that is needed to aggregate more than 90% of swcnts dispersed by ( dt)30 . this difference is consistent with a higher surface charge density for swcnt cooh than swcnt/(dt)30 , as revealed by the zeta potential measurement for these swcnts before addition of counterions ( figure 1c bottom panel ) . the higher surface charge may afford a tighter binding for the same counterions on swcnt surface , which leads to the difference in threshold concentrations . the above results suggest that it is the binding of counterions to swcnts instead of dissociation of charged dispersant molecules that triggers aggregation when the surface charge of swcnt is neutralized beyond a threshold . to further test this hypothesis , we dispersed swcnts using uncharged molecules pluronic f 108 , and titrated the dispersed swcnts with various concentrations of nacl , mgcl2 , cacl2 , or fecl3 . as shown in figure 1d , none of these reagents induced aggregation of swcnts throughout the concentrations investigated , except fecl3 , which induced 10% aggregation at 1 mm fecl3 but this aggregation was less than < 20% even at 1 m fecl3 tested . consistent with these observations , the surface charge of swcnts did not change over the range of salt concentrations investigated , indicating no binding of ions to swcnt surfaces . because pluronic f 108 does not carry any charges , these results reinforce the idea that the aggregation phenomenon we observed in figure 1a c is due to electrostatic interactions between swcnts and counterions in solution . these results support the idea that direct binding of counterions to swcnt due to electrostatic interactions triggers aggregation when the surface charge is neutralized beyond a threshold . if the above hypothesis is true , it should also be applicable to swcnts that carry positive instead of negative charges , and negatively charged counterions can bind swcnt surface that eventually leads to swcnt aggregation . to test this directly , we dispersed swcnts using positively - charged molecules that we described recently . our prediction is that upon titration of negatively - charged counterions , these swcnts will undergo aggregation mediated by counterion binding . as expected , for swcnts dispersed by rb , cv or pll , titration of nacl , na2so4 , na2co3 , and na3po4 induced aggregation of swcnts . as shown in figure 2 , the fraction of dispersed swcnts that remained in solution did not change significantly until the concentration of the added electrolyte reached a certain threshold , which resulted in sharp transitions in these curves as we increased the concentrations of the electrolytes . this threshold behavior depends on the type of the electrolytes added , which roughly follows the order of po4 < co3 < so4 < cl . in contrast to this threshold behavior , the zeta potential of the swcnts displayed a gradual instead of sharp decrease for all cases ( figure 2 , bottom panels ) , suggesting the direct binding of counterions to swcnt surface . comparison between figure 2 top and bottom panels further indicates that aggregation of the swcnts does not occur until the surface charge is neutralized beyond a certain threshold . moreover , this charge neutralization never reached 100% , even when all the swcnts in solution were aggregated . all these observations were consistent with our expectations and thus further support our hypothesis that counterion binding induces partial charge neutralization on swcnt surface , which leads to swcnt aggregation . aggregation of ( a ) swcnt / rb ( b ) swcnt / cv , and ( c ) swcnt / pll upon titration with various electrolytes and the zeta potential changes associated with the process . to quantitatively examine the dependence of swcnt aggregation on their surface charge status , we plotted the fraction of swcnts that remained in solution as a function of the measured zeta potential on swcnt surfaces for each charged dispersant molecule that we have investigated ( figure 3 ) . within each panel , the responses from the addition of various electrolytes were plotted for the same dispersant molecules . notably , these plots all clustered closely to each other despite the differences in the counterions added . this result indicates that regardless of the type of counterions , swcnts undergo aggregation when their surface charge is neutralized beyond a threshold . the apparent plateaus in these plots permit quantitation of the zeta potential at which swcnts are fully aggregated . as shown in supporting information table s1 , for various charged dispersant molecules or groups , swcnts are fully aggregated when the surface charge was neutralized to an average of 80% , varying from 74 to 86% . this result is similar to dna condensation induced by multivalent counterions , which occurs when 8990% of the dna phosphate charges were neutralized by condensed counterions . this apparent attraction instead of repulsion between dna molecules of the same charge is due to electrostatic correlation between screening counterions instead of van der waals attractions . thus , our results reveal the similarity between dna condensation and swcnts aggregation , and swcnts dispersed by various charged dispersant molecules simply behave as polyelectrolytes . this property was conferred by the charges on the dispersant molecules ( figure s1 ) . the essential feature in this model is that the aggregation of swcnts dispersed by these charged dispersant is driven by electrostatic interactions , instead of van der waals or hydrophobic interactions among swcnts . electrostatic attractions between individual swcnts of the same charge can develop as a result of correlation between screening counterions . these electrostatic interactions as mediated by the charged dispersant and counterions lead to swcnt aggregation in solution . fraction of individually dispersed ( a ) swcnt/(dt)30 , ( b ) swcnt / fitc , ( c ) swcnt cooh , ( d ) swcnt / rb , ( e ) swcnt / cv , and ( f ) swcnt / pll that remained in solution as a function of zeta potential on the swcnt surface . the above model of swcnts aggregation indicates that interaction of counterions with dispersant groups on swcnt surfaces eventually leads to swcnt aggregation . the model implies that removal of bound counterions from aggregated swcnts surface may lead to redispersion of swcnts . to test this hypothesis , we induced aggregation of swcnts dispersed with negatively charged dispersant molecules ( ( dt)30 and fitc ) through addition of mg , ca , or fe , and then titrated in edta that can chelate these metal ions . as shown in figure 4a , b , we plotted the fraction of swcnts that remained in solution as a function of added edta expressed as the ratio between [ edta ] and the concentration of corresponding metal ions . all the aggregates can be redispersed into solution upon addition of edta above certain threshold , consistent with the threshold phenomenon observed in aggregation experiments and suggest that this process might be fully reversible under these conditions . a camera shot for this process is shown in figure 4c , where the middle test tube showed the formation of swcnt aggregates , which instantaneously disappeared upon addition of edta above a threshold level . examination of the uv vis nir absorbance spectra of the dispersed hipco swcnts before aggregation and after redispersion revealed only small changes in peak position and very similar features for ad and cvd swcnts we have compared . the peak features of these spectra closely resemble those we published previously , suggesting that the above aggregation and redispersion process are reversible ( figure 4d ) . we confirmed that our dispersion procedure did not induce oxidation of swcnts through raman spectra measurement . thus , the e11 peaks displayed for hipco ( blue spectra ) may be related to the quality of the swcnts from the manufacturer . control experiments using aggregates of swcnt / rb or swcnt / cv induced by addition of cacl2 ( aggregation due to cl binding to positively charged swcnt surface ) showed that addition of edta did not induce any redispersion of these aggregates , consistent with our expectation that only chelation of bound counterions on the surface of swcnts resulted in the dispersion of swcnts aggregates ( figure s2 ) . we note that the threshold concentration of edta required to fully disperse the aggregates vary with the type of metal ions and dispersant molecules . for example , for swcnt/(dt)30 , almost stoichiometric amount of edta is sufficient to redisperse all the aggregates induced by addition of ca or mg , but almost 10-fold higher concentration is needed to redisperse the aggregates induced by addition of fe . these concentrations are not consistent with the binding constants for edta with these metal ions . rather , it suggests that the potential packing structures of these aggregates and the resulting accessibility of metal ions in these aggregates may partially determine the concentration of edta needed to redisperse them . consistent with this view , the aggregates of swcnt / fitc in general require a higher concentration of edta to redisperse than swcnt/(dt)30 ( figure 4b ) , even for the aggregates induced by the same metal ions . this result suggests that the overall structure of swcnt / fitc aggregates may be more compact than that of swcnt/(dt)30 . these experiments , altogether , further support our model that the aggregation is due to binding of counterions to swcnt surface , and sequestration of the bound counterions can lead to the complete redispersion of swcnts in solution . previous study has revealed that swcnts dispersed by pll can be aggregated and redispersed depending on the ph , which is resulted from the change in the charge status of pll in response to ph . although ph is a different trigger for swcnts aggregation and redispersion compared to counterions in current studies , both employ similar mechanisms of electrostatic interactions to control the aggregation status of swcnts . edta - mediated redispersion of aggregated ( a ) swcnt/(dt)30 and ( b ) swcnt / fitc . the concentrations of metal ions used to induce aggregation are 0.5 mm fecl3 , 5 mm cacl2 , and 5 mm mgcl2 for swcnt/(dt)30 and 1 mm fecl3 , 5 mm cacl2 , and 5 mm mgcl2 for swcnt / fitc . ( d ) visible nir absorbance spectra of various dispersed swcnts before and after aggregation and redispersion . in all the above studies , the counterions we studied were monomeric in their chemical structures , i.e. , all the charges were carried in a single functional group within the molecule . under physiological conditions , electrolytes that carry multiple charged groups in a single molecule exist , such as polyamines . to examine whether these polyamines can induce aggregation of swcnts through similar mechanisms as we demonstrated above , we prepared either swcnt/(dt)30 or swcnt cooh dispersion , and then tested spermidine , spermine , together with 1,6-diaminohexane ( dh ) and cystamine dihydrochloride ( cd ) for their effects on dispersed swcnts . as shown in figure 5a for swcnt/(dt)30 , as we titrated spermidine or spermine , swcnts underwent aggregation as we expected ; however , as we further increase the concentration of these polyamines above 1 mm , the aggregates transiently disappeared . further addition of polyamines led to aggregation again , so that at 1 m polyamine , almost all swcnts were aggregated . this phenomenon was highly reproducible for swcnt/(dt)30 ( figure 5a ) or swcnt cooh ( figure 5b ) . notably , the zeta potential we measured for swcnts also varied with the concentration of polyamine in phase : surface charge was neutralized to about 80% when swcnts were fully aggregated ; the zeta potential recovered to original values when aggregates became redispersed . control experiments using swcnts dispersed by pluronic f 108 did not yield any aggregation or redispersion ( figure 5c ) , suggesting once again that this complex phenomenon of aggregation , redispersion , and reaggregation was caused by polyamine binding , dissociation , and rebinding . spermine and spermidine concentration - dependent aggregation and redispersion of ( a ) swcnt/(dt)30 , ( b ) swcnt cooh , and ( c ) swcnt / pluronic together with the zeta potential changes associated with the process . in contrast to these observations in figure 5a , b , experiments using either dh or cd produced simpler results . as shown in figure 6a , b , swcnts underwent aggregation as we titrated either dh or cd into swcnt dispersion . almost all swcnts were aggregated at 100 mm dh or cd , and no transient redispersion was observed throughout this process . control experiments using swcnt / pluronic showed no aggregation throughout the concentrations of dh or cd tested ( figure 6c ) . because dh and spermidine only differ slightly in their chemical structures : spermidine has one extra ch2 and one extra nh group that carried one more positive charge than dh under current solution conditions ( chart s1 ) , we reasoned that the above process of aggregation started with the direct binding of the positively charged amino groups at the ends of these molecules to swcnt surfaces . this is possible because these positive charges serve as counterions for the negative charges on the surface of swcnts , either due to the dna phosphate backbone or cooh group that was covalently attached to the swcnt surface . however , more importantly , these molecules can induce swcnt aggregation through bridges mediated by the charged groups at both ends of these chain molecules . this bridging effect is favored by entropy , as these chain molecules have more degrees of freedom upon bridging between two individual swcnts , in contrast to the binding of both ends of the molecule to the same tube . for both spermidine and spermine but not dh or cd , due to the presence of additional positive charges , the increasing concentration of polyamines in solution increases the ionic strength of the solution , so that these entropic bridge interactions are weakened and collapsed ( as shown in figure s3 ) , consistent with the recovery of surface charge monitored by zeta potential ( figure 5a , b bottom panel ) . this collapse does not occur to either dh or cd due to their lower charge status as compared to either spermidine or spermine . cystamine dihydrochloride ( cd ) and diaminohexane ( dh ) mediated aggregation of ( a ) swcnt/(dt)30 , ( b ) swcnt cooh , and ( c ) swcnt / pluronic . error bars represent standard deviation from three independent repeats of the same experiments . to directly test this model of swcnt aggregation induced by addition of polyelectrolytes , we first focused on the bridging effect . we used cd and dh to induce aggregation of either swcnt/(dt)30 or swcnt cooh . if the bridging effect is responsible for swcnt aggregation , we would expect a redispersion of swcnt aggregates formed with cd upon addition of either dtt or 2-mercaptoethanol , because both reducing agents can reduce cd and thus break the molecule into two separate parts . in contrast , aggregates formed with dh should remain intact because dh contains no disulfide bonds that can be reduced . as we expected , more than 80% of the aggregates formed with cd could be redispersed upon addition of either dtt or 2-mercaptoethanol . this was true for both swcnt/(dt)30 and swcnt cooh ( figure 7 ) . in contrast , the aggregates formed with dh remain intact throughout the concentrations of both reducing agents used ( figure s4 ) . these results directly support our hypothesis that the initial swcnt aggregates formed in the presence of these polyelectrolytes are mediated by bridging interactions . disulfide bond reducing agents , dtt or 2-mecaptoethanol ( bme ) , mediated redispersion of ( a ) swcnt/(dt)30 , ( b ) swcnt cooh , and ( c ) swcnt / fitc aggregates induced by addition of cd . the corresponding zeta potential changes associated with the process were shown in bottom panels . we then focused on the transient redispersion of swcnt aggregates upon addition of medium concentrations of spermidine or spermine , which was absent for either cd or dh . to directly examine the transient nature of this redispersion we then titrated the mixture with increasing concentrations of nacl ( [ nacl ] ) . interestingly , increasing [ nacl ] first reduced aggregation . at 200 mm nacl , these observations were very similar to the redispersion and aggregation of swcnts induced by either spermidine or spermine as shown in figure 5 and suggests that the transient redispersion is due to increased ionic strength in solution that weakened the entropic bridge interactions . the zeta potential first decreased upon addition of nacl , indicating dissociation of bound polyamine molecules . this was then followed by a steady increase in zeta potential , which resulted from the reassociation of counterions to swcnts at high concentrations and induced reaggregation of swcnts . this bridging effect as we observed for polyelectrolytes suggests a possible application of these molecules to induce reversible aggregation and redispersion of swcnts that can be controlled through chain breaking . to test this possibility , we synthesized oligopeptides that are flanked by either two positive lysine residues or two negative glutamic acid residues on the two ends of the peptides ( chart s1 ) . as expected , the positively charged peptide ka8k can induce aggregation of either swcnt/(dt)30 or swcnt cooh , while the negatively charged peptide ea8e can induce aggregation of either swcnt / cv or swcnt / pll , as shown in figure 9 together with the zeta potential measurement results for this titration process . under conditions where we induced full aggregation of swcnts , we treated the swcnt aggregates with either trypsin that can cleave the ka8k peptide or protease k that can cleave the ea8e peptide . as shown in figure 10 , greater than 50% of swcnt aggregates could be redispersed upon addition of these protease enzymes for swcnts initially dispersed with various molecules . this redispersion was due to protease action because inclusion of either trypsin inhibitor or pmsf ( which inhibits protease k ) completely blocked the redispersion of swcnt aggregates ( figure 10 ) . fraction of individual ( a ) negatively charged swcnts and ( b ) positively charged swcnts that remained in solution after addition of polypeptides ( ka8k for negatively charged and ea8e for positively charged swcnts ) . enzyme mediated redispersion of ( a ) swcnt/(dt)30 and swcnt cooh aggregated by polypeptide ( ka8k ) and ( b ) swcnt / pll and swcnt / cv aggregated by polypeptide ( ea8e ) and the inhibition of this redispersion by enzyme inhibitors . the above results on swcnt aggregation , as we observed for both monomeric electrolyte and polyelectrolyte molecules , bear direct relevance for the potential applications of swcnts as delivery vehicles for genes into the cells . when swcnts are conjugated with nucleic acids , they can undergo aggregation and redispersion as a result of interactions with their counterions . notably , the concentrations of counterions that are required to induce aggregation of swcnts under current experimental conditions are close to the concentrations of these ions in vivo . thus , swcnts conjugated with dna or other charged molecules may well undergo aggregation in vivo that could lead to toxicity in cells and tissues . to test this aggregation , as expected , majorities of swcnts precipitated out of the solution with 50% culture media in the solution ( figure s5 ) . in contrast , controls using swcnts dispersed with pluronic f 108 underwent little aggregation even when the solution contains 80% of culture media . thus , our results suggest that the application of swcnts in tissue culture experiments has to consider the potential aggregation due to interactions with counterions . here we have investigated the properties of aggregation and redispersion of swcnts in aqueous medium . for swcnts that are dispersed into aqueous medium assisted by charged molecules , addition of electrolytes can induce their aggregation that is reversible under certain conditions . neutralization of the swcnt surface charge by 74 to 86% leads to aggregation of swcnts . this aggregation is driven by electrostatic attractions instead of van der waals or hydrophobic interactions due to correlations between screening counterions , similar to the mechanisms of dna condensation induced by multivalent cations . polyelectrolyte can induce swcnt aggregation through molecular bridging , which can be utilized to engineer the aggregation and redipersion of swcnts in solution by exploiting various chain breaking mechanisms . our data suggest that swcnts can be aggregated during in vitro as well as in vivo applications as gene delivery vehicles , which may lead to toxicity of these nanomaterials in vivo . our method of redispersing aggregated swcnts could be potentially used to control the aggregation status of swcnts within biological systems . the mechanisms that we identified for swcnt aggregation have broad implications on various applications of swcnts in water .

single - walled carbon nanotubes ( swcnts ) dispersed in aqueous medium have many potential applications in chemistry , biology , and medicine . reversible aggregation of swcnts dispersed in water has been frequently reported , but the mechanisms behind are not well understood . here we show that swcnts dispersed into aqueous medium assisted by various charged molecules can be reversibly aggregated by a variety of electrolytes with two distinct mechanisms . direct binding of counterions to swcnts leads to aggregation when the surface charge is neutralized from 74 to 86% . this aggregation is driven by electrostatic instead of van der waals interactions , thus showing similarity to that of dna condensation induced by multivalent cations . sequestration of counterions by chelating reagents leads to the redispersion of swcnt aggregates . in contrast to various metal ions , polyelectrolytes have the unique ability to induce swcnt aggregation by bridging between individual swcnts . aggregation through the latter mechanism can be engineered to be reversible by exploiting various mechanisms of chain breaking , including reduction of disulfide bond in the polymer chain , and the cleavage action of proteolytic enzymes . these findings clarify the mechanisms of swcnt aggregation , and have broad implications in various applications of swcnts in water .

Introduction Experimental Section Results and Discussion Conclusions

single - walled carbon nanotubes ( swcnts ) dispersed in aqueous media hold great promise for exciting applications in chemistry , biology , and nanomedicine . various reagents or perturbations have been applied to control the aggregation status of swcnts , such as solution ph , light , oxidation and reduction of dispersants , temperature , and addition of salt . the fundamental question that we concern is what the molecular interactions that drive this aggregation process are in aqueous medium . although the surface of swcnts is hydrophobic in nature , which can in principle promote their self - association and aggregation through hydrophobic and van der waals interactions , this is not true for swcnts that have been dispersed into aqueous media assisted by various charged dispersants . for swcnts dispersed with charged molecules , we hypothesize that their aggregation in aqueous medium is mediated by electrostatic instead of van der waals interactions , and modulation of these electrostatic interactions can lead to reversion of swcnt aggregates . herein we test this hypothesis by systematically investigating swcnt aggregation in aqueous medium mediated by a variety of dispersant molecules . we measure the surface charge status of swcnts during this aggregation process through measurement of the zeta potential , and correlate the extent of swcnt aggregation with zeta potential measurement . these results allow us to clarify the mechanisms of swcnt aggregation in aqueous medium . by further exploring these mechanisms , we demonstrate various strategies that can be used to reverse the aggregation of swcnts in solution . these mechanisms that we identified also apply to typical cell culture conditions and are thus relevant to the potential biological applications of swcnts in vivo . we adjusted concentration of dispersed swcnts in aqueous medium as 0.02 g/l for consistency throughout the experiments . the rest of the samples were centrifuged for 30 min at 17 000 g , and supernatants were collected for ultraviolet visible near - infrared ( uv vis nir ) absorbance measurement ( shimadzu uv-1800 , kyoto , japan ) to determine the fraction of swcnts that remained in solution relative to the original quantity of dispersed swcnts . for ethylenediaminetetraacetic acid ( edta)- , dithiothreitol ( dtt)- , 2-mercaptoethanol- , or nacl - mediated redispersion of various swcnt aggregates , we first induced aggregation of swcnts with various electrolytes at designated concentrations by agitation at a speed of 200 rpm for 30 min at room temperature ( 22 c ) . the rest of the samples were centrifuged for 30 min at 17 000 g , and supernatants were collected for uv vis nir absorbance measurement to determine the fraction of swcnts that were redispersed back to solution relative to the original quantity of dispersed swcnts . to determine the visible - nir absorbance spectra before and after inducing aggregation and redispersion , ad swcnts , chemical vapor deposition ( cvd ) swcnts ( ses research , richardson , tx ) and high pressure carbon monoxide ( hipco ) swcnts ( super purified grade , unidym , sunnyvale , ca ) were dispersed in the presence of ( dt)30 and agitated for 30 min with 5 mm of cacl2 in order to induce aggregation . after agitation , the samples were centrifuged for 30 min at 17 000 g , and supernatants were collected for uv vis nir absorbance measurement to determine the fraction of swcnts that were redispersed back to solution relative to the original quantity of dispersed swcnts . trypsin and proteinase k were then added to swcnt aggregates to monitor the redispersion of swcnts as described above . supernatants were collected after centrifugation , and the fraction of individual swcnts that remained in solution was measured with uv vis previous studies on swcnts dispersed in h2o using sodium dodecyl sulfate ( sds ) showed that swcnts could undergo selective aggregation upon addition of salt . this phenomenon has been interpreted as aggregation mediated by van der waals attractions . when the concentration of counterions was increased , the surface of individually dispersed swcnts could be completely neutralized , at which point the adjacent swcnts would come into contact due to van der waals attractions . this leads to aggregation of swcnts upon addition of salt . we then titrated swcnt/(dt)30 with various electrolytes , and monitored the surface charge status of swcnts by measuring the zeta potential of the swcnts after addition of the electrolytes at various concentrations . in contrast to this threshold behavior , the zeta potential of the swcnts displayed a gradual instead of a sharp transition for all cases ( figure 1a , bottom panel ) . upon addition of various electrolytes , the surface charge of swcnts diminished monotonically , suggesting either the binding of counterions to swcnts that lead to charge neutralization or perhaps , dissociation of ( dt)30 from swcnt surface . comparison between figure 1a top and bottom panels suggests that , upon addition of electrolytes , aggregation of the swcnts did not occur until the overall surface charge was reduced below a certain threshold . throughout this paper , aggregation and zeta potential profiles of ad swcnts the explanation offered for the aggregation of swcnts dispersed by sds upon salt addition is that salt reduces the solubility of sds in water , and the loss of sds from swcnt surface leads to charge depletion and aggregation . because cooh groups were covalently attached to swcnts , this result indicates that it is the binding of counterions to swcnt surfaces that partially neutralizes surface charge , which leads to aggregation of swcnts when the surface charge is neutralized beyond a threshold . for swcnt cooh , only 0.01 mm fecl3 is needed to aggregate more than 90% of swcnts , in contrast to 0.5 mm fecl3 that is needed to aggregate more than 90% of swcnts dispersed by ( dt)30 . the higher surface charge may afford a tighter binding for the same counterions on swcnt surface , which leads to the difference in threshold concentrations . the above results suggest that it is the binding of counterions to swcnts instead of dissociation of charged dispersant molecules that triggers aggregation when the surface charge of swcnt is neutralized beyond a threshold . as shown in figure 1d , none of these reagents induced aggregation of swcnts throughout the concentrations investigated , except fecl3 , which induced 10% aggregation at 1 mm fecl3 but this aggregation was less than < 20% even at 1 m fecl3 tested . consistent with these observations , the surface charge of swcnts did not change over the range of salt concentrations investigated , indicating no binding of ions to swcnt surfaces . these results support the idea that direct binding of counterions to swcnt due to electrostatic interactions triggers aggregation when the surface charge is neutralized beyond a threshold . if the above hypothesis is true , it should also be applicable to swcnts that carry positive instead of negative charges , and negatively charged counterions can bind swcnt surface that eventually leads to swcnt aggregation . as expected , for swcnts dispersed by rb , cv or pll , titration of nacl , na2so4 , na2co3 , and na3po4 induced aggregation of swcnts . in contrast to this threshold behavior , the zeta potential of the swcnts displayed a gradual instead of sharp decrease for all cases ( figure 2 , bottom panels ) , suggesting the direct binding of counterions to swcnt surface . comparison between figure 2 top and bottom panels further indicates that aggregation of the swcnts does not occur until the surface charge is neutralized beyond a certain threshold . aggregation of ( a ) swcnt / rb ( b ) swcnt / cv , and ( c ) swcnt / pll upon titration with various electrolytes and the zeta potential changes associated with the process . to quantitatively examine the dependence of swcnt aggregation on their surface charge status , we plotted the fraction of swcnts that remained in solution as a function of the measured zeta potential on swcnt surfaces for each charged dispersant molecule that we have investigated ( figure 3 ) . this result indicates that regardless of the type of counterions , swcnts undergo aggregation when their surface charge is neutralized beyond a threshold . as shown in supporting information table s1 , for various charged dispersant molecules or groups , swcnts are fully aggregated when the surface charge was neutralized to an average of 80% , varying from 74 to 86% . this result is similar to dna condensation induced by multivalent counterions , which occurs when 8990% of the dna phosphate charges were neutralized by condensed counterions . this apparent attraction instead of repulsion between dna molecules of the same charge is due to electrostatic correlation between screening counterions instead of van der waals attractions . thus , our results reveal the similarity between dna condensation and swcnts aggregation , and swcnts dispersed by various charged dispersant molecules simply behave as polyelectrolytes . the essential feature in this model is that the aggregation of swcnts dispersed by these charged dispersant is driven by electrostatic interactions , instead of van der waals or hydrophobic interactions among swcnts . the above model of swcnts aggregation indicates that interaction of counterions with dispersant groups on swcnt surfaces eventually leads to swcnt aggregation . to test this hypothesis , we induced aggregation of swcnts dispersed with negatively charged dispersant molecules ( ( dt)30 and fitc ) through addition of mg , ca , or fe , and then titrated in edta that can chelate these metal ions . as shown in figure 4a , b , we plotted the fraction of swcnts that remained in solution as a function of added edta expressed as the ratio between [ edta ] and the concentration of corresponding metal ions . control experiments using aggregates of swcnt / rb or swcnt / cv induced by addition of cacl2 ( aggregation due to cl binding to positively charged swcnt surface ) showed that addition of edta did not induce any redispersion of these aggregates , consistent with our expectation that only chelation of bound counterions on the surface of swcnts resulted in the dispersion of swcnts aggregates ( figure s2 ) . consistent with this view , the aggregates of swcnt / fitc in general require a higher concentration of edta to redisperse than swcnt/(dt)30 ( figure 4b ) , even for the aggregates induced by the same metal ions . these experiments , altogether , further support our model that the aggregation is due to binding of counterions to swcnt surface , and sequestration of the bound counterions can lead to the complete redispersion of swcnts in solution . previous study has revealed that swcnts dispersed by pll can be aggregated and redispersed depending on the ph , which is resulted from the change in the charge status of pll in response to ph . although ph is a different trigger for swcnts aggregation and redispersion compared to counterions in current studies , both employ similar mechanisms of electrostatic interactions to control the aggregation status of swcnts . the concentrations of metal ions used to induce aggregation are 0.5 mm fecl3 , 5 mm cacl2 , and 5 mm mgcl2 for swcnt/(dt)30 and 1 mm fecl3 , 5 mm cacl2 , and 5 mm mgcl2 for swcnt / fitc . to examine whether these polyamines can induce aggregation of swcnts through similar mechanisms as we demonstrated above , we prepared either swcnt/(dt)30 or swcnt cooh dispersion , and then tested spermidine , spermine , together with 1,6-diaminohexane ( dh ) and cystamine dihydrochloride ( cd ) for their effects on dispersed swcnts . control experiments using swcnts dispersed by pluronic f 108 did not yield any aggregation or redispersion ( figure 5c ) , suggesting once again that this complex phenomenon of aggregation , redispersion , and reaggregation was caused by polyamine binding , dissociation , and rebinding . this is possible because these positive charges serve as counterions for the negative charges on the surface of swcnts , either due to the dna phosphate backbone or cooh group that was covalently attached to the swcnt surface . however , more importantly , these molecules can induce swcnt aggregation through bridges mediated by the charged groups at both ends of these chain molecules . this bridging effect is favored by entropy , as these chain molecules have more degrees of freedom upon bridging between two individual swcnts , in contrast to the binding of both ends of the molecule to the same tube . to directly test this model of swcnt aggregation induced by addition of polyelectrolytes , we first focused on the bridging effect . if the bridging effect is responsible for swcnt aggregation , we would expect a redispersion of swcnt aggregates formed with cd upon addition of either dtt or 2-mercaptoethanol , because both reducing agents can reduce cd and thus break the molecule into two separate parts . disulfide bond reducing agents , dtt or 2-mecaptoethanol ( bme ) , mediated redispersion of ( a ) swcnt/(dt)30 , ( b ) swcnt cooh , and ( c ) swcnt / fitc aggregates induced by addition of cd . we then focused on the transient redispersion of swcnt aggregates upon addition of medium concentrations of spermidine or spermine , which was absent for either cd or dh . at 200 mm nacl , these observations were very similar to the redispersion and aggregation of swcnts induced by either spermidine or spermine as shown in figure 5 and suggests that the transient redispersion is due to increased ionic strength in solution that weakened the entropic bridge interactions . this was then followed by a steady increase in zeta potential , which resulted from the reassociation of counterions to swcnts at high concentrations and induced reaggregation of swcnts . this bridging effect as we observed for polyelectrolytes suggests a possible application of these molecules to induce reversible aggregation and redispersion of swcnts that can be controlled through chain breaking . under conditions where we induced full aggregation of swcnts , we treated the swcnt aggregates with either trypsin that can cleave the ka8k peptide or protease k that can cleave the ea8e peptide . this redispersion was due to protease action because inclusion of either trypsin inhibitor or pmsf ( which inhibits protease k ) completely blocked the redispersion of swcnt aggregates ( figure 10 ) . enzyme mediated redispersion of ( a ) swcnt/(dt)30 and swcnt cooh aggregated by polypeptide ( ka8k ) and ( b ) swcnt / pll and swcnt / cv aggregated by polypeptide ( ea8e ) and the inhibition of this redispersion by enzyme inhibitors . the above results on swcnt aggregation , as we observed for both monomeric electrolyte and polyelectrolyte molecules , bear direct relevance for the potential applications of swcnts as delivery vehicles for genes into the cells . notably , the concentrations of counterions that are required to induce aggregation of swcnts under current experimental conditions are close to the concentrations of these ions in vivo . to test this aggregation , as expected , majorities of swcnts precipitated out of the solution with 50% culture media in the solution ( figure s5 ) . in contrast , controls using swcnts dispersed with pluronic f 108 underwent little aggregation even when the solution contains 80% of culture media . here we have investigated the properties of aggregation and redispersion of swcnts in aqueous medium . for swcnts that are dispersed into aqueous medium assisted by charged molecules , addition of electrolytes can induce their aggregation that is reversible under certain conditions . neutralization of the swcnt surface charge by 74 to 86% leads to aggregation of swcnts . this aggregation is driven by electrostatic attractions instead of van der waals or hydrophobic interactions due to correlations between screening counterions , similar to the mechanisms of dna condensation induced by multivalent cations . polyelectrolyte can induce swcnt aggregation through molecular bridging , which can be utilized to engineer the aggregation and redipersion of swcnts in solution by exploiting various chain breaking mechanisms . the mechanisms that we identified for swcnt aggregation have broad implications on various applications of swcnts in water .

although the surface of swcnts is hydrophobic in nature , which can in principle promote their self - association and aggregation through hydrophobic and van der waals interactions , this is not true for swcnts that have been dispersed into aqueous media assisted by various charged dispersants . for these dispersed swcnts , the charge status of these dispersant molecules on swcnt surface will have profound impact on the physical properties of swcnts in water . for swcnts dispersed with charged molecules , we hypothesize that their aggregation in aqueous medium is mediated by electrostatic instead of van der waals interactions , and modulation of these electrostatic interactions can lead to reversion of swcnt aggregates . herein we test this hypothesis by systematically investigating swcnt aggregation in aqueous medium mediated by a variety of dispersant molecules . we measure the surface charge status of swcnts during this aggregation process through measurement of the zeta potential , and correlate the extent of swcnt aggregation with zeta potential measurement . by further exploring these mechanisms , we demonstrate various strategies that can be used to reverse the aggregation of swcnts in solution . these mechanisms that we identified also apply to typical cell culture conditions and are thus relevant to the potential biological applications of swcnts in vivo . one milligram of arc - discharge ( ad ) swcnts ( helix materials solution , tx ) were dispersed in 1 ml of distilled and deionized water ( ddh2o , synergy uv , millipore corporation , billerica , ma ) with 1 h sonication in the presence of various dispersing reagents in a tip sonicator ( ultrasonic processor s-4000 , misonix , farmingdale , ny ) as described previously to obtain singly dispersed tubes . the concentrations of dispersing reagents are 1 mg / ml for dna oligo ( dt)30 , fluorescein isothiocyanate ( fitc ) , rhodamine b ( rb ) , crystal violet ( cv ) and poly - l - lysine ( pll ) , and 40 mg / ml for pluronic f 108 . seven hundred microliters of individually dispersed swcnts was agitated in an orbital shaker ( excella e-24r incubator shakers , new brunswick scientific co , edison , nj ) in the presence of different concentrations of various electrolytes at 20 c for 30 min at 200 rpm . the rest of the samples were centrifuged for 30 min at 17 000 g , and supernatants were collected for ultraviolet visible near - infrared ( uv vis nir ) absorbance measurement ( shimadzu uv-1800 , kyoto , japan ) to determine the fraction of swcnts that remained in solution relative to the original quantity of dispersed swcnts . throughout this paper , aggregation and zeta potential profiles of ad swcnts all the starting materials for aggregation experiments are supernatants of swcnts after centrifugation to remove nondispersed swcnts . for ethylenediaminetetraacetic acid ( edta)- , dithiothreitol ( dtt)- , 2-mercaptoethanol- , or nacl - mediated redispersion of various swcnt aggregates , we first induced aggregation of swcnts with various electrolytes at designated concentrations by agitation at a speed of 200 rpm for 30 min at room temperature ( 22 c ) . the rest of the samples were centrifuged for 30 min at 17 000 g , and supernatants were collected for uv vis nir absorbance measurement to determine the fraction of swcnts that were redispersed back to solution relative to the original quantity of dispersed swcnts . to determine the visible - nir absorbance spectra before and after inducing aggregation and redispersion , ad swcnts , chemical vapor deposition ( cvd ) swcnts ( ses research , richardson , tx ) and high pressure carbon monoxide ( hipco ) swcnts ( super purified grade , unidym , sunnyvale , ca ) were dispersed in the presence of ( dt)30 and agitated for 30 min with 5 mm of cacl2 in order to induce aggregation . we first induced aggregation of negatively charged swcnts with 1 mm ka8k and positively charged swcnts with 5 mm ea8e ( custom synthesized polypeptides , 95% purity , pierce protein , il . ) for inhibition of protease activity as a control experiment , swcnt aggregates induced by polypeptides ka8k or ea8e were treated with either 0.2 mg / ml trypsin inhibitor ( from soybean ) or 10 mm pmsf , respectively . supernatants were collected after centrifugation , and the fraction of individual swcnts that remained in solution was measured with uv vis previous studies on swcnts dispersed in h2o using sodium dodecyl sulfate ( sds ) showed that swcnts could undergo selective aggregation upon addition of salt . when the concentration of counterions was increased , the surface of individually dispersed swcnts could be completely neutralized , at which point the adjacent swcnts would come into contact due to van der waals attractions . we then titrated swcnt/(dt)30 with various electrolytes , and monitored the surface charge status of swcnts by measuring the zeta potential of the swcnts after addition of the electrolytes at various concentrations . as shown in figure 1a , top panel , the fraction of dispersed swcnts that remained in solution did not change significantly until the concentration of the added electrolyte reached a certain threshold , which resulted in very sharp transitions in these curves as we progressively increased the concentrations of the electrolytes . this threshold behavior depends on the type of the electrolytes added , so that the higher the valence for the counterion , the lower the threshold concentration , which follows the order of fe < mg ca < na . in contrast to this threshold behavior , the zeta potential of the swcnts displayed a gradual instead of a sharp transition for all cases ( figure 1a , bottom panel ) . upon addition of various electrolytes , the surface charge of swcnts diminished monotonically , suggesting either the binding of counterions to swcnts that lead to charge neutralization or perhaps , dissociation of ( dt)30 from swcnt surface . fraction of individual ( a ) swcnt/(dt)30 , ( b ) swcnt / fitc , ( c ) swcnt cooh , and ( d ) swcnt / pluronic that remained in solution upon titration with various electrolytes ( upper panel ) and the zeta potential changes on swcnts associated with this process ( lower panel ) . throughout this paper , aggregation and zeta potential profiles of ad swcnts the explanation offered for the aggregation of swcnts dispersed by sds upon salt addition is that salt reduces the solubility of sds in water , and the loss of sds from swcnt surface leads to charge depletion and aggregation . as shown in figure 1c , the fraction of dispersed swcnts that remained in solution decreased sharply when the concentration of the added electrolyte reached a certain threshold . because cooh groups were covalently attached to swcnts , this result indicates that it is the binding of counterions to swcnt surfaces that partially neutralizes surface charge , which leads to aggregation of swcnts when the surface charge is neutralized beyond a threshold . the above results suggest that it is the binding of counterions to swcnts instead of dissociation of charged dispersant molecules that triggers aggregation when the surface charge of swcnt is neutralized beyond a threshold . as shown in figure 1d , none of these reagents induced aggregation of swcnts throughout the concentrations investigated , except fecl3 , which induced 10% aggregation at 1 mm fecl3 but this aggregation was less than < 20% even at 1 m fecl3 tested . consistent with these observations , the surface charge of swcnts did not change over the range of salt concentrations investigated , indicating no binding of ions to swcnt surfaces . because pluronic f 108 does not carry any charges , these results reinforce the idea that the aggregation phenomenon we observed in figure 1a c is due to electrostatic interactions between swcnts and counterions in solution . as shown in figure 2 , the fraction of dispersed swcnts that remained in solution did not change significantly until the concentration of the added electrolyte reached a certain threshold , which resulted in sharp transitions in these curves as we increased the concentrations of the electrolytes . in contrast to this threshold behavior , the zeta potential of the swcnts displayed a gradual instead of sharp decrease for all cases ( figure 2 , bottom panels ) , suggesting the direct binding of counterions to swcnt surface . aggregation of ( a ) swcnt / rb ( b ) swcnt / cv , and ( c ) swcnt / pll upon titration with various electrolytes and the zeta potential changes associated with the process . to quantitatively examine the dependence of swcnt aggregation on their surface charge status , we plotted the fraction of swcnts that remained in solution as a function of the measured zeta potential on swcnt surfaces for each charged dispersant molecule that we have investigated ( figure 3 ) . thus , our results reveal the similarity between dna condensation and swcnts aggregation , and swcnts dispersed by various charged dispersant molecules simply behave as polyelectrolytes . the essential feature in this model is that the aggregation of swcnts dispersed by these charged dispersant is driven by electrostatic interactions , instead of van der waals or hydrophobic interactions among swcnts . fraction of individually dispersed ( a ) swcnt/(dt)30 , ( b ) swcnt / fitc , ( c ) swcnt cooh , ( d ) swcnt / rb , ( e ) swcnt / cv , and ( f ) swcnt / pll that remained in solution as a function of zeta potential on the swcnt surface . to test this hypothesis , we induced aggregation of swcnts dispersed with negatively charged dispersant molecules ( ( dt)30 and fitc ) through addition of mg , ca , or fe , and then titrated in edta that can chelate these metal ions . as shown in figure 4a , b , we plotted the fraction of swcnts that remained in solution as a function of added edta expressed as the ratio between [ edta ] and the concentration of corresponding metal ions . examination of the uv vis nir absorbance spectra of the dispersed hipco swcnts before aggregation and after redispersion revealed only small changes in peak position and very similar features for ad and cvd swcnts we have compared . control experiments using aggregates of swcnt / rb or swcnt / cv induced by addition of cacl2 ( aggregation due to cl binding to positively charged swcnt surface ) showed that addition of edta did not induce any redispersion of these aggregates , consistent with our expectation that only chelation of bound counterions on the surface of swcnts resulted in the dispersion of swcnts aggregates ( figure s2 ) . rather , it suggests that the potential packing structures of these aggregates and the resulting accessibility of metal ions in these aggregates may partially determine the concentration of edta needed to redisperse them . consistent with this view , the aggregates of swcnt / fitc in general require a higher concentration of edta to redisperse than swcnt/(dt)30 ( figure 4b ) , even for the aggregates induced by the same metal ions . these experiments , altogether , further support our model that the aggregation is due to binding of counterions to swcnt surface , and sequestration of the bound counterions can lead to the complete redispersion of swcnts in solution . previous study has revealed that swcnts dispersed by pll can be aggregated and redispersed depending on the ph , which is resulted from the change in the charge status of pll in response to ph . although ph is a different trigger for swcnts aggregation and redispersion compared to counterions in current studies , both employ similar mechanisms of electrostatic interactions to control the aggregation status of swcnts . to examine whether these polyamines can induce aggregation of swcnts through similar mechanisms as we demonstrated above , we prepared either swcnt/(dt)30 or swcnt cooh dispersion , and then tested spermidine , spermine , together with 1,6-diaminohexane ( dh ) and cystamine dihydrochloride ( cd ) for their effects on dispersed swcnts . as shown in figure 5a for swcnt/(dt)30 , as we titrated spermidine or spermine , swcnts underwent aggregation as we expected ; however , as we further increase the concentration of these polyamines above 1 mm , the aggregates transiently disappeared . notably , the zeta potential we measured for swcnts also varied with the concentration of polyamine in phase : surface charge was neutralized to about 80% when swcnts were fully aggregated ; the zeta potential recovered to original values when aggregates became redispersed . spermine and spermidine concentration - dependent aggregation and redispersion of ( a ) swcnt/(dt)30 , ( b ) swcnt cooh , and ( c ) swcnt / pluronic together with the zeta potential changes associated with the process . because dh and spermidine only differ slightly in their chemical structures : spermidine has one extra ch2 and one extra nh group that carried one more positive charge than dh under current solution conditions ( chart s1 ) , we reasoned that the above process of aggregation started with the direct binding of the positively charged amino groups at the ends of these molecules to swcnt surfaces . this is possible because these positive charges serve as counterions for the negative charges on the surface of swcnts , either due to the dna phosphate backbone or cooh group that was covalently attached to the swcnt surface . for both spermidine and spermine but not dh or cd , due to the presence of additional positive charges , the increasing concentration of polyamines in solution increases the ionic strength of the solution , so that these entropic bridge interactions are weakened and collapsed ( as shown in figure s3 ) , consistent with the recovery of surface charge monitored by zeta potential ( figure 5a , b bottom panel ) . if the bridging effect is responsible for swcnt aggregation , we would expect a redispersion of swcnt aggregates formed with cd upon addition of either dtt or 2-mercaptoethanol , because both reducing agents can reduce cd and thus break the molecule into two separate parts . at 200 mm nacl , these observations were very similar to the redispersion and aggregation of swcnts induced by either spermidine or spermine as shown in figure 5 and suggests that the transient redispersion is due to increased ionic strength in solution that weakened the entropic bridge interactions . this bridging effect as we observed for polyelectrolytes suggests a possible application of these molecules to induce reversible aggregation and redispersion of swcnts that can be controlled through chain breaking . as expected , the positively charged peptide ka8k can induce aggregation of either swcnt/(dt)30 or swcnt cooh , while the negatively charged peptide ea8e can induce aggregation of either swcnt / cv or swcnt / pll , as shown in figure 9 together with the zeta potential measurement results for this titration process . under conditions where we induced full aggregation of swcnts , we treated the swcnt aggregates with either trypsin that can cleave the ka8k peptide or protease k that can cleave the ea8e peptide . fraction of individual ( a ) negatively charged swcnts and ( b ) positively charged swcnts that remained in solution after addition of polypeptides ( ka8k for negatively charged and ea8e for positively charged swcnts ) . enzyme mediated redispersion of ( a ) swcnt/(dt)30 and swcnt cooh aggregated by polypeptide ( ka8k ) and ( b ) swcnt / pll and swcnt / cv aggregated by polypeptide ( ea8e ) and the inhibition of this redispersion by enzyme inhibitors . the above results on swcnt aggregation , as we observed for both monomeric electrolyte and polyelectrolyte molecules , bear direct relevance for the potential applications of swcnts as delivery vehicles for genes into the cells . our data suggest that swcnts can be aggregated during in vitro as well as in vivo applications as gene delivery vehicles , which may lead to toxicity of these nanomaterials in vivo .

sleep behaviors are among the most common concerns that bring parents of young children to their physicians . a child who goes to bed unwillingly or wakes frequently during the night can be highly disruptive to a family . as a rule , the frequency of night waking starts in 100 percent of newborns and tails off to approximately 20 - 30 percent in six - month - olds . it is estimated that sleep problems are experienced by 25 - 30 percent of children and adolescents , regardless of age . thus , while sleep occupies a major portion of the childhood years , childhood sleep problems constitute a major parental concern . sleep problems , which can include inadequate , disrupted , poor quality , or non - restful sleep , are one of the most common complaints raised by parents to pediatricians and practitioners . in contrast , the relationship between insufficient or disturbed sleep and the many manifestations of daytime sleepiness , such as mood and behavior problems , although less frequently recognized by parents , has a major impact on quality of life of children and adolescents . although many sleep problems in infants and children are transient and self - limited in nature , certain intrinsic and extrinsic risk factors such as difficult temperament , chronic illness , and maternal depression may predispose some children to develop more chronic sleep disturbances . despite the magnitude and clinical importance of sleep issues , several studies have documented that there is a low level of recognition of sleep disorders by primary care physicians in children[56 ] . for example , in a survey of over 600 community pediatricians , approximately 20% of the respondents did not routinely screen for sleep problems in school - aged children in well - child visit , about 25 percent of routinely screened toddlers and pre - schoolers for snoring , and less than 40 percent questioned adolescents directly about sleep habits , despite the respondents knowledge of the importance of sleep 's impact on health , behavior , and school performance . in another study a validated pediatric sleep questionnaire used to identify a series of children with sleep - related symptoms at two community based general pediatrics clinics and reviewed medical chart notes for the previous 2 years to determine how often sleep problems had been addressed . fewer than 15 percent of patients had current chart notes that mentioned some of the questionnaire - defined sleep problems ; diagnoses were mentioned for two of 86 patients and no treatments were discussed . the consequences of untreated sleep problems may include significant emotional , behavioral , and cognitive dysfunction[711 ] . the impact of childhood sleep problems is further intensified by their direct effect on parents sleep , resulting in parental daytime fatigue , mood disturbances , and a decreased level of effective parenting . after the school entrance such sleep problems impact adversely on behavior , school functioning , and health - related quality of life . surprisingly little attention has been paid to the impact of sleep problems in the vital preschool years and there have not been enough studies investigating etiologic factors of these problems[7 , 12 ] . preschool and school children with habitual snoring were more likely to have sleep - related daytime and nighttime symptoms . but the studies determined no significant association between habitual snoring and poor school performance[7 , 10 ] . a recent study from nigeria concluded that snoring is an important health problem among preschool and school children as a significant percentage of them snore and most of whom are between third and sixth year of life . most of the studies regarding sleep habits in children are from the west ; however , a few asian studies[1214 ] are available and these studies emphasize the effect of culture . in a previous study in iran , the authors first determined the reliability and validity of persian version of the well - known bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring ( bears ) pediatric sleep questionnaire and then designed a pilot study to describe sleep patterns and sleep problems among pre - school and school - aged children in two primary care pediatric clinics in tehran . the purpose of the tehran 's children sleep study ( tcss ) was to investigate the possible relationship between gender and school entrance on sleep complaints . the authors supposed that these mentioned factors might be of value for both practitioners and parents to plan for tailored and more effective interventions to combat sleep problems in boys and girls and in pre - school ( 2 - 6 years of age ) versus school - age children ( 7 - 12 years of age ) . the subjects were pre - school - aged and school - going children between the ages 2 and 12 years who were selected by clustered randomization of families in tehran based on their zip codes . subjects were included randomly if their parents agreed to cooperate in filling bears sleep screening questionnaire ( see the procedure ) . a total of 746 subjects were entered in the study : 325 pre - school - aged children ( 2 - 6 years old group ) and 421 primary school - aged children ( 7 - 12 years old group ) . our study protocol was approved by the ethical board committee of ent , head and neck research center of tehran university of medical sciences , in tehran . the bears is a screening tool developed by the investigators of brown university school of medicine , rhode island hospital , usa . it was designed to address the most common sleep issues in toddlers , pre - schoolers , and school - aged children . it is an acronym and incorporates five basic sleep domains : bedtime problems , including difficulty going to bed and falling asleep ; excessive daytime sleepiness , which includes behaviors typically associated with daytime somnolence in children ; awakenings during the night ; regularity of sleep / wake cycles ( bedtime , wake time ) and average sleep duration ; and snoring . reliability and validity of the persian version of the bears questionnaire was assessed in a previous study . , all parents were informed about the importance of sleep problems screening in children and they were asked to sign the consent to participate in the study . then , the persian version of the bears questionnaire with five domains was asked by general pediatricians . questions for pre - school children were answered by their parents ; but for school - age children , some questions were asked from the children themselves . data were entered into the statistical package for the social sciences ( spss ) software version 11.5 . descriptive statistics were used to report frequency counts , percentages and means ( standard deviations ) . proportions ( prevalence ) of sleep complaints in each b - e - a - r - s category were calculated for pre - school and school - age groups , totally and stratified by gender . the same test was used to assess if there was any difference between boys and girls in each age category . two - sided p - values were reported and an alpha level of 0.05 was considered as the threshold of statistical significance . as the effect measure , the odds ratio ( or ) plus its 95 percent confidence interval ( 95%ci ) was calculated to compare the effect size of gender on sleep complaints difference in each b - e - a - r - s category . again , the same approach was used to calculate the effects of school attendance on sleep problems . for comparing mean sleep duration between two groups , the subjects were pre - school - aged and school - going children between the ages 2 and 12 years who were selected by clustered randomization of families in tehran based on their zip codes . subjects were included randomly if their parents agreed to cooperate in filling bears sleep screening questionnaire ( see the procedure ) . a total of 746 subjects were entered in the study : 325 pre - school - aged children ( 2 - 6 years old group ) and 421 primary school - aged children ( 7 - 12 years old group ) . our study protocol was approved by the ethical board committee of ent , head and neck research center of tehran university of medical sciences , in tehran . the bears is a screening tool developed by the investigators of brown university school of medicine , rhode island hospital , usa . it was designed to address the most common sleep issues in toddlers , pre - schoolers , and school - aged children . it is an acronym and incorporates five basic sleep domains : bedtime problems , including difficulty going to bed and falling asleep ; excessive daytime sleepiness , which includes behaviors typically associated with daytime somnolence in children ; awakenings during the night ; regularity of sleep / wake cycles ( bedtime , wake time ) and average sleep duration ; and snoring . reliability and validity of the persian version of the bears questionnaire was assessed in a previous study . , all parents were informed about the importance of sleep problems screening in children and they were asked to sign the consent to participate in the study . then , the persian version of the bears questionnaire with five domains was asked by general pediatricians . questions for pre - school children were answered by their parents ; but for school - age children , some questions were asked from the children themselves . data were entered into the statistical package for the social sciences ( spss ) software version 11.5 . descriptive statistics were used to report frequency counts , percentages and means ( standard deviations ) . proportions ( prevalence ) of sleep complaints in each b - e - a - r - s category were calculated for pre - school and school - age groups , totally and stratified by gender . the same test was used to assess if there was any difference between boys and girls in each age category . two - sided p - values were reported and an alpha level of 0.05 was considered as the threshold of statistical significance . as the effect measure , the odds ratio ( or ) plus its 95 percent confidence interval ( 95%ci ) was calculated to compare the effect size of gender on sleep complaints difference in each b - e - a - r - s category . again , the same approach was used to calculate the effects of school attendance on sleep problems . for comparing mean sleep duration between two groups , bears questionnaire was completed for a total of 746 ( 2 - 12 years old ) children ; 325 consisting of 142 ( 43.7% ) females and 183 ( 56.3% ) males were in pre - school - age group ( 2 - 6 years old ) and 421 with 173 ( 41.1% ) females and 248 ( 58.9% ) males were in primary school - age group ( 7 - 12 years old ) . male to female ratio was not different between the two age groups ( p=0.4 ) . the average age was 3.93 ( 0.16 ) years in pre - school - age group and 9.63 ( 0.16 ) years in primary school - age group . table 1 shows the prevalence of probable or definite problems in each domain of bears , where screening questionnaire and also the comparison of percentages between pre - school and school - aged groups is shown . the most common screening problem in both pre - school and school - aged group was excessive daytime sleepiness ( 64.9% and 62.9% respectively ) . the least common problem in both groups was sleep disordered breathing ( 7.1% and 11.9% respectively ) . bedtime problems and also regularity and duration of sleep were significantly more prevalent in pre - school - age group ( p<0.0002 ; or=1.98 ; 95% ci : 1.98 - 4.20 ; and or=2.00 ; 95%ci : 1.41 - 2.84 , respectively ) . however , sleep - disordered breathing was lower in pre - school children ( p=0.029 ; or=0.57 ; 95%ci : 0.34 - 0.94 ) . other bears domains - excessive daytime sleepiness and awakening during the night - did not differ significantly between the two groups . comparison of the prevalence of sleep problems in each domain of bears questionnaire among pre - school and school - aged groups odds ratio for the effect of school entrance ; ci : confidence interval ; bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring for pre - school children , mean time for going to bed at night was 22:54 ( sd 1.20 hrs ) , mean wakeup time in the morning was 08:43 ( sd 1.20 hrs ) , and mean sleep duration was 9.81 hours ( sd 1.13 ) . for school - aged children , mean time for going to bed at night in school days was 22:36 ( sd 1.12 hrs ) , mean wakeup time in the morning in school days was 07:11 ( sd 1.12 hrs ) , and mean sleep duration was 8.59 hours ( sd 1.33 ) . the difference between mean sleep duration between pre - school age and school - age groups was statistically significant ( p<0.0001 ) . table 2 shows the comparison between boys and girls for sleep problems in both pre - school and school - aged groups . pre - school boys showed significantly less bedtime problems than pre - school girls ( p=0.003 ; or=0.49 ; 0.30 - 0.79 ) . however , in school - aged group all items were statistically the same between girls and boys . comparison of the prevalence of sleep problems in each domain of bears questionnaire among boys and girls in both pre - school and school - aged groups bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring tables 3 and 4 reveal the comparison of sleep problems between pre - school and school - aged boys and also between pre - school and school - aged girls . among both genders , bedtime problems , and regularity and duration of sleep problems were significantly more prevalent in pre - school - age group ( p=0.0002 and 0.04 respectively ) , ( p=0.003 and 0.0005 respectively ) . comparison of the prevalence of sleep problems in each domain of bears questionnaire among pre - school and school - aged groups in boys odds ratio for the effect of school entrance in boys ; ci : confidence interval ; bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring comparison of the prevalence of sleep problems in each domain of bears questionnaire among pre - school and school - aged groups in girls odds ratio for the effect of school entrance in girls ; bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring awakening times during the night and sleep - disordered breathing were more prevalent in school - aged boys ( p=0.0003 and 0.04 respectively ) . it seems that the prevalence of sleep problems according to bears questionnaire is relatively high in our study especially in excessive daytime sleeping domain . the overall findings in different bears domains are in agreement with authors previous pilot study ; but in excessive daytime sleeping domain we came to a much larger figure . the prevalence estimates of sleep problems are very diverse in multiple old and new studies and in different parts of the world and it ranges from 20.7% in a 1989 - 90 survey in headington , oxford by ali et al to almost 40% in a 2010 study in australian indigenous and non - indigenous children by blunden et al . but most of these studies have not used the bears questionnaire as a screening tool for sleep problems assessment . owens et al have reported the following prevalence estimates in their leading study : bedtime issues 16.3% ; nighttime awakenings 18.4% ; and snoring 10.7% ( our relevant estimates are 28.9% , 27.7% , and 7.1% in pre - school children and 12.4% , 44.2% , and 11.9% in school - aged children respectively ) . the more important thing here is the regular assessment of the prevalence estimates at different times using reliable and valid tools to become able to perform trend analyses . the prevalence of sleep problems in these groups of children in our study was also nearly comparable to percentages found in other studies[3 , 18 ] . we thought of gender and school attendance as possible factors affecting sleep problems in children . the reason for the first one is that especially in school - aged children , boys and girls attend separate schools in most parts of our country especially in tehran . some studies also have evaluated the effects of gender on sleep related problems and have found interesting points . for example , gau et al reported that girls sleep fewer hours than boys and do not show an increase in daytime sleepiness . simola et al showed that age and gender were related to phenotype of the sleeping problems while there were no gender differences in any sleep parameters in another study conducted by seo et al . few studies have estimated if there is any difference between boys and girls on sleep problems , and as shown in table 2 , there is just a statistically significant difference in one bears domain ( bedtime problems ) between boys and girls . in a study by van litsenburg et al , parents reported that girls experienced more sleep problems than boys , notably for sleep onset delay and daytime sleepiness , while for the child self - reports , no gender difference was found . secondly , few studies guided us to consider school attendance as a possible factor affecting sleep problems[1923 ] . because the usual starting year for school attendance is 7 years of age , we divided the age range of our subjects to two broad categories shown in tables . considering total subjects , bedtime problems regularity and duration of sleep , and sleep - disordered breathing had statistically significant difference between pre - school and school - aged children . the added value of calculating odds ratio as an effect measure helps us better understand the consequence of going to school on sleep . school entrance seems to exacerbate sleep problems in bedtime problems and also regularity and duration of sleep domains and plays the role of a risk factor . the opposite effect is seen for sleep - disordered breathing domain ( table 1 ) . considering males and females separately ( tables 3 and 4 ) , same pattern is seen for males and females in bedtime problems and regularity and duration of sleep ; but , school attendance seems to have a protective effect in awakening during the night domain in both boys and girls . school entrance plays a protective role for sleep - disordered breathing in boys but not in girls . seo et al demonstrated that extracurricular academic activities , is related to the children 's sleep duration and that the older children sleep less than younger children . they thought that the main reason is late bedtimes due to socio - cultural factors , high levels of nighttime and recreational activities , and/or excessive academic activities . quach et al showed that sleep problems during school transition are common and associated with poorer child outcomes . they conducted the first study , to their knowledge , to examine the natural history of sleep problems in children over the transition from preschool to school . liu et al believed that unique school schedules and sleep practices may contribute to the differences in the sleep patterns and sleep problems of children from the united states and china . gau et al hypothesized that little sleep at night made the students feel sleepy in the daytime and tired , drowsy , moody and difficult at arising in the morning . compared with our study showing no significant difference of sleep - quality between girls and boys , japanese findings support the hypothesis that during the junior high school period , the majority of sleep - quality indicators in japanese schoolchildren were better for girls than for boys . boys sleep was less efficient and more fragmented during the entire week in comparison to that of the girls . few studies such as the one conducted by kahn et al , have assessed the effect of poor sleeping on school performance and have found interesting results such as the point that among the poor sleepers , 21% had failed 1 or more years at school . school achievement difficulties were encountered significantly more often among the poor sleepers than among the children without sleep problems ( p=.001 ) . although this point of view has not been considered as our hypothesis of research , there seems to be diverse effects from school attendance on different sleep patterns and behaviors that need specific and more rigorous exploration . no study ( including ours ) has assessed the possibility of correlation between the results of answers to five bears domains ; the same fact is also true for correlation between parent - answered questions and child - answered ones . although van litsenburg et al used different questionnaires to study sleep habits and sleep disturbances in dutch children , they were concerned that correlations between parental and self - assessments were low to moderate and this fact may have an effect on bears questionnaire too . maybe the work done by iwasaki et al should be repeated for bears as well . they studied the utility of subjective sleep assessment tools for healthy preschool children and compared sleep logs , questionnaires , and actigraphy ; their results showed that sleep schedule variables in the parental reports generally correlated well with actigraphic assessment of sleep patterns and although the daily sleep log was better correlated with actigraphy , the brief questionnaire showed a good correlation with sleep pattern on weekday actigraphic assessments . we hypothesized that high prevalence of sleep problems in school aged and pre - school aged children of tehran is due to bad sleep time that we think is caused by our culture and loss of enough family attention and related ignorance on the harmfulness of poor sleep of children . moreover , the poor sleep quality of iranian preschool children is probably due to cultural characteristics , climate differences , or harmful sleep habits . the difference between mean sleep duration between pre - school age and school - age groups is important ( as it is statistically significant in our study ) because it affects the risk of becoming overweight in later years . ochiai et al reported that short sleep duration increases the risk of becoming overweight and this relationship differs between the two sexes . newer population - based researches may elucidate this kind of relationship in iranian children . against this strength , our study had some limitations . cross sectional design limited our causative assessment for sleep problems , more- over we did not include some familial factors such as socioeconomic factors and psychological problems in the families . we have categorized the subjects to two groups according to their age and took the whole 2 - 6 years as one group and the 7 - 12 years as another . this type of unification may have an effect on the final results , but we have tried to conduct a comparable scenario to the work of owens et al , and also to our previous pilot study . the other fact is that socioeconomic status ( ses ) of families was not assessed in our study . higher ses might have negative impact on total sleep duration , sleep hygiene and also on children 's psychological well - being . the present survey shows that the prevalence of sleep problems is relatively high especially in excessive daytime sleeping domain of preschool - aged and school children . we demonstrated that there is a significant relationship between school entrance and lower bedtime as well as regularity problems . our findings suggest a development of a more tailored sleep - related health service to help both preschool and school children and also assist parents to know and tackle sleep troubles more effectively and thoroughly . the authors recommend using results of regularly conducted sleep studies to design , implement , and evaluate effective interventions to downsize the harmful effects of bad sleep habits .

objectivesleep problems are experienced by 25 - 30 percent of children and adolescents , regardless of age . the purpose of this study was to investigate if there is any relationship between gender or school entrance and sleep complaints.methodsfrom june 2008 to may 2009 children aged 2 to 12 years were selected by clustered randomization of families . the persian version of the bears questionnaire ( bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring ) with five domains was filled out by general pediatricians . prevalence of sleep complaints in each b - e - a - r - s category was calculated and compared for pre - school and school - age groups.findingsbears questionnaire was completed for a total of 746 children ( 2 - 12 years old ) ; 325 in pre - school - age group ( 2 - 6 years old ) ( 142 females [ 43.7% ] and 183 males [ 56.3% ] ) and 421 in primary school - age group ( 7 - 12 years old ) with the average age of 3.93 ( 0.16 ) years and 9.63 ( 0.16 ) years respectively . the most common screening problem in both groups was excessive daytime sleepiness ( 64.9% and 62.9% respectively ) . bedtime problems and also regularity and duration of sleep were significantly more prevalent in pre - school - age group ( p<0.0002 ; odds ratio [ or ] = 1.98 ; 95% confidence interval [ 95%ci ] : 1.98 - 4.20 ; and or=2.00 ; 95%ci : 1.41- 2.84 respectively ) . the difference between mean sleep duration between pre - school age and school - age groups was statistically significant ( p<0.0001).conclusionthe current survey shows that different types of sleep problems are relatively high especially in the form of excessive daytime sleeping domain in preschool- and school - aged children . bedtime problems and regularity problems were significantly more prevalent in pre - school - age group . school entrance seems to play a positive role for bedtime problems , and sleep - disordered breathing .

Introduction Subjects and Methods Study Design and Sampling Procedure Statistical Analysis Findings Discussion Conclusion Conflict of Interest

sleep behaviors are among the most common concerns that bring parents of young children to their physicians . a child who goes to bed unwillingly or wakes frequently during the night can be highly disruptive to a family . as a rule , the frequency of night waking starts in 100 percent of newborns and tails off to approximately 20 - 30 percent in six - month - olds . it is estimated that sleep problems are experienced by 25 - 30 percent of children and adolescents , regardless of age . thus , while sleep occupies a major portion of the childhood years , childhood sleep problems constitute a major parental concern . sleep problems , which can include inadequate , disrupted , poor quality , or non - restful sleep , are one of the most common complaints raised by parents to pediatricians and practitioners . in contrast , the relationship between insufficient or disturbed sleep and the many manifestations of daytime sleepiness , such as mood and behavior problems , although less frequently recognized by parents , has a major impact on quality of life of children and adolescents . although many sleep problems in infants and children are transient and self - limited in nature , certain intrinsic and extrinsic risk factors such as difficult temperament , chronic illness , and maternal depression may predispose some children to develop more chronic sleep disturbances . despite the magnitude and clinical importance of sleep issues , several studies have documented that there is a low level of recognition of sleep disorders by primary care physicians in children[56 ] . for example , in a survey of over 600 community pediatricians , approximately 20% of the respondents did not routinely screen for sleep problems in school - aged children in well - child visit , about 25 percent of routinely screened toddlers and pre - schoolers for snoring , and less than 40 percent questioned adolescents directly about sleep habits , despite the respondents knowledge of the importance of sleep 's impact on health , behavior , and school performance . in another study a validated pediatric sleep questionnaire used to identify a series of children with sleep - related symptoms at two community based general pediatrics clinics and reviewed medical chart notes for the previous 2 years to determine how often sleep problems had been addressed . fewer than 15 percent of patients had current chart notes that mentioned some of the questionnaire - defined sleep problems ; diagnoses were mentioned for two of 86 patients and no treatments were discussed . the consequences of untreated sleep problems may include significant emotional , behavioral , and cognitive dysfunction[711 ] . the impact of childhood sleep problems is further intensified by their direct effect on parents sleep , resulting in parental daytime fatigue , mood disturbances , and a decreased level of effective parenting . after the school entrance such sleep problems impact adversely on behavior , school functioning , and health - related quality of life . surprisingly little attention has been paid to the impact of sleep problems in the vital preschool years and there have not been enough studies investigating etiologic factors of these problems[7 , 12 ] . preschool and school children with habitual snoring were more likely to have sleep - related daytime and nighttime symptoms . a recent study from nigeria concluded that snoring is an important health problem among preschool and school children as a significant percentage of them snore and most of whom are between third and sixth year of life . most of the studies regarding sleep habits in children are from the west ; however , a few asian studies[1214 ] are available and these studies emphasize the effect of culture . in a previous study in iran , the authors first determined the reliability and validity of persian version of the well - known bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring ( bears ) pediatric sleep questionnaire and then designed a pilot study to describe sleep patterns and sleep problems among pre - school and school - aged children in two primary care pediatric clinics in tehran . the purpose of the tehran 's children sleep study ( tcss ) was to investigate the possible relationship between gender and school entrance on sleep complaints . the authors supposed that these mentioned factors might be of value for both practitioners and parents to plan for tailored and more effective interventions to combat sleep problems in boys and girls and in pre - school ( 2 - 6 years of age ) versus school - age children ( 7 - 12 years of age ) . the subjects were pre - school - aged and school - going children between the ages 2 and 12 years who were selected by clustered randomization of families in tehran based on their zip codes . subjects were included randomly if their parents agreed to cooperate in filling bears sleep screening questionnaire ( see the procedure ) . a total of 746 subjects were entered in the study : 325 pre - school - aged children ( 2 - 6 years old group ) and 421 primary school - aged children ( 7 - 12 years old group ) . the bears is a screening tool developed by the investigators of brown university school of medicine , rhode island hospital , usa . it was designed to address the most common sleep issues in toddlers , pre - schoolers , and school - aged children . it is an acronym and incorporates five basic sleep domains : bedtime problems , including difficulty going to bed and falling asleep ; excessive daytime sleepiness , which includes behaviors typically associated with daytime somnolence in children ; awakenings during the night ; regularity of sleep / wake cycles ( bedtime , wake time ) and average sleep duration ; and snoring . reliability and validity of the persian version of the bears questionnaire was assessed in a previous study . , all parents were informed about the importance of sleep problems screening in children and they were asked to sign the consent to participate in the study . then , the persian version of the bears questionnaire with five domains was asked by general pediatricians . questions for pre - school children were answered by their parents ; but for school - age children , some questions were asked from the children themselves . proportions ( prevalence ) of sleep complaints in each b - e - a - r - s category were calculated for pre - school and school - age groups , totally and stratified by gender . the same test was used to assess if there was any difference between boys and girls in each age category . as the effect measure , the odds ratio ( or ) plus its 95 percent confidence interval ( 95%ci ) was calculated to compare the effect size of gender on sleep complaints difference in each b - e - a - r - s category . again , the same approach was used to calculate the effects of school attendance on sleep problems . for comparing mean sleep duration between two groups , the subjects were pre - school - aged and school - going children between the ages 2 and 12 years who were selected by clustered randomization of families in tehran based on their zip codes . a total of 746 subjects were entered in the study : 325 pre - school - aged children ( 2 - 6 years old group ) and 421 primary school - aged children ( 7 - 12 years old group ) . the bears is a screening tool developed by the investigators of brown university school of medicine , rhode island hospital , usa . it was designed to address the most common sleep issues in toddlers , pre - schoolers , and school - aged children . it is an acronym and incorporates five basic sleep domains : bedtime problems , including difficulty going to bed and falling asleep ; excessive daytime sleepiness , which includes behaviors typically associated with daytime somnolence in children ; awakenings during the night ; regularity of sleep / wake cycles ( bedtime , wake time ) and average sleep duration ; and snoring . reliability and validity of the persian version of the bears questionnaire was assessed in a previous study . , all parents were informed about the importance of sleep problems screening in children and they were asked to sign the consent to participate in the study . then , the persian version of the bears questionnaire with five domains was asked by general pediatricians . questions for pre - school children were answered by their parents ; but for school - age children , some questions were asked from the children themselves . proportions ( prevalence ) of sleep complaints in each b - e - a - r - s category were calculated for pre - school and school - age groups , totally and stratified by gender . the same test was used to assess if there was any difference between boys and girls in each age category . as the effect measure , the odds ratio ( or ) plus its 95 percent confidence interval ( 95%ci ) was calculated to compare the effect size of gender on sleep complaints difference in each b - e - a - r - s category . for comparing mean sleep duration between two groups , bears questionnaire was completed for a total of 746 ( 2 - 12 years old ) children ; 325 consisting of 142 ( 43.7% ) females and 183 ( 56.3% ) males were in pre - school - age group ( 2 - 6 years old ) and 421 with 173 ( 41.1% ) females and 248 ( 58.9% ) males were in primary school - age group ( 7 - 12 years old ) . male to female ratio was not different between the two age groups ( p=0.4 ) . the average age was 3.93 ( 0.16 ) years in pre - school - age group and 9.63 ( 0.16 ) years in primary school - age group . table 1 shows the prevalence of probable or definite problems in each domain of bears , where screening questionnaire and also the comparison of percentages between pre - school and school - aged groups is shown . the most common screening problem in both pre - school and school - aged group was excessive daytime sleepiness ( 64.9% and 62.9% respectively ) . the least common problem in both groups was sleep disordered breathing ( 7.1% and 11.9% respectively ) . bedtime problems and also regularity and duration of sleep were significantly more prevalent in pre - school - age group ( p<0.0002 ; or=1.98 ; 95% ci : 1.98 - 4.20 ; and or=2.00 ; 95%ci : 1.41 - 2.84 , respectively ) . however , sleep - disordered breathing was lower in pre - school children ( p=0.029 ; or=0.57 ; 95%ci : 0.34 - 0.94 ) . other bears domains - excessive daytime sleepiness and awakening during the night - did not differ significantly between the two groups . comparison of the prevalence of sleep problems in each domain of bears questionnaire among pre - school and school - aged groups odds ratio for the effect of school entrance ; ci : confidence interval ; bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring for pre - school children , mean time for going to bed at night was 22:54 ( sd 1.20 hrs ) , mean wakeup time in the morning was 08:43 ( sd 1.20 hrs ) , and mean sleep duration was 9.81 hours ( sd 1.13 ) . for school - aged children , mean time for going to bed at night in school days was 22:36 ( sd 1.12 hrs ) , mean wakeup time in the morning in school days was 07:11 ( sd 1.12 hrs ) , and mean sleep duration was 8.59 hours ( sd 1.33 ) . the difference between mean sleep duration between pre - school age and school - age groups was statistically significant ( p<0.0001 ) . table 2 shows the comparison between boys and girls for sleep problems in both pre - school and school - aged groups . pre - school boys showed significantly less bedtime problems than pre - school girls ( p=0.003 ; or=0.49 ; 0.30 - 0.79 ) . however , in school - aged group all items were statistically the same between girls and boys . comparison of the prevalence of sleep problems in each domain of bears questionnaire among boys and girls in both pre - school and school - aged groups bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring tables 3 and 4 reveal the comparison of sleep problems between pre - school and school - aged boys and also between pre - school and school - aged girls . among both genders , bedtime problems , and regularity and duration of sleep problems were significantly more prevalent in pre - school - age group ( p=0.0002 and 0.04 respectively ) , ( p=0.003 and 0.0005 respectively ) . comparison of the prevalence of sleep problems in each domain of bears questionnaire among pre - school and school - aged groups in boys odds ratio for the effect of school entrance in boys ; ci : confidence interval ; bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring comparison of the prevalence of sleep problems in each domain of bears questionnaire among pre - school and school - aged groups in girls odds ratio for the effect of school entrance in girls ; bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring awakening times during the night and sleep - disordered breathing were more prevalent in school - aged boys ( p=0.0003 and 0.04 respectively ) . it seems that the prevalence of sleep problems according to bears questionnaire is relatively high in our study especially in excessive daytime sleeping domain . the overall findings in different bears domains are in agreement with authors previous pilot study ; but in excessive daytime sleeping domain we came to a much larger figure . the prevalence estimates of sleep problems are very diverse in multiple old and new studies and in different parts of the world and it ranges from 20.7% in a 1989 - 90 survey in headington , oxford by ali et al to almost 40% in a 2010 study in australian indigenous and non - indigenous children by blunden et al . but most of these studies have not used the bears questionnaire as a screening tool for sleep problems assessment . owens et al have reported the following prevalence estimates in their leading study : bedtime issues 16.3% ; nighttime awakenings 18.4% ; and snoring 10.7% ( our relevant estimates are 28.9% , 27.7% , and 7.1% in pre - school children and 12.4% , 44.2% , and 11.9% in school - aged children respectively ) . the more important thing here is the regular assessment of the prevalence estimates at different times using reliable and valid tools to become able to perform trend analyses . the prevalence of sleep problems in these groups of children in our study was also nearly comparable to percentages found in other studies[3 , 18 ] . we thought of gender and school attendance as possible factors affecting sleep problems in children . the reason for the first one is that especially in school - aged children , boys and girls attend separate schools in most parts of our country especially in tehran . some studies also have evaluated the effects of gender on sleep related problems and have found interesting points . for example , gau et al reported that girls sleep fewer hours than boys and do not show an increase in daytime sleepiness . simola et al showed that age and gender were related to phenotype of the sleeping problems while there were no gender differences in any sleep parameters in another study conducted by seo et al . few studies have estimated if there is any difference between boys and girls on sleep problems , and as shown in table 2 , there is just a statistically significant difference in one bears domain ( bedtime problems ) between boys and girls . in a study by van litsenburg et al , parents reported that girls experienced more sleep problems than boys , notably for sleep onset delay and daytime sleepiness , while for the child self - reports , no gender difference was found . considering total subjects , bedtime problems regularity and duration of sleep , and sleep - disordered breathing had statistically significant difference between pre - school and school - aged children . the added value of calculating odds ratio as an effect measure helps us better understand the consequence of going to school on sleep . school entrance seems to exacerbate sleep problems in bedtime problems and also regularity and duration of sleep domains and plays the role of a risk factor . the opposite effect is seen for sleep - disordered breathing domain ( table 1 ) . considering males and females separately ( tables 3 and 4 ) , same pattern is seen for males and females in bedtime problems and regularity and duration of sleep ; but , school attendance seems to have a protective effect in awakening during the night domain in both boys and girls . school entrance plays a protective role for sleep - disordered breathing in boys but not in girls . quach et al showed that sleep problems during school transition are common and associated with poorer child outcomes . they conducted the first study , to their knowledge , to examine the natural history of sleep problems in children over the transition from preschool to school . liu et al believed that unique school schedules and sleep practices may contribute to the differences in the sleep patterns and sleep problems of children from the united states and china . gau et al hypothesized that little sleep at night made the students feel sleepy in the daytime and tired , drowsy , moody and difficult at arising in the morning . compared with our study showing no significant difference of sleep - quality between girls and boys , japanese findings support the hypothesis that during the junior high school period , the majority of sleep - quality indicators in japanese schoolchildren were better for girls than for boys . boys sleep was less efficient and more fragmented during the entire week in comparison to that of the girls . school achievement difficulties were encountered significantly more often among the poor sleepers than among the children without sleep problems ( p=.001 ) . although this point of view has not been considered as our hypothesis of research , there seems to be diverse effects from school attendance on different sleep patterns and behaviors that need specific and more rigorous exploration . although van litsenburg et al used different questionnaires to study sleep habits and sleep disturbances in dutch children , they were concerned that correlations between parental and self - assessments were low to moderate and this fact may have an effect on bears questionnaire too . they studied the utility of subjective sleep assessment tools for healthy preschool children and compared sleep logs , questionnaires , and actigraphy ; their results showed that sleep schedule variables in the parental reports generally correlated well with actigraphic assessment of sleep patterns and although the daily sleep log was better correlated with actigraphy , the brief questionnaire showed a good correlation with sleep pattern on weekday actigraphic assessments . we hypothesized that high prevalence of sleep problems in school aged and pre - school aged children of tehran is due to bad sleep time that we think is caused by our culture and loss of enough family attention and related ignorance on the harmfulness of poor sleep of children . the difference between mean sleep duration between pre - school age and school - age groups is important ( as it is statistically significant in our study ) because it affects the risk of becoming overweight in later years . cross sectional design limited our causative assessment for sleep problems , more- over we did not include some familial factors such as socioeconomic factors and psychological problems in the families . we have categorized the subjects to two groups according to their age and took the whole 2 - 6 years as one group and the 7 - 12 years as another . this type of unification may have an effect on the final results , but we have tried to conduct a comparable scenario to the work of owens et al , and also to our previous pilot study . higher ses might have negative impact on total sleep duration , sleep hygiene and also on children 's psychological well - being . the present survey shows that the prevalence of sleep problems is relatively high especially in excessive daytime sleeping domain of preschool - aged and school children . we demonstrated that there is a significant relationship between school entrance and lower bedtime as well as regularity problems . our findings suggest a development of a more tailored sleep - related health service to help both preschool and school children and also assist parents to know and tackle sleep troubles more effectively and thoroughly . the authors recommend using results of regularly conducted sleep studies to design , implement , and evaluate effective interventions to downsize the harmful effects of bad sleep habits .

sleep behaviors are among the most common concerns that bring parents of young children to their physicians . as a rule , the frequency of night waking starts in 100 percent of newborns and tails off to approximately 20 - 30 percent in six - month - olds . it is estimated that sleep problems are experienced by 25 - 30 percent of children and adolescents , regardless of age . thus , while sleep occupies a major portion of the childhood years , childhood sleep problems constitute a major parental concern . sleep problems , which can include inadequate , disrupted , poor quality , or non - restful sleep , are one of the most common complaints raised by parents to pediatricians and practitioners . in contrast , the relationship between insufficient or disturbed sleep and the many manifestations of daytime sleepiness , such as mood and behavior problems , although less frequently recognized by parents , has a major impact on quality of life of children and adolescents . although many sleep problems in infants and children are transient and self - limited in nature , certain intrinsic and extrinsic risk factors such as difficult temperament , chronic illness , and maternal depression may predispose some children to develop more chronic sleep disturbances . despite the magnitude and clinical importance of sleep issues , several studies have documented that there is a low level of recognition of sleep disorders by primary care physicians in children[56 ] . for example , in a survey of over 600 community pediatricians , approximately 20% of the respondents did not routinely screen for sleep problems in school - aged children in well - child visit , about 25 percent of routinely screened toddlers and pre - schoolers for snoring , and less than 40 percent questioned adolescents directly about sleep habits , despite the respondents knowledge of the importance of sleep 's impact on health , behavior , and school performance . in another study a validated pediatric sleep questionnaire used to identify a series of children with sleep - related symptoms at two community based general pediatrics clinics and reviewed medical chart notes for the previous 2 years to determine how often sleep problems had been addressed . fewer than 15 percent of patients had current chart notes that mentioned some of the questionnaire - defined sleep problems ; diagnoses were mentioned for two of 86 patients and no treatments were discussed . the consequences of untreated sleep problems may include significant emotional , behavioral , and cognitive dysfunction[711 ] . the impact of childhood sleep problems is further intensified by their direct effect on parents sleep , resulting in parental daytime fatigue , mood disturbances , and a decreased level of effective parenting . after the school entrance such sleep problems impact adversely on behavior , school functioning , and health - related quality of life . surprisingly little attention has been paid to the impact of sleep problems in the vital preschool years and there have not been enough studies investigating etiologic factors of these problems[7 , 12 ] . preschool and school children with habitual snoring were more likely to have sleep - related daytime and nighttime symptoms . but the studies determined no significant association between habitual snoring and poor school performance[7 , 10 ] . a recent study from nigeria concluded that snoring is an important health problem among preschool and school children as a significant percentage of them snore and most of whom are between third and sixth year of life . most of the studies regarding sleep habits in children are from the west ; however , a few asian studies[1214 ] are available and these studies emphasize the effect of culture . in a previous study in iran , the authors first determined the reliability and validity of persian version of the well - known bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring ( bears ) pediatric sleep questionnaire and then designed a pilot study to describe sleep patterns and sleep problems among pre - school and school - aged children in two primary care pediatric clinics in tehran . the purpose of the tehran 's children sleep study ( tcss ) was to investigate the possible relationship between gender and school entrance on sleep complaints . the authors supposed that these mentioned factors might be of value for both practitioners and parents to plan for tailored and more effective interventions to combat sleep problems in boys and girls and in pre - school ( 2 - 6 years of age ) versus school - age children ( 7 - 12 years of age ) . the subjects were pre - school - aged and school - going children between the ages 2 and 12 years who were selected by clustered randomization of families in tehran based on their zip codes . subjects were included randomly if their parents agreed to cooperate in filling bears sleep screening questionnaire ( see the procedure ) . a total of 746 subjects were entered in the study : 325 pre - school - aged children ( 2 - 6 years old group ) and 421 primary school - aged children ( 7 - 12 years old group ) . our study protocol was approved by the ethical board committee of ent , head and neck research center of tehran university of medical sciences , in tehran . it was designed to address the most common sleep issues in toddlers , pre - schoolers , and school - aged children . it is an acronym and incorporates five basic sleep domains : bedtime problems , including difficulty going to bed and falling asleep ; excessive daytime sleepiness , which includes behaviors typically associated with daytime somnolence in children ; awakenings during the night ; regularity of sleep / wake cycles ( bedtime , wake time ) and average sleep duration ; and snoring . reliability and validity of the persian version of the bears questionnaire was assessed in a previous study . , all parents were informed about the importance of sleep problems screening in children and they were asked to sign the consent to participate in the study . questions for pre - school children were answered by their parents ; but for school - age children , some questions were asked from the children themselves . proportions ( prevalence ) of sleep complaints in each b - e - a - r - s category were calculated for pre - school and school - age groups , totally and stratified by gender . as the effect measure , the odds ratio ( or ) plus its 95 percent confidence interval ( 95%ci ) was calculated to compare the effect size of gender on sleep complaints difference in each b - e - a - r - s category . again , the same approach was used to calculate the effects of school attendance on sleep problems . for comparing mean sleep duration between two groups , the subjects were pre - school - aged and school - going children between the ages 2 and 12 years who were selected by clustered randomization of families in tehran based on their zip codes . subjects were included randomly if their parents agreed to cooperate in filling bears sleep screening questionnaire ( see the procedure ) . a total of 746 subjects were entered in the study : 325 pre - school - aged children ( 2 - 6 years old group ) and 421 primary school - aged children ( 7 - 12 years old group ) . our study protocol was approved by the ethical board committee of ent , head and neck research center of tehran university of medical sciences , in tehran . it was designed to address the most common sleep issues in toddlers , pre - schoolers , and school - aged children . it is an acronym and incorporates five basic sleep domains : bedtime problems , including difficulty going to bed and falling asleep ; excessive daytime sleepiness , which includes behaviors typically associated with daytime somnolence in children ; awakenings during the night ; regularity of sleep / wake cycles ( bedtime , wake time ) and average sleep duration ; and snoring . reliability and validity of the persian version of the bears questionnaire was assessed in a previous study . , all parents were informed about the importance of sleep problems screening in children and they were asked to sign the consent to participate in the study . questions for pre - school children were answered by their parents ; but for school - age children , some questions were asked from the children themselves . proportions ( prevalence ) of sleep complaints in each b - e - a - r - s category were calculated for pre - school and school - age groups , totally and stratified by gender . as the effect measure , the odds ratio ( or ) plus its 95 percent confidence interval ( 95%ci ) was calculated to compare the effect size of gender on sleep complaints difference in each b - e - a - r - s category . again , the same approach was used to calculate the effects of school attendance on sleep problems . for comparing mean sleep duration between two groups , bears questionnaire was completed for a total of 746 ( 2 - 12 years old ) children ; 325 consisting of 142 ( 43.7% ) females and 183 ( 56.3% ) males were in pre - school - age group ( 2 - 6 years old ) and 421 with 173 ( 41.1% ) females and 248 ( 58.9% ) males were in primary school - age group ( 7 - 12 years old ) . male to female ratio was not different between the two age groups ( p=0.4 ) . the average age was 3.93 ( 0.16 ) years in pre - school - age group and 9.63 ( 0.16 ) years in primary school - age group . table 1 shows the prevalence of probable or definite problems in each domain of bears , where screening questionnaire and also the comparison of percentages between pre - school and school - aged groups is shown . the most common screening problem in both pre - school and school - aged group was excessive daytime sleepiness ( 64.9% and 62.9% respectively ) . bedtime problems and also regularity and duration of sleep were significantly more prevalent in pre - school - age group ( p<0.0002 ; or=1.98 ; 95% ci : 1.98 - 4.20 ; and or=2.00 ; 95%ci : 1.41 - 2.84 , respectively ) . however , sleep - disordered breathing was lower in pre - school children ( p=0.029 ; or=0.57 ; 95%ci : 0.34 - 0.94 ) . other bears domains - excessive daytime sleepiness and awakening during the night - did not differ significantly between the two groups . comparison of the prevalence of sleep problems in each domain of bears questionnaire among pre - school and school - aged groups odds ratio for the effect of school entrance ; ci : confidence interval ; bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring for pre - school children , mean time for going to bed at night was 22:54 ( sd 1.20 hrs ) , mean wakeup time in the morning was 08:43 ( sd 1.20 hrs ) , and mean sleep duration was 9.81 hours ( sd 1.13 ) . for school - aged children , mean time for going to bed at night in school days was 22:36 ( sd 1.12 hrs ) , mean wakeup time in the morning in school days was 07:11 ( sd 1.12 hrs ) , and mean sleep duration was 8.59 hours ( sd 1.33 ) . the difference between mean sleep duration between pre - school age and school - age groups was statistically significant ( p<0.0001 ) . table 2 shows the comparison between boys and girls for sleep problems in both pre - school and school - aged groups . pre - school boys showed significantly less bedtime problems than pre - school girls ( p=0.003 ; or=0.49 ; 0.30 - 0.79 ) . however , in school - aged group all items were statistically the same between girls and boys . comparison of the prevalence of sleep problems in each domain of bears questionnaire among boys and girls in both pre - school and school - aged groups bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring tables 3 and 4 reveal the comparison of sleep problems between pre - school and school - aged boys and also between pre - school and school - aged girls . among both genders , bedtime problems , and regularity and duration of sleep problems were significantly more prevalent in pre - school - age group ( p=0.0002 and 0.04 respectively ) , ( p=0.003 and 0.0005 respectively ) . comparison of the prevalence of sleep problems in each domain of bears questionnaire among pre - school and school - aged groups in boys odds ratio for the effect of school entrance in boys ; ci : confidence interval ; bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring comparison of the prevalence of sleep problems in each domain of bears questionnaire among pre - school and school - aged groups in girls odds ratio for the effect of school entrance in girls ; bears : bedtime problems , excessive sleepiness , awakenings during the night , regularity of sleep , snoring awakening times during the night and sleep - disordered breathing were more prevalent in school - aged boys ( p=0.0003 and 0.04 respectively ) . it seems that the prevalence of sleep problems according to bears questionnaire is relatively high in our study especially in excessive daytime sleeping domain . the prevalence estimates of sleep problems are very diverse in multiple old and new studies and in different parts of the world and it ranges from 20.7% in a 1989 - 90 survey in headington , oxford by ali et al to almost 40% in a 2010 study in australian indigenous and non - indigenous children by blunden et al . owens et al have reported the following prevalence estimates in their leading study : bedtime issues 16.3% ; nighttime awakenings 18.4% ; and snoring 10.7% ( our relevant estimates are 28.9% , 27.7% , and 7.1% in pre - school children and 12.4% , 44.2% , and 11.9% in school - aged children respectively ) . the more important thing here is the regular assessment of the prevalence estimates at different times using reliable and valid tools to become able to perform trend analyses . the prevalence of sleep problems in these groups of children in our study was also nearly comparable to percentages found in other studies[3 , 18 ] . the reason for the first one is that especially in school - aged children , boys and girls attend separate schools in most parts of our country especially in tehran . some studies also have evaluated the effects of gender on sleep related problems and have found interesting points . simola et al showed that age and gender were related to phenotype of the sleeping problems while there were no gender differences in any sleep parameters in another study conducted by seo et al . few studies have estimated if there is any difference between boys and girls on sleep problems , and as shown in table 2 , there is just a statistically significant difference in one bears domain ( bedtime problems ) between boys and girls . in a study by van litsenburg et al , parents reported that girls experienced more sleep problems than boys , notably for sleep onset delay and daytime sleepiness , while for the child self - reports , no gender difference was found . because the usual starting year for school attendance is 7 years of age , we divided the age range of our subjects to two broad categories shown in tables . considering total subjects , bedtime problems regularity and duration of sleep , and sleep - disordered breathing had statistically significant difference between pre - school and school - aged children . the added value of calculating odds ratio as an effect measure helps us better understand the consequence of going to school on sleep . school entrance seems to exacerbate sleep problems in bedtime problems and also regularity and duration of sleep domains and plays the role of a risk factor . considering males and females separately ( tables 3 and 4 ) , same pattern is seen for males and females in bedtime problems and regularity and duration of sleep ; but , school attendance seems to have a protective effect in awakening during the night domain in both boys and girls . school entrance plays a protective role for sleep - disordered breathing in boys but not in girls . seo et al demonstrated that extracurricular academic activities , is related to the children 's sleep duration and that the older children sleep less than younger children . they thought that the main reason is late bedtimes due to socio - cultural factors , high levels of nighttime and recreational activities , and/or excessive academic activities . they conducted the first study , to their knowledge , to examine the natural history of sleep problems in children over the transition from preschool to school . liu et al believed that unique school schedules and sleep practices may contribute to the differences in the sleep patterns and sleep problems of children from the united states and china . gau et al hypothesized that little sleep at night made the students feel sleepy in the daytime and tired , drowsy , moody and difficult at arising in the morning . compared with our study showing no significant difference of sleep - quality between girls and boys , japanese findings support the hypothesis that during the junior high school period , the majority of sleep - quality indicators in japanese schoolchildren were better for girls than for boys . boys sleep was less efficient and more fragmented during the entire week in comparison to that of the girls . few studies such as the one conducted by kahn et al , have assessed the effect of poor sleeping on school performance and have found interesting results such as the point that among the poor sleepers , 21% had failed 1 or more years at school . school achievement difficulties were encountered significantly more often among the poor sleepers than among the children without sleep problems ( p=.001 ) . although this point of view has not been considered as our hypothesis of research , there seems to be diverse effects from school attendance on different sleep patterns and behaviors that need specific and more rigorous exploration . no study ( including ours ) has assessed the possibility of correlation between the results of answers to five bears domains ; the same fact is also true for correlation between parent - answered questions and child - answered ones . although van litsenburg et al used different questionnaires to study sleep habits and sleep disturbances in dutch children , they were concerned that correlations between parental and self - assessments were low to moderate and this fact may have an effect on bears questionnaire too . they studied the utility of subjective sleep assessment tools for healthy preschool children and compared sleep logs , questionnaires , and actigraphy ; their results showed that sleep schedule variables in the parental reports generally correlated well with actigraphic assessment of sleep patterns and although the daily sleep log was better correlated with actigraphy , the brief questionnaire showed a good correlation with sleep pattern on weekday actigraphic assessments . we hypothesized that high prevalence of sleep problems in school aged and pre - school aged children of tehran is due to bad sleep time that we think is caused by our culture and loss of enough family attention and related ignorance on the harmfulness of poor sleep of children . moreover , the poor sleep quality of iranian preschool children is probably due to cultural characteristics , climate differences , or harmful sleep habits . the difference between mean sleep duration between pre - school age and school - age groups is important ( as it is statistically significant in our study ) because it affects the risk of becoming overweight in later years . ochiai et al reported that short sleep duration increases the risk of becoming overweight and this relationship differs between the two sexes . cross sectional design limited our causative assessment for sleep problems , more- over we did not include some familial factors such as socioeconomic factors and psychological problems in the families . we have categorized the subjects to two groups according to their age and took the whole 2 - 6 years as one group and the 7 - 12 years as another . this type of unification may have an effect on the final results , but we have tried to conduct a comparable scenario to the work of owens et al , and also to our previous pilot study . higher ses might have negative impact on total sleep duration , sleep hygiene and also on children 's psychological well - being . the present survey shows that the prevalence of sleep problems is relatively high especially in excessive daytime sleeping domain of preschool - aged and school children . our findings suggest a development of a more tailored sleep - related health service to help both preschool and school children and also assist parents to know and tackle sleep troubles more effectively and thoroughly .

health services and health policy research can be based on qualitative research methods , especially when they deal with a rapid change and develop a more fully integrated theory base and research agenda . however , the field must be with the best traditions and techniques of qualitative methods and should distinguish the essentiality of special training and experience in applying these methods . health knowledge must also include interpretive action to maintain scientific quality when research methods are applied . qualitative and quantitative strategies should be seen as complementary rather than being thought of as incompatible . although the procedures of interpreting texts are different from those of statistical analysis , due to their different type of data and questions to be answered , the underlying scientific principles are very much the same . while working for more than a decade as qualitative designer , khankeh faced a lot of challenges in conducting qualitative research in the field of health which occupied the mind of other health researchers . therefore , this article contributes to the discussion of challenges related to qualitative research in healthcare in the light of personal experiences of a researcher conducting purely qualitative health research . qualitative research methods involve systematic collection , organizing , and interpretation of material in textual form derived from talk or observations . they are useful to explore the meanings of social phenomena as experienced by individuals in their natural context . the health community still looks at qualitative research with skepticism and accuses it for the subjective nature and absence of facts . scientific standards , criteria and checklists do exist and the adequacy of guidelines has been vigorously debated within this cross - disciplinary field . clinical knowledge consists of interpretive action and interaction factors that involve communication , shared opinions , and experiences . the current quantitative research methods indicate a confined access to clinical knowledge , since they insert only the questions and phenomena that can be controlled , measured , and are countable where it is necessary to investigate , share and contest the tacit knowledge of an experienced practitioner . it is concerned with increased understanding of the meaning of certain conditions for health professionals and patients , and how their relationships are built in a particular social context . these kinds of research allow exploration of the social events as experienced by individuals in their natural context . qualitative inquiry could contribute to a broader understanding of health science considering the substantial congruence between the core elements of health practice and the principles underpinning qualitative research . corbin ( 2008 ) reported that in the past 10 years , the interest in qualitative methods in general and grounded theory in particular has burgeoned according to a review of the literature and dissertation abstracts . a researcher engaged in qualitative research will be confronted with a number of challenges . identifying the research problem and forming the research question are some of the initial challenges that researchers encounter in the early stages of a qualitative research project . researchers and students sometimes fail to understand that adopting a qualitative approach is only the first stage in the process of selecting an appropriate research methodology . once the initial research question has been identified , the crucial decision to be made is on the selection of an appropriate method , such as content analysis , ethnography , or grounded theory , and selecting the research design as well . subsequent arrangements would be on the proper methods of data collection , participants , and the research setting , according to the methodology and the research question . qualitative researchers should also handle other important concerns such as data analysis , ethical issues , and rigor methods of results . in this paper , we are going to discuss important practical challenges of qualitative inquiry in health and the challenges faced by researchers using interpretive research methodologies . it is important to provide an honest and concise appreciation of the essential characteristics of the qualitative research before discussing the challenges of the interpretive research approach to studies in health . qualitative research does not promise a clear or direct and orderly method of tackling research problems in health studies . it does not provide researchers with a set of rules to be followed or give them a comforting sense of security and safety backup against possible mistakes on the road to knowledge . this research method depends on the power of words and images , but does not offer the assimilated meanings such as numbers and equations ; it is rather an attentive search of meaning and understanding and an attempt for profound comprehension and awareness of the problems and phenomena . the essentially diagnostic and exploratory nature of qualitative research is invaluable in developing conceptualizations in health as an evolving discipline . it tenders the possible tap into the sea of complex interactions in health that can be as follows . researchers launch the quest for new theories in health which should acknowledge that qualitative research is an approach rather than a particular set of techniques , and its appropriateness derives from the nature of the social phenomena to be explored . in qualitative research , knowledge derives from the context - specific perspective on the experienced phenomena , interpretations , and explanation of social experiences . the approach is helpful in understanding human experiences , which is important for health professionals who focus on caring , communication , and interaction . many potential researchers intend to find the answer to the questions about a problem or a major issue in clinical practice or quantitative research can not verify them . in fact , they choose qualitative research for some significant reasons : the emotions , perceptions , and actions of people who suffer from a medical condition can be understood by qualitative researchthe meanings of health professions will only be uncovered through observing the interactions of professionals with clients and interviewing about their experience . this is also applicable to the students destined for the healthcare fieldqualitative research is individualized ; hence , researchers consider the participants as whole human beings , not as a bunch of physical compartmentsobservation and asking people are the only ways to understand the causes of particular behaviors . therefore , this type of research can develop health or education policies ; policies for altering health behavior can only be effective if the behavior 's basis is clearly understood . the emotions , perceptions , and actions of people who suffer from a medical condition can be understood by qualitative research the meanings of health professions will only be uncovered through observing the interactions of professionals with clients and interviewing about their experience . this is also applicable to the students destined for the healthcare field qualitative research is individualized ; hence , researchers consider the participants as whole human beings , not as a bunch of physical compartments observation and asking people are the only ways to understand the causes of particular behaviors . therefore , this type of research can develop health or education policies ; policies for altering health behavior can only be effective if the behavior 's basis is clearly understood . before adhering to a distinct research methodology , researchers have to exactly understand the nature and character of their inquiries and the knowledge they choose to create . however , all defects and challenges of qualitative research should be realized rather than discarded as a compelling way to knowledge structure . new endeavors in excellent academic achievement and building new tradition of qualitative research in health can be facilitated through acknowledging traps and clarifying the real practical challenges . finally , qualitative research provides investigators with the tools to study the health phenomena from the perspective of those experiencing them . this approach is especially applied in situ ations that have not been previously studied , where major gaps exists in research field , and when there is a need for a new perspective to be identified for the arena of health care intervention . based on corbin and strauss ( 2008 ) , committed qualitative researchers lean toward qualitative work because they are drawn to the fluid , evolving , and dynamic nature of this approach in contrast to the more rigid and structured format of quantitative methods . it is the endless possibilities to learn more about people that qualitative researchers resonate to . it is not distance that qualitative researchers want between themselves and their participants , but the opportunity to connect with them at a human level ( epistemology ) . qualitative researchers have a natural curiosity that leads them to study worlds that interest them and that they otherwise might not have access to . furthermore , qualitative researchers enjoy playing with words , making order out of seeming disorder , and thinking in terms of complex relationships . for them , doing qualitative research is a challenge that brings the whole self into the process . researchers select approaches and methodology based on some scientific logics , not on being easy or interesting . the nature and type of the research question or problem ; the researcher 's epistemological stance , capabilities , knowledge , skills , and training ; and the resources available for the research project are the criteria upon which adopting methodology and procedures depend . inconsistency between research question and methodology , insufficient methodological knowledge , and lack of attention on philosophical underpinning of qualitative methodology can be mentioned as some important challenges here . there are several different ways of qualitative research and researchers will have to select between various approaches . the qualitative research is based on the theoretical and philosophical assumptions that researchers try to understand . then , the research methodology and process should be chosen to be consistent with these basic assumptions and the research question as well . some researchers believe that there is no need to study the methodology and methods before beginning the research . many researchers neglect to gain this knowledge because they are not aware of the qualitative inquiry complexities which make them go wrong . for instance , lack of information about interview , qualitative data analysis , or sampling is very common . my experience shows that lack of knowledge , experience , and skills in a research team to do qualitative research can hinder the formation of original knowledge and improvement in understanding the phenomenon under study . the result of such a study will not be new and interesting , and even the study process will be very mechanical without good interpretation or enough exploration . sometimes there is an inconsistency between research question , research methodology , and basic philosophical assumptions , and the researchers fail to justify their methods of choice in line with the research question and the ontological and epidemiological assumptions . finally , the researcher 's intentions , the aims of the research question / inquiry , and the chosen approach are regarded as the most important reasons to select a qualitative research method consistent with them and their underpinning philosophical assumptions as well . qualitative research is exciting because it asks questions about people 's everyday lives and experiences . a qualitative researcher will have the chance of discovering the significant truths in the lives of people . that is a wonderful privilege , but you need to get those questions right if you dig into people 's lives and ask about their real experiences . an adequate and explicit research question , or a set of interrelated questions , builds the basis for a good research . but excellent research questions are not easy to write at all . a good research requires a good research question as well because it allows us to identify what we really want to know . however , at the beginning of a project , researchers may be uncertain about what exactly they intend to know , so vague questions can lead to an unfocused project . common problems coming up with a research question include : deciding about the research area among a range of issues that are heeded in your field of interestnot capable of pointing toward any interesting area or topic sufficient to focus a major piece of work onknowing about the area you want to concentrate on ( e.g. emergency ) , but not a certain topicknowing what area and topic is specifically difficult to articulate a clear question . deciding about the research area among a range of issues that are heeded in your field of interest not capable of pointing toward any interesting area or topic sufficient to focus a major piece of work on knowing about the area you want to concentrate on ( e.g. emergency ) , but not a certain topic knowing what area and topic is specifically difficult to articulate a clear question . having identified a research area , your next step will be to identify a topic within that interesting area . suggestions for future work at the end of a paper you have found interesting . moreover , you can search for some verifiable gaps through literature review , or based on your personal or professional experience and expert opinion , which should be studied . therefore , all the previous studies that have already been conducted in the area are considered as important . in this way , you do not run the risk of asking a research question that has already been addressed and/or answered . based on my experience , novice researchers have some problems finding the right topics in their field of interest because they do not perform a broad literature review to find the gaps and problems suitable to be investigated . sometimes their field of interest is different from that of their supervisors or there are no experts to help them in this regard . although the topic may retain your interest and you may be committed to undertake such a study , it is important to recognize that some topics of personal relevance may also be deeply significant and difficult to research . finally you need to make sure that your topic of interest is the one that you can actually study within the project constraints such as time and fund . once you have identified your interesting topic for research ( according to a broad literature review , personal and professional experience , and/or expert opinion ) , you can begin to create a research question . forming the research question is one of the initial challenges that researchers encounter in the early stages of a research project . therefore , it acquires significance by the very fact that it provides brief , but nevertheless , important information on the research topic that allows the reader to decide if the topic is relevant , researchable , and a remarkable issue . furthermore , the research question in qualitative studies has an additional significance as it determines the manner of conducting the study . the qualitative research question delineates the procedures that are executed in the study and provides a map to the readers by which they can trail the researcher 's intentions and actions in the study . therefore , special attention is needed on how a qualitative research question will specifically be structured , organized , and formed in the way to quote the necessary information and elements that allow the readers to assess and evaluate the study . the formation of a qualitative research question acquires a basic conducting role for the study and a fundamental function to develop an audit trail that can empower the readers to judge the value , rigor , and validity of the whole research project . hence , researchers should not only pay special attention toward developing a significant and relevant question , but also formulate it properly . the qualitative research question must be provided in such a way as to impart , reflect , and conjoin the theoretical and abstract assumptions with the practical and pragmatic means of attaining them . in plain words , a good qualitative research question implicates particular phrasing , whereas the order of words should make the topic of interest amenable to the qualitative quest . the researcher has to concentrate on how the content of the research topic is understood when phrasing the qualitative research questions , adhering to the topic with the philosophical / theoretical suggestions and to the structure of the study which requires compounding specific principal elements . also , the content provides a brief focus on the issue to be investigated , but does not define the exact relationship of the variables to make these relationships flexible in emanating from the study according to the qualitative research theory . the qualitative research question incepts necessarily with an active verb like understanding , exploring , interpreting , constructing , explaining , describing , etc . , to reflect the paradigm / philosophy underpinning the qualitative study . consequently , specific nouns that represent the aims of qualitative studies , such as experiences , feelings , views , perspectives , knowledge , etc . finally , the methodology or method should appear in the qualitative research question coherent with them . meanwhile , the structure of a good qualitative research question will address five of the following six : who , when , where , what , how , and why , and the entire research question should devise the sixth element . for instance , exploring the experiences of self - immolated women regarding their motives for attempting suicide : a qualitative content analysis study in kermanshah iran make sure that your research question is consistent with the approach you are adopting . it is like an easy trap if you decide about the research question before considering the proper way by which you are intending to make assumptions and analyze your data . my experiences show that novice researchers formulate their research question without considering the approach of their study in a proper way and usually their research questions are very broad , unclear , and vague . since the intention of their studies is not completely clear at the beginning , they can not decide about the research approach ; also , they have to change their research question and take different directions in the course of study or they will end up without adequate results that can help readers or consumers improve their understanding or solve the problem . although a researcher initiates a study with a general question and topic , the interesting aspect of qualitative research is that the questions , which are more specific and can help in further data collection and analysis , arise during the course of the study . thus , a qualitative research question can be broadly , rather than narrowly , focused in the beginning . qualitative research is cyclic , which means that the research question in this approach immerses gradually into the topic . it means that when you come to know more and more about your topic , your ideas develop about what to focus , either through reading , thinking about what you have read , or in early stages of data analysis . finally , it is literature review , general reading , and discussion with an expert supervisor that can help you find the right topic . if the background knowledge is poor at the beginning of the study , broad but clear research question can be reasonable . research question may become more focused or develop in a different direction according to more reading and/or preliminary data analysis . a clear and focused research question is articulated and used to conduct further analysis and any future literature reviews necessary for the final write - up . however , it is very important to take time to choose a research question , because it can be a very challenging exercise . actually , the ultimate success of the project depends on selecting a clear and convenient question . the question should be appropriate for the qualitative research and for the specific approach you choose which must be grounded in research . it must ask precisely what you want to find out and be articulated and clear . knowing this will help you plan your project . crucial decisions need to be made about an appropriate methodology , such as ethnography or grounded theory , after identifying the initial research question . the main concern of novice researchers is to find the reason and appropriate design to do the research , and proper methodology to answer the question . researchers ought to figure out about the planning of qualitative research and how to choose the methodology . researchers sometimes fail to understand that in the process of selecting an adequate research methodology , adopting a qualitative approach is only the first stage . students , and sometimes researchers , choose qualitative research because they think it is easier to use than the other methodologies . but this reasoning is fumble since qualitative research is a complex methodology where data collection and analysis can be mostly challenging . sometimes lack of planning and inadequate attention paid to the properness of the selected approach considering the purpose of research will be problematic . for new qualitative researchers , it often seems that the researcher should totally concentrate on the dual process of data collection and data analysis . it is very important to consider thorough planning in all stages of the research process , from developing the question to the final write - up of the findings for publication . the research design and methodology must be adequate to address the selected topics and the research question . researchers have to identify , describe , and justify the methodology they chose , besides the strategies and procedures involved . so , it is pivotal to find the proper method for the research question . it should be noticed that some of the details of a qualitative research project can not be ascertained in advance and may be specified as they arise during the research process . an important problem for novice researchers is the little acknowledgement of different approaches that address different kinds and levels of questions and take a different stance on the kind of phenomena which is focused upon . the need for consistency and coherence becomes more obvious when we consider the risk of something called method - slurring . each approach has to demonstrate its consistency to its foundations and will reflect them in data collection , analysis , and knowledge claim . it may be important to acknowledge the distinctive features by specific approaches such as phenomenology or grounded theory at some levels such as the type of question they are suited to answer , data collection methods they are consistent with , and also the kinds of analysis and presentation of the results that fit within the approach such as goodness of fit or logical staged linking and can be referred to as consistency . if such consistency occurs , then the whole thing hangs together as coherent ; that is , the kind of knowledge generated in the results or presentation section doing what is said it would do following the aims of the project . in order to consider these criteria of consistency and coherence in greater detail , we need to look at the distinctive differences between qualitative approaches in the following : the aims of the research approach , its roots in different disciplines and ideologies , the knowledge claims linked to it , and to a lesser extent , the data collection and analysis specific to each approach . my experience shows that novice researchers have some problems to justify their methodology of choice and sometimes they experience some degree of methodological slurring . they do not have any clear understanding of the research process in terms of data gathering strategies , data analysis method , and even appropriate sampling plan , which should be indentified based on philosophical and methodological principles . finally , besides the above - mentioned problems , regarding research design , there are two common problems encountered especially by students who want to do qualitative study ; sometimes researchers and research team try to identify everything , even the sample size , in advance when they design their study because they have a strong background of quantitative research , and this is completely in contrast with the flexible nature and explorative approach of qualitative research . the other problem is the examination committee and the format of proposal of grant sites and funding agencies , which are based on the principles of quantitative study . so , flexibility is regarded as the most important credibility criterion in all kinds of qualitative research and it should be considered when designing the study and following its process . qualitative research does not promise a clear or direct and orderly method of tackling research problems in health studies . it does not provide researchers with a set of rules to be followed or give them a comforting sense of security and safety backup against possible mistakes on the road to knowledge . this research method depends on the power of words and images , but does not offer the assimilated meanings such as numbers and equations ; it is rather an attentive search of meaning and understanding and an attempt for profound comprehension and awareness of the problems and phenomena . the essentially diagnostic and exploratory nature of qualitative research is invaluable in developing conceptualizations in health as an evolving discipline . it tenders the possible tap into the sea of complex interactions in health that can be as follows . researchers launch the quest for new theories in health which should acknowledge that qualitative research is an approach rather than a particular set of techniques , and its appropriateness derives from the nature of the social phenomena to be explored . in qualitative research , knowledge derives from the context - specific perspective on the experienced phenomena , interpretations , and explanation of social experiences . the approach is helpful in understanding human experiences , which is important for health professionals who focus on caring , communication , and interaction . many potential researchers intend to find the answer to the questions about a problem or a major issue in clinical practice or quantitative research can not verify them . in fact , they choose qualitative research for some significant reasons : the emotions , perceptions , and actions of people who suffer from a medical condition can be understood by qualitative researchthe meanings of health professions will only be uncovered through observing the interactions of professionals with clients and interviewing about their experience . this is also applicable to the students destined for the healthcare fieldqualitative research is individualized ; hence , researchers consider the participants as whole human beings , not as a bunch of physical compartmentsobservation and asking people are the only ways to understand the causes of particular behaviors . therefore , this type of research can develop health or education policies ; policies for altering health behavior can only be effective if the behavior 's basis is clearly understood . the emotions , perceptions , and actions of people who suffer from a medical condition can be understood by qualitative research the meanings of health professions will only be uncovered through observing the interactions of professionals with clients and interviewing about their experience . this is also applicable to the students destined for the healthcare field qualitative research is individualized ; hence , researchers consider the participants as whole human beings , not as a bunch of physical compartments observation and asking people are the only ways to understand the causes of particular behaviors . therefore , this type of research can develop health or education policies ; policies for altering health behavior can only be effective if the behavior 's basis is clearly understood . before adhering to a distinct research methodology , researchers have to exactly understand the nature and character of their inquiries and the knowledge they choose to create . however , all defects and challenges of qualitative research should be realized rather than discarded as a compelling way to knowledge structure . new endeavors in excellent academic achievement and building new tradition of qualitative research in health can be facilitated through acknowledging traps and clarifying the real practical challenges . finally , qualitative research provides investigators with the tools to study the health phenomena from the perspective of those experiencing them . this approach is especially applied in situ ations that have not been previously studied , where major gaps exists in research field , and when there is a need for a new perspective to be identified for the arena of health care intervention . based on corbin and strauss ( 2008 ) , committed qualitative researchers lean toward qualitative work because they are drawn to the fluid , evolving , and dynamic nature of this approach in contrast to the more rigid and structured format of quantitative methods . it is the endless possibilities to learn more about people that qualitative researchers resonate to . it is not distance that qualitative researchers want between themselves and their participants , but the opportunity to connect with them at a human level ( epistemology ) . qualitative researchers have a natural curiosity that leads them to study worlds that interest them and that they otherwise might not have access to . furthermore , qualitative researchers enjoy playing with words , making order out of seeming disorder , and thinking in terms of complex relationships . for them , doing qualitative research is a challenge that brings the whole self into the process . researchers select approaches and methodology based on some scientific logics , not on being easy or interesting . the nature and type of the research question or problem ; the researcher 's epistemological stance , capabilities , knowledge , skills , and training ; and the resources available for the research project are the criteria upon which adopting methodology and procedures depend . inconsistency between research question and methodology , insufficient methodological knowledge , and lack of attention on philosophical underpinning of qualitative methodology can be mentioned as some important challenges here . there are several different ways of qualitative research and researchers will have to select between various approaches . the qualitative research is based on the theoretical and philosophical assumptions that researchers try to understand . then , the research methodology and process should be chosen to be consistent with these basic assumptions and the research question as well . some researchers believe that there is no need to study the methodology and methods before beginning the research . many researchers neglect to gain this knowledge because they are not aware of the qualitative inquiry complexities which make them go wrong . for instance , lack of information about interview , qualitative data analysis , or sampling is very common . my experience shows that lack of knowledge , experience , and skills in a research team to do qualitative research can hinder the formation of original knowledge and improvement in understanding the phenomenon under study . the result of such a study will not be new and interesting , and even the study process will be very mechanical without good interpretation or enough exploration . sometimes there is an inconsistency between research question , research methodology , and basic philosophical assumptions , and the researchers fail to justify their methods of choice in line with the research question and the ontological and epidemiological assumptions . finally , the researcher 's intentions , the aims of the research question / inquiry , and the chosen approach are regarded as the most important reasons to select a qualitative research method consistent with them and their underpinning philosophical assumptions as well . qualitative research is exciting because it asks questions about people 's everyday lives and experiences . a qualitative researcher will have the chance of discovering the significant truths in the lives of people . that is a wonderful privilege , but you need to get those questions right if you dig into people 's lives and ask about their real experiences . an adequate and explicit research question , or a set of interrelated questions , builds the basis for a good research . but excellent research questions are not easy to write at all . a good research requires a good research question as well because it allows us to identify what we really want to know . however , at the beginning of a project , researchers may be uncertain about what exactly they intend to know , so vague questions can lead to an unfocused project . common problems coming up with a research question include : deciding about the research area among a range of issues that are heeded in your field of interestnot capable of pointing toward any interesting area or topic sufficient to focus a major piece of work onknowing about the area you want to concentrate on ( e.g. emergency ) , but not a certain topicknowing what area and topic is specifically difficult to articulate a clear question . deciding about the research area among a range of issues that are heeded in your field of interest not capable of pointing toward any interesting area or topic sufficient to focus a major piece of work on knowing about the area you want to concentrate on ( e.g. emergency ) , but not a certain topic knowing what area and topic is specifically difficult to articulate a clear question . having identified a research area , your next step will be to identify a topic within that interesting area . suggestions for future work at the end of a paper you have found interesting . moreover , you can search for some verifiable gaps through literature review , or based on your personal or professional experience and expert opinion , which should be studied . therefore , all the previous studies that have already been conducted in the area are considered as important . in this way , you do not run the risk of asking a research question that has already been addressed and/or answered . based on my experience , novice researchers have some problems finding the right topics in their field of interest because they do not perform a broad literature review to find the gaps and problems suitable to be investigated . sometimes their field of interest is different from that of their supervisors or there are no experts to help them in this regard . although the topic may retain your interest and you may be committed to undertake such a study , it is important to recognize that some topics of personal relevance may also be deeply significant and difficult to research . finally you need to make sure that your topic of interest is the one that you can actually study within the project constraints such as time and fund . once you have identified your interesting topic for research ( according to a broad literature review , personal and professional experience , and/or expert opinion ) , you can begin to create a research question . forming the research question is one of the initial challenges that researchers encounter in the early stages of a research project . therefore , it acquires significance by the very fact that it provides brief , but nevertheless , important information on the research topic that allows the reader to decide if the topic is relevant , researchable , and a remarkable issue . furthermore , the research question in qualitative studies has an additional significance as it determines the manner of conducting the study . the qualitative research question delineates the procedures that are executed in the study and provides a map to the readers by which they can trail the researcher 's intentions and actions in the study . therefore , special attention is needed on how a qualitative research question will specifically be structured , organized , and formed in the way to quote the necessary information and elements that allow the readers to assess and evaluate the study . the formation of a qualitative research question acquires a basic conducting role for the study and a fundamental function to develop an audit trail that can empower the readers to judge the value , rigor , and validity of the whole research project . hence , researchers should not only pay special attention toward developing a significant and relevant question , but also formulate it properly . the qualitative research question must be provided in such a way as to impart , reflect , and conjoin the theoretical and abstract assumptions with the practical and pragmatic means of attaining them . in plain words , a good qualitative research question implicates particular phrasing , whereas the order of words should make the topic of interest amenable to the qualitative quest . the researcher has to concentrate on how the content of the research topic is understood when phrasing the qualitative research questions , adhering to the topic with the philosophical / theoretical suggestions and to the structure of the study which requires compounding specific principal elements . also , the content provides a brief focus on the issue to be investigated , but does not define the exact relationship of the variables to make these relationships flexible in emanating from the study according to the qualitative research theory . the qualitative research question incepts necessarily with an active verb like understanding , exploring , interpreting , constructing , explaining , describing , etc . consequently , specific nouns that represent the aims of qualitative studies , such as experiences , feelings , views , perspectives , knowledge , etc . finally , the methodology or method should appear in the qualitative research question coherent with them . meanwhile , the structure of a good qualitative research question will address five of the following six : who , when , where , what , how , and why , and the entire research question should devise the sixth element . for instance , exploring the experiences of self - immolated women regarding their motives for attempting suicide : a qualitative content analysis study in kermanshah iran make sure that your research question is consistent with the approach you are adopting . it is like an easy trap if you decide about the research question before considering the proper way by which you are intending to make assumptions and analyze your data . my experiences show that novice researchers formulate their research question without considering the approach of their study in a proper way and usually their research questions are very broad , unclear , and vague . since the intention of their studies is not completely clear at the beginning , they can not decide about the research approach ; also , they have to change their research question and take different directions in the course of study or they will end up without adequate results that can help readers or consumers improve their understanding or solve the problem . although a researcher initiates a study with a general question and topic , the interesting aspect of qualitative research is that the questions , which are more specific and can help in further data collection and analysis , arise during the course of the study . thus , a qualitative research question can be broadly , rather than narrowly , focused in the beginning . qualitative research is cyclic , which means that the research question in this approach immerses gradually into the topic . it means that when you come to know more and more about your topic , your ideas develop about what to focus , either through reading , thinking about what you have read , or in early stages of data analysis . finally , it is literature review , general reading , and discussion with an expert supervisor that can help you find the right topic . if the background knowledge is poor at the beginning of the study , broad but clear research question can be reasonable . research question may become more focused or develop in a different direction according to more reading and/or preliminary data analysis . a clear and focused research question is articulated and used to conduct further analysis and any future literature reviews necessary for the final write - up . however , it is very important to take time to choose a research question , because it can be a very challenging exercise . actually , the ultimate success of the project depends on selecting a clear and convenient question . the question should be appropriate for the qualitative research and for the specific approach you choose which must be grounded in research . it must ask precisely what you want to find out and be articulated and clear . knowing this will help you plan your project . having identified a research area , your next step will be to identify a topic within that interesting area . suggestions for future work at the end of a paper you have found interesting . moreover , you can search for some verifiable gaps through literature review , or based on your personal or professional experience and expert opinion , which should be studied . therefore , all the previous studies that have already been conducted in the area are considered as important . in this way , you do not run the risk of asking a research question that has already been addressed and/or answered . based on my experience , novice researchers have some problems finding the right topics in their field of interest because they do not perform a broad literature review to find the gaps and problems suitable to be investigated . sometimes their field of interest is different from that of their supervisors or there are no experts to help them in this regard . although the topic may retain your interest and you may be committed to undertake such a study , it is important to recognize that some topics of personal relevance may also be deeply significant and difficult to research . finally you need to make sure that your topic of interest is the one that you can actually study within the project constraints such as time and fund . once you have identified your interesting topic for research ( according to a broad literature review , personal and professional experience , and/or expert opinion ) , you can begin to create a research question . forming the research question is one of the initial challenges that researchers encounter in the early stages of a research project . therefore , it acquires significance by the very fact that it provides brief , but nevertheless , important information on the research topic that allows the reader to decide if the topic is relevant , researchable , and a remarkable issue . furthermore , the research question in qualitative studies has an additional significance as it determines the manner of conducting the study . the qualitative research question delineates the procedures that are executed in the study and provides a map to the readers by which they can trail the researcher 's intentions and actions in the study . therefore , special attention is needed on how a qualitative research question will specifically be structured , organized , and formed in the way to quote the necessary information and elements that allow the readers to assess and evaluate the study . the formation of a qualitative research question acquires a basic conducting role for the study and a fundamental function to develop an audit trail that can empower the readers to judge the value , rigor , and validity of the whole research project . hence , researchers should not only pay special attention toward developing a significant and relevant question , but also formulate it properly . the qualitative research question must be provided in such a way as to impart , reflect , and conjoin the theoretical and abstract assumptions with the practical and pragmatic means of attaining them . in plain words , a good qualitative research question implicates particular phrasing , whereas the order of words should make the topic of interest amenable to the qualitative quest . the researcher has to concentrate on how the content of the research topic is understood when phrasing the qualitative research questions , adhering to the topic with the philosophical / theoretical suggestions and to the structure of the study which requires compounding specific principal elements . also , the content provides a brief focus on the issue to be investigated , but does not define the exact relationship of the variables to make these relationships flexible in emanating from the study according to the qualitative research theory . the qualitative research question incepts necessarily with an active verb like understanding , exploring , interpreting , constructing , explaining , describing , etc . , to reflect the paradigm / philosophy underpinning the qualitative study . consequently , specific nouns that represent the aims of qualitative studies , such as experiences , feelings , views , perspectives , knowledge , etc . finally , the methodology or method should appear in the qualitative research question coherent with them . meanwhile , the structure of a good qualitative research question will address five of the following six : who , when , where , what , how , and why , and the entire research question should devise the sixth element . for instance , exploring the experiences of self - immolated women regarding their motives for attempting suicide : a qualitative content analysis study in kermanshah iran make sure that your research question is consistent with the approach you are adopting . it is like an easy trap if you decide about the research question before considering the proper way by which you are intending to make assumptions and analyze your data . my experiences show that novice researchers formulate their research question without considering the approach of their study in a proper way and usually their research questions are very broad , unclear , and vague . since the intention of their studies is not completely clear at the beginning , they can not decide about the research approach ; also , they have to change their research question and take different directions in the course of study or they will end up without adequate results that can help readers or consumers improve their understanding or solve the problem . although a researcher initiates a study with a general question and topic , the interesting aspect of qualitative research is that the questions , which are more specific and can help in further data collection and analysis , arise during the course of the study . thus , a qualitative research question can be broadly , rather than narrowly , focused in the beginning . qualitative research is cyclic , which means that the research question in this approach immerses gradually into the topic . it means that when you come to know more and more about your topic , your ideas develop about what to focus , either through reading , thinking about what you have read , or in early stages of data analysis . finally , it is literature review , general reading , and discussion with an expert supervisor that can help you find the right topic . if the background knowledge is poor at the beginning of the study , broad but clear research question can be reasonable . research question may become more focused or develop in a different direction according to more reading and/or preliminary data analysis . a clear and focused research question is articulated and used to conduct further analysis and any future literature reviews necessary for the final write - up . however , it is very important to take time to choose a research question , because it can be a very challenging exercise . actually , the ultimate success of the project depends on selecting a clear and convenient question . the question should be appropriate for the qualitative research and for the specific approach you choose which must be grounded in research . it must ask precisely what you want to find out and be articulated and clear . knowing this will help you plan your project . crucial decisions need to be made about an appropriate methodology , such as ethnography or grounded theory , after identifying the initial research question . the main concern of novice researchers is to find the reason and appropriate design to do the research , and proper methodology to answer the question . researchers ought to figure out about the planning of qualitative research and how to choose the methodology . researchers sometimes fail to understand that in the process of selecting an adequate research methodology , adopting a qualitative approach is only the first stage . students , and sometimes researchers , choose qualitative research because they think it is easier to use than the other methodologies . but this reasoning is fumble since qualitative research is a complex methodology where data collection and analysis can be mostly challenging . sometimes lack of planning and inadequate attention paid to the properness of the selected approach considering the purpose of research will be problematic . for new qualitative researchers , it often seems that the researcher should totally concentrate on the dual process of data collection and data analysis . it is very important to consider thorough planning in all stages of the research process , from developing the question to the final write - up of the findings for publication . the research design and methodology must be adequate to address the selected topics and the research question . researchers have to identify , describe , and justify the methodology they chose , besides the strategies and procedures involved . it should be noticed that some of the details of a qualitative research project can not be ascertained in advance and may be specified as they arise during the research process . an important problem for novice researchers is the little acknowledgement of different approaches that address different kinds and levels of questions and take a different stance on the kind of phenomena which is focused upon . the need for consistency and coherence becomes more obvious when we consider the risk of something called method - slurring . each approach has to demonstrate its consistency to its foundations and will reflect them in data collection , analysis , and knowledge claim . it may be important to acknowledge the distinctive features by specific approaches such as phenomenology or grounded theory at some levels such as the type of question they are suited to answer , data collection methods they are consistent with , and also the kinds of analysis and presentation of the results that fit within the approach such as goodness of fit or logical staged linking and can be referred to as consistency . if such consistency occurs , then the whole thing hangs together as coherent ; that is , the kind of knowledge generated in the results or presentation section doing what is said it would do following the aims of the project . in order to consider these criteria of consistency and coherence in greater detail , we need to look at the distinctive differences between qualitative approaches in the following : the aims of the research approach , its roots in different disciplines and ideologies , the knowledge claims linked to it , and to a lesser extent , the data collection and analysis specific to each approach . my experience shows that novice researchers have some problems to justify their methodology of choice and sometimes they experience some degree of methodological slurring . they do not have any clear understanding of the research process in terms of data gathering strategies , data analysis method , and even appropriate sampling plan , which should be indentified based on philosophical and methodological principles . finally , besides the above - mentioned problems , regarding research design , there are two common problems encountered especially by students who want to do qualitative study ; sometimes researchers and research team try to identify everything , even the sample size , in advance when they design their study because they have a strong background of quantitative research , and this is completely in contrast with the flexible nature and explorative approach of qualitative research . the other problem is the examination committee and the format of proposal of grant sites and funding agencies , which are based on the principles of quantitative study . so , flexibility is regarded as the most important credibility criterion in all kinds of qualitative research and it should be considered when designing the study and following its process . qualitative research focuses on social world and provides investigators with the tools to study health phenomena from the perspective of those experiencing them . identifying the research problem , forming the research question , and selecting an appropriate methodology and research design are some of the initial challenges that researchers encounter in the early stages of a qualitative research project . once the research problem and the initial research question are identified , the crucial decision has to be made in selecting the appropriate methodology . subsequent arrangements would be on the proper methods of data collection , and choosing the participants and the research setting according to the methodology and the research question . it is highly recommended that the researchers exactly understand the nature and character of their inquiries and the knowledge they choose to create before adhering to a distinct research methodology based on scientific knowledge . the essence and type of the research question or problem , the researcher 's epistemological stance , capabilities , knowledge , skills and training , and the resources available for the research project are the criteria upon which the adopting methodology and procedures depend . inconsistency between research question and methodology , insufficient methodological knowledge , and lack of attention to the philosophical underpinning of qualitative methodology are some important challenges . lack of knowledge , experience , and skills to do qualitative research can hinder the formation of original knowledge and improvement in understanding the phenomenon under study . the result of such a study will not be new and interesting , and even the study process will be very mechanical without good interpretation or enough exploration . a good research requires a good research question as well because it allows us to identify what we really want to know . however , at the beginning of a project , researchers may be wavering about what they exactly intend to know ; so , vague questions can lead to an unfocused project . broad literature review , personal and professional experience , and/or expert opinion can be regarded as the main sources to identify interesting research topics and research questions as well . forming the research question is one of the initial challenges that researchers encounter in the early stages of a research project . therefore , it acquires significance by the very fact that it provides brief , but nevertheless , important information on the research topic that allows the reader to decide if the topic is relevant , researchable , and a remarkable issue that can help the researcher to determine the manner of conducting the study . then crucial decisions need to be made about an appropriate methodology . the main concern of novice researchers is to find the reason and appropriate design to do the research and the proper methodology to answer the question . researchers first ought to figure out the planning of qualitative research and how to choose the methodology . it is very important to consider thorough planning in all stages of the research process , from developing the question to final write - up of the findings for publication . it is worth knowing that some of the details of a qualitative research project can not be ascertained in advance and may be specified as they arise during the research process . for a novice researcher , more discussions and debates method - slurring is another common problem , which means the act of blurring distinctions between qualitative approaches . each approach has to demonstrate its consistency to its foundations and will reflect them in data collection , analysis , and knowledge claim . it is not rare to find that researchers and research team try to identify everything , even sample size , in advance when they design their qualitative study because of the strong background they have about the quantitative research . this is completely in contrast with the flexible nature and explorative approach of qualitative research ; as these kinds of researches are completely explorative , the mentioned issues such as sample size should be clarified in the course of the study . the other problem is the examination committee and the format of proposal in the grant sites and funding agencies , which is based on the principles of quantitative study . therefore , flexibility is actually the most important credibility criterion in all qualitative researches that should be considered when a study is designed and the study process is followed . as the final word , the researcher should make sure that he / she gives serious consideration to the chosen area as the basis of research and that a qualitative project is relevant and possible . thus , forming the research question in a proper way and selecting appropriate methodology can guarantee original , interesting , and applied knowledge , which at least can increase our understanding about the meaning of certain conditions for professionals and patients and how their relationships are built in a particular social context .

background : qualitative research focuses on social world and provides the tools to study health phenomena from the perspective of those experiencing them . identifying the problem , forming the question , and selecting an appropriate methodology and design are some of the initial challenges that researchers encounter in the early stages of any research project . these problems are particularly common for novices.materials and methods : this article describes the practical challenges of using qualitative inquiry in the field of health and the challenges of performing an interpretive research based on professional experience as a qualitative researcher and on available literature.results:one of the main topics discussed is the nature of qualitative research , its inherent challenges , and how to overcome them . some of those highlighted here include : identification of the research problem , formation of the research question / aim , and selecting an appropriate methodology and research design , which are the main concerns of qualitative researchers and need to be handled properly . insights from real - life experiences in conducting qualitative research in health reveal these issues.conclusions:the paper provides personal comments on the experiences of a researcher in conducting pure qualitative research in the field of health . it offers insights into the practical difficulties encountered when performing qualitative studies and offers solutions and alternatives applied by these authors , which may be of use to others .

I A M U Virtues of qualitative research Why qualitative research in the health professions? Choosing an approach for health research Research question and aim Just make sure that you give serious consideration to the chosen area as the basis of your research and that a qualitative project is relevant and possible The content of a good qualitative research question takes the form of a declarative rather than an interrogative statement Choosing the right methodology and research design C

while working for more than a decade as qualitative designer , khankeh faced a lot of challenges in conducting qualitative research in the field of health which occupied the mind of other health researchers . therefore , this article contributes to the discussion of challenges related to qualitative research in healthcare in the light of personal experiences of a researcher conducting purely qualitative health research . qualitative inquiry could contribute to a broader understanding of health science considering the substantial congruence between the core elements of health practice and the principles underpinning qualitative research . identifying the research problem and forming the research question are some of the initial challenges that researchers encounter in the early stages of a qualitative research project . researchers and students sometimes fail to understand that adopting a qualitative approach is only the first stage in the process of selecting an appropriate research methodology . once the initial research question has been identified , the crucial decision to be made is on the selection of an appropriate method , such as content analysis , ethnography , or grounded theory , and selecting the research design as well . subsequent arrangements would be on the proper methods of data collection , participants , and the research setting , according to the methodology and the research question . in this paper , we are going to discuss important practical challenges of qualitative inquiry in health and the challenges faced by researchers using interpretive research methodologies . it is important to provide an honest and concise appreciation of the essential characteristics of the qualitative research before discussing the challenges of the interpretive research approach to studies in health . the essentially diagnostic and exploratory nature of qualitative research is invaluable in developing conceptualizations in health as an evolving discipline . researchers launch the quest for new theories in health which should acknowledge that qualitative research is an approach rather than a particular set of techniques , and its appropriateness derives from the nature of the social phenomena to be explored . in qualitative research , knowledge derives from the context - specific perspective on the experienced phenomena , interpretations , and explanation of social experiences . the emotions , perceptions , and actions of people who suffer from a medical condition can be understood by qualitative research the meanings of health professions will only be uncovered through observing the interactions of professionals with clients and interviewing about their experience . however , all defects and challenges of qualitative research should be realized rather than discarded as a compelling way to knowledge structure . new endeavors in excellent academic achievement and building new tradition of qualitative research in health can be facilitated through acknowledging traps and clarifying the real practical challenges . finally , qualitative research provides investigators with the tools to study the health phenomena from the perspective of those experiencing them . based on corbin and strauss ( 2008 ) , committed qualitative researchers lean toward qualitative work because they are drawn to the fluid , evolving , and dynamic nature of this approach in contrast to the more rigid and structured format of quantitative methods . the nature and type of the research question or problem ; the researcher 's epistemological stance , capabilities , knowledge , skills , and training ; and the resources available for the research project are the criteria upon which adopting methodology and procedures depend . the qualitative research is based on the theoretical and philosophical assumptions that researchers try to understand . then , the research methodology and process should be chosen to be consistent with these basic assumptions and the research question as well . some researchers believe that there is no need to study the methodology and methods before beginning the research . sometimes there is an inconsistency between research question , research methodology , and basic philosophical assumptions , and the researchers fail to justify their methods of choice in line with the research question and the ontological and epidemiological assumptions . finally , the researcher 's intentions , the aims of the research question / inquiry , and the chosen approach are regarded as the most important reasons to select a qualitative research method consistent with them and their underpinning philosophical assumptions as well . common problems coming up with a research question include : deciding about the research area among a range of issues that are heeded in your field of interestnot capable of pointing toward any interesting area or topic sufficient to focus a major piece of work onknowing about the area you want to concentrate on ( e.g. forming the research question is one of the initial challenges that researchers encounter in the early stages of a research project . the qualitative research question delineates the procedures that are executed in the study and provides a map to the readers by which they can trail the researcher 's intentions and actions in the study . therefore , special attention is needed on how a qualitative research question will specifically be structured , organized , and formed in the way to quote the necessary information and elements that allow the readers to assess and evaluate the study . the formation of a qualitative research question acquires a basic conducting role for the study and a fundamental function to develop an audit trail that can empower the readers to judge the value , rigor , and validity of the whole research project . the qualitative research question must be provided in such a way as to impart , reflect , and conjoin the theoretical and abstract assumptions with the practical and pragmatic means of attaining them . also , the content provides a brief focus on the issue to be investigated , but does not define the exact relationship of the variables to make these relationships flexible in emanating from the study according to the qualitative research theory . finally , the methodology or method should appear in the qualitative research question coherent with them . meanwhile , the structure of a good qualitative research question will address five of the following six : who , when , where , what , how , and why , and the entire research question should devise the sixth element . for instance , exploring the experiences of self - immolated women regarding their motives for attempting suicide : a qualitative content analysis study in kermanshah iran make sure that your research question is consistent with the approach you are adopting . since the intention of their studies is not completely clear at the beginning , they can not decide about the research approach ; also , they have to change their research question and take different directions in the course of study or they will end up without adequate results that can help readers or consumers improve their understanding or solve the problem . although a researcher initiates a study with a general question and topic , the interesting aspect of qualitative research is that the questions , which are more specific and can help in further data collection and analysis , arise during the course of the study . thus , a qualitative research question can be broadly , rather than narrowly , focused in the beginning . qualitative research is cyclic , which means that the research question in this approach immerses gradually into the topic . crucial decisions need to be made about an appropriate methodology , such as ethnography or grounded theory , after identifying the initial research question . the main concern of novice researchers is to find the reason and appropriate design to do the research , and proper methodology to answer the question . researchers ought to figure out about the planning of qualitative research and how to choose the methodology . it is very important to consider thorough planning in all stages of the research process , from developing the question to the final write - up of the findings for publication . the research design and methodology must be adequate to address the selected topics and the research question . it should be noticed that some of the details of a qualitative research project can not be ascertained in advance and may be specified as they arise during the research process . in order to consider these criteria of consistency and coherence in greater detail , we need to look at the distinctive differences between qualitative approaches in the following : the aims of the research approach , its roots in different disciplines and ideologies , the knowledge claims linked to it , and to a lesser extent , the data collection and analysis specific to each approach . they do not have any clear understanding of the research process in terms of data gathering strategies , data analysis method , and even appropriate sampling plan , which should be indentified based on philosophical and methodological principles . finally , besides the above - mentioned problems , regarding research design , there are two common problems encountered especially by students who want to do qualitative study ; sometimes researchers and research team try to identify everything , even the sample size , in advance when they design their study because they have a strong background of quantitative research , and this is completely in contrast with the flexible nature and explorative approach of qualitative research . the other problem is the examination committee and the format of proposal of grant sites and funding agencies , which are based on the principles of quantitative study . the essentially diagnostic and exploratory nature of qualitative research is invaluable in developing conceptualizations in health as an evolving discipline . researchers launch the quest for new theories in health which should acknowledge that qualitative research is an approach rather than a particular set of techniques , and its appropriateness derives from the nature of the social phenomena to be explored . in qualitative research , knowledge derives from the context - specific perspective on the experienced phenomena , interpretations , and explanation of social experiences . however , all defects and challenges of qualitative research should be realized rather than discarded as a compelling way to knowledge structure . new endeavors in excellent academic achievement and building new tradition of qualitative research in health can be facilitated through acknowledging traps and clarifying the real practical challenges . finally , qualitative research provides investigators with the tools to study the health phenomena from the perspective of those experiencing them . based on corbin and strauss ( 2008 ) , committed qualitative researchers lean toward qualitative work because they are drawn to the fluid , evolving , and dynamic nature of this approach in contrast to the more rigid and structured format of quantitative methods . the nature and type of the research question or problem ; the researcher 's epistemological stance , capabilities , knowledge , skills , and training ; and the resources available for the research project are the criteria upon which adopting methodology and procedures depend . the qualitative research is based on the theoretical and philosophical assumptions that researchers try to understand . then , the research methodology and process should be chosen to be consistent with these basic assumptions and the research question as well . some researchers believe that there is no need to study the methodology and methods before beginning the research . sometimes there is an inconsistency between research question , research methodology , and basic philosophical assumptions , and the researchers fail to justify their methods of choice in line with the research question and the ontological and epidemiological assumptions . finally , the researcher 's intentions , the aims of the research question / inquiry , and the chosen approach are regarded as the most important reasons to select a qualitative research method consistent with them and their underpinning philosophical assumptions as well . common problems coming up with a research question include : deciding about the research area among a range of issues that are heeded in your field of interestnot capable of pointing toward any interesting area or topic sufficient to focus a major piece of work onknowing about the area you want to concentrate on ( e.g. moreover , you can search for some verifiable gaps through literature review , or based on your personal or professional experience and expert opinion , which should be studied . forming the research question is one of the initial challenges that researchers encounter in the early stages of a research project . furthermore , the research question in qualitative studies has an additional significance as it determines the manner of conducting the study . the qualitative research question delineates the procedures that are executed in the study and provides a map to the readers by which they can trail the researcher 's intentions and actions in the study . therefore , special attention is needed on how a qualitative research question will specifically be structured , organized , and formed in the way to quote the necessary information and elements that allow the readers to assess and evaluate the study . the formation of a qualitative research question acquires a basic conducting role for the study and a fundamental function to develop an audit trail that can empower the readers to judge the value , rigor , and validity of the whole research project . the qualitative research question must be provided in such a way as to impart , reflect , and conjoin the theoretical and abstract assumptions with the practical and pragmatic means of attaining them . also , the content provides a brief focus on the issue to be investigated , but does not define the exact relationship of the variables to make these relationships flexible in emanating from the study according to the qualitative research theory . meanwhile , the structure of a good qualitative research question will address five of the following six : who , when , where , what , how , and why , and the entire research question should devise the sixth element . for instance , exploring the experiences of self - immolated women regarding their motives for attempting suicide : a qualitative content analysis study in kermanshah iran make sure that your research question is consistent with the approach you are adopting . since the intention of their studies is not completely clear at the beginning , they can not decide about the research approach ; also , they have to change their research question and take different directions in the course of study or they will end up without adequate results that can help readers or consumers improve their understanding or solve the problem . although a researcher initiates a study with a general question and topic , the interesting aspect of qualitative research is that the questions , which are more specific and can help in further data collection and analysis , arise during the course of the study . thus , a qualitative research question can be broadly , rather than narrowly , focused in the beginning . qualitative research is cyclic , which means that the research question in this approach immerses gradually into the topic . moreover , you can search for some verifiable gaps through literature review , or based on your personal or professional experience and expert opinion , which should be studied . forming the research question is one of the initial challenges that researchers encounter in the early stages of a research project . the qualitative research question delineates the procedures that are executed in the study and provides a map to the readers by which they can trail the researcher 's intentions and actions in the study . therefore , special attention is needed on how a qualitative research question will specifically be structured , organized , and formed in the way to quote the necessary information and elements that allow the readers to assess and evaluate the study . the formation of a qualitative research question acquires a basic conducting role for the study and a fundamental function to develop an audit trail that can empower the readers to judge the value , rigor , and validity of the whole research project . the qualitative research question must be provided in such a way as to impart , reflect , and conjoin the theoretical and abstract assumptions with the practical and pragmatic means of attaining them . also , the content provides a brief focus on the issue to be investigated , but does not define the exact relationship of the variables to make these relationships flexible in emanating from the study according to the qualitative research theory . finally , the methodology or method should appear in the qualitative research question coherent with them . meanwhile , the structure of a good qualitative research question will address five of the following six : who , when , where , what , how , and why , and the entire research question should devise the sixth element . for instance , exploring the experiences of self - immolated women regarding their motives for attempting suicide : a qualitative content analysis study in kermanshah iran make sure that your research question is consistent with the approach you are adopting . since the intention of their studies is not completely clear at the beginning , they can not decide about the research approach ; also , they have to change their research question and take different directions in the course of study or they will end up without adequate results that can help readers or consumers improve their understanding or solve the problem . although a researcher initiates a study with a general question and topic , the interesting aspect of qualitative research is that the questions , which are more specific and can help in further data collection and analysis , arise during the course of the study . thus , a qualitative research question can be broadly , rather than narrowly , focused in the beginning . qualitative research is cyclic , which means that the research question in this approach immerses gradually into the topic . crucial decisions need to be made about an appropriate methodology , such as ethnography or grounded theory , after identifying the initial research question . the main concern of novice researchers is to find the reason and appropriate design to do the research , and proper methodology to answer the question . researchers ought to figure out about the planning of qualitative research and how to choose the methodology . it is very important to consider thorough planning in all stages of the research process , from developing the question to the final write - up of the findings for publication . the research design and methodology must be adequate to address the selected topics and the research question . it should be noticed that some of the details of a qualitative research project can not be ascertained in advance and may be specified as they arise during the research process . in order to consider these criteria of consistency and coherence in greater detail , we need to look at the distinctive differences between qualitative approaches in the following : the aims of the research approach , its roots in different disciplines and ideologies , the knowledge claims linked to it , and to a lesser extent , the data collection and analysis specific to each approach . they do not have any clear understanding of the research process in terms of data gathering strategies , data analysis method , and even appropriate sampling plan , which should be indentified based on philosophical and methodological principles . finally , besides the above - mentioned problems , regarding research design , there are two common problems encountered especially by students who want to do qualitative study ; sometimes researchers and research team try to identify everything , even the sample size , in advance when they design their study because they have a strong background of quantitative research , and this is completely in contrast with the flexible nature and explorative approach of qualitative research . the other problem is the examination committee and the format of proposal of grant sites and funding agencies , which are based on the principles of quantitative study . qualitative research focuses on social world and provides investigators with the tools to study health phenomena from the perspective of those experiencing them . identifying the research problem , forming the research question , and selecting an appropriate methodology and research design are some of the initial challenges that researchers encounter in the early stages of a qualitative research project . once the research problem and the initial research question are identified , the crucial decision has to be made in selecting the appropriate methodology . subsequent arrangements would be on the proper methods of data collection , and choosing the participants and the research setting according to the methodology and the research question . the essence and type of the research question or problem , the researcher 's epistemological stance , capabilities , knowledge , skills and training , and the resources available for the research project are the criteria upon which the adopting methodology and procedures depend . inconsistency between research question and methodology , insufficient methodological knowledge , and lack of attention to the philosophical underpinning of qualitative methodology are some important challenges . forming the research question is one of the initial challenges that researchers encounter in the early stages of a research project . then crucial decisions need to be made about an appropriate methodology . the main concern of novice researchers is to find the reason and appropriate design to do the research and the proper methodology to answer the question . researchers first ought to figure out the planning of qualitative research and how to choose the methodology . it is very important to consider thorough planning in all stages of the research process , from developing the question to final write - up of the findings for publication . it is worth knowing that some of the details of a qualitative research project can not be ascertained in advance and may be specified as they arise during the research process . it is not rare to find that researchers and research team try to identify everything , even sample size , in advance when they design their qualitative study because of the strong background they have about the quantitative research . this is completely in contrast with the flexible nature and explorative approach of qualitative research ; as these kinds of researches are completely explorative , the mentioned issues such as sample size should be clarified in the course of the study . the other problem is the examination committee and the format of proposal in the grant sites and funding agencies , which is based on the principles of quantitative study . thus , forming the research question in a proper way and selecting appropriate methodology can guarantee original , interesting , and applied knowledge , which at least can increase our understanding about the meaning of certain conditions for professionals and patients and how their relationships are built in a particular social context .

however , the field must be with the best traditions and techniques of qualitative methods and should distinguish the essentiality of special training and experience in applying these methods . while working for more than a decade as qualitative designer , khankeh faced a lot of challenges in conducting qualitative research in the field of health which occupied the mind of other health researchers . therefore , this article contributes to the discussion of challenges related to qualitative research in healthcare in the light of personal experiences of a researcher conducting purely qualitative health research . the current quantitative research methods indicate a confined access to clinical knowledge , since they insert only the questions and phenomena that can be controlled , measured , and are countable where it is necessary to investigate , share and contest the tacit knowledge of an experienced practitioner . it is concerned with increased understanding of the meaning of certain conditions for health professionals and patients , and how their relationships are built in a particular social context . qualitative inquiry could contribute to a broader understanding of health science considering the substantial congruence between the core elements of health practice and the principles underpinning qualitative research . corbin ( 2008 ) reported that in the past 10 years , the interest in qualitative methods in general and grounded theory in particular has burgeoned according to a review of the literature and dissertation abstracts . identifying the research problem and forming the research question are some of the initial challenges that researchers encounter in the early stages of a qualitative research project . once the initial research question has been identified , the crucial decision to be made is on the selection of an appropriate method , such as content analysis , ethnography , or grounded theory , and selecting the research design as well . subsequent arrangements would be on the proper methods of data collection , participants , and the research setting , according to the methodology and the research question . in this paper , we are going to discuss important practical challenges of qualitative inquiry in health and the challenges faced by researchers using interpretive research methodologies . it is important to provide an honest and concise appreciation of the essential characteristics of the qualitative research before discussing the challenges of the interpretive research approach to studies in health . this research method depends on the power of words and images , but does not offer the assimilated meanings such as numbers and equations ; it is rather an attentive search of meaning and understanding and an attempt for profound comprehension and awareness of the problems and phenomena . researchers launch the quest for new theories in health which should acknowledge that qualitative research is an approach rather than a particular set of techniques , and its appropriateness derives from the nature of the social phenomena to be explored . in qualitative research , knowledge derives from the context - specific perspective on the experienced phenomena , interpretations , and explanation of social experiences . the approach is helpful in understanding human experiences , which is important for health professionals who focus on caring , communication , and interaction . in fact , they choose qualitative research for some significant reasons : the emotions , perceptions , and actions of people who suffer from a medical condition can be understood by qualitative researchthe meanings of health professions will only be uncovered through observing the interactions of professionals with clients and interviewing about their experience . this is also applicable to the students destined for the healthcare fieldqualitative research is individualized ; hence , researchers consider the participants as whole human beings , not as a bunch of physical compartmentsobservation and asking people are the only ways to understand the causes of particular behaviors . therefore , this type of research can develop health or education policies ; policies for altering health behavior can only be effective if the behavior 's basis is clearly understood . the emotions , perceptions , and actions of people who suffer from a medical condition can be understood by qualitative research the meanings of health professions will only be uncovered through observing the interactions of professionals with clients and interviewing about their experience . this is also applicable to the students destined for the healthcare field qualitative research is individualized ; hence , researchers consider the participants as whole human beings , not as a bunch of physical compartments observation and asking people are the only ways to understand the causes of particular behaviors . therefore , this type of research can develop health or education policies ; policies for altering health behavior can only be effective if the behavior 's basis is clearly understood . new endeavors in excellent academic achievement and building new tradition of qualitative research in health can be facilitated through acknowledging traps and clarifying the real practical challenges . this approach is especially applied in situ ations that have not been previously studied , where major gaps exists in research field , and when there is a need for a new perspective to be identified for the arena of health care intervention . based on corbin and strauss ( 2008 ) , committed qualitative researchers lean toward qualitative work because they are drawn to the fluid , evolving , and dynamic nature of this approach in contrast to the more rigid and structured format of quantitative methods . the nature and type of the research question or problem ; the researcher 's epistemological stance , capabilities , knowledge , skills , and training ; and the resources available for the research project are the criteria upon which adopting methodology and procedures depend . inconsistency between research question and methodology , insufficient methodological knowledge , and lack of attention on philosophical underpinning of qualitative methodology can be mentioned as some important challenges here . my experience shows that lack of knowledge , experience , and skills in a research team to do qualitative research can hinder the formation of original knowledge and improvement in understanding the phenomenon under study . sometimes there is an inconsistency between research question , research methodology , and basic philosophical assumptions , and the researchers fail to justify their methods of choice in line with the research question and the ontological and epidemiological assumptions . finally , the researcher 's intentions , the aims of the research question / inquiry , and the chosen approach are regarded as the most important reasons to select a qualitative research method consistent with them and their underpinning philosophical assumptions as well . common problems coming up with a research question include : deciding about the research area among a range of issues that are heeded in your field of interestnot capable of pointing toward any interesting area or topic sufficient to focus a major piece of work onknowing about the area you want to concentrate on ( e.g. deciding about the research area among a range of issues that are heeded in your field of interest not capable of pointing toward any interesting area or topic sufficient to focus a major piece of work on knowing about the area you want to concentrate on ( e.g. therefore , it acquires significance by the very fact that it provides brief , but nevertheless , important information on the research topic that allows the reader to decide if the topic is relevant , researchable , and a remarkable issue . the qualitative research question delineates the procedures that are executed in the study and provides a map to the readers by which they can trail the researcher 's intentions and actions in the study . therefore , special attention is needed on how a qualitative research question will specifically be structured , organized , and formed in the way to quote the necessary information and elements that allow the readers to assess and evaluate the study . the formation of a qualitative research question acquires a basic conducting role for the study and a fundamental function to develop an audit trail that can empower the readers to judge the value , rigor , and validity of the whole research project . the researcher has to concentrate on how the content of the research topic is understood when phrasing the qualitative research questions , adhering to the topic with the philosophical / theoretical suggestions and to the structure of the study which requires compounding specific principal elements . also , the content provides a brief focus on the issue to be investigated , but does not define the exact relationship of the variables to make these relationships flexible in emanating from the study according to the qualitative research theory . consequently , specific nouns that represent the aims of qualitative studies , such as experiences , feelings , views , perspectives , knowledge , etc . meanwhile , the structure of a good qualitative research question will address five of the following six : who , when , where , what , how , and why , and the entire research question should devise the sixth element . for instance , exploring the experiences of self - immolated women regarding their motives for attempting suicide : a qualitative content analysis study in kermanshah iran make sure that your research question is consistent with the approach you are adopting . since the intention of their studies is not completely clear at the beginning , they can not decide about the research approach ; also , they have to change their research question and take different directions in the course of study or they will end up without adequate results that can help readers or consumers improve their understanding or solve the problem . although a researcher initiates a study with a general question and topic , the interesting aspect of qualitative research is that the questions , which are more specific and can help in further data collection and analysis , arise during the course of the study . it is very important to consider thorough planning in all stages of the research process , from developing the question to the final write - up of the findings for publication . an important problem for novice researchers is the little acknowledgement of different approaches that address different kinds and levels of questions and take a different stance on the kind of phenomena which is focused upon . it may be important to acknowledge the distinctive features by specific approaches such as phenomenology or grounded theory at some levels such as the type of question they are suited to answer , data collection methods they are consistent with , and also the kinds of analysis and presentation of the results that fit within the approach such as goodness of fit or logical staged linking and can be referred to as consistency . in order to consider these criteria of consistency and coherence in greater detail , we need to look at the distinctive differences between qualitative approaches in the following : the aims of the research approach , its roots in different disciplines and ideologies , the knowledge claims linked to it , and to a lesser extent , the data collection and analysis specific to each approach . finally , besides the above - mentioned problems , regarding research design , there are two common problems encountered especially by students who want to do qualitative study ; sometimes researchers and research team try to identify everything , even the sample size , in advance when they design their study because they have a strong background of quantitative research , and this is completely in contrast with the flexible nature and explorative approach of qualitative research . this research method depends on the power of words and images , but does not offer the assimilated meanings such as numbers and equations ; it is rather an attentive search of meaning and understanding and an attempt for profound comprehension and awareness of the problems and phenomena . in qualitative research , knowledge derives from the context - specific perspective on the experienced phenomena , interpretations , and explanation of social experiences . in fact , they choose qualitative research for some significant reasons : the emotions , perceptions , and actions of people who suffer from a medical condition can be understood by qualitative researchthe meanings of health professions will only be uncovered through observing the interactions of professionals with clients and interviewing about their experience . this is also applicable to the students destined for the healthcare fieldqualitative research is individualized ; hence , researchers consider the participants as whole human beings , not as a bunch of physical compartmentsobservation and asking people are the only ways to understand the causes of particular behaviors . the emotions , perceptions , and actions of people who suffer from a medical condition can be understood by qualitative research the meanings of health professions will only be uncovered through observing the interactions of professionals with clients and interviewing about their experience . this is also applicable to the students destined for the healthcare field qualitative research is individualized ; hence , researchers consider the participants as whole human beings , not as a bunch of physical compartments observation and asking people are the only ways to understand the causes of particular behaviors . therefore , this type of research can develop health or education policies ; policies for altering health behavior can only be effective if the behavior 's basis is clearly understood . new endeavors in excellent academic achievement and building new tradition of qualitative research in health can be facilitated through acknowledging traps and clarifying the real practical challenges . this approach is especially applied in situ ations that have not been previously studied , where major gaps exists in research field , and when there is a need for a new perspective to be identified for the arena of health care intervention . based on corbin and strauss ( 2008 ) , committed qualitative researchers lean toward qualitative work because they are drawn to the fluid , evolving , and dynamic nature of this approach in contrast to the more rigid and structured format of quantitative methods . the nature and type of the research question or problem ; the researcher 's epistemological stance , capabilities , knowledge , skills , and training ; and the resources available for the research project are the criteria upon which adopting methodology and procedures depend . my experience shows that lack of knowledge , experience , and skills in a research team to do qualitative research can hinder the formation of original knowledge and improvement in understanding the phenomenon under study . finally , the researcher 's intentions , the aims of the research question / inquiry , and the chosen approach are regarded as the most important reasons to select a qualitative research method consistent with them and their underpinning philosophical assumptions as well . common problems coming up with a research question include : deciding about the research area among a range of issues that are heeded in your field of interestnot capable of pointing toward any interesting area or topic sufficient to focus a major piece of work onknowing about the area you want to concentrate on ( e.g. deciding about the research area among a range of issues that are heeded in your field of interest not capable of pointing toward any interesting area or topic sufficient to focus a major piece of work on knowing about the area you want to concentrate on ( e.g. the qualitative research question delineates the procedures that are executed in the study and provides a map to the readers by which they can trail the researcher 's intentions and actions in the study . therefore , special attention is needed on how a qualitative research question will specifically be structured , organized , and formed in the way to quote the necessary information and elements that allow the readers to assess and evaluate the study . the formation of a qualitative research question acquires a basic conducting role for the study and a fundamental function to develop an audit trail that can empower the readers to judge the value , rigor , and validity of the whole research project . the researcher has to concentrate on how the content of the research topic is understood when phrasing the qualitative research questions , adhering to the topic with the philosophical / theoretical suggestions and to the structure of the study which requires compounding specific principal elements . also , the content provides a brief focus on the issue to be investigated , but does not define the exact relationship of the variables to make these relationships flexible in emanating from the study according to the qualitative research theory . meanwhile , the structure of a good qualitative research question will address five of the following six : who , when , where , what , how , and why , and the entire research question should devise the sixth element . for instance , exploring the experiences of self - immolated women regarding their motives for attempting suicide : a qualitative content analysis study in kermanshah iran make sure that your research question is consistent with the approach you are adopting . since the intention of their studies is not completely clear at the beginning , they can not decide about the research approach ; also , they have to change their research question and take different directions in the course of study or they will end up without adequate results that can help readers or consumers improve their understanding or solve the problem . although a researcher initiates a study with a general question and topic , the interesting aspect of qualitative research is that the questions , which are more specific and can help in further data collection and analysis , arise during the course of the study . the qualitative research question delineates the procedures that are executed in the study and provides a map to the readers by which they can trail the researcher 's intentions and actions in the study . therefore , special attention is needed on how a qualitative research question will specifically be structured , organized , and formed in the way to quote the necessary information and elements that allow the readers to assess and evaluate the study . the formation of a qualitative research question acquires a basic conducting role for the study and a fundamental function to develop an audit trail that can empower the readers to judge the value , rigor , and validity of the whole research project . the researcher has to concentrate on how the content of the research topic is understood when phrasing the qualitative research questions , adhering to the topic with the philosophical / theoretical suggestions and to the structure of the study which requires compounding specific principal elements . also , the content provides a brief focus on the issue to be investigated , but does not define the exact relationship of the variables to make these relationships flexible in emanating from the study according to the qualitative research theory . meanwhile , the structure of a good qualitative research question will address five of the following six : who , when , where , what , how , and why , and the entire research question should devise the sixth element . for instance , exploring the experiences of self - immolated women regarding their motives for attempting suicide : a qualitative content analysis study in kermanshah iran make sure that your research question is consistent with the approach you are adopting . since the intention of their studies is not completely clear at the beginning , they can not decide about the research approach ; also , they have to change their research question and take different directions in the course of study or they will end up without adequate results that can help readers or consumers improve their understanding or solve the problem . although a researcher initiates a study with a general question and topic , the interesting aspect of qualitative research is that the questions , which are more specific and can help in further data collection and analysis , arise during the course of the study . an important problem for novice researchers is the little acknowledgement of different approaches that address different kinds and levels of questions and take a different stance on the kind of phenomena which is focused upon . it may be important to acknowledge the distinctive features by specific approaches such as phenomenology or grounded theory at some levels such as the type of question they are suited to answer , data collection methods they are consistent with , and also the kinds of analysis and presentation of the results that fit within the approach such as goodness of fit or logical staged linking and can be referred to as consistency . in order to consider these criteria of consistency and coherence in greater detail , we need to look at the distinctive differences between qualitative approaches in the following : the aims of the research approach , its roots in different disciplines and ideologies , the knowledge claims linked to it , and to a lesser extent , the data collection and analysis specific to each approach . finally , besides the above - mentioned problems , regarding research design , there are two common problems encountered especially by students who want to do qualitative study ; sometimes researchers and research team try to identify everything , even the sample size , in advance when they design their study because they have a strong background of quantitative research , and this is completely in contrast with the flexible nature and explorative approach of qualitative research . the essence and type of the research question or problem , the researcher 's epistemological stance , capabilities , knowledge , skills and training , and the resources available for the research project are the criteria upon which the adopting methodology and procedures depend . therefore , it acquires significance by the very fact that it provides brief , but nevertheless , important information on the research topic that allows the reader to decide if the topic is relevant , researchable , and a remarkable issue that can help the researcher to determine the manner of conducting the study . this is completely in contrast with the flexible nature and explorative approach of qualitative research ; as these kinds of researches are completely explorative , the mentioned issues such as sample size should be clarified in the course of the study . thus , forming the research question in a proper way and selecting appropriate methodology can guarantee original , interesting , and applied knowledge , which at least can increase our understanding about the meaning of certain conditions for professionals and patients and how their relationships are built in a particular social context .

following endodontic treatment , there is a decrease in the fracture resistance of the teeth . this is also seen after the preparation of wide mesio - occluso - distal ( mod ) cavities . an ideal restoration for a tooth is able to maintain the aesthetics and function , preserve the remaining tooth structure and prevent the microleakage . indirect restorative procedures such as metallic post and core fabrication followed by full crown are customary . however , there may be cases where the tooth is still erupting , or where the tooth or root canal therapy has a questionable prognosis , or where the clinician wants to wait and evaluate the healing of a periapical lesion before proceeding with full crown restorations . to prevent the failure of root canal treatment , a simple , quick , high strength adhesive technology is advancing by leaps and bounds every day , making it possible to create conservative and highly aesthetic restorations with direct bonding to the teeth . a significant increase in the fracture resistance of root filled teeth was observed when they were intracoronally restored with a resin composite material . reinforcing composites with polyethylene fibres and glass fibres has successfully provided superior results.[57 ] however , there are no studies comparing the two in direct composite restorations . the aim of this study was to evaluate the fracture resistance of endodontically treated maxillary premolars with wide mesio - occluso - distal ( mod ) cavities restored with either composite resin , composite resin reinforced with a polyethylene fibre , or composite resin reinforced with a composite impregnated glass fibre . all the specimens were scaled to remove the adhering soft tissue and calculus , and were stored in physiologic saline . five intact teeth were used as positive controls and endodontic access cavities were prepared in forty five teeth using a water cooled diamond bur ( endo access bur ; dentsply ) with an airotor hand piece , and the pulp tissue was removed with barbed broaches . a size 15 k - file ( mani prime dental pvt . ltd . ) the canals were prepared with protaper ( dentsply maillefer ) nickel titanium ( ni - ti ) instruments using a 1:64 reduction handpiece ( anthogyr niti control ; dentsply ) at a speed of 300 revolutions per minute . canals were shaped till the working length using the s1 and s2 files , and the apical third of the canal was finished using finishing file f1 and later f2 . copious irrigation using 5 ml of 5.25% sodium hypochlorite was carried out throughout the procedure . obturation was donewith protaper gutta percha points and ah plus root canal sealer ( dentsply de trey , konstanz , germany ) using a cold lateral condensation technique . mod cavities were prepared in the teeth using an airotor hand piece with a long straight fissure diamond point ( sf-12c ; mani dia burs ) . the dimensions of the cavity were decided with the help of a periodontal probe to standardize the remaining tooth structure . the remaining thickness of the buccal and lingual wall of the teeth was 2.5 mm from the height of contour of the buccal and lingual surface till the buccal and lingual cavity walls , and the gingival cavosurface margin was 1.5 mm above the cement - enamel junction ( cej ) [ figure 1a ] . ( a ) standardized dimensions of mesio - occluso - distal cavity ( b ) preparation of bucco - occluso - lingual groove subsequently , the teeth were randomly divided into three groups ( n = 15 ) : group a - preparations restored with composite , without fibre ( n = 15)group b - preparations restored with composite impregnated with glass fibre ( n = 15)group c - preparations restored with composite impregnated with polyethylene fibre ( n = 15)positive control - intact teeth ( n = 5 ) group a - preparations restored with composite , without fibre ( n = 15 ) group b - preparations restored with composite impregnated with glass fibre ( n = 15 ) group c - preparations restored with composite impregnated with polyethylene fibre ( n = 15 ) positive control - intact teeth ( n = 5 ) cavities were cleaned , dried , and etched with 35% phosphoric acid ( scotch bond multi purpose etchant ; 3 m espe , st . the tooth surfaces were then rinsed for 10 seconds and gently dried for 1 - 2 seconds . subsequently , a single bottle total etch adhesive ( adper single bond 2 ; 3 m espe , st . paul , mn , usa ) was applied and photocured using light - emitting diode ( led ) ( ledition ; ivoclar vivadent ) at 600 mw / cm for 10 seconds . light intensity output was verified after every 10 samples using a digital read out light meter . tofflemire retainer and matrix band were applied and the preparations were restored with a nanocomposite ( filtek z350 xt ; 3 m espe , st . the layers were placed at a 1.5 mm thickness and cured for 40 seconds according to the manufacturer 's instruction . after restoring the preparation as described for group a , a groove 2 mm wide and 1 mm deep was prepared buccolingually on the occlusal surface of the restoration and cusp tips . the ends of the groove were on the occlusal third of the buccal and lingual surfaces of the tooth [ figure 1b ] . after etching the grooves , single bottle total etch adhesive was applied and cured for 10 seconds . flowable composite with 10 mm of composite impregnated glass fibre ( interlig ; angelus , londrina pr , brazil ) was placed on the floor of the groove and light cured for 20 seconds . a layer of the nanocomposite was added to cover the exposed fibre surface and cured from the occlusal direction for 40 seconds according to manufacturer 's instructions . buccolingual grooves were prepared , etched and bonded in a manner similar to group b. flowable composite resin with 10 mm of polyethylene fibre ( ribbond ; ribbond inc . , seattle , wa , usa ) which had been saturated with the unfilled resin ( scotch bond multi purpose plus ; 3 m espe , st . paul , mn , usa ) was added to the groove and cured from occlusal direction for 20 seconds . the exposed surface of the fibre was covered with a layer of the nanocomposite and cured for 40 seconds from the occlusal direction according to the manufacturer 's instructions . the teeth were then mounted in a block of cold cure acrylic measuring 2 cm in diameter , at cej . a layer of polyvinyl siloxane was adapted around the root surfaces to simulate the periodontal ligament . thermocycling was carried out for 500 cycles at 5 to 55c , 30 second dwell time and 5 second transfer time . all the specimens were stored in an incubator ( ibod ) at 37c and 100% humidity for 24 hours . the fracture resistance of the teeth was measured using a universal testing machine ( lr 50k ; lloyd instruments ) , under a compressive force at a strain rate of 0.5 mm / min . a 5 mm diameter round stainless steel ball was positioned parallel to the long axis of the teeth and centered over the teeth until the bar just contacted the occlusal surface of the restoration and the buccal and lingual cusps . the force needed to fracture each tooth was recorded in newtons.[57 ] the study design was a three arm comparative study where the outcome variable was fracture resistance . statistical analysis was performed using descriptive statistical methods , one way anova test and the post - hoc tukey test . the statistical software namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment ver.2.11.1 were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables etc . cavities were cleaned , dried , and etched with 35% phosphoric acid ( scotch bond multi purpose etchant ; 3 m espe , st . the tooth surfaces were then rinsed for 10 seconds and gently dried for 1 - 2 seconds . subsequently , a single bottle total etch adhesive ( adper single bond 2 ; 3 m espe , st . paul , mn , usa ) was applied and photocured using light - emitting diode ( led ) ( ledition ; ivoclar vivadent ) at 600 mw / cm for 10 seconds . light intensity output was verified after every 10 samples using a digital read out light meter . tofflemire retainer and matrix band were applied and the preparations were restored with a nanocomposite ( filtek z350 xt ; 3 m espe , st . the layers were placed at a 1.5 mm thickness and cured for 40 seconds according to the manufacturer 's instruction . after restoring the preparation as described for group a , a groove 2 mm wide and 1 mm deep was prepared buccolingually on the occlusal surface of the restoration and cusp tips . the ends of the groove were on the occlusal third of the buccal and lingual surfaces of the tooth [ figure 1b ] . after etching the grooves , single bottle total etch adhesive was applied and cured for 10 seconds . flowable composite with 10 mm of composite impregnated glass fibre ( interlig ; angelus , londrina pr , brazil ) was placed on the floor of the groove and light cured for 20 seconds . a layer of the nanocomposite was added to cover the exposed fibre surface and cured from the occlusal direction for 40 seconds according to manufacturer 's instructions . buccolingual grooves were prepared , etched and bonded in a manner similar to group b. flowable composite resin with 10 mm of polyethylene fibre ( ribbond ; ribbond inc . , seattle , wa , usa ) which had been saturated with the unfilled resin ( scotch bond multi purpose plus ; 3 m espe , st . paul , mn , usa ) was added to the groove and cured from occlusal direction for 20 seconds . the exposed surface of the fibre was covered with a layer of the nanocomposite and cured for 40 seconds from the occlusal direction according to the manufacturer 's instructions . the teeth were then mounted in a block of cold cure acrylic measuring 2 cm in diameter , at cej . a layer of polyvinyl siloxane was adapted around the root surfaces to simulate the periodontal ligament . thermocycling was carried out for 500 cycles at 5 to 55c , 30 second dwell time and 5 second transfer time . all the specimens were stored in an incubator ( ibod ) at 37c and 100% humidity for 24 hours . the fracture resistance of the teeth was measured using a universal testing machine ( lr 50k ; lloyd instruments ) , under a compressive force at a strain rate of 0.5 mm / min . a 5 mm diameter round stainless steel ball was positioned parallel to the long axis of the teeth and centered over the teeth until the bar just contacted the occlusal surface of the restoration and the buccal and lingual cusps . the force needed to fracture each tooth was recorded in newtons.[57 ] the study design was a three arm comparative study where the outcome variable was fracture resistance . statistical analysis was performed using descriptive statistical methods , one way anova test and the post - hoc tukey test . the statistical software namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment ver.2.11.1 were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables etc . cavities were cleaned , dried , and etched with 35% phosphoric acid ( scotch bond multi purpose etchant ; 3 m espe , st . the tooth surfaces were then rinsed for 10 seconds and gently dried for 1 - 2 seconds . subsequently , a single bottle total etch adhesive ( adper single bond 2 ; 3 m espe , st . paul , mn , usa ) was applied and photocured using light - emitting diode ( led ) ( ledition ; ivoclar vivadent ) at 600 mw / cm for 10 seconds . light intensity output was verified after every 10 samples using a digital read out light meter . tofflemire retainer and matrix band were applied and the preparations were restored with a nanocomposite ( filtek z350 xt ; 3 m espe , st . the layers were placed at a 1.5 mm thickness and cured for 40 seconds according to the manufacturer 's instruction . after restoring the preparation as described for group a , a groove 2 mm wide and 1 mm deep was prepared buccolingually on the occlusal surface of the restoration and cusp tips . the ends of the groove were on the occlusal third of the buccal and lingual surfaces of the tooth [ figure 1b ] . after etching the grooves , single bottle total etch adhesive was applied and cured for 10 seconds . flowable composite with 10 mm of composite impregnated glass fibre ( interlig ; angelus , londrina pr , brazil ) was placed on the floor of the groove and light cured for 20 seconds . a layer of the nanocomposite was added to cover the exposed fibre surface and cured from the occlusal direction for 40 seconds according to manufacturer 's instructions . buccolingual grooves were prepared , etched and bonded in a manner similar to group b. flowable composite resin with 10 mm of polyethylene fibre ( ribbond ; ribbond inc . , seattle , wa , usa ) which had been saturated with the unfilled resin ( scotch bond multi purpose plus ; 3 m espe , st . paul , mn , usa ) was added to the groove and cured from occlusal direction for 20 seconds . the exposed surface of the fibre was covered with a layer of the nanocomposite and cured for 40 seconds from the occlusal direction according to the manufacturer 's instructions . the teeth were then mounted in a block of cold cure acrylic measuring 2 cm in diameter , at cej . a layer of polyvinyl siloxane was adapted around the root surfaces to simulate the periodontal ligament . thermocycling was carried out for 500 cycles at 5 to 55c , 30 second dwell time and 5 second transfer time . all the specimens were stored in an incubator ( ibod ) at 37c and 100% humidity for 24 hours . the fracture resistance of the teeth was measured using a universal testing machine ( lr 50k ; lloyd instruments ) , under a compressive force at a strain rate of 0.5 mm / min . a 5 mm diameter round stainless steel ball was positioned parallel to the long axis of the teeth and centered over the teeth until the bar just contacted the occlusal surface of the restoration and the buccal and lingual cusps . the force needed to fracture each tooth was recorded in newtons.[57 ] the study design was a three arm comparative study where the outcome variable was fracture resistance . statistical analysis was performed using descriptive statistical methods , one way anova test and the post - hoc tukey test . the statistical software namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment ver.2.11.1 were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables etc . highest mean fracture resistance was observed with positive control followed by group b and group a respectively . one way anova test revealed a significant difference ( p = 0.001 ) between the groups [ table 1 ] [ figure 2 ] . on intergroup comparison , post - hoc tukey test revealed a moderately significant difference between control and group b ( p = 0.034 ) whereas a strongly significant difference was recorded between control and group a ( p = 0.002 ) and between control and group c ( p = 0.001 ) . no significant difference was recorded between any of the experimental groups ( group a , b and c ) ( p > 0.1 ) [ table 2 ] . mean fracture resistance in newton ( n ) in all four groups as seen in the study graph depicting mean fracture resistance values pair wise comparison : difference and p value in the study restoration of root filled teeth , despite posing a major challenge to the clinician , is an essential final step of root canal treatment . the purpose of the restoration is not only to repair , reinforce and strengthen the tooth , but also to provide an effective seal between the canal system and the mouth . the gold standard of post endodontic restorations is the full crown ; however , there are situations where the clinician may choose to delay the delivery of the full crown . in such cases , an efficient , direct , high strength adhesive restorations allow a more efficient transfer and distribution of functional stresses across the bonding interface to the tooth structure . the smaller size of the fillers allows them to have excellent optical properties along with good mechanical properties . despite the advances in material sciences , original ribbond ( group c ) consists of cold plasma treated , leno weave ultra high modulus ( lwuhmw ) polyethylene fibres , which are arranged in a lock - stitch design . interlig ( group b ) consists of glass fibres pre - impregnated with light curable composite resin arranged in a braided design . fibres were placed occlusal to the restoration following a technique given by oskoee et al . , and belli et al .. placing fibres on the occlusal surface keeps buccal and lingual cusps together and protects the natural cusps resulting in higher fracture resistance . in addition , fracture resistance increases when fibres are placed close to the point where the force is exerted because it leads to a shorter working arm according to levers principle . the results of the present study revealed that the positive control group had highest mean fracture resistance ( 811.90 n ) . this is in accordance with the study by beli et al . , and also denotes that endodontic and restorative treatment have a detrimental effect on fracture resistance of teeth . among the experimental groups , group b had the highest mean fracture resistance ( 600.49 n ) followed by group a ( 516.96 n ) , and group c ( 514.64 n ) [ table 1 ] [ figure 2 ] . the one way anova test done in the present study revealed a statistically significant difference ( p = 0.001 ) between all the groups [ table 1 ] . post - hoc tukey test revealed a moderately significant difference between control and group b ( p = 0.034 ) and a strongly significant difference between control and group a ( p = 0.002 ) , and between control and group c ( p = 0.001 ) . no significant difference was found between any of the experimental groups ( group a , b and c ) ( p > 0.1 ) [ table 2 ] . group a showed an acceptable fracture resistance due to the use of nanocomposites in the present study . the high filler loading enables nanocomposites to demonstrate good physical and mechanical properties and reinforce the tooth structure well . single bottle total etch system gave evidence of better bond strength than the newer self- etch systems . the presence of the adhesive layer under the restoration acts as a stress buffer . a study by ausiello et al . , has shown that an optimal adhesive layer thickness leads to maximum stress release while preserving interface integrity . the fracture resistance of group a can be attributed to the increase in strain capacity of the adhesive resin and the improved physical and mechanical properties of nanocomposites . this study recorded that group b had the highest fracture resistance amongst the experimental groups . the superior performance of group b may be because of the pre - fabricated resin impregnation of these fibres by the manufacturers . also , glass fibres have very high tensile strength , density and percentage of elongation allowing them to withstand high stresses without fracturing . results of this study were in agreement with the study by kolbeck et al . , who reported the glass fibres performed better than polyethylene fibres due to pre - impregnation with light cured composite which ensures a good bond with the composite resin . polyethylene fibres have shown high fracture resistance in previous studies . according to the manufacturer original ribbond fibres are woven using the lock - stitch leno weave which prevents slipping of fibres within resin matrix , prevents micro - cracks from propagating to form larger cracks and reinforces the restoration in multiple directions . beli et al . , found that insertion of polyethylene fibre in the occlusal third of a composite restoration increased the fracture resistance of root filled teeth compared to teeth restored with composite resin alone . however , the present study showed that insertion of polyethylene fibre did not significantly increase the fracture resistance of a root filled tooth as compared to teeth restored with composite resin alone ( group a ) ( p > 0.1 ) . the causes for lower fracture resistance of group c may include the following : non uniform wetting of fibre with unfilled resin - manual wetting of original ribbond ( group c ) with unfilled adhesive was done as suggested by the manufacturer . this may lead to areas of non - uniform wetting in the fibre which will affect the adhesion of fibre to resin matrix . the poorly impregnated regions may become areas of increased water sorption , hence causing deterioration of mechanical properties of the composite . such voids may also act as oxygen reserves hence preventing complete polymerisation of composite resin.the lwuhmw polyethylene fibre is treated with cold gas plasma to convert the material from hydrophobic to hydrophilic . this treatment is meant to make the fibre surface more receptive to bonding with the resin however ; it makes the fibre very technique sensitive . the fibres frayed on cutting , and became very stiff after the wetting procedure.an additional reason for its poor performance may be lower tensile strength , density and elongation compared to glass fibres . non uniform wetting of fibre with unfilled resin - manual wetting of original ribbond ( group c ) with unfilled adhesive was done as suggested by the manufacturer . this may lead to areas of non - uniform wetting in the fibre which will affect the adhesion of fibre to resin matrix . the poorly impregnated regions may become areas of increased water sorption , hence causing deterioration of mechanical properties of the composite . such voids may also act as oxygen reserves hence preventing complete polymerisation of composite resin . the lwuhmw polyethylene fibre is treated with cold gas plasma to convert the material from hydrophobic to hydrophilic . this treatment is meant to make the fibre surface more receptive to bonding with the resin however ; it makes the fibre very technique sensitive . an additional reason for its poor performance may be lower tensile strength , density and elongation compared to glass fibres . though it was not among the objectives of the study it was observed that the majority of failures in the fibre reinforced groups were favourable in nature . this can prove immensely beneficial to clinicians as despite fracturing , the tooth remains repairable with techniques such as post and core followed by full crown . additionally , the study done by reeh et al . , showed a relative loss of stiffness of 5% due to endodontic procedures and 63% in mod cavity preparation whereas the present study found a relatively smaller decrease of 25 - 35% in fracture resistance of root filled teeth with mod cavity preparation restored with nanocomposites or nanocomposites restored with different fibres . despite this decrease in fracture resistance all the experimental groups demonstrated results much higher than the average normal biting force of human maxillary premolars ( 100 - 300n ) . forces generated intraorally during function vary in magnitude , speed of application and direction , whereas the forces applied to the teeth in this study were at a constant direction and speed and they increased continuously until fracture occurred . further in vivo studies should be done to test the reinforcement effect of fibres in clinical situations . root canal treatment and mod cavity preparation significantly reduced fracture resistance of root filled maxillary premolars ( p = 0.001).even though there was no statistically significant difference between the test groups , the fracture resistance of the composite impregnated glass fibre reinforced group was much higher . root canal treatment and mod cavity preparation significantly reduced fracture resistance of root filled maxillary premolars ( p = 0.001 ) . even though there was no statistically significant difference between the test groups , the fracture resistance of the composite impregnated glass fibre reinforced group was much higher .

aim : the aim of this study was to evaluate the fracture resistance of endodontically treated maxillary premolars with wide mesio - occluso - distal ( mod ) cavities restored with either composite resin , or composite resin reinforced with different types of fibres . materials and methods : fifty human maxillary premolars were selected . five intact teeth served as positive controls . endodontic therapy was carried out in the remaining forty - five teeth . standardized mod cavities were prepared in all the teeth . the teeth were restored with a nanocomposite using an incremental technique . these forty five teeth were randomly divided into three experimental groups ( group a , b and c ) ( n = 15 ) . the teeth in group a did not undergo any further procedures . the teeth in group b and c were reinforced with composite impregnated glass fibre and polyethylene fibre , respectively . fracture resistance was measured in newtons ( n).results : the positive controls showed the highest mean fracture resistance ( 811.90 n ) , followed by group b ( 600.49n ) , group a ( 516.96n ) and group c ( 514.64n ) , respectively . one way analysis of variance ( anova ) test revealed a statistically significant difference between all the groups ( p = 0.001 ) . post - hoc tukey test revealed a moderately significant difference ( p = 0.034 ) between control and group b , and a strongly significant difference between control and group a ( p = 0.002 ) , and control and group c ( p = 0.001).conclusions : endodontic therapy and mod cavity preparation significantly reduced the fracture resistance of endodontically treated maxillary premolars ( p = 0.001 ) . no statistically significant difference was found between the experimental groups ( group a , b and c ) ( p > 0.1 ) . however , the fracture resistance of the composite impregnated glass fibre reinforced group was much higher than the others .

INTRODUCTION MATERIALS AND METHODS Preparation of specimens Group A (No fibre group) Group B (Composite impregnated glass fibre) Group C (Polyethylene fibre) RESULTS DISCUSSION CONCLUSIONS

following endodontic treatment , there is a decrease in the fracture resistance of the teeth . this is also seen after the preparation of wide mesio - occluso - distal ( mod ) cavities . an ideal restoration for a tooth is able to maintain the aesthetics and function , preserve the remaining tooth structure and prevent the microleakage . indirect restorative procedures such as metallic post and core fabrication followed by full crown are customary . however , there may be cases where the tooth is still erupting , or where the tooth or root canal therapy has a questionable prognosis , or where the clinician wants to wait and evaluate the healing of a periapical lesion before proceeding with full crown restorations . to prevent the failure of root canal treatment , a simple , quick , high strength adhesive technology is advancing by leaps and bounds every day , making it possible to create conservative and highly aesthetic restorations with direct bonding to the teeth . a significant increase in the fracture resistance of root filled teeth was observed when they were intracoronally restored with a resin composite material . [57 ] however , there are no studies comparing the two in direct composite restorations . the aim of this study was to evaluate the fracture resistance of endodontically treated maxillary premolars with wide mesio - occluso - distal ( mod ) cavities restored with either composite resin , composite resin reinforced with a polyethylene fibre , or composite resin reinforced with a composite impregnated glass fibre . all the specimens were scaled to remove the adhering soft tissue and calculus , and were stored in physiologic saline . five intact teeth were used as positive controls and endodontic access cavities were prepared in forty five teeth using a water cooled diamond bur ( endo access bur ; dentsply ) with an airotor hand piece , and the pulp tissue was removed with barbed broaches . the canals were prepared with protaper ( dentsply maillefer ) nickel titanium ( ni - ti ) instruments using a 1:64 reduction handpiece ( anthogyr niti control ; dentsply ) at a speed of 300 revolutions per minute . canals were shaped till the working length using the s1 and s2 files , and the apical third of the canal was finished using finishing file f1 and later f2 . copious irrigation using 5 ml of 5.25% sodium hypochlorite was carried out throughout the procedure . mod cavities were prepared in the teeth using an airotor hand piece with a long straight fissure diamond point ( sf-12c ; mani dia burs ) . the dimensions of the cavity were decided with the help of a periodontal probe to standardize the remaining tooth structure . the remaining thickness of the buccal and lingual wall of the teeth was 2.5 mm from the height of contour of the buccal and lingual surface till the buccal and lingual cavity walls , and the gingival cavosurface margin was 1.5 mm above the cement - enamel junction ( cej ) [ figure 1a ] . ( a ) standardized dimensions of mesio - occluso - distal cavity ( b ) preparation of bucco - occluso - lingual groove subsequently , the teeth were randomly divided into three groups ( n = 15 ) : group a - preparations restored with composite , without fibre ( n = 15)group b - preparations restored with composite impregnated with glass fibre ( n = 15)group c - preparations restored with composite impregnated with polyethylene fibre ( n = 15)positive control - intact teeth ( n = 5 ) group a - preparations restored with composite , without fibre ( n = 15 ) group b - preparations restored with composite impregnated with glass fibre ( n = 15 ) group c - preparations restored with composite impregnated with polyethylene fibre ( n = 15 ) positive control - intact teeth ( n = 5 ) cavities were cleaned , dried , and etched with 35% phosphoric acid ( scotch bond multi purpose etchant ; 3 m espe , st . tofflemire retainer and matrix band were applied and the preparations were restored with a nanocomposite ( filtek z350 xt ; 3 m espe , st . after restoring the preparation as described for group a , a groove 2 mm wide and 1 mm deep was prepared buccolingually on the occlusal surface of the restoration and cusp tips . the ends of the groove were on the occlusal third of the buccal and lingual surfaces of the tooth [ figure 1b ] . flowable composite with 10 mm of composite impregnated glass fibre ( interlig ; angelus , londrina pr , brazil ) was placed on the floor of the groove and light cured for 20 seconds . buccolingual grooves were prepared , etched and bonded in a manner similar to group b. flowable composite resin with 10 mm of polyethylene fibre ( ribbond ; ribbond inc . the exposed surface of the fibre was covered with a layer of the nanocomposite and cured for 40 seconds from the occlusal direction according to the manufacturer 's instructions . the teeth were then mounted in a block of cold cure acrylic measuring 2 cm in diameter , at cej . thermocycling was carried out for 500 cycles at 5 to 55c , 30 second dwell time and 5 second transfer time . all the specimens were stored in an incubator ( ibod ) at 37c and 100% humidity for 24 hours . the fracture resistance of the teeth was measured using a universal testing machine ( lr 50k ; lloyd instruments ) , under a compressive force at a strain rate of 0.5 mm / min . a 5 mm diameter round stainless steel ball was positioned parallel to the long axis of the teeth and centered over the teeth until the bar just contacted the occlusal surface of the restoration and the buccal and lingual cusps . the force needed to fracture each tooth was recorded in newtons. [57 ] the study design was a three arm comparative study where the outcome variable was fracture resistance . statistical analysis was performed using descriptive statistical methods , one way anova test and the post - hoc tukey test . the statistical software namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment ver.2.11.1 were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables etc . cavities were cleaned , dried , and etched with 35% phosphoric acid ( scotch bond multi purpose etchant ; 3 m espe , st . paul , mn , usa ) was applied and photocured using light - emitting diode ( led ) ( ledition ; ivoclar vivadent ) at 600 mw / cm for 10 seconds . tofflemire retainer and matrix band were applied and the preparations were restored with a nanocomposite ( filtek z350 xt ; 3 m espe , st . after restoring the preparation as described for group a , a groove 2 mm wide and 1 mm deep was prepared buccolingually on the occlusal surface of the restoration and cusp tips . the ends of the groove were on the occlusal third of the buccal and lingual surfaces of the tooth [ figure 1b ] . flowable composite with 10 mm of composite impregnated glass fibre ( interlig ; angelus , londrina pr , brazil ) was placed on the floor of the groove and light cured for 20 seconds . a layer of the nanocomposite was added to cover the exposed fibre surface and cured from the occlusal direction for 40 seconds according to manufacturer 's instructions . buccolingual grooves were prepared , etched and bonded in a manner similar to group b. flowable composite resin with 10 mm of polyethylene fibre ( ribbond ; ribbond inc . the exposed surface of the fibre was covered with a layer of the nanocomposite and cured for 40 seconds from the occlusal direction according to the manufacturer 's instructions . the teeth were then mounted in a block of cold cure acrylic measuring 2 cm in diameter , at cej . thermocycling was carried out for 500 cycles at 5 to 55c , 30 second dwell time and 5 second transfer time . all the specimens were stored in an incubator ( ibod ) at 37c and 100% humidity for 24 hours . the fracture resistance of the teeth was measured using a universal testing machine ( lr 50k ; lloyd instruments ) , under a compressive force at a strain rate of 0.5 mm / min . a 5 mm diameter round stainless steel ball was positioned parallel to the long axis of the teeth and centered over the teeth until the bar just contacted the occlusal surface of the restoration and the buccal and lingual cusps . statistical analysis was performed using descriptive statistical methods , one way anova test and the post - hoc tukey test . the statistical software namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment ver.2.11.1 were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables etc . cavities were cleaned , dried , and etched with 35% phosphoric acid ( scotch bond multi purpose etchant ; 3 m espe , st . paul , mn , usa ) was applied and photocured using light - emitting diode ( led ) ( ledition ; ivoclar vivadent ) at 600 mw / cm for 10 seconds . tofflemire retainer and matrix band were applied and the preparations were restored with a nanocomposite ( filtek z350 xt ; 3 m espe , st . after restoring the preparation as described for group a , a groove 2 mm wide and 1 mm deep was prepared buccolingually on the occlusal surface of the restoration and cusp tips . the ends of the groove were on the occlusal third of the buccal and lingual surfaces of the tooth [ figure 1b ] . flowable composite with 10 mm of composite impregnated glass fibre ( interlig ; angelus , londrina pr , brazil ) was placed on the floor of the groove and light cured for 20 seconds . buccolingual grooves were prepared , etched and bonded in a manner similar to group b. flowable composite resin with 10 mm of polyethylene fibre ( ribbond ; ribbond inc . the exposed surface of the fibre was covered with a layer of the nanocomposite and cured for 40 seconds from the occlusal direction according to the manufacturer 's instructions . the teeth were then mounted in a block of cold cure acrylic measuring 2 cm in diameter , at cej . thermocycling was carried out for 500 cycles at 5 to 55c , 30 second dwell time and 5 second transfer time . all the specimens were stored in an incubator ( ibod ) at 37c and 100% humidity for 24 hours . the fracture resistance of the teeth was measured using a universal testing machine ( lr 50k ; lloyd instruments ) , under a compressive force at a strain rate of 0.5 mm / min . a 5 mm diameter round stainless steel ball was positioned parallel to the long axis of the teeth and centered over the teeth until the bar just contacted the occlusal surface of the restoration and the buccal and lingual cusps . statistical analysis was performed using descriptive statistical methods , one way anova test and the post - hoc tukey test . the statistical software namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment ver.2.11.1 were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables etc . highest mean fracture resistance was observed with positive control followed by group b and group a respectively . one way anova test revealed a significant difference ( p = 0.001 ) between the groups [ table 1 ] [ figure 2 ] . on intergroup comparison , post - hoc tukey test revealed a moderately significant difference between control and group b ( p = 0.034 ) whereas a strongly significant difference was recorded between control and group a ( p = 0.002 ) and between control and group c ( p = 0.001 ) . no significant difference was recorded between any of the experimental groups ( group a , b and c ) ( p > 0.1 ) [ table 2 ] . mean fracture resistance in newton ( n ) in all four groups as seen in the study graph depicting mean fracture resistance values pair wise comparison : difference and p value in the study restoration of root filled teeth , despite posing a major challenge to the clinician , is an essential final step of root canal treatment . the purpose of the restoration is not only to repair , reinforce and strengthen the tooth , but also to provide an effective seal between the canal system and the mouth . the gold standard of post endodontic restorations is the full crown ; however , there are situations where the clinician may choose to delay the delivery of the full crown . the smaller size of the fillers allows them to have excellent optical properties along with good mechanical properties . despite the advances in material sciences , original ribbond ( group c ) consists of cold plasma treated , leno weave ultra high modulus ( lwuhmw ) polyethylene fibres , which are arranged in a lock - stitch design . interlig ( group b ) consists of glass fibres pre - impregnated with light curable composite resin arranged in a braided design . , and belli et al .. placing fibres on the occlusal surface keeps buccal and lingual cusps together and protects the natural cusps resulting in higher fracture resistance . in addition , fracture resistance increases when fibres are placed close to the point where the force is exerted because it leads to a shorter working arm according to levers principle . the results of the present study revealed that the positive control group had highest mean fracture resistance ( 811.90 n ) . , and also denotes that endodontic and restorative treatment have a detrimental effect on fracture resistance of teeth . among the experimental groups , group b had the highest mean fracture resistance ( 600.49 n ) followed by group a ( 516.96 n ) , and group c ( 514.64 n ) [ table 1 ] [ figure 2 ] . the one way anova test done in the present study revealed a statistically significant difference ( p = 0.001 ) between all the groups [ table 1 ] . post - hoc tukey test revealed a moderately significant difference between control and group b ( p = 0.034 ) and a strongly significant difference between control and group a ( p = 0.002 ) , and between control and group c ( p = 0.001 ) . no significant difference was found between any of the experimental groups ( group a , b and c ) ( p > 0.1 ) [ table 2 ] . group a showed an acceptable fracture resistance due to the use of nanocomposites in the present study . single bottle total etch system gave evidence of better bond strength than the newer self- etch systems . the presence of the adhesive layer under the restoration acts as a stress buffer . the fracture resistance of group a can be attributed to the increase in strain capacity of the adhesive resin and the improved physical and mechanical properties of nanocomposites . this study recorded that group b had the highest fracture resistance amongst the experimental groups . the superior performance of group b may be because of the pre - fabricated resin impregnation of these fibres by the manufacturers . results of this study were in agreement with the study by kolbeck et al . , who reported the glass fibres performed better than polyethylene fibres due to pre - impregnation with light cured composite which ensures a good bond with the composite resin . polyethylene fibres have shown high fracture resistance in previous studies . according to the manufacturer original ribbond fibres are woven using the lock - stitch leno weave which prevents slipping of fibres within resin matrix , prevents micro - cracks from propagating to form larger cracks and reinforces the restoration in multiple directions . , found that insertion of polyethylene fibre in the occlusal third of a composite restoration increased the fracture resistance of root filled teeth compared to teeth restored with composite resin alone . however , the present study showed that insertion of polyethylene fibre did not significantly increase the fracture resistance of a root filled tooth as compared to teeth restored with composite resin alone ( group a ) ( p > 0.1 ) . the causes for lower fracture resistance of group c may include the following : non uniform wetting of fibre with unfilled resin - manual wetting of original ribbond ( group c ) with unfilled adhesive was done as suggested by the manufacturer . this may lead to areas of non - uniform wetting in the fibre which will affect the adhesion of fibre to resin matrix . the poorly impregnated regions may become areas of increased water sorption , hence causing deterioration of mechanical properties of the composite . such voids may also act as oxygen reserves hence preventing complete polymerisation of composite resin.the lwuhmw polyethylene fibre is treated with cold gas plasma to convert the material from hydrophobic to hydrophilic . non uniform wetting of fibre with unfilled resin - manual wetting of original ribbond ( group c ) with unfilled adhesive was done as suggested by the manufacturer . the poorly impregnated regions may become areas of increased water sorption , hence causing deterioration of mechanical properties of the composite . such voids may also act as oxygen reserves hence preventing complete polymerisation of composite resin . the lwuhmw polyethylene fibre is treated with cold gas plasma to convert the material from hydrophobic to hydrophilic . though it was not among the objectives of the study it was observed that the majority of failures in the fibre reinforced groups were favourable in nature . this can prove immensely beneficial to clinicians as despite fracturing , the tooth remains repairable with techniques such as post and core followed by full crown . additionally , the study done by reeh et al . , showed a relative loss of stiffness of 5% due to endodontic procedures and 63% in mod cavity preparation whereas the present study found a relatively smaller decrease of 25 - 35% in fracture resistance of root filled teeth with mod cavity preparation restored with nanocomposites or nanocomposites restored with different fibres . despite this decrease in fracture resistance all the experimental groups demonstrated results much higher than the average normal biting force of human maxillary premolars ( 100 - 300n ) . forces generated intraorally during function vary in magnitude , speed of application and direction , whereas the forces applied to the teeth in this study were at a constant direction and speed and they increased continuously until fracture occurred . further in vivo studies should be done to test the reinforcement effect of fibres in clinical situations . root canal treatment and mod cavity preparation significantly reduced fracture resistance of root filled maxillary premolars ( p = 0.001).even though there was no statistically significant difference between the test groups , the fracture resistance of the composite impregnated glass fibre reinforced group was much higher . root canal treatment and mod cavity preparation significantly reduced fracture resistance of root filled maxillary premolars ( p = 0.001 ) . even though there was no statistically significant difference between the test groups , the fracture resistance of the composite impregnated glass fibre reinforced group was much higher .

following endodontic treatment , there is a decrease in the fracture resistance of the teeth . this is also seen after the preparation of wide mesio - occluso - distal ( mod ) cavities . an ideal restoration for a tooth is able to maintain the aesthetics and function , preserve the remaining tooth structure and prevent the microleakage . however , there may be cases where the tooth is still erupting , or where the tooth or root canal therapy has a questionable prognosis , or where the clinician wants to wait and evaluate the healing of a periapical lesion before proceeding with full crown restorations . to prevent the failure of root canal treatment , a simple , quick , high strength adhesive technology is advancing by leaps and bounds every day , making it possible to create conservative and highly aesthetic restorations with direct bonding to the teeth . a significant increase in the fracture resistance of root filled teeth was observed when they were intracoronally restored with a resin composite material . [57 ] however , there are no studies comparing the two in direct composite restorations . the aim of this study was to evaluate the fracture resistance of endodontically treated maxillary premolars with wide mesio - occluso - distal ( mod ) cavities restored with either composite resin , composite resin reinforced with a polyethylene fibre , or composite resin reinforced with a composite impregnated glass fibre . all the specimens were scaled to remove the adhering soft tissue and calculus , and were stored in physiologic saline . five intact teeth were used as positive controls and endodontic access cavities were prepared in forty five teeth using a water cooled diamond bur ( endo access bur ; dentsply ) with an airotor hand piece , and the pulp tissue was removed with barbed broaches . the canals were prepared with protaper ( dentsply maillefer ) nickel titanium ( ni - ti ) instruments using a 1:64 reduction handpiece ( anthogyr niti control ; dentsply ) at a speed of 300 revolutions per minute . canals were shaped till the working length using the s1 and s2 files , and the apical third of the canal was finished using finishing file f1 and later f2 . obturation was donewith protaper gutta percha points and ah plus root canal sealer ( dentsply de trey , konstanz , germany ) using a cold lateral condensation technique . mod cavities were prepared in the teeth using an airotor hand piece with a long straight fissure diamond point ( sf-12c ; mani dia burs ) . the dimensions of the cavity were decided with the help of a periodontal probe to standardize the remaining tooth structure . the remaining thickness of the buccal and lingual wall of the teeth was 2.5 mm from the height of contour of the buccal and lingual surface till the buccal and lingual cavity walls , and the gingival cavosurface margin was 1.5 mm above the cement - enamel junction ( cej ) [ figure 1a ] . ( a ) standardized dimensions of mesio - occluso - distal cavity ( b ) preparation of bucco - occluso - lingual groove subsequently , the teeth were randomly divided into three groups ( n = 15 ) : group a - preparations restored with composite , without fibre ( n = 15)group b - preparations restored with composite impregnated with glass fibre ( n = 15)group c - preparations restored with composite impregnated with polyethylene fibre ( n = 15)positive control - intact teeth ( n = 5 ) group a - preparations restored with composite , without fibre ( n = 15 ) group b - preparations restored with composite impregnated with glass fibre ( n = 15 ) group c - preparations restored with composite impregnated with polyethylene fibre ( n = 15 ) positive control - intact teeth ( n = 5 ) cavities were cleaned , dried , and etched with 35% phosphoric acid ( scotch bond multi purpose etchant ; 3 m espe , st . subsequently , a single bottle total etch adhesive ( adper single bond 2 ; 3 m espe , st . paul , mn , usa ) was applied and photocured using light - emitting diode ( led ) ( ledition ; ivoclar vivadent ) at 600 mw / cm for 10 seconds . tofflemire retainer and matrix band were applied and the preparations were restored with a nanocomposite ( filtek z350 xt ; 3 m espe , st . after restoring the preparation as described for group a , a groove 2 mm wide and 1 mm deep was prepared buccolingually on the occlusal surface of the restoration and cusp tips . the ends of the groove were on the occlusal third of the buccal and lingual surfaces of the tooth [ figure 1b ] . after etching the grooves , single bottle total etch adhesive was applied and cured for 10 seconds . flowable composite with 10 mm of composite impregnated glass fibre ( interlig ; angelus , londrina pr , brazil ) was placed on the floor of the groove and light cured for 20 seconds . a layer of the nanocomposite was added to cover the exposed fibre surface and cured from the occlusal direction for 40 seconds according to manufacturer 's instructions . buccolingual grooves were prepared , etched and bonded in a manner similar to group b. flowable composite resin with 10 mm of polyethylene fibre ( ribbond ; ribbond inc . , seattle , wa , usa ) which had been saturated with the unfilled resin ( scotch bond multi purpose plus ; 3 m espe , st . paul , mn , usa ) was added to the groove and cured from occlusal direction for 20 seconds . the exposed surface of the fibre was covered with a layer of the nanocomposite and cured for 40 seconds from the occlusal direction according to the manufacturer 's instructions . a layer of polyvinyl siloxane was adapted around the root surfaces to simulate the periodontal ligament . all the specimens were stored in an incubator ( ibod ) at 37c and 100% humidity for 24 hours . the fracture resistance of the teeth was measured using a universal testing machine ( lr 50k ; lloyd instruments ) , under a compressive force at a strain rate of 0.5 mm / min . a 5 mm diameter round stainless steel ball was positioned parallel to the long axis of the teeth and centered over the teeth until the bar just contacted the occlusal surface of the restoration and the buccal and lingual cusps . [57 ] the study design was a three arm comparative study where the outcome variable was fracture resistance . the statistical software namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment ver.2.11.1 were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables etc . subsequently , a single bottle total etch adhesive ( adper single bond 2 ; 3 m espe , st . paul , mn , usa ) was applied and photocured using light - emitting diode ( led ) ( ledition ; ivoclar vivadent ) at 600 mw / cm for 10 seconds . tofflemire retainer and matrix band were applied and the preparations were restored with a nanocomposite ( filtek z350 xt ; 3 m espe , st . after restoring the preparation as described for group a , a groove 2 mm wide and 1 mm deep was prepared buccolingually on the occlusal surface of the restoration and cusp tips . the ends of the groove were on the occlusal third of the buccal and lingual surfaces of the tooth [ figure 1b ] . after etching the grooves , single bottle total etch adhesive was applied and cured for 10 seconds . flowable composite with 10 mm of composite impregnated glass fibre ( interlig ; angelus , londrina pr , brazil ) was placed on the floor of the groove and light cured for 20 seconds . a layer of the nanocomposite was added to cover the exposed fibre surface and cured from the occlusal direction for 40 seconds according to manufacturer 's instructions . buccolingual grooves were prepared , etched and bonded in a manner similar to group b. flowable composite resin with 10 mm of polyethylene fibre ( ribbond ; ribbond inc . , seattle , wa , usa ) which had been saturated with the unfilled resin ( scotch bond multi purpose plus ; 3 m espe , st . paul , mn , usa ) was added to the groove and cured from occlusal direction for 20 seconds . the exposed surface of the fibre was covered with a layer of the nanocomposite and cured for 40 seconds from the occlusal direction according to the manufacturer 's instructions . a layer of polyvinyl siloxane was adapted around the root surfaces to simulate the periodontal ligament . all the specimens were stored in an incubator ( ibod ) at 37c and 100% humidity for 24 hours . the fracture resistance of the teeth was measured using a universal testing machine ( lr 50k ; lloyd instruments ) , under a compressive force at a strain rate of 0.5 mm / min . a 5 mm diameter round stainless steel ball was positioned parallel to the long axis of the teeth and centered over the teeth until the bar just contacted the occlusal surface of the restoration and the buccal and lingual cusps . [57 ] the study design was a three arm comparative study where the outcome variable was fracture resistance . the statistical software namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment ver.2.11.1 were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables etc . subsequently , a single bottle total etch adhesive ( adper single bond 2 ; 3 m espe , st . paul , mn , usa ) was applied and photocured using light - emitting diode ( led ) ( ledition ; ivoclar vivadent ) at 600 mw / cm for 10 seconds . tofflemire retainer and matrix band were applied and the preparations were restored with a nanocomposite ( filtek z350 xt ; 3 m espe , st . after restoring the preparation as described for group a , a groove 2 mm wide and 1 mm deep was prepared buccolingually on the occlusal surface of the restoration and cusp tips . the ends of the groove were on the occlusal third of the buccal and lingual surfaces of the tooth [ figure 1b ] . flowable composite with 10 mm of composite impregnated glass fibre ( interlig ; angelus , londrina pr , brazil ) was placed on the floor of the groove and light cured for 20 seconds . a layer of the nanocomposite was added to cover the exposed fibre surface and cured from the occlusal direction for 40 seconds according to manufacturer 's instructions . buccolingual grooves were prepared , etched and bonded in a manner similar to group b. flowable composite resin with 10 mm of polyethylene fibre ( ribbond ; ribbond inc . paul , mn , usa ) was added to the groove and cured from occlusal direction for 20 seconds . the exposed surface of the fibre was covered with a layer of the nanocomposite and cured for 40 seconds from the occlusal direction according to the manufacturer 's instructions . a layer of polyvinyl siloxane was adapted around the root surfaces to simulate the periodontal ligament . the fracture resistance of the teeth was measured using a universal testing machine ( lr 50k ; lloyd instruments ) , under a compressive force at a strain rate of 0.5 mm / min . a 5 mm diameter round stainless steel ball was positioned parallel to the long axis of the teeth and centered over the teeth until the bar just contacted the occlusal surface of the restoration and the buccal and lingual cusps . [57 ] the study design was a three arm comparative study where the outcome variable was fracture resistance . the statistical software namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment ver.2.11.1 were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables etc . highest mean fracture resistance was observed with positive control followed by group b and group a respectively . one way anova test revealed a significant difference ( p = 0.001 ) between the groups [ table 1 ] [ figure 2 ] . on intergroup comparison , post - hoc tukey test revealed a moderately significant difference between control and group b ( p = 0.034 ) whereas a strongly significant difference was recorded between control and group a ( p = 0.002 ) and between control and group c ( p = 0.001 ) . no significant difference was recorded between any of the experimental groups ( group a , b and c ) ( p > 0.1 ) [ table 2 ] . mean fracture resistance in newton ( n ) in all four groups as seen in the study graph depicting mean fracture resistance values pair wise comparison : difference and p value in the study restoration of root filled teeth , despite posing a major challenge to the clinician , is an essential final step of root canal treatment . the purpose of the restoration is not only to repair , reinforce and strengthen the tooth , but also to provide an effective seal between the canal system and the mouth . the gold standard of post endodontic restorations is the full crown ; however , there are situations where the clinician may choose to delay the delivery of the full crown . in such cases , an efficient , direct , high strength adhesive restorations allow a more efficient transfer and distribution of functional stresses across the bonding interface to the tooth structure . the smaller size of the fillers allows them to have excellent optical properties along with good mechanical properties . despite the advances in material sciences , original ribbond ( group c ) consists of cold plasma treated , leno weave ultra high modulus ( lwuhmw ) polyethylene fibres , which are arranged in a lock - stitch design . interlig ( group b ) consists of glass fibres pre - impregnated with light curable composite resin arranged in a braided design . , and belli et al .. placing fibres on the occlusal surface keeps buccal and lingual cusps together and protects the natural cusps resulting in higher fracture resistance . the results of the present study revealed that the positive control group had highest mean fracture resistance ( 811.90 n ) . , and also denotes that endodontic and restorative treatment have a detrimental effect on fracture resistance of teeth . among the experimental groups , group b had the highest mean fracture resistance ( 600.49 n ) followed by group a ( 516.96 n ) , and group c ( 514.64 n ) [ table 1 ] [ figure 2 ] . the one way anova test done in the present study revealed a statistically significant difference ( p = 0.001 ) between all the groups [ table 1 ] . post - hoc tukey test revealed a moderately significant difference between control and group b ( p = 0.034 ) and a strongly significant difference between control and group a ( p = 0.002 ) , and between control and group c ( p = 0.001 ) . no significant difference was found between any of the experimental groups ( group a , b and c ) ( p > 0.1 ) [ table 2 ] . group a showed an acceptable fracture resistance due to the use of nanocomposites in the present study . the high filler loading enables nanocomposites to demonstrate good physical and mechanical properties and reinforce the tooth structure well . the fracture resistance of group a can be attributed to the increase in strain capacity of the adhesive resin and the improved physical and mechanical properties of nanocomposites . this study recorded that group b had the highest fracture resistance amongst the experimental groups . the superior performance of group b may be because of the pre - fabricated resin impregnation of these fibres by the manufacturers . also , glass fibres have very high tensile strength , density and percentage of elongation allowing them to withstand high stresses without fracturing . , who reported the glass fibres performed better than polyethylene fibres due to pre - impregnation with light cured composite which ensures a good bond with the composite resin . according to the manufacturer original ribbond fibres are woven using the lock - stitch leno weave which prevents slipping of fibres within resin matrix , prevents micro - cracks from propagating to form larger cracks and reinforces the restoration in multiple directions . , found that insertion of polyethylene fibre in the occlusal third of a composite restoration increased the fracture resistance of root filled teeth compared to teeth restored with composite resin alone . however , the present study showed that insertion of polyethylene fibre did not significantly increase the fracture resistance of a root filled tooth as compared to teeth restored with composite resin alone ( group a ) ( p > 0.1 ) . the causes for lower fracture resistance of group c may include the following : non uniform wetting of fibre with unfilled resin - manual wetting of original ribbond ( group c ) with unfilled adhesive was done as suggested by the manufacturer . this may lead to areas of non - uniform wetting in the fibre which will affect the adhesion of fibre to resin matrix . this treatment is meant to make the fibre surface more receptive to bonding with the resin however ; it makes the fibre very technique sensitive . this may lead to areas of non - uniform wetting in the fibre which will affect the adhesion of fibre to resin matrix . this treatment is meant to make the fibre surface more receptive to bonding with the resin however ; it makes the fibre very technique sensitive . though it was not among the objectives of the study it was observed that the majority of failures in the fibre reinforced groups were favourable in nature . , showed a relative loss of stiffness of 5% due to endodontic procedures and 63% in mod cavity preparation whereas the present study found a relatively smaller decrease of 25 - 35% in fracture resistance of root filled teeth with mod cavity preparation restored with nanocomposites or nanocomposites restored with different fibres . despite this decrease in fracture resistance all the experimental groups demonstrated results much higher than the average normal biting force of human maxillary premolars ( 100 - 300n ) . forces generated intraorally during function vary in magnitude , speed of application and direction , whereas the forces applied to the teeth in this study were at a constant direction and speed and they increased continuously until fracture occurred . root canal treatment and mod cavity preparation significantly reduced fracture resistance of root filled maxillary premolars ( p = 0.001).even though there was no statistically significant difference between the test groups , the fracture resistance of the composite impregnated glass fibre reinforced group was much higher . root canal treatment and mod cavity preparation significantly reduced fracture resistance of root filled maxillary premolars ( p = 0.001 ) . even though there was no statistically significant difference between the test groups , the fracture resistance of the composite impregnated glass fibre reinforced group was much higher .

blood - questing mosquitoes mainly rely on olfactory cues to locate their hosts ( takken , 1991 ; costantini et al . , 1996 ; takken and knols , 1999 ) . funestus giles , these cues are strongly reminiscent of those released by humans i.e. , the principal source of the mosquitoes blood meals ( gillies and coetzee , 1987 ) . odorants from human skin and carbon dioxide from breath are particularly important ( costantini et al . , 1996 ; mukabana et al . , 2004 ; spitzen et al . , this difference in host preference is expressed clearly in odor - guided behavior , where an . these behavioral differences also are reflected in the role of these species as malaria vectors , an . arabiensis is equally susceptible to the plasmodium parasite but , due to its different feeding behavior , is of less importance as a vector . host preference , and hence the selection of hosts based on their odorants , is of principal importance to understand the role of these mosquitoes in malaria epidemiology . given the strong association of an . gambiae with humans , unraveling the odor cues that mediate this behavior is of scientific as well as practical importance . for an . gambiae the principal olfactory cues of humans originate from the feet ( de jong and knols , 1995 ) , and recent work has demonstrated that these cues are partially produced by the microbial flora present on the feet ( verhulst et al . , 2010 ) . from these studies , several chemical compounds have been identified that play a critical role in the odor - mediated behavior of an . detailed information on the role of these compounds allows for the development of synthetic blends that can be used to better understand the host - seeking behavior of this mosquito . such blends also have the potential to be used for mosquito surveillance or intervention through mass trapping . studies on the development of kairomones for malaria vectors have demonstrated strong behavioral responses to synthetic blends of human odorant compounds ( smallegange et al . , 2010a ) . in laboratory and semi - field studies , these blends attract a large proportion of host - seeking mosquitoes , but when tested against a natural human host , or against natural human odorants released from a nylon matrix , these blends demonstrate poor competitive characters compared to the natural odorants . this suggests that either the concentration of the odorants in the blend was insufficient or that one or more compounds to make the blend sufficiently competitive were missing ( smallegange et al . , 2005 , 2010a ; verhulst et al . , 2011a ) . recent progress , however , has demonstrated the development of odor blends that approach the attractiveness of natural human skin odorants ( okumu et al . , 2010b ) . in addition , there is sometimes a mismatch between laboratory behavioral results and field trials , which can be caused by differences in concentration or spatial effects ( verhulst et al . , 2009 , 2011a ) . assessment of existing synthetic attractant compounds under semi - field and field conditions provides a potential for the development of technologies that can be used for sampling and control of malaria mosquitoes ( kline , 2006 ; jawara et al . the current study was designed to evaluate the attractiveness of selected synthetic blends and human hosts to host - seeking mosquitoes in western kenya , with emphasis on the malaria vectors an . thus , the comparative trapping efficacy of the attractant blends and the physiological status of the mosquitoes trapped were investigated . behavioral responses of mosquitoes to synthetic attractants were evaluated under field and semi - field conditions . the semi - field experiments utilized a laboratory colony of the mbita strain of an . aquatic stages of the mosquitoes were reared under ambient atmospheric conditions in screen - walled greenhouses at the thomas risley odhiambo ( tro ) campus of the international centre of insect physiology and ecology ( icipe ) located near mbita point township in western kenya . all larval instars were fed on tetramin baby fish food supplied three times a day . pupae were collected daily , transferred to adult holding rooms , and placed in mesh - covered cages ( 30 30 30 cm ) prior to adult emergence . adult mosquitoes were fed on 6% glucose solution through wicks made from adsorbent tissue paper . mosquitoes used for semi - field experiments were placed in mosquito - gauze covered plastic cups and starved for 8 hr . . only water , availed on wet cotton towels placed on top of mosquito holding cups , was provided during starvation . all semi - field experiments were carried out at night ( 20300630 h ) inside the screenhouses ( verhulst et al . , 2011b ) . all chemicals used to constitute the synthetic attractant blends in this study , with the exception of carbon dioxide , water , sugar , and yeast , were purchased from sigma - aldrich chemie gmbh ( germany ) . the chemicals included propionic acid ( 99.6% ) , butanoic acid ( 99.9% ) , pentanoic acid ( > 99% ) , heptanoic acid ( 98% ) , octanoic acid ( 99% ) , tetradecanoic acid ( 99% ) , ammonia solution ( purity 25% ) , ( s)-lactic acid ( 85% ) , isovaleric acid ( 99.8% ) , 4,5-dimethylthiazole ( 97% ) , 2-methyl-1-butanol ( 99% ; a racemic mixture of the r and s isomers of unknown ratio ) , and 3-methyl-1-butanol ( purity 98.5% ) . carbon dioxide was produced by mixing 250 g sucrose ( sony sugar company limited , kenya ) , 17.5 g dry yeast ( angel yeast company limited , china ) , and water ( 2 l ) as described in smallegange et al . field studies were carried out in lwanda and kigoche villages of homa bay and kisumu counties of western kenya , respectively . lwanda village is located on the southern shore of the winam gulf of lake victoria ( 002828s , 341722e ) at an altitude of 1,169 m above sea level ( verhulst et al . , 2011a ) . hoof prints of cattle and night - grazing hippopotami provide excellent mosquito breeding sites in lwanda . kigoche village lies adjacent to the ahero rice irrigation scheme ( 000819s , 345550e ) at an altitude of 1,160 m above sea level . kigoche has an average annual rainfall of 1,0001,800 mm and an average relative humidity of 65% . most houses in the two villages are mud - walled with open eaves , have corrugated iron - sheet roofs , have no ceiling , and are either single- or double - roomed . eaves , about one foot wide , increase ventilation in the houses and form the predominant entry points for mosquitoes ( snow , 1987 ; lindsay and snow , 1988 ) . malaria caused by plasmodium falciparum is endemic in the two villages . the villages experience two rainy seasons : between april june and september october . during these periods , cattle , goats , chicken , dogs , cats , and a few sheep constitute the domestic animal population , with cattle being most abundant . maize , millet and sorghum are cultivated at subsistence level in lwanda , whereas rice is a main cash crop in kigoche . in both villages , trapped mosquitoes were morphologically identified using the keys published by gillies and coetzee ( 1987 ) , counted , and the data entered in ms excel spreadsheets . abdominal statuses of female mosquitoes were categorized as unfed , blood - fed , or gravid . gambiae complex were identified to species using a ribosomal dna polymerase chain reaction assay ( scott et al . , 1993 ) . prototype blends were made by adding components to a standard attractive blend ( standard blend , sb ) consisting of ammonia , ( s)-lactic acid , and tetradecanoic acid ( smallegange et al . the standard blend was augmented with locally - made carbon dioxide plus individual additional candidate compounds or groups of 24 of these compounds . these compounds were selected following two studies in which a total of 39 chemical compounds were evaluated for their attractiveness to an . gambiae ( verhulst et al . , 2011b ; smallegange et al . , 2012 ) . all compounds except carbon dioxide were delivered to experimental mosquitoes by using low density polyethylene sachets ( ldpe ) . to prepare a sachet , a tube foil of ldpe material was cut ( 35 30 mm ) , thermally sealed at one end before pipetting 1,000 l of a pure compound through the open end of the sachet . the sachet was subsequently sealed , confining the candidate compounds within an area of 25 25 mm . the wall thickness of the ldpe material used was 0.1 mm ( isovaleric acid and 3-methyl-1-butanol ) , 0.2 mm ( 2-methyl-1-butanol ) , 0.03 mm ( ammonia solution , 4,5 dimethylthiazole , and tetradecanoic acid ) , and 0.05 mm ( ( s)-lactic acid ) . several sachets , varying in number according to the number of chemical compounds constituting a specific prototype blend , were placed onto a hook ( verhulst et al . , 2009 ) , which was subsequently inserted inside the outlet tube of a mm - x trap ( american biophysics cooperation , ri , usa ) . dual - choice tests comparing mosquito behavioral responses to a total of 15 prototype blends , with different combinations of the four candidate compounds + sb vs. sb alone then were conducted in the semi - field screenhouse facility by placing mm - x traps in diagonal corners ( verhulst et al . the experiments were replicated four times . the prototype blend attracting the highest numbers of an . gambiae mosquitoes in the screenhouse was evaluated in lwanda village and compared with the attractiveness of other blends , which we had tested in previous field experiments ( okumu et al . , 2010b ) . the number of mosquitoes attracted to each one of three blends including ifakara blend 1 ( ib1 ; okumu et al . , 2005 , 2009 ) and a best , newly - formulated prototype blend namely mbita blend ( mb ) was recorded . the textile composition was 90% polyamide and 10% spandex ( bata shoe company , kenya ) . the nylon strips were cut into narrow pieces each measuring 26.5 cm long and 1.0 cm wide . treatments included blend ib1 dispensed via nylon strips , ib1 dispensed via ldpe sachets , sb dispensed via nylon strips , sb dispensed via ldpe sachets , and mb dispensed via ldpe sachets . blend ib1 consisted of propionic acid ( 0.01% ; ldpe thickness 0.2 mm ) , butanoic acid ( 1% ; ldpe 0.2 mm ) , pentanoic acid ( 0.0001% ; ldpe 0.2 mm ) , 3-methyl butanoic acid ( 0.000001% ; ldpe 0.2 mm ) , heptanoic acid ( 0.0001% ; ldpe 0.1 mm ) , octanoic acid ( 0.0001% ; ldpe 0.1 mm ) , tetradecanoic acid ( 0.00025% ; ldpe 0.03 mm ) , ammonia ( 2.5% ; ldpe 0.03 mm ) , ( s)lactic acid ( 85% ; ldpe 0.05 mm ) , distilled water ( ldpe 0.2 mm ) , and carbon dioxide ( 63.23 2.82 ml / min ) . an unbaited mm - x trap acted as the negative control . all blends were dispensed from mm - x traps hung outside the bedroom window ( for details : see verhulst et al . a total of eight village houses were selected and experiments carried out from 1830 to 0630 h each night for 40 nights . the selected houses were mud - walled , had open eaves and corrugated iron sheet roofs . the houses , occupied by owners ( one person per house ) throughout the night , were located at least 25 m. apart ( hill et al . , 2007 ) . the blend that attracted most mosquitoes compared to the standard blend was selected and adapted for release on nylon strips , which are known to yield higher mosquito catches than ldpe sachets ( okumu et al . the blend , named mbita blend or mb consisted of 3-methyl-1-butanol , tetradecanoic acid , ammonia solution , ( s)-lactic acid , and carbon dioxide . the most effective dilutions for tetradecanoic acid ( 0.00025% ) , ammonia ( 2.5% ) , ( s)-lactic acid ( 85% ) had been determined previously ( okumu et al . thus , the optimal dilution for dispensing 3-methyl-1-butanol on nylon strips was determined experimentally under semi - field conditions using mm - x traps . binary assays evaluating mosquito behavioral responses to sb with all constituents availed at their optimally attractive concentrations vs. sb plus 3-methyl-1-butanol offered at variable dilutions ( 100% , 10% , 1% , 0.01% and 0.0001% and 0.000001% v / v ) were run . the efficacy of attracting host - seeking mosquitoes using mb was evaluated by testing it against ifakara blend 1 ( ib1 ) , the standard blend ( sb ) , and a control ( unbaited trap ) under semi - field conditions . all blends were dispensed on nylon strips by pipetting 1,000 l of each component of a blend on a separate strip ( measuring 26.5 1.0 cm ) . the 3-methyl-1-butanol component of mb was used at a dilution of 0.000001% . in kigoche village , five houses spaced apart at a distance of at least 25 m and at least 100 m away from rice paddies were selected for the study . a 5 5 latin square experimental design preceded by a 5 d trial period was run to assess the potential of mb in attracting malaria and other mosquito vectors . the treatments , assigned to each of the five houses on rotational basis per night , included a human host , ib1 , sb , mb , and an empty house i.e. , all components of the synthetic attractants , with the exception of carbon dioxide , were delivered via nylon strips ( okumu et al . , 2010a ) and dispensed using mm - x traps . the traps were suspended 15 cm above a bed inside unimpregnated mosquito nets , and were operated using 12 volt batteries . one of the fans on the mm - x trap was disabled to prevent it from trapping mosquitoes . mosquitoes were trapped by hanging an unlit cdc miniature light trap ( model 512 ; john w. hock company , gainesville fl . , usa ) operated on 6 v batteries ( gaston battery industry ltd , china ) beside and at 15 cm ( jawara et al . , 2009 ) above the odor outlet tube of the mm - x trap , outside the bed net . both mm - x and cdc light traps were hung on the foot end region of the beds in all cases ( mboera et al . , 2000 ) . only one mm - x trap and one cdc light trap were used in houses where mosquito catches were based on synthetic attractants . the volunteer slept inside a well tucked - in bed net with a cdc light trap suspended besides it . whereas a total of five adult men aged 1825 years volunteered to participate in the study , only one individual participated per night . each experimental night lasted from 1900 to 0630 h. after the experiments traps were disconnected from the batteries , the trapped mosquitoes were taken to the laboratory , where they were killed by freezing at 4c . consent for houses to be used in the study was obtained from the household heads and the local administration prior to the start of the study . dispersion tests , performed to establish whether means equaled variances ( grafen and rosie , 2005 ) , were employed to determine if the count data indeed assumed a poisson distribution . henceforth , the number of mosquitoes attracted to the different sources of behavioral stimuli ( human subjects , control , or the synthetic attractants ) was modeled as a proportion of the total number of mosquitoes recovered from the different treatments . all statistical analyses were carried out using generalized linear models ( agresti , 1990 ) in which data were transformed to assume linearity using a logarithmic link function . all fitted models were followed by post hoc t - tests , to assess levels of statistical difference between pairs of competing blends assessed through the binary assays . all data were analyzed using the general statistical software program ( genstat discovery edition 3 ) ( payne , 1986 ) . adding 3-methyl-1-butanol to the standard blend of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide either singly or in combination with 2-methyl-1-butanol formed the two most potent attractant blends for host - seeking an . gambiae mosquitoes ( catching 72% of those released ) relative to the standard blend on its own under semi - field conditions ( table 1 ) . neither adding 2-methyl-1-butanol to the basic blend , nor adding it to 3-methyl-1-butanol enhanced attractiveness . the blend containing 3-methyl-1-butanol plus components of the standard blend was considered the most potent synthetic attractant for an . the prototype product , termed mbita blend ( mb ) , consisted of 3-methyl-1-butanol ( released in 0.1 mm - ldpe sachets ) , tetradecanoic acid ( 0.03 mm - ldpe ) , ammonia solution ( 0.03 mm - ldpe ) , ( s)-lactic acid ( 0.05 mm - ldpe ) , and carbon dioxide ( 130 ml / min ) . as adding isovaleric acid and 4,5 dimethyl - thiazole to the standard blend diminished the numbers of an . gambiae attracted ( table 1 ) , these compounds were excluded from the prototype blend.table 1mean ( se ) mosquito catches per night and levels of statistical difference ( p - value ) between 15 prototype synthetic blends vs. a standard blend ( sb ) of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide . each of the compounds except carbon dioxide was dispensed from a ldpe - sachet in pure form . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped out of a total of 800 released.% response equals n/800 . compound 1 , 2 , 3 , and 4 are isovaleric acid , 4,5 dimethyl - thiazole , 2-methyl-1-butanol , and 3-methyl-1-butanol , respectivelydescription of synthetic blendnn% responsemosquito trap catches ( mean se)p - valuestandard blend ( sb)synthetic blend1 . sb + compound 1&2&3&442192728.00 9.8125.75 13.490.350 mean ( se ) mosquito catches per night and levels of statistical difference ( p - value ) between 15 prototype synthetic blends vs. a standard blend ( sb ) of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide . each of the compounds except carbon dioxide was dispensed from a ldpe - sachet in pure form . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped out of a total of 800 released.% response equals n/800 . compound 1 , 2 , 3 , and 4 are isovaleric acid , 4,5 dimethyl - thiazole , 2-methyl-1-butanol , and 3-methyl-1-butanol , respectively a total of 1,286 mosquitoes were trapped in lwanda village over a period of 40 d from 28 april 2010 to 11 june 2010 . funestus ( n = 207 ) , culex species ( n = 544 ) , mansonia species ( n = 112 ) , aedes species ( n = 43 ) , and other mosquito species ( n = 121 ) ( table 2 ) . gambiae s.l . than the unbaited traps , no blend attracted significantly more mosquitoes of this species than the other ( table 2 ) . however , the standard blend dispensed from nylon strips attracted comparatively higher numbers of mansonia spp , aedes spp . and an . ifakara blend 1 ( ib1 ) dispensed using nylon strips attracted comparatively higher numbers of culex spp . and was performed before the optimal dilution for releasing mb on nylon strips was determined , it was not possible to fully evaluate its competitiveness against other attractants in lwanda village . out of the 107 specimens of an . , mansonia spp . , aedes spp . and other mosquito species collected indoors per night in lwanda village , western kenya . mosquitoes were attracted to various synthetic attractants dispensed from nylon strips or ldpe sachets mounted in mm - x traps placed under a bed net . n = number of replicates ( nights ) ; n is total number of mosquitoes per taxon caught over 40 nights . numbers followed by different letter superscripts in the same column differ significantly ( p < 0.05)treatment ( delivery)nan . spp.aedes spp.other spp.empty ( control)400.650.552.280.250.100.43ib1 ( ldpe)401.300.531.850.530.150.43ib1 ( nylon strips)401.431.152.980.550.130.58sb ( ldpe)401.050.582.480.330.250.38sb ( nylon strips)401.031.731.880.600.380.40 mb ( ldpe)401.030.582.150.550.080.83total mosquito catches ( n)25920754411243121 mean numbers of anopheles gambiae s.l . , an . , aedes spp . and other mosquito species collected indoors per night in lwanda village , western kenya . mosquitoes were attracted to various synthetic attractants dispensed from nylon strips or ldpe sachets mounted in mm - x traps placed under a bed net . n = number of replicates ( nights ) ; n is total number of mosquitoes per taxon caught over 40 nights . numbers followed by different letter superscripts in the same column differ significantly ( p < 0.05 ) the overall combined response of the mosquitoes to the blends ranged from 39% to 68% ( table 3 ) . dilutions of 3-methyl-1-bunanol from pure compound to 10,000 times were significantly ( p < 0.001 ) in favor of the standard blend , except for the 1,000-fold dilution , for which no difference in attractiveness between the two blends was found . only with the highest dilution ( 100,000 ) did significantly more ( p < 0.05 ) mosquitoes respond to the augmented blend than to the standard blend . this blend ( mb ) subsequently was chosen for further evaluation.table 3mean number se of anopheles gambiae caught per night in mm - x traps baited with a synthetic blend containing various dilutions of 3-methyl-1-butanol plus a standard blend vs. the standard blend alone , dispensed from nylon strips , under semi - field conditions . n is the number of replicates ( nights ) and n the number of mosquitoes trappeddilution ( % ) nn% responsemosquito trap catches ( mean se)p - valuesbsynthetic blendpure compound ( 99.9)45476877.50 16.9759.25 2.140.00210.043404352.00 18.4833.00 11.200.0011.044745968.25 13.1250.25 17.800.0010.144125253.25 4.29049.75 14.420.4900.0143073945.25 6.2431.50 4.050.0020.00143794742.25 12.5952.25 8.090.040 mean number se of anopheles gambiae caught per night in mm - x traps baited with a synthetic blend containing various dilutions of 3-methyl-1-butanol plus a standard blend vs. the standard blend alone , dispensed from nylon strips , under semi - field conditions . n is the number of replicates ( nights ) and n the number of mosquitoes trapped the attraction efficacy of mb vs. ib1 , sb , and a control ( unbaited trap ) was evaluated over a period of 16 nights i.e. , from 30 november 2010 to 16 december 2010 . out of the total of 3,200 mosquitoes used for these experiments , there was a significant effect ( glm ; p < 0.001 ) of treatment on mosquito trap catches . whereas all the synthetic blends attracted significantly more mosquitoes than the unbaited control ( p = 0.001 ) , mb attracted more mosquitoes than blends ib1 ( p = 0.001 ) and sb ( p = 0.001 ) . results of these experiments are shown in table 4.table 4mean number se of anopheles gambiae caught per night in mm - x traps baited with different synthetic blends under semi - field conditions . numbers with different letter superscripts in the same column differ significantly ( glm ; p < 0.001 ) . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped . the effect of treatment ( p values ) on overall mosquito responses is also shownblend ( delivery)nnmosquito trap catches ( mean se)treatment ( p - value)control ( no odors)16583.62 0.810.001ib11669143.2 4.40.001sb1652532.8 5.40.001mb1687854.9 8.10.001 mean number se of anopheles gambiae caught per night in mm - x traps baited with different synthetic blends under semi - field conditions . numbers with different letter superscripts in the same column differ significantly ( glm ; p < 0.001 ) . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped . the effect of treatment ( p values ) on overall mosquito responses is also shown the competitiveness of mb over other baits in attracting malaria and other wild mosquitoes was evaluated in kigoche village for 30 d spanning the period 0130 april , 2011 . funestus , 201 ( 9.9% ) other anopheles species and 283 ( 14% ) culex species were collected . the collections also included one aedes and one mansonia mosquito representing 0.1% of the collection . gambiae s.l . collected included 34 male and 1,071 female ( 92.8% unfed ; 5.3% blood fed , and 1.9% gravid ) mosquitoes . funestus species of mosquitoes included 56 male and 377 female ( 96.8% unfed ; 2.1% blood fed , and 1.1% gravid ) mosquitoes . the culex sub - sample consisted of 196 ( 97.5% ) unfed and 5 ( 2.5% ) blood fed mosquitoes . analysis of the data revealed that houses baited with mb caught 31.5% ( n = 637 ) of the mosquitoes ( males and females ) while those with a human being , ib1 , sb , or no host cue collected 26.9% ( n = 545 ) , 19.6% ( n = 397 ) , 17.4% ( n = 352 ) , and 4.6% ( n = 93 ) of the mosquitoes , respectively . ( p < 0.042 ) , an . funestus ( p < 0.014 ) and culex species ( p < the mean number of female mosquitoes with the exception of culex species collected from the five traditional houses did not differ significantly . the numbers of mosquitoes of all species collected from the empty house were significantly lower than those collected from houses baited with mb ( p = 0.001 ) , a human being , ib1 ( p = 0.001 ) , or sb ( p = 0.001 ) . funestus and culex species than a human being ( p = 0.002 and 0.001 , respectively ) or sb ( p = 0.001 and 0.005 , respectively ) . mosquitoes than ib1 ( p = 0.001 ) and sb ( p = 0.001 ) , these numbers did not differ significantly from those attracted by a human being ( p = 0.121 ) . furthermore , although mb attracted more an . funestus mosquitoes then ib1 ( p = 0.001 ) , the number of culex mosquitoes attracted to mb were not different from those attracted to blend ib1 ( p = 0.140)table 5total pooled number of female mosquitoes and mean number se per species per night trapped in response to different synthetic attractant blends ( dispensed form nylon strips ) and a human host in kigoche village , western kenya . numbers with different letter superscripts in the same column differ significantly . the number of replicates ( n ) is showntreatmentntotal no . of female mosquitoes collectedmean ( se ) of female mosquito catches per nightan . funestusculex spp.aedes spp.mansonia spp.other anophelinescontrol30811.2 0.410.8 0.280.33 0.15000.37 0.021human305279.9 2.12.93 0.651.17 0.28003.57 0.96ib1303757.13 2.902.0 0.391.7 0.390.03 0.030.03 0.031.60 0.49 sb303336.27 1.402.03 0.59 1.27 0.33001.53 0.41mb3061811.2 2.14.8 1.262.23 0.72002.37 0.80 total pooled number of female mosquitoes and mean number se per species per night trapped in response to different synthetic attractant blends ( dispensed form nylon strips ) and a human host in kigoche village , western kenya . numbers with different letter superscripts in the same column differ significantly . the mosquito catches in the screenhouse and the village houses consistently showed a higher number of an . gambiae when traps were baited with the mbita blend compared to the ifakara blend 1 and the standard blend . in kigoche village , significantly more an . gambiae s.l . funestus , the main malaria vectors in western kenya , were caught with mb than with the other two blends , and catches were similar to ( an . funestus ) attracted to a human host . in a previous study in tanzania , that used experimental huts , okumu et al . we conclude , therefore , that synthetic blends can be effectively used for sampling african malaria mosquitoes . the odor - baited technology has a distinct advantage over the widely - used cdc trap + human - under - a - bed - net method introduced by garrett - jones and magayuka ( 1975 ) , which has since been used as the standard method for assessing entomological inoculation rates and other relevant epidemiological parameters for malaria ( smith et al . , 2006 ; bousema et al . , 2010 ) ( 1991 ) and found to sample approximately two - thirds of the mosquitoes attracted to a human host . later assessments of the cdc trap reported a similar efficiency but with local variations ( costantini et al . , 1998 ) . given the natural variation in attractiveness of humans to mosquitoes ( smith , 1956 ; brouwer , 1960 ; knols et al . , 1995 ; qiu et al . , 2006 ) , caused by the variation in human skin odorants and breath ( mukabana et al . , 2004 ; the need for a human host to be present during the all - night collections , the cdc trap plus bed net combination for mosquito trapping has obvious disadvantages . these disadvantages can be overcome by replacing the human host with synthetic bait ( over a time frame of at least 1 week ) , which produces a consistent and constant blend of odorants and hence avoids the variance caused by the human - dependent method of mosquito sampling . natural variance in human odorants is likely to affect trap catches , and may potentially lead to stronger attractiveness of the natural host than the synthetic blend , but also to considerably lower attractiveness . odor - baited traps also can be used in large numbers simultaneously across a study area , which avoids bias caused by spatial effects . in this study , we examined the use of ldpe as a material for the slow release of odorants , analogous to the successful use of ldpe for attractants of tsetse flies ( green , 1994 ; torr et al . , 1995 ) . odorants attractive to tsetse flies and released through ldpe remain active for many months and have been used widely for the mass trapping of tsetse flies in remote areas . although odorants released through ldpe used in our study proved attractive to anopheline and other mosquitoes , we found that releasing the odorants from nylon strips caused a significantly higher attraction of several of the mosquito species . it seems likely that the size of the ldpe sachets we employed was too small to provide a sufficiently large surface to release the quantity of odorants needed for optimal attraction of the mosquitoes . it is also possible that the carboxylic acids present in the blend disperse in smaller quantities through ldpe than from the nylon strips . the odor baits used for tsetse flies consist of entirely different chemicals ( mostly 1-octen-3-ol , 4-methyl phenol , and 3-n - propyl phenol ) ( bursell et al . , 1988 ; vale , 1993 ) , which may disperse more readily through ldpe than the mosquito kairomones . from our high throughput work on candidate attractants of an . gambiae ( verhulst et al . , 2011a ; smallegange et al . , 2012 ) , we selected isovaleric acid , 4,5-dimethylthiazole , 2-methyl-1-butanol , and 3-methyl-1-butanol because of the increased attractiveness expressed when these compounds were added to the standard blend . it was , therefore , surprising that in contrast to previous results , here under semi - field conditions both isovaleric acid and 4,5-dimethylthiazole caused an inhibitory effect . even more so , when two or more of these compounds were added to the standard blend , the effect of each compound individually was cancelled out , or the entire blend seemed repellent , causing most mosquitoes to avoid entering any of the traps . with all four compounds added to the standard blend , the overall response of the mosquitoes was significantly reduced . it is possible that when employing higher dilutions of each of these compounds when studying the effect of multiple compounds , the kairomonal potency of the blend might be further enhanced . this compound is known to exist of two isomers ( r and s ) . in our study , we used a racemic mixture . it is possible that only one of the isomers would have evoked a behavioral effect in the mosquito , which should be followed up in a future study . the results from the dose - effect study with 3-methyl-1-butanol show that highly diluted concentrations cause attraction of an . for example , the mosquito repellent deet is repellent over a wide range of concentrations , but becomes attractive at very low concentrations ( mehr et al . , 1990 ) , and this principle of concentration - dependent dual action might apply to many other odorant cues that affect insect behavior ( smallegange and takken , 2010 ) . we examined the response of mosquitoes to candidate odorant blends in traps placed outdoors , next to a house , and traps placed indoors . a recent study showed that catches from indoor and outdoor mm - x traps were comparable ( jawara et al . , 2009 ) , suggesting that the mosquitoes perceived the odorant cues from a distance , and would either be caught while approaching the house or after house entry . although lwanda and kigoche are approximately 120 km apart , with different ecologies , the mean number of an . odor - baited traps caught significantly more mosquitoes than unbaited traps , thus demonstrating the attractive effect of the blends under investigation . on average , the trap placed next to a human - occupied bed net caught similar numbers of mosquitoes as the trap next to the dispenser baited with the mbita blend . unlike in the tanzania study , where ib1 was 23 times as attractive as the human - odor baited trap ( okumu et al . , 2010b ) , here , ib1 was less attractive than the human - baited trap . this difference between these studies may have been caused by different attractiveness of the human volunteers , environmental differences , or both . the differences were small , though , and the overall result shows that the three synthetic blends approached the attractiveness of the human host ( table 5 ) , with mb being the most attractive blend . funestus ( 96.8% ) collected from kigoche village was unfed implies that the blends , more so mb that attracted the highest numbers of mosquitoes , mainly target host - seeking mosquitoes . it is not surprising , therefore , that the numbers of mosquitoes attracted to mb were physiologically not significantly different from those attracted to human subjects . funestus than the trap next to the human - occupied bed net , the catches of mosquitoes displaying this physiological status as well as gravid ones were small . anopheles gambiae s.s . was the species used in the semi - field study , whereas in the field study an . gambiae s.s . have declined dramatically , presumably as a result of widespread bed net use , whereas populations of an . the latter species is less affected by the bed nets as it is more exophilic , and less anthropophilic . arabiensis is attracted strongly to the synthetic blends , as shown by the okumu et al . ( 2010b ) study as well as by the current study ( tables 2 and 5 ) . because an . arabiensis can be an important malaria vector and is regularly found in human landing catches ( port et al . , 1980 ) , the synthetic blends used here can provide a tool for the sampling of this species . funestus was also caught in both villages , responding significantly more strongly to the mbita blend than to the other blends or the human host . as other , non - anopheline mosquitoes were also collected , the synthetic blends , notably mb , should be considered as attractants for a wide range of mosquito species , including other disease vectors . we conclude that the multiple - component odorant blends described in this study can be considered to replace traditional malaria vector sampling tools such as cdc light traps and the human landing catch . host , used in multiple locations simultaneously over a study area , is a distinct advantage in providing unbiased data on relative mosquito densities . this will be of advantage for malaria epidemiological studies , but also cost effective . because the baits are also effective outdoors , they open up a novel avenue for sampling of outdoor biting malaria vectors , which have recently been reported to be a major source of malaria transmission ( reddy et al . , 2011 ; russell et al . ,

estimating the biting fraction of mosquitoes is of critical importance for risk assessment of malaria transmission . here , we present a novel odor - based tool that has been rigorously assessed in semi - field assays and traditional african villages for estimating the number of mosquitoes that enter houses in search of a blood meal . a standard synthetic blend ( sb ) consisting of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide was complemented with isovaleric acid , 4,5 dimethylthiazole , 2-methyl-1-butanol , and 3-methyl-1-butanol in various combinations and concentrations , and tested for attractiveness to the malaria mosquito anopheles gambiae . compounds were released through low density polyethylene ( ldpe ) material or from nylon strips ( nylon ) . studies were done in a semi - field facility and two traditional villages in western kenya . the alcohol 3-methyl-1-butanol significantly increased the attraction of sb . the other compounds proved less effective or inhibitory . tested in a village , 3-methyl-1-butanol , released from ldpe , increased the attraction of sb . further studies showed a significantly enhanced attraction of adding 3-methyl-1-butanol to sb compared to previously - published attractive blends both under semi - field and village conditions . other mosquito species with relevance for public health were collected with this blend in significantly higher numbers as well . these results demonstrate the advent of a novel , reliable odor - based sampling tool for the collection of malaria and other mosquitoes . the advantage of this odor - based tool over existing mosquito sampling tools is its reproducibility , objectiveness , and relatively low cost compared to current standards of cdc light traps or the human landing catch .

Introduction Methods and Materials Results Discussion

arabiensis is equally susceptible to the plasmodium parasite but , due to its different feeding behavior , is of less importance as a vector . detailed information on the role of these compounds allows for the development of synthetic blends that can be used to better understand the host - seeking behavior of this mosquito . in laboratory and semi - field studies , these blends attract a large proportion of host - seeking mosquitoes , but when tested against a natural human host , or against natural human odorants released from a nylon matrix , these blends demonstrate poor competitive characters compared to the natural odorants . assessment of existing synthetic attractant compounds under semi - field and field conditions provides a potential for the development of technologies that can be used for sampling and control of malaria mosquitoes ( kline , 2006 ; jawara et al . the current study was designed to evaluate the attractiveness of selected synthetic blends and human hosts to host - seeking mosquitoes in western kenya , with emphasis on the malaria vectors an . behavioral responses of mosquitoes to synthetic attractants were evaluated under field and semi - field conditions . aquatic stages of the mosquitoes were reared under ambient atmospheric conditions in screen - walled greenhouses at the thomas risley odhiambo ( tro ) campus of the international centre of insect physiology and ecology ( icipe ) located near mbita point township in western kenya . pupae were collected daily , transferred to adult holding rooms , and placed in mesh - covered cages ( 30 30 30 cm ) prior to adult emergence . mosquitoes used for semi - field experiments were placed in mosquito - gauze covered plastic cups and starved for 8 hr . all semi - field experiments were carried out at night ( 20300630 h ) inside the screenhouses ( verhulst et al . all chemicals used to constitute the synthetic attractant blends in this study , with the exception of carbon dioxide , water , sugar , and yeast , were purchased from sigma - aldrich chemie gmbh ( germany ) . the chemicals included propionic acid ( 99.6% ) , butanoic acid ( 99.9% ) , pentanoic acid ( > 99% ) , heptanoic acid ( 98% ) , octanoic acid ( 99% ) , tetradecanoic acid ( 99% ) , ammonia solution ( purity 25% ) , ( s)-lactic acid ( 85% ) , isovaleric acid ( 99.8% ) , 4,5-dimethylthiazole ( 97% ) , 2-methyl-1-butanol ( 99% ; a racemic mixture of the r and s isomers of unknown ratio ) , and 3-methyl-1-butanol ( purity 98.5% ) . carbon dioxide was produced by mixing 250 g sucrose ( sony sugar company limited , kenya ) , 17.5 g dry yeast ( angel yeast company limited , china ) , and water ( 2 l ) as described in smallegange et al . field studies were carried out in lwanda and kigoche villages of homa bay and kisumu counties of western kenya , respectively . most houses in the two villages are mud - walled with open eaves , have corrugated iron - sheet roofs , have no ceiling , and are either single- or double - roomed . prototype blends were made by adding components to a standard attractive blend ( standard blend , sb ) consisting of ammonia , ( s)-lactic acid , and tetradecanoic acid ( smallegange et al . all compounds except carbon dioxide were delivered to experimental mosquitoes by using low density polyethylene sachets ( ldpe ) . the wall thickness of the ldpe material used was 0.1 mm ( isovaleric acid and 3-methyl-1-butanol ) , 0.2 mm ( 2-methyl-1-butanol ) , 0.03 mm ( ammonia solution , 4,5 dimethylthiazole , and tetradecanoic acid ) , and 0.05 mm ( ( s)-lactic acid ) . several sachets , varying in number according to the number of chemical compounds constituting a specific prototype blend , were placed onto a hook ( verhulst et al . dual - choice tests comparing mosquito behavioral responses to a total of 15 prototype blends , with different combinations of the four candidate compounds + sb vs. sb alone then were conducted in the semi - field screenhouse facility by placing mm - x traps in diagonal corners ( verhulst et al . the number of mosquitoes attracted to each one of three blends including ifakara blend 1 ( ib1 ; okumu et al . treatments included blend ib1 dispensed via nylon strips , ib1 dispensed via ldpe sachets , sb dispensed via nylon strips , sb dispensed via ldpe sachets , and mb dispensed via ldpe sachets . blend ib1 consisted of propionic acid ( 0.01% ; ldpe thickness 0.2 mm ) , butanoic acid ( 1% ; ldpe 0.2 mm ) , pentanoic acid ( 0.0001% ; ldpe 0.2 mm ) , 3-methyl butanoic acid ( 0.000001% ; ldpe 0.2 mm ) , heptanoic acid ( 0.0001% ; ldpe 0.1 mm ) , octanoic acid ( 0.0001% ; ldpe 0.1 mm ) , tetradecanoic acid ( 0.00025% ; ldpe 0.03 mm ) , ammonia ( 2.5% ; ldpe 0.03 mm ) , ( s)lactic acid ( 85% ; ldpe 0.05 mm ) , distilled water ( ldpe 0.2 mm ) , and carbon dioxide ( 63.23 2.82 ml / min ) . the blend that attracted most mosquitoes compared to the standard blend was selected and adapted for release on nylon strips , which are known to yield higher mosquito catches than ldpe sachets ( okumu et al . the blend , named mbita blend or mb consisted of 3-methyl-1-butanol , tetradecanoic acid , ammonia solution , ( s)-lactic acid , and carbon dioxide . the most effective dilutions for tetradecanoic acid ( 0.00025% ) , ammonia ( 2.5% ) , ( s)-lactic acid ( 85% ) had been determined previously ( okumu et al . thus , the optimal dilution for dispensing 3-methyl-1-butanol on nylon strips was determined experimentally under semi - field conditions using mm - x traps . the efficacy of attracting host - seeking mosquitoes using mb was evaluated by testing it against ifakara blend 1 ( ib1 ) , the standard blend ( sb ) , and a control ( unbaited trap ) under semi - field conditions . all blends were dispensed on nylon strips by pipetting 1,000 l of each component of a blend on a separate strip ( measuring 26.5 1.0 cm ) . in kigoche village , five houses spaced apart at a distance of at least 25 m and at least 100 m away from rice paddies were selected for the study . a 5 5 latin square experimental design preceded by a 5 d trial period was run to assess the potential of mb in attracting malaria and other mosquito vectors . , all components of the synthetic attractants , with the exception of carbon dioxide , were delivered via nylon strips ( okumu et al . both mm - x and cdc light traps were hung on the foot end region of the beds in all cases ( mboera et al . henceforth , the number of mosquitoes attracted to the different sources of behavioral stimuli ( human subjects , control , or the synthetic attractants ) was modeled as a proportion of the total number of mosquitoes recovered from the different treatments . adding 3-methyl-1-butanol to the standard blend of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide either singly or in combination with 2-methyl-1-butanol formed the two most potent attractant blends for host - seeking an . gambiae mosquitoes ( catching 72% of those released ) relative to the standard blend on its own under semi - field conditions ( table 1 ) . the prototype product , termed mbita blend ( mb ) , consisted of 3-methyl-1-butanol ( released in 0.1 mm - ldpe sachets ) , tetradecanoic acid ( 0.03 mm - ldpe ) , ammonia solution ( 0.03 mm - ldpe ) , ( s)-lactic acid ( 0.05 mm - ldpe ) , and carbon dioxide ( 130 ml / min ) . gambiae attracted ( table 1 ) , these compounds were excluded from the prototype blend.table 1mean ( se ) mosquito catches per night and levels of statistical difference ( p - value ) between 15 prototype synthetic blends vs. a standard blend ( sb ) of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped out of a total of 800 released.% response equals n/800 . compound 1 , 2 , 3 , and 4 are isovaleric acid , 4,5 dimethyl - thiazole , 2-methyl-1-butanol , and 3-methyl-1-butanol , respectivelydescription of synthetic blendnn% responsemosquito trap catches ( mean se)p - valuestandard blend ( sb)synthetic blend1 . sb + compound 1&2&3&442192728.00 9.8125.75 13.490.350 mean ( se ) mosquito catches per night and levels of statistical difference ( p - value ) between 15 prototype synthetic blends vs. a standard blend ( sb ) of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide . each of the compounds except carbon dioxide was dispensed from a ldpe - sachet in pure form . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped out of a total of 800 released.% response equals n/800 . compound 1 , 2 , 3 , and 4 are isovaleric acid , 4,5 dimethyl - thiazole , 2-methyl-1-butanol , and 3-methyl-1-butanol , respectively a total of 1,286 mosquitoes were trapped in lwanda village over a period of 40 d from 28 april 2010 to 11 june 2010 . funestus ( n = 207 ) , culex species ( n = 544 ) , mansonia species ( n = 112 ) , aedes species ( n = 43 ) , and other mosquito species ( n = 121 ) ( table 2 ) . however , the standard blend dispensed from nylon strips attracted comparatively higher numbers of mansonia spp , aedes spp . and other mosquito species collected indoors per night in lwanda village , western kenya . mosquitoes were attracted to various synthetic attractants dispensed from nylon strips or ldpe sachets mounted in mm - x traps placed under a bed net . n = number of replicates ( nights ) ; n is total number of mosquitoes per taxon caught over 40 nights . and other mosquito species collected indoors per night in lwanda village , western kenya . this blend ( mb ) subsequently was chosen for further evaluation.table 3mean number se of anopheles gambiae caught per night in mm - x traps baited with a synthetic blend containing various dilutions of 3-methyl-1-butanol plus a standard blend vs. the standard blend alone , dispensed from nylon strips , under semi - field conditions . n is the number of replicates ( nights ) and n the number of mosquitoes trappeddilution ( % ) nn% responsemosquito trap catches ( mean se)p - valuesbsynthetic blendpure compound ( 99.9)45476877.50 16.9759.25 2.140.00210.043404352.00 18.4833.00 11.200.0011.044745968.25 13.1250.25 17.800.0010.144125253.25 4.29049.75 14.420.4900.0143073945.25 6.2431.50 4.050.0020.00143794742.25 12.5952.25 8.090.040 mean number se of anopheles gambiae caught per night in mm - x traps baited with a synthetic blend containing various dilutions of 3-methyl-1-butanol plus a standard blend vs. the standard blend alone , dispensed from nylon strips , under semi - field conditions . n is the number of replicates ( nights ) and n the number of mosquitoes trapped the attraction efficacy of mb vs. ib1 , sb , and a control ( unbaited trap ) was evaluated over a period of 16 nights i.e. results of these experiments are shown in table 4.table 4mean number se of anopheles gambiae caught per night in mm - x traps baited with different synthetic blends under semi - field conditions . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped . the effect of treatment ( p values ) on overall mosquito responses is also shownblend ( delivery)nnmosquito trap catches ( mean se)treatment ( p - value)control ( no odors)16583.62 0.810.001ib11669143.2 4.40.001sb1652532.8 5.40.001mb1687854.9 8.10.001 mean number se of anopheles gambiae caught per night in mm - x traps baited with different synthetic blends under semi - field conditions . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped . the effect of treatment ( p values ) on overall mosquito responses is also shown the competitiveness of mb over other baits in attracting malaria and other wild mosquitoes was evaluated in kigoche village for 30 d spanning the period 0130 april , 2011 . funestus species of mosquitoes included 56 male and 377 female ( 96.8% unfed ; 2.1% blood fed , and 1.1% gravid ) mosquitoes . funestus mosquitoes then ib1 ( p = 0.001 ) , the number of culex mosquitoes attracted to mb were not different from those attracted to blend ib1 ( p = 0.140)table 5total pooled number of female mosquitoes and mean number se per species per night trapped in response to different synthetic attractant blends ( dispensed form nylon strips ) and a human host in kigoche village , western kenya . the number of replicates ( n ) is showntreatmentntotal no . funestusculex spp.aedes spp.mansonia spp.other anophelinescontrol30811.2 0.410.8 0.280.33 0.15000.37 0.021human305279.9 2.12.93 0.651.17 0.28003.57 0.96ib1303757.13 2.902.0 0.391.7 0.390.03 0.030.03 0.031.60 0.49 sb303336.27 1.402.03 0.59 1.27 0.33001.53 0.41mb3061811.2 2.14.8 1.262.23 0.72002.37 0.80 total pooled number of female mosquitoes and mean number se per species per night trapped in response to different synthetic attractant blends ( dispensed form nylon strips ) and a human host in kigoche village , western kenya . funestus , the main malaria vectors in western kenya , were caught with mb than with the other two blends , and catches were similar to ( an . in this study , we examined the use of ldpe as a material for the slow release of odorants , analogous to the successful use of ldpe for attractants of tsetse flies ( green , 1994 ; torr et al . odorants attractive to tsetse flies and released through ldpe remain active for many months and have been used widely for the mass trapping of tsetse flies in remote areas . although odorants released through ldpe used in our study proved attractive to anopheline and other mosquitoes , we found that releasing the odorants from nylon strips caused a significantly higher attraction of several of the mosquito species . , 2012 ) , we selected isovaleric acid , 4,5-dimethylthiazole , 2-methyl-1-butanol , and 3-methyl-1-butanol because of the increased attractiveness expressed when these compounds were added to the standard blend . it was , therefore , surprising that in contrast to previous results , here under semi - field conditions both isovaleric acid and 4,5-dimethylthiazole caused an inhibitory effect . even more so , when two or more of these compounds were added to the standard blend , the effect of each compound individually was cancelled out , or the entire blend seemed repellent , causing most mosquitoes to avoid entering any of the traps . we examined the response of mosquitoes to candidate odorant blends in traps placed outdoors , next to a house , and traps placed indoors . on average , the trap placed next to a human - occupied bed net caught similar numbers of mosquitoes as the trap next to the dispenser baited with the mbita blend . , 2010b ) , here , ib1 was less attractive than the human - baited trap . funestus than the trap next to the human - occupied bed net , the catches of mosquitoes displaying this physiological status as well as gravid ones were small . , 1980 ) , the synthetic blends used here can provide a tool for the sampling of this species . funestus was also caught in both villages , responding significantly more strongly to the mbita blend than to the other blends or the human host . we conclude that the multiple - component odorant blends described in this study can be considered to replace traditional malaria vector sampling tools such as cdc light traps and the human landing catch . because the baits are also effective outdoors , they open up a novel avenue for sampling of outdoor biting malaria vectors , which have recently been reported to be a major source of malaria transmission ( reddy et al .

arabiensis is equally susceptible to the plasmodium parasite but , due to its different feeding behavior , is of less importance as a vector . host preference , and hence the selection of hosts based on their odorants , is of principal importance to understand the role of these mosquitoes in malaria epidemiology . gambiae with humans , unraveling the odor cues that mediate this behavior is of scientific as well as practical importance . gambiae the principal olfactory cues of humans originate from the feet ( de jong and knols , 1995 ) , and recent work has demonstrated that these cues are partially produced by the microbial flora present on the feet ( verhulst et al . from these studies , several chemical compounds have been identified that play a critical role in the odor - mediated behavior of an . detailed information on the role of these compounds allows for the development of synthetic blends that can be used to better understand the host - seeking behavior of this mosquito . studies on the development of kairomones for malaria vectors have demonstrated strong behavioral responses to synthetic blends of human odorant compounds ( smallegange et al . in laboratory and semi - field studies , these blends attract a large proportion of host - seeking mosquitoes , but when tested against a natural human host , or against natural human odorants released from a nylon matrix , these blends demonstrate poor competitive characters compared to the natural odorants . this suggests that either the concentration of the odorants in the blend was insufficient or that one or more compounds to make the blend sufficiently competitive were missing ( smallegange et al . recent progress , however , has demonstrated the development of odor blends that approach the attractiveness of natural human skin odorants ( okumu et al . assessment of existing synthetic attractant compounds under semi - field and field conditions provides a potential for the development of technologies that can be used for sampling and control of malaria mosquitoes ( kline , 2006 ; jawara et al . the current study was designed to evaluate the attractiveness of selected synthetic blends and human hosts to host - seeking mosquitoes in western kenya , with emphasis on the malaria vectors an . thus , the comparative trapping efficacy of the attractant blends and the physiological status of the mosquitoes trapped were investigated . behavioral responses of mosquitoes to synthetic attractants were evaluated under field and semi - field conditions . aquatic stages of the mosquitoes were reared under ambient atmospheric conditions in screen - walled greenhouses at the thomas risley odhiambo ( tro ) campus of the international centre of insect physiology and ecology ( icipe ) located near mbita point township in western kenya . all chemicals used to constitute the synthetic attractant blends in this study , with the exception of carbon dioxide , water , sugar , and yeast , were purchased from sigma - aldrich chemie gmbh ( germany ) . the chemicals included propionic acid ( 99.6% ) , butanoic acid ( 99.9% ) , pentanoic acid ( > 99% ) , heptanoic acid ( 98% ) , octanoic acid ( 99% ) , tetradecanoic acid ( 99% ) , ammonia solution ( purity 25% ) , ( s)-lactic acid ( 85% ) , isovaleric acid ( 99.8% ) , 4,5-dimethylthiazole ( 97% ) , 2-methyl-1-butanol ( 99% ; a racemic mixture of the r and s isomers of unknown ratio ) , and 3-methyl-1-butanol ( purity 98.5% ) . lwanda village is located on the southern shore of the winam gulf of lake victoria ( 002828s , 341722e ) at an altitude of 1,169 m above sea level ( verhulst et al . hoof prints of cattle and night - grazing hippopotami provide excellent mosquito breeding sites in lwanda . most houses in the two villages are mud - walled with open eaves , have corrugated iron - sheet roofs , have no ceiling , and are either single- or double - roomed . prototype blends were made by adding components to a standard attractive blend ( standard blend , sb ) consisting of ammonia , ( s)-lactic acid , and tetradecanoic acid ( smallegange et al . the wall thickness of the ldpe material used was 0.1 mm ( isovaleric acid and 3-methyl-1-butanol ) , 0.2 mm ( 2-methyl-1-butanol ) , 0.03 mm ( ammonia solution , 4,5 dimethylthiazole , and tetradecanoic acid ) , and 0.05 mm ( ( s)-lactic acid ) . several sachets , varying in number according to the number of chemical compounds constituting a specific prototype blend , were placed onto a hook ( verhulst et al . dual - choice tests comparing mosquito behavioral responses to a total of 15 prototype blends , with different combinations of the four candidate compounds + sb vs. sb alone then were conducted in the semi - field screenhouse facility by placing mm - x traps in diagonal corners ( verhulst et al . gambiae mosquitoes in the screenhouse was evaluated in lwanda village and compared with the attractiveness of other blends , which we had tested in previous field experiments ( okumu et al . blend ib1 consisted of propionic acid ( 0.01% ; ldpe thickness 0.2 mm ) , butanoic acid ( 1% ; ldpe 0.2 mm ) , pentanoic acid ( 0.0001% ; ldpe 0.2 mm ) , 3-methyl butanoic acid ( 0.000001% ; ldpe 0.2 mm ) , heptanoic acid ( 0.0001% ; ldpe 0.1 mm ) , octanoic acid ( 0.0001% ; ldpe 0.1 mm ) , tetradecanoic acid ( 0.00025% ; ldpe 0.03 mm ) , ammonia ( 2.5% ; ldpe 0.03 mm ) , ( s)lactic acid ( 85% ; ldpe 0.05 mm ) , distilled water ( ldpe 0.2 mm ) , and carbon dioxide ( 63.23 2.82 ml / min ) . the blend that attracted most mosquitoes compared to the standard blend was selected and adapted for release on nylon strips , which are known to yield higher mosquito catches than ldpe sachets ( okumu et al . binary assays evaluating mosquito behavioral responses to sb with all constituents availed at their optimally attractive concentrations vs. sb plus 3-methyl-1-butanol offered at variable dilutions ( 100% , 10% , 1% , 0.01% and 0.0001% and 0.000001% v / v ) were run . the efficacy of attracting host - seeking mosquitoes using mb was evaluated by testing it against ifakara blend 1 ( ib1 ) , the standard blend ( sb ) , and a control ( unbaited trap ) under semi - field conditions . , all components of the synthetic attractants , with the exception of carbon dioxide , were delivered via nylon strips ( okumu et al . whereas a total of five adult men aged 1825 years volunteered to participate in the study , only one individual participated per night . consent for houses to be used in the study was obtained from the household heads and the local administration prior to the start of the study . henceforth , the number of mosquitoes attracted to the different sources of behavioral stimuli ( human subjects , control , or the synthetic attractants ) was modeled as a proportion of the total number of mosquitoes recovered from the different treatments . adding 3-methyl-1-butanol to the standard blend of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide either singly or in combination with 2-methyl-1-butanol formed the two most potent attractant blends for host - seeking an . the prototype product , termed mbita blend ( mb ) , consisted of 3-methyl-1-butanol ( released in 0.1 mm - ldpe sachets ) , tetradecanoic acid ( 0.03 mm - ldpe ) , ammonia solution ( 0.03 mm - ldpe ) , ( s)-lactic acid ( 0.05 mm - ldpe ) , and carbon dioxide ( 130 ml / min ) . gambiae attracted ( table 1 ) , these compounds were excluded from the prototype blend.table 1mean ( se ) mosquito catches per night and levels of statistical difference ( p - value ) between 15 prototype synthetic blends vs. a standard blend ( sb ) of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped out of a total of 800 released.% response equals n/800 . compound 1 , 2 , 3 , and 4 are isovaleric acid , 4,5 dimethyl - thiazole , 2-methyl-1-butanol , and 3-methyl-1-butanol , respectivelydescription of synthetic blendnn% responsemosquito trap catches ( mean se)p - valuestandard blend ( sb)synthetic blend1 . sb + compound 1&2&3&442192728.00 9.8125.75 13.490.350 mean ( se ) mosquito catches per night and levels of statistical difference ( p - value ) between 15 prototype synthetic blends vs. a standard blend ( sb ) of ammonia , ( s)-lactic acid , tetradecanoic acid , and carbon dioxide . n is the number of replicates ( nights ) and n the total number of mosquitoes trapped out of a total of 800 released.% response equals n/800 . compound 1 , 2 , 3 , and 4 are isovaleric acid , 4,5 dimethyl - thiazole , 2-methyl-1-butanol , and 3-methyl-1-butanol , respectively a total of 1,286 mosquitoes were trapped in lwanda village over a period of 40 d from 28 april 2010 to 11 june 2010 . funestus ( n = 207 ) , culex species ( n = 544 ) , mansonia species ( n = 112 ) , aedes species ( n = 43 ) , and other mosquito species ( n = 121 ) ( table 2 ) . than the unbaited traps , no blend attracted significantly more mosquitoes of this species than the other ( table 2 ) . numbers followed by different letter superscripts in the same column differ significantly ( p < 0.05 ) the overall combined response of the mosquitoes to the blends ranged from 39% to 68% ( table 3 ) . dilutions of 3-methyl-1-bunanol from pure compound to 10,000 times were significantly ( p < 0.001 ) in favor of the standard blend , except for the 1,000-fold dilution , for which no difference in attractiveness between the two blends was found . this blend ( mb ) subsequently was chosen for further evaluation.table 3mean number se of anopheles gambiae caught per night in mm - x traps baited with a synthetic blend containing various dilutions of 3-methyl-1-butanol plus a standard blend vs. the standard blend alone , dispensed from nylon strips , under semi - field conditions . n is the number of replicates ( nights ) and n the number of mosquitoes trappeddilution ( % ) nn% responsemosquito trap catches ( mean se)p - valuesbsynthetic blendpure compound ( 99.9)45476877.50 16.9759.25 2.140.00210.043404352.00 18.4833.00 11.200.0011.044745968.25 13.1250.25 17.800.0010.144125253.25 4.29049.75 14.420.4900.0143073945.25 6.2431.50 4.050.0020.00143794742.25 12.5952.25 8.090.040 mean number se of anopheles gambiae caught per night in mm - x traps baited with a synthetic blend containing various dilutions of 3-methyl-1-butanol plus a standard blend vs. the standard blend alone , dispensed from nylon strips , under semi - field conditions . n is the number of replicates ( nights ) and n the number of mosquitoes trapped the attraction efficacy of mb vs. ib1 , sb , and a control ( unbaited trap ) was evaluated over a period of 16 nights i.e. out of the total of 3,200 mosquitoes used for these experiments , there was a significant effect ( glm ; p < 0.001 ) of treatment on mosquito trap catches . whereas all the synthetic blends attracted significantly more mosquitoes than the unbaited control ( p = 0.001 ) , mb attracted more mosquitoes than blends ib1 ( p = 0.001 ) and sb ( p = 0.001 ) . results of these experiments are shown in table 4.table 4mean number se of anopheles gambiae caught per night in mm - x traps baited with different synthetic blends under semi - field conditions . the effect of treatment ( p values ) on overall mosquito responses is also shownblend ( delivery)nnmosquito trap catches ( mean se)treatment ( p - value)control ( no odors)16583.62 0.810.001ib11669143.2 4.40.001sb1652532.8 5.40.001mb1687854.9 8.10.001 mean number se of anopheles gambiae caught per night in mm - x traps baited with different synthetic blends under semi - field conditions . the effect of treatment ( p values ) on overall mosquito responses is also shown the competitiveness of mb over other baits in attracting malaria and other wild mosquitoes was evaluated in kigoche village for 30 d spanning the period 0130 april , 2011 . analysis of the data revealed that houses baited with mb caught 31.5% ( n = 637 ) of the mosquitoes ( males and females ) while those with a human being , ib1 , sb , or no host cue collected 26.9% ( n = 545 ) , 19.6% ( n = 397 ) , 17.4% ( n = 352 ) , and 4.6% ( n = 93 ) of the mosquitoes , respectively . funestus ( p < 0.014 ) and culex species ( p < the mean number of female mosquitoes with the exception of culex species collected from the five traditional houses did not differ significantly . the numbers of mosquitoes of all species collected from the empty house were significantly lower than those collected from houses baited with mb ( p = 0.001 ) , a human being , ib1 ( p = 0.001 ) , or sb ( p = 0.001 ) . funestus and culex species than a human being ( p = 0.002 and 0.001 , respectively ) or sb ( p = 0.001 and 0.005 , respectively ) . mosquitoes than ib1 ( p = 0.001 ) and sb ( p = 0.001 ) , these numbers did not differ significantly from those attracted by a human being ( p = 0.121 ) . funestus mosquitoes then ib1 ( p = 0.001 ) , the number of culex mosquitoes attracted to mb were not different from those attracted to blend ib1 ( p = 0.140)table 5total pooled number of female mosquitoes and mean number se per species per night trapped in response to different synthetic attractant blends ( dispensed form nylon strips ) and a human host in kigoche village , western kenya . funestusculex spp.aedes spp.mansonia spp.other anophelinescontrol30811.2 0.410.8 0.280.33 0.15000.37 0.021human305279.9 2.12.93 0.651.17 0.28003.57 0.96ib1303757.13 2.902.0 0.391.7 0.390.03 0.030.03 0.031.60 0.49 sb303336.27 1.402.03 0.59 1.27 0.33001.53 0.41mb3061811.2 2.14.8 1.262.23 0.72002.37 0.80 total pooled number of female mosquitoes and mean number se per species per night trapped in response to different synthetic attractant blends ( dispensed form nylon strips ) and a human host in kigoche village , western kenya . the odor - baited technology has a distinct advantage over the widely - used cdc trap + human - under - a - bed - net method introduced by garrett - jones and magayuka ( 1975 ) , which has since been used as the standard method for assessing entomological inoculation rates and other relevant epidemiological parameters for malaria ( smith et al . , 2004 ; the need for a human host to be present during the all - night collections , the cdc trap plus bed net combination for mosquito trapping has obvious disadvantages . these disadvantages can be overcome by replacing the human host with synthetic bait ( over a time frame of at least 1 week ) , which produces a consistent and constant blend of odorants and hence avoids the variance caused by the human - dependent method of mosquito sampling . natural variance in human odorants is likely to affect trap catches , and may potentially lead to stronger attractiveness of the natural host than the synthetic blend , but also to considerably lower attractiveness . in this study , we examined the use of ldpe as a material for the slow release of odorants , analogous to the successful use of ldpe for attractants of tsetse flies ( green , 1994 ; torr et al . odorants attractive to tsetse flies and released through ldpe remain active for many months and have been used widely for the mass trapping of tsetse flies in remote areas . it seems likely that the size of the ldpe sachets we employed was too small to provide a sufficiently large surface to release the quantity of odorants needed for optimal attraction of the mosquitoes . it is possible that when employing higher dilutions of each of these compounds when studying the effect of multiple compounds , the kairomonal potency of the blend might be further enhanced . the differences were small , though , and the overall result shows that the three synthetic blends approached the attractiveness of the human host ( table 5 ) , with mb being the most attractive blend . funestus than the trap next to the human - occupied bed net , the catches of mosquitoes displaying this physiological status as well as gravid ones were small . as other , non - anopheline mosquitoes were also collected , the synthetic blends , notably mb , should be considered as attractants for a wide range of mosquito species , including other disease vectors . we conclude that the multiple - component odorant blends described in this study can be considered to replace traditional malaria vector sampling tools such as cdc light traps and the human landing catch .

acute liver failure ( alf ) is characterized by an initial devastating hepatic insult followed by gross parenchymal dysfunction , which leads to a multitude of systemic organ failures due to the missing metabolic homeostasis normally provided by the healthy liver . the most common causes of alf are viral hepatitis , idiosyncratic side reactions , chronic liver diseases , autoimmune hepatitis , and dose - dependent drug - induced alf . the disease occurs rapidly and in general requires intensive care with the known high risk of mortality . whole liver transplantation very often is the only therapy option of choice ( ostapowicz and lee , 2000 ; gill and sterling , 2001 ; rahman and hodgson , 2001 ; ogrady , 2005 ) . the incidence of acetaminophen ( paracetamol)-induced alf is rather high in the us and in the uk related both to therapy - associated and suicide - driven overdosage of the drug ( reuben et al . , 2010 ; lee et al . , 2011 ) . in the liver acetaminophen is metabolized by the cytochrome p450 enzyme system located predominantly in the hepatocytes surrounding the distal branches of the liver sinusoids , the so - called perivenous hepatocytes ( jungermann and kietzmann , 2000 ; benhamouche et al . , 2006 ; burke and tosh , 2006 ; hailfinger et al . , 2006 ; there are two principle ways of detoxification : ( 1 ) conjugation by sulfation and/or glucuronidation followed by elimination and ( 2 ) cytochrome p450-dependent oxidation and formation of n - acetyl - p - benzoquinonimine ( napqi ) , which is then conjugated to glutathione and finally eliminated with the bile . yet , sustained napqi formation eventually causes depletion of glutathione , which then in turn leads to formation of protein adducts as well as reactive nitrogen and oxygen species ( figure 1 ) . very likely mitochondrial dysfunction and increased permeability of the mitochondrial membranes contribute to the formation of reactive nitrogen and oxygen metabolites such as peroxynitrate and hydrogen peroxide besides others , which in turn mediate protein nitration and oxidative stress ( jaeschke et al . , 2002 ; james et al . , 2003 ; jaeschke and bajt , 2006 ; doi and ishida , 2009 ) . obviously , besides the hepatocytes non - parenchymal cells such as kupffer cells and sinusoidal endothelial cells seem to be involved in the generation of reactive nitrogen and oxygen species thus augmenting protein and lipid peroxidation . since these reactions are ultimately mediated by the perivenous cytochrome p450 enzyme system , apoptotic cell death followed by centrilobular necrosis is a hallmark of acetaminophen - induced hepatotoxicity ( figure 2 ) . the inflammatory environment produced during alf is also responsible for the activation of hepatic stellate cells probably mediated by il1 , which respond with an increase in expression of -smooth muscle actin and matrix metalloproteinases , mainly mmp9 . this seems to favor the remodeling of the extracellular matrix , thus augmenting hepatocyte cell death ( yan et al . acetaminophen ( paracetamol ) is detoxified in the liver by conjugation or cytochrome p450-dependent oxidation followed by conjugation to glutathione ( gsh ) . depletion of gsh leads to formation of reactive nitrogen and oxygen species , which in turn causes cell death . rats were treated with a repeated oral dose of 4 g / kg body weight of acetaminophen . dashed lines exemplify initial ( area 1 ) and final necrotic perivenous areas ( area 2 , cv , central vein ) of the liver tissue . please note that areas around the portal vein ( pv ) are void of tissue damage . in the normal healthy liver tissue turnover is in the range of 0.01% . without any challenge this rather low regenerative rate would reconstitute the whole liver parenchyma within about 1 year ( steiner et al . , 1966 ; koniaris et al . , 2003 ) . one might suspect then that the liver had a poor regenerative potential , which is also corroborated by the fact that after partial hepatectomy the liver is rebuilt to the original organ size , only . after 2/3 partial hepatectomy this would be accomplished by the 1.5-times cell division of the remaining hepatocytes . however , this situation does not reflect the real regenerative potential of hepatocytes . it has been shown in serial transplantation experiments in the albumin promoter - urokinase plasminogen activator ( upa ) transgenic mouse that hepatocytes feature a nearly unlimited regenerative capacity . in this model , the intracellular activation of the protease plasmin causes hepatocyte damage and perinatal lethality ( heckel et al . , 1990 ) . eventually , mice survived due to the substitution of hepatocytes bearing the transgene by healthy hepatocytes , which obviously had a survival advantage . transplantation of these hepatocytes having escaped the lethal phenotype into the livers of transgenic mice revealed the efficient repopulation of the diseased host liver by the donor hepatocytes , thus rescuing the lethal phenotype . this indicates an enormous mitotic potential of hepatocytes ( sandgren et al . , 1991 ; rhim et al . , , the knockout of fumarylacetoacetate hydrolase ( fah ) leads to the accumulation of tyrosine intermediates , which cause toxic insult of hepatocytes . transplanted healthy hepatocytes display a proliferative advantage over the diseased host hepatocytes , thus achieving nearly complete replacement of the original transgenic hepatocytes by the transplanted cells . in this model , serial transplantation of hepatocytes derived from mutant livers colonized with transplanted wildtype cells revealed that 6 rounds of liver repopulation required a minimum of 69 cell divisions ( overturf et al . this in turn means , that they have the potential of self - renewal and of functional tissue formation in vivo , which are ultimate stem cell characteristics . experimentally , alf might be triggered by the use of chemical noxious compounds such as carbon tetrachloride or acetaminophen as mentioned above . as long as the hepatocytes dispose of sufficient metabolic capacity to detoxify the drugs no obvious tissue lesions emerge . yet , the production of reactive metabolites followed by covalent protein and lipid modification due to metabolic overload as mentioned above finally results in cellular dysfunction , initial cell damage and tissue injury . depending on the dose applied tissue damage proceeds . the initial insult resulting in injury progression is the mitotic challenge for the hepatocytes to restore the tissue loss by functional hepatocyte progeny . again , dependent on the dose of the noxious compounds the regenerative potential of the liver is either sufficient for injury regression or overwhelmed resulting in injury progression followed by alf ( mehendale , 2005 ; palmes et al . tissue regeneration is accomplished by the hepatocytes themselves as long as a minimal liver tissue mass is compliant with a certain threshold of functional tissue loss . yet , if this threshold is surpassed the regenerative capacity of the hepatocytes does not suffice for functional tissue restoration . in this case a progenitor cell compartment is activated giving rise to so - called oval cells in rodents , which are agreed upon to be the progeny of adult hepatic stem cells in the liver ( sell , 2001 ; kofman et al . , 2005 ) . oval cells appear in the periportal areas after massive liver injury adjacent to the canals of hering , structural links between the terminal biliary branches and the periportal hepatocytes ( fausto , 2004 ; santoni - rugiu et al . it is noteworthy that both hepatocytes and hepatic progenitor cells may differentiate into hepatocytes and biliary cells as well indicating their bipotent differentiation capacity . hence , both cell types meet the minimal definition criteria of a stem cell , i.e. , the potential of self - renewal to maintain the stem cell reserve , and a multiple differentiation potential giving rise to progeny of at least two different lineages . in the latter case it is self - evident that proliferation and differentiation of the offspring cells provide the functional backup for tissue repair after injury . the regenerative response of the liver after acute intoxication is triggered by the emergence of initial tissue damage and progression . dependding on the dose of the noxa and the regenerative capacity of the hepatocytes injury regression and regeneration or progression and alf develop . in alf , liver transplantation is the gold standard of treatment . however , about one third of patients on the waiting list for liver transplantation in europe do not profit because of the unavailability of suitable donor organs . the hepatocyte is the smallest functional unit of the liver executing the complete metabolic orchestra , which is provided by the liver as a whole . therefore , transplantation of hepatocytes might be possible to substitute for the functional tissue loss in alf provided the donor cells take over hepatocyte functions in the deteriorated host parenchyma for at least the critical period in time either required to bridge to organ transplantation or to allow for tissue recovery from the toxic insult ( najimi and sokal , 2008 ; oertel and shafritz , 2008 ; smets et al . , 2008 ; ito et al . , 2009 ; puppi and dhawan , 2009 ) . technically , in rodent small animal models the cells are delivered to the liver either after intraportal or intrasplenic injection . it is assumed that the cells distribute homogeneously in the liver by passage with the blood stream where they are entrapped in the sinusoids and eventually penetrate the endothelia , integrate , proliferate , and spread into the host parenchyma . this concept has been verified in various rodent animal models of alf ( for recent reviews , see fox and roy - chowdhury , 2004 ; shafritz et al . , 2006 ; seppen et al . , 2009 ; weber et al . , 2009 ) . there is one major constraint , which probably seriously hampers the clinical translation of hepatocyte transplantation in alf . under non - stimulating conditions the repopulation of an acutely injured liver by transplanted hepatocytes is rather low , i.e. , in the range of 15% of the total liver mass ( ponder et al . , 1991 ; , 1999 ; fox and roy - chowdhury , 2004 ; fisher and strom , 2006 ) . however , if the recipient liver is challenged by a growth stimulus and the proliferation of host hepatocytes is impaired then a significant repopulation by transplanted hepatocytes is achieved . there is an elegant animal model available allowing for the identification of the transplanted cells in the host parenchyma . in this rat model the natural mutation in the cd26 gene leads to the expression of a non - functional protein , however , without obvious pathophysiological consequences . transplanted wildtype donor cells may then be identified histologically in the host parenchyma by the detection of cd26 . providing selective pressure conditions by partial hepatectomy as a mitotic stimulus and pre - treatment with alkaloids such as retrorsine to inhibit host hepatocyte proliferation a repopulation rate for up to nearly 100% may be achieved in this rat model ( laconi et al . , 1998 , 1999 ) . similarly , high rates were obtained using rat fetal liver epithelial cells but without applying selective growth conditions for the transplanted cells ( sandhu et al . . acute liver failure in mice and rats may be induced under various experimental settings , the most common in use are those acutely applying paracetamol or carbon tetrachloride . in general , when adult hepatocytes or oval cells isolated from donor livers under various inducing conditions are used for transplantation without further selective pressure repopulation of the host liver by the transplanted cells is poor , i.e. , in the range of less than 5% . however , cells are functional and survive long - term in the recipient liver indicating support of liver regeneration after acute hepatotoxic injury . if in addition to the acute injury regeneration by host hepatocytes is abrogated by the beforehand treatment with mitotoxins such as the pyrrolizidine alkaloid retrorsine much higher repopulation rates may be achieved , which clearly suffice to substitute for the loss of metabolic capacity due to the toxic parenchymal damage . similar results were obtained using fetal ( ed12.5 ) rat hepatoblasts . yet , using ed14 mouse hepatoblasts 10- to 20-fold higher repopulation rates were achieved without applying selective repopulation conditions . a comprehensive summary of models and conditions used to study liver repopulation by transplanted hepatocytes or hepatocyte progenitor cells under normal and injury conditions is available ( sancho - bru et al . , 2009 ; shafritz and oertel , 2011 ) . to summarize , transplanted cells integrate into the host parenchyma and even at low repopulation rates display hepatocyte functions . thus , hepatocyte transplantation in alf aims at tissue substitution of the recipient liver in order to functionally reconstitute the injured parenchyma by healthy donor cells . in respect to the clinical application of hepatocyte transplantation in alf the major hurdle is probably the scarcity of donor organs to isolate human hepatocytes in sufficient quality and quantity . therefore , one feasible alternative to human adult hepatocytes is the use of stem cell - derived hepatocytes . the bone marrow harbors adult stem cells , both hematopoietic and non - hematopoietic , which are clearly superior in choice over embryonic stem cells for clinical application because of their less ethical constraints and the lack of teratoma formation after tissue implantation . adult stem cells may differentiate into hepatocyte - like cells . in the mouse model of fah deficiency , hematopoietic bone marrow derived cells rescued the diseased phenotype by complementation of the defective fah gene with the wildtype gene in the transplanted cells ( lagasse et al . it is an open question as to whether hepatocytes derived from the bone marrow are the product of differentiation from hematopoietic stem cells or of the fusion with host hepatocytes ( alvarez - dolado et al . , 2003 ; newsome et al . , 2003 ; vassilopoulos et al . , 2003 ; wang et al . , 2003 ; camargo et al . , 2004 ; jang et al . , 2004 ) . in recent years studies in rats ( wang et al . , 2004 ; lange et al . , 2005 ) , mice ( jiang et al . , 2002 ) , and humans ( schwartz et al . , 2002 ; lee et al . , 2004 ; , 2005 ; seo et al . , 2005 ; talns - visconti et al . , 2007 ; banas et al . , 2007 ) verified that mesenchymal stem cells ( msc ) from various tissues like bone marrow , umbilical cord blood , or adipose tissue may differentiate into hepatocyte - like cells following specified growth and differentiation regimens in vitro . yet , under acute injury conditions causing either periportal liver damage induced by allyl alcohol ( sato et al . , 2005 ) or perivenous damage by the use of carbon tetrachloride ( seo et al . , 2005 ; banas et al , 2007 ; yukawa et al . , 2009 ) or acetaminophen ( stock et al . , 2009 ) , msc - derived hepatocyte - like cells integrated into the diseased host liver , though repopulation rates were rather low , i.e. , in the range of 1% of the total liver mass . reasoning that msc feature immunomodulatory functions in that they are able to suppress the immune response mediated through t and b cells , dendritic cells and other immune cells ( chamberlain et al . . , 2007 ; le blanc and ringden , 2007 ) it might not be surprising that the action of msc in alf is rather paracrine than direct tissue support by the transplanted cells . d - galactosamine - induced fulminant hepatic failure in rats was attenuated by msc - derived molecules through inhibition of apoptosis , stimulation of hepatocyte proliferation , and minimization of the inflammatory response ( parekkadan et al . . the paracrine mode of action of msc was also corroborated by the amelioration of systemic inflammation induced by lps or burn indicating in addition pleiotropic effects of the msc ( yagi et al . ectopic recruitment of msc from the bone marrow to the liver has been shown in mice challenged by acute intoxication with carbon tetrachloride or 2-acetylaminofluorene indicating chemotactic activation of the msc very likely mediated by stromal cell - derived factor-1 ( jin et al . , 2009 ; our own data substantiated that msc are able to home to and integrate into an acutely injured liver . msc derived from rat peritoneal adipose tissue were pre - differentiated into hepatocyte - like cells according to our standard protocol ( stock et al . , 2010 ) and then the cells were administered to the diseased animals via tail vein injection . eighteen hours after cell delivery donor - derived cells were detected in the liver ( unpublished ) where they significantly decreased acetaminophen - induced apoptosis as shown immunohistochemically by the tunel assay and stimulated proliferation of host hepatocytes as shown by ki67 staining ( figure 4 ) to regenerate the liver tissue after acute injury ( unpublished ) . besides their anti - inflammatory and immunomodulatory impact msc seem also to communicate with target cells by the exchange of mrna or mirna molecules ( collino et al . thus , genetic material is exchanged , which then might affect the regenerative response of the host tissue cells on the one and the differentiation of donor msc at the site of their engraftment into the host tissue on the other hand . this , however , means that transplanted cells might be imprinted by their target tissue and the molecular microenvironment induced by a specific type of injury . acute liver injury may trigger paracrine effects due to the inflammatory environment of the diseased liver , whereas liver regeneration after , e.g. , partial hepatectomy is achieved by the engraftment and functional tissue replacement by the msc differentiated into hepatocytes at the site of their engraftment . this potential pleiotropic mode of action makes msc ideal candidates for stem cell therapy of different liver diseases ( enns and millan , 2008 ; haridass et al . , 2008 ; alison et al . , 2009 ; flohr et al . , 2009 ; soto - gutierrez et al . , anti - apoptotic and pro - proliferative action of msc after acetaminophen intoxication of the rat liver . rats were treated with a repeated dose of 4 g / kg body weight of acetaminophen . eighteen hours after the last dose the animals were sacrificed and the livers prepared for the detection of apoptotic cells ( dark nuclei ) by the tunel assay ( lower panels ) or proliferating cells ( dark nuclei ) by the ki67 stain ( upper panels ) . where indicated animals received adipose tissue - derived rat msc pre - differentiated into hepatocyte - like cells ( rmsc - hc ) 6 h after the last dose of acetaminophen . it is obvious that the number of apoptotic cells was significantly lower but of proliferating cells was higher in the livers with msc ( right panels ) indicating the anti - apoptotic and pro - proliferative action of the msc . it is obvious that experimental settings in animal models aimed to enhance liver repopulation by transplanted hepatocytes are not suited for clinical translation . thus , the lack of a survival and/or a proliferative advantage of donor vs. host hepatocytes is probably the mechanistical reason for the poor clinical progress of hepatocyte transplantation . the low success rate is augmented by the fact that human hepatocytes are isolated from marginal donor livers not allocated for transplantation . yet , as outlined above msc might be an alternate cell resource to generate hepatocyte - like cells . msc display hepatocyte differentiation potential , which was substantiated both in vitro and in vivo . even if biological and biochemical differences might exist between msc from various tissues they share typical msc characteristics like marker expression , multiple differentiation capacity , and growth on plastic surfaces , which finally determine quantitative , not qualitative , variability in their hepatocyte differentiation potential . it is feasible to suppose that in respect to ethical , technical and biological aspects the transplantation of stem cell - derived hepatocytes follows the principles of hepatocyte transplantation ( fisher and strom , 2006 ) . msc might even open a broader spectrum of activity compared with primary hepatocytes because of their versatile properties such as low immunogenicity as well as their anti - inflammatory , anti - apoptotic , and pro - proliferative activities , which not only substitute the tissue damaged but also actively might temper the inflammatory response , e.g. , after toxic or chronic injury . recently , a couple of clinical trials most in china has been initiated or even completed to demonstrate safety and efficacy of the site of application of mscs concentrating on autologous stem cell transplantation in patients suffering from chronic liver failure ( table 1 ) or acute decompensation after ample liver resection . yet , so far no published results are available . in these studies , undifferentiated cells have been used bearing a potential tumor - promoting risk ( karnoub et al . , 2007 ) , which , however , has not been verified . summary of clinical trials involving mesenchymal stem cells of different tissue sources for the treatment of chronic liver diseases . taking ethical considerations into account these clinical conditions may be adequate to assess safety of hepatic msc transplantation . however , to take advantage of the cells immunomodulatory , chemotactic , and anti - inflammatory properties , alf offering a highly inflammatory environment in the liver may be the disease situation of choice for the use of msc . in this case even the use of allogeneic cell sources may not be a serious problem since only the short - term beneficial actions of the msc might warrant support of liver regeneration in the critical phase of acute poisoning . immunosuppression may be applied from the beginning of treatment on or even continued as long as the recovering of the liver is ongoing but then may be ceased , thus avoiding the theoretical risks of potential sensitization of the host for future organ grafts or promoting life - threatening septic episodes during long - term stay in the intensive care units . to overcome the shortage of donor organs for liver transplantation in alf cell therapy approaches seem to be feasible , which must achieve two principle goals . ( 1 ) the loss of metabolic capacity must be substituted by the healthy donor cells , and ( 2 ) the emergence of the inflammatory environment in alf must be decelerated in order to protect hepatocytes from progression into cell death . it is obvious that the first goal might best be reached using primary hepatocytes , which , however , do not have a survival advantage in the deteriorated alf liver . the second goal might best be met by the use of msc taking advantage of their anti - inflammatory and apoptotic as well as pro - proliferative features , which , however , promises no therapeutic benefit in the case that tissue damage has surpassed the lower threshold needed to maintain body metabolic homeostasis . thus , it might be worthwhile thinking whether a combination of hepatocytes and msc might be the cell therapeutic of best choice . indeed , there is evidence that the performance of hepatocytes is improved in co - culture with msc ( ijima et al . , 2008 ; chen et al . , 2012 ) , and vice versa msc differentiation into hepatocyte - like cells is promoted by inflammatory liver injury conditions ( dong et al . recent data even demonstrated that not msc themselves but as yet unequivocally unidentified soluble factors secreted by msc exert the beneficial effects on hepatocytes under alf conditions in mice and rats ( parekkadan et al . , 2007b ; van poll et al . , 2008 ; zagoura et al . , the anti - inflammatory cytokine il10 secreted by msc seemed to play a major role in alleviating liver damage after acute injury induced by carbon tetrachloride in the nod / scid mouse model ( zagoura et al . , 2011 ) . thus , the identification of these factors might open even cell - free therapeutical options for the treatment of alf with msc - derived molecules . animal models for cell therapy approaches to treat alf as described above enable us to earn knowledge on the mechanisms of interactions between donor and host cells both on the molecular and cellular level , to identify the hepatotropic effects esp . mediated by msc and their impact on the noxious challenge in order to optimize integration of transplanted cells into the recipient tissue thereby to support efficacy of cell transplantation and thus optimize the therapeutical outcome . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

without therapeutic intervention acute liver failure ( alf ) is the consequence of a progredient destruction of the liver parenchyma due to metabolic exhaustion of the hepatocytes . perivenous hepatocytes are responsible for the detoxification of noxious compounds via the cytochrome p450 enzyme system . liver transplantation is the only remaining therapeutic option in the end - stage of the disease . assuming that metabolic capacity could be provided by healthy hepatocytes and thus substitute for the genuine parenchymal cells hepatocyte transplantation since quite some time is considered to be an alternative to whole liver transplantation . while this hypothesis achieved proof - of - concept in animal trials clinical breakthrough is still awaiting success , the reasons of which are ongoing matter of debate . in recent times mesenchymal stem cells ( msc ) came into focus as a transplantable cell source to treat alf . interestingly , as demonstrated in various rodent animal models their mode of action is rather based on trophic support of hepatocytes remaining in the damaged host parenchyma rather than substitution of tissue loss . mechanistically , either direct or indirect paracrine effects from the transplanted cells acting pro - proliferative , anti - apoptotic , and anti - inflammatory seem to trigger the regenerative response of the residual healthy hepatocytes in the otherwise lethally injured liver parenchyma . thus , allogeneic msc may be the best choice for the treatment of alf taking advantage of their short - term benefit to sustain the critical phase of the acute insult avoiding long - term immunosuppression .

Molecular Principles of Tissue Toxicity in ALF Induced by Paracetamol Regenerative Response to Acute Liver Injury Hepatocyte Transplantation in ALF Stem Cell-Derived Hepatocyte Transplantation in ALF Clinical Implications Conclusion Conflict of Interest Statement

acute liver failure ( alf ) is characterized by an initial devastating hepatic insult followed by gross parenchymal dysfunction , which leads to a multitude of systemic organ failures due to the missing metabolic homeostasis normally provided by the healthy liver . the most common causes of alf are viral hepatitis , idiosyncratic side reactions , chronic liver diseases , autoimmune hepatitis , and dose - dependent drug - induced alf . the disease occurs rapidly and in general requires intensive care with the known high risk of mortality . whole liver transplantation very often is the only therapy option of choice ( ostapowicz and lee , 2000 ; gill and sterling , 2001 ; rahman and hodgson , 2001 ; ogrady , 2005 ) . the incidence of acetaminophen ( paracetamol)-induced alf is rather high in the us and in the uk related both to therapy - associated and suicide - driven overdosage of the drug ( reuben et al . in the liver acetaminophen is metabolized by the cytochrome p450 enzyme system located predominantly in the hepatocytes surrounding the distal branches of the liver sinusoids , the so - called perivenous hepatocytes ( jungermann and kietzmann , 2000 ; benhamouche et al . very likely mitochondrial dysfunction and increased permeability of the mitochondrial membranes contribute to the formation of reactive nitrogen and oxygen metabolites such as peroxynitrate and hydrogen peroxide besides others , which in turn mediate protein nitration and oxidative stress ( jaeschke et al . obviously , besides the hepatocytes non - parenchymal cells such as kupffer cells and sinusoidal endothelial cells seem to be involved in the generation of reactive nitrogen and oxygen species thus augmenting protein and lipid peroxidation . since these reactions are ultimately mediated by the perivenous cytochrome p450 enzyme system , apoptotic cell death followed by centrilobular necrosis is a hallmark of acetaminophen - induced hepatotoxicity ( figure 2 ) . the inflammatory environment produced during alf is also responsible for the activation of hepatic stellate cells probably mediated by il1 , which respond with an increase in expression of -smooth muscle actin and matrix metalloproteinases , mainly mmp9 . this seems to favor the remodeling of the extracellular matrix , thus augmenting hepatocyte cell death ( yan et al . acetaminophen ( paracetamol ) is detoxified in the liver by conjugation or cytochrome p450-dependent oxidation followed by conjugation to glutathione ( gsh ) . dashed lines exemplify initial ( area 1 ) and final necrotic perivenous areas ( area 2 , cv , central vein ) of the liver tissue . please note that areas around the portal vein ( pv ) are void of tissue damage . in the normal healthy liver tissue turnover is in the range of 0.01% . without any challenge this rather low regenerative rate would reconstitute the whole liver parenchyma within about 1 year ( steiner et al . one might suspect then that the liver had a poor regenerative potential , which is also corroborated by the fact that after partial hepatectomy the liver is rebuilt to the original organ size , only . after 2/3 partial hepatectomy this would be accomplished by the 1.5-times cell division of the remaining hepatocytes . however , this situation does not reflect the real regenerative potential of hepatocytes . it has been shown in serial transplantation experiments in the albumin promoter - urokinase plasminogen activator ( upa ) transgenic mouse that hepatocytes feature a nearly unlimited regenerative capacity . in this model , the intracellular activation of the protease plasmin causes hepatocyte damage and perinatal lethality ( heckel et al . eventually , mice survived due to the substitution of hepatocytes bearing the transgene by healthy hepatocytes , which obviously had a survival advantage . transplantation of these hepatocytes having escaped the lethal phenotype into the livers of transgenic mice revealed the efficient repopulation of the diseased host liver by the donor hepatocytes , thus rescuing the lethal phenotype . , , the knockout of fumarylacetoacetate hydrolase ( fah ) leads to the accumulation of tyrosine intermediates , which cause toxic insult of hepatocytes . transplanted healthy hepatocytes display a proliferative advantage over the diseased host hepatocytes , thus achieving nearly complete replacement of the original transgenic hepatocytes by the transplanted cells . in this model , serial transplantation of hepatocytes derived from mutant livers colonized with transplanted wildtype cells revealed that 6 rounds of liver repopulation required a minimum of 69 cell divisions ( overturf et al . experimentally , alf might be triggered by the use of chemical noxious compounds such as carbon tetrachloride or acetaminophen as mentioned above . as long as the hepatocytes dispose of sufficient metabolic capacity to detoxify the drugs no obvious tissue lesions emerge . yet , the production of reactive metabolites followed by covalent protein and lipid modification due to metabolic overload as mentioned above finally results in cellular dysfunction , initial cell damage and tissue injury . the initial insult resulting in injury progression is the mitotic challenge for the hepatocytes to restore the tissue loss by functional hepatocyte progeny . again , dependent on the dose of the noxious compounds the regenerative potential of the liver is either sufficient for injury regression or overwhelmed resulting in injury progression followed by alf ( mehendale , 2005 ; palmes et al . tissue regeneration is accomplished by the hepatocytes themselves as long as a minimal liver tissue mass is compliant with a certain threshold of functional tissue loss . yet , if this threshold is surpassed the regenerative capacity of the hepatocytes does not suffice for functional tissue restoration . in this case a progenitor cell compartment is activated giving rise to so - called oval cells in rodents , which are agreed upon to be the progeny of adult hepatic stem cells in the liver ( sell , 2001 ; kofman et al . it is noteworthy that both hepatocytes and hepatic progenitor cells may differentiate into hepatocytes and biliary cells as well indicating their bipotent differentiation capacity . hence , both cell types meet the minimal definition criteria of a stem cell , i.e. , the potential of self - renewal to maintain the stem cell reserve , and a multiple differentiation potential giving rise to progeny of at least two different lineages . in the latter case it is self - evident that proliferation and differentiation of the offspring cells provide the functional backup for tissue repair after injury . the regenerative response of the liver after acute intoxication is triggered by the emergence of initial tissue damage and progression . dependding on the dose of the noxa and the regenerative capacity of the hepatocytes injury regression and regeneration or progression and alf develop . in alf , liver transplantation is the gold standard of treatment . however , about one third of patients on the waiting list for liver transplantation in europe do not profit because of the unavailability of suitable donor organs . the hepatocyte is the smallest functional unit of the liver executing the complete metabolic orchestra , which is provided by the liver as a whole . therefore , transplantation of hepatocytes might be possible to substitute for the functional tissue loss in alf provided the donor cells take over hepatocyte functions in the deteriorated host parenchyma for at least the critical period in time either required to bridge to organ transplantation or to allow for tissue recovery from the toxic insult ( najimi and sokal , 2008 ; oertel and shafritz , 2008 ; smets et al . technically , in rodent small animal models the cells are delivered to the liver either after intraportal or intrasplenic injection . it is assumed that the cells distribute homogeneously in the liver by passage with the blood stream where they are entrapped in the sinusoids and eventually penetrate the endothelia , integrate , proliferate , and spread into the host parenchyma . this concept has been verified in various rodent animal models of alf ( for recent reviews , see fox and roy - chowdhury , 2004 ; shafritz et al . under non - stimulating conditions the repopulation of an acutely injured liver by transplanted hepatocytes is rather low , i.e. , in the range of 15% of the total liver mass ( ponder et al . there is an elegant animal model available allowing for the identification of the transplanted cells in the host parenchyma . in this rat model the natural mutation in the cd26 gene leads to the expression of a non - functional protein , however , without obvious pathophysiological consequences . transplanted wildtype donor cells may then be identified histologically in the host parenchyma by the detection of cd26 . providing selective pressure conditions by partial hepatectomy as a mitotic stimulus and pre - treatment with alkaloids such as retrorsine to inhibit host hepatocyte proliferation a repopulation rate for up to nearly 100% may be achieved in this rat model ( laconi et al . similarly , high rates were obtained using rat fetal liver epithelial cells but without applying selective growth conditions for the transplanted cells ( sandhu et al . acute liver failure in mice and rats may be induced under various experimental settings , the most common in use are those acutely applying paracetamol or carbon tetrachloride . in general , when adult hepatocytes or oval cells isolated from donor livers under various inducing conditions are used for transplantation without further selective pressure repopulation of the host liver by the transplanted cells is poor , i.e. , in the range of less than 5% . however , cells are functional and survive long - term in the recipient liver indicating support of liver regeneration after acute hepatotoxic injury . if in addition to the acute injury regeneration by host hepatocytes is abrogated by the beforehand treatment with mitotoxins such as the pyrrolizidine alkaloid retrorsine much higher repopulation rates may be achieved , which clearly suffice to substitute for the loss of metabolic capacity due to the toxic parenchymal damage . to summarize , transplanted cells integrate into the host parenchyma and even at low repopulation rates display hepatocyte functions . thus , hepatocyte transplantation in alf aims at tissue substitution of the recipient liver in order to functionally reconstitute the injured parenchyma by healthy donor cells . in respect to the clinical application of hepatocyte transplantation in alf the major hurdle is probably the scarcity of donor organs to isolate human hepatocytes in sufficient quality and quantity . therefore , one feasible alternative to human adult hepatocytes is the use of stem cell - derived hepatocytes . the bone marrow harbors adult stem cells , both hematopoietic and non - hematopoietic , which are clearly superior in choice over embryonic stem cells for clinical application because of their less ethical constraints and the lack of teratoma formation after tissue implantation . adult stem cells may differentiate into hepatocyte - like cells . in the mouse model of fah deficiency , hematopoietic bone marrow derived cells rescued the diseased phenotype by complementation of the defective fah gene with the wildtype gene in the transplanted cells ( lagasse et al . it is an open question as to whether hepatocytes derived from the bone marrow are the product of differentiation from hematopoietic stem cells or of the fusion with host hepatocytes ( alvarez - dolado et al . in recent years studies in rats ( wang et al . , 2007 ) verified that mesenchymal stem cells ( msc ) from various tissues like bone marrow , umbilical cord blood , or adipose tissue may differentiate into hepatocyte - like cells following specified growth and differentiation regimens in vitro . , in the range of 1% of the total liver mass . , 2007 ; le blanc and ringden , 2007 ) it might not be surprising that the action of msc in alf is rather paracrine than direct tissue support by the transplanted cells . d - galactosamine - induced fulminant hepatic failure in rats was attenuated by msc - derived molecules through inhibition of apoptosis , stimulation of hepatocyte proliferation , and minimization of the inflammatory response ( parekkadan et al . the paracrine mode of action of msc was also corroborated by the amelioration of systemic inflammation induced by lps or burn indicating in addition pleiotropic effects of the msc ( yagi et al . ectopic recruitment of msc from the bone marrow to the liver has been shown in mice challenged by acute intoxication with carbon tetrachloride or 2-acetylaminofluorene indicating chemotactic activation of the msc very likely mediated by stromal cell - derived factor-1 ( jin et al . , 2009 ; our own data substantiated that msc are able to home to and integrate into an acutely injured liver . eighteen hours after cell delivery donor - derived cells were detected in the liver ( unpublished ) where they significantly decreased acetaminophen - induced apoptosis as shown immunohistochemically by the tunel assay and stimulated proliferation of host hepatocytes as shown by ki67 staining ( figure 4 ) to regenerate the liver tissue after acute injury ( unpublished ) . besides their anti - inflammatory and immunomodulatory impact msc seem also to communicate with target cells by the exchange of mrna or mirna molecules ( collino et al . thus , genetic material is exchanged , which then might affect the regenerative response of the host tissue cells on the one and the differentiation of donor msc at the site of their engraftment into the host tissue on the other hand . this , however , means that transplanted cells might be imprinted by their target tissue and the molecular microenvironment induced by a specific type of injury . acute liver injury may trigger paracrine effects due to the inflammatory environment of the diseased liver , whereas liver regeneration after , e.g. , partial hepatectomy is achieved by the engraftment and functional tissue replacement by the msc differentiated into hepatocytes at the site of their engraftment . this potential pleiotropic mode of action makes msc ideal candidates for stem cell therapy of different liver diseases ( enns and millan , 2008 ; haridass et al . , anti - apoptotic and pro - proliferative action of msc after acetaminophen intoxication of the rat liver . eighteen hours after the last dose the animals were sacrificed and the livers prepared for the detection of apoptotic cells ( dark nuclei ) by the tunel assay ( lower panels ) or proliferating cells ( dark nuclei ) by the ki67 stain ( upper panels ) . where indicated animals received adipose tissue - derived rat msc pre - differentiated into hepatocyte - like cells ( rmsc - hc ) 6 h after the last dose of acetaminophen . it is obvious that the number of apoptotic cells was significantly lower but of proliferating cells was higher in the livers with msc ( right panels ) indicating the anti - apoptotic and pro - proliferative action of the msc . it is obvious that experimental settings in animal models aimed to enhance liver repopulation by transplanted hepatocytes are not suited for clinical translation . thus , the lack of a survival and/or a proliferative advantage of donor vs. host hepatocytes is probably the mechanistical reason for the poor clinical progress of hepatocyte transplantation . the low success rate is augmented by the fact that human hepatocytes are isolated from marginal donor livers not allocated for transplantation . yet , as outlined above msc might be an alternate cell resource to generate hepatocyte - like cells . even if biological and biochemical differences might exist between msc from various tissues they share typical msc characteristics like marker expression , multiple differentiation capacity , and growth on plastic surfaces , which finally determine quantitative , not qualitative , variability in their hepatocyte differentiation potential . it is feasible to suppose that in respect to ethical , technical and biological aspects the transplantation of stem cell - derived hepatocytes follows the principles of hepatocyte transplantation ( fisher and strom , 2006 ) . msc might even open a broader spectrum of activity compared with primary hepatocytes because of their versatile properties such as low immunogenicity as well as their anti - inflammatory , anti - apoptotic , and pro - proliferative activities , which not only substitute the tissue damaged but also actively might temper the inflammatory response , e.g. recently , a couple of clinical trials most in china has been initiated or even completed to demonstrate safety and efficacy of the site of application of mscs concentrating on autologous stem cell transplantation in patients suffering from chronic liver failure ( table 1 ) or acute decompensation after ample liver resection . summary of clinical trials involving mesenchymal stem cells of different tissue sources for the treatment of chronic liver diseases . taking ethical considerations into account these clinical conditions may be adequate to assess safety of hepatic msc transplantation . however , to take advantage of the cells immunomodulatory , chemotactic , and anti - inflammatory properties , alf offering a highly inflammatory environment in the liver may be the disease situation of choice for the use of msc . in this case even the use of allogeneic cell sources may not be a serious problem since only the short - term beneficial actions of the msc might warrant support of liver regeneration in the critical phase of acute poisoning . immunosuppression may be applied from the beginning of treatment on or even continued as long as the recovering of the liver is ongoing but then may be ceased , thus avoiding the theoretical risks of potential sensitization of the host for future organ grafts or promoting life - threatening septic episodes during long - term stay in the intensive care units . to overcome the shortage of donor organs for liver transplantation in alf cell therapy approaches seem to be feasible , which must achieve two principle goals . ( 1 ) the loss of metabolic capacity must be substituted by the healthy donor cells , and ( 2 ) the emergence of the inflammatory environment in alf must be decelerated in order to protect hepatocytes from progression into cell death . it is obvious that the first goal might best be reached using primary hepatocytes , which , however , do not have a survival advantage in the deteriorated alf liver . the second goal might best be met by the use of msc taking advantage of their anti - inflammatory and apoptotic as well as pro - proliferative features , which , however , promises no therapeutic benefit in the case that tissue damage has surpassed the lower threshold needed to maintain body metabolic homeostasis . thus , it might be worthwhile thinking whether a combination of hepatocytes and msc might be the cell therapeutic of best choice . indeed , there is evidence that the performance of hepatocytes is improved in co - culture with msc ( ijima et al . , the anti - inflammatory cytokine il10 secreted by msc seemed to play a major role in alleviating liver damage after acute injury induced by carbon tetrachloride in the nod / scid mouse model ( zagoura et al . thus , the identification of these factors might open even cell - free therapeutical options for the treatment of alf with msc - derived molecules . animal models for cell therapy approaches to treat alf as described above enable us to earn knowledge on the mechanisms of interactions between donor and host cells both on the molecular and cellular level , to identify the hepatotropic effects esp . mediated by msc and their impact on the noxious challenge in order to optimize integration of transplanted cells into the recipient tissue thereby to support efficacy of cell transplantation and thus optimize the therapeutical outcome . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

acute liver failure ( alf ) is characterized by an initial devastating hepatic insult followed by gross parenchymal dysfunction , which leads to a multitude of systemic organ failures due to the missing metabolic homeostasis normally provided by the healthy liver . the most common causes of alf are viral hepatitis , idiosyncratic side reactions , chronic liver diseases , autoimmune hepatitis , and dose - dependent drug - induced alf . whole liver transplantation very often is the only therapy option of choice ( ostapowicz and lee , 2000 ; gill and sterling , 2001 ; rahman and hodgson , 2001 ; ogrady , 2005 ) . the incidence of acetaminophen ( paracetamol)-induced alf is rather high in the us and in the uk related both to therapy - associated and suicide - driven overdosage of the drug ( reuben et al . in the liver acetaminophen is metabolized by the cytochrome p450 enzyme system located predominantly in the hepatocytes surrounding the distal branches of the liver sinusoids , the so - called perivenous hepatocytes ( jungermann and kietzmann , 2000 ; benhamouche et al . , 2006 ; there are two principle ways of detoxification : ( 1 ) conjugation by sulfation and/or glucuronidation followed by elimination and ( 2 ) cytochrome p450-dependent oxidation and formation of n - acetyl - p - benzoquinonimine ( napqi ) , which is then conjugated to glutathione and finally eliminated with the bile . yet , sustained napqi formation eventually causes depletion of glutathione , which then in turn leads to formation of protein adducts as well as reactive nitrogen and oxygen species ( figure 1 ) . very likely mitochondrial dysfunction and increased permeability of the mitochondrial membranes contribute to the formation of reactive nitrogen and oxygen metabolites such as peroxynitrate and hydrogen peroxide besides others , which in turn mediate protein nitration and oxidative stress ( jaeschke et al . obviously , besides the hepatocytes non - parenchymal cells such as kupffer cells and sinusoidal endothelial cells seem to be involved in the generation of reactive nitrogen and oxygen species thus augmenting protein and lipid peroxidation . since these reactions are ultimately mediated by the perivenous cytochrome p450 enzyme system , apoptotic cell death followed by centrilobular necrosis is a hallmark of acetaminophen - induced hepatotoxicity ( figure 2 ) . the inflammatory environment produced during alf is also responsible for the activation of hepatic stellate cells probably mediated by il1 , which respond with an increase in expression of -smooth muscle actin and matrix metalloproteinases , mainly mmp9 . this seems to favor the remodeling of the extracellular matrix , thus augmenting hepatocyte cell death ( yan et al . depletion of gsh leads to formation of reactive nitrogen and oxygen species , which in turn causes cell death . rats were treated with a repeated oral dose of 4 g / kg body weight of acetaminophen . dashed lines exemplify initial ( area 1 ) and final necrotic perivenous areas ( area 2 , cv , central vein ) of the liver tissue . in the normal healthy liver tissue turnover is in the range of 0.01% . one might suspect then that the liver had a poor regenerative potential , which is also corroborated by the fact that after partial hepatectomy the liver is rebuilt to the original organ size , only . after 2/3 partial hepatectomy this would be accomplished by the 1.5-times cell division of the remaining hepatocytes . it has been shown in serial transplantation experiments in the albumin promoter - urokinase plasminogen activator ( upa ) transgenic mouse that hepatocytes feature a nearly unlimited regenerative capacity . in this model , the intracellular activation of the protease plasmin causes hepatocyte damage and perinatal lethality ( heckel et al . eventually , mice survived due to the substitution of hepatocytes bearing the transgene by healthy hepatocytes , which obviously had a survival advantage . transplantation of these hepatocytes having escaped the lethal phenotype into the livers of transgenic mice revealed the efficient repopulation of the diseased host liver by the donor hepatocytes , thus rescuing the lethal phenotype . , , the knockout of fumarylacetoacetate hydrolase ( fah ) leads to the accumulation of tyrosine intermediates , which cause toxic insult of hepatocytes . transplanted healthy hepatocytes display a proliferative advantage over the diseased host hepatocytes , thus achieving nearly complete replacement of the original transgenic hepatocytes by the transplanted cells . in this model , serial transplantation of hepatocytes derived from mutant livers colonized with transplanted wildtype cells revealed that 6 rounds of liver repopulation required a minimum of 69 cell divisions ( overturf et al . this in turn means , that they have the potential of self - renewal and of functional tissue formation in vivo , which are ultimate stem cell characteristics . experimentally , alf might be triggered by the use of chemical noxious compounds such as carbon tetrachloride or acetaminophen as mentioned above . as long as the hepatocytes dispose of sufficient metabolic capacity to detoxify the drugs no obvious tissue lesions emerge . yet , the production of reactive metabolites followed by covalent protein and lipid modification due to metabolic overload as mentioned above finally results in cellular dysfunction , initial cell damage and tissue injury . the initial insult resulting in injury progression is the mitotic challenge for the hepatocytes to restore the tissue loss by functional hepatocyte progeny . again , dependent on the dose of the noxious compounds the regenerative potential of the liver is either sufficient for injury regression or overwhelmed resulting in injury progression followed by alf ( mehendale , 2005 ; palmes et al . tissue regeneration is accomplished by the hepatocytes themselves as long as a minimal liver tissue mass is compliant with a certain threshold of functional tissue loss . yet , if this threshold is surpassed the regenerative capacity of the hepatocytes does not suffice for functional tissue restoration . in this case a progenitor cell compartment is activated giving rise to so - called oval cells in rodents , which are agreed upon to be the progeny of adult hepatic stem cells in the liver ( sell , 2001 ; kofman et al . oval cells appear in the periportal areas after massive liver injury adjacent to the canals of hering , structural links between the terminal biliary branches and the periportal hepatocytes ( fausto , 2004 ; santoni - rugiu et al . it is noteworthy that both hepatocytes and hepatic progenitor cells may differentiate into hepatocytes and biliary cells as well indicating their bipotent differentiation capacity . hence , both cell types meet the minimal definition criteria of a stem cell , i.e. , the potential of self - renewal to maintain the stem cell reserve , and a multiple differentiation potential giving rise to progeny of at least two different lineages . in the latter case it is self - evident that proliferation and differentiation of the offspring cells provide the functional backup for tissue repair after injury . the regenerative response of the liver after acute intoxication is triggered by the emergence of initial tissue damage and progression . dependding on the dose of the noxa and the regenerative capacity of the hepatocytes injury regression and regeneration or progression and alf develop . however , about one third of patients on the waiting list for liver transplantation in europe do not profit because of the unavailability of suitable donor organs . the hepatocyte is the smallest functional unit of the liver executing the complete metabolic orchestra , which is provided by the liver as a whole . therefore , transplantation of hepatocytes might be possible to substitute for the functional tissue loss in alf provided the donor cells take over hepatocyte functions in the deteriorated host parenchyma for at least the critical period in time either required to bridge to organ transplantation or to allow for tissue recovery from the toxic insult ( najimi and sokal , 2008 ; oertel and shafritz , 2008 ; smets et al . technically , in rodent small animal models the cells are delivered to the liver either after intraportal or intrasplenic injection . it is assumed that the cells distribute homogeneously in the liver by passage with the blood stream where they are entrapped in the sinusoids and eventually penetrate the endothelia , integrate , proliferate , and spread into the host parenchyma . there is one major constraint , which probably seriously hampers the clinical translation of hepatocyte transplantation in alf . , in the range of 15% of the total liver mass ( ponder et al . however , if the recipient liver is challenged by a growth stimulus and the proliferation of host hepatocytes is impaired then a significant repopulation by transplanted hepatocytes is achieved . there is an elegant animal model available allowing for the identification of the transplanted cells in the host parenchyma . in this rat model the natural mutation in the cd26 gene leads to the expression of a non - functional protein , however , without obvious pathophysiological consequences . transplanted wildtype donor cells may then be identified histologically in the host parenchyma by the detection of cd26 . providing selective pressure conditions by partial hepatectomy as a mitotic stimulus and pre - treatment with alkaloids such as retrorsine to inhibit host hepatocyte proliferation a repopulation rate for up to nearly 100% may be achieved in this rat model ( laconi et al . similarly , high rates were obtained using rat fetal liver epithelial cells but without applying selective growth conditions for the transplanted cells ( sandhu et al . acute liver failure in mice and rats may be induced under various experimental settings , the most common in use are those acutely applying paracetamol or carbon tetrachloride . in general , when adult hepatocytes or oval cells isolated from donor livers under various inducing conditions are used for transplantation without further selective pressure repopulation of the host liver by the transplanted cells is poor , i.e. however , cells are functional and survive long - term in the recipient liver indicating support of liver regeneration after acute hepatotoxic injury . if in addition to the acute injury regeneration by host hepatocytes is abrogated by the beforehand treatment with mitotoxins such as the pyrrolizidine alkaloid retrorsine much higher repopulation rates may be achieved , which clearly suffice to substitute for the loss of metabolic capacity due to the toxic parenchymal damage . a comprehensive summary of models and conditions used to study liver repopulation by transplanted hepatocytes or hepatocyte progenitor cells under normal and injury conditions is available ( sancho - bru et al . to summarize , transplanted cells integrate into the host parenchyma and even at low repopulation rates display hepatocyte functions . thus , hepatocyte transplantation in alf aims at tissue substitution of the recipient liver in order to functionally reconstitute the injured parenchyma by healthy donor cells . in respect to the clinical application of hepatocyte transplantation in alf the major hurdle is probably the scarcity of donor organs to isolate human hepatocytes in sufficient quality and quantity . therefore , one feasible alternative to human adult hepatocytes is the use of stem cell - derived hepatocytes . the bone marrow harbors adult stem cells , both hematopoietic and non - hematopoietic , which are clearly superior in choice over embryonic stem cells for clinical application because of their less ethical constraints and the lack of teratoma formation after tissue implantation . in the mouse model of fah deficiency , hematopoietic bone marrow derived cells rescued the diseased phenotype by complementation of the defective fah gene with the wildtype gene in the transplanted cells ( lagasse et al . it is an open question as to whether hepatocytes derived from the bone marrow are the product of differentiation from hematopoietic stem cells or of the fusion with host hepatocytes ( alvarez - dolado et al . , 2007 ) verified that mesenchymal stem cells ( msc ) from various tissues like bone marrow , umbilical cord blood , or adipose tissue may differentiate into hepatocyte - like cells following specified growth and differentiation regimens in vitro . , 2009 ) , msc - derived hepatocyte - like cells integrated into the diseased host liver , though repopulation rates were rather low , i.e. reasoning that msc feature immunomodulatory functions in that they are able to suppress the immune response mediated through t and b cells , dendritic cells and other immune cells ( chamberlain et al . , 2007 ; le blanc and ringden , 2007 ) it might not be surprising that the action of msc in alf is rather paracrine than direct tissue support by the transplanted cells . d - galactosamine - induced fulminant hepatic failure in rats was attenuated by msc - derived molecules through inhibition of apoptosis , stimulation of hepatocyte proliferation , and minimization of the inflammatory response ( parekkadan et al . the paracrine mode of action of msc was also corroborated by the amelioration of systemic inflammation induced by lps or burn indicating in addition pleiotropic effects of the msc ( yagi et al . ectopic recruitment of msc from the bone marrow to the liver has been shown in mice challenged by acute intoxication with carbon tetrachloride or 2-acetylaminofluorene indicating chemotactic activation of the msc very likely mediated by stromal cell - derived factor-1 ( jin et al . eighteen hours after cell delivery donor - derived cells were detected in the liver ( unpublished ) where they significantly decreased acetaminophen - induced apoptosis as shown immunohistochemically by the tunel assay and stimulated proliferation of host hepatocytes as shown by ki67 staining ( figure 4 ) to regenerate the liver tissue after acute injury ( unpublished ) . besides their anti - inflammatory and immunomodulatory impact msc seem also to communicate with target cells by the exchange of mrna or mirna molecules ( collino et al . thus , genetic material is exchanged , which then might affect the regenerative response of the host tissue cells on the one and the differentiation of donor msc at the site of their engraftment into the host tissue on the other hand . this , however , means that transplanted cells might be imprinted by their target tissue and the molecular microenvironment induced by a specific type of injury . acute liver injury may trigger paracrine effects due to the inflammatory environment of the diseased liver , whereas liver regeneration after , e.g. , partial hepatectomy is achieved by the engraftment and functional tissue replacement by the msc differentiated into hepatocytes at the site of their engraftment . this potential pleiotropic mode of action makes msc ideal candidates for stem cell therapy of different liver diseases ( enns and millan , 2008 ; haridass et al . , anti - apoptotic and pro - proliferative action of msc after acetaminophen intoxication of the rat liver . eighteen hours after the last dose the animals were sacrificed and the livers prepared for the detection of apoptotic cells ( dark nuclei ) by the tunel assay ( lower panels ) or proliferating cells ( dark nuclei ) by the ki67 stain ( upper panels ) . where indicated animals received adipose tissue - derived rat msc pre - differentiated into hepatocyte - like cells ( rmsc - hc ) 6 h after the last dose of acetaminophen . it is obvious that the number of apoptotic cells was significantly lower but of proliferating cells was higher in the livers with msc ( right panels ) indicating the anti - apoptotic and pro - proliferative action of the msc . thus , the lack of a survival and/or a proliferative advantage of donor vs. host hepatocytes is probably the mechanistical reason for the poor clinical progress of hepatocyte transplantation . even if biological and biochemical differences might exist between msc from various tissues they share typical msc characteristics like marker expression , multiple differentiation capacity , and growth on plastic surfaces , which finally determine quantitative , not qualitative , variability in their hepatocyte differentiation potential . it is feasible to suppose that in respect to ethical , technical and biological aspects the transplantation of stem cell - derived hepatocytes follows the principles of hepatocyte transplantation ( fisher and strom , 2006 ) . msc might even open a broader spectrum of activity compared with primary hepatocytes because of their versatile properties such as low immunogenicity as well as their anti - inflammatory , anti - apoptotic , and pro - proliferative activities , which not only substitute the tissue damaged but also actively might temper the inflammatory response , e.g. recently , a couple of clinical trials most in china has been initiated or even completed to demonstrate safety and efficacy of the site of application of mscs concentrating on autologous stem cell transplantation in patients suffering from chronic liver failure ( table 1 ) or acute decompensation after ample liver resection . summary of clinical trials involving mesenchymal stem cells of different tissue sources for the treatment of chronic liver diseases . however , to take advantage of the cells immunomodulatory , chemotactic , and anti - inflammatory properties , alf offering a highly inflammatory environment in the liver may be the disease situation of choice for the use of msc . in this case even the use of allogeneic cell sources may not be a serious problem since only the short - term beneficial actions of the msc might warrant support of liver regeneration in the critical phase of acute poisoning . immunosuppression may be applied from the beginning of treatment on or even continued as long as the recovering of the liver is ongoing but then may be ceased , thus avoiding the theoretical risks of potential sensitization of the host for future organ grafts or promoting life - threatening septic episodes during long - term stay in the intensive care units . to overcome the shortage of donor organs for liver transplantation in alf cell therapy approaches seem to be feasible , which must achieve two principle goals . ( 1 ) the loss of metabolic capacity must be substituted by the healthy donor cells , and ( 2 ) the emergence of the inflammatory environment in alf must be decelerated in order to protect hepatocytes from progression into cell death . the second goal might best be met by the use of msc taking advantage of their anti - inflammatory and apoptotic as well as pro - proliferative features , which , however , promises no therapeutic benefit in the case that tissue damage has surpassed the lower threshold needed to maintain body metabolic homeostasis . thus , it might be worthwhile thinking whether a combination of hepatocytes and msc might be the cell therapeutic of best choice . , the anti - inflammatory cytokine il10 secreted by msc seemed to play a major role in alleviating liver damage after acute injury induced by carbon tetrachloride in the nod / scid mouse model ( zagoura et al . thus , the identification of these factors might open even cell - free therapeutical options for the treatment of alf with msc - derived molecules . animal models for cell therapy approaches to treat alf as described above enable us to earn knowledge on the mechanisms of interactions between donor and host cells both on the molecular and cellular level , to identify the hepatotropic effects esp . mediated by msc and their impact on the noxious challenge in order to optimize integration of transplanted cells into the recipient tissue thereby to support efficacy of cell transplantation and thus optimize the therapeutical outcome .

the genus pseudoalteromonas was described by gauthier et al . and represents a clade of marine bacteria defined on the basis of 16s rrna gene sequence data . originally many of the pseudoalteromonas species that are discussed in this review were members of the genus alteromonas [ 5 , 77 ] but alteromonas was taxonomically re - organized based on phylogenetic analysis . since 1995 , 22 additional pseudoalteromonas species have been described ( details available at http://www.bacterio.cict.fr/p/pseudoalteromonas.html ) . more recently two pseudoalteromonas species have been moved into the genus algicola [ 50 , 74 ] . 2000/2001 ) are covered in the 2 edition of bergey s manual of systematic bacteriology . possessing gram - negative cell walls , all members of genus pseudoalteromonas require na ions , form rod - shaped cells , are motile via sheathed polar and/or unsheathed lateral flagella and possess a strictly aerobic , chemoheterotrophic metabolism . alteromonad that could be potentially used to now describe other marine genera within class gammaproteobacteria . genus pseudoalteromonas groups within a larger clade of marine taxa , located under the umbrella of order alteromonadales that inhabit all known non - geothermal marine biomes . one interesting feature of pseudoalteromonas is that the genus can be divided relatively cleanly into pigmented and non - pigmented species clades ( fig . the non - pigmented species form a relatively distinct clade typified by phylogenetic shallowness between most member species . pigmented species show greater sequence divergence and are concentrated within the other 2 major clades making up the genus ( fig . 1 ) . with more than 2000 pseudoalteromonas 16s rrna gene sequences available on nucleotide sequence databases ( e.g. www.ncbi.nlm.nih.gov ) studies by holmstrm et al . found deeply pigmented strains within a marine bacterial isolate collection were effective in the inhibition of the settlement of various fouling invertebrates and algae . based on subsequent analyses some of these isolates lead to the general conclusion that pigmented pseudoalteromonas species possess a broad range of bioactivity associated with the secretion of extracellular compounds , several of which include pigment compounds . this realization provided greater credence to earlier studies of alteromonads that this group of marine bacteria represents a rich source of biologically active substances . non - pigmented species of pseudoalteromonas do not appear to share the same extensiveness of bioactive compound synthesis and this may reflect their econiche distribution , which is admittedly still poorly defined . within the limits of existing knowledge non - pigmented clade species tend to possess unusual and diverse enzymatic activities ( carrageenases , chitinases , alginases , cold - active enzymes ) , generally broader environmental tolerance ranges ( temperature , water activity and ph ) and substantially greater nutritional versatility compared to the pigmented species . the pigmented species also tend to have more exacting growth requirements ( some require amino acids ) and have peroxidase activity rather than catalase activity . marine biofilms influence the settlement of a variety of marine invertebrates and algae and may promote cellular metamorphosis . as biofilms can be quite variable in distribution and in species and chemical composition the influence they can have is complex . the activity of marine flora and fauna have been shown to be clearly positively or negatively influenced by experimental monospecific biofilms [ 17 , 82 , 98 ] . this activity is believed to be due mainly to the production of antibiotic compounds as well as stimulatory chemical cues that are so far mostly uncharacterized . several known pseudoalteromonas species and likely many other described and undescribed bacterial species can influence the reproductive success of various marine fauna and flora due to the production of natural anti - fouling substances ( table 1 ) . a survey by holmstrm et al . found that the effectiveness of different pseudoalteromonas species to prevent settlement of fouling invertebrate larvae and algal spores varies considerably ( figure 2 ) . it was generally observed that algal dwelling species were particularly effective in preventing biofouling suggesting the ability is important for their survival in the marine ecosystem and likely required for effective colonization of various surfaces , especially the surfaces of macroalgal species . p. luteoviolacea , p. aurantia ; p. citrea [ 31 , 79 ] , p. tunicata , and p. ulvae [ 2022 , 45 ] are of particular interest as the host of compounds these species form are essentially produced to prevent biofilm residents becoming overwhelmed by other colonizing , potentially fouling species [ 26 , 38 ] . species resistant to natural antibiotics have an advantage in colonization though presumably due to the array of compounds that may be formed no one species is likely to have such an edge that they always become numerically dominant in a given econiche . the result could be that antibiotic - producing strains and development of synergisms within biofilm communities may actually encourage the formation of multi - species biofilms . this protects the biofilm community from being overgrown by a single species as well as reducing invasion by other species . the complex community formed has the advantage in that there is an inherent increase in the efficiency of nutrient acquisition , enhanced tolerance to toxic compounds and physicochemical stresses , and presumably excellent opportunities for genetic exchange . ironically , pseudoalteromonas species are highly effective biofilm formers and thus may potentially cause biofouling issues , however it is still unknown whether their presence controls the type and accumulation level of biofouling species beyond specific habitats such as the surface of marine algal species . quorum - sensing mechanisms may play an important role in the influencing settlement and subsequent biofilm formation [ 19 , 46 ] though it is unknown to what extent quorum sensing molecules influence antibiotic production . pseudoalteromonas species have been found to inhibit the settlement , germination , growth or even directly lyse the cells of various algal species . thus the interactions pseudoalteromonas strains are involved is intricate dictated by the type of ecosystem involved ( planktonic , benthic , surficial etc . ) , physiological state populations of the species involved , and prevailing environmental chemical parameters . at this point in time specific aspects of these ecological networks remain largely unexplored . to make the ecological connections even more manifestly complex strains of pseudoalteromonas have also been shown to have promotive affects on various marine eukaryotes in regards to their potential reproductive success . an example of an inhibitory - oriented interaction involves unidentified strains of pseudoalteromonas [ 18 , 62 ] that were found to inhibit the growth but not kill various diatom species due to production of antibiotic compounds . one of these compounds was identified as 2-heptyl-4-quinolinol ( fig 6i ) that have been found to possess antibacterial activity . 4 produced unknown substances that impeded the motility and surface attachment of a navicula sp . and this ability could be blocked by enhancing lectin formation by the diatoms leading to stronger biofilm attachment . a further different example involves strain y , closely related to pseudoalteromonas piscicida , which produces unidentified brominated antibiotic compounds as well as an unknown low molecular weight compound that can completely lyse algal cells within a matter of hours [ 64 , 92 , 93 ] . this more aggressive activity was found to be pronounced on bloom - forming toxin - producing dinoflagellates of the genera gymnodinium , chatonella and heterosigma species , however only ecdysis was observed for alexandrium species while diatom and other algal species tested were effectively immune . it was also observed that cells were attracted in a swarm to lysed cells providing the concept that the compound production is used as a means to generate nutrients in a predatory - like manner . subsequently it was determined that production of the unknown algicidal factor was triggered by an autoinducer quorum sensing system , possibly an ai-2-type peptide and that it is likely that algicidal activity is maximal during the peak of algal blooms and may thus contribute to bloom dissolution . pseudoalteromonas clearly also positively interacts with higher eukaryotes inducing invertebrate larval settlement and subsequent metamorphosis . p. espejiana was observed to induce the settlement and metamorphosis of the hydrozoan hydractinia echinata possibly due to induction of caspases , suggesting pseudoalteromonas strains may have apoptosis - inducing effects in some marine eukaryotes . pseudoalteromonas sp . a3 , interestingly closely related to p. piscicida , isolated from the crustose coralline algae hydrolithon onkodes clearly encouraged coral larvae settlement and subsequently induced metamorphosis in the planula of the coral acropora . the presumed chemical cue for this induction has not yet been discovered , however methanolic extracts of a3 cultures provided induction that was highly variable . a further example includes an extensive survey of marine bacterial isolates from coralline algae and larvae , from which 12 of 17 pseudoalteromonas strains were found to be effective in inducing the larval settlement of the sea urchin heliocidaris erythrogramma . it was also observed that the larvae metamorphosed in higher numbers on biofilms consisting of p. luteoviolacea a species known to produce several antibiotic and bioactive compounds . huang et al . demonstrated that biofilms strongly promoted the settlement of hydroides elegans larvae suggesting that when in the biofilm state chemical cues are actively formed . the direct detection of antibiotic compounds formed from artificial laboratory biofilms has been challenging possibly due to lack of replicability of natural biofilms as well as due to the variable abundance of antibiotic - producing taxa . these examples suggest that chemical substance production may help various pseudoalteromonas species compete in quite different situations . the advantages derived could include increased access to space for growth or for nutrient access and potentially can occur in marine biofilms or planktonically . it is not clear how significant or prevalent these interactions are for planktonic and benthic sediment associated cells or for cells ensconced in sinking marine aggregates . mechanistically the processes involved are also poorly understood and how these activities contribute to larger networks of ecological interactions . thus the questions that involve pseudoalteromonas are manifold . keeping with the theme of antibiotics , a popular subject in microbiology , the significance of cell - to - cell proximity , dilution rate and inherent antibiotic resistance within natural settings in regards to natural antibiotics are still open questions . in marine biology a greater interest may exist on what are the different broadcasted chemical cues produced by bacteria , including pseudoalteromonas that may affect the development or growth of eukaryotes as well as other prokaryotes . assuming chemical cues can be identified we still need know what they do exactly , why and how . the contribution of many different factors suggests that understanding the ecological ramifications and impact of natural bioactive compounds can only be approached at first by specific , controlled experiments focusing on model species , a necessary simplification that allows important specific ecologically to better conceived and tested . one such model species that has appeared within the last few years is p. tunicata , a species that possesses an unusually extensive capacity for inhibiting other types of microorganisms and larger eukaryotes and is the focus of the prof . staffan kjelleberg research team at the university of new south wales , sydney , australia . p. tunicata possesses the highest and broadest range of anti - fouling activities observed to date and was first isolated from a tunicate off the coast of sweden and subsequently found to dwell on a number of marine species , especially macroalgae [ 20 , 44 ] . transposon mutagenesis analysis demonstrated the pigment production by the species is linked to this activity [ 22 , 23 ] and includes both purple and yellow pigments that give p. tunicata a distinctly dark green - color . the purple pigments were found to prevent the settlement of invertebrate larvae ( balanus amphrtite , hydroides elegans ) and algal spores ( ulva lactuca , polysiphonia sp . ) ( figure 2 ) but remains unidentified . the species p. ulvae , isolated from the surface of the seaweed ulva lactuca , was found to also inhibit the settlement of invertebrate larvae ( balanus amphritite ) and inhibit algal spore germination and settlement [ 20 , 21 ] . the purple pigment formed by p. ulvae may be chemically similar to the compound(s ) found in p. tunicata and other pseudoalteromonas species . the anti - fouling ability of p. tunicata is linked to its ability to produce a range of inhibitory substances ( more details below ) including a toxic antibiotic protein ( alpp ) ; a heat labile anti - algal substance ; violet - colored polar compound(s ) of < 500 da ; an antifungal yellow pigment compound as well as small molecules active against protists and diatoms ( unpublished data ) . wmpr , similar to the vibrio cholerae cholera toxin regulator toxr , was found to act as a response regulator activating several genes in p. tunicata including pigment and antifouling compound production , biofilm formation , type iv pili , some ubiquinone and amino acid synthetic genes , a putative fe complex tonb - type transporter and also a non - ribosomal peptide synthetase that possibly is involved in fe siderophore synthesis . the wild type strain survives iron starvation better than a wmpr mutant suggesting that the tendency of p. tunicata to colonize surfaces and produce antifoulants is controlled by nutrient related responses , perhaps acquisition of trace iron [ 23 , 96 ] . p. tunicata appeared to be most effective in colonization when it had access to an exogenous carbon source associated with the colonization surface e.g. cellobiose derived from degradation of the cellulose surface polymer of the macroalgal species . this may suggest the types and levels of nutrient available for growth may also drive colonization . population estimates of antifouling species p. tunicata and p. ulvae , able to form toxic protein alpp , has been made by using real - time pcr targeting pseudoalteromonas - specific and p.tunicata/p.ulvae-specific 16s rrna genes as well as detection of the gene coding alpp . it was found that from various samples collected off the danish coast that pseudoalteromonas are abundant , especially in seawater ( figure 4 ) . p. tunicata and p. ulvae , however prefer colonization of various cellulose - producing algal species as suggested by the specific abundance of anti - fouling pseudoalteromonas spp , positive detection of the alpp gene ( fig . almost all of the antifouling species found were related to p. tunicata and p. ulvae but these only made up about 1% of the total pseudoalteromonas abundance . this may suggest that other species await isolation that could also have anti - fouling roles on the hosts sampled or the specific populations change over time as the biofilm matures . based on the compiled data shown in figure 2 the variation in settlement inhibition assays is highly species - specific ( and possibly strain - specific ) and that marine macrophyta and marine fauna may play host to their own specific anti - fouling microorganisms . antibiotic substances linked to the genus pseudoalteromonas go back to the very early days of marine natural product discovery . over the years several pseudoalteromonas strains that have been investigated in this regard have been misidentified or misclassified as pseudomonas , chromobacterium , alteromonas etc . with the current rate of bacterial genus and species descriptions from marine samples , it is difficult if not impossible to be certain of genus identities ( let alone species ) from the limited taxonomic analyses applied in the times prior to routine sequence based bacterial identification . the information below is based on strains that on the basis of existing data can be attributed at a high level of confidence to the genus pseudoalteromonas . early discoveries of marine - derived antibiotics included the identification of brominated pyrroles from purple - pigmented alteromonads . several low weight antibacterial substances were obtained from p. luteoviolacea misclassified initially as chromobacterium marinum . these included 2,3,4,5-tetrabromopyrrole ( figure 5a ) , 2,2,3,3,4,4-hexabromobipyrrole ( figure 5b ) , 2,3,4-tribromo-5(2-hydroxy-3,5-dibromophenyl)pyrrole ( also called pentabromopseudilin ) ( figure 5c ) , 2,4-dibromo-6-chlorophenol ( figure 5d ) , 4-hydroxybenzaldehyde , and n - propyl-4-hydroxybenzoate [ 2 , 11 , 24 , 32 , 34 , 37 , 85 , 86 ] . one or more of these substances also seem to also have activity against protists . the seawater species p. phenolica was also found to form a brominated biphenyl compound , 3,3,5,5-tetrabromo-2,2-diphenyldiol ( figure 5e ) that was inhibitory to methicillin resistant staphylococcus aureus strains . the yellow pigment of p. tunicata has anti - fungal activity and was identified as a tambjamine - like alkaloid , designated yp1 ( figure 6a ) . tambjamines are 4-methoxypyrrole - containing bioactive compounds previously isolated from marine invertebrates and possess antimicrobial , anti - tumorigenic , immunosuppressive , anti - proliferation and ichthyodeterrent activities and are likely produced as natural defensive compounds against predators . most evidence points to bacteria , colonizing the surface of higher organisms , as the source of these compounds . indeed , the biosynthetic pathway for yp1 was elucidated in p. tunicata by burke et al . and is coded by a cluster of 19 genes ( tama to tams ) that code proteins with homology to prodigiosin biosynthetic genes in various other bacteria , including the formation of a bipyrrole precursor , 4-methoxybipyrrole-5-carbaldehyde ( mpc ) from proline , malonyl - coa and serine . yp1 is formed by condensation of mpc with dodec-3-en-1-amine . a related compound to yp1 acts as an ionophore . the bright red pigments of p. denitrificans and p. rubra identified as cycloprodigiosin hcl ( figure 6f ) [ 36 , 56 ] also acts as an ionophore blocking proton / chloride ion symporters but have been shown to generally uncouple proton translocation . it has significant potential pharmaceutical impact able to suppress immunoproliferation , has anti - malarial activity and is able to induce apoptosis in several cancer cell lines [ 13 , 80 ] . in synergy with other drugs substituted phenylalkenoic acids referred to as rubrenoic acids ( figure 6e ) have also been purified from p. rubra and shown to have bronchodilatatoric activity . the species p. tetraodonis ( as well as a number of other bacterial species ) , isolated from the skin slime of the puffer fish fugu poecilonorus , was shown to form the notorious tetrodotoxin ( figure 6j ) [ 27 , 91 ] , which is an exceptionally potent blocker of voltage - gated , na ion membrane channels . production of tetrodotoxin by p. tetraodonis was dramatically stimulated under phosphate - limiting growth conditions . korormycin ( figure 6 g ) , produced by an unidentified pseudoalteromonas sp . , is a heptylated hydroxyquinonolone that has antibiotic activity against various marine bacteria , especially vibrio spp . it was found to strongly inhibit the respiratory chain - linked na - ion translocating nadh - quinone reductase of vibrio alginolyticus . butanol extracts of the algal associated species p. issachenkonii cultures were found to inhibit candida albicans and cause hemolysis . analysis of ethyl acetate extracts revealed that isatin ( indole-2,3-dione ) ( figure 6d ) was responsible for the anti - fungal activity . a red - brown haemolytic pigment of 269 da and corresponding to c9h7n3os3 was also discovered and still awaits complete structural analysis . a possibly misidentified pseudoalteromonas species designated alteromonas sp . p7 , isolated from the larva of penaeus monodon was found to be vibriostatic with the antibacterial substance recovered in ethyl acetate but not the culture supernatant suggesting the compound may also be a substituted aromatic compound . a potentially misclassified pigmented pseudoalteromonas - like species called alteromonas rava was found to form a substituted 6-amino-4h - dithiolo[4,3-b]pyrrol-5-one called thiomarinol a ( figure 6h ) . various thiomarinol derivatives have been also synthesized that exhibit potent antibacterial activity [ 89 , 90 ] . the yellow - pigmented species p. piscicida has been described as being able to produce a neuromuscular toxin able to kill a variety of fish and crab species [ 7 , 39 , 72 ] , however no recent studies have re - examined or confirmed this property and the toxin compound has never been detected . p. maricaloris , a close relative of p. piscicida isolated from the coral sea sponge fascaplysinopsis reticulata , forms yellow colored dibromo- and bromo - alterochomides ( figure 5f ) that possess antimicrobial activity and as well as cytotoxicity against the larvae of a sea urchin . the algicidal strain y , also closely related to p. piscicida , forms broad spectrum antimicrobial yellow brominated substances that lc - ms analysis revealed to comprise 12 different but likely structurally similar compounds . nmr spectral analysis revealed the presence of bromine , aromatic rings , hydroxyl and carbonyl groups . these still so far unidentified substances may also represent other cyclic or acyclic depsipeptide brominated compounds and may be relevant to the observation of other low molecular weight peptide like substances potentially contributing to anti - fouling activity . a variety of diketopiperazines ( dkp ) are produced by an antarctic pseudoalteromonas haloplanktis isolate including a novel compound , cyclo-(d - pipecolinyl - l - isoleucine ) ( figure 6c ) . another novel dkp , cyclo-(l - phenylalanyl-4r - hydroxyl - l - proline ) ( figure 6b ) is produced by p. luteoviolacea that exhibited antibacterial activity . the continued examination of peptide like pigment and non - pigment substances from pseudoalteromonas appears to be necessary as these compounds seem to have a potentially important impact in biological interactions . several pseudoalteromonas species have been found to secrete proteins and other soluble high molecular weight substances with antibacterial activities that are also autotoxic in nature . this included a 100 kda protein produced by p. luteoviolacea , which was likely also observed in the study of kamei et al . . the synthesis of this protein appeared to be induced during late exponential to stationary growth . p. rubra was found to form a strongly anthrone - reactive polyanionic substance , possibly a glycoprotein or polysaccharide , which had antibacterial activity , especially to species that did not naturally possess catalase activity . subsequently it was found that growth inhibition by the antibiotic was due to induction of oxidative stress in target cells through increased o2 uptake and accumulation of hydrogen peroxide [ 29 , 30 ] . an alteromonad , designated ncimb 2144 , was found to produce an autotoxic , antibacterial 90 kda thermolabile glycoprotein that could be analogous to that found in p. rubra . this protein was found to inhibit a wide range of marine and medically significant bacteria but is particularly potent against strain d2 itself ( mic 4 g ml ) . the autoxicity of alpp appears to provide a dispersal mechanism to biofilm dwelling p. tunicata , working through a process akin to programmed cell death . the alpp - autolysis of cells within biofilm substructures seems to provide nutrients for a subpopulation of cells resistant to alpp with the lysis causing sections of the biofilm to also detach . survivor cells are actively motile and presumably are dispersed in the water flow and thus can recolonize new surfaces [ 69 , 70 ] . observations in other bacteria suggest dispersal mechanisms analogous to this could be relatively common amongst biofilm forming species . an isolate related to p. piscicida , designated x153 has been proposed as an effective probiont , protecting commercial shellfish species from pathogenic vibrio species . x153 was shown to produce a , unstable tetrameric 280 kda vibriostatic protein that also appeared to have broad spectrum inhibitory activity against marine bacteria . p. haloplanktis and p. undina strains have also been found to provide probiotic benefits [ 66 , 84 ] though it is unknown if antibiotic factors are involved . some species of pseudoalteromonas have been rarely implicated as opportunistic pathogens of marine animals including fish and crustacea [ 14 , 76 , 81 ] . this pathogenicity may relate to lipopolysaccharide and other heat labile factors and other virulence determinants such as hemolysins and thrombolysins . the structure of o - specific polysaccharides has been determined for a number of pseudoalteromonas species , which are notable in being acidic and containing a variety of substituted sugar and non - sugar subunits . it is unknown if these substances have antibiotic or cytotoxic properties in the marine environment . early discoveries of marine - derived antibiotics included the identification of brominated pyrroles from purple - pigmented alteromonads . several low weight antibacterial substances were obtained from p. luteoviolacea misclassified initially as chromobacterium marinum . these included 2,3,4,5-tetrabromopyrrole ( figure 5a ) , 2,2,3,3,4,4-hexabromobipyrrole ( figure 5b ) , 2,3,4-tribromo-5(2-hydroxy-3,5-dibromophenyl)pyrrole ( also called pentabromopseudilin ) ( figure 5c ) , 2,4-dibromo-6-chlorophenol ( figure 5d ) , 4-hydroxybenzaldehyde , and n - propyl-4-hydroxybenzoate [ 2 , 11 , 24 , 32 , 34 , 37 , 85 , 86 ] . one or more of these substances also seem to also have activity against protists . the seawater species p. phenolica was also found to form a brominated biphenyl compound , 3,3,5,5-tetrabromo-2,2-diphenyldiol ( figure 5e ) that was inhibitory to methicillin resistant staphylococcus aureus strains . the yellow pigment of p. tunicata has anti - fungal activity and was identified as a tambjamine - like alkaloid , designated yp1 ( figure 6a ) . tambjamines are 4-methoxypyrrole - containing bioactive compounds previously isolated from marine invertebrates and possess antimicrobial , anti - tumorigenic , immunosuppressive , anti - proliferation and ichthyodeterrent activities and are likely produced as natural defensive compounds against predators . most evidence points to bacteria , colonizing the surface of higher organisms , as the source of these compounds . indeed , the biosynthetic pathway for yp1 was elucidated in p. tunicata by burke et al . and is coded by a cluster of 19 genes ( tama to tams ) that code proteins with homology to prodigiosin biosynthetic genes in various other bacteria , including the formation of a bipyrrole precursor , 4-methoxybipyrrole-5-carbaldehyde ( mpc ) from proline , malonyl - coa and serine . yp1 is formed by condensation of mpc with dodec-3-en-1-amine . a related compound to yp1 acts as an ionophore . the bright red pigments of p. denitrificans and p. rubra identified as cycloprodigiosin hcl ( figure 6f ) [ 36 , 56 ] also acts as an ionophore blocking proton / chloride ion symporters but have been shown to generally uncouple proton translocation . it has significant potential pharmaceutical impact able to suppress immunoproliferation , has anti - malarial activity and is able to induce apoptosis in several cancer cell lines [ 13 , 80 ] . in synergy with other drugs substituted phenylalkenoic acids referred to as rubrenoic acids ( figure 6e ) have also been purified from p. rubra and shown to have bronchodilatatoric activity . the species p. tetraodonis ( as well as a number of other bacterial species ) , isolated from the skin slime of the puffer fish fugu poecilonorus , was shown to form the notorious tetrodotoxin ( figure 6j ) [ 27 , 91 ] , which is an exceptionally potent blocker of voltage - gated , na ion membrane channels . production of tetrodotoxin by p. tetraodonis was dramatically stimulated under phosphate - limiting growth conditions . korormycin ( figure 6 g ) , produced by an unidentified pseudoalteromonas sp . , is a heptylated hydroxyquinonolone that has antibiotic activity against various marine bacteria , especially vibrio spp . it was found to strongly inhibit the respiratory chain - linked na - ion translocating nadh - quinone reductase of vibrio alginolyticus . butanol extracts of the algal associated species p. issachenkonii cultures were found to inhibit candida albicans and cause hemolysis . analysis of ethyl acetate extracts revealed that isatin ( indole-2,3-dione ) ( figure 6d ) was responsible for the anti - fungal activity . a red - brown haemolytic pigment of 269 da and corresponding to c9h7n3os3 was also discovered and still awaits complete structural analysis . a possibly misidentified pseudoalteromonas species designated alteromonas sp . p7 , isolated from the larva of penaeus monodon was found to be vibriostatic with the antibacterial substance recovered in ethyl acetate but not the culture supernatant suggesting the compound may also be a substituted aromatic compound . a potentially misclassified pigmented pseudoalteromonas - like species called alteromonas rava was found to form a substituted 6-amino-4h - dithiolo[4,3-b]pyrrol-5-one called thiomarinol a ( figure 6h ) . various thiomarinol derivatives have been also synthesized that exhibit potent antibacterial activity [ 89 , 90 ] . the yellow - pigmented species p. piscicida has been described as being able to produce a neuromuscular toxin able to kill a variety of fish and crab species [ 7 , 39 , 72 ] , however no recent studies have re - examined or confirmed this property and the toxin compound has never been detected . p. maricaloris , a close relative of p. piscicida isolated from the coral sea sponge fascaplysinopsis reticulata , forms yellow colored dibromo- and bromo - alterochomides ( figure 5f ) that possess antimicrobial activity and as well as cytotoxicity against the larvae of a sea urchin . the algicidal strain y , also closely related to p. piscicida , forms broad spectrum antimicrobial yellow brominated substances that lc - ms analysis revealed to comprise 12 different but likely structurally similar compounds . nmr spectral analysis revealed the presence of bromine , aromatic rings , hydroxyl and carbonyl groups . these still so far unidentified substances may also represent other cyclic or acyclic depsipeptide brominated compounds and may be relevant to the observation of other low molecular weight peptide like substances potentially contributing to anti - fouling activity . a variety of diketopiperazines ( dkp ) are produced by an antarctic pseudoalteromonas haloplanktis isolate including a novel compound , cyclo-(d - pipecolinyl - l - isoleucine ) ( figure 6c ) . another novel dkp , cyclo-(l - phenylalanyl-4r - hydroxyl - l - proline ) ( figure 6b ) is produced by p. luteoviolacea that exhibited antibacterial activity . the continued examination of peptide like pigment and non - pigment substances from pseudoalteromonas appears to be necessary as these compounds seem to have a potentially important impact in biological interactions . several pseudoalteromonas species have been found to secrete proteins and other soluble high molecular weight substances with antibacterial activities that are also autotoxic in nature . this included a 100 kda protein produced by p. luteoviolacea , which was likely also observed in the study of kamei et al . . the synthesis of this protein appeared to be induced during late exponential to stationary growth . p. rubra was found to form a strongly anthrone - reactive polyanionic substance , possibly a glycoprotein or polysaccharide , which had antibacterial activity , especially to species that did not naturally possess catalase activity . subsequently it was found that growth inhibition by the antibiotic was due to induction of oxidative stress in target cells through increased o2 uptake and accumulation of hydrogen peroxide [ 29 , 30 ] . an alteromonad , designated ncimb 2144 , was found to produce an autotoxic , antibacterial 90 kda thermolabile glycoprotein that could be analogous to that found in p. rubra . this protein was found to inhibit a wide range of marine and medically significant bacteria but is particularly potent against strain d2 itself ( mic 4 g ml ) . the autoxicity of alpp appears to provide a dispersal mechanism to biofilm dwelling p. tunicata , working through a process akin to programmed cell death . the alpp - autolysis of cells within biofilm substructures seems to provide nutrients for a subpopulation of cells resistant to alpp with the lysis causing sections of the biofilm to also detach . survivor cells are actively motile and presumably are dispersed in the water flow and thus can recolonize new surfaces [ 69 , 70 ] . observations in other bacteria suggest dispersal mechanisms analogous to this could be relatively common amongst biofilm forming species . an isolate related to p. piscicida , designated x153 has been proposed as an effective probiont , protecting commercial shellfish species from pathogenic vibrio species . x153 was shown to produce a , unstable tetrameric 280 kda vibriostatic protein that also appeared to have broad spectrum inhibitory activity against marine bacteria . undina strains have also been found to provide probiotic benefits [ 66 , 84 ] though it is unknown if antibiotic factors are involved . some species of pseudoalteromonas have been rarely implicated as opportunistic pathogens of marine animals including fish and crustacea [ 14 , 76 , 81 ] . this pathogenicity may relate to lipopolysaccharide and other heat labile factors and other virulence determinants such as hemolysins and thrombolysins . the structure of o - specific polysaccharides has been determined for a number of pseudoalteromonas species , which are notable in being acidic and containing a variety of substituted sugar and non - sugar subunits . it is unknown if these substances have antibiotic or cytotoxic properties in the marine environment . pseudoalteromonas though it appears to be only one of many marine bacterial genera ( either cultured or uncultured ) clearly has been shown to possess ecologically- and pharmaceutically - relevant features that are both unusual and intriguing . greater understanding of the activities of this genus in the marine ecosystem would represent a boon to microbial and marine ecology . more knowledge of the biologically active chemicals formed by pseudoalteromonas species would also be potentially pharmacologically beneficial . such research could be performed from a marine biology point of view in order determine the relationships of pseudoalteromonas to the reproductive success of many invertebrates and algal species . detection and identification of new chemical cues could be valuable in understanding the interactive ecology of marine fauna and their associated microbiota . negri and colleagues proposed there is a possibility that broadcast chemical producing bacteria like pseudoalteromonas sp . a3 could be used as a means to re - seed reefs with coral species owing to its positive effects on coral larvae settlement and metamorphosis . it is unknown if any of these undiscovered compounds would have pharmaceutical benefit too or would have other applications . in that respect several broad spectrum antibiotic compounds including pentabromopseudilin , korormycin , thiomarinol a , bromo - alterochromides a and b , tambjamine yp1 etc . various strains also form compounds with intriguing pharmaceutical properties such as the red pigment cycloprodigiosine hcl . since these substances have been derived from only a small subset of strains the potential pool of natural product diversity possessed by genus pseudoalteromonas is likely mostly unrealized . proteases from various strains of pseudoalteromonas , in particular from a strain of p. issachenkonii were recently found to be effective in reducing biofouling by the bryozoan bugula neritina . the innovative practical application of small and large antibiotic compounds as well as proteinaceous enzymatic substances to combat marine biofouling is progressing through the use of paints and gel - encapsulation [ 19 , 102 ] . thus the extensive enzymatic ability of various pseudoalteromonas ( as suggested for some species in table 1 ) is also worth noting as this generally unexplored facet can be potentially useful in a practical sense to combat biofouling as well as an ecologically relevant feature .

the genus pseudoalteromonas is a marine group of bacteria belonging to the class gammaproteobacteria that has come to attention in the natural product and microbial ecology science fields in the last decade . pigmented species of the genus have been shown to produce an array of low and high molecular weight compounds with antimicrobial , anti - fouling , algicidal and various pharmaceutically - relevant activities . compounds formed include toxic proteins , polyanionic exopolymers , substituted phenolic and pyrolle - containing alkaloids , cyclic peptides and a range of bromine - substituted compounds . ecologically , pseudoalteromonas appears significant and to date has been shown to influence biofilm formation in various marine econiches ; involved in predator - like interactions within the microbial loop ; influence settlement , germination and metamorphosis of various invertebrate and algal species ; and may also be adopted by marine flora and fauna as defensive agents . studies have been so far limited to a relatively small subset of strains compared to the known diversity of the genus suggesting that many more discoveries of novel natural products as well as ecological connections these may have in the marine ecosystem remain to be made .

1. Introduction 2. Ecological Significance 3 4. Natural product compound production by 4.1. Low molecular weight substances 4.2 High molecular weight substances 5. Concluding Remarks

the genus pseudoalteromonas was described by gauthier et al . originally many of the pseudoalteromonas species that are discussed in this review were members of the genus alteromonas [ 5 , 77 ] but alteromonas was taxonomically re - organized based on phylogenetic analysis . more recently two pseudoalteromonas species have been moved into the genus algicola [ 50 , 74 ] . 2000/2001 ) are covered in the 2 edition of bergey s manual of systematic bacteriology . one interesting feature of pseudoalteromonas is that the genus can be divided relatively cleanly into pigmented and non - pigmented species clades ( fig . the non - pigmented species form a relatively distinct clade typified by phylogenetic shallowness between most member species . pigmented species show greater sequence divergence and are concentrated within the other 2 major clades making up the genus ( fig . found deeply pigmented strains within a marine bacterial isolate collection were effective in the inhibition of the settlement of various fouling invertebrates and algae . based on subsequent analyses some of these isolates lead to the general conclusion that pigmented pseudoalteromonas species possess a broad range of bioactivity associated with the secretion of extracellular compounds , several of which include pigment compounds . non - pigmented species of pseudoalteromonas do not appear to share the same extensiveness of bioactive compound synthesis and this may reflect their econiche distribution , which is admittedly still poorly defined . within the limits of existing knowledge non - pigmented clade species tend to possess unusual and diverse enzymatic activities ( carrageenases , chitinases , alginases , cold - active enzymes ) , generally broader environmental tolerance ranges ( temperature , water activity and ph ) and substantially greater nutritional versatility compared to the pigmented species . marine biofilms influence the settlement of a variety of marine invertebrates and algae and may promote cellular metamorphosis . the activity of marine flora and fauna have been shown to be clearly positively or negatively influenced by experimental monospecific biofilms [ 17 , 82 , 98 ] . this activity is believed to be due mainly to the production of antibiotic compounds as well as stimulatory chemical cues that are so far mostly uncharacterized . several known pseudoalteromonas species and likely many other described and undescribed bacterial species can influence the reproductive success of various marine fauna and flora due to the production of natural anti - fouling substances ( table 1 ) . it was generally observed that algal dwelling species were particularly effective in preventing biofouling suggesting the ability is important for their survival in the marine ecosystem and likely required for effective colonization of various surfaces , especially the surfaces of macroalgal species . species resistant to natural antibiotics have an advantage in colonization though presumably due to the array of compounds that may be formed no one species is likely to have such an edge that they always become numerically dominant in a given econiche . this protects the biofilm community from being overgrown by a single species as well as reducing invasion by other species . ironically , pseudoalteromonas species are highly effective biofilm formers and thus may potentially cause biofouling issues , however it is still unknown whether their presence controls the type and accumulation level of biofouling species beyond specific habitats such as the surface of marine algal species . quorum - sensing mechanisms may play an important role in the influencing settlement and subsequent biofilm formation [ 19 , 46 ] though it is unknown to what extent quorum sensing molecules influence antibiotic production . pseudoalteromonas species have been found to inhibit the settlement , germination , growth or even directly lyse the cells of various algal species . to make the ecological connections even more manifestly complex strains of pseudoalteromonas have also been shown to have promotive affects on various marine eukaryotes in regards to their potential reproductive success . a further different example involves strain y , closely related to pseudoalteromonas piscicida , which produces unidentified brominated antibiotic compounds as well as an unknown low molecular weight compound that can completely lyse algal cells within a matter of hours [ 64 , 92 , 93 ] . this more aggressive activity was found to be pronounced on bloom - forming toxin - producing dinoflagellates of the genera gymnodinium , chatonella and heterosigma species , however only ecdysis was observed for alexandrium species while diatom and other algal species tested were effectively immune . subsequently it was determined that production of the unknown algicidal factor was triggered by an autoinducer quorum sensing system , possibly an ai-2-type peptide and that it is likely that algicidal activity is maximal during the peak of algal blooms and may thus contribute to bloom dissolution . p. espejiana was observed to induce the settlement and metamorphosis of the hydrozoan hydractinia echinata possibly due to induction of caspases , suggesting pseudoalteromonas strains may have apoptosis - inducing effects in some marine eukaryotes . a3 , interestingly closely related to p. piscicida , isolated from the crustose coralline algae hydrolithon onkodes clearly encouraged coral larvae settlement and subsequently induced metamorphosis in the planula of the coral acropora . a further example includes an extensive survey of marine bacterial isolates from coralline algae and larvae , from which 12 of 17 pseudoalteromonas strains were found to be effective in inducing the larval settlement of the sea urchin heliocidaris erythrogramma . demonstrated that biofilms strongly promoted the settlement of hydroides elegans larvae suggesting that when in the biofilm state chemical cues are actively formed . the direct detection of antibiotic compounds formed from artificial laboratory biofilms has been challenging possibly due to lack of replicability of natural biofilms as well as due to the variable abundance of antibiotic - producing taxa . in marine biology a greater interest may exist on what are the different broadcasted chemical cues produced by bacteria , including pseudoalteromonas that may affect the development or growth of eukaryotes as well as other prokaryotes . one such model species that has appeared within the last few years is p. tunicata , a species that possesses an unusually extensive capacity for inhibiting other types of microorganisms and larger eukaryotes and is the focus of the prof . p. tunicata possesses the highest and broadest range of anti - fouling activities observed to date and was first isolated from a tunicate off the coast of sweden and subsequently found to dwell on a number of marine species , especially macroalgae [ 20 , 44 ] . the species p. ulvae , isolated from the surface of the seaweed ulva lactuca , was found to also inhibit the settlement of invertebrate larvae ( balanus amphritite ) and inhibit algal spore germination and settlement [ 20 , 21 ] . the anti - fouling ability of p. tunicata is linked to its ability to produce a range of inhibitory substances ( more details below ) including a toxic antibiotic protein ( alpp ) ; a heat labile anti - algal substance ; violet - colored polar compound(s ) of < 500 da ; an antifungal yellow pigment compound as well as small molecules active against protists and diatoms ( unpublished data ) . wmpr , similar to the vibrio cholerae cholera toxin regulator toxr , was found to act as a response regulator activating several genes in p. tunicata including pigment and antifouling compound production , biofilm formation , type iv pili , some ubiquinone and amino acid synthetic genes , a putative fe complex tonb - type transporter and also a non - ribosomal peptide synthetase that possibly is involved in fe siderophore synthesis . population estimates of antifouling species p. tunicata and p. ulvae , able to form toxic protein alpp , has been made by using real - time pcr targeting pseudoalteromonas - specific and p.tunicata/p.ulvae-specific 16s rrna genes as well as detection of the gene coding alpp . p. tunicata and p. ulvae , however prefer colonization of various cellulose - producing algal species as suggested by the specific abundance of anti - fouling pseudoalteromonas spp , positive detection of the alpp gene ( fig . almost all of the antifouling species found were related to p. tunicata and p. ulvae but these only made up about 1% of the total pseudoalteromonas abundance . this may suggest that other species await isolation that could also have anti - fouling roles on the hosts sampled or the specific populations change over time as the biofilm matures . based on the compiled data shown in figure 2 the variation in settlement inhibition assays is highly species - specific ( and possibly strain - specific ) and that marine macrophyta and marine fauna may play host to their own specific anti - fouling microorganisms . antibiotic substances linked to the genus pseudoalteromonas go back to the very early days of marine natural product discovery . over the years several pseudoalteromonas strains that have been investigated in this regard have been misidentified or misclassified as pseudomonas , chromobacterium , alteromonas etc . with the current rate of bacterial genus and species descriptions from marine samples , it is difficult if not impossible to be certain of genus identities ( let alone species ) from the limited taxonomic analyses applied in the times prior to routine sequence based bacterial identification . the information below is based on strains that on the basis of existing data can be attributed at a high level of confidence to the genus pseudoalteromonas . early discoveries of marine - derived antibiotics included the identification of brominated pyrroles from purple - pigmented alteromonads . the yellow pigment of p. tunicata has anti - fungal activity and was identified as a tambjamine - like alkaloid , designated yp1 ( figure 6a ) . tambjamines are 4-methoxypyrrole - containing bioactive compounds previously isolated from marine invertebrates and possess antimicrobial , anti - tumorigenic , immunosuppressive , anti - proliferation and ichthyodeterrent activities and are likely produced as natural defensive compounds against predators . and is coded by a cluster of 19 genes ( tama to tams ) that code proteins with homology to prodigiosin biosynthetic genes in various other bacteria , including the formation of a bipyrrole precursor , 4-methoxybipyrrole-5-carbaldehyde ( mpc ) from proline , malonyl - coa and serine . the bright red pigments of p. denitrificans and p. rubra identified as cycloprodigiosin hcl ( figure 6f ) [ 36 , 56 ] also acts as an ionophore blocking proton / chloride ion symporters but have been shown to generally uncouple proton translocation . it has significant potential pharmaceutical impact able to suppress immunoproliferation , has anti - malarial activity and is able to induce apoptosis in several cancer cell lines [ 13 , 80 ] . in synergy with other drugs substituted phenylalkenoic acids referred to as rubrenoic acids ( figure 6e ) have also been purified from p. rubra and shown to have bronchodilatatoric activity . the species p. tetraodonis ( as well as a number of other bacterial species ) , isolated from the skin slime of the puffer fish fugu poecilonorus , was shown to form the notorious tetrodotoxin ( figure 6j ) [ 27 , 91 ] , which is an exceptionally potent blocker of voltage - gated , na ion membrane channels . , is a heptylated hydroxyquinonolone that has antibiotic activity against various marine bacteria , especially vibrio spp . p7 , isolated from the larva of penaeus monodon was found to be vibriostatic with the antibacterial substance recovered in ethyl acetate but not the culture supernatant suggesting the compound may also be a substituted aromatic compound . the yellow - pigmented species p. piscicida has been described as being able to produce a neuromuscular toxin able to kill a variety of fish and crab species [ 7 , 39 , 72 ] , however no recent studies have re - examined or confirmed this property and the toxin compound has never been detected . p. maricaloris , a close relative of p. piscicida isolated from the coral sea sponge fascaplysinopsis reticulata , forms yellow colored dibromo- and bromo - alterochomides ( figure 5f ) that possess antimicrobial activity and as well as cytotoxicity against the larvae of a sea urchin . these still so far unidentified substances may also represent other cyclic or acyclic depsipeptide brominated compounds and may be relevant to the observation of other low molecular weight peptide like substances potentially contributing to anti - fouling activity . the continued examination of peptide like pigment and non - pigment substances from pseudoalteromonas appears to be necessary as these compounds seem to have a potentially important impact in biological interactions . several pseudoalteromonas species have been found to secrete proteins and other soluble high molecular weight substances with antibacterial activities that are also autotoxic in nature . this included a 100 kda protein produced by p. luteoviolacea , which was likely also observed in the study of kamei et al . an alteromonad , designated ncimb 2144 , was found to produce an autotoxic , antibacterial 90 kda thermolabile glycoprotein that could be analogous to that found in p. rubra . this protein was found to inhibit a wide range of marine and medically significant bacteria but is particularly potent against strain d2 itself ( mic 4 g ml ) . x153 was shown to produce a , unstable tetrameric 280 kda vibriostatic protein that also appeared to have broad spectrum inhibitory activity against marine bacteria . some species of pseudoalteromonas have been rarely implicated as opportunistic pathogens of marine animals including fish and crustacea [ 14 , 76 , 81 ] . it is unknown if these substances have antibiotic or cytotoxic properties in the marine environment . the yellow pigment of p. tunicata has anti - fungal activity and was identified as a tambjamine - like alkaloid , designated yp1 ( figure 6a ) . tambjamines are 4-methoxypyrrole - containing bioactive compounds previously isolated from marine invertebrates and possess antimicrobial , anti - tumorigenic , immunosuppressive , anti - proliferation and ichthyodeterrent activities and are likely produced as natural defensive compounds against predators . and is coded by a cluster of 19 genes ( tama to tams ) that code proteins with homology to prodigiosin biosynthetic genes in various other bacteria , including the formation of a bipyrrole precursor , 4-methoxybipyrrole-5-carbaldehyde ( mpc ) from proline , malonyl - coa and serine . the bright red pigments of p. denitrificans and p. rubra identified as cycloprodigiosin hcl ( figure 6f ) [ 36 , 56 ] also acts as an ionophore blocking proton / chloride ion symporters but have been shown to generally uncouple proton translocation . the species p. tetraodonis ( as well as a number of other bacterial species ) , isolated from the skin slime of the puffer fish fugu poecilonorus , was shown to form the notorious tetrodotoxin ( figure 6j ) [ 27 , 91 ] , which is an exceptionally potent blocker of voltage - gated , na ion membrane channels . , is a heptylated hydroxyquinonolone that has antibiotic activity against various marine bacteria , especially vibrio spp . p7 , isolated from the larva of penaeus monodon was found to be vibriostatic with the antibacterial substance recovered in ethyl acetate but not the culture supernatant suggesting the compound may also be a substituted aromatic compound . the yellow - pigmented species p. piscicida has been described as being able to produce a neuromuscular toxin able to kill a variety of fish and crab species [ 7 , 39 , 72 ] , however no recent studies have re - examined or confirmed this property and the toxin compound has never been detected . p. maricaloris , a close relative of p. piscicida isolated from the coral sea sponge fascaplysinopsis reticulata , forms yellow colored dibromo- and bromo - alterochomides ( figure 5f ) that possess antimicrobial activity and as well as cytotoxicity against the larvae of a sea urchin . these still so far unidentified substances may also represent other cyclic or acyclic depsipeptide brominated compounds and may be relevant to the observation of other low molecular weight peptide like substances potentially contributing to anti - fouling activity . the continued examination of peptide like pigment and non - pigment substances from pseudoalteromonas appears to be necessary as these compounds seem to have a potentially important impact in biological interactions . several pseudoalteromonas species have been found to secrete proteins and other soluble high molecular weight substances with antibacterial activities that are also autotoxic in nature . the synthesis of this protein appeared to be induced during late exponential to stationary growth . an alteromonad , designated ncimb 2144 , was found to produce an autotoxic , antibacterial 90 kda thermolabile glycoprotein that could be analogous to that found in p. rubra . x153 was shown to produce a , unstable tetrameric 280 kda vibriostatic protein that also appeared to have broad spectrum inhibitory activity against marine bacteria . some species of pseudoalteromonas have been rarely implicated as opportunistic pathogens of marine animals including fish and crustacea [ 14 , 76 , 81 ] . it is unknown if these substances have antibiotic or cytotoxic properties in the marine environment . pseudoalteromonas though it appears to be only one of many marine bacterial genera ( either cultured or uncultured ) clearly has been shown to possess ecologically- and pharmaceutically - relevant features that are both unusual and intriguing . greater understanding of the activities of this genus in the marine ecosystem would represent a boon to microbial and marine ecology . more knowledge of the biologically active chemicals formed by pseudoalteromonas species would also be potentially pharmacologically beneficial . such research could be performed from a marine biology point of view in order determine the relationships of pseudoalteromonas to the reproductive success of many invertebrates and algal species . since these substances have been derived from only a small subset of strains the potential pool of natural product diversity possessed by genus pseudoalteromonas is likely mostly unrealized . the innovative practical application of small and large antibiotic compounds as well as proteinaceous enzymatic substances to combat marine biofouling is progressing through the use of paints and gel - encapsulation [ 19 , 102 ] . thus the extensive enzymatic ability of various pseudoalteromonas ( as suggested for some species in table 1 ) is also worth noting as this generally unexplored facet can be potentially useful in a practical sense to combat biofouling as well as an ecologically relevant feature .

originally many of the pseudoalteromonas species that are discussed in this review were members of the genus alteromonas [ 5 , 77 ] but alteromonas was taxonomically re - organized based on phylogenetic analysis . possessing gram - negative cell walls , all members of genus pseudoalteromonas require na ions , form rod - shaped cells , are motile via sheathed polar and/or unsheathed lateral flagella and possess a strictly aerobic , chemoheterotrophic metabolism . one interesting feature of pseudoalteromonas is that the genus can be divided relatively cleanly into pigmented and non - pigmented species clades ( fig . pigmented species show greater sequence divergence and are concentrated within the other 2 major clades making up the genus ( fig . found deeply pigmented strains within a marine bacterial isolate collection were effective in the inhibition of the settlement of various fouling invertebrates and algae . based on subsequent analyses some of these isolates lead to the general conclusion that pigmented pseudoalteromonas species possess a broad range of bioactivity associated with the secretion of extracellular compounds , several of which include pigment compounds . non - pigmented species of pseudoalteromonas do not appear to share the same extensiveness of bioactive compound synthesis and this may reflect their econiche distribution , which is admittedly still poorly defined . within the limits of existing knowledge non - pigmented clade species tend to possess unusual and diverse enzymatic activities ( carrageenases , chitinases , alginases , cold - active enzymes ) , generally broader environmental tolerance ranges ( temperature , water activity and ph ) and substantially greater nutritional versatility compared to the pigmented species . marine biofilms influence the settlement of a variety of marine invertebrates and algae and may promote cellular metamorphosis . as biofilms can be quite variable in distribution and in species and chemical composition the influence they can have is complex . the activity of marine flora and fauna have been shown to be clearly positively or negatively influenced by experimental monospecific biofilms [ 17 , 82 , 98 ] . several known pseudoalteromonas species and likely many other described and undescribed bacterial species can influence the reproductive success of various marine fauna and flora due to the production of natural anti - fouling substances ( table 1 ) . it was generally observed that algal dwelling species were particularly effective in preventing biofouling suggesting the ability is important for their survival in the marine ecosystem and likely required for effective colonization of various surfaces , especially the surfaces of macroalgal species . p. luteoviolacea , p. aurantia ; p. citrea [ 31 , 79 ] , p. tunicata , and p. ulvae [ 2022 , 45 ] are of particular interest as the host of compounds these species form are essentially produced to prevent biofilm residents becoming overwhelmed by other colonizing , potentially fouling species [ 26 , 38 ] . species resistant to natural antibiotics have an advantage in colonization though presumably due to the array of compounds that may be formed no one species is likely to have such an edge that they always become numerically dominant in a given econiche . the result could be that antibiotic - producing strains and development of synergisms within biofilm communities may actually encourage the formation of multi - species biofilms . the complex community formed has the advantage in that there is an inherent increase in the efficiency of nutrient acquisition , enhanced tolerance to toxic compounds and physicochemical stresses , and presumably excellent opportunities for genetic exchange . ironically , pseudoalteromonas species are highly effective biofilm formers and thus may potentially cause biofouling issues , however it is still unknown whether their presence controls the type and accumulation level of biofouling species beyond specific habitats such as the surface of marine algal species . quorum - sensing mechanisms may play an important role in the influencing settlement and subsequent biofilm formation [ 19 , 46 ] though it is unknown to what extent quorum sensing molecules influence antibiotic production . thus the interactions pseudoalteromonas strains are involved is intricate dictated by the type of ecosystem involved ( planktonic , benthic , surficial etc . ) this more aggressive activity was found to be pronounced on bloom - forming toxin - producing dinoflagellates of the genera gymnodinium , chatonella and heterosigma species , however only ecdysis was observed for alexandrium species while diatom and other algal species tested were effectively immune . it was also observed that cells were attracted in a swarm to lysed cells providing the concept that the compound production is used as a means to generate nutrients in a predatory - like manner . subsequently it was determined that production of the unknown algicidal factor was triggered by an autoinducer quorum sensing system , possibly an ai-2-type peptide and that it is likely that algicidal activity is maximal during the peak of algal blooms and may thus contribute to bloom dissolution . p. espejiana was observed to induce the settlement and metamorphosis of the hydrozoan hydractinia echinata possibly due to induction of caspases , suggesting pseudoalteromonas strains may have apoptosis - inducing effects in some marine eukaryotes . a3 , interestingly closely related to p. piscicida , isolated from the crustose coralline algae hydrolithon onkodes clearly encouraged coral larvae settlement and subsequently induced metamorphosis in the planula of the coral acropora . a further example includes an extensive survey of marine bacterial isolates from coralline algae and larvae , from which 12 of 17 pseudoalteromonas strains were found to be effective in inducing the larval settlement of the sea urchin heliocidaris erythrogramma . it was also observed that the larvae metamorphosed in higher numbers on biofilms consisting of p. luteoviolacea a species known to produce several antibiotic and bioactive compounds . demonstrated that biofilms strongly promoted the settlement of hydroides elegans larvae suggesting that when in the biofilm state chemical cues are actively formed . the direct detection of antibiotic compounds formed from artificial laboratory biofilms has been challenging possibly due to lack of replicability of natural biofilms as well as due to the variable abundance of antibiotic - producing taxa . keeping with the theme of antibiotics , a popular subject in microbiology , the significance of cell - to - cell proximity , dilution rate and inherent antibiotic resistance within natural settings in regards to natural antibiotics are still open questions . in marine biology a greater interest may exist on what are the different broadcasted chemical cues produced by bacteria , including pseudoalteromonas that may affect the development or growth of eukaryotes as well as other prokaryotes . the contribution of many different factors suggests that understanding the ecological ramifications and impact of natural bioactive compounds can only be approached at first by specific , controlled experiments focusing on model species , a necessary simplification that allows important specific ecologically to better conceived and tested . one such model species that has appeared within the last few years is p. tunicata , a species that possesses an unusually extensive capacity for inhibiting other types of microorganisms and larger eukaryotes and is the focus of the prof . p. tunicata possesses the highest and broadest range of anti - fouling activities observed to date and was first isolated from a tunicate off the coast of sweden and subsequently found to dwell on a number of marine species , especially macroalgae [ 20 , 44 ] . transposon mutagenesis analysis demonstrated the pigment production by the species is linked to this activity [ 22 , 23 ] and includes both purple and yellow pigments that give p. tunicata a distinctly dark green - color . the purple pigments were found to prevent the settlement of invertebrate larvae ( balanus amphrtite , hydroides elegans ) and algal spores ( ulva lactuca , polysiphonia sp . ) the species p. ulvae , isolated from the surface of the seaweed ulva lactuca , was found to also inhibit the settlement of invertebrate larvae ( balanus amphritite ) and inhibit algal spore germination and settlement [ 20 , 21 ] . the anti - fouling ability of p. tunicata is linked to its ability to produce a range of inhibitory substances ( more details below ) including a toxic antibiotic protein ( alpp ) ; a heat labile anti - algal substance ; violet - colored polar compound(s ) of < 500 da ; an antifungal yellow pigment compound as well as small molecules active against protists and diatoms ( unpublished data ) . wmpr , similar to the vibrio cholerae cholera toxin regulator toxr , was found to act as a response regulator activating several genes in p. tunicata including pigment and antifouling compound production , biofilm formation , type iv pili , some ubiquinone and amino acid synthetic genes , a putative fe complex tonb - type transporter and also a non - ribosomal peptide synthetase that possibly is involved in fe siderophore synthesis . the wild type strain survives iron starvation better than a wmpr mutant suggesting that the tendency of p. tunicata to colonize surfaces and produce antifoulants is controlled by nutrient related responses , perhaps acquisition of trace iron [ 23 , 96 ] . p. tunicata appeared to be most effective in colonization when it had access to an exogenous carbon source associated with the colonization surface e.g. population estimates of antifouling species p. tunicata and p. ulvae , able to form toxic protein alpp , has been made by using real - time pcr targeting pseudoalteromonas - specific and p.tunicata/p.ulvae-specific 16s rrna genes as well as detection of the gene coding alpp . p. tunicata and p. ulvae , however prefer colonization of various cellulose - producing algal species as suggested by the specific abundance of anti - fouling pseudoalteromonas spp , positive detection of the alpp gene ( fig . almost all of the antifouling species found were related to p. tunicata and p. ulvae but these only made up about 1% of the total pseudoalteromonas abundance . based on the compiled data shown in figure 2 the variation in settlement inhibition assays is highly species - specific ( and possibly strain - specific ) and that marine macrophyta and marine fauna may play host to their own specific anti - fouling microorganisms . over the years several pseudoalteromonas strains that have been investigated in this regard have been misidentified or misclassified as pseudomonas , chromobacterium , alteromonas etc . with the current rate of bacterial genus and species descriptions from marine samples , it is difficult if not impossible to be certain of genus identities ( let alone species ) from the limited taxonomic analyses applied in the times prior to routine sequence based bacterial identification . these included 2,3,4,5-tetrabromopyrrole ( figure 5a ) , 2,2,3,3,4,4-hexabromobipyrrole ( figure 5b ) , 2,3,4-tribromo-5(2-hydroxy-3,5-dibromophenyl)pyrrole ( also called pentabromopseudilin ) ( figure 5c ) , 2,4-dibromo-6-chlorophenol ( figure 5d ) , 4-hydroxybenzaldehyde , and n - propyl-4-hydroxybenzoate [ 2 , 11 , 24 , 32 , 34 , 37 , 85 , 86 ] . tambjamines are 4-methoxypyrrole - containing bioactive compounds previously isolated from marine invertebrates and possess antimicrobial , anti - tumorigenic , immunosuppressive , anti - proliferation and ichthyodeterrent activities and are likely produced as natural defensive compounds against predators . and is coded by a cluster of 19 genes ( tama to tams ) that code proteins with homology to prodigiosin biosynthetic genes in various other bacteria , including the formation of a bipyrrole precursor , 4-methoxybipyrrole-5-carbaldehyde ( mpc ) from proline , malonyl - coa and serine . the bright red pigments of p. denitrificans and p. rubra identified as cycloprodigiosin hcl ( figure 6f ) [ 36 , 56 ] also acts as an ionophore blocking proton / chloride ion symporters but have been shown to generally uncouple proton translocation . the species p. tetraodonis ( as well as a number of other bacterial species ) , isolated from the skin slime of the puffer fish fugu poecilonorus , was shown to form the notorious tetrodotoxin ( figure 6j ) [ 27 , 91 ] , which is an exceptionally potent blocker of voltage - gated , na ion membrane channels . it was found to strongly inhibit the respiratory chain - linked na - ion translocating nadh - quinone reductase of vibrio alginolyticus . butanol extracts of the algal associated species p. issachenkonii cultures were found to inhibit candida albicans and cause hemolysis . p7 , isolated from the larva of penaeus monodon was found to be vibriostatic with the antibacterial substance recovered in ethyl acetate but not the culture supernatant suggesting the compound may also be a substituted aromatic compound . the yellow - pigmented species p. piscicida has been described as being able to produce a neuromuscular toxin able to kill a variety of fish and crab species [ 7 , 39 , 72 ] , however no recent studies have re - examined or confirmed this property and the toxin compound has never been detected . p. maricaloris , a close relative of p. piscicida isolated from the coral sea sponge fascaplysinopsis reticulata , forms yellow colored dibromo- and bromo - alterochomides ( figure 5f ) that possess antimicrobial activity and as well as cytotoxicity against the larvae of a sea urchin . a variety of diketopiperazines ( dkp ) are produced by an antarctic pseudoalteromonas haloplanktis isolate including a novel compound , cyclo-(d - pipecolinyl - l - isoleucine ) ( figure 6c ) . another novel dkp , cyclo-(l - phenylalanyl-4r - hydroxyl - l - proline ) ( figure 6b ) is produced by p. luteoviolacea that exhibited antibacterial activity . subsequently it was found that growth inhibition by the antibiotic was due to induction of oxidative stress in target cells through increased o2 uptake and accumulation of hydrogen peroxide [ 29 , 30 ] . the alpp - autolysis of cells within biofilm substructures seems to provide nutrients for a subpopulation of cells resistant to alpp with the lysis causing sections of the biofilm to also detach . the structure of o - specific polysaccharides has been determined for a number of pseudoalteromonas species , which are notable in being acidic and containing a variety of substituted sugar and non - sugar subunits . tambjamines are 4-methoxypyrrole - containing bioactive compounds previously isolated from marine invertebrates and possess antimicrobial , anti - tumorigenic , immunosuppressive , anti - proliferation and ichthyodeterrent activities and are likely produced as natural defensive compounds against predators . and is coded by a cluster of 19 genes ( tama to tams ) that code proteins with homology to prodigiosin biosynthetic genes in various other bacteria , including the formation of a bipyrrole precursor , 4-methoxybipyrrole-5-carbaldehyde ( mpc ) from proline , malonyl - coa and serine . the bright red pigments of p. denitrificans and p. rubra identified as cycloprodigiosin hcl ( figure 6f ) [ 36 , 56 ] also acts as an ionophore blocking proton / chloride ion symporters but have been shown to generally uncouple proton translocation . the species p. tetraodonis ( as well as a number of other bacterial species ) , isolated from the skin slime of the puffer fish fugu poecilonorus , was shown to form the notorious tetrodotoxin ( figure 6j ) [ 27 , 91 ] , which is an exceptionally potent blocker of voltage - gated , na ion membrane channels . it was found to strongly inhibit the respiratory chain - linked na - ion translocating nadh - quinone reductase of vibrio alginolyticus . butanol extracts of the algal associated species p. issachenkonii cultures were found to inhibit candida albicans and cause hemolysis . p7 , isolated from the larva of penaeus monodon was found to be vibriostatic with the antibacterial substance recovered in ethyl acetate but not the culture supernatant suggesting the compound may also be a substituted aromatic compound . the yellow - pigmented species p. piscicida has been described as being able to produce a neuromuscular toxin able to kill a variety of fish and crab species [ 7 , 39 , 72 ] , however no recent studies have re - examined or confirmed this property and the toxin compound has never been detected . p. maricaloris , a close relative of p. piscicida isolated from the coral sea sponge fascaplysinopsis reticulata , forms yellow colored dibromo- and bromo - alterochomides ( figure 5f ) that possess antimicrobial activity and as well as cytotoxicity against the larvae of a sea urchin . a variety of diketopiperazines ( dkp ) are produced by an antarctic pseudoalteromonas haloplanktis isolate including a novel compound , cyclo-(d - pipecolinyl - l - isoleucine ) ( figure 6c ) . another novel dkp , cyclo-(l - phenylalanyl-4r - hydroxyl - l - proline ) ( figure 6b ) is produced by p. luteoviolacea that exhibited antibacterial activity . subsequently it was found that growth inhibition by the antibiotic was due to induction of oxidative stress in target cells through increased o2 uptake and accumulation of hydrogen peroxide [ 29 , 30 ] . the alpp - autolysis of cells within biofilm substructures seems to provide nutrients for a subpopulation of cells resistant to alpp with the lysis causing sections of the biofilm to also detach . detection and identification of new chemical cues could be valuable in understanding the interactive ecology of marine fauna and their associated microbiota . proteases from various strains of pseudoalteromonas , in particular from a strain of p. issachenkonii were recently found to be effective in reducing biofouling by the bryozoan bugula neritina . thus the extensive enzymatic ability of various pseudoalteromonas ( as suggested for some species in table 1 ) is also worth noting as this generally unexplored facet can be potentially useful in a practical sense to combat biofouling as well as an ecologically relevant feature .

we constructed a decision tree cost - effectiveness model for a target population of 163,000 women who at the time of the intervention are sexually active with a male partner , fertile , not desiring pregnancy within the next 12 months , and not using permanent contraception methods ( e.g. , tubal ligation and vasectomy ) ( technical appendix table and figure 1 ) . in the no intervention scenario , no changes in contraceptive use distributions from the status quo are expected to occur . in the intervention scenario , women in puerto rico are assumed to have same - day access to contraception methods , including larc , with no out - of - pocket costs . in addition , healthcare providers would be trained to provide client - centered contraceptive counseling and outreach so that women have the information they need to make an informed choice on the contraception method that is best for them . the model specifies contraceptive method use distribution , unintended pregnancy events , and the frequency of zam ( technical appendix figure 1 ) . we assumed an intervention in place throughout a year - long zika virus outbreak in puerto rico . we evaluated the costs and outcomes of increased access to contraception compared with no intervention ( i.e. , status quo ) . output measures included numbers of zam cases prevented , including stillbirths , elective terminations , and live - born infants , and healthy life years ( hly ) gained . economic benefits of the intervention included avoided costs from zam cases prevented and costs avoided for monitoring for zika virus exposed pregnancies and infants born from zika virus infected mothers . in addition , the avoided cost of prenatal , delivery , postpartum , and neonatal care associated with avoided unwanted pregnancies was considered an economic benefit . in cost - effectiveness analyses , if total avoided cost exceeds the cost of an intervention that improves health , the intervention is considered cost - saving . for scenarios with positive net costs , we reported the incremental cost - effectiveness ratio ( icer ) , which is the net cost per hly gained in comparison to the status quo . independent of zika virus exposed pregnancies and zam , unintended pregnancy is associated with adverse maternal and child health outcomes . because roughly 60% of unintended pregnancies are classified as mistimed , which might result in a delayed rather than avoided pregnancy , with the same costs occurring later ( 7 ) , we only estimated avoided medical costs from prevention of the 40% of unintended pregnancies presumed to be not desired at a later time irrespective of zika virus infection . we estimated the inputs for the decision - tree model and their sources ( table 1 ) . in the no intervention scenario , we took the distribution of women in the target population by use of different types of reversible contraceptives ( or no use ) from a 2002 survey administered in puerto rico and adjusted it to reflect the 36% decrease in fertility rates in puerto rico during 20022015 ( 8,23,24 ) . * brfss , behavior risk surveillance system ; cdc , centers for disease control and prevention ; hly ; healthy life years ; iud , intrauterine device ; larc , long - acting reversible contraceptive ; nsfg , national survey of family growth ; ondieh , office of noncommunicable diseases , injury and environmental health ; zam , zika virus associated microcephaly . authors calculation based on 2015 puerto rico total population size , 2015 birth rate , and adjusted contraception usage in 2002 brfss in puerto rico ( online technical appendix table , http://wwwnc.cdc.gov/eid/article/23/1/16-1322-techapp1.pdf ) . less - effective methods include condoms , spermicides , fertility awareness methods , withdrawal , sponge , and diaphragm . moderately effective methods include oral contraceptive pills , patches , vaginal rings , and injectable contraceptives . the contraception use distribution at baseline ( without intervention ) is based on adjusting the distribution reported in the 2002 brfss in puerto rico excluding women using permanent contraception by assuming 5 percentage points fewer women using no contraception than in 2002 , which is based on a 36% reduction of birth rate among women 1544 years of age in puerto rico from 2002 to 2015 ( national vital statistics report for 2002 and 2015 unpublished birth data from puerto rico ) , and the reported reasons for us teen pregnancy reduction ( http://www.cdc.gov/nchhstp/newsroom/docs/factsheets/yrbs-fact-sheet-final-508.pdf ) ; the decline in teen pregnancies was the fastest of any age group in puerto rico during 20102014 , as reported in national vital statistics reports . data from title x clinics in puerto rico also show that the percentage of women of reproductive age served in title x clinics increased from 2.2% in 2006 to 10% in 2015 . we also assume that new contraception users have the same contraception method distribution as contraception users as reported in the 2002 brfss survey ( 8) . we assume 22% dual use among moderately effective method users at baseline ( based on nsfg 20112013 data and unpublished analyses supplied by karen pazol , ondieh , cdc ; we accounted for the effectiveness of dual - method use in preventing pregnancy but not for preventing sexual transmission of zika virus ) . we calculated method - specific annual pregnancy rates by multiplying the failure rates of contraception methods under typical use by a calculated correction factor of 0.88 to adjust for the model over - predicting the number of unintended births in 2015 using the typical failure rates only . multiplying method - specific contraceptive failure rates by numbers of women in each method category typically results in more predicted pregnancies and births than are actually observed , in part because of heterogeneity in sexual behaviors ( 19 ) . assuming 50% of no contraception users , 60% of less - effective contraception users , and 100% of moderately effective contraception users will visit a healthcare provider for contraception services under intervention . based on the nsfg 20112013 , among women of reproductive age who did not intend to become pregnant and not using permanent contraceptive methods , 21% of no contraception users , 33% less - effective method users , 97% moderately effective method users had at least 1 visit for contraception services in the last 12 months ( personal communication , karen pazol , ondieh , cdc , 2016 ) . contraceptive services include receiving a birth control method or a prescription , receiving a checkup for birth control , receiving counseling about birth control , receiving a sterilizing operation , receiving counseling about a sterilizing operation , receiving emergency contraception , or receiving counseling about emergency contraception . we assume the intervention will increase the percentage of women visiting their provider for contraception counseling . in the contraceptive choice project ( 9 ) , 67% of participants who wished to avoid pregnancy chose to use highly effective methods , and 33% chose to use moderately effective methods . for the main scenario , we applied those estimates to 40% of the target population , assuming that 40% of unintended pregnancies are unwanted , and assumed that 20% of the remaining 60% of switchers would choose highly effective methods . # ellington et al . ( 5 ) estimated 180 cases ( interquartile range 100270 cases ) of congenital microcephaly associated with zika virus in puerto rico among an estimated 31,272 births ( unpublished birth data , puerto rico department of health , 2015 ) . on the basis of those estimates , we estimated the prevalence of congenital microcephaly : 58/10,000 births ( interquartile range 32/10,00086/10,000 ) . * * the pregnancy loss rate in the us zika pregnancy registry as of july 21 , 2016 , was 35% ( 14 ) . the termination rate is calculated as the overall pregnancy loss rate minus the assumed stillbirth rate . a range of the main value 20% was used to create upper and lower bounds used in sensitivity analyses . includes cost for devices and costs for 1 year of injections , pills , patches , rings , condoms , and the cost for related services in the first year . weighted average cost for hormonal iud ( 50% , $ 659 ) , copper iud ( 25% , $ 598 ) , and implant ( 25% , $ 659 ) , based on contraception choice study ( 9 ) and commonly used devices in puerto rico . weighted average cost for generic contraceptive pill ( 78% , $ 370/y ) , injectable ( 14% , $ 240/y plus $ 130 for consultation for the year ) , and ring or patch ( 8% , $ 964/y ) , with the distribution of moderately effective methods based on nsfg , 20112013 . # # weighted average cost of moderately effective method plus cost of less - effective method . * * * male condom . including the cost for checking the placement of iud in the first year of insertion . weighted average for vaginal and c - section delivery , including prenatal care , delivery , postpartum , and neonatal cost at delivery and in the first 3 months . assumes additional costs related to repeated zika virus testing by igm ( 12 tests ) for all pregnant women , 4 extra detailed ultrasound examinations , and 25% of women getting amniocentesis during all zika - positive pregnancies ( 25% infection rate at baseline , range 10%70% ) ( 5 ) , based on oduyebo et al . assumes 2 zika virus tests ( igm and pcr ) for serum and placenta , cranial ultrasound , and eye examination for all infants born to zika virus positive mothers based on fleming - dutra et al . # # # three pcr tests for zika virus using placenta , cord , and brain tissues of the fetus based on martines et al . ( 22 ) . * * * * assuming all prenatal care cost , including extra cost of zika virus associated testing and monitoring during pregnancy and extra cost for zika virus associated testing for fetus . assuming half of the prenatal care cost , including extra cost of zika virus associated testing and monitoring during pregnancy and extra cost for zika virus associated testing for fetus . cost of prenatal care and delivery and extra cost of zika virus associated testing and monitoring during pregnancy and testing of infants for zika virus . present value of cumulative medical and supportive care cost for infant with zam , discounted at 3% annually and taking into account mortality . expenditures by employer - sponsored health plans for privately insured children with combined diagnoses of microcephaly and congenital cytomegalovirus enrolled during the first 4 years of life were used to project medical costs for cases of zam . for the main intervention scenario , we assumed that 50% of no contraception users , 60% of less - effective contraceptive method users , and 100% of moderately effective contraceptive method users would visit a healthcare provider during the intervention period and be counseled about contraception use ( table 1 ) . the first 2 percentages are roughly twice the percentages of women reported in the 20112013 us national survey on family growth to have received contraceptive services ( contraception or counseling ) within the past year because we assumed that , during the zika virus outbreak , more women and providers would discuss contraception ; virtually all moderately effective method users were assumed to see providers to obtain contraceptive prescriptions . for the main scenario , we also assumed , optimistically , that 50% of women in the target population who receive contraceptive services during the zika virus outbreak would be willing to change to a more effective contraceptive method , evenly divided between moderately effective and highly effective methods . 33% used moderately effective methods ) ( 9 ) to the 40% of women assumed to not want to be pregnant ; we assumed 20% of other women not intending pregnancy would use larc . we further assumed that 30% of moderately effective contraception users would also choose to use condoms ( dual - method use ) under the intervention , based on a study reporting dual - method use among persons at risk for hiv ( 25 ) . we calculated method - specific annual pregnancy rates by applying failure rates of contraception methods under typical use ( 10 ) , in combination with information on estimated numbers of unintended pregnancies , to adjust for other factors influencing pregnancy risk ( 19 ) . we estimated the proportion of fetal losses among unintended pregnancies from data for the caribbean region , including puerto rico ( 12 ) , and calculated the proportion of induced abortion among unintended pregnancies from a survey conducted in puerto rico in 2001 ( the latest year for which data were available ) ( 11 ) . we assumed that the distribution of fetal loss and induced abortions in unintended pregnancies unaffected by zam would not be altered by the zika virus outbreak or the intervention . for adverse pregnancy and birth outcomes associated with zika virus , we only considered zam and associated brain anomalies , including live births , stillbirths , and terminations attributable to prenatal diagnosis . although zika virus can cause brain lesions and dysfunction in fetuses and newborns who do not have microcephaly ( 26 ) , we lacked the data to model their prevalence and cost . in the main analysis , we assumed 58 cases of zam per 10,000 live births ( range 3286/10,000 ) based on a modeling study that considered data from other mosquitoborne illnesses in puerto rico and zika virus outbreaks in other locations ( 5 ) . we assumed a pregnancy loss rate of 35% among zika virus exposed fetuses with diagnosed birth defects based on cases in the us zika pregnancy registry as of july 21 , 2016 ( 14 ) . we projected gains in hly by multiplying total cases of zam prevented by 30.0 , which is the average number of quality - adjusted life - years at birth in the united states for an infant without severe microcephaly ( 15 ) and the estimated loss in disability - adjusted life years from microcephaly ( 27 ) . we multiplied 30.0 by the sum of live births and fetal losses associated with zam to calculate gains in hly . we included fetal losses in the hly calculations because in the absence of zam those pregnancies would have resulted in live births , with the same healthy life expectancy as other children ( 15 ) . we conducted the analysis from a healthcare system perspective that includes direct medically related costs regardless of payer . we used payments from private insurance because payments from medicaid might underestimate the cost of healthcare ( 28 ) . intervention costs included program costs of training providers , patient educational materials , outreach / media campaigns on the availability of contraceptives services , and program coordination and the incremental costs of family planning services . the latter comprised the costs of contraception methods and related office visits and services ( e.g. , insertion and removal of larc for new method users resulting from the intervention and the cost of more intensive counseling for all women receiving contraceptive services during the intervention ) . we took the 1-year costs for contraception methods from the literature ( 16,29 ) and based the other program costs on the estimated costs for a pilot program planned to increase access to contraception in puerto rico as part of the current zika virus outbreak response ( 30 ) . we did not apply a discount rate to intervention costs because of the time horizon of 12 months . zika virus related costs prevented by this intervention were in 2 parts : 1 ) costs for zika virus testing and monitoring for zika virus exposed pregnancies and infants , and 2 ) costs of zam cases ( table 1 ) . the cost estimates for testing and monitoring presumed 100% adherence by clinicians and patients to recommendations ( 2022 ) . the lifetime cost per live - born infant with zam includes direct medical and nonmedical costs . zam is among the most severe types of microcephaly and is associated with loss of brain tissue volume , increased fluid spaces , and intracranial calcifications . all 3 cases of live - born infants with zam in french polynesia demonstrated severe neurologic outcomes with delayed cognitive development ( 26 ) . on the basis of expert opinion , infants with zam who survive the neonatal period would be expected to have neurologic dysfunction consistent with severe cerebral palsy within 12 years of birth . as a proxy for the medical cost of zam , we used the estimated cost of treating infants with microcephaly associated with a diagnosis of symptomatic congenital cytomegalovirus ( cmv ) . we used the marketscan commercial database ( truven health analytics ) with a sample of 100 million us residents covered by employer - sponsored insurance at any time during 20092014 . we used average costs for 4 newborn infants with diagnoses of microcephaly and cmv who survived and were enrolled in a health plan for > 3 years . for the direct nonmedical cost of zam , we used the estimated cost for supportive care for children with severe congenital brain injury , both paid care and unpaid care . the total lifetime cost for surviving infants with zam was estimated at $ 3.8 million per infant , taking into account infant and child mortality and discounting of costs in future years at a 3% rate per year ; the sum of undiscounted costs for children who survive to adulthood might reach $ 10 million . zika virus related medical costs associated with women s prenatal care , labor and delivery , and postpartum care for pregnancies ending in live birth and neonatal care from a study of us commercial health plan expenditures ( 17 ) . estimates for costs associated with pregnancies ending in induced abortion were based on our analyses of commercial claims data ( table 1 ) . because many parameters used in the model are uncertain , we conducted sensitivity analyses on selected parameters , including different scenarios for the baseline and postintervention contraception use distributions in puerto rico . we tested alternate baseline contraception use distributions in puerto rico for women at risk for unintended pregnancy by using the actual distribution of method use reported in 2002 ( 8) and among women attending title x clinics in puerto rico in 2014 ( 31 ) . for the postintervention contraception use distribution , we tested scenarios assuming different proportions of women receiving contraceptive services from a healthcare provider , different levels of willingness to switch to a more effective method , and different shares of moderately effective and highly effective methods among switchers . other parameters evaluated during sensitivity analysis included the incidence of zam during the zika virus outbreak in puerto rico , percentage of pregnancies with zam terminated , the cost of caring for a live - born infant with microcephaly , and the cost of the intervention . we conducted sensitivity analyses in which we altered selected assumptions . in one , we annualized the cost of larc devices considering the expected duration of method use . in another , we adjusted observed data on us healthcare and supportive care costs to the generally lower levels of prices in puerto rico market by applying conversion factors of ratios of healthcare spending per capita and wages of nurse assistants between the united states and puerto rico ( 32,33 ) . we also conducted a probabilistic sensitivity analysis by using monte carlo simulation ( 10,000 draws ) that assumed different distributions for all the parameters used in the model ( table 1 ) . all analyses were conducted using treeage pro 2016 software ( treeage software , williamstown , ma , usa ) and excel 2013 ( microsoft , redmond , wa , usa ) . all costs were adjusted to 2014 us dollars by using the health component of the personal consumption expenditures price index ( 34 ) . we estimated the inputs for the decision - tree model and their sources ( table 1 ) . in the no intervention scenario , we took the distribution of women in the target population by use of different types of reversible contraceptives ( or no use ) from a 2002 survey administered in puerto rico and adjusted it to reflect the 36% decrease in fertility rates in puerto rico during 20022015 ( 8,23,24 ) . * brfss , behavior risk surveillance system ; cdc , centers for disease control and prevention ; hly ; healthy life years ; iud , intrauterine device ; larc , long - acting reversible contraceptive ; nsfg , national survey of family growth ; ondieh , office of noncommunicable diseases , injury and environmental health ; zam , zika virus associated microcephaly . authors calculation based on 2015 puerto rico total population size , 2015 birth rate , and adjusted contraception usage in 2002 brfss in puerto rico ( online technical appendix table , http://wwwnc.cdc.gov/eid/article/23/1/16-1322-techapp1.pdf ) . less - effective methods include condoms , spermicides , fertility awareness methods , withdrawal , sponge , and diaphragm . moderately effective methods include oral contraceptive pills , patches , vaginal rings , and injectable contraceptives . the contraception use distribution at baseline ( without intervention ) is based on adjusting the distribution reported in the 2002 brfss in puerto rico excluding women using permanent contraception by assuming 5 percentage points fewer women using no contraception than in 2002 , which is based on a 36% reduction of birth rate among women 1544 years of age in puerto rico from 2002 to 2015 ( national vital statistics report for 2002 and 2015 unpublished birth data from puerto rico ) , and the reported reasons for us teen pregnancy reduction ( http://www.cdc.gov/nchhstp/newsroom/docs/factsheets/yrbs-fact-sheet-final-508.pdf ) ; the decline in teen pregnancies was the fastest of any age group in puerto rico during 20102014 , as reported in national vital statistics reports . data from title x clinics in puerto rico also show that the percentage of women of reproductive age served in title x clinics increased from 2.2% in 2006 to 10% in 2015 . we also assume that new contraception users have the same contraception method distribution as contraception users as reported in the 2002 brfss survey ( 8) . we assume 22% dual use among moderately effective method users at baseline ( based on nsfg 20112013 data and unpublished analyses supplied by karen pazol , ondieh , cdc ; we accounted for the effectiveness of dual - method use in preventing pregnancy but not for preventing sexual transmission of zika virus ) . we calculated method - specific annual pregnancy rates by multiplying the failure rates of contraception methods under typical use by a calculated correction factor of 0.88 to adjust for the model over - predicting the number of unintended births in 2015 using the typical failure rates only . multiplying method - specific contraceptive failure rates by numbers of women in each method category typically results in more predicted pregnancies and births than are actually observed , in part because of heterogeneity in sexual behaviors ( 19 ) . assuming 50% of no contraception users , 60% of less - effective contraception users , and 100% of moderately effective contraception users will visit a healthcare provider for contraception services under intervention . based on the nsfg 20112013 , among women of reproductive age who did not intend to become pregnant and not using permanent contraceptive methods , 21% of no contraception users , 33% less - effective method users , 97% moderately effective method users had at least 1 visit for contraception services in the last 12 months ( personal communication , karen pazol , ondieh , cdc , 2016 ) . contraceptive services include receiving a birth control method or a prescription , receiving a checkup for birth control , receiving counseling about birth control , receiving a sterilizing operation , receiving counseling about a sterilizing operation , receiving emergency contraception , or receiving counseling about emergency contraception . we assume the intervention will increase the percentage of women visiting their provider for contraception counseling . in the contraceptive choice project ( 9 ) , 67% of participants who wished to avoid pregnancy chose to use highly effective methods , and 33% chose to use moderately effective methods . for the main scenario , we applied those estimates to 40% of the target population , assuming that 40% of unintended pregnancies are unwanted , and assumed that 20% of the remaining 60% of switchers would choose highly effective methods . # ellington et al . ( 5 ) estimated 180 cases ( interquartile range 100270 cases ) of congenital microcephaly associated with zika virus in puerto rico among an estimated 31,272 births ( unpublished birth data , puerto rico department of health , 2015 ) . on the basis of those estimates , we estimated the prevalence of congenital microcephaly : 58/10,000 births ( interquartile range 32/10,00086/10,000 ) . * * the pregnancy loss rate in the us zika pregnancy registry as of july 21 , 2016 , was 35% ( 14 ) . the termination rate is calculated as the overall pregnancy loss rate minus the assumed stillbirth rate . a range of the main value 20% was used to create upper and lower bounds used in sensitivity analyses . includes cost for devices and costs for 1 year of injections , pills , patches , rings , condoms , and the cost for related services in the first year . weighted average cost for hormonal iud ( 50% , $ 659 ) , copper iud ( 25% , $ 598 ) , and implant ( 25% , $ 659 ) , based on contraception choice study ( 9 ) and commonly used devices in puerto rico . weighted average cost for generic contraceptive pill ( 78% , $ 370/y ) , injectable ( 14% , $ 240/y plus $ 130 for consultation for the year ) , and ring or patch ( 8% , $ 964/y ) , with the distribution of moderately effective methods based on nsfg , 20112013 . # # weighted average cost of moderately effective method plus cost of less - effective method . * * * male condom . including the cost for checking the placement of iud in the first year of insertion . weighted average for vaginal and c - section delivery , including prenatal care , delivery , postpartum , and neonatal cost at delivery and in the first 3 months . assumes additional costs related to repeated zika virus testing by igm ( 12 tests ) for all pregnant women , 4 extra detailed ultrasound examinations , and 25% of women getting amniocentesis during all zika - positive pregnancies ( 25% infection rate at baseline , range 10%70% ) ( 5 ) , based on oduyebo et al . assumes 2 zika virus tests ( igm and pcr ) for serum and placenta , cranial ultrasound , and eye examination for all infants born to zika virus positive mothers based on fleming - dutra et al . # # # three pcr tests for zika virus using placenta , cord , and brain tissues of the fetus based on martines et al . ( 22 ) . * * * * assuming all prenatal care cost , including extra cost of zika virus associated testing and monitoring during pregnancy and extra cost for zika virus associated testing for fetus . assuming half of the prenatal care cost , including extra cost of zika virus associated testing and monitoring during pregnancy and extra cost for zika virus associated testing for fetus . cost of prenatal care and delivery and extra cost of zika virus associated testing and monitoring during pregnancy and testing of infants for zika virus . present value of cumulative medical and supportive care cost for infant with zam , discounted at 3% annually and taking into account mortality . expenditures by employer - sponsored health plans for privately insured children with combined diagnoses of microcephaly and congenital cytomegalovirus enrolled during the first 4 years of life were used to project medical costs for cases of zam . for the main intervention scenario , we assumed that 50% of no contraception users , 60% of less - effective contraceptive method users , and 100% of moderately effective contraceptive method users would visit a healthcare provider during the intervention period and be counseled about contraception use ( table 1 ) . the first 2 percentages are roughly twice the percentages of women reported in the 20112013 us national survey on family growth to have received contraceptive services ( contraception or counseling ) within the past year because we assumed that , during the zika virus outbreak , more women and providers would discuss contraception ; virtually all moderately effective method users were assumed to see providers to obtain contraceptive prescriptions . for the main scenario , we also assumed , optimistically , that 50% of women in the target population who receive contraceptive services during the zika virus outbreak would be willing to change to a more effective contraceptive method , evenly divided between moderately effective and highly effective methods . 33% used moderately effective methods ) ( 9 ) to the 40% of women assumed to not want to be pregnant ; we assumed 20% of other women not intending pregnancy would use larc . we further assumed that 30% of moderately effective contraception users would also choose to use condoms ( dual - method use ) under the intervention , based on a study reporting dual - method use among persons at risk for hiv ( 25 ) . we calculated method - specific annual pregnancy rates by applying failure rates of contraception methods under typical use ( 10 ) , in combination with information on estimated numbers of unintended pregnancies , to adjust for other factors influencing pregnancy risk ( 19 ) . we estimated the proportion of fetal losses among unintended pregnancies from data for the caribbean region , including puerto rico ( 12 ) , and calculated the proportion of induced abortion among unintended pregnancies from a survey conducted in puerto rico in 2001 ( the latest year for which data were available ) ( 11 ) . we assumed that the distribution of fetal loss and induced abortions in unintended pregnancies unaffected by zam would not be altered by the zika virus outbreak or the intervention . for adverse pregnancy and birth outcomes associated with zika virus , we only considered zam and associated brain anomalies , including live births , stillbirths , and terminations attributable to prenatal diagnosis . although zika virus can cause brain lesions and dysfunction in fetuses and newborns who do not have microcephaly ( 26 ) , we lacked the data to model their prevalence and cost . in the main analysis , we assumed 58 cases of zam per 10,000 live births ( range 3286/10,000 ) based on a modeling study that considered data from other mosquitoborne illnesses in puerto rico and zika virus outbreaks in other locations ( 5 ) . we assumed a pregnancy loss rate of 35% among zika virus exposed fetuses with diagnosed birth defects based on cases in the us zika pregnancy registry as of july 21 , 2016 ( 14 ) . we projected gains in hly by multiplying total cases of zam prevented by 30.0 , which is the average number of quality - adjusted life - years at birth in the united states for an infant without severe microcephaly ( 15 ) and the estimated loss in disability - adjusted life years from microcephaly ( 27 ) . we multiplied 30.0 by the sum of live births and fetal losses associated with zam to calculate gains in hly . we included fetal losses in the hly calculations because in the absence of zam those pregnancies would have resulted in live births , with the same healthy life expectancy as other children ( 15 ) . we conducted the analysis from a healthcare system perspective that includes direct medically related costs regardless of payer . we used payments from private insurance because payments from medicaid might underestimate the cost of healthcare ( 28 ) . intervention costs included program costs of training providers , patient educational materials , outreach / media campaigns on the availability of contraceptives services , and program coordination and the incremental costs of family planning services . the latter comprised the costs of contraception methods and related office visits and services ( e.g. , insertion and removal of larc for new method users resulting from the intervention and the cost of more intensive counseling for all women receiving contraceptive services during the intervention ) . we took the 1-year costs for contraception methods from the literature ( 16,29 ) and based the other program costs on the estimated costs for a pilot program planned to increase access to contraception in puerto rico as part of the current zika virus outbreak response ( 30 ) . we did not apply a discount rate to intervention costs because of the time horizon of 12 months . zika virus related costs prevented by this intervention were in 2 parts : 1 ) costs for zika virus testing and monitoring for zika virus exposed pregnancies and infants , and 2 ) costs of zam cases ( table 1 ) . the cost estimates for testing and monitoring presumed 100% adherence by clinicians and patients to recommendations ( 2022 ) . the lifetime cost per live - born infant with zam includes direct medical and nonmedical costs . zam is among the most severe types of microcephaly and is associated with loss of brain tissue volume , increased fluid spaces , and intracranial calcifications . all 3 cases of live - born infants with zam in french polynesia demonstrated severe neurologic outcomes with delayed cognitive development ( 26 ) . on the basis of expert opinion , infants with zam who survive the neonatal period would be expected to have neurologic dysfunction consistent with severe cerebral palsy within 12 years of birth . as a proxy for the medical cost of zam , we used the estimated cost of treating infants with microcephaly associated with a diagnosis of symptomatic congenital cytomegalovirus ( cmv ) . we used the marketscan commercial database ( truven health analytics ) with a sample of 100 million us residents covered by employer - sponsored insurance at any time during 20092014 . we used average costs for 4 newborn infants with diagnoses of microcephaly and cmv who survived and were enrolled in a health plan for > 3 years . for the direct nonmedical cost of zam , we used the estimated cost for supportive care for children with severe congenital brain injury , both paid care and unpaid care . the total lifetime cost for surviving infants with zam was estimated at $ 3.8 million per infant , taking into account infant and child mortality and discounting of costs in future years at a 3% rate per year ; the sum of undiscounted costs for children who survive to adulthood might reach $ 10 million . zika virus related medical costs associated with women s prenatal care , labor and delivery , and postpartum care for pregnancies ending in live birth and neonatal care from a study of us commercial health plan expenditures ( 17 ) . estimates for costs associated with pregnancies ending in induced abortion were based on our analyses of commercial claims data ( table 1 ) . because many parameters used in the model are uncertain , we conducted sensitivity analyses on selected parameters , including different scenarios for the baseline and postintervention contraception use distributions in puerto rico . we tested alternate baseline contraception use distributions in puerto rico for women at risk for unintended pregnancy by using the actual distribution of method use reported in 2002 ( 8) and among women attending title x clinics in puerto rico in 2014 ( 31 ) . for the postintervention contraception use distribution , we tested scenarios assuming different proportions of women receiving contraceptive services from a healthcare provider , different levels of willingness to switch to a more effective method , and different shares of moderately effective and highly effective methods among switchers . other parameters evaluated during sensitivity analysis included the incidence of zam during the zika virus outbreak in puerto rico , percentage of pregnancies with zam terminated , the cost of caring for a live - born infant with microcephaly , and the cost of the intervention . in one , we annualized the cost of larc devices considering the expected duration of method use . in another , we adjusted observed data on us healthcare and supportive care costs to the generally lower levels of prices in puerto rico market by applying conversion factors of ratios of healthcare spending per capita and wages of nurse assistants between the united states and puerto rico ( 32,33 ) . we also conducted a probabilistic sensitivity analysis by using monte carlo simulation ( 10,000 draws ) that assumed different distributions for all the parameters used in the model ( table 1 ) . all analyses were conducted using treeage pro 2016 software ( treeage software , williamstown , ma , usa ) and excel 2013 ( microsoft , redmond , wa , usa ) . all costs were adjusted to 2014 us dollars by using the health component of the personal consumption expenditures price index ( 34 ) . in the main scenario , we predict the intervention would prevent 25 cases of zam among unintended pregnancies avoided , of which 16 would have resulted in live births ( table 2 ) . the incremental intervention cost of us $ 33.5 million ( i.e. , $ 206 per member of target population ) relative to no intervention ( status quo ) is more than offset by $ 65.2 million in avoided zika virus associated costs , $ 2.8 million from extra testing and monitoring for pregnant women and infants for zika virus exposed pregnancies avoided , and $ 62.3 million from zam cases prevented . the net savings from zika virus the numbers in the columns and rows might not exactly match because of rounding . target population size : 163,000 women who do not intend to become pregnant during zika virus outbreak . women of reproductive age in puerto rico who are sexually active with a male partner , fertile , not desiring pregnancy , and not using permanent contraception methods ( e.g. , tubal ligation and vasectomy ) . only including cost of testing for zika virus and monitoring for exposed infants without zam ; testing costs for infants with zam are included in the direct costs of zam . from healthcare system perspective , includes direct medical and medical - related costs , including supportive care for persons with zam , even if the cost might not be paid by healthcare payers or delivered by healthcare providers . # total zika virus associated cost avoided ( absolute value ) minus the additional cost of family planning service under intervention compared with no intervention . * * unwanted pregnancies which are not desired in the future ( assuming 60% of unintended pregnancies are mistimed ) , irrespective of zika virus infection absolute value of net medical cost for unwanted pregnancy plus absolute value of net cost savings from zika virus associated costs avoided . the number of zam cases prevented and zika virus associated costs avoided are sensitive to the proportion of women receiving contraceptive services and the proportion of those women willing to switch to a more effective contraception method during the zika virus outbreak ( figure ; table 3 ) . if the proportions of women receiving contraception services are assumed to be the same as estimated for the continental united states in the national survey of family growth for 20112013 ( i.e. , 21% among no contraception users , 33% among less - effective method users , and 97% among all moderately effective method users ) , 16 cases of zam are prevented , and the net savings is $ 15.4 million ( table 3 ) . if 10% of women receiving contraceptive services switch to a more effective method , 6 cases of zam are prevented , and net saving is $ 2.8 million . if the intervention only shifts users of moderately effective methods to a highly effective method ( no change in non - use or use of less - effective methods ) , 7 zam cases are prevented , with an icer of $ 24,608/hly gained . increasing the proportion of dual - method users increases the number of cases of zam prevented and net savings attributable to higher contraception effectiveness . the results are also sensitive to the prevalence of zam among mid - trimester pregnancies , the percentage of zam cases resulting in live - born infants , lifetime cost per live - born infant with zam , and the intervention cost . if we adjust us cost estimates for lower prices in puerto rico while keeping intervention costs at us prices , net savings are $ 1.7 million . in all but 1 of the scenarios sensitivity analysis indicating the effect of changes of assumptions on the number of zam cases prevented in a proposed intervention to increase access to contraception to women during the zika virus outbreak , puerto rico , 2016 . larc , long - acting reversible contraceptive ; zam , zika virus associated microcephaly . * brfss , behavioral risk factor surveillance system ; cn , cost - neutral ; cs , cost - saving ; hly , healthy life years ; larc , long - acting reversible contraceptive ; nsfg , national survey of family growth ; up , unwanted pregnancy ; zam , zika virus associated microcephaly . total incremental cost is the additional cost of contraception minus zika virus associated cost avoided . 30% of no contraception users , 60% of less - effective contraceptive method users , 100% of moderately effective contraceptive method users seeking contraceptive services from healthcare provider during the zika virus outbreak . based on nsfg 20112013 , among women of reproductive age who are sexually active , did not intend to become pregnant , and were not using permanent contraceptive methods , 21% of no contraception users , 33% of less - effective contraceptive method users , 97% of moderately effective contraceptive method users , and 94% of dual - method users had at least 1 contraceptive service visit in the last 12 months ( in total 50% ) . based on title x family planning annual report for 20072015 in colorado , 30% of clients who visited title x clinics switched to a new method . # eighteen percentage points of users of moderately effective methods are assumed to switch to highly effective methods , of whom 21% were dual - method users . * * contraception distribution in puerto rico in 2002 15.9% no method , 41.6% less - effective methods , 40.2% moderately effective methods , and 2.4% highly effective methods . 20% of women at risk for unintended pregnancy used less - effective methods , 77% used moderately effective methods , and 2% used highly effective methods . intervention cost equals to the medical savings from zam cases prevented . conversion factor of 0.36 applied to pregnancy and zam medical costs based on the ratio of per capita medical expenditure in puerto rico and in the united states in 2012 as in portela et al . 2015 ( 32 ) ; conversion factor of 0.72 applied to costs of supportive care for live - born infants with zam , based on the ratio of annual salary for assistant nurses in puerto rico and in the united states ( 33 ) . a probabilistic sensitivity analysis scatter graph shows that most of the model simulations result in icers in the lower right quadrant with lower costs and better health outcomes ( technical appendix figure 2 ) . specifically , the intervention is cost - saving in 92.11% of the 10,000 iterations , and in 98.10% of the iterations , the intervention has an icer of < $ 20,000/hly gained . the intervention is also predicted to prevent $ 40.4 million in medical costs from unwanted pregnancies avoided in the main scenario ( table 2 ) . in many sensitivity analyses , the cost avoided from these unwanted pregnancies prevented alone is greater than the intervention cost . the larger the numbers of no contraception users and less - effective method users receiving contraceptive services and willing to switch to more effective methods , the greater the magnitude of cost savings from unwanted pregnancies avoided ( table 3 ) . the results of our modeling analysis suggest that increasing access to effective contraception in the context of the 2016 zika virus outbreak for women in puerto rico who do not intend to become pregnant could proportionally reduce the number of unintended pregnancies and cases of zam by 25% . the intervention is cost - saving ( negative net cost ) when considering the benefits from preventing zam and avoiding zika virus exposed pregnancy costs in the main scenarios and in most of the scenarios we tested . in scenarios in which the intervention is not cost - saving , it is still cost - effective relative to accepted cost - effectiveness thresholds ( 35 ) . the world health organization suggests that interventions that cost <3 times the gross domestic product per capita per hly ( equivalent to $ 150,000 in the united states and $ 60,000 in puerto rico ) are cost - effective and those costing less than gross domestic product per capita are highly cost - effective ( 36 ) . when considering additional benefits from preventing unintended pregnancies not desired at a later time , the intervention is cost - saving in all scenarios . previous studies have shown that expanding access to contraception , especially larc , is cost - saving ( 16,37,38 ) . likewise , our findings suggest that this intervention could be cost - saving or cost - effective within the context of a public health emergency response . first , we project the effects of a hypothetical intervention in place in puerto rico during the 2016 zika virus outbreak . however second , the baseline contraception use distribution is based on a 2002 survey ; the current distribution in puerto rico might be different . third , uncertainty exists about the effect of the proposed intervention on postintervention contraceptive use distribution ; however , the sensitivity analyses indicate that different distributions of larc types among switchers does not have a substantial influence on the results . fourth , our study assumes that women have full access to healthcare providers . in areas with limited access to providers , the effectiveness of the intervention might be lower , although puerto rico has a similar ratio of physicians to population as the united states as a whole ( 39 ) , and despite a loss of physicians in recent years , puerto rico has a network of providers , federally qualified health clinics , and title x providers in rural and urban areas . we lack data on miscarriage and induced abortion rates in puerto rico and so did not have sufficient data to model uncertainty in these parameters . the rates of stillbirth and pregnancy termination among pregnancies with zam in puerto rico are also unknown . our assumed percentage of live births among pregnancies with recognized zam ( 65% ) compares with a 38% rate reported in french polynesia during the 2013 zika virus outbreak ( 11 ) . sixth , pregnancy intentions and use of contraception among women in puerto rico might differ during the zika virus outbreak compared to preoutbreak periods . seventh , our analysis does not consider possibly higher rates of fetal loss and induced abortion among women infected by zika virus during early pregnancy or brain abnormalities or conditions related to zika virus not involving microcephaly . eighth , the assumed zika virus testing costs assume 100% adherence to recommended testing practices ; the actual cost savings taking nonadherence into account would be lower . ninth , the cost estimates of zam cases in live - born infants do not include costs of managing mental health conditions among parents of affected infants . however , if the cost of zam exceeds $ 1.9 million , the intervention is still cost - saving . finally , if efforts to prevent transmission of zika virus in puerto rico are effective , the rate of infection in pregnancy and the incidence of zam relative to that projected could be reduced . despite its limitations , second , the contraception scenarios are based on real - world programs and have resulted from consultation with subject matter experts . third , expenditure data from a large sample of us residents with commercial health insurance were used to calculate the potential medical cost of zam on the basis of combinations of diagnostic codes for virus - associated microcephaly , although costs might be lower for similar children with public insurance . finally , sensitivity analyses give consistent results indicating expected net cost savings associated with an intervention that would increase access to contraception in response to the zika virus outbreak in puerto rico . zika virus can cause devastating birth defects , and infants born with zam and their families will require lifelong support . avoiding unintended pregnancies is a critical intervention to mitigate the effects of zam . efforts to prevent adverse zika virus related pregnancy outcomes in puerto rico are especially important because of limited resources ( 40 ) . our analyses suggest that increasing access to a full range of contraception among women in puerto rico who want to delay or avoid becoming pregnant during a zika virus outbreak would be a cost - saving strategy to reduce the effects of zam . the magnitude of cost savings is even greater when considering the avoided cost of unwanted pregnancies prevented . process for deriving the size of the target population , decision tree structure , and probabilistic sensitivity analysis of cost - effectiveness for a proposed intervention to increase access to contraception to women during the zika virus outbreak , puerto rico , 2016 .

we modeled the potential cost - effectiveness of increasing access to contraception in puerto rico during a zika virus outbreak . the intervention is projected to cost an additional $ 33.5 million in family planning services and is likely to be cost - saving for the healthcare system overall . it could reduce zika virus related costs by $ 65.2 million ( $ 2.8 million from less zika virus testing and monitoring and $ 62.3 million from avoided costs of zika virus associated microcephaly [ zam ] ) . the estimates are influenced by the contraception methods used , the frequency of zam , and the lifetime incremental cost of zam . accounting for unwanted pregnancies that are prevented , irrespective of zika virus infection , an additional $ 40.4 million in medical costs would be avoided through the intervention . increasing contraceptive access for women who want to delay or avoid pregnancy in puerto rico during a zika virus outbreak can substantially reduce the number of cases of zam and healthcare costs .

Methods Contraception Use with and without the Intervention Epidemiologic Model Input Parameters Cost Parameters Sensitivity Analyses Results Discussion None

we constructed a decision tree cost - effectiveness model for a target population of 163,000 women who at the time of the intervention are sexually active with a male partner , fertile , not desiring pregnancy within the next 12 months , and not using permanent contraception methods ( e.g. in the intervention scenario , women in puerto rico are assumed to have same - day access to contraception methods , including larc , with no out - of - pocket costs . the model specifies contraceptive method use distribution , unintended pregnancy events , and the frequency of zam ( technical appendix figure 1 ) . we assumed an intervention in place throughout a year - long zika virus outbreak in puerto rico . output measures included numbers of zam cases prevented , including stillbirths , elective terminations , and live - born infants , and healthy life years ( hly ) gained . in addition , the avoided cost of prenatal , delivery , postpartum , and neonatal care associated with avoided unwanted pregnancies was considered an economic benefit . in cost - effectiveness analyses , if total avoided cost exceeds the cost of an intervention that improves health , the intervention is considered cost - saving . because roughly 60% of unintended pregnancies are classified as mistimed , which might result in a delayed rather than avoided pregnancy , with the same costs occurring later ( 7 ) , we only estimated avoided medical costs from prevention of the 40% of unintended pregnancies presumed to be not desired at a later time irrespective of zika virus infection . * brfss , behavior risk surveillance system ; cdc , centers for disease control and prevention ; hly ; healthy life years ; iud , intrauterine device ; larc , long - acting reversible contraceptive ; nsfg , national survey of family growth ; ondieh , office of noncommunicable diseases , injury and environmental health ; zam , zika virus associated microcephaly . the contraception use distribution at baseline ( without intervention ) is based on adjusting the distribution reported in the 2002 brfss in puerto rico excluding women using permanent contraception by assuming 5 percentage points fewer women using no contraception than in 2002 , which is based on a 36% reduction of birth rate among women 1544 years of age in puerto rico from 2002 to 2015 ( national vital statistics report for 2002 and 2015 unpublished birth data from puerto rico ) , and the reported reasons for us teen pregnancy reduction ( http://www.cdc.gov/nchhstp/newsroom/docs/factsheets/yrbs-fact-sheet-final-508.pdf ) ; the decline in teen pregnancies was the fastest of any age group in puerto rico during 20102014 , as reported in national vital statistics reports . we assume 22% dual use among moderately effective method users at baseline ( based on nsfg 20112013 data and unpublished analyses supplied by karen pazol , ondieh , cdc ; we accounted for the effectiveness of dual - method use in preventing pregnancy but not for preventing sexual transmission of zika virus ) . we calculated method - specific annual pregnancy rates by multiplying the failure rates of contraception methods under typical use by a calculated correction factor of 0.88 to adjust for the model over - predicting the number of unintended births in 2015 using the typical failure rates only . * * * * assuming all prenatal care cost , including extra cost of zika virus associated testing and monitoring during pregnancy and extra cost for zika virus associated testing for fetus . assuming half of the prenatal care cost , including extra cost of zika virus associated testing and monitoring during pregnancy and extra cost for zika virus associated testing for fetus . cost of prenatal care and delivery and extra cost of zika virus associated testing and monitoring during pregnancy and testing of infants for zika virus . for the main intervention scenario , we assumed that 50% of no contraception users , 60% of less - effective contraceptive method users , and 100% of moderately effective contraceptive method users would visit a healthcare provider during the intervention period and be counseled about contraception use ( table 1 ) . for the main scenario , we also assumed , optimistically , that 50% of women in the target population who receive contraceptive services during the zika virus outbreak would be willing to change to a more effective contraceptive method , evenly divided between moderately effective and highly effective methods . we estimated the proportion of fetal losses among unintended pregnancies from data for the caribbean region , including puerto rico ( 12 ) , and calculated the proportion of induced abortion among unintended pregnancies from a survey conducted in puerto rico in 2001 ( the latest year for which data were available ) ( 11 ) . we assumed that the distribution of fetal loss and induced abortions in unintended pregnancies unaffected by zam would not be altered by the zika virus outbreak or the intervention . in the main analysis , we assumed 58 cases of zam per 10,000 live births ( range 3286/10,000 ) based on a modeling study that considered data from other mosquitoborne illnesses in puerto rico and zika virus outbreaks in other locations ( 5 ) . we projected gains in hly by multiplying total cases of zam prevented by 30.0 , which is the average number of quality - adjusted life - years at birth in the united states for an infant without severe microcephaly ( 15 ) and the estimated loss in disability - adjusted life years from microcephaly ( 27 ) . intervention costs included program costs of training providers , patient educational materials , outreach / media campaigns on the availability of contraceptives services , and program coordination and the incremental costs of family planning services . we took the 1-year costs for contraception methods from the literature ( 16,29 ) and based the other program costs on the estimated costs for a pilot program planned to increase access to contraception in puerto rico as part of the current zika virus outbreak response ( 30 ) . zika virus related costs prevented by this intervention were in 2 parts : 1 ) costs for zika virus testing and monitoring for zika virus exposed pregnancies and infants , and 2 ) costs of zam cases ( table 1 ) . as a proxy for the medical cost of zam , we used the estimated cost of treating infants with microcephaly associated with a diagnosis of symptomatic congenital cytomegalovirus ( cmv ) . for the direct nonmedical cost of zam , we used the estimated cost for supportive care for children with severe congenital brain injury , both paid care and unpaid care . zika virus related medical costs associated with women s prenatal care , labor and delivery , and postpartum care for pregnancies ending in live birth and neonatal care from a study of us commercial health plan expenditures ( 17 ) . other parameters evaluated during sensitivity analysis included the incidence of zam during the zika virus outbreak in puerto rico , percentage of pregnancies with zam terminated , the cost of caring for a live - born infant with microcephaly , and the cost of the intervention . in the no intervention scenario , we took the distribution of women in the target population by use of different types of reversible contraceptives ( or no use ) from a 2002 survey administered in puerto rico and adjusted it to reflect the 36% decrease in fertility rates in puerto rico during 20022015 ( 8,23,24 ) . * brfss , behavior risk surveillance system ; cdc , centers for disease control and prevention ; hly ; healthy life years ; iud , intrauterine device ; larc , long - acting reversible contraceptive ; nsfg , national survey of family growth ; ondieh , office of noncommunicable diseases , injury and environmental health ; zam , zika virus associated microcephaly . the contraception use distribution at baseline ( without intervention ) is based on adjusting the distribution reported in the 2002 brfss in puerto rico excluding women using permanent contraception by assuming 5 percentage points fewer women using no contraception than in 2002 , which is based on a 36% reduction of birth rate among women 1544 years of age in puerto rico from 2002 to 2015 ( national vital statistics report for 2002 and 2015 unpublished birth data from puerto rico ) , and the reported reasons for us teen pregnancy reduction ( http://www.cdc.gov/nchhstp/newsroom/docs/factsheets/yrbs-fact-sheet-final-508.pdf ) ; the decline in teen pregnancies was the fastest of any age group in puerto rico during 20102014 , as reported in national vital statistics reports . we assume 22% dual use among moderately effective method users at baseline ( based on nsfg 20112013 data and unpublished analyses supplied by karen pazol , ondieh , cdc ; we accounted for the effectiveness of dual - method use in preventing pregnancy but not for preventing sexual transmission of zika virus ) . we calculated method - specific annual pregnancy rates by multiplying the failure rates of contraception methods under typical use by a calculated correction factor of 0.88 to adjust for the model over - predicting the number of unintended births in 2015 using the typical failure rates only . ( 5 ) estimated 180 cases ( interquartile range 100270 cases ) of congenital microcephaly associated with zika virus in puerto rico among an estimated 31,272 births ( unpublished birth data , puerto rico department of health , 2015 ) . * * * * assuming all prenatal care cost , including extra cost of zika virus associated testing and monitoring during pregnancy and extra cost for zika virus associated testing for fetus . assuming half of the prenatal care cost , including extra cost of zika virus associated testing and monitoring during pregnancy and extra cost for zika virus associated testing for fetus . cost of prenatal care and delivery and extra cost of zika virus associated testing and monitoring during pregnancy and testing of infants for zika virus . expenditures by employer - sponsored health plans for privately insured children with combined diagnoses of microcephaly and congenital cytomegalovirus enrolled during the first 4 years of life were used to project medical costs for cases of zam . for the main scenario , we also assumed , optimistically , that 50% of women in the target population who receive contraceptive services during the zika virus outbreak would be willing to change to a more effective contraceptive method , evenly divided between moderately effective and highly effective methods . we estimated the proportion of fetal losses among unintended pregnancies from data for the caribbean region , including puerto rico ( 12 ) , and calculated the proportion of induced abortion among unintended pregnancies from a survey conducted in puerto rico in 2001 ( the latest year for which data were available ) ( 11 ) . we assumed that the distribution of fetal loss and induced abortions in unintended pregnancies unaffected by zam would not be altered by the zika virus outbreak or the intervention . in the main analysis , we assumed 58 cases of zam per 10,000 live births ( range 3286/10,000 ) based on a modeling study that considered data from other mosquitoborne illnesses in puerto rico and zika virus outbreaks in other locations ( 5 ) . we projected gains in hly by multiplying total cases of zam prevented by 30.0 , which is the average number of quality - adjusted life - years at birth in the united states for an infant without severe microcephaly ( 15 ) and the estimated loss in disability - adjusted life years from microcephaly ( 27 ) . intervention costs included program costs of training providers , patient educational materials , outreach / media campaigns on the availability of contraceptives services , and program coordination and the incremental costs of family planning services . we took the 1-year costs for contraception methods from the literature ( 16,29 ) and based the other program costs on the estimated costs for a pilot program planned to increase access to contraception in puerto rico as part of the current zika virus outbreak response ( 30 ) . zika virus related costs prevented by this intervention were in 2 parts : 1 ) costs for zika virus testing and monitoring for zika virus exposed pregnancies and infants , and 2 ) costs of zam cases ( table 1 ) . as a proxy for the medical cost of zam , we used the estimated cost of treating infants with microcephaly associated with a diagnosis of symptomatic congenital cytomegalovirus ( cmv ) . for the direct nonmedical cost of zam , we used the estimated cost for supportive care for children with severe congenital brain injury , both paid care and unpaid care . zika virus related medical costs associated with women s prenatal care , labor and delivery , and postpartum care for pregnancies ending in live birth and neonatal care from a study of us commercial health plan expenditures ( 17 ) . other parameters evaluated during sensitivity analysis included the incidence of zam during the zika virus outbreak in puerto rico , percentage of pregnancies with zam terminated , the cost of caring for a live - born infant with microcephaly , and the cost of the intervention . the incremental intervention cost of us $ 33.5 million ( i.e. , $ 206 per member of target population ) relative to no intervention ( status quo ) is more than offset by $ 65.2 million in avoided zika virus associated costs , $ 2.8 million from extra testing and monitoring for pregnant women and infants for zika virus exposed pregnancies avoided , and $ 62.3 million from zam cases prevented . women of reproductive age in puerto rico who are sexually active with a male partner , fertile , not desiring pregnancy , and not using permanent contraception methods ( e.g. only including cost of testing for zika virus and monitoring for exposed infants without zam ; testing costs for infants with zam are included in the direct costs of zam . # total zika virus associated cost avoided ( absolute value ) minus the additional cost of family planning service under intervention compared with no intervention . * * unwanted pregnancies which are not desired in the future ( assuming 60% of unintended pregnancies are mistimed ) , irrespective of zika virus infection absolute value of net medical cost for unwanted pregnancy plus absolute value of net cost savings from zika virus associated costs avoided . the number of zam cases prevented and zika virus associated costs avoided are sensitive to the proportion of women receiving contraceptive services and the proportion of those women willing to switch to a more effective contraception method during the zika virus outbreak ( figure ; table 3 ) . , 21% among no contraception users , 33% among less - effective method users , and 97% among all moderately effective method users ) , 16 cases of zam are prevented , and the net savings is $ 15.4 million ( table 3 ) . if 10% of women receiving contraceptive services switch to a more effective method , 6 cases of zam are prevented , and net saving is $ 2.8 million . increasing the proportion of dual - method users increases the number of cases of zam prevented and net savings attributable to higher contraception effectiveness . the results are also sensitive to the prevalence of zam among mid - trimester pregnancies , the percentage of zam cases resulting in live - born infants , lifetime cost per live - born infant with zam , and the intervention cost . in all but 1 of the scenarios sensitivity analysis indicating the effect of changes of assumptions on the number of zam cases prevented in a proposed intervention to increase access to contraception to women during the zika virus outbreak , puerto rico , 2016 . larc , long - acting reversible contraceptive ; zam , zika virus associated microcephaly . * brfss , behavioral risk factor surveillance system ; cn , cost - neutral ; cs , cost - saving ; hly , healthy life years ; larc , long - acting reversible contraceptive ; nsfg , national survey of family growth ; up , unwanted pregnancy ; zam , zika virus associated microcephaly . total incremental cost is the additional cost of contraception minus zika virus associated cost avoided . 2015 ( 32 ) ; conversion factor of 0.72 applied to costs of supportive care for live - born infants with zam , based on the ratio of annual salary for assistant nurses in puerto rico and in the united states ( 33 ) . specifically , the intervention is cost - saving in 92.11% of the 10,000 iterations , and in 98.10% of the iterations , the intervention has an icer of < $ 20,000/hly gained . the intervention is also predicted to prevent $ 40.4 million in medical costs from unwanted pregnancies avoided in the main scenario ( table 2 ) . the results of our modeling analysis suggest that increasing access to effective contraception in the context of the 2016 zika virus outbreak for women in puerto rico who do not intend to become pregnant could proportionally reduce the number of unintended pregnancies and cases of zam by 25% . the intervention is cost - saving ( negative net cost ) when considering the benefits from preventing zam and avoiding zika virus exposed pregnancy costs in the main scenarios and in most of the scenarios we tested . in scenarios in which the intervention is not cost - saving , it is still cost - effective relative to accepted cost - effectiveness thresholds ( 35 ) . the world health organization suggests that interventions that cost <3 times the gross domestic product per capita per hly ( equivalent to $ 150,000 in the united states and $ 60,000 in puerto rico ) are cost - effective and those costing less than gross domestic product per capita are highly cost - effective ( 36 ) . when considering additional benefits from preventing unintended pregnancies not desired at a later time , the intervention is cost - saving in all scenarios . previous studies have shown that expanding access to contraception , especially larc , is cost - saving ( 16,37,38 ) . first , we project the effects of a hypothetical intervention in place in puerto rico during the 2016 zika virus outbreak . however second , the baseline contraception use distribution is based on a 2002 survey ; the current distribution in puerto rico might be different . in areas with limited access to providers , the effectiveness of the intervention might be lower , although puerto rico has a similar ratio of physicians to population as the united states as a whole ( 39 ) , and despite a loss of physicians in recent years , puerto rico has a network of providers , federally qualified health clinics , and title x providers in rural and urban areas . sixth , pregnancy intentions and use of contraception among women in puerto rico might differ during the zika virus outbreak compared to preoutbreak periods . eighth , the assumed zika virus testing costs assume 100% adherence to recommended testing practices ; the actual cost savings taking nonadherence into account would be lower . however , if the cost of zam exceeds $ 1.9 million , the intervention is still cost - saving . finally , if efforts to prevent transmission of zika virus in puerto rico are effective , the rate of infection in pregnancy and the incidence of zam relative to that projected could be reduced . third , expenditure data from a large sample of us residents with commercial health insurance were used to calculate the potential medical cost of zam on the basis of combinations of diagnostic codes for virus - associated microcephaly , although costs might be lower for similar children with public insurance . finally , sensitivity analyses give consistent results indicating expected net cost savings associated with an intervention that would increase access to contraception in response to the zika virus outbreak in puerto rico . efforts to prevent adverse zika virus related pregnancy outcomes in puerto rico are especially important because of limited resources ( 40 ) . our analyses suggest that increasing access to a full range of contraception among women in puerto rico who want to delay or avoid becoming pregnant during a zika virus outbreak would be a cost - saving strategy to reduce the effects of zam . process for deriving the size of the target population , decision tree structure , and probabilistic sensitivity analysis of cost - effectiveness for a proposed intervention to increase access to contraception to women during the zika virus outbreak , puerto rico , 2016 .

we constructed a decision tree cost - effectiveness model for a target population of 163,000 women who at the time of the intervention are sexually active with a male partner , fertile , not desiring pregnancy within the next 12 months , and not using permanent contraception methods ( e.g. for scenarios with positive net costs , we reported the incremental cost - effectiveness ratio ( icer ) , which is the net cost per hly gained in comparison to the status quo . because roughly 60% of unintended pregnancies are classified as mistimed , which might result in a delayed rather than avoided pregnancy , with the same costs occurring later ( 7 ) , we only estimated avoided medical costs from prevention of the 40% of unintended pregnancies presumed to be not desired at a later time irrespective of zika virus infection . in the no intervention scenario , we took the distribution of women in the target population by use of different types of reversible contraceptives ( or no use ) from a 2002 survey administered in puerto rico and adjusted it to reflect the 36% decrease in fertility rates in puerto rico during 20022015 ( 8,23,24 ) . * brfss , behavior risk surveillance system ; cdc , centers for disease control and prevention ; hly ; healthy life years ; iud , intrauterine device ; larc , long - acting reversible contraceptive ; nsfg , national survey of family growth ; ondieh , office of noncommunicable diseases , injury and environmental health ; zam , zika virus associated microcephaly . authors calculation based on 2015 puerto rico total population size , 2015 birth rate , and adjusted contraception usage in 2002 brfss in puerto rico ( online technical appendix table , http://wwwnc.cdc.gov/eid/article/23/1/16-1322-techapp1.pdf ) . the contraception use distribution at baseline ( without intervention ) is based on adjusting the distribution reported in the 2002 brfss in puerto rico excluding women using permanent contraception by assuming 5 percentage points fewer women using no contraception than in 2002 , which is based on a 36% reduction of birth rate among women 1544 years of age in puerto rico from 2002 to 2015 ( national vital statistics report for 2002 and 2015 unpublished birth data from puerto rico ) , and the reported reasons for us teen pregnancy reduction ( http://www.cdc.gov/nchhstp/newsroom/docs/factsheets/yrbs-fact-sheet-final-508.pdf ) ; the decline in teen pregnancies was the fastest of any age group in puerto rico during 20102014 , as reported in national vital statistics reports . we assume 22% dual use among moderately effective method users at baseline ( based on nsfg 20112013 data and unpublished analyses supplied by karen pazol , ondieh , cdc ; we accounted for the effectiveness of dual - method use in preventing pregnancy but not for preventing sexual transmission of zika virus ) . we calculated method - specific annual pregnancy rates by multiplying the failure rates of contraception methods under typical use by a calculated correction factor of 0.88 to adjust for the model over - predicting the number of unintended births in 2015 using the typical failure rates only . based on the nsfg 20112013 , among women of reproductive age who did not intend to become pregnant and not using permanent contraceptive methods , 21% of no contraception users , 33% less - effective method users , 97% moderately effective method users had at least 1 visit for contraception services in the last 12 months ( personal communication , karen pazol , ondieh , cdc , 2016 ) . for the main scenario , we applied those estimates to 40% of the target population , assuming that 40% of unintended pregnancies are unwanted , and assumed that 20% of the remaining 60% of switchers would choose highly effective methods . on the basis of those estimates , we estimated the prevalence of congenital microcephaly : 58/10,000 births ( interquartile range 32/10,00086/10,000 ) . for the main intervention scenario , we assumed that 50% of no contraception users , 60% of less - effective contraceptive method users , and 100% of moderately effective contraceptive method users would visit a healthcare provider during the intervention period and be counseled about contraception use ( table 1 ) . the first 2 percentages are roughly twice the percentages of women reported in the 20112013 us national survey on family growth to have received contraceptive services ( contraception or counseling ) within the past year because we assumed that , during the zika virus outbreak , more women and providers would discuss contraception ; virtually all moderately effective method users were assumed to see providers to obtain contraceptive prescriptions . for the main scenario , we also assumed , optimistically , that 50% of women in the target population who receive contraceptive services during the zika virus outbreak would be willing to change to a more effective contraceptive method , evenly divided between moderately effective and highly effective methods . we calculated method - specific annual pregnancy rates by applying failure rates of contraception methods under typical use ( 10 ) , in combination with information on estimated numbers of unintended pregnancies , to adjust for other factors influencing pregnancy risk ( 19 ) . we estimated the proportion of fetal losses among unintended pregnancies from data for the caribbean region , including puerto rico ( 12 ) , and calculated the proportion of induced abortion among unintended pregnancies from a survey conducted in puerto rico in 2001 ( the latest year for which data were available ) ( 11 ) . for adverse pregnancy and birth outcomes associated with zika virus , we only considered zam and associated brain anomalies , including live births , stillbirths , and terminations attributable to prenatal diagnosis . we projected gains in hly by multiplying total cases of zam prevented by 30.0 , which is the average number of quality - adjusted life - years at birth in the united states for an infant without severe microcephaly ( 15 ) and the estimated loss in disability - adjusted life years from microcephaly ( 27 ) . we took the 1-year costs for contraception methods from the literature ( 16,29 ) and based the other program costs on the estimated costs for a pilot program planned to increase access to contraception in puerto rico as part of the current zika virus outbreak response ( 30 ) . as a proxy for the medical cost of zam , we used the estimated cost of treating infants with microcephaly associated with a diagnosis of symptomatic congenital cytomegalovirus ( cmv ) . the total lifetime cost for surviving infants with zam was estimated at $ 3.8 million per infant , taking into account infant and child mortality and discounting of costs in future years at a 3% rate per year ; the sum of undiscounted costs for children who survive to adulthood might reach $ 10 million . zika virus related medical costs associated with women s prenatal care , labor and delivery , and postpartum care for pregnancies ending in live birth and neonatal care from a study of us commercial health plan expenditures ( 17 ) . we tested alternate baseline contraception use distributions in puerto rico for women at risk for unintended pregnancy by using the actual distribution of method use reported in 2002 ( 8) and among women attending title x clinics in puerto rico in 2014 ( 31 ) . for the postintervention contraception use distribution , we tested scenarios assuming different proportions of women receiving contraceptive services from a healthcare provider , different levels of willingness to switch to a more effective method , and different shares of moderately effective and highly effective methods among switchers . other parameters evaluated during sensitivity analysis included the incidence of zam during the zika virus outbreak in puerto rico , percentage of pregnancies with zam terminated , the cost of caring for a live - born infant with microcephaly , and the cost of the intervention . in another , we adjusted observed data on us healthcare and supportive care costs to the generally lower levels of prices in puerto rico market by applying conversion factors of ratios of healthcare spending per capita and wages of nurse assistants between the united states and puerto rico ( 32,33 ) . in the no intervention scenario , we took the distribution of women in the target population by use of different types of reversible contraceptives ( or no use ) from a 2002 survey administered in puerto rico and adjusted it to reflect the 36% decrease in fertility rates in puerto rico during 20022015 ( 8,23,24 ) . * brfss , behavior risk surveillance system ; cdc , centers for disease control and prevention ; hly ; healthy life years ; iud , intrauterine device ; larc , long - acting reversible contraceptive ; nsfg , national survey of family growth ; ondieh , office of noncommunicable diseases , injury and environmental health ; zam , zika virus associated microcephaly . authors calculation based on 2015 puerto rico total population size , 2015 birth rate , and adjusted contraception usage in 2002 brfss in puerto rico ( online technical appendix table , http://wwwnc.cdc.gov/eid/article/23/1/16-1322-techapp1.pdf ) . the contraception use distribution at baseline ( without intervention ) is based on adjusting the distribution reported in the 2002 brfss in puerto rico excluding women using permanent contraception by assuming 5 percentage points fewer women using no contraception than in 2002 , which is based on a 36% reduction of birth rate among women 1544 years of age in puerto rico from 2002 to 2015 ( national vital statistics report for 2002 and 2015 unpublished birth data from puerto rico ) , and the reported reasons for us teen pregnancy reduction ( http://www.cdc.gov/nchhstp/newsroom/docs/factsheets/yrbs-fact-sheet-final-508.pdf ) ; the decline in teen pregnancies was the fastest of any age group in puerto rico during 20102014 , as reported in national vital statistics reports . we assume 22% dual use among moderately effective method users at baseline ( based on nsfg 20112013 data and unpublished analyses supplied by karen pazol , ondieh , cdc ; we accounted for the effectiveness of dual - method use in preventing pregnancy but not for preventing sexual transmission of zika virus ) . we calculated method - specific annual pregnancy rates by multiplying the failure rates of contraception methods under typical use by a calculated correction factor of 0.88 to adjust for the model over - predicting the number of unintended births in 2015 using the typical failure rates only . based on the nsfg 20112013 , among women of reproductive age who did not intend to become pregnant and not using permanent contraceptive methods , 21% of no contraception users , 33% less - effective method users , 97% moderately effective method users had at least 1 visit for contraception services in the last 12 months ( personal communication , karen pazol , ondieh , cdc , 2016 ) . for the main scenario , we applied those estimates to 40% of the target population , assuming that 40% of unintended pregnancies are unwanted , and assumed that 20% of the remaining 60% of switchers would choose highly effective methods . for the main intervention scenario , we assumed that 50% of no contraception users , 60% of less - effective contraceptive method users , and 100% of moderately effective contraceptive method users would visit a healthcare provider during the intervention period and be counseled about contraception use ( table 1 ) . the first 2 percentages are roughly twice the percentages of women reported in the 20112013 us national survey on family growth to have received contraceptive services ( contraception or counseling ) within the past year because we assumed that , during the zika virus outbreak , more women and providers would discuss contraception ; virtually all moderately effective method users were assumed to see providers to obtain contraceptive prescriptions . for the main scenario , we also assumed , optimistically , that 50% of women in the target population who receive contraceptive services during the zika virus outbreak would be willing to change to a more effective contraceptive method , evenly divided between moderately effective and highly effective methods . we calculated method - specific annual pregnancy rates by applying failure rates of contraception methods under typical use ( 10 ) , in combination with information on estimated numbers of unintended pregnancies , to adjust for other factors influencing pregnancy risk ( 19 ) . we estimated the proportion of fetal losses among unintended pregnancies from data for the caribbean region , including puerto rico ( 12 ) , and calculated the proportion of induced abortion among unintended pregnancies from a survey conducted in puerto rico in 2001 ( the latest year for which data were available ) ( 11 ) . we projected gains in hly by multiplying total cases of zam prevented by 30.0 , which is the average number of quality - adjusted life - years at birth in the united states for an infant without severe microcephaly ( 15 ) and the estimated loss in disability - adjusted life years from microcephaly ( 27 ) . we took the 1-year costs for contraception methods from the literature ( 16,29 ) and based the other program costs on the estimated costs for a pilot program planned to increase access to contraception in puerto rico as part of the current zika virus outbreak response ( 30 ) . as a proxy for the medical cost of zam , we used the estimated cost of treating infants with microcephaly associated with a diagnosis of symptomatic congenital cytomegalovirus ( cmv ) . the total lifetime cost for surviving infants with zam was estimated at $ 3.8 million per infant , taking into account infant and child mortality and discounting of costs in future years at a 3% rate per year ; the sum of undiscounted costs for children who survive to adulthood might reach $ 10 million . zika virus related medical costs associated with women s prenatal care , labor and delivery , and postpartum care for pregnancies ending in live birth and neonatal care from a study of us commercial health plan expenditures ( 17 ) . we tested alternate baseline contraception use distributions in puerto rico for women at risk for unintended pregnancy by using the actual distribution of method use reported in 2002 ( 8) and among women attending title x clinics in puerto rico in 2014 ( 31 ) . for the postintervention contraception use distribution , we tested scenarios assuming different proportions of women receiving contraceptive services from a healthcare provider , different levels of willingness to switch to a more effective method , and different shares of moderately effective and highly effective methods among switchers . other parameters evaluated during sensitivity analysis included the incidence of zam during the zika virus outbreak in puerto rico , percentage of pregnancies with zam terminated , the cost of caring for a live - born infant with microcephaly , and the cost of the intervention . in another , we adjusted observed data on us healthcare and supportive care costs to the generally lower levels of prices in puerto rico market by applying conversion factors of ratios of healthcare spending per capita and wages of nurse assistants between the united states and puerto rico ( 32,33 ) . , $ 206 per member of target population ) relative to no intervention ( status quo ) is more than offset by $ 65.2 million in avoided zika virus associated costs , $ 2.8 million from extra testing and monitoring for pregnant women and infants for zika virus exposed pregnancies avoided , and $ 62.3 million from zam cases prevented . the number of zam cases prevented and zika virus associated costs avoided are sensitive to the proportion of women receiving contraceptive services and the proportion of those women willing to switch to a more effective contraception method during the zika virus outbreak ( figure ; table 3 ) . , 21% among no contraception users , 33% among less - effective method users , and 97% among all moderately effective method users ) , 16 cases of zam are prevented , and the net savings is $ 15.4 million ( table 3 ) . if the intervention only shifts users of moderately effective methods to a highly effective method ( no change in non - use or use of less - effective methods ) , 7 zam cases are prevented , with an icer of $ 24,608/hly gained . the results are also sensitive to the prevalence of zam among mid - trimester pregnancies , the percentage of zam cases resulting in live - born infants , lifetime cost per live - born infant with zam , and the intervention cost . in all but 1 of the scenarios sensitivity analysis indicating the effect of changes of assumptions on the number of zam cases prevented in a proposed intervention to increase access to contraception to women during the zika virus outbreak , puerto rico , 2016 . * brfss , behavioral risk factor surveillance system ; cn , cost - neutral ; cs , cost - saving ; hly , healthy life years ; larc , long - acting reversible contraceptive ; nsfg , national survey of family growth ; up , unwanted pregnancy ; zam , zika virus associated microcephaly . based on nsfg 20112013 , among women of reproductive age who are sexually active , did not intend to become pregnant , and were not using permanent contraceptive methods , 21% of no contraception users , 33% of less - effective contraceptive method users , 97% of moderately effective contraceptive method users , and 94% of dual - method users had at least 1 contraceptive service visit in the last 12 months ( in total 50% ) . 2015 ( 32 ) ; conversion factor of 0.72 applied to costs of supportive care for live - born infants with zam , based on the ratio of annual salary for assistant nurses in puerto rico and in the united states ( 33 ) . a probabilistic sensitivity analysis scatter graph shows that most of the model simulations result in icers in the lower right quadrant with lower costs and better health outcomes ( technical appendix figure 2 ) . specifically , the intervention is cost - saving in 92.11% of the 10,000 iterations , and in 98.10% of the iterations , the intervention has an icer of < $ 20,000/hly gained . the larger the numbers of no contraception users and less - effective method users receiving contraceptive services and willing to switch to more effective methods , the greater the magnitude of cost savings from unwanted pregnancies avoided ( table 3 ) . the results of our modeling analysis suggest that increasing access to effective contraception in the context of the 2016 zika virus outbreak for women in puerto rico who do not intend to become pregnant could proportionally reduce the number of unintended pregnancies and cases of zam by 25% . the world health organization suggests that interventions that cost <3 times the gross domestic product per capita per hly ( equivalent to $ 150,000 in the united states and $ 60,000 in puerto rico ) are cost - effective and those costing less than gross domestic product per capita are highly cost - effective ( 36 ) . third , uncertainty exists about the effect of the proposed intervention on postintervention contraceptive use distribution ; however , the sensitivity analyses indicate that different distributions of larc types among switchers does not have a substantial influence on the results . in areas with limited access to providers , the effectiveness of the intervention might be lower , although puerto rico has a similar ratio of physicians to population as the united states as a whole ( 39 ) , and despite a loss of physicians in recent years , puerto rico has a network of providers , federally qualified health clinics , and title x providers in rural and urban areas . third , expenditure data from a large sample of us residents with commercial health insurance were used to calculate the potential medical cost of zam on the basis of combinations of diagnostic codes for virus - associated microcephaly , although costs might be lower for similar children with public insurance . our analyses suggest that increasing access to a full range of contraception among women in puerto rico who want to delay or avoid becoming pregnant during a zika virus outbreak would be a cost - saving strategy to reduce the effects of zam . process for deriving the size of the target population , decision tree structure , and probabilistic sensitivity analysis of cost - effectiveness for a proposed intervention to increase access to contraception to women during the zika virus outbreak , puerto rico , 2016 .

we evaluated a subset of 1,759 from 2,031 look ahead ( action for health in diabetes ) participants generally corresponding to the first half of enrollees from 15 of 16 participating clinic sites who had hs - crp determinations and fitness data at baseline and 1 year . loss to follow - up at 1 year was very low in our eligible participant pool ( 3.2% ) , as in the overall look ahead cohort ( 3.6% ) . the study design , methods , and subject characteristics of look ahead , an ongoing multicenter clinical trial examining whether a behavioral lifestyle intervention targeting weight loss will reduce cardiovascular events and overall mortality in overweight / obese subjects with type 2 diabetes , have been described previously ( 8) . in brief , subjects were randomly assigned to an intensive lifestyle intervention ( ili ) arm aiming for a 7% weight loss from baseline or to a diabetes , support , and education ( dse ) arm , which served as the control . ili participants attended frequent group and individual sessions in support of behavioral change to increase moderate - intensity exercise progressively to 175 min / week , reduce caloric and saturated fat intake , and change macronutrient composition to improve glycemic control . look ahead and this ancillary study were approved by the institutional review boards of the participating centers . a latex particle - enhanced immunoturbidimetric assay ( equal diagnostics / genzyme ) determination of fitness , using submaximal effort on a graded exercise stress test , and procedures for obtaining anthropometrics , a1c , glucose , and lipids in look ahead have been published previously ( 9,10 ) . hs - crp changes at 1 year were reported as median change and percent change from baseline . quartiles of change in bmi , fitness , and parameters of glucose and lipid control were examined against change in log hs - crp in an exploratory approach to evaluate linearity . the associations between changes in variables of interest and hs - crp change ( log - transformed to correct for a nonnormal distribution ) were examined using multiple linear regression analyses after excluding collinearity ( defined as a correlation coefficient > 0.6 ) . a dichotomous indicator for treatment group ( ili vs. dse ) was included in all models to examine the significance of treatment effect ( shown alone in model a ) . changes in each of the metabolic variables and/or fitness were entered into a separate regression model , alone ( models b j ) or in combination with other predictors ( models k q ) , in the presence of the dichotomous treatment group indicator , to evaluate their contributions . all models were adjusted for the effects of demographics , clinic site , history of cvd , current smoking , and treatment with statins and thiazolidinediones ( tzds ) . treatment effect ( ili ) and statin and insulin use ( tested separately ) were evaluated in the full model with the use of interaction terms to evaluate whether either pharmacological therapy modulated the association of ili with change in hs - crp . analyses were performed using sas ( version 9.2 ; sas institute , cary , nc ) . a latex particle - enhanced immunoturbidimetric assay ( equal diagnostics / genzyme ) was used to measure hs - crp . determination of fitness , using submaximal effort on a graded exercise stress test , and procedures for obtaining anthropometrics , a1c , glucose , and lipids in look ahead have been published previously ( 9,10 ) . hs - crp changes at 1 year were reported as median change and percent change from baseline . quartiles of change in bmi , fitness , and parameters of glucose and lipid control were examined against change in log hs - crp in an exploratory approach to evaluate linearity . the associations between changes in variables of interest and hs - crp change ( log - transformed to correct for a nonnormal distribution ) were examined using multiple linear regression analyses after excluding collinearity ( defined as a correlation coefficient > 0.6 ) . a dichotomous indicator for treatment group ( ili vs. dse ) was included in all models to examine the significance of treatment effect ( shown alone in model a ) . changes in each of the metabolic variables and/or fitness were entered into a separate regression model , alone ( models b j ) or in combination with other predictors ( models k q ) , in the presence of the dichotomous treatment group indicator , to evaluate their contributions . all models were adjusted for the effects of demographics , clinic site , history of cvd , current smoking , and treatment with statins and thiazolidinediones ( tzds ) . treatment effect ( ili ) and statin and insulin use ( tested separately ) were evaluated in the full model with the use of interaction terms to evaluate whether either pharmacological therapy modulated the association of ili with change in hs - crp . analyses were performed using sas ( version 9.2 ; sas institute , cary , nc ) . mg / l ) than in men ( 2.4 [ iqr 1.24.7 ] mg / l ) . because of the change in age eligibility criteria during the 2nd year of recruitment in look ahead , subject characteristics in this ancillary study differ slightly from those of the remaining participants ; 12% had cvd and 40% used statins , compared with 15 and 45% , respectively , for the remainder of look ahead enrollees ( 11 ) . baseline characteristics data are means sd , n ( % ) , or median ( iqr ) . self - reported prior myocardial infarction , stroke , transient ischemic attack , angioplasty / stent , coronary artery bypass graft , carotid endarterectomy , abdominal aortic aneurysm , or heart failure . as reported for the overall look ahead sample ( 10 ) , subjects in the ili arm in this study had significant improvements in glucose control and weight loss at 1 year compared with those in the dse arm . a1c decreased by 0.7% with ili and by 0.2% with dse ( p < 0.001 ) . subjects in the ili arm had mean weight and bmi reductions of 9 kg and 3.2 kg / m ( 8.8% of baseline ) , respectively , compared with respective reductions of 0.8 kg and 0.3 kg / m ( 0.8% of baseline ) in the dse arm ( p < 0.001 ) . ili participants had a greater improvement in fitness , with a 19% increase from baseline compared with a 5.9% increase in the dse arm ( p < 0.001 ) ( table 2 ) . hdl cholesterol and triglycerides improved with ili compared with dse , but ldl cholesterol change was not different between arms . changes in metabolic variables , fitness , and hs - crp at 1 year data are means sd or median ( iqr ) unless otherwise indicated . for difference between ili and dse for change in variable from baseline to 1 year . median hs - crp at 1 year dropped by 43.6% from baseline in the ili group , compared with a 16.7% decrease in the dse group ( p < 0.001 for difference in median change between ili and dse ) ( table 2 ) . women , who had a higher hs - crp level than men at baseline , had a greater absolute change in median hs - crp level with ili at 1 year but a similar proportional drop in hs - crp levels compared with that in men ( table 2 ) . quartiles of change representing greater improvements in adiposity , fitness , and glucose and lipid control were associated with greater decreases in hs - crp . pearson correlation coefficients between variable changes ( except between measures of adiposity and between fasting glucose and a1c ) were all < 0.46 . change in hs - crp ( median and iqr ) at 1 year in the dse arm vs. the ili arm by quartiles ( q ) of change in bmi ( top ) and a1c ( bottom ) . change in hs - crp is a proportional change , with numbers < 1.0 indicating a decrease in hs - crp and those > 1.0 an increase in hs - crp and the horizontal line representing overall mean change . regression analysis , accounting for potential differences between treatment arms in demographics , smoking , and tzd and statin use , among others , confirmed that each of the ili - induced improvements in adiposity ( bmi , weight , and waist circumference ) , glucose control ( a1c and fasting glucose ) , triglycerides , hdl cholesterol , and fitness predicted a decrease in hs - crp at 1 year ( analyzed as log hs - crp , p < 0.001 for all ) ( table 3 , models b j ) . change in waist circumference with ili contributed slightly less to hs - crp change ( r = 0.096 ) than did change in weight ( r = 0.114 ) or change in bmi ( r = 0.115 ) with ili . interestingly , the improvement in glucose control with ili contributed to hs - crp change to an extent similar to that for the reduction in adiposity ( r = 0.112 and 0.100 for fasting glucose and a1c , respectively ) . improvements in fitness with ili explained slightly less ( r = 0.086 ) of the variance in hs - crp change at 1 year than did changes in adiposity or glucose control . both change in hdl cholesterol and change in triglyceride levels , but not change in ldl cholesterol , predicted hs - crp change with ili at 1 year . metabolic variables as predictors of hs - crp change with 1-year ili using multiple variable regression analysis each model ( a q ) was analyzed independently and adjusted for age , sex , ethnicity , clinic site , history of cvd , smoking , and tzd and statin use . the outcome variable , change in hs - crp , was log - transformed to correct for a nonnormal distribution . when change in fitness was evaluated in the regression model with change in a1c ( model k ) , we found that each predicted hs - crp change . however , when change in bmi was added to the model ( model l ) , fitness was no longer a significant predictor , suggesting that the change in adiposity associated with improved fitness partially explained the decline in hs - crp with ili at 1 year . on the other hand , when change in bmi or change in fitness was added to a model containing hdl cholesterol ( models m and o , respectively ) or triglycerides ( models n and p , respectively ) , both lipid variables remained significant predictors of hs - crp change . a final model ( model q ) , including changes in bmi , a1c , hdl cholesterol , triglycerides , and fitness , revealed that , of the metabolic variables studied , only improvements in glucose control and adiposity could independently account for the decrease in hs - crp at 1 year ( p < 0.001 ) . the beneficial effects of changes in fitness , hdl cholesterol , and triglycerides on hs - crp were weakened and no longer statistically significant ( p = 0.095 , 0.106 , and 0.068 , respectively ) when tested in the full model . statin and insulin use did not modify the association of ili and hs - crp when each was tested separately with the use of an interaction term ( statin use ili and insulin use ili ) in the full model ( p = 0.43 and 0.50 , respectively ) . mg / l ) than in men ( 2.4 [ iqr 1.24.7 ] mg / l ) . because of the change in age eligibility criteria during the 2nd year of recruitment in look ahead , subject characteristics in this ancillary study differ slightly from those of the remaining participants ; 12% had cvd and 40% used statins , compared with 15 and 45% , respectively , for the remainder of look ahead enrollees ( 11 ) . baseline characteristics data are means sd , n ( % ) , or median ( iqr ) . self - reported prior myocardial infarction , stroke , transient ischemic attack , angioplasty / stent , coronary artery bypass graft , carotid endarterectomy , abdominal aortic aneurysm , or heart failure . as reported for the overall look ahead sample ( 10 ) , subjects in the ili arm in this study had significant improvements in glucose control and weight loss at 1 year compared with those in the dse arm . a1c decreased by 0.7% with ili and by 0.2% with dse ( p < 0.001 ) . subjects in the ili arm had mean weight and bmi reductions of 9 kg and 3.2 kg / m ( 8.8% of baseline ) , respectively , compared with respective reductions of 0.8 kg and 0.3 kg / m ( 0.8% of baseline ) in the dse arm ( p < 0.001 ) . ili participants had a greater improvement in fitness , with a 19% increase from baseline compared with a 5.9% increase in the dse arm ( p < 0.001 ) ( table 2 ) . hdl cholesterol and triglycerides improved with ili compared with dse , but ldl cholesterol change was not different between arms . changes in metabolic variables , fitness , and hs - crp at 1 year data are means sd or median ( iqr ) unless otherwise indicated . * for difference between ili and dse for change in variable from baseline to 1 year . median hs - crp at 1 year dropped by 43.6% from baseline in the ili group , compared with a 16.7% decrease in the dse group ( p < 0.001 for difference in median change between ili and dse ) ( table 2 ) . women , who had a higher hs - crp level than men at baseline , had a greater absolute change in median hs - crp level with ili at 1 year but a similar proportional drop in hs - crp levels compared with that in men ( table 2 ) . quartiles of change representing greater improvements in adiposity , fitness , and glucose and lipid control were associated with greater decreases in hs - crp . pearson correlation coefficients between variable changes ( except between measures of adiposity and between fasting glucose and a1c ) were all < 0.46 . change in hs - crp ( median and iqr ) at 1 year in the dse arm vs. the ili arm by quartiles ( q ) of change in bmi ( top ) and a1c ( bottom ) . change in hs - crp is a proportional change , with numbers < 1.0 indicating a decrease in hs - crp and those > 1.0 an increase in hs - crp and the horizontal line representing overall mean change . regression analysis , accounting for potential differences between treatment arms in demographics , smoking , and tzd and statin use , among others , confirmed that each of the ili - induced improvements in adiposity ( bmi , weight , and waist circumference ) , glucose control ( a1c and fasting glucose ) , triglycerides , hdl cholesterol , and fitness predicted a decrease in hs - crp at 1 year ( analyzed as log hs - crp , p < 0.001 for all ) ( table 3 , models b j ) . change in waist circumference with ili contributed slightly less to hs - crp change ( r = 0.096 ) than did change in weight ( r = 0.114 ) or change in bmi ( r = 0.115 ) with ili . interestingly , the improvement in glucose control with ili contributed to hs - crp change to an extent similar to that for the reduction in adiposity ( r = 0.112 and 0.100 for fasting glucose and a1c , respectively ) . improvements in fitness with ili explained slightly less ( r = 0.086 ) of the variance in hs - crp change at 1 year than did changes in adiposity or glucose control . both change in hdl cholesterol and change in triglyceride levels , but not change in ldl cholesterol , predicted hs - crp change with ili at 1 year . metabolic variables as predictors of hs - crp change with 1-year ili using multiple variable regression analysis each model ( a q ) was analyzed independently and adjusted for age , sex , ethnicity , clinic site , history of cvd , smoking , and tzd and statin use . the outcome variable , change in hs - crp , was log - transformed to correct for a nonnormal distribution . when change in fitness was evaluated in the regression model with change in a1c ( model k ) , we found that each predicted hs - crp change . however , when change in bmi was added to the model ( model l ) , fitness was no longer a significant predictor , suggesting that the change in adiposity associated with improved fitness partially explained the decline in hs - crp with ili at 1 year . on the other hand , when change in bmi or change in fitness was added to a model containing hdl cholesterol ( models m and o , respectively ) or triglycerides ( models n and p , respectively ) , both lipid variables remained significant predictors of hs - crp change . a final model ( model q ) , including changes in bmi , a1c , hdl cholesterol , triglycerides , and fitness , revealed that , of the metabolic variables studied , only improvements in glucose control and adiposity could independently account for the decrease in hs - crp at 1 year ( p < 0.001 ) . the beneficial effects of changes in fitness , hdl cholesterol , and triglycerides on hs - crp were weakened and no longer statistically significant ( p = 0.095 , 0.106 , and 0.068 , respectively ) when tested in the full model . statin and insulin use did not modify the association of ili and hs - crp when each was tested separately with the use of an interaction term ( statin use ili and insulin use ili ) in the full model ( p = 0.43 and 0.50 , respectively ) . this report contributes information on the effects of a 1-year lifestyle intervention for weight loss on hs - crp in the setting of what is , to our knowledge , the largest randomized clinical trial of its kind in individuals with type 2 diabetes . most studies evaluating cardiovascular risk reduction in individuals with diabetes have focused on the effects of statins and found a substantial benefit ( 12 ) . statins not only decrease ldl cholesterol but they also have anti - inflammatory activity and have been shown to decrease cardiovascular mortality in individuals without diabetes who have elevated hs - crp ( 4 ) . our report showed that in obese men and women with type 2 diabetes , 1 year of lifestyle intervention ( in addition to usual care ) , which led to an 8.8% reduction in baseline weight and a 0.7% drop in a1c , resulted in a 43.6% decrease in median hs - crp , whereas usual care alone , which led to reductions of 0.8% in baseline weight and 0.2% in a1c , resulted in a 16.7% decrease in median hs - crp . the improvement in hs - crp achieved with ili in look ahead is comparable to hs - crp reductions with statins in people without diabetes ( 4 ) . esposito et al . ( 5 ) were the first to present compelling evidence on the benefit of weight loss achieved with lifestyle behavior changes on markers of inflammation . in a 2-year interventional study in 160 obese women without diabetes , they reported a 14% decrease in mean weight and a 34% decrease in median hs - crp from baseline ( compared with decreases of 3 and 9% , respectively , in the control group ) . in the diabetes prevention program ( dpp ) , in which 1,000 of > 3,000 obese participants at risk for diabetes were randomly assigned to a lifestyle intervention arm , behavioral changes in physical activity and diet resulted in a 7.2% decrease in baseline weight and 30% decrease in median hs - crp at 1 year ; hs - crp in the placebo group increased by 5% in men and did not change in women ( 6 ) . the few studies that investigated the effects of weight loss on hs - crp in individuals with diabetes were small , achieved minimal weight reductions , and did not adjust for changes in both fitness and glucose control ( 13 ) . our study indicates that moderate weight loss in obese individuals with type 2 diabetes is associated with a substantial reduction in hs - crp levels and that decreased adiposity is an independent predictor of hs - crp reduction after accounting for improvements in fitness , glucose , and lipid control . debate continues on whether fitness and weight loss have independent effects on inflammation ( 14 ) . this is of particular interest in the care of obese sedentary individuals with diabetes , in whom an increase in physical activity may occur without associated weight loss . mechanisms are emerging that explain how increased fitness , via associated improvements in autonomic nervous system function , may decrease macrophage proinflammatory cytokine production independently of weight loss ( 15 ) . dpp evaluated physical activity , obtained from participant self - report , and concluded that weight loss , not physical activity , accounted for the changes in hs - crp at 1 year ; however , fitness was not assessed ( 6 ) . our study showed that the moderate improvement in fitness observed with ili in our generally obese and sedentary participants with type 2 diabetes was associated with a reduction in hs - crp , but the effects were attenuated ( p = 0.06 ) when weight loss was taken into account . our findings do not exclude the possibility that greater changes in fitness could have a stronger effect on hs - crp change or that the same change in fitness in less obese individuals with diabetes could be associated with hs - crp change independently of weight loss . the predominant role of adiposity on the regulation of the inflammatory response is not surprising . adipose tissue is in itself a source of crp and a major producer of interleukin-6 , a key stimulator of crp secretion ( 16 ) . in obesity , adipose tissue contains an increased number of resident macrophages and t cells , which interact closely with adipocytes to modulate the inflammatory response ( 17,18 ) . it was interesting to find that the associations between improvements in hdl cholesterol and triglyceride levels and the decrease in hs - crp with ili were independent of improved fitness , glucose control , and weight loss . hdl is known to bind to adipocytes ( 19 ) and to possess anti - inflammatory properties , including an inhibitory effect on monocyte chemoattractant protein-1 ( 20 ) , an important player in macrophage recruitment to adipose tissue ( 21 ) . elevated levels of hs - crp have been found in individuals with familial hypoalphalipoproteinemia , in whom hdl cholesterol levels are low and the risk of coronary disease is high ( 22 ) . triglyceride - rich lipoproteins and nonesterified fatty acids are taken up by neutrophils and monocytes , with generation of reactive oxygen species and production of cytokines ( 23 ) . in our study , the effects of hdl cholesterol and triglyceride change on hs - crp variance were attenuated and no longer significant when both were included in the same model ( model q ) . the pearson correlation coefficient for change in hdl cholesterol and change in triglycerides ( 0.26 ) suggests that this attenuation was not the result of collinearity . the effects of improved glucose control with lifestyle on hs - crp were of particular interest to us in light of the recent study by pradhan et al . ( 7 ) in individuals with type 2 diabetes , in whom a 14-week course of insulin glargine was shown to abrogate the hs - crp reduction seen in the placebo group . the mechanisms that would explain this finding are difficult to determine , given that the effects were reported to be independent of weight gain and because it has been previously shown that hyperglycemia stimulates inflammatory cytokine production ( 24 ) . the report suggested that the deleterious effect of insulin therapy on the inflammatory state could explain the lack of benefit of improved glycemic control on incident cardiovascular events found in recent clinical trials . our study showed that improved glycemic control with ili was associated with a reduction in hs - crp at 1 year . the favorable association of improved glycemia and hs - crp change was independent of changes in adiposity and persisted after accounting for multiple covariates , including statin and tzd use , and was not affected by changes in insulin therapy with ili ( p = 0.50 ) . our results , in agreement with a previous small study in subjects with diabetes ( 13 ) and with experimental evidence linking hyperglycemia with increased cytokine production , indicate that improved glucose control per se does not worsen the inflammatory state in individuals with diabetes . a cross - sectional observation in which the correlation between glycemia and hs - crp was not significant after adjustment for bmi ( 3 ) seems to contradict the robust effect of improved glucose control with ili on hs - crp observed in our study . however , findings between studies can not be compared ; the former study evaluated correlation at baseline , whereas this report evaluated variable changes at 1 year . lowering glucose with improved dietary and physical activity behaviors , as observed in look ahead , may reflect the disruption of the paracrine loop between adipocytes and macrophages that promotes inflammation , both locally and systemically , and insulin resistance ( 25 ) . our report supports a substantial benefit of lifestyle intervention for weight loss on the chronic inflammatory state characteristic of diabetes and highlights the contribution of improved glycemic control achieved with lifestyle changes to the reduction of elevated hs - crp levels in obese sedentary individuals with diabetes . follow - up of cardiovascular outcomes in look ahead will confirm whether the improvement in hs - crp with behavioral changes in lifestyle will translate into a reduction of cardiovascular events .

objectivewe examined whether a 1-year intensive lifestyle intervention ( ili ) for weight loss reduced elevated high - sensitivity c - reactive protein ( hs - crp ) levels in obese individuals with diabetes and identified metabolic and fitness predictors of hs - crp change.research design and methodslook ahead ( action for health in diabetes ) is an ongoing multicenter clinical trial examining the effects of weight loss achieved through ili on cardiovascular events and overall mortality in obese / overweight adults with type 2 diabetes . we report on 1,759 look ahead participants who had hs - crp and fitness data at baseline and 1 year . subjects were randomly assigned to ili or to usual care ( diabetes support and education [ dse ] ) . ili involved frequent counseling to increase moderate - intensity exercise to 175 min / week , reduce caloric and saturated fat intake , and change macronutrient composition to improve glycemic control.resultsili reduced median hs - crp by 43.6% from baseline to 1 year , compared with a 16.7% reduction with dse ( p < 0.001 ) . ili decreased weight ( 8.8% ) , a1c ( 0.7% ) , and triglycerides ( 17% ) and increased fitness ( 19% ) and hdl cholesterol ( 7.5% ) ( p < 0.0001 vs. changes with dse ) . changes in adiposity and glucose control with ili remained independent predictors of hs - crp change at 1 year ( p < 0.0001 for each ) after adjustment for demographics , smoking , cardiovascular history , statin and thiazolidinedione use , and changes in fitness and lipid control . neither statin nor insulin therapy modified the association between ili and hs-crp.conclusionsa 1-year lifestyle intervention for weight loss in obese individuals with diabetes was associated with substantial reductions in hs - crp . improved glycemic control and reduced adiposity had comparable effects on hs - crp change .

RESEARCH DESIGN AND METHODS Laboratory, anthropometric, and fitness determinations Statistical analysis RESULTS Baseline Changes in metabolic variables and hs-CRP at 1 year Metabolic predictors of change in hs-CRP at 1 year CONCLUSIONS Supplementary Material

we evaluated a subset of 1,759 from 2,031 look ahead ( action for health in diabetes ) participants generally corresponding to the first half of enrollees from 15 of 16 participating clinic sites who had hs - crp determinations and fitness data at baseline and 1 year . loss to follow - up at 1 year was very low in our eligible participant pool ( 3.2% ) , as in the overall look ahead cohort ( 3.6% ) . the study design , methods , and subject characteristics of look ahead , an ongoing multicenter clinical trial examining whether a behavioral lifestyle intervention targeting weight loss will reduce cardiovascular events and overall mortality in overweight / obese subjects with type 2 diabetes , have been described previously ( 8) . in brief , subjects were randomly assigned to an intensive lifestyle intervention ( ili ) arm aiming for a 7% weight loss from baseline or to a diabetes , support , and education ( dse ) arm , which served as the control . ili participants attended frequent group and individual sessions in support of behavioral change to increase moderate - intensity exercise progressively to 175 min / week , reduce caloric and saturated fat intake , and change macronutrient composition to improve glycemic control . hs - crp changes at 1 year were reported as median change and percent change from baseline . quartiles of change in bmi , fitness , and parameters of glucose and lipid control were examined against change in log hs - crp in an exploratory approach to evaluate linearity . the associations between changes in variables of interest and hs - crp change ( log - transformed to correct for a nonnormal distribution ) were examined using multiple linear regression analyses after excluding collinearity ( defined as a correlation coefficient > 0.6 ) . all models were adjusted for the effects of demographics , clinic site , history of cvd , current smoking , and treatment with statins and thiazolidinediones ( tzds ) . treatment effect ( ili ) and statin and insulin use ( tested separately ) were evaluated in the full model with the use of interaction terms to evaluate whether either pharmacological therapy modulated the association of ili with change in hs - crp . hs - crp changes at 1 year were reported as median change and percent change from baseline . quartiles of change in bmi , fitness , and parameters of glucose and lipid control were examined against change in log hs - crp in an exploratory approach to evaluate linearity . the associations between changes in variables of interest and hs - crp change ( log - transformed to correct for a nonnormal distribution ) were examined using multiple linear regression analyses after excluding collinearity ( defined as a correlation coefficient > 0.6 ) . all models were adjusted for the effects of demographics , clinic site , history of cvd , current smoking , and treatment with statins and thiazolidinediones ( tzds ) . treatment effect ( ili ) and statin and insulin use ( tested separately ) were evaluated in the full model with the use of interaction terms to evaluate whether either pharmacological therapy modulated the association of ili with change in hs - crp . as reported for the overall look ahead sample ( 10 ) , subjects in the ili arm in this study had significant improvements in glucose control and weight loss at 1 year compared with those in the dse arm . a1c decreased by 0.7% with ili and by 0.2% with dse ( p < 0.001 ) . subjects in the ili arm had mean weight and bmi reductions of 9 kg and 3.2 kg / m ( 8.8% of baseline ) , respectively , compared with respective reductions of 0.8 kg and 0.3 kg / m ( 0.8% of baseline ) in the dse arm ( p < 0.001 ) . ili participants had a greater improvement in fitness , with a 19% increase from baseline compared with a 5.9% increase in the dse arm ( p < 0.001 ) ( table 2 ) . hdl cholesterol and triglycerides improved with ili compared with dse , but ldl cholesterol change was not different between arms . changes in metabolic variables , fitness , and hs - crp at 1 year data are means sd or median ( iqr ) unless otherwise indicated . for difference between ili and dse for change in variable from baseline to 1 year . median hs - crp at 1 year dropped by 43.6% from baseline in the ili group , compared with a 16.7% decrease in the dse group ( p < 0.001 for difference in median change between ili and dse ) ( table 2 ) . women , who had a higher hs - crp level than men at baseline , had a greater absolute change in median hs - crp level with ili at 1 year but a similar proportional drop in hs - crp levels compared with that in men ( table 2 ) . quartiles of change representing greater improvements in adiposity , fitness , and glucose and lipid control were associated with greater decreases in hs - crp . change in hs - crp ( median and iqr ) at 1 year in the dse arm vs. the ili arm by quartiles ( q ) of change in bmi ( top ) and a1c ( bottom ) . regression analysis , accounting for potential differences between treatment arms in demographics , smoking , and tzd and statin use , among others , confirmed that each of the ili - induced improvements in adiposity ( bmi , weight , and waist circumference ) , glucose control ( a1c and fasting glucose ) , triglycerides , hdl cholesterol , and fitness predicted a decrease in hs - crp at 1 year ( analyzed as log hs - crp , p < 0.001 for all ) ( table 3 , models b j ) . change in waist circumference with ili contributed slightly less to hs - crp change ( r = 0.096 ) than did change in weight ( r = 0.114 ) or change in bmi ( r = 0.115 ) with ili . interestingly , the improvement in glucose control with ili contributed to hs - crp change to an extent similar to that for the reduction in adiposity ( r = 0.112 and 0.100 for fasting glucose and a1c , respectively ) . improvements in fitness with ili explained slightly less ( r = 0.086 ) of the variance in hs - crp change at 1 year than did changes in adiposity or glucose control . both change in hdl cholesterol and change in triglyceride levels , but not change in ldl cholesterol , predicted hs - crp change with ili at 1 year . metabolic variables as predictors of hs - crp change with 1-year ili using multiple variable regression analysis each model ( a q ) was analyzed independently and adjusted for age , sex , ethnicity , clinic site , history of cvd , smoking , and tzd and statin use . when change in fitness was evaluated in the regression model with change in a1c ( model k ) , we found that each predicted hs - crp change . however , when change in bmi was added to the model ( model l ) , fitness was no longer a significant predictor , suggesting that the change in adiposity associated with improved fitness partially explained the decline in hs - crp with ili at 1 year . on the other hand , when change in bmi or change in fitness was added to a model containing hdl cholesterol ( models m and o , respectively ) or triglycerides ( models n and p , respectively ) , both lipid variables remained significant predictors of hs - crp change . a final model ( model q ) , including changes in bmi , a1c , hdl cholesterol , triglycerides , and fitness , revealed that , of the metabolic variables studied , only improvements in glucose control and adiposity could independently account for the decrease in hs - crp at 1 year ( p < 0.001 ) . the beneficial effects of changes in fitness , hdl cholesterol , and triglycerides on hs - crp were weakened and no longer statistically significant ( p = 0.095 , 0.106 , and 0.068 , respectively ) when tested in the full model . statin and insulin use did not modify the association of ili and hs - crp when each was tested separately with the use of an interaction term ( statin use ili and insulin use ili ) in the full model ( p = 0.43 and 0.50 , respectively ) . as reported for the overall look ahead sample ( 10 ) , subjects in the ili arm in this study had significant improvements in glucose control and weight loss at 1 year compared with those in the dse arm . a1c decreased by 0.7% with ili and by 0.2% with dse ( p < 0.001 ) . subjects in the ili arm had mean weight and bmi reductions of 9 kg and 3.2 kg / m ( 8.8% of baseline ) , respectively , compared with respective reductions of 0.8 kg and 0.3 kg / m ( 0.8% of baseline ) in the dse arm ( p < 0.001 ) . ili participants had a greater improvement in fitness , with a 19% increase from baseline compared with a 5.9% increase in the dse arm ( p < 0.001 ) ( table 2 ) . hdl cholesterol and triglycerides improved with ili compared with dse , but ldl cholesterol change was not different between arms . changes in metabolic variables , fitness , and hs - crp at 1 year data are means sd or median ( iqr ) unless otherwise indicated . * for difference between ili and dse for change in variable from baseline to 1 year . median hs - crp at 1 year dropped by 43.6% from baseline in the ili group , compared with a 16.7% decrease in the dse group ( p < 0.001 for difference in median change between ili and dse ) ( table 2 ) . women , who had a higher hs - crp level than men at baseline , had a greater absolute change in median hs - crp level with ili at 1 year but a similar proportional drop in hs - crp levels compared with that in men ( table 2 ) . quartiles of change representing greater improvements in adiposity , fitness , and glucose and lipid control were associated with greater decreases in hs - crp . change in hs - crp ( median and iqr ) at 1 year in the dse arm vs. the ili arm by quartiles ( q ) of change in bmi ( top ) and a1c ( bottom ) . change in hs - crp is a proportional change , with numbers < 1.0 indicating a decrease in hs - crp and those > 1.0 an increase in hs - crp and the horizontal line representing overall mean change . regression analysis , accounting for potential differences between treatment arms in demographics , smoking , and tzd and statin use , among others , confirmed that each of the ili - induced improvements in adiposity ( bmi , weight , and waist circumference ) , glucose control ( a1c and fasting glucose ) , triglycerides , hdl cholesterol , and fitness predicted a decrease in hs - crp at 1 year ( analyzed as log hs - crp , p < 0.001 for all ) ( table 3 , models b j ) . change in waist circumference with ili contributed slightly less to hs - crp change ( r = 0.096 ) than did change in weight ( r = 0.114 ) or change in bmi ( r = 0.115 ) with ili . interestingly , the improvement in glucose control with ili contributed to hs - crp change to an extent similar to that for the reduction in adiposity ( r = 0.112 and 0.100 for fasting glucose and a1c , respectively ) . improvements in fitness with ili explained slightly less ( r = 0.086 ) of the variance in hs - crp change at 1 year than did changes in adiposity or glucose control . both change in hdl cholesterol and change in triglyceride levels , but not change in ldl cholesterol , predicted hs - crp change with ili at 1 year . metabolic variables as predictors of hs - crp change with 1-year ili using multiple variable regression analysis each model ( a q ) was analyzed independently and adjusted for age , sex , ethnicity , clinic site , history of cvd , smoking , and tzd and statin use . when change in fitness was evaluated in the regression model with change in a1c ( model k ) , we found that each predicted hs - crp change . however , when change in bmi was added to the model ( model l ) , fitness was no longer a significant predictor , suggesting that the change in adiposity associated with improved fitness partially explained the decline in hs - crp with ili at 1 year . on the other hand , when change in bmi or change in fitness was added to a model containing hdl cholesterol ( models m and o , respectively ) or triglycerides ( models n and p , respectively ) , both lipid variables remained significant predictors of hs - crp change . a final model ( model q ) , including changes in bmi , a1c , hdl cholesterol , triglycerides , and fitness , revealed that , of the metabolic variables studied , only improvements in glucose control and adiposity could independently account for the decrease in hs - crp at 1 year ( p < 0.001 ) . the beneficial effects of changes in fitness , hdl cholesterol , and triglycerides on hs - crp were weakened and no longer statistically significant ( p = 0.095 , 0.106 , and 0.068 , respectively ) when tested in the full model . statin and insulin use did not modify the association of ili and hs - crp when each was tested separately with the use of an interaction term ( statin use ili and insulin use ili ) in the full model ( p = 0.43 and 0.50 , respectively ) . this report contributes information on the effects of a 1-year lifestyle intervention for weight loss on hs - crp in the setting of what is , to our knowledge , the largest randomized clinical trial of its kind in individuals with type 2 diabetes . most studies evaluating cardiovascular risk reduction in individuals with diabetes have focused on the effects of statins and found a substantial benefit ( 12 ) . our report showed that in obese men and women with type 2 diabetes , 1 year of lifestyle intervention ( in addition to usual care ) , which led to an 8.8% reduction in baseline weight and a 0.7% drop in a1c , resulted in a 43.6% decrease in median hs - crp , whereas usual care alone , which led to reductions of 0.8% in baseline weight and 0.2% in a1c , resulted in a 16.7% decrease in median hs - crp . the improvement in hs - crp achieved with ili in look ahead is comparable to hs - crp reductions with statins in people without diabetes ( 4 ) . in a 2-year interventional study in 160 obese women without diabetes , they reported a 14% decrease in mean weight and a 34% decrease in median hs - crp from baseline ( compared with decreases of 3 and 9% , respectively , in the control group ) . in the diabetes prevention program ( dpp ) , in which 1,000 of > 3,000 obese participants at risk for diabetes were randomly assigned to a lifestyle intervention arm , behavioral changes in physical activity and diet resulted in a 7.2% decrease in baseline weight and 30% decrease in median hs - crp at 1 year ; hs - crp in the placebo group increased by 5% in men and did not change in women ( 6 ) . the few studies that investigated the effects of weight loss on hs - crp in individuals with diabetes were small , achieved minimal weight reductions , and did not adjust for changes in both fitness and glucose control ( 13 ) . our study indicates that moderate weight loss in obese individuals with type 2 diabetes is associated with a substantial reduction in hs - crp levels and that decreased adiposity is an independent predictor of hs - crp reduction after accounting for improvements in fitness , glucose , and lipid control . dpp evaluated physical activity , obtained from participant self - report , and concluded that weight loss , not physical activity , accounted for the changes in hs - crp at 1 year ; however , fitness was not assessed ( 6 ) . our study showed that the moderate improvement in fitness observed with ili in our generally obese and sedentary participants with type 2 diabetes was associated with a reduction in hs - crp , but the effects were attenuated ( p = 0.06 ) when weight loss was taken into account . our findings do not exclude the possibility that greater changes in fitness could have a stronger effect on hs - crp change or that the same change in fitness in less obese individuals with diabetes could be associated with hs - crp change independently of weight loss . it was interesting to find that the associations between improvements in hdl cholesterol and triglyceride levels and the decrease in hs - crp with ili were independent of improved fitness , glucose control , and weight loss . elevated levels of hs - crp have been found in individuals with familial hypoalphalipoproteinemia , in whom hdl cholesterol levels are low and the risk of coronary disease is high ( 22 ) . in our study , the effects of hdl cholesterol and triglyceride change on hs - crp variance were attenuated and no longer significant when both were included in the same model ( model q ) . the pearson correlation coefficient for change in hdl cholesterol and change in triglycerides ( 0.26 ) suggests that this attenuation was not the result of collinearity . the effects of improved glucose control with lifestyle on hs - crp were of particular interest to us in light of the recent study by pradhan et al . ( 7 ) in individuals with type 2 diabetes , in whom a 14-week course of insulin glargine was shown to abrogate the hs - crp reduction seen in the placebo group . our study showed that improved glycemic control with ili was associated with a reduction in hs - crp at 1 year . the favorable association of improved glycemia and hs - crp change was independent of changes in adiposity and persisted after accounting for multiple covariates , including statin and tzd use , and was not affected by changes in insulin therapy with ili ( p = 0.50 ) . our results , in agreement with a previous small study in subjects with diabetes ( 13 ) and with experimental evidence linking hyperglycemia with increased cytokine production , indicate that improved glucose control per se does not worsen the inflammatory state in individuals with diabetes . a cross - sectional observation in which the correlation between glycemia and hs - crp was not significant after adjustment for bmi ( 3 ) seems to contradict the robust effect of improved glucose control with ili on hs - crp observed in our study . our report supports a substantial benefit of lifestyle intervention for weight loss on the chronic inflammatory state characteristic of diabetes and highlights the contribution of improved glycemic control achieved with lifestyle changes to the reduction of elevated hs - crp levels in obese sedentary individuals with diabetes . follow - up of cardiovascular outcomes in look ahead will confirm whether the improvement in hs - crp with behavioral changes in lifestyle will translate into a reduction of cardiovascular events .

we evaluated a subset of 1,759 from 2,031 look ahead ( action for health in diabetes ) participants generally corresponding to the first half of enrollees from 15 of 16 participating clinic sites who had hs - crp determinations and fitness data at baseline and 1 year . the study design , methods , and subject characteristics of look ahead , an ongoing multicenter clinical trial examining whether a behavioral lifestyle intervention targeting weight loss will reduce cardiovascular events and overall mortality in overweight / obese subjects with type 2 diabetes , have been described previously ( 8) . in brief , subjects were randomly assigned to an intensive lifestyle intervention ( ili ) arm aiming for a 7% weight loss from baseline or to a diabetes , support , and education ( dse ) arm , which served as the control . ili participants attended frequent group and individual sessions in support of behavioral change to increase moderate - intensity exercise progressively to 175 min / week , reduce caloric and saturated fat intake , and change macronutrient composition to improve glycemic control . a latex particle - enhanced immunoturbidimetric assay ( equal diagnostics / genzyme ) determination of fitness , using submaximal effort on a graded exercise stress test , and procedures for obtaining anthropometrics , a1c , glucose , and lipids in look ahead have been published previously ( 9,10 ) . quartiles of change in bmi , fitness , and parameters of glucose and lipid control were examined against change in log hs - crp in an exploratory approach to evaluate linearity . the associations between changes in variables of interest and hs - crp change ( log - transformed to correct for a nonnormal distribution ) were examined using multiple linear regression analyses after excluding collinearity ( defined as a correlation coefficient > 0.6 ) . changes in each of the metabolic variables and/or fitness were entered into a separate regression model , alone ( models b j ) or in combination with other predictors ( models k q ) , in the presence of the dichotomous treatment group indicator , to evaluate their contributions . all models were adjusted for the effects of demographics , clinic site , history of cvd , current smoking , and treatment with statins and thiazolidinediones ( tzds ) . treatment effect ( ili ) and statin and insulin use ( tested separately ) were evaluated in the full model with the use of interaction terms to evaluate whether either pharmacological therapy modulated the association of ili with change in hs - crp . quartiles of change in bmi , fitness , and parameters of glucose and lipid control were examined against change in log hs - crp in an exploratory approach to evaluate linearity . the associations between changes in variables of interest and hs - crp change ( log - transformed to correct for a nonnormal distribution ) were examined using multiple linear regression analyses after excluding collinearity ( defined as a correlation coefficient > 0.6 ) . changes in each of the metabolic variables and/or fitness were entered into a separate regression model , alone ( models b j ) or in combination with other predictors ( models k q ) , in the presence of the dichotomous treatment group indicator , to evaluate their contributions . all models were adjusted for the effects of demographics , clinic site , history of cvd , current smoking , and treatment with statins and thiazolidinediones ( tzds ) . treatment effect ( ili ) and statin and insulin use ( tested separately ) were evaluated in the full model with the use of interaction terms to evaluate whether either pharmacological therapy modulated the association of ili with change in hs - crp . because of the change in age eligibility criteria during the 2nd year of recruitment in look ahead , subject characteristics in this ancillary study differ slightly from those of the remaining participants ; 12% had cvd and 40% used statins , compared with 15 and 45% , respectively , for the remainder of look ahead enrollees ( 11 ) . self - reported prior myocardial infarction , stroke , transient ischemic attack , angioplasty / stent , coronary artery bypass graft , carotid endarterectomy , abdominal aortic aneurysm , or heart failure . as reported for the overall look ahead sample ( 10 ) , subjects in the ili arm in this study had significant improvements in glucose control and weight loss at 1 year compared with those in the dse arm . subjects in the ili arm had mean weight and bmi reductions of 9 kg and 3.2 kg / m ( 8.8% of baseline ) , respectively , compared with respective reductions of 0.8 kg and 0.3 kg / m ( 0.8% of baseline ) in the dse arm ( p < 0.001 ) . ili participants had a greater improvement in fitness , with a 19% increase from baseline compared with a 5.9% increase in the dse arm ( p < 0.001 ) ( table 2 ) . median hs - crp at 1 year dropped by 43.6% from baseline in the ili group , compared with a 16.7% decrease in the dse group ( p < 0.001 for difference in median change between ili and dse ) ( table 2 ) . women , who had a higher hs - crp level than men at baseline , had a greater absolute change in median hs - crp level with ili at 1 year but a similar proportional drop in hs - crp levels compared with that in men ( table 2 ) . quartiles of change representing greater improvements in adiposity , fitness , and glucose and lipid control were associated with greater decreases in hs - crp . change in hs - crp ( median and iqr ) at 1 year in the dse arm vs. the ili arm by quartiles ( q ) of change in bmi ( top ) and a1c ( bottom ) . change in hs - crp is a proportional change , with numbers < 1.0 indicating a decrease in hs - crp and those > 1.0 an increase in hs - crp and the horizontal line representing overall mean change . regression analysis , accounting for potential differences between treatment arms in demographics , smoking , and tzd and statin use , among others , confirmed that each of the ili - induced improvements in adiposity ( bmi , weight , and waist circumference ) , glucose control ( a1c and fasting glucose ) , triglycerides , hdl cholesterol , and fitness predicted a decrease in hs - crp at 1 year ( analyzed as log hs - crp , p < 0.001 for all ) ( table 3 , models b j ) . change in waist circumference with ili contributed slightly less to hs - crp change ( r = 0.096 ) than did change in weight ( r = 0.114 ) or change in bmi ( r = 0.115 ) with ili . interestingly , the improvement in glucose control with ili contributed to hs - crp change to an extent similar to that for the reduction in adiposity ( r = 0.112 and 0.100 for fasting glucose and a1c , respectively ) . improvements in fitness with ili explained slightly less ( r = 0.086 ) of the variance in hs - crp change at 1 year than did changes in adiposity or glucose control . both change in hdl cholesterol and change in triglyceride levels , but not change in ldl cholesterol , predicted hs - crp change with ili at 1 year . metabolic variables as predictors of hs - crp change with 1-year ili using multiple variable regression analysis each model ( a q ) was analyzed independently and adjusted for age , sex , ethnicity , clinic site , history of cvd , smoking , and tzd and statin use . when change in fitness was evaluated in the regression model with change in a1c ( model k ) , we found that each predicted hs - crp change . however , when change in bmi was added to the model ( model l ) , fitness was no longer a significant predictor , suggesting that the change in adiposity associated with improved fitness partially explained the decline in hs - crp with ili at 1 year . on the other hand , when change in bmi or change in fitness was added to a model containing hdl cholesterol ( models m and o , respectively ) or triglycerides ( models n and p , respectively ) , both lipid variables remained significant predictors of hs - crp change . a final model ( model q ) , including changes in bmi , a1c , hdl cholesterol , triglycerides , and fitness , revealed that , of the metabolic variables studied , only improvements in glucose control and adiposity could independently account for the decrease in hs - crp at 1 year ( p < 0.001 ) . the beneficial effects of changes in fitness , hdl cholesterol , and triglycerides on hs - crp were weakened and no longer statistically significant ( p = 0.095 , 0.106 , and 0.068 , respectively ) when tested in the full model . statin and insulin use did not modify the association of ili and hs - crp when each was tested separately with the use of an interaction term ( statin use ili and insulin use ili ) in the full model ( p = 0.43 and 0.50 , respectively ) . because of the change in age eligibility criteria during the 2nd year of recruitment in look ahead , subject characteristics in this ancillary study differ slightly from those of the remaining participants ; 12% had cvd and 40% used statins , compared with 15 and 45% , respectively , for the remainder of look ahead enrollees ( 11 ) . self - reported prior myocardial infarction , stroke , transient ischemic attack , angioplasty / stent , coronary artery bypass graft , carotid endarterectomy , abdominal aortic aneurysm , or heart failure . as reported for the overall look ahead sample ( 10 ) , subjects in the ili arm in this study had significant improvements in glucose control and weight loss at 1 year compared with those in the dse arm . subjects in the ili arm had mean weight and bmi reductions of 9 kg and 3.2 kg / m ( 8.8% of baseline ) , respectively , compared with respective reductions of 0.8 kg and 0.3 kg / m ( 0.8% of baseline ) in the dse arm ( p < 0.001 ) . ili participants had a greater improvement in fitness , with a 19% increase from baseline compared with a 5.9% increase in the dse arm ( p < 0.001 ) ( table 2 ) . median hs - crp at 1 year dropped by 43.6% from baseline in the ili group , compared with a 16.7% decrease in the dse group ( p < 0.001 for difference in median change between ili and dse ) ( table 2 ) . women , who had a higher hs - crp level than men at baseline , had a greater absolute change in median hs - crp level with ili at 1 year but a similar proportional drop in hs - crp levels compared with that in men ( table 2 ) . quartiles of change representing greater improvements in adiposity , fitness , and glucose and lipid control were associated with greater decreases in hs - crp . change in hs - crp ( median and iqr ) at 1 year in the dse arm vs. the ili arm by quartiles ( q ) of change in bmi ( top ) and a1c ( bottom ) . change in hs - crp is a proportional change , with numbers < 1.0 indicating a decrease in hs - crp and those > 1.0 an increase in hs - crp and the horizontal line representing overall mean change . regression analysis , accounting for potential differences between treatment arms in demographics , smoking , and tzd and statin use , among others , confirmed that each of the ili - induced improvements in adiposity ( bmi , weight , and waist circumference ) , glucose control ( a1c and fasting glucose ) , triglycerides , hdl cholesterol , and fitness predicted a decrease in hs - crp at 1 year ( analyzed as log hs - crp , p < 0.001 for all ) ( table 3 , models b j ) . change in waist circumference with ili contributed slightly less to hs - crp change ( r = 0.096 ) than did change in weight ( r = 0.114 ) or change in bmi ( r = 0.115 ) with ili . interestingly , the improvement in glucose control with ili contributed to hs - crp change to an extent similar to that for the reduction in adiposity ( r = 0.112 and 0.100 for fasting glucose and a1c , respectively ) . improvements in fitness with ili explained slightly less ( r = 0.086 ) of the variance in hs - crp change at 1 year than did changes in adiposity or glucose control . metabolic variables as predictors of hs - crp change with 1-year ili using multiple variable regression analysis each model ( a q ) was analyzed independently and adjusted for age , sex , ethnicity , clinic site , history of cvd , smoking , and tzd and statin use . when change in fitness was evaluated in the regression model with change in a1c ( model k ) , we found that each predicted hs - crp change . however , when change in bmi was added to the model ( model l ) , fitness was no longer a significant predictor , suggesting that the change in adiposity associated with improved fitness partially explained the decline in hs - crp with ili at 1 year . on the other hand , when change in bmi or change in fitness was added to a model containing hdl cholesterol ( models m and o , respectively ) or triglycerides ( models n and p , respectively ) , both lipid variables remained significant predictors of hs - crp change . a final model ( model q ) , including changes in bmi , a1c , hdl cholesterol , triglycerides , and fitness , revealed that , of the metabolic variables studied , only improvements in glucose control and adiposity could independently account for the decrease in hs - crp at 1 year ( p < 0.001 ) . the beneficial effects of changes in fitness , hdl cholesterol , and triglycerides on hs - crp were weakened and no longer statistically significant ( p = 0.095 , 0.106 , and 0.068 , respectively ) when tested in the full model . statin and insulin use did not modify the association of ili and hs - crp when each was tested separately with the use of an interaction term ( statin use ili and insulin use ili ) in the full model ( p = 0.43 and 0.50 , respectively ) . this report contributes information on the effects of a 1-year lifestyle intervention for weight loss on hs - crp in the setting of what is , to our knowledge , the largest randomized clinical trial of its kind in individuals with type 2 diabetes . our report showed that in obese men and women with type 2 diabetes , 1 year of lifestyle intervention ( in addition to usual care ) , which led to an 8.8% reduction in baseline weight and a 0.7% drop in a1c , resulted in a 43.6% decrease in median hs - crp , whereas usual care alone , which led to reductions of 0.8% in baseline weight and 0.2% in a1c , resulted in a 16.7% decrease in median hs - crp . in a 2-year interventional study in 160 obese women without diabetes , they reported a 14% decrease in mean weight and a 34% decrease in median hs - crp from baseline ( compared with decreases of 3 and 9% , respectively , in the control group ) . in the diabetes prevention program ( dpp ) , in which 1,000 of > 3,000 obese participants at risk for diabetes were randomly assigned to a lifestyle intervention arm , behavioral changes in physical activity and diet resulted in a 7.2% decrease in baseline weight and 30% decrease in median hs - crp at 1 year ; hs - crp in the placebo group increased by 5% in men and did not change in women ( 6 ) . the few studies that investigated the effects of weight loss on hs - crp in individuals with diabetes were small , achieved minimal weight reductions , and did not adjust for changes in both fitness and glucose control ( 13 ) . our study indicates that moderate weight loss in obese individuals with type 2 diabetes is associated with a substantial reduction in hs - crp levels and that decreased adiposity is an independent predictor of hs - crp reduction after accounting for improvements in fitness , glucose , and lipid control . dpp evaluated physical activity , obtained from participant self - report , and concluded that weight loss , not physical activity , accounted for the changes in hs - crp at 1 year ; however , fitness was not assessed ( 6 ) . our study showed that the moderate improvement in fitness observed with ili in our generally obese and sedentary participants with type 2 diabetes was associated with a reduction in hs - crp , but the effects were attenuated ( p = 0.06 ) when weight loss was taken into account . our findings do not exclude the possibility that greater changes in fitness could have a stronger effect on hs - crp change or that the same change in fitness in less obese individuals with diabetes could be associated with hs - crp change independently of weight loss . it was interesting to find that the associations between improvements in hdl cholesterol and triglyceride levels and the decrease in hs - crp with ili were independent of improved fitness , glucose control , and weight loss . elevated levels of hs - crp have been found in individuals with familial hypoalphalipoproteinemia , in whom hdl cholesterol levels are low and the risk of coronary disease is high ( 22 ) . in our study , the effects of hdl cholesterol and triglyceride change on hs - crp variance were attenuated and no longer significant when both were included in the same model ( model q ) . the report suggested that the deleterious effect of insulin therapy on the inflammatory state could explain the lack of benefit of improved glycemic control on incident cardiovascular events found in recent clinical trials . the favorable association of improved glycemia and hs - crp change was independent of changes in adiposity and persisted after accounting for multiple covariates , including statin and tzd use , and was not affected by changes in insulin therapy with ili ( p = 0.50 ) . our results , in agreement with a previous small study in subjects with diabetes ( 13 ) and with experimental evidence linking hyperglycemia with increased cytokine production , indicate that improved glucose control per se does not worsen the inflammatory state in individuals with diabetes . a cross - sectional observation in which the correlation between glycemia and hs - crp was not significant after adjustment for bmi ( 3 ) seems to contradict the robust effect of improved glucose control with ili on hs - crp observed in our study . lowering glucose with improved dietary and physical activity behaviors , as observed in look ahead , may reflect the disruption of the paracrine loop between adipocytes and macrophages that promotes inflammation , both locally and systemically , and insulin resistance ( 25 ) . our report supports a substantial benefit of lifestyle intervention for weight loss on the chronic inflammatory state characteristic of diabetes and highlights the contribution of improved glycemic control achieved with lifestyle changes to the reduction of elevated hs - crp levels in obese sedentary individuals with diabetes .

in 2014 , researchers and medical personnel celebrated the 25th anniversary of the discovery of hepatitis c virus ( hcv ) . the first publication on the new virus genome appeared in 1989.1 after that , comprehensive study of new hcv and related disease began , including diagnostics , pathogenesis , and epidemiology . the official registration of acute hcv ( ahcv ) infection in the russian federation began in 1994 . the standard case definition for ahcv was adapted from world health organization recommendations.2 it is necessary to diagnose ahcv based on epidemiological and clinicobiochemical findings , such as the presence of newly identified markers of hcv antibodies to hcv ( anti - hcv ) and hcv rna . anti - hcv detection has a special diagnostic value for acute hepatitis discrimination in disease dynamic ( after 46 weeks ) after negative results of this marker in the early stage of the disease . the presence of hcv rna in the phase of serological window is an important diagnostic criterion . five years later ( 1999 ) , the registration of chronic hcv ( chcv ) cases also began in the russian federation . moreover , since the beginning , all laboratories dealing with virology and clinical diagnostics reported on the testing for anti - hcv and registered all positive results . the aim of the current review was to summarize available epidemiological data and to reveal the main manifestation of the epidemic process of hcv infection in the russian federation in the modern period . having a territory of 17,098,246 km , russia is the largest country in the world , covering more than one - eighth of the earth s inhabited land area . russia is also the world s ninth most populous nation with 143 million people as of 2012 . according to the national constitution,3 the country comprises 85 federal subjects , including 46 oblasts ( provinces ) , 22 republics , and nine krais ( same as oblasts ) . designation is historic , originally given to frontier regions and later also to administrative units that comprised autonomous all federal subjects are grouped into eight federal regions on geographical and economical principles : north - west , central , southern , north - caucasus , volga , ural , siberian , far east . it is important to analyze all types of infection by territories to define the scale of disease distribution and risk regions . we carried out the retrospective epidemiological analysis of morbidity rate ( the incidence per 100,000 ) for ahcv and chcv infections . more than 200,000 cases of ahcv infection and 700,000 cases of chcv infection that were registered in 19942013 in the country were included in the epidemiological analysis . we studied the incidence by year , distribution among different federal territories , age group , risk group , the main route of transmission , and the factors of risk . for analysis , data from the state statistical accounts4,5 of infectious morbidity rate ( incidence ) in the russian federation and analytical tables sent from the territories for ahcv and chcv were used . for chronic cases , the mean incidence of newly detected patients who were registered in the current year was calculated . for chcv cases , the prevalence rates for different years , age groups , and territories were also calculated . this article also contains characteristics of hcv distribution in the russian federation based on the results of testing of different risk groups for anti - hcv in 20112013 . for testing of the risk groups for anti - hcv , commercial hcv genotyping tests have been available in the russian federation since 2000 . in this review the dynamics of ahcv cases in russia during 19942013 could be characterized by different trends ( figure 1 ) , in different periods . for example , in 19942001 , the epidemic curve rose , which reflected the active spread of hcv and increase in acute clinical cases . the maximum incidence rate was seen in 2001 reaching 22.2/100,000 . from 2001 up to now , the epidemic curve looks like a descending line that characterizes the annual decrease in incidence due to a significant reduction of hcv infection . in general , dynamics of ahcv incidence in different federal territories of russia was absolutely synchronous during 19942013 . the difference was only in the level of incidence and the year of maximal incidence . during 19992000 , the maximal incidence was registered in north - west ( 44.5/100,000 ) and ural ( 37.0/100,000 ) federal territories ( figure 2 ) . meaning that both territories had similar active risk factors for virus spread . after 2007 , the maximum incidence was reported by ural with comparatively high rates reaching 3.33.5/100,000 . minimal ahcv incidence rates were reported by provinces located in the southern federal region during all years . in these territories , the maximum of ahcv reported was registered in 2001 ( 10/100,000 ) , ie , three to four times less than in north - west and ural . since the middle of 2000 the incidence data are available only for the past 4 years and rates were lower than in all other federal regions during this period ( 20102013 ) . during all years , the ahcv incidence in children aged 014 years was significantly lower in comparison with rates in teenagers ( 1519 years ) and adults . for instance , in the period of active hcv spread in 19992000 , the national incidence of ahcv in persons older than 15 years was 25/100,000 , but in children less than 14 years this rate was only 3/100,000 . in the present time , the age distribution of ahcv is characterized by very low incidence ( < 1/100,000 ) in children and in adults older than 50 years . it is important to note that the third ranking position in the recorded incidence of hcv among children younger than 14 years of age takes into account children up to 1 year . in 2009 , the incidence rate in this age group was 4.2/100,000 , in 2012 2.8/100,000 , and in 2013 , it fell to 1.5/100,000 . it should be noted that the proportion of children younger than 1 year in all cases of ahcv among children younger than 14 years does not fall < 50% in recent years . it should be emphasized that the diagnosis of a child younger than 1 year can be established only with the appropriate clinical and biochemical parameters and viral rna detection . . the maximal level of ahcv in 2013 was registered in two age groups ; 2029 years and 3039 years ( figure 3 ) . owing to such age distribution of ahcv cases , it is important to analyze the possible routes of virus transmission . the information on routes of hcv transmission / risk factors is summarized in figure 4 and includes data on 6,800 patients registered in 20112013 as ahcv cases . in children aged 014 years , the principal significance has been the transmission of hcv from the mother with chcv to her child during pregnancy or labor . the analysis of data about likely routes routes of hcv infection and risk factors the 3 year period showed that the main route of transmission in children younger than 14 years is vertical , which is caused by children younger than 1 year . these materials confirm that the problem of hcv vertical transmission exists on site and medical professional should pay attention to it . in adults the most important risk factor is sexual contact with probable source of infection ( 32.6% ) ; the second significant risk was connected with sharing needles and syringes during intravenous drug use ( 21.7% ) ; and the third risk factor was mainly related to cosmetic procedures , including tattooing and piercing ( 7.6% ) . it is important to note that in 37.5% of patients , risk factors for hcv transmission remained unknown . this percentage of patients was more than among the children 014 years ( 21.1% ) . the poorest results of epidemiological investigations were in adults older than 40 years because risk factors were not determined in 66.7% of cases . in this age group , the most significant risk factors were sexual contacts ( 17.7% ) and cosmetic procedures ( 9.0% ) . it should be emphasized that drug use as a risk factor in this age group was reported only in 3.8% of cases almost six times less than in the younger adult group of 2039 years . in general , medical manipulations did not play any visible role in hcv transmission in the modern time . the biggest proportion of this risk factor reaching 2.9% was found in persons older than 40 years . thus , it is important to emphasize , probable risk factors of hcv transmission in children were vertical transmission , involving children aged younger than 1 year in the russian federation in 20112013 . it required careful justification using modern molecular - biological methods of diagnosis , and the obtained data confirm that the problem of hcv vertical transmission existed in russia . in adults , the most significant risk factor was intravenous drug use in the age group of 2039 years . iatrogenic transmission played a minimal role and was more visible in the group of persons older than 40 years . the study of hcv routes of transmission and risk factors among children aged 014 years and adults is only preliminary , and requires detailed analysis over a longer period of time . the dynamics of registration of newly diagnosed chcv cases , unlike ahcv , is characterized by an ascending trend since 1999 . the registration rate ( incidence ) for chcv increased in the russian federation from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times ( figure 1 ) . in general , the morbidity rate maintained at 3940/100,000 in the russian federation since 2008 . probably meaning that the maximum detection of new chcv cases is already reached . the dynamic of newly detected chcv cases in different federal territories varied significantly by rates and trends of morbidity . in three federal territories ( far east , ural , and north - west ) , the incidence of chcv reached 47.2/100,000 ( far east ) , 48.3/100,000 ( ural ) , and 67.8/100,000 ( north - west ; figure 5 ) . the registration curve in the north - west has manifested ascending type , but in the ural , the increase was interrupted in 2009 and then the decreasing trend became clear . during the last 2 years ( 20122013 ) , chcv registration the same as in ural tendency was also registered in the far east territory . trends of chcv registration in the volga and siberia federal territories are similar to those in the whole country and could be characterized as ascending . in 2013 , three other federal territories had chcv level less than the level in the whole country , but dynamic curves look different . for instance , the trend in the central federal territory was ascending with chcv incidence in 2013 equal to 32.6/100,000 . the curve in the southern territory could be described as plateaued with weak tendency to decrease ( 22.9/100,000 in 2013 ) . rates in north - caucasus ( data available for analysis only for the last 4 years ) were stable , 1213/100,000 . the age distribution of newly registered chcv cases is characterized by maximal rates in the age group 2049 years ( figure 6 ) . the highest incidence reaching 96.3/100,000 two age groups ( 2029 years and 4049 years old ) have shown the same rates 53/100,000 . such age distribution of chronic cases reflects the epidemic situation of hcv spread at the end of 1990s . these data support the idea that hcv is not a children s infection . in comparison with newly detected chcv cases ( incidence ) , the disease prevalence ( accumulated cases ) gives more complete vision of epidemiological situation in the territory . in 2013 , the chcv prevalence in the country reached 335.8/100,000 ( figure 7 ) . the highest chcv prevalence was registered in the north - west region , ural , and far east ( 670.4/100,000 , 587.5/100,000 , and 585.6/100,000 , respectively ) . the chcv prevalence in the volga federal region was higher than the average country level ( 425.9/100,000 ) and in the remaining four regions , it was lower than the prevalence found in the whole country ( range from 243.8/100,000 in siberia to 142.4/100,000 in north - caucasus ) . in most administrative territories , the maximal chcv prevalence was reported in the age group 3039 years in 2013 ( figure 8) . this correlates with ahcv age - specific incidence rates in 19992000 showing the highest levels in the age group 1529 years . in age - specific structure of prevalence in all the main territories in the country , the proportion of persons in the age group 3039 years was the highest among all the chcv cases . the average proportion of this age group was 24% in the whole country with fluctuations from 20% to 29.5% . the far east and north - caucasus regions could be excluded from the mentioned common age - specific structure because in these territories , the second rank among chcv patients belonged to persons in the age group 4049 years . in other territories , the persons of age group 4049 years had only third rank with rates varying from 17% to 23% . the proportion of children up to and including 14 years of age and teenagers 1519 years of age with chcv did not exceed 3% in total . age - specific rates of chcv designated as prevalence per 100,000 of population look unlike the age structure of cases . in 2013 , the highest prevalence was reported in the age group 3039 years . the average prevalence in this age group in the country reached 684/100,000 , but in three regions , these rates were even higher : ural region 1,257/100,000 , north - west region 1,179/100,000 , and volga region 944/100,000 . such prevalence means that 1% of the population in this age group was infected with hcv . the second rank in half of the territories ( north - west , far east , south , north - caucasus ) in 2013 belonged to adults 4049 years of age , but in other four territories ( ural , volga , siberia , and central ) to the age group of 2029 years . such distribution of territories by age - specific prevalence gives evidence concerning intensity of involvement of different age groups in hcv transmission during previous years . it should be noted that the chcv prevalence in the age group 3039 years was maximal in ural and north - west regions . at the same time , the second rank in the ural region belonged to the age group of 2029 years ( 842/100,000 ) , but in the north - west region to the age group of 4049 years ( 878/100,000 ) . it is logical to assume that in the ural region , the intensive spread of hcv took place in younger persons than in the north - west region in previous years . the fourth rank by chcv prevalence belonged to persons of 5059 years of age , but with different levels of rates . relatively high levels similar to the average rate in the whole territory in the age group of 5059 years were demonstrated by north - west ( 567/100,000 ) , far east ( 531/100,000 ) , and south ( 298/100,000 ) regions . annually , 1516 million tests for anti - hcv are performed in 20 different population risk groups in the russian federation . four risk groups had the highest anti - hcv rates in 20112012 ( figure 9 ) : newborns from chcv infected women ( 10.3%14.3% ) , persons from correctional facilities ( 11.8%10.6% ) , patients with chronic liver diseases ( 6.9%6.1% ) , and clients from clinics for sexually transmitted disease patients and drug users ( 5.8%5.2% ) . relatively high anti - hcv rates were detected in patients from hemodialysis settings , units of renal transplantation , cardiovascular and lung surgery , and hematology ( 2.6%2.9% ) . this is rather a big group of patients who are under high risk of hcv transmission . in this group , special attention in one independent study revealed that 25%40% of patients in hemodialysis units were anti - hcv positive.6 also patients with different kinds of chronic pathology had high proportion of anti - hcv positivity reaching 2.9%3.1% . relatively high anti - hcv rates were detected in household contacts of chcv patients ( 1.8%1.9% ) and in patients who were admitted to hospital for planned surgery ( 1.6% ) . pregnant women infected with hcv represent a special risk group because of possible virus transmission to newborns . overall , in the country , anti - hcv was detected in 1.3% of pregnant women in 20112012 . this rate was similar to the level in patients admitted to hospital for planned surgery or in household contact with chcv patients . it could be suggested that anti - hcv rate of 1% reflected an average frequency of chronic asymptomatic hcv infection in the population of the russian federation . in different territories , for example , high anti - hcv rates ( > 2% ) were reported in 15 territories of the country in 2010 . these territories also reported chcv incidence and prevalence rates higher than the average rate in the country . it can indirectly indicate the existence of correlation between frequency of anti - hcv and chcv prevalence . possibly , the rising probability of transmitting hcv from mothers to their newborn children can not be excluded . it is also very interesting to analyze results of anti - hcv tests in persons admitted to hospital for planned surgery . in 20112013 , it is important to note that this index was two times higher than the frequency of hepatitis b surface antigen ( hbsag ) in the same group of patients . perhaps , it can be explained by accumulation of more patients chronically infected with hcv in comparison with patients with chronic hepatitis b virus ( hbv ) infection because of the higher potential of hcv to produce chronic infection . in 20112013 , the highest levels of anti - hcv in planned surgery patients were found in the central federal territory ( 1.7% ) , far east ( 2.1% ) , and ural ( 3.3% ) . one of the first important studies concerning hcv genotypes distribution in russia was published by researchers from moscow institute of virology in 1996.7 they demonstrated that five virus subtypes ( 1a , 1b , 2a , 2b , and 3a ) circulated in the russian federation with significant domination of hcv1b ( 68.9% ) . these data were confirmed in 1997 by another independent group that revealed hcv1b subtype in 76% of hcv patients who were tested.8 a special study of hcv genotypes in st petersburg in 19992000 showed statistically significant differences in hcv subtype distribution between hcv cases in patients attending infectious diseases clinics and in dialysis patients.6 five subtypes ( 3a , 1b , 1a , 2a , and 4a [ in order of frequency ] ) were found in outpatients , while three subtypes ( 1b , 3a , and 2c ) circulated in dialysis patients . subtype 3a was the dominant subtype in outpatients ( 51% of cases ) , followed by subtype 1b ( 40% of cases ) . it was noted that in st petersburg , 56% of patients with hcv who attended infectious disease clinics were between 11 years and 20 years of age , 63% of whom were infected with subtype 3a and only 33% with subtype 1b . however , among patients older than 20 years , subtype 1b was prevalent ( 49% ) , followed by subtype 3a ( 36% ) . these data were also confirmed by other authors.9 subtype 1b was dominant in all dialysis patients and accounted for 89% of cases . later , researchers from st petersburg demonstrated the recombination of natural intergenotypes between subtypes 2k and 1b , designated as rf_2k/1b.10 the finding of a 2k/1b recombinant strain in 5% of hcv - infected patients in st petersburg has shown that this particular recombinant hcv strain was not only viable but had also spread in the city . this hcv strain was also revealed in different regions of russia and other countries evidenced its wide spread.1114 the latest data on distribution of hcv genotypes published in 2012 confirmed results of previous research,15 ie , the domination of hcv1b subtype in general population ( 57.1% ) as in hemodialysis patients ( 83.6% ) . the second rank belonged to hcv3a in both studied groups ( 29.7% and 16.4% , respectively ) . the dynamics of ahcv cases in russia during 19942013 could be characterized by different trends ( figure 1 ) , in different periods . for example , in 19942001 , the epidemic curve rose , which reflected the active spread of hcv and increase in acute clinical cases . the maximum incidence rate was seen in 2001 reaching 22.2/100,000 . from 2001 up to now , the epidemic curve looks like a descending line that characterizes the annual decrease in incidence due to a significant reduction of hcv infection . in general , dynamics of ahcv incidence in different federal territories of russia was absolutely synchronous during 19942013 . the difference was only in the level of incidence and the year of maximal incidence . during 19992000 , the maximal incidence was registered in north - west ( 44.5/100,000 ) and ural ( 37.0/100,000 ) federal territories ( figure 2 ) . meaning that both territories had similar active risk factors for virus spread . after 2007 , the maximum incidence was reported by ural with comparatively high rates reaching 3.33.5/100,000 . minimal ahcv incidence rates were reported by provinces located in the southern federal region during all years . in these territories , the maximum of ahcv reported was registered in 2001 ( 10/100,000 ) , ie , three to four times less than in north - west and ural . since the middle of 2000 , the incidence data are available only for the past 4 years and rates were lower than in all other federal regions during this period ( 20102013 ) . during all years , the ahcv incidence in children aged 014 years was significantly lower in comparison with rates in teenagers ( 1519 years ) and adults . for instance , in the period of active hcv spread in 19992000 , the national incidence of ahcv in persons older than 15 years was 25/100,000 , but in children less than 14 years this rate was only 3/100,000 . in the present time , the age distribution of ahcv is characterized by very low incidence ( < 1/100,000 ) in children and in adults older than 50 years . it is important to note that the third ranking position in the recorded incidence of hcv among children younger than 14 years of age takes into account children up to 1 year . in 2009 , the incidence rate in this age group was 4.2/100,000 , in 2012 2.8/100,000 , and in 2013 , it fell to 1.5/100,000 . it should be noted that the proportion of children younger than 1 year in all cases of ahcv among children younger than 14 years does not fall < 50% in recent years . it should be emphasized that the diagnosis of a child younger than 1 year can be established only with the appropriate clinical and biochemical parameters and viral rna detection . . the maximal level of ahcv in 2013 was registered in two age groups ; 2029 years and 3039 years ( figure 3 ) . owing to such age distribution of ahcv cases , it is important to analyze the possible routes of virus transmission . the information on routes of hcv transmission / risk factors is summarized in figure 4 and includes data on 6,800 patients registered in 20112013 as ahcv cases . in children aged 014 years , the principal significance has been the transmission of hcv from the mother with chcv to her child during pregnancy or labor . the analysis of data about likely routes routes of hcv infection and risk factors the 3 year period showed that the main route of transmission in children younger than 14 years is vertical , which is caused by children younger than 1 year . these materials confirm that the problem of hcv vertical transmission exists on site and medical professional should pay attention to it . in adults the most important risk factor is sexual contact with probable source of infection ( 32.6% ) ; the second significant risk was connected with sharing needles and syringes during intravenous drug use ( 21.7% ) ; and the third risk factor was mainly related to cosmetic procedures , including tattooing and piercing ( 7.6% ) . it is important to note that in 37.5% of patients , risk factors for hcv transmission remained unknown . this percentage of patients was more than among the children 014 years ( 21.1% ) . the poorest results of epidemiological investigations were in adults older than 40 years because risk factors were not determined in 66.7% of cases . in this age group , the most significant risk factors were sexual contacts ( 17.7% ) and cosmetic procedures ( 9.0% ) . it should be emphasized that drug use as a risk factor in this age group was reported only in 3.8% of cases almost six times less than in the younger adult group of 2039 years . in general , medical manipulations did not play any visible role in hcv transmission in the modern time . the proportion of medical manipulations as risk factor was only 1% of cases . the biggest proportion of this risk factor reaching 2.9% was found in persons older than 40 years . thus , it is important to emphasize , probable risk factors of hcv transmission in children were vertical transmission , involving children aged younger than 1 year in the russian federation in 20112013 . it required careful justification using modern molecular - biological methods of diagnosis , and the obtained data confirm that the problem of hcv vertical transmission existed in russia . in adults , the most significant risk factor was intravenous drug use in the age group of 2039 years . iatrogenic transmission played a minimal role and was more visible in the group of persons older than 40 years . the study of hcv routes of transmission and risk factors among children aged 014 years and adults is only preliminary , and requires detailed analysis over a longer period of time . the dynamics of registration of newly diagnosed chcv cases , unlike ahcv , is characterized by an ascending trend since 1999 . the registration rate ( incidence ) for chcv increased in the russian federation from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times ( figure 1 ) . in general , the morbidity rate maintained at 3940/100,000 in the russian federation since 2008 . probably meaning that the maximum detection of new chcv cases is already reached . the dynamic of newly detected chcv cases in different federal territories varied significantly by rates and trends of morbidity . in three federal territories ( far east , ural , and north - west ) , the chcv incidence was higher than the average level in the country . the incidence of chcv reached 47.2/100,000 ( far east ) , 48.3/100,000 ( ural ) , and 67.8/100,000 ( north - west ; figure 5 ) . the registration curve in the north - west has manifested ascending type , but in the ural , the increase was interrupted in 2009 and then the decreasing trend became clear . during the last 2 years ( 20122013 ) , chcv registration the same as in ural tendency was also registered in the far east territory . trends of chcv registration in the volga and siberia federal territories are similar to those in the whole country and could be characterized as ascending . in 2013 , three other federal territories had chcv level less than the level in the whole country , but dynamic curves look different . for instance , the trend in the central federal territory was ascending with chcv incidence in 2013 equal to 32.6/100,000 . the curve in the southern territory could be described as plateaued with weak tendency to decrease ( 22.9/100,000 in 2013 ) . rates in north - caucasus ( data available for analysis only for the last 4 years ) were stable , 1213/100,000 . the age distribution of newly registered chcv cases is characterized by maximal rates in the age group 2049 years ( figure 6 ) . the highest incidence reaching 96.3/100,000 two age groups ( 2029 years and 4049 years old ) have shown the same rates 53/100,000 . such age distribution of chronic cases reflects the epidemic situation of hcv spread at the end of 1990s . it should be noted that chcv incidence in children is very low one to three cases per 100,000 . these data support the idea that hcv is not a children s infection . in comparison with newly detected chcv cases ( incidence ) , the disease prevalence ( accumulated cases ) gives more complete vision of epidemiological situation in the territory . in 2013 , the highest chcv prevalence was registered in the north - west region , ural , and far east ( 670.4/100,000 , 587.5/100,000 , and 585.6/100,000 , respectively ) . the chcv prevalence in the volga federal region was higher than the average country level ( 425.9/100,000 ) and in the remaining four regions , it was lower than the prevalence found in the whole country ( range from 243.8/100,000 in siberia to 142.4/100,000 in north - caucasus ) . in most administrative territories , the maximal chcv prevalence was reported in the age group 3039 years in 2013 ( figure 8) . this correlates with ahcv age - specific incidence rates in 19992000 showing the highest levels in the age group 1529 years . in age - specific structure of prevalence in all the main territories in the country , the proportion of persons in the age group 3039 years was the highest among all the chcv cases . the average proportion of this age group was 24% in the whole country with fluctuations from 20% to 29.5% . the far east and north - caucasus regions could be excluded from the mentioned common age - specific structure because in these territories , the second rank among chcv patients belonged to persons in the age group 4049 years . in other territories , the persons of age group 4049 years had only third rank with rates varying from 17% to 23% . the proportion of children up to and including 14 years of age and teenagers 1519 years of age with chcv did not exceed 3% in total . age - specific rates of chcv designated as prevalence per 100,000 of population look unlike the age structure of cases . in 2013 , the highest prevalence was reported in the age group 3039 years . the average prevalence in this age group in the country reached 684/100,000 , but in three regions , these rates were even higher : ural region 1,257/100,000 , north - west region 1,179/100,000 , and volga region 944/100,000 . such prevalence means that 1% of the population in this age group was infected with hcv . the second rank in half of the territories ( north - west , far east , south , north - caucasus ) in 2013 belonged to adults 4049 years of age , but in other four territories ( ural , volga , siberia , and central ) to the age group of 2029 years . such distribution of territories by age - specific prevalence gives evidence concerning intensity of involvement of different age groups in hcv transmission during previous years . it should be noted that the chcv prevalence in the age group 3039 years was maximal in ural and north - west regions . at the same time , the second rank in the ural region belonged to the age group of 2029 years ( 842/100,000 ) , but in the north - west region to the age group of 4049 years ( 878/100,000 ) . it is logical to assume that in the ural region , the intensive spread of hcv took place in younger persons than in the north - west region in previous years . the fourth rank by chcv prevalence belonged to persons of 5059 years of age , but with different levels of rates . relatively high levels similar to the average rate in the whole territory in the age group of 5059 years were demonstrated by north - west ( 567/100,000 ) , far east ( 531/100,000 ) , and south ( 298/100,000 ) regions . annually , 1516 million tests for anti - hcv are performed in 20 different population risk groups in the russian federation . four risk groups had the highest anti - hcv rates in 20112012 ( figure 9 ) : newborns from chcv infected women ( 10.3%14.3% ) , persons from correctional facilities ( 11.8%10.6% ) , patients with chronic liver diseases ( 6.9%6.1% ) , and clients from clinics for sexually transmitted disease patients and drug users ( 5.8%5.2% ) . relatively high anti - hcv rates were detected in patients from hemodialysis settings , units of renal transplantation , cardiovascular and lung surgery , and hematology ( 2.6%2.9% ) . this is rather a big group of patients who are under high risk of hcv transmission . in this group , special attention in one independent study revealed that 25%40% of patients in hemodialysis units were anti - hcv positive.6 also patients with different kinds of chronic pathology had high proportion of anti - hcv positivity reaching 2.9%3.1% . relatively high anti - hcv rates were detected in household contacts of chcv patients ( 1.8%1.9% ) and in patients who were admitted to hospital for planned surgery ( 1.6% ) . pregnant women infected with hcv represent a special risk group because of possible virus transmission to newborns . overall , in the country , anti - hcv was detected in 1.3% of pregnant women in 20112012 . this rate was similar to the level in patients admitted to hospital for planned surgery or in household contact with chcv patients . it could be suggested that anti - hcv rate of 1% reflected an average frequency of chronic asymptomatic hcv infection in the population of the russian federation . in different territories , for example , high anti - hcv rates ( > 2% ) were reported in 15 territories of the country in 2010 . these territories also reported chcv incidence and prevalence rates higher than the average rate in the country . it can indirectly indicate the existence of correlation between frequency of anti - hcv and chcv prevalence . possibly , the rising probability of transmitting hcv from mothers to their newborn children can not be excluded . it is also very interesting to analyze results of anti - hcv tests in persons admitted to hospital for planned surgery . in 20112013 , it is important to note that this index was two times higher than the frequency of hepatitis b surface antigen ( hbsag ) in the same group of patients . perhaps , it can be explained by accumulation of more patients chronically infected with hcv in comparison with patients with chronic hepatitis b virus ( hbv ) infection because of the higher potential of hcv to produce chronic infection . in 20112013 , the highest levels of anti - hcv in planned surgery patients were found in the central federal territory ( 1.7% ) , far east ( 2.1% ) , and ural ( 3.3% ) . one of the first important studies concerning hcv genotypes distribution in russia was published by researchers from moscow institute of virology in 1996.7 they demonstrated that five virus subtypes ( 1a , 1b , 2a , 2b , and 3a ) circulated in the russian federation with significant domination of hcv1b ( 68.9% ) . these data were confirmed in 1997 by another independent group that revealed hcv1b subtype in 76% of hcv patients who were tested.8 a special study of hcv genotypes in st petersburg in 19992000 showed statistically significant differences in hcv subtype distribution between hcv cases in patients attending infectious diseases clinics and in dialysis patients.6 five subtypes ( 3a , 1b , 1a , 2a , and 4a [ in order of frequency ] ) were found in outpatients , while three subtypes ( 1b , 3a , and 2c ) circulated in dialysis patients . subtype 3a was the dominant subtype in outpatients ( 51% of cases ) , followed by subtype 1b ( 40% of cases ) . it was noted that in st petersburg , 56% of patients with hcv who attended infectious disease clinics were between 11 years and 20 years of age , 63% of whom were infected with subtype 3a and only 33% with subtype 1b . however , among patients older than 20 years , subtype 1b was prevalent ( 49% ) , followed by subtype 3a ( 36% ) . these data were also confirmed by other authors.9 subtype 1b was dominant in all dialysis patients and accounted for 89% of cases . later , researchers from st petersburg demonstrated the recombination of natural intergenotypes between subtypes 2k and 1b , designated as rf_2k/1b.10 the finding of a 2k/1b recombinant strain in 5% of hcv - infected patients in st petersburg has shown that this particular recombinant hcv strain was not only viable but had also spread in the city . this hcv strain was also revealed in different regions of russia and other countries evidenced its wide spread.1114 the latest data on distribution of hcv genotypes published in 2012 confirmed results of previous research,15 ie , the domination of hcv1b subtype in general population ( 57.1% ) as in hemodialysis patients ( 83.6% ) . the second rank belonged to hcv3a in both studied groups ( 29.7% and 16.4% , respectively ) . hcv continues to remain one of the main problems in the public health sector in the russian federation . currently , the manifestations of an epidemic process of hcv based on data of registers of acute and chronic forms of hcv has undergone significant changes . it was established that hcv epidemiological features are characterized by : low incidence of ahcv cases in all territories of the country ( from 22.2/100,000 in 2001 to 1.5/100,000 in 2013 , ie , more than 14 times); increase in registration ( incidence ) of newly detected chronic cases of infection ( from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times); a significant increase in cumulative rate of chcv cases ( prevalence ) that reached 335.8/100,00 and in most administrative territories , higher in specific age groups ( 3049 years); low hcv incidence and prevalence in children in comparison with adults; a rise in hcv rates in specific population groups ( newborns from chronically infected mothers , persons from correctional facilities , patients with chronic liver diseases , and clients from clinics for std patients and drug users); the use of molecular - genetic methods in epidemiological diagnostics for hcv , that will make it possible to reliably establish the source of the infection , routes , and factors of transmission ; and domination of hcv1b virus subtype and rising importance of hcv3a subtype and newly designated recombinant rf_2k/1b . low incidence of ahcv cases in all territories of the country ( from 22.2/100,000 in 2001 to 1.5/100,000 in 2013 , ie , more than 14 times ) ; increase in registration ( incidence ) of newly detected chronic cases of infection ( from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times ) ; a significant increase in cumulative rate of chcv cases ( prevalence ) that reached 335.8/100,00 and in most administrative territories , higher in specific age groups ( 3049 years ) ; low hcv incidence and prevalence in children in comparison with adults ; a rise in hcv rates in specific population groups ( newborns from chronically infected mothers , persons from correctional facilities , patients with chronic liver diseases , and clients from clinics for std patients and drug users ) ; the use of molecular - genetic methods in epidemiological diagnostics for hcv , that will make it possible to reliably establish the source of the infection , routes , and factors of transmission ; and domination of hcv1b virus subtype and rising importance of hcv3a subtype and newly designated recombinant rf_2k/1b . all the aforementioned data are extremely important for planning and implementation of primary , secondary , and tertiary preventive measures for the control of hcv infection in the russian federation .

hepatitis c virus ( hcv ) infection is one of the most prevalent health problems in the world . official registration of hcv infections in the russian federation started in 1994 . two clinical forms of infection acute and chronic hepatitis c are registered separately . moreover , the hcv national surveillance system also includes reports from laboratories on results from testing 20 population risk groups for antibodies to hcv ; approximately 1516 million tests are performed annually . modern epidemiological features of hcv infection in the russian federation are characterized by low incidence of the acute form of infection ( acute hcv ; one to two per 100,000 ) and a dramatic increase in chronic hcv ( chcv ) cases . in 2013 , the average nationwide rate of newly detected chcv cases was 39.3/100,000 . in the same year , the prevalence of chcv demonstrating an accumulation of chronically infected patients in the country was much higher 335.8/100,000 . four risk groups were identified as greatly affected by hcv , which were demonstrated by a high prevalence of antibodies to hcv : newborns from chronically infected women , persons from correctional facilities , patients with chronic liver diseases , and clients from clinics for sexually transmitted disease patients and drug users . it was found that several hcv genotypes circulated in different regions of the country ; hcv1b had a prevalence of 55%80% in almost every part of the country . however , in st petersburg during the final decade of the last century and from 20012005 , hcv3a subtype expanded circulation among young people due to increased intravenous drug addiction . intravenous drug users were the major cause of a higher registration of double infection , with two different virus subtypes , and the appearance in russia of new recombinant virus rf_2k/1b . it can be concluded that chcv infection should be a focus of the health care system in russia because serious epidemics of liver cirrhosis and hepatocellular carcinoma will be seen in the near future that will require urgent preventive and therapeutic measures .

Introduction Materials and methods AHCV in the Russian Federation CHCV in the Russian Federation Frequency of anti-HCV in different risk groups Molecular epidemiology Discussion and conclusion

in 2014 , researchers and medical personnel celebrated the 25th anniversary of the discovery of hepatitis c virus ( hcv ) . the official registration of acute hcv ( ahcv ) infection in the russian federation began in 1994 . the standard case definition for ahcv was adapted from world health organization recommendations.2 it is necessary to diagnose ahcv based on epidemiological and clinicobiochemical findings , such as the presence of newly identified markers of hcv antibodies to hcv ( anti - hcv ) and hcv rna . five years later ( 1999 ) , the registration of chronic hcv ( chcv ) cases also began in the russian federation . the aim of the current review was to summarize available epidemiological data and to reveal the main manifestation of the epidemic process of hcv infection in the russian federation in the modern period . more than 200,000 cases of ahcv infection and 700,000 cases of chcv infection that were registered in 19942013 in the country were included in the epidemiological analysis . we studied the incidence by year , distribution among different federal territories , age group , risk group , the main route of transmission , and the factors of risk . for chronic cases , the mean incidence of newly detected patients who were registered in the current year was calculated . for chcv cases , the prevalence rates for different years , age groups , and territories were also calculated . this article also contains characteristics of hcv distribution in the russian federation based on the results of testing of different risk groups for anti - hcv in 20112013 . for testing of the risk groups for anti - hcv , commercial hcv genotyping tests have been available in the russian federation since 2000 . in this review the dynamics of ahcv cases in russia during 19942013 could be characterized by different trends ( figure 1 ) , in different periods . for example , in 19942001 , the epidemic curve rose , which reflected the active spread of hcv and increase in acute clinical cases . from 2001 up to now , the epidemic curve looks like a descending line that characterizes the annual decrease in incidence due to a significant reduction of hcv infection . for instance , in the period of active hcv spread in 19992000 , the national incidence of ahcv in persons older than 15 years was 25/100,000 , but in children less than 14 years this rate was only 3/100,000 . in the present time , the age distribution of ahcv is characterized by very low incidence ( < 1/100,000 ) in children and in adults older than 50 years . in 2009 , the incidence rate in this age group was 4.2/100,000 , in 2012 2.8/100,000 , and in 2013 , it fell to 1.5/100,000 . in adults the most important risk factor is sexual contact with probable source of infection ( 32.6% ) ; the second significant risk was connected with sharing needles and syringes during intravenous drug use ( 21.7% ) ; and the third risk factor was mainly related to cosmetic procedures , including tattooing and piercing ( 7.6% ) . thus , it is important to emphasize , probable risk factors of hcv transmission in children were vertical transmission , involving children aged younger than 1 year in the russian federation in 20112013 . it required careful justification using modern molecular - biological methods of diagnosis , and the obtained data confirm that the problem of hcv vertical transmission existed in russia . in adults , the most significant risk factor was intravenous drug use in the age group of 2039 years . the dynamics of registration of newly diagnosed chcv cases , unlike ahcv , is characterized by an ascending trend since 1999 . the registration rate ( incidence ) for chcv increased in the russian federation from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times ( figure 1 ) . in general , the morbidity rate maintained at 3940/100,000 in the russian federation since 2008 . the dynamic of newly detected chcv cases in different federal territories varied significantly by rates and trends of morbidity . in three federal territories ( far east , ural , and north - west ) , the incidence of chcv reached 47.2/100,000 ( far east ) , 48.3/100,000 ( ural ) , and 67.8/100,000 ( north - west ; figure 5 ) . during the last 2 years ( 20122013 ) , chcv registration the same as in ural tendency was also registered in the far east territory . the age distribution of newly registered chcv cases is characterized by maximal rates in the age group 2049 years ( figure 6 ) . in comparison with newly detected chcv cases ( incidence ) , the disease prevalence ( accumulated cases ) gives more complete vision of epidemiological situation in the territory . in 2013 , the chcv prevalence in the country reached 335.8/100,000 ( figure 7 ) . the chcv prevalence in the volga federal region was higher than the average country level ( 425.9/100,000 ) and in the remaining four regions , it was lower than the prevalence found in the whole country ( range from 243.8/100,000 in siberia to 142.4/100,000 in north - caucasus ) . in age - specific structure of prevalence in all the main territories in the country , the proportion of persons in the age group 3039 years was the highest among all the chcv cases . in 2013 , the highest prevalence was reported in the age group 3039 years . the average prevalence in this age group in the country reached 684/100,000 , but in three regions , these rates were even higher : ural region 1,257/100,000 , north - west region 1,179/100,000 , and volga region 944/100,000 . relatively high levels similar to the average rate in the whole territory in the age group of 5059 years were demonstrated by north - west ( 567/100,000 ) , far east ( 531/100,000 ) , and south ( 298/100,000 ) regions . annually , 1516 million tests for anti - hcv are performed in 20 different population risk groups in the russian federation . four risk groups had the highest anti - hcv rates in 20112012 ( figure 9 ) : newborns from chcv infected women ( 10.3%14.3% ) , persons from correctional facilities ( 11.8%10.6% ) , patients with chronic liver diseases ( 6.9%6.1% ) , and clients from clinics for sexually transmitted disease patients and drug users ( 5.8%5.2% ) . it could be suggested that anti - hcv rate of 1% reflected an average frequency of chronic asymptomatic hcv infection in the population of the russian federation . perhaps , it can be explained by accumulation of more patients chronically infected with hcv in comparison with patients with chronic hepatitis b virus ( hbv ) infection because of the higher potential of hcv to produce chronic infection . one of the first important studies concerning hcv genotypes distribution in russia was published by researchers from moscow institute of virology in 1996.7 they demonstrated that five virus subtypes ( 1a , 1b , 2a , 2b , and 3a ) circulated in the russian federation with significant domination of hcv1b ( 68.9% ) . these data were confirmed in 1997 by another independent group that revealed hcv1b subtype in 76% of hcv patients who were tested.8 a special study of hcv genotypes in st petersburg in 19992000 showed statistically significant differences in hcv subtype distribution between hcv cases in patients attending infectious diseases clinics and in dialysis patients.6 five subtypes ( 3a , 1b , 1a , 2a , and 4a [ in order of frequency ] ) were found in outpatients , while three subtypes ( 1b , 3a , and 2c ) circulated in dialysis patients . it was noted that in st petersburg , 56% of patients with hcv who attended infectious disease clinics were between 11 years and 20 years of age , 63% of whom were infected with subtype 3a and only 33% with subtype 1b . later , researchers from st petersburg demonstrated the recombination of natural intergenotypes between subtypes 2k and 1b , designated as rf_2k/1b.10 the finding of a 2k/1b recombinant strain in 5% of hcv - infected patients in st petersburg has shown that this particular recombinant hcv strain was not only viable but had also spread in the city . this hcv strain was also revealed in different regions of russia and other countries evidenced its wide spread.1114 the latest data on distribution of hcv genotypes published in 2012 confirmed results of previous research,15 ie , the domination of hcv1b subtype in general population ( 57.1% ) as in hemodialysis patients ( 83.6% ) . the dynamics of ahcv cases in russia during 19942013 could be characterized by different trends ( figure 1 ) , in different periods . for example , in 19942001 , the epidemic curve rose , which reflected the active spread of hcv and increase in acute clinical cases . from 2001 up to now , the epidemic curve looks like a descending line that characterizes the annual decrease in incidence due to a significant reduction of hcv infection . for instance , in the period of active hcv spread in 19992000 , the national incidence of ahcv in persons older than 15 years was 25/100,000 , but in children less than 14 years this rate was only 3/100,000 . in the present time , the age distribution of ahcv is characterized by very low incidence ( < 1/100,000 ) in children and in adults older than 50 years . in 2009 , the incidence rate in this age group was 4.2/100,000 , in 2012 2.8/100,000 , and in 2013 , it fell to 1.5/100,000 . in adults the most important risk factor is sexual contact with probable source of infection ( 32.6% ) ; the second significant risk was connected with sharing needles and syringes during intravenous drug use ( 21.7% ) ; and the third risk factor was mainly related to cosmetic procedures , including tattooing and piercing ( 7.6% ) . thus , it is important to emphasize , probable risk factors of hcv transmission in children were vertical transmission , involving children aged younger than 1 year in the russian federation in 20112013 . it required careful justification using modern molecular - biological methods of diagnosis , and the obtained data confirm that the problem of hcv vertical transmission existed in russia . in adults , the most significant risk factor was intravenous drug use in the age group of 2039 years . the dynamics of registration of newly diagnosed chcv cases , unlike ahcv , is characterized by an ascending trend since 1999 . the registration rate ( incidence ) for chcv increased in the russian federation from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times ( figure 1 ) . in general , the morbidity rate maintained at 3940/100,000 in the russian federation since 2008 . the dynamic of newly detected chcv cases in different federal territories varied significantly by rates and trends of morbidity . in three federal territories ( far east , ural , and north - west ) , the chcv incidence was higher than the average level in the country . during the last 2 years ( 20122013 ) , chcv registration the same as in ural tendency was also registered in the far east territory . the age distribution of newly registered chcv cases is characterized by maximal rates in the age group 2049 years ( figure 6 ) . it should be noted that chcv incidence in children is very low one to three cases per 100,000 . in comparison with newly detected chcv cases ( incidence ) , the disease prevalence ( accumulated cases ) gives more complete vision of epidemiological situation in the territory . in 2013 , the highest chcv prevalence was registered in the north - west region , ural , and far east ( 670.4/100,000 , 587.5/100,000 , and 585.6/100,000 , respectively ) . the chcv prevalence in the volga federal region was higher than the average country level ( 425.9/100,000 ) and in the remaining four regions , it was lower than the prevalence found in the whole country ( range from 243.8/100,000 in siberia to 142.4/100,000 in north - caucasus ) . in age - specific structure of prevalence in all the main territories in the country , the proportion of persons in the age group 3039 years was the highest among all the chcv cases . in 2013 , the highest prevalence was reported in the age group 3039 years . the average prevalence in this age group in the country reached 684/100,000 , but in three regions , these rates were even higher : ural region 1,257/100,000 , north - west region 1,179/100,000 , and volga region 944/100,000 . at the same time , the second rank in the ural region belonged to the age group of 2029 years ( 842/100,000 ) , but in the north - west region to the age group of 4049 years ( 878/100,000 ) . relatively high levels similar to the average rate in the whole territory in the age group of 5059 years were demonstrated by north - west ( 567/100,000 ) , far east ( 531/100,000 ) , and south ( 298/100,000 ) regions . annually , 1516 million tests for anti - hcv are performed in 20 different population risk groups in the russian federation . four risk groups had the highest anti - hcv rates in 20112012 ( figure 9 ) : newborns from chcv infected women ( 10.3%14.3% ) , persons from correctional facilities ( 11.8%10.6% ) , patients with chronic liver diseases ( 6.9%6.1% ) , and clients from clinics for sexually transmitted disease patients and drug users ( 5.8%5.2% ) . it could be suggested that anti - hcv rate of 1% reflected an average frequency of chronic asymptomatic hcv infection in the population of the russian federation . perhaps , it can be explained by accumulation of more patients chronically infected with hcv in comparison with patients with chronic hepatitis b virus ( hbv ) infection because of the higher potential of hcv to produce chronic infection . one of the first important studies concerning hcv genotypes distribution in russia was published by researchers from moscow institute of virology in 1996.7 they demonstrated that five virus subtypes ( 1a , 1b , 2a , 2b , and 3a ) circulated in the russian federation with significant domination of hcv1b ( 68.9% ) . these data were confirmed in 1997 by another independent group that revealed hcv1b subtype in 76% of hcv patients who were tested.8 a special study of hcv genotypes in st petersburg in 19992000 showed statistically significant differences in hcv subtype distribution between hcv cases in patients attending infectious diseases clinics and in dialysis patients.6 five subtypes ( 3a , 1b , 1a , 2a , and 4a [ in order of frequency ] ) were found in outpatients , while three subtypes ( 1b , 3a , and 2c ) circulated in dialysis patients . it was noted that in st petersburg , 56% of patients with hcv who attended infectious disease clinics were between 11 years and 20 years of age , 63% of whom were infected with subtype 3a and only 33% with subtype 1b . later , researchers from st petersburg demonstrated the recombination of natural intergenotypes between subtypes 2k and 1b , designated as rf_2k/1b.10 the finding of a 2k/1b recombinant strain in 5% of hcv - infected patients in st petersburg has shown that this particular recombinant hcv strain was not only viable but had also spread in the city . this hcv strain was also revealed in different regions of russia and other countries evidenced its wide spread.1114 the latest data on distribution of hcv genotypes published in 2012 confirmed results of previous research,15 ie , the domination of hcv1b subtype in general population ( 57.1% ) as in hemodialysis patients ( 83.6% ) . hcv continues to remain one of the main problems in the public health sector in the russian federation . currently , the manifestations of an epidemic process of hcv based on data of registers of acute and chronic forms of hcv has undergone significant changes . it was established that hcv epidemiological features are characterized by : low incidence of ahcv cases in all territories of the country ( from 22.2/100,000 in 2001 to 1.5/100,000 in 2013 , ie , more than 14 times); increase in registration ( incidence ) of newly detected chronic cases of infection ( from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times); a significant increase in cumulative rate of chcv cases ( prevalence ) that reached 335.8/100,00 and in most administrative territories , higher in specific age groups ( 3049 years); low hcv incidence and prevalence in children in comparison with adults; a rise in hcv rates in specific population groups ( newborns from chronically infected mothers , persons from correctional facilities , patients with chronic liver diseases , and clients from clinics for std patients and drug users); the use of molecular - genetic methods in epidemiological diagnostics for hcv , that will make it possible to reliably establish the source of the infection , routes , and factors of transmission ; and domination of hcv1b virus subtype and rising importance of hcv3a subtype and newly designated recombinant rf_2k/1b . low incidence of ahcv cases in all territories of the country ( from 22.2/100,000 in 2001 to 1.5/100,000 in 2013 , ie , more than 14 times ) ; increase in registration ( incidence ) of newly detected chronic cases of infection ( from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times ) ; a significant increase in cumulative rate of chcv cases ( prevalence ) that reached 335.8/100,00 and in most administrative territories , higher in specific age groups ( 3049 years ) ; low hcv incidence and prevalence in children in comparison with adults ; a rise in hcv rates in specific population groups ( newborns from chronically infected mothers , persons from correctional facilities , patients with chronic liver diseases , and clients from clinics for std patients and drug users ) ; the use of molecular - genetic methods in epidemiological diagnostics for hcv , that will make it possible to reliably establish the source of the infection , routes , and factors of transmission ; and domination of hcv1b virus subtype and rising importance of hcv3a subtype and newly designated recombinant rf_2k/1b . all the aforementioned data are extremely important for planning and implementation of primary , secondary , and tertiary preventive measures for the control of hcv infection in the russian federation .

the first publication on the new virus genome appeared in 1989.1 after that , comprehensive study of new hcv and related disease began , including diagnostics , pathogenesis , and epidemiology . the standard case definition for ahcv was adapted from world health organization recommendations.2 it is necessary to diagnose ahcv based on epidemiological and clinicobiochemical findings , such as the presence of newly identified markers of hcv antibodies to hcv ( anti - hcv ) and hcv rna . anti - hcv detection has a special diagnostic value for acute hepatitis discrimination in disease dynamic ( after 46 weeks ) after negative results of this marker in the early stage of the disease . the aim of the current review was to summarize available epidemiological data and to reveal the main manifestation of the epidemic process of hcv infection in the russian federation in the modern period . designation is historic , originally given to frontier regions and later also to administrative units that comprised autonomous all federal subjects are grouped into eight federal regions on geographical and economical principles : north - west , central , southern , north - caucasus , volga , ural , siberian , far east . we studied the incidence by year , distribution among different federal territories , age group , risk group , the main route of transmission , and the factors of risk . for analysis , data from the state statistical accounts4,5 of infectious morbidity rate ( incidence ) in the russian federation and analytical tables sent from the territories for ahcv and chcv were used . this article also contains characteristics of hcv distribution in the russian federation based on the results of testing of different risk groups for anti - hcv in 20112013 . in this review the dynamics of ahcv cases in russia during 19942013 could be characterized by different trends ( figure 1 ) , in different periods . during all years , the ahcv incidence in children aged 014 years was significantly lower in comparison with rates in teenagers ( 1519 years ) and adults . for instance , in the period of active hcv spread in 19992000 , the national incidence of ahcv in persons older than 15 years was 25/100,000 , but in children less than 14 years this rate was only 3/100,000 . in the present time , the age distribution of ahcv is characterized by very low incidence ( < 1/100,000 ) in children and in adults older than 50 years . it is important to note that the third ranking position in the recorded incidence of hcv among children younger than 14 years of age takes into account children up to 1 year . it should be noted that the proportion of children younger than 1 year in all cases of ahcv among children younger than 14 years does not fall < 50% in recent years . the analysis of data about likely routes routes of hcv infection and risk factors the 3 year period showed that the main route of transmission in children younger than 14 years is vertical , which is caused by children younger than 1 year . in adults the most important risk factor is sexual contact with probable source of infection ( 32.6% ) ; the second significant risk was connected with sharing needles and syringes during intravenous drug use ( 21.7% ) ; and the third risk factor was mainly related to cosmetic procedures , including tattooing and piercing ( 7.6% ) . it should be emphasized that drug use as a risk factor in this age group was reported only in 3.8% of cases almost six times less than in the younger adult group of 2039 years . thus , it is important to emphasize , probable risk factors of hcv transmission in children were vertical transmission , involving children aged younger than 1 year in the russian federation in 20112013 . the study of hcv routes of transmission and risk factors among children aged 014 years and adults is only preliminary , and requires detailed analysis over a longer period of time . in three federal territories ( far east , ural , and north - west ) , the incidence of chcv reached 47.2/100,000 ( far east ) , 48.3/100,000 ( ural ) , and 67.8/100,000 ( north - west ; figure 5 ) . the registration curve in the north - west has manifested ascending type , but in the ural , the increase was interrupted in 2009 and then the decreasing trend became clear . in comparison with newly detected chcv cases ( incidence ) , the disease prevalence ( accumulated cases ) gives more complete vision of epidemiological situation in the territory . the highest chcv prevalence was registered in the north - west region , ural , and far east ( 670.4/100,000 , 587.5/100,000 , and 585.6/100,000 , respectively ) . the chcv prevalence in the volga federal region was higher than the average country level ( 425.9/100,000 ) and in the remaining four regions , it was lower than the prevalence found in the whole country ( range from 243.8/100,000 in siberia to 142.4/100,000 in north - caucasus ) . in age - specific structure of prevalence in all the main territories in the country , the proportion of persons in the age group 3039 years was the highest among all the chcv cases . the far east and north - caucasus regions could be excluded from the mentioned common age - specific structure because in these territories , the second rank among chcv patients belonged to persons in the age group 4049 years . the average prevalence in this age group in the country reached 684/100,000 , but in three regions , these rates were even higher : ural region 1,257/100,000 , north - west region 1,179/100,000 , and volga region 944/100,000 . the second rank in half of the territories ( north - west , far east , south , north - caucasus ) in 2013 belonged to adults 4049 years of age , but in other four territories ( ural , volga , siberia , and central ) to the age group of 2029 years . at the same time , the second rank in the ural region belonged to the age group of 2029 years ( 842/100,000 ) , but in the north - west region to the age group of 4049 years ( 878/100,000 ) . relatively high levels similar to the average rate in the whole territory in the age group of 5059 years were demonstrated by north - west ( 567/100,000 ) , far east ( 531/100,000 ) , and south ( 298/100,000 ) regions . four risk groups had the highest anti - hcv rates in 20112012 ( figure 9 ) : newborns from chcv infected women ( 10.3%14.3% ) , persons from correctional facilities ( 11.8%10.6% ) , patients with chronic liver diseases ( 6.9%6.1% ) , and clients from clinics for sexually transmitted disease patients and drug users ( 5.8%5.2% ) . relatively high anti - hcv rates were detected in patients from hemodialysis settings , units of renal transplantation , cardiovascular and lung surgery , and hematology ( 2.6%2.9% ) . in this group , special attention in one independent study revealed that 25%40% of patients in hemodialysis units were anti - hcv positive.6 also patients with different kinds of chronic pathology had high proportion of anti - hcv positivity reaching 2.9%3.1% . relatively high anti - hcv rates were detected in household contacts of chcv patients ( 1.8%1.9% ) and in patients who were admitted to hospital for planned surgery ( 1.6% ) . perhaps , it can be explained by accumulation of more patients chronically infected with hcv in comparison with patients with chronic hepatitis b virus ( hbv ) infection because of the higher potential of hcv to produce chronic infection . in 20112013 , the highest levels of anti - hcv in planned surgery patients were found in the central federal territory ( 1.7% ) , far east ( 2.1% ) , and ural ( 3.3% ) . one of the first important studies concerning hcv genotypes distribution in russia was published by researchers from moscow institute of virology in 1996.7 they demonstrated that five virus subtypes ( 1a , 1b , 2a , 2b , and 3a ) circulated in the russian federation with significant domination of hcv1b ( 68.9% ) . these data were confirmed in 1997 by another independent group that revealed hcv1b subtype in 76% of hcv patients who were tested.8 a special study of hcv genotypes in st petersburg in 19992000 showed statistically significant differences in hcv subtype distribution between hcv cases in patients attending infectious diseases clinics and in dialysis patients.6 five subtypes ( 3a , 1b , 1a , 2a , and 4a [ in order of frequency ] ) were found in outpatients , while three subtypes ( 1b , 3a , and 2c ) circulated in dialysis patients . it was noted that in st petersburg , 56% of patients with hcv who attended infectious disease clinics were between 11 years and 20 years of age , 63% of whom were infected with subtype 3a and only 33% with subtype 1b . later , researchers from st petersburg demonstrated the recombination of natural intergenotypes between subtypes 2k and 1b , designated as rf_2k/1b.10 the finding of a 2k/1b recombinant strain in 5% of hcv - infected patients in st petersburg has shown that this particular recombinant hcv strain was not only viable but had also spread in the city . this hcv strain was also revealed in different regions of russia and other countries evidenced its wide spread.1114 the latest data on distribution of hcv genotypes published in 2012 confirmed results of previous research,15 ie , the domination of hcv1b subtype in general population ( 57.1% ) as in hemodialysis patients ( 83.6% ) . during all years , the ahcv incidence in children aged 014 years was significantly lower in comparison with rates in teenagers ( 1519 years ) and adults . for instance , in the period of active hcv spread in 19992000 , the national incidence of ahcv in persons older than 15 years was 25/100,000 , but in children less than 14 years this rate was only 3/100,000 . in the present time , the age distribution of ahcv is characterized by very low incidence ( < 1/100,000 ) in children and in adults older than 50 years . the analysis of data about likely routes routes of hcv infection and risk factors the 3 year period showed that the main route of transmission in children younger than 14 years is vertical , which is caused by children younger than 1 year . in adults the most important risk factor is sexual contact with probable source of infection ( 32.6% ) ; the second significant risk was connected with sharing needles and syringes during intravenous drug use ( 21.7% ) ; and the third risk factor was mainly related to cosmetic procedures , including tattooing and piercing ( 7.6% ) . thus , it is important to emphasize , probable risk factors of hcv transmission in children were vertical transmission , involving children aged younger than 1 year in the russian federation in 20112013 . in 2013 , the highest chcv prevalence was registered in the north - west region , ural , and far east ( 670.4/100,000 , 587.5/100,000 , and 585.6/100,000 , respectively ) . the chcv prevalence in the volga federal region was higher than the average country level ( 425.9/100,000 ) and in the remaining four regions , it was lower than the prevalence found in the whole country ( range from 243.8/100,000 in siberia to 142.4/100,000 in north - caucasus ) . in age - specific structure of prevalence in all the main territories in the country , the proportion of persons in the age group 3039 years was the highest among all the chcv cases . the far east and north - caucasus regions could be excluded from the mentioned common age - specific structure because in these territories , the second rank among chcv patients belonged to persons in the age group 4049 years . the average prevalence in this age group in the country reached 684/100,000 , but in three regions , these rates were even higher : ural region 1,257/100,000 , north - west region 1,179/100,000 , and volga region 944/100,000 . the second rank in half of the territories ( north - west , far east , south , north - caucasus ) in 2013 belonged to adults 4049 years of age , but in other four territories ( ural , volga , siberia , and central ) to the age group of 2029 years . at the same time , the second rank in the ural region belonged to the age group of 2029 years ( 842/100,000 ) , but in the north - west region to the age group of 4049 years ( 878/100,000 ) . relatively high levels similar to the average rate in the whole territory in the age group of 5059 years were demonstrated by north - west ( 567/100,000 ) , far east ( 531/100,000 ) , and south ( 298/100,000 ) regions . four risk groups had the highest anti - hcv rates in 20112012 ( figure 9 ) : newborns from chcv infected women ( 10.3%14.3% ) , persons from correctional facilities ( 11.8%10.6% ) , patients with chronic liver diseases ( 6.9%6.1% ) , and clients from clinics for sexually transmitted disease patients and drug users ( 5.8%5.2% ) . relatively high anti - hcv rates were detected in patients from hemodialysis settings , units of renal transplantation , cardiovascular and lung surgery , and hematology ( 2.6%2.9% ) . in this group , special attention in one independent study revealed that 25%40% of patients in hemodialysis units were anti - hcv positive.6 also patients with different kinds of chronic pathology had high proportion of anti - hcv positivity reaching 2.9%3.1% . relatively high anti - hcv rates were detected in household contacts of chcv patients ( 1.8%1.9% ) and in patients who were admitted to hospital for planned surgery ( 1.6% ) . perhaps , it can be explained by accumulation of more patients chronically infected with hcv in comparison with patients with chronic hepatitis b virus ( hbv ) infection because of the higher potential of hcv to produce chronic infection . in 20112013 , the highest levels of anti - hcv in planned surgery patients were found in the central federal territory ( 1.7% ) , far east ( 2.1% ) , and ural ( 3.3% ) . one of the first important studies concerning hcv genotypes distribution in russia was published by researchers from moscow institute of virology in 1996.7 they demonstrated that five virus subtypes ( 1a , 1b , 2a , 2b , and 3a ) circulated in the russian federation with significant domination of hcv1b ( 68.9% ) . these data were confirmed in 1997 by another independent group that revealed hcv1b subtype in 76% of hcv patients who were tested.8 a special study of hcv genotypes in st petersburg in 19992000 showed statistically significant differences in hcv subtype distribution between hcv cases in patients attending infectious diseases clinics and in dialysis patients.6 five subtypes ( 3a , 1b , 1a , 2a , and 4a [ in order of frequency ] ) were found in outpatients , while three subtypes ( 1b , 3a , and 2c ) circulated in dialysis patients . it was noted that in st petersburg , 56% of patients with hcv who attended infectious disease clinics were between 11 years and 20 years of age , 63% of whom were infected with subtype 3a and only 33% with subtype 1b . later , researchers from st petersburg demonstrated the recombination of natural intergenotypes between subtypes 2k and 1b , designated as rf_2k/1b.10 the finding of a 2k/1b recombinant strain in 5% of hcv - infected patients in st petersburg has shown that this particular recombinant hcv strain was not only viable but had also spread in the city . this hcv strain was also revealed in different regions of russia and other countries evidenced its wide spread.1114 the latest data on distribution of hcv genotypes published in 2012 confirmed results of previous research,15 ie , the domination of hcv1b subtype in general population ( 57.1% ) as in hemodialysis patients ( 83.6% ) . it was established that hcv epidemiological features are characterized by : low incidence of ahcv cases in all territories of the country ( from 22.2/100,000 in 2001 to 1.5/100,000 in 2013 , ie , more than 14 times); increase in registration ( incidence ) of newly detected chronic cases of infection ( from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times); a significant increase in cumulative rate of chcv cases ( prevalence ) that reached 335.8/100,00 and in most administrative territories , higher in specific age groups ( 3049 years); low hcv incidence and prevalence in children in comparison with adults; a rise in hcv rates in specific population groups ( newborns from chronically infected mothers , persons from correctional facilities , patients with chronic liver diseases , and clients from clinics for std patients and drug users); the use of molecular - genetic methods in epidemiological diagnostics for hcv , that will make it possible to reliably establish the source of the infection , routes , and factors of transmission ; and domination of hcv1b virus subtype and rising importance of hcv3a subtype and newly designated recombinant rf_2k/1b . low incidence of ahcv cases in all territories of the country ( from 22.2/100,000 in 2001 to 1.5/100,000 in 2013 , ie , more than 14 times ) ; increase in registration ( incidence ) of newly detected chronic cases of infection ( from 12.9/100,000 in 1999 to 39.3/100,000 in 2013 , ie , more than three times ) ; a significant increase in cumulative rate of chcv cases ( prevalence ) that reached 335.8/100,00 and in most administrative territories , higher in specific age groups ( 3049 years ) ; low hcv incidence and prevalence in children in comparison with adults ; a rise in hcv rates in specific population groups ( newborns from chronically infected mothers , persons from correctional facilities , patients with chronic liver diseases , and clients from clinics for std patients and drug users ) ; the use of molecular - genetic methods in epidemiological diagnostics for hcv , that will make it possible to reliably establish the source of the infection , routes , and factors of transmission ; and domination of hcv1b virus subtype and rising importance of hcv3a subtype and newly designated recombinant rf_2k/1b .

over the past decade , dementia with lewy bodies ( dlbs ) has arguably become the second most common form of neurodegenerative dementia behind alzheimer 's disease ( ad ) . in addition to progressive decline in cognition , dlb is characterized by fluctuations in cognition with variations in attention and alertness , recurrent formed visual hallucinations , visuospatial dysfunction , and spontaneous parkinsonism . often , dlb patients also exhibit neuroleptic sensitivity , transient loss of consciousness , falls , and rapid eye movement ( rem- ) sleep behavior disorder . the clinical separation of dlb from other similar disorders is often difficult resulting in poor diagnostic accuracy , but the relative temporal co - occurrence of parkinsonian features with the typical dlb cognitive and behavioral symptoms such as visuospatial disturbance strongly suggests the diagnosis . clinical presentation of dlb is also influenced by the amount of ad tau pathology , further complicating the diagnosis . approximately 2040% of parkinson 's disease ( pd ) patients also eventually develop a progressive dementing illness designated as parkinson 's disease dementia ( pdd ) characterized by a frontal - subcortical clinical presentation . dlb / diffuse lewy body disease ( dlbd ) , lewy body variant of ad ( lbv ) , and pdd comprise an emerging spectrum of clinical phenotypes from relatively pure motor pd to the more predominant cognitive and behavioral disturbance observed in pdd and dlb , yet the reason for the variability remains unknown . despite the heterogeneity of their clinical phenotypes , a significant neuropathological overlap is observed among these diseases , hence the term lewy body disorders ( lbds ) to collectively describe conditions in which lewy bodies ( lbs ) and lewy neurites ( lns ) predominate as the hallmark histological lesions . variation in the distribution of lewy body pathology is present among lbds , with more neocortical and limbic system lb in both dlb and pdd compared to the brains of pd patients without cognitive symptoms and greater neuronal loss in substantia nigra in pdd than dlb . yet , dlb and overlap disorders such as lbv , more so than pdd , have -amyloid pathology and basal forebrain cholinergic deficit similar to ad patients . as with other forms of dementia , the pathobiological changes in lbd likely occur decades prior to the onset of clinical symptoms and correspond to widespread irreversible neurodegeneration [ 6 , 7 ] . it is increasingly clear from the ad therapeutic experience that by the time widespread neuronal injury ensues , symptomatic cholinergic treatments are minimally effective at best , and disease - modifying therapeutic approaches in trials have thus far proven ineffective at altering disease course or in rescuing diseased brain [ 8 , 9 ] . to demonstrate efficacy , any potential disease - modifying therapy in neurodegenerative dementia must be initiated prior to the expression of the clinical phenotype during the initial molecular pathogenetic events before irreversible neuronal damage has occurred . at present , accurately predicting those individuals at risk for developing neurodegenerative dementia is challenging , and this places greater urgency on developing earlier methods of disease detection . critically important is not only distinguishing the lbd from ad and other forms of dementia , but also separating dlbs and other lbds . although there are many promising candidates for lbd , no biomarkers have yet been validated for clinical diagnostic use , and thus many opportunities exist to develop such tests . here , we highlight from the perspective of how major genetic discoveries in the lbd and their corresponding biomolecular processes might translate into useful disease markers in biological fluids . because of many common pathogenetic features among the lbds , the emerging genetic influences found in pd have readily been translated to dlbs and other lbds , providing clues to rational approaches for selecting future dlb and lbd biomarker targets for exploration . this paper will highlight several pd and dlb genes and their protein products as candidates for biological disease markers ( table 1 ) . a , a key component of neuritic plaques in ad brain , is overproduced leading to various degrees of amyloid aggregation and synaptic and neuronal toxicity . as indicated previously , amyloid pathology in the form of neuritic and diffuse plaques can be also found in varying degrees in the brain tissue of patients with dlb , which may interact with lb or synuclein pathology or influence the clinical features of lbds [ 4 , 11 ] . the genetic mechanisms of a overproduction in ad are well established ; the app gene ( chromosome 21 ) , the first identified ad susceptibility gene , encodes a transmembranous protein ranging from 695 to 770 residues , which undergoes a process of regulated intramembranous proteolysis ultimately releasing a peptides , primarily a42 and a40 , as well as other fragments . a is generated by the concerted action of -secretase and -secretase complex , while the -secretase pathway precludes a formation by cleaving app at a site within the a sequence . genetic analysis of early - onset familial ad cases revealed numerous mutations in the app gene as well as presenilin 1 ( ps1 ; chromosome 14 ) and presenilin 2 ( ps2 ; chromosome 1 ) genes , all of which accelerate the processing of app , leading to increased a generation . specifically , the app km670/671nl ( swedish ) mutation affects the -secretase site , a692 g ( flemish ) mutation alters the -secretase site , and both v717f ( indiana ) and v717i ( london ) mutations affect the -secretase processing , leading to elevated a levels . also important in the notch developmental signaling pathway which is analogous to app processing , the presenilins are thought to be a component of the -secretase enzyme complex , which suggests that missense mutations in the presenilins mechanistically lead to accelerated app processing to a . therefore , the abnormal proteolytic cleavage of app leads to elevated brain a deposition , and as a result , diminished peripheral levels of a. reflecting a shift from soluble a to insoluble brain deposits , significant decreases in csf a42 levels have been demonstrated in ad and more recently in dlb cases . parnetti et al . found that dlb , compared with pd , pdd , and ad patients , showed the lowest csf levels of a42 and , when combined with csf tau , differentiated dlb from pd and pdd , but not from ad . also , spies and colleagues showed a greater decrease in a40 in clinical dlb and vascular dementia patients compared with control levels and even with ad . differentiation of non - ad dementias such as vascular dementia and dlb was improved by comparing the ratio of a42 and a40 . more recently , the detection of amyloid in dementia patients has been greatly enhanced by the use of amyloid - binding agents such as pittsburgh compound b , which also demonstrated amyloid burden in dlb . an australian study reported more variable cortical pib binding in dlb patients than in ad , whereas a subsequent examination of pib binding in lbds including dlb , pdd , and pd , compared with ad and normal patients , showed higher amyloid burden in dlb and ad than in pdd , pd , or nc patients . amyloid load was highest in lbd patients in the parietal and posterior cingulate regions , corresponding to visuospatial impairments on neuropsychological testing , suggesting that amyloid deposition could partly contribute to the clinical presentation of lbds . mutations in the tau gene on chromosome 17 may also present with phenotypic features of pdd or dlb , but they differ pathologically from these disorders in that lbs are generally absent . tau - bearing neurofibrillary tangles remain one of the pathological hallmarks of ad but are also central to a diverse group of disorders termed tauopathies which include progressive supranuclear palsy , corticobasal ganglionic degeneration , frontotemporal dementia ( ftd ) with parkinsonism linked to chromosome 17 , and other disorders . tau is a microtubule binding protein , which acts to stabilize tubulin polymerization in microtubules critical for axonal cytoskeletal integrity and function . in disease , tau protein truncation at glu 391 or hyperphosphorylation causes microtubule destabilization and aggregation of unbound tau into paired helical filaments ( phfs ) leading to characteristic tangle formations . unlike the tauopathies , no direct pathogenetic tau mutations have been identified in lbds , but tau pathology appears to be a consistent feature among neurodegenerative dementias including ad and lbds , and given the pathological overlap , they might share similar pathogenetic pathways ( reviewed in stoothoff and johnson ) . the ser / thr kinase and glycogen synthase kinase-3 ( gsk3 ) , in concert with other molecules such as fyn kinase , normally regulate tau function but with aberrant activation accelerate the hyperphosphorylation of tau in neurodegenerative disease . similarly , the cell cycle family kinase and cyclin - dependent kinase 5 ( cdk5/p35 ) , active during normal brain development and involved in regulatory tau phosphorylation during mitosis , may also contribute to phf formation . consequently , both total tau and hyperphosphorylated forms have been widely investigated and detected in csf , but not serum , by enzyme - linked immunosorbent assay methods . in the differentiation of dementia types , arai et al . initially reported elevated total csf tau levels in ad but not in pd , but subsequently , they showed that total tau was also increased in dlb at similar levels to ad . yet , others have found differences for both total and phospho - tau ( p - tau ) in differentiating dlb from ad , and levels of total tau and p - tau 181 were significantly increased in autopsy - confirmed dlb patients . in clinically diagnosed dementia cases , csf p - tau 231 discriminated ad from non - ad dementias as a group , where levels were significantly higher in ad patients compared with dlb , ftd , vascular dementia , other disorders , and control subjects . separation of dlb from ad , however , was less robust , provided that csf p - tau 231 levels were also increased in dlb . clinically diagnosed dlb cases also showed elevated levels of csf p - tau 181 compared with controls , and hampel et al . reported that p - tau 181 provided the best discrimination of dlb from ad yielding a sensitivity of 94% and specificity of 64% . in autopsy - confirmed dlb and ad patients , however , sensitivity decreased to 75% and specificity to 61% , with a diagnostic accuracy reported as 73% . lbs are filamentous inclusions consisting primarily of the presynaptic protein -synuclein ( -syn ) , which might have several roles in vivo . studies demonstrate that it is localized to multiple neural tissues , including high expression in neocortex and hippocampus , and that expression increases during acquisition - related synaptic plasticity . interaction with tubulin suggests -syn could be a microtubule - associated protein similar to tau [ 32 , 33 ] , and it is highly active in various membrane lipid bilayers such as in presynaptic vesicles acting as a chaperone for soluble nsf attachment protein receptor ( snare ) complex formation , in neuronal golgi apparatus influencing protein trafficking and in the inner membrane of neuronal mitochondrial . the synucleins might act to preserve membrane stability , provide antioxidant function , and assist with membrane turnover , although the actual role of synucleins remains elusive [ 37 , 38 ] . because of its association with lb and the tendency to self - aggregate into pathological oligomers and ultimately fibrillar structures , -syn plays a central role in the pathogenesis of lbd , hence the alternate designation synucleinopathies . the degree of -syn immunoreactivity in cortical lbs correlates with cognitive severity and disease progression in pdd and dlb [ 4 , 40 ] . also , the protein can be recovered from filaments in purified lewy bodies from pdd and dlb brain , and recombinant -syn tends to form lewy body - like fibrillar structures in vitro . in the past decade , tremendous advances have been made in understanding the genetic factors influencing the pathogenesis of lewy body disorders . compelling evidence for a genetic basis for pd and dlb followed the discovery of mutations in the -syn gene ( park1/4 ) in patients with autosomal dominant familial parkinson 's disease , and subsequently , mutations were identified in patients with both sporadic and familial dlbs . from a susceptibility marker on chromosome 4q21 - 23 that segregated with the pd phenotype in italian and greek kindreds , a53 t and a30p were the first two missense mutations in -syn associated with familial parkinson 's disease . clinical analysis of the italian a53 t mutation revealed phenotypic variability over the disease course with several individuals demonstrating moderate to severe dementia . subsequently , a case of clinically and pathologically well - characterized dlbd in the united states and a greek proband of dlb with a family history of pd were both determined to have the a53 t -syn mutation [ 46 , 47 ] . another mutation , e46k , was discovered in a spanish family presenting with autosomal dominant dlb , and in genetic studies of a large family with the spectrum of lewy body phenotype ranging from pd to dlb , -syn gene triplication was described , causing -syn overproduction similar to the trisomy effect observed in down syndrome patients . autosomal dominant point mutations are shown to affect the aggregative properties of -syn , which has mechanistic implications for the pathogenesis of lbd . compared to wild - type -syn , biophysical analyses reveal that -syn aggregation is folding state dependent , where a53 t and a30p mutated proteins cause increased aggregation only from the partially folded intermediate state and not the monomeric state . a53 t -syn transgenic mice have increased oligomerization of the protein in brain regions devoid of inclusions as well as those areas with more abundant lesions and neurodegeneration , and consistent with prior biophysical findings , -syn toxicity in these mice was dependent on the conformation of intermediate species . in fact , the e46k mutation , as well as the others not only increase the tendency toward aggregation , but also promote formation of annular protofibrillar structures , causes pore formation in various membranes and neuronal damage . -syn is a member of a larger family of synuclein proteins which also includes -synuclein ( -syn ) and -synuclein ( -syn ) . -syn has recently been implicated in pd and dlb pathogenesis , but its precise role in disease is still emerging . despite having strong homology with -syn , it is not clearly amyloidogenic , but is highly localized to presynaptic sites in neocortex , hippocampus , and thalamus like -syn [ 53 , 54 ] . normal -syn may act as a biological negative regulator of -syn . in bigenic -syn/-syn - overexpressing mice and in doubly transfected cultured cells , -syn ameliorated amyloidogenicity , neurodegenerative changes , and motor deficits induced by -syn overexpression alone . on the other hand , mutated -syn leads to neuronal damage and disease and augments neurodegeneration , perhaps through a loss of its natural regulator function . two novel -syn point mutations , p123h and v70 m , were found in highly conserved regions of the -syn gene in respective familial ( p123h ) and sporadic ( v70 m ) dlb index cases , where abundant lb pathology and -syn aggregation was present without -syn aggregation . p123h -syn overexpression in transgenic mice resulted in axonal damage , gliosis , profound memory , and behavioral deficits . these phenomena may involve -syn , since bigenic mice overexpressing -syn with p123h -syn show greater deficits compared with monogenic mice and compared with p123h -syn expressed with -syn knockout , implying that the p123h mutation has a synergistic effect with other synucleinopathies to cause neurodegeneration . p123h as well as v70 m -syn mutations might also injure neurons by disrupting normal lysosomal pathways and corresponding cellular autophagic processes . unlike the other synuclein family members , -syn or persyn is largely expressed in the cell bodies and axons of primary sensory neurons , sympathetic neurons , and motor neurons as well as in brain . in cancer biology , -syn is associated with abnormally altering cellular mitotic checkpoints in various types of malignancies , making them more aggressively metastatic , but as far as neurodegeneration , it is the most recent synuclein member to be linked to lbd neuropathology and the least well understood . single - nucleotide polymorphisms in all three synucleins have been associated with sporadic dlbd , most prominently -syn , and in sporadic pd , dlb , and lbv patients , -syn antibodies , as well as -syn and -syn reveal unique hippocampal axonal pathology . in vivo , -syn overexpression in transgenic mice shows age- and dose - dependent neuronal loss throughout the neuraxis , especially in spinal motor neurons , where -syn - bearing inclusions , gliosis , and alterations in heat shock protein and neurofilament structure are found , perhaps suggesting relevance to motor neuron disease associated with dementia . in vitro evidence further supports a cytoskeletal role for -syn in maintaining neurofilament structure ; -syn overexpression in cultured neurons causes disruption of the neurofilament network by destabilizing the structural integrity of neurofilament - h allowing degradation by calcium - dependent proteases , which has implications for neurodegeneration . synucleins are known as intracellular molecules , but they also appear in extracellular and peripheral fluids from active and passive processes . evidence suggests that turnover and secretion of these proteins might occur during normal cellular processing , releasing synucleins into extracellular space and hence into peripheral sites . in transfected and untransfected cultured neuroblastoma cells , 15 kda -syn is released into surrounding media , and furthermore , not only monomeric -syn but also aggregated forms are secreted in an unconventional exocytic manner into extracellular fluid in response to proteasomal and mitochondrial dysfunction . . recently showed that neuronally secreted -syn can also be taken in endocytically by other neurons or glia as a means of transmitting pathology . secreted -syn interacts with various molecules such enzymes ; in cultures , matrix metalloproteinase-3 cleaves native -syn to smaller proteolytic fragments that enhance its aggregative properties . whether -syn and -syn also undergo unconventional exocytosis and secretion remains unknown , but given structural and functional similarity to -syn , the possibility exists . certainly , synaptic and axonal damage reflecting neurodegeneration may also allow release of synucleins into the extracellular millieu and access to peripheral fluids such as csf and blood . multiple forms of -syn are released into cerebrospinal fluid ( csf ) and other biological fluids . full - length -syn has been recovered from lumbar csf from living normal control , pd and dlb patients [ 69 , 70 ] , and also from postmortem csf from dlb and other neurodegenerative diseases . comparative findings regarding differences in csf -syn levels among various neurodegenerative diseases , however , are difficult to interpret because of inconsistent observations . in pd , a smaller early study showed that no differences in full - length csf 19 kda -syn have been found in relation to control individuals , but a recent effort using a new luminex assay in a larger sample controlling for extraneous influences showed significantly decreased levels in pd compared to controls with 92% disease sensitivity and 58% specificity . elevated -syn levels , however , were found in dlb , ad , and vascular dementia with no differences among them . perhaps more intriguing , higher - molecular weight aggregated -syn species in csf might be associated with pd and dlb . reduced levels of a 24 kd -syn - immunoreactive band were found in dlb csf and correlated directly with declining cognition . moreover , using a specific enzyme - linked immunosorbent assay ( elisa ) , soluble aggregated -syn oligomers in csf were significantly increased in pd patients compared against control subjects , ad and progressive supranuclear palsy , and specificity ranged from approximately 85 to 87% , while sensitivity was about 5375% range . plasma -syn detected by immunoblotting was decreased in pd compared with age - matched control subjects , and those pd patients with age - at - onset prior to 55 years ( early - onset ) had significantly lower levels than those with onset after 55 years of age ( late - onset ) . in addition , soluble oligomeric -syn detected by specific elisa was significantly elevated in plasma from pd . this test demonstrated a specificity of approximately 85% , a sensitivity of 53% , and a positive predictive value of 0.818 . although measurement of plasma -syn appears interesting as a biomarker , it was reported that skin cells and platelets are also sources for -syn , and their levels did not correlate with disease presence or severity . moreover , red blood cells are also a major source of -syn , and thus , plasma could be contaminated by -syn not originating from brain , which might render interpretation of results difficult . one promising consideration for the future exploration of -syn as an lbd biomarker will be the development of novel imaging compounds and techniques , similar to amyloid imaging , to specifically target and visualize -syn distribution in the pd and lbd brain . the availability of such methods will be a significant advance in biomarkers for synucleinopathies . due to their increasing importance in lbd pathogenesis , -syn and -syn , as much as -syn as such , levels of these synucleins might be altered in the csf of patients with pd / pdd and dlb , reflecting the underlying degenerative processes in brain . no studies to date have examined -syn levels in peripheral fluids in relation to neurodegenerative disease , but a small study reported elevated postmortem ventricular csf -syn levels in dlb , ad , and vascular dementia patients , with the highest levels seen in dlb patients . more detailed examination of both -syn and -syn as a peripheral disease markers in well - characterized populations of pd , dlb , and other disorders is warranted to determine their specificity and sensitivity in the synucleinopathies . recently , dj-1 ( park 7 ) has emerged as a significant molecular target of interest in lbd principally because of its genetic association with pd and its increasing importance in cellular oxidative neuroprotection . although its exact role is unknown , multiple functions have been assigned to the dj-1 protein ras - related signaling pathways , it shares structural homology with the carboxy - terminal domain of escherichia coli hpii catalase and is reported to possess catalase activity which reduces oxidative stress in cultured cells . it also binds to and regulates the pias sumo-1 ligase and is itself posttranslationally modified by sumoylation [ 81 , 82 ] . of relevance to lewy body formation and neurotoxicity , dj-1 displays redox - dependent chaperone activity conferring proper protein folding and thermal stability , which in fact , also inhibits -syn aggregation . the overexpression of dj-1 in rats protects nigral dopaminergic neurons against degeneration involving 6-hydroxydopamine , while mutant dj-1 in mice causes abnormal dopamine reuptake and susceptibility to 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) toxicity . deletion of dj-1 homologs in drosophila renders them sensitive to h2o2 , paraquat , and rotenone toxicity . no less than 13 gene mutations have been identified in dj-1 in atypical younger - onset pd patients , but their significance to idiopathic late - onset pd remains uncertain . in autosomal recessive early - onset pd from consanguineous families , a complete dj-1 deletion in a dutch family and a point mutation l166p in an italian case were identified . when expressed in cultured cells , l166p appears to be a loss - of - function mutation which leads to dj-1 functional instability , degradation by the proteasome system [ 86 , 87 ] , abnormal translocation of dj-1 to mitochondria , and loss of chaperone activity . the importance of dj-1 gene alterations in dementia and dlb , however , is uncertain . one report found no impact on dementia risk of the dj-1 14 kb deletion , and analysis of an insertion / deletion variant ( g.168_185del ) in dj-1 in a larger sample of patients also showed no association with either pd or dlb compared to control patients . given these early negative findings , the relevance of dj-1 genetic mutations to dlb and other lbd is not known . at present , no patient harboring a dj-1 mutation has come to autopsy , so the precise pathology is not known . although dj-1 mutant cases may ultimately not be lbds , it is possible that alterations in dj-1 may somehow influence the aggregation of -syn and lb formation or contribute to pathogenesis by other molecular pathways . dj-1 is found in brain across a wide range of neurodegenerative diseases including pd , ftd , ad , dlb , and lbvad , and demonstrates striking association with neuropil threads and neurofibrillary pathology in neocortex and subcortical brain regions in these disorders . interestingly , this association with tau pathology was seen in dlb and lbv brains , suggesting that as a chaperone molecule , dj-1 may be involved in tangle formation , and the binding of dj-1 with these lesions could abolish the normally protective effect of dj-1 , enhancing oxidative neurotoxicity . wang et al . observed that dj-1 knockout mice have markedly abnormal hippocampal long - term depression accompanied by a less severe abnormality in long - term potentiation , which was reversed by the d2/3 agonist quinpirole , indicating that dj-1 has a role in dopamine - dependent signaling in hippocampal plasticity . given its pathogenetic significance , dj-1 could be a candidate biological marker for dlb and lb and might serve as a means of monitoring in vivo oxidative damage and protein misfolding . although intracellular and mitochondrial in localization , dj-1 is presumed to be secreted perhaps specifically under disease conditions which induce oxidative damage . using semiquantitative immunoblotting , we previously identified dj-1 in csf of sporadic pd patients , where levels were significantly elevated compared with controls . levels were higher in the earlier stage pd cohort ( hoehn - yahr stages i - ii ) than in the more severe patients ( hoehn - yahr stages iii - iv ) . similarly , plasma dj-1 levels in pd patients were markedly increased compared to controls , but unlike csf , levels were relatively higher in late stage ( iii - iv ) rather than early stage pd ( i - ii ) . the reason for this difference between plasma and csf dj-1 is unknown , but we surmised previously that since csf dj-1 originates from a central source produced mainly by reactive glia , early increases in csf dj-1 levels probably represent an early protective response to damage , whereas plasma dj-1 , like other plasma disease markers , likely represents peripheral oxidative stress damage . in fact , dj-1 is secreted into blood in breast cancer , melanoma , familial amyloid neuropathy , and stroke [ 9496 ] . in the largest study to date , hong et al . developed a more sensitive and quantitative luminex assay for csf dj-1 to complement immunoblotting mass spectrometric and chromatographic analysis methods and found decreasing rather than increasing levels of dj-1 in pd csf compared with control patients . the 90% disease sensitivity and 70% disease specificity for pd using this method approaches minimal desired parameters for a clinically useful biomarker for pd . importantly , the study highlighted the fact that dj-1 levels are greatly influenced by such variables as the extent of blood contamination and patient age , which could account for some of the variability across studies . of note , dj-1 is also subject to oxidative modifications in pd and ad brain tissue , and this might be measured in peripheral fluids as well , as another monitor of oxidative damage . csf dj-1 remains a promising and perhaps clinically useful biomarker for pd , but as far as dlb and other lbd , it is unknown whether csf levels of dj-1 are altered . since plasma dj-1 is increased in dlb , it is hypothesized that csf dj-1 may also be elevated . further investigation will be necessary to clarify the utility of dj-1 as a biomarker in dlb and lbd . many clinicopathologic parallels can be drawn between the lysosomal storage disorders , such as niemann - pick , sandhoff 's , tay - sachs disease and others , and the age - related neurodegenerative disorders , when considering the aberrant accumulation of pathological substances ( e.g. , lysosomal sphingomyelin in niemann - pick disease versus synucleins in pd and dlb ) and the phenotypes of neuronal loss and cognitive deterioration found in both . common to these diseases are abnormalities in lysosomal and autophagic mechanisms as part of a larger disruption of cellular proteostasis leading to abnormal storage / accumulation of toxic materials and neuronal damage . in the past few years , an altogether unexpected pathogenetic relationship emerged between gaucher 's disease ( gd ) , a prototypic storage disease , and the synucleinopathies . despite its overall rarity , gd is the most common inherited lysosomal storage disease , especially in the ashkenazi jewish population . it is caused by autosomal recessive gene mutations in the glucocerebrosidase ( gba ) gene ( chromosome 1q21 ) , leading to either partial or complete deficiency of gba , and hence , toxic lysosomal accumulation of its substrate , glucosylceramide , in multiple cell types including neurons . recent reports documented an increased incidence of pd in heterozygous relatives of patients with gd [ 99 , 100 ] , but interest in this phenomenon was propelled by the finding that gba mutations were in fact more common in pd patients of ashkenazi background compared with ad patients and pd patients in the general population [ 101103 ] . moreover , more severe gba mutations such as 84 dupl g and ivs 2 + 1 were associated with a greater degree of pd risk , compared with less severe gba mutations such as n370s . the relationship between pd and gba has now been replicated in much larger international studies with the most common mutations being l444p and n370s , and about 28 gba mutations are presently recognized . interestingly , in a study of british patients with pd and gba mutations , all 17 carrier patients demonstrated abundant -syn neuropathology with braak stage 5 - 6 severity and common neocortical lb pathology . clinically , these patients had earlier age at onset , and hallucinations were present in 45% of patients , while 48% had cognitive impairment or dementia consistent with pdd . greater severity of gba mutation also predicted the presence of cognitive impairment in pd patients ; 56% of severe gba mutation carriers had cognitive impairment compared to 25% of mild mutation carriers . these observations suggest a much broader link between gba mutations and the dementia phenotype of lbd . in fact , examination of gba gene alterations in dlb patients , with and without concomitant lbv - type ad pathology , showed that the majority of gba mutations were found in dlb patients rather than in pd , with a mutation rate in dlb ranging from 18 to 23% overall [ 108 , 109 ] . the proportion of dlb patients with gba mutations was higher in those with pure neocortical lb pathology compared to those with mixed lb and ad pathology and to those with predominantly brainstem lb . a significant association was also found between gba mutation status and the presence of lb , indicating that altered gba might play a role in their formation and in synucleinopathy . important in neurodegeneration , disrupted cellular proteostasis represents a state in which an imbalance exists between effective functioning of the innate cytoprotective machinery and excessive accumulation and aggregation of abnormally misfolded proteins , leading to neurotoxicity . it is increasingly apparent that chaperone - mediated autophagy ( cma ) and lysosomal degradation pathways are important in maintaining cellular proteostasis as part of a larger network of cellular actions , with particular relevance for neurodegenerative diseases . recently , as evidence for cma dysfunction in synucleinopathies , a significant decrease in autophagy markers was reported in substantia nigra from pd brain . soluble forms of -syn , including monomers , oligomers , and even protofibrils , are normally cleared through the cma / lysosomal degradation by interacting with the chaperone , heat shock cognate-70 , and becoming internalized into lysosomes via the lamp-2a membrane receptor [ 111 , 112 ] . studies have indicated that -syn shares a common pentapeptide structure with other lysosomal substrates , designating it as a target for removal by this pathway , and the lysosomal structure is critical to maintaining the internal acidic environment , allowing lysosomal hydrolases to degrade -syn into peptides released into the cytosol . mutant gba could therefore disrupt lysosomal activity leading to abnormal accumulation of nondegraded -syn , which then aggregates to toxic soluble oligomers and protofibrils . also , abnormalities in the ubiquitin - proteasome system ( ups ) are present in ad and pd , and gba alterations might secondarily overwhelm the ability of ups to remove accumulated -syn , promoting aggregation and neurotoxicity . pathologically , in gd with parkinsonism , -syn - positive inclusions were observed in neurons in hippocampal ca2 - 4 regions , while cortical synuclein pathology was identified in other gd cases . further , parkin , an e3 ubiquitin ligase also implicated in pd , has been shown to affect the stability of mutant gba and increase its degradation causing further lysosomal dysfunction . because of the importance of mutant gba function to pd and dlb pathogenesis , the issue arises as to whether the measurement of gba activity , or a perhaps other related molecules , might be utilized as a biological marker . the activity of peripherally secreted gba was measured in plasma and csf in a 10-month - old female with gd with the aim of monitoring the effect of experimental cerezyme replacement therapy . baseline gba activity was detected in both plasma ( 2.7 10 u/l ) and csf ( 0.096 10 u/l ) , although csf activity was several magnitudes lower than plasma . intravenous cerezyme , a macrophage - targeted gba , rapidly raised the plasma activity within 1 hour and csf activity by 2.3-fold at 3 hours , both returning to baseline after 24 hours . this study suggests the intriguing possibility that gba activity , especially in csf and plasma , might be useful in monitoring the efficacy of novel therapies involving cma and lysosomal function . to extend this observation , balducci et al . determined that multiple lysosomal hydrolases , including gba , are significantly decreased in the lumbar csf of pd patients , perhaps supporting a more widespread lysosomal dysfunction in pd not limited to gba alone . in this regard , other lysosomal enzymes such as mannosidase and -hexosaminidase might be important additional biomarker targets for neurodegeneration . moreover , in dlb , ad , and ftd patients , lysosomal enzyme activities in csf demonstrated a very specific pattern of decrease , in which only dlb showed significant decreases in csf activity of -mannosidase , -mannosidase , gba , galactosidase , and -hexosaminidase , whereas in ad and ftd , only csf -mannosidase activity was significantly diminished . in dlb , csf gba activity showed the greatest magnitude of decrease , reinforcing its importance in the lbd , but also noteworthy is the fact that ad and ftd showed decreased -mannosidase activity , suggesting that this might be another important factor in lysosomal dysfunction in neurodegeneration . indeed , these promising candidates need to be investigated further to establish diagnostic accuracy in terms of disease specificity and sensitivity in cohorts of pd , dlb , and other dementing disorders . polymorphisms in proinflammatory cytokine genes including il-1 , il-1 , and tnf- are associated with increased risk in ad . in pd , several case control genetic analyses have demonstrated that homozygous carriers of the il-1 511 and tnf- 308 promoter region variants have increased disease risk [ 120 , 121 ] , and that earlier age at onset in pd was associated with il-1 511 homozygosity at allele 1 . but as yet , no such genetic alterations in cytokines genes have been reported in dlb . similar to a-induced upregulation of inflammatory cytokines in ad , soluble secreted -syn in the extracellular space in lbd might also induce the production of a variety of neuroinflammatory mediators into the extracellular fluid . for instance , microglial activation in response to stimulation by secreted -syn from cultured cells and from overexpression in transgenic mouse models occurs in a dose - dependent manner , causing release tnf- , il-1 , and il-6 . because secreted cns cytokines are readily detected in csf , they have been extensively examined as potential disease biomarkers . il-1 , il-2 , il-6 , and tnf- are all upregulated in pd brain , as well as in csf from pd patients [ 124126 ] , and chen et al . showed that plasma il-6 , but not il-1 , tnf- , or other acute phase reactants , predicted risk for future pd in males . in terms of dlb , csf il-1 levels , which were relatively low , did not differ compared to ad or normal controls and could not distinguish them apart . comparable increases in csf il-6 levels were found in ad and dlb , but again not significantly different from each other to be of diagnostic value . indeed , the neuroinflammatory cytokines may be important as a pathogenetic response to cns injury caused by accumulation of amyloidogenic proteins , but their role as biomarkers for the lbd , especially for dlb , is still unclear . disorganization and breakdown in the cytoskeletal network occurs in various lbds and other neurodegenerative diseases , and as discussed , gamma - synuclein and proteolytic degradation of the cytoskeleton may be involved . as a result , a failure of normal axonal transport results from the accumulation of disrupted neurofilament molecules within the neuropil , causing neuronal demise . recently , a mutation in the nefm gene encoding the rod domain 2b of neurofilament m ( nf - m ) which causes aberrant nf assembly was identified in a single early - onset pd patient . it is recognized that in addition to -syn , three types of nf protein also comprise the structure of lewy bodies . upon cell death or axonal damage , accumulated neurofilament leaks into the extracellular space , subsequently appearing in csf and perhaps other peripheral fluids . elevated csf nf protein was reported in msa and psp , but not in pd , and this was suggested to clinically aid in differentiating parkinsonian syndromes . csf nf protein was also measured in dementia , and although increased levels were observed in dlb , late - onset ad , and ftd , there were no differences among them . therefore , because cytoskeletal abnormalities are present in many neurodegenerative dementias as well as in pd , nf protein may be more a reflection of nonspecific alterations in neuronal and axonal function , which does not appear to able to clinically separate dlb from other disorders . severe cortical cholinergic deficits originating from deficiencies in the nucleus basalis of meynert are characteristic of ad brain , but studies have shown that cholinergic deficits are perhaps more severe in dlb brain . this suggests that measurement of cholinergic activity and/or acetylcholine ( ach ) might be developed into a potential biomarker for the lbds . indeed , early attempts to quantify ach or its major metabolite , choline , have shown baseline levels to be low and perhaps difficult to measure accurately . in ad , csf ach was reported to be significantly lower than control levels , while in pd and huntington 's disease patients , despite some cholinergic deficit , lumbar csf ach and choline levels did not differ from normal . no studies have directly examined csf cholinergic levels in dlb or lbds , but recently , shimada and colleagues employed positron emission tomography ( pet ) mapping of brain ach activity in dlb and pdd patients and normal controls and demonstrated a marked reduction in cholinergic activity in medial occipital cortex of dlb and pdd , greater than that observed in pd patients without dementia . some correlation of mapped cholinergic activity with cognitive decline measured by the mini - mental state exam was also found . although preliminary , this has potential to be a more practical and sensitive cholinergic biomarker for lbd . because of similar nigrostriatal loss to pd , a relative dopaminergic deficiency also exists in dlb and lbds . csf dopamine ( da ) and its metabolites have been investigated previously in pd , and recently , lunardi et al . showed differences in csf da and its metabolites , homovanillic acid ( hva ) and dihydroxyphenylacetic acid ( dopac ) , in pd patients , demonstrating early - stage dopaminergic loss and a correlation with the development of dyskinesia . in dlb , similar to cholinergic activity , imaging modalities may also contribute to the assessment of dopaminergic function in the lbds . in a small study , striatal da uptake as measured by f - fluorodopa pet was decreased in both caudate and putamen in dlb as compared with ad patients and controls . also , da transporter loss was determined across multiple studies using i-2-carbometoxy-3-(4-iodophenyl)-n-(3-fluoropropyl ) nortropane ligand with single - photon emission computed tomography ( i - fp cit spect ) and demonstrated significant loss of caudate and putaminal da transport compared with ad and control levels [ 139141 ] . a larger phase iii , multicenter study of i - fp cit spect in possible and probable dlb patients and non - dlb comparators ( mostly ad ) demonstrated a mean sensitivity of 77.7% for detecting clinically probable dlb , with a specificity of 90.4% and 85.7% overall diagnostic accuracy . i - fp cit spect da transporter imaging greatly enhanced diagnostic accuracy for dlb over clinical diagnosis alone when coupled with autopsy confirmation , raising sensitivity for dlb from 75% to 88% and specificity from 42% to 100% . furthermore , da transporter loss in the caudate may also be inversely associated with depression , apathy , and delusions in dlb patients . autonomic failure is a common clinical finding in lbd , including pd and dlb , but not in non - lbd dementias , and therefore it has been investigated as an alternative biomarker for the diagnostic separation of dlb from other dementias . abnormal autonomic function can be determined using cardiac i - meta - iodobenzyl guanidine ( i - mibg ) imaging , a technique which assesses cardiac sympathetic nerve function in both cardiac and neurological disorders by measuring the uptake of i - mibg , a norepinephrine analogue . in the last decade , a series of japanese studies consistently demonstrated delayed heart to mediastinum ratio ( h / m ) of i - mibg uptake in dlb compared with ad and controls [ 143146 ] . i - mibg imaging distinguished dlb from other dementias with a sensitivity of 94% , specificity of 96% , and a diagnostic accuracy of 95% . finally , consistent with autonomic dysfunction in dlb , both early and delayed h / m i - mibg uptake were significantly associated with the presence of orthostatic hypotension in dlb patients and discriminated dlb from ad even in the absence of parkinsonism . various magnetic resonance ( mr ) imaging modalities have been explored in dlb and pdd , including volumetric imaging , diffusion tensor imaging , and proton magnetic resonance spectroscopy ( reviewed in watson et al . ) , and although not directly useful as biomarkers at present , they have revealed insights in the pathobiology of lbds . using conventional mri techniques such as voxel - based morphometry and region of interest analysis atrophy has been rated at 1.4% per year in dlb brain , 1.31% per year in pdd , and 0.31% per year in pd . not surprising is the fact that unlike ad brain , medial temporal structures are relatively preserved in dlb and pdd , with global hippocampal loss at about 1020% compared with controls and about 2125% in ad . diffusion tensor imaging , an mr technique mapping brain microdiffusion of water in the direction of white matter tracts , has shown decreased fractional anisotropy of water movement in dlb in the precuneus and posterior cingulate areas , perhaps highlighting their role in dlb pathogenesis . brain perfusion spect ( tc - hmpao spect ) has been evaluated in its ability to diagnostically separate dlb from ad , and in ad , reduced relative cerebral blood flow ( rcbf ) in the frontal , and medial temporal regions is characteristic , whereas in dlb , occipital hypoperfusion is often observed . applied statistical parametric mapping to spect imaging of dlb patients , more precisely showing large perfusion deficits in the left medial occipital gyrus and the bilateral central , inferior parietal , precuneate , superior frontal and cingulate regions on the brain , which are functionally consistent with frontal - executive and visuospatial deficits in dlb . across studies , sensitivity ranged from 65 to 85% and specificity from 8587% , which appears less robust as a potential imaging marker compared with other methods . aside from -syn and dj-1 , numerous other mutations have been associated with familial early - onset pd and possibly lbd ( table 1 ) . among these gene products are parkin ( park 2 ) , uchl-1 ( park 5 ) , pink1 ( pten - induced putative kinase 1 ; park 6 ) , and lrrk2/dardarin ( park 8) . indeed , none of these mutations have yet been associated with prototypic lbd pathology , and it remains to be determined whether they actually represent lbds or separate diseases with parkinsonian phenotype . furthermore , no studies have addressed their role as biological markers of disease , but since both synucleins and dj-1 are detected in csf and peripheral fluids , it seems plausible that the protein products of other dominant genes in pd could be peripheral biomarker candidates for dlb and other lbd . parkin , uchl-1 , and pink1 genes , like dj-1 , all encode proteins important in neuroprotection in terms of maintaining protein homeostasis and preventing stress - related cellular damage , and mutations in these genes cause a loss of these critical functions . leucine - rich repeat kinase 2 ( lrrk2/dardarin ) , on the contrary , is linked with autosomal - dominant late - onset pd , and mutations result in a toxic gain of function . lrrk2/dardarin is a kinase consisting of multiple functional domains , and recent evidence suggests that physiologically , its principal function may be to regulate neurite outgrowth . expression in cultured neurons of several lrrk2 mutations associated with familial pd , such as g2019s , increased kinase activity and significantly reduced neurite outgrowth , whereas expression of a dominant - negative mutation , k1906 m , markedly increased neurite length . pd - associated mutations also generated tau - positive axonal inclusions in cultured neurons , suggesting that lrrk2 may be linked to abnormalities in tau . indeed , expression of mutant g2019s lrrk2 in drosophila caused activation of the drosophila gsk-3 homolog and promoted tau hyperphosphorylation leading to microtubule fragmentation and dendritic pathology . similar tau hyperphosphorylation was also present in transgenic mice expressing g2019s lrrk2 , and expression of both wild - type human lrrk2 and g2019s mutant lrrk2 caused abnormal dopaminergic transmission . lrrk2 may also interact with -syn , another dominantly inherited pd gene , to exert its effect . lin et al . showed that overexpression of lrrk2 with a53 t mutant -syn in transgenic mice worsened neurodegeneration , while ablation of lrrk2 expression suppressed -syn aggregation and pathology , and -syn also activates gsk-3 in mice causing tau hyperphosphorylation , indicating that lrrk2 , -syn , and tau alterations may all be linked in the same pathway , perhaps with lrrk2 upstream of these events . although early , evidence has indicated that lrrk2 is also a component of lb in pd and dlb brains , and that lrrk2 and -syn interact in dlb brain and coimmunoprecipitate in cultured cells after oxidative stress challenge , suggesting that the lrrk2 may also be important in dlb pathogenesis . interestingly , genome - wide association studies ( gwass ) in a european cohort demonstrated that lrrk2 , -syn , and tau are loci associated with pd risk , but examination of tau in a japanese gwas cohort failed to identify it as a pd risk locus , showing a population difference with regard to this locus . certainly , population differences might apply to all risk loci examined for pd and lbd , and it is important to determine whether the relationship among lrrk2 , -syn , and tau in pd , dlb , and other lbd is also influenced by population differences . these findings make lrrk2/dardarin an attractive candidate for examination as a potential biomarker , and if identified in csf or peripheral fluids , they might be used with -syn and tau as combined biomarkers . furthermore , emerging evidence is redefining the roles of pink1 and parkin in pd pathogenesis . because energy generation is critical for cellular function , mammalian cells are highly dependent on mitochondria . depolarization and morphological defects characterize damaged or impaired mitochondria which are targeted for removal through mitophagy , a highly specialized form of autophagy in which parkin and pink1 play a crucial role ( reviewed by vives - bauza and przedborski ) . in this process , pink1 cleavage is inhibited by the loss of mitochondrial membrane potential , causing its lengthening and the recruitment of cytosolic parkin [ 170 , 171 ] . voltage - dependent anion channel 1 and other outer mitochondrial membrane proteins are then ubiquitinated in a parkin - dependent manner , and this in turn recruits the binding of adapter proteins such as p62 and histone deacetylase 6 to initiate autophagosome assembly around the damaged mitochondrion and subsequent removal . of relevance to pd , mutant pink1 and mutant parkin both cause motor dysfunction , dopaminergic loss , and abnormal mitochondrial morphology in drosophila . in this paradigm , loss of function pink1 mutants are rescued by concurrent overexpression with wild - type parkin but not vice versa , indicating that parkin specifically acts downstream of pink1 . also , parkin mutations have been shown to interfere with ubiquitination and the downstream steps in normal mitophagy . thus , pd , and possibly related dementias , might be a result , to some extent , of defective mitophagy due to loss of function in pink1 and parkin such as found in autosomal dominant early - onset pd . although lrrk2 , parkin , pink1 , and uchl-1 have not yet been identified in peripheral fluids , pink1 and parkin may be a promising candidates . unexpectedly , both pink1 and parkin , which are normally cytosolic or targeted to mitochondria , were localized extracellularly in ad and multiple sclerosis brain , and colocalized with amyloid plaques , reactive astrocytes , as well as amyloid - affected vessels [ 174 , 175 ] . this suggests that both pink1 and parkin are actively released from neurons and glia in response to injury and might be upregulated in csf and peripheral fluids during neurodegeneration . interestingly , given a role in mitophagy , they might also be a csf or peripheral reflection of mitochondrial health and turnover . it remains to be seen whether these gene products can be detected in biological fluids such as csf as potential biomarkers in pd and lbd . as detailed above , traditional methods for molecular biomarker determination have been derived from targeted analyses of candidate genes / mutations and corresponding proteins in brain and body fluids such as csf and blood , with the subsequent exploration of mechanisms in cell culture and animal models . an emerging alternate approach has been to evaluate genomes and proteomes with regard to specific neurodegenerative diseases and their components in an unbiased manner to yield a number of potential pathogenetic , therapeutic , and biomarker targets for further validation . with regard to the genomic analysis of the lbds , scherzer et al . , for instance , examined transgenic drosophila expressing the human -syn gene and performed temporal profiling of resultant gene expression . they demonstrated a number of changes , including a downregulation of phospholipase a2 and other lipid genes , downregulation of several mitochondrial respiratory chain molecules , and alteration in membrane transport and energy genes such as voltage - gated calcium channel and lysosomal atpase , suggesting that mitochondrial integrity might be affected by -syn overexpression . in parkinson 's disease brain , rna from populations of mesencephalic dopaminergic neurons with and without lb interestingly , upregulation of the ubiquitin - specific protease 8 in lb - containing neurons indicated cellular damage and increased levels of ubiquitination in lb , whereas non - lb - bearing neurons showed increased expression of novel cytoprotective genes such as bullous pemphigoid antigen 1 , an hsp-70-like gene ( stch ) and kelch - like 1 . although promising , further genomic profiling studies in dlb , pdd , and other lbd are needed to expand the range of novel gene targets for examination and validation . as a complement to gene expression profiling and genomic methods , proteomic profiling has also assumed a greater importance in biomarker discovery for neurodegeneration with relevance to the lbd . advances in methodologies such as 2-dimensional gel electrophoresis ( 2-d ge ) , liquid chromatography ( lc ) , high - resolution mass spectrometry ( ms ) , and quantitative proteomics allow analysis of static or condition - dependent protein structure and function associated with pd and lbd in a variety of sample types such as brain or body fluids ( reviewed in shi et al . 2009 ) . in mice treated with mptp , a specific mitochondrial toxin , isotope - coded affinity tag assay of brain tissue followed by ms analysis revealed 100 proteins with significantly altered levels including many mitochondrial and metabolic molecules , app and dj-1 . first examined the proteome of the substantia nigra from parkinson 's disease brain and age - matched controls . using 2d ge and peptide fingerprinting , of the 44 expressed proteins , 9 proteins differed in pd versus controls , including oxidative and mitochondrial proteins such as peroxiredoxin ii , mitochondrial complex iii , calcium channel , and others . a subsequent study in pd brain showed decreased frontal cortex levels of mortalin , a novel mitochondrial chaperone protein with roles in energy generation . in addition , lbs isolated by laser - capture microdissection , were analyzed by lc / ms and ultimately demonstrated 156 candidate proteins involved in ubiquitin - proteasome system and synaptic function , from which the heat shock cognate-71 , a chaperone involved in neurodegenerative disease , was identified and validated as a candidate target . abdi and colleagues carried out proteomic evaluation of csf from ad , pd , and dlb patients and normal control individuals , using chromatography , ms , and isobaric tagging for relative and absolute quantification ( itraq ) , identifying numerous candidate proteins related to pd and dlb , such as lipoproteins apoc1 and apoh . lastly , using surface - enhanced laser desorption / ionization - time of flight ( seldi - tof ) ms analysis of serum from dlb patients compared to ad , a combination of protein peaks provided the ability to separate dlb from non - dlb cases , with a sensitivity of 83.3% and a specificity of 95.8% . given promising findings , further exploration of the proteomics of the lbds is warranted , and perhaps consideration should be given to determining whether combining various genomic and proteomic methods will be of value . over the last decade , tremendous advances have been made in understanding the pathogenetics of pd , pdd , and dlb , which has revealed not only the genetic basis of these disorders , but also related mechanisms common to all the lbd . in parallel , these discoveries have been a catalyst for translating and developing many of the involved proteins into promising biomarkers for disease . a common theme centers on genes that drive a complex network of synergistic and opposing cellular actions underlying pathogenesis . aggregation of -syn , the main constituent of intracellular lbs , results in toxic oligomers and protofibrils which not only act intracellularly , but also are actively and passively released into the extracellular environment causing damage to surrounding tissue . on the contrary , dj-1 , pink1 , parkin , and perhaps others molecules are upregulated to oppose cellular protein misfolding and oxidative stress and maintain mitochondrial function , while autophagy mechanisms attempt to limit the toxic effect of synucleins and other toxins by lysosomal engulfment and digestion . much of this is reminiscent of a relatively new concept applied to infectious diseases and mechanical tissue injury termed damage - associated molecular patterning ( damp ) , which is an evolved system to recognize , contain , and repair damage to cells and tissues . it is characterized by the abnormal release of molecules normally confined and operating within healthy cells or from foreign pathogenic agents , that when released into the extracellular space activate receptors and pathways leading to inflammation and multiplying cellular damage ( reviewed by bianchi ) . in this regard , events in the pathogenesis of pd , dlb , and related disorders may represent a novel variation of the damp response , and in a sense , biological fluid markers are therefore a measurement of damp activity as it relates to neurodegeneration . despite progress in developing biological markers for pd , pdd , and dlb , clinical diagnosis of this spectrum of disorders remains challenging . the need for highly sensitive and specific biomarkers that accurately mirror the underlying pathogenetic features of these disorders demands not only that more advanced detection methods be devised and validated in large sample populations , but also that novel biomarker candidates be selected for evaluation based on rational selection from the multiple - associated gene - mechanism associations in the lbd . gene products including - and -synuclein , gba , parkin , and pink1 need to be examined in csf , blood , and even urine to confirm their presence in biological fluids and threshold of detection . alterations in the levels of these putative biomarker candidates in csf and blood can provide further insight into the role these mechanisms may play in disease and also the ability of the potential biomarker to reflect resulting cns changes . to better understand the relationship of gene mutations , mechanisms , and disease biomarkers in lbd , it would be of great interest to determine whether the levels of these putative biomarkers in csf and peripheral fluids are altered in patients with known pd , dlb , and lbd mutations . it is likely that combinations of multiple peripheral biomarkers could be needed to monitor the various mechanistic aspects underlying the lbd , but the optimal combination has yet to be determined . furthermore , both existing imaging modalities as well as novel imaging techniques to detect specific molecular biomarker targets will greatly complement peripheral biomarkers . new specific therapies for the lbd yet to be developed will probably target one or more of the multiple pathways described above , and indeed , this could determine which biomarker or combination of biomarkers would be appropriate as a therapeutic endpoint . studies are also needed to establish which biomarkers will fulfill the criteria of minimum sensitivity and specificity for the lbd for consistent and reproducible diagnostic use in presymptomatic disease detection and also serve as robust tracking tools and endpoints in monitoring the efficacy of future lbd therapies .

recent advances have been made in defining the genetic and molecular basis of dementia with lewy bodies ( dlbs ) and related neurodegenerative disorders such as parkinson 's disease ( pd ) and parkinson 's disease dementia ( pdd ) which comprise the spectrum of lewy body disorders ( lbds ) . the genetic alterations and underlying disease mechanisms in the lbd overlap substantially , suggesting common disease mechanisms . as with the other neurodegenerative dementias , early diagnosis in lbd or even identification prior to symptom onset is key to developing effective therapeutic strategies , but this is dependent upon the development of robust , specific , and sensitive biomarkers as diagnostic tools and therapeutic endpoints . recently identified mutations in the synucleins and other relevant genes in pd and dlb as well as related biomolecular pathways suggest candidate markers from biological fluids and imaging modalities that reflect the underlying disease mechanisms . in this context , several promising biomarkers for the lbd have already been identified and examined , while other intriguing possible candidates have recently emerged . challenges remain in defining their correlation with pathological processes and their ability to detect dlb and related disorders , and perhaps a combined array of biomarkers may be needed to distinguish various lbds .

1. Introduction 2. Amyloid and Tau in Lewy Body Disorders 3. Synucleins: Genetics to Biomarkers in the Lewy Body Disorders 4. DJ-1 in the Lewy Body Disorders 5. Glucocerebrosidase as a Novel Biomarker for Lewy Body Disorders 6. Miscellanous Candidate Biomarkers 7. Unbiased Methods in LBD Biomarker Discovery 8. Conclusions

over the past decade , dementia with lewy bodies ( dlbs ) has arguably become the second most common form of neurodegenerative dementia behind alzheimer 's disease ( ad ) . approximately 2040% of parkinson 's disease ( pd ) patients also eventually develop a progressive dementing illness designated as parkinson 's disease dementia ( pdd ) characterized by a frontal - subcortical clinical presentation . dlb / diffuse lewy body disease ( dlbd ) , lewy body variant of ad ( lbv ) , and pdd comprise an emerging spectrum of clinical phenotypes from relatively pure motor pd to the more predominant cognitive and behavioral disturbance observed in pdd and dlb , yet the reason for the variability remains unknown . despite the heterogeneity of their clinical phenotypes , a significant neuropathological overlap is observed among these diseases , hence the term lewy body disorders ( lbds ) to collectively describe conditions in which lewy bodies ( lbs ) and lewy neurites ( lns ) predominate as the hallmark histological lesions . variation in the distribution of lewy body pathology is present among lbds , with more neocortical and limbic system lb in both dlb and pdd compared to the brains of pd patients without cognitive symptoms and greater neuronal loss in substantia nigra in pdd than dlb . as with other forms of dementia , the pathobiological changes in lbd likely occur decades prior to the onset of clinical symptoms and correspond to widespread irreversible neurodegeneration [ 6 , 7 ] . critically important is not only distinguishing the lbd from ad and other forms of dementia , but also separating dlbs and other lbds . here , we highlight from the perspective of how major genetic discoveries in the lbd and their corresponding biomolecular processes might translate into useful disease markers in biological fluids . this paper will highlight several pd and dlb genes and their protein products as candidates for biological disease markers ( table 1 ) . the genetic mechanisms of a overproduction in ad are well established ; the app gene ( chromosome 21 ) , the first identified ad susceptibility gene , encodes a transmembranous protein ranging from 695 to 770 residues , which undergoes a process of regulated intramembranous proteolysis ultimately releasing a peptides , primarily a42 and a40 , as well as other fragments . genetic analysis of early - onset familial ad cases revealed numerous mutations in the app gene as well as presenilin 1 ( ps1 ; chromosome 14 ) and presenilin 2 ( ps2 ; chromosome 1 ) genes , all of which accelerate the processing of app , leading to increased a generation . amyloid load was highest in lbd patients in the parietal and posterior cingulate regions , corresponding to visuospatial impairments on neuropsychological testing , suggesting that amyloid deposition could partly contribute to the clinical presentation of lbds . tau - bearing neurofibrillary tangles remain one of the pathological hallmarks of ad but are also central to a diverse group of disorders termed tauopathies which include progressive supranuclear palsy , corticobasal ganglionic degeneration , frontotemporal dementia ( ftd ) with parkinsonism linked to chromosome 17 , and other disorders . unlike the tauopathies , no direct pathogenetic tau mutations have been identified in lbds , but tau pathology appears to be a consistent feature among neurodegenerative dementias including ad and lbds , and given the pathological overlap , they might share similar pathogenetic pathways ( reviewed in stoothoff and johnson ) . also , the protein can be recovered from filaments in purified lewy bodies from pdd and dlb brain , and recombinant -syn tends to form lewy body - like fibrillar structures in vitro . in the past decade , tremendous advances have been made in understanding the genetic factors influencing the pathogenesis of lewy body disorders . compelling evidence for a genetic basis for pd and dlb followed the discovery of mutations in the -syn gene ( park1/4 ) in patients with autosomal dominant familial parkinson 's disease , and subsequently , mutations were identified in patients with both sporadic and familial dlbs . from a susceptibility marker on chromosome 4q21 - 23 that segregated with the pd phenotype in italian and greek kindreds , a53 t and a30p were the first two missense mutations in -syn associated with familial parkinson 's disease . another mutation , e46k , was discovered in a spanish family presenting with autosomal dominant dlb , and in genetic studies of a large family with the spectrum of lewy body phenotype ranging from pd to dlb , -syn gene triplication was described , causing -syn overproduction similar to the trisomy effect observed in down syndrome patients . a53 t -syn transgenic mice have increased oligomerization of the protein in brain regions devoid of inclusions as well as those areas with more abundant lesions and neurodegeneration , and consistent with prior biophysical findings , -syn toxicity in these mice was dependent on the conformation of intermediate species . -syn has recently been implicated in pd and dlb pathogenesis , but its precise role in disease is still emerging . unlike the other synuclein family members , -syn or persyn is largely expressed in the cell bodies and axons of primary sensory neurons , sympathetic neurons , and motor neurons as well as in brain . single - nucleotide polymorphisms in all three synucleins have been associated with sporadic dlbd , most prominently -syn , and in sporadic pd , dlb , and lbv patients , -syn antibodies , as well as -syn and -syn reveal unique hippocampal axonal pathology . multiple forms of -syn are released into cerebrospinal fluid ( csf ) and other biological fluids . full - length -syn has been recovered from lumbar csf from living normal control , pd and dlb patients [ 69 , 70 ] , and also from postmortem csf from dlb and other neurodegenerative diseases . plasma -syn detected by immunoblotting was decreased in pd compared with age - matched control subjects , and those pd patients with age - at - onset prior to 55 years ( early - onset ) had significantly lower levels than those with onset after 55 years of age ( late - onset ) . one promising consideration for the future exploration of -syn as an lbd biomarker will be the development of novel imaging compounds and techniques , similar to amyloid imaging , to specifically target and visualize -syn distribution in the pd and lbd brain . due to their increasing importance in lbd pathogenesis , -syn and -syn , as much as -syn as such , levels of these synucleins might be altered in the csf of patients with pd / pdd and dlb , reflecting the underlying degenerative processes in brain . more detailed examination of both -syn and -syn as a peripheral disease markers in well - characterized populations of pd , dlb , and other disorders is warranted to determine their specificity and sensitivity in the synucleinopathies . no less than 13 gene mutations have been identified in dj-1 in atypical younger - onset pd patients , but their significance to idiopathic late - onset pd remains uncertain . interestingly , this association with tau pathology was seen in dlb and lbv brains , suggesting that as a chaperone molecule , dj-1 may be involved in tangle formation , and the binding of dj-1 with these lesions could abolish the normally protective effect of dj-1 , enhancing oxidative neurotoxicity . of note , dj-1 is also subject to oxidative modifications in pd and ad brain tissue , and this might be measured in peripheral fluids as well , as another monitor of oxidative damage . csf dj-1 remains a promising and perhaps clinically useful biomarker for pd , but as far as dlb and other lbd , it is unknown whether csf levels of dj-1 are altered . many clinicopathologic parallels can be drawn between the lysosomal storage disorders , such as niemann - pick , sandhoff 's , tay - sachs disease and others , and the age - related neurodegenerative disorders , when considering the aberrant accumulation of pathological substances ( e.g. , lysosomal sphingomyelin in niemann - pick disease versus synucleins in pd and dlb ) and the phenotypes of neuronal loss and cognitive deterioration found in both . in the past few years , an altogether unexpected pathogenetic relationship emerged between gaucher 's disease ( gd ) , a prototypic storage disease , and the synucleinopathies . recent reports documented an increased incidence of pd in heterozygous relatives of patients with gd [ 99 , 100 ] , but interest in this phenomenon was propelled by the finding that gba mutations were in fact more common in pd patients of ashkenazi background compared with ad patients and pd patients in the general population [ 101103 ] . in dlb , csf gba activity showed the greatest magnitude of decrease , reinforcing its importance in the lbd , but also noteworthy is the fact that ad and ftd showed decreased -mannosidase activity , suggesting that this might be another important factor in lysosomal dysfunction in neurodegeneration . in pd , several case control genetic analyses have demonstrated that homozygous carriers of the il-1 511 and tnf- 308 promoter region variants have increased disease risk [ 120 , 121 ] , and that earlier age at onset in pd was associated with il-1 511 homozygosity at allele 1 . il-1 , il-2 , il-6 , and tnf- are all upregulated in pd brain , as well as in csf from pd patients [ 124126 ] , and chen et al . indeed , the neuroinflammatory cytokines may be important as a pathogenetic response to cns injury caused by accumulation of amyloidogenic proteins , but their role as biomarkers for the lbd , especially for dlb , is still unclear . disorganization and breakdown in the cytoskeletal network occurs in various lbds and other neurodegenerative diseases , and as discussed , gamma - synuclein and proteolytic degradation of the cytoskeleton may be involved . it is recognized that in addition to -syn , three types of nf protein also comprise the structure of lewy bodies . elevated csf nf protein was reported in msa and psp , but not in pd , and this was suggested to clinically aid in differentiating parkinsonian syndromes . therefore , because cytoskeletal abnormalities are present in many neurodegenerative dementias as well as in pd , nf protein may be more a reflection of nonspecific alterations in neuronal and axonal function , which does not appear to able to clinically separate dlb from other disorders . in ad , csf ach was reported to be significantly lower than control levels , while in pd and huntington 's disease patients , despite some cholinergic deficit , lumbar csf ach and choline levels did not differ from normal . no studies have directly examined csf cholinergic levels in dlb or lbds , but recently , shimada and colleagues employed positron emission tomography ( pet ) mapping of brain ach activity in dlb and pdd patients and normal controls and demonstrated a marked reduction in cholinergic activity in medial occipital cortex of dlb and pdd , greater than that observed in pd patients without dementia . csf dopamine ( da ) and its metabolites have been investigated previously in pd , and recently , lunardi et al . showed differences in csf da and its metabolites , homovanillic acid ( hva ) and dihydroxyphenylacetic acid ( dopac ) , in pd patients , demonstrating early - stage dopaminergic loss and a correlation with the development of dyskinesia . autonomic failure is a common clinical finding in lbd , including pd and dlb , but not in non - lbd dementias , and therefore it has been investigated as an alternative biomarker for the diagnostic separation of dlb from other dementias . various magnetic resonance ( mr ) imaging modalities have been explored in dlb and pdd , including volumetric imaging , diffusion tensor imaging , and proton magnetic resonance spectroscopy ( reviewed in watson et al . ) using conventional mri techniques such as voxel - based morphometry and region of interest analysis atrophy has been rated at 1.4% per year in dlb brain , 1.31% per year in pdd , and 0.31% per year in pd . brain perfusion spect ( tc - hmpao spect ) has been evaluated in its ability to diagnostically separate dlb from ad , and in ad , reduced relative cerebral blood flow ( rcbf ) in the frontal , and medial temporal regions is characteristic , whereas in dlb , occipital hypoperfusion is often observed . furthermore , no studies have addressed their role as biological markers of disease , but since both synucleins and dj-1 are detected in csf and peripheral fluids , it seems plausible that the protein products of other dominant genes in pd could be peripheral biomarker candidates for dlb and other lbd . although early , evidence has indicated that lrrk2 is also a component of lb in pd and dlb brains , and that lrrk2 and -syn interact in dlb brain and coimmunoprecipitate in cultured cells after oxidative stress challenge , suggesting that the lrrk2 may also be important in dlb pathogenesis . certainly , population differences might apply to all risk loci examined for pd and lbd , and it is important to determine whether the relationship among lrrk2 , -syn , and tau in pd , dlb , and other lbd is also influenced by population differences . voltage - dependent anion channel 1 and other outer mitochondrial membrane proteins are then ubiquitinated in a parkin - dependent manner , and this in turn recruits the binding of adapter proteins such as p62 and histone deacetylase 6 to initiate autophagosome assembly around the damaged mitochondrion and subsequent removal . thus , pd , and possibly related dementias , might be a result , to some extent , of defective mitophagy due to loss of function in pink1 and parkin such as found in autosomal dominant early - onset pd . unexpectedly , both pink1 and parkin , which are normally cytosolic or targeted to mitochondria , were localized extracellularly in ad and multiple sclerosis brain , and colocalized with amyloid plaques , reactive astrocytes , as well as amyloid - affected vessels [ 174 , 175 ] . it remains to be seen whether these gene products can be detected in biological fluids such as csf as potential biomarkers in pd and lbd . as detailed above , traditional methods for molecular biomarker determination have been derived from targeted analyses of candidate genes / mutations and corresponding proteins in brain and body fluids such as csf and blood , with the subsequent exploration of mechanisms in cell culture and animal models . they demonstrated a number of changes , including a downregulation of phospholipase a2 and other lipid genes , downregulation of several mitochondrial respiratory chain molecules , and alteration in membrane transport and energy genes such as voltage - gated calcium channel and lysosomal atpase , suggesting that mitochondrial integrity might be affected by -syn overexpression . in parkinson 's disease brain , rna from populations of mesencephalic dopaminergic neurons with and without lb interestingly , upregulation of the ubiquitin - specific protease 8 in lb - containing neurons indicated cellular damage and increased levels of ubiquitination in lb , whereas non - lb - bearing neurons showed increased expression of novel cytoprotective genes such as bullous pemphigoid antigen 1 , an hsp-70-like gene ( stch ) and kelch - like 1 . although promising , further genomic profiling studies in dlb , pdd , and other lbd are needed to expand the range of novel gene targets for examination and validation . advances in methodologies such as 2-dimensional gel electrophoresis ( 2-d ge ) , liquid chromatography ( lc ) , high - resolution mass spectrometry ( ms ) , and quantitative proteomics allow analysis of static or condition - dependent protein structure and function associated with pd and lbd in a variety of sample types such as brain or body fluids ( reviewed in shi et al . using 2d ge and peptide fingerprinting , of the 44 expressed proteins , 9 proteins differed in pd versus controls , including oxidative and mitochondrial proteins such as peroxiredoxin ii , mitochondrial complex iii , calcium channel , and others . abdi and colleagues carried out proteomic evaluation of csf from ad , pd , and dlb patients and normal control individuals , using chromatography , ms , and isobaric tagging for relative and absolute quantification ( itraq ) , identifying numerous candidate proteins related to pd and dlb , such as lipoproteins apoc1 and apoh . over the last decade , tremendous advances have been made in understanding the pathogenetics of pd , pdd , and dlb , which has revealed not only the genetic basis of these disorders , but also related mechanisms common to all the lbd . in parallel , these discoveries have been a catalyst for translating and developing many of the involved proteins into promising biomarkers for disease . on the contrary , dj-1 , pink1 , parkin , and perhaps others molecules are upregulated to oppose cellular protein misfolding and oxidative stress and maintain mitochondrial function , while autophagy mechanisms attempt to limit the toxic effect of synucleins and other toxins by lysosomal engulfment and digestion . in this regard , events in the pathogenesis of pd , dlb , and related disorders may represent a novel variation of the damp response , and in a sense , biological fluid markers are therefore a measurement of damp activity as it relates to neurodegeneration . the need for highly sensitive and specific biomarkers that accurately mirror the underlying pathogenetic features of these disorders demands not only that more advanced detection methods be devised and validated in large sample populations , but also that novel biomarker candidates be selected for evaluation based on rational selection from the multiple - associated gene - mechanism associations in the lbd . gene products including - and -synuclein , gba , parkin , and pink1 need to be examined in csf , blood , and even urine to confirm their presence in biological fluids and threshold of detection . it is likely that combinations of multiple peripheral biomarkers could be needed to monitor the various mechanistic aspects underlying the lbd , but the optimal combination has yet to be determined . furthermore , both existing imaging modalities as well as novel imaging techniques to detect specific molecular biomarker targets will greatly complement peripheral biomarkers . new specific therapies for the lbd yet to be developed will probably target one or more of the multiple pathways described above , and indeed , this could determine which biomarker or combination of biomarkers would be appropriate as a therapeutic endpoint . studies are also needed to establish which biomarkers will fulfill the criteria of minimum sensitivity and specificity for the lbd for consistent and reproducible diagnostic use in presymptomatic disease detection and also serve as robust tracking tools and endpoints in monitoring the efficacy of future lbd therapies .

dlb / diffuse lewy body disease ( dlbd ) , lewy body variant of ad ( lbv ) , and pdd comprise an emerging spectrum of clinical phenotypes from relatively pure motor pd to the more predominant cognitive and behavioral disturbance observed in pdd and dlb , yet the reason for the variability remains unknown . despite the heterogeneity of their clinical phenotypes , a significant neuropathological overlap is observed among these diseases , hence the term lewy body disorders ( lbds ) to collectively describe conditions in which lewy bodies ( lbs ) and lewy neurites ( lns ) predominate as the hallmark histological lesions . variation in the distribution of lewy body pathology is present among lbds , with more neocortical and limbic system lb in both dlb and pdd compared to the brains of pd patients without cognitive symptoms and greater neuronal loss in substantia nigra in pdd than dlb . it is increasingly clear from the ad therapeutic experience that by the time widespread neuronal injury ensues , symptomatic cholinergic treatments are minimally effective at best , and disease - modifying therapeutic approaches in trials have thus far proven ineffective at altering disease course or in rescuing diseased brain [ 8 , 9 ] . to demonstrate efficacy , any potential disease - modifying therapy in neurodegenerative dementia must be initiated prior to the expression of the clinical phenotype during the initial molecular pathogenetic events before irreversible neuronal damage has occurred . because of many common pathogenetic features among the lbds , the emerging genetic influences found in pd have readily been translated to dlbs and other lbds , providing clues to rational approaches for selecting future dlb and lbd biomarker targets for exploration . as indicated previously , amyloid pathology in the form of neuritic and diffuse plaques can be also found in varying degrees in the brain tissue of patients with dlb , which may interact with lb or synuclein pathology or influence the clinical features of lbds [ 4 , 11 ] . the genetic mechanisms of a overproduction in ad are well established ; the app gene ( chromosome 21 ) , the first identified ad susceptibility gene , encodes a transmembranous protein ranging from 695 to 770 residues , which undergoes a process of regulated intramembranous proteolysis ultimately releasing a peptides , primarily a42 and a40 , as well as other fragments . genetic analysis of early - onset familial ad cases revealed numerous mutations in the app gene as well as presenilin 1 ( ps1 ; chromosome 14 ) and presenilin 2 ( ps2 ; chromosome 1 ) genes , all of which accelerate the processing of app , leading to increased a generation . specifically , the app km670/671nl ( swedish ) mutation affects the -secretase site , a692 g ( flemish ) mutation alters the -secretase site , and both v717f ( indiana ) and v717i ( london ) mutations affect the -secretase processing , leading to elevated a levels . also important in the notch developmental signaling pathway which is analogous to app processing , the presenilins are thought to be a component of the -secretase enzyme complex , which suggests that missense mutations in the presenilins mechanistically lead to accelerated app processing to a . therefore , the abnormal proteolytic cleavage of app leads to elevated brain a deposition , and as a result , diminished peripheral levels of a. reflecting a shift from soluble a to insoluble brain deposits , significant decreases in csf a42 levels have been demonstrated in ad and more recently in dlb cases . found that dlb , compared with pd , pdd , and ad patients , showed the lowest csf levels of a42 and , when combined with csf tau , differentiated dlb from pd and pdd , but not from ad . an australian study reported more variable cortical pib binding in dlb patients than in ad , whereas a subsequent examination of pib binding in lbds including dlb , pdd , and pd , compared with ad and normal patients , showed higher amyloid burden in dlb and ad than in pdd , pd , or nc patients . tau - bearing neurofibrillary tangles remain one of the pathological hallmarks of ad but are also central to a diverse group of disorders termed tauopathies which include progressive supranuclear palsy , corticobasal ganglionic degeneration , frontotemporal dementia ( ftd ) with parkinsonism linked to chromosome 17 , and other disorders . unlike the tauopathies , no direct pathogenetic tau mutations have been identified in lbds , but tau pathology appears to be a consistent feature among neurodegenerative dementias including ad and lbds , and given the pathological overlap , they might share similar pathogenetic pathways ( reviewed in stoothoff and johnson ) . interaction with tubulin suggests -syn could be a microtubule - associated protein similar to tau [ 32 , 33 ] , and it is highly active in various membrane lipid bilayers such as in presynaptic vesicles acting as a chaperone for soluble nsf attachment protein receptor ( snare ) complex formation , in neuronal golgi apparatus influencing protein trafficking and in the inner membrane of neuronal mitochondrial . compelling evidence for a genetic basis for pd and dlb followed the discovery of mutations in the -syn gene ( park1/4 ) in patients with autosomal dominant familial parkinson 's disease , and subsequently , mutations were identified in patients with both sporadic and familial dlbs . another mutation , e46k , was discovered in a spanish family presenting with autosomal dominant dlb , and in genetic studies of a large family with the spectrum of lewy body phenotype ranging from pd to dlb , -syn gene triplication was described , causing -syn overproduction similar to the trisomy effect observed in down syndrome patients . a53 t -syn transgenic mice have increased oligomerization of the protein in brain regions devoid of inclusions as well as those areas with more abundant lesions and neurodegeneration , and consistent with prior biophysical findings , -syn toxicity in these mice was dependent on the conformation of intermediate species . in cancer biology , -syn is associated with abnormally altering cellular mitotic checkpoints in various types of malignancies , making them more aggressively metastatic , but as far as neurodegeneration , it is the most recent synuclein member to be linked to lbd neuropathology and the least well understood . single - nucleotide polymorphisms in all three synucleins have been associated with sporadic dlbd , most prominently -syn , and in sporadic pd , dlb , and lbv patients , -syn antibodies , as well as -syn and -syn reveal unique hippocampal axonal pathology . in vivo , -syn overexpression in transgenic mice shows age- and dose - dependent neuronal loss throughout the neuraxis , especially in spinal motor neurons , where -syn - bearing inclusions , gliosis , and alterations in heat shock protein and neurofilament structure are found , perhaps suggesting relevance to motor neuron disease associated with dementia . in vitro evidence further supports a cytoskeletal role for -syn in maintaining neurofilament structure ; -syn overexpression in cultured neurons causes disruption of the neurofilament network by destabilizing the structural integrity of neurofilament - h allowing degradation by calcium - dependent proteases , which has implications for neurodegeneration . in pd , a smaller early study showed that no differences in full - length csf 19 kda -syn have been found in relation to control individuals , but a recent effort using a new luminex assay in a larger sample controlling for extraneous influences showed significantly decreased levels in pd compared to controls with 92% disease sensitivity and 58% specificity . plasma -syn detected by immunoblotting was decreased in pd compared with age - matched control subjects , and those pd patients with age - at - onset prior to 55 years ( early - onset ) had significantly lower levels than those with onset after 55 years of age ( late - onset ) . although its exact role is unknown , multiple functions have been assigned to the dj-1 protein ras - related signaling pathways , it shares structural homology with the carboxy - terminal domain of escherichia coli hpii catalase and is reported to possess catalase activity which reduces oxidative stress in cultured cells . when expressed in cultured cells , l166p appears to be a loss - of - function mutation which leads to dj-1 functional instability , degradation by the proteasome system [ 86 , 87 ] , abnormal translocation of dj-1 to mitochondria , and loss of chaperone activity . dj-1 is found in brain across a wide range of neurodegenerative diseases including pd , ftd , ad , dlb , and lbvad , and demonstrates striking association with neuropil threads and neurofibrillary pathology in neocortex and subcortical brain regions in these disorders . it is caused by autosomal recessive gene mutations in the glucocerebrosidase ( gba ) gene ( chromosome 1q21 ) , leading to either partial or complete deficiency of gba , and hence , toxic lysosomal accumulation of its substrate , glucosylceramide , in multiple cell types including neurons . recent reports documented an increased incidence of pd in heterozygous relatives of patients with gd [ 99 , 100 ] , but interest in this phenomenon was propelled by the finding that gba mutations were in fact more common in pd patients of ashkenazi background compared with ad patients and pd patients in the general population [ 101103 ] . in fact , examination of gba gene alterations in dlb patients , with and without concomitant lbv - type ad pathology , showed that the majority of gba mutations were found in dlb patients rather than in pd , with a mutation rate in dlb ranging from 18 to 23% overall [ 108 , 109 ] . moreover , in dlb , ad , and ftd patients , lysosomal enzyme activities in csf demonstrated a very specific pattern of decrease , in which only dlb showed significant decreases in csf activity of -mannosidase , -mannosidase , gba , galactosidase , and -hexosaminidase , whereas in ad and ftd , only csf -mannosidase activity was significantly diminished . in dlb , csf gba activity showed the greatest magnitude of decrease , reinforcing its importance in the lbd , but also noteworthy is the fact that ad and ftd showed decreased -mannosidase activity , suggesting that this might be another important factor in lysosomal dysfunction in neurodegeneration . in pd , several case control genetic analyses have demonstrated that homozygous carriers of the il-1 511 and tnf- 308 promoter region variants have increased disease risk [ 120 , 121 ] , and that earlier age at onset in pd was associated with il-1 511 homozygosity at allele 1 . no studies have directly examined csf cholinergic levels in dlb or lbds , but recently , shimada and colleagues employed positron emission tomography ( pet ) mapping of brain ach activity in dlb and pdd patients and normal controls and demonstrated a marked reduction in cholinergic activity in medial occipital cortex of dlb and pdd , greater than that observed in pd patients without dementia . a larger phase iii , multicenter study of i - fp cit spect in possible and probable dlb patients and non - dlb comparators ( mostly ad ) demonstrated a mean sensitivity of 77.7% for detecting clinically probable dlb , with a specificity of 90.4% and 85.7% overall diagnostic accuracy . abnormal autonomic function can be determined using cardiac i - meta - iodobenzyl guanidine ( i - mibg ) imaging , a technique which assesses cardiac sympathetic nerve function in both cardiac and neurological disorders by measuring the uptake of i - mibg , a norepinephrine analogue . finally , consistent with autonomic dysfunction in dlb , both early and delayed h / m i - mibg uptake were significantly associated with the presence of orthostatic hypotension in dlb patients and discriminated dlb from ad even in the absence of parkinsonism . diffusion tensor imaging , an mr technique mapping brain microdiffusion of water in the direction of white matter tracts , has shown decreased fractional anisotropy of water movement in dlb in the precuneus and posterior cingulate areas , perhaps highlighting their role in dlb pathogenesis . brain perfusion spect ( tc - hmpao spect ) has been evaluated in its ability to diagnostically separate dlb from ad , and in ad , reduced relative cerebral blood flow ( rcbf ) in the frontal , and medial temporal regions is characteristic , whereas in dlb , occipital hypoperfusion is often observed . applied statistical parametric mapping to spect imaging of dlb patients , more precisely showing large perfusion deficits in the left medial occipital gyrus and the bilateral central , inferior parietal , precuneate , superior frontal and cingulate regions on the brain , which are functionally consistent with frontal - executive and visuospatial deficits in dlb . furthermore , no studies have addressed their role as biological markers of disease , but since both synucleins and dj-1 are detected in csf and peripheral fluids , it seems plausible that the protein products of other dominant genes in pd could be peripheral biomarker candidates for dlb and other lbd . expression in cultured neurons of several lrrk2 mutations associated with familial pd , such as g2019s , increased kinase activity and significantly reduced neurite outgrowth , whereas expression of a dominant - negative mutation , k1906 m , markedly increased neurite length . indeed , expression of mutant g2019s lrrk2 in drosophila caused activation of the drosophila gsk-3 homolog and promoted tau hyperphosphorylation leading to microtubule fragmentation and dendritic pathology . showed that overexpression of lrrk2 with a53 t mutant -syn in transgenic mice worsened neurodegeneration , while ablation of lrrk2 expression suppressed -syn aggregation and pathology , and -syn also activates gsk-3 in mice causing tau hyperphosphorylation , indicating that lrrk2 , -syn , and tau alterations may all be linked in the same pathway , perhaps with lrrk2 upstream of these events . although early , evidence has indicated that lrrk2 is also a component of lb in pd and dlb brains , and that lrrk2 and -syn interact in dlb brain and coimmunoprecipitate in cultured cells after oxidative stress challenge , suggesting that the lrrk2 may also be important in dlb pathogenesis . interestingly , genome - wide association studies ( gwass ) in a european cohort demonstrated that lrrk2 , -syn , and tau are loci associated with pd risk , but examination of tau in a japanese gwas cohort failed to identify it as a pd risk locus , showing a population difference with regard to this locus . certainly , population differences might apply to all risk loci examined for pd and lbd , and it is important to determine whether the relationship among lrrk2 , -syn , and tau in pd , dlb , and other lbd is also influenced by population differences . voltage - dependent anion channel 1 and other outer mitochondrial membrane proteins are then ubiquitinated in a parkin - dependent manner , and this in turn recruits the binding of adapter proteins such as p62 and histone deacetylase 6 to initiate autophagosome assembly around the damaged mitochondrion and subsequent removal . they demonstrated a number of changes , including a downregulation of phospholipase a2 and other lipid genes , downregulation of several mitochondrial respiratory chain molecules , and alteration in membrane transport and energy genes such as voltage - gated calcium channel and lysosomal atpase , suggesting that mitochondrial integrity might be affected by -syn overexpression . in parkinson 's disease brain , rna from populations of mesencephalic dopaminergic neurons with and without lb interestingly , upregulation of the ubiquitin - specific protease 8 in lb - containing neurons indicated cellular damage and increased levels of ubiquitination in lb , whereas non - lb - bearing neurons showed increased expression of novel cytoprotective genes such as bullous pemphigoid antigen 1 , an hsp-70-like gene ( stch ) and kelch - like 1 . advances in methodologies such as 2-dimensional gel electrophoresis ( 2-d ge ) , liquid chromatography ( lc ) , high - resolution mass spectrometry ( ms ) , and quantitative proteomics allow analysis of static or condition - dependent protein structure and function associated with pd and lbd in a variety of sample types such as brain or body fluids ( reviewed in shi et al . in addition , lbs isolated by laser - capture microdissection , were analyzed by lc / ms and ultimately demonstrated 156 candidate proteins involved in ubiquitin - proteasome system and synaptic function , from which the heat shock cognate-71 , a chaperone involved in neurodegenerative disease , was identified and validated as a candidate target . abdi and colleagues carried out proteomic evaluation of csf from ad , pd , and dlb patients and normal control individuals , using chromatography , ms , and isobaric tagging for relative and absolute quantification ( itraq ) , identifying numerous candidate proteins related to pd and dlb , such as lipoproteins apoc1 and apoh . lastly , using surface - enhanced laser desorption / ionization - time of flight ( seldi - tof ) ms analysis of serum from dlb patients compared to ad , a combination of protein peaks provided the ability to separate dlb from non - dlb cases , with a sensitivity of 83.3% and a specificity of 95.8% . over the last decade , tremendous advances have been made in understanding the pathogenetics of pd , pdd , and dlb , which has revealed not only the genetic basis of these disorders , but also related mechanisms common to all the lbd . in this regard , events in the pathogenesis of pd , dlb , and related disorders may represent a novel variation of the damp response , and in a sense , biological fluid markers are therefore a measurement of damp activity as it relates to neurodegeneration . the need for highly sensitive and specific biomarkers that accurately mirror the underlying pathogenetic features of these disorders demands not only that more advanced detection methods be devised and validated in large sample populations , but also that novel biomarker candidates be selected for evaluation based on rational selection from the multiple - associated gene - mechanism associations in the lbd . alterations in the levels of these putative biomarker candidates in csf and blood can provide further insight into the role these mechanisms may play in disease and also the ability of the potential biomarker to reflect resulting cns changes . to better understand the relationship of gene mutations , mechanisms , and disease biomarkers in lbd , it would be of great interest to determine whether the levels of these putative biomarkers in csf and peripheral fluids are altered in patients with known pd , dlb , and lbd mutations . studies are also needed to establish which biomarkers will fulfill the criteria of minimum sensitivity and specificity for the lbd for consistent and reproducible diagnostic use in presymptomatic disease detection and also serve as robust tracking tools and endpoints in monitoring the efficacy of future lbd therapies .

the amount of body fat may significantly change over the seasons , particularly in latitudes away from the equator , where seasonal changes in climate , temperature and duration of daylight are greater . these lead to changes in availability of certain foods and in individuals feeding habits and outdoor activity ( resulting e.g. in picnics being more common in summer ) ( reilly and peiser 2006 ) . plasqui and westerterp ( 2004 ) have shown that there is a significant seasonal variation in physical activity and total energy expenditure , with lower amounts in winter , in young dutch adults . the biological significance of such seasonal and daily environmental rhythms has long been appreciated ( reinberg 1972 ) and is best seen in seasonal animals and hibernators , which adjust their physiology both in preparation for , and in response to , changing demands of the environment ( ebling and barrett 2008 ) . however , in humans living in modern societies , the impact of seasonality has somewhat diminished following the introduction of artificial lighting and heating and air - conditioning systems . the use of these artificial aids reduces the exposure of individuals to fluctuations in ambient temperature and light , and this is more convenient for practising a modern lifestyle . however , these natural fluctuations contribute to the normal adjustment of the body clock to a 24-h period ; their extensive use ( artificial aids ) will lessen this synchronization and may increase the risk of developing mismatches between the natural environment and the body clock ( similar to the problems observed after a time - zone transition or during night work ) . it has been claimed that these misalignments may lead to alterations in metabolism and thermoregulation that promote obesity ( johnson et al . while seasonal rhythmicity in energy storage and expenditure is significantly influenced by changes in the external environment ( reilly and peiser 2006 ) , the nature of daily rhythms in metabolism is more complex . in this respect , humans possess internal timing mechanisms which can act independently of daily changes in the environment . all cells show a genetic potential for daily rhythmicity , but in practice , this rhythmicity is manifested in only some regions of the body . these regions include the liver ( which possesses a food - entrainable oscillator ) and the suprachiasmatic nucleus ( scn ) paired structures in the base of the hypothalamus . the scn normally coordinates rhythmic activity throughout the body ( acting via the autonomic nervous system , temperature regulation , hormone secretion , sleep , and feeding behaviour ) and is known as the body clock . evidence is accumulating to suggest that the disruption of these body clocks may contribute to metabolic disorders and predispose to obesity ( eckel - mahan and sassone - corsi 2013 ) . rhythmicity seen in many processes , including metabolism , reflects both personal habits ( e.g. sleep , activity and mealtimes ) and the impact of internal body clocks . humans , like other organisms , possess a timing system that consists of self - sustained oscillators that are reset by various synchronizers . in the absence of time cues , the dominant component of this system free runs with a period of 2425 h , giving rise to a so - called circadian rhythm ( from the latin : circa diem about a day ) . normally , this rhythm is entrained or synchronized to a 24-h cycle ( called daily in this review ) , predominantly by natural light dark cycles and , to lesser extent , by cycles of rest and activity or feeding and fasting . external stimuli that can synchronize the body clock to a 24-h cycle are called zeitgebers ( from german : time givers ) ( reilly and peiser 2006 ) . the molecular mechanisms that underlie the function of cellular clocks are the oscillating post - translational modifications of proteins ( e.g. phosphorylation ) and the autoregulatory feedback loops that control gene transcription and translation . the main loop comprises a positive and a negative limb that are interconnected ( albrecht 2012 ) . it consists of the transcriptional activators clock and bmal1 and their target genes per ( period ) and cry ( cryptochrome ) . products of these genes accumulate gradually and inhibit clock - bmal1 transcription . in turn , the feedback loop controlling bmal1 involves nuclear receptors rev - erb and ror/ that inhibit and activate bmal1 transcription , respectively . in addition , the activity of rev - erb links metabolism to the clock system ( liu et al . administration of rev - erb ligands in mice has been found to alter expression of both the clock genes in the hypothalamus and the metabolic genes in the liver , skeletal muscles and adipose tissue , resulting in increased energy expenditure ( solt et al . the reader is referred to recent excellent reviews ( albrecht 2012 ; bass 2012 ; bass and takahashi 2010 ; mohawk et al . the observation that all cells , tissues and organs contain the molecular potential to manifest a clock gave rise to the concept of peripheral and central ( master ) oscillators , the former normally being subservient to the latter . the central pacemaker in mammals is located in two hypothalamic clusters of neurons , the scn . these centres control behavioural , metabolic and physiological rhythms and can synchronize the peripheral oscillators ( welsh et al . an important peripheral oscillator is the food - entrainable oscillator ( feo ) , which controls food - anticipatory activity ( faa ) in rodents , the exact location of which is unknown ( mieda et al . faa in rodents is an increase in activity just before the food is regularly available . daily rhythms of locomotor activity , body temperature and corticosterone secretion can thus synchronize with the rhythm of food availability ( stephan 2002 ) , even when food is presented during the resting phase or when the central oscillator is destroyed ( mistlberger 2011 ) . these observations indicate that the feo ( and , possibly , peripheral clocks in general ) can act independently of the scn , at least in some animal . this independence of activity may become important when the master oscillator and lifestyle become misaligned . in normal circumstances , the master clock , the environment and peripheral clocks are synchronized to one another ( reilly and peiser 2006 ) . whereas periodicity of the master clock is controlled mainly through the light dark cycle ( acting as a zeitgeber ) , peripheral oscillators are affected either by behaviour ( cycles of rest and activity or feeding and fasting ) or by fluctuations in the levels of circulating hormones , such as catecholamines ( dibner et al . for example , peripheral oscillators in the pancreas and the liver ( marcheva et al . 2010 ) can be adjusted by regular food intake , even if this intake is timed unusually . the scn produces the endogenous component of the observed rhythm , and the exogenous component is superimposed upon it and corresponds to the environment and lifestyle ( e.g. inactivity and fasting when asleep ) . in practice , therefore , rhythms measured in the presence of an exogenous component may not give clear information regarding the activity of the internal oscillators , and the implications of this will be considered at the end of this review . however , normally , the endogenous and exogenous components are in phase but may become desynchronized ( e.g. by night work ) , because the scn is rather slow to adjust ( reilly and peiser 2006 ) . under such circumstances , the environment and lifestyle may also adjust the timing of the master oscillator ( e.g. by changing the light dark cycle ) and of the peripheral oscillators ( e.g. by changing feeding times , which will impact on feo ) . food intake in humans shows not only a daily rhythm ( daytime rather than nocturnal eating ) but also a rhythm related to the intake of individual meals with a period of about 45 h. this rhythm is ultradian , having a period of oscillation less than 20 h. such rhythms are probably seen in the activity of all hormones associated with food metabolism . while they are linked directly to food intake ( and , as such , can be considered exogenous ) , other rhythms are likely to be derived from internal oscillators . episodic release of hormones may exhibit yet another periodicity that lasts for minutes and reflects pulsatile release of a hormone followed by its removal and breakdown . there are also infradian rhythms ( with periodicity greater than 28 h ) including seasonal rhythms . since there is no clear evidence that endogenous circannual oscillators exists in humans , seasonal rhythms observed are attributed rather to exogenous factors . as indicated earlier , they include seasonal variations in food intake , physical activity , ambient temperature and the duration of daylight . this lack of information is partly due to the obvious fact that such studies demand a more elaborate protocol which covers the four seasons . moreover , if any interactions between daily rhythms and seasonal rhythms are sought , then the a full set of data covering the 24 h must be collected four times per year . daily food intake in humans usually consists of two to three main meals consumed at times that depend largely on lifestyle and social factors . it has been observed that food intake in the morning is more satiating than the same meal eaten in the evening ( de castro 2009 ) ; it has also been observed that the amount of food eaten shows seasonal variations , with increased meal size and total calorie intake occurring in the autumn ( de castro 1991 ) . the hunger and satiety centres in the hypothalamus contain receptors for mediators that affect feeding behaviour . these substances are either orexigenic ( stimulate feeding ) or anorexigenic ( inhibit feeding ) ( naslund and hellstrom 2007 ) . short - term regulation of food intake involves cholecystokinin ( little et al . 2005 ) , peptide yy , glucagon - like peptide and insulin ( suzuki et al . 2012 ) , all of which act anorexigenically , and ghrelin , which stimulates appetite ( cummings 2006 ) . these mediators display daily and ultradian rhythms in phase with food intake and some of them ( ghrelin , leptin , insulin ) are also involved in long - term regulation of body weight and energy homeostasis ( stutz et al . the mechanisms controlling seasonal food intake and energy balance in seasonal animals and hibernators differ across the species and reflect different strategies employed to survive in a harsh environment ( florant and healy 2012 ) . the seasonal changes are executed through fluctuations in humoral signals , including leptin and ghrelin ( adam and mercer 2004 ; florant and healy 2012 ) . interestingly , while leptin concentrations in humans do not exhibit consistent seasonal changes , it has been observed that the levels of leptin , cholesterol and triglycerides in obese males are significantly higher in winter ( kanikowska et al . 2013 ) . the digestive system shows rhythmicity in many functions , including basal gastric acid secretion , epithelial cell proliferation and gastrointestinal motility ( ekmekcioglu and touitou 2011 ) . it is attributed primarily to the timing and the size of meals and exhibits daily and ultradian components . however , some aspects of this rhythmicity may reflect the function of a peripheral clock . for example , it has been demonstrated in animals that rhythmic expression of clock genes within the neurons of the myenteric plexus modulates colonic motility by controlling the expression of neuronal nitric oxide synthetase ( nnos ) and vasoactive intestinal peptide ( vip ) ( hoogerwerf 2010 ) . indirect evidence for a role of biological clocks in gastrointestinal functions in humans comes from observations of night workers who have altered appetites and a higher prevalence of constipation , diarrhoea and abdominal discomfort ( nojkov et al . 2010 ) . metabolism of absorbed foodstuffs shows rhythmicity that reflects changes in the release of endocrine regulators . hormones associated with metabolism ( including glucagon , insulin , catecholamines , glucocorticoids and thyroid hormones ) show both circadian and ultradian oscillations . these rhythms are dominated by food intake ( and as such are exogenous and ultradian ) but may also be influenced by the scn that produces daily fluctuations in sympathetic nervous system outflow which may affect hormone secretion ( e.g. insulin ) . rhythmicity in clock gene expression and adipocytokine release is seen also in white and brown adipose tissues ( wat and bat , respectively ) ( gavrila et al . 2003 ) . body temperature in adult humans is about 1 c higher during the day , which is attributed both to the sleep - wake cycle ( exogenous component ) and to the scn - generated rhythmicity in metabolism and peripheral vasculature tone ( endogenous component ) . in babies , however , these rhythms of temperature regulation are not fully developed , and bat plays an important role in heat production . 2006 ) and shows a 24-h rhythm of glucose uptake ( van der veen et al . 2012 ) . moreover , bat activity is regulated via the sympathetic nervous system and by several hormones , all of which express daily variations ( kriegsfeld and silver 2006 ) . seasonal changes in bat have been observed in humans , with bat growth induced by exposure to cold and associated with the shorter hours of daylight in the autumn and winter ( au - yong et al . it is now clear that several aspects of metabolism show daily rhythmicity related to scn function ( bass 2012 ; bass and takahashi 2010 ; marcheva et al . however , the scn is also adjusted by feeding behaviour and metabolic products , which results in additional ultradian rhythmicity . moreover , many organs involved in food metabolism , such as the liver ( balsalobre et al . 2000 ) , pancreas ( sadacca et al . 2011 ) , intestine and stomach ( bostwick et al . 2010 ) and the adipose tissue ( johnston 2012 ) , have autonomous peripheral oscillators ( fig . 1 ) . therefore , it is not surprising that factors altering these interactions might adversely impact on metabolism and be associated with an increased risk of obesity . in this respect , it has recently been shown that the impairment of peripheral clocks may be associated with the development of diabetes ( pappa et al . 1interactions between the master clock ( scn ) , peripheral oscillators and the environment interactions between the master clock ( scn ) , peripheral oscillators and the environment however , other lifestyle - related factors that have been implicated in obesity ( such as sleep duration , eating habits , shift work ) have not always received enough attention ( chaput et al . . short sleep ( defined as 6 h of sleep per day ) and sleep disorders have been associated with lower concentrations of leptin and higher levels of ghrelin and with increased hunger and appetite ( taheri et al . in addition , it has been demonstrated that sleep deprivation may contribute to obesity through modulating plasma levels of leptin ( mullington et al . 2003 ) . also , the lack of orexin , a hypothalamic wakefulness - inducing neuropeptide ( sakurai et al . 1998 ) , affects sleep , feeding and metabolism ( adamantidis and de lecea 2008 ) and is associated with increased likelihood of developing obesity ( funato et al . narcolepsy , when patients suffer from extreme daytime sleepiness due to the loss of orexin - producing neurons ( tsujino and sakurai 2013 ) , has also been linked with increased body mass and obesity ( kotagal et al . patients with night eating syndrome , in whom the patterns of sleep and eating are disrupted , are often obese ( colles et al . 2007 ) and have altered rhythms of plasma leptin , insulin , cortisol , ghrelin , melatonin and glucose ( goel et al . it appears that most overweight and obese people sleep less than normal , and therefore , they have more time to eat ( chaput et al . 2011 ) and snack ( nedeltcheva et al . 2009b ) , and they are also at increased risk of insulin resistance and type 2 diabetes ( chao et al . interestingly , sleep architecture changes with seasons , with increased rapid eye movement ( rem ) sleep during the winter ( kohsaka et al . 2possible effects of altered sleep - wake cycles on metabolic hormones and body weight possible effects of altered sleep - wake cycles on metabolic hormones and body weight the exact mechanism linking sleep disturbances and it has been postulated that loss of sleep reduces glucose tolerance and increases insulin resistance ( nedeltcheva et al . on the other hand , sleep deprivation may increase food intake and appetite ( brondel et al . 2010 ) by decreasing leptin or by increasing ghrelin ( taheri et al . 2004 ) and orexin ( zeitzer 2013 ) . since sleep is believed to allow the brain to replenish energy stores , it has been speculated that altered sleeping habits might through the autonomic nervous system and hypothalamic - pituitary - adrenal axis impact on the release of metabolic hormones and the control of food intake ( spiegel et al . eating patterns when to eat , the amount of food eaten and the circumstances leading to finishing a meal affect energy intake ( blundell and cooling 2000 ) . an important study on the effect of meal frequency / pattern on body fat and metabolic functions in humans was by fbry and tepperman ( 1970 ) , who found that excessive weight , hypercholesterolemia , impaired glucose tolerance and ischemic heart disease were more common among persons who ate larger meals less frequently rather than smaller meals more often . grazers , who eat smaller meals throughout the daytime , may be metabolically advantaged compared to gorgers ( who eat fewer but larger meals ) , since larger meals may lead to increased fat synthesis and storage ( verboeket - van de venne and westerterp 1991 ) . it has been demonstrated that the same meal eaten at different times of the day may exert different metabolic effects ; thus , it appears that a morning meal is associated with better control of body mass than when the same meal is eaten later in the day ( keim et al . such an effect may be related to the amount of physical work performed during the daytime and endocrine responses to food intake ( with the insulin response to food intake being time - of - day dependent , for example ) . in this respect , it has recently been demonstrated in mice that a feeding regimen that restricted feeding time but not calorie intake showed improved nutrient utilization and energy expenditure ( hatori et al . 2012 ) . patterns of food intake can be determined by the social context , time availability and night work . binge eating involves food intake greatly in excess of metabolic requirements , often with the loss of control over the amount eaten . it has been observed that breakfast was the least , and dinner the most , common meal associated with this practice , and binge eating was often associated with evening snacking ( harvey et al . 2011 ; stunkard and allison 2003 ) . a positive relationship between appetite and food intake no longer exists for meals eaten during the working day ; in these circumstances , a proper meal is often replaced by fast food eaten at amounts reflecting time availability rather than appetite . by contrast , if there is plenty of time and one is with friends , food intake is often in excess of metabolic requirements , particularly if alcohol is drunk as part of the occasion s conviviality ( de castro 2009 ; waterhouse et al . the type of food eaten varies also over the day with greater intake of carbohydrates at breakfast and of fat at dinner ( westerterp - plantenga et al . short and irregular sleeps are associated with consuming more fats , fast foods and sweet beverages , and less vegetables ( baron et al . 2011 ) , as well as with more palatable foods high in sugar , fat and salt rather than rich in protein or roughage ( st - onge et al . these problems are particularly evident when snacking late at night or during a nocturnal waking episode . the mechanisms linking frequency of food intake and the type of food eaten remain unclear , but it has been suggested that lower leptin and higher ghrelin concentrations are involved ( taheri et al . for example , meals with a high fat - to - carbohydrate ratio decrease plasma concentrations of leptin ( havel et al . 1999 ) and ghrelin ( monteleone et al . 2003 ) concentrations . shift and night work may disturb 24-h rhythms , including endocrine rhythms ( morris et al . night work is associated with an increased risk of metabolic syndrome ( esquirol et al . 2009 ) , obesity ( pandalai et al . 2013 ) and sleep disturbances ( ohayon et al . 2003 ) and tend to snack ( on foods high in salt and carbohydrate ) rather than eat a full meal during the shift . in addition , attempts to eat with their families whenever possible often mean that total daily food intake increases . the combination of these factors increases the likelihood of developing indigestion , obesity and metabolic disorders ( karlsson et al . lack of synchrony between body clocks and lifestyle may change the secretory profiles of metabolic hormones , including ghrelin and leptin ( crispim et al . 2011 ) , which may contribute to increased appetite and higher energy intake ( spiegel et al . 2004 ) . gaining weight is more likely if night workers also experience social disturbances due to their abnormal lifestyle ( waterhouse et al . 2005 ) . thus , disturbed expression of clock genes in wat was detected in genetically obese mice of the kk and kk - a(y ) strains ( ando et al . these mice showed also abnormal leptin secretion , sleep disturbances , altered locomotor activity and changed rhythms of adiponectin and resistin release . also , mice with adipose tissue - targeted deletion of bmal1 displayed increased adiposity and body weight , impairment of feeding rhythms and alterations in the expression of hypothalamic neuropeptides that regulate appetite ( paschos et al . similarly , obese humans were found to differ in the expression patterns of several clock and metabolic genes in adipose tissue ( garaulet et al . 2011 ) , in the rhythms of plasma adipokines ( saad et al . 1998 ) and in daily rhythms of leptin and ghrelin secretion ( heptulla et al . 2001 ; yildiz et al . in addition to being a key component of the body clock , adipocyte bmal1 has also been implicated in adipose tissue differentiation and lipogenesis ( shimba et al . 2005 ) . moreover , the expression of uncoupling protein-1 ( ucp1 ) that is in involved in bat thermogenesis was found to associate with winter accumulation of visceral fat ( nakayama et al . the central and peripheral clocks act to coordinate behavioural and metabolic responses with the environment . light dark cycles entrain the central clock in the scn , which then synchronizes peripheral clocks and the rest of the body through autonomic innervation , body temperature , endocrine signalling and feeding - related cues . feeding can regulate peripheral clocks independent of the central clock through local metabolites and signalling pathways . increasing evidence suggests that this harmony may become disturbed either through behavioural misalignment ( such as shift work or jet lag ) or metabolic challenges ( e.g. high - fat feeding ) ( bass and takahashi 2010 ) . this may lead to further weakening of links between the clocks and result in abnormalities that promote weight gain , obesity and development of metabolic syndrome . future research will need to elucidate the exact molecular mechanisms linking biological clocks with metabolic homeostasis and nutrient state . ultimately , such studies may help to prevent metabolic derangement and obesity in individuals with sleeping disorders or working on shifts and to optimize weight loss regimens . also , whilst there is considerable evidence that altered daily rhythms are associated with obesity and allied problems , there is an interpretive problem associated with such results . as already mentioned , a measured daily rhythm reflects not only the activity of the central and peripheral oscillators controlling metabolism but also exogenous effects directly due to the pattern of food intake ( including the period of fasting during sleep ) . that is , a measured rhythm is not necessarily an accurate reflection of the activities of the internal mechanisms ( the oscillators ) ; it might be masked by the exogenous component . it is important to distinguish between these two causes of a rhythm if detailed information regarding the interactions between the internal oscillators and metabolic processes in obesity is sought . one way to separate the effects of these two causes is to minimize the exogenous component ; this could be achieved by giving identical meals at equal time intervals throughout the 24 h , also prohibiting sleep . the rhythms observed in these circumstances ( when the patterns of food intake and the sleep - wake cycle have been removed ) would then reflect those of the internal processes far more closely . such studies are of fundamental importance to a better understanding of obesity and need to be performed in future research .

while the significance of obesity as a serious health problem is well recognized , little is known about whether and how biometerological factors and biorhythms causally contribute to obesity . obesity is often associated with altered seasonal and daily rhythmicity in food intake , metabolism and adipose tissue function . environmental stimuli affect both seasonal and daily rhythms , and the latter are under additional control of internal molecular oscillators , or body clocks . modifications of clock genes in animals and changes to normal daily rhythms in humans ( as in shift work and sleep deprivation ) result in metabolic dysregulation that favours weight gain . here , we briefly review the potential links between biorhythms and obesity in humans .

Seasonal and daily influence on metabolism and body weight Basic principles of chronobiology Peripheral and central oscillators The rhythmic organism Rhythmicity of food intake, gut function and metabolism Factors associated with increased risk of obesity Conclusions and perspectives

the biological significance of such seasonal and daily environmental rhythms has long been appreciated ( reinberg 1972 ) and is best seen in seasonal animals and hibernators , which adjust their physiology both in preparation for , and in response to , changing demands of the environment ( ebling and barrett 2008 ) . however , in humans living in modern societies , the impact of seasonality has somewhat diminished following the introduction of artificial lighting and heating and air - conditioning systems . the use of these artificial aids reduces the exposure of individuals to fluctuations in ambient temperature and light , and this is more convenient for practising a modern lifestyle . however , these natural fluctuations contribute to the normal adjustment of the body clock to a 24-h period ; their extensive use ( artificial aids ) will lessen this synchronization and may increase the risk of developing mismatches between the natural environment and the body clock ( similar to the problems observed after a time - zone transition or during night work ) . it has been claimed that these misalignments may lead to alterations in metabolism and thermoregulation that promote obesity ( johnson et al . while seasonal rhythmicity in energy storage and expenditure is significantly influenced by changes in the external environment ( reilly and peiser 2006 ) , the nature of daily rhythms in metabolism is more complex . all cells show a genetic potential for daily rhythmicity , but in practice , this rhythmicity is manifested in only some regions of the body . these regions include the liver ( which possesses a food - entrainable oscillator ) and the suprachiasmatic nucleus ( scn ) paired structures in the base of the hypothalamus . the scn normally coordinates rhythmic activity throughout the body ( acting via the autonomic nervous system , temperature regulation , hormone secretion , sleep , and feeding behaviour ) and is known as the body clock . evidence is accumulating to suggest that the disruption of these body clocks may contribute to metabolic disorders and predispose to obesity ( eckel - mahan and sassone - corsi 2013 ) . rhythmicity seen in many processes , including metabolism , reflects both personal habits ( e.g. sleep , activity and mealtimes ) and the impact of internal body clocks . in the absence of time cues , the dominant component of this system free runs with a period of 2425 h , giving rise to a so - called circadian rhythm ( from the latin : circa diem about a day ) . the molecular mechanisms that underlie the function of cellular clocks are the oscillating post - translational modifications of proteins ( e.g. phosphorylation ) and the autoregulatory feedback loops that control gene transcription and translation . it consists of the transcriptional activators clock and bmal1 and their target genes per ( period ) and cry ( cryptochrome ) . in addition , the activity of rev - erb links metabolism to the clock system ( liu et al . administration of rev - erb ligands in mice has been found to alter expression of both the clock genes in the hypothalamus and the metabolic genes in the liver , skeletal muscles and adipose tissue , resulting in increased energy expenditure ( solt et al . the observation that all cells , tissues and organs contain the molecular potential to manifest a clock gave rise to the concept of peripheral and central ( master ) oscillators , the former normally being subservient to the latter . the central pacemaker in mammals is located in two hypothalamic clusters of neurons , the scn . daily rhythms of locomotor activity , body temperature and corticosterone secretion can thus synchronize with the rhythm of food availability ( stephan 2002 ) , even when food is presented during the resting phase or when the central oscillator is destroyed ( mistlberger 2011 ) . whereas periodicity of the master clock is controlled mainly through the light dark cycle ( acting as a zeitgeber ) , peripheral oscillators are affected either by behaviour ( cycles of rest and activity or feeding and fasting ) or by fluctuations in the levels of circulating hormones , such as catecholamines ( dibner et al . for example , peripheral oscillators in the pancreas and the liver ( marcheva et al . 2010 ) can be adjusted by regular food intake , even if this intake is timed unusually . the scn produces the endogenous component of the observed rhythm , and the exogenous component is superimposed upon it and corresponds to the environment and lifestyle ( e.g. inactivity and fasting when asleep ) . in practice , therefore , rhythms measured in the presence of an exogenous component may not give clear information regarding the activity of the internal oscillators , and the implications of this will be considered at the end of this review . however , normally , the endogenous and exogenous components are in phase but may become desynchronized ( e.g. by night work ) , because the scn is rather slow to adjust ( reilly and peiser 2006 ) . under such circumstances , the environment and lifestyle may also adjust the timing of the master oscillator ( e.g. by changing feeding times , which will impact on feo ) . food intake in humans shows not only a daily rhythm ( daytime rather than nocturnal eating ) but also a rhythm related to the intake of individual meals with a period of about 45 h. this rhythm is ultradian , having a period of oscillation less than 20 h. such rhythms are probably seen in the activity of all hormones associated with food metabolism . while they are linked directly to food intake ( and , as such , can be considered exogenous ) , other rhythms are likely to be derived from internal oscillators . episodic release of hormones may exhibit yet another periodicity that lasts for minutes and reflects pulsatile release of a hormone followed by its removal and breakdown . there are also infradian rhythms ( with periodicity greater than 28 h ) including seasonal rhythms . since there is no clear evidence that endogenous circannual oscillators exists in humans , seasonal rhythms observed are attributed rather to exogenous factors . as indicated earlier , they include seasonal variations in food intake , physical activity , ambient temperature and the duration of daylight . this lack of information is partly due to the obvious fact that such studies demand a more elaborate protocol which covers the four seasons . moreover , if any interactions between daily rhythms and seasonal rhythms are sought , then the a full set of data covering the 24 h must be collected four times per year . daily food intake in humans usually consists of two to three main meals consumed at times that depend largely on lifestyle and social factors . it has been observed that food intake in the morning is more satiating than the same meal eaten in the evening ( de castro 2009 ) ; it has also been observed that the amount of food eaten shows seasonal variations , with increased meal size and total calorie intake occurring in the autumn ( de castro 1991 ) . the hunger and satiety centres in the hypothalamus contain receptors for mediators that affect feeding behaviour . these substances are either orexigenic ( stimulate feeding ) or anorexigenic ( inhibit feeding ) ( naslund and hellstrom 2007 ) . short - term regulation of food intake involves cholecystokinin ( little et al . 2005 ) , peptide yy , glucagon - like peptide and insulin ( suzuki et al . 2012 ) , all of which act anorexigenically , and ghrelin , which stimulates appetite ( cummings 2006 ) . these mediators display daily and ultradian rhythms in phase with food intake and some of them ( ghrelin , leptin , insulin ) are also involved in long - term regulation of body weight and energy homeostasis ( stutz et al . the mechanisms controlling seasonal food intake and energy balance in seasonal animals and hibernators differ across the species and reflect different strategies employed to survive in a harsh environment ( florant and healy 2012 ) . the seasonal changes are executed through fluctuations in humoral signals , including leptin and ghrelin ( adam and mercer 2004 ; florant and healy 2012 ) . interestingly , while leptin concentrations in humans do not exhibit consistent seasonal changes , it has been observed that the levels of leptin , cholesterol and triglycerides in obese males are significantly higher in winter ( kanikowska et al . the digestive system shows rhythmicity in many functions , including basal gastric acid secretion , epithelial cell proliferation and gastrointestinal motility ( ekmekcioglu and touitou 2011 ) . it is attributed primarily to the timing and the size of meals and exhibits daily and ultradian components . however , some aspects of this rhythmicity may reflect the function of a peripheral clock . for example , it has been demonstrated in animals that rhythmic expression of clock genes within the neurons of the myenteric plexus modulates colonic motility by controlling the expression of neuronal nitric oxide synthetase ( nnos ) and vasoactive intestinal peptide ( vip ) ( hoogerwerf 2010 ) . indirect evidence for a role of biological clocks in gastrointestinal functions in humans comes from observations of night workers who have altered appetites and a higher prevalence of constipation , diarrhoea and abdominal discomfort ( nojkov et al . 2010 ) . hormones associated with metabolism ( including glucagon , insulin , catecholamines , glucocorticoids and thyroid hormones ) show both circadian and ultradian oscillations . these rhythms are dominated by food intake ( and as such are exogenous and ultradian ) but may also be influenced by the scn that produces daily fluctuations in sympathetic nervous system outflow which may affect hormone secretion ( e.g. insulin ) . rhythmicity in clock gene expression and adipocytokine release is seen also in white and brown adipose tissues ( wat and bat , respectively ) ( gavrila et al . 2003 ) . body temperature in adult humans is about 1 c higher during the day , which is attributed both to the sleep - wake cycle ( exogenous component ) and to the scn - generated rhythmicity in metabolism and peripheral vasculature tone ( endogenous component ) . in babies , however , these rhythms of temperature regulation are not fully developed , and bat plays an important role in heat production . moreover , bat activity is regulated via the sympathetic nervous system and by several hormones , all of which express daily variations ( kriegsfeld and silver 2006 ) . seasonal changes in bat have been observed in humans , with bat growth induced by exposure to cold and associated with the shorter hours of daylight in the autumn and winter ( au - yong et al . it is now clear that several aspects of metabolism show daily rhythmicity related to scn function ( bass 2012 ; bass and takahashi 2010 ; marcheva et al . however , the scn is also adjusted by feeding behaviour and metabolic products , which results in additional ultradian rhythmicity . moreover , many organs involved in food metabolism , such as the liver ( balsalobre et al . 2010 ) and the adipose tissue ( johnston 2012 ) , have autonomous peripheral oscillators ( fig . 1 ) . therefore , it is not surprising that factors altering these interactions might adversely impact on metabolism and be associated with an increased risk of obesity . in this respect , it has recently been shown that the impairment of peripheral clocks may be associated with the development of diabetes ( pappa et al . 1interactions between the master clock ( scn ) , peripheral oscillators and the environment interactions between the master clock ( scn ) , peripheral oscillators and the environment however , other lifestyle - related factors that have been implicated in obesity ( such as sleep duration , eating habits , shift work ) have not always received enough attention ( chaput et al . . short sleep ( defined as 6 h of sleep per day ) and sleep disorders have been associated with lower concentrations of leptin and higher levels of ghrelin and with increased hunger and appetite ( taheri et al . in addition , it has been demonstrated that sleep deprivation may contribute to obesity through modulating plasma levels of leptin ( mullington et al . also , the lack of orexin , a hypothalamic wakefulness - inducing neuropeptide ( sakurai et al . 1998 ) , affects sleep , feeding and metabolism ( adamantidis and de lecea 2008 ) and is associated with increased likelihood of developing obesity ( funato et al . narcolepsy , when patients suffer from extreme daytime sleepiness due to the loss of orexin - producing neurons ( tsujino and sakurai 2013 ) , has also been linked with increased body mass and obesity ( kotagal et al . patients with night eating syndrome , in whom the patterns of sleep and eating are disrupted , are often obese ( colles et al . 2007 ) and have altered rhythms of plasma leptin , insulin , cortisol , ghrelin , melatonin and glucose ( goel et al . it appears that most overweight and obese people sleep less than normal , and therefore , they have more time to eat ( chaput et al . 2009b ) , and they are also at increased risk of insulin resistance and type 2 diabetes ( chao et al . interestingly , sleep architecture changes with seasons , with increased rapid eye movement ( rem ) sleep during the winter ( kohsaka et al . 2possible effects of altered sleep - wake cycles on metabolic hormones and body weight possible effects of altered sleep - wake cycles on metabolic hormones and body weight the exact mechanism linking sleep disturbances and it has been postulated that loss of sleep reduces glucose tolerance and increases insulin resistance ( nedeltcheva et al . on the other hand , sleep deprivation may increase food intake and appetite ( brondel et al . 2010 ) by decreasing leptin or by increasing ghrelin ( taheri et al . since sleep is believed to allow the brain to replenish energy stores , it has been speculated that altered sleeping habits might through the autonomic nervous system and hypothalamic - pituitary - adrenal axis impact on the release of metabolic hormones and the control of food intake ( spiegel et al . eating patterns when to eat , the amount of food eaten and the circumstances leading to finishing a meal affect energy intake ( blundell and cooling 2000 ) . an important study on the effect of meal frequency / pattern on body fat and metabolic functions in humans was by fbry and tepperman ( 1970 ) , who found that excessive weight , hypercholesterolemia , impaired glucose tolerance and ischemic heart disease were more common among persons who ate larger meals less frequently rather than smaller meals more often . it has been demonstrated that the same meal eaten at different times of the day may exert different metabolic effects ; thus , it appears that a morning meal is associated with better control of body mass than when the same meal is eaten later in the day ( keim et al . such an effect may be related to the amount of physical work performed during the daytime and endocrine responses to food intake ( with the insulin response to food intake being time - of - day dependent , for example ) . patterns of food intake can be determined by the social context , time availability and night work . binge eating involves food intake greatly in excess of metabolic requirements , often with the loss of control over the amount eaten . it has been observed that breakfast was the least , and dinner the most , common meal associated with this practice , and binge eating was often associated with evening snacking ( harvey et al . a positive relationship between appetite and food intake no longer exists for meals eaten during the working day ; in these circumstances , a proper meal is often replaced by fast food eaten at amounts reflecting time availability rather than appetite . by contrast , if there is plenty of time and one is with friends , food intake is often in excess of metabolic requirements , particularly if alcohol is drunk as part of the occasion s conviviality ( de castro 2009 ; waterhouse et al . short and irregular sleeps are associated with consuming more fats , fast foods and sweet beverages , and less vegetables ( baron et al . these problems are particularly evident when snacking late at night or during a nocturnal waking episode . the mechanisms linking frequency of food intake and the type of food eaten remain unclear , but it has been suggested that lower leptin and higher ghrelin concentrations are involved ( taheri et al . 1999 ) and ghrelin ( monteleone et al . shift and night work may disturb 24-h rhythms , including endocrine rhythms ( morris et al . night work is associated with an increased risk of metabolic syndrome ( esquirol et al . 2013 ) and sleep disturbances ( ohayon et al . in addition , attempts to eat with their families whenever possible often mean that total daily food intake increases . the combination of these factors increases the likelihood of developing indigestion , obesity and metabolic disorders ( karlsson et al . lack of synchrony between body clocks and lifestyle may change the secretory profiles of metabolic hormones , including ghrelin and leptin ( crispim et al . 2011 ) , which may contribute to increased appetite and higher energy intake ( spiegel et al . 2005 ) . thus , disturbed expression of clock genes in wat was detected in genetically obese mice of the kk and kk - a(y ) strains ( ando et al . these mice showed also abnormal leptin secretion , sleep disturbances , altered locomotor activity and changed rhythms of adiponectin and resistin release . also , mice with adipose tissue - targeted deletion of bmal1 displayed increased adiposity and body weight , impairment of feeding rhythms and alterations in the expression of hypothalamic neuropeptides that regulate appetite ( paschos et al . similarly , obese humans were found to differ in the expression patterns of several clock and metabolic genes in adipose tissue ( garaulet et al . 2011 ) , in the rhythms of plasma adipokines ( saad et al . 1998 ) and in daily rhythms of leptin and ghrelin secretion ( heptulla et al . in addition to being a key component of the body clock , adipocyte bmal1 has also been implicated in adipose tissue differentiation and lipogenesis ( shimba et al . moreover , the expression of uncoupling protein-1 ( ucp1 ) that is in involved in bat thermogenesis was found to associate with winter accumulation of visceral fat ( nakayama et al . light dark cycles entrain the central clock in the scn , which then synchronizes peripheral clocks and the rest of the body through autonomic innervation , body temperature , endocrine signalling and feeding - related cues . feeding can regulate peripheral clocks independent of the central clock through local metabolites and signalling pathways . increasing evidence suggests that this harmony may become disturbed either through behavioural misalignment ( such as shift work or jet lag ) or metabolic challenges ( e.g. high - fat feeding ) ( bass and takahashi 2010 ) . this may lead to further weakening of links between the clocks and result in abnormalities that promote weight gain , obesity and development of metabolic syndrome . ultimately , such studies may help to prevent metabolic derangement and obesity in individuals with sleeping disorders or working on shifts and to optimize weight loss regimens . also , whilst there is considerable evidence that altered daily rhythms are associated with obesity and allied problems , there is an interpretive problem associated with such results . as already mentioned , a measured daily rhythm reflects not only the activity of the central and peripheral oscillators controlling metabolism but also exogenous effects directly due to the pattern of food intake ( including the period of fasting during sleep ) . it is important to distinguish between these two causes of a rhythm if detailed information regarding the interactions between the internal oscillators and metabolic processes in obesity is sought . the rhythms observed in these circumstances ( when the patterns of food intake and the sleep - wake cycle have been removed ) would then reflect those of the internal processes far more closely . such studies are of fundamental importance to a better understanding of obesity and need to be performed in future research .

the amount of body fat may significantly change over the seasons , particularly in latitudes away from the equator , where seasonal changes in climate , temperature and duration of daylight are greater . plasqui and westerterp ( 2004 ) have shown that there is a significant seasonal variation in physical activity and total energy expenditure , with lower amounts in winter , in young dutch adults . the biological significance of such seasonal and daily environmental rhythms has long been appreciated ( reinberg 1972 ) and is best seen in seasonal animals and hibernators , which adjust their physiology both in preparation for , and in response to , changing demands of the environment ( ebling and barrett 2008 ) . however , in humans living in modern societies , the impact of seasonality has somewhat diminished following the introduction of artificial lighting and heating and air - conditioning systems . the use of these artificial aids reduces the exposure of individuals to fluctuations in ambient temperature and light , and this is more convenient for practising a modern lifestyle . however , these natural fluctuations contribute to the normal adjustment of the body clock to a 24-h period ; their extensive use ( artificial aids ) will lessen this synchronization and may increase the risk of developing mismatches between the natural environment and the body clock ( similar to the problems observed after a time - zone transition or during night work ) . while seasonal rhythmicity in energy storage and expenditure is significantly influenced by changes in the external environment ( reilly and peiser 2006 ) , the nature of daily rhythms in metabolism is more complex . in this respect , humans possess internal timing mechanisms which can act independently of daily changes in the environment . all cells show a genetic potential for daily rhythmicity , but in practice , this rhythmicity is manifested in only some regions of the body . these regions include the liver ( which possesses a food - entrainable oscillator ) and the suprachiasmatic nucleus ( scn ) paired structures in the base of the hypothalamus . the scn normally coordinates rhythmic activity throughout the body ( acting via the autonomic nervous system , temperature regulation , hormone secretion , sleep , and feeding behaviour ) and is known as the body clock . evidence is accumulating to suggest that the disruption of these body clocks may contribute to metabolic disorders and predispose to obesity ( eckel - mahan and sassone - corsi 2013 ) . sleep , activity and mealtimes ) and the impact of internal body clocks . humans , like other organisms , possess a timing system that consists of self - sustained oscillators that are reset by various synchronizers . in the absence of time cues , the dominant component of this system free runs with a period of 2425 h , giving rise to a so - called circadian rhythm ( from the latin : circa diem about a day ) . normally , this rhythm is entrained or synchronized to a 24-h cycle ( called daily in this review ) , predominantly by natural light dark cycles and , to lesser extent , by cycles of rest and activity or feeding and fasting . the molecular mechanisms that underlie the function of cellular clocks are the oscillating post - translational modifications of proteins ( e.g. phosphorylation ) and the autoregulatory feedback loops that control gene transcription and translation . it consists of the transcriptional activators clock and bmal1 and their target genes per ( period ) and cry ( cryptochrome ) . in turn , the feedback loop controlling bmal1 involves nuclear receptors rev - erb and ror/ that inhibit and activate bmal1 transcription , respectively . in addition , the activity of rev - erb links metabolism to the clock system ( liu et al . administration of rev - erb ligands in mice has been found to alter expression of both the clock genes in the hypothalamus and the metabolic genes in the liver , skeletal muscles and adipose tissue , resulting in increased energy expenditure ( solt et al . the observation that all cells , tissues and organs contain the molecular potential to manifest a clock gave rise to the concept of peripheral and central ( master ) oscillators , the former normally being subservient to the latter . the central pacemaker in mammals is located in two hypothalamic clusters of neurons , the scn . these centres control behavioural , metabolic and physiological rhythms and can synchronize the peripheral oscillators ( welsh et al . an important peripheral oscillator is the food - entrainable oscillator ( feo ) , which controls food - anticipatory activity ( faa ) in rodents , the exact location of which is unknown ( mieda et al . daily rhythms of locomotor activity , body temperature and corticosterone secretion can thus synchronize with the rhythm of food availability ( stephan 2002 ) , even when food is presented during the resting phase or when the central oscillator is destroyed ( mistlberger 2011 ) . these observations indicate that the feo ( and , possibly , peripheral clocks in general ) can act independently of the scn , at least in some animal . whereas periodicity of the master clock is controlled mainly through the light dark cycle ( acting as a zeitgeber ) , peripheral oscillators are affected either by behaviour ( cycles of rest and activity or feeding and fasting ) or by fluctuations in the levels of circulating hormones , such as catecholamines ( dibner et al . for example , peripheral oscillators in the pancreas and the liver ( marcheva et al . 2010 ) can be adjusted by regular food intake , even if this intake is timed unusually . the scn produces the endogenous component of the observed rhythm , and the exogenous component is superimposed upon it and corresponds to the environment and lifestyle ( e.g. in practice , therefore , rhythms measured in the presence of an exogenous component may not give clear information regarding the activity of the internal oscillators , and the implications of this will be considered at the end of this review . however , normally , the endogenous and exogenous components are in phase but may become desynchronized ( e.g. by changing the light dark cycle ) and of the peripheral oscillators ( e.g. food intake in humans shows not only a daily rhythm ( daytime rather than nocturnal eating ) but also a rhythm related to the intake of individual meals with a period of about 45 h. this rhythm is ultradian , having a period of oscillation less than 20 h. such rhythms are probably seen in the activity of all hormones associated with food metabolism . while they are linked directly to food intake ( and , as such , can be considered exogenous ) , other rhythms are likely to be derived from internal oscillators . episodic release of hormones may exhibit yet another periodicity that lasts for minutes and reflects pulsatile release of a hormone followed by its removal and breakdown . since there is no clear evidence that endogenous circannual oscillators exists in humans , seasonal rhythms observed are attributed rather to exogenous factors . as indicated earlier , they include seasonal variations in food intake , physical activity , ambient temperature and the duration of daylight . moreover , if any interactions between daily rhythms and seasonal rhythms are sought , then the a full set of data covering the 24 h must be collected four times per year . daily food intake in humans usually consists of two to three main meals consumed at times that depend largely on lifestyle and social factors . it has been observed that food intake in the morning is more satiating than the same meal eaten in the evening ( de castro 2009 ) ; it has also been observed that the amount of food eaten shows seasonal variations , with increased meal size and total calorie intake occurring in the autumn ( de castro 1991 ) . the hunger and satiety centres in the hypothalamus contain receptors for mediators that affect feeding behaviour . short - term regulation of food intake involves cholecystokinin ( little et al . 2005 ) , peptide yy , glucagon - like peptide and insulin ( suzuki et al . 2012 ) , all of which act anorexigenically , and ghrelin , which stimulates appetite ( cummings 2006 ) . these mediators display daily and ultradian rhythms in phase with food intake and some of them ( ghrelin , leptin , insulin ) are also involved in long - term regulation of body weight and energy homeostasis ( stutz et al . the mechanisms controlling seasonal food intake and energy balance in seasonal animals and hibernators differ across the species and reflect different strategies employed to survive in a harsh environment ( florant and healy 2012 ) . the seasonal changes are executed through fluctuations in humoral signals , including leptin and ghrelin ( adam and mercer 2004 ; florant and healy 2012 ) . interestingly , while leptin concentrations in humans do not exhibit consistent seasonal changes , it has been observed that the levels of leptin , cholesterol and triglycerides in obese males are significantly higher in winter ( kanikowska et al . the digestive system shows rhythmicity in many functions , including basal gastric acid secretion , epithelial cell proliferation and gastrointestinal motility ( ekmekcioglu and touitou 2011 ) . it is attributed primarily to the timing and the size of meals and exhibits daily and ultradian components . however , some aspects of this rhythmicity may reflect the function of a peripheral clock . for example , it has been demonstrated in animals that rhythmic expression of clock genes within the neurons of the myenteric plexus modulates colonic motility by controlling the expression of neuronal nitric oxide synthetase ( nnos ) and vasoactive intestinal peptide ( vip ) ( hoogerwerf 2010 ) . indirect evidence for a role of biological clocks in gastrointestinal functions in humans comes from observations of night workers who have altered appetites and a higher prevalence of constipation , diarrhoea and abdominal discomfort ( nojkov et al . metabolism of absorbed foodstuffs shows rhythmicity that reflects changes in the release of endocrine regulators . hormones associated with metabolism ( including glucagon , insulin , catecholamines , glucocorticoids and thyroid hormones ) show both circadian and ultradian oscillations . these rhythms are dominated by food intake ( and as such are exogenous and ultradian ) but may also be influenced by the scn that produces daily fluctuations in sympathetic nervous system outflow which may affect hormone secretion ( e.g. rhythmicity in clock gene expression and adipocytokine release is seen also in white and brown adipose tissues ( wat and bat , respectively ) ( gavrila et al . body temperature in adult humans is about 1 c higher during the day , which is attributed both to the sleep - wake cycle ( exogenous component ) and to the scn - generated rhythmicity in metabolism and peripheral vasculature tone ( endogenous component ) . in babies , however , these rhythms of temperature regulation are not fully developed , and bat plays an important role in heat production . moreover , bat activity is regulated via the sympathetic nervous system and by several hormones , all of which express daily variations ( kriegsfeld and silver 2006 ) . seasonal changes in bat have been observed in humans , with bat growth induced by exposure to cold and associated with the shorter hours of daylight in the autumn and winter ( au - yong et al . it is now clear that several aspects of metabolism show daily rhythmicity related to scn function ( bass 2012 ; bass and takahashi 2010 ; marcheva et al . however , the scn is also adjusted by feeding behaviour and metabolic products , which results in additional ultradian rhythmicity . moreover , many organs involved in food metabolism , such as the liver ( balsalobre et al . 2010 ) and the adipose tissue ( johnston 2012 ) , have autonomous peripheral oscillators ( fig . therefore , it is not surprising that factors altering these interactions might adversely impact on metabolism and be associated with an increased risk of obesity . in this respect , it has recently been shown that the impairment of peripheral clocks may be associated with the development of diabetes ( pappa et al . 1interactions between the master clock ( scn ) , peripheral oscillators and the environment interactions between the master clock ( scn ) , peripheral oscillators and the environment however , other lifestyle - related factors that have been implicated in obesity ( such as sleep duration , eating habits , shift work ) have not always received enough attention ( chaput et al . short sleep ( defined as 6 h of sleep per day ) and sleep disorders have been associated with lower concentrations of leptin and higher levels of ghrelin and with increased hunger and appetite ( taheri et al . in addition , it has been demonstrated that sleep deprivation may contribute to obesity through modulating plasma levels of leptin ( mullington et al . also , the lack of orexin , a hypothalamic wakefulness - inducing neuropeptide ( sakurai et al . 1998 ) , affects sleep , feeding and metabolism ( adamantidis and de lecea 2008 ) and is associated with increased likelihood of developing obesity ( funato et al . narcolepsy , when patients suffer from extreme daytime sleepiness due to the loss of orexin - producing neurons ( tsujino and sakurai 2013 ) , has also been linked with increased body mass and obesity ( kotagal et al . patients with night eating syndrome , in whom the patterns of sleep and eating are disrupted , are often obese ( colles et al . 2007 ) and have altered rhythms of plasma leptin , insulin , cortisol , ghrelin , melatonin and glucose ( goel et al . it appears that most overweight and obese people sleep less than normal , and therefore , they have more time to eat ( chaput et al . 2009b ) , and they are also at increased risk of insulin resistance and type 2 diabetes ( chao et al . interestingly , sleep architecture changes with seasons , with increased rapid eye movement ( rem ) sleep during the winter ( kohsaka et al . 2possible effects of altered sleep - wake cycles on metabolic hormones and body weight possible effects of altered sleep - wake cycles on metabolic hormones and body weight the exact mechanism linking sleep disturbances and it has been postulated that loss of sleep reduces glucose tolerance and increases insulin resistance ( nedeltcheva et al . on the other hand , sleep deprivation may increase food intake and appetite ( brondel et al . since sleep is believed to allow the brain to replenish energy stores , it has been speculated that altered sleeping habits might through the autonomic nervous system and hypothalamic - pituitary - adrenal axis impact on the release of metabolic hormones and the control of food intake ( spiegel et al . eating patterns when to eat , the amount of food eaten and the circumstances leading to finishing a meal affect energy intake ( blundell and cooling 2000 ) . an important study on the effect of meal frequency / pattern on body fat and metabolic functions in humans was by fbry and tepperman ( 1970 ) , who found that excessive weight , hypercholesterolemia , impaired glucose tolerance and ischemic heart disease were more common among persons who ate larger meals less frequently rather than smaller meals more often . grazers , who eat smaller meals throughout the daytime , may be metabolically advantaged compared to gorgers ( who eat fewer but larger meals ) , since larger meals may lead to increased fat synthesis and storage ( verboeket - van de venne and westerterp 1991 ) . it has been demonstrated that the same meal eaten at different times of the day may exert different metabolic effects ; thus , it appears that a morning meal is associated with better control of body mass than when the same meal is eaten later in the day ( keim et al . such an effect may be related to the amount of physical work performed during the daytime and endocrine responses to food intake ( with the insulin response to food intake being time - of - day dependent , for example ) . in this respect , it has recently been demonstrated in mice that a feeding regimen that restricted feeding time but not calorie intake showed improved nutrient utilization and energy expenditure ( hatori et al . patterns of food intake can be determined by the social context , time availability and night work . binge eating involves food intake greatly in excess of metabolic requirements , often with the loss of control over the amount eaten . it has been observed that breakfast was the least , and dinner the most , common meal associated with this practice , and binge eating was often associated with evening snacking ( harvey et al . a positive relationship between appetite and food intake no longer exists for meals eaten during the working day ; in these circumstances , a proper meal is often replaced by fast food eaten at amounts reflecting time availability rather than appetite . by contrast , if there is plenty of time and one is with friends , food intake is often in excess of metabolic requirements , particularly if alcohol is drunk as part of the occasion s conviviality ( de castro 2009 ; waterhouse et al . the type of food eaten varies also over the day with greater intake of carbohydrates at breakfast and of fat at dinner ( westerterp - plantenga et al . 2011 ) , as well as with more palatable foods high in sugar , fat and salt rather than rich in protein or roughage ( st - onge et al . the mechanisms linking frequency of food intake and the type of food eaten remain unclear , but it has been suggested that lower leptin and higher ghrelin concentrations are involved ( taheri et al . night work is associated with an increased risk of metabolic syndrome ( esquirol et al . the combination of these factors increases the likelihood of developing indigestion , obesity and metabolic disorders ( karlsson et al . lack of synchrony between body clocks and lifestyle may change the secretory profiles of metabolic hormones , including ghrelin and leptin ( crispim et al . thus , disturbed expression of clock genes in wat was detected in genetically obese mice of the kk and kk - a(y ) strains ( ando et al . these mice showed also abnormal leptin secretion , sleep disturbances , altered locomotor activity and changed rhythms of adiponectin and resistin release . also , mice with adipose tissue - targeted deletion of bmal1 displayed increased adiposity and body weight , impairment of feeding rhythms and alterations in the expression of hypothalamic neuropeptides that regulate appetite ( paschos et al . similarly , obese humans were found to differ in the expression patterns of several clock and metabolic genes in adipose tissue ( garaulet et al . in addition to being a key component of the body clock , adipocyte bmal1 has also been implicated in adipose tissue differentiation and lipogenesis ( shimba et al . moreover , the expression of uncoupling protein-1 ( ucp1 ) that is in involved in bat thermogenesis was found to associate with winter accumulation of visceral fat ( nakayama et al . light dark cycles entrain the central clock in the scn , which then synchronizes peripheral clocks and the rest of the body through autonomic innervation , body temperature , endocrine signalling and feeding - related cues . this may lead to further weakening of links between the clocks and result in abnormalities that promote weight gain , obesity and development of metabolic syndrome . also , whilst there is considerable evidence that altered daily rhythms are associated with obesity and allied problems , there is an interpretive problem associated with such results . as already mentioned , a measured daily rhythm reflects not only the activity of the central and peripheral oscillators controlling metabolism but also exogenous effects directly due to the pattern of food intake ( including the period of fasting during sleep ) . that is , a measured rhythm is not necessarily an accurate reflection of the activities of the internal mechanisms ( the oscillators ) ; it might be masked by the exogenous component . the rhythms observed in these circumstances ( when the patterns of food intake and the sleep - wake cycle have been removed ) would then reflect those of the internal processes far more closely .

since the clinical severity ranges from mild to potentially life - threatening , major efforts are currently being made to develop methods to detect patients with these diseases in the neonatal period . in fact , making a diagnosis before vaccines or blood products are administered , as well as before the infection develops , allows the patient to proceed to haematopoietic stem cell transplantation , and receive enzyme replacement and gene therapies . indeed , the best outcome for the most severe form of primary immunodeficiency , as with many other conditions for which newborn screenings are now performed , is achieved when treatment is given in the first months of life , ideally before clinical presentation . the pilot studies of newborn screenings for severe combined immunodeficiency have demonstrated their cost - effectiveness and have also proved successful in terms of public health resulting from improved quality of life and survival of children with these diseases . the goal of newborn blood screening ( nbs ) is to identify pre - symptomatic newborns with potentially serious or fatal disorders which could be successfully treated , leading to significant reductions in morbidity and mortality . nbs debuted as a public health programme in the us in the early 1960s , and has expanded to countries around the world , with different testing options in each country . nbs has progressively evolved since 1963 , when guthrie and susi demonstrated that postnatal testing of dried blood spots on filter paper led to the identification and treatment of infants prior to the development of the cognitive deficits associated with phenylketonuria . more recently , the advent of dna - based technologies has again shifted the paradigm of nbs , offering the potential of screening for numerous disorders at the same time . the american college of medical genetics recommends a uniform panel of 29 disorders that all infants born in every state should be screened for . therefore , every year , millions of babies in the us are routinely screened for congenital deafness and , using a few drops of blood from the newborn s heel , for blood cell disorders ( sickle cell anaemia , sickle cell disease , and hb s / beta - thalassaemia ) , inborn errors of amino acid metabolism ( tyrosinaemia i , argininosuccinic aciduria , citrullinaemia , phenylketonuria , maple syrup urine disease , and homocystinuria ) , inborn errors of organic acid metabolism ( glutaric acidaemia type i , hydroxymethylglutaryl lyase deficiency , isovaleric acidaemia , 3-methylcrotonyl - coa carboxylase deficiency , methylmalonyl - coa mutase deficiency , methylmalonic aciduria , cbla and cblb forms , beta - ketothiolase deficiency , propionic acidaemia , and multiple - coa carboxylase deficiency ) , inborn errors of fatty acid metabolism ( long - chain hydroxyacyl - coa dehydrogenase deficiency , medium - chain acyl - coa dehydrogenase deficiency , very - long - chain acyl - coa dehydrogenase deficiency , trifunctional protein deficiency , and carnitine uptake defect ) and miscellaneous multisystemic diseases ( cystic fibrosis , congenital hypothyroidism , biotinidase deficiency , congenital adrenal hyperplasia , and classical galactosaemia ) . in addition , expanded screening programmes have been developed in different states in the us , with some programmes covering almost 50 conditions . in europe , some programmes screen for only one or two conditions , whereas others screen for up to a few dozen . for example , the uk and france national screening committees recommend that all babies in their countries should be screened for phenylketonuria , congenital hypothyroidism , sickle cell disease , and cystic fibrosis , with the addition of medium - chain acyl - coa dehydrogenase deficiency in the uk and congenital adrenal hyperplasia in france . in italy , the application of the nbs programme application differs widely between regions . in some regions , only the three tests listed in the national programme are performed while other regions screen for up to 47 rare metabolic diseases . while developing countries face additional challenges related to poor economies , unstable governments , unique local cultures , geographical extremes , and different public health priorities , all other countries face challenges in implementing nbs because of the identification of the causes of genetic disorders , advances in detection technologies , and the development of better treatment regimens . one of the last nbs tests to be introduced measures the presence of t - cell deficiencies using t - cell receptor excision circle ( trec ) quantification . they typically manifest during infancy and childhood as abnormally frequent / recurrent or unusual infections , often accompanied by immunoregulatory defects . the vast majority of patients with pid have decreased t- and/or b - cell functions that grossly impair immunity . b - cell defects lead to antibody deficiencies . as a result of a mutation in the btk gene , a subgroup of these patients have x - linked agammaglobulinaemia ( xla ) due to a b - cell differentiation arrest in the bone marrow and the consequent absence of mature b cells and serum immunoglobulins ( ig ) . more than 30% of these patients develop irreversible organ damage in childhood or early adulthood , mainly in the lungs . non - xla is characterised by hypo - gammaglobulinaemia with decreased b - cell counts ( less than 2% mature b cells ) in the absence of the btk gene mutation . in patients with xla and non - xla , recurrent infections appear between three and 18 months of age , whereas the mean age at diagnosis is three years . this delayed diagnosis results in frequent hospitalisations because of pneumonia , sepsis , meningitis , and other bacterial infections , which frequently require the intravenous administration of antibiotics and can be fatal . thus , early diagnosis and treatment , including periodic intravenous ig replacement therapy , are essential to improve the prognosis and quality of life of these patients . t - cell defects result in combined immunodeficiencies , affecting both cellular and humoral immunity , with severe combined immunodeficiency ( scid ) being the most serious and lethal form . scid , which was reported for the first time more than 60 years ago , comprises a heterogeneous group of diseases , characterised by profound deficiencies of t- and b - cell functions , and , in some types , also of natural killer ( nk ) cells . the overall frequency of scid is estimated to be between 1:50,000 and 1:100,000 live births . these infants develop failure to thrive , chronic diarrhoea , and infections in the first months of life . infections can occur with common pathogens , but most of the time the infants also suffer from opportunistic infections , such as thrush or pneumocystis jiroveci pneumonia . graft - versus - host disease caused by maternal t - cell engraftment may occur in these patients , who also show skin rashes and organomegaly . scid is now known to be caused in humans by mutations in several different genes , such as cytokine receptor genes , antigen receptor genes and others , but there are many other probable causes yet to be discovered . an updated classification of scid is based on the underlying genetics and prevalent molecular pathogenetic mechanisms and includes the following : impaired cytokine - mediated signalling , alterations in v(d)j recombination , impaired signalling through the pre - t cell receptor ( tcr ) , increased lymphocyte apoptosis , absence of the thymus , alterations in thymus embryogenesis , impaired calcium flux , and other mechanisms . they all have a profound t - cell deficiency , and those t cells that are present are usually of maternal origin , having crossed the placenta . b cells can be elevated , normal or absent , depending on the type of scid , and the nk - cell number is variable . therefore , according to the presence or absence of the t , b and nk lymphocytes , scid can also be phenotypically categorised as the tbnk , tbnk , tbnk , and tbnk ( with minus meaning absence or severely reduced counts ) subtypes . for example , tbnkscid , accounting for up to 50% of scid cases , is predominantly x - linked , and is caused by mutations in the il2rg gene encoding the interleukin 2 receptor gamma chain . the early recognition of scid should be considered a paediatric emergency because a diagnosis before live vaccines or prior to the development of infections permits lifesaving unfractionated hla - identical or t - cell depleted haploidentical non - ablative haematopoietic stem cell transplantation , enzyme replacement therapy , or gene therapy . however , these infants often appear normal at birth and have no family history of immunodeficiency , and consequently , many of them are not identified until a life - threatening infection occurs . this is important because the long - term prognosis of infants with scid and other serious immunodeficiencies can be markedly improved if the diagnosis is made early , before the onset of severe infections . indeed , if scid is not detected until the infant is older , there is a much higher likelihood of death from live vaccines , graft - versus - host disease from non - irradiated blood products , or infections , before a successful definitive therapy can be adopted . because scid is not apparent at birth and early recognition is essential for life - saving treatment , it has , for many years now , been recognised as a candidate for nbs . indeed , the disease satisfies the criteria that the secretary s advisory committee on heritable disorders in newborns and children recommends for routine inclusion in nbs . these criteria are that : i ) the frequency of the disease in the population is high enough to warrant screening ; ii ) the untreated natural history of the disease is well - defined ; iii ) the untreated disease clearly confers significant morbidity and/or mortality ; iv ) the disease is treatable , and treatment significantly improves outcome ; v ) disease testing is safe , simple , and sufficiently sensitive to detect all cases ; vi ) specific confirmatory testing is available for the disease ; and vii ) the test , treatment , and treatment outcomes are cost - effective with respect to the non - treatment of the disease.36 an initial screening test for scid was based on the detection of peripheral blood lymphopenia . however , a complete blood count can not be performed using filter paper - dried blood spots , making this test unsuitable for nbs . in addition , the quantification of peripheral blood lymphocytes may fail to identify some infants with scid due to the presence of normal b - cell or nk - cell numbers , the maternal engraftment of t cells , and/or the expansion of a few t - cell clonotypes . therefore , no laboratory test on newborn dried blood spots was available to detect t - cell lymphopenia in infants with scid until 2005 when real - time polymerase chain reaction ( pcr ) for the quantification of trecs was developed and validated for population - based screening . trecs are created during the process of lymphocyte differentiation ( figure 1a ) when tcr rearrangements occur within the thymus . the rearrangement of the tcr alpha ( tcra ) chain has the peculiarity of involving the excision of delta - coding segments , which encode the delta chain of gamma / delta t cells that , being nestled in the tcra locus between the variable ( v ) and junctional ( j ) gene segments , must be excised to generate the tcra chain . several properties were established for trecs that make them useful markers of thymic output : they are stable , do not degrade easily over time , are not replicated when a cell divides , and are ( almost ) exclusively of thymic origin , without extrathymic sources of tcr rearrangements . after the demonstration that the trec assay detects scid patients regardless of the genetic cause , the test was modified and refined so it could be performed using blood from dried spots , was highly sensitive and specific for scid , and was cost - effective and reproducible , thereby becoming amenable to high - throughput population - based testing . at that point , pilot studies of nbs for scid , integrated with plans for its definitive diagnosis and management , were established in some us states . in 2008 , wisconsin became the first state to implement mandatory nbs for t - cell deficiency , followed by massachusetts in february 2009 , and later by new york , the navajo nation , california , puerto rico and louisiana . with the exception of california , where a trec assay kit under development by a medical technology company is being used , all the other states are using in - house modifications of the trec assay . by april 30 2011 , these pilot studies had been performed on 961,925 newborns , and had identified 14 cases of scid , 6 cases of scid variants and 40 cases of t - cell lymphopenia that were not related to scid . all infants diagnosed with scid in wisconsin and massachusetts have undergone transplantation or enzyme replacement therapy and no deaths have been reported . taking advantage of the introduction of the k - deleting recombination excision circle ( krec ) method to study the replication history of b cells , efforts have recently been made to establish a nbs for b - cell maturation defects . during the process of b - cell maturation , krecs are produced by recombination events that determine the allelic and isotypic exclusion of the kappa chain . indeed , during b - cell development , krecs are produced in those b lymphocytes that , after the ig heavy chain rearrangement , have failed to productively rearrange the ig light kappa chain genes ( igk ) on one or both alleles . in these cells , the igk locus becomes non - functional through the deletion of the constant k - gene segment ( igkc ) by recombination of the k - deleting element ( kde ) , which is a sequence located approximately 24 kb downstream of the constant k - gene segment , with one of the two upstream recombination signal sequences . when the kde recombines with the recombination signal sequence located in the intron between the junction and constant k - gene segments , portions of the dna are deleted , and the ligation of the excised recombination - signal ends generates the circularised dna elements defined as krecs ( figure 2a ) . these products persist in the cell , are unable to replicate , and are diluted as a result of cell division . the frequency of these recombination events in human b - cell malignancies ranged from 31% to 69% ; approximately 50% of transformed b cells should contain krecs . therefore , the quantification of krecs was initially used to determine the number of developing b lymphocytes in the bone marrow of children with b - precursor acute lymphoblastic leukaemia treated with allogeneic human stem cell transplantation . however , as igk gene deletion occurs physiologically in all b lymphocytes that fail to productively rearrange the igk genes on one or both alleles , the number of krecs has been be proposed to be a quantitative marker of bone marrow output in all individuals . furthermore , because krecs are not supposed to be produced in xla and non - xla patients because their b - cell maturation defects occur before k - deleting recombination occurs ( figure 2b ) , the krec measurement can potentially be applied to identify these types of b - cell deficiencies , which account for approximately 20% of all b - cell defects . in addition , some other types of combined immunodeficiencies show an arrest at the b - cell maturation stage and can also be identified using krec detection . indeed , more than 200 krec copies/g dna were present in the dried blood spots of healthy children , whereas no krecs were detected in 30 xla and 5 non - xla patients . this suggests that the measurement of krecs in neonatal dried blood spots could be used in an nbs that identifies neonates with early b - cell maturation defects . this early identification of children at risk should improve their future quality of life and help reduce health care costs , considering that the clinical phenotype of agammaglobulinaemia is milder than that of scid . therefore , the disease would be underdiagnosed in the first years of life in the absence of a screening programme , with patients developing serious organ damage before an accurate diagnosis could be made . in 2010 , we improved the methods for trec and krec quantification by developing a duplex quantitative real - time pcr protocol that allows an accurate and simultaneous assessment of both targets . for their absolute quantification , a standard curve is created using serial dilutions of trec and krec signal joint constructs cloned in a bacterial plasmid , together with a control gene , the tcra constant gene ( tcrac ) . the primers and probes used for the duplex real - time pcr assay are listed in table 1 . the main advantage of the combined trec / krec assay is that the variability related to dna quantification is eliminated by the use of the triple - insert plasmid in which the trec , krec and tcrac gene fragments are present in a 1:1:1 ratio . furthermore , the simultaneous quantification of the two targets in the same reaction contains the reagent costs . the number of trecs or krecs per 10 peripheral blood mononuclear cells ( pbmcs ) is calculated by dividing the mean quantity of trecs or krecs by the mean number of tcrac gene copies , which has to be divided by 2 because there are two tcrac gene copies in each cell ( i.e. one for each chromosome ) and then multiplied by 10 . this value , together with the lymphocyte plus monocyte count ( which are the cells obtained in the pbmc preparation ) in 1 ml of blood , was used to calculate the number of trecs or krecs per ml of blood as follows : using this strategy , we found that in healthy subjects , the number of trecs and krecs is correlated to age ( figure 3 ) and that , in particular , it significantly decreases over time , with a steeper slope in the first three years of life . with this assay , we demonstrated that the t- and b - cell reconstitution in children with different pids who underwent human stem cell transplantation or were cured with enzyme replacement therapy using pegylated bovine adenosine deaminase involves the mobilisation of both t and b cells from the respective production and maturation sites , and that the increase in trecs and krecs can be either strictly associated with or independent from one another . these results demonstrated that knowledge of the numbers of trecs and krecs , obtained with an assay that can be easily introduced into routine laboratory practice , is highly informative and can be used for a more precise identification of patients with pid or for their therapeutic monitoring . accordingly , this method has been recently validated in a cohort of 2560 anonymised nbs cards and in 49 originally stored guthrie cards from patients diagnosed with different pids , allowing the identification of patients with scid , xla , ataxia - telangiectasia ( at ) and nijmegen - breakage syndrome . therefore , the set up of a single assay for the simultaneous quantification of trecs and krecs will permit nbs for both scid and agammaglobulinaemia , which together have a combined estimated incidence of 1:30,000 - 1:50,000 births . furthermore , this test allows the separation of t - cell deficient cases into b - cell positive and b - cell negative subgroups , guiding additional diagnostics toward either tb or tb scid . the use of the trec assay has identified a large number of subjects that do not meet the criteria for scid . some of these other conditions , such as digeorge syndrome and down syndrome , are known to result in t - cell lymphopenia , whereas others were unexpected and many remain undefined . in particular , premature babies have been shown to have a lower number of trecs than full - term babies , despite not being immunodeficient . moreover , the trec and krec assay may identify other forms of immunodeficiency , some of them severe , for which a definitive treatment is not available , e.g. at . therefore , it is important to emphasise that the identification of scid and agammaglobulinaemia at birth must be based on a two - tier assay , with trec / krec determination only being the first tier . appropriate second - tier assays must be used to define the disease ( if any ) that results in the low trec / krec values . for some disorders , such as at , defining the disease at birth may indeed prove challenging because the values of alphafetoprotein in newborns are elevated . this requirement of a two - tiered system may represent an important limitation of the use of the trec / krec assay for the population - based screening of genetic conditions in newborns . other principal obstacles to the full implementation of nbs for pid could be the costs of setting up the screening test and the possibility that the combined measurement of trecs and krecs may generate higher call rates for newborns to be on clinical follow up , which in turn has an impact on the total screening costs . however , the introduction of the scid nbs in the pilot studies had an approximate cost of less than 1 million dollars , whereas in the us , the cost of treating one baby with scid that is not diagnosed until he or she has a serious infection can easily exceed 2 million dollars . there is also a striking difference with regard to the costs for transplantation in scid infants under 3.5 months of age ( approx . 1 million dollars ) and the costs for those aged over 3.5 months ( more than 4 million dollars ) . an initial study , performed before the introduction of the trec assay on dried blood spots , demonstrated that a scid screening test that cost approximately 5 us dollars would be considered cost - effective . however , the study incorporated limited information about the care costs or outcome differences between infants identified before and after becoming symptomatic . this limitation was bypassed by a recent study that evaluated the cost - effectiveness of universal screening using a model incorporating the impact of early detection on the natural history of scid . the authors demonstrated that assuming a scid incidence of 1:75,000 births and test specificity and sensitivity of 0.99 each , the screen remained cost - effective up to a maximum cost of 15 dollars per infant screened . therefore , considering that the trec assay is relatively inexpensive , with a cost of approximately 5.50 dollars per assay , that the incidence of t - cell lymphopenia is relatively high , and that better health outcomes and lower costs are associated with earlier stem cell transplantation , nbs for scid meets the reasonable standards of willingness - to - pay and could be a worthwhile addition to nbs panels . despite the favourable evaluation of scid based upon the nbs guidelines , national implementation is pending in most countries which have long - lasting traditions of their own nbs programmes . the inclusion of krec detection in the test only slightly increases the cost because it only involves the addition of a single reagent to the assay . thus , a single assay capable of screening for both scid and agammaglobulinaemia , which together have a combined estimated incidence of 1:30,000 - 1:50,000 births , should further improve the cost - effectiveness of nbs . additional preventive medical research for other severe pids , including , but not limited to , inherited agammaglobulinaemias and haemophagocytic syndromes , would be advisable . the implications of nbs for pid in countries other than the us can not be easily established as the attributed value for a quality life - year considerably differs among countries , particularly among the us , europe and other regions . indeed , although pids are mentioned within those disorders that might be considered for the gradual expansion of nbs in the member states of the european union , developing countries will probably face additional challenges related to poor economies , unstable governments , unique local cultures , geographical extremes , and different public health priorities . newborn blood screening for scid and other t - cell deficiencies , based on trec quantification , was the first screening introduced for pid , but with the advent of modern molecular technologies , screening for other immune system defects will probably be available soon . one of the most promising candidates for nbs protocols is the combined quantification of trecs and krecs that could support early diagnosis , classification of patient subgroups , and evaluation of stem cell replacement therapy for both t- and b - cell defects . in order to achieve this , clinicians have to play an active role in promoting nbs for pid and other t- and b - lymphocyte abnormalities , and work closely with public health officials and nbs committees . they also have to offer their expertise to advise laboratories that are developing the new techniques . they will have an even more important role in establishing an accurate diagnosis for those infants who have nbs - positive results and in ensuring that these children are given the best possible and most appropriate treatment .

since its introduction as a public health programme in the united states in the early 1960s , newborn blood screening ( nbs ) has evolved from the detection of phenylalanine levels on filter paper to the application of dna - based technologies to identify t - cell lymphopenia in infants with severe combined immunodeficiency . this latter use of nbs has required the development of an assay for t - cell lymphopenia based on the quantification of t - cell receptor excision circles ( trecs ) that could be performed on dried blood spots routinely collected from newborn infants . the trec - based nbs was developed six years ago , and there have already been 7 successful pilot studies since then . similarly , efforts are now being made to establish a screen for b - cell defects , in particular agammaglobulinaemia , taking advantage of the introduction of the method for the quantification of k - deleting recombination excision circles ( krecs ) . a further achievement of nbs could be the simultaneous recognition of t- and b - cell defects using the combined quantification of trecs and krecs from guthrie card blood spots . this approach may help the early identification of infants with t- and b - cell deficiencies so that they can then be referred to specialised paediatric centres , where a precise diagnosis of severe combined immunodeficiency and agammaglobulinaemia can be performed , and where then they can immediately receive specific therapy . simultaneous trec and krec quantification should also allow classification of patients into subgroups and help identify children with less serious primary immunodeficiencies . this would help avoid the opportunistic infections and frequent hospitalisations that result from a late or lack of diagnosis .

Significance for public health Newborn screenings Primary immunodeficiencies Pilot studies for newborn blood screening for severe combined immunodeficiency using T-cell receptor excision circles quantification Identification of patients with agammaglobulinaemia using K-deleting recombination excision circle quantification Simultaneous quantification of T- and B-cell defects using the T-cell receptor excision circles/K-deleting recombination excision circles assay Limitations of the introduction of newborn blood screening for primary immunodeficiencies Conclusions

since the clinical severity ranges from mild to potentially life - threatening , major efforts are currently being made to develop methods to detect patients with these diseases in the neonatal period . indeed , the best outcome for the most severe form of primary immunodeficiency , as with many other conditions for which newborn screenings are now performed , is achieved when treatment is given in the first months of life , ideally before clinical presentation . the pilot studies of newborn screenings for severe combined immunodeficiency have demonstrated their cost - effectiveness and have also proved successful in terms of public health resulting from improved quality of life and survival of children with these diseases . the goal of newborn blood screening ( nbs ) is to identify pre - symptomatic newborns with potentially serious or fatal disorders which could be successfully treated , leading to significant reductions in morbidity and mortality . nbs debuted as a public health programme in the us in the early 1960s , and has expanded to countries around the world , with different testing options in each country . nbs has progressively evolved since 1963 , when guthrie and susi demonstrated that postnatal testing of dried blood spots on filter paper led to the identification and treatment of infants prior to the development of the cognitive deficits associated with phenylketonuria . more recently , the advent of dna - based technologies has again shifted the paradigm of nbs , offering the potential of screening for numerous disorders at the same time . therefore , every year , millions of babies in the us are routinely screened for congenital deafness and , using a few drops of blood from the newborn s heel , for blood cell disorders ( sickle cell anaemia , sickle cell disease , and hb s / beta - thalassaemia ) , inborn errors of amino acid metabolism ( tyrosinaemia i , argininosuccinic aciduria , citrullinaemia , phenylketonuria , maple syrup urine disease , and homocystinuria ) , inborn errors of organic acid metabolism ( glutaric acidaemia type i , hydroxymethylglutaryl lyase deficiency , isovaleric acidaemia , 3-methylcrotonyl - coa carboxylase deficiency , methylmalonyl - coa mutase deficiency , methylmalonic aciduria , cbla and cblb forms , beta - ketothiolase deficiency , propionic acidaemia , and multiple - coa carboxylase deficiency ) , inborn errors of fatty acid metabolism ( long - chain hydroxyacyl - coa dehydrogenase deficiency , medium - chain acyl - coa dehydrogenase deficiency , very - long - chain acyl - coa dehydrogenase deficiency , trifunctional protein deficiency , and carnitine uptake defect ) and miscellaneous multisystemic diseases ( cystic fibrosis , congenital hypothyroidism , biotinidase deficiency , congenital adrenal hyperplasia , and classical galactosaemia ) . in addition , expanded screening programmes have been developed in different states in the us , with some programmes covering almost 50 conditions . for example , the uk and france national screening committees recommend that all babies in their countries should be screened for phenylketonuria , congenital hypothyroidism , sickle cell disease , and cystic fibrosis , with the addition of medium - chain acyl - coa dehydrogenase deficiency in the uk and congenital adrenal hyperplasia in france . in italy , the application of the nbs programme application differs widely between regions . in some regions , only the three tests listed in the national programme are performed while other regions screen for up to 47 rare metabolic diseases . while developing countries face additional challenges related to poor economies , unstable governments , unique local cultures , geographical extremes , and different public health priorities , all other countries face challenges in implementing nbs because of the identification of the causes of genetic disorders , advances in detection technologies , and the development of better treatment regimens . one of the last nbs tests to be introduced measures the presence of t - cell deficiencies using t - cell receptor excision circle ( trec ) quantification . the vast majority of patients with pid have decreased t- and/or b - cell functions that grossly impair immunity . b - cell defects lead to antibody deficiencies . as a result of a mutation in the btk gene , a subgroup of these patients have x - linked agammaglobulinaemia ( xla ) due to a b - cell differentiation arrest in the bone marrow and the consequent absence of mature b cells and serum immunoglobulins ( ig ) . non - xla is characterised by hypo - gammaglobulinaemia with decreased b - cell counts ( less than 2% mature b cells ) in the absence of the btk gene mutation . this delayed diagnosis results in frequent hospitalisations because of pneumonia , sepsis , meningitis , and other bacterial infections , which frequently require the intravenous administration of antibiotics and can be fatal . t - cell defects result in combined immunodeficiencies , affecting both cellular and humoral immunity , with severe combined immunodeficiency ( scid ) being the most serious and lethal form . scid , which was reported for the first time more than 60 years ago , comprises a heterogeneous group of diseases , characterised by profound deficiencies of t- and b - cell functions , and , in some types , also of natural killer ( nk ) cells . these infants develop failure to thrive , chronic diarrhoea , and infections in the first months of life . infections can occur with common pathogens , but most of the time the infants also suffer from opportunistic infections , such as thrush or pneumocystis jiroveci pneumonia . graft - versus - host disease caused by maternal t - cell engraftment may occur in these patients , who also show skin rashes and organomegaly . an updated classification of scid is based on the underlying genetics and prevalent molecular pathogenetic mechanisms and includes the following : impaired cytokine - mediated signalling , alterations in v(d)j recombination , impaired signalling through the pre - t cell receptor ( tcr ) , increased lymphocyte apoptosis , absence of the thymus , alterations in thymus embryogenesis , impaired calcium flux , and other mechanisms . they all have a profound t - cell deficiency , and those t cells that are present are usually of maternal origin , having crossed the placenta . b cells can be elevated , normal or absent , depending on the type of scid , and the nk - cell number is variable . therefore , according to the presence or absence of the t , b and nk lymphocytes , scid can also be phenotypically categorised as the tbnk , tbnk , tbnk , and tbnk ( with minus meaning absence or severely reduced counts ) subtypes . for example , tbnkscid , accounting for up to 50% of scid cases , is predominantly x - linked , and is caused by mutations in the il2rg gene encoding the interleukin 2 receptor gamma chain . the early recognition of scid should be considered a paediatric emergency because a diagnosis before live vaccines or prior to the development of infections permits lifesaving unfractionated hla - identical or t - cell depleted haploidentical non - ablative haematopoietic stem cell transplantation , enzyme replacement therapy , or gene therapy . this is important because the long - term prognosis of infants with scid and other serious immunodeficiencies can be markedly improved if the diagnosis is made early , before the onset of severe infections . these criteria are that : i ) the frequency of the disease in the population is high enough to warrant screening ; ii ) the untreated natural history of the disease is well - defined ; iii ) the untreated disease clearly confers significant morbidity and/or mortality ; iv ) the disease is treatable , and treatment significantly improves outcome ; v ) disease testing is safe , simple , and sufficiently sensitive to detect all cases ; vi ) specific confirmatory testing is available for the disease ; and vii ) the test , treatment , and treatment outcomes are cost - effective with respect to the non - treatment of the disease.36 an initial screening test for scid was based on the detection of peripheral blood lymphopenia . however , a complete blood count can not be performed using filter paper - dried blood spots , making this test unsuitable for nbs . in addition , the quantification of peripheral blood lymphocytes may fail to identify some infants with scid due to the presence of normal b - cell or nk - cell numbers , the maternal engraftment of t cells , and/or the expansion of a few t - cell clonotypes . therefore , no laboratory test on newborn dried blood spots was available to detect t - cell lymphopenia in infants with scid until 2005 when real - time polymerase chain reaction ( pcr ) for the quantification of trecs was developed and validated for population - based screening . the rearrangement of the tcr alpha ( tcra ) chain has the peculiarity of involving the excision of delta - coding segments , which encode the delta chain of gamma / delta t cells that , being nestled in the tcra locus between the variable ( v ) and junctional ( j ) gene segments , must be excised to generate the tcra chain . after the demonstration that the trec assay detects scid patients regardless of the genetic cause , the test was modified and refined so it could be performed using blood from dried spots , was highly sensitive and specific for scid , and was cost - effective and reproducible , thereby becoming amenable to high - throughput population - based testing . at that point , pilot studies of nbs for scid , integrated with plans for its definitive diagnosis and management , were established in some us states . in 2008 , wisconsin became the first state to implement mandatory nbs for t - cell deficiency , followed by massachusetts in february 2009 , and later by new york , the navajo nation , california , puerto rico and louisiana . with the exception of california , where a trec assay kit under development by a medical technology company is being used , all the other states are using in - house modifications of the trec assay . by april 30 2011 , these pilot studies had been performed on 961,925 newborns , and had identified 14 cases of scid , 6 cases of scid variants and 40 cases of t - cell lymphopenia that were not related to scid . taking advantage of the introduction of the k - deleting recombination excision circle ( krec ) method to study the replication history of b cells , efforts have recently been made to establish a nbs for b - cell maturation defects . during the process of b - cell maturation , krecs are produced by recombination events that determine the allelic and isotypic exclusion of the kappa chain . indeed , during b - cell development , krecs are produced in those b lymphocytes that , after the ig heavy chain rearrangement , have failed to productively rearrange the ig light kappa chain genes ( igk ) on one or both alleles . in these cells , the igk locus becomes non - functional through the deletion of the constant k - gene segment ( igkc ) by recombination of the k - deleting element ( kde ) , which is a sequence located approximately 24 kb downstream of the constant k - gene segment , with one of the two upstream recombination signal sequences . when the kde recombines with the recombination signal sequence located in the intron between the junction and constant k - gene segments , portions of the dna are deleted , and the ligation of the excised recombination - signal ends generates the circularised dna elements defined as krecs ( figure 2a ) . these products persist in the cell , are unable to replicate , and are diluted as a result of cell division . the frequency of these recombination events in human b - cell malignancies ranged from 31% to 69% ; approximately 50% of transformed b cells should contain krecs . therefore , the quantification of krecs was initially used to determine the number of developing b lymphocytes in the bone marrow of children with b - precursor acute lymphoblastic leukaemia treated with allogeneic human stem cell transplantation . furthermore , because krecs are not supposed to be produced in xla and non - xla patients because their b - cell maturation defects occur before k - deleting recombination occurs ( figure 2b ) , the krec measurement can potentially be applied to identify these types of b - cell deficiencies , which account for approximately 20% of all b - cell defects . indeed , more than 200 krec copies/g dna were present in the dried blood spots of healthy children , whereas no krecs were detected in 30 xla and 5 non - xla patients . this suggests that the measurement of krecs in neonatal dried blood spots could be used in an nbs that identifies neonates with early b - cell maturation defects . this early identification of children at risk should improve their future quality of life and help reduce health care costs , considering that the clinical phenotype of agammaglobulinaemia is milder than that of scid . in 2010 , we improved the methods for trec and krec quantification by developing a duplex quantitative real - time pcr protocol that allows an accurate and simultaneous assessment of both targets . for their absolute quantification , a standard curve is created using serial dilutions of trec and krec signal joint constructs cloned in a bacterial plasmid , together with a control gene , the tcra constant gene ( tcrac ) . the main advantage of the combined trec / krec assay is that the variability related to dna quantification is eliminated by the use of the triple - insert plasmid in which the trec , krec and tcrac gene fragments are present in a 1:1:1 ratio . furthermore , the simultaneous quantification of the two targets in the same reaction contains the reagent costs . this value , together with the lymphocyte plus monocyte count ( which are the cells obtained in the pbmc preparation ) in 1 ml of blood , was used to calculate the number of trecs or krecs per ml of blood as follows : using this strategy , we found that in healthy subjects , the number of trecs and krecs is correlated to age ( figure 3 ) and that , in particular , it significantly decreases over time , with a steeper slope in the first three years of life . with this assay , we demonstrated that the t- and b - cell reconstitution in children with different pids who underwent human stem cell transplantation or were cured with enzyme replacement therapy using pegylated bovine adenosine deaminase involves the mobilisation of both t and b cells from the respective production and maturation sites , and that the increase in trecs and krecs can be either strictly associated with or independent from one another . these results demonstrated that knowledge of the numbers of trecs and krecs , obtained with an assay that can be easily introduced into routine laboratory practice , is highly informative and can be used for a more precise identification of patients with pid or for their therapeutic monitoring . accordingly , this method has been recently validated in a cohort of 2560 anonymised nbs cards and in 49 originally stored guthrie cards from patients diagnosed with different pids , allowing the identification of patients with scid , xla , ataxia - telangiectasia ( at ) and nijmegen - breakage syndrome . therefore , the set up of a single assay for the simultaneous quantification of trecs and krecs will permit nbs for both scid and agammaglobulinaemia , which together have a combined estimated incidence of 1:30,000 - 1:50,000 births . furthermore , this test allows the separation of t - cell deficient cases into b - cell positive and b - cell negative subgroups , guiding additional diagnostics toward either tb or tb scid . the use of the trec assay has identified a large number of subjects that do not meet the criteria for scid . some of these other conditions , such as digeorge syndrome and down syndrome , are known to result in t - cell lymphopenia , whereas others were unexpected and many remain undefined . moreover , the trec and krec assay may identify other forms of immunodeficiency , some of them severe , for which a definitive treatment is not available , e.g. therefore , it is important to emphasise that the identification of scid and agammaglobulinaemia at birth must be based on a two - tier assay , with trec / krec determination only being the first tier . appropriate second - tier assays must be used to define the disease ( if any ) that results in the low trec / krec values . this requirement of a two - tiered system may represent an important limitation of the use of the trec / krec assay for the population - based screening of genetic conditions in newborns . other principal obstacles to the full implementation of nbs for pid could be the costs of setting up the screening test and the possibility that the combined measurement of trecs and krecs may generate higher call rates for newborns to be on clinical follow up , which in turn has an impact on the total screening costs . however , the introduction of the scid nbs in the pilot studies had an approximate cost of less than 1 million dollars , whereas in the us , the cost of treating one baby with scid that is not diagnosed until he or she has a serious infection can easily exceed 2 million dollars . an initial study , performed before the introduction of the trec assay on dried blood spots , demonstrated that a scid screening test that cost approximately 5 us dollars would be considered cost - effective . therefore , considering that the trec assay is relatively inexpensive , with a cost of approximately 5.50 dollars per assay , that the incidence of t - cell lymphopenia is relatively high , and that better health outcomes and lower costs are associated with earlier stem cell transplantation , nbs for scid meets the reasonable standards of willingness - to - pay and could be a worthwhile addition to nbs panels . the inclusion of krec detection in the test only slightly increases the cost because it only involves the addition of a single reagent to the assay . thus , a single assay capable of screening for both scid and agammaglobulinaemia , which together have a combined estimated incidence of 1:30,000 - 1:50,000 births , should further improve the cost - effectiveness of nbs . indeed , although pids are mentioned within those disorders that might be considered for the gradual expansion of nbs in the member states of the european union , developing countries will probably face additional challenges related to poor economies , unstable governments , unique local cultures , geographical extremes , and different public health priorities . newborn blood screening for scid and other t - cell deficiencies , based on trec quantification , was the first screening introduced for pid , but with the advent of modern molecular technologies , screening for other immune system defects will probably be available soon . one of the most promising candidates for nbs protocols is the combined quantification of trecs and krecs that could support early diagnosis , classification of patient subgroups , and evaluation of stem cell replacement therapy for both t- and b - cell defects . in order to achieve this , clinicians have to play an active role in promoting nbs for pid and other t- and b - lymphocyte abnormalities , and work closely with public health officials and nbs committees .

in fact , making a diagnosis before vaccines or blood products are administered , as well as before the infection develops , allows the patient to proceed to haematopoietic stem cell transplantation , and receive enzyme replacement and gene therapies . indeed , the best outcome for the most severe form of primary immunodeficiency , as with many other conditions for which newborn screenings are now performed , is achieved when treatment is given in the first months of life , ideally before clinical presentation . the pilot studies of newborn screenings for severe combined immunodeficiency have demonstrated their cost - effectiveness and have also proved successful in terms of public health resulting from improved quality of life and survival of children with these diseases . the goal of newborn blood screening ( nbs ) is to identify pre - symptomatic newborns with potentially serious or fatal disorders which could be successfully treated , leading to significant reductions in morbidity and mortality . nbs debuted as a public health programme in the us in the early 1960s , and has expanded to countries around the world , with different testing options in each country . nbs has progressively evolved since 1963 , when guthrie and susi demonstrated that postnatal testing of dried blood spots on filter paper led to the identification and treatment of infants prior to the development of the cognitive deficits associated with phenylketonuria . more recently , the advent of dna - based technologies has again shifted the paradigm of nbs , offering the potential of screening for numerous disorders at the same time . therefore , every year , millions of babies in the us are routinely screened for congenital deafness and , using a few drops of blood from the newborn s heel , for blood cell disorders ( sickle cell anaemia , sickle cell disease , and hb s / beta - thalassaemia ) , inborn errors of amino acid metabolism ( tyrosinaemia i , argininosuccinic aciduria , citrullinaemia , phenylketonuria , maple syrup urine disease , and homocystinuria ) , inborn errors of organic acid metabolism ( glutaric acidaemia type i , hydroxymethylglutaryl lyase deficiency , isovaleric acidaemia , 3-methylcrotonyl - coa carboxylase deficiency , methylmalonyl - coa mutase deficiency , methylmalonic aciduria , cbla and cblb forms , beta - ketothiolase deficiency , propionic acidaemia , and multiple - coa carboxylase deficiency ) , inborn errors of fatty acid metabolism ( long - chain hydroxyacyl - coa dehydrogenase deficiency , medium - chain acyl - coa dehydrogenase deficiency , very - long - chain acyl - coa dehydrogenase deficiency , trifunctional protein deficiency , and carnitine uptake defect ) and miscellaneous multisystemic diseases ( cystic fibrosis , congenital hypothyroidism , biotinidase deficiency , congenital adrenal hyperplasia , and classical galactosaemia ) . for example , the uk and france national screening committees recommend that all babies in their countries should be screened for phenylketonuria , congenital hypothyroidism , sickle cell disease , and cystic fibrosis , with the addition of medium - chain acyl - coa dehydrogenase deficiency in the uk and congenital adrenal hyperplasia in france . while developing countries face additional challenges related to poor economies , unstable governments , unique local cultures , geographical extremes , and different public health priorities , all other countries face challenges in implementing nbs because of the identification of the causes of genetic disorders , advances in detection technologies , and the development of better treatment regimens . one of the last nbs tests to be introduced measures the presence of t - cell deficiencies using t - cell receptor excision circle ( trec ) quantification . the vast majority of patients with pid have decreased t- and/or b - cell functions that grossly impair immunity . as a result of a mutation in the btk gene , a subgroup of these patients have x - linked agammaglobulinaemia ( xla ) due to a b - cell differentiation arrest in the bone marrow and the consequent absence of mature b cells and serum immunoglobulins ( ig ) . more than 30% of these patients develop irreversible organ damage in childhood or early adulthood , mainly in the lungs . non - xla is characterised by hypo - gammaglobulinaemia with decreased b - cell counts ( less than 2% mature b cells ) in the absence of the btk gene mutation . in patients with xla and non - xla , recurrent infections appear between three and 18 months of age , whereas the mean age at diagnosis is three years . this delayed diagnosis results in frequent hospitalisations because of pneumonia , sepsis , meningitis , and other bacterial infections , which frequently require the intravenous administration of antibiotics and can be fatal . thus , early diagnosis and treatment , including periodic intravenous ig replacement therapy , are essential to improve the prognosis and quality of life of these patients . t - cell defects result in combined immunodeficiencies , affecting both cellular and humoral immunity , with severe combined immunodeficiency ( scid ) being the most serious and lethal form . scid , which was reported for the first time more than 60 years ago , comprises a heterogeneous group of diseases , characterised by profound deficiencies of t- and b - cell functions , and , in some types , also of natural killer ( nk ) cells . these infants develop failure to thrive , chronic diarrhoea , and infections in the first months of life . graft - versus - host disease caused by maternal t - cell engraftment may occur in these patients , who also show skin rashes and organomegaly . scid is now known to be caused in humans by mutations in several different genes , such as cytokine receptor genes , antigen receptor genes and others , but there are many other probable causes yet to be discovered . an updated classification of scid is based on the underlying genetics and prevalent molecular pathogenetic mechanisms and includes the following : impaired cytokine - mediated signalling , alterations in v(d)j recombination , impaired signalling through the pre - t cell receptor ( tcr ) , increased lymphocyte apoptosis , absence of the thymus , alterations in thymus embryogenesis , impaired calcium flux , and other mechanisms . they all have a profound t - cell deficiency , and those t cells that are present are usually of maternal origin , having crossed the placenta . b cells can be elevated , normal or absent , depending on the type of scid , and the nk - cell number is variable . therefore , according to the presence or absence of the t , b and nk lymphocytes , scid can also be phenotypically categorised as the tbnk , tbnk , tbnk , and tbnk ( with minus meaning absence or severely reduced counts ) subtypes . for example , tbnkscid , accounting for up to 50% of scid cases , is predominantly x - linked , and is caused by mutations in the il2rg gene encoding the interleukin 2 receptor gamma chain . the early recognition of scid should be considered a paediatric emergency because a diagnosis before live vaccines or prior to the development of infections permits lifesaving unfractionated hla - identical or t - cell depleted haploidentical non - ablative haematopoietic stem cell transplantation , enzyme replacement therapy , or gene therapy . this is important because the long - term prognosis of infants with scid and other serious immunodeficiencies can be markedly improved if the diagnosis is made early , before the onset of severe infections . indeed , if scid is not detected until the infant is older , there is a much higher likelihood of death from live vaccines , graft - versus - host disease from non - irradiated blood products , or infections , before a successful definitive therapy can be adopted . because scid is not apparent at birth and early recognition is essential for life - saving treatment , it has , for many years now , been recognised as a candidate for nbs . these criteria are that : i ) the frequency of the disease in the population is high enough to warrant screening ; ii ) the untreated natural history of the disease is well - defined ; iii ) the untreated disease clearly confers significant morbidity and/or mortality ; iv ) the disease is treatable , and treatment significantly improves outcome ; v ) disease testing is safe , simple , and sufficiently sensitive to detect all cases ; vi ) specific confirmatory testing is available for the disease ; and vii ) the test , treatment , and treatment outcomes are cost - effective with respect to the non - treatment of the disease.36 an initial screening test for scid was based on the detection of peripheral blood lymphopenia . in addition , the quantification of peripheral blood lymphocytes may fail to identify some infants with scid due to the presence of normal b - cell or nk - cell numbers , the maternal engraftment of t cells , and/or the expansion of a few t - cell clonotypes . therefore , no laboratory test on newborn dried blood spots was available to detect t - cell lymphopenia in infants with scid until 2005 when real - time polymerase chain reaction ( pcr ) for the quantification of trecs was developed and validated for population - based screening . the rearrangement of the tcr alpha ( tcra ) chain has the peculiarity of involving the excision of delta - coding segments , which encode the delta chain of gamma / delta t cells that , being nestled in the tcra locus between the variable ( v ) and junctional ( j ) gene segments , must be excised to generate the tcra chain . several properties were established for trecs that make them useful markers of thymic output : they are stable , do not degrade easily over time , are not replicated when a cell divides , and are ( almost ) exclusively of thymic origin , without extrathymic sources of tcr rearrangements . after the demonstration that the trec assay detects scid patients regardless of the genetic cause , the test was modified and refined so it could be performed using blood from dried spots , was highly sensitive and specific for scid , and was cost - effective and reproducible , thereby becoming amenable to high - throughput population - based testing . in 2008 , wisconsin became the first state to implement mandatory nbs for t - cell deficiency , followed by massachusetts in february 2009 , and later by new york , the navajo nation , california , puerto rico and louisiana . by april 30 2011 , these pilot studies had been performed on 961,925 newborns , and had identified 14 cases of scid , 6 cases of scid variants and 40 cases of t - cell lymphopenia that were not related to scid . taking advantage of the introduction of the k - deleting recombination excision circle ( krec ) method to study the replication history of b cells , efforts have recently been made to establish a nbs for b - cell maturation defects . during the process of b - cell maturation , krecs are produced by recombination events that determine the allelic and isotypic exclusion of the kappa chain . indeed , during b - cell development , krecs are produced in those b lymphocytes that , after the ig heavy chain rearrangement , have failed to productively rearrange the ig light kappa chain genes ( igk ) on one or both alleles . in these cells , the igk locus becomes non - functional through the deletion of the constant k - gene segment ( igkc ) by recombination of the k - deleting element ( kde ) , which is a sequence located approximately 24 kb downstream of the constant k - gene segment , with one of the two upstream recombination signal sequences . when the kde recombines with the recombination signal sequence located in the intron between the junction and constant k - gene segments , portions of the dna are deleted , and the ligation of the excised recombination - signal ends generates the circularised dna elements defined as krecs ( figure 2a ) . therefore , the quantification of krecs was initially used to determine the number of developing b lymphocytes in the bone marrow of children with b - precursor acute lymphoblastic leukaemia treated with allogeneic human stem cell transplantation . however , as igk gene deletion occurs physiologically in all b lymphocytes that fail to productively rearrange the igk genes on one or both alleles , the number of krecs has been be proposed to be a quantitative marker of bone marrow output in all individuals . furthermore , because krecs are not supposed to be produced in xla and non - xla patients because their b - cell maturation defects occur before k - deleting recombination occurs ( figure 2b ) , the krec measurement can potentially be applied to identify these types of b - cell deficiencies , which account for approximately 20% of all b - cell defects . in addition , some other types of combined immunodeficiencies show an arrest at the b - cell maturation stage and can also be identified using krec detection . indeed , more than 200 krec copies/g dna were present in the dried blood spots of healthy children , whereas no krecs were detected in 30 xla and 5 non - xla patients . this early identification of children at risk should improve their future quality of life and help reduce health care costs , considering that the clinical phenotype of agammaglobulinaemia is milder than that of scid . therefore , the disease would be underdiagnosed in the first years of life in the absence of a screening programme , with patients developing serious organ damage before an accurate diagnosis could be made . in 2010 , we improved the methods for trec and krec quantification by developing a duplex quantitative real - time pcr protocol that allows an accurate and simultaneous assessment of both targets . the main advantage of the combined trec / krec assay is that the variability related to dna quantification is eliminated by the use of the triple - insert plasmid in which the trec , krec and tcrac gene fragments are present in a 1:1:1 ratio . the number of trecs or krecs per 10 peripheral blood mononuclear cells ( pbmcs ) is calculated by dividing the mean quantity of trecs or krecs by the mean number of tcrac gene copies , which has to be divided by 2 because there are two tcrac gene copies in each cell ( i.e. this value , together with the lymphocyte plus monocyte count ( which are the cells obtained in the pbmc preparation ) in 1 ml of blood , was used to calculate the number of trecs or krecs per ml of blood as follows : using this strategy , we found that in healthy subjects , the number of trecs and krecs is correlated to age ( figure 3 ) and that , in particular , it significantly decreases over time , with a steeper slope in the first three years of life . with this assay , we demonstrated that the t- and b - cell reconstitution in children with different pids who underwent human stem cell transplantation or were cured with enzyme replacement therapy using pegylated bovine adenosine deaminase involves the mobilisation of both t and b cells from the respective production and maturation sites , and that the increase in trecs and krecs can be either strictly associated with or independent from one another . these results demonstrated that knowledge of the numbers of trecs and krecs , obtained with an assay that can be easily introduced into routine laboratory practice , is highly informative and can be used for a more precise identification of patients with pid or for their therapeutic monitoring . accordingly , this method has been recently validated in a cohort of 2560 anonymised nbs cards and in 49 originally stored guthrie cards from patients diagnosed with different pids , allowing the identification of patients with scid , xla , ataxia - telangiectasia ( at ) and nijmegen - breakage syndrome . therefore , the set up of a single assay for the simultaneous quantification of trecs and krecs will permit nbs for both scid and agammaglobulinaemia , which together have a combined estimated incidence of 1:30,000 - 1:50,000 births . furthermore , this test allows the separation of t - cell deficient cases into b - cell positive and b - cell negative subgroups , guiding additional diagnostics toward either tb or tb scid . therefore , it is important to emphasise that the identification of scid and agammaglobulinaemia at birth must be based on a two - tier assay , with trec / krec determination only being the first tier . other principal obstacles to the full implementation of nbs for pid could be the costs of setting up the screening test and the possibility that the combined measurement of trecs and krecs may generate higher call rates for newborns to be on clinical follow up , which in turn has an impact on the total screening costs . however , the introduction of the scid nbs in the pilot studies had an approximate cost of less than 1 million dollars , whereas in the us , the cost of treating one baby with scid that is not diagnosed until he or she has a serious infection can easily exceed 2 million dollars . this limitation was bypassed by a recent study that evaluated the cost - effectiveness of universal screening using a model incorporating the impact of early detection on the natural history of scid . therefore , considering that the trec assay is relatively inexpensive , with a cost of approximately 5.50 dollars per assay , that the incidence of t - cell lymphopenia is relatively high , and that better health outcomes and lower costs are associated with earlier stem cell transplantation , nbs for scid meets the reasonable standards of willingness - to - pay and could be a worthwhile addition to nbs panels . despite the favourable evaluation of scid based upon the nbs guidelines , national implementation is pending in most countries which have long - lasting traditions of their own nbs programmes . thus , a single assay capable of screening for both scid and agammaglobulinaemia , which together have a combined estimated incidence of 1:30,000 - 1:50,000 births , should further improve the cost - effectiveness of nbs . the implications of nbs for pid in countries other than the us can not be easily established as the attributed value for a quality life - year considerably differs among countries , particularly among the us , europe and other regions . indeed , although pids are mentioned within those disorders that might be considered for the gradual expansion of nbs in the member states of the european union , developing countries will probably face additional challenges related to poor economies , unstable governments , unique local cultures , geographical extremes , and different public health priorities . newborn blood screening for scid and other t - cell deficiencies , based on trec quantification , was the first screening introduced for pid , but with the advent of modern molecular technologies , screening for other immune system defects will probably be available soon . one of the most promising candidates for nbs protocols is the combined quantification of trecs and krecs that could support early diagnosis , classification of patient subgroups , and evaluation of stem cell replacement therapy for both t- and b - cell defects . in order to achieve this , clinicians have to play an active role in promoting nbs for pid and other t- and b - lymphocyte abnormalities , and work closely with public health officials and nbs committees .

embryonic development relies upon the precise control of genetic programs to create complex tissues and organs . mutagenesis screens and the sequencing of multiple genomes have revealed an extensive list of patterning genes , many of which are expressed in a tissue - specific manner within the developing embryo . one of the remaining challenges in developmental biology is to understand how these genes act in space and time to modulate cell proliferation , migration , and differentiation in a stereotypic manner . toward that goal , several genetic approaches for conditional gene regulation have been developed , such as the flp / frt , cre / lox , and tet - on / tet - off systems , and these technologies have provided key insights into the molecular mechanisms that underlie tissue patterning and function.(1 ) chemical technologies are also required for surmounting this challenge , especially in biological systems for which reverse - genetic methods are limited . for example , the zebrafish is ideally suited for visualizing vertebrate ontogeny , since its embryos and larvae are optically transparent and develop rapidly ex utero.(2 ) however , methods for regulating endogenous gene function in zebrafish are underdeveloped relative to those for other model organisms ; targeted gene knockouts by homologous recombination and inducible rna interference technologies have not yet been achieved.(3 ) in lieu of these approaches , synthetic oligonucleotides such as morpholinos ( mos ) and negatively charged peptide nucleic acids ( ncpnas ) have been employed as antisense reagents in zebrafish embryos ( figure 1 ) . mo nucleoside analogues display dna bases from a morpholine ring system and are connected by a phosphorodiamidate backbone , while ncpna monomers are composed of alternating trans-4-hydroxy - l - proline / phosphonate polyamides , each functionalized with a dna base . because of these non - natural structures , both mos and ncpnas are resistant to nucleases and persist for up to 4 days in zebrafish embryos . thus , when mo or ncpna oligomers are injected into zebrafish prior to the eight - cell stage , they become uniformly distributed throughout the embryo and constitutively block either rna splicing or translation , depending on the targeted sequence . in the case of mos , oligomers containing 25 bases are typically used , while ncpna oligomers are limited to 18 bases . unlike rna interference technologies , mos and ncpnas do not promote rna degradation.(6 ) structures of dna , mo , and ncpna oligonucleotides . while mos and ncpnas have been used to interrogate gene function in zebrafish and other model organisms , the utility of these reagents is restricted by their constitutive activity . gene expression is inhibited immediately after introduction of the antisense oligomers , and because they are typically injected into early - stage embryos , this blockade persists in most , if not all , cell lineages . conventional mos and ncpnas therefore are less effective for studying genes that are required for embryonic survival and/or have pleiotropic functions at different developmental stages or in distinct tissues . to overcome this limitation , we recently developed caged morpholinos ( cmos ) that can be activated by light , taking advantage of the transparency of zebrafish embryos and larvae.(7 ) this was achieved by tethering a complementary mo - derived inhibitor to the 25-base targeting sequence through a dimethoxynitrobenzyl ( dmnb)-based photocleavable linker , resulting in a hairpin structure ( figure 2 ) . the intramolecular duplex suppresses binding of the targeting sequence to its complementary rna , and the stemloop configuration is significantly less active in vivo . after linker cleavage with 360-nm light , the inhibitor dissociates from the 25-base mo , allowing the antisense reagent to prevent splicing or translation of its rna target . similar caging strategies have previously been described for regulating dna duplex formation and rna function in vitro , and this general approach differs from complementary oligonucleotide - caging technologies that target individual nucleoside bases , the phosphate backbone , or oligonucleotide termini.(17 ) schematic representation of the hairpin cmo strategy . as a proof of principle , we first used this methodology to conditionally silence a t - box transcription factor called no tail - a ( ntla ) , which is required for the differentiation of axial mesodermal cells into a transient , chordate - specific organ called the notochord . embryos lacking ntla function exhibit clear morphological phenotypes in a cell - autonomous manner , providing an ideal system for evaluating the efficacy of cmos in vivo . in particular , ntla mutants or morphants ( as mo - injected embryos are commonly called ) lack a notochord , are posteriorly truncated , and exhibit u - shaped rather than v - shaped somites . the latter defect is collateral to notochord ablation , since the notochord secretes morphogens to pattern the flanking myotome.(20 ) mutants or morphants lacking ntla function also exhibit an ectopic medial floor plate , a ventral region of the developing spinal cord , and it is believed that ntla acts as a transcriptional switch between notochord and medial floor plate cell fates . by varying the developmental stage at which we activated the ntla cmo , we found that this transcription factor is required not only for specification of the mesoderm toward notochord cell fates but also for the maturation of notochord progenitors into a highly vacuolated tissue.(7 ) we also demonstrated our ability to silence ntla expression in a subset of mesodermal cells by activating the ntla cmo in a spatially restricted manner , selectively redirecting these populations to differentiate into medial floor plate cells . a similar caging approach has been applied to ncpnas targeting the chordin ( chd ) and dharma ( dha ) genes , although the use of these photoactivatable ncpna hairpins to spatially control gene expression has not been reported to date.(22 ) while our results established the general principle of using caged oligonucleotides to conditionally regulate in vivo gene expression , there are limitations associated with these initial investigations . first , our previous studies required preparation of the inhibitory oligomer and its appendant dmnb linker through solid - phase chemistries , since conventional mos amenable to terminal hydroxyl modifications were not commercially available at that time . these procedures are laborious and time - consuming , hindering the synthesis and evaluation of other cmos . second , guidelines for the design of other hairpin cmos were not evident from our findings , as only one inhibitory sequence and structural configuration was tested . nor could general rules for caged oligonucleotide design be derived from the ncpna study , which limited its analysis to single chd- and dha - targeting reagents with divergent structures and dosages.(22 ) finally , the reliance of both classes of caged oligonucleotides on nitrobenzyl - based chromophores restricts their use to single - photon irradiation with uv light , which can induce dna lesions and has limited spatial resolution . photoactivatable mos and ncpnas that are compatible with two - photon excitation could be activated with less coincident damage , deeper tissue penetration , and greater spatial precision.(23 ) we report herein our resolution of these issues through the development of new hairpin cmos . we have designed and synthesized a dmnb - based bifunctional linker that can be used to conjugate the targeting mo and its complementary inhibitor in only three steps , starting with appropriately functionalized mo oligomers that are now commercially available . we have utilized this optimized synthetic route to prepare a series of ntla - targeting cmos with differing structural configurations , thereby allowing us to systematically analyze how the in vivo efficacy of these reagents correlates with their biophysical properties . through these studies , we have demonstrated that cmo activity in vivo can be modeled in simplified thermodynamic terms , and we have established guidelines for the preparation of other cmos . these design criteria have enabled us to prepare photoactivatable reagents targeting the zebrafish genes heart of glass ( heg),(24)floating head ( flh),(25 ) and endothelial - specific variant gene 2 ( etv2).(26 ) we have further demonstrated the versatility of this approach by replacing the dmnb chromophore with a bromohydroxyquinoline ( bhq ) group , which has a significantly greater cross section for two - photon excitation . these studies represent the first comprehensive analysis of the structure and in vivo function of hairpin - caged oligonucleotides , providing a foundation upon which future chemical technologies for conditional gene regulation can be based . since our current cmo design involves the conjugation of a targeting mo to a complementary inhibitor , modifications of the hydroxyl- and amine - functionalized termini of each oligomer ( designated as the 5 and 3 ends , respectively ) are necessary . at the time of our initial studies , mos functionalized with 3 primary amines were commercially available , but 5-amine oligomers were not . we therefore prepared an inhibitory oligonucleotide conjugated at its 5 end to a photocleavable linker ( 1 ; chart 1 ) using solid - phase chemistry . this required the synthesis of all four mo bases , a linker - modified mo monomer , and multiple rounds of solid - phase coupling ( scheme s1 in the supporting information).(7 ) addition of the linker - functionalized inhibitor to the targeting mo was then achieved through a cu(i)-catalyzed huisgen 1,3-dipolar cycloaddition ( click chemistry(29 ) ) . although this approach is ultimately effective , its utilization of time - consuming and labor - intensive procedures hinders the application of hairpin cmos to other developmental genes . to streamline the synthesis of cmos , we developed a new strategy that takes advantage of the recent commercial availability of 5-amine mos . our goal was to prepare a bifunctional linker that contains the dmnb chromophore , an n - hydroxysuccinimide ester for amine conjugation , and a terminal alkyne for azide cycloaddition through click chemistry . with appropriately functionalized targeting and inhibitory mos , the cmo we first synthesized the bifunctional , photocleavable cross - linker 2a ( chart 1 and scheme 1 ) . the dmnb amino alcohol 3a(7 ) was selectively acylated with methyl adipoyl chloride to give amide 4a , which was then activated by 1,1-carbonyldiimidazole and coupled with 6-amino - n-(prop-2-ynyl)hexanamide(30 ) to provide the propargyl intermediate 5a . saponification of 5a and re - esterification of the corresponding acid with disuccinimidyl carbonate then yielded linker 2a , which is orthogonally reactive with azides and amines . reagents and conditions : ( a ) methyl adipoyl chloride , dipea , ch2cl2 , 86% ; ( b ) 1,1-carbonyldiimidazole , dmf , 88% ; ( c ) 6-amino - n-(prop-2-ynyl)hexanamide , dipea , ch2cl2 , 64% ; ( d ) 2 m naoh(aq ) , thf , meoh , 93% ; ( e ) disuccinimidyl carbonate , pyridine , acetonitrile , 75% . yields are those obtained for the dmnb derivatives . to prepare a ntla cmo through this synthetic approach , a targeting mo ( 5-gacttgaggcagacatatttccgat-3 ) was functionalized with 3-azidopropionic acid succinimidyl ester to yield the azide derivative 6 ( scheme 2 ) . linker 2a was then reacted with the commercially available 5-amine- and 3-fluorescein - functionalized mo ( 5-tatgtctgcc-3 ) in 0.1 m sodium borate buffer ( ph 8.5 ) to generate linker - derivatized inhibitory oligomer 7a , and the two mo oligomers were coupled through click chemistry in 0.1 m potassium phosphate buffer ( ph 8) containing sodium ascorbate , cui , and tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine ( tbta ) . purification of the final product by ion - exchange hplc using naclo4 as the eluent then yielded the ntla cmo 8a . reagents and conditions : ( a ) 2,5-dioxopyrrolidin-1-yl 3-azidopropanoate , 0.1 m sodium borate ( ph 8.5)/3:1 ( v / v ) dmso , 98% ; ( b ) bifunctional dmnb linker 2a or 2b , 0.1 m sodium borate ( ph 8.5)/dmso 6:1 ( v / v ) , 7090% ; ( c ) sodium ascorbate , tbta , cui , 0.1 m potassium phosphate ( ph 8.0 ) , dmso , 1025% after hplc . the ntla cmos 8ah , 8a , and 8e were prepared through this synthetic route , using the ntla - targeting mo ( 5-gacttgaggcagacatatttccgat-5 ) and inhibitory mos having various lengths and sequences ( table 1 ) . to evaluate the in vivo efficacy of this reagent , we next injected 8a into wild - type zebrafish embryos at the one - cell stage and globally irradiated approximately half of the embryos with 360-nm light ( 13 mw / cm ) for 10 s at 3 h post fertilization ( hpf ) . the remaining embryos were cultured in the dark as negative controls , and the resulting phenotypes at 1 day post fertilization ( dpf ) were then scored . as we have described previously , ntla loss - of - function phenotypes can be categorized into four classes according to their severity : class i = a fully penetrant ntla mutant phenotype characterized by no notochord , u - shaped somites , and a lack of posterior structures ; class ii = no notochord , u - shaped somites , and some posterior somites ; class iii = incompletely vacuolated notochord , v - shaped somites , and a shortened anteriorposterior axis ; and class iv = wild - type phenotype ( figure 3a).(7 ) these phenotypes can be recapitulated by injecting early - stage embryos with varying doses of the conventional ntla mo , and classes iiv correspond respectively to doses of 115 , 57 , 28 , and 14 fmol / embryo ( figure 3b ) . since the class i phenotype can be achieved with ntla mo levels 115 fmol / embryo , we utilized 8a at this minimum dose in this experiment . to our surprise , 8a induced class - ii and class - iii phenotypes in the absence of irradiation ( figure 3c ) , while our original ntla cmo exhibited little activity prior to uncaging.(7 ) since the targeting and inhibitory mos used in this reagent are identical to those in our previous study , this difference in caging efficiency must reflect changes in the linker structure . somitic ( s ) , medial floor plate ( mfp ) , notochord ( nc ) , and yolk extension ( ye ) tissues are labeled . bright - field ( left ) and differential interference contrast ( right ) images of 1 dpf embryos are shown . ( b ) distribution of phenotypes according to the dose ( per embryo ) of a conventional ntla mo . ( c ) distribution of phenotypes for ntla cmo 8a at a dose of 115 fmol / embryo with and without photoactivation . reasoning that the hairpin cmo efficacy depends on the interplay between inhibitor length , stemloop configuration , and linker formats , we used linker 2a to prepare ntla cmos with differing structures ( 8ah ; figure 4 and table 1 ) . in particular , we varied the length of the inhibitory mo ( 10 , 12 , 14 , and 16 bases ) and evaluated both blunt and staggered stemloops . each ntla cmo was synthesized , purified by ion - exchange hplc , injected into one - cell - stage zebrafish embryos , and photoactivated as before , except that a dose of 230 fmol / embryo was evaluated in each case . this higher cmo concentration was used for these studies to maximize our ability to identify caged configurations with minimum basal activity . the leftmost panels show schematic representations of staggered ( top ) and blunt ( bottom ) cmo configurations ( n = number of bases ) , and the other panels are distributions of phenotypes for each cmo configuration 8ah ( see table 1 and figure 3 ) at a dose of 230 fmol / embryo . the resulting phenotypes confirmed that hairpin cmo activity varies significantly with inhibitor length and stemloop structure . within a stemloop configuration , increasing the number of bases in the inhibitory oligomer decreased the cmo activity under both basal and photoactivated conditions . system failed to achieve an adequate activity differential between the caged and uncaged forms under any of the conditions we tested . as described above , cmo 8a still exhibited gene - silencing activity in its caged form , even though it successfully induced class - i phenotypes upon photoactivation . the other staggered cmos ( 8bd ) had lower basal activities , but their uncaged forms failed to yield strong ntla mutant phenotypes . stemloop design provided greater caging efficiency , and two cmos , 8e and 8f , exhibited dynamic ranges appropriate for conditional gene silencing : these two reagents did not induce mutant phenotypes in their caged forms , and photoactivation of the cmos yielded fully penetrant phenotypes in most embryos . since our linker-1-based ntla cmo utilized the same inhibitory oligomer as the staggered reagent 8a,(7 ) these results underscore the importance of matching linker and stemloop structures for optimum cmo activity . to better understand how cmo structure dictates in vivo activity we first determined the binding energies for the ntla mo / rna duplex , each ntla mo / inhibitor heterodimer , and various stemloop structures . thermal denaturation curves for the ntla mo / rna duplex and ntla mo / inhibitor heterodimers were acquired by mixing the ntla - targeting mo with the complementary oligomers in a 1:1 ratio and measuring their temperature - dependent changes in hypochromicity at 260 nm ( figure 5 and tables 13 ) . for these studies , we used the commercially available amine - functionalized ntla - targeting and inhibitory mo oligomers prior to their modification for cmo synthesis . the thermal denaturation curves were fit to a two - state oligomer binding model(31 ) to provide the corresponding g values at 28 c , the standard temperature for culturing zebrafish embryos . these analyses indicated that the binding free energy for ntla mo and its complementary 25-base rna is 28.1 kcal / mol , while binding free energies for the various ntla mo / inhibitor duplexes range from 10.7 to 16.2 kcal / mol . the g values for intermolecular ntla mo / inhibitor complexes correlated with the length and sequence content of the inhibitory mos , independent of the region of complementarity ( corresponding to blunt vs staggered hairpins ) . to assess binding energies for ntla mo / inhibitor interactions within an intramolecular stemloop , we next conducted hypochromicity measurements of blunt and staggered ntla mo hairpins . since the 260-nm absorbance measurements would photolyze the ntla cmos ( 8ah ) , we synthesized two ntla mo / inhibitor hairpins ( 8a and 8e ) ( scheme 2 ) using the bifunctional but nonphotolabile cross - linker 2b , which was prepared from 3-(methylamino)propan-1-ol ( 3b ) in analogy to the dmnb - based reagent 2a ( scheme 2 ) . derivation of the g values from these thermal denaturation curves using a hairpin - binding model(31 ) revealed that the intramolecular binding free energies of 8a and 8e were 4.9 and 6.9 kcal / mol , respectively ( table 4 ) . representative thermal denaturation curves for mo duplexes corresponding to ntla cmos 8ah ( ntla - targeting and inhibitory mos , 0.5 m each ) and noncleavable hairpins 8a and 8e. dimers of mo and inhibitory mo oligomers with 3 and 5 amine modifications , respectively . melting temperature of the mo / inhibitor dimer . predicted melting temperature according to eq 10 , which is based upon the tm and g values for 8ah . binding free energy of the dimer at 28 c . tm and g values were determined from sigmoidal fits of the thermal denaturation curves using the non - self - complementary algorithm in meltwin 3.0b software . dimers of 25-mer mo and rna oligomers . binding free energy of the mo / rna dimer at 28 c . melting temperature of the noncleavable mo hairpin . binding free energy of the hairpin at 28 c . tm and ghairpin values were determined from sigmoidal fits of the thermal denaturation curves using the hairpin algorithm in meltwin 3.0b software . taken together , intermolecular ntla mo / inhibitor interactions with g values lower than 12 kcal / mol are required for low basal activities , and hairpins exhibit higher caging efficiencies than their staggered counterparts because of their greater stabilization of the intramolecular mo / inhibitor duplex ( table 2 and figure 4 ) . surprisingly , the g values for the blunt and staggered ntla mo / inhibitor duplexes correlate with the inhibitor length and sequence in a similar manner , yet the activities of their corresponding ntla cmos upon photoactivation diverge . ntla cmos 8e and 8f have g values of 12.3 and 13.5 kcal / mol , respectively , and activated forms of these cmos produce strong ntla mutant phenotypes . the other two blunt ntla cmos ( 8 g and 8h ) have g values of 15.5 and 16.2 kcal / mol , respectively , and both induce only partial loss - of - function phenotypes upon linker photolysis . an in vitro ntla mo / inhibitor g value between 12 and 14 kcal / mol therefore represents the optimal balance of basal and induced activities for the blunt ntla cmos . however , the staggered ntla mos ( 8a and 8b ) fail to induce full ntla mutant phenotypes after uncaging , even though their corresponding ntla mo / inhibitor duplexes have g values of 10.7 and 13.3 kcal / mol . our original solid - phase - chemistry - derived ntla mo(7 ) and the staggered cmo 8a have identical targeting and inhibitory mo sequences , suggesting that linker elements may contribute to cmo activity after uncaging . in addition , the photolysis products of ntla cmos 8a and 8e are functionalized with linker substituents that are not present in the oligomers used in our binding energy measurements ; the targeting mo liberated upon cmo activation is 3-functionalized with a 1,2,3-triazole and an aliphatic amine , while the inhibitory oligomer is 5-functionalized with an aliphatic chain and the dmnb - derived chromophore . we therefore irradiated 8a and 8e with 360-nm light and obtained thermal denaturation curves for the resulting ntla mo / inhibitor heterodimers . analysis of the photolysis products by hplc confirmed that the two hairpin oligonucleotides are uncaged with comparable efficacies ( 75% conversion ) , and the g values for the resulting linker - functionalized ntla mo / inhibitor complexes are similar to those observed for their amine - functionalized counterparts ( figure s1 and table s1 in the supporting information ) . since we were unable to discern any thermodynamic differences between the intermolecular mo / inhibitor duplexes corresponding to 8a and 8e , we next interrogated whether rna strand exchange rates might account for their divergent activities in vivo . we incubated each ntla mo / inhibitor duplex with complementary 25-base rna for different lengths of time and resolved the resulting mo / rna duplexes by polyacrylamide gel electrophoresis ( figure s2 in the supporting information ) . the rna exchange rates for the staggered and blunt mo / inhibitor duplexes were indistinguishable in this assay ; mo / rna hybridization was complete in both cases within the time frame of rna addition . thus , the activity differences between 8a and 8e can not be explained by in vitro mo / inhibitor thermodynamics or kinetics alone , and the two cmos might exhibit divergent photolysis , inhibitor dissociation , or rna exchange rates in vivo . while the correlation between mo / inhibitor interaction strength and cmo activity provides qualitative guidelines for reagent design , a quantitative thermodynamic analysis is necessary to predict cmo efficacy with greater precision ( figure 6 ) . prior to photolysis , cmos adopt open and closed states according to the equilibrium constant khairpin ( eq 1 ) : with the assumption that the total concentration of cmo prior to uncaging ( [ cmo]t ) significantly exceeds that of its rna target ( [ rna]t ) , the basal level of rna bound by the nonphotolyzed cmo , given by ( [ cmoopenrna]/[rna]t ) , can be described as a function of [ cmo]t , khairpin , and the dissociation constant for the mo / rna duplex ( kdmorna ) ( eq 2 ) : upon photoactivation , the fraction of rna complexed with the released targeting mo is a function of [ cmo]t , kdmorna , and the dissociation constant for the mo / inhibitor complex ( kdmoinh ) ( eqs 3 and 4 ) : setting [ cmo]t to a value of 4.6 m , which approximates the embryonic concentration of the ntla cmo ( 230 fmol / embryo , 50 nl embryonic volume at 5 hpf ) , and using our ntla cmo 8e data to establish g values of 28.1 , 12.3 , and 6.9 kcal / mol for the mo / rna duplex , intermolecular mo / inhibitor duplex , and intramolecular mo / inhibitor hairpin , respectively , led to the prediction that essentially all of the ntla rna is mo - complexed before and after cmo photolysis . this conclusion clearly deviates from the phenotypes we observed with ntla cmo 8e - injected embryos . schematic representation of cmo / rna , mo / inhibitor ( inh ) , and mo / rna equilibria . since these predictions do not take into account the complexity of mo and rna interactions in live organisms , we sought to empirically derive the activity profiles of mos in vivo . we first investigated whether the relationship between ntla rna activity and total ntla mo concentration can still be described as a two - state equilibrium , even though the apparent equilibrium constant for in vivo mo / rna interactions ( kappmorna , analogous to kdmorna in figure 6 ) would include contributions from oligonucleotide - binding proteins , rna secondary structure , and other embryonic factors . we injected the targeting mo into one - cell - stage zebrafish at doses of 0 , 14 , 28 , 57 , and 115 fmol / embryo ( approximate final concentrations of 0 , 0.28 , 0.56 , 1.1 , and 2.3 m , respectively ) , lysed the embryos at 10 hpf , and then detected the ntla protein by quantitative immunoblotting ( figure 7a ) . the ntla mo reduced the ntla protein levels in a dose - dependent manner that can be modeled as two - state thermodynamic interaction , with the fraction of wild - type rna activity remaining in mo - injected embryos ( rnamoact / rnawtact ) described as a function of total mo concentration ( [ mo]t ) and kappmorna ( figure 7b and eq 5 ) : through this analysis , an apparent free - energy value ( gappmorna ) of 8.7 kcal / mol for embryonic ntla mo / rna interactions was obtained . ( a ) ntla protein knockdown in zebrafish embryos injected at the one - cell stage with various ntla mo doses . ( b ) these data can be modeled as a two - state equilibrium ( solid line ) , yielding an apparent free energy value ( gappmorna = 8.7 kcal / mol ) that describes mo / rna interactions in live embryos . ( c ) ntla protein knockdown in embryos injected at the one - cell stage with ntla mo ( 115 fmol / embryo ) and various doses of the 14-base inhibitory oligomer corresponding to ntla cmo 8 g . ntla and -actin levels at 10 hpf were detected by immunoblotting and quantified as above . ( d ) modeling of the 8 g data in ( c ) as a three - state , competitive equilibrium ( solid line ) yields an apparent free - energy value ( gappmoinh = 7.3 kcal / mol ) that describes 8 g mo / inhibitor interactions in vivo . the graphical data are means of triplicate samples with error bars representing the standard deviations . it is important to note that this gappmorna value does not reflect the actual binding constant for the ntla mo / rna duplex in zebrafish embryos but instead is an aggregate descriptor of mo / rna affinity , rna accessibility , mo / protein interactions , and the influence of other embryonic factors on mo efficacy . indeed , the 19.4 kcal / mol difference between gappmorna and the corresponding in vitro g value underscores how significant these other variables can be . our empirical data , however , suggest that this thermodynamic description can have predictive value , even though it does not explicitly consider mo and rna interactions with other cellular components or possible kinetic contributions to in vivo function . thus , mo - induced gene silencing can be modeled in these simplified thermodynamic terms . to determine whether cmo activity can also be modeled in simple thermodynamic terms , we next analyzed mo / inhibitor interactions in vivo . we injected zebrafish embryos with the ntla mo ( 115 fmol / embryo ; 2.3 m ) and various doses of the 14-base inhibitor corresponding to ntla cmo 8 g ( 0 , 150 , 450 , and 1350 fmol / embryo ; 0 , 3 , 9 , and 27 m , respectively ) . the resulting ntla protein levels at 10 hpf were then quantified as before ( figure 7c ) . the inhibitory oligomer repressed the ntla mo activity in a concentration - dependent manner that can be modeled as a three - state , competitive equilibrium involving mo , inhibitor , and rna interactions ( figure 7d ) . the fraction of wild - type rna activity associated with each mo and inhibitor dose , rnamo , inhact / rnawtact , can be expressed as a function of the apparent equilibrium constant for mo / inhibitor interactions ( kappmoinh ; analogous to kdmoinh in figure 6 ) , the total concentration of the inhibitory oligomer ( [ inh]t ) , and [ mo]t ( eqs 6 and 7 ) : through this analysis , we derived an apparent gappmoinh value of 7.3 kcal / mol for 8 g mo / inhibitor interactions . as with the gappmorna value we determined for mo - dependent ntla silencing , this apparent free - energy value does not reflect the actual binding constant for the ntla mo / inhibitor duplex in vivo but instead integrates other interactions between these synthetic oligonucleotides and cellular components . since the 8.2 kcal / mol difference between gappmoinh and the corresponding in vitro g value is significantly smaller than the 19.4 kcal / mol difference we observed for mo / rna interactions ( see table 3 and figure 7b ) , cellular factors appear to impact rna activity to a greater extent than mo function . to assess the validity of modeling in vivo mo , rna , and inhibitor interactions in these simplified terms , we investigated whether the apparent gappmorna and gappmoinh values can be used to predict how cmo gene - silencing activity will be influenced by changes in inhibitor structure . as discussed above , the fraction of total rna activity inhibited by a complementary mo and the mitigating influence of an inhibitory oligomer can be described by eqs 57 . in the case of a cmo , [ mo]t and [ inh]t will be equal after photoactivation , and the fraction of wild - type rna activity remaining in the presence of photoactivated cmo ( rnacmoact / rnawtact ) is therefore a function of kappmorna , kappmoinh , and [ cmo]t ( eqs 8 and 9 ) : in the case of our ntla cmo experiments shown in figure 4 , kappmorna and [ cmo]t can be approximated as 0.48 m ( gappmorna = 8.7 kcal / mol ) and 4.6 m , respectively . if it is assumed that blunt mo / inhibitor interactions generally exhibit a 8.2 kcal / mol difference between gappmoinh and the corresponding in vitro g , the kappmoinh values for mo / inhibitor interactions associated with ntla cmos 8e , 8f , 8 g , and 8h can be estimated as 1100 , 140 , 50 , and 1.6 m , respectively . using these parameters in the model described by eqs 8 and 9 leads to the prediction that ntla cmos 8e and 8f will be similar in efficacy to the conventional ntla mo , achieving at least a 90% knockdown of ntla protein expression levels ( red lines in figure 8a , b ) . in contrast , ntla cmos 8 g and 8h are predicted to exhibit weaker efficacies at the same embryonic dose , since the inhibitory oligomer interacts more strongly with the targeting mo ( red lines in figure 8c , d ) . photoactivated cmos and mos inhibit their rna targets with different efficacies , which diverge as the mo / rna interaction strength decreases . rna activity curves for mos and photoactivated cmos associated with in vivo mo / rna interaction energies of ( red ) 8.7 and ( blue ) 7.3 kcal / mol are shown for intermolecular mo / inhibitor interaction energies estimated for the blunt ntla cmos ( a ) 8e , ( b ) 8f , ( c ) 8 g , and ( d ) 8h , assuming complete uncaging upon irradiation photoactivated cmo and conventional mo activity profiles are drawn as solid and dashed colored lines , respectively . the embryonic ntla cmo concentration used in our structureactivity studies ( see figure 4 ) is indicated in each panel by the vertical black line . our observations in vivo match these predictions . photoactivated 8e and 8f yielded the strongest mutant phenotypes of the ntla cmo configurations we tested . moreover , the predicted efficacies of 8 g and 8h are consistent with their photoinduced phenotypes and the relationship between ntla rna activity and phenotypic class ( compare figures 3b , 4 , 7a , b , and 8c , d ) , especially considering that uv light penetrance and therefore cmo uncaging efficiencies in vivo will be attenuated to some degree . on the basis of our in vitro uncaging results ( see figure s1 in the supporting information ) and the observed phenotypes for irradiated ntla cmo - injected embryos , we estimate that our whole - organism irradiation procedure achieves cmo photoactivation yields of 5075% . our model also predicts that cmo efficacy will be increasingly compromised as gappmorna increases ( compare the blue and red lines in figure 8 , which represent a 10-fold change in mo / rna interaction strength ) , since the concentration of photoactivated cmo required to achieve a given level of rna silencing increases in a manner disproportionate to that of a conventional mo . this latter issue is of particular importance because the mo doses required to induce mutant phenotypes vary significantly between genes , reflecting different rna activity thresholds for wild - type physiology and variable rna sequence accessibilities . in addition , mo doses greater than 1000 fmol / embryo ( 20 m ) are generally avoided to minimize cytotoxicity . in contrast , the mo / inhibitor interaction strength associated with optimum cmo efficacy should not vary significantly between genes . the 25-base targeting mos are typically designed to avoid secondary structures , and they are not known to interact in a sequence - specific manner with dna- or rna - binding proteins . rather , the binding free energies for the intra- and intermolecular mo / inhibitor duplexes dictate the fraction of targeting mo that is free to anneal to its rna target , thereby establishing the basal and photoinduced activities of a given cmo . our findings suggest that cmos should have gappmoinh values of approximately 5 kcal / mol or greater for their corresponding intermolecular mo / inhibitor duplexes , as this interaction strength in vivo would allow efficient gene - silencing upon cmo photoactivation for a broad range of targeting mo potencies . maximizing the cmo activity in this manner , however , is counterbalanced by the need to maintain the closed cmo hairpin state prior to photoactivation . in fact , the gappmoinh value of 4.1 kcal / mol predicted for ntla cmo 8e may represent the optimum thermodynamic parameter for cmo efficacy . further attenuation of the mo / inhibitor interaction will eventually yield undesirable levels of basal activity , and a small fraction of embryos injected with the ntla cmo 8e exhibited weak mutant phenotypes without irradiation ( see figure 4 ) . taken together , our results demonstrate that cmo activity can be modeled as a competitive three - state equilibrium , even though this approach does not explicitly consider how mo activity and rna accessibility are influenced by cellular proteins , rna structure , and other embryonic factors . moreover , this analysis does not require the determination of actual mo / rna or mo / inhibitor binding affinities in whole embryos . we therefore sought to establish simple guidelines for the preparation of cmos targeting other genes and to empirically test their validity . such design criteria would significantly advance the use of cmos in chemical and developmental biology research , especially considering the financial and time investments associated with these studies . the disparate efficacies of the two stemloop structures indicate that blunt cmo hairpins are preferable to staggered configurations , and within our series of blunt ntla cmos 8eh , in vitro mo / inhibitor g values between 12 and 14 kcal / mol yield an optimum balance between caged and uncaged activities . our modeling of cmo activity in vivo further suggests that the higher mo / inhibitor g value associated with ntla cmo 8e should be preferred , since it would maximize the inducible gene - silencing activity over a broader range of targeting mo efficacies . this binding energy corresponds to a duplex melting temperature of 45 c ( see figure 5 and table 2 ) . to facilitate the design of thermodynamically equivalent cmos against other genes , we first determined the relationship between mo duplex sequence and thermal stability . by multiple - regression analysis of the ntla mo / inhibitor pairs listed in table 2 , we determined that the melting temperature of mo duplexes ( tmmo , in c ) is correlated with sequence content according to eq 10 and can be empirically related to its in vitro g value ( in kcal / mol at 28 c ) by eq 11 : in eq 10 , each nj ( j = a , t , g , c ) is the number of times the corresponding base appears in the mo sequence . using this equation , one can identify an appropriate inhibitor for a given targeting mo , which ideally would generate a blunt this empirically derived algorithm should be most accurate with mo duplexes similar in length to those in this study , since it does not take into account the contribution of nearest - neighbor effects . we next tested the validity of our design criteria by targeting four other zebrafish genes : heg , flh , etv2 , and spadetail / tbx16 ( spt ) . the transmembrane protein heg is expressed in the endocardium during embryogenesis , mediating a signal that is required for concentric growth of the heart.(24 ) embryos lacking heg function exhibit abnormally large heart chambers with walls that are only one cell thick and therefore incapable of sustaining blood circulation . these defects are apparent by 2 dpf and can be recapitulated with the heg - targeting mo ( 5-gtaatcgtacttgcagcaggtgaca-3 ) at doses of 230 fmol / embryo ( 4.6 m ) or higher ( 79% , n = 48 ) . by 4 dpf , heg mutants and morphants exhibit severe cardiac edema ( figure 9a ) . to generate a heg cmo ( 9a ) , we conjugated the targeting mo to an inhibitory oligomer 5-caagtacgattac-3 using linker 2a ( tables 13 ) . we then injected wild - type zebrafish embryos with 9a at the one - cell stage ( 460 fmol / embryo ; 9.2 m ) and either irradiated the embryos with 360-nm light at 2 hpf or cultured them in the dark . by 4 dpf , the irradiated embryos had no blood circulation and exhibited cardiac edema ( 86% , n = 21 ) , while the nonirradiated zebrafish had normal cardiac patterning and function ( 98% , n = 44 ) ( figure 9b , c and figure s3 in the supporting information ) . to further test the predictive value of our cmo design criteria , we evaluated two additional heg cmos : one contained a staggered inhibitor ( 9b ; inhibitor sequence 5-gctgcaagtac-3 ) with an intermolecular mo / inhibitor g value comparable to that for 9a , and the other contained a shorter blunt inhibitor ( 9c ; inhibitor sequence 5-gtacgattac-3 ) with a weaker mo / inhibitor interaction strength ( tables 1 and 2 ) . in comparison to 9a , these alternative heg cmos were less effective in vivo . the majority of zebrafish embryos injected with an equivalent dose of either 9b or 9c exhibited heg mutant phenotypes in the absence of irradiation ( 9b : 66% , n = 53 ; 9c : 62% , n = 45 ) , indicating that these structural configurations do not adequately cage the mo activity ( figure s3 ) . thus , the design parameters established through our ntla cmo structureactivity relationship studies are transferable to other genes . ( a ) embryos injected with a conventional heg mo recapitulate heg mutant phenotypes , including enlarged heart chambers , no blood circulation , and cardiac edema . ( b , c ) embryos injected with heg cmo 9a exhibit heg mutant phenotypes upon photoactivation . ( d ) embryos injected with a conventional flh mo recapitulate flh mutant phenotypes , including notochord ablation and somite fusion through the trunk midline . ( e , f ) embryos injected with flh cmo 10 exhibit flh mutant phenotypes upon photoactivation . ( g ) embryos injected with a conventional spt mo recapitulate spt mutant phenotypes , including a loss of trunk somitic tissue and mislocalized mesodermal progenitors in the posterior ( spadetail morphology ) . ( h , i ) embryos injected with spt cmo 12 exhibit partial spt mutant phenotypes upon photoactivation . developmental stages : ( ac ) 4 dpf ; ( df , gi ) 1 dpf . scale bars : ( ac ) 400 m ; ( df , gi ) 200 m . the flh homeobox transcription factor is coexpressed with ntla in the zebrafish mesoderm , and loss of flh expression also causes ablation of the notochord . however , mesodermal progenitors normally fated to become the notochord do not transform into medial floor plate cells in flh mutants ; rather , these populations differentiate into ectopic muscle to create fused somites . since no flh - targeting mo had yet been described , we first identified a flh - blocking oligonucleotide ( 5-gggaatctgcatggcgtctgtttag-3 ) and demonstrated its efficacy in zebrafish embryos ( figure 9d ) . this antisense reagent is a potent inhibitor of flh function , and a dose of 60 fmol / embryo ( 1.2 m ) is sufficient to cause replacement of the notochord with axial muscle cells in 1 dpf zebrafish ( 89% , n = 19 ) . on the basis of our cmo design criteria , we synthesized a hairpin flh cmo ( 10 ) using linker 2a and a 5-gcagattccc-3 oligomer as the mo inhibitor ( tables 13 ) . wild - type zebrafish were injected at the one - cell stage with 10 at a dose of 100 fmol / embryo ( 2 m ) and either globally irradiated with 360-nm light for 10 s at 2 hpf or cultured in the dark . as expected , the majority of nonirradiated flh cmo - injected embryos developed normally ( 89% , n = 28 ) , while the irradiated embryos lacked a notochord and had fused somites ( 100% , n = 11 ) , matching the mutant phenotype ( figure 9e , f ) . the etv2 transcription factor is expressed in the lateral mesoderm during early somitogenesis and then in vascular endothelial cells of the axial , head , and intersomitic vessels.(26 ) it is believed that these early etv2-expressing cells are endothelial precursors , and etv2 mutants or embryos injected with an etv2-blocking mo ( 5-cactgagtccttatttcactatatc-3 ) exhibit disrupted blood - vessel formation and lack circulation.(26 ) mo doses of 115 fmol / embryo ( 2.3 m ) or higher are sufficient to induce a fully penetrant mutant phenotype ( data not shown ) . we therefore synthesized a hairpin etv2 cmo ( 11 ) containing a 5-ggactcagtg-3 inhibitory oligomer and injected it into wild - type zebrafish at the one - cell stage ( 230 fmol / embryo ; 4.6 m ) ( tables 13 ) . embryos then irradiated with 360 nm light for 10 s at 3 hpf had limited or no blood circulation by 2.5 dpf ( 100% , n = 12 ; see supplementary movie 1 in the supporting information ) , but most of the etv2 cmo - injected embryos cultured in the dark exhibited normal blood flow ( 87% , n = 15 ; see supplementary movie 2 in the supporting information ) . finally , we designed and synthesized a cmo targeting spt , which is another mesodermal t - box transcription factor . the ntla and spt genes are expressed in overlapping domains during early embryogenesis and then become restricted to the axial and paraxial mesoderm , respectively . cells with ntla function become the notochord , while the spt - expressing cells contribute to the skeletal muscle in the flanking somites . a loss of spt function therefore causes a severe deficit in trunk somitic mesoderm as well as a gross mislocalization of the corresponding progenitor cells to posterior regions ( hence the spadetail these phenotypes can be recapitulated with a spt - targeting mo 12 ( 5-gcttgaggtctctgatagcctgcat-3)(34 ) at doses of 345 fmol / embryo ( 6.9 m ) or higher ( 61% , n = 23 ) ( figure 9 g ) . as with the other cmos described above , we prepared the corresponding spt cmo hairpin using linker 2a and the inhibitory oligomer 5-gacctcaagc-3 ( tables 13 ) . wild - type embryos at the one - cell stage were injected with 12 , and a subset was globally irradiated with 360 nm light for 10 s at 2 hpf . the majority of embryos cultured in the dark developed normally ( 87% , n = 24 ) . zebrafish injected with a dose of 700 fmol / embryo ( 14 m ) exhibited a loss of trunk mesoderm but not posteriorly mislocalized progenitor cells upon cmo photoactivation ( 31% , n = 16 ) and also showed nonspecific developmental defects due to general mo toxicity ( 62% , n = 16 ) ( figure 9h , i ) . these phenotypes indicate that the photoactivated spt cmo 12 is not able to fully recapitulate the activity of the conventional mo and that this reagent also exhibits greater nonspecific toxicity . thus , although our design criteria were successful for cmos targeting ntla , heg , flh , and etv2 , there can be unforeseen mo or inhibitor interactions with embryonic factors that reduce cmo efficacy in certain situations . in the case of the spt cmo , one possibility is that a 700 fmol / embryo dose of the 35-base reagent approaches the toxicity level of a 1000 fmol / embryo dose of the conventional 25-base mo . to conclude our studies , we investigated the ability of our hairpin cmo design to incorporate other photocleavable groups . while nitrobenzyl - based chromophores such as the dmnb group in our ntla , flh , heg , etv2 , and spt cmos have been widely used in biological applications , the uv light required for their photolysis is potentially damaging and difficult to restrict in three - dimensional space . two - photon excitation typically uses wavelengths greater than 700 nm and affords greater spatial resolution , but the dmnb group and most other caging moieties have small two - photon cross sections and are therefore inefficiently cleaved under these conditions . two notable exceptions are the bromohydroxycoumarin ( bhc)(23 ) and bromohydroxyquinoline ( bhq)(27 ) groups . since the low fluorescence of bhq chromophores makes them particularly useful for biological applications , we designed a bhq - based bifunctional cross - linker ( 13 , scheme 3 ) for the preparation of cmos . as with our dmnb - based linker 2a , this two - photon - sensitive linker incorporates an n - hydroxysuccinimide ester and a terminal alkyne to enable orthogonal coupling to appropriately modified mo oligomers . reagents and conditions : ( a ) benzenesulfonyl chloride , dipea , ch2cl2 , 97% ; ( b ) seo2 , dioxane , 80 c , 91% ; ( c ) in powder , allyl bromide , nh4cl(aq ) , thf , 96% ; ( d ) k2oso42h2o , lutidine , dioxane , h2o , then naio4 , 75% ; ( e ) methylamine(aq ) , nabh(oac)3 , meoh , h2o , 81% ; ( f ) methyl adipoyl chloride , dipea , ch2cl2 , 59% ; ( g ) carbonyldiimidazole , dmf then 6-amino - n-(prop-2-ynyl)hexanamide , dipea , dmf , 81% ; ( h ) 0.2 m naoh(aq ) , thf , meoh , 82% ; ( i ) n - hydroxysuccinimide , edci , dmf , 48% . to synthesize linker 13 , we first prepared bhq derivative 14 as previously described(27 ) and then protected its phenolic hydroxyl group with a benzenesulfonyl moiety to give 15 . the 2-methyl group of 15 was then oxidized with selenium dioxide , and the resulting aldehyde 16 was allylated to give alcohol 17 . oxidative cleavage of 17 yielded aldehyde 18,(35 ) which was reductively aminated with aqueous methylamine in the presence of nabh(oac)3 to give the amino alcohol intermediate 19 . in analogy to scheme 1 , compound 19 was acylated to form the amide 20 , and 1,1-carbonyldiimidazole - mediated coupling of this product with 6-amino - n-(prop-2-ynyl)hexanamide afforded carbamate 21 . phenol deprotection and ester saponification of 21 were simultaneously accomplished with 0.2 m naoh to give an acid intermediate , which was re - esterified with n - hydroxysuccinimide in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to give the two - photon - sensitive linker 13 . the bifunctional linker was then used to assemble fluorescein - conjugated and nonfluorescent ntla cmos ( 22a and 22b , respectively ; chart 2 ) using the corresponding inhibitory oligomers ( 23a and 23b ) , which are analogous to that in the optimum dmnb - containing ntla cmo 8e ( figure 4 and table 1 ) . since the bhq group has not been used previously with carbamates and other bhq - caged compounds have not been tested in cultured cells or live organisms , we first determined whether the bhq carbamate is stable in vivo and can be efficiently photolyzed . we injected ntla cmo 22a into wild - type zebrafish embryos at the one - cell stage ( 230 fmol / embryo ) and irradiated a subset of them with 360-nm light for 10 s at 3 hpf . the embryos were then cultured for another day and scored according to the four phenotypic classes described above . as we hoped , the bhq - based ntla cmo exhibited activity essentially identical to that of the dmnb - based reagent 8e ; embryos injected with this reagent developed normally when cultured in the dark but displayed a ntla phenotype upon irradiation ( figure s4 in the supporting information ) . we then examined whether the bhq - based ntla cmo 22b could be activated in targeted regions of the zebrafish embryo using two - photon excitation . these experiments followed studies conducted previously in our laboratory , in which we uncaged a dmnb - based ntla cmo in a subset of mesodermal progenitor cells , selectively inducing them to differentiate into medial floor plate cells rather than the notochord.(7 ) to identify the irradiated cells in this earlier investigation , we coinjected the zebrafish embryos with mrna encoding kaede fluorescent protein , which undergoes a green - to - red photoconversion upon one - photon excitation . because kaede is inefficiently converted by two - photon excitation , we utilized a caged coumarin fluorophore conjugated to dextran ( dextranhcc - npe ) as a cell - autonomous photoactivatable tracer ( figure s5 in the supporting information ) . this new class of caged coumarins is highly sensitive to two - photon irradiation , and dextranhcc - npe has demonstrated efficacy in vivo.(38 ) we injected dextranhcc - npe into one - cell - stage embryos with either the bhq - based ntla cmo 22b or the bhq - conjugated inhibitory oligomer 23b . an 80 m 60 m 50 m region of the posterior , axial mesoderm at the end of gastrulation was then subjected to two - photon irradiation for 2 min ( figure 10ad ) . these cells normally differentiate into vacuolated notochord cells by 1 dpf , and the embryos injected with the coumarin tracer and bhq - functionalized ntla mo inhibitor 23b contained a cluster of blue - fluorescent notochord cells toward the end of the yolk extension ( figure 10a , e ) . in contrast , embryos injected with both the photoactivatable tracer and ntla cmo 22b exhibited medial floor plate cells with blue fluorescence and few , if any , fluorescently labeled notochord cells ( figure 10f ) . these observations confirm the sensitivity of the bhq - based reagent to two - photon photolysis and illustrate the versatility of the hairpin cmo architecture . ( a ) schematic representation of embryos injected with the dextranhcc - npe photoactivatable tracer and then two - photon - irradiated within the posterior axial mesoderm at the 10 hpf stage ( red square , dorsal view ) . by 1 dpf , these irradiated cells normally differentiate into vacuolated notochord cells ( red rectangle , lateral view ) toward the end of the yolk extension . ( b ) ir gradient contrast image of the posterior axial mesoderm with the targeted irradiation area indicated by the dashed red box ( dorsal view ) . ( c ) fluorescent image of the same region prior to irradiation ( excitation , 820 nm ; emission , 525/70 nm ) . ( d ) fluorescent image post - irradiation ( same conditions as in c ) . ( e ) embryos injected with the dextranhcc - npe and oligomer 23b contained a cluster of blue - fluorescent notochord ( nc ) cells by 1 dpf ( lateral view ; excitation , 436/20 nm ; emission , 480/40 nm ) . ( f ) embryos injected with dextranhcc - npe and ntla cmo 22b exhibited medial floor plate ( mfp ) cells with blue fluorescence and few fluorescently labeled notochord cells ( same conditions as in e ) . photoactivatable mos are valuable probes of embryonic patterning mechanisms , and their implementation by the developmental biology community will require efficient synthetic procedures and general principles for cmo design . to advance these goals , we have devised a bifunctional linker for the synthesis of hairpin cmos that enables these probes to be prepared in three steps from commercially available reagents . this optimized synthetic procedure has enabled us to prepare several ntla - targeting cmos of differing configurations , and we have used these structural variants to determine how the thermodynamic properties of cmos correlate with their in vivo functions . our studies reveal that hairpin cmo function is highly dependent on the sequence and location of the complementary inhibitor as well as the linker structure . we further observed that mo / mo and mo / rna binding free energies in vitro differ dramatically from the apparent free energies of their functional interactions in vivo , undoubtedly reflecting the influence of oligonucleotide - binding proteins , rna secondary structure , and other embryonic factors . although the apparent free - energy values obtained through our in vivo studies can not accurately describe the true mo and rna binding affinities in zebrafish embryos , our findings demonstrate that these thermodynamic descriptors can be used to predict mo efficacy . thus , cmo activity in vivo can be modeled as a competitive three - state equilibrium , and the complex interactions between mos and embryonic factors can be treated as an aggregate phenomenon . our findings establish a metric for cmo design , which we have successfully applied to cmos that target the flh , heg , and etv2 genes , and these empirically derived principles will help guide future applications of cmos in functional genomic studies . our insights should also be applicable to other caged hairpin reagents , including those based upon ncpnas and 2-o - methyl rnas.(22 ) although these oligonucleotide analogues interact with complementary oligomers with distinct in vitro and in vivo free energy values , their design and biological application are subject to the same principles outlined in this study . as illustrated by the partial phenotypes we observed with our spt cmo , however , the activity of caged hairpin oligonucleotides can be attenuated in vivo through mechanisms that are not yet understood . we anticipate that our guidelines for cmo design will become more refined as this chemical technology is more broadly utilized in embryological research . finally , the modularity of our hairpin cmos provides an opportunity for future improvements to this technology , as illustrated by our development of a bhq - based cmo that is compatible with two - photon excitation . for example , linkers that minimize the entropic loss associated with stemloop formation could increase the energetic difference between inter- and intramolecular mo / inhibitor duplexes , thereby improving the dynamic range of basal and photoinduced activities . alternative approaches might involve incorporating the photocleavable bond within the inhibitory oligomer or caging the mo with two short complementary oligomers rather than one long inhibitor . investigating these possibilities through organic synthesis will expand the utility of cmo technologies and help shed new light on the molecular mechanisms of embryonic development . all of the reactions were carried out in flame - dried glassware under an argon atmosphere using commercial reagents without further purification , unless otherwise indicated . reactions were magnetically stirred and monitored by thin - layer chromatography ( tlc ) using glass - backed silica gel 60f254 ( merck , 250 m thickness ) . sio2 chromatography was carried out with em science silica gel ( 60 , 70230 mesh ) as a stationary phase . h nmr and c nmr spectra were acquired on varian 300 , 400 , and 500 mhz spectrometers and standardized to the nmr solvent peak . electrospray mass spectrometry ( esi - ms ) was performed using a micromass zq single - quadrupole liquid chromatographmass spectrometer ( lcms ) and a micromass q - tof hybrid quadrupole lcms . an mo oligomer ( 5-gcctcaagtc-3 ) with 5-amine and 3-fluorescein modifications was purchased from gene - tools , llc and used without further purification . this oligomer ( 100 nmol ) was dissolved in borate buffer [ 0.1 m na2b4o7 ( ph 8.5 ) , 100 l ] and combined with linker 2a ( 0.76 mg , 1.5 mol ) in dmso ( 15 l ) . the resulting yellow gum was dissolved in water ( 0.5 ml ) , washed three times with chcl3 ( 0.5 ml ) , and diluted to 1.5 ml with water . the yellow solution was loaded onto toyopearl super - q resin ( 400 l ) , washed three times with wash solution [ aqueous 2.5 mm na2b4o7 ( ph 8.5 ) , 50% acetonitrile ( acn ) ] and two times with water . fluoresceinated oligomers were eluted from the resin with 600 l of aqueous 5% hoac/50% acn , washed three times with chcl3 ( 0.6 ml ) , and neutralized with 10% nh4oh(aq ) ( 0.3 ml ) . solvent was removed in vacuo , and the remaining nh4oac was removed by repeated aqueous solubilization and lyophilization , affording 7e as a yellow solid ( 70 nmol , 70% ) . ms - esi for 7e [ m + h ] ( m / z ) : calcd for c184h264n69o61p10 , 4728 ; found , 4728 . the inhibitory oligomer 7e ( 50 nmol ) and azide - functionalized ntla mo(7)6 ( 50 nmol ) were dissolved in phosphate buffer [ kh2po4 ( ph 8.0 ) , 230 l ] . to this mixture was added sodium ascorbate ( 99.0 g , 500 nmol ) in 25 l of water followed by tbta ( 265 g , 500 nmol ) and cui ( 95.2 g , 500 nmol ) in 50 l of dmso . the reaction mixture was briefly sonicated and then stirred overnight at room temperature in the dark . precipitate was removed from the reaction mixture by centrifugation , and the supernatant was split and desalted over two zeba desalt size - exclusion columns ( pierce ) according to the manufacturer s instructions . the desired product was purified from the reaction mixture by adjusting the solution ph to 11.5 with 1 m naoh(aq ) and loading it onto a dnapac pa-100 ion - exchange hplc column ( dionex , 9 mm 250 mm ) . aqueous running buffers were ( a ) 0.02 m naoh , 1% acn and ( b ) 0.375 m naclo4 in 0.02 m naoh and 1% acn . a stepwise gradient was used to separate the product and starting materials , with specific conditions determined by column capacity . a representative purification gradient was the following : 7 to 19% b in 5 min , 19 to 22% b in 10 min , 22 to 50% b in 1 min , and 50% b for 9 min ( flow rate 4 ml / min ) . elution fractions were collected with the uvvis flow - cell lamp turned off to prevent photolysis . fractions ( 1 ml ) were collected every 15 s and buffered with 1 m nh4oac(aq ) ( ph 5.0 ) ( 40 l ) . the fractions containing fluoresceinated product were combined and desalted over a zeba size - exclusion column . the eluent volume was reduced in vacuo to 50 l , and the cmos were precipitated with acetone ( 400 l ) . after centrifugation , the supernatant was discarded , and the cmo pellet was washed with acn ( 100 l ) and briefly lyophilized , affording 8e as a yellow solid ( 7 nmol , 14% ) . ms - esi for 8e [ m + h ] ( m / z ) : calcd for c488h737n219o164p35 , 13379 ; found , 13380 . adult zebrafish ( wild - type ab strain ) were acquired from the zebrafish international resource center . embryos used in these studies were obtained by natural matings and cultured in e3 embryo medium at 28.5 c according to standard procedures.(39 ) various mo , mo / inhibitor duplex , and cmo solutions containing 0.1% ( w / v ) phenol red were prepared and microinjected at 1 or 2 nl / embryo . for example , to inject 115 fmol of mo , 2 nl of a 57.5 m solution containing 0.1% ( w / v ) phenol red was injected into each zebrafish embryo at the one - cell stage . all embryo injections were done according to standard procedures , and the embryos were subsequently cultured in e3 medium at 28.5 c . for two - photon experiments , solutions containing 1.25 mm dextranhcc - npe and 0.1% ( w / v ) phenol red with or without 57.5 m cmo 22b were injected at 2 nl / embryo . photolysis reactions were performed by dissolving 1 nmol of cmo hairpin in water ( 2 l ) and irradiating for 1 min using a leica dm4500b compound microscope equipped with an a4 filter cube ( ex : 360 nm , 40 nm bandpass ) and a 20 water - immersion objective ( 0.50 na , 13 mw / cm intensity at 360 nm ) . the solutions were then adjusted to ph 11.5 with 0.02 m naoh and analyzed by hplc using a dnapac pa-200 ion - exchange column ( dionex , 4 mm 250 mm ) . aqueous running buffers were ( a ) 0.02 m naoh , 1% acn and ( b ) 0.375 m naclo4 in 0.02 m naoh and 1% acn . the hplc gradient was 7 to 50% b in 27 min at 1.2 ml / min . zebrafish embryos between the 64- and 256-cell stages were arrayed in an agarose microinjection template . mercury lamp light was focused onto the individual embryos for 10 s using a leica dm4500b compound microscope equipped with an a4 filtercube and a 20 water - immersion objective . two - photon cmo photoactivation in zebrafish embryos was performed on an upright two - photon confocal microscope ( ultima xy , prairie technologies , inc . , middleton , wi ) equipped with two ti : sapphire lasers ( mai tai hp , spectra physics , mountain view , ca ) and a 40 ( 0.8 na ) water - immersion objective ( lumplanfl / ir , olympus america , center valley , pa ) . the 820-nm illumination from the first laser ( 10 mw at the back focal plane of the objective ) was used to collect two initial images for each embryo : an epifluorescence image ( bandpass : 525 nm center , 70 nm fwhm ) and an ir gradient contrast image ( 820-nm illumination).(40 ) using the gradient contrast image , an 80 m 60 m 50 m region of interest ( roi ) was selected for photoactivation . the roi was then illuminated for 2 min at 750 nm ( 65 mw at the back focal plane of the objective ) with the second laser . following photoactivation , the embryo was reimaged with 820-nm illumination . following two - photon irradiation , chorions were manually removed from 1 dpf embryos , which were immobilized in e3 medium containing 0.7% ( w / v ) low - melt agarose and 0.05% ( w / v ) tricaine . bright - field images were obtained at 5 with a leica mzfliii fluorescence stereoscope equipped with a leica dc300f digital camera . differential interference contrast images and time - lapse movies were obtained with a leica dm4500b fluorescence microscope equipped with a 10 ( 0.25 na ) objective and a qimaging retiga - srv digital camera . fluorescence images were also obtained with this equipment and a cfp filterset ( excitation , 436/20 nm ; emission , 480/40 nm ) . for intermolecular mo duplexes , the complementary oligomers ( 0.5 m , 1:1 molar ratio ) in buffer [ 100 mm kcl , 20 mm hepes , 10 mm mgcl2 , 0.1 mm edta ( ph 7.0 ) , 1 ml ] were denatured at 95 c for 5 min . thermal denaturation curves were obtained by monitoring temperature - dependent changes in the absorbance of 260-nm light using a varian cary 300 spectrophotometer ( annealing at 0.5 c / min ) . the hypochromicity curves were fitted to a sigmoidal function , and thermodynamic parameters were calculated using the non - self - complementary algorithm in meltwin 3.0b software . for intermolecular mo / rna duplexes , the complementary mo and rna oligomers were used in a 2:1 molar ratio to minimize hypochromicity changes due to rna self - annealing . for intramolecular duplexes , thermodynamic parameters were calculated using the hairpin algorithm in meltwin 3.0b software . at bud stage ( 10 hpf ) , wild - type and mo - injected embryos were dechorinated with pronase ( 1 mg / ml ) for 10 min at 28 c . the embryos were transferred to microcentrifuge tubes and homogenized with a pipet in tm1 buffer [ 180 l / sample ; 100 mm nacl , 5 mm kcl , 5 mm hepes ( ph 7.0 ) , 1% ( w / v ) peg-20000 ] containing protease inhibitors [ 1 mm pmsf , 5 mg / ml complete mini protease inhibitor cocktail , edta - free ( roche ) ] to remove yolks . following centrifugation ( 500 g , 5 min , 4 c ) , the tm1 solution was replaced , and the pelleted cells were homogenized again with a pipet and recentrifuged . eighteen deyolked embryos from each set of experimental conditions were vortexed in sds - page loading buffer [ 50 l / sample ; 100 mm tris - hcl ( ph 6.8 ) , 330 mm 2-mercaptoethanol , 4% ( w / v ) sds , 20% ( v / v ) glycerol , 100 mm dtt ] , sonicated for 1 min , and heated to 100 c for 5 min . the lysates were resolved on a 412% bis - tris gradient acrylamide gel ( five embryos / lane ) and blotted onto nitrocellulose according to standard protocols . anti - ntla antibody was used at a 1:2000 dilution in 1 phosphate - buffered saline containing 0.1% ( v / v ) tween 20 and 0.2% ( w / v ) i - block ( roche ) . the anti - ntla antibody was then detected using a horseradish peroxidase - conjugated anti - rabbit igg antibodies ( ge ) at 1:10000 dilution and the supersignal west dura extended duration substrate kit ( pierce ) according to the manufacturer s instructions . the chemiluminescence from the membrane was digitally imaged ( chemidoc xrs , biorad ) , and the band intensity was measured with quantity one 4.5 software . the nitrocellulose membranes were then reprobed with mouse anti--actin [ sc-8432 ( santa cruz biotechnology ) at a 1:250 dilution or clone ac-15 ( sigma ) at a 1:10000 dilution ] and horseradish peroxidase - conjugated anti - mouse igg antibodies ( ge ) at 1:10000 dilution to normalize for loading differences between lanes . targeting and inhibitory oligomers corresponding to ntla cmos 8a and 8e ( 5 m , 1:1 molar ratio ) in buffer [ 100 mm kcl , 20 mm hepes , 10 mm mgcl2 , 0.1 mm edta ( ph 7.0 ) , 28 l ] were denatured at 95 c for 2 min and annealed by cooling to 28 c over 15 min . the complementary , 3-fluoresceinated 25-base rna ( 3 m in the above buffer , 2 l ) was then added to the annealed mo duplex solution to achieve a final rna concentration of 0.2 m . the mixture of oligomers was incubated at 28 c for either 1 or 10 min and then chilled to 4 c on ice . sample was prepared by adding the rna to the annealed mo duplex solution at 4 c , and the mo / rna duplex was prepared by heat denaturation and annealing , as was done for the mo / inhibitor duplexes . all of the samples were then immediately mixed with chilled loading dye [ 6 stock : 60% glycerol , 0.1 m tris - hcl , 90 mm boric acid , 1 mm edta , 0.9 mm xylene cyanol ( ph 8.4 ) ] and resolved on a 15% tris - borateedta acrylamide gel at 200 v for 20 min at 4 c . after electrophoresis , the acrylamide gel was analyzed with a ge typhoon imager ( 488 nm excitation , 580 nm emission ) .

embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner , and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision . chemical technologies can enable such genetic manipulations , as exemplified by the use of caged morpholino ( cmo ) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control . here we report optimized methods for the design and synthesis of hairpin cmos incorporating a dimethoxynitrobenzyl ( dmnb)-based bifunctional linker that permits cmo assembly in only three steps from commercially available reagents . using this simplified procedure , we have systematically prepared cmos with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities . through these studies , we have established general principles for cmo design and successfully applied them to several developmental genes . our optimized synthetic and design methodologies have also enabled us to prepare a next - generation cmo that contains a bromohydroxyquinoline ( bhq)-based linker for two - photon uncaging . collectively , these advances establish the generality of cmo technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies .

Introduction Results and Discussion Conclusion Experimental Section

one of the remaining challenges in developmental biology is to understand how these genes act in space and time to modulate cell proliferation , migration , and differentiation in a stereotypic manner . (2 ) however , methods for regulating endogenous gene function in zebrafish are underdeveloped relative to those for other model organisms ; targeted gene knockouts by homologous recombination and inducible rna interference technologies have not yet been achieved. because of these non - natural structures , both mos and ncpnas are resistant to nucleases and persist for up to 4 days in zebrafish embryos . while mos and ncpnas have been used to interrogate gene function in zebrafish and other model organisms , the utility of these reagents is restricted by their constitutive activity . (7 ) this was achieved by tethering a complementary mo - derived inhibitor to the 25-base targeting sequence through a dimethoxynitrobenzyl ( dmnb)-based photocleavable linker , resulting in a hairpin structure ( figure 2 ) . as a proof of principle , we first used this methodology to conditionally silence a t - box transcription factor called no tail - a ( ntla ) , which is required for the differentiation of axial mesodermal cells into a transient , chordate - specific organ called the notochord . embryos lacking ntla function exhibit clear morphological phenotypes in a cell - autonomous manner , providing an ideal system for evaluating the efficacy of cmos in vivo . by varying the developmental stage at which we activated the ntla cmo , we found that this transcription factor is required not only for specification of the mesoderm toward notochord cell fates but also for the maturation of notochord progenitors into a highly vacuolated tissue. (7 ) we also demonstrated our ability to silence ntla expression in a subset of mesodermal cells by activating the ntla cmo in a spatially restricted manner , selectively redirecting these populations to differentiate into medial floor plate cells . a similar caging approach has been applied to ncpnas targeting the chordin ( chd ) and dharma ( dha ) genes , although the use of these photoactivatable ncpna hairpins to spatially control gene expression has not been reported to date. second , guidelines for the design of other hairpin cmos were not evident from our findings , as only one inhibitory sequence and structural configuration was tested . (22 ) finally , the reliance of both classes of caged oligonucleotides on nitrobenzyl - based chromophores restricts their use to single - photon irradiation with uv light , which can induce dna lesions and has limited spatial resolution . photoactivatable mos and ncpnas that are compatible with two - photon excitation could be activated with less coincident damage , deeper tissue penetration , and greater spatial precision. (23 ) we report herein our resolution of these issues through the development of new hairpin cmos . we have designed and synthesized a dmnb - based bifunctional linker that can be used to conjugate the targeting mo and its complementary inhibitor in only three steps , starting with appropriately functionalized mo oligomers that are now commercially available . we have utilized this optimized synthetic route to prepare a series of ntla - targeting cmos with differing structural configurations , thereby allowing us to systematically analyze how the in vivo efficacy of these reagents correlates with their biophysical properties . through these studies , we have demonstrated that cmo activity in vivo can be modeled in simplified thermodynamic terms , and we have established guidelines for the preparation of other cmos . these design criteria have enabled us to prepare photoactivatable reagents targeting the zebrafish genes heart of glass ( heg),(24)floating head ( flh),(25 ) and endothelial - specific variant gene 2 ( etv2). (26 ) we have further demonstrated the versatility of this approach by replacing the dmnb chromophore with a bromohydroxyquinoline ( bhq ) group , which has a significantly greater cross section for two - photon excitation . these studies represent the first comprehensive analysis of the structure and in vivo function of hairpin - caged oligonucleotides , providing a foundation upon which future chemical technologies for conditional gene regulation can be based . at the time of our initial studies , mos functionalized with 3 primary amines were commercially available , but 5-amine oligomers were not . this required the synthesis of all four mo bases , a linker - modified mo monomer , and multiple rounds of solid - phase coupling ( scheme s1 in the supporting information). although this approach is ultimately effective , its utilization of time - consuming and labor - intensive procedures hinders the application of hairpin cmos to other developmental genes . to streamline the synthesis of cmos , we developed a new strategy that takes advantage of the recent commercial availability of 5-amine mos . our goal was to prepare a bifunctional linker that contains the dmnb chromophore , an n - hydroxysuccinimide ester for amine conjugation , and a terminal alkyne for azide cycloaddition through click chemistry . to prepare a ntla cmo through this synthetic approach , a targeting mo ( 5-gacttgaggcagacatatttccgat-3 ) was functionalized with 3-azidopropionic acid succinimidyl ester to yield the azide derivative 6 ( scheme 2 ) . to evaluate the in vivo efficacy of this reagent , we next injected 8a into wild - type zebrafish embryos at the one - cell stage and globally irradiated approximately half of the embryos with 360-nm light ( 13 mw / cm ) for 10 s at 3 h post fertilization ( hpf ) . reasoning that the hairpin cmo efficacy depends on the interplay between inhibitor length , stemloop configuration , and linker formats , we used linker 2a to prepare ntla cmos with differing structures ( 8ah ; figure 4 and table 1 ) . to better understand how cmo structure dictates in vivo activity we first determined the binding energies for the ntla mo / rna duplex , each ntla mo / inhibitor heterodimer , and various stemloop structures . thermal denaturation curves for the ntla mo / rna duplex and ntla mo / inhibitor heterodimers were acquired by mixing the ntla - targeting mo with the complementary oligomers in a 1:1 ratio and measuring their temperature - dependent changes in hypochromicity at 260 nm ( figure 5 and tables 13 ) . for these studies , we used the commercially available amine - functionalized ntla - targeting and inhibitory mo oligomers prior to their modification for cmo synthesis . surprisingly , the g values for the blunt and staggered ntla mo / inhibitor duplexes correlate with the inhibitor length and sequence in a similar manner , yet the activities of their corresponding ntla cmos upon photoactivation diverge . an in vitro ntla mo / inhibitor g value between 12 and 14 kcal / mol therefore represents the optimal balance of basal and induced activities for the blunt ntla cmos . thus , the activity differences between 8a and 8e can not be explained by in vitro mo / inhibitor thermodynamics or kinetics alone , and the two cmos might exhibit divergent photolysis , inhibitor dissociation , or rna exchange rates in vivo . prior to photolysis , cmos adopt open and closed states according to the equilibrium constant khairpin ( eq 1 ) : with the assumption that the total concentration of cmo prior to uncaging ( [ cmo]t ) significantly exceeds that of its rna target ( [ rna]t ) , the basal level of rna bound by the nonphotolyzed cmo , given by ( [ cmoopenrna]/[rna]t ) , can be described as a function of [ cmo]t , khairpin , and the dissociation constant for the mo / rna duplex ( kdmorna ) ( eq 2 ) : upon photoactivation , the fraction of rna complexed with the released targeting mo is a function of [ cmo]t , kdmorna , and the dissociation constant for the mo / inhibitor complex ( kdmoinh ) ( eqs 3 and 4 ) : setting [ cmo]t to a value of 4.6 m , which approximates the embryonic concentration of the ntla cmo ( 230 fmol / embryo , 50 nl embryonic volume at 5 hpf ) , and using our ntla cmo 8e data to establish g values of 28.1 , 12.3 , and 6.9 kcal / mol for the mo / rna duplex , intermolecular mo / inhibitor duplex , and intramolecular mo / inhibitor hairpin , respectively , led to the prediction that essentially all of the ntla rna is mo - complexed before and after cmo photolysis . we first investigated whether the relationship between ntla rna activity and total ntla mo concentration can still be described as a two - state equilibrium , even though the apparent equilibrium constant for in vivo mo / rna interactions ( kappmorna , analogous to kdmorna in figure 6 ) would include contributions from oligonucleotide - binding proteins , rna secondary structure , and other embryonic factors . the ntla mo reduced the ntla protein levels in a dose - dependent manner that can be modeled as two - state thermodynamic interaction , with the fraction of wild - type rna activity remaining in mo - injected embryos ( rnamoact / rnawtact ) described as a function of total mo concentration ( [ mo]t ) and kappmorna ( figure 7b and eq 5 ) : through this analysis , an apparent free - energy value ( gappmorna ) of 8.7 kcal / mol for embryonic ntla mo / rna interactions was obtained . it is important to note that this gappmorna value does not reflect the actual binding constant for the ntla mo / rna duplex in zebrafish embryos but instead is an aggregate descriptor of mo / rna affinity , rna accessibility , mo / protein interactions , and the influence of other embryonic factors on mo efficacy . the fraction of wild - type rna activity associated with each mo and inhibitor dose , rnamo , inhact / rnawtact , can be expressed as a function of the apparent equilibrium constant for mo / inhibitor interactions ( kappmoinh ; analogous to kdmoinh in figure 6 ) , the total concentration of the inhibitory oligomer ( [ inh]t ) , and [ mo]t ( eqs 6 and 7 ) : through this analysis , we derived an apparent gappmoinh value of 7.3 kcal / mol for 8 g mo / inhibitor interactions . to assess the validity of modeling in vivo mo , rna , and inhibitor interactions in these simplified terms , we investigated whether the apparent gappmorna and gappmoinh values can be used to predict how cmo gene - silencing activity will be influenced by changes in inhibitor structure . if it is assumed that blunt mo / inhibitor interactions generally exhibit a 8.2 kcal / mol difference between gappmoinh and the corresponding in vitro g , the kappmoinh values for mo / inhibitor interactions associated with ntla cmos 8e , 8f , 8 g , and 8h can be estimated as 1100 , 140 , 50 , and 1.6 m , respectively . rna activity curves for mos and photoactivated cmos associated with in vivo mo / rna interaction energies of ( red ) 8.7 and ( blue ) 7.3 kcal / mol are shown for intermolecular mo / inhibitor interaction energies estimated for the blunt ntla cmos ( a ) 8e , ( b ) 8f , ( c ) 8 g , and ( d ) 8h , assuming complete uncaging upon irradiation photoactivated cmo and conventional mo activity profiles are drawn as solid and dashed colored lines , respectively . moreover , the predicted efficacies of 8 g and 8h are consistent with their photoinduced phenotypes and the relationship between ntla rna activity and phenotypic class ( compare figures 3b , 4 , 7a , b , and 8c , d ) , especially considering that uv light penetrance and therefore cmo uncaging efficiencies in vivo will be attenuated to some degree . on the basis of our in vitro uncaging results ( see figure s1 in the supporting information ) and the observed phenotypes for irradiated ntla cmo - injected embryos , we estimate that our whole - organism irradiation procedure achieves cmo photoactivation yields of 5075% . our findings suggest that cmos should have gappmoinh values of approximately 5 kcal / mol or greater for their corresponding intermolecular mo / inhibitor duplexes , as this interaction strength in vivo would allow efficient gene - silencing upon cmo photoactivation for a broad range of targeting mo potencies . maximizing the cmo activity in this manner , however , is counterbalanced by the need to maintain the closed cmo hairpin state prior to photoactivation . taken together , our results demonstrate that cmo activity can be modeled as a competitive three - state equilibrium , even though this approach does not explicitly consider how mo activity and rna accessibility are influenced by cellular proteins , rna structure , and other embryonic factors . such design criteria would significantly advance the use of cmos in chemical and developmental biology research , especially considering the financial and time investments associated with these studies . the disparate efficacies of the two stemloop structures indicate that blunt cmo hairpins are preferable to staggered configurations , and within our series of blunt ntla cmos 8eh , in vitro mo / inhibitor g values between 12 and 14 kcal / mol yield an optimum balance between caged and uncaged activities . our modeling of cmo activity in vivo further suggests that the higher mo / inhibitor g value associated with ntla cmo 8e should be preferred , since it would maximize the inducible gene - silencing activity over a broader range of targeting mo efficacies . to facilitate the design of thermodynamically equivalent cmos against other genes , we first determined the relationship between mo duplex sequence and thermal stability . to further test the predictive value of our cmo design criteria , we evaluated two additional heg cmos : one contained a staggered inhibitor ( 9b ; inhibitor sequence 5-gctgcaagtac-3 ) with an intermolecular mo / inhibitor g value comparable to that for 9a , and the other contained a shorter blunt inhibitor ( 9c ; inhibitor sequence 5-gtacgattac-3 ) with a weaker mo / inhibitor interaction strength ( tables 1 and 2 ) . to conclude our studies , we investigated the ability of our hairpin cmo design to incorporate other photocleavable groups . two - photon excitation typically uses wavelengths greater than 700 nm and affords greater spatial resolution , but the dmnb group and most other caging moieties have small two - photon cross sections and are therefore inefficiently cleaved under these conditions . since the low fluorescence of bhq chromophores makes them particularly useful for biological applications , we designed a bhq - based bifunctional cross - linker ( 13 , scheme 3 ) for the preparation of cmos . phenol deprotection and ester saponification of 21 were simultaneously accomplished with 0.2 m naoh to give an acid intermediate , which was re - esterified with n - hydroxysuccinimide in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to give the two - photon - sensitive linker 13 . since the bhq group has not been used previously with carbamates and other bhq - caged compounds have not been tested in cultured cells or live organisms , we first determined whether the bhq carbamate is stable in vivo and can be efficiently photolyzed . we then examined whether the bhq - based ntla cmo 22b could be activated in targeted regions of the zebrafish embryo using two - photon excitation . because kaede is inefficiently converted by two - photon excitation , we utilized a caged coumarin fluorophore conjugated to dextran ( dextranhcc - npe ) as a cell - autonomous photoactivatable tracer ( figure s5 in the supporting information ) . this new class of caged coumarins is highly sensitive to two - photon irradiation , and dextranhcc - npe has demonstrated efficacy in vivo. ( a ) schematic representation of embryos injected with the dextranhcc - npe photoactivatable tracer and then two - photon - irradiated within the posterior axial mesoderm at the 10 hpf stage ( red square , dorsal view ) . photoactivatable mos are valuable probes of embryonic patterning mechanisms , and their implementation by the developmental biology community will require efficient synthetic procedures and general principles for cmo design . to advance these goals , we have devised a bifunctional linker for the synthesis of hairpin cmos that enables these probes to be prepared in three steps from commercially available reagents . this optimized synthetic procedure has enabled us to prepare several ntla - targeting cmos of differing configurations , and we have used these structural variants to determine how the thermodynamic properties of cmos correlate with their in vivo functions . we further observed that mo / mo and mo / rna binding free energies in vitro differ dramatically from the apparent free energies of their functional interactions in vivo , undoubtedly reflecting the influence of oligonucleotide - binding proteins , rna secondary structure , and other embryonic factors . our findings establish a metric for cmo design , which we have successfully applied to cmos that target the flh , heg , and etv2 genes , and these empirically derived principles will help guide future applications of cmos in functional genomic studies . (22 ) although these oligonucleotide analogues interact with complementary oligomers with distinct in vitro and in vivo free energy values , their design and biological application are subject to the same principles outlined in this study . as illustrated by the partial phenotypes we observed with our spt cmo , however , the activity of caged hairpin oligonucleotides can be attenuated in vivo through mechanisms that are not yet understood . finally , the modularity of our hairpin cmos provides an opportunity for future improvements to this technology , as illustrated by our development of a bhq - based cmo that is compatible with two - photon excitation . investigating these possibilities through organic synthesis will expand the utility of cmo technologies and help shed new light on the molecular mechanisms of embryonic development . for two - photon experiments , solutions containing 1.25 mm dextranhcc - npe and 0.1% ( w / v ) phenol red with or without 57.5 m cmo 22b were injected at 2 nl / embryo . two - photon cmo photoactivation in zebrafish embryos was performed on an upright two - photon confocal microscope ( ultima xy , prairie technologies , inc . sample was prepared by adding the rna to the annealed mo duplex solution at 4 c , and the mo / rna duplex was prepared by heat denaturation and annealing , as was done for the mo / inhibitor duplexes .

toward that goal , several genetic approaches for conditional gene regulation have been developed , such as the flp / frt , cre / lox , and tet - on / tet - off systems , and these technologies have provided key insights into the molecular mechanisms that underlie tissue patterning and function. as a proof of principle , we first used this methodology to conditionally silence a t - box transcription factor called no tail - a ( ntla ) , which is required for the differentiation of axial mesodermal cells into a transient , chordate - specific organ called the notochord . by varying the developmental stage at which we activated the ntla cmo , we found that this transcription factor is required not only for specification of the mesoderm toward notochord cell fates but also for the maturation of notochord progenitors into a highly vacuolated tissue. (7 ) we also demonstrated our ability to silence ntla expression in a subset of mesodermal cells by activating the ntla cmo in a spatially restricted manner , selectively redirecting these populations to differentiate into medial floor plate cells . a similar caging approach has been applied to ncpnas targeting the chordin ( chd ) and dharma ( dha ) genes , although the use of these photoactivatable ncpna hairpins to spatially control gene expression has not been reported to date. to evaluate the in vivo efficacy of this reagent , we next injected 8a into wild - type zebrafish embryos at the one - cell stage and globally irradiated approximately half of the embryos with 360-nm light ( 13 mw / cm ) for 10 s at 3 h post fertilization ( hpf ) . as we have described previously , ntla loss - of - function phenotypes can be categorized into four classes according to their severity : class i = a fully penetrant ntla mutant phenotype characterized by no notochord , u - shaped somites , and a lack of posterior structures ; class ii = no notochord , u - shaped somites , and some posterior somites ; class iii = incompletely vacuolated notochord , v - shaped somites , and a shortened anteriorposterior axis ; and class iv = wild - type phenotype ( figure 3a). reasoning that the hairpin cmo efficacy depends on the interplay between inhibitor length , stemloop configuration , and linker formats , we used linker 2a to prepare ntla cmos with differing structures ( 8ah ; figure 4 and table 1 ) . the leftmost panels show schematic representations of staggered ( top ) and blunt ( bottom ) cmo configurations ( n = number of bases ) , and the other panels are distributions of phenotypes for each cmo configuration 8ah ( see table 1 and figure 3 ) at a dose of 230 fmol / embryo . to better understand how cmo structure dictates in vivo activity we first determined the binding energies for the ntla mo / rna duplex , each ntla mo / inhibitor heterodimer , and various stemloop structures . thermal denaturation curves for the ntla mo / rna duplex and ntla mo / inhibitor heterodimers were acquired by mixing the ntla - targeting mo with the complementary oligomers in a 1:1 ratio and measuring their temperature - dependent changes in hypochromicity at 260 nm ( figure 5 and tables 13 ) . since the 260-nm absorbance measurements would photolyze the ntla cmos ( 8ah ) , we synthesized two ntla mo / inhibitor hairpins ( 8a and 8e ) ( scheme 2 ) using the bifunctional but nonphotolabile cross - linker 2b , which was prepared from 3-(methylamino)propan-1-ol ( 3b ) in analogy to the dmnb - based reagent 2a ( scheme 2 ) . taken together , intermolecular ntla mo / inhibitor interactions with g values lower than 12 kcal / mol are required for low basal activities , and hairpins exhibit higher caging efficiencies than their staggered counterparts because of their greater stabilization of the intramolecular mo / inhibitor duplex ( table 2 and figure 4 ) . in addition , the photolysis products of ntla cmos 8a and 8e are functionalized with linker substituents that are not present in the oligomers used in our binding energy measurements ; the targeting mo liberated upon cmo activation is 3-functionalized with a 1,2,3-triazole and an aliphatic amine , while the inhibitory oligomer is 5-functionalized with an aliphatic chain and the dmnb - derived chromophore . analysis of the photolysis products by hplc confirmed that the two hairpin oligonucleotides are uncaged with comparable efficacies ( 75% conversion ) , and the g values for the resulting linker - functionalized ntla mo / inhibitor complexes are similar to those observed for their amine - functionalized counterparts ( figure s1 and table s1 in the supporting information ) . thus , the activity differences between 8a and 8e can not be explained by in vitro mo / inhibitor thermodynamics or kinetics alone , and the two cmos might exhibit divergent photolysis , inhibitor dissociation , or rna exchange rates in vivo . prior to photolysis , cmos adopt open and closed states according to the equilibrium constant khairpin ( eq 1 ) : with the assumption that the total concentration of cmo prior to uncaging ( [ cmo]t ) significantly exceeds that of its rna target ( [ rna]t ) , the basal level of rna bound by the nonphotolyzed cmo , given by ( [ cmoopenrna]/[rna]t ) , can be described as a function of [ cmo]t , khairpin , and the dissociation constant for the mo / rna duplex ( kdmorna ) ( eq 2 ) : upon photoactivation , the fraction of rna complexed with the released targeting mo is a function of [ cmo]t , kdmorna , and the dissociation constant for the mo / inhibitor complex ( kdmoinh ) ( eqs 3 and 4 ) : setting [ cmo]t to a value of 4.6 m , which approximates the embryonic concentration of the ntla cmo ( 230 fmol / embryo , 50 nl embryonic volume at 5 hpf ) , and using our ntla cmo 8e data to establish g values of 28.1 , 12.3 , and 6.9 kcal / mol for the mo / rna duplex , intermolecular mo / inhibitor duplex , and intramolecular mo / inhibitor hairpin , respectively , led to the prediction that essentially all of the ntla rna is mo - complexed before and after cmo photolysis . we first investigated whether the relationship between ntla rna activity and total ntla mo concentration can still be described as a two - state equilibrium , even though the apparent equilibrium constant for in vivo mo / rna interactions ( kappmorna , analogous to kdmorna in figure 6 ) would include contributions from oligonucleotide - binding proteins , rna secondary structure , and other embryonic factors . we injected the targeting mo into one - cell - stage zebrafish at doses of 0 , 14 , 28 , 57 , and 115 fmol / embryo ( approximate final concentrations of 0 , 0.28 , 0.56 , 1.1 , and 2.3 m , respectively ) , lysed the embryos at 10 hpf , and then detected the ntla protein by quantitative immunoblotting ( figure 7a ) . the ntla mo reduced the ntla protein levels in a dose - dependent manner that can be modeled as two - state thermodynamic interaction , with the fraction of wild - type rna activity remaining in mo - injected embryos ( rnamoact / rnawtact ) described as a function of total mo concentration ( [ mo]t ) and kappmorna ( figure 7b and eq 5 ) : through this analysis , an apparent free - energy value ( gappmorna ) of 8.7 kcal / mol for embryonic ntla mo / rna interactions was obtained . ( d ) modeling of the 8 g data in ( c ) as a three - state , competitive equilibrium ( solid line ) yields an apparent free - energy value ( gappmoinh = 7.3 kcal / mol ) that describes 8 g mo / inhibitor interactions in vivo . we injected zebrafish embryos with the ntla mo ( 115 fmol / embryo ; 2.3 m ) and various doses of the 14-base inhibitor corresponding to ntla cmo 8 g ( 0 , 150 , 450 , and 1350 fmol / embryo ; 0 , 3 , 9 , and 27 m , respectively ) . the fraction of wild - type rna activity associated with each mo and inhibitor dose , rnamo , inhact / rnawtact , can be expressed as a function of the apparent equilibrium constant for mo / inhibitor interactions ( kappmoinh ; analogous to kdmoinh in figure 6 ) , the total concentration of the inhibitory oligomer ( [ inh]t ) , and [ mo]t ( eqs 6 and 7 ) : through this analysis , we derived an apparent gappmoinh value of 7.3 kcal / mol for 8 g mo / inhibitor interactions . as with the gappmorna value we determined for mo - dependent ntla silencing , this apparent free - energy value does not reflect the actual binding constant for the ntla mo / inhibitor duplex in vivo but instead integrates other interactions between these synthetic oligonucleotides and cellular components . since the 8.2 kcal / mol difference between gappmoinh and the corresponding in vitro g value is significantly smaller than the 19.4 kcal / mol difference we observed for mo / rna interactions ( see table 3 and figure 7b ) , cellular factors appear to impact rna activity to a greater extent than mo function . to assess the validity of modeling in vivo mo , rna , and inhibitor interactions in these simplified terms , we investigated whether the apparent gappmorna and gappmoinh values can be used to predict how cmo gene - silencing activity will be influenced by changes in inhibitor structure . in the case of a cmo , [ mo]t and [ inh]t will be equal after photoactivation , and the fraction of wild - type rna activity remaining in the presence of photoactivated cmo ( rnacmoact / rnawtact ) is therefore a function of kappmorna , kappmoinh , and [ cmo]t ( eqs 8 and 9 ) : in the case of our ntla cmo experiments shown in figure 4 , kappmorna and [ cmo]t can be approximated as 0.48 m ( gappmorna = 8.7 kcal / mol ) and 4.6 m , respectively . if it is assumed that blunt mo / inhibitor interactions generally exhibit a 8.2 kcal / mol difference between gappmoinh and the corresponding in vitro g , the kappmoinh values for mo / inhibitor interactions associated with ntla cmos 8e , 8f , 8 g , and 8h can be estimated as 1100 , 140 , 50 , and 1.6 m , respectively . using these parameters in the model described by eqs 8 and 9 leads to the prediction that ntla cmos 8e and 8f will be similar in efficacy to the conventional ntla mo , achieving at least a 90% knockdown of ntla protein expression levels ( red lines in figure 8a , b ) . rna activity curves for mos and photoactivated cmos associated with in vivo mo / rna interaction energies of ( red ) 8.7 and ( blue ) 7.3 kcal / mol are shown for intermolecular mo / inhibitor interaction energies estimated for the blunt ntla cmos ( a ) 8e , ( b ) 8f , ( c ) 8 g , and ( d ) 8h , assuming complete uncaging upon irradiation photoactivated cmo and conventional mo activity profiles are drawn as solid and dashed colored lines , respectively . moreover , the predicted efficacies of 8 g and 8h are consistent with their photoinduced phenotypes and the relationship between ntla rna activity and phenotypic class ( compare figures 3b , 4 , 7a , b , and 8c , d ) , especially considering that uv light penetrance and therefore cmo uncaging efficiencies in vivo will be attenuated to some degree . on the basis of our in vitro uncaging results ( see figure s1 in the supporting information ) and the observed phenotypes for irradiated ntla cmo - injected embryos , we estimate that our whole - organism irradiation procedure achieves cmo photoactivation yields of 5075% . our model also predicts that cmo efficacy will be increasingly compromised as gappmorna increases ( compare the blue and red lines in figure 8 , which represent a 10-fold change in mo / rna interaction strength ) , since the concentration of photoactivated cmo required to achieve a given level of rna silencing increases in a manner disproportionate to that of a conventional mo . rather , the binding free energies for the intra- and intermolecular mo / inhibitor duplexes dictate the fraction of targeting mo that is free to anneal to its rna target , thereby establishing the basal and photoinduced activities of a given cmo . taken together , our results demonstrate that cmo activity can be modeled as a competitive three - state equilibrium , even though this approach does not explicitly consider how mo activity and rna accessibility are influenced by cellular proteins , rna structure , and other embryonic factors . the disparate efficacies of the two stemloop structures indicate that blunt cmo hairpins are preferable to staggered configurations , and within our series of blunt ntla cmos 8eh , in vitro mo / inhibitor g values between 12 and 14 kcal / mol yield an optimum balance between caged and uncaged activities . our modeling of cmo activity in vivo further suggests that the higher mo / inhibitor g value associated with ntla cmo 8e should be preferred , since it would maximize the inducible gene - silencing activity over a broader range of targeting mo efficacies . by multiple - regression analysis of the ntla mo / inhibitor pairs listed in table 2 , we determined that the melting temperature of mo duplexes ( tmmo , in c ) is correlated with sequence content according to eq 10 and can be empirically related to its in vitro g value ( in kcal / mol at 28 c ) by eq 11 : in eq 10 , each nj ( j = a , t , g , c ) is the number of times the corresponding base appears in the mo sequence . to further test the predictive value of our cmo design criteria , we evaluated two additional heg cmos : one contained a staggered inhibitor ( 9b ; inhibitor sequence 5-gctgcaagtac-3 ) with an intermolecular mo / inhibitor g value comparable to that for 9a , and the other contained a shorter blunt inhibitor ( 9c ; inhibitor sequence 5-gtacgattac-3 ) with a weaker mo / inhibitor interaction strength ( tables 1 and 2 ) . the majority of zebrafish embryos injected with an equivalent dose of either 9b or 9c exhibited heg mutant phenotypes in the absence of irradiation ( 9b : 66% , n = 53 ; 9c : 62% , n = 45 ) , indicating that these structural configurations do not adequately cage the mo activity ( figure s3 ) . a loss of spt function therefore causes a severe deficit in trunk somitic mesoderm as well as a gross mislocalization of the corresponding progenitor cells to posterior regions ( hence the spadetail these phenotypes can be recapitulated with a spt - targeting mo 12 ( 5-gcttgaggtctctgatagcctgcat-3)(34 ) at doses of 345 fmol / embryo ( 6.9 m ) or higher ( 61% , n = 23 ) ( figure 9 g ) . zebrafish injected with a dose of 700 fmol / embryo ( 14 m ) exhibited a loss of trunk mesoderm but not posteriorly mislocalized progenitor cells upon cmo photoactivation ( 31% , n = 16 ) and also showed nonspecific developmental defects due to general mo toxicity ( 62% , n = 16 ) ( figure 9h , i ) . reagents and conditions : ( a ) benzenesulfonyl chloride , dipea , ch2cl2 , 97% ; ( b ) seo2 , dioxane , 80 c , 91% ; ( c ) in powder , allyl bromide , nh4cl(aq ) , thf , 96% ; ( d ) k2oso42h2o , lutidine , dioxane , h2o , then naio4 , 75% ; ( e ) methylamine(aq ) , nabh(oac)3 , meoh , h2o , 81% ; ( f ) methyl adipoyl chloride , dipea , ch2cl2 , 59% ; ( g ) carbonyldiimidazole , dmf then 6-amino - n-(prop-2-ynyl)hexanamide , dipea , dmf , 81% ; ( h ) 0.2 m naoh(aq ) , thf , meoh , 82% ; ( i ) n - hydroxysuccinimide , edci , dmf , 48% . phenol deprotection and ester saponification of 21 were simultaneously accomplished with 0.2 m naoh to give an acid intermediate , which was re - esterified with n - hydroxysuccinimide in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to give the two - photon - sensitive linker 13 . as we hoped , the bhq - based ntla cmo exhibited activity essentially identical to that of the dmnb - based reagent 8e ; embryos injected with this reagent developed normally when cultured in the dark but displayed a ntla phenotype upon irradiation ( figure s4 in the supporting information ) . these experiments followed studies conducted previously in our laboratory , in which we uncaged a dmnb - based ntla cmo in a subset of mesodermal progenitor cells , selectively inducing them to differentiate into medial floor plate cells rather than the notochord. (7 ) to identify the irradiated cells in this earlier investigation , we coinjected the zebrafish embryos with mrna encoding kaede fluorescent protein , which undergoes a green - to - red photoconversion upon one - photon excitation . we further observed that mo / mo and mo / rna binding free energies in vitro differ dramatically from the apparent free energies of their functional interactions in vivo , undoubtedly reflecting the influence of oligonucleotide - binding proteins , rna secondary structure , and other embryonic factors .

the effects of smoking habits of mother during pregnancy have been studied in the past century or so in various communities and different cultures . one of the earliest studies ( 1 ) in birmingham , uk , reported in 1959 shows that infants of mothers who smoked regularly during pregnancy were on the average more than 170 g lighter than the infants of those who never smoked during pregnancy . despite the association of the children s health with the smoking habits of mother during pregnancy have been investigated for many years and well - documented , the topic is still being studied by various research groups . ( 2 ) estimated that about 5% of infant deaths in the united states are attributable to maternal smoking while pregnant , with variations by race / ethnicity . a recently published study ( 3 ) reported that an experimental citywide smoking ban in pueblo , colorado , usa , improved maternal and fetal outcomes . ( 4 ) discussed not only the relationship of smoking habits of mothers and fetal risks , but the importance of negative impact of smoking on the health of mothers as well . they suggested that smokers have a higher chance of experiencing cardiovascular problems ( deep vein thrombosis , myocardial infarction , and stroke ) and pulmonary disorders ( bronchitis , asthma , pneumonia , influenza ) than non - smokers . in the united states , mateja et al . ( 5 ) investigated the data from the pregnancy risk assessment monitoring survey ( prams ) for nine states over a 10-year period ( 19962005 ) , and revealed the risk factor of maternal alcohol use and smoking habits , in early pregnancy , and congenital heart defects . ( 6 ) also suggested the importance of preconception prevention efforts for women who are at dual risk for alcohol and smoking habits . ( 7 ) investigated the healthy lifestyle behaviors during pregnancy , from 2001 to 2009 , in a survey from 22,604 american pregnant women aged 1844 years , and pointed out the importance of reducing alcohol use , binge drinking , and smoking and increasing fruit and vegetable consumption during pregnancy . this paper describes the association of maternal smoking habits with stillbirths , abortions , neonatal deaths , birth weights , placental weights and the outcomes on the 28 day of life . during 2001 and 2002 , the first author had a fellowship at the division of clinical and metabolic genetics , hospital for sick children , toronto , canada , in the late prof . a questionnaire was developed and completed with the hospitals recorded data which have been collected over a period of 5 years from about 47,000 babies born in several hospitals in ontario . the designed questionnaire covered a comprehensive range of information on both mothers and their babies health , which were and will be the source for a series of other articles . the babies , whose records had missing data on any of the maternal or infant variables under study , were excluded from the analyses . for the purpose of this study , the mothers were classified into four categories : non - smokers , light smokers ( less than 10 cigarettes per day ) , moderate smokers ( between 10 and 19 cigarettes per day ) and heavy smokers ( 20 or more cigarettes per day ) . other groups such as sportive smoking , ex - smokers and mothers who have smoked before pregnancy and ceased during pregnancy were out of the scope of present study . throughout this study statistical analysis was performed using the spss statistical package , version 15.0 for windows ( spss , chicago , usa ) and the chi - square test was used to estimate the probable association between the variables , and a p value less than 0.05 was considered statistically significant for all the tables . the odds ratios ( or ) the present analysis is a cohort study where the maternal smokers during pregnancy are compared with the maternal non - smokers during pregnancy . during 2001 and 2002 , the first author had a fellowship at the division of clinical and metabolic genetics , hospital for sick children , toronto , canada , in the late prof . a questionnaire was developed and completed with the hospitals recorded data which have been collected over a period of 5 years from about 47,000 babies born in several hospitals in ontario . the designed questionnaire covered a comprehensive range of information on both mothers and their babies health , which were and will be the source for a series of other articles . the babies , whose records had missing data on any of the maternal or infant variables under study , were excluded from the analyses . for the purpose of this study , the mothers were classified into four categories : non - smokers , light smokers ( less than 10 cigarettes per day ) , moderate smokers ( between 10 and 19 cigarettes per day ) and heavy smokers ( 20 or more cigarettes per day ) . other groups such as sportive smoking , ex - smokers and mothers who have smoked before pregnancy and ceased during pregnancy were out of the scope of present study . throughout this study statistical analysis was performed using the spss statistical package , version 15.0 for windows ( spss , chicago , usa ) and the chi - square test was used to estimate the probable association between the variables , and a p value less than 0.05 was considered statistically significant for all the tables . the odds ratios ( or ) the present analysis is a cohort study where the maternal smokers during pregnancy are compared with the maternal non - smokers during pregnancy . previous stillbirths were consisted of three subgroups of no previous stillbirths , 1 previous stillbirth and 2 or more previous stillbirths . previous abortions were divided into four subgroups of no previous abortions , 1 previous abortion , 2 previous abortions and 3 or more previous abortions . table 1 shows the incidence of previous stillbirths and previous abortions in regard to smoking habits . it was found , that mothers who smoke have significantly more previous stillbirths ( = 60.98 , p < 0.0001 ) and also previous abortions ( = 290.77 , p < 0.0001 ) than mothers who do not smoke . our data shows that there is no significant difference between non - smokers and light smokers with regard to the number of previous stillbirths ( = 2.38 , p > 0.30 ) and also to the number of previous abortions ( = 8.51 , p > 0.036 ) . the frequency of previous neonatal deaths in relation to smoking habits is given in table 2 . in this study neonatal deaths were scored as being absent or 1 , 2 or more . early neonatal mortality refers to a death of a live - born baby within the first 7 days of life . late neonatal mortality refers to a death of a live - born baby after 7 days until before 28 days of life . our results revealed that the proportion of previous neonatal deaths was significantly higher amongst smoker mothers than non - smokers ( = 45.02 , p < 0.0001 ) . the results show that there is no significant difference between no - smokers and light smokers in regard to previous neonatal deaths ( = 1.22 , p > 0.50 ) . birth weight was consisted of six subgroups of 2.5 kg or less , ( 2.6 2.9 ) kg , ( 3.0 3.3 ) kg , ( 3.4 3.7 ) kg , ( 3.8 4.1 ) kg , and 4.2 kg or more . the data from the present study suggest that mothers who smoke have lighter babies than mothers who do not smoke . the differences were statistically highly significant ( = 1689.11 , p < 0.0001 ) . placental weight was divided into four subgroups of 400 g or less , ( 400 599 ) g , ( 600 799 ) g , and 800 g or more . the pattern of results is similar to that found for birth weight results and indicates that those babies born to smoking mothers have significantly lighter placental weight than those born to non - smokers ( = 145.20 , p < 0.0001 ) . the result of smoking habits and outcome with respect to mortality up to 28th days after birth is given in table 5 . this outcome mortality consisted of the perinatal deaths plus the deaths after 7 days until 28th days after birth . it was found that babies born to heavy smokers have the highest mortality rate , and the rate , gradually increases as smoking increases . our results show that there is no significant difference between non - smokers and light smokers ( = 0.01 , p > 0.90 ) and also no significant difference between moderate and heavy smokers ( = 0.15 , p > 0.60 ) . kllen ( 8) reported that maternal smoking during pregnancy is associated with a number of effects such as pre - term birth , intrauterine growth retardation , a small head circumference , and stillbirths and neonatal deaths . the smoking habits of the mothers were recorded in three categories : non - smokers , < 10 cigarettes / day and 10 cigarettes / day . the results showed the stillbirths risk estimates are 0.42% and 0.32% for smokers and non - smokers , respectively . hgberg and cnattingius ( 9 ) reported that maternal smoking during pregnancy is causally associated with stillbirth risk . they showed that compared with nonsmokers , women who smoked during the first pregnancy but not during the second do not have an increased risk of stillbirth ( or 1.02 ; 95% ci 0.791.30 ) , while corresponding risk among women who smoked during both pregnancies was 1.35 ( 95% ci 1.151.58 ) . gray et al . ( 10 ) studied the contribution of smoking during pregnancy to inequalities in stillbirth in scotland from 1994 to 2003 and reported that smoking during pregnancy accounted for 38% of the inequality in stillbirths . it was found that ( 1 ) . there is no significant difference between moderate and heavy smoking mothers with respect to previous stillbirths where the odds ratios vary from 1.20 ( 95% ci : 0.64 2.25 ) to 1.42 ( 95% ci : 0.69 2.94 ) for 2 or more previous stillbirths , and ( 2 ) there was no significant difference between non - smokers and light smokers ( < 10 cigs / day ) with any previous stillbirths.( = 2.38 , p > 0.30 ) in a study ( 11 ) conducted in montreal , quebec , canada , the researchers interviewed approximately 56,000 women who had had a delivery or a spontaneous abortion in 11 montreal hospitals during the two - year period 19821984.the researchers found that 22% of the women had had previous pregnancies that ended in spontaneous abortion . compared with women who abstained during pregnancy , women who smoked cigarettes had a 20% increase in spontaneous abortion ( odds ratio of 1.20 ) for each 10 cigarettes smoked per day . ( 12 ) shows that they found no association between maternal smoking and the risk of spontaneous abortion . nielsen et al . ( 13 ) has also reported that smoking status was only weakly related to spontaneous abortion after adjustment for maternal age . the results of the present study show that the previous abortion is a significant factor for women who smoke 20 or more cigarettes per day . the odds ratios for the previous number of abortions of 1 , 2 and 3 are 1.54 ( 95% ci , 1.42 1.67 ) , 1.97 ( 95% ci , 1.70 2.28 ) and 2.86 ( 95% ci , 2.30 3.55 ) , respectively . no significant difference was observed between the light smokers ( < 10 cigs / day ) and non - smokers ( = 8.51 , p > 0.036 ) . one of the early studies on smoking and pregnancy ( 14 ) reports the prospective survey of almost 7000 women from 11 paris hospitals between 1963 and 1969 . the results show while smokers had a stillbirth rate more than three times as high ( 28 versus 8 per 1000 births ) they found that the neonatal death rates were almost the same for smokers and nonsmokers . in a study ( 15 ) conducted in sweden between 1983 and 1985 , significant relative risks for early neonatal mortality were found for multiple births ( 4.9 ) and smoking ( 1.2 ) . they concluded that maternal cigarette smoking may be the most important preventable risk factor for late fetal death . the study ( 16 ) based on the number of stillbirths or neonatal deaths among infants born in sweden from 1983 to 1995 showed that maternal smoking seems to aggravate the placental abruption because the death risk in the presence of abruption increases when mother smoked ( or = 1.74 , 95% ci 1.45 2.08 ) . the present results show that there is no significant difference between non - smokers and light smokers with < 10 cigarettes per day ( = 1.22 , p > 0.50 ) . for moderate and heavy smokers the odds ratios are up to or = 1.84 , 95% ci 1.05 3.23 . in a study reported by dsouza ( 17 ) a total of 452 mothers who attended antenatal clinics regularly at st mary s hospital , manchester , uk , were selected with a normal singleton pregnancy . at each visit the mothers were asked about the number of cigarettes smoked per day , and they were grouped as follows : ( a ) non - smokers , ( b ) light - to - moderate smokers , ( 114 cigarettes / day ) , and ( c ) heavy smokers ( 15 or more cigarettes/ day ) . in both sexes babies born to non - smokers were heavier , longer , and had larger head circumferences than those born to heavy smokers . smoking during pregnancy appears to have caused a general retardation in intrauterine growth , resulting in babies born with lower birth weights , shorter lengths , and smaller head circumferences . dickute ( 18 ) reported a case - control study involved 851 newborns with low birth weight ( < 2500 g ) and 851 normal weight newborns . the study started 1 february 2001 and ended 31 october 2002 in six main maternity hospitals in lithuania . the results showed that smoking during pregnancy in combination with the socioeconomic inequalities is significantly associated with the higher risk of low birth weight . the association of socioeconomic status with heath and birth weight has been discussed by some authors ( 1922 ) . in a study in illinois , usa , keeton et al . ( 23 ) suggested that the family case management program may be effective in reducing very low birth weight and low birth weight rates among infants born to low - income women . ( 24 ) reported the prenatal smoking status of west virginia , usa , women and the associated changes in infant birth weights . a population - based secondary data analysis was conducted for all singleton infant siblings born between 1989 and 2006 . they found that infants born to women who smoked during pregnancy had significantly lower birth weights than infants born to non - smokers . the results showed that maternal prenatal smoking was the strongest predictor of low birth weight ( < 2500 g ) with an odds ratio of 3.29 ( 95% ci 2.87 , 3.77 ) for smoking during the recent pregnancy . the odds ratio is significantly reduced from the birth weight of < 2.5 kg to = > 4.2 kg for smoking mothers . the variation of odds ratio is from 0.95 ( 95% ci 0.85 1.06 ) to 0.18 ( 95% ci 0.14 0.23 ) . jones et al . ( 25 ) reported a study with the objective of determining whether maternal smoking during pregnancy is associated with bone mass and other growth variables in pre - pubertal children in tasmania , australia . they showed that the mothers who smoked during pregnancy had lower placental weight ( 56 g , 95% ci 95 to 17 ) . ( 26 ) reported the study conducted in the county of north jutland , denmark . they examined the association between mothers smoking habits during pregnancy , taking the sex of the offspring into consideration , and the risk and prognosis of placental abruption , placenta previa , and preeclampsia . the results showed that smoking was associated with the risk of placental abruption ( or=1.99 , 95% ci 1.722.30 ) and placenta previa ( or=1.88 , 95% ci 1.153.07 ) . the effect of maternal smoking on placental volume was studied ( 27 ) in 80 pregnancies categorized according to cigarette consumption : group a never smoked , b smoking < 10 cigarettes / day , c smoking 1020 cigarettes / day , and d smoking > 20 cigarettes / day . the three - dimensional power doppler ultrasonography of the placenta was performed in this study and the results showed no differences in placental volume among different groups . the odds ratios for heavy smokers ( > 20 cigs / day ) are 0.81 ( 95% ci : 0.69 0.95 ) , 0.67 ( 95% ci : 0.57 0.79 ) and 0.59 ( 95% ci : 0.49 0.71 ) for the placental weight groups of 400 599 g , 600 799 g and = > 800 g , respectively . the perinatal mortality ( pnm ) has been defined ( 28 ) as the total number of stillbirths and deaths in the first week of life ( early neonatal deaths ) . the sudden infant death syndrome ( sids ) happens usually with no known illness and typically affecting sleeping infants between the ages of two weeks to six months . schoendorf and kiely ( 29 ) reported the results of a case - control analysis performed on data from the 1998 us national maternal and infant health survey ( nmihs ) . they showed that sudden infant death syndrome ( sids ) is associated with maternal smoking during pregnancy . ( 30 ) have shown that maternal smoking during pregnancy remains the most important risk factor for sids in sweden . in the present study we defined the outcome of pregnancy as whether the infant was alive on the 28 day of life . we considered the total number of infants deaths as the sum of stillbirths and neonatal deaths ( early neonatal : within the first 7 days of life and late neonatal : after 7 days until 28 day ) . the outcomes on the 28 day were 45923 ( 97.66% ) lives and 1100 ( 2.34% ) deaths . the odds ratios were 1.01 ( 95% ci 0.82 1.24 ) , 1.43 ( 95% ci 1.23 1.66 ) and 1.49 ( 95% ci 1.24 1.79 ) for light smokers ( < 10 cigs / day ) , moderate smokers ( 10 19 cigs / day ) and heavy smokers ( 20 cigs / day ) , respectively . it was found , firstly , that babies born to moderate and heavy smokers have the highest mortality rate . secondly , there were no significant differences between non - smokers and light smokers ( = 0.01 , p > 0.90 ) or between moderate and heavy smokers ( = 0.15 , p > 0.60 ) . kllen ( 8) reported that maternal smoking during pregnancy is associated with a number of effects such as pre - term birth , intrauterine growth retardation , a small head circumference , and stillbirths and neonatal deaths . the smoking habits of the mothers were recorded in three categories : non - smokers , < 10 cigarettes / day and 10 cigarettes / day . the results showed the stillbirths risk estimates are 0.42% and 0.32% for smokers and non - smokers , respectively . hgberg and cnattingius ( 9 ) reported that maternal smoking during pregnancy is causally associated with stillbirth risk . they showed that compared with nonsmokers , women who smoked during the first pregnancy but not during the second do not have an increased risk of stillbirth ( or 1.02 ; 95% ci 0.791.30 ) , while corresponding risk among women who smoked during both pregnancies was 1.35 ( 95% ci 1.151.58 ) . gray et al . ( 10 ) studied the contribution of smoking during pregnancy to inequalities in stillbirth in scotland from 1994 to 2003 and reported that smoking during pregnancy accounted for 38% of the inequality in stillbirths . it was found that ( 1 ) . there is no significant difference between moderate and heavy smoking mothers with respect to previous stillbirths where the odds ratios vary from 1.20 ( 95% ci : 0.64 2.25 ) to 1.42 ( 95% ci : 0.69 2.94 ) for 2 or more previous stillbirths , and ( 2 ) there was no significant difference between non - smokers and light smokers ( < 10 cigs / day ) with any previous stillbirths.( = 2.38 , p > 0.30 ) in a study ( 11 ) conducted in montreal , quebec , canada , the researchers interviewed approximately 56,000 women who had had a delivery or a spontaneous abortion in 11 montreal hospitals during the two - year period 19821984.the researchers found that 22% of the women had had previous pregnancies that ended in spontaneous abortion . compared with women who abstained during pregnancy , women who smoked cigarettes had a 20% increase in spontaneous abortion ( odds ratio of 1.20 ) for each 10 cigarettes smoked per day . ( 12 ) shows that they found no association between maternal smoking and the risk of spontaneous abortion . nielsen et al . ( 13 ) has also reported that smoking status was only weakly related to spontaneous abortion after adjustment for maternal age . the results of the present study show that the previous abortion is a significant factor for women who smoke 20 or more cigarettes per day . the odds ratios for the previous number of abortions of 1 , 2 and 3 are 1.54 ( 95% ci , 1.42 1.67 ) , 1.97 ( 95% ci , 1.70 2.28 ) and 2.86 ( 95% ci , 2.30 3.55 ) , respectively . no significant difference was observed between the light smokers ( < 10 cigs / day ) and non - smokers ( = 8.51 , p > 0.036 ) . one of the early studies on smoking and pregnancy ( 14 ) reports the prospective survey of almost 7000 women from 11 paris hospitals between 1963 and 1969 . the results show while smokers had a stillbirth rate more than three times as high ( 28 versus 8 per 1000 births ) they found that the neonatal death rates were almost the same for smokers and nonsmokers . in a study ( 15 ) conducted in sweden between 1983 and 1985 , significant relative risks for early neonatal mortality were found for multiple births ( 4.9 ) and smoking ( 1.2 ) . they concluded that maternal cigarette smoking may be the most important preventable risk factor for late fetal death . the study ( 16 ) based on the number of stillbirths or neonatal deaths among infants born in sweden from 1983 to 1995 showed that maternal smoking seems to aggravate the placental abruption because the death risk in the presence of abruption increases when mother smoked ( or = 1.74 , 95% ci 1.45 2.08 ) . the present results show that there is no significant difference between non - smokers and light smokers with < 10 cigarettes per day ( = 1.22 , p > 0.50 ) . for moderate and heavy smokers the odds ratios are up to or = 1.84 , 95% ci 1.05 3.23 . in a study reported by dsouza et al . ( 17 ) a total of 452 mothers who attended antenatal clinics regularly at st mary s hospital , manchester , uk , were selected with a normal singleton pregnancy . at each visit the mothers were asked about the number of cigarettes smoked per day , and they were grouped as follows : ( a ) non - smokers , ( b ) light - to - moderate smokers , ( 114 cigarettes / day ) , and ( c ) heavy smokers ( 15 or more cigarettes/ day ) . in both sexes babies born to non - smokers were heavier , longer , and had larger head circumferences than those born to heavy smokers . smoking during pregnancy appears to have caused a general retardation in intrauterine growth , resulting in babies born with lower birth weights , shorter lengths , and smaller head circumferences . dickute ( 18 ) reported a case - control study involved 851 newborns with low birth weight ( < 2500 g ) and 851 normal weight newborns . the study started 1 february 2001 and ended 31 october 2002 in six main maternity hospitals in lithuania . the results showed that smoking during pregnancy in combination with the socioeconomic inequalities is significantly associated with the higher risk of low birth weight . the association of socioeconomic status with heath and birth weight has been discussed by some authors ( 1922 ) . in a study in illinois , usa , keeton et al . ( 23 ) suggested that the family case management program may be effective in reducing very low birth weight and low birth weight rates among infants born to low - income women . ( 24 ) reported the prenatal smoking status of west virginia , usa , women and the associated changes in infant birth weights . a population - based secondary data analysis was conducted for all singleton infant siblings born between 1989 and 2006 . they found that infants born to women who smoked during pregnancy had significantly lower birth weights than infants born to non - smokers . the results showed that maternal prenatal smoking was the strongest predictor of low birth weight ( < 2500 g ) with an odds ratio of 3.29 ( 95% ci 2.87 , 3.77 ) for smoking during the recent pregnancy . the odds ratio is significantly reduced from the birth weight of < 2.5 kg to = > 4.2 kg for smoking mothers . the variation of odds ratio is from 0.95 ( 95% ci 0.85 1.06 ) to 0.18 ( 95% ci 0.14 0.23 ) . jones et al . ( 25 ) reported a study with the objective of determining whether maternal smoking during pregnancy is associated with bone mass and other growth variables in pre - pubertal children in tasmania , australia . they showed that the mothers who smoked during pregnancy had lower placental weight ( 56 g , 95% ci 95 to 17 ) . ( 26 ) reported the study conducted in the county of north jutland , denmark . they examined the association between mothers smoking habits during pregnancy , taking the sex of the offspring into consideration , and the risk and prognosis of placental abruption , placenta previa , and preeclampsia . the results showed that smoking was associated with the risk of placental abruption ( or=1.99 , 95% ci 1.722.30 ) and placenta previa ( or=1.88 , 95% ci 1.153.07 ) . the effect of maternal smoking on placental volume was studied ( 27 ) in 80 pregnancies categorized according to cigarette consumption : group a never smoked , b smoking < 10 cigarettes / day , c smoking 1020 cigarettes / day , and d smoking > 20 cigarettes / day . the three - dimensional power doppler ultrasonography of the placenta was performed in this study and the results showed no differences in placental volume among different groups . the odds ratios for heavy smokers ( > 20 cigs / day ) are 0.81 ( 95% ci : 0.69 0.95 ) , 0.67 ( 95% ci : 0.57 0.79 ) and 0.59 ( 95% ci : 0.49 0.71 ) for the placental weight groups of 400 599 g , 600 799 g and = > 800 g , respectively . the perinatal mortality ( pnm ) has been defined ( 28 ) as the total number of stillbirths and deaths in the first week of life ( early neonatal deaths ) . the sudden infant death syndrome ( sids ) happens usually with no known illness and typically affecting sleeping infants between the ages of two weeks to six months . schoendorf and kiely ( 29 ) reported the results of a case - control analysis performed on data from the 1998 us national maternal and infant health survey ( nmihs ) . they showed that sudden infant death syndrome ( sids ) is associated with maternal smoking during pregnancy . ( 30 ) have shown that maternal smoking during pregnancy remains the most important risk factor for sids in sweden . in the present study we defined the outcome of pregnancy as whether the infant was alive on the 28 day of life . we considered the total number of infants deaths as the sum of stillbirths and neonatal deaths ( early neonatal : within the first 7 days of life and late neonatal : after 7 days until 28 day ) . the outcomes on the 28 day were 45923 ( 97.66% ) lives and 1100 ( 2.34% ) deaths . the odds ratios were 1.01 ( 95% ci 0.82 1.24 ) , 1.43 ( 95% ci 1.23 1.66 ) and 1.49 ( 95% ci 1.24 1.79 ) for light smokers ( < 10 cigs / day ) , moderate smokers ( 10 19 cigs / day ) and heavy smokers ( 20 cigs / day ) , respectively . it was found , firstly , that babies born to moderate and heavy smokers have the highest mortality rate . secondly , there were no significant differences between non - smokers and light smokers ( = 0.01 , p > 0.90 ) or between moderate and heavy smokers ( = 0.15 , p > 0.60 ) . the association of smoking habits of mother during pregnancy investigated with the stillbirths , abortions , neonatal deaths , birth weights , placental weights and the outcomes on the 28 day of life . the smoker mothers were classified as light smokers ( less than 10 cigarettes per day ) , moderate smokers ( between 10 and 19 cigarettes per day ) and heavy smokers ( 20 or more cigarettes per day ) . the results show that even the light smoking habit has an effect on the birth weight and the placental weight but for other characteristics , stillbirth , abortion , and the outcomes on the 28 day of life , there is no significant difference between light smokers and non - smokers . while quit smoking must be the ultimate goal for any smoker , the present study concludes that moderate and heavy smokers should reduce their number of cigarettes per day to at least the level of light smokers to achieve the same results for non - smokers . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

background : the objectives of this work were to study the association of maternal smoking habits with stillbirths , abortions , neonatal deaths , birth weights , placental weights and the outcomes on the 28th day of life.methods:a questionnaire was developed and completed with the hospitals recorded data collected over a period of 5 years from 47,000 babies born in several hospitals in ontario , canada . the mothers were classified into four categories : non - smokers , light smokers ( less than 10 cigarettes per day ) , moderate smokers ( between 10 and 19 cigarettes per day ) and heavy smokers ( 20 or more cigarettes per day ) . the population surveyed was of mixed ethnicity from both rural and urban areas . statistical analysis was performed using the spss statistical package.results:even the light smoking habit has an effect on the birth weight and the placental weight but for other characteristics , stillbirth , abortion , and the outcomes on the 28th day of life , no significant difference observed between light smokers and non-smokers.conclusion:while quit smoking must be the ultimate goal for any smoker , the present study concludes that moderate and heavy smokers , if they will not be able to quit , they should reduce their number of cigarettes per day to at least the level of light smokers to achieve the same results for non - smokers . all characteristics show significant difference between non - smokers and moderate and heavy smokers .

Introduction Materials and Methods Research Design Data Collection Statistical Analysis Results Discussion Smoking Habits and Previous Stillbirths and Previous Abortions Smoking Habits and Previous Neonatal Deaths Smoking Habits and Birth Weight Smoking Habits and Placental Weight Smoking Habits and Outcomes on the 28 Conclusions Ethical considerations

despite the association of the children s health with the smoking habits of mother during pregnancy have been investigated for many years and well - documented , the topic is still being studied by various research groups . ( 4 ) discussed not only the relationship of smoking habits of mothers and fetal risks , but the importance of negative impact of smoking on the health of mothers as well . they suggested that smokers have a higher chance of experiencing cardiovascular problems ( deep vein thrombosis , myocardial infarction , and stroke ) and pulmonary disorders ( bronchitis , asthma , pneumonia , influenza ) than non - smokers . ( 5 ) investigated the data from the pregnancy risk assessment monitoring survey ( prams ) for nine states over a 10-year period ( 19962005 ) , and revealed the risk factor of maternal alcohol use and smoking habits , in early pregnancy , and congenital heart defects . this paper describes the association of maternal smoking habits with stillbirths , abortions , neonatal deaths , birth weights , placental weights and the outcomes on the 28 day of life . a questionnaire was developed and completed with the hospitals recorded data which have been collected over a period of 5 years from about 47,000 babies born in several hospitals in ontario . for the purpose of this study , the mothers were classified into four categories : non - smokers , light smokers ( less than 10 cigarettes per day ) , moderate smokers ( between 10 and 19 cigarettes per day ) and heavy smokers ( 20 or more cigarettes per day ) . other groups such as sportive smoking , ex - smokers and mothers who have smoked before pregnancy and ceased during pregnancy were out of the scope of present study . throughout this study statistical analysis was performed using the spss statistical package , version 15.0 for windows ( spss , chicago , usa ) and the chi - square test was used to estimate the probable association between the variables , and a p value less than 0.05 was considered statistically significant for all the tables . the odds ratios ( or ) the present analysis is a cohort study where the maternal smokers during pregnancy are compared with the maternal non - smokers during pregnancy . during 2001 and 2002 , the first author had a fellowship at the division of clinical and metabolic genetics , hospital for sick children , toronto , canada , in the late prof . a questionnaire was developed and completed with the hospitals recorded data which have been collected over a period of 5 years from about 47,000 babies born in several hospitals in ontario . for the purpose of this study , the mothers were classified into four categories : non - smokers , light smokers ( less than 10 cigarettes per day ) , moderate smokers ( between 10 and 19 cigarettes per day ) and heavy smokers ( 20 or more cigarettes per day ) . other groups such as sportive smoking , ex - smokers and mothers who have smoked before pregnancy and ceased during pregnancy were out of the scope of present study . throughout this study statistical analysis was performed using the spss statistical package , version 15.0 for windows ( spss , chicago , usa ) and the chi - square test was used to estimate the probable association between the variables , and a p value less than 0.05 was considered statistically significant for all the tables . the odds ratios ( or ) the present analysis is a cohort study where the maternal smokers during pregnancy are compared with the maternal non - smokers during pregnancy . previous abortions were divided into four subgroups of no previous abortions , 1 previous abortion , 2 previous abortions and 3 or more previous abortions . our data shows that there is no significant difference between non - smokers and light smokers with regard to the number of previous stillbirths ( = 2.38 , p > 0.30 ) and also to the number of previous abortions ( = 8.51 , p > 0.036 ) . our results revealed that the proportion of previous neonatal deaths was significantly higher amongst smoker mothers than non - smokers ( = 45.02 , p < 0.0001 ) . the results show that there is no significant difference between no - smokers and light smokers in regard to previous neonatal deaths ( = 1.22 , p > 0.50 ) . birth weight was consisted of six subgroups of 2.5 kg or less , ( 2.6 2.9 ) kg , ( 3.0 3.3 ) kg , ( 3.4 3.7 ) kg , ( 3.8 4.1 ) kg , and 4.2 kg or more . the data from the present study suggest that mothers who smoke have lighter babies than mothers who do not smoke . placental weight was divided into four subgroups of 400 g or less , ( 400 599 ) g , ( 600 799 ) g , and 800 g or more . the pattern of results is similar to that found for birth weight results and indicates that those babies born to smoking mothers have significantly lighter placental weight than those born to non - smokers ( = 145.20 , p < 0.0001 ) . it was found that babies born to heavy smokers have the highest mortality rate , and the rate , gradually increases as smoking increases . our results show that there is no significant difference between non - smokers and light smokers ( = 0.01 , p > 0.90 ) and also no significant difference between moderate and heavy smokers ( = 0.15 , p > 0.60 ) . kllen ( 8) reported that maternal smoking during pregnancy is associated with a number of effects such as pre - term birth , intrauterine growth retardation , a small head circumference , and stillbirths and neonatal deaths . the smoking habits of the mothers were recorded in three categories : non - smokers , < 10 cigarettes / day and 10 cigarettes / day . the results showed the stillbirths risk estimates are 0.42% and 0.32% for smokers and non - smokers , respectively . there is no significant difference between moderate and heavy smoking mothers with respect to previous stillbirths where the odds ratios vary from 1.20 ( 95% ci : 0.64 2.25 ) to 1.42 ( 95% ci : 0.69 2.94 ) for 2 or more previous stillbirths , and ( 2 ) there was no significant difference between non - smokers and light smokers ( < 10 cigs / day ) with any previous stillbirths. compared with women who abstained during pregnancy , women who smoked cigarettes had a 20% increase in spontaneous abortion ( odds ratio of 1.20 ) for each 10 cigarettes smoked per day . the results of the present study show that the previous abortion is a significant factor for women who smoke 20 or more cigarettes per day . the odds ratios for the previous number of abortions of 1 , 2 and 3 are 1.54 ( 95% ci , 1.42 1.67 ) , 1.97 ( 95% ci , 1.70 2.28 ) and 2.86 ( 95% ci , 2.30 3.55 ) , respectively . no significant difference was observed between the light smokers ( < 10 cigs / day ) and non - smokers ( = 8.51 , p > 0.036 ) . the results show while smokers had a stillbirth rate more than three times as high ( 28 versus 8 per 1000 births ) they found that the neonatal death rates were almost the same for smokers and nonsmokers . the study ( 16 ) based on the number of stillbirths or neonatal deaths among infants born in sweden from 1983 to 1995 showed that maternal smoking seems to aggravate the placental abruption because the death risk in the presence of abruption increases when mother smoked ( or = 1.74 , 95% ci 1.45 2.08 ) . the present results show that there is no significant difference between non - smokers and light smokers with < 10 cigarettes per day ( = 1.22 , p > 0.50 ) . for moderate and heavy smokers the odds ratios are up to or = 1.84 , 95% ci 1.05 3.23 . at each visit the mothers were asked about the number of cigarettes smoked per day , and they were grouped as follows : ( a ) non - smokers , ( b ) light - to - moderate smokers , ( 114 cigarettes / day ) , and ( c ) heavy smokers ( 15 or more cigarettes/ day ) . in both sexes babies born to non - smokers were heavier , longer , and had larger head circumferences than those born to heavy smokers . smoking during pregnancy appears to have caused a general retardation in intrauterine growth , resulting in babies born with lower birth weights , shorter lengths , and smaller head circumferences . the results showed that smoking during pregnancy in combination with the socioeconomic inequalities is significantly associated with the higher risk of low birth weight . the association of socioeconomic status with heath and birth weight has been discussed by some authors ( 1922 ) . ( 23 ) suggested that the family case management program may be effective in reducing very low birth weight and low birth weight rates among infants born to low - income women . ( 24 ) reported the prenatal smoking status of west virginia , usa , women and the associated changes in infant birth weights . they found that infants born to women who smoked during pregnancy had significantly lower birth weights than infants born to non - smokers . the odds ratio is significantly reduced from the birth weight of < 2.5 kg to = > 4.2 kg for smoking mothers . ( 25 ) reported a study with the objective of determining whether maternal smoking during pregnancy is associated with bone mass and other growth variables in pre - pubertal children in tasmania , australia . they showed that the mothers who smoked during pregnancy had lower placental weight ( 56 g , 95% ci 95 to 17 ) . they examined the association between mothers smoking habits during pregnancy , taking the sex of the offspring into consideration , and the risk and prognosis of placental abruption , placenta previa , and preeclampsia . the results showed that smoking was associated with the risk of placental abruption ( or=1.99 , 95% ci 1.722.30 ) and placenta previa ( or=1.88 , 95% ci 1.153.07 ) . the effect of maternal smoking on placental volume was studied ( 27 ) in 80 pregnancies categorized according to cigarette consumption : group a never smoked , b smoking < 10 cigarettes / day , c smoking 1020 cigarettes / day , and d smoking > 20 cigarettes / day . the odds ratios for heavy smokers ( > 20 cigs / day ) are 0.81 ( 95% ci : 0.69 0.95 ) , 0.67 ( 95% ci : 0.57 0.79 ) and 0.59 ( 95% ci : 0.49 0.71 ) for the placental weight groups of 400 599 g , 600 799 g and = > 800 g , respectively . the perinatal mortality ( pnm ) has been defined ( 28 ) as the total number of stillbirths and deaths in the first week of life ( early neonatal deaths ) . in the present study we defined the outcome of pregnancy as whether the infant was alive on the 28 day of life . we considered the total number of infants deaths as the sum of stillbirths and neonatal deaths ( early neonatal : within the first 7 days of life and late neonatal : after 7 days until 28 day ) . the outcomes on the 28 day were 45923 ( 97.66% ) lives and 1100 ( 2.34% ) deaths . the odds ratios were 1.01 ( 95% ci 0.82 1.24 ) , 1.43 ( 95% ci 1.23 1.66 ) and 1.49 ( 95% ci 1.24 1.79 ) for light smokers ( < 10 cigs / day ) , moderate smokers ( 10 19 cigs / day ) and heavy smokers ( 20 cigs / day ) , respectively . it was found , firstly , that babies born to moderate and heavy smokers have the highest mortality rate . secondly , there were no significant differences between non - smokers and light smokers ( = 0.01 , p > 0.90 ) or between moderate and heavy smokers ( = 0.15 , p > 0.60 ) . kllen ( 8) reported that maternal smoking during pregnancy is associated with a number of effects such as pre - term birth , intrauterine growth retardation , a small head circumference , and stillbirths and neonatal deaths . the smoking habits of the mothers were recorded in three categories : non - smokers , < 10 cigarettes / day and 10 cigarettes / day . the results showed the stillbirths risk estimates are 0.42% and 0.32% for smokers and non - smokers , respectively . there is no significant difference between moderate and heavy smoking mothers with respect to previous stillbirths where the odds ratios vary from 1.20 ( 95% ci : 0.64 2.25 ) to 1.42 ( 95% ci : 0.69 2.94 ) for 2 or more previous stillbirths , and ( 2 ) there was no significant difference between non - smokers and light smokers ( < 10 cigs / day ) with any previous stillbirths. ( = 2.38 , p > 0.30 ) in a study ( 11 ) conducted in montreal , quebec , canada , the researchers interviewed approximately 56,000 women who had had a delivery or a spontaneous abortion in 11 montreal hospitals during the two - year period 19821984.the researchers found that 22% of the women had had previous pregnancies that ended in spontaneous abortion . compared with women who abstained during pregnancy , women who smoked cigarettes had a 20% increase in spontaneous abortion ( odds ratio of 1.20 ) for each 10 cigarettes smoked per day . the results of the present study show that the previous abortion is a significant factor for women who smoke 20 or more cigarettes per day . the odds ratios for the previous number of abortions of 1 , 2 and 3 are 1.54 ( 95% ci , 1.42 1.67 ) , 1.97 ( 95% ci , 1.70 2.28 ) and 2.86 ( 95% ci , 2.30 3.55 ) , respectively . no significant difference was observed between the light smokers ( < 10 cigs / day ) and non - smokers ( = 8.51 , p > 0.036 ) . the study ( 16 ) based on the number of stillbirths or neonatal deaths among infants born in sweden from 1983 to 1995 showed that maternal smoking seems to aggravate the placental abruption because the death risk in the presence of abruption increases when mother smoked ( or = 1.74 , 95% ci 1.45 2.08 ) . the present results show that there is no significant difference between non - smokers and light smokers with < 10 cigarettes per day ( = 1.22 , p > 0.50 ) . for moderate and heavy smokers the odds ratios are up to or = 1.84 , 95% ci 1.05 3.23 . at each visit the mothers were asked about the number of cigarettes smoked per day , and they were grouped as follows : ( a ) non - smokers , ( b ) light - to - moderate smokers , ( 114 cigarettes / day ) , and ( c ) heavy smokers ( 15 or more cigarettes/ day ) . in both sexes babies born to non - smokers were heavier , longer , and had larger head circumferences than those born to heavy smokers . smoking during pregnancy appears to have caused a general retardation in intrauterine growth , resulting in babies born with lower birth weights , shorter lengths , and smaller head circumferences . the results showed that smoking during pregnancy in combination with the socioeconomic inequalities is significantly associated with the higher risk of low birth weight . the association of socioeconomic status with heath and birth weight has been discussed by some authors ( 1922 ) . ( 23 ) suggested that the family case management program may be effective in reducing very low birth weight and low birth weight rates among infants born to low - income women . they found that infants born to women who smoked during pregnancy had significantly lower birth weights than infants born to non - smokers . the odds ratio is significantly reduced from the birth weight of < 2.5 kg to = > 4.2 kg for smoking mothers . they examined the association between mothers smoking habits during pregnancy , taking the sex of the offspring into consideration , and the risk and prognosis of placental abruption , placenta previa , and preeclampsia . the effect of maternal smoking on placental volume was studied ( 27 ) in 80 pregnancies categorized according to cigarette consumption : group a never smoked , b smoking < 10 cigarettes / day , c smoking 1020 cigarettes / day , and d smoking > 20 cigarettes / day . the three - dimensional power doppler ultrasonography of the placenta was performed in this study and the results showed no differences in placental volume among different groups . the odds ratios for heavy smokers ( > 20 cigs / day ) are 0.81 ( 95% ci : 0.69 0.95 ) , 0.67 ( 95% ci : 0.57 0.79 ) and 0.59 ( 95% ci : 0.49 0.71 ) for the placental weight groups of 400 599 g , 600 799 g and = > 800 g , respectively . the perinatal mortality ( pnm ) has been defined ( 28 ) as the total number of stillbirths and deaths in the first week of life ( early neonatal deaths ) . in the present study we defined the outcome of pregnancy as whether the infant was alive on the 28 day of life . we considered the total number of infants deaths as the sum of stillbirths and neonatal deaths ( early neonatal : within the first 7 days of life and late neonatal : after 7 days until 28 day ) . the outcomes on the 28 day were 45923 ( 97.66% ) lives and 1100 ( 2.34% ) deaths . the odds ratios were 1.01 ( 95% ci 0.82 1.24 ) , 1.43 ( 95% ci 1.23 1.66 ) and 1.49 ( 95% ci 1.24 1.79 ) for light smokers ( < 10 cigs / day ) , moderate smokers ( 10 19 cigs / day ) and heavy smokers ( 20 cigs / day ) , respectively . it was found , firstly , that babies born to moderate and heavy smokers have the highest mortality rate . secondly , there were no significant differences between non - smokers and light smokers ( = 0.01 , p > 0.90 ) or between moderate and heavy smokers ( = 0.15 , p > 0.60 ) . the association of smoking habits of mother during pregnancy investigated with the stillbirths , abortions , neonatal deaths , birth weights , placental weights and the outcomes on the 28 day of life . the smoker mothers were classified as light smokers ( less than 10 cigarettes per day ) , moderate smokers ( between 10 and 19 cigarettes per day ) and heavy smokers ( 20 or more cigarettes per day ) . the results show that even the light smoking habit has an effect on the birth weight and the placental weight but for other characteristics , stillbirth , abortion , and the outcomes on the 28 day of life , there is no significant difference between light smokers and non - smokers . while quit smoking must be the ultimate goal for any smoker , the present study concludes that moderate and heavy smokers should reduce their number of cigarettes per day to at least the level of light smokers to achieve the same results for non - smokers .

one of the earliest studies ( 1 ) in birmingham , uk , reported in 1959 shows that infants of mothers who smoked regularly during pregnancy were on the average more than 170 g lighter than the infants of those who never smoked during pregnancy . despite the association of the children s health with the smoking habits of mother during pregnancy have been investigated for many years and well - documented , the topic is still being studied by various research groups . ( 4 ) discussed not only the relationship of smoking habits of mothers and fetal risks , but the importance of negative impact of smoking on the health of mothers as well . they suggested that smokers have a higher chance of experiencing cardiovascular problems ( deep vein thrombosis , myocardial infarction , and stroke ) and pulmonary disorders ( bronchitis , asthma , pneumonia , influenza ) than non - smokers . ( 5 ) investigated the data from the pregnancy risk assessment monitoring survey ( prams ) for nine states over a 10-year period ( 19962005 ) , and revealed the risk factor of maternal alcohol use and smoking habits , in early pregnancy , and congenital heart defects . ( 7 ) investigated the healthy lifestyle behaviors during pregnancy , from 2001 to 2009 , in a survey from 22,604 american pregnant women aged 1844 years , and pointed out the importance of reducing alcohol use , binge drinking , and smoking and increasing fruit and vegetable consumption during pregnancy . this paper describes the association of maternal smoking habits with stillbirths , abortions , neonatal deaths , birth weights , placental weights and the outcomes on the 28 day of life . during 2001 and 2002 , the first author had a fellowship at the division of clinical and metabolic genetics , hospital for sick children , toronto , canada , in the late prof . the designed questionnaire covered a comprehensive range of information on both mothers and their babies health , which were and will be the source for a series of other articles . for the purpose of this study , the mothers were classified into four categories : non - smokers , light smokers ( less than 10 cigarettes per day ) , moderate smokers ( between 10 and 19 cigarettes per day ) and heavy smokers ( 20 or more cigarettes per day ) . other groups such as sportive smoking , ex - smokers and mothers who have smoked before pregnancy and ceased during pregnancy were out of the scope of present study . throughout this study statistical analysis was performed using the spss statistical package , version 15.0 for windows ( spss , chicago , usa ) and the chi - square test was used to estimate the probable association between the variables , and a p value less than 0.05 was considered statistically significant for all the tables . the odds ratios ( or ) the present analysis is a cohort study where the maternal smokers during pregnancy are compared with the maternal non - smokers during pregnancy . during 2001 and 2002 , the first author had a fellowship at the division of clinical and metabolic genetics , hospital for sick children , toronto , canada , in the late prof . the designed questionnaire covered a comprehensive range of information on both mothers and their babies health , which were and will be the source for a series of other articles . for the purpose of this study , the mothers were classified into four categories : non - smokers , light smokers ( less than 10 cigarettes per day ) , moderate smokers ( between 10 and 19 cigarettes per day ) and heavy smokers ( 20 or more cigarettes per day ) . other groups such as sportive smoking , ex - smokers and mothers who have smoked before pregnancy and ceased during pregnancy were out of the scope of present study . throughout this study statistical analysis was performed using the spss statistical package , version 15.0 for windows ( spss , chicago , usa ) and the chi - square test was used to estimate the probable association between the variables , and a p value less than 0.05 was considered statistically significant for all the tables . the odds ratios ( or ) the present analysis is a cohort study where the maternal smokers during pregnancy are compared with the maternal non - smokers during pregnancy . it was found , that mothers who smoke have significantly more previous stillbirths ( = 60.98 , p < 0.0001 ) and also previous abortions ( = 290.77 , p < 0.0001 ) than mothers who do not smoke . our data shows that there is no significant difference between non - smokers and light smokers with regard to the number of previous stillbirths ( = 2.38 , p > 0.30 ) and also to the number of previous abortions ( = 8.51 , p > 0.036 ) . our results revealed that the proportion of previous neonatal deaths was significantly higher amongst smoker mothers than non - smokers ( = 45.02 , p < 0.0001 ) . the results show that there is no significant difference between no - smokers and light smokers in regard to previous neonatal deaths ( = 1.22 , p > 0.50 ) . birth weight was consisted of six subgroups of 2.5 kg or less , ( 2.6 2.9 ) kg , ( 3.0 3.3 ) kg , ( 3.4 3.7 ) kg , ( 3.8 4.1 ) kg , and 4.2 kg or more . the pattern of results is similar to that found for birth weight results and indicates that those babies born to smoking mothers have significantly lighter placental weight than those born to non - smokers ( = 145.20 , p < 0.0001 ) . our results show that there is no significant difference between non - smokers and light smokers ( = 0.01 , p > 0.90 ) and also no significant difference between moderate and heavy smokers ( = 0.15 , p > 0.60 ) . kllen ( 8) reported that maternal smoking during pregnancy is associated with a number of effects such as pre - term birth , intrauterine growth retardation , a small head circumference , and stillbirths and neonatal deaths . the smoking habits of the mothers were recorded in three categories : non - smokers , < 10 cigarettes / day and 10 cigarettes / day . the results showed the stillbirths risk estimates are 0.42% and 0.32% for smokers and non - smokers , respectively . they showed that compared with nonsmokers , women who smoked during the first pregnancy but not during the second do not have an increased risk of stillbirth ( or 1.02 ; 95% ci 0.791.30 ) , while corresponding risk among women who smoked during both pregnancies was 1.35 ( 95% ci 1.151.58 ) . ( 10 ) studied the contribution of smoking during pregnancy to inequalities in stillbirth in scotland from 1994 to 2003 and reported that smoking during pregnancy accounted for 38% of the inequality in stillbirths . there is no significant difference between moderate and heavy smoking mothers with respect to previous stillbirths where the odds ratios vary from 1.20 ( 95% ci : 0.64 2.25 ) to 1.42 ( 95% ci : 0.69 2.94 ) for 2 or more previous stillbirths , and ( 2 ) there was no significant difference between non - smokers and light smokers ( < 10 cigs / day ) with any previous stillbirths. ( = 2.38 , p > 0.30 ) in a study ( 11 ) conducted in montreal , quebec , canada , the researchers interviewed approximately 56,000 women who had had a delivery or a spontaneous abortion in 11 montreal hospitals during the two - year period 19821984.the researchers found that 22% of the women had had previous pregnancies that ended in spontaneous abortion . compared with women who abstained during pregnancy , women who smoked cigarettes had a 20% increase in spontaneous abortion ( odds ratio of 1.20 ) for each 10 cigarettes smoked per day . the results of the present study show that the previous abortion is a significant factor for women who smoke 20 or more cigarettes per day . the odds ratios for the previous number of abortions of 1 , 2 and 3 are 1.54 ( 95% ci , 1.42 1.67 ) , 1.97 ( 95% ci , 1.70 2.28 ) and 2.86 ( 95% ci , 2.30 3.55 ) , respectively . no significant difference was observed between the light smokers ( < 10 cigs / day ) and non - smokers ( = 8.51 , p > 0.036 ) . the results show while smokers had a stillbirth rate more than three times as high ( 28 versus 8 per 1000 births ) they found that the neonatal death rates were almost the same for smokers and nonsmokers . in a study ( 15 ) conducted in sweden between 1983 and 1985 , significant relative risks for early neonatal mortality were found for multiple births ( 4.9 ) and smoking ( 1.2 ) . the study ( 16 ) based on the number of stillbirths or neonatal deaths among infants born in sweden from 1983 to 1995 showed that maternal smoking seems to aggravate the placental abruption because the death risk in the presence of abruption increases when mother smoked ( or = 1.74 , 95% ci 1.45 2.08 ) . the present results show that there is no significant difference between non - smokers and light smokers with < 10 cigarettes per day ( = 1.22 , p > 0.50 ) . at each visit the mothers were asked about the number of cigarettes smoked per day , and they were grouped as follows : ( a ) non - smokers , ( b ) light - to - moderate smokers , ( 114 cigarettes / day ) , and ( c ) heavy smokers ( 15 or more cigarettes/ day ) . the results showed that smoking during pregnancy in combination with the socioeconomic inequalities is significantly associated with the higher risk of low birth weight . ( 23 ) suggested that the family case management program may be effective in reducing very low birth weight and low birth weight rates among infants born to low - income women . the results showed that maternal prenatal smoking was the strongest predictor of low birth weight ( < 2500 g ) with an odds ratio of 3.29 ( 95% ci 2.87 , 3.77 ) for smoking during the recent pregnancy . the odds ratio is significantly reduced from the birth weight of < 2.5 kg to = > 4.2 kg for smoking mothers . the variation of odds ratio is from 0.95 ( 95% ci 0.85 1.06 ) to 0.18 ( 95% ci 0.14 0.23 ) . ( 25 ) reported a study with the objective of determining whether maternal smoking during pregnancy is associated with bone mass and other growth variables in pre - pubertal children in tasmania , australia . they examined the association between mothers smoking habits during pregnancy , taking the sex of the offspring into consideration , and the risk and prognosis of placental abruption , placenta previa , and preeclampsia . the results showed that smoking was associated with the risk of placental abruption ( or=1.99 , 95% ci 1.722.30 ) and placenta previa ( or=1.88 , 95% ci 1.153.07 ) . the effect of maternal smoking on placental volume was studied ( 27 ) in 80 pregnancies categorized according to cigarette consumption : group a never smoked , b smoking < 10 cigarettes / day , c smoking 1020 cigarettes / day , and d smoking > 20 cigarettes / day . the three - dimensional power doppler ultrasonography of the placenta was performed in this study and the results showed no differences in placental volume among different groups . the odds ratios for heavy smokers ( > 20 cigs / day ) are 0.81 ( 95% ci : 0.69 0.95 ) , 0.67 ( 95% ci : 0.57 0.79 ) and 0.59 ( 95% ci : 0.49 0.71 ) for the placental weight groups of 400 599 g , 600 799 g and = > 800 g , respectively . the perinatal mortality ( pnm ) has been defined ( 28 ) as the total number of stillbirths and deaths in the first week of life ( early neonatal deaths ) . in the present study we defined the outcome of pregnancy as whether the infant was alive on the 28 day of life . we considered the total number of infants deaths as the sum of stillbirths and neonatal deaths ( early neonatal : within the first 7 days of life and late neonatal : after 7 days until 28 day ) . the odds ratios were 1.01 ( 95% ci 0.82 1.24 ) , 1.43 ( 95% ci 1.23 1.66 ) and 1.49 ( 95% ci 1.24 1.79 ) for light smokers ( < 10 cigs / day ) , moderate smokers ( 10 19 cigs / day ) and heavy smokers ( 20 cigs / day ) , respectively . secondly , there were no significant differences between non - smokers and light smokers ( = 0.01 , p > 0.90 ) or between moderate and heavy smokers ( = 0.15 , p > 0.60 ) . kllen ( 8) reported that maternal smoking during pregnancy is associated with a number of effects such as pre - term birth , intrauterine growth retardation , a small head circumference , and stillbirths and neonatal deaths . they showed that compared with nonsmokers , women who smoked during the first pregnancy but not during the second do not have an increased risk of stillbirth ( or 1.02 ; 95% ci 0.791.30 ) , while corresponding risk among women who smoked during both pregnancies was 1.35 ( 95% ci 1.151.58 ) . ( 10 ) studied the contribution of smoking during pregnancy to inequalities in stillbirth in scotland from 1994 to 2003 and reported that smoking during pregnancy accounted for 38% of the inequality in stillbirths . there is no significant difference between moderate and heavy smoking mothers with respect to previous stillbirths where the odds ratios vary from 1.20 ( 95% ci : 0.64 2.25 ) to 1.42 ( 95% ci : 0.69 2.94 ) for 2 or more previous stillbirths , and ( 2 ) there was no significant difference between non - smokers and light smokers ( < 10 cigs / day ) with any previous stillbirths. ( = 2.38 , p > 0.30 ) in a study ( 11 ) conducted in montreal , quebec , canada , the researchers interviewed approximately 56,000 women who had had a delivery or a spontaneous abortion in 11 montreal hospitals during the two - year period 19821984.the researchers found that 22% of the women had had previous pregnancies that ended in spontaneous abortion . the odds ratios for the previous number of abortions of 1 , 2 and 3 are 1.54 ( 95% ci , 1.42 1.67 ) , 1.97 ( 95% ci , 1.70 2.28 ) and 2.86 ( 95% ci , 2.30 3.55 ) , respectively . no significant difference was observed between the light smokers ( < 10 cigs / day ) and non - smokers ( = 8.51 , p > 0.036 ) . the study ( 16 ) based on the number of stillbirths or neonatal deaths among infants born in sweden from 1983 to 1995 showed that maternal smoking seems to aggravate the placental abruption because the death risk in the presence of abruption increases when mother smoked ( or = 1.74 , 95% ci 1.45 2.08 ) . the present results show that there is no significant difference between non - smokers and light smokers with < 10 cigarettes per day ( = 1.22 , p > 0.50 ) . at each visit the mothers were asked about the number of cigarettes smoked per day , and they were grouped as follows : ( a ) non - smokers , ( b ) light - to - moderate smokers , ( 114 cigarettes / day ) , and ( c ) heavy smokers ( 15 or more cigarettes/ day ) . the results showed that smoking during pregnancy in combination with the socioeconomic inequalities is significantly associated with the higher risk of low birth weight . the results showed that maternal prenatal smoking was the strongest predictor of low birth weight ( < 2500 g ) with an odds ratio of 3.29 ( 95% ci 2.87 , 3.77 ) for smoking during the recent pregnancy . the odds ratio is significantly reduced from the birth weight of < 2.5 kg to = > 4.2 kg for smoking mothers . ( 25 ) reported a study with the objective of determining whether maternal smoking during pregnancy is associated with bone mass and other growth variables in pre - pubertal children in tasmania , australia . they examined the association between mothers smoking habits during pregnancy , taking the sex of the offspring into consideration , and the risk and prognosis of placental abruption , placenta previa , and preeclampsia . the results showed that smoking was associated with the risk of placental abruption ( or=1.99 , 95% ci 1.722.30 ) and placenta previa ( or=1.88 , 95% ci 1.153.07 ) . the effect of maternal smoking on placental volume was studied ( 27 ) in 80 pregnancies categorized according to cigarette consumption : group a never smoked , b smoking < 10 cigarettes / day , c smoking 1020 cigarettes / day , and d smoking > 20 cigarettes / day . the odds ratios for heavy smokers ( > 20 cigs / day ) are 0.81 ( 95% ci : 0.69 0.95 ) , 0.67 ( 95% ci : 0.57 0.79 ) and 0.59 ( 95% ci : 0.49 0.71 ) for the placental weight groups of 400 599 g , 600 799 g and = > 800 g , respectively . the perinatal mortality ( pnm ) has been defined ( 28 ) as the total number of stillbirths and deaths in the first week of life ( early neonatal deaths ) . we considered the total number of infants deaths as the sum of stillbirths and neonatal deaths ( early neonatal : within the first 7 days of life and late neonatal : after 7 days until 28 day ) . the odds ratios were 1.01 ( 95% ci 0.82 1.24 ) , 1.43 ( 95% ci 1.23 1.66 ) and 1.49 ( 95% ci 1.24 1.79 ) for light smokers ( < 10 cigs / day ) , moderate smokers ( 10 19 cigs / day ) and heavy smokers ( 20 cigs / day ) , respectively . secondly , there were no significant differences between non - smokers and light smokers ( = 0.01 , p > 0.90 ) or between moderate and heavy smokers ( = 0.15 , p > 0.60 ) . the association of smoking habits of mother during pregnancy investigated with the stillbirths , abortions , neonatal deaths , birth weights , placental weights and the outcomes on the 28 day of life . the results show that even the light smoking habit has an effect on the birth weight and the placental weight but for other characteristics , stillbirth , abortion , and the outcomes on the 28 day of life , there is no significant difference between light smokers and non - smokers . while quit smoking must be the ultimate goal for any smoker , the present study concludes that moderate and heavy smokers should reduce their number of cigarettes per day to at least the level of light smokers to achieve the same results for non - smokers .

over the last 2 decades , various molecular imaging technologies , including positron emission tomography ( pet ) , computed tomography ( ct ) , single photon emission computed tomography ( spect ) , magnetic resonance imaging ( mri ) , ultrasound , and fluorescence reflectance imaging , have revolutionized the way that we investigate complex biochemical phenomena . along with the rapid advances in molecular and cell biology , molecular imaging can greatly enhance the ability for researchers and clinicians to identify novel molecular targets and biomarkers , especially those involved in disease ( particularly cancer ) initiation , progression , and treatment response . detection of such biomarkers can lead to faster diagnosis and treatment , better prognosis and staging , and improved management . molecular imaging is defined as the noninvasive visualization , characterization , and measurement of biological processes at the cellular and molecular level in humans and other living systems . because molecular imaging provides both anatomical and physiological information , it has become an essential tool in bench - side research , clinical trials , and medical practice . one of the central challenges for molecular imaging is the development of specific imaging probes that have a high target - to - background ratio and improved contrast in vivo . the ideal imaging probe should possess high affinity and specificity for target , adequate retention in the target , low nonspecific uptake , and efficient capillary permeability . to date , many ligand - mediated targeting probes have been explored , and some of them have been approved for clinical use . a few examples are the cyclic octapeptide octreotide , a peptide that targets the somatostain receptor , trastuzumab ( herceptin ) , an antibody that binds to the antiepidermal growth factor receptor 2 ( erbb2 , her2 ) receptor , and bevacizumab ( avastin ) , an antibody that binds to the extracellular vascular endothelial growth factor a ( vegf - a ) . many diseases , especially inflammatory disorders and cancer , result from complex interactions between disease - mediated ligands and growth - promoting receptors . the crosstalk with other signaling pathways complicates the use of ligand - based probes for molecular imaging . thus , accurate knowledge of the receptor s role in the interaction between cells and their microenvironment is important . solid tumors , for instance , are usually composed of an assemblage of distinct cell types ( e.g. , endothelial cells , pericytes , immune inflammatory cells , cancer - associated fibroblasts , cancer cells , cancer stem cells , etc . ) that interact through the reciprocal heterotypic signaling pathway to maintain and orchestrate the tumor microenvironment . for example , epidermal growth factor ( egf ) , vegf , other proangiogenic factors ( e.g. , fibroblast growth factor 2 ( fgf2 ) , chemokines , and cytokines ) can amplify the inflammatory state and serve as effectors of tumor progression . additionally , tumor heterogeneity and binding site barriers between ligand and receptor can limit the targeting and therapeutic efficiency of ligands because they are typically monospecific . in fact , mounting evidence has demonstrated that acquired resistance to antibody therapy can occur if the antibody is against a single receptor , and this resistance is often related to pathway switching between receptors . consequently , multiple targeting , or the ability to bind multiple targets simultaneously , has become a more advantageous approach for the development of ligand - based imaging probes and therapeutics . over the past few decades , dual targeting with bispecific peptides or antibodies has been explored in clinical trials as an alternative combination therapy for cancer patients ( with over 50 ongoing or completed trials listed at clinicaltrials.gov ) . heterodimers ligands are composed of two covalently linked targeting subunits and are a simple , beneficial model for the investigation of dual targeting . recently , many bispecific heterodimers , summarized in table 1 , have been developed . compared with monoreceptor targeting compounds , bispecific heterodimers have several advantages including increased affinity , avidity , and efficacy , which establishes them as strong applicants for use in molecular imaging . in this review , we will discuss the design of bispecific peptide and antibody heterodimers and their applications in molecular targeting and imaging , with special emphasis on antibody heterodimers . we will also briefly discuss the design and application of bispecific heterodimer - conjugated nanomaterials . two major strategies for the design of heterodimers exist . in the first strategy , the heterodimer is formed by cross - linking two ligands that target two receptors from different cells at a given location ( figure 1a ) . this strategy is commonly used in the design of peptide heterodimers . in the second strategy , this strategy is more applicable in the design of protein - based heterodimers in which the structural integrity is of great concern during their development . clear advantages of this strategy include greater flexibility , higher production yield , and lower binding affinity loss . three primary approaches are readily adopted in the production of protein heterodimer : the first approach involves gene fusion and expression in escherichia coli to produce protein heterodimers in a tandem manner ( figure 1a ) . the second approach uses somatic hybridization by two protein - secreting cells ( e.g. , hybridomas ) along with affinity chromatography purification and is employed in the production of bispecific antibodies . the third approach is comparatively rare ; it introduces mutations into monospecific proteins and screens bispecific candidates out from the mutant library ( figure 1a ) . in which they introduced an integrin 3 binding capacity into the single - chain vegf ( scvegf ) by a yeast - displayed mutant library to generate a dual - specific scvegf mutant with high affinity to both vegfr2 and integrin 3 . compared with monospecific mutants that bind only to vegfr2 or integrin 3 , the dual - specific scvegf proteins demonstrated more effective inhibition of vegf - mediated receptor phosphorylation , endothelial cell proliferation , and blood vessel formation both in vitro and in vivo . in the following text ( a ) synthesis strategies of bispecific heterodimer based on chemical coupling , mutation / screening , and gene fusion . ( b ) interactions between bispecific heterodimer / monospecific ligand and cellular receptors during the molecular imaging process . after the cross - linking of two peptides , evaluation of binding affinity and specificity is essential for their imaging applications . generally speaking , there are two primary approaches to evaluate these parameters : the first approach can be carried out in two different cell types in which one cell type overexpresses a single receptor and the other cell type overexpresses both target receptors . in the second approach , binding affinity / specificity is examined in one cell type with high expression of both receptors ( figure 1b ) . ligands with strong affinity for each individual receptor compete with the heterodimer during its interaction with the target cells . in both of these approaches , the key point is to confirm that the peptide heterodimer has satisfactory a binding affinity and high specificity for each of its target receptors . on the basis of these strategies , heterobivalent ligands ( htbvls ) were developed that contain both melanocyte - stimulating hormone ( msh ) and cholecystokinin ( cck ) peptide ligands tethered with linkers of different rigidity and length . these heterodimers could simultaneously bind melanocortin-4 receptor ( mc4r ) and cck-2 receptor ( cck-2r ) , which are overexpressed in multiple cancer types including pancreatic cancer . the monovalent binding capacity of these ligands was evaluated in hek293 cells transfected with either mc4r , cck-2r , or both . the binding affinity of the optimized heterodimer to cells expressing both mc4r and cck-2r was over 20-fold higher than for cells expressing only mc4r . more recently , the same research group assessed the in vivo targeting efficacy of one heterodimer compound ( named htbvl1 ) composed of similar peptide ligands and optimized the linker between the two ligands . flow cytometry analysis indicated that cells expressing both receptors had higher cellular uptake of heterodimer than those expressing either receptor at a concentration of 50 nm . after systemic injection of cy5-labeled htbvl1 in tumor - bearing mice , higher uptake and longer retention were observed in tumors that overexpressed both receptors compared with single - receptor - positive tumors . blocking with msh , cck , or both reduced the uptake of each target tumor significantly ( figure 2a ) . these studies provide valuable insights into the design of heterobivalent ligands with high avidity : the length and conformation of the linker can be very crucial during the design of peptide heterodimers . because the binding of one pharmacophore to its corresponding site at the target brings the second pharmacophore in close proximity to that target , the enhanced tumor affinity from heterodimers mainly arises from increases in local ligand concentration . when the pharmacophores of peptide heterodimers overlap , simultaneous binding of two peptide ligands to two different receptors is impossible . ( a ) molecular structure of cy5-labeled heterobivalent ligand 1 ( htbvl1 ) and representative in vivo fluorescence images showing its specific uptake in target tumor ( right flank , target tumor with mc1r and cck-2r expression ; left flank , control tumor with only mc1r expression ) . ( b ) structure and spect / ct images of i - cmbp - click - c(rgdyk ) heterodimer in u87 mg tumor ( c - met and integrin 3 positive ) at 1 ( upper panel ) and 4 h p.i . ( lower left image ) . blocking with crgdyk ( lower middle image ) or cmbp ( lower right image ) t , tumor ; b , bladder ; thy , thyroid ; and k , kidney . adapted with permission from ref ( 28 ) . ( c ) pet images of ga - nota - rgd - bbn , ga - nota - bbn , and ga - nota - rgd at 1 h p.i . in pc-3 tumor - bearing mice . adapted with permission from ref ( 80 ) . another important factor for peptide heterodimer design is the careful evaluation of the receptors of interest . the selection of targets is important for the development of bispecific heterodimers with improved tumor uptake . epithelial transition factor ( c - met ) binding peptide ( cmbp ) was conjugated with cyclic rgd ( c(rgdyk ) ) through a click reaction to form a cmbp - click - c(rgdyk ) peptide heterodimer . this heterodimer was designed to possess the ability to recognize both c - met and integrin 3 receptors simultaneously . however , biodistribution studies and spect / ct imaging showed that despite the uptake of i - cmbp - click - c(rgdyk ) in a u87 mg tumor ( positive for both c - met and integrin 3 ) that could be blocked partially by cmbp or c(rgdyk ) ( figure 2b ) , it did not demonstrate any improvement to that of a cmbp - scrambled peptide . monoclonal antibodies are well - established workhorses for therapeutic and diagnostic purposes , particularly in oncology . because of their exceptional ability to recognize specific antigens , monoclonal antibodies play a central role in targeted therapeutics . however , targeting only one antigen is usually insufficient in oncology , where tumors can progress after a latency period during antibody treatment . compared with classic monospecific antibodies , bispecific antibodies can further improve the specificity for particular antigens and serve as more powerful tools for studying the molecular mechanisms of disease and developing more potent therapeutics . by artificial manipulation of antibody genes , bispecific antibody heterodimers are being developed to enable targeting of different epitopes on the same cell surface receptors , targeting two different receptors simultaneously , and enhancing cell cell interactions . bispecific antibodies can be produced by three main methods : chemical conjugation , hybridoma cell line fusion , and protein engineering involving recombinant dna . chemical cross - linking of two different fragment - antigen - binding ( f(ab ) ) fragments was the first strategy introduced in 1980s to generate bispecific antibodies . in this method , two different f(ab ) a number of bispecific f(ab)2 fragments heterodimers has been produced in this manner , including anti - cea / anti - indium - dtpa , anti - id / anti - hsg , anti - cea / anti - dtpa - in , and anti - mlc1/anti - cd90 . however , chemical modification may cause the inactivation of antibody binding sites or dysfunction of the effector agents . furthermore , chemical cross - linking requires extra purification compared to homodimer formation and often results in poor dimer stability . the resulting hybrid hybridoma secretes a heterogeneous population of antibodies , including bispecific antibodies . however , this technology also requires extensive purification procedures , and the production efficiency of bispecific antibodies is comparatively low . by far , recombinant dna technology is the most frequently used and trustworthy method for producing bispecific antibodies . this method can produce bispecific antibodies in large quantities , does not involve chemical linkage , and requires minimum purification process . a variety of antibody - based heterodimers , such as knobs - into - holes structure , bispecific f(ab)2 , heterodimeric scfv , and heterodimeric fab , have been produced using recombinant dna technology . detailed examples of bispecific antibody production will be discussed in the following paragraphs . linear fusion of genes encoding different antibody single - chain variable ( scfv ) fragments was the initially used technology to produce bispecific antibodies . a recombinant linear cd3/egp-2-directed bispecific monoclonal antibody , bis-1 f(ab)2 , was produced by this method . to improve the production yield of heterodimers , leucine zipper sequences were introduced into the c terminus of two different scfv fragments . bispecific anti - cd3/anti - tac f(ab-zipper)2 heterodimers were produced by this method and demonstrated high efficacy for cytotoxic t cell recruitment . a variety of other linkers have also been used to produce heterodimers , such as the cd3/17 - 1a bispecific antibody and anti - her3/anti - her2 bispecific scfv . by fusing with a helical dimerization domain ( e.g. , cysteine - containing peptide , helix loop helix motif , and barnse dibarnase domain ) , the length and 3d structure of the linker between scfv s are closely associated with the expression abundance and immunogenicity of the final heterodimers . at the same time , this gene fusion strategy can also be applicable in the formation of bispecific antibody fragments such as diabody ( db ) , a dimeric antibody fragment composed of the variable region of igg heavy and light chains ( vh and vl ) connected with a peptide linker . coexpression of vh vl fusions in the periplasm of e. coli enables the stable production of bispecific diabodies . however , the comparatively short linker between vh and vl inside diabodies may restrict their ability to simultaneously access two antigens on two different cells . complementary fragments from antibodies can also be used as heterodimerization scaffolds for the production of recombinant fab , spontaneous interactions between heavy chain constant domains 1 ( ch1 ) and light chain constant domains ( cl ) can result in heterodimerization that forms a covalently linked heterodimer named a chcl miniantibody . one humanized immunoglobulin ( igg ) ch1 was connected with the cl domains of another antibody in this manner to form a bispecific anti - egfr / anti - cd2 heterodimer that has high avidity for both egfr and cd2 as well as low immunogenicity . when both domains were coexpressed in e. coli , 63% of the total proteins formed were bispecific . another option is the interaction between fd and l chains of fab and the c terminus of a scfv molecule ; in this way , bispecific fab scfv and trispecific fab(scfv)2 can be generated with up to a 90% production yield . the engineering of an antibody s fragment crystallizable ( fc ) region is another means to produce bispecific antibody heterodimers . in antibodies , fab the fc region of igg mediates antibody effector functions through interactions with fc receptors and serves as an important factor for an antibody s long serum half - life in vivo through interactions with neonatal fc receptors . the fc is often used to generate antibody - like fusion proteins because of its inherent dimeric nature and can be used to create complex heterogeneous antibody mixtures . methods based on fc engineering , such as knobs - into - holes ( kih ) , have been frequently adopted to generate heterodimeric antibody assemblies . in the kih method , amino acids are mutated within the ch3 domains of antibody heavy chains , forcing complementary heterodimeric assembly between two different heavy chains . a bispecific antibody heterodimer targeting both c - met and vegfr-2 was created by this method and exhibited potent antitumor efficacy in gastric cancer . mutations induced via the kih method change the charge complementarity at the ch3 domain interface , promoting fc heterodimer formation and suppressing the formation of knob knob or hole unfortunately , the kih method sometimes produces bispecific antibodies with unnatural domain junctions and a loss of natural antibody architecture . correct association of the light chains and their cognate heavy chains can be achieved by exchange of heavy - chain and light - chain domains within the fab of one - half of the bispecific antibody . another strategy is to utilize an igg4 antibody , which readily engages in fab - arm exchange with other igg4 antibodies . the sequences essential for arm exchange are present only in the ch3 and core hinge regions of the igg4 isotype . to facilitate arm exchange in antibodies of these isotypes , minimal point mutations were introduced into the ch3 and core hinge sequences of igg1 and igg2 antibodies . the generated igg1 bispecific antibodies had faster clearance than the parental igg1 antibodies in rats , but the impact of these modifications on immunogenicity was not investigated . more recently , a modified kih method that relied on coculture of two bacterial strains ( one expressing the knob and the other expressing the hole half of the antibody ) was developed for the generation of nonimmunogenic , stable bispecific antibodies . after inoculating with an appropriate ratio of bacteria expressing anti - egfr and anti - met , respectively , the purified bispecific antibodies demonstrated similar monomeric stability and heterodimer purity as the bispecific antibody produced by the half antibody redox method . the resulting bispecific antibody against met and egfr could bind both targets monovalently , inhibit their signaling , and suppress met and egfr - driven cell and tumor growth . molecular imaging of cancer with peptide ligands has attracted widespread research attention because they have relatively high affinity and excellent tissue penetration . a number of different peptide receptors are massively overexpressed in numerous cancers ; examples include , but are not limited to , somatostatin receptor , gastrin - releasing peptide receptor ( grpr ) , cck2/cck - b , glucagon - like peptide-1 receptor ( glp-1 ) , and integrin 3 . measuring receptor expression is crucial for accurate diagnosis as well as for monitoring the response to therapy . several peptide - based radiotracers have shown promising results in animal studies , and some of them have been investigated in clinical trials . chemically linked peptide heterodimers , which bind to two different receptors , can increase the functional affinity and binding specificity of the probe . recently , an integrin 3 and grpr dual - targeted peptide ( rgd - bbn heterodimer ) was developed and utilized in pet imaging of cancer . aspartate ( rgd ) peptides can specifically target integrin 3 , a molecular marker of angiogenesis , and have been successfully used for imaging by pet and spect techniques . bombesin ( bbn ) is an amphibian homologue of mammalian grp , which can specifically bind to grpr , and has been extensively investigated for the diagnosis and treatment of grpr - positive tumors . used a glutamate linker to bridge cyclic rgd and bbn and radiolabeled the fused peptide with f ( t1/2 : 110 min , , 100% ) to investigate its dual - receptor - targeting ability in pc-3 prostate tumor xenografts ( integrin 3 positive ; grpr positive ) . they found that tumor uptake of f - fb - rgd - bbn was significantly higher than that of f - fb - bbn and f - fb - rgd , respectively . compared with f - fb - bbn and f - fb - rgd , f - fb one limitation of this study is that the heterodimer was merged by a short glutamate linker , which compromised its simultaneous binding capacity to grpr and integrin 3 . to overcome this limitation , the same group further modified the structure of rgd - bbn by using orthogonally protected fmoc - glu - oall . the optimized rgd - bbn heterodimer was radiolabeled with ga ( t1/2 : 68 min ; , 89% ) for pet imaging . biodistribution studies showed that pc-3 tumor uptake of ga - nota - rgd - bbn was significantly higher than that of f - fb - rgd - bbn . the tumor uptake of ga - nota - rgd - bbn was also evaluated in two different cell types : pc-3 and mda - mb-435 ( grpr negative , integrin 3 positive ) . in the pc-3 tumor model , the tumor uptake of ga - nota - rgd - bbn was slightly higher than that of ga - nota - bbn and significantly higher than that of ga - nota - rgd at 1 h p.i . mda - mb-435 tumors had significantly lower tumor uptake of the heterodimer compared with pc-3 tumors . in blocking studies with rgd or bbn alone ( figure 2c ) , a partial decrease in tumor uptake was observed . when blocking with both rgd and bbn , tumor uptake of the tracer was reduced to background levels . these results indicated that the dual - targeting tracer could still bind to one available receptor while the other receptor was blocked . several groups have used the radiolabeled chemotactic peptide cflflfk and its analogues to detect neutrophils as they target the formyl peptide receptor ( fpr ) on leukocytes . however , poor pharmacokinetic parameters and low detection sensitivity have limited their utility . to address these issues , a heterobivalent peptide was designed using cflflf and tkppr connected with a peg linker . the resulting cflflf-(peg)12-tkppr - tc was able to target the fpr and tuftsin receptor simultaneously . high expression of these two receptors activates polymorphonuclear leukocytes ( pmns ) , and sites of inflammation can be monitored by imaging of pmns with cflflf-(peg)12-tkppr - tc . spect / ct imaging showed that the accumulation of cflflf-(peg)12-tkppr - tc in the inflamed tissue was 3.15-fold higher than in the control tissue . bispecific antibody or antibody fragments - based heterodimers have shown potential for the molecular imaging of cancer . the anti - her2 monoclonal antibodies trastuzumab and pertuzumab can inhibit the proliferation of breast cancer by preventing receptor dimerization . in one study , the tumor - targeting efficacy of anti - c - her2 741f8 - 1 ( sfv)2 homodimers was compared with that of 741f8/26 - 10 ( sfv)2 heterodimers , which has specificity for digoxin and related cardiac glycosides . skov-3 tumor accumulation of i-741f8(sfv)2 was significantly higher than that of i-741f8/26 - 10 ( sfv)2 at 24 h p.i .. the difference in tumor retention between 741f8 - 1 ( sfv)2 and 741f8/26 - 10 ( sfv)2 might have been caused by the greater effective affinity for 741f8 - 1 ( sfv)2 compared with 741f8/2610 ( sfv)2 . later , another bispecific anti - her3/her2 a5-linker - ml3.9 bs - scfv ( alm ) was engineered with similar selective binding capacity to both target antigens in tumor cells . the accumulation of i - alm in skov-3 tumor xenografts ( her2 positive / her3 positive ) was significantly higher than that in either the mvm2 tumors ( her2 positive / her3 negative ) or mda - mb-468 tumors ( her2 negative / her3 positive ) . at the same time , skov-3 tumor uptake of i - alm was statistically higher than either that of a5 scfv or ml3.9 scfv . together , these results indicate that the similar binding affinity and expression levels of both targeting tumor - associated antigens are important for increasing the overall tumor retention of bispecific antibodies . antibody fragments fused with other nonantibody proteins is another important category of heterodimers for molecular targeting and imaging . recently , a novel bispecific radioimmunoconjugate ( bsric ) consisting of trastuzumab fab fragments and human heregulin-1 ( hrg ) was developed with the goal of imaging her2/her3 heterodimers selectively . hrg is the targeting ligand of her3 that promotes the recruitment of her2 to the complex . found that tumor uptake of the bsric in - dtpa - fab - peg24-hrg in bt-474 human breast cancer xenografts ( her2 positive / her3 positive ) was higher than that of in - dtpa - hrg in mda - mb-468 xenografts ( her2 negative / her3 positive ) ( figure 3a ) . excessive hrg or trastuzumab fab blocking decreased the uptake of in - bsrics in mda - mb-468 tumors , which demonstrated the specificity of in - bsrics for her2 and her3 in vivo . using a similar strategy , trastuzumab fab fragments were chemically cross - linked with human egf to synthesize bsrics that recognize her2 and egfr . however , the different binding affinity of fab and egf to 231-h2n ( her2 positive / egfr positive ) human breast cancer xenografts and skov-3 ( her2 negative / egfr positive ) human ovarian cancer xenografts resulted in similar tumor uptake of in - bsrics in both tumor models . future studies with bispecific heterodimers need to focus on choosing the correct flexible linker , ligands with similar binding affinities , and receptors that have appropriate expression levels . ( a ) whole - body spect / ct images of in - fab - peg24-hrg in cd1 athymic mice bearing bt-474 ( her2/her3 ) , skov-3 ( her2/her3 ) , or mda - mb-468 ( her2/her3 ) tumors ( arrows ) at 48 h. adapted with permission from ref ( 90 ) . ( b ) structure of diphtheria toxin ( dt390)/anti - cd19 and anti - cd22 scfv conjugates ( dt2219arl ) and serial bioluminescence images of mice bearing raji - luc burkitt s lymphomas treated with or without dt2219arl . bispecific antibodies can also cross - link different target antigens on two different cells and have been used to retarget immune effector cells to tumor cells . multiple successful studies have demonstrated the capacity of bispecific antibodies to enhance the interactions between malignant cells and cytotoxic t cells ( ctls ) , macrophages , or natural killer ( nk ) cells . if one binding site specifically recognizes the tumor - associated antigens and the other binding site is oriented against a marker for effector cells of the immune system ( e.g. , cd3 on t cells and cd16 on nk cells ) , then immune effector cell retargeting can be achieved . for instance , cd3-directed bispecific antibodies have proven to be beneficial for redirected tumor therapies . de jonge et al . developed an anti - cd3/anti - idiotype ( i d , a tumor - specific antigen ) bispecific scfv that could retarget ctls toward bcl1 lymphoma cells and exhibited antitumor activity toward bcl1 . although bispecific antibodies can trigger direct killing of tumor cells , i d variants are not always tumor exclusive and can lead to destructive immune response . therefore , a humanized bispecific f(ab)2-her2 cd3 was further developed to retarget cytotoxic cd8 nkt cells for the immunotherapy of her2-expressing tumors . f(ab)2-her2 cd3 was found to substantially enhance cytotoxic activity of cd8 nkt cells . to directly assess the specific cytotoxic activity of cd8 nkt cells in vivo , genetically modified skov-3 tumor cells expressing luciferase were used for monitoring tumor growth and the response to therapy . they found that the bioluminescence from tumors treated with cd8 nkt cells redirected with f(ab)2-her2 cd3 was significantly weaker than that of tumors treated with cd8 nkt cells alone or f(ab)2-her2 cd3 . additionally , animals treated with cd8 nkt cells redirected with f(ab)2-her2 cd3 had the highest survival rate at week 21 . the cd19 antigen is expressed in virtually all b - cell malignancies . to treat leukemia and malignant lymphomas , a bispecific heterodimeric diabody , cd3 cd19 , specific for the -chain of the cd3/tcr complex and cd19 on b cells , was constructed . the cd3 cd19 diabody could specifically interact with both cd3-positive and cd19-positive cells and inhibited the growth of b lymphoma xenografts in immunodeficient mice before preactivated human peripheral blood lymphocytes could . to enhance the selective killing efficiency of tumor cells , antibodies can be also coupled with immunotoxins . anti - cd19 immunotoxins have reported anticancer effects , and anti - cd22 immunotoxins have been successfully used to treat rare hairy cell leukemia . however , toxin - related side effects limited their clinical application . to address these issues , vallera et al . fused diphtheria toxin ( dt390 ) with anti - cd19 and anti - cd22 scfv to generate a novel bispecific fusion protein dt2219 , which had broader reactivity in recognizing and inhibiting b - cell malignancies . to increase the targeting ability of dt2219 , reverse - oriented vh vl domains of anti - cd19 and anti - cd22 sfv seventy five percent of the dt2219arl - treated mice were found to be completely tumor free on day 87 after intravenous injection with raji - luc burkitt s lymphoma ( cd22 positive / cd19 positive ) . additionally , luciferase bioluminescent imaging of untreated mice showed tumor present in the lung , bone marrow , and spinal cord on day 21 ( figure 3b ) . these data indicate that dt2219arl can prevent and kill malignant b cells in vivo . by retargeting immune effector cells or targeted delivery of immunotoxins to tumor cells , bispecific antibody heterodimers can be used in tumor therapy , and this therapy can be monitored using molecular imaging . although a number of successful studies have been carried out with bispecific antibodies and antibody fragments , molecular targeting / imaging with bispecific antibody heterodimers is still in its infancy . the careful choice of targets , optimization of protein fusion technology , and improved binding capacity for both of the targets will always be needed during the development of bispecific antibody - based molecular targeting / imaging agents . to meet these requirements , nanomaterials nanoparticles offer the ability to deliver a larger therapeutic payload per target recognition event than traditional probes and are able to carry multiple targeting agents for therapy or imaging of tumor cells . these multispecific nanoparticles can be divided into two categories : nanomaterials conjugated with two different ligands that target their individual receptors or nanomaterials conjugated with bispecific ligands . although dual - ligand modification of nanomaterials is not readily achievable , mounting attempts have already been devoted to produce bispecific nanomaterials for drug delivery , gene delivery , or combination therapy of cancer . however , molecular imaging research with those types of bispecific nanomaterials is extremely limited , and so far , there is only one report using a bispecific liposome to target integrin v3 and neurokinin-1 receptor in glioblastoma . unfortunately , there was no observable tumor - uptake enhancement compared with that of an unconjugated liposome in this study , which was monitored by spect / ct ( figure 4a ) . combined peptide - grafting and phage - display techniques to generate a high - affinity bispecific antibody fragment that can be strongly absorbed onto gold nanoparticles . they designed and constructed multispecific antibodies by joining gold - binding and egfr - binding antibody fragments ; these antibodies were used to enhance the surface plasmon resonance ( spr ) scattering signal from gold nanoparticles followed by their use for spr imaging of cancer cells ( figure 4b ) . using a similar strategy , an anti - gold antibody fragment , a14p - b2 , was fused with an anti - hen egg white lysozyme antibody fragment , hyhel10 fv , to generate a bispecific diabody . the resulting diabody enabled the functionalization of gold nanoparticles and allowed for selective protein accumulation on a gold - patterned silicon substrate . ( a ) spect / ct images of u87 mg tumor - bearing mice 4 h p.i . of in - labeled rgd - liposome , rgd / substance p - liposome ( bispecific ) , and nontargeted liposome . adapted with permission from ref ( 109 ) . ( b ) diagram depicting the bottom - up assembly of the zno - binding e32 vhh dimer and surface plasmon resonance ( spr ) images of a431 cells treated with the gold - binding e32 vhh fragment . adapted from ref ( 110 ) . ( c ) schematic illustration of nanoparticle - mediated coupling between a malignant b cell and a dc and fluorescence image of bjab cells ( green cytoplasm ) attached to dcs ( blue nuclei ) . another application for heterodimer - modified nanoparticles is to redirect the immune cells to recognize and eliminate the tumor cells . bispecific antibody ( anti - cd20/anti - cd86)-conjugated gold nanoparticles were recently designed to selectively attach malignant cells ( burkitt lymphoma b cells ; bjab ) to antigen - presenting cells ( human monocyte - derived dendritic cells ; dcs ) ( figure 4c ) . the resulting nanoparticles caused widespread cell fusion and the formation of hybrid cells after femtosecond pulse irradiation . a relatively uniform distribution of the individual gold nanoparticles on the plasma membranes of both cells was observed . after mixing bjab and dc in a 1:1 ratio , the cells formed pairs or small clusters in the bispecific nanoparticle - treated group at levels that were more than 4-fold higher than the single antibody nanoparticle or nonspecific anti - egfr - coated nanoparticle - treated groups . thus , the anti - cd20/anti - cd86-conjugated gold nanoparticles offer a simple and effective method to boost specific fusion / interaction between different cells . as an interdisciplinary field involving physics , chemistry , engineering , biology , and medicine , nanotechnology has the potential to improve the early detection , accurate diagnosis , and personalized treatment of various diseases , especially cancer . , heterodimers can assist in the functionalization of certain nanoparticles as well as link different nanomaterials , allowing for the generation of new nanomaterials with novel characteristics . on the other hand , nanomaterials provide a versatile platform to enhance the applicability of different types of heterodimers for drug delivery and theranostic purposes . given the wide range of physiological processes involved in disease progression , a number of promising molecular targets exist for the development of molecular imaging probes . powerful probes with optimal in vivo biodistribution and imaging characteristics are required for such technologies . bispecific ligand - based heterodimers have potent binding affinity and efficacy compared to traditional probes , making them promising candidates for molecular targeting and imaging applications . on the basis of the quantitative data acquired from different imaging studies , bispecific heterodimers usually display a significantly improved target - to - background ratio ( the ultimate holy grail pursued in molecular imaging ) compared with their monospecific peers because of the enhanced specificity brought by the binding of two targets . bispecific peptide heterodimers can be easily prepared by chemical conjugation of two peptides that bind different targets via a flexible linker . however , the length and rigidity of the linkers play an important role in the in vitro and in vivo characteristics of peptide heterodimers . thus , the choice of appropriate linkers is crucial for their design and screening . apart from this , careful analysis of the receptor expression patterns and selection of appropriate imaging labels are prerequisites for the development of suitable bispecific peptide heterodimers for a certain disease . in many scenarios , the cell signaling pathways of two targeted receptors are interconnected , which provides the foundation for bispecific peptides to trigger additive or synergistic biological effects in vivo . following these guidelines , a number of bispecific heterodimers have demonstrated excellent tumor - targeting capability . compared with bispecific peptide heterodimers , most bispecific antibodies possess higher affinities for both targets and are readily applicable for the treatment of cancer and inflammatory diseases . although the use of bispecific antibody heterodimers in molecular imaging is still in a very preliminary stage , their enhanced affinity and ability to target different epitopes make them promising imaging probes . recent studies have shown that molecules with molecular weights of approximately 60100 kda ( i.e. , diabodies , triabodies , or tetramers ) are ideal for tumor targeting because of their increased tumor penetration and fast clearance . these probes have the potential to make same - day imaging possible for clinical applications . exploring suitable truncated forms is necessary for the future development and optimization of bispecific antibodies for a variety of molecular applications . for the success of bispecific heterodimers in molecular imaging applications , understanding a receptor s expression pattern and its role in the cross - talk between tumor cells and their microenvironment is crucial . fine tuning the heterodimer s properties ( e.g. , size , fusion types , specific amino acid mutations , pharmacokinetic adjustment , etc . ) can also impact its stability , biodistribution , and tumor - to - background ratio . the affinity between ligand(s ) and receptor(s ) is dependent on a series of parameters such as charge , polarity , aromaticity , residue volume , surface area , or solvent accessibility . inside a bispecific heterodimer molecule , a ligand with lower affinity could still serve to further improve the binding capacity of a ligand with higher affinity as long as the relevant physical / chemical properties of the high - affinity ligand can be optimized . through careful analysis of these parameters , bispecific heterodimers with optimized pharmacokinetic and imaging characteristics can be developed , improving both the management of patient s with various diseases and disease - related bench - side research .

ligand - based molecular imaging probes have been designed with high affinity and specificity for monitoring biological process and responses . single - target recognition by traditional probes can limit their applicability for disease detection and therapy because synergistic action between disease mediators and different receptors is often involved in disease progression . consequently , probes that can recognize multiple targets should demonstrate higher targeting efficacy and specificity than their monospecific peers . this concept has been validated by multiple bispecific heterodimer - based imaging probes that have demonstrated promising results in several animal models . this review summarizes the design strategies for bispecific peptide- and antibody - based heterodimers and their applications in molecular targeting and imaging . the design and application of bispecific heterodimer - conjugated nanomaterials are also discussed .

Introduction Design of Bispecific Heterodimers Molecular Imaging with Bispecific Heterodimers Conclusions

over the last 2 decades , various molecular imaging technologies , including positron emission tomography ( pet ) , computed tomography ( ct ) , single photon emission computed tomography ( spect ) , magnetic resonance imaging ( mri ) , ultrasound , and fluorescence reflectance imaging , have revolutionized the way that we investigate complex biochemical phenomena . along with the rapid advances in molecular and cell biology , molecular imaging can greatly enhance the ability for researchers and clinicians to identify novel molecular targets and biomarkers , especially those involved in disease ( particularly cancer ) initiation , progression , and treatment response . molecular imaging is defined as the noninvasive visualization , characterization , and measurement of biological processes at the cellular and molecular level in humans and other living systems . because molecular imaging provides both anatomical and physiological information , it has become an essential tool in bench - side research , clinical trials , and medical practice . one of the central challenges for molecular imaging is the development of specific imaging probes that have a high target - to - background ratio and improved contrast in vivo . the ideal imaging probe should possess high affinity and specificity for target , adequate retention in the target , low nonspecific uptake , and efficient capillary permeability . to date , many ligand - mediated targeting probes have been explored , and some of them have been approved for clinical use . many diseases , especially inflammatory disorders and cancer , result from complex interactions between disease - mediated ligands and growth - promoting receptors . the crosstalk with other signaling pathways complicates the use of ligand - based probes for molecular imaging . thus , accurate knowledge of the receptor s role in the interaction between cells and their microenvironment is important . additionally , tumor heterogeneity and binding site barriers between ligand and receptor can limit the targeting and therapeutic efficiency of ligands because they are typically monospecific . in fact , mounting evidence has demonstrated that acquired resistance to antibody therapy can occur if the antibody is against a single receptor , and this resistance is often related to pathway switching between receptors . consequently , multiple targeting , or the ability to bind multiple targets simultaneously , has become a more advantageous approach for the development of ligand - based imaging probes and therapeutics . over the past few decades , dual targeting with bispecific peptides or antibodies has been explored in clinical trials as an alternative combination therapy for cancer patients ( with over 50 ongoing or completed trials listed at clinicaltrials.gov ) . heterodimers ligands are composed of two covalently linked targeting subunits and are a simple , beneficial model for the investigation of dual targeting . recently , many bispecific heterodimers , summarized in table 1 , have been developed . compared with monoreceptor targeting compounds , bispecific heterodimers have several advantages including increased affinity , avidity , and efficacy , which establishes them as strong applicants for use in molecular imaging . in this review , we will discuss the design of bispecific peptide and antibody heterodimers and their applications in molecular targeting and imaging , with special emphasis on antibody heterodimers . we will also briefly discuss the design and application of bispecific heterodimer - conjugated nanomaterials . two major strategies for the design of heterodimers exist . in the first strategy , the heterodimer is formed by cross - linking two ligands that target two receptors from different cells at a given location ( figure 1a ) . this strategy is commonly used in the design of peptide heterodimers . in the second strategy , this strategy is more applicable in the design of protein - based heterodimers in which the structural integrity is of great concern during their development . clear advantages of this strategy include greater flexibility , higher production yield , and lower binding affinity loss . , hybridomas ) along with affinity chromatography purification and is employed in the production of bispecific antibodies . in which they introduced an integrin 3 binding capacity into the single - chain vegf ( scvegf ) by a yeast - displayed mutant library to generate a dual - specific scvegf mutant with high affinity to both vegfr2 and integrin 3 . in the following text ( a ) synthesis strategies of bispecific heterodimer based on chemical coupling , mutation / screening , and gene fusion . ( b ) interactions between bispecific heterodimer / monospecific ligand and cellular receptors during the molecular imaging process . after the cross - linking of two peptides , evaluation of binding affinity and specificity is essential for their imaging applications . in the second approach , binding affinity / specificity is examined in one cell type with high expression of both receptors ( figure 1b ) . in both of these approaches , the key point is to confirm that the peptide heterodimer has satisfactory a binding affinity and high specificity for each of its target receptors . more recently , the same research group assessed the in vivo targeting efficacy of one heterodimer compound ( named htbvl1 ) composed of similar peptide ligands and optimized the linker between the two ligands . after systemic injection of cy5-labeled htbvl1 in tumor - bearing mice , higher uptake and longer retention were observed in tumors that overexpressed both receptors compared with single - receptor - positive tumors . these studies provide valuable insights into the design of heterobivalent ligands with high avidity : the length and conformation of the linker can be very crucial during the design of peptide heterodimers . when the pharmacophores of peptide heterodimers overlap , simultaneous binding of two peptide ligands to two different receptors is impossible . the selection of targets is important for the development of bispecific heterodimers with improved tumor uptake . compared with classic monospecific antibodies , bispecific antibodies can further improve the specificity for particular antigens and serve as more powerful tools for studying the molecular mechanisms of disease and developing more potent therapeutics . by artificial manipulation of antibody genes , bispecific antibody heterodimers are being developed to enable targeting of different epitopes on the same cell surface receptors , targeting two different receptors simultaneously , and enhancing cell cell interactions . in this method , two different f(ab ) a number of bispecific f(ab)2 fragments heterodimers has been produced in this manner , including anti - cea / anti - indium - dtpa , anti - id / anti - hsg , anti - cea / anti - dtpa - in , and anti - mlc1/anti - cd90 . furthermore , chemical cross - linking requires extra purification compared to homodimer formation and often results in poor dimer stability . however , this technology also requires extensive purification procedures , and the production efficiency of bispecific antibodies is comparatively low . a variety of antibody - based heterodimers , such as knobs - into - holes structure , bispecific f(ab)2 , heterodimeric scfv , and heterodimeric fab , have been produced using recombinant dna technology . detailed examples of bispecific antibody production will be discussed in the following paragraphs . linear fusion of genes encoding different antibody single - chain variable ( scfv ) fragments was the initially used technology to produce bispecific antibodies . a recombinant linear cd3/egp-2-directed bispecific monoclonal antibody , bis-1 f(ab)2 , was produced by this method . by fusing with a helical dimerization domain ( e.g. , cysteine - containing peptide , helix loop helix motif , and barnse dibarnase domain ) , the length and 3d structure of the linker between scfv s are closely associated with the expression abundance and immunogenicity of the final heterodimers . at the same time , this gene fusion strategy can also be applicable in the formation of bispecific antibody fragments such as diabody ( db ) , a dimeric antibody fragment composed of the variable region of igg heavy and light chains ( vh and vl ) connected with a peptide linker . coexpression of vh vl fusions in the periplasm of e. coli enables the stable production of bispecific diabodies . the fc is often used to generate antibody - like fusion proteins because of its inherent dimeric nature and can be used to create complex heterogeneous antibody mixtures . methods based on fc engineering , such as knobs - into - holes ( kih ) , have been frequently adopted to generate heterodimeric antibody assemblies . mutations induced via the kih method change the charge complementarity at the ch3 domain interface , promoting fc heterodimer formation and suppressing the formation of knob knob or hole unfortunately , the kih method sometimes produces bispecific antibodies with unnatural domain junctions and a loss of natural antibody architecture . correct association of the light chains and their cognate heavy chains can be achieved by exchange of heavy - chain and light - chain domains within the fab of one - half of the bispecific antibody . to facilitate arm exchange in antibodies of these isotypes , minimal point mutations were introduced into the ch3 and core hinge sequences of igg1 and igg2 antibodies . the generated igg1 bispecific antibodies had faster clearance than the parental igg1 antibodies in rats , but the impact of these modifications on immunogenicity was not investigated . molecular imaging of cancer with peptide ligands has attracted widespread research attention because they have relatively high affinity and excellent tissue penetration . measuring receptor expression is crucial for accurate diagnosis as well as for monitoring the response to therapy . several peptide - based radiotracers have shown promising results in animal studies , and some of them have been investigated in clinical trials . chemically linked peptide heterodimers , which bind to two different receptors , can increase the functional affinity and binding specificity of the probe . aspartate ( rgd ) peptides can specifically target integrin 3 , a molecular marker of angiogenesis , and have been successfully used for imaging by pet and spect techniques . bombesin ( bbn ) is an amphibian homologue of mammalian grp , which can specifically bind to grpr , and has been extensively investigated for the diagnosis and treatment of grpr - positive tumors . they found that tumor uptake of f - fb - rgd - bbn was significantly higher than that of f - fb - bbn and f - fb - rgd , respectively . to address these issues , a heterobivalent peptide was designed using cflflf and tkppr connected with a peg linker . bispecific antibody or antibody fragments - based heterodimers have shown potential for the molecular imaging of cancer . in one study , the tumor - targeting efficacy of anti - c - her2 741f8 - 1 ( sfv)2 homodimers was compared with that of 741f8/26 - 10 ( sfv)2 heterodimers , which has specificity for digoxin and related cardiac glycosides . skov-3 tumor accumulation of i-741f8(sfv)2 was significantly higher than that of i-741f8/26 - 10 ( sfv)2 at 24 h p.i .. the difference in tumor retention between 741f8 - 1 ( sfv)2 and 741f8/26 - 10 ( sfv)2 might have been caused by the greater effective affinity for 741f8 - 1 ( sfv)2 compared with 741f8/2610 ( sfv)2 . together , these results indicate that the similar binding affinity and expression levels of both targeting tumor - associated antigens are important for increasing the overall tumor retention of bispecific antibodies . antibody fragments fused with other nonantibody proteins is another important category of heterodimers for molecular targeting and imaging . future studies with bispecific heterodimers need to focus on choosing the correct flexible linker , ligands with similar binding affinities , and receptors that have appropriate expression levels . ( b ) structure of diphtheria toxin ( dt390)/anti - cd19 and anti - cd22 scfv conjugates ( dt2219arl ) and serial bioluminescence images of mice bearing raji - luc burkitt s lymphomas treated with or without dt2219arl . bispecific antibodies can also cross - link different target antigens on two different cells and have been used to retarget immune effector cells to tumor cells . multiple successful studies have demonstrated the capacity of bispecific antibodies to enhance the interactions between malignant cells and cytotoxic t cells ( ctls ) , macrophages , or natural killer ( nk ) cells . to directly assess the specific cytotoxic activity of cd8 nkt cells in vivo , genetically modified skov-3 tumor cells expressing luciferase were used for monitoring tumor growth and the response to therapy . they found that the bioluminescence from tumors treated with cd8 nkt cells redirected with f(ab)2-her2 cd3 was significantly weaker than that of tumors treated with cd8 nkt cells alone or f(ab)2-her2 cd3 . anti - cd19 immunotoxins have reported anticancer effects , and anti - cd22 immunotoxins have been successfully used to treat rare hairy cell leukemia . by retargeting immune effector cells or targeted delivery of immunotoxins to tumor cells , bispecific antibody heterodimers can be used in tumor therapy , and this therapy can be monitored using molecular imaging . although a number of successful studies have been carried out with bispecific antibodies and antibody fragments , molecular targeting / imaging with bispecific antibody heterodimers is still in its infancy . the careful choice of targets , optimization of protein fusion technology , and improved binding capacity for both of the targets will always be needed during the development of bispecific antibody - based molecular targeting / imaging agents . to meet these requirements , nanomaterials nanoparticles offer the ability to deliver a larger therapeutic payload per target recognition event than traditional probes and are able to carry multiple targeting agents for therapy or imaging of tumor cells . however , molecular imaging research with those types of bispecific nanomaterials is extremely limited , and so far , there is only one report using a bispecific liposome to target integrin v3 and neurokinin-1 receptor in glioblastoma . unfortunately , there was no observable tumor - uptake enhancement compared with that of an unconjugated liposome in this study , which was monitored by spect / ct ( figure 4a ) . combined peptide - grafting and phage - display techniques to generate a high - affinity bispecific antibody fragment that can be strongly absorbed onto gold nanoparticles . another application for heterodimer - modified nanoparticles is to redirect the immune cells to recognize and eliminate the tumor cells . on the other hand , nanomaterials provide a versatile platform to enhance the applicability of different types of heterodimers for drug delivery and theranostic purposes . given the wide range of physiological processes involved in disease progression , a number of promising molecular targets exist for the development of molecular imaging probes . powerful probes with optimal in vivo biodistribution and imaging characteristics are required for such technologies . bispecific ligand - based heterodimers have potent binding affinity and efficacy compared to traditional probes , making them promising candidates for molecular targeting and imaging applications . on the basis of the quantitative data acquired from different imaging studies , bispecific heterodimers usually display a significantly improved target - to - background ratio ( the ultimate holy grail pursued in molecular imaging ) compared with their monospecific peers because of the enhanced specificity brought by the binding of two targets . thus , the choice of appropriate linkers is crucial for their design and screening . in many scenarios , the cell signaling pathways of two targeted receptors are interconnected , which provides the foundation for bispecific peptides to trigger additive or synergistic biological effects in vivo . following these guidelines , a number of bispecific heterodimers have demonstrated excellent tumor - targeting capability . compared with bispecific peptide heterodimers , most bispecific antibodies possess higher affinities for both targets and are readily applicable for the treatment of cancer and inflammatory diseases . although the use of bispecific antibody heterodimers in molecular imaging is still in a very preliminary stage , their enhanced affinity and ability to target different epitopes make them promising imaging probes . these probes have the potential to make same - day imaging possible for clinical applications . exploring suitable truncated forms is necessary for the future development and optimization of bispecific antibodies for a variety of molecular applications . for the success of bispecific heterodimers in molecular imaging applications , understanding a receptor s expression pattern and its role in the cross - talk between tumor cells and their microenvironment is crucial . inside a bispecific heterodimer molecule , a ligand with lower affinity could still serve to further improve the binding capacity of a ligand with higher affinity as long as the relevant physical / chemical properties of the high - affinity ligand can be optimized . through careful analysis of these parameters , bispecific heterodimers with optimized pharmacokinetic and imaging characteristics can be developed , improving both the management of patient s with various diseases and disease - related bench - side research .

over the last 2 decades , various molecular imaging technologies , including positron emission tomography ( pet ) , computed tomography ( ct ) , single photon emission computed tomography ( spect ) , magnetic resonance imaging ( mri ) , ultrasound , and fluorescence reflectance imaging , have revolutionized the way that we investigate complex biochemical phenomena . along with the rapid advances in molecular and cell biology , molecular imaging can greatly enhance the ability for researchers and clinicians to identify novel molecular targets and biomarkers , especially those involved in disease ( particularly cancer ) initiation , progression , and treatment response . one of the central challenges for molecular imaging is the development of specific imaging probes that have a high target - to - background ratio and improved contrast in vivo . a few examples are the cyclic octapeptide octreotide , a peptide that targets the somatostain receptor , trastuzumab ( herceptin ) , an antibody that binds to the antiepidermal growth factor receptor 2 ( erbb2 , her2 ) receptor , and bevacizumab ( avastin ) , an antibody that binds to the extracellular vascular endothelial growth factor a ( vegf - a ) . over the past few decades , dual targeting with bispecific peptides or antibodies has been explored in clinical trials as an alternative combination therapy for cancer patients ( with over 50 ongoing or completed trials listed at clinicaltrials.gov ) . in this review , we will discuss the design of bispecific peptide and antibody heterodimers and their applications in molecular targeting and imaging , with special emphasis on antibody heterodimers . three primary approaches are readily adopted in the production of protein heterodimer : the first approach involves gene fusion and expression in escherichia coli to produce protein heterodimers in a tandem manner ( figure 1a ) . in which they introduced an integrin 3 binding capacity into the single - chain vegf ( scvegf ) by a yeast - displayed mutant library to generate a dual - specific scvegf mutant with high affinity to both vegfr2 and integrin 3 . compared with monospecific mutants that bind only to vegfr2 or integrin 3 , the dual - specific scvegf proteins demonstrated more effective inhibition of vegf - mediated receptor phosphorylation , endothelial cell proliferation , and blood vessel formation both in vitro and in vivo . generally speaking , there are two primary approaches to evaluate these parameters : the first approach can be carried out in two different cell types in which one cell type overexpresses a single receptor and the other cell type overexpresses both target receptors . on the basis of these strategies , heterobivalent ligands ( htbvls ) were developed that contain both melanocyte - stimulating hormone ( msh ) and cholecystokinin ( cck ) peptide ligands tethered with linkers of different rigidity and length . more recently , the same research group assessed the in vivo targeting efficacy of one heterodimer compound ( named htbvl1 ) composed of similar peptide ligands and optimized the linker between the two ligands . these studies provide valuable insights into the design of heterobivalent ligands with high avidity : the length and conformation of the linker can be very crucial during the design of peptide heterodimers . because the binding of one pharmacophore to its corresponding site at the target brings the second pharmacophore in close proximity to that target , the enhanced tumor affinity from heterodimers mainly arises from increases in local ligand concentration . ( a ) molecular structure of cy5-labeled heterobivalent ligand 1 ( htbvl1 ) and representative in vivo fluorescence images showing its specific uptake in target tumor ( right flank , target tumor with mc1r and cck-2r expression ; left flank , control tumor with only mc1r expression ) . ( b ) structure and spect / ct images of i - cmbp - click - c(rgdyk ) heterodimer in u87 mg tumor ( c - met and integrin 3 positive ) at 1 ( upper panel ) and 4 h p.i . however , biodistribution studies and spect / ct imaging showed that despite the uptake of i - cmbp - click - c(rgdyk ) in a u87 mg tumor ( positive for both c - met and integrin 3 ) that could be blocked partially by cmbp or c(rgdyk ) ( figure 2b ) , it did not demonstrate any improvement to that of a cmbp - scrambled peptide . in this method , two different f(ab ) a number of bispecific f(ab)2 fragments heterodimers has been produced in this manner , including anti - cea / anti - indium - dtpa , anti - id / anti - hsg , anti - cea / anti - dtpa - in , and anti - mlc1/anti - cd90 . , cysteine - containing peptide , helix loop helix motif , and barnse dibarnase domain ) , the length and 3d structure of the linker between scfv s are closely associated with the expression abundance and immunogenicity of the final heterodimers . at the same time , this gene fusion strategy can also be applicable in the formation of bispecific antibody fragments such as diabody ( db ) , a dimeric antibody fragment composed of the variable region of igg heavy and light chains ( vh and vl ) connected with a peptide linker . complementary fragments from antibodies can also be used as heterodimerization scaffolds for the production of recombinant fab , spontaneous interactions between heavy chain constant domains 1 ( ch1 ) and light chain constant domains ( cl ) can result in heterodimerization that forms a covalently linked heterodimer named a chcl miniantibody . another option is the interaction between fd and l chains of fab and the c terminus of a scfv molecule ; in this way , bispecific fab scfv and trispecific fab(scfv)2 can be generated with up to a 90% production yield . mutations induced via the kih method change the charge complementarity at the ch3 domain interface , promoting fc heterodimer formation and suppressing the formation of knob knob or hole unfortunately , the kih method sometimes produces bispecific antibodies with unnatural domain junctions and a loss of natural antibody architecture . correct association of the light chains and their cognate heavy chains can be achieved by exchange of heavy - chain and light - chain domains within the fab of one - half of the bispecific antibody . more recently , a modified kih method that relied on coculture of two bacterial strains ( one expressing the knob and the other expressing the hole half of the antibody ) was developed for the generation of nonimmunogenic , stable bispecific antibodies . a number of different peptide receptors are massively overexpressed in numerous cancers ; examples include , but are not limited to , somatostatin receptor , gastrin - releasing peptide receptor ( grpr ) , cck2/cck - b , glucagon - like peptide-1 receptor ( glp-1 ) , and integrin 3 . used a glutamate linker to bridge cyclic rgd and bbn and radiolabeled the fused peptide with f ( t1/2 : 110 min , , 100% ) to investigate its dual - receptor - targeting ability in pc-3 prostate tumor xenografts ( integrin 3 positive ; grpr positive ) . they found that tumor uptake of f - fb - rgd - bbn was significantly higher than that of f - fb - bbn and f - fb - rgd , respectively . compared with f - fb - bbn and f - fb - rgd , f - fb one limitation of this study is that the heterodimer was merged by a short glutamate linker , which compromised its simultaneous binding capacity to grpr and integrin 3 . biodistribution studies showed that pc-3 tumor uptake of ga - nota - rgd - bbn was significantly higher than that of f - fb - rgd - bbn . the tumor uptake of ga - nota - rgd - bbn was also evaluated in two different cell types : pc-3 and mda - mb-435 ( grpr negative , integrin 3 positive ) . in the pc-3 tumor model , the tumor uptake of ga - nota - rgd - bbn was slightly higher than that of ga - nota - bbn and significantly higher than that of ga - nota - rgd at 1 h p.i . spect / ct imaging showed that the accumulation of cflflf-(peg)12-tkppr - tc in the inflamed tissue was 3.15-fold higher than in the control tissue . in one study , the tumor - targeting efficacy of anti - c - her2 741f8 - 1 ( sfv)2 homodimers was compared with that of 741f8/26 - 10 ( sfv)2 heterodimers , which has specificity for digoxin and related cardiac glycosides . skov-3 tumor accumulation of i-741f8(sfv)2 was significantly higher than that of i-741f8/26 - 10 ( sfv)2 at 24 h p.i .. the difference in tumor retention between 741f8 - 1 ( sfv)2 and 741f8/26 - 10 ( sfv)2 might have been caused by the greater effective affinity for 741f8 - 1 ( sfv)2 compared with 741f8/2610 ( sfv)2 . the accumulation of i - alm in skov-3 tumor xenografts ( her2 positive / her3 positive ) was significantly higher than that in either the mvm2 tumors ( her2 positive / her3 negative ) or mda - mb-468 tumors ( her2 negative / her3 positive ) . together , these results indicate that the similar binding affinity and expression levels of both targeting tumor - associated antigens are important for increasing the overall tumor retention of bispecific antibodies . recently , a novel bispecific radioimmunoconjugate ( bsric ) consisting of trastuzumab fab fragments and human heregulin-1 ( hrg ) was developed with the goal of imaging her2/her3 heterodimers selectively . found that tumor uptake of the bsric in - dtpa - fab - peg24-hrg in bt-474 human breast cancer xenografts ( her2 positive / her3 positive ) was higher than that of in - dtpa - hrg in mda - mb-468 xenografts ( her2 negative / her3 positive ) ( figure 3a ) . excessive hrg or trastuzumab fab blocking decreased the uptake of in - bsrics in mda - mb-468 tumors , which demonstrated the specificity of in - bsrics for her2 and her3 in vivo . however , the different binding affinity of fab and egf to 231-h2n ( her2 positive / egfr positive ) human breast cancer xenografts and skov-3 ( her2 negative / egfr positive ) human ovarian cancer xenografts resulted in similar tumor uptake of in - bsrics in both tumor models . ( a ) whole - body spect / ct images of in - fab - peg24-hrg in cd1 athymic mice bearing bt-474 ( her2/her3 ) , skov-3 ( her2/her3 ) , or mda - mb-468 ( her2/her3 ) tumors ( arrows ) at 48 h. adapted with permission from ref ( 90 ) . ( b ) structure of diphtheria toxin ( dt390)/anti - cd19 and anti - cd22 scfv conjugates ( dt2219arl ) and serial bioluminescence images of mice bearing raji - luc burkitt s lymphomas treated with or without dt2219arl . multiple successful studies have demonstrated the capacity of bispecific antibodies to enhance the interactions between malignant cells and cytotoxic t cells ( ctls ) , macrophages , or natural killer ( nk ) cells . if one binding site specifically recognizes the tumor - associated antigens and the other binding site is oriented against a marker for effector cells of the immune system ( e.g. developed an anti - cd3/anti - idiotype ( i d , a tumor - specific antigen ) bispecific scfv that could retarget ctls toward bcl1 lymphoma cells and exhibited antitumor activity toward bcl1 . to directly assess the specific cytotoxic activity of cd8 nkt cells in vivo , genetically modified skov-3 tumor cells expressing luciferase were used for monitoring tumor growth and the response to therapy . they found that the bioluminescence from tumors treated with cd8 nkt cells redirected with f(ab)2-her2 cd3 was significantly weaker than that of tumors treated with cd8 nkt cells alone or f(ab)2-her2 cd3 . fused diphtheria toxin ( dt390 ) with anti - cd19 and anti - cd22 scfv to generate a novel bispecific fusion protein dt2219 , which had broader reactivity in recognizing and inhibiting b - cell malignancies . to increase the targeting ability of dt2219 , reverse - oriented vh vl domains of anti - cd19 and anti - cd22 sfv seventy five percent of the dt2219arl - treated mice were found to be completely tumor free on day 87 after intravenous injection with raji - luc burkitt s lymphoma ( cd22 positive / cd19 positive ) . by retargeting immune effector cells or targeted delivery of immunotoxins to tumor cells , bispecific antibody heterodimers can be used in tumor therapy , and this therapy can be monitored using molecular imaging . the careful choice of targets , optimization of protein fusion technology , and improved binding capacity for both of the targets will always be needed during the development of bispecific antibody - based molecular targeting / imaging agents . however , molecular imaging research with those types of bispecific nanomaterials is extremely limited , and so far , there is only one report using a bispecific liposome to target integrin v3 and neurokinin-1 receptor in glioblastoma . unfortunately , there was no observable tumor - uptake enhancement compared with that of an unconjugated liposome in this study , which was monitored by spect / ct ( figure 4a ) . they designed and constructed multispecific antibodies by joining gold - binding and egfr - binding antibody fragments ; these antibodies were used to enhance the surface plasmon resonance ( spr ) scattering signal from gold nanoparticles followed by their use for spr imaging of cancer cells ( figure 4b ) . ( b ) diagram depicting the bottom - up assembly of the zno - binding e32 vhh dimer and surface plasmon resonance ( spr ) images of a431 cells treated with the gold - binding e32 vhh fragment . ( c ) schematic illustration of nanoparticle - mediated coupling between a malignant b cell and a dc and fluorescence image of bjab cells ( green cytoplasm ) attached to dcs ( blue nuclei ) . bispecific antibody ( anti - cd20/anti - cd86)-conjugated gold nanoparticles were recently designed to selectively attach malignant cells ( burkitt lymphoma b cells ; bjab ) to antigen - presenting cells ( human monocyte - derived dendritic cells ; dcs ) ( figure 4c ) . after mixing bjab and dc in a 1:1 ratio , the cells formed pairs or small clusters in the bispecific nanoparticle - treated group at levels that were more than 4-fold higher than the single antibody nanoparticle or nonspecific anti - egfr - coated nanoparticle - treated groups . as an interdisciplinary field involving physics , chemistry , engineering , biology , and medicine , nanotechnology has the potential to improve the early detection , accurate diagnosis , and personalized treatment of various diseases , especially cancer . on the basis of the quantitative data acquired from different imaging studies , bispecific heterodimers usually display a significantly improved target - to - background ratio ( the ultimate holy grail pursued in molecular imaging ) compared with their monospecific peers because of the enhanced specificity brought by the binding of two targets . apart from this , careful analysis of the receptor expression patterns and selection of appropriate imaging labels are prerequisites for the development of suitable bispecific peptide heterodimers for a certain disease . for the success of bispecific heterodimers in molecular imaging applications , understanding a receptor s expression pattern and its role in the cross - talk between tumor cells and their microenvironment is crucial . inside a bispecific heterodimer molecule , a ligand with lower affinity could still serve to further improve the binding capacity of a ligand with higher affinity as long as the relevant physical / chemical properties of the high - affinity ligand can be optimized . through careful analysis of these parameters , bispecific heterodimers with optimized pharmacokinetic and imaging characteristics can be developed , improving both the management of patient s with various diseases and disease - related bench - side research .

the immune system is delicately balanced between immunity and tolerance to protect the host from pathogens while minimizing local damage to tissues . indoleamine 2,3-dioxygenase ( ido ) is an endogenous molecular mechanism that contributes to this immune regulation in a variety of settings . ido seems to be critical in limiting potentially exaggerated inflammatory reactions in response to danger signals and in assisting regulatory t - cell effector function . in addition , ido is an important component of a regulatory system that allows long - term control of immune homeostasis as may be required by tolerance to self or during pregnancy . ido is a major inhibitor of the effector phase of the immune response [ 45 , 50 ] . ido expression can suppress effector t cells directly by degradation of the essential amino acid tryptophan . some of the biological effect of ido is mediated through local depletion of tryptophan , but is in addition mediated via immune modulatory tryptophan metabolites [ 4 , 30 ] . thus , regulation of tryptophan metabolism by ido in dendritic cells ( dc ) is a highly adaptable modulator of immunity . when ido dc are injected in vivo , they create suppression and anergy in antigen - specific t cells in the ln draining the injection site [ 3 , 25 ] . effector t cells starved of tryptophan are unable to proliferate and go into g1 cell cycle arrest . an ido - responsive signaling system in t cells has been identified , comprising the stress kinase gc non - derepressing 2 kinase ( gcn2 ) . gcn2 responds to elevations in uncharged trna , as would occur if the t cell were deprived of tryptophan . another effect of ido is mediated through enhancement of local treg - mediated immune suppression . constitutive ido expression in dc provides t cells with regulatory properties that block t - cell responses to antigenic stimulation . the b7 receptors on ido dc bind to ctla4 on tregs causing them to proliferate and induce antigen - specific anergy . thus , ido does not only suppress effector t cells directly but also influence tregs bystander suppressor activity [ 2 , 32 , 39 ] . it has been described that exposure of tregs to pro - inflammatory cytokines like il-6 induce reprogramming of mature tregs to acquire a phenotype resembling pro - inflammatory th17 cells [ 6 , 49 , 51 ] . ido stimulates treg bystander suppressor activity and simultaneously blocks the il-6 production that is required to convert tregs into th17-like t cells [ 2 , 39 ] . the phenotype of reprogrammed tregs after ido - blocking have been described as similar to that of polyfunctional t - helper cells co - expressing il-17 , il-22 , il-2 as well as tnf- . thus , ido suppression of pro - inflammatory processes may dominantly block effector t - cell responses to antigens encountered . conversely , absence of ido activity may not elicit local treg suppression even when strong pro - inflammatory stimuli are present . finally , it was recently shown that ido has a non - enzymic function that contributes to tgf- driven tolerance in non - inflammatory contexts . ido may contribute in a critical manner to inhibit or terminate inflammation and are highly overexpressed in cancer [ 14 , 22 ] . in cancer patients , ido elevation occurs in a subset of plasmacytoid dc in tumor - draining lymph nodes . in addition , ido may be expressed within the tumor by tumor cells as well as tumor stromal cells , where it inhibits the effector phase of immune responses . activation of ido in either tumor cells or nodal regulatory dc each appears to be sufficient to facilitate immune escape of tumors . in this regard , it has been described that expression of ido in tumor cells is associated with an impaired prognosis . in a murine model , it was observed that tumor cells transfected with ido became resistant to immune eradication , even in mice in which a fully protective immune response had been established by immunization . ido - expressing cd19 plasmacytoid dc isolated from tumor - draining ln mediate profound immune suppression and t - cell anergy in vivo [ 25 , 37 ] , whereas plasmacytoid dc from normal lns and spleen do not express ido . in this respect , it should be noted that very few cells constitutively express ido in normal lymphoid tissue except in the gut . it is believed that constitutive ido expression in dc in tumor - draining ln is induced by stimulation from tregs migrating from the tumor to the draining ln . the induction of ido converts the tumor - draining ln from an immunizing into a tolerizing milieu . all in all , ido is a critical cellular factor contributing to immune suppression and as such is a crucial mechanism in cancer . hence , ido has become a very attractive target for the design of new anticancer drugs and several ido inhibitors are under investigation in preclinical as well as in clinical studies . in particular , the compound 1-methyl - tryptophan ( 1mt ) has been widely studied as an inhibitor of ido activity . interestingly , recent studies have shown that the racemer d-1-mt has superior antitumor activity compared to the racemer l-1-mt . ido2 functions like ido in tryptophan catabolism , but it has been found that d-1mt but not the l-1mt isomer selectively and potently inhibits ido2 activity suggesting that ido2 activity may have a role in the inhibition of immune responses to tumors . in this respect , ido2 expression has been found in human tumors , including gastric , colon , renal , and in pancreatic tumors ido2 expression have been found both in tumor cells as well as in immune cells in tumor - draining ln . it is not yet known to what extent each isoform of ido contributes to tumor - related immune suppression and how much clinical benefit ( or autoimmune toxicity ) targeting one isoform over another confers . despite the fact that neoplastic transformation is associated with the expression of immunogenic antigens , the immune system often fails to respond effectively and becomes tolerant toward these antigens . as described above ido plays a critical role in the tolerance induction and immune suppression of anti - cancer immune responses . we sat out to determine if and how ido itself serve as target for specific t - cell responses , which may be exploited for immune therapy . this was done by identifying and characterizing specific t cells spontaneously present among peripheral blood mononuclear cells ( pbmc ) isolated from cancer patients of different origin . in this regard , we described that peptides comprised in the ido protein sequence are spontaneously recognized by cytotoxic t cells ( ctl ) in cancer patients ( fig . 1principle of the processing pathway of ido peptides by ido - expressing cells ( red ) , for example , tumor cells or dendritic cells and the subsequent recognition by specific cd8 t cells ( green ; here entitled a supporter t cell ( tsup).the epitopes recognized by the t cells are short ido - derived peptides resulting from the degradation of intracellular ido protein , which are presented on the cell surface of hla molecules . t cells receive an activation signal through their t - cell receptor complex , leading to a variety of functional consequences , including release of cytokines and cytotoxic molecules principle of the processing pathway of ido peptides by ido - expressing cells ( red ) , for example , tumor cells or dendritic cells and the subsequent recognition by specific cd8 t cells ( green ; here entitled a supporter t cell ( tsup).the epitopes recognized by the t cells are short ido - derived peptides resulting from the degradation of intracellular ido protein , which are presented on the cell surface of hla molecules . t cells receive an activation signal through their t - cell receptor complex , leading to a variety of functional consequences , including release of cytokines and cytotoxic molecules first , we identified hla - restricted peptides within the ido protein to which spontaneous t - cell reactivity were detected in patients suffering from unrelated tumor types , i.e. , melanoma , renal cell carcinoma and breast cancer by flow cytometry using hla / peptide tetramers as well as in elispot assays after in vitro stimulation but also in direct ex vivo assays . such ido - reactive cd8 t cells were peptide - specific , cytotoxic effector cells . thus , ido - specific t cells effectively lysed ido cancer cell lines of different origin , such us colon carcinoma , melanoma , and breast cancer as well as directly ex vivo enriched leukemia cells . ido driven immune suppression is a general mechanism that has been described in a variety of human cancers and the immune responses against ido seem likewise to be relevant in cancers of unrelated origin , which emphasize the immunotherapeutic potential of ido . however , even more distinctive was our finding that ido - specific ctl recognized and killed ido , mature dc ; hence , ido - specific t cells were in addition able to kill immune - regulatory cells . we could at first not detect spontaneous responses against ido in the control group of healthy individuals . thus , although ido has immune suppressive functions , the constitutive up regulation of ido expression in cancer patients seemed to induce ido - specific t - cell responses . ido is playing a crucial role in immune regulation and is inducible under normal physiological conditions . thus , we found the apparent lack of tolerance against ido intriguing , since it suggested a more general role of ido - specific t cells in the regulation of the immune system . we hypothesized that such cells could take part in the control of immune homeostasis ; ido - specific cd8 t cells could play an important role by eliminating ido cells thereby suppressing and/or delaying local immune suppression . hence , we continued our search for possible ido - specific t - cell responses in healthy donors and found that circulating ido - specific , cytotoxic cd8 t cells indeed were present in healthy donors although not as frequent as in patients with cancer . furthermore , we were able to directly link the up regulation of ido with ido - specific t cells by showing that the addition of ido - inducing mediators like ifn- and cpg odn generated measurable numbers of cd8 ido - specific t cells among pbmc . to examine a possible immune - regulatory effect of ido - specific t cells , we examined their effect on t - cell immunity against viral or tumor - associated antigens . in this respect , we found that the presence of ido - specific cd8 t cells boosted cd8 t - cell responses against other antigens probably by eliminating ido suppressive cells ( fig . 2 ) . supporter t cells ( tsup ) due to their immune enhancing function .fig . 2ido - specific t cells are able to boost specific immunity against virus or tumor antigens in human pbmc . a when stimulating pbmc with a known hla - restricted t - cell virus epitope and il-2 , epitope - specific t cells begin to expand due to activation by antigen presenting cells ( apc ) . in response to the subsequent production of cytokines like inf- , ido expression is induced and ido - expressing apc inhibit further expansion of virus - specific t cells both directly and indirectly through activation of tregs . b the addition of cytotoxic , ido - specific t cells ( tsup ) removes immune suppressive cells from the pbmc culture thereby facilitating further expansion of virus - specific t cells ido - specific t cells are able to boost specific immunity against virus or tumor antigens in human pbmc . a when stimulating pbmc with a known hla - restricted t - cell virus epitope and il-2 , epitope - specific t cells begin to expand due to activation by antigen presenting cells ( apc ) . in response to the subsequent production of cytokines like inf- , ido expression is induced and ido - expressing apc inhibit further expansion of virus - specific t cells both directly and indirectly through activation of tregs . b the addition of cytotoxic , ido - specific t cells ( tsup ) removes immune suppressive cells from the pbmc culture thereby facilitating further expansion of virus - specific t cells ido expression contributes to the strength and duration of a given immune response due to its inflammation - induced counter - regulatory function . thus , any supportive effect of ido - specific t cells on other immune cells may well be mediated in several direct and indirect manners . in this respect , the level of tryptophan was elevated , the frequency of tregs decreased , and the frequency of il-17 producing cells increased when ido - specific t cells were present , which taken together suggest an overall decrease in ido activity . furthermore , ido - specific t cells increased the overall production of both il-6 as well as the other pro - inflammatory cytokine tnf-. in contrast , we observed a decrease in il-10 . another possible effect of ido - specific t cells could be mediated through the metabolites of tryptophan , which have been shown to be directly toxic to cd8 t cells and cd4 th1 cells , but not th2 cells . hence , increased ido activity seems to tilt helper t - cell polarization toward a th2 phenotype . the presence of activated ido - specific , cytotoxic t cells may screw the th - response in a th1-direction . finally , it should be noted that ido cells may be immune suppressive by other means than by the expression of ido . hence , the same cells might express , for example , arginase , pd - l1 or immune - regulatory cytokines ( e.g. , il-10 and tgf- ) . hence , ido - specific , cytotoxic t cells may not only reduce ido - mediated suppression directly but in addition further immune suppression mediated by ido regulatory cells . recently , we identified spontaneous cd8 t - cell reactivity against the ido analogue ido2 in peripheral blood of both healthy donors and cancer patients . furthermore , we confirmed that ido2-reactive cd8 t cells were peptide - specific , cytotoxic effector t cells . hence , isolated and expanded ido2-specific t cells effectively lysed cancer cell lines of different origin , that is , colon carcinoma cells as well as breast cancer cells . however , ido2-specific t cells did not seem to kill melanoma cells although they expressed ido2 . at least , we did not observe killing of three different ido2 melanoma cell lines . likewise , ido2-specific t cells did not seem to support other immune responses in the same way as ido - specific , cytotoxic t cells . hence , the function and potential role of the ido2-specific class - i - restricted lymphocytes present in peripheral blood still need to be resolved . we speculated that cd4 ido - specific t cells releasing pro - inflammatory cytokines may play a role in the early phases of an immune response as a counter - response to the induced immune suppression facilitated by ido cells . hence , ido - specific th1-cells may delay local immune suppression if the activation of an ido - specific cd4 th1-response could overcome the immune suppressive actions of the ido protein , which are otherwise a result of the early expression of ido in maturing dc or macrophages . indeed , identified detectable numbers of specific cd4 t cells both in cancer patients as well as healthy individuals . we found that such ido - specific cd4 t cells released inf- as well as tnf-. although , we were able to detect both inf- and tnf- response toward ido in healthy donors , the responses were more frequent in cancer patients . the cancer relevance of these cd4 t cells were further underlined , since ido - reacting t cells in addition react toward dc pulsed with ido tumor lysates . interestingly , we detected a correlation between patients harboring cd4 and cd8 responses against ido , which that class - i- and class - ii - restricted ido responses co - develop . furthermore , we detected frequent ido - specific cd4 t - cell responses when examining for il-17 release upon stimulation with the ido - derived cd4 epitope . il-17 has been the focus of great interest recently since the production of il-17 is characterized to a subset of cd4 t - helper cells ( th17 cells ) . one of the main roles of th17 cells is believed to be promoting host defense against infectious agents . th17 cells are thought to be particularly important in maintaining barrier immunity at mucosal surfaces such as in the lungs , gut , and skin . interestingly , ido is expressed at high levels in the gastrointestinal tract , although its precise role in intestinal immunity is not well understood . one could speculate that a fraction of the th17 that are highly prevalent at the mucosal tissues of healthy individuals is recognizing ido ; however , this is yet to be established . , th17 cells might have a protective role in tumor immunopathology by promoting antitumor immunity . tumor - infiltrating th17 cells express other cytokines in addition to il-17 , which might be functionally relevant . a large fraction of th17 cells produce high levels of effector cytokines such as il-2 , inf- as well as tnf . ido - specific th17 cells seemed to exhibit a similar effector t - cell cytokine profile . we could in contrast not detect any release of the th2 cytokine il-4 in response to the ido - derived peptide . it was recently suggested that the foxp3 treg cell lineage in addition to immune suppression have an unappreciated helper role . th17-like effector cells were distinguished by their unique ability to deliver help immediately and spontaneously , without needing prior priming or pre - activation . it was suggested that these cd4 lineage cells correspond to a pool of constitutively primed first responder cells . ido plays an important role in this conversion of foxp3 tregs to th17-like effector cells [ 2 , 39 ] . thus , it is possible that ido - specific t cells could in addition belong to a foxp3 lineage of constitutively primed first responder th17-like t cells ; however , it should be strengthen that this is speculation . naturally , some cd4-positive ido - specific t cells could in addition be immune suppressive tregs . it would be obvious that ido - specific tregs may enhance the ido - mediated immune suppression protecting cells from an immune attack . in this regard , we have previously described specific regulatory cd8 t cells in cancer patients , which recognized the immune suppressive heme oxygenase-1 . il-10 is mainly expressed by tregs that have been defined as a specialized subpopulation of t cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self - antigens [ 34 , 35 ] . we could in addition in some donors detect il-10 release in response to the ido - derived cd4 epitope peptide . hence , the role of ido - specific cd4 t cells in immune - regulatory networks may be a complex balance between activation and inhibition depending on the microenvironment . interestingly , in some donors we detected background il-10 release in in vitro pre - stimulated elispot assays . this enabled us to recognize that stimulation with the ido - derived peptide in two healthy donors triggered an overall suppression of il-10 . in this regard , we have previously observed a decrease in il-10 when ido - specific cd8 t cells were present . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as well as in the effector phases ( at the site of the tumor ) . with regard to the latter , ido may even by induced as an inflammation - induced counter - regulatory mechanism . counter - regulatory responses are important in the immune system as they help to limit the intensity and extent of immune responses , which otherwise could cause damage to the host . however , with regard to anti - cancer immunotherapy , counter - regulatory responses antagonize the ability to create an intense immune response against the tumor . counter - regulation differs from tolerance in the sense that counter - regulation is a secondary event , elicited only in response to immune activation . ido is known to be induced by both type i and ii interferons , which are likely to be found at sites of immune activation and inflammation [ 31 , 36 ] . in this respect , it should be mentioned that the susceptibility of tumor cells to lysis by ido - reactive t cells were increased by pre - incubation with ifn- . hence , in cancer immune therapy , the boosting of ido - specific immunity could have both direct and indirect effects ( fig . first of all , ido - specific , cytotoxic t cells are able to directly recognize and kill ido cancer cells . in fact , it may even be speculated that the measurable reactivity to this antigen in normal individuals contributes to immune surveillance against cancer . furthermore , the induction of ido - specific immune responses by therapeutic measures could function highly synergistic with additional anti - cancer immune therapy not only by eliminating cancer cells but in addition immune suppressive cells . by definition , anti - cancer immune therapies aim at the induction of an immunological activation and inflammation . the therapy aims to induce as much immune activation as possible ( within the limits of acceptable toxicity ) , and , accordingly , immune suppressive counter - regulation is not desired.fig . 3vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells adoptive transfer of ex vivo expanded tumor - infiltrating lymphocytes ( til ) after host lymphodepletion has the potential to significantly improve the prognosis of patients with metastatic melanoma . the impressive clinical responses associated with adoptive transfer of til urge that this strategy is pursued and investigated for the treatment of other types of cancer . in this regard , patient ido - specific t cells isolated and expanded from pbmc may well be an interesting supplement to the ongoing adaptive t - cell transfer strategies . it goes without saying that the possible introduction of autoimmunity and toxicity are the major worries when targeting a molecule like ido . however , the circulation of a measurable number of ido - specific t cells did not seem to cause autoimmunity . furthermore , since ido - specific t cells can be introduced by ifn or cpg this appears to be under solid control . in this regard , an interesting aspect of ido is that systemic inactivation at the organism level , either pharmacologically or genetically , does not appear to cause autoimmunity . we believe that the findings that presented here justified and warranted clinical testing to evaluate the efficiency and safety of ido - based vaccinations . hence , we initiated a phase i vaccination study , which is ongoing ( from june 2010 ) at center for caner immune therapy , copenhagen university hospital , herlev , in which patients with non - small cell lung cancer ( nsclc ) are vaccinated with a ido - derived peptide with montanide adjuvant ( www.clinicaltrials.gov ; nct01219348 ) . it has been suggested that ido may rather be involved in tolerance to non - self - antigens than self - antigens in situations where immune non - responsiveness may be important , for example , during pregnancy . in this respect , induction of ido immune - regulatory dendritic cells ( dc ) have been described to occur during infection of dcs with viruses and intracellular pathogens . in listeria monocytogenes infections , such ido dc seems to be involved in protection of the host from granuloma breakdown and pathogen dissemination in advanced human listeriosis . likewise , it was recently described that ido is increased in lymph nodes in cutaneous leishmania major infection . ido is implicated in suppressing t - cell immunity to parasite antigens and ido inhibition reduced local inflammation and parasite burdens , which suggest that ido were of benefit for the pathogen , not the host . during hiv infection , multiple mechanisms involving both viral and cellular components contribute to enhance ido expression and activity in an uncontrolled manner . among others , furthermore , it was recently described that ido is increased in hemodialysis ( hd ) patients compared to healthy donors . furthermore , ido suppresses adaptive immunity in hd patients as it is assessed by the response to hbv vaccination . hence , the targeting of ido could have synergistic effects in anti - viral immune therapy , for example , in hepatitis b vaccines . the fact that ido may be involved in tolerance to non - self - antigens might have major implications for ido - based immune therapy as boosting immunity to neoantigens , but not normal self - antigens , by triggering ido - specific t cells is very attractive . since ido - expressing cells might antagonize the desired effects of other immunotherapeutic approaches targeting ido - expressing cells by vaccination however , it was recently described that although ido might play biologically important roles in the host response to diverse intracellular infections like toxoplasma gondii , leishmaniasis , and herpes simplex virus , the nature of this role that being antimicrobial or immunoregulatory might depend on the pathogen . this should naturally been taken into account when exploring the possible use of ido - specific t cells in the clinic . finally , it should be mentioned that cd14 monocytes are major cmv target cells in vivo . cmv is the most immune dominant antigen to be encountered by the human immune system . monocytes are responsible for dissemination of the virus throughout the body during acute and late phase of infection . cmv has been shown to induce ido expression in monocytes , which has been suggested to confer an advantage to cmv - infected monocytes to escape t - cell responses . the cd8 t - cell response to cmv typically comprises a sizeable percentage of the cd8 t - cell repertoire in cmv - seropositive individuals . in light of this , it is possible that ido - specific t cells might function as support for the constitutive anti - cmv cd8 t - cell response . naturally , this can only be speculation , but notably we found that the presence of ido - specific cd4 t - cell responses correlated to the presence of cmv - responses . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as well as in the effector phases ( at the site of the tumor ) . with regard to the latter , ido may even by induced as an inflammation - induced counter - regulatory mechanism . counter - regulatory responses are important in the immune system as they help to limit the intensity and extent of immune responses , which otherwise could cause damage to the host . however , with regard to anti - cancer immunotherapy , counter - regulatory responses antagonize the ability to create an intense immune response against the tumor . counter - regulation differs from tolerance in the sense that counter - regulation is a secondary event , elicited only in response to immune activation . ido is known to be induced by both type i and ii interferons , which are likely to be found at sites of immune activation and inflammation [ 31 , 36 ] . in this respect , it should be mentioned that the susceptibility of tumor cells to lysis by ido - reactive t cells were increased by pre - incubation with ifn- . hence , in cancer immune therapy , the boosting of ido - specific immunity could have both direct and indirect effects ( fig . first of all , ido - specific , cytotoxic t cells are able to directly recognize and kill ido cancer cells . in fact , it may even be speculated that the measurable reactivity to this antigen in normal individuals contributes to immune surveillance against cancer . furthermore , the induction of ido - specific immune responses by therapeutic measures could function highly synergistic with additional anti - cancer immune therapy not only by eliminating cancer cells but in addition immune suppressive cells . by definition , anti - cancer immune therapies aim at the induction of an immunological activation and inflammation . the therapy aims to induce as much immune activation as possible ( within the limits of acceptable toxicity ) , and , accordingly , immune suppressive counter - regulation is not desired.fig . 3vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells adoptive transfer of ex vivo expanded tumor - infiltrating lymphocytes ( til ) after host lymphodepletion has the potential to significantly improve the prognosis of patients with metastatic melanoma . the impressive clinical responses associated with adoptive transfer of til urge that this strategy is pursued and investigated for the treatment of other types of cancer . in this regard , patient ido - specific t cells isolated and expanded from pbmc may well be an interesting supplement to the ongoing adaptive t - cell transfer strategies . it goes without saying that the possible introduction of autoimmunity and toxicity are the major worries when targeting a molecule like ido . however , the circulation of a measurable number of ido - specific t cells did not seem to cause autoimmunity . furthermore , since ido - specific t cells can be introduced by ifn or cpg this appears to be under solid control . in this regard , an interesting aspect of ido is that systemic inactivation at the organism level , either pharmacologically or genetically , does not appear to cause autoimmunity . we believe that the findings that presented here justified and warranted clinical testing to evaluate the efficiency and safety of ido - based vaccinations . hence , we initiated a phase i vaccination study , which is ongoing ( from june 2010 ) at center for caner immune therapy , copenhagen university hospital , herlev , in which patients with non - small cell lung cancer ( nsclc ) are vaccinated with a ido - derived peptide with montanide adjuvant ( www.clinicaltrials.gov ; nct01219348 ) . it has been suggested that ido may rather be involved in tolerance to non - self - antigens than self - antigens in situations where immune non - responsiveness may be important , for example , during pregnancy . in this respect , induction of ido immune - regulatory dendritic cells ( dc ) have been described to occur during infection of dcs with viruses and intracellular pathogens . in listeria monocytogenes infections , such ido dc seems to be involved in protection of the host from granuloma breakdown and pathogen dissemination in advanced human listeriosis . likewise , it was recently described that ido is increased in lymph nodes in cutaneous leishmania major infection . ido is implicated in suppressing t - cell immunity to parasite antigens and ido inhibition reduced local inflammation and parasite burdens , which suggest that ido were of benefit for the pathogen , not the host . during hiv infection , multiple mechanisms involving both viral and cellular components contribute to enhance ido expression and activity in an uncontrolled manner . among others , furthermore , it was recently described that ido is increased in hemodialysis ( hd ) patients compared to healthy donors . furthermore , ido suppresses adaptive immunity in hd patients as it is assessed by the response to hbv vaccination . hence , the targeting of ido could have synergistic effects in anti - viral immune therapy , for example , in hepatitis b vaccines . the fact that ido may be involved in tolerance to non - self - antigens might have major implications for ido - based immune therapy as boosting immunity to neoantigens , but not normal self - antigens , by triggering ido - specific t cells is very attractive . since ido - expressing cells might antagonize the desired effects of other immunotherapeutic approaches targeting ido - expressing cells by vaccination however , it was recently described that although ido might play biologically important roles in the host response to diverse intracellular infections like toxoplasma gondii , leishmaniasis , and herpes simplex virus , the nature of this role that being antimicrobial or immunoregulatory might depend on the pathogen . this should naturally been taken into account when exploring the possible use of ido - specific t cells in the clinic . finally , it should be mentioned that cd14 monocytes are major cmv target cells in vivo . cmv is the most immune dominant antigen to be encountered by the human immune system . monocytes are responsible for dissemination of the virus throughout the body during acute and late phase of infection . cmv has been shown to induce ido expression in monocytes , which has been suggested to confer an advantage to cmv - infected monocytes to escape t - cell responses . the cd8 t - cell response to cmv typically comprises a sizeable percentage of the cd8 t - cell repertoire in cmv - seropositive individuals . in light of this , it is possible that ido - specific t cells might function as support for the constitutive anti - cmv cd8 t - cell response . naturally , this can only be speculation , but notably we found that the presence of ido - specific cd4 t - cell responses correlated to the presence of cmv - responses .

indoleamine 2,3-dioxygenase ( ido ) is an immunoregulatory enzyme that is implicated in suppressing t - cell immunity in many settings including cancer . in recent years , we have described spontaneous cd8 + as well as cd4 + t - cell reactivity against ido in the tumor microenvironment of different cancer patients as well as in the peripheral blood of both cancer patients and to a lesser extent in healthy donors . we have demonstrated that ido - reactive cd8 + t cells were peptide - specific , cytotoxic effector cells , which are able to recognize and kill ido - expressing cells including tumor cells as well as dendritic cells . consequently , ido may serve as a widely applicable target for immunotherapeutic strategies with a completely different function as well as expression pattern compared to previously described antigens . ido constitutes a significant counter - regulatory mechanism induced by pro - inflammatory signals , and ido - based immunotherapy may consequently be synergistic with additional immunotherapy . in this regard , we have shown that the presence of ido - specific t cells boosted immunity against cmv and tumor antigens by eliminating ido+ suppressive cells and changing the regulatory microenvironment . the current review summarizes current knowledge of ido as a t - cell antigen , reports the initial results that are suggesting a general function of ido - specific t cells in immunoregulation , and discusses future opportunities .

IDO and immune suppression IDO and cancer CD8 responses against IDO CD4 responses against IDO Clinical perspectives Cancer Additional pathogenic settings

indoleamine 2,3-dioxygenase ( ido ) is an endogenous molecular mechanism that contributes to this immune regulation in a variety of settings . when ido dc are injected in vivo , they create suppression and anergy in antigen - specific t cells in the ln draining the injection site [ 3 , 25 ] . the phenotype of reprogrammed tregs after ido - blocking have been described as similar to that of polyfunctional t - helper cells co - expressing il-17 , il-22 , il-2 as well as tnf- . thus , ido suppression of pro - inflammatory processes may dominantly block effector t - cell responses to antigens encountered . in addition , ido may be expressed within the tumor by tumor cells as well as tumor stromal cells , where it inhibits the effector phase of immune responses . in this regard , it has been described that expression of ido in tumor cells is associated with an impaired prognosis . ido - expressing cd19 plasmacytoid dc isolated from tumor - draining ln mediate profound immune suppression and t - cell anergy in vivo [ 25 , 37 ] , whereas plasmacytoid dc from normal lns and spleen do not express ido . hence , ido has become a very attractive target for the design of new anticancer drugs and several ido inhibitors are under investigation in preclinical as well as in clinical studies . interestingly , recent studies have shown that the racemer d-1-mt has superior antitumor activity compared to the racemer l-1-mt . in this respect , ido2 expression has been found in human tumors , including gastric , colon , renal , and in pancreatic tumors ido2 expression have been found both in tumor cells as well as in immune cells in tumor - draining ln . we sat out to determine if and how ido itself serve as target for specific t - cell responses , which may be exploited for immune therapy . this was done by identifying and characterizing specific t cells spontaneously present among peripheral blood mononuclear cells ( pbmc ) isolated from cancer patients of different origin . in this regard , we described that peptides comprised in the ido protein sequence are spontaneously recognized by cytotoxic t cells ( ctl ) in cancer patients ( fig . 1principle of the processing pathway of ido peptides by ido - expressing cells ( red ) , for example , tumor cells or dendritic cells and the subsequent recognition by specific cd8 t cells ( green ; here entitled a supporter t cell ( tsup).the epitopes recognized by the t cells are short ido - derived peptides resulting from the degradation of intracellular ido protein , which are presented on the cell surface of hla molecules . t cells receive an activation signal through their t - cell receptor complex , leading to a variety of functional consequences , including release of cytokines and cytotoxic molecules principle of the processing pathway of ido peptides by ido - expressing cells ( red ) , for example , tumor cells or dendritic cells and the subsequent recognition by specific cd8 t cells ( green ; here entitled a supporter t cell ( tsup).the epitopes recognized by the t cells are short ido - derived peptides resulting from the degradation of intracellular ido protein , which are presented on the cell surface of hla molecules . t cells receive an activation signal through their t - cell receptor complex , leading to a variety of functional consequences , including release of cytokines and cytotoxic molecules first , we identified hla - restricted peptides within the ido protein to which spontaneous t - cell reactivity were detected in patients suffering from unrelated tumor types , i.e. such ido - reactive cd8 t cells were peptide - specific , cytotoxic effector cells . thus , ido - specific t cells effectively lysed ido cancer cell lines of different origin , such us colon carcinoma , melanoma , and breast cancer as well as directly ex vivo enriched leukemia cells . however , even more distinctive was our finding that ido - specific ctl recognized and killed ido , mature dc ; hence , ido - specific t cells were in addition able to kill immune - regulatory cells . thus , although ido has immune suppressive functions , the constitutive up regulation of ido expression in cancer patients seemed to induce ido - specific t - cell responses . thus , we found the apparent lack of tolerance against ido intriguing , since it suggested a more general role of ido - specific t cells in the regulation of the immune system . we hypothesized that such cells could take part in the control of immune homeostasis ; ido - specific cd8 t cells could play an important role by eliminating ido cells thereby suppressing and/or delaying local immune suppression . hence , we continued our search for possible ido - specific t - cell responses in healthy donors and found that circulating ido - specific , cytotoxic cd8 t cells indeed were present in healthy donors although not as frequent as in patients with cancer . furthermore , we were able to directly link the up regulation of ido with ido - specific t cells by showing that the addition of ido - inducing mediators like ifn- and cpg odn generated measurable numbers of cd8 ido - specific t cells among pbmc . to examine a possible immune - regulatory effect of ido - specific t cells , we examined their effect on t - cell immunity against viral or tumor - associated antigens . in this respect , we found that the presence of ido - specific cd8 t cells boosted cd8 t - cell responses against other antigens probably by eliminating ido suppressive cells ( fig . 2ido - specific t cells are able to boost specific immunity against virus or tumor antigens in human pbmc . a when stimulating pbmc with a known hla - restricted t - cell virus epitope and il-2 , epitope - specific t cells begin to expand due to activation by antigen presenting cells ( apc ) . in response to the subsequent production of cytokines like inf- , ido expression is induced and ido - expressing apc inhibit further expansion of virus - specific t cells both directly and indirectly through activation of tregs . b the addition of cytotoxic , ido - specific t cells ( tsup ) removes immune suppressive cells from the pbmc culture thereby facilitating further expansion of virus - specific t cells ido - specific t cells are able to boost specific immunity against virus or tumor antigens in human pbmc . a when stimulating pbmc with a known hla - restricted t - cell virus epitope and il-2 , epitope - specific t cells begin to expand due to activation by antigen presenting cells ( apc ) . in response to the subsequent production of cytokines like inf- , ido expression is induced and ido - expressing apc inhibit further expansion of virus - specific t cells both directly and indirectly through activation of tregs . b the addition of cytotoxic , ido - specific t cells ( tsup ) removes immune suppressive cells from the pbmc culture thereby facilitating further expansion of virus - specific t cells ido expression contributes to the strength and duration of a given immune response due to its inflammation - induced counter - regulatory function . thus , any supportive effect of ido - specific t cells on other immune cells may well be mediated in several direct and indirect manners . in this respect , the level of tryptophan was elevated , the frequency of tregs decreased , and the frequency of il-17 producing cells increased when ido - specific t cells were present , which taken together suggest an overall decrease in ido activity . furthermore , ido - specific t cells increased the overall production of both il-6 as well as the other pro - inflammatory cytokine tnf-. another possible effect of ido - specific t cells could be mediated through the metabolites of tryptophan , which have been shown to be directly toxic to cd8 t cells and cd4 th1 cells , but not th2 cells . the presence of activated ido - specific , cytotoxic t cells may screw the th - response in a th1-direction . hence , ido - specific , cytotoxic t cells may not only reduce ido - mediated suppression directly but in addition further immune suppression mediated by ido regulatory cells . recently , we identified spontaneous cd8 t - cell reactivity against the ido analogue ido2 in peripheral blood of both healthy donors and cancer patients . furthermore , we confirmed that ido2-reactive cd8 t cells were peptide - specific , cytotoxic effector t cells . hence , isolated and expanded ido2-specific t cells effectively lysed cancer cell lines of different origin , that is , colon carcinoma cells as well as breast cancer cells . likewise , ido2-specific t cells did not seem to support other immune responses in the same way as ido - specific , cytotoxic t cells . we speculated that cd4 ido - specific t cells releasing pro - inflammatory cytokines may play a role in the early phases of an immune response as a counter - response to the induced immune suppression facilitated by ido cells . hence , ido - specific th1-cells may delay local immune suppression if the activation of an ido - specific cd4 th1-response could overcome the immune suppressive actions of the ido protein , which are otherwise a result of the early expression of ido in maturing dc or macrophages . indeed , identified detectable numbers of specific cd4 t cells both in cancer patients as well as healthy individuals . we found that such ido - specific cd4 t cells released inf- as well as tnf-. although , we were able to detect both inf- and tnf- response toward ido in healthy donors , the responses were more frequent in cancer patients . the cancer relevance of these cd4 t cells were further underlined , since ido - reacting t cells in addition react toward dc pulsed with ido tumor lysates . furthermore , we detected frequent ido - specific cd4 t - cell responses when examining for il-17 release upon stimulation with the ido - derived cd4 epitope . ido - specific th17 cells seemed to exhibit a similar effector t - cell cytokine profile . thus , it is possible that ido - specific t cells could in addition belong to a foxp3 lineage of constitutively primed first responder th17-like t cells ; however , it should be strengthen that this is speculation . in this regard , we have previously described specific regulatory cd8 t cells in cancer patients , which recognized the immune suppressive heme oxygenase-1 . hence , the role of ido - specific cd4 t cells in immune - regulatory networks may be a complex balance between activation and inhibition depending on the microenvironment . in this regard , we have previously observed a decrease in il-10 when ido - specific cd8 t cells were present . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as well as in the effector phases ( at the site of the tumor ) . with regard to the latter , ido may even by induced as an inflammation - induced counter - regulatory mechanism . counter - regulatory responses are important in the immune system as they help to limit the intensity and extent of immune responses , which otherwise could cause damage to the host . in this respect , it should be mentioned that the susceptibility of tumor cells to lysis by ido - reactive t cells were increased by pre - incubation with ifn- . first of all , ido - specific , cytotoxic t cells are able to directly recognize and kill ido cancer cells . furthermore , the induction of ido - specific immune responses by therapeutic measures could function highly synergistic with additional anti - cancer immune therapy not only by eliminating cancer cells but in addition immune suppressive cells . 3vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells adoptive transfer of ex vivo expanded tumor - infiltrating lymphocytes ( til ) after host lymphodepletion has the potential to significantly improve the prognosis of patients with metastatic melanoma . in this regard , patient ido - specific t cells isolated and expanded from pbmc may well be an interesting supplement to the ongoing adaptive t - cell transfer strategies . however , the circulation of a measurable number of ido - specific t cells did not seem to cause autoimmunity . furthermore , since ido - specific t cells can be introduced by ifn or cpg this appears to be under solid control . in this regard , an interesting aspect of ido is that systemic inactivation at the organism level , either pharmacologically or genetically , does not appear to cause autoimmunity . ido is implicated in suppressing t - cell immunity to parasite antigens and ido inhibition reduced local inflammation and parasite burdens , which suggest that ido were of benefit for the pathogen , not the host . the fact that ido may be involved in tolerance to non - self - antigens might have major implications for ido - based immune therapy as boosting immunity to neoantigens , but not normal self - antigens , by triggering ido - specific t cells is very attractive . since ido - expressing cells might antagonize the desired effects of other immunotherapeutic approaches targeting ido - expressing cells by vaccination however , it was recently described that although ido might play biologically important roles in the host response to diverse intracellular infections like toxoplasma gondii , leishmaniasis , and herpes simplex virus , the nature of this role that being antimicrobial or immunoregulatory might depend on the pathogen . this should naturally been taken into account when exploring the possible use of ido - specific t cells in the clinic . in light of this , it is possible that ido - specific t cells might function as support for the constitutive anti - cmv cd8 t - cell response . naturally , this can only be speculation , but notably we found that the presence of ido - specific cd4 t - cell responses correlated to the presence of cmv - responses . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as well as in the effector phases ( at the site of the tumor ) . with regard to the latter , ido may even by induced as an inflammation - induced counter - regulatory mechanism . in this respect , it should be mentioned that the susceptibility of tumor cells to lysis by ido - reactive t cells were increased by pre - incubation with ifn- . first of all , ido - specific , cytotoxic t cells are able to directly recognize and kill ido cancer cells . furthermore , the induction of ido - specific immune responses by therapeutic measures could function highly synergistic with additional anti - cancer immune therapy not only by eliminating cancer cells but in addition immune suppressive cells . 3vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells adoptive transfer of ex vivo expanded tumor - infiltrating lymphocytes ( til ) after host lymphodepletion has the potential to significantly improve the prognosis of patients with metastatic melanoma . in this regard , patient ido - specific t cells isolated and expanded from pbmc may well be an interesting supplement to the ongoing adaptive t - cell transfer strategies . however , the circulation of a measurable number of ido - specific t cells did not seem to cause autoimmunity . hence , we initiated a phase i vaccination study , which is ongoing ( from june 2010 ) at center for caner immune therapy , copenhagen university hospital , herlev , in which patients with non - small cell lung cancer ( nsclc ) are vaccinated with a ido - derived peptide with montanide adjuvant ( www.clinicaltrials.gov ; nct01219348 ) . in this respect , induction of ido immune - regulatory dendritic cells ( dc ) have been described to occur during infection of dcs with viruses and intracellular pathogens . ido is implicated in suppressing t - cell immunity to parasite antigens and ido inhibition reduced local inflammation and parasite burdens , which suggest that ido were of benefit for the pathogen , not the host . the fact that ido may be involved in tolerance to non - self - antigens might have major implications for ido - based immune therapy as boosting immunity to neoantigens , but not normal self - antigens , by triggering ido - specific t cells is very attractive . since ido - expressing cells might antagonize the desired effects of other immunotherapeutic approaches targeting ido - expressing cells by vaccination however , it was recently described that although ido might play biologically important roles in the host response to diverse intracellular infections like toxoplasma gondii , leishmaniasis , and herpes simplex virus , the nature of this role that being antimicrobial or immunoregulatory might depend on the pathogen . this should naturally been taken into account when exploring the possible use of ido - specific t cells in the clinic . in light of this , it is possible that ido - specific t cells might function as support for the constitutive anti - cmv cd8 t - cell response . naturally , this can only be speculation , but notably we found that the presence of ido - specific cd4 t - cell responses correlated to the presence of cmv - responses .

in addition , ido is an important component of a regulatory system that allows long - term control of immune homeostasis as may be required by tolerance to self or during pregnancy . some of the biological effect of ido is mediated through local depletion of tryptophan , but is in addition mediated via immune modulatory tryptophan metabolites [ 4 , 30 ] . when ido dc are injected in vivo , they create suppression and anergy in antigen - specific t cells in the ln draining the injection site [ 3 , 25 ] . an ido - responsive signaling system in t cells has been identified , comprising the stress kinase gc non - derepressing 2 kinase ( gcn2 ) . constitutive ido expression in dc provides t cells with regulatory properties that block t - cell responses to antigenic stimulation . it has been described that exposure of tregs to pro - inflammatory cytokines like il-6 induce reprogramming of mature tregs to acquire a phenotype resembling pro - inflammatory th17 cells [ 6 , 49 , 51 ] . the phenotype of reprogrammed tregs after ido - blocking have been described as similar to that of polyfunctional t - helper cells co - expressing il-17 , il-22 , il-2 as well as tnf- . in a murine model , it was observed that tumor cells transfected with ido became resistant to immune eradication , even in mice in which a fully protective immune response had been established by immunization . ido - expressing cd19 plasmacytoid dc isolated from tumor - draining ln mediate profound immune suppression and t - cell anergy in vivo [ 25 , 37 ] , whereas plasmacytoid dc from normal lns and spleen do not express ido . it is believed that constitutive ido expression in dc in tumor - draining ln is induced by stimulation from tregs migrating from the tumor to the draining ln . hence , ido has become a very attractive target for the design of new anticancer drugs and several ido inhibitors are under investigation in preclinical as well as in clinical studies . ido2 functions like ido in tryptophan catabolism , but it has been found that d-1mt but not the l-1mt isomer selectively and potently inhibits ido2 activity suggesting that ido2 activity may have a role in the inhibition of immune responses to tumors . in this respect , ido2 expression has been found in human tumors , including gastric , colon , renal , and in pancreatic tumors ido2 expression have been found both in tumor cells as well as in immune cells in tumor - draining ln . despite the fact that neoplastic transformation is associated with the expression of immunogenic antigens , the immune system often fails to respond effectively and becomes tolerant toward these antigens . as described above ido plays a critical role in the tolerance induction and immune suppression of anti - cancer immune responses . we sat out to determine if and how ido itself serve as target for specific t - cell responses , which may be exploited for immune therapy . in this regard , we described that peptides comprised in the ido protein sequence are spontaneously recognized by cytotoxic t cells ( ctl ) in cancer patients ( fig . 1principle of the processing pathway of ido peptides by ido - expressing cells ( red ) , for example , tumor cells or dendritic cells and the subsequent recognition by specific cd8 t cells ( green ; here entitled a supporter t cell ( tsup).the epitopes recognized by the t cells are short ido - derived peptides resulting from the degradation of intracellular ido protein , which are presented on the cell surface of hla molecules . t cells receive an activation signal through their t - cell receptor complex , leading to a variety of functional consequences , including release of cytokines and cytotoxic molecules principle of the processing pathway of ido peptides by ido - expressing cells ( red ) , for example , tumor cells or dendritic cells and the subsequent recognition by specific cd8 t cells ( green ; here entitled a supporter t cell ( tsup).the epitopes recognized by the t cells are short ido - derived peptides resulting from the degradation of intracellular ido protein , which are presented on the cell surface of hla molecules . t cells receive an activation signal through their t - cell receptor complex , leading to a variety of functional consequences , including release of cytokines and cytotoxic molecules first , we identified hla - restricted peptides within the ido protein to which spontaneous t - cell reactivity were detected in patients suffering from unrelated tumor types , i.e. , melanoma , renal cell carcinoma and breast cancer by flow cytometry using hla / peptide tetramers as well as in elispot assays after in vitro stimulation but also in direct ex vivo assays . thus , ido - specific t cells effectively lysed ido cancer cell lines of different origin , such us colon carcinoma , melanoma , and breast cancer as well as directly ex vivo enriched leukemia cells . ido driven immune suppression is a general mechanism that has been described in a variety of human cancers and the immune responses against ido seem likewise to be relevant in cancers of unrelated origin , which emphasize the immunotherapeutic potential of ido . however , even more distinctive was our finding that ido - specific ctl recognized and killed ido , mature dc ; hence , ido - specific t cells were in addition able to kill immune - regulatory cells . thus , although ido has immune suppressive functions , the constitutive up regulation of ido expression in cancer patients seemed to induce ido - specific t - cell responses . thus , we found the apparent lack of tolerance against ido intriguing , since it suggested a more general role of ido - specific t cells in the regulation of the immune system . we hypothesized that such cells could take part in the control of immune homeostasis ; ido - specific cd8 t cells could play an important role by eliminating ido cells thereby suppressing and/or delaying local immune suppression . hence , we continued our search for possible ido - specific t - cell responses in healthy donors and found that circulating ido - specific , cytotoxic cd8 t cells indeed were present in healthy donors although not as frequent as in patients with cancer . furthermore , we were able to directly link the up regulation of ido with ido - specific t cells by showing that the addition of ido - inducing mediators like ifn- and cpg odn generated measurable numbers of cd8 ido - specific t cells among pbmc . to examine a possible immune - regulatory effect of ido - specific t cells , we examined their effect on t - cell immunity against viral or tumor - associated antigens . in this respect , we found that the presence of ido - specific cd8 t cells boosted cd8 t - cell responses against other antigens probably by eliminating ido suppressive cells ( fig . a when stimulating pbmc with a known hla - restricted t - cell virus epitope and il-2 , epitope - specific t cells begin to expand due to activation by antigen presenting cells ( apc ) . in response to the subsequent production of cytokines like inf- , ido expression is induced and ido - expressing apc inhibit further expansion of virus - specific t cells both directly and indirectly through activation of tregs . b the addition of cytotoxic , ido - specific t cells ( tsup ) removes immune suppressive cells from the pbmc culture thereby facilitating further expansion of virus - specific t cells ido - specific t cells are able to boost specific immunity against virus or tumor antigens in human pbmc . a when stimulating pbmc with a known hla - restricted t - cell virus epitope and il-2 , epitope - specific t cells begin to expand due to activation by antigen presenting cells ( apc ) . in response to the subsequent production of cytokines like inf- , ido expression is induced and ido - expressing apc inhibit further expansion of virus - specific t cells both directly and indirectly through activation of tregs . b the addition of cytotoxic , ido - specific t cells ( tsup ) removes immune suppressive cells from the pbmc culture thereby facilitating further expansion of virus - specific t cells ido expression contributes to the strength and duration of a given immune response due to its inflammation - induced counter - regulatory function . in this respect , the level of tryptophan was elevated , the frequency of tregs decreased , and the frequency of il-17 producing cells increased when ido - specific t cells were present , which taken together suggest an overall decrease in ido activity . furthermore , ido - specific t cells increased the overall production of both il-6 as well as the other pro - inflammatory cytokine tnf-. another possible effect of ido - specific t cells could be mediated through the metabolites of tryptophan , which have been shown to be directly toxic to cd8 t cells and cd4 th1 cells , but not th2 cells . recently , we identified spontaneous cd8 t - cell reactivity against the ido analogue ido2 in peripheral blood of both healthy donors and cancer patients . hence , isolated and expanded ido2-specific t cells effectively lysed cancer cell lines of different origin , that is , colon carcinoma cells as well as breast cancer cells . hence , the function and potential role of the ido2-specific class - i - restricted lymphocytes present in peripheral blood still need to be resolved . we speculated that cd4 ido - specific t cells releasing pro - inflammatory cytokines may play a role in the early phases of an immune response as a counter - response to the induced immune suppression facilitated by ido cells . hence , ido - specific th1-cells may delay local immune suppression if the activation of an ido - specific cd4 th1-response could overcome the immune suppressive actions of the ido protein , which are otherwise a result of the early expression of ido in maturing dc or macrophages . interestingly , we detected a correlation between patients harboring cd4 and cd8 responses against ido , which that class - i- and class - ii - restricted ido responses co - develop . furthermore , we detected frequent ido - specific cd4 t - cell responses when examining for il-17 release upon stimulation with the ido - derived cd4 epitope . thus , it is possible that ido - specific t cells could in addition belong to a foxp3 lineage of constitutively primed first responder th17-like t cells ; however , it should be strengthen that this is speculation . in this regard , we have previously described specific regulatory cd8 t cells in cancer patients , which recognized the immune suppressive heme oxygenase-1 . il-10 is mainly expressed by tregs that have been defined as a specialized subpopulation of t cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self - antigens [ 34 , 35 ] . hence , the role of ido - specific cd4 t cells in immune - regulatory networks may be a complex balance between activation and inhibition depending on the microenvironment . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as well as in the effector phases ( at the site of the tumor ) . counter - regulatory responses are important in the immune system as they help to limit the intensity and extent of immune responses , which otherwise could cause damage to the host . hence , in cancer immune therapy , the boosting of ido - specific immunity could have both direct and indirect effects ( fig . furthermore , the induction of ido - specific immune responses by therapeutic measures could function highly synergistic with additional anti - cancer immune therapy not only by eliminating cancer cells but in addition immune suppressive cells . the therapy aims to induce as much immune activation as possible ( within the limits of acceptable toxicity ) , and , accordingly , immune suppressive counter - regulation is not desired.fig . 3vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells adoptive transfer of ex vivo expanded tumor - infiltrating lymphocytes ( til ) after host lymphodepletion has the potential to significantly improve the prognosis of patients with metastatic melanoma . the impressive clinical responses associated with adoptive transfer of til urge that this strategy is pursued and investigated for the treatment of other types of cancer . hence , we initiated a phase i vaccination study , which is ongoing ( from june 2010 ) at center for caner immune therapy , copenhagen university hospital , herlev , in which patients with non - small cell lung cancer ( nsclc ) are vaccinated with a ido - derived peptide with montanide adjuvant ( www.clinicaltrials.gov ; nct01219348 ) . it has been suggested that ido may rather be involved in tolerance to non - self - antigens than self - antigens in situations where immune non - responsiveness may be important , for example , during pregnancy . in this respect , induction of ido immune - regulatory dendritic cells ( dc ) have been described to occur during infection of dcs with viruses and intracellular pathogens . in listeria monocytogenes infections , such ido dc seems to be involved in protection of the host from granuloma breakdown and pathogen dissemination in advanced human listeriosis . ido is implicated in suppressing t - cell immunity to parasite antigens and ido inhibition reduced local inflammation and parasite burdens , which suggest that ido were of benefit for the pathogen , not the host . the fact that ido may be involved in tolerance to non - self - antigens might have major implications for ido - based immune therapy as boosting immunity to neoantigens , but not normal self - antigens , by triggering ido - specific t cells is very attractive . since ido - expressing cells might antagonize the desired effects of other immunotherapeutic approaches targeting ido - expressing cells by vaccination however , it was recently described that although ido might play biologically important roles in the host response to diverse intracellular infections like toxoplasma gondii , leishmaniasis , and herpes simplex virus , the nature of this role that being antimicrobial or immunoregulatory might depend on the pathogen . naturally , this can only be speculation , but notably we found that the presence of ido - specific cd4 t - cell responses correlated to the presence of cmv - responses . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as well as in the effector phases ( at the site of the tumor ) . hence , in cancer immune therapy , the boosting of ido - specific immunity could have both direct and indirect effects ( fig . furthermore , the induction of ido - specific immune responses by therapeutic measures could function highly synergistic with additional anti - cancer immune therapy not only by eliminating cancer cells but in addition immune suppressive cells . 3vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells vaccine induced ido - specific t cells might kill ido suppressive antigen presenting cells ( apc ) as well as ido cancer cells both at the tumor site and in the draining lymph nodes . ido may exhibit its immune inhibitory functions both in the activation phases ( in the draining lymph node ) as wells as in the effector phases ( at the site of the tumor ) . hence , an ido - based cancer vaccine might work directly at the tumor site by the attack of cancer cells as well as stromal cells as well as in the draining lymph node by the attack of ido - expressing regulatory cells adoptive transfer of ex vivo expanded tumor - infiltrating lymphocytes ( til ) after host lymphodepletion has the potential to significantly improve the prognosis of patients with metastatic melanoma . the impressive clinical responses associated with adoptive transfer of til urge that this strategy is pursued and investigated for the treatment of other types of cancer . hence , we initiated a phase i vaccination study , which is ongoing ( from june 2010 ) at center for caner immune therapy , copenhagen university hospital , herlev , in which patients with non - small cell lung cancer ( nsclc ) are vaccinated with a ido - derived peptide with montanide adjuvant ( www.clinicaltrials.gov ; nct01219348 ) . it has been suggested that ido may rather be involved in tolerance to non - self - antigens than self - antigens in situations where immune non - responsiveness may be important , for example , during pregnancy . in this respect , induction of ido immune - regulatory dendritic cells ( dc ) have been described to occur during infection of dcs with viruses and intracellular pathogens . ido is implicated in suppressing t - cell immunity to parasite antigens and ido inhibition reduced local inflammation and parasite burdens , which suggest that ido were of benefit for the pathogen , not the host . the fact that ido may be involved in tolerance to non - self - antigens might have major implications for ido - based immune therapy as boosting immunity to neoantigens , but not normal self - antigens , by triggering ido - specific t cells is very attractive . since ido - expressing cells might antagonize the desired effects of other immunotherapeutic approaches targeting ido - expressing cells by vaccination however , it was recently described that although ido might play biologically important roles in the host response to diverse intracellular infections like toxoplasma gondii , leishmaniasis , and herpes simplex virus , the nature of this role that being antimicrobial or immunoregulatory might depend on the pathogen . naturally , this can only be speculation , but notably we found that the presence of ido - specific cd4 t - cell responses correlated to the presence of cmv - responses .

the flaviviridae family includes approximately 80 members divided into four genera : flavivirus , pestivirus , pegivirus , and hepacivirus . the flavivirus genus can be further divided into four categories : mosquito - borne , tick - borne , no known vector ( nkv ) , and insect - specific ( isf ) viruses . mosquito- and tick - borne flaviviruses , including dengue ( with four serotypes ) , japanese encephalitis virus ( jev ) , yellow fever virus ( yfv ) , saint louis encephalitis virus ( slev ) , west nile virus ( wnv ) , murray valley encephalitis virus ( mve ) , and tick - borne encephalitis virus ( tbev ) , are important pathogens responsible for human diseases , such as encephalitis , fever , and haemorrhagic fever . isf viruses are restricted to mosquitoes , such as culex and aedes , and include the aedes flavivirus ( aefv ) , calbertado virus , cell - fusing agent virus ( cfav ) , chaoyang virus , culex flavivirus ( cxfv ) , culex theileri flavivirus ( ctfv ) , kamiti river virus ( krv ) , lammi virus , nakiwogo virus ( nakv ) , nounane virus , quang binh virus ( qbv ) , and palm creek virus ( pcv ) [ 312 ] . mosquito- and tick - borne flaviviruses are transmitted to humans through haematophagous insects during blood meal feeding . viruses obtained from vertebrate host initially replicate in the midgut within 57 minutes of exposure . after escaping the midgut , the virus spreads to other tissues via haemolymph and can be transmitted through infected salivary glands and saliva [ 1315 ] . the period from the initial infection in the midgut to when the vector transmits the virus is termed the extrinsic incubation period ( eip ) , and this time period varies from 7 to 14 days [ 14 , 15 ] . however , viruses have been detected in the salivary glands of denv - infected aedes aegypti as early as 24 hours . thus , the eip depends on the virus , the mosquito , and certain environmental factors [ 14 , 15 ] . these viruses are approximately 11 kb in length , with a single open reading frame ( orf ) encoding a polyprotein that is co- and posttranslationally processed through cellular and viral proteases into three structural ( c , m , and e ) and seven nonstructural ( ns1 , ns2a , ns2b , ns3 , ns4a , ns4b , and ns5 ) proteins . the orf is flanked at the 5 and 3 ends by two untranslated regions ( 5 and 3 utrs ) that are important in viral translation and replication . the capsid ( c ) protein interacts with the viral genome to form the nucleocapsid , which is surrounded by a lipid bilayer containing the membrane ( m ) and envelope ( e ) proteins . interestingly , flavivirus infections in humans are self - limiting episodes with or without pathological consequences , despite a life - long or persistent infection in the vector , without apparent pathological effects [ 13 , 1820 ] . in contrast to acute infections , where the virus is eventually eliminated through the destruction of the host or the immune response , in persistent infections , the virus remains in the host cells for long periods of time , can be transmitted to other cells , and is not cleared . mosquito cell cultures have been used as suitable models to study flavivirus persistence ( table 1 ) because these models are easy to manage , they have several variables and can be controlled , and their results are typically easy to interpret . one of the common features observed in c6/36 persistently infected with denv [ 2224 ] is the presence of a cytopathic effect ( cpe ) during the first 47 weeks of infection , which becomes progressively imperceptible throughout the infection [ 23 , 24 ] . however , the viral proteins and genome are clearly detected in infected cells [ 22 , 25 ] . these findings are consistent with the nonpathogenic flavivirus infection in mosquitoes , suggesting the presence of a balance between virus replication and the antiviral host response [ 26 , 27 ] , which results in an infection that is controlled , but not eliminated , and not lethal [ 21 , 28 ] . the mechanisms underlying the establishment and maintenance of persistent flaviviral infection in insects are not completely understood , but apparently both host and viral factors are involved [ 2830 ] . for example , viral titres in the supernatants of c6/36 [ 22 , 24 , 31 ] or tra-171 cells persistently infected with denv display an oscillation pattern during persistent infection , suggesting the presence of defective interfering particles ( dis ) , one of the major self - controlling mechanisms for viral replication . because dis are more evident after several passages in cell lines , these particles are frequently observed in persistently infected cultures , and the oscillating phenomenon observed in long - term or persistently infected cell cultures reflects a mutual interaction between the parental virus , required for the replication of both defective and standard viruses , and the viral interference induced through dis in the viral titres . a more detailed description of dis is included in a subsequent section of this review . among the host factors that might participate in the maintenance of persistent infection are those associated with cell antiviral mechanisms , particularly rna interference ( rnai ) and the innate immune responses that control but do not eliminate viral infections . viral interference is a phenomenon whereby the infection with one virus ( primary ) inhibits infection with other viruses ( secondary ) . superinfection exclusion occurs when the primary virus inhibits the infection of the secondary virus . additionally , when both viruses reciprocally inhibit their infections , this is referred to as intrinsic interference [ 35 , 36 ] . when both viruses belong to the same family , the interference is referred to as homologous viral interference [ 3739 ] . heterotypic interference is a variation of this type of interference , and this phenomenon is observed when the viruses involved have different serotypes but belong to the same species , that is , denv serotypes . heterologous viral interference describes a negative interaction between viruses from different families . in some cases , the infection with two different viruses does not result in viral interference , and both infectious agents can coexist in the same cells . this phenomenon is called viral accommodation . based on the time of infection , mixed infections can be classified as coinfections when the two viruses interact with the host at the same time or superinfections when one virus invades the host prior to the second virus . because flaviviral infection persists for the life of the vector , the opportunities for competition or viral interference in the vector are higher than in humans , where the infection is only transient and the evaluation of viral interference during flavivirus infection is relatively easy to detect in cell lines , and it has primarily been examined in mosquito c6/36 cells ( from aedes albopictus ) [ 12 , 23 , 30 , 31 , 35 , 39 , 4148 ] , tra-171 ( from toxorhynchites amboinensis ) , sf9 ( from spodoptera frugiperda ) , c7 - 10 , and u4.4 cells ( from aedes albopictus ) [ 29 , 35 ] . homologous or heterotypic , but not heterologous , viral interference is frequently observed during superinfections ( table 2 ) , and this condition is particularly evident in persistently infected cells [ 23 , 31 , 32 , 44 , 49 ] . however , some exceptions have been documented [ 32 , 39 , 41 , 44 ] . for example , sinv inhibits denv replication in c6/36 cells infected 1 hour prior to denv-4 . the same cells persistently infected with aal dnv and reinfected with denv-2 showed an important reduction in the severity and mortality of the denv-2 infection compared with those of noninfected cells , and denv-2 titres were lower than in nave cells . interestingly , c6/36 cells persistently infected with three different viruses , including two flaviviruses , denv-2 and jev , result in a stable coinfection with the three viruses without apparent viral interference . these discrepancies indicate that the interference might vary among different flaviviruses and might be influenced through both the type of virus and the cell line used . it has been shown that the interval between the primary and secondary viral infections has an important effect on viral interference . the primary infection of c6/36 cells with either denv-2 or denv-4 , followed by a secondary infection 1 or 6 hours later with the opposite virus at the same multiplicity of infection ( moi ) , showed a stronger reduction in the virus titres of the secondary virus when the second infection was performed 6 hours after the first infection . it is likely that denv-4 infection requires more than 1 hour to establish conditions in which this virus will not be affected by sinv . some studies have reported that cxfv - infected c6/36 cells were reinfected with wnv 48 hours later and display significantly reduced titres of the secondary virus at 108 hours postinfection , indicating the presence of homologous viral interference . however , other studies have reported that when the same cxfv - infected cells were reinfected with wnv two days later , homologous viral interference was not observed . these differences could reflect the time of the secondary infections , but more experiments will be necessary to clarify this point . flavivirus coinfection experiments have primarily been performed using c6/36 cells [ 30 , 4143 , 46 , 48 , 50 ] , and recently aag2 cells have been used ; these infections typically result in homologous or heterologous viral interference ( table 3 ) . experiments in mosquitoes have revealed some similarities to the findings obtained using mosquito cell lines . for example , c6/36 cells infected with denv display heterotypic viral interference [ 30 , 31 , 42 , 43 , 50 , 51 ] . aedes aegypti mosquitoes orally infected with denv-2 and denv-3 simultaneously showed higher amounts of denv-2 viral rna than denv-3 viral rna , suggesting that the replicative advantage of denv-2 observed in c6/36 also applies to the vector . aedes aegypti mosquitoes orally coinfected with two clades of denv-2 ( ni-1 and ni-2b ) showed a higher replicative index for the ni-2b clade than the ni-1 clade . homologous viral interference has been observed in aedes aegypti mosquitoes infected with denv and yfv or mve and denv , culex tritaeniorhynchus mosquitoes infected with jev and mve , and culex quinquefasciatus mosquitoes infected with other flaviviruses , such as wnv and slev . the absence of heterologous viral interference between denv and chikv has been documented in c6/36 cells [ 31 , 46 ] , and apparently this phenomenon also occurs in mosquitoes . aedes albopictus mosquitoes intrathoracically inoculated with denv-1 and subsequently with chikv via the oral route at 7 or 13 days later showed the presence of the two viruses , without superinfection exclusion . mosquitoes orally coinfected with both viruses showed the same results , and the viruses could also be detected in saliva , indicating the absence of heterologous viral interference . however , studies have shown that aedes aegypti mosquitoes orally fed denv and chikv did not exhibit dual infection , either in the same pool or in individual mosquitoes , suggesting the presence of heterologous viral interference . the discrepancy of these results requires further investigation but apparently could reflect the virus strain used in these studies . interestingly , aedes albopictus mosquitoes coinfected with denv-4 and sinv display a reduction in both the infection and population dissemination rates compared with mosquitoes infected with denv-4 alone , even when the vector used exhibits a low sinv infection rate . although contradictory results have been reported for wnv and cxfv infections in c6/36 cells , the findings reported for these two groups in mosquitoes are consistent . culex quinquefasciatus mosquitoes inoculated with cxfv via an intrathoracic route and fed a blood meal containing wnv seven days later ( superinfection ) do not display superinfection exclusion . when both viruses were simultaneously inoculated ( coinfection ) , both viruses were detected in the same mosquito tissues through immunofluorescence , indicating a physical interaction between these infectious agents . in another study , culex pipiens mosquitoes persistently infected with cxfv and orally challenged with wnv displayed a reduction in the dissemination rates only during the early stages of the infection ( 7 days ) , but not during the late stages ( 14 days ) , with no effects on the transmission rates . although both are flaviviruses , the absence of homologous viral interference could reflect differences between these viruses . for example , although the mosquito rnai response might represent an antiviral response , the similarity between both viral genomes is not sufficient to generate a cross - reaction , particularly because the rnai response is based on a highly specific - sequence mechanism . the absence of competition for cellular factors could also be a contributing factor because the 5 and 3 utrs of cxfv have some differences compared with wnv . moreover , the mechanism of transmission between both viruses could also contribute to the absence of viral interference , as wnv is transmitted to the vertebrate host through saliva , and cxfv is maintained among the vector population through vertical transmission . unfortunately , the relevance of the viral interference in naturally infected vectors remains unknown , as several studies have demonstrated the presence of flavivirus coinfections that typically compete in both cell lines and mosquitoes . for example , aedes albopictus or aedes aegypti mosquitoes are naturally coinfected with more than one denv serotype [ 5557 ] . although several studies have been performed using pools of mosquitoes where the coinfection in the same individual is difficult to determine [ 55 , 57 ] , other studies have clearly established that one individual can be infected with both denv serotypes for example , the absence of viral interference between chikv and denv in cell lines has been confirmed through the evidence of dual chikv and denv-1 viral infections in aedes aegypti mosquitoes and in a single aedes albopictus mosquito . il ( usa ) , were naturally coinfected with cxfv and wnv , suggesting that in this vector homologous viral interference does not occur between these two flaviviruses , which are not closely related . the mechanisms involved in viral interference remain elusive , but the inhibition could occur at different levels of the viral replicative cycle , such as binding , entry , replication , and morphogenesis [ 19 , 37 ] . however , some studies have implicated several factors , such as dis and the rnai response , in homologous viral interference , and the competition for cellular replication factors and the innate immune response for heterotypic viral interference . the heterotypic viral interference against denv-3 observed in c6/36 cells with an acute denv-1 infection is affected through treatments with puromycin ( an inhibitor of the protein synthesis ) but not actinomycin d ( an inhibitor of the cellular transcription ) , suggesting that the viral interference is predominantly mediated through the virus instead of cellular factors . the competitive success of one virus could reflect the appropriation of the host cellular machinery for replication , which is directly related to the density of the viral genomes in the infected cells . this idea might explain why the heterotypic viral interference between two different denv serotypes is stronger when the interval between the primary and secondary infections is longer ( e.g. , between 1 and 6 hours ) and weaker in a coinfection compared with superinfection . this idea might also explain why this type of viral interference is observed in persistently infected cells . these virions contain a partially deleted genome , encoding generally normal viral structural proteins , with enough genomic information for replication and incorporation into mature virions ; however , these viruses can not perform their own replication . therefore , they require the assistance of a standard helper virus for this process . because the genome is shorter , dis are apparently preferentially replicated ; therefore , these viruses obtain the viral genome density necessary to specifically interfere with the replication of the parental virus [ 3335 ] . thus , dis represent a major self - controlling mechanism for viral replication , and these particles have been implicated in the establishment and maintenance of persistent viral infections [ 25 , 33 ] . the generation of dis is a common feature among viruses and has been observed in both rna and dna viruses [ 20 , 33 ] . the most commonly accepted mechanism for the generation of dis is the participation of the viral polymerase , particularly in rna - dependent viruses that lack proofreading activity . more recently , it has been proposed that drosophila melanogaster cells persistently infected with several nonflavivirus rna viruses generated cdnas from the genomes of defective interfering particles through cellular retrotransposon reverse transcriptase - mediated retrotranscription ( figure 1 ) , but this finding requires further investigation . defective viral genomes have been detected in mosquito cells persistently infected with flaviviruses , such as sle , jev , and denv , and also those that occur in nature , circulating between mosquitoes and human populations . these defective genomes have been implicated as cofactors in reducing the prevalence of denv and the severity of the disease in specific geographic areas . in addition to the participation of defective interfering particles / genomes in viral interference , some mutations have recently been associated with this phenomenon . culex quinquefasciatus mosquitoes orally superinfected with wnv containing a mutation in the 2k peptide ( v9 m ) overcome the homologous viral interference typically observed in superinfections . the 2k peptide is a 23-amino - acid peptide located between the ns4a and ns4b proteins , and this peptide is anchored to the membrane of the endoplasmic reticulum ( er ) . the 2k peptide has been implicated in virus replication and the evasion of the rnase l - mediated antiviral response . this point mutation likely confers replication advantages , protection against rna degradation , and/or the ability to compete with wild - type viruses under some circumstances . recent transcriptomic studies in aedes aegypti mosquitoes infected with denv-2 [ 16 , 6569 ] and culex pipiens quinquefasciatus infected with wnv have shown that these flaviviruses induce important and complex changes in gene expression . although these studies have been performed with denv and wnv , some preliminary experiments suggest that the expression of several genes is commonly activated through mosquito - borne flaviviruses [ 71 , 72 ] . although different results have been obtained , several studies have suggested that during flaviviral infection genes associated with specific pathways are activated to maintain the proper condition of the cell for viral replication , such as metabolism ( nucleotide , lipid , amino acid , and energy ) [ 6568 ] , oxidative stress [ 67 , 68 ] and transcription / translation [ 6568 ] , and other pathways associated with the antiviral response [ 16 , 65 , 67 , 68 , 73 ] . actually , some genes have been associated with the susceptibility or refractoriness to denv infection in aedes mosquitoes . for example , genes associated with the inhibition of the apoptosis were identified in susceptible and proapoptotic genes in refractory mosquitoes . some genes associated with the immune response are upregulated in refractory mosquito strains [ 68 , 73 ] . the requirement for cellular factors for viral replication could represent a homologous / heterotypic viral interference mechanism . the primary virus might sequester the host factors essential for the replication of the secondary virus [ 35 , 37 ] , which might result in viral interference , particularly during a superinfection ( figure 1 ) . the absence of heterologous viral interference among nonrelated viruses might reflect the requirements for different cellular and viral factors to complete the viral replicative cycles . for example , denv-2 is an rna virus that assembles in the cytoplasm , while aal dnv is a dna virus that assembles in the nucleus . when the viruses involved in the infection are closely related , the requirements for host factors are the same and the viral interference is stronger . accordingly , c6/36 cells with a primary nhuv infection and a superinfection with wnv , slev , or jev displayed a significant reduction in the secondary virus titre ( homologous interference through superinfection ) . an analysis of the secondary structure of the nhuv 3 utr revealed similarities with the yfv and jev serogroups and viruses in the tick - borne flavivirus clade , suggesting that the requirements for cellular factors and the mechanism for viral translation / replication might be similar among these viruses , thereby establishing conditions for competition . in aag2 cells coinfected with two denv-2 clades from nicaragua , clade ni-2b showed a replicative preference over clade ni-1 . both viruses exhibited differences in some amino acids in two structural and four nonstructural proteins and showed four nucleotide variations in the 5 and 3 utrs . these changes could influence some steps of the viral replicative cycle , resulting in advantages of one virus over the other . interestingly , although aag2 cells are persistently infected with cfav , the denv infection is not affected , probably by the same cause that cxfv does not interfere with wnv replication in mosquitoes [ 45 , 47 ] . because members of the flavivirus genus have ssrna+ , these viruses use a common strategy similar to other rna viruses to translate the genome ( revised in ) . these viruses release viral rna into the cytoplasm for recognition by both the viral replication apparatus and the translational cell machinery to assemble the rna replication complex on cellular membranes [ 7678 ] . viral translation and replication can not occur at the same time because the ribosome moves from the 5 end towards the 3 end of the rna to translate the proteins , whereas the rna viral polymerase generally binds to the 3 end of the same rna molecule to initiate replication . thus , it is necessary to identify a balance between these two viral events [ 79 , 80 ] . this balance or switch is performed through ribonucleoprotein complexes ( rnps ) located in subcellular membranes , as described above . the flavivirus rna viral genome contains two utrs that have various functions , such as initiating and regulating viral translation , as well as viral complex replication and assembly at membrane fractions , through interactions with host cellular factors and nonstructural proteins that form rnps ( revised in ) . most of these factors have been identified in mammalian cells , including bhk21 ( for baby hamster kidney ) , vero ( green monkey kidney ) , hek293a ( human embryonic kidney-293 ) , k562 ( human erythroleukaemia cells ) , and u937 ( a human monocytic cell line derived from a patient with generalized histiocytic lymphoma ) cells . these cellular factors have been identified as elongation factor 1 ( ef1 ) , polypyrimidine tract binding ( ptb ) protein , the autoantigen - la ( la protein ) , calreticulin , and nuclear factor 90 ( nf90 ) [ 8285 ] . however , little information is available about the discovery of the cellular factors in mosquito cells , such as c6/36 cells , compared with mammalian cells . notably , ef1 , the la protein , eukaryotic initiation factor 5 ( eif5 ) , 40s ribosomal protein s6 , and 60s ribosomal protein l4 ( table 4 ) have been implicated in viral replication in mosquitoes [ 72 , 83 , 86 , 87 ] . the cellular factors mentioned above , such as ef1 ( highly conserved between different host species as mammals , chicken , and mosquitoes ) , translation initiation factor eif5 , and ribosomal proteins s6 and l4 , participate in several steps of the translation process [ 8891 ] , except autoantigen - la , a nuclear protein involved in rna polymerase iii transcription termination [ 92 , 93 ] and small rna biogenesis , which acts as a chaperone and contributes to the retention of nascent rna in the nucleus or stabilizes the rna structure . the relocalization of this protein to the cytoplasm has been observed in several rna viral infections , including flavivirus infections . additionally , eif5 functions in cell proliferation , cell viability , and cell - cycle progression , and it is essential for cell survival . a recent study reported that eif5 possesses a higher similarity and shorter evolutionary distance in insects than in other organisms , suggesting that this protein plays an important and common physiological role . the ribosomal protein s6 ( rps6 ) is a component of the 40s ribosomal subunit , while the ribosomal protein l4 ( rpl4 or rpl1 ) is a component of the 60s ribosomal subunit . while the precise function of rps6 is currently under investigation , studies have shown that this protein is involved in regulating cell size , cell proliferation , and glucose homeostasis through the selective translation of particular classes of mrnas . both ribosomal proteins were discovered using tandem affinity purification assays in mosquito cells infected with wnv and denv . ef1 is involved in wnv and denv replication and could act as a chaperone targeting the rna to the viral replication compartments , as this protein is associated with the er membrane fraction where rna viral replication complexes are assembled [ 86 , 88 ] . in infected mammalian cells , ef1 was colocalized with the wnv and denv ns3 and ns5 ( the viral rna - dependent rna polymerase - rdrp ) proteins . this evidence suggests that ef1 is important for minus - strand rna synthesis through interactions with the viral rna and the replication complex proteins , including ns3 and ns5 [ 90 , 97 ] , and these interactions might be similar in mosquito cells . furthermore , the shih group results suggested that eif5 is upregulated in denv-2-infected c6/36 cells , and this upregulated expression might play a role in preventing mosquito cell death in response to the viral infection . thus , eif5 facilitates continued viral growth and potentially persistent infection in mosquito cells , without affecting viral replication . this finding is supported by results in c6/36 cells treated with ciclopirox olamine ( cpo ) , an eif5 inhibitor , which did not affect the viral titres . these results provide evidence that eif5 plays a role in the interactions between the viruses and components of the host cells . however , the interaction between the ribosomal proteins s6 and l4 and the ns2a and ns4b nonstructural viral proteins is likely involved in flavivirus rna translation . this report provided the first evidence of the binding of flaviviral proteins to either 40s or 60s ribosomal proteins . barr virus noncoding rna eber1 , which recruits ribosomal protein l22 during infection , presumably , to aid in viral replication , or the hepatitis c ( hcv ) ires region , which associates with the ribosomal s5 protein to position the hcv rna on the 40s ribosomal subunit during translation . the requirement of these translation factors during the viral genome processing or viral replication suggests that flaviviral proteins might have evolved mechanisms to bind and localize the proteins in appropriate compartments of the cell . moreover , the human la protein ( 47 kda ) in mosquito cells has a molecular weight of 50 kda and potentially binds to the sinv rna . because the la protein might function as an rna chaperone , the human la protein interacts with the viral 3 sl rna , and sirna - mediated downregulation represses jev replication . in addition , in denv , the human la protein interacts with two viral nonstructural proteins that form the denv replicase complex : ns3 and ns5 . however , the mosquito la protein also binds to the 3 utr of positive and negative polarity denv rnas and relocates to the cytoplasm in c6/36-infected cells . these findings suggest a role for the human la protein in flaviviral replication as a component of the rnp . for example , denv can be grown in human , primate , and mosquito cells but first requires viral growth in mosquitoes . therefore , characterizing the cellular and viral proteins required for denv translation and replication in mosquito cells is essential to understand the replicative cycle of the virus . after analyzing this information , we proposed that the association of ef1 and the la protein , which function as chaperones , with eif5 and ribosomal proteins s6 and l4 , components of the translational machinery , could stimulate flavivirus translation in a favourable context . denv infections induce the drastic rearrangement of the er membranes , resulting in complex membranous structures that promote the switch from translation to viral replication once viral and/or cellular factors facilitate communication of the utrs , thereby maximizing translation and replication as global processes . in addition to stabilizing rna , the formation of viral ribonucleoprotein complexes ( vrnps ) between the host cell factors and/or the viral proteins , as described above , likely regulates various steps of the viral life cycle . the formation of vrnps might establish the genomic architecture and facilitate various processes in the viral life cycle , and the fact that flaviviruses share several cellular factors for translation / replication might explain the homologous viral interference frequently observed in both co- and superinfections in mosquitoes and mosquito - derived cell lines . the rna interference ( rnai ) response is one of the most important antiviral mechanisms in insects . this response includes three main pathways to generate small rnas that regulate gene expression : small interfering rna ( sirna ) , microrna ( mirna ) , and piwirna . the sirnas are induced through the presence of double - stranded rna ( e.g. , as a result of replicative intermediates or secondary structure of genome of rna viruses ) , which is processed through the rnase iii - like enzymes dicer-1 or dicer-2 ( primarily dicer-2 ) , to generate small dsrna ( 21 to 25 nt ) . with the assistance of the r2d2 protein , these small rnas are loaded into the rna - induced silencing complex ( risc ) involving the argonaute proteins ( ago-1 or ago-2 ) . the synthesized sirnas perfectly compliment the target rna , resulting in rna degradation through the exosome or the exoribonuclease xrn1/pacman [ 100 , 102 ] . the participation of the sirna response in the regulation of flavivirus infection has been studied in both mosquito cells lines [ 74 , 103107 ] , aedes aegypti , culex pipiens quinquefasciatus , and even in drosophila melanogaster . several studies have indicated that the sirna response is activated during denv [ 74 , 104 , 106 , 107 ] or wnv [ 103 , 105 , 108 ] infections , and the susceptibility to these flaviviruses increases when components of the sirna pathway are silenced [ 103 , 104 , 106 ] . moreover , c6/36 cells , which have a deficiency in the activity of the dicer-2 enzyme , are more susceptible to denv infection than dicer-2 competent cells [ 74 , 105 , 107 ] , and the natural polymorphism of the dicer-2 gene has been associated with the susceptibility of aedes aegypti mosquitoes to some isolates of denv-1 . although mosquitoes have a competent rnai pathway to degrade viral rna , flaviviruses have mechanisms to evade the rnai response and establish a persistent and noncytopathic infection in the vector . one of these mechanisms might be the sequestration of the viral replicative complex into endoplasmic reticulum - derived vesicles ; thus , the double - stranded viral rnas generated during the viral replication can not be sensed through dicer-2 [ 73 , 103 , 104 , 110 ] . additionally , flaviviruses mutate at high rates , reflecting the absence of proofreading activity or rna polymerases . because the rnai response is highly sequence - specific , these mutations in the viral genome might be another mechanism to evade this response . for example , mosquito and mammal cells infected with flaviviruses generate subgenomic rna corresponding to the 3 utr of the viral genome through the participation of the cellular exoribonuclease xrn1 , and this subgenomic rna binds to dicer-2 and dicer-1 , inhibiting the activity of these enzymes in vitro [ 1 , 102 ] . in the case of denv moreover , the dis in the sirna pathway could be responsible for the homologous / heterotypic viral interference and persistent infection . drosophila melanogaster cells persistently infected with several nonflavivirus rna viruses generated cdnas from the genomes of dis through cellular ltr - retrotransposon reverse transcriptase - mediated retrotranscription . these cdnas are apparently transcribed through the cellular transcription machinery to generate small double - stranded rnas via several mechanisms , which are the source of the dicer-2-risc - produced interfering rnas ( rnai ) [ 21 , 28 ] that control viral replication ( figure 1 ) . interesting similar results were obtained with the sindbis virus , a member of togaviridae , which shares similarities in the replicative cycle and genome type with members of the flaviviridae family . this finding suggests that a similar process might occur with flaviviruses ; however , this idea requires further investigation . furthermore , several retrotransposons with retrotranscriptase activity have been detected in aedes aegypti [ 112114 ] , aedes albopictus , and aedes polynesiensis , suggesting that the same mechanism could operate in mosquitoes infected with flaviviruses ; however , this hypothesis requires further investigation . the immune response in the mosquito might determine the susceptibility of these insects to arboviruses [ 115117 ] and could be implicated in both the viral interference phenomenon and persistence . the immune response in mosquitoes is primarily regulated through three signalling pathways : immune deficiency ( imd ) , which mediates the production of antimicrobial peptides with activity against gram - negative bacteria ; the toll - mediated pathway , which is involved in the defence against viruses , gram - positive bacteria , and fungi and also stimulates the secretion of some antimicrobial peptides ; and the janus kinase - signal transducer and activator of transcription ( jak - stat ) pathway , which has been strongly associated with the antiviral response in aedes mosquitoes [ 118120 ] . among the three main pathways involved in the immune response in mosquitoes , the toll [ 14 , 15 , 72 , 115 , 116 , 120 ] and jak - stat [ 15 , 72 , 115 , 116 , 119 ] pathways are apparently more important for controlling flavivirus infection . however , recent studies have suggested that the imd response could play a secondary role [ 15 , 120 ] . the participation of the immune response during flavivirus infection has primarily been studied with denv [ 1416 , 115 , 116 , 119121 ] in aedes aegypti mosquitoes [ 1416 , 115 , 116 , 119 ] , and this response has recently been characterized in the aag2 aedes aegypti cell line . however , the transcriptome analysis of aedes aegypti mosquitoes infected with denv , yfv , or wnv showed similar overall gene expression , indicating a conserved transcriptome signature . during infections with denv and other mosquito - borne flaviviruses , the first virus - vector interaction occurs in the midgut , where the immune response , known as the local immune response , is initially activated [ 15 , 115 , 119 ] . however , the systemic immune response represented by the abdominal fat body has also been implicated . more recently , it has been reported that denv upregulates the expression of salivary gland genes in aedes aegypti mosquitoes that encode for proteins involved in the immune response and also induces the expression of a putative antibacterial , cecropin - like peptide , which exhibits activity against the four denv serotypes and chikv . although the participation of the mosquito immune system has been clearly demonstrated during single flavivirus infection , there are no studies concerning the participation of this response in viral interference during co- or superinfections . moreover , it is not clear whether immune system activation through a primary virus blocks the infection of a secondary virus . however , there is some indirect evidence suggesting the participation of the immune response in the viral interference phenomenon . culex quinquefasciatus mosquitoes sequentially infected with wnv and slev through an oral route displayed lowered susceptibility to infection and lower dissemination rates of the second virus , but similar infection rates to the first virus . some mosquitoes become infected with two viruses , but only one virus escapes to the midgut , suggesting the participation of the midgut infection barrier during secondary infection . because wnv and slev belong to the same antigenic complex and both viruses are primarily transmitted through culex spp . , the primary virus likely blocks the infection of the secondary virus through the induction of the host antiviral response . however , studies using the mosquito bacterial microflora have suggested that prior immune system activation influences the course of viral infection . the eradication of the endogenous bacterial flora in the midgut of aedes aegypti mosquitoes treated with an antibiotic increased the viral titres in the mosquitoes infected with denv-2 compared with a nontreated group . this effect is coincident with the ability of the bacterial flora to activate the toll pathway and produce several antimicrobial peptides , such as cecropin , defensin , attacin , and gramicidin , in the midgut [ 15 , 121 ] . a similar result was obtained with mosquitoes previously fed with either blood or sugar meals contaminated with proteus sp . and paenibacillus sp . . apparently , immune system activation through the microflora is not only limited to the midgut but also present in the abdominal fat body . interestingly , denv-2 apparently reduces the microbial load in the midgut through the secretion of lysozyme c and cecropin g , indicating an interrelationship between the virus- and bacteria - induced immune responses . a similar mechanism might operate during co- and superinfection with different flaviviruses and in homologous or heterotypic viral interference . however , as previously discussed , equilibrium between the viral replication and the antiviral response exists during persistent infection in mosquito cells [ 26 , 27 ] , and the precise participation of the immune response in persistent infection in mosquito cells and the mechanism used by the flaviviruses to circumvent this response remain unknown . although 80% of c6/36 cells persistently infected with jev were positive for the ns5 protein based on immunofluorescence analysis , there is typically little correlation between the number of cells positive for the viral antigen and the release of infectious virus particles . this finding has been reported in c6/36 cells persistently infected with denv and in different mosquito cells lines persistently infected with slev , reflecting the inhibition of virus particle production [ 22 , 123 ] or the fact that initially all cells in culture support virus replication , but some cells stop replicating the virus and become resistant to superinfection during the late stages of infection . eventually , the cells regain sensitivity to reinfection by the virus in the medium or the residual viral rna in the cell , and thus the culture remains persistently infected . however , another explanation is that a soluble antiviral factor is secreted from persistently infected cells . pretreatment of tra-171 cells with the supernatant from the same cells persistently infected with denv reduces the viral titres of the four denv serotypes . similarly , c6/36 cells pretreated with filtered culture medium from the same cells persistently infected with the four serotypes of denv become resistant to reinfection with the same denv viruses ( heterotypic viral interference ) but remain susceptible to infection with chikv . this effect is eliminated when the culture medium is preheated at 56c for 30 minutes , suggesting the presence of a soluble , thermolabile , anti - denv - specific factor . consistent with these findings , interferon and interferon - like substances are involved in heterologous interference , and vago , a peptide with antiviral activity , has recently been identified and shown to be secreted from wnv - infected culex mosquito cells , culex pipiens mosquitoes , and denv - infected rml12 cells ( from aedes albopictus ) [ 124 , 125 ] . vago induction is dicer-2-dependent , but rnai - independent through the activation of traf , which cleaves the rel2 protein ( a homologue of mammalian nfb ) and facilitates the translocation of this protein to the nucleus to activate the vago gene . however , the antiviral activity of vago is mediated through the jak - stat pathway . although vago expression is decreased in culex pipiens mosquitoes at 8 days postinfection with wnv , the participation of vago in viral interference during persistent infection can not be excluded and will require further investigation . flaviviral infection of the vectors is long - lived or persistent , and the mechanisms that participate in the establishment of this type of infection remain unknown . the present data suggest that this type of infection is a multifactorial phenomenon involving factors from the virus , such as defective interfering particles / genomes , and the host , such as the immune response , rna interference , and cellular factors . the characterization of the mechanisms that participate in viral persistence is important to obtain a better understanding of the complex interactions between flaviviruses and mosquito cells to develop new strategies for control . the cocirculation of two or more flaviviruses / alphaviruses in the same geographic area increases the opportunity for interviral interactions , and in most cases , the interaction between two mosquito - borne flaviviruses results in homologous viral interference , indicating that these viruses share mechanisms that regulate replication and several cellular factors required for viral translation / replication , a common requirement for different flaviviruses , which favours the conditions for competition . the studies of viral interference in cell lines typically correspond to the results observed in mosquitoes , but the relevance of these findings to the dynamics of viral infection and transmission in nature requires additional studies . the viral interference experiments have primarily been performed with the aedes albopictus mosquito cell line c6/36 , a traditional cell line used as a model to study flavivirus infection in mosquitoes . however , recent studies have shown that this cell line contains a defective rnai pathway , reflecting a defect in the dicer-2 protein [ 74 , 105 ] . although the rnai pathway is not the only factor that might participate in maintaining a persistent infection or explaining viral interference , these data indicate that these types of experiments should be performed using other cell lines , such as ccl-125 , u4.4 , and c7 - 10 cells , and vectors , such as aedes and culex mosquitoes . moreover , these cell lines should be used to characterize the cellular factors involved in flavivirus replication and the pathways involved in the immune response to provide a cellular model to study the relevance of these phenomena in the transmission and epidemiology of mosquito - borne flaviviruses .

mosquito - borne flaviviruses are important pathogens for humans , and the detection of two or more flaviviruses cocirculating in the same geographic area has often been reported . however , the epidemiological impact remains to be determined . mosquito - borne flaviviruses are primarily transmitted through aedes and culex mosquitoes ; these viruses establish a life - long or persistent infection without apparent pathological effects . this establishment requires a balance between virus replication and the antiviral host response . viral interference is a phenomenon whereby one virus inhibits the replication of other viruses , and this condition is frequently associated with persistent infections . viral interference and persistent infection are determined by several factors , such as defective interfering particles , competition for cellular factors required for translation / replication , and the host antiviral response . the interaction between two flaviviruses typically results in viral interference , indicating that these viruses share common features during the replicative cycle in the vector . the potential mechanisms involved in these processes are reviewed here .

1. Introduction 2. Persistence 3. Viral Interference 4. Defective Interfering Particles 5. Cellular Factors 6. RNA Interference 7. The Immune Response in Mosquitoes 8. Concluding Remarks

the flavivirus genus can be further divided into four categories : mosquito - borne , tick - borne , no known vector ( nkv ) , and insect - specific ( isf ) viruses . mosquito- and tick - borne flaviviruses , including dengue ( with four serotypes ) , japanese encephalitis virus ( jev ) , yellow fever virus ( yfv ) , saint louis encephalitis virus ( slev ) , west nile virus ( wnv ) , murray valley encephalitis virus ( mve ) , and tick - borne encephalitis virus ( tbev ) , are important pathogens responsible for human diseases , such as encephalitis , fever , and haemorrhagic fever . isf viruses are restricted to mosquitoes , such as culex and aedes , and include the aedes flavivirus ( aefv ) , calbertado virus , cell - fusing agent virus ( cfav ) , chaoyang virus , culex flavivirus ( cxfv ) , culex theileri flavivirus ( ctfv ) , kamiti river virus ( krv ) , lammi virus , nakiwogo virus ( nakv ) , nounane virus , quang binh virus ( qbv ) , and palm creek virus ( pcv ) [ 312 ] . mosquito- and tick - borne flaviviruses are transmitted to humans through haematophagous insects during blood meal feeding . the period from the initial infection in the midgut to when the vector transmits the virus is termed the extrinsic incubation period ( eip ) , and this time period varies from 7 to 14 days [ 14 , 15 ] . interestingly , flavivirus infections in humans are self - limiting episodes with or without pathological consequences , despite a life - long or persistent infection in the vector , without apparent pathological effects [ 13 , 1820 ] . in contrast to acute infections , where the virus is eventually eliminated through the destruction of the host or the immune response , in persistent infections , the virus remains in the host cells for long periods of time , can be transmitted to other cells , and is not cleared . these findings are consistent with the nonpathogenic flavivirus infection in mosquitoes , suggesting the presence of a balance between virus replication and the antiviral host response [ 26 , 27 ] , which results in an infection that is controlled , but not eliminated , and not lethal [ 21 , 28 ] . for example , viral titres in the supernatants of c6/36 [ 22 , 24 , 31 ] or tra-171 cells persistently infected with denv display an oscillation pattern during persistent infection , suggesting the presence of defective interfering particles ( dis ) , one of the major self - controlling mechanisms for viral replication . because dis are more evident after several passages in cell lines , these particles are frequently observed in persistently infected cultures , and the oscillating phenomenon observed in long - term or persistently infected cell cultures reflects a mutual interaction between the parental virus , required for the replication of both defective and standard viruses , and the viral interference induced through dis in the viral titres . among the host factors that might participate in the maintenance of persistent infection are those associated with cell antiviral mechanisms , particularly rna interference ( rnai ) and the innate immune responses that control but do not eliminate viral infections . viral interference is a phenomenon whereby the infection with one virus ( primary ) inhibits infection with other viruses ( secondary ) . when both viruses belong to the same family , the interference is referred to as homologous viral interference [ 3739 ] . heterotypic interference is a variation of this type of interference , and this phenomenon is observed when the viruses involved have different serotypes but belong to the same species , that is , denv serotypes . in some cases , the infection with two different viruses does not result in viral interference , and both infectious agents can coexist in the same cells . based on the time of infection , mixed infections can be classified as coinfections when the two viruses interact with the host at the same time or superinfections when one virus invades the host prior to the second virus . because flaviviral infection persists for the life of the vector , the opportunities for competition or viral interference in the vector are higher than in humans , where the infection is only transient and the evaluation of viral interference during flavivirus infection is relatively easy to detect in cell lines , and it has primarily been examined in mosquito c6/36 cells ( from aedes albopictus ) [ 12 , 23 , 30 , 31 , 35 , 39 , 4148 ] , tra-171 ( from toxorhynchites amboinensis ) , sf9 ( from spodoptera frugiperda ) , c7 - 10 , and u4.4 cells ( from aedes albopictus ) [ 29 , 35 ] . homologous or heterotypic , but not heterologous , viral interference is frequently observed during superinfections ( table 2 ) , and this condition is particularly evident in persistently infected cells [ 23 , 31 , 32 , 44 , 49 ] . interestingly , c6/36 cells persistently infected with three different viruses , including two flaviviruses , denv-2 and jev , result in a stable coinfection with the three viruses without apparent viral interference . however , other studies have reported that when the same cxfv - infected cells were reinfected with wnv two days later , homologous viral interference was not observed . flavivirus coinfection experiments have primarily been performed using c6/36 cells [ 30 , 4143 , 46 , 48 , 50 ] , and recently aag2 cells have been used ; these infections typically result in homologous or heterologous viral interference ( table 3 ) . homologous viral interference has been observed in aedes aegypti mosquitoes infected with denv and yfv or mve and denv , culex tritaeniorhynchus mosquitoes infected with jev and mve , and culex quinquefasciatus mosquitoes infected with other flaviviruses , such as wnv and slev . mosquitoes orally coinfected with both viruses showed the same results , and the viruses could also be detected in saliva , indicating the absence of heterologous viral interference . however , studies have shown that aedes aegypti mosquitoes orally fed denv and chikv did not exhibit dual infection , either in the same pool or in individual mosquitoes , suggesting the presence of heterologous viral interference . when both viruses were simultaneously inoculated ( coinfection ) , both viruses were detected in the same mosquito tissues through immunofluorescence , indicating a physical interaction between these infectious agents . the absence of competition for cellular factors could also be a contributing factor because the 5 and 3 utrs of cxfv have some differences compared with wnv . moreover , the mechanism of transmission between both viruses could also contribute to the absence of viral interference , as wnv is transmitted to the vertebrate host through saliva , and cxfv is maintained among the vector population through vertical transmission . although several studies have been performed using pools of mosquitoes where the coinfection in the same individual is difficult to determine [ 55 , 57 ] , other studies have clearly established that one individual can be infected with both denv serotypes for example , the absence of viral interference between chikv and denv in cell lines has been confirmed through the evidence of dual chikv and denv-1 viral infections in aedes aegypti mosquitoes and in a single aedes albopictus mosquito . the mechanisms involved in viral interference remain elusive , but the inhibition could occur at different levels of the viral replicative cycle , such as binding , entry , replication , and morphogenesis [ 19 , 37 ] . however , some studies have implicated several factors , such as dis and the rnai response , in homologous viral interference , and the competition for cellular replication factors and the innate immune response for heterotypic viral interference . the heterotypic viral interference against denv-3 observed in c6/36 cells with an acute denv-1 infection is affected through treatments with puromycin ( an inhibitor of the protein synthesis ) but not actinomycin d ( an inhibitor of the cellular transcription ) , suggesting that the viral interference is predominantly mediated through the virus instead of cellular factors . the competitive success of one virus could reflect the appropriation of the host cellular machinery for replication , which is directly related to the density of the viral genomes in the infected cells . these virions contain a partially deleted genome , encoding generally normal viral structural proteins , with enough genomic information for replication and incorporation into mature virions ; however , these viruses can not perform their own replication . because the genome is shorter , dis are apparently preferentially replicated ; therefore , these viruses obtain the viral genome density necessary to specifically interfere with the replication of the parental virus [ 3335 ] . thus , dis represent a major self - controlling mechanism for viral replication , and these particles have been implicated in the establishment and maintenance of persistent viral infections [ 25 , 33 ] . defective viral genomes have been detected in mosquito cells persistently infected with flaviviruses , such as sle , jev , and denv , and also those that occur in nature , circulating between mosquitoes and human populations . in addition to the participation of defective interfering particles / genomes in viral interference , some mutations have recently been associated with this phenomenon . the 2k peptide is a 23-amino - acid peptide located between the ns4a and ns4b proteins , and this peptide is anchored to the membrane of the endoplasmic reticulum ( er ) . the 2k peptide has been implicated in virus replication and the evasion of the rnase l - mediated antiviral response . although these studies have been performed with denv and wnv , some preliminary experiments suggest that the expression of several genes is commonly activated through mosquito - borne flaviviruses [ 71 , 72 ] . although different results have been obtained , several studies have suggested that during flaviviral infection genes associated with specific pathways are activated to maintain the proper condition of the cell for viral replication , such as metabolism ( nucleotide , lipid , amino acid , and energy ) [ 6568 ] , oxidative stress [ 67 , 68 ] and transcription / translation [ 6568 ] , and other pathways associated with the antiviral response [ 16 , 65 , 67 , 68 , 73 ] . the primary virus might sequester the host factors essential for the replication of the secondary virus [ 35 , 37 ] , which might result in viral interference , particularly during a superinfection ( figure 1 ) . when the viruses involved in the infection are closely related , the requirements for host factors are the same and the viral interference is stronger . an analysis of the secondary structure of the nhuv 3 utr revealed similarities with the yfv and jev serogroups and viruses in the tick - borne flavivirus clade , suggesting that the requirements for cellular factors and the mechanism for viral translation / replication might be similar among these viruses , thereby establishing conditions for competition . the flavivirus rna viral genome contains two utrs that have various functions , such as initiating and regulating viral translation , as well as viral complex replication and assembly at membrane fractions , through interactions with host cellular factors and nonstructural proteins that form rnps ( revised in ) . these cellular factors have been identified as elongation factor 1 ( ef1 ) , polypyrimidine tract binding ( ptb ) protein , the autoantigen - la ( la protein ) , calreticulin , and nuclear factor 90 ( nf90 ) [ 8285 ] . however , little information is available about the discovery of the cellular factors in mosquito cells , such as c6/36 cells , compared with mammalian cells . the cellular factors mentioned above , such as ef1 ( highly conserved between different host species as mammals , chicken , and mosquitoes ) , translation initiation factor eif5 , and ribosomal proteins s6 and l4 , participate in several steps of the translation process [ 8891 ] , except autoantigen - la , a nuclear protein involved in rna polymerase iii transcription termination [ 92 , 93 ] and small rna biogenesis , which acts as a chaperone and contributes to the retention of nascent rna in the nucleus or stabilizes the rna structure . this evidence suggests that ef1 is important for minus - strand rna synthesis through interactions with the viral rna and the replication complex proteins , including ns3 and ns5 [ 90 , 97 ] , and these interactions might be similar in mosquito cells . furthermore , the shih group results suggested that eif5 is upregulated in denv-2-infected c6/36 cells , and this upregulated expression might play a role in preventing mosquito cell death in response to the viral infection . however , the interaction between the ribosomal proteins s6 and l4 and the ns2a and ns4b nonstructural viral proteins is likely involved in flavivirus rna translation . the formation of vrnps might establish the genomic architecture and facilitate various processes in the viral life cycle , and the fact that flaviviruses share several cellular factors for translation / replication might explain the homologous viral interference frequently observed in both co- and superinfections in mosquitoes and mosquito - derived cell lines . moreover , c6/36 cells , which have a deficiency in the activity of the dicer-2 enzyme , are more susceptible to denv infection than dicer-2 competent cells [ 74 , 105 , 107 ] , and the natural polymorphism of the dicer-2 gene has been associated with the susceptibility of aedes aegypti mosquitoes to some isolates of denv-1 . although mosquitoes have a competent rnai pathway to degrade viral rna , flaviviruses have mechanisms to evade the rnai response and establish a persistent and noncytopathic infection in the vector . in the case of denv moreover , the dis in the sirna pathway could be responsible for the homologous / heterotypic viral interference and persistent infection . interesting similar results were obtained with the sindbis virus , a member of togaviridae , which shares similarities in the replicative cycle and genome type with members of the flaviviridae family . furthermore , several retrotransposons with retrotranscriptase activity have been detected in aedes aegypti [ 112114 ] , aedes albopictus , and aedes polynesiensis , suggesting that the same mechanism could operate in mosquitoes infected with flaviviruses ; however , this hypothesis requires further investigation . the immune response in mosquitoes is primarily regulated through three signalling pathways : immune deficiency ( imd ) , which mediates the production of antimicrobial peptides with activity against gram - negative bacteria ; the toll - mediated pathway , which is involved in the defence against viruses , gram - positive bacteria , and fungi and also stimulates the secretion of some antimicrobial peptides ; and the janus kinase - signal transducer and activator of transcription ( jak - stat ) pathway , which has been strongly associated with the antiviral response in aedes mosquitoes [ 118120 ] . among the three main pathways involved in the immune response in mosquitoes , the toll [ 14 , 15 , 72 , 115 , 116 , 120 ] and jak - stat [ 15 , 72 , 115 , 116 , 119 ] pathways are apparently more important for controlling flavivirus infection . the participation of the immune response during flavivirus infection has primarily been studied with denv [ 1416 , 115 , 116 , 119121 ] in aedes aegypti mosquitoes [ 1416 , 115 , 116 , 119 ] , and this response has recently been characterized in the aag2 aedes aegypti cell line . during infections with denv and other mosquito - borne flaviviruses , the first virus - vector interaction occurs in the midgut , where the immune response , known as the local immune response , is initially activated [ 15 , 115 , 119 ] . more recently , it has been reported that denv upregulates the expression of salivary gland genes in aedes aegypti mosquitoes that encode for proteins involved in the immune response and also induces the expression of a putative antibacterial , cecropin - like peptide , which exhibits activity against the four denv serotypes and chikv . because wnv and slev belong to the same antigenic complex and both viruses are primarily transmitted through culex spp . , the primary virus likely blocks the infection of the secondary virus through the induction of the host antiviral response . this effect is coincident with the ability of the bacterial flora to activate the toll pathway and produce several antimicrobial peptides , such as cecropin , defensin , attacin , and gramicidin , in the midgut [ 15 , 121 ] . however , as previously discussed , equilibrium between the viral replication and the antiviral response exists during persistent infection in mosquito cells [ 26 , 27 ] , and the precise participation of the immune response in persistent infection in mosquito cells and the mechanism used by the flaviviruses to circumvent this response remain unknown . this finding has been reported in c6/36 cells persistently infected with denv and in different mosquito cells lines persistently infected with slev , reflecting the inhibition of virus particle production [ 22 , 123 ] or the fact that initially all cells in culture support virus replication , but some cells stop replicating the virus and become resistant to superinfection during the late stages of infection . eventually , the cells regain sensitivity to reinfection by the virus in the medium or the residual viral rna in the cell , and thus the culture remains persistently infected . consistent with these findings , interferon and interferon - like substances are involved in heterologous interference , and vago , a peptide with antiviral activity , has recently been identified and shown to be secreted from wnv - infected culex mosquito cells , culex pipiens mosquitoes , and denv - infected rml12 cells ( from aedes albopictus ) [ 124 , 125 ] . however , the antiviral activity of vago is mediated through the jak - stat pathway . although vago expression is decreased in culex pipiens mosquitoes at 8 days postinfection with wnv , the participation of vago in viral interference during persistent infection can not be excluded and will require further investigation . flaviviral infection of the vectors is long - lived or persistent , and the mechanisms that participate in the establishment of this type of infection remain unknown . the present data suggest that this type of infection is a multifactorial phenomenon involving factors from the virus , such as defective interfering particles / genomes , and the host , such as the immune response , rna interference , and cellular factors . the cocirculation of two or more flaviviruses / alphaviruses in the same geographic area increases the opportunity for interviral interactions , and in most cases , the interaction between two mosquito - borne flaviviruses results in homologous viral interference , indicating that these viruses share mechanisms that regulate replication and several cellular factors required for viral translation / replication , a common requirement for different flaviviruses , which favours the conditions for competition . although the rnai pathway is not the only factor that might participate in maintaining a persistent infection or explaining viral interference , these data indicate that these types of experiments should be performed using other cell lines , such as ccl-125 , u4.4 , and c7 - 10 cells , and vectors , such as aedes and culex mosquitoes . moreover , these cell lines should be used to characterize the cellular factors involved in flavivirus replication and the pathways involved in the immune response to provide a cellular model to study the relevance of these phenomena in the transmission and epidemiology of mosquito - borne flaviviruses .

mosquito- and tick - borne flaviviruses , including dengue ( with four serotypes ) , japanese encephalitis virus ( jev ) , yellow fever virus ( yfv ) , saint louis encephalitis virus ( slev ) , west nile virus ( wnv ) , murray valley encephalitis virus ( mve ) , and tick - borne encephalitis virus ( tbev ) , are important pathogens responsible for human diseases , such as encephalitis , fever , and haemorrhagic fever . isf viruses are restricted to mosquitoes , such as culex and aedes , and include the aedes flavivirus ( aefv ) , calbertado virus , cell - fusing agent virus ( cfav ) , chaoyang virus , culex flavivirus ( cxfv ) , culex theileri flavivirus ( ctfv ) , kamiti river virus ( krv ) , lammi virus , nakiwogo virus ( nakv ) , nounane virus , quang binh virus ( qbv ) , and palm creek virus ( pcv ) [ 312 ] . these viruses are approximately 11 kb in length , with a single open reading frame ( orf ) encoding a polyprotein that is co- and posttranslationally processed through cellular and viral proteases into three structural ( c , m , and e ) and seven nonstructural ( ns1 , ns2a , ns2b , ns3 , ns4a , ns4b , and ns5 ) proteins . in contrast to acute infections , where the virus is eventually eliminated through the destruction of the host or the immune response , in persistent infections , the virus remains in the host cells for long periods of time , can be transmitted to other cells , and is not cleared . one of the common features observed in c6/36 persistently infected with denv [ 2224 ] is the presence of a cytopathic effect ( cpe ) during the first 47 weeks of infection , which becomes progressively imperceptible throughout the infection [ 23 , 24 ] . these findings are consistent with the nonpathogenic flavivirus infection in mosquitoes , suggesting the presence of a balance between virus replication and the antiviral host response [ 26 , 27 ] , which results in an infection that is controlled , but not eliminated , and not lethal [ 21 , 28 ] . for example , viral titres in the supernatants of c6/36 [ 22 , 24 , 31 ] or tra-171 cells persistently infected with denv display an oscillation pattern during persistent infection , suggesting the presence of defective interfering particles ( dis ) , one of the major self - controlling mechanisms for viral replication . because dis are more evident after several passages in cell lines , these particles are frequently observed in persistently infected cultures , and the oscillating phenomenon observed in long - term or persistently infected cell cultures reflects a mutual interaction between the parental virus , required for the replication of both defective and standard viruses , and the viral interference induced through dis in the viral titres . among the host factors that might participate in the maintenance of persistent infection are those associated with cell antiviral mechanisms , particularly rna interference ( rnai ) and the innate immune responses that control but do not eliminate viral infections . based on the time of infection , mixed infections can be classified as coinfections when the two viruses interact with the host at the same time or superinfections when one virus invades the host prior to the second virus . because flaviviral infection persists for the life of the vector , the opportunities for competition or viral interference in the vector are higher than in humans , where the infection is only transient and the evaluation of viral interference during flavivirus infection is relatively easy to detect in cell lines , and it has primarily been examined in mosquito c6/36 cells ( from aedes albopictus ) [ 12 , 23 , 30 , 31 , 35 , 39 , 4148 ] , tra-171 ( from toxorhynchites amboinensis ) , sf9 ( from spodoptera frugiperda ) , c7 - 10 , and u4.4 cells ( from aedes albopictus ) [ 29 , 35 ] . the same cells persistently infected with aal dnv and reinfected with denv-2 showed an important reduction in the severity and mortality of the denv-2 infection compared with those of noninfected cells , and denv-2 titres were lower than in nave cells . the primary infection of c6/36 cells with either denv-2 or denv-4 , followed by a secondary infection 1 or 6 hours later with the opposite virus at the same multiplicity of infection ( moi ) , showed a stronger reduction in the virus titres of the secondary virus when the second infection was performed 6 hours after the first infection . homologous viral interference has been observed in aedes aegypti mosquitoes infected with denv and yfv or mve and denv , culex tritaeniorhynchus mosquitoes infected with jev and mve , and culex quinquefasciatus mosquitoes infected with other flaviviruses , such as wnv and slev . in another study , culex pipiens mosquitoes persistently infected with cxfv and orally challenged with wnv displayed a reduction in the dissemination rates only during the early stages of the infection ( 7 days ) , but not during the late stages ( 14 days ) , with no effects on the transmission rates . moreover , the mechanism of transmission between both viruses could also contribute to the absence of viral interference , as wnv is transmitted to the vertebrate host through saliva , and cxfv is maintained among the vector population through vertical transmission . although several studies have been performed using pools of mosquitoes where the coinfection in the same individual is difficult to determine [ 55 , 57 ] , other studies have clearly established that one individual can be infected with both denv serotypes for example , the absence of viral interference between chikv and denv in cell lines has been confirmed through the evidence of dual chikv and denv-1 viral infections in aedes aegypti mosquitoes and in a single aedes albopictus mosquito . the heterotypic viral interference against denv-3 observed in c6/36 cells with an acute denv-1 infection is affected through treatments with puromycin ( an inhibitor of the protein synthesis ) but not actinomycin d ( an inhibitor of the cellular transcription ) , suggesting that the viral interference is predominantly mediated through the virus instead of cellular factors . more recently , it has been proposed that drosophila melanogaster cells persistently infected with several nonflavivirus rna viruses generated cdnas from the genomes of defective interfering particles through cellular retrotransposon reverse transcriptase - mediated retrotranscription ( figure 1 ) , but this finding requires further investigation . although different results have been obtained , several studies have suggested that during flaviviral infection genes associated with specific pathways are activated to maintain the proper condition of the cell for viral replication , such as metabolism ( nucleotide , lipid , amino acid , and energy ) [ 6568 ] , oxidative stress [ 67 , 68 ] and transcription / translation [ 6568 ] , and other pathways associated with the antiviral response [ 16 , 65 , 67 , 68 , 73 ] . an analysis of the secondary structure of the nhuv 3 utr revealed similarities with the yfv and jev serogroups and viruses in the tick - borne flavivirus clade , suggesting that the requirements for cellular factors and the mechanism for viral translation / replication might be similar among these viruses , thereby establishing conditions for competition . viral translation and replication can not occur at the same time because the ribosome moves from the 5 end towards the 3 end of the rna to translate the proteins , whereas the rna viral polymerase generally binds to the 3 end of the same rna molecule to initiate replication . the flavivirus rna viral genome contains two utrs that have various functions , such as initiating and regulating viral translation , as well as viral complex replication and assembly at membrane fractions , through interactions with host cellular factors and nonstructural proteins that form rnps ( revised in ) . most of these factors have been identified in mammalian cells , including bhk21 ( for baby hamster kidney ) , vero ( green monkey kidney ) , hek293a ( human embryonic kidney-293 ) , k562 ( human erythroleukaemia cells ) , and u937 ( a human monocytic cell line derived from a patient with generalized histiocytic lymphoma ) cells . these cellular factors have been identified as elongation factor 1 ( ef1 ) , polypyrimidine tract binding ( ptb ) protein , the autoantigen - la ( la protein ) , calreticulin , and nuclear factor 90 ( nf90 ) [ 8285 ] . notably , ef1 , the la protein , eukaryotic initiation factor 5 ( eif5 ) , 40s ribosomal protein s6 , and 60s ribosomal protein l4 ( table 4 ) have been implicated in viral replication in mosquitoes [ 72 , 83 , 86 , 87 ] . the cellular factors mentioned above , such as ef1 ( highly conserved between different host species as mammals , chicken , and mosquitoes ) , translation initiation factor eif5 , and ribosomal proteins s6 and l4 , participate in several steps of the translation process [ 8891 ] , except autoantigen - la , a nuclear protein involved in rna polymerase iii transcription termination [ 92 , 93 ] and small rna biogenesis , which acts as a chaperone and contributes to the retention of nascent rna in the nucleus or stabilizes the rna structure . ef1 is involved in wnv and denv replication and could act as a chaperone targeting the rna to the viral replication compartments , as this protein is associated with the er membrane fraction where rna viral replication complexes are assembled [ 86 , 88 ] . this evidence suggests that ef1 is important for minus - strand rna synthesis through interactions with the viral rna and the replication complex proteins , including ns3 and ns5 [ 90 , 97 ] , and these interactions might be similar in mosquito cells . however , the interaction between the ribosomal proteins s6 and l4 and the ns2a and ns4b nonstructural viral proteins is likely involved in flavivirus rna translation . barr virus noncoding rna eber1 , which recruits ribosomal protein l22 during infection , presumably , to aid in viral replication , or the hepatitis c ( hcv ) ires region , which associates with the ribosomal s5 protein to position the hcv rna on the 40s ribosomal subunit during translation . after analyzing this information , we proposed that the association of ef1 and the la protein , which function as chaperones , with eif5 and ribosomal proteins s6 and l4 , components of the translational machinery , could stimulate flavivirus translation in a favourable context . denv infections induce the drastic rearrangement of the er membranes , resulting in complex membranous structures that promote the switch from translation to viral replication once viral and/or cellular factors facilitate communication of the utrs , thereby maximizing translation and replication as global processes . the formation of vrnps might establish the genomic architecture and facilitate various processes in the viral life cycle , and the fact that flaviviruses share several cellular factors for translation / replication might explain the homologous viral interference frequently observed in both co- and superinfections in mosquitoes and mosquito - derived cell lines . , as a result of replicative intermediates or secondary structure of genome of rna viruses ) , which is processed through the rnase iii - like enzymes dicer-1 or dicer-2 ( primarily dicer-2 ) , to generate small dsrna ( 21 to 25 nt ) . with the assistance of the r2d2 protein , these small rnas are loaded into the rna - induced silencing complex ( risc ) involving the argonaute proteins ( ago-1 or ago-2 ) . the participation of the sirna response in the regulation of flavivirus infection has been studied in both mosquito cells lines [ 74 , 103107 ] , aedes aegypti , culex pipiens quinquefasciatus , and even in drosophila melanogaster . moreover , c6/36 cells , which have a deficiency in the activity of the dicer-2 enzyme , are more susceptible to denv infection than dicer-2 competent cells [ 74 , 105 , 107 ] , and the natural polymorphism of the dicer-2 gene has been associated with the susceptibility of aedes aegypti mosquitoes to some isolates of denv-1 . one of these mechanisms might be the sequestration of the viral replicative complex into endoplasmic reticulum - derived vesicles ; thus , the double - stranded viral rnas generated during the viral replication can not be sensed through dicer-2 [ 73 , 103 , 104 , 110 ] . for example , mosquito and mammal cells infected with flaviviruses generate subgenomic rna corresponding to the 3 utr of the viral genome through the participation of the cellular exoribonuclease xrn1 , and this subgenomic rna binds to dicer-2 and dicer-1 , inhibiting the activity of these enzymes in vitro [ 1 , 102 ] . drosophila melanogaster cells persistently infected with several nonflavivirus rna viruses generated cdnas from the genomes of dis through cellular ltr - retrotransposon reverse transcriptase - mediated retrotranscription . these cdnas are apparently transcribed through the cellular transcription machinery to generate small double - stranded rnas via several mechanisms , which are the source of the dicer-2-risc - produced interfering rnas ( rnai ) [ 21 , 28 ] that control viral replication ( figure 1 ) . furthermore , several retrotransposons with retrotranscriptase activity have been detected in aedes aegypti [ 112114 ] , aedes albopictus , and aedes polynesiensis , suggesting that the same mechanism could operate in mosquitoes infected with flaviviruses ; however , this hypothesis requires further investigation . the immune response in mosquitoes is primarily regulated through three signalling pathways : immune deficiency ( imd ) , which mediates the production of antimicrobial peptides with activity against gram - negative bacteria ; the toll - mediated pathway , which is involved in the defence against viruses , gram - positive bacteria , and fungi and also stimulates the secretion of some antimicrobial peptides ; and the janus kinase - signal transducer and activator of transcription ( jak - stat ) pathway , which has been strongly associated with the antiviral response in aedes mosquitoes [ 118120 ] . among the three main pathways involved in the immune response in mosquitoes , the toll [ 14 , 15 , 72 , 115 , 116 , 120 ] and jak - stat [ 15 , 72 , 115 , 116 , 119 ] pathways are apparently more important for controlling flavivirus infection . the participation of the immune response during flavivirus infection has primarily been studied with denv [ 1416 , 115 , 116 , 119121 ] in aedes aegypti mosquitoes [ 1416 , 115 , 116 , 119 ] , and this response has recently been characterized in the aag2 aedes aegypti cell line . during infections with denv and other mosquito - borne flaviviruses , the first virus - vector interaction occurs in the midgut , where the immune response , known as the local immune response , is initially activated [ 15 , 115 , 119 ] . more recently , it has been reported that denv upregulates the expression of salivary gland genes in aedes aegypti mosquitoes that encode for proteins involved in the immune response and also induces the expression of a putative antibacterial , cecropin - like peptide , which exhibits activity against the four denv serotypes and chikv . although the participation of the mosquito immune system has been clearly demonstrated during single flavivirus infection , there are no studies concerning the participation of this response in viral interference during co- or superinfections . the eradication of the endogenous bacterial flora in the midgut of aedes aegypti mosquitoes treated with an antibiotic increased the viral titres in the mosquitoes infected with denv-2 compared with a nontreated group . this effect is coincident with the ability of the bacterial flora to activate the toll pathway and produce several antimicrobial peptides , such as cecropin , defensin , attacin , and gramicidin , in the midgut [ 15 , 121 ] . however , as previously discussed , equilibrium between the viral replication and the antiviral response exists during persistent infection in mosquito cells [ 26 , 27 ] , and the precise participation of the immune response in persistent infection in mosquito cells and the mechanism used by the flaviviruses to circumvent this response remain unknown . this finding has been reported in c6/36 cells persistently infected with denv and in different mosquito cells lines persistently infected with slev , reflecting the inhibition of virus particle production [ 22 , 123 ] or the fact that initially all cells in culture support virus replication , but some cells stop replicating the virus and become resistant to superinfection during the late stages of infection . similarly , c6/36 cells pretreated with filtered culture medium from the same cells persistently infected with the four serotypes of denv become resistant to reinfection with the same denv viruses ( heterotypic viral interference ) but remain susceptible to infection with chikv . consistent with these findings , interferon and interferon - like substances are involved in heterologous interference , and vago , a peptide with antiviral activity , has recently been identified and shown to be secreted from wnv - infected culex mosquito cells , culex pipiens mosquitoes , and denv - infected rml12 cells ( from aedes albopictus ) [ 124 , 125 ] . the present data suggest that this type of infection is a multifactorial phenomenon involving factors from the virus , such as defective interfering particles / genomes , and the host , such as the immune response , rna interference , and cellular factors . the characterization of the mechanisms that participate in viral persistence is important to obtain a better understanding of the complex interactions between flaviviruses and mosquito cells to develop new strategies for control . the cocirculation of two or more flaviviruses / alphaviruses in the same geographic area increases the opportunity for interviral interactions , and in most cases , the interaction between two mosquito - borne flaviviruses results in homologous viral interference , indicating that these viruses share mechanisms that regulate replication and several cellular factors required for viral translation / replication , a common requirement for different flaviviruses , which favours the conditions for competition . although the rnai pathway is not the only factor that might participate in maintaining a persistent infection or explaining viral interference , these data indicate that these types of experiments should be performed using other cell lines , such as ccl-125 , u4.4 , and c7 - 10 cells , and vectors , such as aedes and culex mosquitoes . moreover , these cell lines should be used to characterize the cellular factors involved in flavivirus replication and the pathways involved in the immune response to provide a cellular model to study the relevance of these phenomena in the transmission and epidemiology of mosquito - borne flaviviruses .

los intentos a la hora de ayudar a dejar de fumar rara vez resultan mejores que simplemente efectivos parcialmente . describir un estudio observacional de un programa ya existente y de gran xito para dejar de fumar , ofrecido por un consejero de salud como intervencin primaria . los componentes bsicos del diseo del programa y la informacin se presentan y pueden servir como modelo en otros centros de salud pblicos . se presentan tres componentes complementarios del programa y un consejero de salud ( auriculoterapia , estimulacin elctrica de ondas alfa y tcnicas de relajacin ) . las tasas de abandono a los 6 meses de 161 pacientes a lo largo de 3 aos se presentan por cada 30 das de abstinencia y en valores de intencin de tratamiento . se proporcionan comparaciones de las consultas telefnicas frente a consultas mdicas en la clnica , de libre eleccin frente a la participacin obligatoria y se facilitan los costes del programa . la prevalencia puntual de la tasa de abandono fue de un 88,7 % mientras que la tasa de abandono ms conservadora del grupo con intencin de tratamiento fue de un 51,6 % . la asistencia sanitaria telefnica y la asistencia en la clnica no fueron significativamente diferentes en ningn momento . las tasas de abstinencia total al tabaco a los 6 y 12 meses fueron de 76,9 % y 63,2 % , respectivamente . se observa una prevalencia puntual ( 30 das ) por encima de un 80 % y un efecto duradero en el tiempo . las cargas personales y sociales ( de salud y econmicas ) implcitas en el tabaco podran verse considerablemente afectadas si dichos programas se pusiesen en prctica a mayor escala . between 2009 and 2011 , 161 patients ( 87 male and 74 females , mean [ sd ] age : 44.4 11.8 y ) entered the mtcp . patients were included in a treatment cohort if they had smoked at least five cigarettes ( or chewed tobacco 30 min ) daily for at least 6 months . these patients , largely employees of ( n=82 ) or dependents of employees ( n=41 ) of the mercy hospital system , were supported by an outside employer ( n = 26 ) or came through word - of - mouth referral ( n = 9 ) . some mtcp patients participated because of an offer by their employer to achieve a $ 600 insurance premium reduction . participants reported average smoking of 18.9 ( 11.5 ) cigarettes / day at the outset of treatment and had smoked for an average of 23.8 years ( 12.9 ) . most ( 88.1% ) had tried to quit smoking previously , averaging 2.9 ( 2.8 ) quit attempts . on enrolling , each patient initially received a quit plan to consider and a questionnaire asking about tobacco use and motivation for behavior change . data are presented anonymously and were collected as part of routine clinical practice . for these reasons , informed consent was not collected and the mercy springfield hospital institutional review board did not believe retrospective project review was necessary before dissemination . the project design was a single - site , prospective , non - randomized , observational study of an existing practice ( ie , mtcp ) with multiple follow - ups examining the effects of an hc tobacco cessation intervention . there was no control group , but the ineffectiveness of non - coaching tobacco cessation efforts are well known and described later . it made little sense to turn away patients who wanted to quit smoking for the establishment of a control group . the flow of patient enrollment and progress through the mtcp is illustrated in figure 1 . in addition to hc , in - clinic patients received alpha - electrical stimulation ( aes ) , relaxation techniques , and auriculotherapy . of 161 patients , 93 ( 57.8% ) utilized in - clinic services while 68 used only telephonic hc . hc involved establishing an individualized quit plan while acknowledging the participant as the final decision maker in when to quit . after an in - clinic session , each patient continued with in - clinic visits or chose remote treatment using only telephonic health coaching . initial visit . this first session was with an experienced tobacco cessation rn educator and planned to last 45 minutes . patients returned a completed questionnaire on smoking habits , worked on a co - created quit plan , and determined if they would use complementary therapies . hc was delivered by two registered nurses with wellcoaches training ( wellcoaches corp , wellesley , massachusetts ) . this approach emphasizes relationship development , is patient - centered , and encourages a self - discovery process while using mindfulness and motivational interviewing techniques . the initial hc visit was followed by subsequent visits planned to last 20 to 25 minutes . hc could be delivered telephonically or patients could elect for in - clinic , face - to - face appointments . telephonic hc patients were given information about and often participated in relaxation techniques ( see below ) . the amount of hc was patient determined ; however , 6 to 8 sessions over 3 months were recommended . in following a true health coaching paradigm , patients were supported when considering any additional or complementary therapies and were permitted to use them as desired . all patients had the option of using aes , relaxation techniques , and auriculotherapy as complementary treatment to hc ( unless travel distance precluded it ) . before hc , in - clinic patients typically completed 20 minutes of relaxation techniques while receiving aes prior to engaging in a 15-minute session of auriculotherapy . aes enhances alpha - wave activity with the intention of inducing a calmer and more relaxed state . aes is delivered in 10 s waves of 0.5 hz in a pattern range of 100 to 300 micro - amps via earlobe - located electrodes . alpha - stim scs ( electromedical products international , mineral wells , texas ) is a class iia , type b medical device with federal law restricting its use . a physician 's orders were required for including aes in a patient 's quit plan . relaxation techniques involving deep breathing and progressive muscle relaxation [ pmr ] were initially taught to patients by staff . several minutes of deep breathing were followed by several minutes of pmr using standard jacobsen techniques . by the third visit , most patients practiced self - guided relaxation techniques for 20 minutes . auriculotherapy is a micro - current stimulation to reflex and acupuncture points on the ear delivered using the electro medical stim flex ( electro medical inc , tulsa , oklahoma ) . site selection followed the national acupuncture detoxification association ( nada ) 5-point protocol , and these sites are related to treatment for addiction , relaxation , and anxiety . auriculotherapy was administered in 15 to 30minute sessions , and as an acupuncture - like process , is reported to assist with diminishing nicotine addiction . medications ( particularly those acting at the nicotinic receptor ) were a prevalent health coaching topic , but such treatment was a personal choice reached in consultation with a primary care physician . a large minority of mtcp patients ( 43.7% ) used some form of nicotinic receptor therapy ( bupropion hcl [ wellbutrin , 3% ] ; varenicline [ chantix , 17% ] ; and nicotine replacement therapy [ nrt , 24% ] ) during the program . additional treatments , as well as monetary incentive to participate provided by an employer , are potential confounders of the hc effect . confounding variables such as nrt , employer mandate , and the prevalent use of complementary therapies are addressed statistically in the results section . the goal of the mtcp was to follow a true coaching model , employing maximum therapeutic flexibility to empower cessation of tobacco use . participants were defined as those completing at least one hc session and grouped as free choice or mandated ( employer required five visits for insurance premium reduction ) . patients indicated importance of change of tobacco use on a simple 10-point likert scale along with readiness to change . readiness to change is based on the transtheoretical model , which allowed classification of patients into one of five stages ( ie , precontemplation , contemplation , preparation , action , maintenance ) . smoking quit rates were calculated as the number of patients who reported smoking cigarettes at baseline but not at follow - up . self - reported cessation ( 7 mo , 30-d point prevalence ) is currently the standard used by the north american quitline provided a 50% response rate can be achieved . smoke - free rates examined follow - up on those who initially quit and were determined at 1 , 3 , 6 , 12 , 18 , and 24 months after quit date . intent - to - treat was calculated as all patients who quit smoking at 6 months divided by all participants who reported smoking at baseline . patients lost to follow - up were presumed to have relapsed for the conservative intent - to - treat calculation . for 30-day point prevalence calculation , patients reporting that they were smoke - free for 30 days at 7 months determined the numerator while all patients reporting smoking at baseline was the denominator , excluding those not completing at least four hc sessions , those whose participation was mandated , or those who were lost to follow - up . statistical analysis . program impact on tobacco use ( quit and smoke - free rates ) was compared between coaching groups using chi - square . the influence of coaching on quit rates and smoke - free rates was examined in conjunction with motivational variables and covariates ( smoking history and nrt use ) . due to the number of logistic analyses conducted ( one for each time point ) , the criterion for statistical significance was set at p<.007 based on a bonforroni correction ( .05/7 ) . two additional tests were conducted to examine reduction in tobacco use : a t - test was used to examine pre - to - post program changes in tobacco use , and a regression examined predictors ( coaching , motivational variables , history of smoking behavior , and nrt ) on reduced tobacco use . data analyses were conducted in spss v21 ( ibm corp , armonk , new york ) . between 2009 and 2011 , 161 patients ( 87 male and 74 females , mean [ sd ] age : 44.4 11.8 y ) entered the mtcp . patients were included in a treatment cohort if they had smoked at least five cigarettes ( or chewed tobacco 30 min ) daily for at least 6 months . these patients , largely employees of ( n=82 ) or dependents of employees ( n=41 ) of the mercy hospital system , were supported by an outside employer ( n = 26 ) or came through word - of - mouth referral ( n = 9 ) . some mtcp patients participated because of an offer by their employer to achieve a $ 600 insurance premium reduction . participants reported average smoking of 18.9 ( 11.5 ) cigarettes / day at the outset of treatment and had smoked for an average of 23.8 years ( 12.9 ) . most ( 88.1% ) had tried to quit smoking previously , averaging 2.9 ( 2.8 ) quit attempts . on enrolling , each patient initially received a quit plan to consider and a questionnaire asking about tobacco use and motivation for behavior change . data are presented anonymously and were collected as part of routine clinical practice . for these reasons , informed consent was not collected and the mercy springfield hospital institutional review board did not believe retrospective project review was necessary before dissemination . the project design was a single - site , prospective , non - randomized , observational study of an existing practice ( ie , mtcp ) with multiple follow - ups examining the effects of an hc tobacco cessation intervention . there was no control group , but the ineffectiveness of non - coaching tobacco cessation efforts are well known and described later . it made little sense to turn away patients who wanted to quit smoking for the establishment of a control group . the flow of patient enrollment and progress through the mtcp is illustrated in figure 1 . in addition to hc , in - clinic patients received alpha - electrical stimulation ( aes ) , relaxation techniques , and auriculotherapy . of 161 patients , 93 ( 57.8% ) utilized in - clinic services while 68 used only telephonic hc . hc involved establishing an individualized quit plan while acknowledging the participant as the final decision maker in when to quit . after an in - clinic session , each patient continued with in - clinic visits or chose remote treatment using only telephonic health coaching . this first session was with an experienced tobacco cessation rn educator and planned to last 45 minutes . patients returned a completed questionnaire on smoking habits , worked on a co - created quit plan , and determined if they would use complementary therapies . hc was delivered by two registered nurses with wellcoaches training ( wellcoaches corp , wellesley , massachusetts ) . this approach emphasizes relationship development , is patient - centered , and encourages a self - discovery process while using mindfulness and motivational interviewing techniques . the initial hc visit was followed by subsequent visits planned to last 20 to 25 minutes . hc could be delivered telephonically or patients could elect for in - clinic , face - to - face appointments . telephonic hc patients were given information about and often participated in relaxation techniques ( see below ) . the amount of hc was patient determined ; however , 6 to 8 sessions over 3 months were recommended . in following a true health coaching paradigm , patients were supported when considering any additional or complementary therapies and were permitted to use them as desired . all patients had the option of using aes , relaxation techniques , and auriculotherapy as complementary treatment to hc ( unless travel distance precluded it ) . before hc , in - clinic patients typically completed 20 minutes of relaxation techniques while receiving aes prior to engaging in a 15-minute session of auriculotherapy . aes enhances alpha - wave activity with the intention of inducing a calmer and more relaxed state . aes is delivered in 10 s waves of 0.5 hz in a pattern range of 100 to 300 micro - amps via earlobe - located electrodes . alpha - stim scs ( electromedical products international , mineral wells , texas ) is a class iia , type b medical device with federal law restricting its use . a physician 's orders were required for including aes in a patient 's quit plan . relaxation techniques involving deep breathing and progressive muscle relaxation [ pmr ] were initially taught to patients by staff . several minutes of deep breathing were followed by several minutes of pmr using standard jacobsen techniques . by the third visit , most patients practiced self - guided relaxation techniques for 20 minutes . auriculotherapy is a micro - current stimulation to reflex and acupuncture points on the ear delivered using the electro medical stim flex ( electro medical inc , tulsa , oklahoma ) . site selection followed the national acupuncture detoxification association ( nada ) 5-point protocol , and these sites are related to treatment for addiction , relaxation , and anxiety . auriculotherapy was administered in 15 to 30minute sessions , and as an acupuncture - like process , is reported to assist with diminishing nicotine addiction . medications ( particularly those acting at the nicotinic receptor ) were a prevalent health coaching topic , but such treatment was a personal choice reached in consultation with a primary care physician . a large minority of mtcp patients ( 43.7% ) used some form of nicotinic receptor therapy ( bupropion hcl [ wellbutrin , 3% ] ; varenicline [ chantix , 17% ] ; and nicotine replacement therapy [ nrt , 24% ] ) during the program . additional treatments , as well as monetary incentive to participate provided by an employer , are potential confounders of the hc effect . confounding variables such as nrt , employer mandate , and the prevalent use of complementary therapies are addressed statistically in the results section . the goal of the mtcp was to follow a true coaching model , employing maximum therapeutic flexibility to empower cessation of tobacco use . participation and motivational variables . participants were defined as those completing at least one hc session and grouped as free choice or mandated ( employer required five visits for insurance premium reduction ) . patients indicated importance of change of tobacco use on a simple 10-point likert scale along with readiness to change . readiness to change is based on the transtheoretical model , which allowed classification of patients into one of five stages ( ie , precontemplation , contemplation , preparation , action , maintenance ) . smoking quit rates were calculated as the number of patients who reported smoking cigarettes at baseline but not at follow - up . self - reported cessation ( 7 mo , 30-d point prevalence ) is currently the standard used by the north american quitline provided a 50% response rate can be achieved . smoke - free rates examined follow - up on those who initially quit and were determined at 1 , 3 , 6 , 12 , 18 , and 24 months after quit date . intent - to - treat was calculated as all patients who quit smoking at 6 months divided by all participants who reported smoking at baseline . patients lost to follow - up were presumed to have relapsed for the conservative intent - to - treat calculation . for 30-day point prevalence calculation , patients reporting that they were smoke - free for 30 days at 7 months determined the numerator while all patients reporting smoking at baseline was the denominator , excluding those not completing at least four hc sessions , those whose participation was mandated , or those who were lost to follow - up . statistical analysis . program impact on tobacco use ( quit and smoke - free rates ) was compared between coaching groups using chi - square . the influence of coaching on quit rates and smoke - free rates was examined in conjunction with motivational variables and covariates ( smoking history and nrt use ) . due to the number of logistic analyses conducted ( one for each time point ) , the criterion for statistical significance was set at p<.007 based on a bonforroni correction ( .05/7 ) . two additional tests were conducted to examine reduction in tobacco use : a t - test was used to examine pre - to - post program changes in tobacco use , and a regression examined predictors ( coaching , motivational variables , history of smoking behavior , and nrt ) on reduced tobacco use . data analyses were conducted in spss v21 ( ibm corp , armonk , new york ) . for the 161 participants in this study , an average of 6.20 ( 2.71 , range=2 - 15 ) hc sessions were recorded . the initial consult was 42.1 min ( 9.80 ) with 35.8 min ( 13.37 ) per subsequent session . a total of 119 patients ( 73.9% ) demonstrated program adherence of four or more coaching sessions . patients were motivated to change tobacco habits ( 7.862.17 ) and most ( 88.8% ) were at least contemplating change ( precontemplation 11.2% ; contemplation 26.7% ; planning 28% ; action 34.1% ) . there were 65 participants ( 40.4% ) whose participation was considered employer - mandated ( ie , motivated to take part by an insurance premium reduction ) . intent - to - treat quit rate at 6 months was 51.6% ( 83 of 161 ) . thirty - day point prevalence quit rate at 7 months was 88.7% ( 47 of 53 ) . overall smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months were 95.3% ( 102 of 107 ) , 89.9% ( 98 of 109 ) , 76.9% ( 83 of 108 ) , 63.2% ( 67 of 106 ) , 51.8% ( 44 of 85 ) , and 40.5% ( 30 of 74 ) , respectively . figure 2 shows no difference in smoke - free rates between patients receiving in - clinic care ( hc plus complementary therapies ) vs telephonic hc . treatment / coaching style . in - clinic patients received health coaching face - to - face in combination with complementary therapies while telephonic patients remotely received health coaching and no complementary therapies . note that the zero ( 0 ) time point reflects the average smoke - free rate for the subgroups . values below the x - axis reflect the number of patients responding at each time point . the predictors , coaching subgroup ( in - clinic / telephonic ) , motivational factors ( premium reduction mandated participation , importance , and stage of change ) and covariates , smoking history ( average amount smoked per day and years of use ) , and nrt , reliably distinguished between quitters and non - quitters as seen in the table . these variables explained a significant , small - moderate effect in smoking cessation ( (8)=48.81 , p<.001 ; nagelkerke 's r=.39 ; 79% prediction success ) . insurance mandate and stage of change significantly contributed to prediction . specifically , when patients participated freely ( ie , without a premium reduction mandate ) , they were 6.26 times more likely to quit smoking ( p<.001 ) . a unit increase in stage of change ( eg , from precontemplation to contemplation ) saw patients 3.18 times more likely to quit smoking ( p<.001 ) . the table also details the predictive ability of this model for smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months . it appears that the farther removed participants are from coaching , the more their initial rates of smoking influence remaining smoke free , with those who smoked the most at baseline having the most difficulty remaining smoke - free . logistic regression predictors of smoking cessation and smoke - free rates abbreviation : nrt , nicotine receptor therapy . participants significantly reduced tobacco use rates to an average of 3.52 ( 7.45 ) cigarettes per day ( t(134 ) = 17.94 , p<.001 ) . tobacco reduction was predicted by one 's history of smoking ( quantity smoked per day b[se ] = 0.27 ( 0.05 ) , p<.001 ; years smoked b[se ] = .09[0.04 ] , p=.04 ; number of previous quit attempts b[se ] = 0.01[.18 ] , p=.97 ; nrt ( b[se ] = 0.33[1.00 ] , p=.74 ; motivational variables ( importance b[se ] = 0.38[0.25 ] , p=.13 ; stage of change b[se ] = 2.26(0.55 ) , p<.001 ; insurance mandate b[se ] = 1.64[.97 ] , p=.10 ; and their coaching group ( in clinic / telephonic [se]=2.73[1.04 ] , p<.01 ) , f(8,155 ) = 11.82 , p<.001 ; r = .39 ) . every additional cigarette smoked at baseline related to .42 more cigarettes smoked at follow - up ; every additional year patients had smoked led to an additional .15 cigarettes smoked ; with every increase in stage of change , .31 fewer cigarettes were smoked ; and patients in the telephonic coaching group smoked .18 cigarettes fewer than those receiving in - clinic coaching . quitting successful quit rates are at a meager 4.4% when examining programs over a 20-year period ( 1991 - 2010 ) . this poor rate of success exists despite well - funded and widely accessible smoking cessation interventions implemented after the 1998 master settlement agreement . cold turkey and unassisted quitting reveal a success rate of about 5% while some programs using behavioral therapy with nrt claim quit rates exceeding 40% . accordingly , the mtcp 30-day point prevalence ( 88.7% ) and intent - to - treat ( 51.6% ) success rates are remarkable . according to longitudinal ( 5 - 8 y ) data , the overall quit rate ( 57% ) achieved by mtcp patients at 12 months is a very good predictor of long - term success . telephonic coaching ( or counseling ) is a process widely used for tobacco cessation programs , particularly by accessible government - supported quit lines ( eg , 1 - 866 ny - quits or 1 - 800-quitnow ) . previous studies of coaching reported quit rates above 30% , but sometimes below 15% . however , the training and background of coaches in previous studies is poorly described and unlikely to meet the hc paradigm recently forwarded by wolever et al . mtcp health coaches were registered nurses with extensive coach training who delivered hc compatible with the wolever et al definition . others have called for further study of such coaching methods because of preliminary success in smoking cessation . we speculate the care and techniques provided by mtcp coaches accounted for the outstanding rate of successfully achieving tobacco independence in the present report . adding complementary therapies did little to enhance mtcp quit rates . telephonic - only coaching quit rates were not significantly different than in - clinic treatment that included aes and auriculotherapy . cranial stimulation and acupuncture are accepted clinical procedures with effectiveness in addiction / smoking cessation . in the present study , however , providing these procedures before each hc session did not improve effects of hc alone on quit rates . while these procedures may be effective , combining them with hc likely removed their influence on the quitting process . it seems that whether hc was administered with or without complementary therapies , it was the primary treatment explaining smoking cessation in these patients . the question of coaching effectiveness when delivered telephonically vs face - to - face is of great interest with ease and cost - effectiveness of remote coaching , making it preferred assuming it is equally effective . the absence of facial expressions and body language are considered shortcomings of the telephonic method . our results , however , demonstrated telephonic coaching to be equally effective as in - clinic , face - to - face sessions . it may be speculated that many patients are less inhibited speaking on the phone than in person , making them inclined to more fully tell their stories , contributing to hc success . our tobacco cessation results are not surprising given that telephonic coaching sessions apparently are also effective for weight loss and treatment of depression . our data lend further credence to remote , telephonic delivery of coaching processes as an effective behavior change intervention . the use of readiness - to - change scores appears extremely valuable for health coaches working with patients attempting to quit smoking . moving a patient one behavior - change category ( eg , from contemplation to preparation ) more than doubled chances of quitting and equally affected chances of remaining smoke - free . with such a large influence on outcome , monitoring , and influencing , stage of behavior change seems to be a critical hc strategy . this is an account of an existing practice ; therefore , these data are not from a prospectively designed , randomized , controlled trial ( rct ) . these data were systematically collected ; however , conclusive assessment of the mtcp coaching process may require an experimentally designed study . while ideal , designing such a project may not be ethically pragmatic given it requires withholding an apparently effective treatment from a ( control ) group of patients who need to be as motivated to quit smoking as those assigned to treatment . as a description of an existing practice and not an rct , this project did not control for other factors that may impact smoking cessation and might have augmented the hc effect . for example , nrt is a controversial intervention sometimes described as effectively assisting with quitting a tobacco habit . our analysis found that hc patients using nrt were no more likely to quit tobacco than patients without nrt . many mtcp patients employed nrt ( 44% at one point or another ) though hc was the only treatment consistently applied for all patients . in standard practice , a health coach using a patient - centered approach encourages patients to seek other strategies , aids , or social supports to optimize positive behavior change . the goal of the mtcp was to follow a true coaching paradigm allowing maximum therapeutic flexibility . undoubtedly , a high level of treatment flexibility was achieved ; however , hc was the common and primary intervention for all patients in this highly successful , tobacco cessation program . smoking has huge economic implications with a $ 96 billion cdc estimated annual us health cost burden shared by smokers and tax - payers in general . this number has recently been translated to a corporate cost of greater than $ 5000 per smoking employee with additional costs for human pain and suffering immeasurable . finding a strategy to incentivize smoking cessation is economically imperative and many employers attempt to provide such motivation . our observations revealed coercing smoking cessation through an insurance premium reduction mandate is not a good strategy . patients required to participate to avoid an insurance - premium penalty were about six times less likely to quit smoking than those participating freely . program compliance ( > 4 visits ) improved success for these mandated participators , but they were still about twice as likely to continue smoking as free - choice patients . nearly twice as many mandated participants were in precontemplation ( ie , not considering quitting ) compared to those freely entering mtcp . providing an insurance premium upcharge apparently may not be a good strategy to augment smoking cessation . however , a recent study indicates other employer - organized programs , using financial incentives , are viable to motivate behavior change . programs for smoking cessation can be expensive while claiming efficacy rates of 25% to 30% . the mtcp in - clinic program had quitting success rates exceeding 70% and smoke - free rate above 60% at 1 year with patient cost of $ 38 per visit ( $ 266 for seven visits ) and total staff labor costs of $ 204 for the average patient who had about seven visits . more impressively , patient cost for the equally effective telephonic - only hc was $ 32 per session with labor costs of only $ 112 for ( on average ) seven sessions . in practice , implementing a hospital - based , hc smoking cessation program is highly feasible . allied healthcare personnel ( existing staff , eg , nurses ) building on their knowledge base , with as little as 50 to 200 hours of training in relational and communication skills , may become qualified as an hc . once an hc staff exists then adding the program into existing departments ( eg , cardiac rehabilitation or employee/ community wellness ) becomes a matter of best logistical fit . given the potential economic and societal benefit , adding an hc - grounded smoking cessation program to the menu of patient treatment options should be a primary consideration for many public healthcare settings . furthermore , government sponsorship of such programs deserves careful investigation and deliberation given the prospects for reducing national healthcare costs . for the 161 participants in this study , an average of 6.20 ( 2.71 , range=2 - 15 ) hc sessions were recorded . the initial consult was 42.1 min ( 9.80 ) with 35.8 min ( 13.37 ) per subsequent session . a total of 119 patients ( 73.9% ) demonstrated program adherence of four or more coaching sessions . patients were motivated to change tobacco habits ( 7.862.17 ) and most ( 88.8% ) were at least contemplating change ( precontemplation 11.2% ; contemplation 26.7% ; planning 28% ; action 34.1% ) . there were 65 participants ( 40.4% ) whose participation was considered employer - mandated ( ie , motivated to take part by an insurance premium reduction ) . intent - to - treat quit rate at 6 months was 51.6% ( 83 of 161 ) . thirty - day point prevalence quit rate at 7 months was 88.7% ( 47 of 53 ) . overall smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months were 95.3% ( 102 of 107 ) , 89.9% ( 98 of 109 ) , 76.9% ( 83 of 108 ) , 63.2% ( 67 of 106 ) , 51.8% ( 44 of 85 ) , and 40.5% ( 30 of 74 ) , respectively . figure 2 shows no difference in smoke - free rates between patients receiving in - clinic care ( hc plus complementary therapies ) vs telephonic hc . treatment / coaching style . in - clinic patients received health coaching face - to - face in combination with complementary therapies while telephonic patients remotely received health coaching and no complementary therapies . note that the zero ( 0 ) time point reflects the average smoke - free rate for the subgroups . values below the x - axis reflect the number of patients responding at each time point . the predictors , coaching subgroup ( in - clinic / telephonic ) , motivational factors ( premium reduction mandated participation , importance , and stage of change ) and covariates , smoking history ( average amount smoked per day and years of use ) , and nrt , reliably distinguished between quitters and non - quitters as seen in the table . these variables explained a significant , small - moderate effect in smoking cessation ( (8)=48.81 , p<.001 ; nagelkerke 's r=.39 ; 79% prediction success ) . insurance mandate and stage of change significantly contributed to prediction . specifically , when patients participated freely ( ie , without a premium reduction mandate ) , they were 6.26 times more likely to quit smoking ( p<.001 ) . a unit increase in stage of change ( eg , from precontemplation to contemplation ) saw patients 3.18 times more likely to quit smoking ( p<.001 ) . the table also details the predictive ability of this model for smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months . it appears that the farther removed participants are from coaching , the more their initial rates of smoking influence remaining smoke free , with those who smoked the most at baseline having the most difficulty remaining smoke - free . logistic regression predictors of smoking cessation and smoke - free rates abbreviation : nrt , nicotine receptor therapy . participants significantly reduced tobacco use rates to an average of 3.52 ( 7.45 ) cigarettes per day ( t(134 ) = 17.94 , p<.001 ) . tobacco reduction was predicted by one 's history of smoking ( quantity smoked per day b[se ] = 0.27 ( 0.05 ) , p<.001 ; years smoked b[se ] = .09[0.04 ] , p=.04 ; number of previous quit attempts b[se ] = 0.01[.18 ] , p=.97 ; nrt ( b[se ] = 0.33[1.00 ] , p=.74 ; motivational variables ( importance b[se ] = 0.38[0.25 ] , p=.13 ; stage of change b[se ] = 2.26(0.55 ) , p<.001 ; insurance mandate b[se ] = 1.64[.97 ] , p=.10 ; and their coaching group ( in clinic / telephonic [se]=2.73[1.04 ] , p<.01 ) , f(8,155 ) = 11.82 , p<.001 ; r = .39 ) . every additional cigarette smoked at baseline related to .42 more cigarettes smoked at follow - up ; every additional year patients had smoked led to an additional .15 cigarettes smoked ; with every increase in stage of change , .31 fewer cigarettes were smoked ; and patients in the telephonic coaching group smoked .18 cigarettes fewer than those receiving in - clinic coaching . successful quit rates are at a meager 4.4% when examining programs over a 20-year period ( 1991 - 2010 ) . this poor rate of success exists despite well - funded and widely accessible smoking cessation interventions implemented after the 1998 master settlement agreement . cold turkey and unassisted quitting reveal a success rate of about 5% while some programs using behavioral therapy with nrt claim quit rates exceeding 40% . accordingly , the mtcp 30-day point prevalence ( 88.7% ) and intent - to - treat ( 51.6% ) success rates are remarkable . according to longitudinal ( 5 - 8 y ) data , the overall quit rate ( 57% ) achieved by mtcp patients at 12 months is a very good predictor of long - term success . telephonic coaching ( or counseling ) is a process widely used for tobacco cessation programs , particularly by accessible government - supported quit lines ( eg , 1 - 866 ny - quits or 1 - 800-quitnow ) . previous studies of coaching reported quit rates above 30% , but sometimes below 15% . however , the training and background of coaches in previous studies is poorly described and unlikely to meet the hc paradigm recently forwarded by wolever et al . mtcp health coaches were registered nurses with extensive coach training who delivered hc compatible with the wolever et al definition . others have called for further study of such coaching methods because of preliminary success in smoking cessation . we speculate the care and techniques provided by mtcp coaches accounted for the outstanding rate of successfully achieving tobacco independence in the present report . adding complementary therapies did little to enhance mtcp quit rates . telephonic - only coaching quit rates were not significantly different than in - clinic treatment that included aes and auriculotherapy . cranial stimulation and acupuncture are accepted clinical procedures with effectiveness in addiction / smoking cessation . in the present study , however , providing these procedures before each hc session did not improve effects of hc alone on quit rates . while these procedures may be effective , combining them with hc likely removed their influence on the quitting process . it seems that whether hc was administered with or without complementary therapies , it was the primary treatment explaining smoking cessation in these patients . the question of coaching effectiveness when delivered telephonically vs face - to - face is of great interest with ease and cost - effectiveness of remote coaching , making it preferred assuming it is equally effective . the absence of facial expressions and body language are considered shortcomings of the telephonic method . our results , however , demonstrated telephonic coaching to be equally effective as in - clinic , face - to - face sessions . it may be speculated that many patients are less inhibited speaking on the phone than in person , making them inclined to more fully tell their stories , contributing to hc success . our tobacco cessation results are not surprising given that telephonic coaching sessions apparently are also effective for weight loss and treatment of depression . our data lend further credence to remote , telephonic delivery of coaching processes as an effective behavior change intervention . the use of readiness - to - change scores appears extremely valuable for health coaches working with patients attempting to quit smoking . moving a patient one behavior - change category ( eg , from contemplation to preparation ) more than doubled chances of quitting and equally affected chances of remaining smoke - free . with such a large influence on outcome , monitoring , and influencing , stage of behavior change seems to be a critical hc strategy . this is an account of an existing practice ; therefore , these data are not from a prospectively designed , randomized , controlled trial ( rct ) . these data were systematically collected ; however , conclusive assessment of the mtcp coaching process may require an experimentally designed study . while ideal , designing such a project may not be ethically pragmatic given it requires withholding an apparently effective treatment from a ( control ) group of patients who need to be as motivated to quit smoking as those assigned to treatment . as a description of an existing practice and not an rct , this project did not control for other factors that may impact smoking cessation and for example , nrt is a controversial intervention sometimes described as effectively assisting with quitting a tobacco habit . our analysis found that hc patients using nrt were no more likely to quit tobacco than patients without nrt . many mtcp patients employed nrt ( 44% at one point or another ) though hc was the only treatment consistently applied for all patients . in standard practice , a health coach using a patient - centered approach encourages patients to seek other strategies , aids , or social supports to optimize positive behavior change . the goal of the mtcp was to follow a true coaching paradigm allowing maximum therapeutic flexibility . undoubtedly , a high level of treatment flexibility was achieved ; however , hc was the common and primary intervention for all patients in this highly successful , tobacco cessation program . smoking has huge economic implications with a $ 96 billion cdc estimated annual us health cost burden shared by smokers and tax - payers in general . this number has recently been translated to a corporate cost of greater than $ 5000 per smoking employee with additional costs for human pain and suffering immeasurable . finding a strategy to incentivize smoking cessation our observations revealed coercing smoking cessation through an insurance premium reduction mandate is not a good strategy . patients required to participate to avoid an insurance - premium penalty were about six times less likely to quit smoking than those participating freely . program compliance ( > 4 visits ) improved success for these mandated participators , but they were still about twice as likely to continue smoking as free - choice patients . nearly twice as many mandated participants were in precontemplation ( ie , not considering quitting ) compared to those freely entering mtcp . providing an insurance premium upcharge apparently may not be a good strategy to augment smoking cessation . however , a recent study indicates other employer - organized programs , using financial incentives , are viable to motivate behavior change . programs for smoking cessation can be expensive while claiming efficacy rates of 25% to 30% . the mtcp in - clinic program had quitting success rates exceeding 70% and smoke - free rate above 60% at 1 year with patient cost of $ 38 per visit ( $ 266 for seven visits ) and total staff labor costs of $ 204 for the average patient who had about seven visits . more impressively , patient cost for the equally effective telephonic - only hc was $ 32 per session with labor costs of only $ 112 for ( on average ) seven sessions . in practice , implementing a hospital - based , hc smoking cessation program is highly feasible . allied healthcare personnel ( existing staff , eg , nurses ) building on their knowledge base , with as little as 50 to 200 hours of training in relational and communication skills , may become qualified as an hc . once an hc staff exists then adding the program into existing departments ( eg , cardiac rehabilitation or employee/ community wellness ) becomes a matter of best logistical fit . given the potential economic and societal benefit , adding an hc - grounded smoking cessation program to the menu of patient treatment options should be a primary consideration for many public healthcare settings . furthermore , government sponsorship of such programs deserves careful investigation and deliberation given the prospects for reducing national healthcare costs . the mtcp features a carefully defined program of hc , and reports a very high quit rate ( 72.7% ) and excellent smoke free rates at 6 mo ( 76.9% ) and 12 mo ( 63.2% ) . habitual tobacco use is a very difficult addiction to overcome , is widely recognized as primary risk factor for chronic diseases , and is a scourge on public health . most smoking cessation programs report quit rates that rarely exceed 30% at 6 mo and unassisted quitting efforts have success rates well below 10% . for scientific purposes , greater experimental controls on our data would be ideal , but these results are highly encouraging and the success of mtcp practices for many individuals that were struggling with tobacco addiction is clear . within the limitations of this study , health coaching ( when defined by strict patient - centered standards ) other clinical and public healthcare settings should consider adapting and implementing this cost efficient model to assist their patients with tobacco abstention .

background : tobacco abuse is a well - recognized scourge on health and healthcare costs . attempts to facilitate tobacco cessation are rarely better than marginally effective.primary objective : to describe an observational trial of an existing and highly successful tobacco cessation program featuring health coaching as the primary intervention . core components of program design and data are presented and may serve as a model for other public health settings.methods:health coaching and three complementary program components ( auriculotherapy , alpha - electrical stimulation , and relaxation techniques ) are presented . quit rates at 6 months for 161 patients over 3 years are provided featuring 30-day point prevalence smoke free and intent - to - treat values . comparisons for telephonic vs in - clinic health coaching , free choice vs mandated participation , and program costs are provided.results:point prevalence quit rate was 88.7% while the more conservative intent - to - treat quit rate was 51.6% . telephonic and in - clinic health coaching were not significantly different at any time point . smoke - free rates at 6 and 12 months were 76.9% and 63.2% , respectively.conclusions:two cost - effective smoking cessation models featuring health coaching are presented . point prevalence ( 30-day ) above 80% and an enduring effect was seen . personal and societal burdens ( health and financial ) of tobacco use might be greatly impacted if such programs were successfully implemented on a larger scale .

Antecedentes: Objetivo principal: Mtodos: Resultados: Conclusiones: METHODS Patients Basic Program Design Data Management and Statistical Analysis RESULTS Participation Quit Rates Tobacco Reduction Discussion SUMMARY AND CONCLUSIONS

between 2009 and 2011 , 161 patients ( 87 male and 74 females , mean [ sd ] age : 44.4 11.8 y ) entered the mtcp . some mtcp patients participated because of an offer by their employer to achieve a $ 600 insurance premium reduction . on enrolling , each patient initially received a quit plan to consider and a questionnaire asking about tobacco use and motivation for behavior change . data are presented anonymously and were collected as part of routine clinical practice . the project design was a single - site , prospective , non - randomized , observational study of an existing practice ( ie , mtcp ) with multiple follow - ups examining the effects of an hc tobacco cessation intervention . in addition to hc , in - clinic patients received alpha - electrical stimulation ( aes ) , relaxation techniques , and auriculotherapy . of 161 patients , 93 ( 57.8% ) utilized in - clinic services while 68 used only telephonic hc . hc involved establishing an individualized quit plan while acknowledging the participant as the final decision maker in when to quit . after an in - clinic session , each patient continued with in - clinic visits or chose remote treatment using only telephonic health coaching . patients returned a completed questionnaire on smoking habits , worked on a co - created quit plan , and determined if they would use complementary therapies . hc could be delivered telephonically or patients could elect for in - clinic , face - to - face appointments . telephonic hc patients were given information about and often participated in relaxation techniques ( see below ) . the amount of hc was patient determined ; however , 6 to 8 sessions over 3 months were recommended . all patients had the option of using aes , relaxation techniques , and auriculotherapy as complementary treatment to hc ( unless travel distance precluded it ) . before hc , in - clinic patients typically completed 20 minutes of relaxation techniques while receiving aes prior to engaging in a 15-minute session of auriculotherapy . alpha - stim scs ( electromedical products international , mineral wells , texas ) is a class iia , type b medical device with federal law restricting its use . auriculotherapy was administered in 15 to 30minute sessions , and as an acupuncture - like process , is reported to assist with diminishing nicotine addiction . the goal of the mtcp was to follow a true coaching model , employing maximum therapeutic flexibility to empower cessation of tobacco use . participants were defined as those completing at least one hc session and grouped as free choice or mandated ( employer required five visits for insurance premium reduction ) . patients indicated importance of change of tobacco use on a simple 10-point likert scale along with readiness to change . smoking quit rates were calculated as the number of patients who reported smoking cigarettes at baseline but not at follow - up . smoke - free rates examined follow - up on those who initially quit and were determined at 1 , 3 , 6 , 12 , 18 , and 24 months after quit date . intent - to - treat was calculated as all patients who quit smoking at 6 months divided by all participants who reported smoking at baseline . patients lost to follow - up were presumed to have relapsed for the conservative intent - to - treat calculation . for 30-day point prevalence calculation , patients reporting that they were smoke - free for 30 days at 7 months determined the numerator while all patients reporting smoking at baseline was the denominator , excluding those not completing at least four hc sessions , those whose participation was mandated , or those who were lost to follow - up . program impact on tobacco use ( quit and smoke - free rates ) was compared between coaching groups using chi - square . the influence of coaching on quit rates and smoke - free rates was examined in conjunction with motivational variables and covariates ( smoking history and nrt use ) . due to the number of logistic analyses conducted ( one for each time point ) , the criterion for statistical significance was set at p<.007 based on a bonforroni correction ( .05/7 ) . two additional tests were conducted to examine reduction in tobacco use : a t - test was used to examine pre - to - post program changes in tobacco use , and a regression examined predictors ( coaching , motivational variables , history of smoking behavior , and nrt ) on reduced tobacco use . between 2009 and 2011 , 161 patients ( 87 male and 74 females , mean [ sd ] age : 44.4 11.8 y ) entered the mtcp . some mtcp patients participated because of an offer by their employer to achieve a $ 600 insurance premium reduction . on enrolling , each patient initially received a quit plan to consider and a questionnaire asking about tobacco use and motivation for behavior change . data are presented anonymously and were collected as part of routine clinical practice . the project design was a single - site , prospective , non - randomized , observational study of an existing practice ( ie , mtcp ) with multiple follow - ups examining the effects of an hc tobacco cessation intervention . in addition to hc , in - clinic patients received alpha - electrical stimulation ( aes ) , relaxation techniques , and auriculotherapy . of 161 patients , 93 ( 57.8% ) utilized in - clinic services while 68 used only telephonic hc . after an in - clinic session , each patient continued with in - clinic visits or chose remote treatment using only telephonic health coaching . patients returned a completed questionnaire on smoking habits , worked on a co - created quit plan , and determined if they would use complementary therapies . this approach emphasizes relationship development , is patient - centered , and encourages a self - discovery process while using mindfulness and motivational interviewing techniques . hc could be delivered telephonically or patients could elect for in - clinic , face - to - face appointments . the amount of hc was patient determined ; however , 6 to 8 sessions over 3 months were recommended . all patients had the option of using aes , relaxation techniques , and auriculotherapy as complementary treatment to hc ( unless travel distance precluded it ) . before hc , in - clinic patients typically completed 20 minutes of relaxation techniques while receiving aes prior to engaging in a 15-minute session of auriculotherapy . alpha - stim scs ( electromedical products international , mineral wells , texas ) is a class iia , type b medical device with federal law restricting its use . site selection followed the national acupuncture detoxification association ( nada ) 5-point protocol , and these sites are related to treatment for addiction , relaxation , and anxiety . the goal of the mtcp was to follow a true coaching model , employing maximum therapeutic flexibility to empower cessation of tobacco use . participants were defined as those completing at least one hc session and grouped as free choice or mandated ( employer required five visits for insurance premium reduction ) . patients indicated importance of change of tobacco use on a simple 10-point likert scale along with readiness to change . smoking quit rates were calculated as the number of patients who reported smoking cigarettes at baseline but not at follow - up . smoke - free rates examined follow - up on those who initially quit and were determined at 1 , 3 , 6 , 12 , 18 , and 24 months after quit date . intent - to - treat was calculated as all patients who quit smoking at 6 months divided by all participants who reported smoking at baseline . patients lost to follow - up were presumed to have relapsed for the conservative intent - to - treat calculation . for 30-day point prevalence calculation , patients reporting that they were smoke - free for 30 days at 7 months determined the numerator while all patients reporting smoking at baseline was the denominator , excluding those not completing at least four hc sessions , those whose participation was mandated , or those who were lost to follow - up . program impact on tobacco use ( quit and smoke - free rates ) was compared between coaching groups using chi - square . the influence of coaching on quit rates and smoke - free rates was examined in conjunction with motivational variables and covariates ( smoking history and nrt use ) . due to the number of logistic analyses conducted ( one for each time point ) , the criterion for statistical significance was set at p<.007 based on a bonforroni correction ( .05/7 ) . two additional tests were conducted to examine reduction in tobacco use : a t - test was used to examine pre - to - post program changes in tobacco use , and a regression examined predictors ( coaching , motivational variables , history of smoking behavior , and nrt ) on reduced tobacco use . intent - to - treat quit rate at 6 months was 51.6% ( 83 of 161 ) . thirty - day point prevalence quit rate at 7 months was 88.7% ( 47 of 53 ) . overall smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months were 95.3% ( 102 of 107 ) , 89.9% ( 98 of 109 ) , 76.9% ( 83 of 108 ) , 63.2% ( 67 of 106 ) , 51.8% ( 44 of 85 ) , and 40.5% ( 30 of 74 ) , respectively . figure 2 shows no difference in smoke - free rates between patients receiving in - clinic care ( hc plus complementary therapies ) vs telephonic hc . in - clinic patients received health coaching face - to - face in combination with complementary therapies while telephonic patients remotely received health coaching and no complementary therapies . note that the zero ( 0 ) time point reflects the average smoke - free rate for the subgroups . the predictors , coaching subgroup ( in - clinic / telephonic ) , motivational factors ( premium reduction mandated participation , importance , and stage of change ) and covariates , smoking history ( average amount smoked per day and years of use ) , and nrt , reliably distinguished between quitters and non - quitters as seen in the table . the table also details the predictive ability of this model for smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months . it appears that the farther removed participants are from coaching , the more their initial rates of smoking influence remaining smoke free , with those who smoked the most at baseline having the most difficulty remaining smoke - free . logistic regression predictors of smoking cessation and smoke - free rates abbreviation : nrt , nicotine receptor therapy . every additional cigarette smoked at baseline related to .42 more cigarettes smoked at follow - up ; every additional year patients had smoked led to an additional .15 cigarettes smoked ; with every increase in stage of change , .31 fewer cigarettes were smoked ; and patients in the telephonic coaching group smoked .18 cigarettes fewer than those receiving in - clinic coaching . this poor rate of success exists despite well - funded and widely accessible smoking cessation interventions implemented after the 1998 master settlement agreement . cold turkey and unassisted quitting reveal a success rate of about 5% while some programs using behavioral therapy with nrt claim quit rates exceeding 40% . accordingly , the mtcp 30-day point prevalence ( 88.7% ) and intent - to - treat ( 51.6% ) success rates are remarkable . according to longitudinal ( 5 - 8 y ) data , the overall quit rate ( 57% ) achieved by mtcp patients at 12 months is a very good predictor of long - term success . telephonic coaching ( or counseling ) is a process widely used for tobacco cessation programs , particularly by accessible government - supported quit lines ( eg , 1 - 866 ny - quits or 1 - 800-quitnow ) . previous studies of coaching reported quit rates above 30% , but sometimes below 15% . telephonic - only coaching quit rates were not significantly different than in - clinic treatment that included aes and auriculotherapy . it seems that whether hc was administered with or without complementary therapies , it was the primary treatment explaining smoking cessation in these patients . the question of coaching effectiveness when delivered telephonically vs face - to - face is of great interest with ease and cost - effectiveness of remote coaching , making it preferred assuming it is equally effective . our results , however , demonstrated telephonic coaching to be equally effective as in - clinic , face - to - face sessions . our tobacco cessation results are not surprising given that telephonic coaching sessions apparently are also effective for weight loss and treatment of depression . the use of readiness - to - change scores appears extremely valuable for health coaches working with patients attempting to quit smoking . moving a patient one behavior - change category ( eg , from contemplation to preparation ) more than doubled chances of quitting and equally affected chances of remaining smoke - free . with such a large influence on outcome , monitoring , and influencing , stage of behavior change seems to be a critical hc strategy . this is an account of an existing practice ; therefore , these data are not from a prospectively designed , randomized , controlled trial ( rct ) . as a description of an existing practice and not an rct , this project did not control for other factors that may impact smoking cessation and might have augmented the hc effect . undoubtedly , a high level of treatment flexibility was achieved ; however , hc was the common and primary intervention for all patients in this highly successful , tobacco cessation program . our observations revealed coercing smoking cessation through an insurance premium reduction mandate is not a good strategy . programs for smoking cessation can be expensive while claiming efficacy rates of 25% to 30% . the mtcp in - clinic program had quitting success rates exceeding 70% and smoke - free rate above 60% at 1 year with patient cost of $ 38 per visit ( $ 266 for seven visits ) and total staff labor costs of $ 204 for the average patient who had about seven visits . in practice , implementing a hospital - based , hc smoking cessation program is highly feasible . given the potential economic and societal benefit , adding an hc - grounded smoking cessation program to the menu of patient treatment options should be a primary consideration for many public healthcare settings . furthermore , government sponsorship of such programs deserves careful investigation and deliberation given the prospects for reducing national healthcare costs . intent - to - treat quit rate at 6 months was 51.6% ( 83 of 161 ) . thirty - day point prevalence quit rate at 7 months was 88.7% ( 47 of 53 ) . overall smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months were 95.3% ( 102 of 107 ) , 89.9% ( 98 of 109 ) , 76.9% ( 83 of 108 ) , 63.2% ( 67 of 106 ) , 51.8% ( 44 of 85 ) , and 40.5% ( 30 of 74 ) , respectively . figure 2 shows no difference in smoke - free rates between patients receiving in - clinic care ( hc plus complementary therapies ) vs telephonic hc . in - clinic patients received health coaching face - to - face in combination with complementary therapies while telephonic patients remotely received health coaching and no complementary therapies . note that the zero ( 0 ) time point reflects the average smoke - free rate for the subgroups . values below the x - axis reflect the number of patients responding at each time point . the predictors , coaching subgroup ( in - clinic / telephonic ) , motivational factors ( premium reduction mandated participation , importance , and stage of change ) and covariates , smoking history ( average amount smoked per day and years of use ) , and nrt , reliably distinguished between quitters and non - quitters as seen in the table . the table also details the predictive ability of this model for smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months . it appears that the farther removed participants are from coaching , the more their initial rates of smoking influence remaining smoke free , with those who smoked the most at baseline having the most difficulty remaining smoke - free . logistic regression predictors of smoking cessation and smoke - free rates abbreviation : nrt , nicotine receptor therapy . every additional cigarette smoked at baseline related to .42 more cigarettes smoked at follow - up ; every additional year patients had smoked led to an additional .15 cigarettes smoked ; with every increase in stage of change , .31 fewer cigarettes were smoked ; and patients in the telephonic coaching group smoked .18 cigarettes fewer than those receiving in - clinic coaching . this poor rate of success exists despite well - funded and widely accessible smoking cessation interventions implemented after the 1998 master settlement agreement . accordingly , the mtcp 30-day point prevalence ( 88.7% ) and intent - to - treat ( 51.6% ) success rates are remarkable . according to longitudinal ( 5 - 8 y ) data , the overall quit rate ( 57% ) achieved by mtcp patients at 12 months is a very good predictor of long - term success . telephonic coaching ( or counseling ) is a process widely used for tobacco cessation programs , particularly by accessible government - supported quit lines ( eg , 1 - 866 ny - quits or 1 - 800-quitnow ) . previous studies of coaching reported quit rates above 30% , but sometimes below 15% . telephonic - only coaching quit rates were not significantly different than in - clinic treatment that included aes and auriculotherapy . it seems that whether hc was administered with or without complementary therapies , it was the primary treatment explaining smoking cessation in these patients . the question of coaching effectiveness when delivered telephonically vs face - to - face is of great interest with ease and cost - effectiveness of remote coaching , making it preferred assuming it is equally effective . our results , however , demonstrated telephonic coaching to be equally effective as in - clinic , face - to - face sessions . the use of readiness - to - change scores appears extremely valuable for health coaches working with patients attempting to quit smoking . moving a patient one behavior - change category ( eg , from contemplation to preparation ) more than doubled chances of quitting and equally affected chances of remaining smoke - free . with such a large influence on outcome , monitoring , and influencing , stage of behavior change seems to be a critical hc strategy . this is an account of an existing practice ; therefore , these data are not from a prospectively designed , randomized , controlled trial ( rct ) . as a description of an existing practice and not an rct , this project did not control for other factors that may impact smoking cessation and for example , nrt is a controversial intervention sometimes described as effectively assisting with quitting a tobacco habit . undoubtedly , a high level of treatment flexibility was achieved ; however , hc was the common and primary intervention for all patients in this highly successful , tobacco cessation program . the mtcp in - clinic program had quitting success rates exceeding 70% and smoke - free rate above 60% at 1 year with patient cost of $ 38 per visit ( $ 266 for seven visits ) and total staff labor costs of $ 204 for the average patient who had about seven visits . in practice , implementing a hospital - based , hc smoking cessation program is highly feasible . given the potential economic and societal benefit , adding an hc - grounded smoking cessation program to the menu of patient treatment options should be a primary consideration for many public healthcare settings . furthermore , government sponsorship of such programs deserves careful investigation and deliberation given the prospects for reducing national healthcare costs . the mtcp features a carefully defined program of hc , and reports a very high quit rate ( 72.7% ) and excellent smoke free rates at 6 mo ( 76.9% ) and 12 mo ( 63.2% ) . habitual tobacco use is a very difficult addiction to overcome , is widely recognized as primary risk factor for chronic diseases , and is a scourge on public health . most smoking cessation programs report quit rates that rarely exceed 30% at 6 mo and unassisted quitting efforts have success rates well below 10% . within the limitations of this study , health coaching ( when defined by strict patient - centered standards ) other clinical and public healthcare settings should consider adapting and implementing this cost efficient model to assist their patients with tobacco abstention .

between 2009 and 2011 , 161 patients ( 87 male and 74 females , mean [ sd ] age : 44.4 11.8 y ) entered the mtcp . these patients , largely employees of ( n=82 ) or dependents of employees ( n=41 ) of the mercy hospital system , were supported by an outside employer ( n = 26 ) or came through word - of - mouth referral ( n = 9 ) . participants reported average smoking of 18.9 ( 11.5 ) cigarettes / day at the outset of treatment and had smoked for an average of 23.8 years ( 12.9 ) . the project design was a single - site , prospective , non - randomized , observational study of an existing practice ( ie , mtcp ) with multiple follow - ups examining the effects of an hc tobacco cessation intervention . there was no control group , but the ineffectiveness of non - coaching tobacco cessation efforts are well known and described later . it made little sense to turn away patients who wanted to quit smoking for the establishment of a control group . in addition to hc , in - clinic patients received alpha - electrical stimulation ( aes ) , relaxation techniques , and auriculotherapy . this approach emphasizes relationship development , is patient - centered , and encourages a self - discovery process while using mindfulness and motivational interviewing techniques . all patients had the option of using aes , relaxation techniques , and auriculotherapy as complementary treatment to hc ( unless travel distance precluded it ) . before hc , in - clinic patients typically completed 20 minutes of relaxation techniques while receiving aes prior to engaging in a 15-minute session of auriculotherapy . aes is delivered in 10 s waves of 0.5 hz in a pattern range of 100 to 300 micro - amps via earlobe - located electrodes . by the third visit , most patients practiced self - guided relaxation techniques for 20 minutes . auriculotherapy is a micro - current stimulation to reflex and acupuncture points on the ear delivered using the electro medical stim flex ( electro medical inc , tulsa , oklahoma ) . medications ( particularly those acting at the nicotinic receptor ) were a prevalent health coaching topic , but such treatment was a personal choice reached in consultation with a primary care physician . a large minority of mtcp patients ( 43.7% ) used some form of nicotinic receptor therapy ( bupropion hcl [ wellbutrin , 3% ] ; varenicline [ chantix , 17% ] ; and nicotine replacement therapy [ nrt , 24% ] ) during the program . confounding variables such as nrt , employer mandate , and the prevalent use of complementary therapies are addressed statistically in the results section . the goal of the mtcp was to follow a true coaching model , employing maximum therapeutic flexibility to empower cessation of tobacco use . readiness to change is based on the transtheoretical model , which allowed classification of patients into one of five stages ( ie , precontemplation , contemplation , preparation , action , maintenance ) . smoking quit rates were calculated as the number of patients who reported smoking cigarettes at baseline but not at follow - up . self - reported cessation ( 7 mo , 30-d point prevalence ) is currently the standard used by the north american quitline provided a 50% response rate can be achieved . smoke - free rates examined follow - up on those who initially quit and were determined at 1 , 3 , 6 , 12 , 18 , and 24 months after quit date . intent - to - treat was calculated as all patients who quit smoking at 6 months divided by all participants who reported smoking at baseline . patients lost to follow - up were presumed to have relapsed for the conservative intent - to - treat calculation . for 30-day point prevalence calculation , patients reporting that they were smoke - free for 30 days at 7 months determined the numerator while all patients reporting smoking at baseline was the denominator , excluding those not completing at least four hc sessions , those whose participation was mandated , or those who were lost to follow - up . program impact on tobacco use ( quit and smoke - free rates ) was compared between coaching groups using chi - square . the influence of coaching on quit rates and smoke - free rates was examined in conjunction with motivational variables and covariates ( smoking history and nrt use ) . due to the number of logistic analyses conducted ( one for each time point ) , the criterion for statistical significance was set at p<.007 based on a bonforroni correction ( .05/7 ) . two additional tests were conducted to examine reduction in tobacco use : a t - test was used to examine pre - to - post program changes in tobacco use , and a regression examined predictors ( coaching , motivational variables , history of smoking behavior , and nrt ) on reduced tobacco use . between 2009 and 2011 , 161 patients ( 87 male and 74 females , mean [ sd ] age : 44.4 11.8 y ) entered the mtcp . these patients , largely employees of ( n=82 ) or dependents of employees ( n=41 ) of the mercy hospital system , were supported by an outside employer ( n = 26 ) or came through word - of - mouth referral ( n = 9 ) . participants reported average smoking of 18.9 ( 11.5 ) cigarettes / day at the outset of treatment and had smoked for an average of 23.8 years ( 12.9 ) . the project design was a single - site , prospective , non - randomized , observational study of an existing practice ( ie , mtcp ) with multiple follow - ups examining the effects of an hc tobacco cessation intervention . there was no control group , but the ineffectiveness of non - coaching tobacco cessation efforts are well known and described later . it made little sense to turn away patients who wanted to quit smoking for the establishment of a control group . in addition to hc , in - clinic patients received alpha - electrical stimulation ( aes ) , relaxation techniques , and auriculotherapy . this approach emphasizes relationship development , is patient - centered , and encourages a self - discovery process while using mindfulness and motivational interviewing techniques . by the third visit , most patients practiced self - guided relaxation techniques for 20 minutes . medications ( particularly those acting at the nicotinic receptor ) were a prevalent health coaching topic , but such treatment was a personal choice reached in consultation with a primary care physician . a large minority of mtcp patients ( 43.7% ) used some form of nicotinic receptor therapy ( bupropion hcl [ wellbutrin , 3% ] ; varenicline [ chantix , 17% ] ; and nicotine replacement therapy [ nrt , 24% ] ) during the program . confounding variables such as nrt , employer mandate , and the prevalent use of complementary therapies are addressed statistically in the results section . the goal of the mtcp was to follow a true coaching model , employing maximum therapeutic flexibility to empower cessation of tobacco use . readiness to change is based on the transtheoretical model , which allowed classification of patients into one of five stages ( ie , precontemplation , contemplation , preparation , action , maintenance ) . smoking quit rates were calculated as the number of patients who reported smoking cigarettes at baseline but not at follow - up . self - reported cessation ( 7 mo , 30-d point prevalence ) is currently the standard used by the north american quitline provided a 50% response rate can be achieved . smoke - free rates examined follow - up on those who initially quit and were determined at 1 , 3 , 6 , 12 , 18 , and 24 months after quit date . intent - to - treat was calculated as all patients who quit smoking at 6 months divided by all participants who reported smoking at baseline . patients lost to follow - up were presumed to have relapsed for the conservative intent - to - treat calculation . for 30-day point prevalence calculation , patients reporting that they were smoke - free for 30 days at 7 months determined the numerator while all patients reporting smoking at baseline was the denominator , excluding those not completing at least four hc sessions , those whose participation was mandated , or those who were lost to follow - up . program impact on tobacco use ( quit and smoke - free rates ) was compared between coaching groups using chi - square . the influence of coaching on quit rates and smoke - free rates was examined in conjunction with motivational variables and covariates ( smoking history and nrt use ) . two additional tests were conducted to examine reduction in tobacco use : a t - test was used to examine pre - to - post program changes in tobacco use , and a regression examined predictors ( coaching , motivational variables , history of smoking behavior , and nrt ) on reduced tobacco use . for the 161 participants in this study , an average of 6.20 ( 2.71 , range=2 - 15 ) hc sessions were recorded . patients were motivated to change tobacco habits ( 7.862.17 ) and most ( 88.8% ) were at least contemplating change ( precontemplation 11.2% ; contemplation 26.7% ; planning 28% ; action 34.1% ) . overall smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months were 95.3% ( 102 of 107 ) , 89.9% ( 98 of 109 ) , 76.9% ( 83 of 108 ) , 63.2% ( 67 of 106 ) , 51.8% ( 44 of 85 ) , and 40.5% ( 30 of 74 ) , respectively . figure 2 shows no difference in smoke - free rates between patients receiving in - clinic care ( hc plus complementary therapies ) vs telephonic hc . in - clinic patients received health coaching face - to - face in combination with complementary therapies while telephonic patients remotely received health coaching and no complementary therapies . the predictors , coaching subgroup ( in - clinic / telephonic ) , motivational factors ( premium reduction mandated participation , importance , and stage of change ) and covariates , smoking history ( average amount smoked per day and years of use ) , and nrt , reliably distinguished between quitters and non - quitters as seen in the table . these variables explained a significant , small - moderate effect in smoking cessation ( (8)=48.81 , p<.001 ; nagelkerke 's r=.39 ; 79% prediction success ) . specifically , when patients participated freely ( ie , without a premium reduction mandate ) , they were 6.26 times more likely to quit smoking ( p<.001 ) . a unit increase in stage of change ( eg , from precontemplation to contemplation ) saw patients 3.18 times more likely to quit smoking ( p<.001 ) . the table also details the predictive ability of this model for smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months . it appears that the farther removed participants are from coaching , the more their initial rates of smoking influence remaining smoke free , with those who smoked the most at baseline having the most difficulty remaining smoke - free . logistic regression predictors of smoking cessation and smoke - free rates abbreviation : nrt , nicotine receptor therapy . tobacco reduction was predicted by one 's history of smoking ( quantity smoked per day b[se ] = 0.27 ( 0.05 ) , p<.001 ; years smoked b[se ] = .09[0.04 ] , p=.04 ; number of previous quit attempts b[se ] = 0.01[.18 ] , p=.97 ; nrt ( b[se ] = 0.33[1.00 ] , p=.74 ; motivational variables ( importance b[se ] = 0.38[0.25 ] , p=.13 ; stage of change b[se ] = 2.26(0.55 ) , p<.001 ; insurance mandate b[se ] = 1.64[.97 ] , p=.10 ; and their coaching group ( in clinic / telephonic [se]=2.73[1.04 ] , p<.01 ) , f(8,155 ) = 11.82 , p<.001 ; r = .39 ) . every additional cigarette smoked at baseline related to .42 more cigarettes smoked at follow - up ; every additional year patients had smoked led to an additional .15 cigarettes smoked ; with every increase in stage of change , .31 fewer cigarettes were smoked ; and patients in the telephonic coaching group smoked .18 cigarettes fewer than those receiving in - clinic coaching . accordingly , the mtcp 30-day point prevalence ( 88.7% ) and intent - to - treat ( 51.6% ) success rates are remarkable . according to longitudinal ( 5 - 8 y ) data , the overall quit rate ( 57% ) achieved by mtcp patients at 12 months is a very good predictor of long - term success . we speculate the care and techniques provided by mtcp coaches accounted for the outstanding rate of successfully achieving tobacco independence in the present report . telephonic - only coaching quit rates were not significantly different than in - clinic treatment that included aes and auriculotherapy . in the present study , however , providing these procedures before each hc session did not improve effects of hc alone on quit rates . the question of coaching effectiveness when delivered telephonically vs face - to - face is of great interest with ease and cost - effectiveness of remote coaching , making it preferred assuming it is equally effective . our results , however , demonstrated telephonic coaching to be equally effective as in - clinic , face - to - face sessions . the use of readiness - to - change scores appears extremely valuable for health coaches working with patients attempting to quit smoking . moving a patient one behavior - change category ( eg , from contemplation to preparation ) more than doubled chances of quitting and equally affected chances of remaining smoke - free . with such a large influence on outcome , monitoring , and influencing , stage of behavior change seems to be a critical hc strategy . our analysis found that hc patients using nrt were no more likely to quit tobacco than patients without nrt . in standard practice , a health coach using a patient - centered approach encourages patients to seek other strategies , aids , or social supports to optimize positive behavior change . undoubtedly , a high level of treatment flexibility was achieved ; however , hc was the common and primary intervention for all patients in this highly successful , tobacco cessation program . the mtcp in - clinic program had quitting success rates exceeding 70% and smoke - free rate above 60% at 1 year with patient cost of $ 38 per visit ( $ 266 for seven visits ) and total staff labor costs of $ 204 for the average patient who had about seven visits . more impressively , patient cost for the equally effective telephonic - only hc was $ 32 per session with labor costs of only $ 112 for ( on average ) seven sessions . allied healthcare personnel ( existing staff , eg , nurses ) building on their knowledge base , with as little as 50 to 200 hours of training in relational and communication skills , may become qualified as an hc . given the potential economic and societal benefit , adding an hc - grounded smoking cessation program to the menu of patient treatment options should be a primary consideration for many public healthcare settings . overall smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months were 95.3% ( 102 of 107 ) , 89.9% ( 98 of 109 ) , 76.9% ( 83 of 108 ) , 63.2% ( 67 of 106 ) , 51.8% ( 44 of 85 ) , and 40.5% ( 30 of 74 ) , respectively . figure 2 shows no difference in smoke - free rates between patients receiving in - clinic care ( hc plus complementary therapies ) vs telephonic hc . in - clinic patients received health coaching face - to - face in combination with complementary therapies while telephonic patients remotely received health coaching and no complementary therapies . the predictors , coaching subgroup ( in - clinic / telephonic ) , motivational factors ( premium reduction mandated participation , importance , and stage of change ) and covariates , smoking history ( average amount smoked per day and years of use ) , and nrt , reliably distinguished between quitters and non - quitters as seen in the table . these variables explained a significant , small - moderate effect in smoking cessation ( (8)=48.81 , p<.001 ; nagelkerke 's r=.39 ; 79% prediction success ) . specifically , when patients participated freely ( ie , without a premium reduction mandate ) , they were 6.26 times more likely to quit smoking ( p<.001 ) . the table also details the predictive ability of this model for smoke - free rates at 1 , 3 , 6 , 12 , 18 , and 24 months . it appears that the farther removed participants are from coaching , the more their initial rates of smoking influence remaining smoke free , with those who smoked the most at baseline having the most difficulty remaining smoke - free . logistic regression predictors of smoking cessation and smoke - free rates abbreviation : nrt , nicotine receptor therapy . tobacco reduction was predicted by one 's history of smoking ( quantity smoked per day b[se ] = 0.27 ( 0.05 ) , p<.001 ; years smoked b[se ] = .09[0.04 ] , p=.04 ; number of previous quit attempts b[se ] = 0.01[.18 ] , p=.97 ; nrt ( b[se ] = 0.33[1.00 ] , p=.74 ; motivational variables ( importance b[se ] = 0.38[0.25 ] , p=.13 ; stage of change b[se ] = 2.26(0.55 ) , p<.001 ; insurance mandate b[se ] = 1.64[.97 ] , p=.10 ; and their coaching group ( in clinic / telephonic [se]=2.73[1.04 ] , p<.01 ) , f(8,155 ) = 11.82 , p<.001 ; r = .39 ) . every additional cigarette smoked at baseline related to .42 more cigarettes smoked at follow - up ; every additional year patients had smoked led to an additional .15 cigarettes smoked ; with every increase in stage of change , .31 fewer cigarettes were smoked ; and patients in the telephonic coaching group smoked .18 cigarettes fewer than those receiving in - clinic coaching . according to longitudinal ( 5 - 8 y ) data , the overall quit rate ( 57% ) achieved by mtcp patients at 12 months is a very good predictor of long - term success . we speculate the care and techniques provided by mtcp coaches accounted for the outstanding rate of successfully achieving tobacco independence in the present report . the question of coaching effectiveness when delivered telephonically vs face - to - face is of great interest with ease and cost - effectiveness of remote coaching , making it preferred assuming it is equally effective . our results , however , demonstrated telephonic coaching to be equally effective as in - clinic , face - to - face sessions . the use of readiness - to - change scores appears extremely valuable for health coaches working with patients attempting to quit smoking . undoubtedly , a high level of treatment flexibility was achieved ; however , hc was the common and primary intervention for all patients in this highly successful , tobacco cessation program . the mtcp in - clinic program had quitting success rates exceeding 70% and smoke - free rate above 60% at 1 year with patient cost of $ 38 per visit ( $ 266 for seven visits ) and total staff labor costs of $ 204 for the average patient who had about seven visits . more impressively , patient cost for the equally effective telephonic - only hc was $ 32 per session with labor costs of only $ 112 for ( on average ) seven sessions .

body mass index ( bmi ) is the most commonly used anthropometric method to define human obesity . bmi is a complex trait affected by many environmental ( eg , diet , age , physical activity ) and genetic factors , with heritability estimates that vary from 4080% in twin studies , 2050% in family studies and 2060% in adoption studies . recent genome - wide association ( gwa ) studies have successfully identified numerous single - nucleotide polymorphisms ( snps ) that are robustly associated with obesity related traits , including bmi . they shed light on the biological basis of obesity and suggest a role for neuronal influences on the regulation of appetite and/or energy balance . however , the identified genetic variants jointly explained only a small proportion of the trait variation and thus had limited predictive value for obesity risk . for example , in a recent meta - analysis ( 249 796 individuals ) 32 identified and replicated snps together explained only 1.45% of the inter - individual variation in bmi where the strongest snp accounted for just 0.34% of the variance . the 32 bmi snps map to 32 different genes that are referred to as bmi loci hereafter . gene gene interactions ( epistasis ) are thought to be potential sources of the unexplained genetic variation , but they remain largely unexplored in the gwa studies conducted for bmi so far . a major hurdle for analysing epistasis in gwa studies was the lack of fast methods to enumerate billions of interaction tests in a full pair - wise genome scan to map different types of epistasis ( eg , with or without main effects ) while keeping false - positive rates under control . another hurdle for studying epistasis is the relatively small sample size in many existing gwa cohorts that may limit the power of detection and replication of epistasis signals unless the epistatic effects to be detected are large . it was showed in simulation that more than 4000 case control pairs were needed to achieve 80% power of detection of epistasis with an odds ratio of 3.0 in complex diseases . for quantitative traits , the major hurdle is gradually easing and full pair - wise genome scans are beginning to be applied to gwa populations individually . meta - analysis of epistasis as applied in gwa studies could be a good way to overcome the sample size hurdle but requires new methods to accommodate imputed snp genotype data . various approaches in search space reduction ( ie , less stringent significance thresholds as result of the much reduced number of tests ) can be applied to improve the power of detection of epistasis in individual gwa populations . testing interactions involving genome - wide significant loci ( of marginal effects ) with a threshold corrected for the actual number of tests has been suggested and applied successfully in recent studies . another approach is to select snps based on existing biological knowledge ( eg , protein protein interactions ) and test interactions among them only . however , cautions should be taken when making the snp selection because biological knowledge may not be directly related to the trait studied and any biases in the pre - identified loci could lead to false - positive epistatic signals . here we demonstrate a different approach to exploit the value of genome - wide analysis of epistasis using multiple populations . first we performed full pair - wise genome scans for epistasis in bmi in four gwa populations to which we had direct access : the scottish orcades , the croatia - vis and croatia - korcula , and the italian micros study cohorts . each of these cohorts has a relatively small sample size and is sampled from distinct european regions with widely differing lifestyles and diets . second , we identified common and potentially important gene gene interactions using the epistasis signals uncovered in each cohort and their gene ontology ( go ) enrichment across populations . in addition , we also identified a set of interactions involving the bmi loci ( as prior knowledge ) in different cohorts . third , we tested the identified interactions in each cohort for replication and then the replicated signals in the northern finland birth cohort 1966 ( nfbc1966 ) . we aim to address the question whether epistasis analysis is of value for the dissection of the genetic regulation of bmi in these study cohorts . , the scottish orcades cohort was recruited from a subgroup of 10 islands of the archipelago of orkney . this study was approved by the nhs orkney research ethics committee and the north of scotland rec . the croatia - vis and croatia - korcula cohorts were recruited from the island of vis and the island of korcula , respectively . both studies were approved by the ethical committee of the medical school , university of zagreb and the multi - centre research ethics committee for scotland . the italian micros cohort was recruited from villages in an isolated highland area of the south tyrol . all participants gave written informed consent and were measured for a number of traits , including weight and height from which bmi values were calculated . dna samples were genotyped with illumina infinium humanhap300v1/v2 ( for croatia - vis by the wellcome trust clinical facility in einburgh , uk ) or humancnv370v1 snp bead microarrays ( for croatia - korcula , orcades and micros by the helmholtz zentrum munchen in munich , germany ) and analysed using the beadstudio software ( illumina ) . quality control of the genotype data was performed for each cohort using the r / genabel package ( version 1.6 - 7 ) based on a common set of criteria : individual call rate at 97% , snp call rate at 95% , p - value for deviation from hardy weinberg equilibrium at 1.0e-10 , minor allele frequency at 2% . the nfbc1966 data were provided by the database of genotype and phenotype ( dbgap ) via specific data use certification and used as the replication cohort . nfbc1966 includes nearly all individuals born in 1966 in the two northernmost finnish provinces that were genotyped with humancnv370v1 snp bead microarrays and was put through the same quality control procedure as above . the summary information of each cohort after quality control and excluding individuals without bmi or age records or with extremely high bmi ( ie , bmi>50 kg / m ) is given in table 1 . the raw bmi data in each of the four study cohorts were corrected for age and sex and normalised using the rntransform function that is implemented in the genabel package performing quantile normalisation of residuals from a generalised linear model analysis . the normalised bmi residuals were then analysed using a linear mixed model to correct for polygenic effects due to relatedness using the polygenic function in the genabel package and the resultant environmental residuals ( ie , pgresidualy in genabel ) were used as the trait to test for association . the original gwa study , in the nfbc1966 cohort individuals with pregnancy and/or self reported bmi measures were excluded , and the raw bmi values were corrected for the sexocpg factor ( recoded according to gender , status of taking oral contraception and pregnancy ) and then normalised and corrected for relatedness as above . a single - snp based gwa scan was performed in each population using a score test method ( based on the additive model ) implemented in the mmscore function in the genabel package . the consensus gwa threshold of 7.3 ( log10(5.0e08 ) ) was applied to identify gwa significant snps . we also performed a full pair - wise genome scan using the regression models described below . considering a pair of snps denoted as snp1 and snp2 , the following genetic models were used to detect epistasis where genotypes of each snp ( ie , homozygote of the minor allele , homozygote of the major allele and heterozygote ) were fitted as fixed factors : where y is the trait of interest , is the model constant , snp1 ( or snp2 ) is a fixed factor with three levels ( genotype classes ) , snp1snp2 is the interaction term , e is the random error term . the f ratio test of model 1 against model 3 evaluates the whole pair effect , including interaction ( ie , fpair , 8 degrees of freedom ) . the f ratio test of model 1 against model 2 evaluates the interaction between the two snps ( ie , fint , 4 degrees of freedom ) . p - values were calculated based on the f distribution with relevant degrees of freedom and transformed to the log10 scale ( ie , log10 ppair for the fpair test , log10 pint for the fint test ) . genome - wide significant thresholds ( all in the log10 scale ) were derived based on bonferroni correction for multiple tests , that is , the 5% nominal p value corrected by the number of tests performed . considering 300 000 snps , a full pair - wise genome scan perform 4.5e+10 association tests and thus the 5% genome - wide threshold is 11.95 ( ie , log10(0.05/4.5e+10 ) ) . after each pair - wise genome scan , results were evaluated using the predefined threshold to identify genome - wide significant interaction signals . each snp in the results was annotated to the nearest gene within a window of 20 kilobases flanking the snp ( based on the physical distance to either the start or end of transcription of a gene ; the distance is considered as zero if the snp is within a gene ) . a go enrichment analysis was conducted for each study cohort using the running mode of two unranked lists of genes ' in gorilla based on the standard hyper geometric statistics , where the annotated epistatic genes were used as the target with the full list of human genes as the background . for simplicity , we chose to use the same log10 p value as the consensus gwa threshold ( ie , log10 pint > 7.3 ) to select snp pairs of each cohort and used their gene annotations as the input for the go enrichment analysis . the go terms enriched ( p<1.0e03 ) were compared across study cohorts to identify firstly common go terms and then their member genes shared by the cohorts . the shared epistatic genes were examined further for biological functions via literature mining and their associated interactions in the retained results of each cohort to identify potentially important interactions for replication tests . the bmi loci involved snp pairs ( log10 pint > 7.3 ) in each study cohort were also identified as potentially important interaction signals for replication tests . genome - wide significant snp pairs and those identified as potentially important interactions were tested for replication across the four study cohorts . each replication test was done at both the snp and region levels . at the snp level , each replicated snp was exactly the same as the corresponding epistatic snp and thus the 5% nominal significance threshold ( ie , log10(0.05)=1.30 ) was used because only one replication test was needed . at the region level , interactions between each of 10 adjacent snps ( ie , five upstream and five downstream ) of the first epistatic snp and each of those of the second were tested , to accommodate the situation where multiple snps may tag a same mutant of a gene . permutation was used to derive significance thresholds for replication of each epistatic pair at the region level , where phenotypes were permuted and the highest log10 pint value of 121 ( ie , 11 11 ) interaction tests was recorded in each of 1000 iterations . the replicated snp pairs were fitted together into the full model to calculate the proportion of phenotypic variance explained in each study cohort . , the scottish orcades cohort was recruited from a subgroup of 10 islands of the archipelago of orkney . this study was approved by the nhs orkney research ethics committee and the north of scotland rec . the croatia - vis and croatia - korcula cohorts were recruited from the island of vis and the island of korcula , respectively . both studies were approved by the ethical committee of the medical school , university of zagreb and the multi - centre research ethics committee for scotland . the italian micros cohort was recruited from villages in an isolated highland area of the south tyrol . all participants gave written informed consent and were measured for a number of traits , including weight and height from which bmi values were calculated . dna samples were genotyped with illumina infinium humanhap300v1/v2 ( for croatia - vis by the wellcome trust clinical facility in einburgh , uk ) or humancnv370v1 snp bead microarrays ( for croatia - korcula , orcades and micros by the helmholtz zentrum munchen in munich , germany ) and analysed using the beadstudio software ( illumina ) . quality control of the genotype data was performed for each cohort using the r / genabel package ( version 1.6 - 7 ) based on a common set of criteria : individual call rate at 97% , snp call rate at 95% , p - value for deviation from hardy weinberg equilibrium at 1.0e-10 , minor allele frequency at 2% . the nfbc1966 data were provided by the database of genotype and phenotype ( dbgap ) via specific data use certification and used as the replication cohort . nfbc1966 includes nearly all individuals born in 1966 in the two northernmost finnish provinces that were genotyped with humancnv370v1 snp bead microarrays and was put through the same quality control procedure as above . the summary information of each cohort after quality control and excluding individuals without bmi or age records or with extremely high bmi ( ie , bmi>50 kg / m ) is given in table 1 . the raw bmi data in each of the four study cohorts were corrected for age and sex and normalised using the rntransform function that is implemented in the genabel package performing quantile normalisation of residuals from a generalised linear model analysis . the normalised bmi residuals were then analysed using a linear mixed model to correct for polygenic effects due to relatedness using the polygenic function in the genabel package and the resultant environmental residuals ( ie , pgresidualy in genabel ) were used as the trait to test for association . the polygenic heritability was estimated at the mixed - model step . following the original gwa study , in the nfbc1966 cohort individuals with pregnancy and/or self reported bmi measures were excluded , and the raw bmi values were corrected for the sexocpg factor ( recoded according to gender , status of taking oral contraception and pregnancy ) and then normalised and corrected for relatedness as above . a single - snp based gwa scan was performed in each population using a score test method ( based on the additive model ) implemented in the mmscore function in the genabel package . the consensus gwa threshold of 7.3 ( log10(5.0e08 ) ) was applied to identify gwa significant snps . we also performed a full pair - wise genome scan using the regression models described below . considering a pair of snps denoted as snp1 and snp2 , the following genetic models were used to detect epistasis where genotypes of each snp ( ie , homozygote of the minor allele , homozygote of the major allele and heterozygote ) were fitted as fixed factors : where y is the trait of interest , is the model constant , snp1 ( or snp2 ) is a fixed factor with three levels ( genotype classes ) , snp1snp2 is the interaction term , e is the random error term . the f ratio test of model 1 against model 3 evaluates the whole pair effect , including interaction ( ie , fpair , 8 degrees of freedom ) . the f ratio test of model 1 against model 2 evaluates the interaction between the two snps ( ie , fint , 4 degrees of freedom ) . p - values were calculated based on the f distribution with relevant degrees of freedom and transformed to the log10 scale ( ie , log10 ppair for the fpair test , log10 pint for the fint test ) . genome - wide significant thresholds ( all in the log10 scale ) were derived based on bonferroni correction for multiple tests , that is , the 5% nominal p value corrected by the number of tests performed . considering 300 000 snps , a full pair - wise genome scan perform 4.5e+10 association tests and thus the 5% genome - wide threshold is 11.95 ( ie , log10(0.05/4.5e+10 ) ) . after each pair - wise genome scan , results were evaluated using the predefined threshold to identify genome - wide significant interaction signals . each snp in the results was annotated to the nearest gene within a window of 20 kilobases flanking the snp ( based on the physical distance to either the start or end of transcription of a gene ; the distance is considered as zero if the snp is within a gene ) . a go enrichment analysis was conducted for each study cohort using the running mode of two unranked lists of genes ' in gorilla based on the standard hyper geometric statistics , where the annotated epistatic genes were used as the target with the full list of human genes as the background . for simplicity , we chose to use the same log10 p value as the consensus gwa threshold ( ie , log10 pint > 7.3 ) to select snp pairs of each cohort and used their gene annotations as the input for the go enrichment analysis . the go terms enriched ( p<1.0e03 ) were compared across study cohorts to identify firstly common go terms and then their member genes shared by the cohorts . the shared epistatic genes were examined further for biological functions via literature mining and their associated interactions in the retained results of each cohort to identify potentially important interactions for replication tests . the bmi loci involved snp pairs ( log10 pint > 7.3 ) in each study cohort were also identified as potentially important interaction signals for replication tests . genome - wide significant snp pairs and those identified as potentially important interactions were tested for replication across the four study cohorts . each replication test was done at both the snp and region levels . at the snp level , each replicated snp was exactly the same as the corresponding epistatic snp and thus the 5% nominal significance threshold ( ie , log10(0.05)=1.30 ) was used because only one replication test was needed . at the region level , interactions between each of 10 adjacent snps ( ie , five upstream and five downstream ) of the first epistatic snp and each of those of the second were tested , to accommodate the situation where multiple snps may tag a same mutant of a gene . permutation was used to derive significance thresholds for replication of each epistatic pair at the region level , where phenotypes were permuted and the highest log10 pint value of 121 ( ie , 11 11 ) interaction tests was recorded in each of 1000 iterations . the replicated snp pairs were fitted together into the full model to calculate the proportion of phenotypic variance explained in each study cohort . the mean bmi was similar across the croatia - vis , croatia - korcula and orcades cohorts but lower in micros ( table 1 ) . the polygenic heritability estimates varied from 0.356 ( croatia - vis ) to 0.514 ( orcades ) . the inflation factor lambda ( computed by regression of observed association p - values against the expected ) of each gwa scan was very close to 1 ( table 1 ) , suggesting the family relatedness in each cohort was well accounted for . only 8 out of the 32 bmi snps previously identified were genotyped in the four study cohorts and none of these showed a strong association with bmi ( supplementary table s1 ) . full pair - wise genome scans found no snp pairs that passed the genome - wide threshold ( log10 pint=11.95 ) in any of the four study cohorts ( figure 1 ) . considering interaction signals with log10 pint > 7.3 , micros had the least number of snp pairs and consequently the least number of annotated genes , whereas the remaining three cohorts had relatively similar numbers of snp pairs and annotated genes ( table 2 ) . five out of the 32 bmi loci ( but not the bmi snps ) were involved in 7 epistatic pairs in croatia - vis : fto , kctd15 , lrp1b , negr1 and prkd1 . similarly , three bmi loci ( negr1 , nrxn3 and prkd1 ) were involved in croatia - korcula , two ( fto and mtch2 ) in orcades and two ( fto and lrp1b ) in micros . go terms enriched by epistatic genes ( log10 pint > 7.3 ) in each cohort were compared ( supplementary table s2 ) and identified 9 common in all four cohorts ( table 3 ) that might indicate common regulation mechanisms ( eg , go:0008038 neuron recognition ) . among the epistatic genes that enriched the 9 go terms , we found 19 epistatic genes shared by the four cohorts of which 15 are previously published gwa loci ( mostly not genome - wide significant ) associating with various phenotypes ( supplementary table s3 ) . most of the 19 shared epistatic genes interacted with one another despite their interactions being relatively weak ( log10 pint<7.3 , supplementary table s4 ) in general , including cdh13 ( cadherin 13 ) associated with height and sorcs2 ( sortilin - related vps10 domain containing receptor 2 ) associated with circulating insulin - like growth factor 1 and insulin - like growth factor - binding protein-3 , which are important for anthropometric traits and risk of cancer and cardiovascular disease . we further tested replication of the snp pairs involving either bmi loci ( 19 , table 2 ) or two shared epistatic genes across the study cohorts ( 50 , supplementary table s4 ) . despite none of the 69 snp pairs being genome - wide significant , eight of them had a replication in one or more cohorts at the snp level ( ie , log10 pint>1.30 ; table 4 ) . the best replicated pairs at the snp level were rs2202167 ( nrxn3 ) rs11150880 ( log10 pint was 8.19 , 1.68 and 1.43 in croatia - korcula , croatia - vis and orcades , respectively ) and rs1474056 ( mtch2 ) - rs7250947 ( plin4 ) ( log10 pint was 8.08 in orcades and 2.44 in croatia - korcula ) . the rs11150880 snp is near the rph3al gene , which is known to have a key role in insulin secretion by pancreatic cells . the eight replicated snp pairs together explained the phenotypic variance of bmi by 4 , 4 , 2 and 0.5% in croatia - vis , croatia - korcula , orcades and micros , respectively . by testing replication at the region level , we found the pair of rs9858278 ( naaladl2 ) - rs7198915 ( cdh13 ) replicated in croatia - vis , croatia - korcula and micros ( exceed the 5% thresholds , table 4 and supplementary table s5 ) . further testing the nine replicated snp pairs in the nfbc1966 cohort found none replicated at either the snp or region levels . however , seven out of the nine pairs had log10 pint>2 , of which three exceed the 20% thresholds ( table 4 and supplementary table s5 ) . gene gene interactions have been suggested as sources of the hidden genetic variations in gwa studies , but the extent of their role in this regard has yet to be demonstrated . one big challenge is that the sample sizes of many gwa data sets are relatively small ( eg , less than 4000 individuals ) and hence the power to detect epistasis could be low . therefore studying epistasis in a single gwa population this is certainly true in our case where exhaustive genome scans in the four study cohorts found no genome - wide significant epistasis associated with bmi . we suggest to tackle the challenge by looking for common ( thus potentially important ) gene gene interactions from sub genome - wide significant epistatic signals ( log10 pint>7.3 ) in multiple gwa populations . we showed that go enrichment analysis could be used to identify common go terms ( ie gene function groups ) enriched by the epistatic signals in the four study cohorts from which 19 shared epistatic genes were identified . most of the 19 shared epistatic genes are previously identified gwa loci associating with phenotypes other than bmi and interacted with one another . their interactions were considered potentially important because they belong to one or multiple commonly enriched go terms . interactions involving at least one of the 32 bmi loci with log10 pint>7.3 were also considered potentially important assuming the bmi loci are likely interactive . being aware of possible noises in those potentially important interactions , we used replication to identify the most reliable epistatic signals across the study cohorts . eight epistatic pairs involving either the bmi loci or two shared epistatic genes showed replication at the snp level in at least one cohort ( table 4 ) . the eight epistatic pairs together could indeed explain a considerable proportion of the bmi variation in each individual cohort . nevertheless , caution is recommended in light of the potential overestimation of the effects due to the winner 's curse ' . besides , none of the eight epistatic pairs were replicated in all of the four study cohorts , or in the replication cohort nfbc1966 . further replication tests in other populations and/or functional assays are useful to confirm whether they are true signals . statistical replication has been used as the golden rule to prevent reporting false positives in gwa studies . this however appears to be far more difficult for epistatic signals than for single snp signals for reasons , including power , minor allele frequency change , and linkage disequilibrium between epistatic snp and mutant for both loci . the moderate log10 pint values of the epistatic pairs tested for replication suggest that the linkage disequilibrium between epistatic snps and mutants is not high so replication of these pairs will be difficult . furthermore , different environments may cause different phenotype distributions in the discovery and replication cohorts . the lack of replication in the nfbc1966 cohort could be due to two important environmental factors of bmi : age ( ie , 31 vs a range between 18 and 90 in the study cohorts ) and diets . gene interactions in multiple gwa populations is an effective solution to the issue of limited power of detection of epistasis . it is just a partial solution though because some ignored interactions may be important as well . comparison of sub genome - wide significant epistatic signals across multiple populations can be made at either the snp , or gene or pathway level and seem more fruitful at the gene or pathway level than the snp level . the approach may become more useful if better annotation methods ( considering only gwa signals without interactions ) can be adapted to epistasis . for example , not all epistatic snps were annotated to genes in the study and hence did not contribute to the enrichment analysis . the approach will likely remain important even once new tools for meta - analysis of epistasis in gwa data sets become available to increase power for detection of epistasis .

we surveyed gene gene interactions ( epistasis ) in human body mass index ( bmi ) in four european populations ( n<1200 ) via exhaustive pair - wise genome scans where interactions were computed as f ratios by testing a linear regression model fitting two single - nucleotide polymorphisms ( snps ) with interactions against the one without . before the association tests , bmi was corrected for sex and age , normalised and adjusted for relatedness . neither single snps nor snp interactions were genome - wide significant in either cohort based on the consensus threshold ( p=5.0e08 ) and a bonferroni corrected threshold ( p=1.1e12 ) , respectively . next we compared sub genome - wide significant snp interactions ( p<5.0e08 ) across cohorts to identify common epistatic signals , where snps were annotated to genes to test for gene ontology ( go ) enrichment . among the epistatic genes contributing to the commonly enriched go terms , 19 were shared across study cohorts of which 15 are previously published genome - wide association loci , including cdh13 ( cadherin 13 ) associated with height and sorcs2 ( sortilin - related vps10 domain containing receptor 2 ) associated with circulating insulin - like growth factor 1 and binding protein 3 . interactions between the 19 shared epistatic genes and those involving bmi candidate loci ( p<5.0e08 ) were tested across cohorts and found eight replicated at the snp level ( p<0.05 ) in at least one cohort , which were further tested and showed limited replication in a separate european population ( n>5000 ) . we conclude that genome - wide analysis of epistasis in multiple populations is an effective approach to provide new insights into the genetic regulation of bmi but requires additional efforts to confirm the findings .

Introduction Materials and methods Study cohorts and ethics statement Statistical analysis Results Discussion Supplementary Material

body mass index ( bmi ) is the most commonly used anthropometric method to define human obesity . bmi is a complex trait affected by many environmental ( eg , diet , age , physical activity ) and genetic factors , with heritability estimates that vary from 4080% in twin studies , 2050% in family studies and 2060% in adoption studies . recent genome - wide association ( gwa ) studies have successfully identified numerous single - nucleotide polymorphisms ( snps ) that are robustly associated with obesity related traits , including bmi . they shed light on the biological basis of obesity and suggest a role for neuronal influences on the regulation of appetite and/or energy balance . gene gene interactions ( epistasis ) are thought to be potential sources of the unexplained genetic variation , but they remain largely unexplored in the gwa studies conducted for bmi so far . a major hurdle for analysing epistasis in gwa studies was the lack of fast methods to enumerate billions of interaction tests in a full pair - wise genome scan to map different types of epistasis ( eg , with or without main effects ) while keeping false - positive rates under control . another hurdle for studying epistasis is the relatively small sample size in many existing gwa cohorts that may limit the power of detection and replication of epistasis signals unless the epistatic effects to be detected are large . for quantitative traits , the major hurdle is gradually easing and full pair - wise genome scans are beginning to be applied to gwa populations individually . meta - analysis of epistasis as applied in gwa studies could be a good way to overcome the sample size hurdle but requires new methods to accommodate imputed snp genotype data . various approaches in search space reduction ( ie , less stringent significance thresholds as result of the much reduced number of tests ) can be applied to improve the power of detection of epistasis in individual gwa populations . testing interactions involving genome - wide significant loci ( of marginal effects ) with a threshold corrected for the actual number of tests has been suggested and applied successfully in recent studies . another approach is to select snps based on existing biological knowledge ( eg , protein protein interactions ) and test interactions among them only . however , cautions should be taken when making the snp selection because biological knowledge may not be directly related to the trait studied and any biases in the pre - identified loci could lead to false - positive epistatic signals . here we demonstrate a different approach to exploit the value of genome - wide analysis of epistasis using multiple populations . first we performed full pair - wise genome scans for epistasis in bmi in four gwa populations to which we had direct access : the scottish orcades , the croatia - vis and croatia - korcula , and the italian micros study cohorts . second , we identified common and potentially important gene gene interactions using the epistasis signals uncovered in each cohort and their gene ontology ( go ) enrichment across populations . in addition , we also identified a set of interactions involving the bmi loci ( as prior knowledge ) in different cohorts . we aim to address the question whether epistasis analysis is of value for the dissection of the genetic regulation of bmi in these study cohorts . the raw bmi data in each of the four study cohorts were corrected for age and sex and normalised using the rntransform function that is implemented in the genabel package performing quantile normalisation of residuals from a generalised linear model analysis . the normalised bmi residuals were then analysed using a linear mixed model to correct for polygenic effects due to relatedness using the polygenic function in the genabel package and the resultant environmental residuals ( ie , pgresidualy in genabel ) were used as the trait to test for association . the original gwa study , in the nfbc1966 cohort individuals with pregnancy and/or self reported bmi measures were excluded , and the raw bmi values were corrected for the sexocpg factor ( recoded according to gender , status of taking oral contraception and pregnancy ) and then normalised and corrected for relatedness as above . a single - snp based gwa scan was performed in each population using a score test method ( based on the additive model ) implemented in the mmscore function in the genabel package . the consensus gwa threshold of 7.3 ( log10(5.0e08 ) ) was applied to identify gwa significant snps . we also performed a full pair - wise genome scan using the regression models described below . considering a pair of snps denoted as snp1 and snp2 , the following genetic models were used to detect epistasis where genotypes of each snp ( ie , homozygote of the minor allele , homozygote of the major allele and heterozygote ) were fitted as fixed factors : where y is the trait of interest , is the model constant , snp1 ( or snp2 ) is a fixed factor with three levels ( genotype classes ) , snp1snp2 is the interaction term , e is the random error term . p - values were calculated based on the f distribution with relevant degrees of freedom and transformed to the log10 scale ( ie , log10 ppair for the fpair test , log10 pint for the fint test ) . genome - wide significant thresholds ( all in the log10 scale ) were derived based on bonferroni correction for multiple tests , that is , the 5% nominal p value corrected by the number of tests performed . considering 300 000 snps , a full pair - wise genome scan perform 4.5e+10 association tests and thus the 5% genome - wide threshold is 11.95 ( ie , log10(0.05/4.5e+10 ) ) . after each pair - wise genome scan , results were evaluated using the predefined threshold to identify genome - wide significant interaction signals . each snp in the results was annotated to the nearest gene within a window of 20 kilobases flanking the snp ( based on the physical distance to either the start or end of transcription of a gene ; the distance is considered as zero if the snp is within a gene ) . a go enrichment analysis was conducted for each study cohort using the running mode of two unranked lists of genes ' in gorilla based on the standard hyper geometric statistics , where the annotated epistatic genes were used as the target with the full list of human genes as the background . for simplicity , we chose to use the same log10 p value as the consensus gwa threshold ( ie , log10 pint > 7.3 ) to select snp pairs of each cohort and used their gene annotations as the input for the go enrichment analysis . the go terms enriched ( p<1.0e03 ) were compared across study cohorts to identify firstly common go terms and then their member genes shared by the cohorts . the shared epistatic genes were examined further for biological functions via literature mining and their associated interactions in the retained results of each cohort to identify potentially important interactions for replication tests . genome - wide significant snp pairs and those identified as potentially important interactions were tested for replication across the four study cohorts . at the snp level , each replicated snp was exactly the same as the corresponding epistatic snp and thus the 5% nominal significance threshold ( ie , log10(0.05)=1.30 ) was used because only one replication test was needed . at the region level , interactions between each of 10 adjacent snps ( ie , five upstream and five downstream ) of the first epistatic snp and each of those of the second were tested , to accommodate the situation where multiple snps may tag a same mutant of a gene . permutation was used to derive significance thresholds for replication of each epistatic pair at the region level , where phenotypes were permuted and the highest log10 pint value of 121 ( ie , 11 11 ) interaction tests was recorded in each of 1000 iterations . the replicated snp pairs were fitted together into the full model to calculate the proportion of phenotypic variance explained in each study cohort . the nfbc1966 data were provided by the database of genotype and phenotype ( dbgap ) via specific data use certification and used as the replication cohort . the raw bmi data in each of the four study cohorts were corrected for age and sex and normalised using the rntransform function that is implemented in the genabel package performing quantile normalisation of residuals from a generalised linear model analysis . the normalised bmi residuals were then analysed using a linear mixed model to correct for polygenic effects due to relatedness using the polygenic function in the genabel package and the resultant environmental residuals ( ie , pgresidualy in genabel ) were used as the trait to test for association . following the original gwa study , in the nfbc1966 cohort individuals with pregnancy and/or self reported bmi measures were excluded , and the raw bmi values were corrected for the sexocpg factor ( recoded according to gender , status of taking oral contraception and pregnancy ) and then normalised and corrected for relatedness as above . a single - snp based gwa scan was performed in each population using a score test method ( based on the additive model ) implemented in the mmscore function in the genabel package . the consensus gwa threshold of 7.3 ( log10(5.0e08 ) ) was applied to identify gwa significant snps . we also performed a full pair - wise genome scan using the regression models described below . considering a pair of snps denoted as snp1 and snp2 , the following genetic models were used to detect epistasis where genotypes of each snp ( ie , homozygote of the minor allele , homozygote of the major allele and heterozygote ) were fitted as fixed factors : where y is the trait of interest , is the model constant , snp1 ( or snp2 ) is a fixed factor with three levels ( genotype classes ) , snp1snp2 is the interaction term , e is the random error term . p - values were calculated based on the f distribution with relevant degrees of freedom and transformed to the log10 scale ( ie , log10 ppair for the fpair test , log10 pint for the fint test ) . genome - wide significant thresholds ( all in the log10 scale ) were derived based on bonferroni correction for multiple tests , that is , the 5% nominal p value corrected by the number of tests performed . considering 300 000 snps , a full pair - wise genome scan perform 4.5e+10 association tests and thus the 5% genome - wide threshold is 11.95 ( ie , log10(0.05/4.5e+10 ) ) . after each pair - wise genome scan , results were evaluated using the predefined threshold to identify genome - wide significant interaction signals . each snp in the results was annotated to the nearest gene within a window of 20 kilobases flanking the snp ( based on the physical distance to either the start or end of transcription of a gene ; the distance is considered as zero if the snp is within a gene ) . a go enrichment analysis was conducted for each study cohort using the running mode of two unranked lists of genes ' in gorilla based on the standard hyper geometric statistics , where the annotated epistatic genes were used as the target with the full list of human genes as the background . for simplicity , we chose to use the same log10 p value as the consensus gwa threshold ( ie , log10 pint > 7.3 ) to select snp pairs of each cohort and used their gene annotations as the input for the go enrichment analysis . the go terms enriched ( p<1.0e03 ) were compared across study cohorts to identify firstly common go terms and then their member genes shared by the cohorts . the shared epistatic genes were examined further for biological functions via literature mining and their associated interactions in the retained results of each cohort to identify potentially important interactions for replication tests . genome - wide significant snp pairs and those identified as potentially important interactions were tested for replication across the four study cohorts . at the snp level , each replicated snp was exactly the same as the corresponding epistatic snp and thus the 5% nominal significance threshold ( ie , log10(0.05)=1.30 ) was used because only one replication test was needed . at the region level , interactions between each of 10 adjacent snps ( ie , five upstream and five downstream ) of the first epistatic snp and each of those of the second were tested , to accommodate the situation where multiple snps may tag a same mutant of a gene . permutation was used to derive significance thresholds for replication of each epistatic pair at the region level , where phenotypes were permuted and the highest log10 pint value of 121 ( ie , 11 11 ) interaction tests was recorded in each of 1000 iterations . the mean bmi was similar across the croatia - vis , croatia - korcula and orcades cohorts but lower in micros ( table 1 ) . the inflation factor lambda ( computed by regression of observed association p - values against the expected ) of each gwa scan was very close to 1 ( table 1 ) , suggesting the family relatedness in each cohort was well accounted for . only 8 out of the 32 bmi snps previously identified were genotyped in the four study cohorts and none of these showed a strong association with bmi ( supplementary table s1 ) . full pair - wise genome scans found no snp pairs that passed the genome - wide threshold ( log10 pint=11.95 ) in any of the four study cohorts ( figure 1 ) . five out of the 32 bmi loci ( but not the bmi snps ) were involved in 7 epistatic pairs in croatia - vis : fto , kctd15 , lrp1b , negr1 and prkd1 . similarly , three bmi loci ( negr1 , nrxn3 and prkd1 ) were involved in croatia - korcula , two ( fto and mtch2 ) in orcades and two ( fto and lrp1b ) in micros . go terms enriched by epistatic genes ( log10 pint > 7.3 ) in each cohort were compared ( supplementary table s2 ) and identified 9 common in all four cohorts ( table 3 ) that might indicate common regulation mechanisms ( eg , go:0008038 neuron recognition ) . among the epistatic genes that enriched the 9 go terms , we found 19 epistatic genes shared by the four cohorts of which 15 are previously published gwa loci ( mostly not genome - wide significant ) associating with various phenotypes ( supplementary table s3 ) . most of the 19 shared epistatic genes interacted with one another despite their interactions being relatively weak ( log10 pint<7.3 , supplementary table s4 ) in general , including cdh13 ( cadherin 13 ) associated with height and sorcs2 ( sortilin - related vps10 domain containing receptor 2 ) associated with circulating insulin - like growth factor 1 and insulin - like growth factor - binding protein-3 , which are important for anthropometric traits and risk of cancer and cardiovascular disease . we further tested replication of the snp pairs involving either bmi loci ( 19 , table 2 ) or two shared epistatic genes across the study cohorts ( 50 , supplementary table s4 ) . despite none of the 69 snp pairs being genome - wide significant , eight of them had a replication in one or more cohorts at the snp level ( ie , log10 pint>1.30 ; table 4 ) . the best replicated pairs at the snp level were rs2202167 ( nrxn3 ) rs11150880 ( log10 pint was 8.19 , 1.68 and 1.43 in croatia - korcula , croatia - vis and orcades , respectively ) and rs1474056 ( mtch2 ) - rs7250947 ( plin4 ) ( log10 pint was 8.08 in orcades and 2.44 in croatia - korcula ) . the eight replicated snp pairs together explained the phenotypic variance of bmi by 4 , 4 , 2 and 0.5% in croatia - vis , croatia - korcula , orcades and micros , respectively . by testing replication at the region level , we found the pair of rs9858278 ( naaladl2 ) - rs7198915 ( cdh13 ) replicated in croatia - vis , croatia - korcula and micros ( exceed the 5% thresholds , table 4 and supplementary table s5 ) . further testing the nine replicated snp pairs in the nfbc1966 cohort found none replicated at either the snp or region levels . gene gene interactions have been suggested as sources of the hidden genetic variations in gwa studies , but the extent of their role in this regard has yet to be demonstrated . one big challenge is that the sample sizes of many gwa data sets are relatively small ( eg , less than 4000 individuals ) and hence the power to detect epistasis could be low . therefore studying epistasis in a single gwa population this is certainly true in our case where exhaustive genome scans in the four study cohorts found no genome - wide significant epistasis associated with bmi . we suggest to tackle the challenge by looking for common ( thus potentially important ) gene gene interactions from sub genome - wide significant epistatic signals ( log10 pint>7.3 ) in multiple gwa populations . we showed that go enrichment analysis could be used to identify common go terms ( ie gene function groups ) enriched by the epistatic signals in the four study cohorts from which 19 shared epistatic genes were identified . most of the 19 shared epistatic genes are previously identified gwa loci associating with phenotypes other than bmi and interacted with one another . their interactions were considered potentially important because they belong to one or multiple commonly enriched go terms . interactions involving at least one of the 32 bmi loci with log10 pint>7.3 were also considered potentially important assuming the bmi loci are likely interactive . being aware of possible noises in those potentially important interactions , we used replication to identify the most reliable epistatic signals across the study cohorts . eight epistatic pairs involving either the bmi loci or two shared epistatic genes showed replication at the snp level in at least one cohort ( table 4 ) . this however appears to be far more difficult for epistatic signals than for single snp signals for reasons , including power , minor allele frequency change , and linkage disequilibrium between epistatic snp and mutant for both loci . the lack of replication in the nfbc1966 cohort could be due to two important environmental factors of bmi : age ( ie , 31 vs a range between 18 and 90 in the study cohorts ) and diets . gene interactions in multiple gwa populations is an effective solution to the issue of limited power of detection of epistasis . comparison of sub genome - wide significant epistatic signals across multiple populations can be made at either the snp , or gene or pathway level and seem more fruitful at the gene or pathway level than the snp level . for example , not all epistatic snps were annotated to genes in the study and hence did not contribute to the enrichment analysis . the approach will likely remain important even once new tools for meta - analysis of epistasis in gwa data sets become available to increase power for detection of epistasis .

bmi is a complex trait affected by many environmental ( eg , diet , age , physical activity ) and genetic factors , with heritability estimates that vary from 4080% in twin studies , 2050% in family studies and 2060% in adoption studies . recent genome - wide association ( gwa ) studies have successfully identified numerous single - nucleotide polymorphisms ( snps ) that are robustly associated with obesity related traits , including bmi . they shed light on the biological basis of obesity and suggest a role for neuronal influences on the regulation of appetite and/or energy balance . however , the identified genetic variants jointly explained only a small proportion of the trait variation and thus had limited predictive value for obesity risk . for example , in a recent meta - analysis ( 249 796 individuals ) 32 identified and replicated snps together explained only 1.45% of the inter - individual variation in bmi where the strongest snp accounted for just 0.34% of the variance . gene gene interactions ( epistasis ) are thought to be potential sources of the unexplained genetic variation , but they remain largely unexplored in the gwa studies conducted for bmi so far . a major hurdle for analysing epistasis in gwa studies was the lack of fast methods to enumerate billions of interaction tests in a full pair - wise genome scan to map different types of epistasis ( eg , with or without main effects ) while keeping false - positive rates under control . another hurdle for studying epistasis is the relatively small sample size in many existing gwa cohorts that may limit the power of detection and replication of epistasis signals unless the epistatic effects to be detected are large . it was showed in simulation that more than 4000 case control pairs were needed to achieve 80% power of detection of epistasis with an odds ratio of 3.0 in complex diseases . for quantitative traits , the major hurdle is gradually easing and full pair - wise genome scans are beginning to be applied to gwa populations individually . various approaches in search space reduction ( ie , less stringent significance thresholds as result of the much reduced number of tests ) can be applied to improve the power of detection of epistasis in individual gwa populations . testing interactions involving genome - wide significant loci ( of marginal effects ) with a threshold corrected for the actual number of tests has been suggested and applied successfully in recent studies . however , cautions should be taken when making the snp selection because biological knowledge may not be directly related to the trait studied and any biases in the pre - identified loci could lead to false - positive epistatic signals . here we demonstrate a different approach to exploit the value of genome - wide analysis of epistasis using multiple populations . first we performed full pair - wise genome scans for epistasis in bmi in four gwa populations to which we had direct access : the scottish orcades , the croatia - vis and croatia - korcula , and the italian micros study cohorts . second , we identified common and potentially important gene gene interactions using the epistasis signals uncovered in each cohort and their gene ontology ( go ) enrichment across populations . in addition , we also identified a set of interactions involving the bmi loci ( as prior knowledge ) in different cohorts . third , we tested the identified interactions in each cohort for replication and then the replicated signals in the northern finland birth cohort 1966 ( nfbc1966 ) . we aim to address the question whether epistasis analysis is of value for the dissection of the genetic regulation of bmi in these study cohorts . the croatia - vis and croatia - korcula cohorts were recruited from the island of vis and the island of korcula , respectively . dna samples were genotyped with illumina infinium humanhap300v1/v2 ( for croatia - vis by the wellcome trust clinical facility in einburgh , uk ) or humancnv370v1 snp bead microarrays ( for croatia - korcula , orcades and micros by the helmholtz zentrum munchen in munich , germany ) and analysed using the beadstudio software ( illumina ) . quality control of the genotype data was performed for each cohort using the r / genabel package ( version 1.6 - 7 ) based on a common set of criteria : individual call rate at 97% , snp call rate at 95% , p - value for deviation from hardy weinberg equilibrium at 1.0e-10 , minor allele frequency at 2% . the nfbc1966 data were provided by the database of genotype and phenotype ( dbgap ) via specific data use certification and used as the replication cohort . the summary information of each cohort after quality control and excluding individuals without bmi or age records or with extremely high bmi ( ie , bmi>50 kg / m ) is given in table 1 . the raw bmi data in each of the four study cohorts were corrected for age and sex and normalised using the rntransform function that is implemented in the genabel package performing quantile normalisation of residuals from a generalised linear model analysis . the normalised bmi residuals were then analysed using a linear mixed model to correct for polygenic effects due to relatedness using the polygenic function in the genabel package and the resultant environmental residuals ( ie , pgresidualy in genabel ) were used as the trait to test for association . the original gwa study , in the nfbc1966 cohort individuals with pregnancy and/or self reported bmi measures were excluded , and the raw bmi values were corrected for the sexocpg factor ( recoded according to gender , status of taking oral contraception and pregnancy ) and then normalised and corrected for relatedness as above . a single - snp based gwa scan was performed in each population using a score test method ( based on the additive model ) implemented in the mmscore function in the genabel package . considering a pair of snps denoted as snp1 and snp2 , the following genetic models were used to detect epistasis where genotypes of each snp ( ie , homozygote of the minor allele , homozygote of the major allele and heterozygote ) were fitted as fixed factors : where y is the trait of interest , is the model constant , snp1 ( or snp2 ) is a fixed factor with three levels ( genotype classes ) , snp1snp2 is the interaction term , e is the random error term . the f ratio test of model 1 against model 3 evaluates the whole pair effect , including interaction ( ie , fpair , 8 degrees of freedom ) . the f ratio test of model 1 against model 2 evaluates the interaction between the two snps ( ie , fint , 4 degrees of freedom ) . p - values were calculated based on the f distribution with relevant degrees of freedom and transformed to the log10 scale ( ie , log10 ppair for the fpair test , log10 pint for the fint test ) . genome - wide significant thresholds ( all in the log10 scale ) were derived based on bonferroni correction for multiple tests , that is , the 5% nominal p value corrected by the number of tests performed . considering 300 000 snps , a full pair - wise genome scan perform 4.5e+10 association tests and thus the 5% genome - wide threshold is 11.95 ( ie , log10(0.05/4.5e+10 ) ) . each snp in the results was annotated to the nearest gene within a window of 20 kilobases flanking the snp ( based on the physical distance to either the start or end of transcription of a gene ; the distance is considered as zero if the snp is within a gene ) . a go enrichment analysis was conducted for each study cohort using the running mode of two unranked lists of genes ' in gorilla based on the standard hyper geometric statistics , where the annotated epistatic genes were used as the target with the full list of human genes as the background . for simplicity , we chose to use the same log10 p value as the consensus gwa threshold ( ie , log10 pint > 7.3 ) to select snp pairs of each cohort and used their gene annotations as the input for the go enrichment analysis . the shared epistatic genes were examined further for biological functions via literature mining and their associated interactions in the retained results of each cohort to identify potentially important interactions for replication tests . genome - wide significant snp pairs and those identified as potentially important interactions were tested for replication across the four study cohorts . at the region level , interactions between each of 10 adjacent snps ( ie , five upstream and five downstream ) of the first epistatic snp and each of those of the second were tested , to accommodate the situation where multiple snps may tag a same mutant of a gene . dna samples were genotyped with illumina infinium humanhap300v1/v2 ( for croatia - vis by the wellcome trust clinical facility in einburgh , uk ) or humancnv370v1 snp bead microarrays ( for croatia - korcula , orcades and micros by the helmholtz zentrum munchen in munich , germany ) and analysed using the beadstudio software ( illumina ) . quality control of the genotype data was performed for each cohort using the r / genabel package ( version 1.6 - 7 ) based on a common set of criteria : individual call rate at 97% , snp call rate at 95% , p - value for deviation from hardy weinberg equilibrium at 1.0e-10 , minor allele frequency at 2% . the nfbc1966 data were provided by the database of genotype and phenotype ( dbgap ) via specific data use certification and used as the replication cohort . the summary information of each cohort after quality control and excluding individuals without bmi or age records or with extremely high bmi ( ie , bmi>50 kg / m ) is given in table 1 . the raw bmi data in each of the four study cohorts were corrected for age and sex and normalised using the rntransform function that is implemented in the genabel package performing quantile normalisation of residuals from a generalised linear model analysis . the normalised bmi residuals were then analysed using a linear mixed model to correct for polygenic effects due to relatedness using the polygenic function in the genabel package and the resultant environmental residuals ( ie , pgresidualy in genabel ) were used as the trait to test for association . following the original gwa study , in the nfbc1966 cohort individuals with pregnancy and/or self reported bmi measures were excluded , and the raw bmi values were corrected for the sexocpg factor ( recoded according to gender , status of taking oral contraception and pregnancy ) and then normalised and corrected for relatedness as above . a single - snp based gwa scan was performed in each population using a score test method ( based on the additive model ) implemented in the mmscore function in the genabel package . considering a pair of snps denoted as snp1 and snp2 , the following genetic models were used to detect epistasis where genotypes of each snp ( ie , homozygote of the minor allele , homozygote of the major allele and heterozygote ) were fitted as fixed factors : where y is the trait of interest , is the model constant , snp1 ( or snp2 ) is a fixed factor with three levels ( genotype classes ) , snp1snp2 is the interaction term , e is the random error term . the f ratio test of model 1 against model 3 evaluates the whole pair effect , including interaction ( ie , fpair , 8 degrees of freedom ) . the f ratio test of model 1 against model 2 evaluates the interaction between the two snps ( ie , fint , 4 degrees of freedom ) . p - values were calculated based on the f distribution with relevant degrees of freedom and transformed to the log10 scale ( ie , log10 ppair for the fpair test , log10 pint for the fint test ) . genome - wide significant thresholds ( all in the log10 scale ) were derived based on bonferroni correction for multiple tests , that is , the 5% nominal p value corrected by the number of tests performed . considering 300 000 snps , a full pair - wise genome scan perform 4.5e+10 association tests and thus the 5% genome - wide threshold is 11.95 ( ie , log10(0.05/4.5e+10 ) ) . each snp in the results was annotated to the nearest gene within a window of 20 kilobases flanking the snp ( based on the physical distance to either the start or end of transcription of a gene ; the distance is considered as zero if the snp is within a gene ) . a go enrichment analysis was conducted for each study cohort using the running mode of two unranked lists of genes ' in gorilla based on the standard hyper geometric statistics , where the annotated epistatic genes were used as the target with the full list of human genes as the background . for simplicity , we chose to use the same log10 p value as the consensus gwa threshold ( ie , log10 pint > 7.3 ) to select snp pairs of each cohort and used their gene annotations as the input for the go enrichment analysis . the shared epistatic genes were examined further for biological functions via literature mining and their associated interactions in the retained results of each cohort to identify potentially important interactions for replication tests . at the region level , interactions between each of 10 adjacent snps ( ie , five upstream and five downstream ) of the first epistatic snp and each of those of the second were tested , to accommodate the situation where multiple snps may tag a same mutant of a gene . the mean bmi was similar across the croatia - vis , croatia - korcula and orcades cohorts but lower in micros ( table 1 ) . the inflation factor lambda ( computed by regression of observed association p - values against the expected ) of each gwa scan was very close to 1 ( table 1 ) , suggesting the family relatedness in each cohort was well accounted for . only 8 out of the 32 bmi snps previously identified were genotyped in the four study cohorts and none of these showed a strong association with bmi ( supplementary table s1 ) . full pair - wise genome scans found no snp pairs that passed the genome - wide threshold ( log10 pint=11.95 ) in any of the four study cohorts ( figure 1 ) . considering interaction signals with log10 pint > 7.3 , micros had the least number of snp pairs and consequently the least number of annotated genes , whereas the remaining three cohorts had relatively similar numbers of snp pairs and annotated genes ( table 2 ) . five out of the 32 bmi loci ( but not the bmi snps ) were involved in 7 epistatic pairs in croatia - vis : fto , kctd15 , lrp1b , negr1 and prkd1 . similarly , three bmi loci ( negr1 , nrxn3 and prkd1 ) were involved in croatia - korcula , two ( fto and mtch2 ) in orcades and two ( fto and lrp1b ) in micros . go terms enriched by epistatic genes ( log10 pint > 7.3 ) in each cohort were compared ( supplementary table s2 ) and identified 9 common in all four cohorts ( table 3 ) that might indicate common regulation mechanisms ( eg , go:0008038 neuron recognition ) . among the epistatic genes that enriched the 9 go terms , we found 19 epistatic genes shared by the four cohorts of which 15 are previously published gwa loci ( mostly not genome - wide significant ) associating with various phenotypes ( supplementary table s3 ) . most of the 19 shared epistatic genes interacted with one another despite their interactions being relatively weak ( log10 pint<7.3 , supplementary table s4 ) in general , including cdh13 ( cadherin 13 ) associated with height and sorcs2 ( sortilin - related vps10 domain containing receptor 2 ) associated with circulating insulin - like growth factor 1 and insulin - like growth factor - binding protein-3 , which are important for anthropometric traits and risk of cancer and cardiovascular disease . we further tested replication of the snp pairs involving either bmi loci ( 19 , table 2 ) or two shared epistatic genes across the study cohorts ( 50 , supplementary table s4 ) . despite none of the 69 snp pairs being genome - wide significant , eight of them had a replication in one or more cohorts at the snp level ( ie , log10 pint>1.30 ; table 4 ) . the best replicated pairs at the snp level were rs2202167 ( nrxn3 ) rs11150880 ( log10 pint was 8.19 , 1.68 and 1.43 in croatia - korcula , croatia - vis and orcades , respectively ) and rs1474056 ( mtch2 ) - rs7250947 ( plin4 ) ( log10 pint was 8.08 in orcades and 2.44 in croatia - korcula ) . the rs11150880 snp is near the rph3al gene , which is known to have a key role in insulin secretion by pancreatic cells . the eight replicated snp pairs together explained the phenotypic variance of bmi by 4 , 4 , 2 and 0.5% in croatia - vis , croatia - korcula , orcades and micros , respectively . by testing replication at the region level , we found the pair of rs9858278 ( naaladl2 ) - rs7198915 ( cdh13 ) replicated in croatia - vis , croatia - korcula and micros ( exceed the 5% thresholds , table 4 and supplementary table s5 ) . gene gene interactions have been suggested as sources of the hidden genetic variations in gwa studies , but the extent of their role in this regard has yet to be demonstrated . therefore studying epistasis in a single gwa population this is certainly true in our case where exhaustive genome scans in the four study cohorts found no genome - wide significant epistasis associated with bmi . we suggest to tackle the challenge by looking for common ( thus potentially important ) gene gene interactions from sub genome - wide significant epistatic signals ( log10 pint>7.3 ) in multiple gwa populations . we showed that go enrichment analysis could be used to identify common go terms ( ie gene function groups ) enriched by the epistatic signals in the four study cohorts from which 19 shared epistatic genes were identified . the moderate log10 pint values of the epistatic pairs tested for replication suggest that the linkage disequilibrium between epistatic snps and mutants is not high so replication of these pairs will be difficult . the lack of replication in the nfbc1966 cohort could be due to two important environmental factors of bmi : age ( ie , 31 vs a range between 18 and 90 in the study cohorts ) and diets . comparison of sub genome - wide significant epistatic signals across multiple populations can be made at either the snp , or gene or pathway level and seem more fruitful at the gene or pathway level than the snp level .

age- or injury - induced muscle weakness leading to frailty is a major public health problem that is predicted to escalate in the future as the number and proportion of older adults increase in the general population ( berger and doherty 2010 ) . there is an unmet need for therapeutic strategies that can slow the effects of aging on muscle function in the frail elderly so as to maintain or improve their quality of life . identifying suitable therapeutic targets and testing candidate drugs for the ability to improve muscle function require cell - based model systems that reliably predict in vivo effects in both pre - clinical rodent models and human patients . a number of useful rodent cell lines such as mouse c2c12 or rat l6 myoblasts are available ( sultan et al . 2006 ) . these cell lines , and others , have been used extensively to explore the molecular mechanisms of muscle differentiation and function ( mcfarlane et al . in addition , they have been used in some drug discovery screens ( baudy et al . however , because immortalized cell lines often are genetically abnormal , and have been maintained under artificial conditions in culture for very long periods of time which can cause them to deviate from normal function , a potentially more predictive screening strategy would use primary human muscle cells . such cells have recently become available from a number of commercial vendors , but only limited characterization of these various primary cells has been reported ( janowski 2011 ) . therefore , we examined several primary human skeletal muscle cell preparations from different vendors and characterized each for their capacity to reproducibly differentiate into multinucleated myotubes . those that reproducibly differentiated into myotubes were further examined for the expression of various markers of skeletal muscle cells such as myogenic differentiation-1 ( myod ) , myocyte enhancer factor 2c ( mef2c ) , myogenin ( myog ) , troponin t type 1 ( tnnt ) , and myosin heavy chain-2 ( myh2 ) . we also tested known inducers of skeletal muscle atrophy ( myostatin and dexamethasone ) and hypertrophy ( insulin - like growth factor-1 ) for effects on the differentiated myotubes derived from the primary cells . in addition , we found that myostatin - induced activity on the myotubes can be blocked by treatment with a soluble myostatin receptor , similar to one that is currently in clinical trials for diseases associated with muscle wasting ( lee et al . finally , we show that both the undifferentiated primary muscle cells as well as the myotubes they give rise to can be infected with adenovirus . this observation suggests the potential for using these cells in genetic screens ( both over - expression and knock - down ) to identify factors that play a role in muscle differentiation and function . hsmm ( catalog # cc-2580 , lot # 6f4528 , sourced from quadriceps muscle of a 16-yr - old male cadaver ) and skmc - l ( catalog # cc-2561 , lot # 6f3791 , sourced from human fetal skeletal muscle ) were purchased from lonza ( walkersville , md ) . skmdc ( catalog # sk-1111 , lot # p101014 - 50m2 , sourced from rectus abdominus muscle of a 50-yr - old male caucasian with a body mass index ( bmi ) of 21 , non - diabetic , smoker for 20 yr ) came from cook myosite ( pittsburgh , pa ) . skmc - p ( catalog # c-12530 , lot # 8121902.17 , sourced from unspecified skeletal muscle of a 21-yr - old male caucasian ) was obtained from promocell ( heidelberg , germany ) . hsmm differentiation medium was prepared by adding 2% horse serum to dmem - f12 medium ( both from invitrogen , grand island , ny ) . the vendor stated that hsmm would differentiate in all horse serum lots , so no pre - screening was required . skmdc differentiation medium ( ready to use ) was supplied by the vendor ( catalog # md-5555 ) . recombinant human myostatin ( mstn ) and actriib - fc were generated in - house as previously described ( thies et al . recombinant human igf-1 and tweak were purchased from r&d systems ( minneapolis , mn ) , while dex was obtained from sigma ( st . hsmm and skmdc were maintained in growth medium with supplements and fetal bovine calf ( fbs ) serum provided by the vendors . skmdc were passaged when they reached 50% confluence , ( about every 23 d ) . when passaging , the culture medium was warmed to 37c and the cells were seeded at 5,0007,500 cells / cm and incubated in a humidified incubator at 37c , 5% co2 . hsmm were passaged when they reached 5070% confluency ( about every 3 d ) . the culture conditions for these cells were the same as those for skmdc except they were seeded at 3,500 cells / cm . for both cell types , cultures were used for experiments after no more than seven population doublings because the cells rate of differentiation declined after higher population doublings . to induce differentiation , cells were plated at 20,000 cells / cm in 12-well polystyrene cell culture plates ( vwr ) and incubated overnight in growth medium in a cell culture incubator ( 37c , 5% co2 ) . the following morning , the growth medium was replaced with differentiation medium and the cultures were incubated for 3 d , during which time myotube differentiation occurred . to examine effects on differentiation , agents to be tested were added at the time of medium change and cultured for 3 d. to examine effects on differentiated myotubes , agents to be tested were added on day 3 of differentiation , after myotube formation had occurred , and then cultures were incubated for another 23 d. for analysis , to unambiguously identify myotubes , the cultures were fixed and immuno - stained with an antibody against myosin heavy chain-2 protein ( myh2 ) , as described below . cells were fixed with 10% formalin for 20 min at room temperature ( rt ) . following 23 rinses with pbs , cells were permeabilized with 0.5% triton x-100 ( sigma ) in pbs for 15 min at rt . non - specific binding was blocked with 2% bsa ( sigma ) , 0.25% triton x-100 in pbs at rt for 30 min . cells were then incubated with anti - skeletal myosin fast primary antibody , my-32 ( sigma ) , diluted 1:200 in blocking buffer for 2 h at rt . after rinsing with blocking buffer without triton 2 times for 10 min each , cells were incubated with alexa 488 ( green ) or alexa 555 ( red ) conjugated anti - mouse igg secondary antibodies ( both from invitrogen ) at 1:400 dilution along with hoechst nuclear stain ( # 33342 ; sigma ) at 1:5,000 dilution in blocking buffer at rt for 1 h. finally , cells were washed with pbs 3 times , 5 min each , before mounting with vectashield containing hoechst stain . images were processed with nis - elements br 3.10 software ( micro video instruments , avon , ma ) . for quantitative analysis of immuno - stained samples , briefly , 5 images were captured from each of 3 replicate wells for each treatment group , for a total of 15 images per condition . a fluorescence intensity threshold was set such that only the myh2-positive areas were measured by the software . the mean area for the untreated group ( media only ) was used to calculate the percent increase or decrease in myotube area for each treatment . this method allows a relatively rapid assessment of a compound s effect on myotube size . for western blot analyses , antibodies against phosphorylated - smad2 ( ser465/467 ) ( psmad2 ) , smad2 , phosphorylated - akt ( ser473 ) ( pakt ) , akt , and alpha - tubulin were purchased from cell signaling technologies ( danvers , ma ) . they were detected with peroxidase - labeled goat anti - rabbit igg from pierce ( rockford , il ) . cells were washed in ice cold pbs before lysis in a buffer comprised of 40 mm mops ph 7 , 4 mm egta , 10 mm edta with 2% triton x-100 , containing protease and phosphatase inhibitors ( 30 mm sodium fluoride , 60 mm -glycerophosphate , 20 mm sodium pyrophosphate , 1 mm sodium orthovanadate , and 1% protease inhibitor cocktail , plus 1 mm phenylmethylsulfonylfluoride ( pmsf ) , all from sigma . supernatants were collected and protein contents were measured using the dc protein assay from biorad ( hercules , ca ) . samples were diluted in sds - page sample buffer and denatured for 5 min at 95c . tris for nupage gels , invitrogen , electrophoresed , and then transferred on to nitrocellulose membranes . membranes were blocked in tris - buffered saline with 0.1% tween 20 ( tbs - t ) with 5% ( wt / vol ) non - fat milk powder . primary antibodies were diluted in tbs - t with 5% bsa and secondary antibodies were diluted in tbs - t with 5% non - fat milk . immuno - reactivity was detected by supersignal west pico chemiluminescent substrate ( thermo scientific , rockford , il ) and exposed to film . rna was isolated using an rneasy kit ( qiagen , valencia , ca ) , following the manufacturer s protocol . quantitative real - time pcr ( rt - pcr ) was performed using an applied biosystems 7900 ht fast real - time pcr system . the human taqman probe sets for myosin heavy chain , myh2 ( hs00430042_m1 ) , troponin t type 1 , tnnt ( hs00162848_m1 ) , myocyte enhancer factor 2c , mef2c ( hs00231149_m1 ) , myogenic differentiation 1 , myod ( hs00159528_m1 ) , myogenin , myog ( hs01072232_m1 ) , and beta-2 microglobulin , b2 m ( # 4333766f ) , were purchased from applied biosystems ( wilmington , de ) . an adenovirus expressing gfp ( adeno - gfp ) was purchased from viraquest ( north liberty , ia ) ( vqad cmv egfp titer : 5.0 10 pfu / ml ) . for infection of myotubes , undifferentiated cells were plated at 20,000 cells / cm into 96-well polystyrene cell culture plates and incubated overnight in growth medium and then switched to differentiation medium . after myotubes appeared ( 23 d ) , infection with adeno - gfp was performed . several different ratios of viral particles to cells were tested ( multiplicity of infection , or moi , of 100 , 250 , 500 , 700 , and 1,000 ) . infections were performed in growth medium and incubated with the cells overnight ( 18 h ) . the following morning , this medium was removed and replaced with fresh growth medium and the cells were cultured for an additional 6 h before switching back to differentiation medium for 48 h. the cells were then fixed for immuno - staining and visualization of gfp . for infection of undifferentiated cells , the cells were plated at 20,000 cells / cm in 96-well tissue culture plates and incubated for 24 h in growth medium . for infection , the same mois and procedure described above were utilized . in some cases , to observe the effects of infection on myotube differentiation , cells were returned to differentiation medium for 6 d after a 6 h recovery period in growth medium . four different primary human muscle cell preparations from three different commercial vendors were screened for the ability to differentiate into multinucleated myotubes in cell culture . we tested human skeletal muscle myoblasts ( hsmm ; lonza ) , human skeletal muscle cells ( skmc - l ; lonza ) , human skeletal muscle - derived cells ( skmdc ; cook myosite ) , and human skeletal muscle cells ( skmc - p ; promocell ) . initial experiments found that two of these preparations ( skmc - l and skmc - p ) showed little , if any , evidence of morphological differentiation into myotubes when cultured according to the recommendations of the suppliers . in addition , skmc - p grew very poorly as undifferentiated cells in growth medium . however , hsmm and skmdc demonstrated robust myotube differentiation , and could be readily cultured as undifferentiated cells in growth medium , using polystyrene cell culture plates . thus , we focused on these two primary cell preparations for the remainder of our studies presented here . hsmm were provided as a stock of myoblasts cryopreserved at passage 2 and characterized by the vendor to display greater than 60% desmin - positive cells at first passage out of cryopreservation . skmdc were characterized as displaying 92% desmin - positive cells and a gene expression analysis provided by the vendor showed that these cells expressed several markers of muscle cell precursors such as pax3 , pax7 , myod1 , myog , and myf5 . this marker analysis therefore suggests that the skmdc population is a committed myoblast cell population . for both hsmm and skmdc , the vendors recommend that these cell populations should be used for experiments at a population doubling ( pd ) less than 10 . in our initial studies , we evaluated cell growth and differentiation of cells through 15 pds . skmdc had a pd time of 24 h for up to 45 passages , after which their doubling time slowed gradually until 15 pds were reached . hsmm were slower to recover from cryopreservation compared to skmdc . for the first 5 d , their pd time was approximately 40 h , after which it decreased to 24 h for about 7 d and then increased to 48 h up to 15 pds . in general , above 10 pds , both cell types took longer to differentiate and their differentiation was not as robust , compared to cells at pd < 10 ( data not shown ) . for the studies described in this paper , we used cells that had undergone no more than 7 pd . cells were also plated on several different extracellular matrix protein ( ecm)-coated culture plates to assess their effect on cell growth and differentiation . the following human ecms were screened using millipore s millicoat ecm - coated strips : fibronectin , vitronectin , laminin , collagen type i , and collagen type iv . in addition , we also screened bd purecoat amine ( bd biosciences , san jose , ca ) and polystyrene plates coated in - house with 2% gelatin ( sigma ) . no obvious improvement in cell growth or differentiation was detected with any of these matrices compared to regular tissue - culture treated polystyrene plates . therefore , standard cell - culture grade polystyrene plates were used throughout these studies . both hsmm and skmdc differentiated into myosin heavy chain ( myh2)-positive multinucleated myotubes within 3 d when cultured on polystyrene cell culture plates in differentiation medium ( fig . in general , the undifferentiated cells cultured in growth medium did not express myh2 , although occasional myh2-positive cells were seen in growing cultures , possibly resulting from cell confluence associated contact - dependent cell differentiation ( see skmdc in fig . 1a ) . to minimize this issue , care was taken to subculture growing cultures of cells before they became confluent . it was noted that the organization of nuclei within the myotubes was different between hsmm and skmdc . in hsmm myotubes , the nuclei tend to be arranged as singlets , or small groups , in linear arrays . in contrast , the nuclei in skmdc myotubes were often clustered together in large groups ( fig . this difference may be caused by differences in the mechanisms of cell fusion during myotube generation , or by differences in the regulation of nuclear dynamics in the myotubes , or other unknown factors . to further characterize the differentiation process of hsmm and skmdc , rna was prepared from cells harvested on day 0 through day 6 of culture in differentiation medium . gene expression analyses were performed using quantitative rt - pcr for various markers of muscle cell differentiation , ( fig . gene expression levels were normalized to beta-2 microglobulin ( b2 m ) expression levels in order to assess changes in relative expression . myod and mef2c , transcription factors that play important roles in specifying the myogenic lineage , represent two early markers of myoblast differentiation into myotubes ( megeney and rudnicki 1995 ; black and olson 1998 ; zammit et al . , there was a transient , modest upregulation of myod at day 1 ( although it was statistically significant for hsmm only ) which returned to baseline levels by day 2 . mef2c was also upregulated by day 1 in both cell populations , reaching statistical significance by day 2 , but unlike myod , the expression of this gene remained elevated throughout the 6-d culture period . myogenin ( myog ) , troponin t ( tnnt ) , and myh2 represent late markers of muscle cell differentiation ( burattini et al . myog was upregulated approximately tenfold by day 1 of differentiation and that expression level remained significantly elevated from day 2 to day 6 of culture . tnnt was significantly upregulated by day 1 of differentiation in hsmm and by day 2 of differentiation for skmdc . tnnt expression remained at least tenfold upregulated through day 6 of differentiation in both cell populations . myh2 was also expressed at least fivefold above control expression levels by day 1 of differentiation for hsmm and by day 2 of differentiation for skmdc . then , myh2 expression reached greater than tenfold above control levels through day 6 of differentiation . thus , hsmm and skmdc undergo morphological differentiation in culture into multinucleated myotubes and express characteristic molecular markers of muscle cell differentiation.figure 1.hsmm and skmdc differentiated into multinucleated myosin heavy chain-2 ( myh2)-positive myotubes . ( a ) representative phase contrast and dark - field images of myh2-immunostained cells ( green ) , with hoechst - labeled nuclei ( blue ) after 1 d in growth medium ( phase contrast images ) or 3 d in differentiation medium ( dark - field images ; magnification 10 ; scale bar = 100 microns ) . plots show gene expression of early ( myod , mef2c ) and late ( myog , tnnt , and myh2 ) muscle cell markers , normalized to b2 m gene expression , relative to expression levels at day 0 ( time at which differentiation media was added to the cells ) ; * p < 0.05 vs. day 0 using student s t test . hsmm and skmdc differentiated into multinucleated myosin heavy chain-2 ( myh2)-positive myotubes . ( a ) representative phase contrast and dark - field images of myh2-immunostained cells ( green ) , with hoechst - labeled nuclei ( blue ) after 1 d in growth medium ( phase contrast images ) or 3 d in differentiation medium ( dark - field images ; magnification 10 ; scale bar = 100 microns ) . plots show gene expression of early ( myod , mef2c ) and late ( myog , tnnt , and myh2 ) muscle cell markers , normalized to b2 m gene expression , relative to expression levels at day 0 ( time at which differentiation media was added to the cells ) ; * p < 0.05 vs. day 0 using student s t test . dexamethasone , a synthetic steroid , and myostatin , a growth factor which is a negative regulator of muscle mass , are well - characterized inducers of myotube atrophy , as shown for c2c12 myotubes ( lee 2004 ; stitt et al . 2004 ) . also , c2c12 myotubes display a hypertrophic response following treatment with insulin - like growth factor-1 ( igf-1 ; semsarian et al . therefore , to examine the response of primary human skeletal muscle cell cultures to these factors , myotubes derived from hsmm and skmdc were treated with various concentrations of dex , mstn , or igf-1 for 48 h and then fixed and immuno - stained for myh2 . representative images of untreated myotubes or those treated with mstn ( 1 g / ml ) or dex ( 50 m ) are shown in fig . 2a and with igf-1 ( 1 g / ml ) are shown in fig . hsmm and skmdc undergo an atrophy - like response to both mstn and dex treatment , while only hsmm display a hypertrophy - like response to igf-1 . ( 2010 ) and results were plotted as the percent decrease or increase in myotube area compared to untreated control cultures ( fig . the plots show that mstn and dex induced statistically significant decreases in myotube area over untreated control cultures at all concentrations ( except for the hsmm 250 ng / ml mstn - treated group which showed decreased myotube area but failed to reach statistical significance ; fig . quantification of igf-1 effects on both cell populations confirmed that only hsmm cultures responded with a hypertrophic response , with significant increases in myotube area at 100 and 1,000 ng / ml igf-1 , ( fig . 2c ) . statistical significance , using student s t test was performed by comparing the total myotube area in multiple untreated cultures with the total myotube area in treated cultures and then assigning statistical significance ( * ) if p < 0.05.figure 2.induction of atrophy and hypertrophy in hsmm and skmdc myotubes . ( a ) differentiated myotubes were treated with a concentration series of mstn or dex for 48 h. representative images of myh2/hoechst - labeled myotubes treated with 1 g / ml mstn or 50 m dex for 48 h. plots of the percent decrease , from untreated cells , in myotube area at each concentration of test agent are shown . ( all plots represent means sem , n = 15 . * p < 0.05 vs. untreated using student s t test . all images at 10 magnification ; scale bar = 100 microns ) . ( b ) differentiated myotubes were treated with a concentration series of igf-1 for 48 h. representative images of myh2/hoechst - labeled myotubes following treatment with 1 g / ml insulin - like growth factor-1 ( igf-1 ) . plots show the percent increase , from untreated cultures , in myotube area at each concentration of igf-1 . ( all plots represent means sem , n = 15 . * p < 0.05 vs. untreated using student s t test . all images at 10 magnification ; scale bar = 100 microns ) . ( c ) western blots of myotube lysates from cultures treated with 1 g / ml mstn , 50 m dex , or 1 g / ml igf-1 for 48 h and probed with antibodies to phosphorylated- and total smad2 and phosphorylated- and total akt . ( a ) differentiated myotubes were treated with a concentration series of mstn or dex for 48 h. representative images of myh2/hoechst - labeled myotubes treated with 1 g / ml mstn or 50 m dex for 48 h. plots of the percent decrease , from untreated cells , in myotube area at each concentration of test agent are shown . ( all plots represent means sem , n = 15 . * p < 0.05 vs. untreated using student s t test . all images at 10 magnification ; scale bar = 100 microns ) . ( b ) differentiated myotubes were treated with a concentration series of igf-1 for 48 h. representative images of myh2/hoechst - labeled myotubes following treatment with 1 g / ml insulin - like growth factor-1 ( igf-1 ) . plots show the percent increase , from untreated cultures , in myotube area at each concentration of igf-1 . ( all plots represent means sem , n = 15 . * p < 0.05 vs. untreated using student s t test . all images at 10 magnification ; scale bar = 100 microns ) . ( c ) western blots of myotube lysates from cultures treated with 1 g / ml mstn , 50 m dex , or 1 g / ml igf-1 for 48 h and probed with antibodies to phosphorylated- and total smad2 and phosphorylated- and total akt . anti - alpha tubulin was used as a loading control . to examine the signaling pathways that were activated during these treatments , cell lysates were prepared at the end of the 48 h treatment period and then probed with antibodies to phosphorylated- and total smad2 , and phosphorylated- and total akt , since these molecules are involved in the earliest stage responses to the mstn and igf-1 ligands ( rebbapragada et al . the myostatin - receptor complex directly phosphorylates the signaling molecules smad2 and smad3 , rebbrapragada et al . in addition , mstn reduced the level of phosphorylated - akt ( pakt ) in hsmm and skmdc ( fig . 2c ) . inhibition of akt phosphorylation is consistent with an atrophic response , since pakt activity is a known positive regulator of muscle protein synthesis and growth ( duan et al . in contrast to myostatin , dex treatment induced downregulation of the control levels of psmad2 in skmdc ( fig . psmad2 was not detectable in untreated hsmm cultures , so it is unclear if dex affected psmad2 levels in these cultures . in our studies overall , these results suggest that at least for skmdc , mstn and dex operate through separate pathways to induce an atrophy - like response . finally , igf-1 binding to its receptor is known to stimulate the phosphorylation of akt , which stimulates protein synthesis and cell growth ( duan et al . igf-1 treatment of skmdc , but not hsmm , increased pakt levels ( fig . these results are puzzling since only hsmm , and not skmdc , showed a morphological response to igf-1 , with a substantial increase in myotube area ( fig . this could be due to the relatively high pakt levels in untreated hsmm compared to skmdc which may help prime the cells hypertrophic response to exogenous igf-1 . it is also possible that igf-1 acts through an akt - independent mechanism to induce a hypertrophy - like response in hsmm and is insufficient to cause such a response in skmdc . in addition to mstn and dex , the cytokine tnf - related weak inducer of apoptosis ( tweak ) has been reported to be a potent inducer of muscle atrophy ( dogra et al . 2007a ) and blocks the differentiation of c2c12 myoblasts into myotubes ( dogra et al . 2006 , 2007b ) . in our studies , treatment of hsmm and skmdc myotubes with tweak had no observable effects ( data not shown ) . however , when added to the culture at the start of myotube differentiation , the differentiation of skmdc was completely blocked , while it had no effect on hsmm differentiation ( fig . expression of the tweak receptor , fn14 , has been demonstrated on human muscle satellite cells ( girgenrath et al . 2006 ) , but it is possible that fn14 is not expressed by hsmm which would explain the absence of an effect of tweak in these cultures . we examined fn14 expression in both cell populations by real - time rt - pcr and found that it was expressed in both myoblasts and myotubes and showed no changes during differentiation ( data not shown).figure 3.effect of tweak on hsmm and skmdc . representative images of myh2/hoechst - labeled myotubes from cultures differentiated for 4 d in differentiation medium with tweak ( 1 g / ml ) . representative images of myh2/hoechst - labeled myotubes from cultures differentiated for 4 d in differentiation medium with tweak ( 1 g / ml ) . as shown above , mstn induced an atrophy - like morphological response in both hsmm and skmdc where the myotubes showed a substantial decrease in size compared to untreated cultures ( fig . we and others are working to target myostatin with inhibitors to block its negative regulation of muscle mass as a treatment for frailty . one such inhibitor consists of a fusion between the ligand - binding domain of actriib , the high affinity receptor for mstn , and the fc region of immunoglobulin g ( actriib - fc ) . this molecule is a potent inhibitor of mstn activity in vivo ( lee et al . 2005 ) . to assess its activity in hsmm and skmdc , myotubes were treated with mstn in the presence of various concentrations of actriib - fc for 48 h. images of myh2-positive myotubes were captured and myotube areas were quantified as described above . as expected , the images showed that mstn alone induced a significant decrease in myotube area , and this atrophy - like response was blocked by actriib - fc in both cell populations ( fig . the rescue of mstn - induced atrophy by actriib - fc was quantified and plotted as the percent change from untreated myotubes ( fig . 50 g / ml actriib - fc induced a significant percent change in area compared to cells treated with mstn ( 1 g / ml ) alone ( 0 g / ml actriib - fc ) . the plots demonstrated that actriib - fc blocked mstn - induced atrophy in these cultures , such that the myotube area was significantly different to that measured in cultures treated with mstn alone . at lower concentrations of actriib - fc , statistically significant effects were shown in skmdc cultures only , suggesting that these cells were more sensitive to the rescue effect . thus , hsmm and skmdc may be used to identify and characterize inhibitors of myostatin activity.figure 4.actriib-fc ( a myostatin inhibitor ) blocks myostatin - induced atrophy in hsmm and skmdc . myh2/hoechst - labeled myotubes differentiated for 3 d and then treated with 1 g / ml myostatin a dilution series of actriib - fc for 48 h. representative images of untreated myotubes and those treated with 1 g / ml mstn alone and 1 g / ml mstn + 50 g / ml actriib - fc . plot shows the mean percent change in myotube area from untreated myotubes sem , n = 15 , * p < 0.05 vs 1 g / ml mstn using student s t test ) . actriib - fc ( a myostatin inhibitor ) blocks myostatin - induced atrophy in hsmm and skmdc . myh2/hoechst - labeled myotubes differentiated for 3 d and then treated with 1 g / ml myostatin a dilution series of actriib - fc for 48 h. representative images of untreated myotubes and those treated with 1 g / ml mstn alone and 1 g / ml mstn + 50 g / ml actriib - fc . plot shows the mean percent change in myotube area from untreated myotubes sem , n = 15 , * p < 0.05 vs 1 g / ml mstn using student s t test ) . the ability to genetically manipulate cells in culture provides powerful tools for analysis . lipid - mediated transfection reagents were largely ineffective ( we estimated that < 20% of the cells were transfected as assessed by the number of gfp - positive cells ) , although hsmm appeared to be slightly more susceptible to transfection by this method than skmdc ( data not shown ) . in addition , we observed that most lipid transfection reagents significantly slowed down the differentiation of myotubes , requiring 6 d for full differentiation compared to 23 d for untreated cultures ( data not shown ) . similar results were obtained using electroporation methods with the undifferentiated cells ( data not shown ) . to investigate an alternative method of gene delivery to hsmm and skmdc , myotubes were identified by staining with anti - myh2 and detected with alexa fluor 555 goat antimouse igg secondary antibody ( red ) , while the infected cells were detected by gfp fluorescence ( green ) . infected myotubes were orange ( red plus green ) , uninfected myotubes were red , and infected , undifferentiated cells were green . although not quantified , it was noted that almost all the myotubes were orange . in addition , most of the undifferentiated ( non - myotube ) cells in the cultures were also successfully infected , displaying mostly gfp - positive cells ( fig . finally , undifferentiated cells were also effectively infected by the adenovirus , although the infection efficiency varied from experiment to experiment from 30% to 80% , quantified by visual inspection only ( data not shown ) . however , infection of the undifferentiated cells slowed down myotube differentiation by about 34 d ( data not shown ) . in general , adenoviral infection was found to be the most efficient method for gene delivery into both cell populations.figure 5.infection of hsmm and skmdc with adenovirus . representative images of myh2/hoechst - labeled myotubes differentiated for 2 d and then infected with adeno - gfp at 500 moi . myh2 stained red ( not infected images ) in this experiment and adeno - gfp is green . the overlap results in a yellow / orange color which demonstrates expression of gfp in myh2-positive myotubes ( ad - gfp images ) . representative images of myh2/hoechst - labeled myotubes differentiated for 2 d and then infected with adeno - gfp at 500 moi . myh2 stained red ( not infected images ) in this experiment and adeno - gfp is green . the overlap results in a yellow / orange color which demonstrates expression of gfp in myh2-positive myotubes ( ad - gfp images ) . these studies were performed to screen several commercially available sources of primary human skeletal myoblasts for their capacity to reproducibly differentiate into myotubes and respond appropriately to inducers of atrophy and hypertrophy . out of the four cell types that were screened , only two demonstrated these features ( hsmm from lonza and skmdc from cook myosite ) . although hsmm and skmdc both differentiate into myotubes , we observed several marked differences between them . first , for hsmm there were notably fewer cell nuclei that were not incorporated into myotubes after culturing in differentiation medium compared to skmdc ( see darkfield images in fig . 1 ) . for example , although both cell populations were plated at the same density , differences in the rate of cell proliferation prior to the induction of differentiation could be responsible , as could different myoblast fusion efficiencies . alternatively , greater cell death during the 3-d differentiation period in the hsmm cultures could cause this effect , or the unincorporated nuclei may represent another cell type that the skmdc can generate during differentiation . second , as noted in the results , the organization of nuclei within the myotubes was different between hsmm and skmdc . for example , skmdc did not undergo a morphological hypertrophy response when treated with igf-1 while hsmm showed distinct increases in myotube size . these cells die when exposed to 100 m dex whereas hsmm do not ( data not shown ) . the finding that dex had no effect on pakt levels in either cell population ( see fig . 2c ) was surprising because it has been shown that dex treatment reduced pakt levels in c2c12 myotubes ( zhao et al . however , in those experiments , c2c12 myotubes were treated with only 100 nm dex overnight . in our studies , myotubes were treated with 50 m dex for 48 h. such culture conditions induced visible and quantifiable myotube atrophy , whereas 100 nm dex failed to induce a visible atrophy . it is possible that the absence of dex - regulated pakt levels in our studies may be due to the higher dex concentration and longer incubation time compared to previous studies with c2c12 myotubes , or that this is a novel feature of primary human skeletal muscle cells . finally , the cytokine tweak inhibited the differentiation of skmdc but not hsmm ( fig . 3 ) and had no effect on differentiated skmdc and hsmm myotubes ( data not shown ) . tweak has been shown to induce atrophy in differentiated c2c12 myotubes ( dogra et al . 2007a ) and prevent the differentiation of c2c12 myoblasts into myotubes ( dogra et al . however , we are not aware that similar effects of tweak on primary human skeletal muscle cells have been examined . our findings with tweak were not explained by the absence of the appropriate tweak receptor ( fn14 ) on these cells , both as myoblasts and myotubes ( data not shown ) . therefore , we are unable to explain the absence of an effect of tweak on the differentiation of hsmm and the absence of tweak - induced atrophy in the myotubes derived from both cell populations at this time . it is possible that all of these differences may be due to the origins of the cells . hsmm were derived from the quadriceps muscle of a young adult male ( 16 yr old ) cadaver , while skmdc came from the rectus abdominus muscle of an older male ( 52 yr old ) living donor . it is possible , even likely , that the precise characteristics of primary human skeletal muscle cells will be affected by numerous donor factors , such as age , gender , and general health . thus , comparison of results from experiments using cells from different donors may be challenging . during our analyses , we noticed differences in the gene expression profile for myod and myog in both cell populations compared to what has been published for primary skeletal muscle cells ( cornelison and wold 1997 ; zammit et al . , it was shown that myod expression is typical of a transcription factor with a rapid significant increase in expression upon commitment of satellite cells to the muscle lineage . this expression is then followed by rapid downregulation of expression , at the same time as myog expression is turned on . in our studies , we failed to demonstrate a significant upregulation of myod after 24 h of culture in differentiation medium . it is possible that myod was upregulated within 10 h and so we were measuring the down regulation of its expression at 24 h. it is also possible that both cell populations represent committed muscle precursors , which already express myod , so the fold change in expression upon switching to differentiation medium would be minimal . indeed , the skmdc vendor ( cook myosite ) has shown that as myoblasts , these cells already express myod ( janowski 2011 ) . in our cultures , myog expression was maximal between 24 and 48 h in differentiation medium . expression of myog has been shown as early as 10 h after switching to differentiation medium , with highest expression levels seen before 48 and 60 h in differentiation medium ( bigot et al . it is possible that our cultures also upregulate myog expression at earlier time - points in differentiation medium , but we did not harvest rna at time - points earlier than 24 h. the prolonged expression profile of myog from the 24 h time - point in our cultures agrees with what has been shown for primary human muscle cells ( bigot et al . we have identified two commercially available sources of primary human skeletal muscle cells that can reproducibly differentiate into multinucleated myotubes in culture . myotube differentiation occurs rapidly upon shifting the cells into differentiation medium , within 23 d , and they express characteristic molecular markers of muscle differentiation . the myotubes derived from these cells can be induced to undergo morphological atrophy- and hypertrophy - like responses . we have also shown that the atrophy response can be blocked with a pharmacological inhibitor of myostatin , a known negative regulator of muscle mass . finally , these myotubes can be efficiently infected with adenoviruses , providing a method for genetic modification . such modifications would potentially allow adenoviral - shrna knock - down screens for muscle atrophy targets which could then be targeted for inhibition by small or large molecule compounds . taken together , our results indicate that these primary human skeletal muscle cells may be a useful system for studying skeletal muscle cell differentiation and may also provide tools for studying new therapeutic molecules such as myostatin inhibitors .

there is a significant unmet need for safe , anabolic muscle therapies to treat diseases and conditions associated with severe muscle weakness and frailty . the identification of such therapies requires appropriate cell - based screening assays to select compounds for further development using animal models . primary human skeletal muscle cells have recently become available from a number of commercial vendors . such cells may be valuable for studying the mechanisms that direct muscle differentiation , and for identifying and characterizing novel therapeutic approaches for the treatment of age- and injury - induced muscle disorders . however , only limited characterization of these cells has been reported to date . therefore , we have examined four primary human muscle cell preparations from three different vendors for their capacity to differentiate into multinucleated myotubes . two of the preparations demonstrated robust myotube formation and expressed characteristic markers of muscle differentiation . furthermore , these myotubes could be induced to undergo morphological atrophy- and hypertrophy - like responses , and atrophy could be blocked with an inhibitor of myostatin signaling , a pathway that is known to negatively regulate muscle mass . finally , the myotubes were efficiently infected with recombinant adenovirus , providing a tool for genetic modification . taken together , our results indicate that primary human muscle cells can be a useful system for studying muscle differentiation , and may also provide tools for studying new therapeutic molecules for the treatment of muscle disease .

Introduction Materials and Methods Results Discussion

age- or injury - induced muscle weakness leading to frailty is a major public health problem that is predicted to escalate in the future as the number and proportion of older adults increase in the general population ( berger and doherty 2010 ) . there is an unmet need for therapeutic strategies that can slow the effects of aging on muscle function in the frail elderly so as to maintain or improve their quality of life . identifying suitable therapeutic targets and testing candidate drugs for the ability to improve muscle function require cell - based model systems that reliably predict in vivo effects in both pre - clinical rodent models and human patients . a number of useful rodent cell lines such as mouse c2c12 or rat l6 myoblasts are available ( sultan et al . these cell lines , and others , have been used extensively to explore the molecular mechanisms of muscle differentiation and function ( mcfarlane et al . however , because immortalized cell lines often are genetically abnormal , and have been maintained under artificial conditions in culture for very long periods of time which can cause them to deviate from normal function , a potentially more predictive screening strategy would use primary human muscle cells . such cells have recently become available from a number of commercial vendors , but only limited characterization of these various primary cells has been reported ( janowski 2011 ) . therefore , we examined several primary human skeletal muscle cell preparations from different vendors and characterized each for their capacity to reproducibly differentiate into multinucleated myotubes . those that reproducibly differentiated into myotubes were further examined for the expression of various markers of skeletal muscle cells such as myogenic differentiation-1 ( myod ) , myocyte enhancer factor 2c ( mef2c ) , myogenin ( myog ) , troponin t type 1 ( tnnt ) , and myosin heavy chain-2 ( myh2 ) . we also tested known inducers of skeletal muscle atrophy ( myostatin and dexamethasone ) and hypertrophy ( insulin - like growth factor-1 ) for effects on the differentiated myotubes derived from the primary cells . in addition , we found that myostatin - induced activity on the myotubes can be blocked by treatment with a soluble myostatin receptor , similar to one that is currently in clinical trials for diseases associated with muscle wasting ( lee et al . finally , we show that both the undifferentiated primary muscle cells as well as the myotubes they give rise to can be infected with adenovirus . this observation suggests the potential for using these cells in genetic screens ( both over - expression and knock - down ) to identify factors that play a role in muscle differentiation and function . when passaging , the culture medium was warmed to 37c and the cells were seeded at 5,0007,500 cells / cm and incubated in a humidified incubator at 37c , 5% co2 . the following morning , the growth medium was replaced with differentiation medium and the cultures were incubated for 3 d , during which time myotube differentiation occurred . to examine effects on differentiation , agents to be tested were added at the time of medium change and cultured for 3 d. to examine effects on differentiated myotubes , agents to be tested were added on day 3 of differentiation , after myotube formation had occurred , and then cultures were incubated for another 23 d. for analysis , to unambiguously identify myotubes , the cultures were fixed and immuno - stained with an antibody against myosin heavy chain-2 protein ( myh2 ) , as described below . cells were washed in ice cold pbs before lysis in a buffer comprised of 40 mm mops ph 7 , 4 mm egta , 10 mm edta with 2% triton x-100 , containing protease and phosphatase inhibitors ( 30 mm sodium fluoride , 60 mm -glycerophosphate , 20 mm sodium pyrophosphate , 1 mm sodium orthovanadate , and 1% protease inhibitor cocktail , plus 1 mm phenylmethylsulfonylfluoride ( pmsf ) , all from sigma . tris for nupage gels , invitrogen , electrophoresed , and then transferred on to nitrocellulose membranes . several different ratios of viral particles to cells were tested ( multiplicity of infection , or moi , of 100 , 250 , 500 , 700 , and 1,000 ) . in some cases , to observe the effects of infection on myotube differentiation , cells were returned to differentiation medium for 6 d after a 6 h recovery period in growth medium . four different primary human muscle cell preparations from three different commercial vendors were screened for the ability to differentiate into multinucleated myotubes in cell culture . we tested human skeletal muscle myoblasts ( hsmm ; lonza ) , human skeletal muscle cells ( skmc - l ; lonza ) , human skeletal muscle - derived cells ( skmdc ; cook myosite ) , and human skeletal muscle cells ( skmc - p ; promocell ) . initial experiments found that two of these preparations ( skmc - l and skmc - p ) showed little , if any , evidence of morphological differentiation into myotubes when cultured according to the recommendations of the suppliers . however , hsmm and skmdc demonstrated robust myotube differentiation , and could be readily cultured as undifferentiated cells in growth medium , using polystyrene cell culture plates . thus , we focused on these two primary cell preparations for the remainder of our studies presented here . skmdc were characterized as displaying 92% desmin - positive cells and a gene expression analysis provided by the vendor showed that these cells expressed several markers of muscle cell precursors such as pax3 , pax7 , myod1 , myog , and myf5 . this marker analysis therefore suggests that the skmdc population is a committed myoblast cell population . in our initial studies , we evaluated cell growth and differentiation of cells through 15 pds . for the studies described in this paper , we used cells that had undergone no more than 7 pd . in addition , we also screened bd purecoat amine ( bd biosciences , san jose , ca ) and polystyrene plates coated in - house with 2% gelatin ( sigma ) . therefore , standard cell - culture grade polystyrene plates were used throughout these studies . in contrast , the nuclei in skmdc myotubes were often clustered together in large groups ( fig . this difference may be caused by differences in the mechanisms of cell fusion during myotube generation , or by differences in the regulation of nuclear dynamics in the myotubes , or other unknown factors . gene expression analyses were performed using quantitative rt - pcr for various markers of muscle cell differentiation , ( fig . myod and mef2c , transcription factors that play important roles in specifying the myogenic lineage , represent two early markers of myoblast differentiation into myotubes ( megeney and rudnicki 1995 ; black and olson 1998 ; zammit et al . mef2c was also upregulated by day 1 in both cell populations , reaching statistical significance by day 2 , but unlike myod , the expression of this gene remained elevated throughout the 6-d culture period . myogenin ( myog ) , troponin t ( tnnt ) , and myh2 represent late markers of muscle cell differentiation ( burattini et al . thus , hsmm and skmdc undergo morphological differentiation in culture into multinucleated myotubes and express characteristic molecular markers of muscle cell differentiation.figure 1.hsmm and skmdc differentiated into multinucleated myosin heavy chain-2 ( myh2)-positive myotubes . plots show gene expression of early ( myod , mef2c ) and late ( myog , tnnt , and myh2 ) muscle cell markers , normalized to b2 m gene expression , relative to expression levels at day 0 ( time at which differentiation media was added to the cells ) ; * p < 0.05 vs. day 0 using student s t test . plots show gene expression of early ( myod , mef2c ) and late ( myog , tnnt , and myh2 ) muscle cell markers , normalized to b2 m gene expression , relative to expression levels at day 0 ( time at which differentiation media was added to the cells ) ; * p < 0.05 vs. day 0 using student s t test . dexamethasone , a synthetic steroid , and myostatin , a growth factor which is a negative regulator of muscle mass , are well - characterized inducers of myotube atrophy , as shown for c2c12 myotubes ( lee 2004 ; stitt et al . therefore , to examine the response of primary human skeletal muscle cell cultures to these factors , myotubes derived from hsmm and skmdc were treated with various concentrations of dex , mstn , or igf-1 for 48 h and then fixed and immuno - stained for myh2 . hsmm and skmdc undergo an atrophy - like response to both mstn and dex treatment , while only hsmm display a hypertrophy - like response to igf-1 . ( a ) differentiated myotubes were treated with a concentration series of mstn or dex for 48 h. representative images of myh2/hoechst - labeled myotubes treated with 1 g / ml mstn or 50 m dex for 48 h. plots of the percent decrease , from untreated cells , in myotube area at each concentration of test agent are shown . ( b ) differentiated myotubes were treated with a concentration series of igf-1 for 48 h. representative images of myh2/hoechst - labeled myotubes following treatment with 1 g / ml insulin - like growth factor-1 ( igf-1 ) . ( a ) differentiated myotubes were treated with a concentration series of mstn or dex for 48 h. representative images of myh2/hoechst - labeled myotubes treated with 1 g / ml mstn or 50 m dex for 48 h. plots of the percent decrease , from untreated cells , in myotube area at each concentration of test agent are shown . ( b ) differentiated myotubes were treated with a concentration series of igf-1 for 48 h. representative images of myh2/hoechst - labeled myotubes following treatment with 1 g / ml insulin - like growth factor-1 ( igf-1 ) . to examine the signaling pathways that were activated during these treatments , cell lysates were prepared at the end of the 48 h treatment period and then probed with antibodies to phosphorylated- and total smad2 , and phosphorylated- and total akt , since these molecules are involved in the earliest stage responses to the mstn and igf-1 ligands ( rebbapragada et al . inhibition of akt phosphorylation is consistent with an atrophic response , since pakt activity is a known positive regulator of muscle protein synthesis and growth ( duan et al . in our studies overall , these results suggest that at least for skmdc , mstn and dex operate through separate pathways to induce an atrophy - like response . finally , igf-1 binding to its receptor is known to stimulate the phosphorylation of akt , which stimulates protein synthesis and cell growth ( duan et al . igf-1 treatment of skmdc , but not hsmm , increased pakt levels ( fig . this could be due to the relatively high pakt levels in untreated hsmm compared to skmdc which may help prime the cells hypertrophic response to exogenous igf-1 . it is also possible that igf-1 acts through an akt - independent mechanism to induce a hypertrophy - like response in hsmm and is insufficient to cause such a response in skmdc . in addition to mstn and dex , the cytokine tnf - related weak inducer of apoptosis ( tweak ) has been reported to be a potent inducer of muscle atrophy ( dogra et al . however , when added to the culture at the start of myotube differentiation , the differentiation of skmdc was completely blocked , while it had no effect on hsmm differentiation ( fig . expression of the tweak receptor , fn14 , has been demonstrated on human muscle satellite cells ( girgenrath et al . as shown above , mstn induced an atrophy - like morphological response in both hsmm and skmdc where the myotubes showed a substantial decrease in size compared to untreated cultures ( fig . we and others are working to target myostatin with inhibitors to block its negative regulation of muscle mass as a treatment for frailty . one such inhibitor consists of a fusion between the ligand - binding domain of actriib , the high affinity receptor for mstn , and the fc region of immunoglobulin g ( actriib - fc ) . this molecule is a potent inhibitor of mstn activity in vivo ( lee et al . to assess its activity in hsmm and skmdc , myotubes were treated with mstn in the presence of various concentrations of actriib - fc for 48 h. images of myh2-positive myotubes were captured and myotube areas were quantified as described above . as expected , the images showed that mstn alone induced a significant decrease in myotube area , and this atrophy - like response was blocked by actriib - fc in both cell populations ( fig . the rescue of mstn - induced atrophy by actriib - fc was quantified and plotted as the percent change from untreated myotubes ( fig . the plots demonstrated that actriib - fc blocked mstn - induced atrophy in these cultures , such that the myotube area was significantly different to that measured in cultures treated with mstn alone . thus , hsmm and skmdc may be used to identify and characterize inhibitors of myostatin activity.figure 4.actriib-fc ( a myostatin inhibitor ) blocks myostatin - induced atrophy in hsmm and skmdc . the ability to genetically manipulate cells in culture provides powerful tools for analysis . lipid - mediated transfection reagents were largely ineffective ( we estimated that < 20% of the cells were transfected as assessed by the number of gfp - positive cells ) , although hsmm appeared to be slightly more susceptible to transfection by this method than skmdc ( data not shown ) . infected myotubes were orange ( red plus green ) , uninfected myotubes were red , and infected , undifferentiated cells were green . although not quantified , it was noted that almost all the myotubes were orange . in addition , most of the undifferentiated ( non - myotube ) cells in the cultures were also successfully infected , displaying mostly gfp - positive cells ( fig . finally , undifferentiated cells were also effectively infected by the adenovirus , although the infection efficiency varied from experiment to experiment from 30% to 80% , quantified by visual inspection only ( data not shown ) . however , infection of the undifferentiated cells slowed down myotube differentiation by about 34 d ( data not shown ) . representative images of myh2/hoechst - labeled myotubes differentiated for 2 d and then infected with adeno - gfp at 500 moi . these studies were performed to screen several commercially available sources of primary human skeletal myoblasts for their capacity to reproducibly differentiate into myotubes and respond appropriately to inducers of atrophy and hypertrophy . out of the four cell types that were screened , only two demonstrated these features ( hsmm from lonza and skmdc from cook myosite ) . although hsmm and skmdc both differentiate into myotubes , we observed several marked differences between them . second , as noted in the results , the organization of nuclei within the myotubes was different between hsmm and skmdc . these cells die when exposed to 100 m dex whereas hsmm do not ( data not shown ) . 2c ) was surprising because it has been shown that dex treatment reduced pakt levels in c2c12 myotubes ( zhao et al . however , in those experiments , c2c12 myotubes were treated with only 100 nm dex overnight . it is possible that the absence of dex - regulated pakt levels in our studies may be due to the higher dex concentration and longer incubation time compared to previous studies with c2c12 myotubes , or that this is a novel feature of primary human skeletal muscle cells . finally , the cytokine tweak inhibited the differentiation of skmdc but not hsmm ( fig . however , we are not aware that similar effects of tweak on primary human skeletal muscle cells have been examined . our findings with tweak were not explained by the absence of the appropriate tweak receptor ( fn14 ) on these cells , both as myoblasts and myotubes ( data not shown ) . therefore , we are unable to explain the absence of an effect of tweak on the differentiation of hsmm and the absence of tweak - induced atrophy in the myotubes derived from both cell populations at this time . it is possible that all of these differences may be due to the origins of the cells . it is possible , even likely , that the precise characteristics of primary human skeletal muscle cells will be affected by numerous donor factors , such as age , gender , and general health . during our analyses , we noticed differences in the gene expression profile for myod and myog in both cell populations compared to what has been published for primary skeletal muscle cells ( cornelison and wold 1997 ; zammit et al . in our studies , we failed to demonstrate a significant upregulation of myod after 24 h of culture in differentiation medium . indeed , the skmdc vendor ( cook myosite ) has shown that as myoblasts , these cells already express myod ( janowski 2011 ) . it is possible that our cultures also upregulate myog expression at earlier time - points in differentiation medium , but we did not harvest rna at time - points earlier than 24 h. the prolonged expression profile of myog from the 24 h time - point in our cultures agrees with what has been shown for primary human muscle cells ( bigot et al . we have identified two commercially available sources of primary human skeletal muscle cells that can reproducibly differentiate into multinucleated myotubes in culture . myotube differentiation occurs rapidly upon shifting the cells into differentiation medium , within 23 d , and they express characteristic molecular markers of muscle differentiation . the myotubes derived from these cells can be induced to undergo morphological atrophy- and hypertrophy - like responses . we have also shown that the atrophy response can be blocked with a pharmacological inhibitor of myostatin , a known negative regulator of muscle mass . finally , these myotubes can be efficiently infected with adenoviruses , providing a method for genetic modification . taken together , our results indicate that these primary human skeletal muscle cells may be a useful system for studying skeletal muscle cell differentiation and may also provide tools for studying new therapeutic molecules such as myostatin inhibitors .

age- or injury - induced muscle weakness leading to frailty is a major public health problem that is predicted to escalate in the future as the number and proportion of older adults increase in the general population ( berger and doherty 2010 ) . there is an unmet need for therapeutic strategies that can slow the effects of aging on muscle function in the frail elderly so as to maintain or improve their quality of life . identifying suitable therapeutic targets and testing candidate drugs for the ability to improve muscle function require cell - based model systems that reliably predict in vivo effects in both pre - clinical rodent models and human patients . these cell lines , and others , have been used extensively to explore the molecular mechanisms of muscle differentiation and function ( mcfarlane et al . therefore , we examined several primary human skeletal muscle cell preparations from different vendors and characterized each for their capacity to reproducibly differentiate into multinucleated myotubes . those that reproducibly differentiated into myotubes were further examined for the expression of various markers of skeletal muscle cells such as myogenic differentiation-1 ( myod ) , myocyte enhancer factor 2c ( mef2c ) , myogenin ( myog ) , troponin t type 1 ( tnnt ) , and myosin heavy chain-2 ( myh2 ) . we also tested known inducers of skeletal muscle atrophy ( myostatin and dexamethasone ) and hypertrophy ( insulin - like growth factor-1 ) for effects on the differentiated myotubes derived from the primary cells . in addition , we found that myostatin - induced activity on the myotubes can be blocked by treatment with a soluble myostatin receptor , similar to one that is currently in clinical trials for diseases associated with muscle wasting ( lee et al . finally , we show that both the undifferentiated primary muscle cells as well as the myotubes they give rise to can be infected with adenovirus . this observation suggests the potential for using these cells in genetic screens ( both over - expression and knock - down ) to identify factors that play a role in muscle differentiation and function . hsmm ( catalog # cc-2580 , lot # 6f4528 , sourced from quadriceps muscle of a 16-yr - old male cadaver ) and skmc - l ( catalog # cc-2561 , lot # 6f3791 , sourced from human fetal skeletal muscle ) were purchased from lonza ( walkersville , md ) . skmdc ( catalog # sk-1111 , lot # p101014 - 50m2 , sourced from rectus abdominus muscle of a 50-yr - old male caucasian with a body mass index ( bmi ) of 21 , non - diabetic , smoker for 20 yr ) came from cook myosite ( pittsburgh , pa ) . skmc - p ( catalog # c-12530 , lot # 8121902.17 , sourced from unspecified skeletal muscle of a 21-yr - old male caucasian ) was obtained from promocell ( heidelberg , germany ) . to examine effects on differentiation , agents to be tested were added at the time of medium change and cultured for 3 d. to examine effects on differentiated myotubes , agents to be tested were added on day 3 of differentiation , after myotube formation had occurred , and then cultures were incubated for another 23 d. for analysis , to unambiguously identify myotubes , the cultures were fixed and immuno - stained with an antibody against myosin heavy chain-2 protein ( myh2 ) , as described below . for western blot analyses , antibodies against phosphorylated - smad2 ( ser465/467 ) ( psmad2 ) , smad2 , phosphorylated - akt ( ser473 ) ( pakt ) , akt , and alpha - tubulin were purchased from cell signaling technologies ( danvers , ma ) . the human taqman probe sets for myosin heavy chain , myh2 ( hs00430042_m1 ) , troponin t type 1 , tnnt ( hs00162848_m1 ) , myocyte enhancer factor 2c , mef2c ( hs00231149_m1 ) , myogenic differentiation 1 , myod ( hs00159528_m1 ) , myogenin , myog ( hs01072232_m1 ) , and beta-2 microglobulin , b2 m ( # 4333766f ) , were purchased from applied biosystems ( wilmington , de ) . several different ratios of viral particles to cells were tested ( multiplicity of infection , or moi , of 100 , 250 , 500 , 700 , and 1,000 ) . in some cases , to observe the effects of infection on myotube differentiation , cells were returned to differentiation medium for 6 d after a 6 h recovery period in growth medium . four different primary human muscle cell preparations from three different commercial vendors were screened for the ability to differentiate into multinucleated myotubes in cell culture . we tested human skeletal muscle myoblasts ( hsmm ; lonza ) , human skeletal muscle cells ( skmc - l ; lonza ) , human skeletal muscle - derived cells ( skmdc ; cook myosite ) , and human skeletal muscle cells ( skmc - p ; promocell ) . initial experiments found that two of these preparations ( skmc - l and skmc - p ) showed little , if any , evidence of morphological differentiation into myotubes when cultured according to the recommendations of the suppliers . thus , we focused on these two primary cell preparations for the remainder of our studies presented here . skmdc were characterized as displaying 92% desmin - positive cells and a gene expression analysis provided by the vendor showed that these cells expressed several markers of muscle cell precursors such as pax3 , pax7 , myod1 , myog , and myf5 . in general , above 10 pds , both cell types took longer to differentiate and their differentiation was not as robust , compared to cells at pd < 10 ( data not shown ) . for the studies described in this paper , we used cells that had undergone no more than 7 pd . this difference may be caused by differences in the mechanisms of cell fusion during myotube generation , or by differences in the regulation of nuclear dynamics in the myotubes , or other unknown factors . to further characterize the differentiation process of hsmm and skmdc , rna was prepared from cells harvested on day 0 through day 6 of culture in differentiation medium . myod and mef2c , transcription factors that play important roles in specifying the myogenic lineage , represent two early markers of myoblast differentiation into myotubes ( megeney and rudnicki 1995 ; black and olson 1998 ; zammit et al . thus , hsmm and skmdc undergo morphological differentiation in culture into multinucleated myotubes and express characteristic molecular markers of muscle cell differentiation.figure 1.hsmm and skmdc differentiated into multinucleated myosin heavy chain-2 ( myh2)-positive myotubes . ( a ) representative phase contrast and dark - field images of myh2-immunostained cells ( green ) , with hoechst - labeled nuclei ( blue ) after 1 d in growth medium ( phase contrast images ) or 3 d in differentiation medium ( dark - field images ; magnification 10 ; scale bar = 100 microns ) . plots show gene expression of early ( myod , mef2c ) and late ( myog , tnnt , and myh2 ) muscle cell markers , normalized to b2 m gene expression , relative to expression levels at day 0 ( time at which differentiation media was added to the cells ) ; * p < 0.05 vs. day 0 using student s t test . ( a ) representative phase contrast and dark - field images of myh2-immunostained cells ( green ) , with hoechst - labeled nuclei ( blue ) after 1 d in growth medium ( phase contrast images ) or 3 d in differentiation medium ( dark - field images ; magnification 10 ; scale bar = 100 microns ) . plots show gene expression of early ( myod , mef2c ) and late ( myog , tnnt , and myh2 ) muscle cell markers , normalized to b2 m gene expression , relative to expression levels at day 0 ( time at which differentiation media was added to the cells ) ; * p < 0.05 vs. day 0 using student s t test . dexamethasone , a synthetic steroid , and myostatin , a growth factor which is a negative regulator of muscle mass , are well - characterized inducers of myotube atrophy , as shown for c2c12 myotubes ( lee 2004 ; stitt et al . therefore , to examine the response of primary human skeletal muscle cell cultures to these factors , myotubes derived from hsmm and skmdc were treated with various concentrations of dex , mstn , or igf-1 for 48 h and then fixed and immuno - stained for myh2 . the plots show that mstn and dex induced statistically significant decreases in myotube area over untreated control cultures at all concentrations ( except for the hsmm 250 ng / ml mstn - treated group which showed decreased myotube area but failed to reach statistical significance ; fig . quantification of igf-1 effects on both cell populations confirmed that only hsmm cultures responded with a hypertrophic response , with significant increases in myotube area at 100 and 1,000 ng / ml igf-1 , ( fig . statistical significance , using student s t test was performed by comparing the total myotube area in multiple untreated cultures with the total myotube area in treated cultures and then assigning statistical significance ( * ) if p < 0.05.figure 2.induction of atrophy and hypertrophy in hsmm and skmdc myotubes . ( a ) differentiated myotubes were treated with a concentration series of mstn or dex for 48 h. representative images of myh2/hoechst - labeled myotubes treated with 1 g / ml mstn or 50 m dex for 48 h. plots of the percent decrease , from untreated cells , in myotube area at each concentration of test agent are shown . ( b ) differentiated myotubes were treated with a concentration series of igf-1 for 48 h. representative images of myh2/hoechst - labeled myotubes following treatment with 1 g / ml insulin - like growth factor-1 ( igf-1 ) . ( c ) western blots of myotube lysates from cultures treated with 1 g / ml mstn , 50 m dex , or 1 g / ml igf-1 for 48 h and probed with antibodies to phosphorylated- and total smad2 and phosphorylated- and total akt . ( a ) differentiated myotubes were treated with a concentration series of mstn or dex for 48 h. representative images of myh2/hoechst - labeled myotubes treated with 1 g / ml mstn or 50 m dex for 48 h. plots of the percent decrease , from untreated cells , in myotube area at each concentration of test agent are shown . ( b ) differentiated myotubes were treated with a concentration series of igf-1 for 48 h. representative images of myh2/hoechst - labeled myotubes following treatment with 1 g / ml insulin - like growth factor-1 ( igf-1 ) . ( c ) western blots of myotube lysates from cultures treated with 1 g / ml mstn , 50 m dex , or 1 g / ml igf-1 for 48 h and probed with antibodies to phosphorylated- and total smad2 and phosphorylated- and total akt . to examine the signaling pathways that were activated during these treatments , cell lysates were prepared at the end of the 48 h treatment period and then probed with antibodies to phosphorylated- and total smad2 , and phosphorylated- and total akt , since these molecules are involved in the earliest stage responses to the mstn and igf-1 ligands ( rebbapragada et al . in addition , mstn reduced the level of phosphorylated - akt ( pakt ) in hsmm and skmdc ( fig . inhibition of akt phosphorylation is consistent with an atrophic response , since pakt activity is a known positive regulator of muscle protein synthesis and growth ( duan et al . in contrast to myostatin , dex treatment induced downregulation of the control levels of psmad2 in skmdc ( fig . in addition to mstn and dex , the cytokine tnf - related weak inducer of apoptosis ( tweak ) has been reported to be a potent inducer of muscle atrophy ( dogra et al . however , when added to the culture at the start of myotube differentiation , the differentiation of skmdc was completely blocked , while it had no effect on hsmm differentiation ( fig . we examined fn14 expression in both cell populations by real - time rt - pcr and found that it was expressed in both myoblasts and myotubes and showed no changes during differentiation ( data not shown).figure 3.effect of tweak on hsmm and skmdc . one such inhibitor consists of a fusion between the ligand - binding domain of actriib , the high affinity receptor for mstn , and the fc region of immunoglobulin g ( actriib - fc ) . to assess its activity in hsmm and skmdc , myotubes were treated with mstn in the presence of various concentrations of actriib - fc for 48 h. images of myh2-positive myotubes were captured and myotube areas were quantified as described above . as expected , the images showed that mstn alone induced a significant decrease in myotube area , and this atrophy - like response was blocked by actriib - fc in both cell populations ( fig . 50 g / ml actriib - fc induced a significant percent change in area compared to cells treated with mstn ( 1 g / ml ) alone ( 0 g / ml actriib - fc ) . the plots demonstrated that actriib - fc blocked mstn - induced atrophy in these cultures , such that the myotube area was significantly different to that measured in cultures treated with mstn alone . at lower concentrations of actriib - fc , statistically significant effects were shown in skmdc cultures only , suggesting that these cells were more sensitive to the rescue effect . thus , hsmm and skmdc may be used to identify and characterize inhibitors of myostatin activity.figure 4.actriib-fc ( a myostatin inhibitor ) blocks myostatin - induced atrophy in hsmm and skmdc . myh2/hoechst - labeled myotubes differentiated for 3 d and then treated with 1 g / ml myostatin a dilution series of actriib - fc for 48 h. representative images of untreated myotubes and those treated with 1 g / ml mstn alone and 1 g / ml mstn + 50 g / ml actriib - fc . myh2/hoechst - labeled myotubes differentiated for 3 d and then treated with 1 g / ml myostatin a dilution series of actriib - fc for 48 h. representative images of untreated myotubes and those treated with 1 g / ml mstn alone and 1 g / ml mstn + 50 g / ml actriib - fc . lipid - mediated transfection reagents were largely ineffective ( we estimated that < 20% of the cells were transfected as assessed by the number of gfp - positive cells ) , although hsmm appeared to be slightly more susceptible to transfection by this method than skmdc ( data not shown ) . in addition , we observed that most lipid transfection reagents significantly slowed down the differentiation of myotubes , requiring 6 d for full differentiation compared to 23 d for untreated cultures ( data not shown ) . to investigate an alternative method of gene delivery to hsmm and skmdc , myotubes were identified by staining with anti - myh2 and detected with alexa fluor 555 goat antimouse igg secondary antibody ( red ) , while the infected cells were detected by gfp fluorescence ( green ) . in addition , most of the undifferentiated ( non - myotube ) cells in the cultures were also successfully infected , displaying mostly gfp - positive cells ( fig . in general , adenoviral infection was found to be the most efficient method for gene delivery into both cell populations.figure 5.infection of hsmm and skmdc with adenovirus . for example , although both cell populations were plated at the same density , differences in the rate of cell proliferation prior to the induction of differentiation could be responsible , as could different myoblast fusion efficiencies . alternatively , greater cell death during the 3-d differentiation period in the hsmm cultures could cause this effect , or the unincorporated nuclei may represent another cell type that the skmdc can generate during differentiation . it is possible that the absence of dex - regulated pakt levels in our studies may be due to the higher dex concentration and longer incubation time compared to previous studies with c2c12 myotubes , or that this is a novel feature of primary human skeletal muscle cells . therefore , we are unable to explain the absence of an effect of tweak on the differentiation of hsmm and the absence of tweak - induced atrophy in the myotubes derived from both cell populations at this time . hsmm were derived from the quadriceps muscle of a young adult male ( 16 yr old ) cadaver , while skmdc came from the rectus abdominus muscle of an older male ( 52 yr old ) living donor . during our analyses , we noticed differences in the gene expression profile for myod and myog in both cell populations compared to what has been published for primary skeletal muscle cells ( cornelison and wold 1997 ; zammit et al . it is possible that myod was upregulated within 10 h and so we were measuring the down regulation of its expression at 24 h. it is also possible that both cell populations represent committed muscle precursors , which already express myod , so the fold change in expression upon switching to differentiation medium would be minimal . it is possible that our cultures also upregulate myog expression at earlier time - points in differentiation medium , but we did not harvest rna at time - points earlier than 24 h. the prolonged expression profile of myog from the 24 h time - point in our cultures agrees with what has been shown for primary human muscle cells ( bigot et al . taken together , our results indicate that these primary human skeletal muscle cells may be a useful system for studying skeletal muscle cell differentiation and may also provide tools for studying new therapeutic molecules such as myostatin inhibitors .

it was estimated that there were 1,500,000 new cancer cases and approximately 560,000 deaths out of cancer in 2009 . chemotherapy is an important treatment option for patients with cancer , however chemotherapy drugs suffer from numerous problems including nonspecific uptake by healthy tissue , poor circulation times , and suboptimal accumulation in the tumor . often , a large percentage of cytotoxic drug administered to the patient does not reach the tumor environment , but rather is distributed throughout the body , resulting in the many toxic effects associated with chemotherapy and a narrowing of the drug 's therapeutic window . the delivery of chemotherapeutic drugs to tumors is still a major hurdle in the eradication of cancer , and the continual development of drug delivery technologies is vital to future breakthroughs in chemotherapy . polymer micelles offer a promising approach to achieving these goals due to their inherent ability to overcome multiple biological barriers , such as avoidance of the reticuloendothelial system ( res ) . due to their unique size range ( 20150 nm ) , micelles are able to avoid renal clearance ( typically less than 20 nm ) and uptake by the liver and spleen ( particles greater than 150 nm ) . these micelles can also preferentially accumulate in solid tumors via the enhanced permeation and retention ( epr ) effect [ 3 , 4 ] . the epr effect is a consequence of the disorganized nature of the tumor vasculature , which results in increased permeability of polymer therapeutics and drug retention at the tumor site . due to these promising aspects , a number of groups have developed various polymer micelle motifs , encapsulating a wide range of therapeutic classes [ 517 ] . colon cancer is the third most common cancer in men and women in most of the developed world . irinotecan , a topoisomerase i inhibitor , is approved in the clinic for colorectal cancer first - line therapy in combination with 5-fluorouracil / leucovorin / oxaliplatin ( folfox ) regimen or for monotherapy in second - line therapy following a failed folfox regimen . sn-38 , the active metabolite of irinotecan , is about 5001000 times more cytotoxic than irinotecan [ 1820 ] . although irinotecan has demonstrated clinical utility , it is highly inefficient in delivering active sn-38 to tumor tissue . studies in humans have shown that only three to four percent of the administered irinotecan is actually converted to sn-38 , which is reliant upon activating carboxylesterase enzymes localized in the liver and gastrointestinal tract . in addition , up to 95% of sn-38 is bound to circulating proteins such as albumin , which drastically reduces its bioavailability . irinotecan treatment also is accompanied by dose - limiting toxicities of grade 3 and 4 diarrhea and neutropenia . these limitations of irinotecan result in poor exposure of sn-38 to the tumor environment and severe side effects in the patient . a major limitation , however , of free sn-38 is that it is hydrophobic and is unable to be used as a free drug in the clinic . several groups have addressed the solubility problem of sn-38 by covalently attaching sn-38 to a polymer or peptide [ 2426 ] . in particular , a polymeric micellar formulation of sn-38 based on peo - poly ( glutamic acid ) block copolymers through chemical conjugation of sn-38 to the free carboxyl groups present on the poly ( glutamic acid ) backbone has been developed . this formulation , known as nk012 , as well as a peglyated sn-38 formulation ( ezn-2208 ) , is currently in clinical trials [ 27 , 28 ] . while polymer - drug conjugates effectively address solubility of hydrophobic drugs , this prodrug approach is dependent on enzymatic or chemical cleavage of the bond to release the active drug . to develop an encapsulated formulation of sn-38 , sn-38 was loaded into a polymer micelle , resulting in aqueous solubility of sn-38 without modification of the drug . this polymer micelle ( termed it-141 ) was evaluated for pharmacokinetics and antitumor activity compared to irinotecan . the data reported herein support it-141 as a promising new antineoplastic agent for the treatment of colorectal cancer . azido - poly(ethylene glycol)-t - butyl carbonate - amine ( n3-peg - nh - boc ) was prepared as described previously . n - carboxy anhydrides ( ncas ) were prepared according to previously published procedures [ 30 , 31 ] . n3-peg12k - nh - boc ( 150 g , 12.5 mmol ) was dissolved into 1 l of ch2cl2/difluoracetic acid ( dfa ) ( 70/30 ) and was allowed to stir at room temperature overnight . the product was precipitated twice in diethyl ether and was recovered as a white powder ( yield 90% ) : h nmr ( d6-dmso ) 7.77 ( 3h ) , 5.97 ( 1h ) , 3.833.21 ( 1050 h ) , 2.98 ( 2h ) ppm . n3-peg10k - nh3/dfa ( 95 g , 7.92 mmol ) was weighed into an oven - dried , 2 l - round - bottom flask and was left under vacuum for three hours before adding the nca . asp(obu ) nca ( 17.04 g , 79.2 mmol ) was added to the flask ; the flask was evacuated under reduced pressure , and subsequently backfilled with nitrogen gas . dry n - methylpyrrolidone ( nmp ) ( 560 ml ) was introduced by cannula , and the solution was heated to 60c . the reaction mixture was allowed to stir for 24 hours at 60c under nitrogen gas . then , d - leu nca ( 24.88 g , 158 mmol ) and tyr ( obzl ) nca ( 47.08 g , 158 mmol ) were dissolved under nitrogen gas into 360 ml of nmp into an oven - dried , round bottom flask , and the mixture was subsequently added to the polymerization reaction via a syringe . the solution was allowed to stir at 60c for another three days at which point the reaction was complete ( as determined by hplc ) . the solution was cooled to room temperature , and diisopropylethylamine ( dipea ) ( 10 ml ) , dimethylaminopyridine ( dmap ) ( 100 mg ) , and acetic anhydride ( 10 ml ) were added . the polymer was precipitated into diethyl ether ( 10 l ) and isolated by filtration . the solid was redissolved in dichloromethane ( 500 ml ) and precipitated into diethyl ether ( 10 l ) . the product was isolated by filtration and dried in vacuo to give the block copolymer as an off - white powder ( 134.6 g , yield = 73% ) : h nmr ( d6-dmso ) 8.437.62 ( 50h ) , 7.35 ( 100h ) , 7.1 ( 40h ) , 6.82 ( 40h ) , 4.96 ( 40h ) , 4.633.99 ( 50h ) , 3.743.2 ( 1500h ) , 3.062.6 ( 60h ) , 1.36 ( 90h ) , 1.270.47 ( 180 ) . n3-peg12 k - b - poly(asp(obu)10)-b - poly(tyr(obzl)20-co - d - leu20)-ac ( 134.6 g , 6.4 mmol ) was dissolved into 1000 ml of a solution of pentamethylbenzene ( pmb , 0.5 m ) in trifluoroacetic acid ( tfa ) . the solution was precipitated into a 10-fold excess of diethyl ether , and the solid was recovered by filtration . the polymer was redissolved into 800 ml of dichloromethane and precipitated into diethyl ether . an off - white polymer was obtained after drying the product overnight in vacuo ( 111.8 g , yield = 93% ) : h nmr ( d6-dmso ) 12.2 ( 10h ) , 9.1 ( 10h ) , 8.517.71 ( 50h ) , 6.96 ( 40h ) , 6.59 ( 40h ) , 4.693.96 ( 60h ) , 3.813.25 ( 1500h ) , 3.062.65 ( 60h ) , 1.00.43 ( 180 ) . h nmr ( d6-dmso ) 171.9 , 171 , 170.5 , 170.3 , 155.9 , 130.6 , 129.6 , 127.9 115.3 , 114.3 , 70.7 , 69.8 , 54.5 , 51.5 , 50 , 49.8 , 49.4 , 36.9 , 36 , 24.3 , 23.3 , 22.3 , 21.2 . ir ( atr ) 3290 , 2882 , 1733 , 1658 , 1342 , 1102 , 962 cm . sn-38-loaded micelles were prepared by dissolving 1 g of itp-101 in 200 ml of water and 100 mg of sn-38 in 8 ml of methanol and 16 ml of toluene . the water was mixed with a silverson lt4r shear mixer at 10,000 rpm at 4c , and the organic solution was added dropwise . the solution was mixed for 30 minutes , then the resulting emulsion gently stirred on a magnetic stir plate overnight , allowing the toluene to evaporate . the sn-38-loaded micelle solution was filtered through a 0.22 m pes filter , then lyophilized to give a slightly yellow powder . the hplc instrumentation consisted of a waters alliance separation module ( w2695 ) equipped with a lichrosphere select b ( 5 m ) , 250 4.6 mm column coupled with a waters multi - wavelength fluorescence detector ( w2475 ) with excitation at 355 nm and emission at 515 nm . mobile phase consisted of a 70 : 30 phosphate buffer ( 10 mm nah2po4 , 0.1% tea , ph 3.5)/acetonitrile . flow rate was isocratic at 0.8 ml / min . elution time for sn-38 was determined to be 11.6 minutes , while camptothecin internal standard was 4.2 minutes . particle sizes were determined using dynamic light scattering on a wyatt dynapro ( santa barbara , calif ) . micelle solutions were prepared at 1 mg / ml in filtered water and were centrifuged at 2,000 rpm to remove any dust prior to analysis . all cells were purchased from american type tissue collection ( atcc ) and maintained in the following media : rpmi 1640 with 10% fbs , 2 mm l - glutamine , and 100 units / ml penicillin / streptomycin ( lncap , pc-3 , mg-63 , bxpc-3 , mcf-7 , and bt-474 ) , dmem with 10% fbs , 2 mm l - glutamine and 100 units / ml penicillin / streptomycin ( mda - mb-453 , mda - mb-231 ) , f12k with 10% fbs , 2 mm l - glutamine and 100 units / ml penicillin / streptomycin ( a549 ) , and mccoy 's 5a with 10% fbs , 2 mm l - glutamine , and 100 units / ml penicillin / streptomycin ( ht-29 and hct116 ) . all media , fbs , and supplements were purchased from mediatech ( manassas , va ) or hyclone . female athymic nude mice weighing about 2025 g were obtained from charles river laboratories ( wilmington , mass ) . for assessing cytotoxicity , cancer cell lines the following day , when the cells were 50% confluent , the cells were treated with it-141 , free sn-38 , or irinotecan in complete growth medium . the drugs remained on the cells for 72 hours without media change . at this timepoint , cell viability was determined using the cell titer glo kit and measured using a luminescent plate reader ( bmg labtech , cary , nc ) . ht-29 cells were subcutaneously injected into the right flank of nude mice at a concentration of 5 million in 0.1 ml pbs . when the tumors were approximately 300 mm , mice were randomly divided into two groups of eight and injected with 30 mg / kg ( sn-38-equivalent ) of it-141 or 30 mg / kg irinotecan . injection occurred by a fast iv bolus into the tail vein in a volume of 0.2 ml . the delivery vehicle for it-141 was isotonic saline and acidified ( ph 3.5 ) isotonic saline for irinotecan . mouse blood was collected at timepoints of 5 minutes , 15 minutes , 1 hour , 4 hours , 12 hours , 24 hours , and 72 hours . plasma was processed for hplc analysis by protein precipitation in ice - cold , acidified methanol ( 10% perchloric acid / methanol ) with 100 ng / ml camptothecin as internal standard , at a ratio of 1 : 4 plasma to methanol . tumors were homogenized in 20 mm ammonium acetate , ph 3.5 and extracted in acidified methanol as described above . samples were vortexed for 10 minutes , centrifuged at 13,000 rpm for 10 minutes , and the supernatant was transferred to hplc vials for analysis . ht-29 cells were subcutaneously injected into the right flank of nude mice at a concentration of 5 million in 0.1 ml pbs . when the tumors were approximately 300 mm , mice were given both single and multidose ( q4d 3 , day 0 , 4 , 8) intravenous injections of it-141 at doses ranging from 1090 mg / kg . the mtd was defined as a dose that caused no greater than a 10% loss in body weight and no treatment - related deaths . ht-29 and hct-116 colon cancer cells were harvested and resuspended in sterile pbs at a concentration of 2 million ( ht-29 ) or 4 million ( hct-116 ) cells per 0.1 ml pbs and injected subcutaneously into the right flank of athymic nude mice . tumors were allowed to establish logarithmic growth ( 714 days ) , and the animals were randomly divided into six to eight mice per group . drug was administered by a fast bolus injection of 0.2 ml into the mouse tail vein on a schedule of q4d 3 . tumor volume was calculated according to the formula : v = ( short diameter)(long diameter)/2 . percent inhibition was calculated using the following formula : ( 1)100vgroupvgroup 0vctlvctl 0100 , where vgroup is the tumor volume on the final day of the study , vgroup 0 is the tumor volume of the group on day 0 , vctl is the tumor volume of the control group on the final day of the study , and vctl 0 is the tumor volume of the control group on day 0 . tumor regression was calculated using the following formula : ( 2 ) vgroup 0vgroup100100 , where vgroup is the tumor volume on the final day of the study and vgroup 0 is the tumor volume of the group on day 0 . statistical differences in tumor volume between groups were calculated using the student 's t - test using microsoft excel , whereby p < 0.05 was considered statistically significant . itp-101 is a triblock copolymer consisting of poly(ethylene glycol)-b - poly(aspartic acid)-b - poly(d - leucine - co - tyrosine ) . the hydrophobic amino acids provide a core region into which a hydrophobic drug can reside , and the amphiphilic peg block forms a protective corona around the micelle , giving the delivery system stealth - like properties to avoid protein opsonization and res uptake ( figure 1 ) . the use of both d and l stereoisomers of amino acids in the leucine / tyrosine core block disrupts the secondary structure of the polypeptide . replacing the rodlike helical nature of the polypeptide with the flexibility of a random coil the middle aspartic acid block allows for a hydrogen - bonding segment which can be further stabilized with the use of metal ions , an aspect that is not utilized for it-141 . it-141 was formulated using itp-101 with various concentrations of sn-38 , ranging from 1 to 14% ( w / w ) , achieving greater than 90% loading efficiency . formulations of it-141 reconstituted in water or saline resulted in a homogeneous solution free of precipitate for up to four days at room temperature , and the lyophilized powder is stable for months . following formulation , the aqueous solubility of sn-38 in it-141 was 30 mg / ml , which is about a 6,000-fold increase in solubility of sn-38 . . dynamic light scattering ( dls ) experiments demonstrated that the micelle size was approximately 130 nm , with a standard deviation of 6 nm . thus , the average size of it-141 falls within the desired range to avoid renal clearance ( above 20 nm ) and escape uptake by the res ( below 150 nm ) . zeta potential measurements from electrophoretic light scattering experiments demonstrated that the surface charge of the micelle is overall neutral , with a range of readings from 5 to 5 mv . the sensitivity of various cancer cell lines to free sn-38 , it-141 , and irinotecan was compared in a cytotoxicity assay . as shown in table 1 , both free sn-38 and it-141 were extremely potent , and the sensitivity of the cells to it-141 was similar to free sn-38 across the cell lines . certain cell lines ( pc-3 , mda - mb-231 , and bt-474 ) were insensitive to both free sn-38 and it-141 . to determine the mtd of it-141 , ht-29 tumor - bearing nude mice were given both single and multidose ( q4d 3 ) intravenous injections of it-141 . these studies demonstrated that the multidose mtd of it-141 in tumor - bearing animals was 45 mg / kg and single dose mtd was 60 mg / kg . using 30 mg / kg of it-141 as a safe dose , the pharmacokinetic ( pk ) profile and tumor accumulation of sn-38 delivered from it-141 then compared to irinotecan in nude mice bearing ht-29 tumors ( table 2 ) . mice receiving a single injection of 30 mg / kg it-141 achieved a significant improvement in sn-38 plasma concentration and exposure compared to 30 mg / kg of irinotecan ( figure 2(a ) , table 2 ) . the cmax for both groups was achieved by the first measured time point of 5 minutes , with > 200-fold higher sn-38 concentration in mice treated with it-141 ( 209 g / ml ) compared to irinotecan ( 1.0 g / ml ) . sn-38 exposure as measured by area under curve ( auc ) from irinotecan was 2.5 ghr / ml , while sn-38 exposure from it-141 was 13.8-fold greater at 34.6 ghr / ml . no data could be obtained for irinotecan plasma concentrations beyond 12 hours as the concentration fell below the limit of detection . the concentration of sn-38 in the tumor over time is plotted in figure 2(b ) . the tumor auc of it-141 was determined to be 16.4 gh / g , which was significantly higher than irinotecan at 1.9 gh / g . it-141 also had a 47-fold higher cmax in the tumor than irinotecan ( 9.4 g / ml versus 0.2 g / ml ) . based on the pharmacokinetic data , it was hypothesized that it-141 would show superior antitumor efficacy in colon cancer xenograft models compared to irinotecan . to test the antitumor efficacy of it-141 , ht-29 tumor - bearing mice were treated with either itp-101 alone at 300 mg / kg , irinotecan at 60 mg / kg , or it-141 at 30 mg / kg ( figure 3(a ) ) . treatment with irinotecan at 60 mg / kg , which is near its mtd on this dosing schedule , did not inhibit ht-29 tumor growth significantly compared to polymer alone [ 26 , 32 ] . however , treatment with it-141 at half the dose of irinotecan induced significant tumor regression by day 18 , ultimately resulting in complete inhibition of tumor growth compared to itp-101 control and 35% regression from initial tumor volume ( p = 0.002 ) . dose - ranging studies were then performed to determine if the antitumor efficacy of it-141 is dose dependent ( figure 3(b ) ) . ht-29 tumor - bearing mice were intravenously administered it-141 at doses of 1 , 5 , 10 , 15 , 30 , and 45 mg / kg via tail vein injection . treatment with 1 , 5 , or 10 mg / kg did not result in a statistically significant inhibition of tumor growth compared to control mice receiving only saline . by day 20 , treatment with 15 mg / kg it-141 resulted in a 54% inhibition of tumor growth , respectively , compared to mice treated with saline ( p = 0.028 ) . treatment with 30 and 45 mg / kg resulted in complete tumor growth inhibition compared to saline control , with tumor regression of 59 and 87% , respectively ( p = 0.005 for both ) . similar results were found using another colon cancer xenograft model , hct116 ( figure 3(c ) ) . in this model , a dose of 5 mg / kg resulted in a 59% inhibition of tumor growth ( p = 0.008 ) compared to the itp-101-treated group . treatment with it-141 at 15 and 30 mg / kg in this model resulted in complete inhibition of tumor growth compared to the itp-101 polymer control , with 15% and 51% regression , respectively ( p = 1.0 e and 8.1 ) . taken together , these data demonstrate that it-141 achieved significantly greater antitumor efficacy , compared to irinotecan , and dose - dependent tumor regression in two colorectal cancer xenograft models of colon cancer , with effective doses between 15 and 30 mg / kg . a final study was performed whereby it-141 formulations with different weight loadings of sn-38 were compared to each other . it-141 formulations were prepared with 11% ( it-141 - 11% ) and 4% ( it-141 - 4% ) sn-38 ( w / w ) , and equivalent doses of sn-38 were administered i.v . in an ht-29 colon cancer xenograft model ( figure 4 ) . there were no statistical differences in efficacy between the two formulations at either 30 mg / kg ( p = 0.292 ) , 15 mg / kg ( p = 0.119 ) , or 5 mg / kg ( p = 0.138 ) . these data demonstrate that the percent loading by weight of sn-38 into the micelles does not affect antitumor activity . in this report , a novel triblock copolymer was used to encapsulate and solubilize the hydrophobic drug , sn-38 , which is the active metabolite of irinotecan . although irinotecan is used in the clinic as a prodrug , its efficacy is reliant upon carboxylesterase enzymes localized in the liver and gastrointestinal tract for conversion to the active metabolite , sn-38 . irinotecan treatment is often followed by late - stage diarrhea with 24% grade 4 incidence and can require antidiarrheal premedication . this limits the dose of irinotecan that can be administered safely in subsequent administrations , thereby reducing response rates in these patients [ 34 , 35 ] . sn-38 is a potent cytotoxic compound that , by itself , can not be used in the clinic due to its extreme hydrophobicity . have effectively addressed the solubility problem of sn-38 by conjugating sn-38 to peg - poly(glutamic acid ) , forming a micelle called nk012 , which is currently in clinical trials . other nanocarriers for sn-38 have been developed involving conjugation of sn-38 to a polymer or peptide [ 24 , 25 ] . as an alternative approach to direct sn-38 conjugation , a novel triblock copolymer was used to encapsulate sn-38 into a polymer micelle , precluding the need to modify the drug and for cleavage of the bond to release the active drug . the itp-101 triblock copolymer was developed to efficiently encapsulate hydrophobic molecules and release them at the site of disease ( in the tumor ) without drug conjugation . encapsulation of sn-38 to create it-141 resulted in a 6,000-fold increase in solubility of sn-38 and a micelle size of 130 nm , which is ideal for accumulation in tumors due to the epr effect . in vitro , it-141 was found to possess potent cytotoxic activity , which was similar to that of free sn-38 but several fold more potent than irinotecan . cell lines that were resistant to killing by it-141 were also resistant to free sn-38 , which may indicate a natural insensitivity of these cell lines to inhibition of topoisomerase i. this could arise through alterations in the expression of , or mutations in , the gene encoding topoisomerase i or the activity of drug efflux pumps . it has been shown that the drug efflux pump abcg2 is overexpressed in cells resistant to sn-38 . the pharmacokinetic profile of it-141 demonstrated significant improvement in exposure and cmax for sn-38 , with a modest improvement in half - life , compared to sn-38 derived from irinotecan . importantly , the plasma auc from it-141 exposure was 14-fold higher than the sn-38 exposure from irinotecan administered at the same dose ( 34.6 ghr / ml versus 2.5 ghr / ml ) . similarly , it-141 demonstrated higher exposure in ht-29 tumors , as measured by auc , than irinotecan . the higher auc of it-141 in the tumor indicated that it would potentially be more efficacious than irinotecan in xenograft models . indeed , it-141 was found to be superior to irinotecan in an ht-29 xenograft model and was potent in dose - range finding studies in both ht-29 and hct-116 xenografts . in both models , tumor regression was observed at 30 mg / kg in the ht-29 model and 15 mg / kg in the hct116 model . during the development of it-141 , it was found that it-141 could be formulated with sn-38 with weight loadings in the range of 114% . different it-141 formulations were prepared with varying weight loadings of sn-38 and were evaluated in an ht-29 xenograft experiment . it was found that it-141 - 4% w / w had equivalent antitumor activity to it-141 - 11% w / w , demonstrating no differences in efficacy between these formulations . it can be speculated , therefore , that despite sn-38 loading differences between the micelle , equivalent or similar overall concentrations of sn-38 are being delivered to these tumors . in summary , it-141 is a novel sn-38-loaded polymer micelle with superior pharmacokinetics and antitumor activity compared to irinotecan . although irinotecan is effective in the clinic , the ability to deliver sn-38 could be a superior treatment option for many patients . it-141 increased the solubility of sn-38 by 6,000-fold and had a diameter of 130 nm . it-141 demonstrated superior pharmacokinetics to irinotecan and potent antitumor activity in ht-29 and hct-116 colorectal cancer xenograft models . in summary , it-141 is a promising new therapeutic agent for colorectal cancer that warrants clinical investigation .

polymer micelles are promising drug delivery vehicles for the delivery of anticancer agents to tumors . often , anticancer drugs display potent cytotoxic effects towards cancer cells but are too hydrophobic to be administered in the clinic as a free drug . to address this problem , a polymer micelle was designed using a triblock copolymer ( itp-101 ) that enables hydrophobic drugs to be encapsulated . an sn-38 encapsulated micelle , it-141 , was prepared that exhibited potent in vitro cytotoxicity against a wide array of cancer cell lines . in a mouse model , pharmacokinetic analysis revealed that it-141 had a much longer circulation time , plasma exposure , and tumor exposure compared to irinotecan . it-141 was also superior to irinotecan in terms of antitumor activity , exhibiting greater tumor inhibition in ht-29 and hct116 colorectal cancer xenograft models at half the dose of irinotecan . the antitumor effect of it-141 was dose - dependent and caused complete growth inhibition and tumor regression at well - tolerated doses . varying the specific concentration of sn-38 within the it-141 micelle had no detectible effect on this antitumor activity , indicating no differences in activity between different it-141 formulations . in summary , it-141 is a potent micelle - based chemotherapy that holds promise for the treatment of colorectal cancer .

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

chemotherapy is an important treatment option for patients with cancer , however chemotherapy drugs suffer from numerous problems including nonspecific uptake by healthy tissue , poor circulation times , and suboptimal accumulation in the tumor . often , a large percentage of cytotoxic drug administered to the patient does not reach the tumor environment , but rather is distributed throughout the body , resulting in the many toxic effects associated with chemotherapy and a narrowing of the drug 's therapeutic window . the delivery of chemotherapeutic drugs to tumors is still a major hurdle in the eradication of cancer , and the continual development of drug delivery technologies is vital to future breakthroughs in chemotherapy . due to these promising aspects , a number of groups have developed various polymer micelle motifs , encapsulating a wide range of therapeutic classes [ 517 ] . irinotecan , a topoisomerase i inhibitor , is approved in the clinic for colorectal cancer first - line therapy in combination with 5-fluorouracil / leucovorin / oxaliplatin ( folfox ) regimen or for monotherapy in second - line therapy following a failed folfox regimen . studies in humans have shown that only three to four percent of the administered irinotecan is actually converted to sn-38 , which is reliant upon activating carboxylesterase enzymes localized in the liver and gastrointestinal tract . in addition , up to 95% of sn-38 is bound to circulating proteins such as albumin , which drastically reduces its bioavailability . irinotecan treatment also is accompanied by dose - limiting toxicities of grade 3 and 4 diarrhea and neutropenia . these limitations of irinotecan result in poor exposure of sn-38 to the tumor environment and severe side effects in the patient . a major limitation , however , of free sn-38 is that it is hydrophobic and is unable to be used as a free drug in the clinic . several groups have addressed the solubility problem of sn-38 by covalently attaching sn-38 to a polymer or peptide [ 2426 ] . in particular , a polymeric micellar formulation of sn-38 based on peo - poly ( glutamic acid ) block copolymers through chemical conjugation of sn-38 to the free carboxyl groups present on the poly ( glutamic acid ) backbone has been developed . while polymer - drug conjugates effectively address solubility of hydrophobic drugs , this prodrug approach is dependent on enzymatic or chemical cleavage of the bond to release the active drug . to develop an encapsulated formulation of sn-38 , sn-38 was loaded into a polymer micelle , resulting in aqueous solubility of sn-38 without modification of the drug . this polymer micelle ( termed it-141 ) was evaluated for pharmacokinetics and antitumor activity compared to irinotecan . the data reported herein support it-141 as a promising new antineoplastic agent for the treatment of colorectal cancer . azido - poly(ethylene glycol)-t - butyl carbonate - amine ( n3-peg - nh - boc ) was prepared as described previously . the product was precipitated twice in diethyl ether and was recovered as a white powder ( yield 90% ) : h nmr ( d6-dmso ) 7.77 ( 3h ) , 5.97 ( 1h ) , 3.833.21 ( 1050 h ) , 2.98 ( 2h ) ppm . asp(obu ) nca ( 17.04 g , 79.2 mmol ) was added to the flask ; the flask was evacuated under reduced pressure , and subsequently backfilled with nitrogen gas . sn-38-loaded micelles were prepared by dissolving 1 g of itp-101 in 200 ml of water and 100 mg of sn-38 in 8 ml of methanol and 16 ml of toluene . the water was mixed with a silverson lt4r shear mixer at 10,000 rpm at 4c , and the organic solution was added dropwise . elution time for sn-38 was determined to be 11.6 minutes , while camptothecin internal standard was 4.2 minutes . all cells were purchased from american type tissue collection ( atcc ) and maintained in the following media : rpmi 1640 with 10% fbs , 2 mm l - glutamine , and 100 units / ml penicillin / streptomycin ( lncap , pc-3 , mg-63 , bxpc-3 , mcf-7 , and bt-474 ) , dmem with 10% fbs , 2 mm l - glutamine and 100 units / ml penicillin / streptomycin ( mda - mb-453 , mda - mb-231 ) , f12k with 10% fbs , 2 mm l - glutamine and 100 units / ml penicillin / streptomycin ( a549 ) , and mccoy 's 5a with 10% fbs , 2 mm l - glutamine , and 100 units / ml penicillin / streptomycin ( ht-29 and hct116 ) . all media , fbs , and supplements were purchased from mediatech ( manassas , va ) or hyclone . for assessing cytotoxicity , cancer cell lines the following day , when the cells were 50% confluent , the cells were treated with it-141 , free sn-38 , or irinotecan in complete growth medium . at this timepoint , cell viability was determined using the cell titer glo kit and measured using a luminescent plate reader ( bmg labtech , cary , nc ) . ht-29 cells were subcutaneously injected into the right flank of nude mice at a concentration of 5 million in 0.1 ml pbs . when the tumors were approximately 300 mm , mice were randomly divided into two groups of eight and injected with 30 mg / kg ( sn-38-equivalent ) of it-141 or 30 mg / kg irinotecan . injection occurred by a fast iv bolus into the tail vein in a volume of 0.2 ml . the delivery vehicle for it-141 was isotonic saline and acidified ( ph 3.5 ) isotonic saline for irinotecan . mouse blood was collected at timepoints of 5 minutes , 15 minutes , 1 hour , 4 hours , 12 hours , 24 hours , and 72 hours . samples were vortexed for 10 minutes , centrifuged at 13,000 rpm for 10 minutes , and the supernatant was transferred to hplc vials for analysis . when the tumors were approximately 300 mm , mice were given both single and multidose ( q4d 3 , day 0 , 4 , 8) intravenous injections of it-141 at doses ranging from 1090 mg / kg . the mtd was defined as a dose that caused no greater than a 10% loss in body weight and no treatment - related deaths . ht-29 and hct-116 colon cancer cells were harvested and resuspended in sterile pbs at a concentration of 2 million ( ht-29 ) or 4 million ( hct-116 ) cells per 0.1 ml pbs and injected subcutaneously into the right flank of athymic nude mice . tumors were allowed to establish logarithmic growth ( 714 days ) , and the animals were randomly divided into six to eight mice per group . statistical differences in tumor volume between groups were calculated using the student 's t - test using microsoft excel , whereby p < 0.05 was considered statistically significant . itp-101 is a triblock copolymer consisting of poly(ethylene glycol)-b - poly(aspartic acid)-b - poly(d - leucine - co - tyrosine ) . the hydrophobic amino acids provide a core region into which a hydrophobic drug can reside , and the amphiphilic peg block forms a protective corona around the micelle , giving the delivery system stealth - like properties to avoid protein opsonization and res uptake ( figure 1 ) . the use of both d and l stereoisomers of amino acids in the leucine / tyrosine core block disrupts the secondary structure of the polypeptide . it-141 was formulated using itp-101 with various concentrations of sn-38 , ranging from 1 to 14% ( w / w ) , achieving greater than 90% loading efficiency . formulations of it-141 reconstituted in water or saline resulted in a homogeneous solution free of precipitate for up to four days at room temperature , and the lyophilized powder is stable for months . following formulation , the aqueous solubility of sn-38 in it-141 was 30 mg / ml , which is about a 6,000-fold increase in solubility of sn-38 . thus , the average size of it-141 falls within the desired range to avoid renal clearance ( above 20 nm ) and escape uptake by the res ( below 150 nm ) . the sensitivity of various cancer cell lines to free sn-38 , it-141 , and irinotecan was compared in a cytotoxicity assay . as shown in table 1 , both free sn-38 and it-141 were extremely potent , and the sensitivity of the cells to it-141 was similar to free sn-38 across the cell lines . certain cell lines ( pc-3 , mda - mb-231 , and bt-474 ) were insensitive to both free sn-38 and it-141 . to determine the mtd of it-141 , ht-29 tumor - bearing nude mice were given both single and multidose ( q4d 3 ) intravenous injections of it-141 . using 30 mg / kg of it-141 as a safe dose , the pharmacokinetic ( pk ) profile and tumor accumulation of sn-38 delivered from it-141 then compared to irinotecan in nude mice bearing ht-29 tumors ( table 2 ) . mice receiving a single injection of 30 mg / kg it-141 achieved a significant improvement in sn-38 plasma concentration and exposure compared to 30 mg / kg of irinotecan ( figure 2(a ) , table 2 ) . the cmax for both groups was achieved by the first measured time point of 5 minutes , with > 200-fold higher sn-38 concentration in mice treated with it-141 ( 209 g / ml ) compared to irinotecan ( 1.0 g / ml ) . sn-38 exposure as measured by area under curve ( auc ) from irinotecan was 2.5 ghr / ml , while sn-38 exposure from it-141 was 13.8-fold greater at 34.6 ghr / ml . the concentration of sn-38 in the tumor over time is plotted in figure 2(b ) . the tumor auc of it-141 was determined to be 16.4 gh / g , which was significantly higher than irinotecan at 1.9 gh / g . it-141 also had a 47-fold higher cmax in the tumor than irinotecan ( 9.4 g / ml versus 0.2 g / ml ) . based on the pharmacokinetic data , it was hypothesized that it-141 would show superior antitumor efficacy in colon cancer xenograft models compared to irinotecan . to test the antitumor efficacy of it-141 , ht-29 tumor - bearing mice were treated with either itp-101 alone at 300 mg / kg , irinotecan at 60 mg / kg , or it-141 at 30 mg / kg ( figure 3(a ) ) . treatment with irinotecan at 60 mg / kg , which is near its mtd on this dosing schedule , did not inhibit ht-29 tumor growth significantly compared to polymer alone [ 26 , 32 ] . however , treatment with it-141 at half the dose of irinotecan induced significant tumor regression by day 18 , ultimately resulting in complete inhibition of tumor growth compared to itp-101 control and 35% regression from initial tumor volume ( p = 0.002 ) . dose - ranging studies were then performed to determine if the antitumor efficacy of it-141 is dose dependent ( figure 3(b ) ) . treatment with 1 , 5 , or 10 mg / kg did not result in a statistically significant inhibition of tumor growth compared to control mice receiving only saline . by day 20 , treatment with 15 mg / kg it-141 resulted in a 54% inhibition of tumor growth , respectively , compared to mice treated with saline ( p = 0.028 ) . treatment with 30 and 45 mg / kg resulted in complete tumor growth inhibition compared to saline control , with tumor regression of 59 and 87% , respectively ( p = 0.005 for both ) . similar results were found using another colon cancer xenograft model , hct116 ( figure 3(c ) ) . in this model , a dose of 5 mg / kg resulted in a 59% inhibition of tumor growth ( p = 0.008 ) compared to the itp-101-treated group . treatment with it-141 at 15 and 30 mg / kg in this model resulted in complete inhibition of tumor growth compared to the itp-101 polymer control , with 15% and 51% regression , respectively ( p = 1.0 e and 8.1 ) . taken together , these data demonstrate that it-141 achieved significantly greater antitumor efficacy , compared to irinotecan , and dose - dependent tumor regression in two colorectal cancer xenograft models of colon cancer , with effective doses between 15 and 30 mg / kg . a final study was performed whereby it-141 formulations with different weight loadings of sn-38 were compared to each other . it-141 formulations were prepared with 11% ( it-141 - 11% ) and 4% ( it-141 - 4% ) sn-38 ( w / w ) , and equivalent doses of sn-38 were administered i.v . there were no statistical differences in efficacy between the two formulations at either 30 mg / kg ( p = 0.292 ) , 15 mg / kg ( p = 0.119 ) , or 5 mg / kg ( p = 0.138 ) . these data demonstrate that the percent loading by weight of sn-38 into the micelles does not affect antitumor activity . in this report , a novel triblock copolymer was used to encapsulate and solubilize the hydrophobic drug , sn-38 , which is the active metabolite of irinotecan . although irinotecan is used in the clinic as a prodrug , its efficacy is reliant upon carboxylesterase enzymes localized in the liver and gastrointestinal tract for conversion to the active metabolite , sn-38 . this limits the dose of irinotecan that can be administered safely in subsequent administrations , thereby reducing response rates in these patients [ 34 , 35 ] . sn-38 is a potent cytotoxic compound that , by itself , can not be used in the clinic due to its extreme hydrophobicity . have effectively addressed the solubility problem of sn-38 by conjugating sn-38 to peg - poly(glutamic acid ) , forming a micelle called nk012 , which is currently in clinical trials . other nanocarriers for sn-38 have been developed involving conjugation of sn-38 to a polymer or peptide [ 24 , 25 ] . as an alternative approach to direct sn-38 conjugation , a novel triblock copolymer was used to encapsulate sn-38 into a polymer micelle , precluding the need to modify the drug and for cleavage of the bond to release the active drug . the itp-101 triblock copolymer was developed to efficiently encapsulate hydrophobic molecules and release them at the site of disease ( in the tumor ) without drug conjugation . encapsulation of sn-38 to create it-141 resulted in a 6,000-fold increase in solubility of sn-38 and a micelle size of 130 nm , which is ideal for accumulation in tumors due to the epr effect . in vitro , it-141 was found to possess potent cytotoxic activity , which was similar to that of free sn-38 but several fold more potent than irinotecan . cell lines that were resistant to killing by it-141 were also resistant to free sn-38 , which may indicate a natural insensitivity of these cell lines to inhibition of topoisomerase i. this could arise through alterations in the expression of , or mutations in , the gene encoding topoisomerase i or the activity of drug efflux pumps . the pharmacokinetic profile of it-141 demonstrated significant improvement in exposure and cmax for sn-38 , with a modest improvement in half - life , compared to sn-38 derived from irinotecan . similarly , it-141 demonstrated higher exposure in ht-29 tumors , as measured by auc , than irinotecan . the higher auc of it-141 in the tumor indicated that it would potentially be more efficacious than irinotecan in xenograft models . indeed , it-141 was found to be superior to irinotecan in an ht-29 xenograft model and was potent in dose - range finding studies in both ht-29 and hct-116 xenografts . in both models , tumor regression was observed at 30 mg / kg in the ht-29 model and 15 mg / kg in the hct116 model . during the development of it-141 , it was found that it-141 could be formulated with sn-38 with weight loadings in the range of 114% . different it-141 formulations were prepared with varying weight loadings of sn-38 and were evaluated in an ht-29 xenograft experiment . it was found that it-141 - 4% w / w had equivalent antitumor activity to it-141 - 11% w / w , demonstrating no differences in efficacy between these formulations . it can be speculated , therefore , that despite sn-38 loading differences between the micelle , equivalent or similar overall concentrations of sn-38 are being delivered to these tumors . in summary , it-141 is a novel sn-38-loaded polymer micelle with superior pharmacokinetics and antitumor activity compared to irinotecan . although irinotecan is effective in the clinic , the ability to deliver sn-38 could be a superior treatment option for many patients . it-141 increased the solubility of sn-38 by 6,000-fold and had a diameter of 130 nm . it-141 demonstrated superior pharmacokinetics to irinotecan and potent antitumor activity in ht-29 and hct-116 colorectal cancer xenograft models . in summary , it-141 is a promising new therapeutic agent for colorectal cancer that warrants clinical investigation .

chemotherapy is an important treatment option for patients with cancer , however chemotherapy drugs suffer from numerous problems including nonspecific uptake by healthy tissue , poor circulation times , and suboptimal accumulation in the tumor . often , a large percentage of cytotoxic drug administered to the patient does not reach the tumor environment , but rather is distributed throughout the body , resulting in the many toxic effects associated with chemotherapy and a narrowing of the drug 's therapeutic window . the delivery of chemotherapeutic drugs to tumors is still a major hurdle in the eradication of cancer , and the continual development of drug delivery technologies is vital to future breakthroughs in chemotherapy . polymer micelles offer a promising approach to achieving these goals due to their inherent ability to overcome multiple biological barriers , such as avoidance of the reticuloendothelial system ( res ) . the epr effect is a consequence of the disorganized nature of the tumor vasculature , which results in increased permeability of polymer therapeutics and drug retention at the tumor site . due to these promising aspects , a number of groups have developed various polymer micelle motifs , encapsulating a wide range of therapeutic classes [ 517 ] . colon cancer is the third most common cancer in men and women in most of the developed world . irinotecan , a topoisomerase i inhibitor , is approved in the clinic for colorectal cancer first - line therapy in combination with 5-fluorouracil / leucovorin / oxaliplatin ( folfox ) regimen or for monotherapy in second - line therapy following a failed folfox regimen . sn-38 , the active metabolite of irinotecan , is about 5001000 times more cytotoxic than irinotecan [ 1820 ] . although irinotecan has demonstrated clinical utility , it is highly inefficient in delivering active sn-38 to tumor tissue . studies in humans have shown that only three to four percent of the administered irinotecan is actually converted to sn-38 , which is reliant upon activating carboxylesterase enzymes localized in the liver and gastrointestinal tract . in addition , up to 95% of sn-38 is bound to circulating proteins such as albumin , which drastically reduces its bioavailability . irinotecan treatment also is accompanied by dose - limiting toxicities of grade 3 and 4 diarrhea and neutropenia . these limitations of irinotecan result in poor exposure of sn-38 to the tumor environment and severe side effects in the patient . a major limitation , however , of free sn-38 is that it is hydrophobic and is unable to be used as a free drug in the clinic . several groups have addressed the solubility problem of sn-38 by covalently attaching sn-38 to a polymer or peptide [ 2426 ] . in particular , a polymeric micellar formulation of sn-38 based on peo - poly ( glutamic acid ) block copolymers through chemical conjugation of sn-38 to the free carboxyl groups present on the poly ( glutamic acid ) backbone has been developed . this formulation , known as nk012 , as well as a peglyated sn-38 formulation ( ezn-2208 ) , is currently in clinical trials [ 27 , 28 ] . while polymer - drug conjugates effectively address solubility of hydrophobic drugs , this prodrug approach is dependent on enzymatic or chemical cleavage of the bond to release the active drug . to develop an encapsulated formulation of sn-38 , sn-38 was loaded into a polymer micelle , resulting in aqueous solubility of sn-38 without modification of the drug . this polymer micelle ( termed it-141 ) was evaluated for pharmacokinetics and antitumor activity compared to irinotecan . the data reported herein support it-141 as a promising new antineoplastic agent for the treatment of colorectal cancer . n3-peg10k - nh3/dfa ( 95 g , 7.92 mmol ) was weighed into an oven - dried , 2 l - round - bottom flask and was left under vacuum for three hours before adding the nca . then , d - leu nca ( 24.88 g , 158 mmol ) and tyr ( obzl ) nca ( 47.08 g , 158 mmol ) were dissolved under nitrogen gas into 360 ml of nmp into an oven - dried , round bottom flask , and the mixture was subsequently added to the polymerization reaction via a syringe . the product was isolated by filtration and dried in vacuo to give the block copolymer as an off - white powder ( 134.6 g , yield = 73% ) : h nmr ( d6-dmso ) 8.437.62 ( 50h ) , 7.35 ( 100h ) , 7.1 ( 40h ) , 6.82 ( 40h ) , 4.96 ( 40h ) , 4.633.99 ( 50h ) , 3.743.2 ( 1500h ) , 3.062.6 ( 60h ) , 1.36 ( 90h ) , 1.270.47 ( 180 ) . n3-peg12 k - b - poly(asp(obu)10)-b - poly(tyr(obzl)20-co - d - leu20)-ac ( 134.6 g , 6.4 mmol ) was dissolved into 1000 ml of a solution of pentamethylbenzene ( pmb , 0.5 m ) in trifluoroacetic acid ( tfa ) . an off - white polymer was obtained after drying the product overnight in vacuo ( 111.8 g , yield = 93% ) : h nmr ( d6-dmso ) 12.2 ( 10h ) , 9.1 ( 10h ) , 8.517.71 ( 50h ) , 6.96 ( 40h ) , 6.59 ( 40h ) , 4.693.96 ( 60h ) , 3.813.25 ( 1500h ) , 3.062.65 ( 60h ) , 1.00.43 ( 180 ) . sn-38-loaded micelles were prepared by dissolving 1 g of itp-101 in 200 ml of water and 100 mg of sn-38 in 8 ml of methanol and 16 ml of toluene . the hplc instrumentation consisted of a waters alliance separation module ( w2695 ) equipped with a lichrosphere select b ( 5 m ) , 250 4.6 mm column coupled with a waters multi - wavelength fluorescence detector ( w2475 ) with excitation at 355 nm and emission at 515 nm . all cells were purchased from american type tissue collection ( atcc ) and maintained in the following media : rpmi 1640 with 10% fbs , 2 mm l - glutamine , and 100 units / ml penicillin / streptomycin ( lncap , pc-3 , mg-63 , bxpc-3 , mcf-7 , and bt-474 ) , dmem with 10% fbs , 2 mm l - glutamine and 100 units / ml penicillin / streptomycin ( mda - mb-453 , mda - mb-231 ) , f12k with 10% fbs , 2 mm l - glutamine and 100 units / ml penicillin / streptomycin ( a549 ) , and mccoy 's 5a with 10% fbs , 2 mm l - glutamine , and 100 units / ml penicillin / streptomycin ( ht-29 and hct116 ) . for assessing cytotoxicity , cancer cell lines the following day , when the cells were 50% confluent , the cells were treated with it-141 , free sn-38 , or irinotecan in complete growth medium . when the tumors were approximately 300 mm , mice were randomly divided into two groups of eight and injected with 30 mg / kg ( sn-38-equivalent ) of it-141 or 30 mg / kg irinotecan . plasma was processed for hplc analysis by protein precipitation in ice - cold , acidified methanol ( 10% perchloric acid / methanol ) with 100 ng / ml camptothecin as internal standard , at a ratio of 1 : 4 plasma to methanol . the mtd was defined as a dose that caused no greater than a 10% loss in body weight and no treatment - related deaths . ht-29 and hct-116 colon cancer cells were harvested and resuspended in sterile pbs at a concentration of 2 million ( ht-29 ) or 4 million ( hct-116 ) cells per 0.1 ml pbs and injected subcutaneously into the right flank of athymic nude mice . tumors were allowed to establish logarithmic growth ( 714 days ) , and the animals were randomly divided into six to eight mice per group . percent inhibition was calculated using the following formula : ( 1)100vgroupvgroup 0vctlvctl 0100 , where vgroup is the tumor volume on the final day of the study , vgroup 0 is the tumor volume of the group on day 0 , vctl is the tumor volume of the control group on the final day of the study , and vctl 0 is the tumor volume of the control group on day 0 . tumor regression was calculated using the following formula : ( 2 ) vgroup 0vgroup100100 , where vgroup is the tumor volume on the final day of the study and vgroup 0 is the tumor volume of the group on day 0 . itp-101 is a triblock copolymer consisting of poly(ethylene glycol)-b - poly(aspartic acid)-b - poly(d - leucine - co - tyrosine ) . the hydrophobic amino acids provide a core region into which a hydrophobic drug can reside , and the amphiphilic peg block forms a protective corona around the micelle , giving the delivery system stealth - like properties to avoid protein opsonization and res uptake ( figure 1 ) . the use of both d and l stereoisomers of amino acids in the leucine / tyrosine core block disrupts the secondary structure of the polypeptide . replacing the rodlike helical nature of the polypeptide with the flexibility of a random coil the middle aspartic acid block allows for a hydrogen - bonding segment which can be further stabilized with the use of metal ions , an aspect that is not utilized for it-141 . it-141 was formulated using itp-101 with various concentrations of sn-38 , ranging from 1 to 14% ( w / w ) , achieving greater than 90% loading efficiency . formulations of it-141 reconstituted in water or saline resulted in a homogeneous solution free of precipitate for up to four days at room temperature , and the lyophilized powder is stable for months . following formulation , the aqueous solubility of sn-38 in it-141 was 30 mg / ml , which is about a 6,000-fold increase in solubility of sn-38 . thus , the average size of it-141 falls within the desired range to avoid renal clearance ( above 20 nm ) and escape uptake by the res ( below 150 nm ) . the sensitivity of various cancer cell lines to free sn-38 , it-141 , and irinotecan was compared in a cytotoxicity assay . as shown in table 1 , both free sn-38 and it-141 were extremely potent , and the sensitivity of the cells to it-141 was similar to free sn-38 across the cell lines . certain cell lines ( pc-3 , mda - mb-231 , and bt-474 ) were insensitive to both free sn-38 and it-141 . to determine the mtd of it-141 , ht-29 tumor - bearing nude mice were given both single and multidose ( q4d 3 ) intravenous injections of it-141 . these studies demonstrated that the multidose mtd of it-141 in tumor - bearing animals was 45 mg / kg and single dose mtd was 60 mg / kg . using 30 mg / kg of it-141 as a safe dose , the pharmacokinetic ( pk ) profile and tumor accumulation of sn-38 delivered from it-141 then compared to irinotecan in nude mice bearing ht-29 tumors ( table 2 ) . mice receiving a single injection of 30 mg / kg it-141 achieved a significant improvement in sn-38 plasma concentration and exposure compared to 30 mg / kg of irinotecan ( figure 2(a ) , table 2 ) . the cmax for both groups was achieved by the first measured time point of 5 minutes , with > 200-fold higher sn-38 concentration in mice treated with it-141 ( 209 g / ml ) compared to irinotecan ( 1.0 g / ml ) . sn-38 exposure as measured by area under curve ( auc ) from irinotecan was 2.5 ghr / ml , while sn-38 exposure from it-141 was 13.8-fold greater at 34.6 ghr / ml . the tumor auc of it-141 was determined to be 16.4 gh / g , which was significantly higher than irinotecan at 1.9 gh / g . based on the pharmacokinetic data , it was hypothesized that it-141 would show superior antitumor efficacy in colon cancer xenograft models compared to irinotecan . to test the antitumor efficacy of it-141 , ht-29 tumor - bearing mice were treated with either itp-101 alone at 300 mg / kg , irinotecan at 60 mg / kg , or it-141 at 30 mg / kg ( figure 3(a ) ) . treatment with irinotecan at 60 mg / kg , which is near its mtd on this dosing schedule , did not inhibit ht-29 tumor growth significantly compared to polymer alone [ 26 , 32 ] . however , treatment with it-141 at half the dose of irinotecan induced significant tumor regression by day 18 , ultimately resulting in complete inhibition of tumor growth compared to itp-101 control and 35% regression from initial tumor volume ( p = 0.002 ) . dose - ranging studies were then performed to determine if the antitumor efficacy of it-141 is dose dependent ( figure 3(b ) ) . treatment with 1 , 5 , or 10 mg / kg did not result in a statistically significant inhibition of tumor growth compared to control mice receiving only saline . by day 20 , treatment with 15 mg / kg it-141 resulted in a 54% inhibition of tumor growth , respectively , compared to mice treated with saline ( p = 0.028 ) . treatment with 30 and 45 mg / kg resulted in complete tumor growth inhibition compared to saline control , with tumor regression of 59 and 87% , respectively ( p = 0.005 for both ) . in this model , a dose of 5 mg / kg resulted in a 59% inhibition of tumor growth ( p = 0.008 ) compared to the itp-101-treated group . treatment with it-141 at 15 and 30 mg / kg in this model resulted in complete inhibition of tumor growth compared to the itp-101 polymer control , with 15% and 51% regression , respectively ( p = 1.0 e and 8.1 ) . taken together , these data demonstrate that it-141 achieved significantly greater antitumor efficacy , compared to irinotecan , and dose - dependent tumor regression in two colorectal cancer xenograft models of colon cancer , with effective doses between 15 and 30 mg / kg . it-141 formulations were prepared with 11% ( it-141 - 11% ) and 4% ( it-141 - 4% ) sn-38 ( w / w ) , and equivalent doses of sn-38 were administered i.v . there were no statistical differences in efficacy between the two formulations at either 30 mg / kg ( p = 0.292 ) , 15 mg / kg ( p = 0.119 ) , or 5 mg / kg ( p = 0.138 ) . these data demonstrate that the percent loading by weight of sn-38 into the micelles does not affect antitumor activity . in this report , a novel triblock copolymer was used to encapsulate and solubilize the hydrophobic drug , sn-38 , which is the active metabolite of irinotecan . although irinotecan is used in the clinic as a prodrug , its efficacy is reliant upon carboxylesterase enzymes localized in the liver and gastrointestinal tract for conversion to the active metabolite , sn-38 . as an alternative approach to direct sn-38 conjugation , a novel triblock copolymer was used to encapsulate sn-38 into a polymer micelle , precluding the need to modify the drug and for cleavage of the bond to release the active drug . the itp-101 triblock copolymer was developed to efficiently encapsulate hydrophobic molecules and release them at the site of disease ( in the tumor ) without drug conjugation . encapsulation of sn-38 to create it-141 resulted in a 6,000-fold increase in solubility of sn-38 and a micelle size of 130 nm , which is ideal for accumulation in tumors due to the epr effect . in vitro , it-141 was found to possess potent cytotoxic activity , which was similar to that of free sn-38 but several fold more potent than irinotecan . cell lines that were resistant to killing by it-141 were also resistant to free sn-38 , which may indicate a natural insensitivity of these cell lines to inhibition of topoisomerase i. this could arise through alterations in the expression of , or mutations in , the gene encoding topoisomerase i or the activity of drug efflux pumps . the pharmacokinetic profile of it-141 demonstrated significant improvement in exposure and cmax for sn-38 , with a modest improvement in half - life , compared to sn-38 derived from irinotecan . importantly , the plasma auc from it-141 exposure was 14-fold higher than the sn-38 exposure from irinotecan administered at the same dose ( 34.6 ghr / ml versus 2.5 ghr / ml ) . indeed , it-141 was found to be superior to irinotecan in an ht-29 xenograft model and was potent in dose - range finding studies in both ht-29 and hct-116 xenografts . in both models , tumor regression was observed at 30 mg / kg in the ht-29 model and 15 mg / kg in the hct116 model . in summary , it-141 is a novel sn-38-loaded polymer micelle with superior pharmacokinetics and antitumor activity compared to irinotecan .

skin aging is a complex process that affects all its layers and structure and changes the functional properties of the intracellular matrix . a wide range of biomimetic peptides with different mechanisms of action can be applied to solve this problem . by imitating the action of naturally occurring growth factors and cytokines , they are able to bind to the specific receptors , regulate gene transcription , and provide a stimulating effect on keratinocytes and fibroblasts . however , the target genes of different peptides are different ; hence , the maximum effect is achieved only by the combined application of several biomimetic peptides.1 caregen co. , ltd . ( anyang - si , gyeonggi - do , south korea ) has developed biomimetic peptides based on extensive studies of growth factors since 2002 . the term biomimetic peptide as used herein relates to a synthetic agonist of naturally occurring growth factors and completely mimics the action of the parental molecules . the biomimetic peptides are oligopeptides consisting of ten to 15 amino acids and can provide clinical benefits similar to recombinant growth factors , reduce costs , and have greater chemical stability . in the production system uses a unique mass production of growth factors provided by transformed bacteria ( escherichia coli system ) , containing synthetic human genes . double - layered encapsulation technology improves penetration of active ingredients into skin and protects the molecules from endogenous proteases , which provides higher efficacy.2 biomimetic peptides have a wide range of applications such as increasing the efficacies of any cosmetic and meso - formulations , including dermal fillers , in antiaging , antipigmentation , hair growth , body fat reduction , and many inflammation - related products , which are also applicable for pharmaceutical purposes.3,4 in addition , in the recent decade , an interest in synthetic oligopeptides and their use in diagnosis and treatment of different pathologies has increased dramatically ; for example , investigation devoted to influence of short peptides on endothelial growth5 or protection of kidney during the cisplatin - induced acute renal failure.6 in this study , we identified several molecules associated with aging and skin regeneration , such as ki-67 , type i procollagen , sirt6 , and ap-1 . in aging , the number , quality , and quantity of collagen fibers are modified ; they become thin , lose their clear organization , and are resorbed . matrix metalloproteinases ( mmp-1 , mmp-2 , and mmp-3 ) are key signaling molecules of collagen expression . many studies have shown that their activity is regulated by ap-1.79 in parallel , with the degradation of the existing collagen , the activation of factor ap-1 is accompanied with a decrease in procollagen types i and iii . ap-1 regulates a number of cellular processes , including differentiation , proliferation , and apoptosis.10 there is a decrease in the proliferative activity of fibroblasts during aging . using the cultivation method , it was shown that fibroblasts of young donors are characterized by twice as many mitoses , whereby one cell ( there are 60% of such type of cells ) is able to form a colony of 256 fibroblasts , while in the case of older donors , only 2% of cells form colonies of similar volume.11 the hayflick phenomenon can be a substantiation of fewer cell divisions , whereby somatic cells without telomerase expression are capable , on average , of only 50 doublings of population , but fibroblasts of older donors have already passed a number of cell cycles prior to isolation in vitro.12 there is a correlation between donor age and the replicative lifespan of human cells in culture.13 for accuracy , it is better to investigate one serially cultured sample at different periods of time than to examine several different cultured samples.14 the protein ki-67 is present in all active phases of the cell cycle ( g1 , s , g2 , and mitosis ) , and it is a recognized marker for determining the growth of the cell population . sirt6 is a recently identified factor associated with skin aging . dna repair and control of proliferation are its main functions in the skin , and this protein also participates in inflammation processes . thus , hyperexpression of sirt6 leads to increase in tnf- synthesis through posttranscriptional mechanisms.15 sirt6 modulates telomeric chromatin by lys-9 deacetylation of histone h3 and thus prevents telomeric dysfunction and early cellular aging.16 the aim of this study was to investigate the influence of biomimetic peptides on the reparation processes in the dermis by the cell culture model in vitro and in vivo . in the human study , we investigated the effect of revofil aquashine ( aq ) that possesses all physical , structural , and biocompatibility properties that are required of a superior gel for use in skin restoring and has a encumbrance certificate . during the clinical study done for encumbrance certification , no major side effects requiring medical attention were observed . aq contains biomimetic peptides ( cg - cgc2 , cg - cgc3 , rejuline , boostrin ) , free hyaluronic acid , amino acids , multivitamins , and minerals . aq was administered to the patients ( n=5 , age : 4548 years ) 1 month prior to the abdominoplasty by means of intradermal microinjections into skin of the right half of anterior abdominal wall below the navel ( in areas of localized fat deposits that are to be subsequently removed ) . the samples were received from caregen research center ( seoul , south korea ) . as a control , pure ( not containing biomimetic peptides ) hyaluronic acid was injected simultaneously into a symmetric area to the left . the treatment and study details were fully explained to the subjects , all of whom signed written informed consent forms pertaining to treatment and participation in this study , which included consent to injection of cosmeceutical product and skin biopsy . a total of 2 ml of aq was expected to hold a single procedure ; this volume was recommended by caregen . aq was injected to a depth of 46 mm ( depending on skin thickness ) and at an angle of 4590 to the surface of the skin . during the abdominoplasty , skin samples were taken from the injected areas of the abdomen and fixed in 10% formalin solution . the study was conducted with caregen research ethics committee approval and patients provided written informed consent . in the cell culture model , we investigated complex of biomimetic peptides that were part of fillers revofil aq and aq brightening ( br ) . the complex of biomimetic peptides was provided by the supplier in ampoules and was clear , colorless aqueous sterile solution . the subcultivation of normal human diploid fibroblasts as a model of aging was used in this study in vitro . the biopsy was taken from a middle - aged donor ( 42 years ) ; when the culture reach 7 passage , we classified it as a mature cells , meaning that all processes as energy exchange , cell growth , biosynthesis are in a highest stage , and passage 15 as a senescent culture , thus all the processes mentioned above regress . the senescent cell line was proved by staining for -galactosidase ( anti--galactosidase antibody ; abcam , cambridge , uk ) as a technique to label senescent cells . isolation of fibroblasts with enzymatic digestion was carried out using dispase ii ( thermo fisher scientific , waltham , ma , usa ) at the concentration of 2.4 iu / ml for 18 hours at 4c , and then the epidermis was mechanically separated from the dermis . to obtain a suspension of fibroblasts , the dermis was minced with scissors into pieces of 34 mm and placed in a solution of type i collagenase ( thermo fisher scientific ) in minimum essential medium ( biolot , saint - petersburg , russia ) for 30 minutes at 37c . the obtained culture was tested to specific molecular fibroblast marker vimentin ( antivimentin monoclonal antibody ; dako denmark a / s , glostrup , denmark ) , which confirmed that the investigated culture was skin fibroblasts . we studied three groups : 1 ) control group ( physiological solution ) ; 2 ) aq group with addition of biomimetic peptide components of aq ( acetyl decapeptide-3 [ rejuline ] , oligopeptide-24 [ cg - egp3 ] , oligopeptide-34 [ cg - tgp2 ] , oligopeptide-72 [ boostrin ] ) in concentration of 2 g / ml ; and 3 ) aq br group with addition of biomimetic peptide components of aq br ( oligopeptide-23 [ flatin ] , oligopeptide-34 [ cg - tgp2 ] , oligopeptide-51 [ purilux ] , oligopeptide-72 [ boostrin ] ) in concentration of 2 g / ml . at passages 7 and 15 , cells were seeded on coverslips ( 15 mm , thermo fisher scientific ) fixed with paraformaldehyde ( sigma - aldrich co. , st louis , mo , usa ) and incubated with antibodies . the confocal microscope olympus fv1000 ( olympus corporation , tokyo , japan ) was used for visualization of the nuclear markers ki-67 and sirt6 in fibroblast culture samples by applying secondary antibodies conjugated with a far red - emitting dye ( alexa fluor 647 ; abcam ) . in both studies ( in vivo and in vitro ) , we used primary monoclonal antibodies to markers ki-67 ( 1:75 ; dako denmark a / s ) , ap-1 ( 1:200 ; sigma - aldrich co. ) , type i procollagen ( 1:100 , lifespan biosciences , seattle , wa , usa ) , and sirt6 ( 1:200 , alexa fluor 647 ) and biotinylated anti - mouse immunoglobulins ( dako denmark a / s ) as secondary antibodies to provide the immunohistochemical reaction . visualization of the reaction was carried out using the envision detection system kit ( dako denmark a / s ) . in both cases , morphometric investigation was carried out by using a system of computer analysis of microscopic images , which includes a microscope olympus bx46 , a digital camera ( olympus corporation ) , and a personal computer based on intel pentium 5 and software morphology 5.2 ( videotest , saint - petersburg , russia ) . we measured the optical density ( in cu ) and the area of immunopositive expression , which was calculated as the ratio of the area occupied by immunopositive cells ( or nuclei ) and the total area of cells ( or nuclei ) in the field of view ( in % ) . statistical analysis of experimental data included calculation of the arithmetic average , the standard deviation , and the confidence interval for each sample , which was performed using the program statistica 8.0 . ( dell ; statistica , round rock , tx , usa ) . to analyze the distribution of species nonparametric procedures , including one - way analysis of variance ( kruskal wallis ) , were used to verify the statistical homogeneity of several samples . in cases where the variance analysis revealed a statistically significant heterogeneity of multiple samples for subsequent detection of heterogeneous groups ( through their pairwise comparisons ) in the human study , we investigated the effect of revofil aquashine ( aq ) that possesses all physical , structural , and biocompatibility properties that are required of a superior gel for use in skin restoring and has a encumbrance certificate . during the clinical study done for encumbrance certification , no major side effects requiring medical attention were observed . aq contains biomimetic peptides ( cg - cgc2 , cg - cgc3 , rejuline , boostrin ) , free hyaluronic acid , amino acids , multivitamins , and minerals . aq was administered to the patients ( n=5 , age : 4548 years ) 1 month prior to the abdominoplasty by means of intradermal microinjections into skin of the right half of anterior abdominal wall below the navel ( in areas of localized fat deposits that are to be subsequently removed ) . the samples were received from caregen research center ( seoul , south korea ) . as a control , pure ( not containing biomimetic peptides ) hyaluronic acid was injected simultaneously into a symmetric area to the left . the treatment and study details were fully explained to the subjects , all of whom signed written informed consent forms pertaining to treatment and participation in this study , which included consent to injection of cosmeceutical product and skin biopsy . a total of 2 ml of aq was expected to hold a single procedure ; this volume was recommended by caregen . aq was injected to a depth of 46 mm ( depending on skin thickness ) and at an angle of 4590 to the surface of the skin . during the abdominoplasty , skin samples were taken from the injected areas of the abdomen and fixed in 10% formalin solution . the study was conducted with caregen research ethics committee approval and patients provided written informed consent . in the cell culture model , we investigated complex of biomimetic peptides that were part of fillers revofil aq and aq brightening ( br ) . the complex of biomimetic peptides was provided by the supplier in ampoules and was clear , colorless aqueous sterile solution . the subcultivation of normal human diploid fibroblasts as a model of aging was used in this study in vitro . the biopsy was taken from a middle - aged donor ( 42 years ) ; when the culture reach 7 passage , we classified it as a mature cells , meaning that all processes as energy exchange , cell growth , biosynthesis are in a highest stage , and passage 15 as a senescent culture , thus all the processes mentioned above regress . the senescent cell line was proved by staining for -galactosidase ( anti--galactosidase antibody ; abcam , cambridge , uk ) as a technique to label senescent cells . isolation of fibroblasts with enzymatic digestion was carried out using dispase ii ( thermo fisher scientific , waltham , ma , usa ) at the concentration of 2.4 iu / ml for 18 hours at 4c , and then the epidermis was mechanically separated from the dermis . to obtain a suspension of fibroblasts , the dermis was minced with scissors into pieces of 34 mm and placed in a solution of type i collagenase ( thermo fisher scientific ) in minimum essential medium ( biolot , saint - petersburg , russia ) for 30 minutes at 37c . the obtained culture was tested to specific molecular fibroblast marker vimentin ( antivimentin monoclonal antibody ; dako denmark a / s , glostrup , denmark ) , which confirmed that the investigated culture was skin fibroblasts . we studied three groups : 1 ) control group ( physiological solution ) ; 2 ) aq group with addition of biomimetic peptide components of aq ( acetyl decapeptide-3 [ rejuline ] , oligopeptide-24 [ cg - egp3 ] , oligopeptide-34 [ cg - tgp2 ] , oligopeptide-72 [ boostrin ] ) in concentration of 2 g / ml ; and 3 ) aq br group with addition of biomimetic peptide components of aq br ( oligopeptide-23 [ flatin ] , oligopeptide-34 [ cg - tgp2 ] , oligopeptide-51 [ purilux ] , oligopeptide-72 [ boostrin ] ) in concentration of 2 g / ml . at passages 7 and 15 , cells were seeded on coverslips ( 15 mm , thermo fisher scientific ) fixed with paraformaldehyde ( sigma - aldrich co. , st louis , mo , usa ) and incubated with antibodies . the confocal microscope olympus fv1000 ( olympus corporation , tokyo , japan ) was used for visualization of the nuclear markers ki-67 and sirt6 in fibroblast culture samples by applying secondary antibodies conjugated with a far red - emitting dye ( alexa fluor 647 ; abcam ) . in both studies ( in vivo and in vitro ) , we used primary monoclonal antibodies to markers ki-67 ( 1:75 ; dako denmark a / s ) , ap-1 ( 1:200 ; sigma - aldrich co. ) , type i procollagen ( 1:100 , lifespan biosciences , seattle , wa , usa ) , and sirt6 ( 1:200 , alexa fluor 647 ) and biotinylated anti - mouse immunoglobulins ( dako denmark a / s ) as secondary antibodies to provide the immunohistochemical reaction . visualization of the reaction was carried out using the envision detection system kit ( dako denmark a / s ) . in both cases , morphometric investigation was carried out by using a system of computer analysis of microscopic images , which includes a microscope olympus bx46 , a digital camera ( olympus corporation ) , and a personal computer based on intel pentium 5 and software morphology 5.2 ( videotest , saint - petersburg , russia ) . we measured the optical density ( in cu ) and the area of immunopositive expression , which was calculated as the ratio of the area occupied by immunopositive cells ( or nuclei ) and the total area of cells ( or nuclei ) in the field of view ( in % ) . statistical analysis of experimental data included calculation of the arithmetic average , the standard deviation , and the confidence interval for each sample , which was performed using the program statistica 8.0 . ( dell ; statistica , round rock , tx , usa ) . to analyze the distribution of species nonparametric procedures , including one - way analysis of variance ( kruskal wallis ) , were used to verify the statistical homogeneity of several samples . in cases where the variance analysis revealed a statistically significant heterogeneity of multiple samples for subsequent detection of heterogeneous groups ( through their pairwise comparisons ) , we used multiple comparison procedures such as the mann whitney u test . it was shown that in a control sample , with the saline injected , the size of the matrix prevailed in relation to the area of collagen and elastic fibers ( figure 1a ) . a moderate increase in the matrix was revealed without any change in the structure of the fibers after administration of hyaluronic acid ( figure 1b ) . during the histological study , it was revealed that the maximum area of the fibers relative to the matrix was observed in the samples after administration of aq ( figure 1c ) that was determined by the increase in the thickness and density of collagen fibers . the study of the extracellular matrix component expression of the type i collagen precursor type i procollagen was conducted to explore the synthetic activity of dermal fibroblasts . the optical density of expression was measured , as it is the most informative for this marker and characterizes the intensity of immunohistochemical reaction in the samples . indirectly , it gives an indication of the amount of test substance in the samples . the maximum value of optical density of the type i procollagen expression was observed in the sample with double injection of aq , which was 0.285 cu , it was also high in the other three samples with a single administration ( 0.260 cu , 0.273 cu , and 0.278 cu , respectively ) , indicating a dose - dependent stimulation of the procollagen synthesis and a noticeable effect with an increase in the number of treatment sessions . the minimum value of optical density was observed in the control group and in the samples treated with hyaluronic acid ( 0.175 cu and 0.194 cu ) . the obtained data were confirmed using the confocal laser scanning microscopy studies . the three - dimensional reconstruction of collagen fibers was formed by means of thick slices of the specimens and fluorescent labels conjugated with antibodies to type i procollagen . by comparing the volume ratios , it was found that the area of newly synthesized type i procollagen with the injection of aq is two times larger than that in the comparison groups ( figure 2 ) . the formation and maintenance of collagen fibers not only promotes the synthesis of procollagen by skin fibroblasts but also slows down the process of collagen degradation . the transcription factor ap-1 is one of the main factors regulating the destruction of collagen fibers . in terms of the area of the transcription factor ap-1 expression , there was a tendency to reduce it in the samples with the aq administration , where it ranged between 2% and 6% , while in the samples with the introduction of hyaluronic acid and in the control group , it was within 7%14% . we studied the protein sirt6 to evaluate the activity of skin protective mechanisms from premature aging . it is known that this protein is involved in many processes in the cell , such as dna repair , telomere elongation , and glycolysis . the area of immunopositive nuclei was assessed by marker for sirt6 in relation to the total number of nuclei in the dermis in the field of view and expressed as percentage . the analysis of the data by the area of immunopositive nuclei expression to sirt6 revealed that there was an increase in the number of immunopositive nuclei compared with that in the control samples even after a single injection of aq . thus , aq could improve skin aging by affecting bioactive molecules such as sirt6 and ap-1 , promoting division of fibroblasts and expression of collagen de novo . the skin on a skin is rejuvenation through reducing wrinkles and improving skin elasticity by generating new skin cells . comparative analysis of the injectable preparation effect on the synthetic activity of fibroblasts of mature and senescent skin models showed a significant increase in the area of type i procollagen expression in groups with the administration of aq and aq br compared to the control group ( table 1 ) . the introduction of the investigated preparations promotes the synthesis of collagen by skin fibroblasts de novo . however , the response of fibroblasts was weaker at passage 15 than in younger passages . these findings were confirmed by experiments in vivo , when the synthesis of collagen in the ultraviolet - protected skin of old people ( 80 years ) was reduced by ~75% in relation to collagen synthesis of young people ( 1829 years).17 stimulating effects of aq and aq br cosmeceutical products on the proliferative activity of human skin fibroblasts were detected in cell culture . even in aging cell cultures , the parameter of the relative number of immunopositive nuclei to ki-67-marker was two times higher than that in the control group ( figure 3 ) . the study of the transcription factor ap-1 in the cell culture of the skin fibroblasts revealed a statistically significant decrease in the area of expression in groups with the introduction of aq and aq br by 2.1 and 3.1 times , respectively , than that in the control group . statistically significant differences were identified only at the effect of aq , indicating dermatogenic protective action of the investigated substances , realized through antidegradation of collagen fibers . the expression of sirt6 protein was revealed by cell cultivation of skin fibroblasts in all the three groups . the maximum amount of immunopositive nuclei was observed in the culture of fibroblasts incubated with aq br at passage 7 , but this value was not statistically different from the data of the group with the introduction of aq . biomimetic peptides contributed to an increase in the area of immunopositive nuclei by 79% after incubation with aq br and by 77% with the introduction of aq ( table 1 ) . this indicates the start of the mechanisms of dna stability maintenance , protection of telomeres , and other processes preventing aging . in the future investigation , it will be interesting to use real - time polymerase chain reaction and enzyme - linked immunosorbent assay methods to confirm data obtained in this study and expand knowledge of action of biomimetic peptides on skin aging processes . the immunohistochemical studies revealed the molecular mechanisms of the clinical effects ( lifting effect , remodeling of the dermis with filling of small wrinkles , dermoprotective effect on the elastic matrix of the skin , improvement in skin resistance to aggressive environmental factors and mutations ) observed with intradermal administration of aq both in vitro ( in cell culture of human fibroblasts ) and in vivo ( in biopsies of human skin ) .

backgroundbiomimetic peptides are synthetic compounds that are identical to amino acid sequence synthesized by an organism and can interact with growth factor receptors and provide antiaging clinical effects.purposethe purpose of this study was to investigate the effects of biomimetic peptides on the repair processes in the dermis using a model of cell cultures and in vivo.patients and methodsfive female volunteers were subjected to the injection of biomimetic peptides 1 month prior to the abdominoplasty procedure . cell culture , immunocytochemistry , and confocal microscopy methods were used in this study.resultsbiomimetic peptides regulate the synthesis of proteins ki-67 , type i procollagen , ap-1 , and sirt6 in cell cultures of human fibroblasts . they contribute to the activation of regeneration processes and initiation of mechanisms that prevent aging . intradermal administration of complex of biomimetic peptides produces a more dense arrangement of collagen fibers in the dermis and increased size of the fibers after 2 weeks . the complex of biomimetic peptides was effective in the in vivo experiments , where an increase in the proliferative and synthetic activities of fibroblasts was observed.conclusionthis investigation showed that the studied peptides have biological effects , testifying the stimulation of reparative processes in the skin under their control .

Introduction Patients and methods Human study design Cell cultivation Immunohistochemical study Statistical analysis Results and discussion Conclusion

a wide range of biomimetic peptides with different mechanisms of action can be applied to solve this problem . by imitating the action of naturally occurring growth factors and cytokines , they are able to bind to the specific receptors , regulate gene transcription , and provide a stimulating effect on keratinocytes and fibroblasts . however , the target genes of different peptides are different ; hence , the maximum effect is achieved only by the combined application of several biomimetic peptides.1 caregen co. , ltd . ( anyang - si , gyeonggi - do , south korea ) has developed biomimetic peptides based on extensive studies of growth factors since 2002 . the biomimetic peptides are oligopeptides consisting of ten to 15 amino acids and can provide clinical benefits similar to recombinant growth factors , reduce costs , and have greater chemical stability . in the production system uses a unique mass production of growth factors provided by transformed bacteria ( escherichia coli system ) , containing synthetic human genes . double - layered encapsulation technology improves penetration of active ingredients into skin and protects the molecules from endogenous proteases , which provides higher efficacy.2 biomimetic peptides have a wide range of applications such as increasing the efficacies of any cosmetic and meso - formulations , including dermal fillers , in antiaging , antipigmentation , hair growth , body fat reduction , and many inflammation - related products , which are also applicable for pharmaceutical purposes.3,4 in addition , in the recent decade , an interest in synthetic oligopeptides and their use in diagnosis and treatment of different pathologies has increased dramatically ; for example , investigation devoted to influence of short peptides on endothelial growth5 or protection of kidney during the cisplatin - induced acute renal failure.6 in this study , we identified several molecules associated with aging and skin regeneration , such as ki-67 , type i procollagen , sirt6 , and ap-1 . in aging , the number , quality , and quantity of collagen fibers are modified ; they become thin , lose their clear organization , and are resorbed . matrix metalloproteinases ( mmp-1 , mmp-2 , and mmp-3 ) are key signaling molecules of collagen expression . many studies have shown that their activity is regulated by ap-1.79 in parallel , with the degradation of the existing collagen , the activation of factor ap-1 is accompanied with a decrease in procollagen types i and iii . ap-1 regulates a number of cellular processes , including differentiation , proliferation , and apoptosis.10 there is a decrease in the proliferative activity of fibroblasts during aging . using the cultivation method , it was shown that fibroblasts of young donors are characterized by twice as many mitoses , whereby one cell ( there are 60% of such type of cells ) is able to form a colony of 256 fibroblasts , while in the case of older donors , only 2% of cells form colonies of similar volume.11 the hayflick phenomenon can be a substantiation of fewer cell divisions , whereby somatic cells without telomerase expression are capable , on average , of only 50 doublings of population , but fibroblasts of older donors have already passed a number of cell cycles prior to isolation in vitro.12 there is a correlation between donor age and the replicative lifespan of human cells in culture.13 for accuracy , it is better to investigate one serially cultured sample at different periods of time than to examine several different cultured samples.14 the protein ki-67 is present in all active phases of the cell cycle ( g1 , s , g2 , and mitosis ) , and it is a recognized marker for determining the growth of the cell population . dna repair and control of proliferation are its main functions in the skin , and this protein also participates in inflammation processes . thus , hyperexpression of sirt6 leads to increase in tnf- synthesis through posttranscriptional mechanisms.15 sirt6 modulates telomeric chromatin by lys-9 deacetylation of histone h3 and thus prevents telomeric dysfunction and early cellular aging.16 the aim of this study was to investigate the influence of biomimetic peptides on the reparation processes in the dermis by the cell culture model in vitro and in vivo . in the human study , we investigated the effect of revofil aquashine ( aq ) that possesses all physical , structural , and biocompatibility properties that are required of a superior gel for use in skin restoring and has a encumbrance certificate . aq contains biomimetic peptides ( cg - cgc2 , cg - cgc3 , rejuline , boostrin ) , free hyaluronic acid , amino acids , multivitamins , and minerals . aq was administered to the patients ( n=5 , age : 4548 years ) 1 month prior to the abdominoplasty by means of intradermal microinjections into skin of the right half of anterior abdominal wall below the navel ( in areas of localized fat deposits that are to be subsequently removed ) . as a control , pure ( not containing biomimetic peptides ) hyaluronic acid was injected simultaneously into a symmetric area to the left . the treatment and study details were fully explained to the subjects , all of whom signed written informed consent forms pertaining to treatment and participation in this study , which included consent to injection of cosmeceutical product and skin biopsy . aq was injected to a depth of 46 mm ( depending on skin thickness ) and at an angle of 4590 to the surface of the skin . during the abdominoplasty , skin samples were taken from the injected areas of the abdomen and fixed in 10% formalin solution . the study was conducted with caregen research ethics committee approval and patients provided written informed consent . in the cell culture model , we investigated complex of biomimetic peptides that were part of fillers revofil aq and aq brightening ( br ) . the complex of biomimetic peptides was provided by the supplier in ampoules and was clear , colorless aqueous sterile solution . the subcultivation of normal human diploid fibroblasts as a model of aging was used in this study in vitro . the biopsy was taken from a middle - aged donor ( 42 years ) ; when the culture reach 7 passage , we classified it as a mature cells , meaning that all processes as energy exchange , cell growth , biosynthesis are in a highest stage , and passage 15 as a senescent culture , thus all the processes mentioned above regress . isolation of fibroblasts with enzymatic digestion was carried out using dispase ii ( thermo fisher scientific , waltham , ma , usa ) at the concentration of 2.4 iu / ml for 18 hours at 4c , and then the epidermis was mechanically separated from the dermis . to obtain a suspension of fibroblasts , the dermis was minced with scissors into pieces of 34 mm and placed in a solution of type i collagenase ( thermo fisher scientific ) in minimum essential medium ( biolot , saint - petersburg , russia ) for 30 minutes at 37c . the confocal microscope olympus fv1000 ( olympus corporation , tokyo , japan ) was used for visualization of the nuclear markers ki-67 and sirt6 in fibroblast culture samples by applying secondary antibodies conjugated with a far red - emitting dye ( alexa fluor 647 ; abcam ) . in both studies ( in vivo and in vitro ) , we used primary monoclonal antibodies to markers ki-67 ( 1:75 ; dako denmark a / s ) , ap-1 ( 1:200 ; sigma - aldrich co. ) , type i procollagen ( 1:100 , lifespan biosciences , seattle , wa , usa ) , and sirt6 ( 1:200 , alexa fluor 647 ) and biotinylated anti - mouse immunoglobulins ( dako denmark a / s ) as secondary antibodies to provide the immunohistochemical reaction . in both cases , morphometric investigation was carried out by using a system of computer analysis of microscopic images , which includes a microscope olympus bx46 , a digital camera ( olympus corporation ) , and a personal computer based on intel pentium 5 and software morphology 5.2 ( videotest , saint - petersburg , russia ) . we measured the optical density ( in cu ) and the area of immunopositive expression , which was calculated as the ratio of the area occupied by immunopositive cells ( or nuclei ) and the total area of cells ( or nuclei ) in the field of view ( in % ) . statistical analysis of experimental data included calculation of the arithmetic average , the standard deviation , and the confidence interval for each sample , which was performed using the program statistica 8.0 . in cases where the variance analysis revealed a statistically significant heterogeneity of multiple samples for subsequent detection of heterogeneous groups ( through their pairwise comparisons ) in the human study , we investigated the effect of revofil aquashine ( aq ) that possesses all physical , structural , and biocompatibility properties that are required of a superior gel for use in skin restoring and has a encumbrance certificate . aq contains biomimetic peptides ( cg - cgc2 , cg - cgc3 , rejuline , boostrin ) , free hyaluronic acid , amino acids , multivitamins , and minerals . aq was administered to the patients ( n=5 , age : 4548 years ) 1 month prior to the abdominoplasty by means of intradermal microinjections into skin of the right half of anterior abdominal wall below the navel ( in areas of localized fat deposits that are to be subsequently removed ) . as a control , pure ( not containing biomimetic peptides ) hyaluronic acid was injected simultaneously into a symmetric area to the left . the treatment and study details were fully explained to the subjects , all of whom signed written informed consent forms pertaining to treatment and participation in this study , which included consent to injection of cosmeceutical product and skin biopsy . aq was injected to a depth of 46 mm ( depending on skin thickness ) and at an angle of 4590 to the surface of the skin . during the abdominoplasty , skin samples were taken from the injected areas of the abdomen and fixed in 10% formalin solution . in the cell culture model , we investigated complex of biomimetic peptides that were part of fillers revofil aq and aq brightening ( br ) . the complex of biomimetic peptides was provided by the supplier in ampoules and was clear , colorless aqueous sterile solution . the subcultivation of normal human diploid fibroblasts as a model of aging was used in this study in vitro . the biopsy was taken from a middle - aged donor ( 42 years ) ; when the culture reach 7 passage , we classified it as a mature cells , meaning that all processes as energy exchange , cell growth , biosynthesis are in a highest stage , and passage 15 as a senescent culture , thus all the processes mentioned above regress . isolation of fibroblasts with enzymatic digestion was carried out using dispase ii ( thermo fisher scientific , waltham , ma , usa ) at the concentration of 2.4 iu / ml for 18 hours at 4c , and then the epidermis was mechanically separated from the dermis . to obtain a suspension of fibroblasts , the dermis was minced with scissors into pieces of 34 mm and placed in a solution of type i collagenase ( thermo fisher scientific ) in minimum essential medium ( biolot , saint - petersburg , russia ) for 30 minutes at 37c . the confocal microscope olympus fv1000 ( olympus corporation , tokyo , japan ) was used for visualization of the nuclear markers ki-67 and sirt6 in fibroblast culture samples by applying secondary antibodies conjugated with a far red - emitting dye ( alexa fluor 647 ; abcam ) . in both studies ( in vivo and in vitro ) , we used primary monoclonal antibodies to markers ki-67 ( 1:75 ; dako denmark a / s ) , ap-1 ( 1:200 ; sigma - aldrich co. ) , type i procollagen ( 1:100 , lifespan biosciences , seattle , wa , usa ) , and sirt6 ( 1:200 , alexa fluor 647 ) and biotinylated anti - mouse immunoglobulins ( dako denmark a / s ) as secondary antibodies to provide the immunohistochemical reaction . in both cases , morphometric investigation was carried out by using a system of computer analysis of microscopic images , which includes a microscope olympus bx46 , a digital camera ( olympus corporation ) , and a personal computer based on intel pentium 5 and software morphology 5.2 ( videotest , saint - petersburg , russia ) . we measured the optical density ( in cu ) and the area of immunopositive expression , which was calculated as the ratio of the area occupied by immunopositive cells ( or nuclei ) and the total area of cells ( or nuclei ) in the field of view ( in % ) . statistical analysis of experimental data included calculation of the arithmetic average , the standard deviation , and the confidence interval for each sample , which was performed using the program statistica 8.0 . it was shown that in a control sample , with the saline injected , the size of the matrix prevailed in relation to the area of collagen and elastic fibers ( figure 1a ) . a moderate increase in the matrix was revealed without any change in the structure of the fibers after administration of hyaluronic acid ( figure 1b ) . during the histological study , it was revealed that the maximum area of the fibers relative to the matrix was observed in the samples after administration of aq ( figure 1c ) that was determined by the increase in the thickness and density of collagen fibers . the study of the extracellular matrix component expression of the type i collagen precursor type i procollagen was conducted to explore the synthetic activity of dermal fibroblasts . indirectly , it gives an indication of the amount of test substance in the samples . the maximum value of optical density of the type i procollagen expression was observed in the sample with double injection of aq , which was 0.285 cu , it was also high in the other three samples with a single administration ( 0.260 cu , 0.273 cu , and 0.278 cu , respectively ) , indicating a dose - dependent stimulation of the procollagen synthesis and a noticeable effect with an increase in the number of treatment sessions . the minimum value of optical density was observed in the control group and in the samples treated with hyaluronic acid ( 0.175 cu and 0.194 cu ) . the three - dimensional reconstruction of collagen fibers was formed by means of thick slices of the specimens and fluorescent labels conjugated with antibodies to type i procollagen . by comparing the volume ratios , it was found that the area of newly synthesized type i procollagen with the injection of aq is two times larger than that in the comparison groups ( figure 2 ) . the formation and maintenance of collagen fibers not only promotes the synthesis of procollagen by skin fibroblasts but also slows down the process of collagen degradation . the transcription factor ap-1 is one of the main factors regulating the destruction of collagen fibers . in terms of the area of the transcription factor ap-1 expression , there was a tendency to reduce it in the samples with the aq administration , where it ranged between 2% and 6% , while in the samples with the introduction of hyaluronic acid and in the control group , it was within 7%14% . it is known that this protein is involved in many processes in the cell , such as dna repair , telomere elongation , and glycolysis . the area of immunopositive nuclei was assessed by marker for sirt6 in relation to the total number of nuclei in the dermis in the field of view and expressed as percentage . the analysis of the data by the area of immunopositive nuclei expression to sirt6 revealed that there was an increase in the number of immunopositive nuclei compared with that in the control samples even after a single injection of aq . thus , aq could improve skin aging by affecting bioactive molecules such as sirt6 and ap-1 , promoting division of fibroblasts and expression of collagen de novo . comparative analysis of the injectable preparation effect on the synthetic activity of fibroblasts of mature and senescent skin models showed a significant increase in the area of type i procollagen expression in groups with the administration of aq and aq br compared to the control group ( table 1 ) . the introduction of the investigated preparations promotes the synthesis of collagen by skin fibroblasts de novo . however , the response of fibroblasts was weaker at passage 15 than in younger passages . these findings were confirmed by experiments in vivo , when the synthesis of collagen in the ultraviolet - protected skin of old people ( 80 years ) was reduced by ~75% in relation to collagen synthesis of young people ( 1829 years).17 stimulating effects of aq and aq br cosmeceutical products on the proliferative activity of human skin fibroblasts were detected in cell culture . even in aging cell cultures , the parameter of the relative number of immunopositive nuclei to ki-67-marker was two times higher than that in the control group ( figure 3 ) . the study of the transcription factor ap-1 in the cell culture of the skin fibroblasts revealed a statistically significant decrease in the area of expression in groups with the introduction of aq and aq br by 2.1 and 3.1 times , respectively , than that in the control group . statistically significant differences were identified only at the effect of aq , indicating dermatogenic protective action of the investigated substances , realized through antidegradation of collagen fibers . the maximum amount of immunopositive nuclei was observed in the culture of fibroblasts incubated with aq br at passage 7 , but this value was not statistically different from the data of the group with the introduction of aq . biomimetic peptides contributed to an increase in the area of immunopositive nuclei by 79% after incubation with aq br and by 77% with the introduction of aq ( table 1 ) . this indicates the start of the mechanisms of dna stability maintenance , protection of telomeres , and other processes preventing aging . in the future investigation , it will be interesting to use real - time polymerase chain reaction and enzyme - linked immunosorbent assay methods to confirm data obtained in this study and expand knowledge of action of biomimetic peptides on skin aging processes . the immunohistochemical studies revealed the molecular mechanisms of the clinical effects ( lifting effect , remodeling of the dermis with filling of small wrinkles , dermoprotective effect on the elastic matrix of the skin , improvement in skin resistance to aggressive environmental factors and mutations ) observed with intradermal administration of aq both in vitro ( in cell culture of human fibroblasts ) and in vivo ( in biopsies of human skin ) .

skin aging is a complex process that affects all its layers and structure and changes the functional properties of the intracellular matrix . by imitating the action of naturally occurring growth factors and cytokines , they are able to bind to the specific receptors , regulate gene transcription , and provide a stimulating effect on keratinocytes and fibroblasts . however , the target genes of different peptides are different ; hence , the maximum effect is achieved only by the combined application of several biomimetic peptides.1 caregen co. , ltd . the term biomimetic peptide as used herein relates to a synthetic agonist of naturally occurring growth factors and completely mimics the action of the parental molecules . the biomimetic peptides are oligopeptides consisting of ten to 15 amino acids and can provide clinical benefits similar to recombinant growth factors , reduce costs , and have greater chemical stability . in the production system uses a unique mass production of growth factors provided by transformed bacteria ( escherichia coli system ) , containing synthetic human genes . double - layered encapsulation technology improves penetration of active ingredients into skin and protects the molecules from endogenous proteases , which provides higher efficacy.2 biomimetic peptides have a wide range of applications such as increasing the efficacies of any cosmetic and meso - formulations , including dermal fillers , in antiaging , antipigmentation , hair growth , body fat reduction , and many inflammation - related products , which are also applicable for pharmaceutical purposes.3,4 in addition , in the recent decade , an interest in synthetic oligopeptides and their use in diagnosis and treatment of different pathologies has increased dramatically ; for example , investigation devoted to influence of short peptides on endothelial growth5 or protection of kidney during the cisplatin - induced acute renal failure.6 in this study , we identified several molecules associated with aging and skin regeneration , such as ki-67 , type i procollagen , sirt6 , and ap-1 . in aging , the number , quality , and quantity of collagen fibers are modified ; they become thin , lose their clear organization , and are resorbed . many studies have shown that their activity is regulated by ap-1.79 in parallel , with the degradation of the existing collagen , the activation of factor ap-1 is accompanied with a decrease in procollagen types i and iii . ap-1 regulates a number of cellular processes , including differentiation , proliferation , and apoptosis.10 there is a decrease in the proliferative activity of fibroblasts during aging . using the cultivation method , it was shown that fibroblasts of young donors are characterized by twice as many mitoses , whereby one cell ( there are 60% of such type of cells ) is able to form a colony of 256 fibroblasts , while in the case of older donors , only 2% of cells form colonies of similar volume.11 the hayflick phenomenon can be a substantiation of fewer cell divisions , whereby somatic cells without telomerase expression are capable , on average , of only 50 doublings of population , but fibroblasts of older donors have already passed a number of cell cycles prior to isolation in vitro.12 there is a correlation between donor age and the replicative lifespan of human cells in culture.13 for accuracy , it is better to investigate one serially cultured sample at different periods of time than to examine several different cultured samples.14 the protein ki-67 is present in all active phases of the cell cycle ( g1 , s , g2 , and mitosis ) , and it is a recognized marker for determining the growth of the cell population . dna repair and control of proliferation are its main functions in the skin , and this protein also participates in inflammation processes . thus , hyperexpression of sirt6 leads to increase in tnf- synthesis through posttranscriptional mechanisms.15 sirt6 modulates telomeric chromatin by lys-9 deacetylation of histone h3 and thus prevents telomeric dysfunction and early cellular aging.16 the aim of this study was to investigate the influence of biomimetic peptides on the reparation processes in the dermis by the cell culture model in vitro and in vivo . in the human study , we investigated the effect of revofil aquashine ( aq ) that possesses all physical , structural , and biocompatibility properties that are required of a superior gel for use in skin restoring and has a encumbrance certificate . aq contains biomimetic peptides ( cg - cgc2 , cg - cgc3 , rejuline , boostrin ) , free hyaluronic acid , amino acids , multivitamins , and minerals . aq was administered to the patients ( n=5 , age : 4548 years ) 1 month prior to the abdominoplasty by means of intradermal microinjections into skin of the right half of anterior abdominal wall below the navel ( in areas of localized fat deposits that are to be subsequently removed ) . the treatment and study details were fully explained to the subjects , all of whom signed written informed consent forms pertaining to treatment and participation in this study , which included consent to injection of cosmeceutical product and skin biopsy . aq was injected to a depth of 46 mm ( depending on skin thickness ) and at an angle of 4590 to the surface of the skin . during the abdominoplasty , skin samples were taken from the injected areas of the abdomen and fixed in 10% formalin solution . in the cell culture model , we investigated complex of biomimetic peptides that were part of fillers revofil aq and aq brightening ( br ) . the complex of biomimetic peptides was provided by the supplier in ampoules and was clear , colorless aqueous sterile solution . the biopsy was taken from a middle - aged donor ( 42 years ) ; when the culture reach 7 passage , we classified it as a mature cells , meaning that all processes as energy exchange , cell growth , biosynthesis are in a highest stage , and passage 15 as a senescent culture , thus all the processes mentioned above regress . isolation of fibroblasts with enzymatic digestion was carried out using dispase ii ( thermo fisher scientific , waltham , ma , usa ) at the concentration of 2.4 iu / ml for 18 hours at 4c , and then the epidermis was mechanically separated from the dermis . to obtain a suspension of fibroblasts , the dermis was minced with scissors into pieces of 34 mm and placed in a solution of type i collagenase ( thermo fisher scientific ) in minimum essential medium ( biolot , saint - petersburg , russia ) for 30 minutes at 37c . we studied three groups : 1 ) control group ( physiological solution ) ; 2 ) aq group with addition of biomimetic peptide components of aq ( acetyl decapeptide-3 [ rejuline ] , oligopeptide-24 [ cg - egp3 ] , oligopeptide-34 [ cg - tgp2 ] , oligopeptide-72 [ boostrin ] ) in concentration of 2 g / ml ; and 3 ) aq br group with addition of biomimetic peptide components of aq br ( oligopeptide-23 [ flatin ] , oligopeptide-34 [ cg - tgp2 ] , oligopeptide-51 [ purilux ] , oligopeptide-72 [ boostrin ] ) in concentration of 2 g / ml . the confocal microscope olympus fv1000 ( olympus corporation , tokyo , japan ) was used for visualization of the nuclear markers ki-67 and sirt6 in fibroblast culture samples by applying secondary antibodies conjugated with a far red - emitting dye ( alexa fluor 647 ; abcam ) . in both studies ( in vivo and in vitro ) , we used primary monoclonal antibodies to markers ki-67 ( 1:75 ; dako denmark a / s ) , ap-1 ( 1:200 ; sigma - aldrich co. ) , type i procollagen ( 1:100 , lifespan biosciences , seattle , wa , usa ) , and sirt6 ( 1:200 , alexa fluor 647 ) and biotinylated anti - mouse immunoglobulins ( dako denmark a / s ) as secondary antibodies to provide the immunohistochemical reaction . in both cases , morphometric investigation was carried out by using a system of computer analysis of microscopic images , which includes a microscope olympus bx46 , a digital camera ( olympus corporation ) , and a personal computer based on intel pentium 5 and software morphology 5.2 ( videotest , saint - petersburg , russia ) . we measured the optical density ( in cu ) and the area of immunopositive expression , which was calculated as the ratio of the area occupied by immunopositive cells ( or nuclei ) and the total area of cells ( or nuclei ) in the field of view ( in % ) . to analyze the distribution of species nonparametric procedures , including one - way analysis of variance ( kruskal wallis ) , were used to verify the statistical homogeneity of several samples . in cases where the variance analysis revealed a statistically significant heterogeneity of multiple samples for subsequent detection of heterogeneous groups ( through their pairwise comparisons ) in the human study , we investigated the effect of revofil aquashine ( aq ) that possesses all physical , structural , and biocompatibility properties that are required of a superior gel for use in skin restoring and has a encumbrance certificate . aq contains biomimetic peptides ( cg - cgc2 , cg - cgc3 , rejuline , boostrin ) , free hyaluronic acid , amino acids , multivitamins , and minerals . aq was administered to the patients ( n=5 , age : 4548 years ) 1 month prior to the abdominoplasty by means of intradermal microinjections into skin of the right half of anterior abdominal wall below the navel ( in areas of localized fat deposits that are to be subsequently removed ) . the treatment and study details were fully explained to the subjects , all of whom signed written informed consent forms pertaining to treatment and participation in this study , which included consent to injection of cosmeceutical product and skin biopsy . aq was injected to a depth of 46 mm ( depending on skin thickness ) and at an angle of 4590 to the surface of the skin . during the abdominoplasty , skin samples were taken from the injected areas of the abdomen and fixed in 10% formalin solution . in the cell culture model , we investigated complex of biomimetic peptides that were part of fillers revofil aq and aq brightening ( br ) . the complex of biomimetic peptides was provided by the supplier in ampoules and was clear , colorless aqueous sterile solution . the biopsy was taken from a middle - aged donor ( 42 years ) ; when the culture reach 7 passage , we classified it as a mature cells , meaning that all processes as energy exchange , cell growth , biosynthesis are in a highest stage , and passage 15 as a senescent culture , thus all the processes mentioned above regress . isolation of fibroblasts with enzymatic digestion was carried out using dispase ii ( thermo fisher scientific , waltham , ma , usa ) at the concentration of 2.4 iu / ml for 18 hours at 4c , and then the epidermis was mechanically separated from the dermis . to obtain a suspension of fibroblasts , the dermis was minced with scissors into pieces of 34 mm and placed in a solution of type i collagenase ( thermo fisher scientific ) in minimum essential medium ( biolot , saint - petersburg , russia ) for 30 minutes at 37c . we studied three groups : 1 ) control group ( physiological solution ) ; 2 ) aq group with addition of biomimetic peptide components of aq ( acetyl decapeptide-3 [ rejuline ] , oligopeptide-24 [ cg - egp3 ] , oligopeptide-34 [ cg - tgp2 ] , oligopeptide-72 [ boostrin ] ) in concentration of 2 g / ml ; and 3 ) aq br group with addition of biomimetic peptide components of aq br ( oligopeptide-23 [ flatin ] , oligopeptide-34 [ cg - tgp2 ] , oligopeptide-51 [ purilux ] , oligopeptide-72 [ boostrin ] ) in concentration of 2 g / ml . the confocal microscope olympus fv1000 ( olympus corporation , tokyo , japan ) was used for visualization of the nuclear markers ki-67 and sirt6 in fibroblast culture samples by applying secondary antibodies conjugated with a far red - emitting dye ( alexa fluor 647 ; abcam ) . in both studies ( in vivo and in vitro ) , we used primary monoclonal antibodies to markers ki-67 ( 1:75 ; dako denmark a / s ) , ap-1 ( 1:200 ; sigma - aldrich co. ) , type i procollagen ( 1:100 , lifespan biosciences , seattle , wa , usa ) , and sirt6 ( 1:200 , alexa fluor 647 ) and biotinylated anti - mouse immunoglobulins ( dako denmark a / s ) as secondary antibodies to provide the immunohistochemical reaction . in both cases , morphometric investigation was carried out by using a system of computer analysis of microscopic images , which includes a microscope olympus bx46 , a digital camera ( olympus corporation ) , and a personal computer based on intel pentium 5 and software morphology 5.2 ( videotest , saint - petersburg , russia ) . we measured the optical density ( in cu ) and the area of immunopositive expression , which was calculated as the ratio of the area occupied by immunopositive cells ( or nuclei ) and the total area of cells ( or nuclei ) in the field of view ( in % ) . it was shown that in a control sample , with the saline injected , the size of the matrix prevailed in relation to the area of collagen and elastic fibers ( figure 1a ) . a moderate increase in the matrix was revealed without any change in the structure of the fibers after administration of hyaluronic acid ( figure 1b ) . during the histological study , it was revealed that the maximum area of the fibers relative to the matrix was observed in the samples after administration of aq ( figure 1c ) that was determined by the increase in the thickness and density of collagen fibers . the study of the extracellular matrix component expression of the type i collagen precursor type i procollagen was conducted to explore the synthetic activity of dermal fibroblasts . the optical density of expression was measured , as it is the most informative for this marker and characterizes the intensity of immunohistochemical reaction in the samples . the maximum value of optical density of the type i procollagen expression was observed in the sample with double injection of aq , which was 0.285 cu , it was also high in the other three samples with a single administration ( 0.260 cu , 0.273 cu , and 0.278 cu , respectively ) , indicating a dose - dependent stimulation of the procollagen synthesis and a noticeable effect with an increase in the number of treatment sessions . the minimum value of optical density was observed in the control group and in the samples treated with hyaluronic acid ( 0.175 cu and 0.194 cu ) . by comparing the volume ratios , it was found that the area of newly synthesized type i procollagen with the injection of aq is two times larger than that in the comparison groups ( figure 2 ) . in terms of the area of the transcription factor ap-1 expression , there was a tendency to reduce it in the samples with the aq administration , where it ranged between 2% and 6% , while in the samples with the introduction of hyaluronic acid and in the control group , it was within 7%14% . we studied the protein sirt6 to evaluate the activity of skin protective mechanisms from premature aging . it is known that this protein is involved in many processes in the cell , such as dna repair , telomere elongation , and glycolysis . the area of immunopositive nuclei was assessed by marker for sirt6 in relation to the total number of nuclei in the dermis in the field of view and expressed as percentage . the analysis of the data by the area of immunopositive nuclei expression to sirt6 revealed that there was an increase in the number of immunopositive nuclei compared with that in the control samples even after a single injection of aq . thus , aq could improve skin aging by affecting bioactive molecules such as sirt6 and ap-1 , promoting division of fibroblasts and expression of collagen de novo . comparative analysis of the injectable preparation effect on the synthetic activity of fibroblasts of mature and senescent skin models showed a significant increase in the area of type i procollagen expression in groups with the administration of aq and aq br compared to the control group ( table 1 ) . the introduction of the investigated preparations promotes the synthesis of collagen by skin fibroblasts de novo . these findings were confirmed by experiments in vivo , when the synthesis of collagen in the ultraviolet - protected skin of old people ( 80 years ) was reduced by ~75% in relation to collagen synthesis of young people ( 1829 years).17 stimulating effects of aq and aq br cosmeceutical products on the proliferative activity of human skin fibroblasts were detected in cell culture . even in aging cell cultures , the parameter of the relative number of immunopositive nuclei to ki-67-marker was two times higher than that in the control group ( figure 3 ) . the study of the transcription factor ap-1 in the cell culture of the skin fibroblasts revealed a statistically significant decrease in the area of expression in groups with the introduction of aq and aq br by 2.1 and 3.1 times , respectively , than that in the control group . statistically significant differences were identified only at the effect of aq , indicating dermatogenic protective action of the investigated substances , realized through antidegradation of collagen fibers . the maximum amount of immunopositive nuclei was observed in the culture of fibroblasts incubated with aq br at passage 7 , but this value was not statistically different from the data of the group with the introduction of aq . in the future investigation , it will be interesting to use real - time polymerase chain reaction and enzyme - linked immunosorbent assay methods to confirm data obtained in this study and expand knowledge of action of biomimetic peptides on skin aging processes . the immunohistochemical studies revealed the molecular mechanisms of the clinical effects ( lifting effect , remodeling of the dermis with filling of small wrinkles , dermoprotective effect on the elastic matrix of the skin , improvement in skin resistance to aggressive environmental factors and mutations ) observed with intradermal administration of aq both in vitro ( in cell culture of human fibroblasts ) and in vivo ( in biopsies of human skin ) .

glaucoma is the second leading cause of blindness globally.1 from 1991 to 1999 , primary open - angle glaucoma prevalence increased from 4.6% to 13.8% among the elderly.2 its treatment is aimed essentially at lowering intraocular pressure ( iop ) by eye drop instillations , reserving surgery or laser surgery for the most severe cases . several classes of medicine are available , ie , prostaglandin analogs , miotics , beta - blockers , alpha - adrenergic agonists , and carbonic anhydrase inhibitors . glaucoma treatment principles and options have been reported by the european glaucoma society.3 successful treatment depends upon strict lifetime adherence to the instillation schedule . thus , higher adherence is associated with better iop control , on average,4 and a lower risk of eventual blindness.5 however , patients perceive few symptoms in the early stages , whereas eye drops ( with potential side effects ) are needed daily and may become a burden , leading to poor treatment adherence.6 adherence to treatment schedules has been examined by numerous studies in glaucoma , using various methods . for example , the medication possession ratio , determining the mean proportion of days during a given period when patients possess medication , was calculated from insurance claims or prescription databases,79 and from electronic devices capturing drop counts.10 alternatively , patients self - declared compliance was obtained from interviews1113 or standardized questionnaires.8,1418 another difference between studies were noncompliance criteria , eg , patients who missed more than two doses per week18 or possessed insufficient drops for the specified period ( medication possession ratio < 1).5 with this array of methodology across different drug classes and countries , compliance rates varied from 59% to 77%.7,11,14,16,1821 more informally , imperfect compliance is consistently reported among glaucoma patients . to improve glaucoma care , it is critical to identify patients who may not adhere to treatment . factors conducive to noncompliance have been explored . for example , complex dosing regimens have an impact on compliance.19,22 however , barriers cited by most studies relate to patients perception and knowledge about their illness and its treatment.7,11,16,18 these considerations prompted the development of an eye - drop satisfaction questionnaire ( edsq ) which asks patients to self - report their satisfaction and compliance with topical ophthalmic treatments.23 replies to these questions should be relevant to an exploration of noncompliance in glaucoma patients . a suitable technique for analyzing such data is a bayesian network ( bn ) which facilitates the representation and manipulation of information . a bn is a directed acyclic graph representing relationships between variables ( nodes in the graph ) with a related set of conditional probability tables that characterize local dependencies between the various nodes . hence , it provides a powerful tool to study interdependencies between complex processes , such as patient behavior . the objective of this study was to identify poorly compliant glaucoma patients by exploring edsq data . the protocol was reviewed and approved by the comit consultatif sur le traitement de linformation en matire de recherche dans le domaine de la sant and the commission nationale informatique et libert.2426 this research was conducted according to the tenets of the declaration of helsinki.27 this multicenter study was conducted in france by 17 ophthalmologists ( at 17 sites ) specializing in the treatment of glaucoma , who also prescribed travalert , a computerized device that reminds patients to instill eye drops , assists with instillations , and records dosing times . the accuracy of travalert for measuring treatment adherence has been described elsewhere.28,29 ophthalmologists recruited patients who used the device and gave informed consent before participating . at the inclusion visit , general comorbidities ( cardiovascular , central nervous system , hepatic diseases , diabetes , pulmonary diseases , digestive diseases , others ) and eye comorbidities ( macular degeneration , diabetic retinopathy , retinal detachment , cataract , uveitis , others ) were collected . patients included used the travalert device for at least eight weeks , were 18 years of age or older , and diagnosed with either primary open - angle glaucoma ( including juvenile glaucoma , exfoliative glaucoma , pigmentary glaucoma , or normal - tension glaucoma ) or high iop . patients with secondary glaucoma ( congenital glaucoma , inflammatory glaucoma , angle - closure or narrow - angle glaucoma following cataract surgery ) , or took an anticoagulant three times per day or more , and patients with chronic eye dryness requiring instillations of more than five drops a day , were excluded . according to a previous clustering analysis , data collected with the travalert device allowed the patient sample to be categorized according to three compliance profiles , ie , high compliance , moderate compliance , or low compliance.4 the edsq is a self - assessment questionnaire that assesses patient satisfaction and compliance with eye - drop treatment for glaucoma or iop . first , patient and clinician interviews were conducted to determine an appropriate conceptual model and obtain patients ratings . second , a comprehension evaluation was undertaken to ensure the questionnaire s clarity , ease of comprehension , and cultural equivalence.23 the questions are scored on a 15 likert scale . a scoring algorithm for the 21-item questionnaire was also developed and shown to have satisfactory validity and reliability.30 the edsq algorithm computes scores ( 0100 ) for six dimensions of attitudes towards eye - drop treatment , ie , treatment concern ( five items ) , disease concern ( two items ) , satisfaction with the patient clinician relationship ( five items ) ; positive beliefs ( three items ) , treatment convenience ( three items ) , and self - declared compliance ( three items ) . edsq data were collected at the follow - up visit when the travalert data were collected from the patient . patients who completed the edsq and provided sufficient travalert data to define their compliance profile were included in the analysis . an oversampling method was adopted to increase the ability of the bn to identify risk factors for noncompliance.31 this involved randomly selecting patients from the smallest group ( low compliance ) as successive replacements to create a sample equal to the largest group ( high and moderate compliance ) . a uniform ( all patients had the same probability to be sampled , without site stratification ) sampling method was used because the small sample size did not authorize strong distribution estimates . the objective was to achieve a ratio of one patient with low compliance to one with high or moderate compliance . a bn is a form of probabilistic graph , representing a joint probability distribution of a set of variables with explicit independency assumptions . it is a directed acyclic graph , ie , without cycles and has edges that are orientated . it is composed of a set of nodes ( each node represents one variable modeling the process of interest ) and a set of conditional probability tables encoding local dependencies between the nodes / variables . bns are often used to identify structure of the information when very few hypotheses could be identified a priori . a bn was constructed to identify poor compliers by discriminating between low - compliance and moderate- or high - compliance patients . center was not included in the bn due to the numbers of patients per center being too small . algorithm to scores derived from the bn learning algorithm.32 a score denoted the minimum description length , and was two fold ; the first part qualified the network with respect to its structural complexity and the second part measured the goodness - of - fit between the network and the data . with a given order of nodes , it is simple to determine the best bn , because each node looks to its best parent , ie , its immediate predecessor amongst all available nodes . the goal of taboo order is to search for the order of nodes resulting in the best bn value ( highest minimum description length score ) , and to this end uses taboo search.33 this algorithm is fully driven by the data , is fully inferential , and with no clinical expert input . missing data were inferred according to the expectation - maximization structural method . because bn requires categoric variables , edsq scores were categorized by dichotomizing ( thresholds were determined while estimating the bn ) them according to a decision tree - based algorithm aimed at maximizing the detection of low compliance . descriptive analyses were performed using sas for windows v9.2 ( sas institute , inc . , cary , nc ) and bn was obtained using bayesialab v4.6.1 ( bayesia , mayenne , france ) . this multicenter study was conducted in france by 17 ophthalmologists ( at 17 sites ) specializing in the treatment of glaucoma , who also prescribed travalert , a computerized device that reminds patients to instill eye drops , assists with instillations , and records dosing times . the accuracy of travalert for measuring treatment adherence has been described elsewhere.28,29 ophthalmologists recruited patients who used the device and gave informed consent before participating . at the inclusion visit , general comorbidities ( cardiovascular , central nervous system , hepatic diseases , diabetes , pulmonary diseases , digestive diseases , others ) and eye comorbidities ( macular degeneration , diabetic retinopathy , retinal detachment , cataract , uveitis , others ) were collected . patients included used the travalert device for at least eight weeks , were 18 years of age or older , and diagnosed with either primary open - angle glaucoma ( including juvenile glaucoma , exfoliative glaucoma , pigmentary glaucoma , or normal - tension glaucoma ) or high iop . patients with secondary glaucoma ( congenital glaucoma , inflammatory glaucoma , angle - closure or narrow - angle glaucoma following cataract surgery ) , or took an anticoagulant three times per day or more , and patients with chronic eye dryness requiring instillations of more than five drops a day , were excluded . according to a previous clustering analysis , data collected with the travalert device allowed the patient sample to be categorized according to three compliance profiles , ie , high compliance , moderate compliance , or low compliance.4 the edsq is a self - assessment questionnaire that assesses patient satisfaction and compliance with eye - drop treatment for glaucoma or iop . first , patient and clinician interviews were conducted to determine an appropriate conceptual model and obtain patients ratings . second , a comprehension evaluation was undertaken to ensure the questionnaire s clarity , ease of comprehension , and cultural equivalence.23 the questions are scored on a 15 likert scale . a scoring algorithm for the 21-item questionnaire was also developed and shown to have satisfactory validity and reliability.30 the edsq algorithm computes scores ( 0100 ) for six dimensions of attitudes towards eye - drop treatment , ie , treatment concern ( two items ) , satisfaction with the patient clinician relationship ( five items ) ; positive beliefs ( three items ) , treatment convenience ( three items ) , and self - declared compliance ( three items ) . edsq data were collected at the follow - up visit when the travalert data were collected from the patient . patients who completed the edsq and provided sufficient travalert data to define their compliance profile were included in the analysis . an oversampling method was adopted to increase the ability of the bn to identify risk factors for noncompliance.31 this involved randomly selecting patients from the smallest group ( low compliance ) as successive replacements to create a sample equal to the largest group ( high and moderate compliance ) . a uniform ( all patients had the same probability to be sampled , without site stratification ) sampling method was used because the small sample size did not authorize strong distribution estimates . the objective was to achieve a ratio of one patient with low compliance to one with high or moderate compliance . a bn is a form of probabilistic graph , representing a joint probability distribution of a set of variables with explicit independency assumptions . it is a directed acyclic graph , ie , without cycles and has edges that are orientated . it is composed of a set of nodes ( each node represents one variable modeling the process of interest ) and a set of conditional probability tables encoding local dependencies between the nodes / variables . bns are often used to identify structure of the information when very few hypotheses could be identified a priori . a bn was constructed to identify poor compliers by discriminating between low - compliance and moderate- or high - compliance patients . center was not included in the bn due to the numbers of patients per center being too small . algorithm to scores derived from the bn learning algorithm.32 a score denoted the minimum description length , and was two fold ; the first part qualified the network with respect to its structural complexity and the second part measured the goodness - of - fit between the network and the data . with a given order of nodes , it is simple to determine the best bn , because each node looks to its best parent , ie , its immediate predecessor amongst all available nodes . the goal of taboo order is to search for the order of nodes resulting in the best bn value ( highest minimum description length score ) , and to this end uses taboo search.33 this algorithm is fully driven by the data , is fully inferential , and with no clinical expert input . missing data were inferred according to the expectation - maximization structural method . because bn requires categoric variables , edsq scores were categorized by dichotomizing ( thresholds were determined while estimating the bn ) them according to a decision tree - based algorithm aimed at maximizing the detection of low compliance . descriptive analyses were performed using sas for windows v9.2 ( sas institute , inc . , cary , nc ) and bn was obtained using bayesialab v4.6.1 ( bayesia , mayenne , france ) . patients who completed the edsq and provided sufficient travalert data to define their compliance profile were included in the analysis . an oversampling method was adopted to increase the ability of the bn to identify risk factors for noncompliance.31 this involved randomly selecting patients from the smallest group ( low compliance ) as successive replacements to create a sample equal to the largest group ( high and moderate compliance ) . a uniform ( all patients had the same probability to be sampled , without site stratification ) sampling method was used because the small sample size did not authorize strong distribution estimates . the objective was to achieve a ratio of one patient with low compliance to one with high or moderate compliance . a bn is a form of probabilistic graph , representing a joint probability distribution of a set of variables with explicit independency assumptions . it is a directed acyclic graph , ie , without cycles and has edges that are orientated . it is composed of a set of nodes ( each node represents one variable modeling the process of interest ) and a set of conditional probability tables encoding local dependencies between the nodes / variables . bns are often used to identify structure of the information when very few hypotheses could be identified a priori . a bn was constructed to identify poor compliers by discriminating between low - compliance and moderate- or high - compliance patients . center was not included in the bn due to the numbers of patients per center being too small . the network structure ( relationships between variables ) was based on data by applying the taboo order algorithm to scores derived from the bn learning algorithm.32 a score denoted the minimum description length , and was two fold ; the first part qualified the network with respect to its structural complexity and the second part measured the goodness - of - fit between the network and the data . with a given order of nodes , it is simple to determine the best bn , because each node looks to its best parent , ie , its immediate predecessor amongst all available nodes . the goal of taboo order is to search for the order of nodes resulting in the best bn value ( highest minimum description length score ) , and to this end uses taboo search.33 this algorithm is fully driven by the data , is fully inferential , and with no clinical expert input . missing data were inferred according to the expectation - maximization structural method . because bn requires categoric variables , edsq scores were categorized by dichotomizing ( thresholds were determined while estimating the bn ) them according to a decision tree - based algorithm aimed at maximizing the detection of low compliance . descriptive analyses were performed using sas for windows v9.2 ( sas institute , inc . , cary , nc ) and bn was obtained using bayesialab v4.6.1 ( bayesia , mayenne , france ) . of the glaucoma specialists , 88.2% were male , 29.4% worked in a public hospital setting , and had qualified on average 16.5 years earlier . they declared 36.7 visits per week for primary open - angle glaucoma and 11.4 for ocular hypertension . among one hundred and forty patients ( 78.2% ) used the travalert , brought it back to the visit , with successful data transfer to the investigator computer . among the 140 transferred files , 113 ( 80.7% ) provided reliable data ( compliance fully documented for at least eight weeks ) sufficient for the classification algorithm . technical criteria to participate to the clustering algorithm have been published elsewhere.4 the characteristics of patients meeting the inclusion criteria are presented in table 1 . the gender ratio was balanced , mean age was 65.1 years , 16.6% had undergone surgery for primary open - angle glaucoma , and 10.1% had received laser therapy . at the inclusion visit , worst eye mean iop was 16.4 mmhg and best eye mean visual acuity was 8.80 . the frequency of high compliance ( 56.6% ) was much greater than that of low compliance ( 22.1% ) . the bn ( figure 1 ) shows three nodes directly related to travalert - identified compliance , ie , patient treatment concern has three drivers , which are disease concern , gender , and positive beliefs , and itself impacts on the patient . two subgroups of patients had a noticeable distribution across groups defined according to treatment concern score ( table 2 ) . 47.018 were markedly more likely to have a treatment concern score of > 24.197 than the total population . second , patients with a disease concern score of < 47.018 and a positive beliefs score > 61.335 were also more likely to have a treatment concern score of > 24.197 . when compared with the total population , a higher percentage of patients with a treatment concern score < 24.197 had a patient more patients with a treatment convenience score of < 69.397 had a positive beliefs score < 61.335 compared with the total analysis population ( table 4 ) . a higher percentage of patients with a positive beliefs score < 61.335 had a self - declared compliance score < 89.583 , as compared with the analyzed population ( table 5 ) . compliance identified by travalert was related to three variables , these being age , patient three different combinations of these characteristics were relevant to low or high compliance , compared with the total sample ( figure 2 ) . low compliance was associated with two combinations ; first , age < 77.5 years , a self - declared compliance score < 89.5 , and a patient clinician relationship score < 67.5 and , second , age > 77.5 years , a self - declared compliance score of > 89.5 , and a patient by contrast , high compliance was associated with age < 77.5 years , a self - declared compliance score > 89.5 , and a patient the gender ratio was balanced , mean age was 65.1 years , 16.6% had undergone surgery for primary open - angle glaucoma , and 10.1% had received laser therapy . at the inclusion visit , worst eye mean iop was 16.4 mmhg and best eye mean visual acuity was 8.80 . the frequency of high compliance ( 56.6% ) was much greater than that of low compliance ( 22.1% ) . the bn ( figure 1 ) shows three nodes directly related to travalert - identified compliance , ie , patient treatment concern has three drivers , which are disease concern , gender , and positive beliefs , and itself impacts on the patient two subgroups of patients had a noticeable distribution across groups defined according to treatment concern score ( table 2 ) . 47.018 were markedly more likely to have a treatment concern score of > 24.197 than the total population . second , patients with a disease concern score of < 47.018 and a positive beliefs score > 61.335 were also more likely to have a treatment concern score of > 24.197 . when compared with the total population , a higher percentage of patients with a treatment concern score < 24.197 had a patient more patients with a treatment convenience score of < 69.397 had a positive beliefs score < 61.335 compared with the total analysis population ( table 4 ) . a higher percentage of patients with a positive beliefs score < 61.335 had a self - declared compliance score < 89.583 , as compared with the analyzed population ( table 5 ) . compliance identified by travalert was related to three variables , these being age , patient three different combinations of these characteristics were relevant to low or high compliance , compared with the total sample ( figure 2 ) . low compliance was associated with two combinations ; first , age < 77.5 years , a self - declared compliance score < 89.5 , and a patient clinician relationship score < 67.5 and , second , age > 77.5 years , a self - declared compliance score of > 89.5 , and a patient by contrast , high compliance was associated with age < 77.5 years , a self - declared compliance score > 89.5 , and a patient the primary objective of this study was to identify glaucoma patients who adhered poorly to treatment by recording their daily instillation times and eye drop counts objectively , with a computerized bottle holder ( travalert ) . in a previous analysis , patient demographics and baseline clinical parameters failed to discriminate between patients showing high , moderate , or low compliance.4 this second attempt enquired about attitudes towards treatment that might characterize poor compliers . this was explored with the edsq that examines patients views about eye drop treatments , with aspects relevant to glaucoma , in order to explore compliance profiles . the analysis identified two subgroups of patients at high risk of poor compliance when measured objectively ; first , patients younger than 77.5 years of age with low self - declared compliance and an unsatisfactory relationship with their physician and , second , patients older than 77.5 years of age with high self - declared compliance and an unsatisfactory relationship with their physician . physician relationship that provides education for patients about glaucoma and its treatment is essential for compliance.8,1113,16 conversely , because patients rely strongly on their doctor for such information , a poor relationship was identified as a major barrier to compliance.8,11,12,1618 interestingly , our results are not fully consistent with previous publications with respect to age and other factors associated with compliance . first , we found a relationship between age and compliance that was not observed in several studies.11,14,18,20,34 however , in contrast with customary statistical methods , we used bn based on association tables and inevitably model interaction effects between variables . consequently , age was included with other factors ( patient clinician relationship and self - declared compliance ) , and was thereby related to objectively identified compliance ( travalert ) . second , trust in the efficacy of treatment ( closely related to our positive beliefs score ) has been previously related to compliance,8,12 as have been concerns about disease5 and treatment convenience.12,34 our bn analysis linked no equivalent edsq item directly to compliance . this difference may be explained by specific features of bn . in our study , the absence of direct links between travalert - identified compliance and previously identified components of compliance can be explained by the notion of conditional independence . clinician relationship scores were linked directly to compliance indicates that compliance was conditionally independent of other bn variables . in fact , bn showed these scores to be indirect components of compliance by linkage through other variables . thus , bn may provide a more precise description of the compliance process , and may show how different conceptions of compliance , finally impact on the behavior of glaucoma patients . the rationale for bn was that patients satisfaction and compliance with treatment is a multidimensional phenomenon calling for methods that take complexity into account . also , an oversampling strategy had to be introduced . the numeric imbalance between compliant patients and poorly compliant patients jeopardized the quality of our results , so we considered possible simple solutions to the problem , ie , oversampling ( artificially increasing the size of the smallest group by random sampling and replacement ) , and undersampling ( reducing the size of the largest group by randomly deleting cases ) . despite evidence that undersampling has previously shown better results35,36 and that oversampling may increase the risk of overfitting,37 the latter method had to be used because of our very small dataset , ie , only 113 patients provided sufficient data to construct a compliance profile , and the low - compliance group included only 25 patients . the age and gender ratios of our sample are close to those of glaucoma - treated patients in france.38 whether these results could apply to other countries ( within or even outside the european union ) is debatable , especially when some factors appeared to be culture - dependent , such as confidence in the doctor . for example , reimbursement status in france is almost independent of retirement , which is not the case in other countries . on the one hand , the edsq was developed according to a conceptual model and it was tested that this model applied in other countries . on the other hand , patient data collection will be requested to check whether this hypothesis will remain empirically valid . also , only 113 ( 63.1% ) patients of 179 were investigated for both edsq and compliance , possibly leading to selection bias , although internal validity ( explored by the bn ) might be less affected . we have not performed sample size estimation for a protocol aimed at confirming our findings without using the oversampling method . microsimulation based on the bn described and accounting for the prevalence of poor compliance might be useful to fix the size of a future experiment . second , we followed up patients who were willing to use travalert , and these patients might have different behavior from that of the general population . third , the results reported were observed in patients treated either by travatan or duotrav , so extrapolation of the bn results to other drugs needs to be documented . the small sample size of our survey did not allow for a test - retest - validation process . hence , the resulting structure should be viewed with caution , and new surveys should be conducted to validate our findings . also , it might well be worthwhile to undertake a validation of the structure , replicating our analysis with other structural learning algorithms and larger datasets . electronic devices have been recognized as an efficient way to assess compliance,19 but they may have limitations . in particular , one reason for poor compliance encountered by some patients is difficulty with instilling eye drops correctly.13,18 because travalert counts only the number of drops dispensed by a bottle , and can not verify that they are correctly instilled , our study could not control this aspect of the research . moreover , patients knew they were being observed , which might have impacted on their behavior . however , in a previous paper , we reported that these poor compliers had a significant increase in iop ( by about 2 mmhg ) in comparison with good compliers . therefore , this observational bias might have underestimated the impact of compliance on iop control . consequently , this bn identified poor compliers who had a lack of iop control of at least 2 mmhg , which is a clinically relevant difference . our sample size was not big enough to explore both the impact of clinician and patient characteristics on identifying poor compliers . in this approach , we favored data collected at a patient level . additional research might be useful for identifying the correct information to be collected at an ophthalmologist level . these limitations warrant a cautious interpretation of our results and further work with a larger sample size and test - retest methodology to confirm the findings . in conclusion , the application of bn to edsq data made it possible to re - examine the complex process of treatment compliance in glaucoma patients and to identify reasons for poor compliance . the crucial message for physicians from our results is that , before switching glaucoma treatments because of poor iop control , they should consider the patient s age , self - declared compliance , and how best to educate the patient about glaucoma and its treatment .

objective : to identify poorly compliant glaucoma patients , using the eye - drop satisfaction questionnaire ( edsq).methods : this was an observational cross - sectional study with compliance data collected by an electronic monitoring device . patients with primary open - angle glaucoma or ocular hypertension completed the edsq , a six - dimension self - reported questionnaire addressing treatment concern , disease concern , patient clinician relationship , positive beliefs , treatment convenience , and self - declared compliance . a bayesian network ( bn ) was applied to explore compliance associations with edsq.results:among 169 patients who completed the edsq , 113 had valid travalert data , of whom 25 ( 22.1% ) demonstrated low compliance . all six edsq dimensions were associated directly , or indirectly , with compliance . two profiles exhibited low compliance , ie , patients aged younger than 77.5 years with a poor patient physician relationship and self - declared poor compliance and patients aged older than 77.5 years with a poor patient physician relationship and self - declared good compliance . the third profile showed high compliance , ie , patients aged younger than 77.5 years with a good patient - physician relationship and self - declared good compliance.conclusion:our results confirm a central role for the patient physician relationship in the compliance process . age , self - declared compliance , and patient satisfaction with the patient physician relationship are all dimensions worth exploring before glaucoma medication is switched or proceeding to laser treatment or surgery .

Introduction Methods and materials Patients and study design Eye-Drop Satisfaction Questionnaire Statistical analyses Population analysis and oversampling strategy Bayesian networks Results Patient characteristics Bayesian networks Discussion Conclusion

glaucoma is the second leading cause of blindness globally.1 from 1991 to 1999 , primary open - angle glaucoma prevalence increased from 4.6% to 13.8% among the elderly.2 its treatment is aimed essentially at lowering intraocular pressure ( iop ) by eye drop instillations , reserving surgery or laser surgery for the most severe cases . several classes of medicine are available , ie , prostaglandin analogs , miotics , beta - blockers , alpha - adrenergic agonists , and carbonic anhydrase inhibitors . thus , higher adherence is associated with better iop control , on average,4 and a lower risk of eventual blindness.5 however , patients perceive few symptoms in the early stages , whereas eye drops ( with potential side effects ) are needed daily and may become a burden , leading to poor treatment adherence.6 adherence to treatment schedules has been examined by numerous studies in glaucoma , using various methods . for example , the medication possession ratio , determining the mean proportion of days during a given period when patients possess medication , was calculated from insurance claims or prescription databases,79 and from electronic devices capturing drop counts.10 alternatively , patients self - declared compliance was obtained from interviews1113 or standardized questionnaires.8,1418 another difference between studies were noncompliance criteria , eg , patients who missed more than two doses per week18 or possessed insufficient drops for the specified period ( medication possession ratio < 1).5 with this array of methodology across different drug classes and countries , compliance rates varied from 59% to 77%.7,11,14,16,1821 more informally , imperfect compliance is consistently reported among glaucoma patients . to improve glaucoma care , it is critical to identify patients who may not adhere to treatment . for example , complex dosing regimens have an impact on compliance.19,22 however , barriers cited by most studies relate to patients perception and knowledge about their illness and its treatment.7,11,16,18 these considerations prompted the development of an eye - drop satisfaction questionnaire ( edsq ) which asks patients to self - report their satisfaction and compliance with topical ophthalmic treatments.23 replies to these questions should be relevant to an exploration of noncompliance in glaucoma patients . a suitable technique for analyzing such data is a bayesian network ( bn ) which facilitates the representation and manipulation of information . a bn is a directed acyclic graph representing relationships between variables ( nodes in the graph ) with a related set of conditional probability tables that characterize local dependencies between the various nodes . the objective of this study was to identify poorly compliant glaucoma patients by exploring edsq data . the protocol was reviewed and approved by the comit consultatif sur le traitement de linformation en matire de recherche dans le domaine de la sant and the commission nationale informatique et libert.2426 this research was conducted according to the tenets of the declaration of helsinki.27 this multicenter study was conducted in france by 17 ophthalmologists ( at 17 sites ) specializing in the treatment of glaucoma , who also prescribed travalert , a computerized device that reminds patients to instill eye drops , assists with instillations , and records dosing times . patients included used the travalert device for at least eight weeks , were 18 years of age or older , and diagnosed with either primary open - angle glaucoma ( including juvenile glaucoma , exfoliative glaucoma , pigmentary glaucoma , or normal - tension glaucoma ) or high iop . patients with secondary glaucoma ( congenital glaucoma , inflammatory glaucoma , angle - closure or narrow - angle glaucoma following cataract surgery ) , or took an anticoagulant three times per day or more , and patients with chronic eye dryness requiring instillations of more than five drops a day , were excluded . according to a previous clustering analysis , data collected with the travalert device allowed the patient sample to be categorized according to three compliance profiles , ie , high compliance , moderate compliance , or low compliance.4 the edsq is a self - assessment questionnaire that assesses patient satisfaction and compliance with eye - drop treatment for glaucoma or iop . second , a comprehension evaluation was undertaken to ensure the questionnaire s clarity , ease of comprehension , and cultural equivalence.23 the questions are scored on a 15 likert scale . a scoring algorithm for the 21-item questionnaire was also developed and shown to have satisfactory validity and reliability.30 the edsq algorithm computes scores ( 0100 ) for six dimensions of attitudes towards eye - drop treatment , ie , treatment concern ( five items ) , disease concern ( two items ) , satisfaction with the patient clinician relationship ( five items ) ; positive beliefs ( three items ) , treatment convenience ( three items ) , and self - declared compliance ( three items ) . edsq data were collected at the follow - up visit when the travalert data were collected from the patient . patients who completed the edsq and provided sufficient travalert data to define their compliance profile were included in the analysis . an oversampling method was adopted to increase the ability of the bn to identify risk factors for noncompliance.31 this involved randomly selecting patients from the smallest group ( low compliance ) as successive replacements to create a sample equal to the largest group ( high and moderate compliance ) . it is a directed acyclic graph , ie , without cycles and has edges that are orientated . with a given order of nodes , it is simple to determine the best bn , because each node looks to its best parent , ie , its immediate predecessor amongst all available nodes . the goal of taboo order is to search for the order of nodes resulting in the best bn value ( highest minimum description length score ) , and to this end uses taboo search.33 this algorithm is fully driven by the data , is fully inferential , and with no clinical expert input . because bn requires categoric variables , edsq scores were categorized by dichotomizing ( thresholds were determined while estimating the bn ) them according to a decision tree - based algorithm aimed at maximizing the detection of low compliance . this multicenter study was conducted in france by 17 ophthalmologists ( at 17 sites ) specializing in the treatment of glaucoma , who also prescribed travalert , a computerized device that reminds patients to instill eye drops , assists with instillations , and records dosing times . patients included used the travalert device for at least eight weeks , were 18 years of age or older , and diagnosed with either primary open - angle glaucoma ( including juvenile glaucoma , exfoliative glaucoma , pigmentary glaucoma , or normal - tension glaucoma ) or high iop . patients with secondary glaucoma ( congenital glaucoma , inflammatory glaucoma , angle - closure or narrow - angle glaucoma following cataract surgery ) , or took an anticoagulant three times per day or more , and patients with chronic eye dryness requiring instillations of more than five drops a day , were excluded . according to a previous clustering analysis , data collected with the travalert device allowed the patient sample to be categorized according to three compliance profiles , ie , high compliance , moderate compliance , or low compliance.4 the edsq is a self - assessment questionnaire that assesses patient satisfaction and compliance with eye - drop treatment for glaucoma or iop . second , a comprehension evaluation was undertaken to ensure the questionnaire s clarity , ease of comprehension , and cultural equivalence.23 the questions are scored on a 15 likert scale . a scoring algorithm for the 21-item questionnaire was also developed and shown to have satisfactory validity and reliability.30 the edsq algorithm computes scores ( 0100 ) for six dimensions of attitudes towards eye - drop treatment , ie , treatment concern ( two items ) , satisfaction with the patient clinician relationship ( five items ) ; positive beliefs ( three items ) , treatment convenience ( three items ) , and self - declared compliance ( three items ) . edsq data were collected at the follow - up visit when the travalert data were collected from the patient . patients who completed the edsq and provided sufficient travalert data to define their compliance profile were included in the analysis . an oversampling method was adopted to increase the ability of the bn to identify risk factors for noncompliance.31 this involved randomly selecting patients from the smallest group ( low compliance ) as successive replacements to create a sample equal to the largest group ( high and moderate compliance ) . it is a directed acyclic graph , ie , without cycles and has edges that are orientated . a bn was constructed to identify poor compliers by discriminating between low - compliance and moderate- or high - compliance patients . with a given order of nodes , it is simple to determine the best bn , because each node looks to its best parent , ie , its immediate predecessor amongst all available nodes . the goal of taboo order is to search for the order of nodes resulting in the best bn value ( highest minimum description length score ) , and to this end uses taboo search.33 this algorithm is fully driven by the data , is fully inferential , and with no clinical expert input . because bn requires categoric variables , edsq scores were categorized by dichotomizing ( thresholds were determined while estimating the bn ) them according to a decision tree - based algorithm aimed at maximizing the detection of low compliance . patients who completed the edsq and provided sufficient travalert data to define their compliance profile were included in the analysis . an oversampling method was adopted to increase the ability of the bn to identify risk factors for noncompliance.31 this involved randomly selecting patients from the smallest group ( low compliance ) as successive replacements to create a sample equal to the largest group ( high and moderate compliance ) . it is a directed acyclic graph , ie , without cycles and has edges that are orientated . a bn was constructed to identify poor compliers by discriminating between low - compliance and moderate- or high - compliance patients . the network structure ( relationships between variables ) was based on data by applying the taboo order algorithm to scores derived from the bn learning algorithm.32 a score denoted the minimum description length , and was two fold ; the first part qualified the network with respect to its structural complexity and the second part measured the goodness - of - fit between the network and the data . with a given order of nodes , it is simple to determine the best bn , because each node looks to its best parent , ie , its immediate predecessor amongst all available nodes . the goal of taboo order is to search for the order of nodes resulting in the best bn value ( highest minimum description length score ) , and to this end uses taboo search.33 this algorithm is fully driven by the data , is fully inferential , and with no clinical expert input . because bn requires categoric variables , edsq scores were categorized by dichotomizing ( thresholds were determined while estimating the bn ) them according to a decision tree - based algorithm aimed at maximizing the detection of low compliance . of the glaucoma specialists , 88.2% were male , 29.4% worked in a public hospital setting , and had qualified on average 16.5 years earlier . they declared 36.7 visits per week for primary open - angle glaucoma and 11.4 for ocular hypertension . among the 140 transferred files , 113 ( 80.7% ) provided reliable data ( compliance fully documented for at least eight weeks ) sufficient for the classification algorithm . the gender ratio was balanced , mean age was 65.1 years , 16.6% had undergone surgery for primary open - angle glaucoma , and 10.1% had received laser therapy . the frequency of high compliance ( 56.6% ) was much greater than that of low compliance ( 22.1% ) . the bn ( figure 1 ) shows three nodes directly related to travalert - identified compliance , ie , patient treatment concern has three drivers , which are disease concern , gender , and positive beliefs , and itself impacts on the patient . second , patients with a disease concern score of < 47.018 and a positive beliefs score > 61.335 were also more likely to have a treatment concern score of > 24.197 . when compared with the total population , a higher percentage of patients with a treatment concern score < 24.197 had a patient more patients with a treatment convenience score of < 69.397 had a positive beliefs score < 61.335 compared with the total analysis population ( table 4 ) . a higher percentage of patients with a positive beliefs score < 61.335 had a self - declared compliance score < 89.583 , as compared with the analyzed population ( table 5 ) . compliance identified by travalert was related to three variables , these being age , patient three different combinations of these characteristics were relevant to low or high compliance , compared with the total sample ( figure 2 ) . low compliance was associated with two combinations ; first , age < 77.5 years , a self - declared compliance score < 89.5 , and a patient clinician relationship score < 67.5 and , second , age > 77.5 years , a self - declared compliance score of > 89.5 , and a patient by contrast , high compliance was associated with age < 77.5 years , a self - declared compliance score > 89.5 , and a patient the gender ratio was balanced , mean age was 65.1 years , 16.6% had undergone surgery for primary open - angle glaucoma , and 10.1% had received laser therapy . the frequency of high compliance ( 56.6% ) was much greater than that of low compliance ( 22.1% ) . the bn ( figure 1 ) shows three nodes directly related to travalert - identified compliance , ie , patient treatment concern has three drivers , which are disease concern , gender , and positive beliefs , and itself impacts on the patient two subgroups of patients had a noticeable distribution across groups defined according to treatment concern score ( table 2 ) . second , patients with a disease concern score of < 47.018 and a positive beliefs score > 61.335 were also more likely to have a treatment concern score of > 24.197 . when compared with the total population , a higher percentage of patients with a treatment concern score < 24.197 had a patient more patients with a treatment convenience score of < 69.397 had a positive beliefs score < 61.335 compared with the total analysis population ( table 4 ) . a higher percentage of patients with a positive beliefs score < 61.335 had a self - declared compliance score < 89.583 , as compared with the analyzed population ( table 5 ) . compliance identified by travalert was related to three variables , these being age , patient three different combinations of these characteristics were relevant to low or high compliance , compared with the total sample ( figure 2 ) . low compliance was associated with two combinations ; first , age < 77.5 years , a self - declared compliance score < 89.5 , and a patient clinician relationship score < 67.5 and , second , age > 77.5 years , a self - declared compliance score of > 89.5 , and a patient by contrast , high compliance was associated with age < 77.5 years , a self - declared compliance score > 89.5 , and a patient the primary objective of this study was to identify glaucoma patients who adhered poorly to treatment by recording their daily instillation times and eye drop counts objectively , with a computerized bottle holder ( travalert ) . in a previous analysis , patient demographics and baseline clinical parameters failed to discriminate between patients showing high , moderate , or low compliance.4 this second attempt enquired about attitudes towards treatment that might characterize poor compliers . this was explored with the edsq that examines patients views about eye drop treatments , with aspects relevant to glaucoma , in order to explore compliance profiles . the analysis identified two subgroups of patients at high risk of poor compliance when measured objectively ; first , patients younger than 77.5 years of age with low self - declared compliance and an unsatisfactory relationship with their physician and , second , patients older than 77.5 years of age with high self - declared compliance and an unsatisfactory relationship with their physician . physician relationship that provides education for patients about glaucoma and its treatment is essential for compliance.8,1113,16 conversely , because patients rely strongly on their doctor for such information , a poor relationship was identified as a major barrier to compliance.8,11,12,1618 interestingly , our results are not fully consistent with previous publications with respect to age and other factors associated with compliance . consequently , age was included with other factors ( patient clinician relationship and self - declared compliance ) , and was thereby related to objectively identified compliance ( travalert ) . second , trust in the efficacy of treatment ( closely related to our positive beliefs score ) has been previously related to compliance,8,12 as have been concerns about disease5 and treatment convenience.12,34 our bn analysis linked no equivalent edsq item directly to compliance . thus , bn may provide a more precise description of the compliance process , and may show how different conceptions of compliance , finally impact on the behavior of glaucoma patients . the numeric imbalance between compliant patients and poorly compliant patients jeopardized the quality of our results , so we considered possible simple solutions to the problem , ie , oversampling ( artificially increasing the size of the smallest group by random sampling and replacement ) , and undersampling ( reducing the size of the largest group by randomly deleting cases ) . despite evidence that undersampling has previously shown better results35,36 and that oversampling may increase the risk of overfitting,37 the latter method had to be used because of our very small dataset , ie , only 113 patients provided sufficient data to construct a compliance profile , and the low - compliance group included only 25 patients . also , only 113 ( 63.1% ) patients of 179 were investigated for both edsq and compliance , possibly leading to selection bias , although internal validity ( explored by the bn ) might be less affected . microsimulation based on the bn described and accounting for the prevalence of poor compliance might be useful to fix the size of a future experiment . second , we followed up patients who were willing to use travalert , and these patients might have different behavior from that of the general population . in particular , one reason for poor compliance encountered by some patients is difficulty with instilling eye drops correctly.13,18 because travalert counts only the number of drops dispensed by a bottle , and can not verify that they are correctly instilled , our study could not control this aspect of the research . our sample size was not big enough to explore both the impact of clinician and patient characteristics on identifying poor compliers . in conclusion , the application of bn to edsq data made it possible to re - examine the complex process of treatment compliance in glaucoma patients and to identify reasons for poor compliance . the crucial message for physicians from our results is that , before switching glaucoma treatments because of poor iop control , they should consider the patient s age , self - declared compliance , and how best to educate the patient about glaucoma and its treatment .

glaucoma is the second leading cause of blindness globally.1 from 1991 to 1999 , primary open - angle glaucoma prevalence increased from 4.6% to 13.8% among the elderly.2 its treatment is aimed essentially at lowering intraocular pressure ( iop ) by eye drop instillations , reserving surgery or laser surgery for the most severe cases . several classes of medicine are available , ie , prostaglandin analogs , miotics , beta - blockers , alpha - adrenergic agonists , and carbonic anhydrase inhibitors . thus , higher adherence is associated with better iop control , on average,4 and a lower risk of eventual blindness.5 however , patients perceive few symptoms in the early stages , whereas eye drops ( with potential side effects ) are needed daily and may become a burden , leading to poor treatment adherence.6 adherence to treatment schedules has been examined by numerous studies in glaucoma , using various methods . for example , the medication possession ratio , determining the mean proportion of days during a given period when patients possess medication , was calculated from insurance claims or prescription databases,79 and from electronic devices capturing drop counts.10 alternatively , patients self - declared compliance was obtained from interviews1113 or standardized questionnaires.8,1418 another difference between studies were noncompliance criteria , eg , patients who missed more than two doses per week18 or possessed insufficient drops for the specified period ( medication possession ratio < 1).5 with this array of methodology across different drug classes and countries , compliance rates varied from 59% to 77%.7,11,14,16,1821 more informally , imperfect compliance is consistently reported among glaucoma patients . to improve glaucoma care , it is critical to identify patients who may not adhere to treatment . for example , complex dosing regimens have an impact on compliance.19,22 however , barriers cited by most studies relate to patients perception and knowledge about their illness and its treatment.7,11,16,18 these considerations prompted the development of an eye - drop satisfaction questionnaire ( edsq ) which asks patients to self - report their satisfaction and compliance with topical ophthalmic treatments.23 replies to these questions should be relevant to an exploration of noncompliance in glaucoma patients . a suitable technique for analyzing such data is a bayesian network ( bn ) which facilitates the representation and manipulation of information . a bn is a directed acyclic graph representing relationships between variables ( nodes in the graph ) with a related set of conditional probability tables that characterize local dependencies between the various nodes . hence , it provides a powerful tool to study interdependencies between complex processes , such as patient behavior . the protocol was reviewed and approved by the comit consultatif sur le traitement de linformation en matire de recherche dans le domaine de la sant and the commission nationale informatique et libert.2426 this research was conducted according to the tenets of the declaration of helsinki.27 this multicenter study was conducted in france by 17 ophthalmologists ( at 17 sites ) specializing in the treatment of glaucoma , who also prescribed travalert , a computerized device that reminds patients to instill eye drops , assists with instillations , and records dosing times . at the inclusion visit , general comorbidities ( cardiovascular , central nervous system , hepatic diseases , diabetes , pulmonary diseases , digestive diseases , others ) and eye comorbidities ( macular degeneration , diabetic retinopathy , retinal detachment , cataract , uveitis , others ) were collected . patients included used the travalert device for at least eight weeks , were 18 years of age or older , and diagnosed with either primary open - angle glaucoma ( including juvenile glaucoma , exfoliative glaucoma , pigmentary glaucoma , or normal - tension glaucoma ) or high iop . patients with secondary glaucoma ( congenital glaucoma , inflammatory glaucoma , angle - closure or narrow - angle glaucoma following cataract surgery ) , or took an anticoagulant three times per day or more , and patients with chronic eye dryness requiring instillations of more than five drops a day , were excluded . according to a previous clustering analysis , data collected with the travalert device allowed the patient sample to be categorized according to three compliance profiles , ie , high compliance , moderate compliance , or low compliance.4 the edsq is a self - assessment questionnaire that assesses patient satisfaction and compliance with eye - drop treatment for glaucoma or iop . second , a comprehension evaluation was undertaken to ensure the questionnaire s clarity , ease of comprehension , and cultural equivalence.23 the questions are scored on a 15 likert scale . a scoring algorithm for the 21-item questionnaire was also developed and shown to have satisfactory validity and reliability.30 the edsq algorithm computes scores ( 0100 ) for six dimensions of attitudes towards eye - drop treatment , ie , treatment concern ( five items ) , disease concern ( two items ) , satisfaction with the patient clinician relationship ( five items ) ; positive beliefs ( three items ) , treatment convenience ( three items ) , and self - declared compliance ( three items ) . edsq data were collected at the follow - up visit when the travalert data were collected from the patient . an oversampling method was adopted to increase the ability of the bn to identify risk factors for noncompliance.31 this involved randomly selecting patients from the smallest group ( low compliance ) as successive replacements to create a sample equal to the largest group ( high and moderate compliance ) . a uniform ( all patients had the same probability to be sampled , without site stratification ) sampling method was used because the small sample size did not authorize strong distribution estimates . bns are often used to identify structure of the information when very few hypotheses could be identified a priori . algorithm to scores derived from the bn learning algorithm.32 a score denoted the minimum description length , and was two fold ; the first part qualified the network with respect to its structural complexity and the second part measured the goodness - of - fit between the network and the data . the goal of taboo order is to search for the order of nodes resulting in the best bn value ( highest minimum description length score ) , and to this end uses taboo search.33 this algorithm is fully driven by the data , is fully inferential , and with no clinical expert input . because bn requires categoric variables , edsq scores were categorized by dichotomizing ( thresholds were determined while estimating the bn ) them according to a decision tree - based algorithm aimed at maximizing the detection of low compliance . this multicenter study was conducted in france by 17 ophthalmologists ( at 17 sites ) specializing in the treatment of glaucoma , who also prescribed travalert , a computerized device that reminds patients to instill eye drops , assists with instillations , and records dosing times . at the inclusion visit , general comorbidities ( cardiovascular , central nervous system , hepatic diseases , diabetes , pulmonary diseases , digestive diseases , others ) and eye comorbidities ( macular degeneration , diabetic retinopathy , retinal detachment , cataract , uveitis , others ) were collected . patients included used the travalert device for at least eight weeks , were 18 years of age or older , and diagnosed with either primary open - angle glaucoma ( including juvenile glaucoma , exfoliative glaucoma , pigmentary glaucoma , or normal - tension glaucoma ) or high iop . patients with secondary glaucoma ( congenital glaucoma , inflammatory glaucoma , angle - closure or narrow - angle glaucoma following cataract surgery ) , or took an anticoagulant three times per day or more , and patients with chronic eye dryness requiring instillations of more than five drops a day , were excluded . according to a previous clustering analysis , data collected with the travalert device allowed the patient sample to be categorized according to three compliance profiles , ie , high compliance , moderate compliance , or low compliance.4 the edsq is a self - assessment questionnaire that assesses patient satisfaction and compliance with eye - drop treatment for glaucoma or iop . second , a comprehension evaluation was undertaken to ensure the questionnaire s clarity , ease of comprehension , and cultural equivalence.23 the questions are scored on a 15 likert scale . a scoring algorithm for the 21-item questionnaire was also developed and shown to have satisfactory validity and reliability.30 the edsq algorithm computes scores ( 0100 ) for six dimensions of attitudes towards eye - drop treatment , ie , treatment concern ( two items ) , satisfaction with the patient clinician relationship ( five items ) ; positive beliefs ( three items ) , treatment convenience ( three items ) , and self - declared compliance ( three items ) . edsq data were collected at the follow - up visit when the travalert data were collected from the patient . an oversampling method was adopted to increase the ability of the bn to identify risk factors for noncompliance.31 this involved randomly selecting patients from the smallest group ( low compliance ) as successive replacements to create a sample equal to the largest group ( high and moderate compliance ) . a uniform ( all patients had the same probability to be sampled , without site stratification ) sampling method was used because the small sample size did not authorize strong distribution estimates . bns are often used to identify structure of the information when very few hypotheses could be identified a priori . algorithm to scores derived from the bn learning algorithm.32 a score denoted the minimum description length , and was two fold ; the first part qualified the network with respect to its structural complexity and the second part measured the goodness - of - fit between the network and the data . with a given order of nodes , it is simple to determine the best bn , because each node looks to its best parent , ie , its immediate predecessor amongst all available nodes . the goal of taboo order is to search for the order of nodes resulting in the best bn value ( highest minimum description length score ) , and to this end uses taboo search.33 this algorithm is fully driven by the data , is fully inferential , and with no clinical expert input . because bn requires categoric variables , edsq scores were categorized by dichotomizing ( thresholds were determined while estimating the bn ) them according to a decision tree - based algorithm aimed at maximizing the detection of low compliance . an oversampling method was adopted to increase the ability of the bn to identify risk factors for noncompliance.31 this involved randomly selecting patients from the smallest group ( low compliance ) as successive replacements to create a sample equal to the largest group ( high and moderate compliance ) . a uniform ( all patients had the same probability to be sampled , without site stratification ) sampling method was used because the small sample size did not authorize strong distribution estimates . bns are often used to identify structure of the information when very few hypotheses could be identified a priori . a bn was constructed to identify poor compliers by discriminating between low - compliance and moderate- or high - compliance patients . the network structure ( relationships between variables ) was based on data by applying the taboo order algorithm to scores derived from the bn learning algorithm.32 a score denoted the minimum description length , and was two fold ; the first part qualified the network with respect to its structural complexity and the second part measured the goodness - of - fit between the network and the data . with a given order of nodes , it is simple to determine the best bn , because each node looks to its best parent , ie , its immediate predecessor amongst all available nodes . the goal of taboo order is to search for the order of nodes resulting in the best bn value ( highest minimum description length score ) , and to this end uses taboo search.33 this algorithm is fully driven by the data , is fully inferential , and with no clinical expert input . because bn requires categoric variables , edsq scores were categorized by dichotomizing ( thresholds were determined while estimating the bn ) them according to a decision tree - based algorithm aimed at maximizing the detection of low compliance . of the glaucoma specialists , 88.2% were male , 29.4% worked in a public hospital setting , and had qualified on average 16.5 years earlier . among one hundred and forty patients ( 78.2% ) used the travalert , brought it back to the visit , with successful data transfer to the investigator computer . technical criteria to participate to the clustering algorithm have been published elsewhere.4 the characteristics of patients meeting the inclusion criteria are presented in table 1 . the gender ratio was balanced , mean age was 65.1 years , 16.6% had undergone surgery for primary open - angle glaucoma , and 10.1% had received laser therapy . the bn ( figure 1 ) shows three nodes directly related to travalert - identified compliance , ie , patient treatment concern has three drivers , which are disease concern , gender , and positive beliefs , and itself impacts on the patient . second , patients with a disease concern score of < 47.018 and a positive beliefs score > 61.335 were also more likely to have a treatment concern score of > 24.197 . when compared with the total population , a higher percentage of patients with a treatment concern score < 24.197 had a patient more patients with a treatment convenience score of < 69.397 had a positive beliefs score < 61.335 compared with the total analysis population ( table 4 ) . a higher percentage of patients with a positive beliefs score < 61.335 had a self - declared compliance score < 89.583 , as compared with the analyzed population ( table 5 ) . low compliance was associated with two combinations ; first , age < 77.5 years , a self - declared compliance score < 89.5 , and a patient clinician relationship score < 67.5 and , second , age > 77.5 years , a self - declared compliance score of > 89.5 , and a patient by contrast , high compliance was associated with age < 77.5 years , a self - declared compliance score > 89.5 , and a patient the gender ratio was balanced , mean age was 65.1 years , 16.6% had undergone surgery for primary open - angle glaucoma , and 10.1% had received laser therapy . the bn ( figure 1 ) shows three nodes directly related to travalert - identified compliance , ie , patient treatment concern has three drivers , which are disease concern , gender , and positive beliefs , and itself impacts on the patient two subgroups of patients had a noticeable distribution across groups defined according to treatment concern score ( table 2 ) . second , patients with a disease concern score of < 47.018 and a positive beliefs score > 61.335 were also more likely to have a treatment concern score of > 24.197 . when compared with the total population , a higher percentage of patients with a treatment concern score < 24.197 had a patient more patients with a treatment convenience score of < 69.397 had a positive beliefs score < 61.335 compared with the total analysis population ( table 4 ) . a higher percentage of patients with a positive beliefs score < 61.335 had a self - declared compliance score < 89.583 , as compared with the analyzed population ( table 5 ) . low compliance was associated with two combinations ; first , age < 77.5 years , a self - declared compliance score < 89.5 , and a patient clinician relationship score < 67.5 and , second , age > 77.5 years , a self - declared compliance score of > 89.5 , and a patient by contrast , high compliance was associated with age < 77.5 years , a self - declared compliance score > 89.5 , and a patient the primary objective of this study was to identify glaucoma patients who adhered poorly to treatment by recording their daily instillation times and eye drop counts objectively , with a computerized bottle holder ( travalert ) . in a previous analysis , patient demographics and baseline clinical parameters failed to discriminate between patients showing high , moderate , or low compliance.4 this second attempt enquired about attitudes towards treatment that might characterize poor compliers . the analysis identified two subgroups of patients at high risk of poor compliance when measured objectively ; first , patients younger than 77.5 years of age with low self - declared compliance and an unsatisfactory relationship with their physician and , second , patients older than 77.5 years of age with high self - declared compliance and an unsatisfactory relationship with their physician . physician relationship that provides education for patients about glaucoma and its treatment is essential for compliance.8,1113,16 conversely , because patients rely strongly on their doctor for such information , a poor relationship was identified as a major barrier to compliance.8,11,12,1618 interestingly , our results are not fully consistent with previous publications with respect to age and other factors associated with compliance . first , we found a relationship between age and compliance that was not observed in several studies.11,14,18,20,34 however , in contrast with customary statistical methods , we used bn based on association tables and inevitably model interaction effects between variables . second , trust in the efficacy of treatment ( closely related to our positive beliefs score ) has been previously related to compliance,8,12 as have been concerns about disease5 and treatment convenience.12,34 our bn analysis linked no equivalent edsq item directly to compliance . in our study , the absence of direct links between travalert - identified compliance and previously identified components of compliance can be explained by the notion of conditional independence . thus , bn may provide a more precise description of the compliance process , and may show how different conceptions of compliance , finally impact on the behavior of glaucoma patients . the numeric imbalance between compliant patients and poorly compliant patients jeopardized the quality of our results , so we considered possible simple solutions to the problem , ie , oversampling ( artificially increasing the size of the smallest group by random sampling and replacement ) , and undersampling ( reducing the size of the largest group by randomly deleting cases ) . despite evidence that undersampling has previously shown better results35,36 and that oversampling may increase the risk of overfitting,37 the latter method had to be used because of our very small dataset , ie , only 113 patients provided sufficient data to construct a compliance profile , and the low - compliance group included only 25 patients . the age and gender ratios of our sample are close to those of glaucoma - treated patients in france.38 whether these results could apply to other countries ( within or even outside the european union ) is debatable , especially when some factors appeared to be culture - dependent , such as confidence in the doctor . however , in a previous paper , we reported that these poor compliers had a significant increase in iop ( by about 2 mmhg ) in comparison with good compliers . in conclusion , the application of bn to edsq data made it possible to re - examine the complex process of treatment compliance in glaucoma patients and to identify reasons for poor compliance . the crucial message for physicians from our results is that , before switching glaucoma treatments because of poor iop control , they should consider the patient s age , self - declared compliance , and how best to educate the patient about glaucoma and its treatment .

the lens of the eye is recognized as one of the most radio - sensitive tissues in the human body , and radiation - induced cataract is a well - known adverse effect . from a review of epidemiologic data , the threshold dose for cataract formation may be judged to be 0.5 gy [ 1 , 2 ] . a recent draft report by the international commission on radiological protection ( icrp ) documented that better techniques for detecting , quantifying and documenting early radiation - associated lens changes , as well as better dosimetry , have potentially contributed to recent findings of radiation cataract risk at lower exposure levels than previously considered . for occupational exposure , a new icrp positional statement recommended that an equivalent dose limit for the lens be reduced from 150 msv / year to 20 msv / year , averaged over defined periods of 5 years , with no single year exceeding 50 msv . the cross - sectional data from astronauts and matched subjects found using an automated anterior eye segment analysis system ( eas-1000 , nidek , aichi , japan ) were analyzed and revealed a small deleterious effect of space radiation for cortical cataracts and possibly for psc cataracts . we hypothesized that interventional radiologists ( irs ) are exposed to low - dose scattered radiation , which may cause radiation - related lens changes well before they would otherwise be appreciated by slit - lamp examination , and may therefore be picked up by directed objective scoring systems [ 5 , 6 ] . even recent studies [ 7 , 8 ] lack quantitative assessment of radiation - induced lens changes utilizing the metric variable of light scattering intensities ( lsis ) . we and others believe that lsis represent focal aggregated proteins that form from various effects , such as radiation exposure , and that an increase in lsis might be predictive of cataract formation [ 9 , 10 ] . to evaluate minimal lsi changes in specific regions in the lens of the eye , we carried out a pilot study on our described exposed subjects and unexposed subjects using eas-1000 . following institutional review board ( irb ) approval , written informed consent was obtained from all participants . all exposed subjects were japanese volunteer male irs in the japanese society of interventional radiology ( jsir ) , and all unexposed subjects were japanese volunteer male employees of medical service companies with no history of occupational radiation exposure . exposed subjects ' examinations were conducted during the annual jsir meeting in osaka , japan , on 1819 may 2006 , and unexposed subjects ' examinations took place in tokyo , japan , 31 may and 1 june 2007 . examiners were aware of the status of the two groups , specifically whether they were exposed or unexposed subjects . eye examinations were performed following a health survey questionnaire inquiring about eye health , general medical health and lifestyle . questions included age , radiation exposure in interventional procedures ( yes / no ) , smoking ( smoking index = cigarettes smoked per day years of smoking ) , drinking ( ever / never ) , wearing glasses ( ever / never ) and solar ultraviolet ray exposure during working hours ( working indoors only , versus working both indoors and outdoors ) . for the exposed subjects of irs the survey also included a self - report of professional experience as an ir . a team of ophthalmologists performed all initial examinations . to screen suitable subjects for pharmacologic mydriasis , an ophthalmologist performed an initial screening slit - lamp examination ( slit lamp bq 900 haag - streit , koeniz , switzerland ) of the left eye anterior eye segment , in addition to intraocular pressure measurement ( non - contact totometer nt-4000 , nidek , aichi , japan ) . one hundred and sixty exposed subjects and 326 unexposed subjects were included following screening and mydriasis safety assessment , and subsequently underwent examination of the left eye with pharmacologic pupillary dilation using a drop of midrin - p ( tropicamide and phenylephrine ; santen pharmaceuticals , japan ) . because the examinations were performed in the daytime while exposed subjects were attending their annual professional society meeting , and during the daytime while the unexposed subjects were also working , only single eye pharmacologic dilations were performed . therefore only examination of the left eye was conducted . the left side was specifically chosen since the ir 's left eye is closer than the right eye to the x - ray tube in the vast majority of instances . in addition a recent study showed that the dose on the side nearest to the x - ray tube was three to five times greater than for the farthest side of the head . for all qualified subjects the lens of the left eye was examined with scheimpflug slit images obtained using eas-1000 , with a representative slit image shown in fig . 1 . scheimpflug photography and densitometric image analysis are techniques applied to light scattering measurement and biometry in the anterior eye segment . they reproducibly characterize the anterior eye and allow discrimination of minimal light scattering changes , using a grading system from otherwise aging , disease or toxic effects . these tools help quantify threshold levels or maximum allowable dosages of physical and chemical noxious factors , which are causative or associative with opacifying ocular pathologies . therefore , most epidemiological studies dealing with ocular pathologies in the lens use either cataract scoring or the scheimpflug technique . fig . 1.scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . the peak light scattering intensity for each segment is demonstrated to the right of each respective layer . scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . the peak light scattering intensity for each segment is demonstrated to the right of each respective layer . one experienced technician performed all examinations using the eas-1000 . the highest quality scheimpflug photograph obtained in one session was recorded , and quantitative analysis of the crystalline lens lsis was provided and saved as an 8-bit digital value . the data of exposed and unexposed subjects were analyzed at the same time , although the technologist was not blinded to the condition of exposure status . the peak lsi value was measured in each of the six layers of the lens automatically and measured points were confirmed manually . these six layers included the anterior capsule , the most transparent layer of the anterior superficial cortex , the anterior adult nucleus , the anterior fetal nucleus , the central clear zone and the posterior subcapsular region ( psr ) ( fig . subjects with cataract risk factors other than occupational radiation exposure were excluded from analysis to increase the comparability of the groups and reduce the influence of group bias risk . such other factors included a history of diabetes mellitus ( exposed subjects n= 4 ; unexposed subjects n= 6 ) , eye trauma ( exposed subjects n= 0 ; unexposed subjects n= 1 ) , ophthalmic surgery ( exposed subjects n= 1 ; unexposed subjects n= 4 ) and ocular inflammation ( exposed subjects n= 0 ; unexposed subjects n= 2 ) . subjects with uncorrectable factors causing poor - quality photographs on the eas-1000 were also excluded ( exposed subjects n= 15 ; unexposed subjects n= 35 ) . poor photographic factors included insufficient pupillary dilation ( exposed subjects n= 44 ; unexposed subjects n= 62 ) , congenital cataracts ( exposed subjects n= 5 ; unexposed subjects n= 9 ) , excessive myopia by a spherical equivalent ( se ) of less than 6.00 diopters ( exposed subjects n= 31 ; unexposed subjects n= 61 ) , excessive hypermetropia by an se larger than 5.25 diopters ( exposed subjects and unexposed subjects n= 0 ) , ocular hypertension ( tonometer pressure > 20 mmhg ) ( exposed subjects n= 8 ; unexposed subjects n= 7 ) , corneal abnormalities ( exposed subjects n= 2 ; unexposed subjects n= 15 ) and miscellaneous contributory congenital ocular abnormalities ( exposed subjects n= 4 ; unexposed subjects n= 7 ) . subjects with an insufficient number of lens images to permit proper tabulation were also excluded . after consideration of the above exclusion criteria , 92 of the 160 exposed subjects and 155 of the 326 unexposed subjects were excluded . in the final count , many excluded subjects had more than one exclusion criterion . the eligible population for quantitative analyses consisted of 68 exposed subjects and 171 unexposed subjects . none of the selected subjects had any visual complaints or any subjective decrease in visual acuity . age - related increases in lsis have been demonstrated in prior investigations as a physiological change . to reduce the effect of aging , the eligible subjects were matched and selected with a chosen ratio of one ir to two randomly selected unexposed subjects within 1-year difference . the age - matched group consisted of 54 exposed subjects and 108 unexposed subjects . for intergroup comparison , student 's t - test was performed for descriptive characteristics and light scattering intensity in the six lens layers . multiple regression analyses were carried out using the stepwise method to evaluate lsis in the six layers of the left lens adjusting for age , smoking , drinking , wearing glasses , sunlight exposure during working hours and scattered radiation exposure during working hours . the selected variables were evaluated with an automatic model selection procedure using a sequence of f - tests in the stepwise method ( the pvalue for additional variables was 0.25 and for removal of variables was 0.10 ) . comparison of the average lsis between the age - matched groups was performing using analysis of variance ( anova ) . following institutional review board ( irb ) approval , written informed consent was obtained from all participants . all exposed subjects were japanese volunteer male irs in the japanese society of interventional radiology ( jsir ) , and all unexposed subjects were japanese volunteer male employees of medical service companies with no history of occupational radiation exposure . exposed subjects ' examinations were conducted during the annual jsir meeting in osaka , japan , on 1819 may 2006 , and unexposed subjects ' examinations took place in tokyo , japan , 31 may and 1 june 2007 . examiners were aware of the status of the two groups , specifically whether they were exposed or unexposed subjects . eye examinations were performed following a health survey questionnaire inquiring about eye health , general medical health and lifestyle . questions included age , radiation exposure in interventional procedures ( yes / no ) , smoking ( smoking index = cigarettes smoked per day years of smoking ) , drinking ( ever / never ) , wearing glasses ( ever / never ) and solar ultraviolet ray exposure during working hours ( working indoors only , versus working both indoors and outdoors ) . for the exposed subjects of irs the survey also included a self - report of professional experience as an ir . a team of ophthalmologists performed all initial examinations . to screen suitable subjects for pharmacologic mydriasis , an ophthalmologist performed an initial screening slit - lamp examination ( slit lamp bq 900 haag - streit , koeniz , switzerland ) of the left eye anterior eye segment , in addition to intraocular pressure measurement ( non - contact totometer nt-4000 , nidek , aichi , japan ) . one hundred and sixty exposed subjects and 326 unexposed subjects were included following screening and mydriasis safety assessment , and subsequently underwent examination of the left eye with pharmacologic pupillary dilation using a drop of midrin - p ( tropicamide and phenylephrine ; santen pharmaceuticals , japan ) . because the examinations were performed in the daytime while exposed subjects were attending their annual professional society meeting , and during the daytime while the unexposed subjects were also working , only single eye pharmacologic dilations were performed . therefore only examination of the left eye was conducted . the left side was specifically chosen since the ir 's left eye is closer than the right eye to the x - ray tube in the vast majority of instances . in addition a recent study showed that the dose on the side nearest to the x - ray tube was three to five times greater than for the farthest side of the head . for all qualified subjects the lens of the left eye was examined with scheimpflug slit images obtained using eas-1000 , with a representative slit image shown in fig . 1 . scheimpflug photography and densitometric image analysis are techniques applied to light scattering measurement and biometry in the anterior eye segment . they reproducibly characterize the anterior eye and allow discrimination of minimal light scattering changes , using a grading system from otherwise aging , disease or toxic effects . these tools help quantify threshold levels or maximum allowable dosages of physical and chemical noxious factors , which are causative or associative with opacifying ocular pathologies . therefore , most epidemiological studies dealing with ocular pathologies in the lens use either cataract scoring or the scheimpflug technique . fig . 1.scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . the peak light scattering intensity for each segment is demonstrated to the right of each respective layer . scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . the peak light scattering intensity for each segment is demonstrated to the right of each respective layer . one experienced technician performed all examinations using the eas-1000 . the highest quality scheimpflug photograph obtained in one session was recorded , and quantitative analysis of the crystalline lens lsis was provided and saved as an 8-bit digital value . the data of exposed and unexposed subjects were analyzed at the same time , although the technologist was not blinded to the condition of exposure status . the peak lsi value was measured in each of the six layers of the lens automatically and measured points were confirmed manually . these six layers included the anterior capsule , the most transparent layer of the anterior superficial cortex , the anterior adult nucleus , the anterior fetal nucleus , the central clear zone and the posterior subcapsular region ( psr ) ( fig . subjects with cataract risk factors other than occupational radiation exposure were excluded from analysis to increase the comparability of the groups and reduce the influence of group bias risk . such other factors included a history of diabetes mellitus ( exposed subjects n= 4 ; unexposed subjects n= 6 ) , eye trauma ( exposed subjects n= 0 ; unexposed subjects n= 1 ) , ophthalmic surgery ( exposed subjects n= 1 ; unexposed subjects n= 4 ) and ocular inflammation ( exposed subjects n= 0 ; unexposed subjects n= 2 ) . subjects with uncorrectable factors causing poor - quality photographs on the eas-1000 were also excluded ( exposed subjects n= 15 ; unexposed subjects n= 35 ) . poor photographic factors included insufficient pupillary dilation ( exposed subjects n= 44 ; unexposed subjects n= 62 ) , congenital cataracts ( exposed subjects n= 5 ; unexposed subjects n= 9 ) , excessive myopia by a spherical equivalent ( se ) of less than 6.00 diopters ( exposed subjects n= 31 ; unexposed subjects n= 61 ) , excessive hypermetropia by an se larger than 5.25 diopters ( exposed subjects and unexposed subjects n= 0 ) , ocular hypertension ( tonometer pressure > 20 mmhg ) ( exposed subjects n= 8 ; unexposed subjects n= 7 ) , corneal abnormalities ( exposed subjects n= 2 ; unexposed subjects n= 15 ) and miscellaneous contributory congenital ocular abnormalities ( exposed subjects n= 4 ; unexposed subjects n= 7 ) . subjects with an insufficient number of lens images to permit proper tabulation were also excluded . after consideration of the above exclusion criteria , 92 of the 160 exposed subjects and 155 of the 326 unexposed subjects were excluded . in the final count , many excluded subjects had more than one exclusion criterion . the eligible population for quantitative analyses consisted of 68 exposed subjects and 171 unexposed subjects . none of the selected subjects had any visual complaints or any subjective decrease in visual acuity . age - related increases in lsis have been demonstrated in prior investigations as a physiological change . to reduce the effect of aging , the eligible subjects were matched and selected with a chosen ratio of one ir to two randomly selected unexposed subjects within 1-year difference . for intergroup comparison , student 's t - test was performed for descriptive characteristics and light scattering intensity in the six lens layers . multiple regression analyses were carried out using the stepwise method to evaluate lsis in the six layers of the left lens adjusting for age , smoking , drinking , wearing glasses , sunlight exposure during working hours and scattered radiation exposure during working hours . the selected variables were evaluated with an automatic model selection procedure using a sequence of f - tests in the stepwise method ( the pvalue for additional variables was 0.25 and for removal of variables was 0.10 ) . comparison of the average lsis between the age - matched groups was performing using analysis of variance ( anova ) . in the screening examination for all subjects without exclusion , slit - lamp examination of the left eye under pupillary dilation showed posterior subcapsular cataract ( psc ) in three ( 1.9% ) of 160 exposed subjects ( 45 , 62 and 63 years ) and one ( 0.3% ) of 326 unexposed subjects ( 51 years ) . one exposed subject showed a dense patchy opacity and two showed vacuoles in the psr on retroillumination images . all four subjects were excluded from quantitative analyses because they met other predetermined exclusion criteria ( two had excessive myopia and one had poor pupillary dilation in the exposed subjects , and one had excessive myopia in the unexposed subjects ) . 8539.8 690.0059smoking ( yes ) , n(%)40 ( 58.8)115 ( 67.2)0.180smoking index1770 28.5211.7 18.00.06drinking ( yes ) , n(%)53 ( 77.9)147 ( 85.9)0.080wearing glasses ( yes ) , n(%)54 ( 79.4)120 ( 70.5)0.101workplace ( indoors only ) , n(%)68 ( 100)82 ( 48.0)<0.0001years of interventional experience,15.6 7.8na median ( minimum maximum)14 ( 132)total number of angiographies,1846.1 1998.6na median ( minimum maximum)1500 ( 010 000)total number of interventions,1982.3 2056.5na median ( minimum maximum)1200 ( 1012 000)irs = interventional radiologists , values are means sd , na = not applicable . descriptive characteristics of the study subjects who met the eligibility criteria irs = interventional radiologists , values are means sd , na = not applicable . the characteristics of the study subjects for quantitative analysis are shown in table 1 . there was a significant difference in the ages of the two groups , with the exposed subjects showing a greater age than the unexposed subjects . there were significant differences in the lsis in five out of six eye layers when comparing exposed subjects with the unexposed subjects . table 2.light scattering intensity in the six lens layers between exposed and unexposed group ( 8-bit grayscale value)layer of lensirscontrolspvalue(n= 68)(n= 171)anterior capsule63.0 7.860.3 7.90.020most transparent layer of the anterior superficial cortex41.1 8.339.5 7.70.157anterior adult nucleus96.6 33.081.3 25.50.0002anterior fetal nucleus50.7 11.547.4 10.90.039central clear zone38.3 9.635.3 8.40.018posterior subcapsular region39.7 light scattering intensity in the six lens layers between exposed and unexposed group ( 8-bit grayscale value ) values are means sd . the results of the stepwise method for lsi assessment in each of the six layers of the eye lens are shown in table 3 . in all six layers of the eye lens , the variable of age was statistically significant for increased lsis . in the layer of the central clear zone , smoking was also significant for increased lsis ( p= 0.002 ) . in the psr , radiation exposure from interventional procedures was significant ( p= 0.012 ) for increased lsis following adjustment for the other variables including age . table 3.evaluation of light scattering intensity in the six lens layers by multiple regression analysis using the stepwise method in all subjects ( n= 239)layer of lensvariableparameter estimate ( 95% ci)pvalueanterior capsuleage0.691 ( 0.6160.766)<0.0001most transparent layer of the anterior superficial cortexage0.153 ( 0.0350.270)0.011workplace0.580 ( 0.2911.452)0.191smoking0.003 ( 0.0070.001)0.094anterior adult nucleusradiation exposure2.307 ( 0.6205.233)0.122age2.644 ( 2.2882.999)<0.0001work place1.793 ( 0.9104.496)0.192anterior fetal nucleusage1.405 ( 1.2351.574)<0.0001smoking0.003 ( 0.0020.008)0.290wearing glasses1.072 ( 2.7850.641)0.219central clear zoneage1.132 ( 1.0571.207)<0.0001smoking0.004 ( 0.0010.006)0.002posterior subcapsular regionradiation exposure2.089 ( 0.4663.711)0.012age0.307 ( 0.528 to 0.086)0.007smoking0.004 ( 0.0100.003)0.277wearing glasses0.925 ( 0.7352.586)0.273ci = confidence interval . evaluation of light scattering intensity in the six lens layers by multiple regression analysis using the stepwise method in all subjects ( n= 239 ) ci = confidence interval . the mean age standard deviation ( sd ) of age - matched exposed subjects and unexposed subjects were 39.5 6.9 and 39.4 6.9 , respectively . in the age - matched comparison , there was a significant difference in lsis in the psr ( table 4 ) . table 4.evaluation of light scattering intensity in the six layers between exposed and unexposed group by age - matched comparison ( 8-bit grayscale value)layer of lensirscontrolspvalue(n= 54)(n= 108)anterior capsule61.3 7.359.4 7.30.10most transparent layer of the anterior superficial cortex41.1 8.838.7 7.60.07anterior adult nucleus87.6 26.982.2 26.90.23anterior fetal nucleus47.7 10.347.2 11.10.80central clear zone35.6 8.435.1 8.60.75posterior subcapsular region40.5 13.434.5 7.20.0031values are means sd . evaluation of light scattering intensity in the six layers between exposed and unexposed group by age - matched comparison ( 8-bit grayscale value ) values are means sd . lens opacities were classified into three types according to their anatomical location : cortical region , nucleus and psr . it has been demonstrated that high - dose ionizing radiation primarily associated with psc can also induce cortical cataract formation [ 12 , 13 ] . in the widely used grading system for radiation - induced cataracts by slit - lamp examination , the early stage of psc shows a lens change consisting of the formation of small dots and vacuoles . in a cross - sectional study in interventional cardiologists , pscs were significantly more frequent among interventional cardiologists than the control subjects . the increased lsis in exposed subjects compared with unexposed subjects analyzed by multiple regression analysis and age - matched comparison suggest a potential adverse effect on the lens with low - dose radiation exposure . in spite of the minimal increased lsis , the functional effect of these increased lsis on visual acuity and undesirable risk is unclear . this phenomenon may be a predictor of a primary effect of low - dose radiation on the eye . smoking is independently recognized to correlate with a higher risk of incident nuclear cataract development . our findings regarding smoking reinforce previous studies and suggest that the increase in lsis in the central clear zone may be an early sign of nuclear cataracts . in our study , the variable of age was significant for lsis in all six layers of the eye lens . we performed multiple regression analyses including exposed subjects and unexposed subjects to adjusting the variables . other factors such as ultraviolet light exposure , diabetes , renal failure , drug intake and malnutrition also individually and collectively contribute over time to further increase light scattering and can not be readily separated from aging . lsis in the six layers of the lens increase in the anterior five layers and decrease in the psr in a linear manner with aging . this decrease in apparent lsis in the psr over time is likely related to age - related reduction in lens transparency . in the exposed subjects , therefore radiation exposure shows a positive relation to lsis in the psr . in the most transparent layer of the anterior superficial cortex , lsis did not show a significant difference between irs and controls , and the parameter estimate of age was relative lower than for the other layers . this may be related to the fact that the age - related cortical cataract began on the outer edge of the lens cortex and slowly extended to the center . regarding ir dose , the average lens dose received by japanese radiologists measured by thermoluminescent dosimeters ( tlds ) during hepatocellular carcinoma embolization has been calculated at an average dose per procedure of approximately 0.04 msv . the average estimated cumulative dose in exposed subjects was 79.3 msv ( mean number of interventional procedure = 1982.3 ) . the estimated annual dose was 5.1 msv ( mean annual number of interventional procedure = 127.1 ) . a multi - center study with continuous 2-month dose readings demonstrated extrapolated annual radiation doses at the collar badge of 48 msv in the mean annual number of interventional procedure of 972 . the estimated average lens dose in interventional cardiologists with estimated cumulative occupational dose considering eye protection was 6.0 6.6 sv ( 0.127 sv ) and 3.7 7.5 gy ( 0.0243 gy ) [ 7 , 8 ] . it is estimated that the lens dose of exposed subjects in our study is lower than that of previous reports . in our screening examination , the detection rate of the psc in the exposed subjects is 1.9% , considerably lower than the 38% and 52% reported in interventional cardiologists [ 7 , 8 ] . this lower incidence of psc may also be a collateral finding of lower radiation exposure . lens dosimetry was not performed in this retrospective study since two categories of information were unavailable ; first , collar - badge readings were not uniformly available for all practitioners and second , existing under - apron individual monitoring data were unreliable due to inconstant usage of personal dosimeter , employment changes and also the fact that full versus part - time employment was not noted , neither was individual positioning and wearing of protection devices . due to the aforementioned challenges , we chose not to include estimated lens dose , which can not be considered in such circumstances as objectively quantifiable , and we therefore chose to simply register presence or absence of radiation exposure status ( yes or no ) as the categorical variable . individual dosimetry remains problematic , and alternative strategies will be needed in order to more precisely evaluate the effects of radiation in specific occupational situations , since strict individual monitoring and strategic management will be necessary . one of the largest study limitations is our choice of a single left - eye examination for the purposes of this study ; the laterality of lsis of the lens could not be assessed . in the oramed ( optimization of radiation protection for medical staff ) project , the dosimeter on the near side eye to the x - ray tube showed a higher dose than the dosimeter in the region between the eyes . and , in the study of dose distribution with ten tlds on the eyebrow ridge , the dose on the side nearest to the x - ray tube was three to five times greater than those on the farthest side . it was recommended that dosimeters for the monitoring of eye dosage should be positioned on the side of the brow ridge adjacent to the x - ray tube . because the majority of exposed subjects were primarily abdominal oncologic vascular interventionalists and the physician 's left eye was commonly closer to the x - ray tube , we believed that the left - sided eye examination in this study was more likely to evaluate the side with the highest exposure . further study is required that includes lens dosimetry and evaluation of lsis on each side . our study limitations include our group choices for exposed subjects and unexposed subjects ; a more comparable control group may have been physicians without radiation exposure . similarly , we had numerous exclusion criteria that were only answered and served as exclusions following initial screening ophthalmologic examination . we felt that rigid adherence to the exclusionary criteria was necessary for the purposes of data integrity , although it resulted in a large number of exclusions reducing the sample size . the questionnaire was also imperfect ; unfortunately questions regarding any medical history of ionizing radiation exposure to the head and neck , such as head computed tomography , were not included . our single meridian cross - sectional analyses by eas-1000 were performed in the mid - sagittal plane of the eye lens , therefore , there were no data obtained for off - center , off - angle and off - axis portions of the lens . as has been previously reported with the eas-1000 , the correlation coefficient in scatter light intensity measurement as measured over a 2-week interval may be greater than or equal to r= 0.9 , additionally interoperator measurement error has previously been measured to range from 8 to 10% . in order to confirm the reproducibility of the eas-1000 , mean individual coefficient of variation was segmentally measured in four different lens regions with a 1-week interval in measurements , and the variation was measured as between 3.6 and 5.1% , in keeping with the previously described examination . as previously alluded to , a potential limitation of our study is also that the technologist was unblinded , although the automated nature of the lsi measurements for data collection , and the reliable proscriptive nature of mid - sagittal plane selection , suggests this is an unlikely cause for concern . in conclusion , we found that there were minimal increased lsis in the psr of the lens in exposed subjects as compared with the unexposed subjects in this pilot study . such findings will need to be longitudinally followed to establish their predictive value as far as cataractogenesis is concerned . further quantitative studies concerning minimal radiation - related lens changes may additionally be required especially in low - dose exposure groups .

to evaluate low - dose x - ray radiation effects on the eye by measuring the amount of light scattering in specific regions of the lens , we compared exposed subjects ( interventional radiologists ) with unexposed subjects ( employees of medical service companies ) , as a pilot study . according to numerous exclusionary rules , subjects with confounding variables contributing to cataract formation were excluded . left eye examinations were performed on 68 exposed subjects and 171 unexposed subjects . the eye examinations consisted of an initial screening examination , followed by scheimpflug imaging of the lens using an anterior eye segment analysis system . the subjects were assessed for the quantity of light scattering intensities found in each of the six layers of the lens . multiple stepwise regression analyses were performed with the stepwise regression for six variables : age , radiation exposure , smoking , drinking , wearing glasses and workplace . in addition , an age - matched comparison between exposed and unexposed subjects was performed . minimal increased light scattering intensity in the posterior subcapsular region showed statistical significance . our results indicate that occupational radiation exposure in interventional radiologists may affect the posterior subcapsular region of the lens . since by its very nature this retrospective study had many limitations , further well - designed studies concerning minimal radiation - related lens changes should be carried out in a low - dose exposure group .

INTRODUCTION MATERIALS AND METHODS Screening examination and subjects Quantitative analyses Selection of subjects Statistical analysis RESULTS DISCUSSION

the lens of the eye is recognized as one of the most radio - sensitive tissues in the human body , and radiation - induced cataract is a well - known adverse effect . the cross - sectional data from astronauts and matched subjects found using an automated anterior eye segment analysis system ( eas-1000 , nidek , aichi , japan ) were analyzed and revealed a small deleterious effect of space radiation for cortical cataracts and possibly for psc cataracts . we hypothesized that interventional radiologists ( irs ) are exposed to low - dose scattered radiation , which may cause radiation - related lens changes well before they would otherwise be appreciated by slit - lamp examination , and may therefore be picked up by directed objective scoring systems [ 5 , 6 ] . even recent studies [ 7 , 8 ] lack quantitative assessment of radiation - induced lens changes utilizing the metric variable of light scattering intensities ( lsis ) . to evaluate minimal lsi changes in specific regions in the lens of the eye , we carried out a pilot study on our described exposed subjects and unexposed subjects using eas-1000 . all exposed subjects were japanese volunteer male irs in the japanese society of interventional radiology ( jsir ) , and all unexposed subjects were japanese volunteer male employees of medical service companies with no history of occupational radiation exposure . exposed subjects ' examinations were conducted during the annual jsir meeting in osaka , japan , on 1819 may 2006 , and unexposed subjects ' examinations took place in tokyo , japan , 31 may and 1 june 2007 . questions included age , radiation exposure in interventional procedures ( yes / no ) , smoking ( smoking index = cigarettes smoked per day years of smoking ) , drinking ( ever / never ) , wearing glasses ( ever / never ) and solar ultraviolet ray exposure during working hours ( working indoors only , versus working both indoors and outdoors ) . to screen suitable subjects for pharmacologic mydriasis , an ophthalmologist performed an initial screening slit - lamp examination ( slit lamp bq 900 haag - streit , koeniz , switzerland ) of the left eye anterior eye segment , in addition to intraocular pressure measurement ( non - contact totometer nt-4000 , nidek , aichi , japan ) . one hundred and sixty exposed subjects and 326 unexposed subjects were included following screening and mydriasis safety assessment , and subsequently underwent examination of the left eye with pharmacologic pupillary dilation using a drop of midrin - p ( tropicamide and phenylephrine ; santen pharmaceuticals , japan ) . because the examinations were performed in the daytime while exposed subjects were attending their annual professional society meeting , and during the daytime while the unexposed subjects were also working , only single eye pharmacologic dilations were performed . in addition a recent study showed that the dose on the side nearest to the x - ray tube was three to five times greater than for the farthest side of the head . for all qualified subjects the lens of the left eye was examined with scheimpflug slit images obtained using eas-1000 , with a representative slit image shown in fig . scheimpflug photography and densitometric image analysis are techniques applied to light scattering measurement and biometry in the anterior eye segment . 1.scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . the data of exposed and unexposed subjects were analyzed at the same time , although the technologist was not blinded to the condition of exposure status . the peak lsi value was measured in each of the six layers of the lens automatically and measured points were confirmed manually . these six layers included the anterior capsule , the most transparent layer of the anterior superficial cortex , the anterior adult nucleus , the anterior fetal nucleus , the central clear zone and the posterior subcapsular region ( psr ) ( fig . subjects with cataract risk factors other than occupational radiation exposure were excluded from analysis to increase the comparability of the groups and reduce the influence of group bias risk . subjects with uncorrectable factors causing poor - quality photographs on the eas-1000 were also excluded ( exposed subjects n= 15 ; unexposed subjects n= 35 ) . poor photographic factors included insufficient pupillary dilation ( exposed subjects n= 44 ; unexposed subjects n= 62 ) , congenital cataracts ( exposed subjects n= 5 ; unexposed subjects n= 9 ) , excessive myopia by a spherical equivalent ( se ) of less than 6.00 diopters ( exposed subjects n= 31 ; unexposed subjects n= 61 ) , excessive hypermetropia by an se larger than 5.25 diopters ( exposed subjects and unexposed subjects n= 0 ) , ocular hypertension ( tonometer pressure > 20 mmhg ) ( exposed subjects n= 8 ; unexposed subjects n= 7 ) , corneal abnormalities ( exposed subjects n= 2 ; unexposed subjects n= 15 ) and miscellaneous contributory congenital ocular abnormalities ( exposed subjects n= 4 ; unexposed subjects n= 7 ) . after consideration of the above exclusion criteria , 92 of the 160 exposed subjects and 155 of the 326 unexposed subjects were excluded . the eligible population for quantitative analyses consisted of 68 exposed subjects and 171 unexposed subjects . the age - matched group consisted of 54 exposed subjects and 108 unexposed subjects . for intergroup comparison , student 's t - test was performed for descriptive characteristics and light scattering intensity in the six lens layers . multiple regression analyses were carried out using the stepwise method to evaluate lsis in the six layers of the left lens adjusting for age , smoking , drinking , wearing glasses , sunlight exposure during working hours and scattered radiation exposure during working hours . all exposed subjects were japanese volunteer male irs in the japanese society of interventional radiology ( jsir ) , and all unexposed subjects were japanese volunteer male employees of medical service companies with no history of occupational radiation exposure . questions included age , radiation exposure in interventional procedures ( yes / no ) , smoking ( smoking index = cigarettes smoked per day years of smoking ) , drinking ( ever / never ) , wearing glasses ( ever / never ) and solar ultraviolet ray exposure during working hours ( working indoors only , versus working both indoors and outdoors ) . to screen suitable subjects for pharmacologic mydriasis , an ophthalmologist performed an initial screening slit - lamp examination ( slit lamp bq 900 haag - streit , koeniz , switzerland ) of the left eye anterior eye segment , in addition to intraocular pressure measurement ( non - contact totometer nt-4000 , nidek , aichi , japan ) . one hundred and sixty exposed subjects and 326 unexposed subjects were included following screening and mydriasis safety assessment , and subsequently underwent examination of the left eye with pharmacologic pupillary dilation using a drop of midrin - p ( tropicamide and phenylephrine ; santen pharmaceuticals , japan ) . because the examinations were performed in the daytime while exposed subjects were attending their annual professional society meeting , and during the daytime while the unexposed subjects were also working , only single eye pharmacologic dilations were performed . the left side was specifically chosen since the ir 's left eye is closer than the right eye to the x - ray tube in the vast majority of instances . in addition a recent study showed that the dose on the side nearest to the x - ray tube was three to five times greater than for the farthest side of the head . for all qualified subjects the lens of the left eye was examined with scheimpflug slit images obtained using eas-1000 , with a representative slit image shown in fig . 1.scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . the data of exposed and unexposed subjects were analyzed at the same time , although the technologist was not blinded to the condition of exposure status . the peak lsi value was measured in each of the six layers of the lens automatically and measured points were confirmed manually . these six layers included the anterior capsule , the most transparent layer of the anterior superficial cortex , the anterior adult nucleus , the anterior fetal nucleus , the central clear zone and the posterior subcapsular region ( psr ) ( fig . subjects with cataract risk factors other than occupational radiation exposure were excluded from analysis to increase the comparability of the groups and reduce the influence of group bias risk . subjects with uncorrectable factors causing poor - quality photographs on the eas-1000 were also excluded ( exposed subjects n= 15 ; unexposed subjects n= 35 ) . poor photographic factors included insufficient pupillary dilation ( exposed subjects n= 44 ; unexposed subjects n= 62 ) , congenital cataracts ( exposed subjects n= 5 ; unexposed subjects n= 9 ) , excessive myopia by a spherical equivalent ( se ) of less than 6.00 diopters ( exposed subjects n= 31 ; unexposed subjects n= 61 ) , excessive hypermetropia by an se larger than 5.25 diopters ( exposed subjects and unexposed subjects n= 0 ) , ocular hypertension ( tonometer pressure > 20 mmhg ) ( exposed subjects n= 8 ; unexposed subjects n= 7 ) , corneal abnormalities ( exposed subjects n= 2 ; unexposed subjects n= 15 ) and miscellaneous contributory congenital ocular abnormalities ( exposed subjects n= 4 ; unexposed subjects n= 7 ) . after consideration of the above exclusion criteria , 92 of the 160 exposed subjects and 155 of the 326 unexposed subjects were excluded . the eligible population for quantitative analyses consisted of 68 exposed subjects and 171 unexposed subjects . for intergroup comparison , student 's t - test was performed for descriptive characteristics and light scattering intensity in the six lens layers . multiple regression analyses were carried out using the stepwise method to evaluate lsis in the six layers of the left lens adjusting for age , smoking , drinking , wearing glasses , sunlight exposure during working hours and scattered radiation exposure during working hours . in the screening examination for all subjects without exclusion , slit - lamp examination of the left eye under pupillary dilation showed posterior subcapsular cataract ( psc ) in three ( 1.9% ) of 160 exposed subjects ( 45 , 62 and 63 years ) and one ( 0.3% ) of 326 unexposed subjects ( 51 years ) . all four subjects were excluded from quantitative analyses because they met other predetermined exclusion criteria ( two had excessive myopia and one had poor pupillary dilation in the exposed subjects , and one had excessive myopia in the unexposed subjects ) . there was a significant difference in the ages of the two groups , with the exposed subjects showing a greater age than the unexposed subjects . there were significant differences in the lsis in five out of six eye layers when comparing exposed subjects with the unexposed subjects . table 2.light scattering intensity in the six lens layers between exposed and unexposed group ( 8-bit grayscale value)layer of lensirscontrolspvalue(n= 68)(n= 171)anterior capsule63.0 7.860.3 7.90.020most transparent layer of the anterior superficial cortex41.1 8.339.5 7.70.157anterior adult nucleus96.6 33.081.3 25.50.0002anterior fetal nucleus50.7 11.547.4 10.90.039central clear zone38.3 9.635.3 8.40.018posterior subcapsular region39.7 light scattering intensity in the six lens layers between exposed and unexposed group ( 8-bit grayscale value ) values are means sd . the results of the stepwise method for lsi assessment in each of the six layers of the eye lens are shown in table 3 . in all six layers of the eye lens , the variable of age was statistically significant for increased lsis . in the psr , radiation exposure from interventional procedures was significant ( p= 0.012 ) for increased lsis following adjustment for the other variables including age . table 3.evaluation of light scattering intensity in the six lens layers by multiple regression analysis using the stepwise method in all subjects ( n= 239)layer of lensvariableparameter estimate ( 95% ci)pvalueanterior capsuleage0.691 ( 0.6160.766)<0.0001most transparent layer of the anterior superficial cortexage0.153 ( 0.0350.270)0.011workplace0.580 ( 0.2911.452)0.191smoking0.003 ( 0.0070.001)0.094anterior adult nucleusradiation exposure2.307 ( 0.6205.233)0.122age2.644 ( 2.2882.999)<0.0001work place1.793 ( 0.9104.496)0.192anterior fetal nucleusage1.405 ( 1.2351.574)<0.0001smoking0.003 ( 0.0020.008)0.290wearing glasses1.072 ( 2.7850.641)0.219central clear zoneage1.132 ( 1.0571.207)<0.0001smoking0.004 ( 0.0010.006)0.002posterior subcapsular regionradiation exposure2.089 ( 0.4663.711)0.012age0.307 ( 0.528 to 0.086)0.007smoking0.004 ( 0.0100.003)0.277wearing glasses0.925 ( 0.7352.586)0.273ci = confidence interval . evaluation of light scattering intensity in the six lens layers by multiple regression analysis using the stepwise method in all subjects ( n= 239 ) ci = confidence interval . the mean age standard deviation ( sd ) of age - matched exposed subjects and unexposed subjects were 39.5 6.9 and 39.4 6.9 , respectively . in the age - matched comparison , there was a significant difference in lsis in the psr ( table 4 ) . table 4.evaluation of light scattering intensity in the six layers between exposed and unexposed group by age - matched comparison ( 8-bit grayscale value)layer of lensirscontrolspvalue(n= 54)(n= 108)anterior capsule61.3 7.359.4 7.30.10most transparent layer of the anterior superficial cortex41.1 8.838.7 7.60.07anterior adult nucleus87.6 26.982.2 26.90.23anterior fetal nucleus47.7 10.347.2 11.10.80central clear zone35.6 8.435.1 8.60.75posterior subcapsular region40.5 13.434.5 7.20.0031values are means sd . evaluation of light scattering intensity in the six layers between exposed and unexposed group by age - matched comparison ( 8-bit grayscale value ) values are means sd . in the widely used grading system for radiation - induced cataracts by slit - lamp examination , the early stage of psc shows a lens change consisting of the formation of small dots and vacuoles . the increased lsis in exposed subjects compared with unexposed subjects analyzed by multiple regression analysis and age - matched comparison suggest a potential adverse effect on the lens with low - dose radiation exposure . this phenomenon may be a predictor of a primary effect of low - dose radiation on the eye . in our study , the variable of age was significant for lsis in all six layers of the eye lens . we performed multiple regression analyses including exposed subjects and unexposed subjects to adjusting the variables . lsis in the six layers of the lens increase in the anterior five layers and decrease in the psr in a linear manner with aging . this may be related to the fact that the age - related cortical cataract began on the outer edge of the lens cortex and slowly extended to the center . in our screening examination , the detection rate of the psc in the exposed subjects is 1.9% , considerably lower than the 38% and 52% reported in interventional cardiologists [ 7 , 8 ] . in the oramed ( optimization of radiation protection for medical staff ) project , the dosimeter on the near side eye to the x - ray tube showed a higher dose than the dosimeter in the region between the eyes . and , in the study of dose distribution with ten tlds on the eyebrow ridge , the dose on the side nearest to the x - ray tube was three to five times greater than those on the farthest side . it was recommended that dosimeters for the monitoring of eye dosage should be positioned on the side of the brow ridge adjacent to the x - ray tube . because the majority of exposed subjects were primarily abdominal oncologic vascular interventionalists and the physician 's left eye was commonly closer to the x - ray tube , we believed that the left - sided eye examination in this study was more likely to evaluate the side with the highest exposure . our study limitations include our group choices for exposed subjects and unexposed subjects ; a more comparable control group may have been physicians without radiation exposure . our single meridian cross - sectional analyses by eas-1000 were performed in the mid - sagittal plane of the eye lens , therefore , there were no data obtained for off - center , off - angle and off - axis portions of the lens . in conclusion , we found that there were minimal increased lsis in the psr of the lens in exposed subjects as compared with the unexposed subjects in this pilot study . further quantitative studies concerning minimal radiation - related lens changes may additionally be required especially in low - dose exposure groups .

the lens of the eye is recognized as one of the most radio - sensitive tissues in the human body , and radiation - induced cataract is a well - known adverse effect . a recent draft report by the international commission on radiological protection ( icrp ) documented that better techniques for detecting , quantifying and documenting early radiation - associated lens changes , as well as better dosimetry , have potentially contributed to recent findings of radiation cataract risk at lower exposure levels than previously considered . for occupational exposure , a new icrp positional statement recommended that an equivalent dose limit for the lens be reduced from 150 msv / year to 20 msv / year , averaged over defined periods of 5 years , with no single year exceeding 50 msv . the cross - sectional data from astronauts and matched subjects found using an automated anterior eye segment analysis system ( eas-1000 , nidek , aichi , japan ) were analyzed and revealed a small deleterious effect of space radiation for cortical cataracts and possibly for psc cataracts . we hypothesized that interventional radiologists ( irs ) are exposed to low - dose scattered radiation , which may cause radiation - related lens changes well before they would otherwise be appreciated by slit - lamp examination , and may therefore be picked up by directed objective scoring systems [ 5 , 6 ] . to evaluate minimal lsi changes in specific regions in the lens of the eye , we carried out a pilot study on our described exposed subjects and unexposed subjects using eas-1000 . questions included age , radiation exposure in interventional procedures ( yes / no ) , smoking ( smoking index = cigarettes smoked per day years of smoking ) , drinking ( ever / never ) , wearing glasses ( ever / never ) and solar ultraviolet ray exposure during working hours ( working indoors only , versus working both indoors and outdoors ) . for the exposed subjects of irs the survey also included a self - report of professional experience as an ir . to screen suitable subjects for pharmacologic mydriasis , an ophthalmologist performed an initial screening slit - lamp examination ( slit lamp bq 900 haag - streit , koeniz , switzerland ) of the left eye anterior eye segment , in addition to intraocular pressure measurement ( non - contact totometer nt-4000 , nidek , aichi , japan ) . one hundred and sixty exposed subjects and 326 unexposed subjects were included following screening and mydriasis safety assessment , and subsequently underwent examination of the left eye with pharmacologic pupillary dilation using a drop of midrin - p ( tropicamide and phenylephrine ; santen pharmaceuticals , japan ) . because the examinations were performed in the daytime while exposed subjects were attending their annual professional society meeting , and during the daytime while the unexposed subjects were also working , only single eye pharmacologic dilations were performed . the left side was specifically chosen since the ir 's left eye is closer than the right eye to the x - ray tube in the vast majority of instances . in addition a recent study showed that the dose on the side nearest to the x - ray tube was three to five times greater than for the farthest side of the head . for all qualified subjects the lens of the left eye was examined with scheimpflug slit images obtained using eas-1000 , with a representative slit image shown in fig . 1.scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . these six layers included the anterior capsule , the most transparent layer of the anterior superficial cortex , the anterior adult nucleus , the anterior fetal nucleus , the central clear zone and the posterior subcapsular region ( psr ) ( fig . subjects with cataract risk factors other than occupational radiation exposure were excluded from analysis to increase the comparability of the groups and reduce the influence of group bias risk . such other factors included a history of diabetes mellitus ( exposed subjects n= 4 ; unexposed subjects n= 6 ) , eye trauma ( exposed subjects n= 0 ; unexposed subjects n= 1 ) , ophthalmic surgery ( exposed subjects n= 1 ; unexposed subjects n= 4 ) and ocular inflammation ( exposed subjects n= 0 ; unexposed subjects n= 2 ) . poor photographic factors included insufficient pupillary dilation ( exposed subjects n= 44 ; unexposed subjects n= 62 ) , congenital cataracts ( exposed subjects n= 5 ; unexposed subjects n= 9 ) , excessive myopia by a spherical equivalent ( se ) of less than 6.00 diopters ( exposed subjects n= 31 ; unexposed subjects n= 61 ) , excessive hypermetropia by an se larger than 5.25 diopters ( exposed subjects and unexposed subjects n= 0 ) , ocular hypertension ( tonometer pressure > 20 mmhg ) ( exposed subjects n= 8 ; unexposed subjects n= 7 ) , corneal abnormalities ( exposed subjects n= 2 ; unexposed subjects n= 15 ) and miscellaneous contributory congenital ocular abnormalities ( exposed subjects n= 4 ; unexposed subjects n= 7 ) . to reduce the effect of aging , the eligible subjects were matched and selected with a chosen ratio of one ir to two randomly selected unexposed subjects within 1-year difference . multiple regression analyses were carried out using the stepwise method to evaluate lsis in the six layers of the left lens adjusting for age , smoking , drinking , wearing glasses , sunlight exposure during working hours and scattered radiation exposure during working hours . questions included age , radiation exposure in interventional procedures ( yes / no ) , smoking ( smoking index = cigarettes smoked per day years of smoking ) , drinking ( ever / never ) , wearing glasses ( ever / never ) and solar ultraviolet ray exposure during working hours ( working indoors only , versus working both indoors and outdoors ) . for the exposed subjects of irs the survey also included a self - report of professional experience as an ir . to screen suitable subjects for pharmacologic mydriasis , an ophthalmologist performed an initial screening slit - lamp examination ( slit lamp bq 900 haag - streit , koeniz , switzerland ) of the left eye anterior eye segment , in addition to intraocular pressure measurement ( non - contact totometer nt-4000 , nidek , aichi , japan ) . one hundred and sixty exposed subjects and 326 unexposed subjects were included following screening and mydriasis safety assessment , and subsequently underwent examination of the left eye with pharmacologic pupillary dilation using a drop of midrin - p ( tropicamide and phenylephrine ; santen pharmaceuticals , japan ) . because the examinations were performed in the daytime while exposed subjects were attending their annual professional society meeting , and during the daytime while the unexposed subjects were also working , only single eye pharmacologic dilations were performed . the left side was specifically chosen since the ir 's left eye is closer than the right eye to the x - ray tube in the vast majority of instances . in addition a recent study showed that the dose on the side nearest to the x - ray tube was three to five times greater than for the farthest side of the head . 1.scheimpflug slit image obtained using an anterior eye segment analysis system digitalized anterior eye segment image is demonstrated at a mid - sagittal section , the front of the cornea is at the top of the image . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . the seven numbers depicted on the axis sequentially indicate the cornea ( 1 ) , anterior capsule ( 2 ) , most transparent layer of the anterior superficial cortex ( 3 ) , anterior adult nucleus ( 4 ) , anterior fetal nucleus ( 5 ) , central clear zone ( 6 ) and posterior subcapsular region ( 7 ) . these six layers included the anterior capsule , the most transparent layer of the anterior superficial cortex , the anterior adult nucleus , the anterior fetal nucleus , the central clear zone and the posterior subcapsular region ( psr ) ( fig . such other factors included a history of diabetes mellitus ( exposed subjects n= 4 ; unexposed subjects n= 6 ) , eye trauma ( exposed subjects n= 0 ; unexposed subjects n= 1 ) , ophthalmic surgery ( exposed subjects n= 1 ; unexposed subjects n= 4 ) and ocular inflammation ( exposed subjects n= 0 ; unexposed subjects n= 2 ) . poor photographic factors included insufficient pupillary dilation ( exposed subjects n= 44 ; unexposed subjects n= 62 ) , congenital cataracts ( exposed subjects n= 5 ; unexposed subjects n= 9 ) , excessive myopia by a spherical equivalent ( se ) of less than 6.00 diopters ( exposed subjects n= 31 ; unexposed subjects n= 61 ) , excessive hypermetropia by an se larger than 5.25 diopters ( exposed subjects and unexposed subjects n= 0 ) , ocular hypertension ( tonometer pressure > 20 mmhg ) ( exposed subjects n= 8 ; unexposed subjects n= 7 ) , corneal abnormalities ( exposed subjects n= 2 ; unexposed subjects n= 15 ) and miscellaneous contributory congenital ocular abnormalities ( exposed subjects n= 4 ; unexposed subjects n= 7 ) . to reduce the effect of aging , the eligible subjects were matched and selected with a chosen ratio of one ir to two randomly selected unexposed subjects within 1-year difference . multiple regression analyses were carried out using the stepwise method to evaluate lsis in the six layers of the left lens adjusting for age , smoking , drinking , wearing glasses , sunlight exposure during working hours and scattered radiation exposure during working hours . in the screening examination for all subjects without exclusion , slit - lamp examination of the left eye under pupillary dilation showed posterior subcapsular cataract ( psc ) in three ( 1.9% ) of 160 exposed subjects ( 45 , 62 and 63 years ) and one ( 0.3% ) of 326 unexposed subjects ( 51 years ) . 8539.8 690.0059smoking ( yes ) , n(%)40 ( 58.8)115 ( 67.2)0.180smoking index1770 28.5211.7 18.00.06drinking ( yes ) , n(%)53 ( 77.9)147 ( 85.9)0.080wearing glasses ( yes ) , n(%)54 ( 79.4)120 ( 70.5)0.101workplace ( indoors only ) , n(%)68 ( 100)82 ( 48.0)<0.0001years of interventional experience,15.6 7.8na median ( minimum maximum)14 ( 132)total number of angiographies,1846.1 1998.6na median ( minimum maximum)1500 ( 010 000)total number of interventions,1982.3 2056.5na median ( minimum maximum)1200 ( 1012 000)irs = interventional radiologists , values are means sd , na = not applicable . there was a significant difference in the ages of the two groups , with the exposed subjects showing a greater age than the unexposed subjects . table 2.light scattering intensity in the six lens layers between exposed and unexposed group ( 8-bit grayscale value)layer of lensirscontrolspvalue(n= 68)(n= 171)anterior capsule63.0 7.860.3 7.90.020most transparent layer of the anterior superficial cortex41.1 8.339.5 7.70.157anterior adult nucleus96.6 33.081.3 25.50.0002anterior fetal nucleus50.7 11.547.4 10.90.039central clear zone38.3 9.635.3 8.40.018posterior subcapsular region39.7 light scattering intensity in the six lens layers between exposed and unexposed group ( 8-bit grayscale value ) values are means sd . table 3.evaluation of light scattering intensity in the six lens layers by multiple regression analysis using the stepwise method in all subjects ( n= 239)layer of lensvariableparameter estimate ( 95% ci)pvalueanterior capsuleage0.691 ( 0.6160.766)<0.0001most transparent layer of the anterior superficial cortexage0.153 ( 0.0350.270)0.011workplace0.580 ( 0.2911.452)0.191smoking0.003 ( 0.0070.001)0.094anterior adult nucleusradiation exposure2.307 ( 0.6205.233)0.122age2.644 ( 2.2882.999)<0.0001work place1.793 ( 0.9104.496)0.192anterior fetal nucleusage1.405 ( 1.2351.574)<0.0001smoking0.003 ( 0.0020.008)0.290wearing glasses1.072 ( 2.7850.641)0.219central clear zoneage1.132 ( 1.0571.207)<0.0001smoking0.004 ( 0.0010.006)0.002posterior subcapsular regionradiation exposure2.089 ( 0.4663.711)0.012age0.307 ( 0.528 to 0.086)0.007smoking0.004 ( 0.0100.003)0.277wearing glasses0.925 ( 0.7352.586)0.273ci = confidence interval . table 4.evaluation of light scattering intensity in the six layers between exposed and unexposed group by age - matched comparison ( 8-bit grayscale value)layer of lensirscontrolspvalue(n= 54)(n= 108)anterior capsule61.3 7.359.4 7.30.10most transparent layer of the anterior superficial cortex41.1 8.838.7 7.60.07anterior adult nucleus87.6 26.982.2 26.90.23anterior fetal nucleus47.7 10.347.2 11.10.80central clear zone35.6 8.435.1 8.60.75posterior subcapsular region40.5 13.434.5 7.20.0031values are means sd . in the widely used grading system for radiation - induced cataracts by slit - lamp examination , the early stage of psc shows a lens change consisting of the formation of small dots and vacuoles . in a cross - sectional study in interventional cardiologists , pscs were significantly more frequent among interventional cardiologists than the control subjects . the increased lsis in exposed subjects compared with unexposed subjects analyzed by multiple regression analysis and age - matched comparison suggest a potential adverse effect on the lens with low - dose radiation exposure . our findings regarding smoking reinforce previous studies and suggest that the increase in lsis in the central clear zone may be an early sign of nuclear cataracts . other factors such as ultraviolet light exposure , diabetes , renal failure , drug intake and malnutrition also individually and collectively contribute over time to further increase light scattering and can not be readily separated from aging . lsis in the six layers of the lens increase in the anterior five layers and decrease in the psr in a linear manner with aging . in the most transparent layer of the anterior superficial cortex , lsis did not show a significant difference between irs and controls , and the parameter estimate of age was relative lower than for the other layers . the estimated average lens dose in interventional cardiologists with estimated cumulative occupational dose considering eye protection was 6.0 6.6 sv ( 0.127 sv ) and 3.7 7.5 gy ( 0.0243 gy ) [ 7 , 8 ] . in our screening examination , the detection rate of the psc in the exposed subjects is 1.9% , considerably lower than the 38% and 52% reported in interventional cardiologists [ 7 , 8 ] . lens dosimetry was not performed in this retrospective study since two categories of information were unavailable ; first , collar - badge readings were not uniformly available for all practitioners and second , existing under - apron individual monitoring data were unreliable due to inconstant usage of personal dosimeter , employment changes and also the fact that full versus part - time employment was not noted , neither was individual positioning and wearing of protection devices . due to the aforementioned challenges , we chose not to include estimated lens dose , which can not be considered in such circumstances as objectively quantifiable , and we therefore chose to simply register presence or absence of radiation exposure status ( yes or no ) as the categorical variable . individual dosimetry remains problematic , and alternative strategies will be needed in order to more precisely evaluate the effects of radiation in specific occupational situations , since strict individual monitoring and strategic management will be necessary . one of the largest study limitations is our choice of a single left - eye examination for the purposes of this study ; the laterality of lsis of the lens could not be assessed . in the oramed ( optimization of radiation protection for medical staff ) project , the dosimeter on the near side eye to the x - ray tube showed a higher dose than the dosimeter in the region between the eyes . and , in the study of dose distribution with ten tlds on the eyebrow ridge , the dose on the side nearest to the x - ray tube was three to five times greater than those on the farthest side . because the majority of exposed subjects were primarily abdominal oncologic vascular interventionalists and the physician 's left eye was commonly closer to the x - ray tube , we believed that the left - sided eye examination in this study was more likely to evaluate the side with the highest exposure . our single meridian cross - sectional analyses by eas-1000 were performed in the mid - sagittal plane of the eye lens , therefore , there were no data obtained for off - center , off - angle and off - axis portions of the lens . in order to confirm the reproducibility of the eas-1000 , mean individual coefficient of variation was segmentally measured in four different lens regions with a 1-week interval in measurements , and the variation was measured as between 3.6 and 5.1% , in keeping with the previously described examination . as previously alluded to , a potential limitation of our study is also that the technologist was unblinded , although the automated nature of the lsi measurements for data collection , and the reliable proscriptive nature of mid - sagittal plane selection , suggests this is an unlikely cause for concern . in conclusion , we found that there were minimal increased lsis in the psr of the lens in exposed subjects as compared with the unexposed subjects in this pilot study .

transgenic zebrafish ( danio rerio ) tg(gfap : egfp)mi2002 and tl strain wild - type zebrafish were maintained using standard husbandry protocols . all procedures were approved by the institutional animal care and use committee ( iacuc ) at the university of michigan and abided by the arvo statement for the use of animals in ophthalmic and visual research . the regenerative response in mller glia was induced as previously described by exposing free - swimming , adult tg(gfap : egfp)mi2002 zebrafish ( 811 months old ) to intense light ( > 120,000 lux ) from an exfo x - cite 120w metal halide lamp ( exfo photonic solutions , quebec city , qc , canada ) for 30 minutes ( fig . this acute light lesion results in the selective death of photoreceptors , with peak cell death response at 24 hpl . retinas from nonlesioned and light - lesioned tg(gfap : egfp)mi2002 zebrafish were surgically removed and dissociated into single cells ( figs . green fluorescent protein ( gfp)-positive ( gfp+ ) mller glia cells were collected by facs ( fig . nonlesioned , wild - type zebrafish were used as gfp - negative ( gfp ) controls for sorting . we collected mller glia at 0 ( nonlesioned ) , 8 , and 16 hpl , prior to their initial asymmetric , stem cell like division . these times were chosen to avoid including the rapidly dividing rpcs in the neurogenic clusters that begin to appear after the initial asymmetric division of the mller glia at 20 to 36 hpl . although the glial - specific promoter ( gfap ) is no longer active in rpcs , they retain the egfp label through perseverance of the fluorescent protein for several days . therefore , gfp+ cells isolated after 20 hpl inevitably include a heterogeneous mixture of mller glia and rpcs ( a ) photoreceptors were ablated in free - swimming tg(gfap : egfp)mi2002 fish using an acute light - lesion paradigm . ( b ) retinas were dissected from unlesioned controls ( 0 hpl ) and 8 and 16 hpl tg(gfap : egfp)mi2002 fish . retinas were also dissected from unlesioned , wild - type ( nontransgenic , gfp ) control fish . ( d ) dissociated samples were subjected to fluorescence - activated cell sorting ( facs ) , and gfp+ cells were collected . the facs plots shown are representative images from the final gating and collection of actual samples . ( e ) rna was isolated from the sorted cells and checked by bioanalyzer for quality and concentration . samples with an rna integrity number ( rin ) above 7.0 were advanced to library preparation . the bioanalyzer electropherogram shown is a representative plot from an actual sample with a rin of 8.6 . the y - axis shows fluorescent units ( fu ) , corresponding to the amount of rna . ( g ) the rna - seq libraries were then sequenced on an illumina gaiix . ( h ) the sequencing data were processed with bioinformatic tools for differential expression analysis , gene ontology analysis , and pathway analysis . rna was extracted from the sorted cells and the quality was checked with bioanalyzer ( agilent technologies , santa clara , ca , usa ) ( fig . . samples with an rna integrity number ( rin ) of acceptable quality ( > 7.0 ) were used for illumina rna - seq library preparation ( fig . rna - seq libraries were subjected to deep sequencing on an illumina gaiix sequencer ( illumina , inc . , san diego , ca , usa ) ( fig sequencing data were analyzed for differential gene expression , gene ontology enrichment , and enriched pathways ( fig . additional methodological details and a complete description of the bioinformatic analysis pipeline are in the supplementary methods . supplementary data s1 , s2 , and s3 contain complete datasets for differential gene expression data , gene ontology enrichment , and enriched pathways , respectively . to identify genes that were differentially expressed at each time point , we used the commonly accepted threshold values for differentially expressed genes of |log2 fold change| ( fc ) 1 and a false discovery rate ( fdr ) 0.05 . this analysis identified 2690 genes differentially expressed in mller glia isolated from light - lesioned zebrafish retinas at 8 or 16 hpl compared with mller glia from control ( unlesioned ) retinas ( fig . the number of genes differentially expressed at 8 hpl ( n = 2221 ) is almost 2.5 times greater than at 16 hpl ( n = 923 ) ( fig . 2a ) , although the total number of genes expressed above a threshold of 1 fpkm ( fragment per kilobase of exon per million reads mapped ) did not differ among the samples ( fig . a venn diagram plot reveals 1767 differentially expressed genes specific to 8 hpl , 469 genes specific to 16 hpl , and 454 genes common to both ( roughly 20% of the genes differentially expressed at 8 hpl remain differentially expressed at 16 hpl ) ( fig . figures 2c and 2d display the log2fc in expression as a function of log2 counts per million ( cpm ) mapped reads from rna - seq data for all differentially expressed genes at 8 hpl and 16 hpl , respectively . the top 20 positively and negatively regulated known zebrafish genes are labeled in blue ( log2fc values for 8 hpl are listed in supplementary table s2 and for 16 hpl in supplementary table s3 ) . ( a ) the total number of expressed and differentially expressed genes at each sample time point . genes considered to be expressed were those with a fragment per kilobase of exon per million reads mapped ( fpkm ) value 1 . ( b ) the number of expressed genes in each group is based on entrez geneid , which were used for further analyses . differentially expressed genes are those with an absolute log2 fold change ( |log2fc| ) 1 and a false discovery rate ( fdr ) 0.05 . ( c , d ) transformed log ratio and mean average ( ma ) plot [ log2fc versus log2 counts per million ( cpm ) ] at 8 hpl and 16 hpl , respectively . dots represent genes : red for genes with a fdr 0.05 and blue for genes of interest . ( e , f ) validation of rna - seq data for selected genes at 8 hpl and 16 hpl , respectively , with reverse transcription quantitative polymerase chain reaction ( rt - qpcr ) . the x - axis shows the target gene and the y - axis shows the log2fc versus unlesioned control . rna - seq log2fc data are denoted by blue bars , and rt - qpcr log2fc data are denoted by red bars . error bars for the rt - qpcr data represent the standard error of the mean for three biological replicates . the table lists the log2fc values for selected differentially expressed genes previously identified in microarray profiling studies and/or implicated in zebrafish retinal regeneration . many of these genes belong to specific categories of biological processes , including stress response ( hspd1 ) , immune response / cytokines ( stat3 , lepb ) , secreted factors ( hb - egf ) , mller glial cell dedifferentiation and acquisition of stem cell properties ( tgif1 , lin28 , apobec2b ) , and cell adhesion ( cdh2 ) . a few of the known regeneration genes with small or below criterion values of differential expression in this rna - seq dataset ( table ) are primarily implicated in processes that occur later than those assayed here , such as cell fate specification ( ascl1a , sox2 ) and progenitor proliferation ( fabp7a , insm1a ) . log2fold changes ( fc ) of selected regeneration - associated genes in mller glia at 8 and 16 hours post lesion ( hpl ) validation of expression levels with rt quantitative pcr ( qpcr ) confirmed the rna - seq data for several genes of interest tgf-induced factor homeobox 1 ( tgif1 ) , cytokine receptor like factor 1a ( crlf1a ) , heat shock protein family d member 1 ( hspd1 ) , complement component 7 ( c7 ) , prostaglandin e synthase ( ptges ) , and matrix metallopeptidase 9 ( mmp9 ) at 8 hpl ( fig . 2e ) and 16 hpl ( fig . 2f)many of which have experimentally confirmed roles in zebrafish retinal regeneration . we next performed gene ontology ( go ) and pathway - level analyses with kegg and reactome tools as described in more detail in the supplementary methods . reactome data agreed with kegg data , yet were highly redundant and thus not included in figures presented here . to provide a global overview of the rapid and dynamic transcriptional changes in mller glia as they respond to photoreceptor loss and prepare to generate retinal progenitors , we discuss the results by classifying these data into eight general , nonexclusive , categories : stress response , prostaglandins , circadian rhythm , wnt signaling , nuclear factorb ( nf-b ) signaling , immune response , cytokines , and pluripotency . to illustrate dynamic transcriptional changes in mller glia as they prepare for an asymmetric stem cell like mitotic division , figure 3 plots the log2fc differential expression of genes in each of these categories at 8 vs. 16 hpl . for each category ( each part of figure ) , log2fc at 8 hpl ( y - axis ) is plotted versus log2fc at 16 hpl ( x - axis ) for top differentially expressed genes ( |log2fc| 1 and fdr 0.05 ) within each category . dashed line represents equivalent values of differential expression of a given gene at both sample times ( slope = 1 and y - intercept = 0 ) . the top positively regulated annotated gene at 8 hpl with 7.75 log2fc is heme - binding protein soul 5 ( soul5 ) , an oxidative stress induced protein ( figs . 2c , 3 ; supplementary table s2 ) . other evidence of a strong oxidative stress response includes elevated expression of biliverdin reductase a ( blvra ) , heat shock protein alpha - crystallin - related b11 ( hspb11 ) , and heat shock 60-kda protein 1 ( hspd1 ) ( figs . the enriched go terms and kegg pathways in figure 4 similarly show strong positive regulation of redox and biological processes and pathways indicative of a stress response . enrichment map of enriched kegg pathways and biological process go terms ( with redundancy reduced ) with fdr 0.05 . circular node color reflects positive ( red ) or negative enrichment ( blue ) ; color intensity reflects the significance of the enrichment ; and area reflects the size of the gene set . lines ( green ) represent significant overlap in genes within linked sets , and line thickness represents degree of overlap . two categories of biological responses that are rapidly activated in mller glia , but have not previously been associated with retinal regeneration , are prostaglandin metabolism and circadian rhythm ( figs . metabolic processes and pathways , in general , are very highly enriched at 8 hpl ( fig . 4 ) ; especially prominent are pathways associated with lipid metabolism , including icosanoids ( also called eicosanoids ) , fatty acid derivatives , and arachidonic acid . these metabolic changes are reflected in the increased expression of zebrafish orthologs of prostaglandin e synthase ( ptges , ptges1 ) , prostaglandin endoperoxide synthase ( ptges , ptges1 ) , and prostaglandin d2 synthase ( ptgdsb ) genes in the biosynthetic pathway for the eicosanoid prostaglandin e2 ( pge2 ) ( fig . somewhat unexpectedly , several core circadian clock genes are included in the top 20 positively regulated genes at 8 hpl ( figs . s2a ; supplementary table s2 ) , and the go terms associated with circadian and biological rhythms are positively enriched ( fig . 4 ) . with the exception of period 1a ( per1a ) , which is negatively regulated ( fig . 2c ; supplementary table s2 ) , several core clock genes are positively regulated ( figs . s2a ) , including period 2 ( per2 ) , cryptochrome 5 ( cry5 ) , neuronal pas domain protein 2 ( npas2 ) , and orthologs ( arntl1a , arntl1b ) of aryl hydrocarbon receptor nuclear translocator - like ( arntl1 ) , also known as bmal1 . enrichment analyses reveal that wnt signaling is negatively regulated at 8 hpl and remains repressed at 16 hpl ( figs . 3 , 4 ) with reduced expression of wnt target genes such as lymphoid enhancer binding factor 1 ( lef1 ) and frizzled class receptors ( fzd7b , fzd9b ) ( fig . activity is likewise dampened in the notch and bone morphogenetic protein ( bmp ) signaling pathways ( fig . 4 ) , and the notch target hairy - related 4 , tandem duplicate 1 ( her4.1 ) is among the top negatively regulated genes ( fig . 2c ; supplementary table s2 ) . negatively expressed genes and negatively enriched go terms and kegg pathways at both 8 and 16 hpl include several indicative of photoreceptors and neural functions , including sensory transduction and synaptic mechanisms ( figs . 2c , 2d , 3 , 4 , 5 ; supplementary tables s2 , s3 ) . some of the negatively regulated photoreceptor - specific genes have relatively high log2cpm , such as the rod photoreceptor gene nuclear subfamily 2 group e member 3 ( nr2e3 ) at 8 hpl ( fig . 2c ) and cone photoreceptor genes g protein subunit alpha 2 ( gnat2 ) and cone opsins ( opn1mw2 , opn1sw2 ) at 16 hpl ( fig . these results suggest that the preparations of facs - isolated mller glia are contaminated with photoreceptor transcripts , as has been noted in previous microarray profiling studies of sorted mller glia from zebrafish ( supplementary figs . s3b , s3c ; supplementary tables s4 , s5 ) , as well as microarray and rna - seq expression profiles of facs - isolated gfp+ mller glia from adult mouse retina ( supplementary figs . even single mller glial cells hand - picked from young and adult mouse retinas can show significant levels of photoreceptor - specific transcripts ( supplementary figs . this unavoidable photoreceptor contamination is likely attributed to the dense network of mller glial processes that enwrap the photoreceptor cells . as this tight physical association would predict , the inverse is also true specific transcripts ( retinaldehyde binding protein 1 ; supplementary table s5 ) are found in facs - isolated rod and cone photoreceptors from adult mouse ( supplementary figs . specific transcripts such as rhodopsin and gnat1 ( supplementary table s5 ) are abundantly present in 97.5% pure cone photoreceptor preparations ( supplementary fig . contaminating transcripts are avoided only when the offending cell type is not present ; for example , rods are absent in the nrl mouse ( supplementary fig . the negative enrichment values of transcripts related to photoreceptors at 8 or 16 hpl versus unlesioned samples in our dataset ( figs . 4 , 5 , sensory perception ) are consistent with a reduction in cross - contamination caused by concurrent degeneration of light - damaged photoreceptors . pathways and biological processes enriched at 16 hpl in zebrafish mller glia . enrichment map of enriched kegg pathways and redundancy - reduced biological process go terms ( with redundancy reduced ) with fdr 0.05 . circular node color reflects positive ( red ) or negative enrichment ( blue ) , color intensity reflects the significance of the enrichment , and area of the circle reflects the size of the gene set . lines ( green ) represent significant overlap in genes within linked sets , and thickness represents degree of overlap . activation of biological processes and pathways indicative of oxidative stress and the regulation of redox homeostasis , which were already evident at 8 hpl ( figs . 3 , 4 ) , continue to be enriched at 16 hpl , but with a clear shift toward more prominent activation of signaling pathways related to inflammation , including both innate and adaptive immune responses ( figs . 3 , 5 ) . the nf-b signaling pathway is activated by both oxidative stress and proinflammatory cytokines , and it regulates cell survival , cell proliferation , and inflammation . 3 ) , including tumor necrosis factor receptor superfamily orthologs ( tnfrsf1a , tnfrsf11a ) , mitogen - activated protein kinase kinase kinase 14 ( map3k14 ) , and growth arrest and dna - damage - inducible 45 beta orthologs ( gadd45ba , gadd45bb ) . notably , two of the upregulated prostaglandin synthetases ( ptgs2a and ptgs2b ) mentioned previously are also in the nf-b signaling pathway ( fig . 3 ) . a number of pathways associated with cytokine - mediated signaling and immune processes , including janus kinase - signal transducer and activator of transcription ( jak - stat ) and tumor necrosis factor ( tnf ) signaling pathways ( fig . 5 ) , are positively enriched at 16 hpl , as reflected by upregulation of genes ( fig . 3 ) such as janus kinase 1 ( jak1 ) , signal transducer and activator of transcription 3 ( stat3 ) , suppressor of cytokine signaling 3b ( socs3b ) , cytokine inducible sh protein orthologs ( cish , cishb ) , and interferon regulator factor 9 ( irf9 ) . activation of immune responses mediated by the complement cascade is also very prominent at 16 hpl : several complement component genes ( c4a , c6 , c7a , c7 ) show a log2fc > 2.0 ( figs . the most highly enriched gene at 16 hpl is leptin b ( lepb ) , a hormone that signals via jak - stat and modulates the immune system . in addition to cytokine signaling and immune responses , the jak - stat signaling pathway is also implicated in regulation of pluripotency in stem cells ( fig . other pluripotency - related enriched genes are associated with several growth factor signaling pathways : fibroblast growth factor receptor 1 orthologs ( fgfr1a , fgfr1b ) , wingless - type mmtv integration site , family member 7a ( wnt7a ) , glycogen synthase kinase 3b ( gsk3b ) , inhibin beta aa ( inhbaa ) , and chromatin modifier polycomb group ring finger 5 orthologs ( pcgf5a , pcgf5b ) . whereas biological processes associated with cell growth and differentiation ( e.g. , cell movement , biological adhesion , tissue development , insulin secretion ) were negatively regulated at 8 hpl ( fig . 4 ) , by 16 hpl evidence for positive enrichment of proliferation processes and pathways ( e.g. , cell proliferation , negative regulation of cell differentiation , regulation of epigenetic gene expression , locomotion and cell migration , regeneration , growth ) was very strong ( fig . 5 ) , suggestive of the transition to cell cycle entry and production of rpcs . consistent with this interpretation is that signaling pathways regulating pluripotency of stem cells were positively enriched at 16 hpl ( fig . 5 ) . to identify transcriptional changes that distinguish regeneration - competent zebrafish mller glia from mammalian mller glia , which lack the ability to regenerate retinal neurons , we analyzed a publicly available microarray dataset of single isolated mouse mller glia from two different mouse models of retinal degeneration , pde6b and rho , which vary in their degeneration kinetics . we used data only from the initial major rod death phase ( postnatal day 13 and postnatal week 8 for pde6b and rho , respectively ) as these were most comparable to our zebrafish dataset , which focused on the initial response of mller glia to photoreceptor loss . we performed a parallel differential expression analysis of biological processes and pathways , as illustrated in figures 6a and 6b and described in more detail in supplementary methods . ( a ) schematic showing the methodology for the pathway analysis based on rna - seq data for the initial stages of mller glia ( b ) schematic showing the methodology for the pathway analysis of microarray data from single mller glia isolated from mouse ( mus musculus ) photoreceptor degeneration models , and the comparison between d. rerio and m. musculus . columns from left to right : signaling pathway or biological process , zebrafish mller glia at 8 hpl , zebrafish mller glia at 16 hpl , mouse mller glia from pde6b retinal degeneration model at postnatal day 13 , mouse mller glia from rho retinal degeneration model at 8 weeks postnatal . arrows and corresponding colors represent the overall direction of regulation according to the legend at the right ; gray dashes represent pathways or processes that were not enriched compared with controls . direction of regulation for 8 and 16 hpl zebrafish mller glia is based on data from figures 4 and 5 , respectively . direction of regulation for pde6b and rho mller glia is based on data from figures 6a and 6b , respectively , with the exception of wnt signaling . pde6b is based on results from reactome analysis ( positive enrichment of formation of the beta - catenin : tcf transactivating complex , supplementary data s3 ) and biological process go terms ( positive enrichment of regulation of canonical wnt signaling pathway , supplementary data s2 ) . rho is based on biological process go term analysis ( negative enrichment of regulation of canonical wnt signaling pathway , supplementary data s2 ) . pathway enrichment analysis in the pde6b retinal degeneration model revealed strong positive regulation of metabolic responses in mouse mller glia ( fig . 7a ) , but in contrast to zebrafish mller glia , the nf-b signaling pathway , cytokine signaling and immune system responses were negatively enriched ( figs . 6c , 7a ) . in the rho mouse retinal degeneration model , a strong metabolic response 7b ) ; but in contrast to the pde6b model , the complement cascade pathway was positively enriched ( fig . genes in the biosynthetic pathway leading to pge2 , which were strongly upregulated in fish mller glia at both 8 and 16 hpl ( figs . 3 , 6c , supplementary fig . s1b ) , although several genes in the arachidonic acid metabolism pathway are differentially but inconsistently regulated in both mouse degeneration models ( supplementary fig . s1b ) . similarly , the positive regulation of core circadian clock genes seen in zebrafish mller glia , including per2 , clock , and arntl ( fig . s2a ) , is not replicated in mouse mller glia ; instead , negative regulators of per1 ( beta - transducin repeat containing e3 ubiquitin protein ligase , btrc , and s - phase kinase - associated protein 1a , skp1a ) are strongly upregulated , and per1 and arntl ( bmal1 ) are downregulated ( fig . 6c , supplementary fig . wnt signaling is repressed in zebrafish mller glia and variably regulated in the degeneration models ( fig . unlike in zebrafish , in the rho model , signaling pathways regulating pluripotency in stem cells were negatively enriched ( figs . enrichment map of kegg pathways with fdr 0.05 in ( a ) pde6b and ( b ) rho degeneration models . circular node color reflects positive ( red ) or negative enrichment ( blue ) ; color intensity reflects the significance of the enrichment ; and area reflects the size of the gene set . lines ( green ) represent significant overlap in genes within linked sets , and line thickness represents degree of overlap . to identify genes that were differentially expressed at each time point , we used the commonly accepted threshold values for differentially expressed genes of |log2 fold change| ( fc ) 1 and a false discovery rate ( fdr ) 0.05 . this analysis identified 2690 genes differentially expressed in mller glia isolated from light - lesioned zebrafish retinas at 8 or 16 hpl compared with mller glia from control ( unlesioned ) retinas ( fig . the number of genes differentially expressed at 8 hpl ( n = 2221 ) is almost 2.5 times greater than at 16 hpl ( n = 923 ) ( fig . 2a ) , although the total number of genes expressed above a threshold of 1 fpkm ( fragment per kilobase of exon per million reads mapped ) did not differ among the samples ( fig . a venn diagram plot reveals 1767 differentially expressed genes specific to 8 hpl , 469 genes specific to 16 hpl , and 454 genes common to both ( roughly 20% of the genes differentially expressed at 8 hpl remain differentially expressed at 16 hpl ) ( fig . figures 2c and 2d display the log2fc in expression as a function of log2 counts per million ( cpm ) mapped reads from rna - seq data for all differentially expressed genes at 8 hpl and 16 hpl , respectively . the top 20 positively and negatively regulated known zebrafish genes are labeled in blue ( log2fc values for 8 hpl are listed in supplementary table s2 and for 16 hpl in supplementary table s3 ) . ( a ) the total number of expressed and differentially expressed genes at each sample time point . genes considered to be expressed were those with a fragment per kilobase of exon per million reads mapped ( fpkm ) value 1 . ( b ) the number of expressed genes in each group is based on entrez geneid , which were used for further analyses . differentially expressed genes are those with an absolute log2 fold change ( |log2fc| ) 1 and a false discovery rate ( fdr ) 0.05 . ( c , d ) transformed log ratio and mean average ( ma ) plot [ log2fc versus log2 counts per million ( cpm ) ] at 8 hpl and 16 hpl , respectively . dots represent genes : red for genes with a fdr 0.05 and blue for genes of interest . ( e , f ) validation of rna - seq data for selected genes at 8 hpl and 16 hpl , respectively , with reverse transcription quantitative polymerase chain reaction ( rt - qpcr ) . the x - axis shows the target gene and the y - axis shows the log2fc versus unlesioned control . rna - seq log2fc data are denoted by blue bars , and rt - qpcr log2fc data are denoted by red bars . error bars for the rt - qpcr data represent the standard error of the mean for three biological replicates . the table lists the log2fc values for selected differentially expressed genes previously identified in microarray profiling studies and/or implicated in zebrafish retinal regeneration . many of these genes belong to specific categories of biological processes , including stress response ( hspd1 ) , immune response / cytokines ( stat3 , lepb ) , secreted factors ( hb - egf ) , mller glial cell dedifferentiation and acquisition of stem cell properties ( tgif1 , lin28 , apobec2b ) , and cell adhesion ( cdh2 ) . a few of the known regeneration genes with small or below criterion values of differential expression in this rna - seq dataset ( table ) are primarily implicated in processes that occur later than those assayed here , such as cell fate specification ( ascl1a , sox2 ) and progenitor proliferation ( fabp7a , insm1a ) . log2fold changes ( fc ) of selected regeneration - associated genes in mller glia at 8 and 16 hours post lesion ( hpl ) validation of expression levels with rt quantitative pcr ( qpcr ) confirmed the rna - seq data for several genes of interest tgf-induced factor homeobox 1 ( tgif1 ) , cytokine receptor like factor 1a ( crlf1a ) , heat shock protein family d member 1 ( hspd1 ) , complement component 7 ( c7 ) , prostaglandin e synthase ( ptges ) , and matrix metallopeptidase 9 ( mmp9 ) at 8 hpl ( fig . 2e ) and 16 hpl ( fig . 2f)many of which have experimentally confirmed roles in zebrafish retinal regeneration . we next performed gene ontology ( go ) and pathway - level analyses with kegg and reactome tools as described in more detail in the supplementary methods . reactome data agreed with kegg data , yet were highly redundant and thus not included in figures presented here . to provide a global overview of the rapid and dynamic transcriptional changes in mller glia as they respond to photoreceptor loss and prepare to generate retinal progenitors , we discuss the results by classifying these data into eight general , nonexclusive , categories : stress response , prostaglandins , circadian rhythm , wnt signaling , nuclear factorb ( nf-b ) signaling , immune response , cytokines , and pluripotency . to illustrate dynamic transcriptional changes in mller glia as they prepare for an asymmetric stem cell like mitotic division , figure 3 plots the log2fc differential expression of genes in each of these categories at 8 vs. 16 hpl . for each category ( each part of figure ) , log2fc at 8 hpl ( y - axis ) is plotted versus log2fc at 16 hpl ( x - axis ) for top differentially expressed genes ( |log2fc| 1 and fdr 0.05 ) within each category . dashed line represents equivalent values of differential expression of a given gene at both sample times ( slope = 1 and y - intercept = 0 ) . the top positively regulated annotated gene at 8 hpl with 7.75 log2fc is heme - binding protein soul 5 ( soul5 ) , an oxidative stress induced protein ( figs . 2c , 3 ; supplementary table s2 ) . other evidence of a strong oxidative stress response includes elevated expression of biliverdin reductase a ( blvra ) , heat shock protein alpha - crystallin - related b11 ( hspb11 ) , and heat shock 60-kda protein 1 ( hspd1 ) ( figs . the enriched go terms and kegg pathways in figure 4 similarly show strong positive regulation of redox and biological processes and pathways indicative of a stress response . enrichment map of enriched kegg pathways and biological process go terms ( with redundancy reduced ) with fdr 0.05 . circular node color reflects positive ( red ) or negative enrichment ( blue ) ; color intensity reflects the significance of the enrichment ; and area reflects the size of the gene set . lines ( green ) represent significant overlap in genes within linked sets , and line thickness represents degree of overlap . two categories of biological responses that are rapidly activated in mller glia , but have not previously been associated with retinal regeneration , are prostaglandin metabolism and circadian rhythm ( figs . metabolic processes and pathways , in general , are very highly enriched at 8 hpl ( fig . 4 ) ; especially prominent are pathways associated with lipid metabolism , including icosanoids ( also called eicosanoids ) , fatty acid derivatives , and arachidonic acid . these metabolic changes are reflected in the increased expression of zebrafish orthologs of prostaglandin e synthase ( ptges , ptges1 ) , prostaglandin endoperoxide synthase ( ptges , ptges1 ) , and prostaglandin d2 synthase ( ptgdsb ) genes in the biosynthetic pathway for the eicosanoid prostaglandin e2 ( pge2 ) ( fig . somewhat unexpectedly , several core circadian clock genes are included in the top 20 positively regulated genes at 8 hpl ( figs . s2a ; supplementary table s2 ) , and the go terms associated with circadian and biological rhythms are positively enriched ( fig . 4 ) . with the exception of period 1a ( per1a ) , which is negatively regulated ( fig . 2c ; supplementary table s2 ) , several core clock genes are positively regulated ( figs . s2a ) , including period 2 ( per2 ) , cryptochrome 5 ( cry5 ) , neuronal pas domain protein 2 ( npas2 ) , and orthologs ( arntl1a , arntl1b ) of aryl hydrocarbon receptor nuclear translocator - like ( arntl1 ) , also known as bmal1 . enrichment analyses reveal that wnt signaling is negatively regulated at 8 hpl and remains repressed at 16 hpl ( figs . 3 , 4 ) with reduced expression of wnt target genes such as lymphoid enhancer binding factor 1 ( lef1 ) and frizzled class receptors ( fzd7b , fzd9b ) ( fig . activity is likewise dampened in the notch and bone morphogenetic protein ( bmp ) signaling pathways ( fig . 4 ) , and the notch target hairy - related 4 , tandem duplicate 1 ( her4.1 ) is among the top negatively regulated genes ( fig . 2c ; supplementary table s2 ) . negatively expressed genes and negatively enriched go terms and kegg pathways at both 8 and 16 hpl include several indicative of photoreceptors and neural functions , including sensory transduction and synaptic mechanisms ( figs . 2c , 2d , 3 , 4 , 5 ; supplementary tables s2 , s3 ) . some of the negatively regulated photoreceptor - specific genes have relatively high log2cpm , such as the rod photoreceptor gene nuclear subfamily 2 group e member 3 ( nr2e3 ) at 8 hpl ( fig . 2c ) and cone photoreceptor genes g protein subunit alpha 2 ( gnat2 ) and cone opsins ( opn1mw2 , opn1sw2 ) at 16 hpl ( fig . these results suggest that the preparations of facs - isolated mller glia are contaminated with photoreceptor transcripts , as has been noted in previous microarray profiling studies of sorted mller glia from zebrafish ( supplementary figs . s3b , s3c ; supplementary tables s4 , s5 ) , as well as microarray and rna - seq expression profiles of facs - isolated gfp+ mller glia from adult mouse retina ( supplementary figs . even single mller glial cells hand - picked from young and adult mouse retinas can show significant levels of photoreceptor - specific transcripts ( supplementary figs . this unavoidable photoreceptor contamination is likely attributed to the dense network of mller glial processes that enwrap the photoreceptor cells . as this tight physical association would predict , the inverse is also true specific transcripts ( retinaldehyde binding protein 1 ; supplementary table s5 ) are found in facs - isolated rod and cone photoreceptors from adult mouse ( supplementary figs . specific transcripts such as rhodopsin and gnat1 ( supplementary table s5 ) are abundantly present in 97.5% pure cone photoreceptor preparations ( supplementary fig . contaminating transcripts are avoided only when the offending cell type is not present ; for example , rods are absent in the nrl mouse ( supplementary fig . the negative enrichment values of transcripts related to photoreceptors at 8 or 16 hpl versus unlesioned samples in our dataset ( figs . 4 , 5 , sensory perception ) are consistent with a reduction in cross - contamination caused by concurrent degeneration of light - damaged photoreceptors . enrichment map of enriched kegg pathways and redundancy - reduced biological process go terms ( with redundancy reduced ) with fdr 0.05 . circular node color reflects positive ( red ) or negative enrichment ( blue ) , color intensity reflects the significance of the enrichment , and area of the circle reflects the size of the gene set . lines ( green ) represent significant overlap in genes within linked sets , and thickness represents degree of overlap . activation of biological processes and pathways indicative of oxidative stress and the regulation of redox homeostasis , which were already evident at 8 hpl ( figs . 3 , 4 ) , continue to be enriched at 16 hpl , but with a clear shift toward more prominent activation of signaling pathways related to inflammation , including both innate and adaptive immune responses ( figs . 3 , 5 ) . the nf-b signaling pathway is activated by both oxidative stress and proinflammatory cytokines , and it regulates cell survival , cell proliferation , and inflammation . positive enrichment of this pathway ( fig . 3 ) , including tumor necrosis factor receptor superfamily orthologs ( tnfrsf1a , tnfrsf11a ) , mitogen - activated protein kinase kinase kinase 14 ( map3k14 ) , and growth arrest and dna - damage - inducible 45 beta orthologs ( gadd45ba , gadd45bb ) . notably , two of the upregulated prostaglandin synthetases ( ptgs2a and ptgs2b ) mentioned previously are also in the nf-b signaling pathway ( fig . 3 ) . a number of pathways associated with cytokine - mediated signaling and immune processes , including janus kinase - signal transducer and activator of transcription ( jak - stat ) and tumor necrosis factor ( tnf ) signaling pathways ( fig . 5 ) , are positively enriched at 16 hpl , as reflected by upregulation of genes ( fig . 3 ) such as janus kinase 1 ( jak1 ) , signal transducer and activator of transcription 3 ( stat3 ) , suppressor of cytokine signaling 3b ( socs3b ) , cytokine inducible sh protein orthologs ( cish , cishb ) , and interferon regulator factor 9 ( irf9 ) . activation of immune responses mediated by the complement cascade is also very prominent at 16 hpl : several complement component genes ( c4a , c6 , c7a , c7 ) show a log2fc > 2.0 ( figs . the most highly enriched gene at 16 hpl is leptin b ( lepb ) , a hormone that signals via jak - stat and modulates the immune system . in addition to cytokine signaling and immune responses , the jak - stat signaling pathway is also implicated in regulation of pluripotency in stem cells ( fig . other pluripotency - related enriched genes are associated with several growth factor signaling pathways : fibroblast growth factor receptor 1 orthologs ( fgfr1a , fgfr1b ) , wingless - type mmtv integration site , family member 7a ( wnt7a ) , glycogen synthase kinase 3b ( gsk3b ) , inhibin beta aa ( inhbaa ) , and chromatin modifier polycomb group ring finger 5 orthologs ( pcgf5a , pcgf5b ) . whereas biological processes associated with cell growth and differentiation ( e.g. , cell movement , biological adhesion , tissue development , insulin secretion ) were negatively regulated at 8 hpl ( fig . 4 ) , by 16 hpl evidence for positive enrichment of proliferation processes and pathways ( e.g. , cell proliferation , negative regulation of cell differentiation , regulation of epigenetic gene expression , locomotion and cell migration , regeneration , growth ) was very strong ( fig . 5 ) , suggestive of the transition to cell cycle entry and production of rpcs . consistent with this interpretation is that signaling pathways regulating pluripotency of stem cells were positively enriched at 16 hpl ( fig . to identify transcriptional changes that distinguish regeneration - competent zebrafish mller glia from mammalian mller glia , which lack the ability to regenerate retinal neurons , we analyzed a publicly available microarray dataset of single isolated mouse mller glia from two different mouse models of retinal degeneration , pde6b and rho , which vary in their degeneration kinetics . we used data only from the initial major rod death phase ( postnatal day 13 and postnatal week 8 for pde6b and rho , respectively ) as these were most comparable to our zebrafish dataset , which focused on the initial response of mller glia to photoreceptor loss . we performed a parallel differential expression analysis of biological processes and pathways , as illustrated in figures 6a and 6b and described in more detail in supplementary methods . ( a ) schematic showing the methodology for the pathway analysis based on rna - seq data for the initial stages of mller glia ( b ) schematic showing the methodology for the pathway analysis of microarray data from single mller glia isolated from mouse ( mus musculus ) photoreceptor degeneration models , and the comparison between d. rerio and m. musculus . columns from left to right : signaling pathway or biological process , zebrafish mller glia at 8 hpl , zebrafish mller glia at 16 hpl , mouse mller glia from pde6b retinal degeneration model at postnatal day 13 , mouse mller glia from rho retinal degeneration model at 8 weeks postnatal . arrows and corresponding colors represent the overall direction of regulation according to the legend at the right ; gray dashes represent pathways or processes that were not enriched compared with controls . direction of regulation for 8 and 16 hpl zebrafish mller glia is based on data from figures 4 and 5 , respectively . direction of regulation for pde6b and rho mller glia is based on data from figures 6a and 6b , respectively , with the exception of wnt signaling . pde6b is based on results from reactome analysis ( positive enrichment of formation of the beta - catenin : tcf transactivating complex , supplementary data s3 ) and biological process go terms ( positive enrichment of regulation of canonical wnt signaling pathway , supplementary data s2 ) . rho is based on biological process go term analysis ( negative enrichment of regulation of canonical wnt signaling pathway , supplementary data s2 ) . pathway enrichment analysis in the pde6b retinal degeneration model revealed strong positive regulation of metabolic responses in mouse mller glia ( fig . 7a ) , but in contrast to zebrafish mller glia , the nf-b signaling pathway , cytokine signaling and immune system responses were negatively enriched ( figs . 6c , 7a ) . in the rho mouse retinal degeneration model , a strong metabolic response was also seen the mller glia ( fig . 7b ) ; but in contrast to the pde6b model , the complement cascade pathway was positively enriched ( fig . genes in the biosynthetic pathway leading to pge2 , which were strongly upregulated in fish mller glia at both 8 and 16 hpl ( figs . 3 , 6c , supplementary fig . s1b ) , although several genes in the arachidonic acid metabolism pathway are differentially but inconsistently regulated in both mouse degeneration models ( supplementary fig . similarly , the positive regulation of core circadian clock genes seen in zebrafish mller glia , including per2 , clock , and arntl ( fig . 6c , supplementary fig . s2a ) , is not replicated in mouse mller glia ; instead , negative regulators of per1 ( beta - transducin repeat containing e3 ubiquitin protein ligase , btrc , and s - phase kinase - associated protein 1a , skp1a ) are strongly upregulated , and per1 and arntl ( bmal1 ) are downregulated ( fig . 6c , supplementary fig . wnt signaling is repressed in zebrafish mller glia and variably regulated in the degeneration models ( fig . 6c ; supplementary material s2 , s3 ) . finally , unlike in zebrafish , in the rho model , signaling pathways regulating pluripotency in stem cells were negatively enriched ( figs . 6c , 7b ) . enrichment map of kegg pathways with fdr 0.05 in ( a ) pde6b and ( b ) rho degeneration models . circular node color reflects positive ( red ) or negative enrichment ( blue ) ; color intensity reflects the significance of the enrichment ; and area reflects the size of the gene set . lines ( green ) represent significant overlap in genes within linked sets , and line thickness represents degree of overlap . the inability of mammals to endogenously regenerate neurons is in marked contrast to the highly regeneration - competent zebrafish . the objectives of the current study were to determine a more complete catalog of transcript expression changes during the early steps of mller glia response to neuronal loss and to compare gene regulatory responses of regeneration - competent zebrafish mller glia with those of mller glia in mouse models of retinal degeneration . to help identify regeneration - relevant factors that are missing from mammalian mller glia , which fail to regenerate retinal neurons , we compared transcriptional changes in zebrafish mller glia to publicly available transcriptome data from single mouse mller glia isolated from two genetic models of retinitis pigmentosa in which rod photoreceptors degenerate . ( 1 ) the photoreceptor injury models were not equivalent ( acute photic damage of cones and rods in zebrafish versus genetically induced rod degeneration in mouse ) . ( 2 ) the methods for isolating mller glia were different ( facs isolation of gfp - labeled zebrafish mller glial cells versus single mouse mller glial cells selected based on morphology ) . ( 3 ) the methods for transcriptome analysis were different ( rna - seq for zebrafish mller glia versus microarray analysis for mouse mller glia ) . despite these limitations , the comparison highlighted several potentially important differences between regeneration - competent mller glia in zebrafish and mller glial responding to retinal degeneration in mice . previous gene expression studies of the zebrafish retinal response to injury have shown a rapid induction of stress response transcripts and proteins , and many of the genes identified in those studies were also present in our dataset . although the transcriptional responses of mller glia from pde6b and rho mice show some evidence of gene expression changes associated with gliosis and stress responses , these pathways were not among those identified by the kegg enrichment analyses of these data . however , single - cell transcriptomic data on this heterogeneous population of mller glia result in a lack of statistical power , which might account for this observation . another potential explanation for this apparent difference in activation of stress response pathways is that the mouse models represent chronic photoreceptor degeneration , whereas the zebrafish model is an acute , traumatic injury . for example , hspb11 is a top upregulated gene at 8 hpl in zebrafish mller glia ; hspb11 is required for hedgehog signaling in mice , and this pathway has been shown to promote the stem cell potential of mller glia in mammalian and chick retinas . constitutive hedgehog signaling in developing zebrafish retinas results in an increase in the number of reactive mller glia , although its role has not been studied in the context of zebrafish photoreceptor regeneration . in the mouse hippo signaling also induces drosophila neural stem cell quiescence , inhibits zebrafish rpc proliferation , and promotes differentiation , but again , this pathway has not been examined in retinal regeneration . another signaling pathway responsive to stress is nf-b signaling pathway , which was strongly activated in zebrafish mller glia , but repressed in pde6b mouse mller glia . inhibition of nf-b signaling in neural stem cells results in symmetric division and accumulation of nestin / sox2/gfap - expressing cells , reminiscent of gliosis . nuclear factorb signaling is reactive oxygen species ( ros ) responsive and stimulates an immune response by activating transcription of cytokines and ligands of known importance in retinal regeneration , such as interleukin-6 ( il-6 ) , tnf - alpha , activin a , and in zebrafish tail fin regeneration , such as mmp9 . several genes in the complement cascade ( c4b , c6 , c7 , c7a ) are also upregulated in zebrafish mller glia , and related complement fragments ( c3a and c5a ) can directly induce retinal regeneration in the embryonic chick model through transdifferentiation of retinal pigmented epithelial cells . complement activation can also induce il-6 and tnf - alpha , resulting in nf-b and stat-3 activation , and stat3 activation is critical for retinal regeneration in zebrafish . the role of the immune system in regeneration is increasingly recognized , and an interaction between stem cells and immune cell derived factors is necessary for effective tissue regeneration . in the zebrafish brain , inflammation is both necessary and sufficient to trigger neurogenesis that resembles a regenerative response , but additional studies are needed to understand the role of the immune system in retinal regeneration . several other signaling pathways whose activity is altered in zebrafish mller glia and that have previously been implicated in regulating retinal regeneration include wnt , notch , and tgf/bmp . both wnt and notch signaling are necessary for zebrafish retinal regeneration , so the early repression of these signaling pathways that we observed is somewhat puzzling , although the requirement for activation of wnt signaling is at a later stage , to promote proliferation of rpcs and regeneration of neurons . activation of notch signaling in the mature retina maintains differentiated mller glial fate , inhibits stat3 and ascl1 expression , and prevents progenitor proliferation ; hence the initial rapid downregulation of notch signaling might promote mller glial dedifferentiation and cell cycle reentry . signaling through the tgf/bmp pathway also promotes mller glia differentiation , and zebrafish mutants with impaired capacity to negatively regulate tgf signaling show reduced rpc proliferation and diminished regenerative capacity . interestingly , inhibitors of tgf can replace two of the classic pluripotency factors , c - myc and sox2 , in reprogramming fibroblasts to ipscs . surprisingly , circadian rhythm related terms and pathways were among the most positively enriched in our dataset . disruption of the circadian - regulated , heparin - binding cytokine , midkine a ( mdka ) , in the injured zebrafish retina results in altered cell cycle kinetics , rpc proliferation , and diminished rod photoreceptor regeneration , but no investigations have directly addressed the role of core clock genes . recent studies of stem cell division and differentiation implicate clock genes in functions that have no clear link to the circadian rhythm itself . for instance , per2 , which is upregulated in our dataset , is nonrhythmically expressed in neural stem cells ( nsc ) in the adult hippocampus , and regulates their proliferation and neurogenesis in mice . recent work showed that mouse and human mller glial cells in vitro have an endogenous circadian clock , but in the microarray dataset from mouse degeneration models that we analyzed , the core clock genes were downregulated . a strong metabolic response to photoreceptor degeneration was evident in both fish and mouse mller glia pathway enrichment analyses . the injury - induced metabolic responses in zebrafish mller glia associated with regeneration are largely unexplored , in contrast to extensive investigations of metabolic profiles associated with reactive gliosis in mammalian mller glia . the essential role of metabolism in directing cell proliferation , differentiation , and cell fate determination is increasingly recognized , and levels of glycolysis , oxidative phosphorylation , and oxidative stress provide a metabolic signature that distinguishes pluripotent stem cells , rapidly dividing progenitor cells , and differentiated cells . the initial metabolic state and energy metabolism of zebrafish mller glia in response to photoreceptor injury includes enhanced oxidative phosphorylation and lipid metabolism , similar to neural progenitors , which are characterized by high levels of aerobic glycolysis , oxidative phosphorylation , ros , and eicosanoid and fatty acid biosynthesis . within a few hours , the profile of zebrafish mller glia shifts to negative regulation of metabolic pathways , decreased glycolysis , and enrichment of the foxo signaling pathway , which more resembles nsc . fox - o signaling mediates ros suppression , and inhibiting foxo - mediated ros suppression results in a depleted nsc population in mice . the rapidly shifting profile of energy and metabolism in zebrafish mller glia toward a stemness - like profile may reflect preparation for the asymmetric , self - renewing division that initiates the regenerative process . an in - depth , comparative metabolomic study of zebrafish mller glia and mammalian mller glia could reveal metabolic pathways that regulate the regenerative response in zebrafish retina , and the relevant enzymes might be excellent therapeutic targets for pharmacologic treatments to enhance endogenous regeneration . of particular interest are changes in fatty acid metabolism , which play an important role in the regulation of neural stem cell proliferation . icosanoid ( also called eicosanoid)-derived metabolites , including pge2 , can regulate nf-b , differentiation , and inflammation in stem cell populations , and inhibitors of pge2-degrading enzymes potentiate tissue regeneration in mice . the addition of pge2 to rat mller glia in vitro enhances their dedifferentiation , stem cell like properties , and proliferative abilities . our transcriptome analysis suggests that pge2 levels in zebrafish , but not in mouse , mller glia are increased after loss of photoreceptors . whether pge2 promotes mller glia dependent retinal regeneration is unknown , but if so , this may be a promising therapeutic target . a final and critical difference is that zebrafish mller glia demonstrate positive enrichment of pluripotency factors and cell proliferation , whereas mouse mller glia show reduced pluripotency factors and no evidence of cell proliferation in response to photoreceptor loss . although mammalian mller glia express some genes associated with rpcs and have latent neurogenic capabilities , these attributes are not enhanced in response to neuronal loss . in conclusion , a comparison of transcriptional profiles from regeneration - competent zebrafish mller glia and gliotic mouse mller glia has revealed several novel , previously unexplored , categories of biological responses that could act to promote endogenous retinal regeneration : nf-b signaling , pge2 synthesis , expression of core clock genes , and signaling pathways and metabolic signatures associated with stem cells . these and other candidates can be explored in future investigations and may provide potential therapeutic targets that could enhance endogenous mller glial dependent regeneration of retinal neurons in humans . the datasets supporting the conclusions of this article are available in the national center for biotechnology information sequence read archive and gene expression omnibus repository ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse86872 ) , and are included within the article or the supplementary material .

purposezebrafish neurons regenerate from mller glia following retinal lesions . genes and signaling pathways important for retinal regeneration in zebrafish have been described , but our understanding of how mller glial stem cell properties are regulated is incomplete . mammalian mller glia possess a latent neurogenic capacity that might be enhanced in regenerative therapies to treat degenerative retinal diseases.methodsto identify transcriptional changes associated with stem cell properties in zebrafish mller glia , we performed a comparative transcriptome analysis from isolated cells at 8 and 16 hours following an acute photic lesion , prior to the asymmetric division that produces retinal progenitors.resultswe report a rapid , dynamic response of zebrafish mller glia , characterized by activation of pathways related to stress , nuclear factorb ( nf-b ) signaling , cytokine signaling , immunity , prostaglandin metabolism , circadian rhythm , and pluripotency , and an initial repression of wnt signaling . when we compared publicly available transcriptomes of isolated mouse mller glia from two retinal degeneration models , we found that mouse mller glia showed evidence of oxidative stress , variable responses associated with immune regulation , and repression of pathways associated with pluripotency , development , and proliferation.conclusionscategories of biological processes / pathways activated following photoreceptor loss in regeneration - competent zebrafish mller glia , which distinguished them from mouse mller glia in retinal degeneration models , included cytokine signaling ( notably nf-b ) , prostaglandin e2 synthesis , expression of core clock genes , and pathways / metabolic states associated with pluripotency . these regulatory mechanisms are relatively unexplored as potential mediators of stem cell properties likely to be important in mller glial cells for successful retinal regeneration .

Methods Results A Large, Rapid, and Dynamic Transcriptional Response in Zebrafish Mller Glia After Photoreceptor Injury With Acute Light Damage Stress Response, Prostaglandin Metabolism, and Circadian Rhythm Pathways Are Enriched and Wnt Signaling Is Repressed in Zebrafish Mller Glia at 8 hpl NF-B Signaling, Immune Responses, Cytokine Signaling, and Pluripotency Processes and Pathways Are Enriched in Zebrafish Mller Glia at 16 hpl Mouse Retinal Degeneration Model Analysis Discussion Availability of Data and Material Supplementary Material

we collected mller glia at 0 ( nonlesioned ) , 8 , and 16 hpl , prior to their initial asymmetric , stem cell like division . this analysis identified 2690 genes differentially expressed in mller glia isolated from light - lesioned zebrafish retinas at 8 or 16 hpl compared with mller glia from control ( unlesioned ) retinas ( fig . many of these genes belong to specific categories of biological processes , including stress response ( hspd1 ) , immune response / cytokines ( stat3 , lepb ) , secreted factors ( hb - egf ) , mller glial cell dedifferentiation and acquisition of stem cell properties ( tgif1 , lin28 , apobec2b ) , and cell adhesion ( cdh2 ) . log2fold changes ( fc ) of selected regeneration - associated genes in mller glia at 8 and 16 hours post lesion ( hpl ) validation of expression levels with rt quantitative pcr ( qpcr ) confirmed the rna - seq data for several genes of interest tgf-induced factor homeobox 1 ( tgif1 ) , cytokine receptor like factor 1a ( crlf1a ) , heat shock protein family d member 1 ( hspd1 ) , complement component 7 ( c7 ) , prostaglandin e synthase ( ptges ) , and matrix metallopeptidase 9 ( mmp9 ) at 8 hpl ( fig . to provide a global overview of the rapid and dynamic transcriptional changes in mller glia as they respond to photoreceptor loss and prepare to generate retinal progenitors , we discuss the results by classifying these data into eight general , nonexclusive , categories : stress response , prostaglandins , circadian rhythm , wnt signaling , nuclear factorb ( nf-b ) signaling , immune response , cytokines , and pluripotency . to illustrate dynamic transcriptional changes in mller glia as they prepare for an asymmetric stem cell like mitotic division , figure 3 plots the log2fc differential expression of genes in each of these categories at 8 vs. 16 hpl . other evidence of a strong oxidative stress response includes elevated expression of biliverdin reductase a ( blvra ) , heat shock protein alpha - crystallin - related b11 ( hspb11 ) , and heat shock 60-kda protein 1 ( hspd1 ) ( figs . two categories of biological responses that are rapidly activated in mller glia , but have not previously been associated with retinal regeneration , are prostaglandin metabolism and circadian rhythm ( figs . 4 ) ; especially prominent are pathways associated with lipid metabolism , including icosanoids ( also called eicosanoids ) , fatty acid derivatives , and arachidonic acid . these metabolic changes are reflected in the increased expression of zebrafish orthologs of prostaglandin e synthase ( ptges , ptges1 ) , prostaglandin endoperoxide synthase ( ptges , ptges1 ) , and prostaglandin d2 synthase ( ptgdsb ) genes in the biosynthetic pathway for the eicosanoid prostaglandin e2 ( pge2 ) ( fig . pathways and biological processes enriched at 16 hpl in zebrafish mller glia . activation of biological processes and pathways indicative of oxidative stress and the regulation of redox homeostasis , which were already evident at 8 hpl ( figs . 3 , 4 ) , continue to be enriched at 16 hpl , but with a clear shift toward more prominent activation of signaling pathways related to inflammation , including both innate and adaptive immune responses ( figs . a number of pathways associated with cytokine - mediated signaling and immune processes , including janus kinase - signal transducer and activator of transcription ( jak - stat ) and tumor necrosis factor ( tnf ) signaling pathways ( fig . 3 ) such as janus kinase 1 ( jak1 ) , signal transducer and activator of transcription 3 ( stat3 ) , suppressor of cytokine signaling 3b ( socs3b ) , cytokine inducible sh protein orthologs ( cish , cishb ) , and interferon regulator factor 9 ( irf9 ) . other pluripotency - related enriched genes are associated with several growth factor signaling pathways : fibroblast growth factor receptor 1 orthologs ( fgfr1a , fgfr1b ) , wingless - type mmtv integration site , family member 7a ( wnt7a ) , glycogen synthase kinase 3b ( gsk3b ) , inhibin beta aa ( inhbaa ) , and chromatin modifier polycomb group ring finger 5 orthologs ( pcgf5a , pcgf5b ) . to identify transcriptional changes that distinguish regeneration - competent zebrafish mller glia from mammalian mller glia , which lack the ability to regenerate retinal neurons , we analyzed a publicly available microarray dataset of single isolated mouse mller glia from two different mouse models of retinal degeneration , pde6b and rho , which vary in their degeneration kinetics . we performed a parallel differential expression analysis of biological processes and pathways , as illustrated in figures 6a and 6b and described in more detail in supplementary methods . ( a ) schematic showing the methodology for the pathway analysis based on rna - seq data for the initial stages of mller glia ( b ) schematic showing the methodology for the pathway analysis of microarray data from single mller glia isolated from mouse ( mus musculus ) photoreceptor degeneration models , and the comparison between d. rerio and m. musculus . columns from left to right : signaling pathway or biological process , zebrafish mller glia at 8 hpl , zebrafish mller glia at 16 hpl , mouse mller glia from pde6b retinal degeneration model at postnatal day 13 , mouse mller glia from rho retinal degeneration model at 8 weeks postnatal . direction of regulation for 8 and 16 hpl zebrafish mller glia is based on data from figures 4 and 5 , respectively . 7a ) , but in contrast to zebrafish mller glia , the nf-b signaling pathway , cytokine signaling and immune system responses were negatively enriched ( figs . similarly , the positive regulation of core circadian clock genes seen in zebrafish mller glia , including per2 , clock , and arntl ( fig . s2a ) , is not replicated in mouse mller glia ; instead , negative regulators of per1 ( beta - transducin repeat containing e3 ubiquitin protein ligase , btrc , and s - phase kinase - associated protein 1a , skp1a ) are strongly upregulated , and per1 and arntl ( bmal1 ) are downregulated ( fig . wnt signaling is repressed in zebrafish mller glia and variably regulated in the degeneration models ( fig . many of these genes belong to specific categories of biological processes , including stress response ( hspd1 ) , immune response / cytokines ( stat3 , lepb ) , secreted factors ( hb - egf ) , mller glial cell dedifferentiation and acquisition of stem cell properties ( tgif1 , lin28 , apobec2b ) , and cell adhesion ( cdh2 ) . log2fold changes ( fc ) of selected regeneration - associated genes in mller glia at 8 and 16 hours post lesion ( hpl ) validation of expression levels with rt quantitative pcr ( qpcr ) confirmed the rna - seq data for several genes of interest tgf-induced factor homeobox 1 ( tgif1 ) , cytokine receptor like factor 1a ( crlf1a ) , heat shock protein family d member 1 ( hspd1 ) , complement component 7 ( c7 ) , prostaglandin e synthase ( ptges ) , and matrix metallopeptidase 9 ( mmp9 ) at 8 hpl ( fig . to provide a global overview of the rapid and dynamic transcriptional changes in mller glia as they respond to photoreceptor loss and prepare to generate retinal progenitors , we discuss the results by classifying these data into eight general , nonexclusive , categories : stress response , prostaglandins , circadian rhythm , wnt signaling , nuclear factorb ( nf-b ) signaling , immune response , cytokines , and pluripotency . to illustrate dynamic transcriptional changes in mller glia as they prepare for an asymmetric stem cell like mitotic division , figure 3 plots the log2fc differential expression of genes in each of these categories at 8 vs. 16 hpl . other evidence of a strong oxidative stress response includes elevated expression of biliverdin reductase a ( blvra ) , heat shock protein alpha - crystallin - related b11 ( hspb11 ) , and heat shock 60-kda protein 1 ( hspd1 ) ( figs . two categories of biological responses that are rapidly activated in mller glia , but have not previously been associated with retinal regeneration , are prostaglandin metabolism and circadian rhythm ( figs . 4 ) ; especially prominent are pathways associated with lipid metabolism , including icosanoids ( also called eicosanoids ) , fatty acid derivatives , and arachidonic acid . these metabolic changes are reflected in the increased expression of zebrafish orthologs of prostaglandin e synthase ( ptges , ptges1 ) , prostaglandin endoperoxide synthase ( ptges , ptges1 ) , and prostaglandin d2 synthase ( ptgdsb ) genes in the biosynthetic pathway for the eicosanoid prostaglandin e2 ( pge2 ) ( fig . activation of biological processes and pathways indicative of oxidative stress and the regulation of redox homeostasis , which were already evident at 8 hpl ( figs . 3 , 4 ) , continue to be enriched at 16 hpl , but with a clear shift toward more prominent activation of signaling pathways related to inflammation , including both innate and adaptive immune responses ( figs . a number of pathways associated with cytokine - mediated signaling and immune processes , including janus kinase - signal transducer and activator of transcription ( jak - stat ) and tumor necrosis factor ( tnf ) signaling pathways ( fig . 3 ) such as janus kinase 1 ( jak1 ) , signal transducer and activator of transcription 3 ( stat3 ) , suppressor of cytokine signaling 3b ( socs3b ) , cytokine inducible sh protein orthologs ( cish , cishb ) , and interferon regulator factor 9 ( irf9 ) . to identify transcriptional changes that distinguish regeneration - competent zebrafish mller glia from mammalian mller glia , which lack the ability to regenerate retinal neurons , we analyzed a publicly available microarray dataset of single isolated mouse mller glia from two different mouse models of retinal degeneration , pde6b and rho , which vary in their degeneration kinetics . we used data only from the initial major rod death phase ( postnatal day 13 and postnatal week 8 for pde6b and rho , respectively ) as these were most comparable to our zebrafish dataset , which focused on the initial response of mller glia to photoreceptor loss . we performed a parallel differential expression analysis of biological processes and pathways , as illustrated in figures 6a and 6b and described in more detail in supplementary methods . ( a ) schematic showing the methodology for the pathway analysis based on rna - seq data for the initial stages of mller glia ( b ) schematic showing the methodology for the pathway analysis of microarray data from single mller glia isolated from mouse ( mus musculus ) photoreceptor degeneration models , and the comparison between d. rerio and m. musculus . columns from left to right : signaling pathway or biological process , zebrafish mller glia at 8 hpl , zebrafish mller glia at 16 hpl , mouse mller glia from pde6b retinal degeneration model at postnatal day 13 , mouse mller glia from rho retinal degeneration model at 8 weeks postnatal . 7a ) , but in contrast to zebrafish mller glia , the nf-b signaling pathway , cytokine signaling and immune system responses were negatively enriched ( figs . similarly , the positive regulation of core circadian clock genes seen in zebrafish mller glia , including per2 , clock , and arntl ( fig . wnt signaling is repressed in zebrafish mller glia and variably regulated in the degeneration models ( fig . the inability of mammals to endogenously regenerate neurons is in marked contrast to the highly regeneration - competent zebrafish . the objectives of the current study were to determine a more complete catalog of transcript expression changes during the early steps of mller glia response to neuronal loss and to compare gene regulatory responses of regeneration - competent zebrafish mller glia with those of mller glia in mouse models of retinal degeneration . to help identify regeneration - relevant factors that are missing from mammalian mller glia , which fail to regenerate retinal neurons , we compared transcriptional changes in zebrafish mller glia to publicly available transcriptome data from single mouse mller glia isolated from two genetic models of retinitis pigmentosa in which rod photoreceptors degenerate . ( 2 ) the methods for isolating mller glia were different ( facs isolation of gfp - labeled zebrafish mller glial cells versus single mouse mller glial cells selected based on morphology ) . ( 3 ) the methods for transcriptome analysis were different ( rna - seq for zebrafish mller glia versus microarray analysis for mouse mller glia ) . despite these limitations , the comparison highlighted several potentially important differences between regeneration - competent mller glia in zebrafish and mller glial responding to retinal degeneration in mice . although the transcriptional responses of mller glia from pde6b and rho mice show some evidence of gene expression changes associated with gliosis and stress responses , these pathways were not among those identified by the kegg enrichment analyses of these data . for example , hspb11 is a top upregulated gene at 8 hpl in zebrafish mller glia ; hspb11 is required for hedgehog signaling in mice , and this pathway has been shown to promote the stem cell potential of mller glia in mammalian and chick retinas . in the mouse hippo signaling also induces drosophila neural stem cell quiescence , inhibits zebrafish rpc proliferation , and promotes differentiation , but again , this pathway has not been examined in retinal regeneration . another signaling pathway responsive to stress is nf-b signaling pathway , which was strongly activated in zebrafish mller glia , but repressed in pde6b mouse mller glia . nuclear factorb signaling is reactive oxygen species ( ros ) responsive and stimulates an immune response by activating transcription of cytokines and ligands of known importance in retinal regeneration , such as interleukin-6 ( il-6 ) , tnf - alpha , activin a , and in zebrafish tail fin regeneration , such as mmp9 . several genes in the complement cascade ( c4b , c6 , c7 , c7a ) are also upregulated in zebrafish mller glia , and related complement fragments ( c3a and c5a ) can directly induce retinal regeneration in the embryonic chick model through transdifferentiation of retinal pigmented epithelial cells . complement activation can also induce il-6 and tnf - alpha , resulting in nf-b and stat-3 activation , and stat3 activation is critical for retinal regeneration in zebrafish . several other signaling pathways whose activity is altered in zebrafish mller glia and that have previously been implicated in regulating retinal regeneration include wnt , notch , and tgf/bmp . both wnt and notch signaling are necessary for zebrafish retinal regeneration , so the early repression of these signaling pathways that we observed is somewhat puzzling , although the requirement for activation of wnt signaling is at a later stage , to promote proliferation of rpcs and regeneration of neurons . disruption of the circadian - regulated , heparin - binding cytokine , midkine a ( mdka ) , in the injured zebrafish retina results in altered cell cycle kinetics , rpc proliferation , and diminished rod photoreceptor regeneration , but no investigations have directly addressed the role of core clock genes . recent studies of stem cell division and differentiation implicate clock genes in functions that have no clear link to the circadian rhythm itself . recent work showed that mouse and human mller glial cells in vitro have an endogenous circadian clock , but in the microarray dataset from mouse degeneration models that we analyzed , the core clock genes were downregulated . the injury - induced metabolic responses in zebrafish mller glia associated with regeneration are largely unexplored , in contrast to extensive investigations of metabolic profiles associated with reactive gliosis in mammalian mller glia . the initial metabolic state and energy metabolism of zebrafish mller glia in response to photoreceptor injury includes enhanced oxidative phosphorylation and lipid metabolism , similar to neural progenitors , which are characterized by high levels of aerobic glycolysis , oxidative phosphorylation , ros , and eicosanoid and fatty acid biosynthesis . within a few hours , the profile of zebrafish mller glia shifts to negative regulation of metabolic pathways , decreased glycolysis , and enrichment of the foxo signaling pathway , which more resembles nsc . the rapidly shifting profile of energy and metabolism in zebrafish mller glia toward a stemness - like profile may reflect preparation for the asymmetric , self - renewing division that initiates the regenerative process . an in - depth , comparative metabolomic study of zebrafish mller glia and mammalian mller glia could reveal metabolic pathways that regulate the regenerative response in zebrafish retina , and the relevant enzymes might be excellent therapeutic targets for pharmacologic treatments to enhance endogenous regeneration . the addition of pge2 to rat mller glia in vitro enhances their dedifferentiation , stem cell like properties , and proliferative abilities . our transcriptome analysis suggests that pge2 levels in zebrafish , but not in mouse , mller glia are increased after loss of photoreceptors . a final and critical difference is that zebrafish mller glia demonstrate positive enrichment of pluripotency factors and cell proliferation , whereas mouse mller glia show reduced pluripotency factors and no evidence of cell proliferation in response to photoreceptor loss . although mammalian mller glia express some genes associated with rpcs and have latent neurogenic capabilities , these attributes are not enhanced in response to neuronal loss . in conclusion , a comparison of transcriptional profiles from regeneration - competent zebrafish mller glia and gliotic mouse mller glia has revealed several novel , previously unexplored , categories of biological responses that could act to promote endogenous retinal regeneration : nf-b signaling , pge2 synthesis , expression of core clock genes , and signaling pathways and metabolic signatures associated with stem cells .

the regenerative response in mller glia was induced as previously described by exposing free - swimming , adult tg(gfap : egfp)mi2002 zebrafish ( 811 months old ) to intense light ( > 120,000 lux ) from an exfo x - cite 120w metal halide lamp ( exfo photonic solutions , quebec city , qc , canada ) for 30 minutes ( fig . many of these genes belong to specific categories of biological processes , including stress response ( hspd1 ) , immune response / cytokines ( stat3 , lepb ) , secreted factors ( hb - egf ) , mller glial cell dedifferentiation and acquisition of stem cell properties ( tgif1 , lin28 , apobec2b ) , and cell adhesion ( cdh2 ) . a few of the known regeneration genes with small or below criterion values of differential expression in this rna - seq dataset ( table ) are primarily implicated in processes that occur later than those assayed here , such as cell fate specification ( ascl1a , sox2 ) and progenitor proliferation ( fabp7a , insm1a ) . log2fold changes ( fc ) of selected regeneration - associated genes in mller glia at 8 and 16 hours post lesion ( hpl ) validation of expression levels with rt quantitative pcr ( qpcr ) confirmed the rna - seq data for several genes of interest tgf-induced factor homeobox 1 ( tgif1 ) , cytokine receptor like factor 1a ( crlf1a ) , heat shock protein family d member 1 ( hspd1 ) , complement component 7 ( c7 ) , prostaglandin e synthase ( ptges ) , and matrix metallopeptidase 9 ( mmp9 ) at 8 hpl ( fig . to provide a global overview of the rapid and dynamic transcriptional changes in mller glia as they respond to photoreceptor loss and prepare to generate retinal progenitors , we discuss the results by classifying these data into eight general , nonexclusive , categories : stress response , prostaglandins , circadian rhythm , wnt signaling , nuclear factorb ( nf-b ) signaling , immune response , cytokines , and pluripotency . other evidence of a strong oxidative stress response includes elevated expression of biliverdin reductase a ( blvra ) , heat shock protein alpha - crystallin - related b11 ( hspb11 ) , and heat shock 60-kda protein 1 ( hspd1 ) ( figs . these metabolic changes are reflected in the increased expression of zebrafish orthologs of prostaglandin e synthase ( ptges , ptges1 ) , prostaglandin endoperoxide synthase ( ptges , ptges1 ) , and prostaglandin d2 synthase ( ptgdsb ) genes in the biosynthetic pathway for the eicosanoid prostaglandin e2 ( pge2 ) ( fig . these results suggest that the preparations of facs - isolated mller glia are contaminated with photoreceptor transcripts , as has been noted in previous microarray profiling studies of sorted mller glia from zebrafish ( supplementary figs . as this tight physical association would predict , the inverse is also true specific transcripts ( retinaldehyde binding protein 1 ; supplementary table s5 ) are found in facs - isolated rod and cone photoreceptors from adult mouse ( supplementary figs . 3 ) , including tumor necrosis factor receptor superfamily orthologs ( tnfrsf1a , tnfrsf11a ) , mitogen - activated protein kinase kinase kinase 14 ( map3k14 ) , and growth arrest and dna - damage - inducible 45 beta orthologs ( gadd45ba , gadd45bb ) . a number of pathways associated with cytokine - mediated signaling and immune processes , including janus kinase - signal transducer and activator of transcription ( jak - stat ) and tumor necrosis factor ( tnf ) signaling pathways ( fig . 3 ) such as janus kinase 1 ( jak1 ) , signal transducer and activator of transcription 3 ( stat3 ) , suppressor of cytokine signaling 3b ( socs3b ) , cytokine inducible sh protein orthologs ( cish , cishb ) , and interferon regulator factor 9 ( irf9 ) . other pluripotency - related enriched genes are associated with several growth factor signaling pathways : fibroblast growth factor receptor 1 orthologs ( fgfr1a , fgfr1b ) , wingless - type mmtv integration site , family member 7a ( wnt7a ) , glycogen synthase kinase 3b ( gsk3b ) , inhibin beta aa ( inhbaa ) , and chromatin modifier polycomb group ring finger 5 orthologs ( pcgf5a , pcgf5b ) . to identify transcriptional changes that distinguish regeneration - competent zebrafish mller glia from mammalian mller glia , which lack the ability to regenerate retinal neurons , we analyzed a publicly available microarray dataset of single isolated mouse mller glia from two different mouse models of retinal degeneration , pde6b and rho , which vary in their degeneration kinetics . we used data only from the initial major rod death phase ( postnatal day 13 and postnatal week 8 for pde6b and rho , respectively ) as these were most comparable to our zebrafish dataset , which focused on the initial response of mller glia to photoreceptor loss . ( a ) schematic showing the methodology for the pathway analysis based on rna - seq data for the initial stages of mller glia ( b ) schematic showing the methodology for the pathway analysis of microarray data from single mller glia isolated from mouse ( mus musculus ) photoreceptor degeneration models , and the comparison between d. rerio and m. musculus . columns from left to right : signaling pathway or biological process , zebrafish mller glia at 8 hpl , zebrafish mller glia at 16 hpl , mouse mller glia from pde6b retinal degeneration model at postnatal day 13 , mouse mller glia from rho retinal degeneration model at 8 weeks postnatal . pde6b is based on results from reactome analysis ( positive enrichment of formation of the beta - catenin : tcf transactivating complex , supplementary data s3 ) and biological process go terms ( positive enrichment of regulation of canonical wnt signaling pathway , supplementary data s2 ) . s2a ) , is not replicated in mouse mller glia ; instead , negative regulators of per1 ( beta - transducin repeat containing e3 ubiquitin protein ligase , btrc , and s - phase kinase - associated protein 1a , skp1a ) are strongly upregulated , and per1 and arntl ( bmal1 ) are downregulated ( fig . many of these genes belong to specific categories of biological processes , including stress response ( hspd1 ) , immune response / cytokines ( stat3 , lepb ) , secreted factors ( hb - egf ) , mller glial cell dedifferentiation and acquisition of stem cell properties ( tgif1 , lin28 , apobec2b ) , and cell adhesion ( cdh2 ) . a few of the known regeneration genes with small or below criterion values of differential expression in this rna - seq dataset ( table ) are primarily implicated in processes that occur later than those assayed here , such as cell fate specification ( ascl1a , sox2 ) and progenitor proliferation ( fabp7a , insm1a ) . log2fold changes ( fc ) of selected regeneration - associated genes in mller glia at 8 and 16 hours post lesion ( hpl ) validation of expression levels with rt quantitative pcr ( qpcr ) confirmed the rna - seq data for several genes of interest tgf-induced factor homeobox 1 ( tgif1 ) , cytokine receptor like factor 1a ( crlf1a ) , heat shock protein family d member 1 ( hspd1 ) , complement component 7 ( c7 ) , prostaglandin e synthase ( ptges ) , and matrix metallopeptidase 9 ( mmp9 ) at 8 hpl ( fig . to provide a global overview of the rapid and dynamic transcriptional changes in mller glia as they respond to photoreceptor loss and prepare to generate retinal progenitors , we discuss the results by classifying these data into eight general , nonexclusive , categories : stress response , prostaglandins , circadian rhythm , wnt signaling , nuclear factorb ( nf-b ) signaling , immune response , cytokines , and pluripotency . other evidence of a strong oxidative stress response includes elevated expression of biliverdin reductase a ( blvra ) , heat shock protein alpha - crystallin - related b11 ( hspb11 ) , and heat shock 60-kda protein 1 ( hspd1 ) ( figs . these metabolic changes are reflected in the increased expression of zebrafish orthologs of prostaglandin e synthase ( ptges , ptges1 ) , prostaglandin endoperoxide synthase ( ptges , ptges1 ) , and prostaglandin d2 synthase ( ptgdsb ) genes in the biosynthetic pathway for the eicosanoid prostaglandin e2 ( pge2 ) ( fig . these results suggest that the preparations of facs - isolated mller glia are contaminated with photoreceptor transcripts , as has been noted in previous microarray profiling studies of sorted mller glia from zebrafish ( supplementary figs . as this tight physical association would predict , the inverse is also true specific transcripts ( retinaldehyde binding protein 1 ; supplementary table s5 ) are found in facs - isolated rod and cone photoreceptors from adult mouse ( supplementary figs . 3 ) , including tumor necrosis factor receptor superfamily orthologs ( tnfrsf1a , tnfrsf11a ) , mitogen - activated protein kinase kinase kinase 14 ( map3k14 ) , and growth arrest and dna - damage - inducible 45 beta orthologs ( gadd45ba , gadd45bb ) . a number of pathways associated with cytokine - mediated signaling and immune processes , including janus kinase - signal transducer and activator of transcription ( jak - stat ) and tumor necrosis factor ( tnf ) signaling pathways ( fig . 3 ) such as janus kinase 1 ( jak1 ) , signal transducer and activator of transcription 3 ( stat3 ) , suppressor of cytokine signaling 3b ( socs3b ) , cytokine inducible sh protein orthologs ( cish , cishb ) , and interferon regulator factor 9 ( irf9 ) . other pluripotency - related enriched genes are associated with several growth factor signaling pathways : fibroblast growth factor receptor 1 orthologs ( fgfr1a , fgfr1b ) , wingless - type mmtv integration site , family member 7a ( wnt7a ) , glycogen synthase kinase 3b ( gsk3b ) , inhibin beta aa ( inhbaa ) , and chromatin modifier polycomb group ring finger 5 orthologs ( pcgf5a , pcgf5b ) . to identify transcriptional changes that distinguish regeneration - competent zebrafish mller glia from mammalian mller glia , which lack the ability to regenerate retinal neurons , we analyzed a publicly available microarray dataset of single isolated mouse mller glia from two different mouse models of retinal degeneration , pde6b and rho , which vary in their degeneration kinetics . we used data only from the initial major rod death phase ( postnatal day 13 and postnatal week 8 for pde6b and rho , respectively ) as these were most comparable to our zebrafish dataset , which focused on the initial response of mller glia to photoreceptor loss . ( a ) schematic showing the methodology for the pathway analysis based on rna - seq data for the initial stages of mller glia ( b ) schematic showing the methodology for the pathway analysis of microarray data from single mller glia isolated from mouse ( mus musculus ) photoreceptor degeneration models , and the comparison between d. rerio and m. musculus . columns from left to right : signaling pathway or biological process , zebrafish mller glia at 8 hpl , zebrafish mller glia at 16 hpl , mouse mller glia from pde6b retinal degeneration model at postnatal day 13 , mouse mller glia from rho retinal degeneration model at 8 weeks postnatal . pde6b is based on results from reactome analysis ( positive enrichment of formation of the beta - catenin : tcf transactivating complex , supplementary data s3 ) and biological process go terms ( positive enrichment of regulation of canonical wnt signaling pathway , supplementary data s2 ) . s2a ) , is not replicated in mouse mller glia ; instead , negative regulators of per1 ( beta - transducin repeat containing e3 ubiquitin protein ligase , btrc , and s - phase kinase - associated protein 1a , skp1a ) are strongly upregulated , and per1 and arntl ( bmal1 ) are downregulated ( fig . the objectives of the current study were to determine a more complete catalog of transcript expression changes during the early steps of mller glia response to neuronal loss and to compare gene regulatory responses of regeneration - competent zebrafish mller glia with those of mller glia in mouse models of retinal degeneration . to help identify regeneration - relevant factors that are missing from mammalian mller glia , which fail to regenerate retinal neurons , we compared transcriptional changes in zebrafish mller glia to publicly available transcriptome data from single mouse mller glia isolated from two genetic models of retinitis pigmentosa in which rod photoreceptors degenerate . although the transcriptional responses of mller glia from pde6b and rho mice show some evidence of gene expression changes associated with gliosis and stress responses , these pathways were not among those identified by the kegg enrichment analyses of these data . for example , hspb11 is a top upregulated gene at 8 hpl in zebrafish mller glia ; hspb11 is required for hedgehog signaling in mice , and this pathway has been shown to promote the stem cell potential of mller glia in mammalian and chick retinas . constitutive hedgehog signaling in developing zebrafish retinas results in an increase in the number of reactive mller glia , although its role has not been studied in the context of zebrafish photoreceptor regeneration . inhibition of nf-b signaling in neural stem cells results in symmetric division and accumulation of nestin / sox2/gfap - expressing cells , reminiscent of gliosis . nuclear factorb signaling is reactive oxygen species ( ros ) responsive and stimulates an immune response by activating transcription of cytokines and ligands of known importance in retinal regeneration , such as interleukin-6 ( il-6 ) , tnf - alpha , activin a , and in zebrafish tail fin regeneration , such as mmp9 . several genes in the complement cascade ( c4b , c6 , c7 , c7a ) are also upregulated in zebrafish mller glia , and related complement fragments ( c3a and c5a ) can directly induce retinal regeneration in the embryonic chick model through transdifferentiation of retinal pigmented epithelial cells . the role of the immune system in regeneration is increasingly recognized , and an interaction between stem cells and immune cell derived factors is necessary for effective tissue regeneration . in the zebrafish brain , inflammation is both necessary and sufficient to trigger neurogenesis that resembles a regenerative response , but additional studies are needed to understand the role of the immune system in retinal regeneration . several other signaling pathways whose activity is altered in zebrafish mller glia and that have previously been implicated in regulating retinal regeneration include wnt , notch , and tgf/bmp . both wnt and notch signaling are necessary for zebrafish retinal regeneration , so the early repression of these signaling pathways that we observed is somewhat puzzling , although the requirement for activation of wnt signaling is at a later stage , to promote proliferation of rpcs and regeneration of neurons . activation of notch signaling in the mature retina maintains differentiated mller glial fate , inhibits stat3 and ascl1 expression , and prevents progenitor proliferation ; hence the initial rapid downregulation of notch signaling might promote mller glial dedifferentiation and cell cycle reentry . disruption of the circadian - regulated , heparin - binding cytokine , midkine a ( mdka ) , in the injured zebrafish retina results in altered cell cycle kinetics , rpc proliferation , and diminished rod photoreceptor regeneration , but no investigations have directly addressed the role of core clock genes . the injury - induced metabolic responses in zebrafish mller glia associated with regeneration are largely unexplored , in contrast to extensive investigations of metabolic profiles associated with reactive gliosis in mammalian mller glia . the essential role of metabolism in directing cell proliferation , differentiation , and cell fate determination is increasingly recognized , and levels of glycolysis , oxidative phosphorylation , and oxidative stress provide a metabolic signature that distinguishes pluripotent stem cells , rapidly dividing progenitor cells , and differentiated cells . the initial metabolic state and energy metabolism of zebrafish mller glia in response to photoreceptor injury includes enhanced oxidative phosphorylation and lipid metabolism , similar to neural progenitors , which are characterized by high levels of aerobic glycolysis , oxidative phosphorylation , ros , and eicosanoid and fatty acid biosynthesis . within a few hours , the profile of zebrafish mller glia shifts to negative regulation of metabolic pathways , decreased glycolysis , and enrichment of the foxo signaling pathway , which more resembles nsc . an in - depth , comparative metabolomic study of zebrafish mller glia and mammalian mller glia could reveal metabolic pathways that regulate the regenerative response in zebrafish retina , and the relevant enzymes might be excellent therapeutic targets for pharmacologic treatments to enhance endogenous regeneration . icosanoid ( also called eicosanoid)-derived metabolites , including pge2 , can regulate nf-b , differentiation , and inflammation in stem cell populations , and inhibitors of pge2-degrading enzymes potentiate tissue regeneration in mice . a final and critical difference is that zebrafish mller glia demonstrate positive enrichment of pluripotency factors and cell proliferation , whereas mouse mller glia show reduced pluripotency factors and no evidence of cell proliferation in response to photoreceptor loss . in conclusion , a comparison of transcriptional profiles from regeneration - competent zebrafish mller glia and gliotic mouse mller glia has revealed several novel , previously unexplored , categories of biological responses that could act to promote endogenous retinal regeneration : nf-b signaling , pge2 synthesis , expression of core clock genes , and signaling pathways and metabolic signatures associated with stem cells .

edible plants ( 100 g ) were minced into 5 mm fragments and extracts were obtained with methanol ( 200 ml ) overnight at room temperature . the extracts were filtered and the residue was re - extracted under the same conditions as above . the combined filtrates were evaporated in a vacuum slightly below 40c in a rotary evaporator . the specimen was dissolved in dmso ( 1.0 ml ) . human umbilical vein endothelial cells ( no . jcrb0408 , health science research resources bank , osaka , japan ) were cultured at 37c in sfm cs - c medium ( ds pharma biomedical , osaka , japan ) supplemented with 1.5 g / ml fungizone under air - co2 ( 95:5 ) ( referred to hereafter as cs - cf medium ) . for screening study purposes , huvecs were seeded in 60 mm culture dishes ( 1.5 10 cells ) . then , huvecs were incubated with 1 g / l ( normal glucose medium ) or 4.5 g / l ( high glucose medium ) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed , one receiving 1 ) continuous 10 l meep , and the other receiving 2 ) continuous 100 l meep . determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer 's protocol ( b - bridge international , inc . , supernatants cultured with each sample were added to elisa plates to measure the esrage content . dna of huvecs was extracted from the homogenates using a dna extractor wb kit ( wako pure chemical industries , osaka , japan ) according to the protocol . the 8-ohdg level was determined by using the 8-ohdg check elisa kit ( japan institute for the control of aging , shizuoka , japan ) according to the manufacturer 's protocol . the recovery rate of 8-ohdg level was expressed as % recovery of the normal glucose medium a450 nm ( experimental a450nm / normal glucose medium a450 nm 100 ) . peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein ( dcdhf ) to 2,7-dichlorofluorescein ( dcf ) was estimated based on the method described by crow . samples or control ( dmso ) were added to 100 mm pbs ( ph 7.4 ) containing 100 m diethylenetriamine - n , n , n , n , n-pentaacetic acid ( dojindo molecular technologies , inc . , japan ) , 5 m sodium peroxynitrite and 100 m dcdhf . the peroxynitrite scavenging activity was expressed as % inhibition of the control a500 nm ( [ control a500 nm experimental a500nm]/control a500 nm 100 ) . because meep were methanolic extracts , we measured their tpc . tpc was measured with a modified version of the folin briefly , 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . we allowed the mixture to sit for 60 min before reading absorbance at 750 nm using a uv-460 spectrophotometer ( nihonbunko ltd . , the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . a statistical comparison between the groups was carried out using either anova or student 's t - test . edible plants ( 100 g ) were minced into 5 mm fragments and extracts were obtained with methanol ( 200 ml ) overnight at room temperature . the extracts were filtered and the residue was re - extracted under the same conditions as above . the combined filtrates were evaporated in a vacuum slightly below 40c in a rotary evaporator . jcrb0408 , health science research resources bank , osaka , japan ) were cultured at 37c in sfm cs - c medium ( ds pharma biomedical , osaka , japan ) supplemented with 1.5 g / ml fungizone under air - co2 ( 95:5 ) ( referred to hereafter as cs - cf medium ) . for screening study purposes , huvecs were seeded in 60 mm culture dishes ( 1.5 10 cells ) . then , huvecs were incubated with 1 g / l ( normal glucose medium ) or 4.5 g / l ( high glucose medium ) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed , one receiving 1 ) continuous 10 l meep , and the other receiving 2 ) continuous 100 l meep . determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer 's protocol ( b - bridge international , inc . , supernatants cultured with each sample were added to elisa plates to measure the esrage content . dna of huvecs was extracted from the homogenates using a dna extractor wb kit ( wako pure chemical industries , osaka , japan ) according to the protocol . the 8-ohdg level was determined by using the 8-ohdg check elisa kit ( japan institute for the control of aging , shizuoka , japan ) according to the manufacturer 's protocol . the recovery rate of 8-ohdg level was expressed as % recovery of the normal glucose medium a450 nm ( experimental a450nm / normal glucose medium a450 nm 100 ) . peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein ( dcdhf ) to 2,7-dichlorofluorescein ( dcf ) was estimated based on the method described by crow . samples or control ( dmso ) were added to 100 mm pbs ( ph 7.4 ) containing 100 m diethylenetriamine - n , n , n , n , n-pentaacetic acid ( dojindo molecular technologies , inc . , japan ) , 5 m sodium peroxynitrite and 100 m dcdhf . the peroxynitrite scavenging activity was expressed as % inhibition of the control a500 nm ( [ control a500 nm experimental a500nm]/control a500 nm 100 ) . because meep were methanolic extracts , we measured their tpc . tpc was measured with a modified version of the folin briefly , 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . we allowed the mixture to sit for 60 min before reading absorbance at 750 nm using a uv-460 spectrophotometer ( nihonbunko ltd . , the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . a statistical comparison between the groups was carried out using either anova or student 's t - test . we examined the esrage - producing ability of 29 meep in high glucose - induced huvecs . the result showed an increase in esrage production in the following gight samples ( eggplant [ solanum melongena ] , carrot peel [ daucus carota subsp . sativus ] , young sweet corn [ zea mays saccharata ] , broad bean [ vicia faba ] , japanese radish [ raphanus sativus var . longipinnatus ] sprout , jew 's marrow [ corchorus olitorius ] and cauliflower [ brassica oleracea var . the results of the esrage production of these eight samples on huvecs are summarized in figure 1 . figure 1a shows the effects of the eight samples ( 10 l ) on esrage production in high glucose - induced huvecs . the esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . genistein ( 10 m ) increased esrage production by approximately 12% , but egcg ( 10 m ) caused no increase . the results for plant samples suggest that white radish sprout , jew 's marrow and cauliflower have esrage production potential on induced endothelial cells . in particular , white radish sprout , which had higher esrage production potential than genistein , significantly ( p < 0.01 ) increased esrage production . in addition , white radish sprout showed an esrage production potential approximately 2.4 times higher than the control ( 4.5 g / l of glucose only ) . on the other hand , in 100 l meep [ figure 1b ] , more samples had a significant influence on esrage production . the results suggest that eggplant , carrot peel , young sweet corn , broad bean , japanese radish , and white radish sprout have esrage production potential on induced endothelial cells . however , different meep seem to have different effects on esrage production in the presence of high glucose . in 100 m concentration , genistein also showed a tendency to increase slightly , but the increase was not seen in egcg . in studies to date , no plant has yet been shown to increase esrage production . therefore , the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . however , it was reported through similar experimental techniques that hibiscus sabdariffa polyphenolic extract inhibited the expression of rage . furthermore , lu et al . indicated that atorvastatin exerted a beneficial effect on diabetic nephropathy with reduced age accumulation , down - regulating rage expression and up - regulating srage in the kidney . from the above - mentioned report , the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . however , although lee and lee show that 5 m egcg down - regulates the rage expression , this may not necessarily indicate an inverse correlation between rage and esrage because in our study 10 m egcg does not increase esrage production . effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products ( esrage ) production in human umbilical vein endothelial cells cultured in high glucose . the esrage productions of control ( dimethyl sulfoxide ) and samples in human umbilical vein endothelial cells cultured in high ( 4.5 two groups of experiments were formed , one receiving continuous 10 l methanolic extracts from edible plants ( meep ) ( a ) and the other receiving continuous 100 l meep ( b ) values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . performed the study to compare concentration of srage and its ligands ( en - rage and hmgb1 ) in type 1 ( t1 dm ) and t2 dm . similarly , en - rage was significantly higher in both diabetic groups and hmgb1 concentrations were elevated in t2 dm patients . they concluded that serum srage and rage ligands concentrations reflect endothelial dysfunction developing in diabetes . therefore , the extract of these eight plants may have an effect on t2 dm disease restraint through esrage production . on the other hand , some reports show esrage and rage to be related to guanosine in dna and peroxynitrite radicals . on other word , showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . in addition , liu et al . showed that administration of euk134 ( peroxynitrite scavenger ) attenuated rage expression , and decreased cardiomyocyte apoptosis in diabetic mice . therefore , we examined the correlation between esrage production , and the two indicators mentioned above ( peroxynitrite and 8-ohdg levels ) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . in our experiment , the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml ( data not shown ) . the formation of 8-ohdg in calf thymus dna by 3-morpholinosydnomine n - ethylcarbamide ( sin-1 ; peroxynitrite donor ) was also inhibited by egcg . in our experiment , egcg ( 100 m ) showed 8-ohdg recovery rates of approximately 90% , but genistein ( 100 m ) caused 8-ohdg recovery rates of approximately 60% . the calculated 8-ohdg recovery rates for white radish sprout , broad bean , young sweet corn , japanese radish , eggplant , jew 's marrow , cauliflower , and carrot peel were 61.9% , 47.7% , 68.9% , 55.5% , 43.8% , 62.0% , 60.2% , and 77.1% , respectively in comparison to the normal glucose medium [ figure 2 ] indicating that the samples are potent inhibitors of 8-ohdg . in particular , carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein , significantly ( p < 0.05 ) decreased 8-ohdg level . in addition , carrot peel showed an 8-ohdg recovery rate in the vicinity of egcg . figure 3 shows the correlation between esrage production and 8-ohdg level of eight plant extracts . the results show that the 8-ohdg level does not correlate with esrage production ( r = 0.086 ) . the 8-ohdg recovery rates of control ( normal glucose medium ) and samples ( high glucose medium ) are indicated by unshaded and shaded columns , respectively . values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls . the correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants figure 4 shows the calculated peroxynitrite scavenging activities of eight extracts . because peroxynitrite has a short half - life , the measurement of the intracellular scavenging activity is difficult . therefore , we performed the scavenging activity measurement of the sample in the test tube . amongst the eight extracts , white radish sprout revealed the highest scavenging activity ( 44.8% ) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . the peroxynitrite level significantly correlated with esrage production ( r = -0.706 , p < 0.10 ) . wu et al . showed that treatment of sinoaortic denervated rats with srage abated aortic oxidative stress . this was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the correlation between esrage production and tpc was analyzed [ figure 6 ] . the correlation coefficient for esrage production was found to be greater than 0.6 ( r = 0.660 , p < 0.10 ) . a positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . the correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . however , the seven meep except for eggplant contain a lot of quercetin . because there was a positive correlation between tpc and esrage [ figure 6 ] , it is very likely to be quercetin that had an effect . the effect of these seven plants may resemble the mechanism that zhang et al . showed . hyperoside , quercetin-3-o - galactoside , is a flavonoid isolated from many medicinal plants . in their studies , quiescent ecv304 ( human endothelial - like ) cells were treated in vitro with advanced glycation end products ( ages ) in the presence or absence of hyperoside . the results demonstrated that ages induced c - jun n - terminal kinases ( jnk ) activation and apoptosis in ecv304 cells . furthermore , hyperoside significantly inhibited rage expression in age - stimulated ecv304 cells , whereas knockdown of rage inhibited age - induced jnk activation . these results suggested that ages may promote jnk activation , leading to viability inhibition of ecv304 cells via the rage signaling pathway . furthermore , from the above - mentioned report , the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes . we examined the esrage - producing ability of 29 meep in high glucose - induced huvecs . the result showed an increase in esrage production in the following gight samples ( eggplant [ solanum melongena ] , carrot peel [ daucus carota subsp . sativus ] , young sweet corn [ zea mays saccharata ] , broad bean [ vicia faba ] , japanese radish [ raphanus sativus var . longipinnatus ] sprout , jew 's marrow [ corchorus olitorius ] and cauliflower [ brassica oleracea var . the results of the esrage production of these eight samples on huvecs are summarized in figure 1 . figure 1a shows the effects of the eight samples ( 10 l ) on esrage production in high glucose - induced huvecs . the esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . genistein ( 10 m ) increased esrage production by approximately 12% , but egcg ( 10 m ) caused no increase . the results for plant samples suggest that white radish sprout , jew 's marrow and cauliflower have esrage production potential on induced endothelial cells . in particular , white radish sprout , which had higher esrage production potential than genistein , significantly ( p < 0.01 ) increased esrage production . in addition , white radish sprout showed an esrage production potential approximately 2.4 times higher than the control ( 4.5 g / l of glucose only ) . on the other hand , in 100 l meep [ figure 1b ] , more samples had a significant influence on esrage production . the results suggest that eggplant , carrot peel , young sweet corn , broad bean , japanese radish , and white radish sprout have esrage production potential on induced endothelial cells . however , different meep seem to have different effects on esrage production in the presence of high glucose . in 100 m concentration , genistein also showed a tendency to increase slightly , but the increase was not seen in egcg . in studies to date , no plant has yet been shown to increase esrage production . therefore , the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . however , it was reported through similar experimental techniques that hibiscus sabdariffa polyphenolic extract inhibited the expression of rage . furthermore , lu et al . indicated that atorvastatin exerted a beneficial effect on diabetic nephropathy with reduced age accumulation , down - regulating rage expression and up - regulating srage in the kidney . from the above - mentioned report , the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . however , although lee and lee show that 5 m egcg down - regulates the rage expression , this may not necessarily indicate an inverse correlation between rage and esrage because in our study 10 m egcg does not increase esrage production . effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products ( esrage ) production in human umbilical vein endothelial cells cultured in high glucose . the esrage productions of control ( dimethyl sulfoxide ) and samples in human umbilical vein endothelial cells cultured in high ( 4.5 two groups of experiments were formed , one receiving continuous 10 l methanolic extracts from edible plants ( meep ) ( a ) and the other receiving continuous 100 l meep ( b ) values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . performed the study to compare concentration of srage and its ligands ( en - rage and hmgb1 ) in type 1 ( t1 dm ) and t2 dm . similarly , en - rage was significantly higher in both diabetic groups and hmgb1 concentrations were elevated in t2 dm patients . they concluded that serum srage and rage ligands concentrations reflect endothelial dysfunction developing in diabetes . therefore , the extract of these eight plants may have an effect on t2 dm disease restraint through esrage production . on the other hand , some reports show esrage and rage to be related to guanosine in dna and peroxynitrite radicals . on other word , showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . in addition , liu et al . showed that administration of euk134 ( peroxynitrite scavenger ) attenuated rage expression , and decreased cardiomyocyte apoptosis in diabetic mice . therefore , we examined the correlation between esrage production , and the two indicators mentioned above ( peroxynitrite and 8-ohdg levels ) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . in our experiment , the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml ( data not shown ) . the formation of 8-ohdg in calf thymus dna by 3-morpholinosydnomine n - ethylcarbamide ( sin-1 ; peroxynitrite donor ) was also inhibited by egcg . in our experiment , egcg ( 100 m ) showed 8-ohdg recovery rates of approximately 90% , but genistein ( 100 m ) caused 8-ohdg recovery rates of approximately 60% . the calculated 8-ohdg recovery rates for white radish sprout , broad bean , young sweet corn , japanese radish , eggplant , jew 's marrow , cauliflower , and carrot peel were 61.9% , 47.7% , 68.9% , 55.5% , 43.8% , 62.0% , 60.2% , and 77.1% , respectively in comparison to the normal glucose medium [ figure 2 ] indicating that the samples are potent inhibitors of 8-ohdg . in particular , carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein , significantly ( p < 0.05 ) decreased 8-ohdg level . in addition , carrot peel showed an 8-ohdg recovery rate in the vicinity of egcg . figure 3 shows the correlation between esrage production and 8-ohdg level of eight plant extracts . the results show that the 8-ohdg level does not correlate with esrage production ( r = 0.086 ) . the 8-ohdg recovery rates of control ( normal glucose medium ) and samples ( high glucose medium ) are indicated by unshaded and shaded columns , respectively . values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls . the correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants because peroxynitrite has a short half - life , the measurement of the intracellular scavenging activity is difficult . therefore , we performed the scavenging activity measurement of the sample in the test tube . amongst the eight extracts , white radish sprout revealed the highest scavenging activity ( 44.8% ) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . the peroxynitrite level significantly correlated with esrage production ( r = -0.706 , p < 0.10 ) . wu et al . showed that treatment of sinoaortic denervated rats with srage abated aortic oxidative stress . this was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the tpc of the eight meep significantly correlated with esrage production . the correlation coefficient for esrage production was found to be greater than 0.6 ( r = 0.660 , p < 0.10 ) . a positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . the correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . because there was a positive correlation between tpc and esrage [ figure 6 ] , it is very likely to be quercetin that had an effect . the effect of these seven plants may resemble the mechanism that zhang et al . showed . hyperoside , quercetin-3-o - galactoside , is a flavonoid isolated from many medicinal plants . in their studies , quiescent ecv304 ( human endothelial - like ) cells were treated in vitro with advanced glycation end products ( ages ) in the presence or absence of hyperoside . the results demonstrated that ages induced c - jun n - terminal kinases ( jnk ) activation and apoptosis in ecv304 cells . furthermore , hyperoside significantly inhibited rage expression in age - stimulated ecv304 cells , whereas knockdown of rage inhibited age - induced jnk activation . these results suggested that ages may promote jnk activation , leading to viability inhibition of ecv304 cells via the rage signaling pathway . furthermore , from the above - mentioned report , the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes .

background : in diabetic populations , endogenous secretory receptor for advanced glycation end products ( esrage ) levels may be related to the degree of diabetic complications or to the protection from diabetic complications.objective:we investigated the impact of 29 methanolic extracts from edible plants on esrage production in human umbilical vein endothelial cells ( huvecs ) cultured in high ( 4.5 g / l ) glucose.materials and methods : edible plants were minced , and extracts were obtained with methanol overnight . the methanolic extracts from 29 edible plants were evaporated in a vacuum . for screening study purposes , huvecs were seeded in culture dishes ( 1.5 105 cells ) . then , huvecs were incubated with 1 g / l or 4.5 g / l of glucose in sfm cs - c medium treated with methanolic extracts from edible plants ( meep ) for 96 h. determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa . the 8-hydroxydeoxyguanosine ( 8-ohdg ) level was determined by using the 8-ohdg check elisa kit . peroxynitrite - dependent oxidation of 2 , 7-dichlorodihydrofluorescein to 2 , 7-dichlorofluorescein was estimated based on the method described by crow . because meep were methanolic extracts , we measured their total phenolic content ( tpc ) . tpc was measured with a modified version of the folin ciocalteu method.results:the results showed eight extracts increased esrage production . the extract from white radish sprouts showed the highest esrage production activity , and then eggplant , carrot peel , young sweet corn , jew 's marrow , broad bean , japanese radish and cauliflower . in order to understand the mechanism of esrage production , the eight extracts were examined for dna damage , peroxynitrite scavenging activity , and tpc in correlation with their esrage production . the results showed esrage production correlates with the peroxynitrite level and tpc.conclusion:this study supports the utilization of these eight extracts in folk medicine for improved treatment of diabetic complications .

Materials and Methods Preparation of methanolic extracts from 29 edible plants Cell culture Measurement of endogenous secretory receptor for advanced glycation end products concentrations in culture media Measurement of 8-hydroxydeoxyguanosine in human umbilical vein endothelial cells DNA Measurement of peroxynitrite level Determination of total phenolic content in methanolic extracts from edible plants Statistical analysis Results and Discussion Effects of methanolic extracts from edible plants on endogenous secretory receptor for advanced glycation end products production in high glucose-induced human umbilical vein endothelial cells Assessment of the relationship between 8-hydroxydeoxyguanosine level and endogenous secretory receptor for advanced glycation end products production Assessment of the relationship between peroxynitrite level and endogenous secretory receptor for advanced glycation end products production Assessment of the relationship between total phenolic content and endogenous secretory receptor for advanced glycation end products production

edible plants ( 100 g ) were minced into 5 mm fragments and extracts were obtained with methanol ( 200 ml ) overnight at room temperature . the combined filtrates were evaporated in a vacuum slightly below 40c in a rotary evaporator . human umbilical vein endothelial cells ( no . jcrb0408 , health science research resources bank , osaka , japan ) were cultured at 37c in sfm cs - c medium ( ds pharma biomedical , osaka , japan ) supplemented with 1.5 g / ml fungizone under air - co2 ( 95:5 ) ( referred to hereafter as cs - cf medium ) . for screening study purposes , huvecs were seeded in 60 mm culture dishes ( 1.5 10 cells ) . then , huvecs were incubated with 1 g / l ( normal glucose medium ) or 4.5 g / l ( high glucose medium ) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed , one receiving 1 ) continuous 10 l meep , and the other receiving 2 ) continuous 100 l meep . determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer 's protocol ( b - bridge international , inc . the 8-ohdg level was determined by using the 8-ohdg check elisa kit ( japan institute for the control of aging , shizuoka , japan ) according to the manufacturer 's protocol . the recovery rate of 8-ohdg level was expressed as % recovery of the normal glucose medium a450 nm ( experimental a450nm / normal glucose medium a450 nm 100 ) . peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein ( dcdhf ) to 2,7-dichlorofluorescein ( dcf ) was estimated based on the method described by crow . the peroxynitrite scavenging activity was expressed as % inhibition of the control a500 nm ( [ control a500 nm experimental a500nm]/control a500 nm 100 ) . because meep were methanolic extracts , we measured their tpc . tpc was measured with a modified version of the folin briefly , 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . , the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . edible plants ( 100 g ) were minced into 5 mm fragments and extracts were obtained with methanol ( 200 ml ) overnight at room temperature . the combined filtrates were evaporated in a vacuum slightly below 40c in a rotary evaporator . jcrb0408 , health science research resources bank , osaka , japan ) were cultured at 37c in sfm cs - c medium ( ds pharma biomedical , osaka , japan ) supplemented with 1.5 g / ml fungizone under air - co2 ( 95:5 ) ( referred to hereafter as cs - cf medium ) . for screening study purposes , huvecs were seeded in 60 mm culture dishes ( 1.5 10 cells ) . then , huvecs were incubated with 1 g / l ( normal glucose medium ) or 4.5 g / l ( high glucose medium ) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed , one receiving 1 ) continuous 10 l meep , and the other receiving 2 ) continuous 100 l meep . determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer 's protocol ( b - bridge international , inc . the 8-ohdg level was determined by using the 8-ohdg check elisa kit ( japan institute for the control of aging , shizuoka , japan ) according to the manufacturer 's protocol . peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein ( dcdhf ) to 2,7-dichlorofluorescein ( dcf ) was estimated based on the method described by crow . the peroxynitrite scavenging activity was expressed as % inhibition of the control a500 nm ( [ control a500 nm experimental a500nm]/control a500 nm 100 ) . because meep were methanolic extracts , we measured their tpc . tpc was measured with a modified version of the folin briefly , 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . , the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . we examined the esrage - producing ability of 29 meep in high glucose - induced huvecs . the result showed an increase in esrage production in the following gight samples ( eggplant [ solanum melongena ] , carrot peel [ daucus carota subsp . sativus ] , young sweet corn [ zea mays saccharata ] , broad bean [ vicia faba ] , japanese radish [ raphanus sativus var . longipinnatus ] sprout , jew 's marrow [ corchorus olitorius ] and cauliflower [ brassica oleracea var . the results of the esrage production of these eight samples on huvecs are summarized in figure 1 . figure 1a shows the effects of the eight samples ( 10 l ) on esrage production in high glucose - induced huvecs . the esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . genistein ( 10 m ) increased esrage production by approximately 12% , but egcg ( 10 m ) caused no increase . the results for plant samples suggest that white radish sprout , jew 's marrow and cauliflower have esrage production potential on induced endothelial cells . in particular , white radish sprout , which had higher esrage production potential than genistein , significantly ( p < 0.01 ) increased esrage production . in addition , white radish sprout showed an esrage production potential approximately 2.4 times higher than the control ( 4.5 g / l of glucose only ) . on the other hand , in 100 l meep [ figure 1b ] , more samples had a significant influence on esrage production . the results suggest that eggplant , carrot peel , young sweet corn , broad bean , japanese radish , and white radish sprout have esrage production potential on induced endothelial cells . however , different meep seem to have different effects on esrage production in the presence of high glucose . therefore , the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . from the above - mentioned report , the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products ( esrage ) production in human umbilical vein endothelial cells cultured in high glucose . the esrage productions of control ( dimethyl sulfoxide ) and samples in human umbilical vein endothelial cells cultured in high ( 4.5 two groups of experiments were formed , one receiving continuous 10 l methanolic extracts from edible plants ( meep ) ( a ) and the other receiving continuous 100 l meep ( b ) values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . therefore , the extract of these eight plants may have an effect on t2 dm disease restraint through esrage production . on the other hand , some reports show esrage and rage to be related to guanosine in dna and peroxynitrite radicals . on other word , showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . showed that administration of euk134 ( peroxynitrite scavenger ) attenuated rage expression , and decreased cardiomyocyte apoptosis in diabetic mice . therefore , we examined the correlation between esrage production , and the two indicators mentioned above ( peroxynitrite and 8-ohdg levels ) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . in our experiment , the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml ( data not shown ) . the calculated 8-ohdg recovery rates for white radish sprout , broad bean , young sweet corn , japanese radish , eggplant , jew 's marrow , cauliflower , and carrot peel were 61.9% , 47.7% , 68.9% , 55.5% , 43.8% , 62.0% , 60.2% , and 77.1% , respectively in comparison to the normal glucose medium [ figure 2 ] indicating that the samples are potent inhibitors of 8-ohdg . in particular , carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein , significantly ( p < 0.05 ) decreased 8-ohdg level . in addition , carrot peel showed an 8-ohdg recovery rate in the vicinity of egcg . the results show that the 8-ohdg level does not correlate with esrage production ( r = 0.086 ) . the correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants figure 4 shows the calculated peroxynitrite scavenging activities of eight extracts . because peroxynitrite has a short half - life , the measurement of the intracellular scavenging activity is difficult . therefore , we performed the scavenging activity measurement of the sample in the test tube . amongst the eight extracts , white radish sprout revealed the highest scavenging activity ( 44.8% ) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . the peroxynitrite level significantly correlated with esrage production ( r = -0.706 , p < 0.10 ) . this was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . * * p < 0.01 , * p < 0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the correlation between esrage production and tpc was analyzed [ figure 6 ] . a positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . the correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . the effect of these seven plants may resemble the mechanism that zhang et al . in their studies , quiescent ecv304 ( human endothelial - like ) cells were treated in vitro with advanced glycation end products ( ages ) in the presence or absence of hyperoside . furthermore , from the above - mentioned report , the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes . the result showed an increase in esrage production in the following gight samples ( eggplant [ solanum melongena ] , carrot peel [ daucus carota subsp . sativus ] , young sweet corn [ zea mays saccharata ] , broad bean [ vicia faba ] , japanese radish [ raphanus sativus var . longipinnatus ] sprout , jew 's marrow [ corchorus olitorius ] and cauliflower [ brassica oleracea var . the results of the esrage production of these eight samples on huvecs are summarized in figure 1 . figure 1a shows the effects of the eight samples ( 10 l ) on esrage production in high glucose - induced huvecs . the esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . genistein ( 10 m ) increased esrage production by approximately 12% , but egcg ( 10 m ) caused no increase . the results for plant samples suggest that white radish sprout , jew 's marrow and cauliflower have esrage production potential on induced endothelial cells . in particular , white radish sprout , which had higher esrage production potential than genistein , significantly ( p < 0.01 ) increased esrage production . in addition , white radish sprout showed an esrage production potential approximately 2.4 times higher than the control ( 4.5 g / l of glucose only ) . on the other hand , in 100 l meep [ figure 1b ] , more samples had a significant influence on esrage production . the results suggest that eggplant , carrot peel , young sweet corn , broad bean , japanese radish , and white radish sprout have esrage production potential on induced endothelial cells . however , different meep seem to have different effects on esrage production in the presence of high glucose . therefore , the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . from the above - mentioned report , the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products ( esrage ) production in human umbilical vein endothelial cells cultured in high glucose . the esrage productions of control ( dimethyl sulfoxide ) and samples in human umbilical vein endothelial cells cultured in high ( 4.5 two groups of experiments were formed , one receiving continuous 10 l methanolic extracts from edible plants ( meep ) ( a ) and the other receiving continuous 100 l meep ( b ) values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . therefore , the extract of these eight plants may have an effect on t2 dm disease restraint through esrage production . on the other hand , some reports show esrage and rage to be related to guanosine in dna and peroxynitrite radicals . on other word , showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . showed that administration of euk134 ( peroxynitrite scavenger ) attenuated rage expression , and decreased cardiomyocyte apoptosis in diabetic mice . therefore , we examined the correlation between esrage production , and the two indicators mentioned above ( peroxynitrite and 8-ohdg levels ) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . in our experiment , the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml ( data not shown ) . the calculated 8-ohdg recovery rates for white radish sprout , broad bean , young sweet corn , japanese radish , eggplant , jew 's marrow , cauliflower , and carrot peel were 61.9% , 47.7% , 68.9% , 55.5% , 43.8% , 62.0% , 60.2% , and 77.1% , respectively in comparison to the normal glucose medium [ figure 2 ] indicating that the samples are potent inhibitors of 8-ohdg . in particular , carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein , significantly ( p < 0.05 ) decreased 8-ohdg level . in addition , carrot peel showed an 8-ohdg recovery rate in the vicinity of egcg . the results show that the 8-ohdg level does not correlate with esrage production ( r = 0.086 ) . the correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants because peroxynitrite has a short half - life , the measurement of the intracellular scavenging activity is difficult . therefore , we performed the scavenging activity measurement of the sample in the test tube . amongst the eight extracts , white radish sprout revealed the highest scavenging activity ( 44.8% ) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . the peroxynitrite level significantly correlated with esrage production ( r = -0.706 , p < 0.10 ) . this was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . * * p < 0.01 , * p < 0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the tpc of the eight meep significantly correlated with esrage production . a positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . the correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . the effect of these seven plants may resemble the mechanism that zhang et al . in their studies , quiescent ecv304 ( human endothelial - like ) cells were treated in vitro with advanced glycation end products ( ages ) in the presence or absence of hyperoside . furthermore , from the above - mentioned report , the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes .

jcrb0408 , health science research resources bank , osaka , japan ) were cultured at 37c in sfm cs - c medium ( ds pharma biomedical , osaka , japan ) supplemented with 1.5 g / ml fungizone under air - co2 ( 95:5 ) ( referred to hereafter as cs - cf medium ) . then , huvecs were incubated with 1 g / l ( normal glucose medium ) or 4.5 g / l ( high glucose medium ) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed , one receiving 1 ) continuous 10 l meep , and the other receiving 2 ) continuous 100 l meep . determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer 's protocol ( b - bridge international , inc . dna of huvecs was extracted from the homogenates using a dna extractor wb kit ( wako pure chemical industries , osaka , japan ) according to the protocol . the recovery rate of 8-ohdg level was expressed as % recovery of the normal glucose medium a450 nm ( experimental a450nm / normal glucose medium a450 nm 100 ) . peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein ( dcdhf ) to 2,7-dichlorofluorescein ( dcf ) was estimated based on the method described by crow . samples or control ( dmso ) were added to 100 mm pbs ( ph 7.4 ) containing 100 m diethylenetriamine - n , n , n , n , n-pentaacetic acid ( dojindo molecular technologies , inc . tpc was measured with a modified version of the folin briefly , 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . , the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . jcrb0408 , health science research resources bank , osaka , japan ) were cultured at 37c in sfm cs - c medium ( ds pharma biomedical , osaka , japan ) supplemented with 1.5 g / ml fungizone under air - co2 ( 95:5 ) ( referred to hereafter as cs - cf medium ) . then , huvecs were incubated with 1 g / l ( normal glucose medium ) or 4.5 g / l ( high glucose medium ) of glucose in cs - cf medium treated with meep for 96 h. two groups of experiments were formed , one receiving 1 ) continuous 10 l meep , and the other receiving 2 ) continuous 100 l meep . determination of esrage production in the cell culture - derived supernatants was performed by colorimetric elisa under the manufacturer 's protocol ( b - bridge international , inc . dna of huvecs was extracted from the homogenates using a dna extractor wb kit ( wako pure chemical industries , osaka , japan ) according to the protocol . the 8-ohdg level was determined by using the 8-ohdg check elisa kit ( japan institute for the control of aging , shizuoka , japan ) according to the manufacturer 's protocol . the recovery rate of 8-ohdg level was expressed as % recovery of the normal glucose medium a450 nm ( experimental a450nm / normal glucose medium a450 nm 100 ) . peroxynitrite - dependent oxidation of 2,7- dichlorodihydrofluorescein ( dcdhf ) to 2,7-dichlorofluorescein ( dcf ) was estimated based on the method described by crow . samples or control ( dmso ) were added to 100 mm pbs ( ph 7.4 ) containing 100 m diethylenetriamine - n , n , n , n , n-pentaacetic acid ( dojindo molecular technologies , inc . tpc was measured with a modified version of the folin briefly , 100 l of the sample or standard were combined with 200 l of folin ciocalteu reagent and 2.0 ml of 2% na2co3 solution . , the concentration of tpc present in meep was determined by comparing it with the absorbance rates of standard chlorogenic acid at different concentrations . we examined the esrage - producing ability of 29 meep in high glucose - induced huvecs . the result showed an increase in esrage production in the following gight samples ( eggplant [ solanum melongena ] , carrot peel [ daucus carota subsp . the results of the esrage production of these eight samples on huvecs are summarized in figure 1 . figure 1a shows the effects of the eight samples ( 10 l ) on esrage production in high glucose - induced huvecs . the esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . the results for plant samples suggest that white radish sprout , jew 's marrow and cauliflower have esrage production potential on induced endothelial cells . in particular , white radish sprout , which had higher esrage production potential than genistein , significantly ( p < 0.01 ) increased esrage production . in addition , white radish sprout showed an esrage production potential approximately 2.4 times higher than the control ( 4.5 g / l of glucose only ) . on the other hand , in 100 l meep [ figure 1b ] , more samples had a significant influence on esrage production . the results suggest that eggplant , carrot peel , young sweet corn , broad bean , japanese radish , and white radish sprout have esrage production potential on induced endothelial cells . however , different meep seem to have different effects on esrage production in the presence of high glucose . in 100 m concentration , genistein also showed a tendency to increase slightly , but the increase was not seen in egcg . therefore , the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . however , it was reported through similar experimental techniques that hibiscus sabdariffa polyphenolic extract inhibited the expression of rage . indicated that atorvastatin exerted a beneficial effect on diabetic nephropathy with reduced age accumulation , down - regulating rage expression and up - regulating srage in the kidney . from the above - mentioned report , the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . however , although lee and lee show that 5 m egcg down - regulates the rage expression , this may not necessarily indicate an inverse correlation between rage and esrage because in our study 10 m egcg does not increase esrage production . effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products ( esrage ) production in human umbilical vein endothelial cells cultured in high glucose . the esrage productions of control ( dimethyl sulfoxide ) and samples in human umbilical vein endothelial cells cultured in high ( 4.5 two groups of experiments were formed , one receiving continuous 10 l methanolic extracts from edible plants ( meep ) ( a ) and the other receiving continuous 100 l meep ( b ) values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . performed the study to compare concentration of srage and its ligands ( en - rage and hmgb1 ) in type 1 ( t1 dm ) and t2 dm . similarly , en - rage was significantly higher in both diabetic groups and hmgb1 concentrations were elevated in t2 dm patients . therefore , the extract of these eight plants may have an effect on t2 dm disease restraint through esrage production . on the other hand , some reports show esrage and rage to be related to guanosine in dna and peroxynitrite radicals . on other word , showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . showed that administration of euk134 ( peroxynitrite scavenger ) attenuated rage expression , and decreased cardiomyocyte apoptosis in diabetic mice . therefore , we examined the correlation between esrage production , and the two indicators mentioned above ( peroxynitrite and 8-ohdg levels ) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . in our experiment , the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml ( data not shown ) . in our experiment , egcg ( 100 m ) showed 8-ohdg recovery rates of approximately 90% , but genistein ( 100 m ) caused 8-ohdg recovery rates of approximately 60% . the calculated 8-ohdg recovery rates for white radish sprout , broad bean , young sweet corn , japanese radish , eggplant , jew 's marrow , cauliflower , and carrot peel were 61.9% , 47.7% , 68.9% , 55.5% , 43.8% , 62.0% , 60.2% , and 77.1% , respectively in comparison to the normal glucose medium [ figure 2 ] indicating that the samples are potent inhibitors of 8-ohdg . in particular , carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein , significantly ( p < 0.05 ) decreased 8-ohdg level . in addition , carrot peel showed an 8-ohdg recovery rate in the vicinity of egcg . figure 3 shows the correlation between esrage production and 8-ohdg level of eight plant extracts . the results show that the 8-ohdg level does not correlate with esrage production ( r = 0.086 ) . the correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants figure 4 shows the calculated peroxynitrite scavenging activities of eight extracts . therefore , we performed the scavenging activity measurement of the sample in the test tube . amongst the eight extracts , white radish sprout revealed the highest scavenging activity ( 44.8% ) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . the peroxynitrite level significantly correlated with esrage production ( r = -0.706 , p < 0.10 ) . this was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . * * p < 0.01 , * p < 0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the correlation between esrage production and tpc was analyzed [ figure 6 ] . the correlation coefficient for esrage production was found to be greater than 0.6 ( r = 0.660 , p < 0.10 ) . a positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . the correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . because there was a positive correlation between tpc and esrage [ figure 6 ] , it is very likely to be quercetin that had an effect . in their studies , quiescent ecv304 ( human endothelial - like ) cells were treated in vitro with advanced glycation end products ( ages ) in the presence or absence of hyperoside . the results demonstrated that ages induced c - jun n - terminal kinases ( jnk ) activation and apoptosis in ecv304 cells . furthermore , hyperoside significantly inhibited rage expression in age - stimulated ecv304 cells , whereas knockdown of rage inhibited age - induced jnk activation . furthermore , from the above - mentioned report , the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes . we examined the esrage - producing ability of 29 meep in high glucose - induced huvecs . the result showed an increase in esrage production in the following gight samples ( eggplant [ solanum melongena ] , carrot peel [ daucus carota subsp . figure 1a shows the effects of the eight samples ( 10 l ) on esrage production in high glucose - induced huvecs . the esrage production resulting from high glucose treatment was induced when cells were treated with 8 plant extracts as compared to control treated with 4.5 g / l of glucose only . the results for plant samples suggest that white radish sprout , jew 's marrow and cauliflower have esrage production potential on induced endothelial cells . in particular , white radish sprout , which had higher esrage production potential than genistein , significantly ( p < 0.01 ) increased esrage production . the results suggest that eggplant , carrot peel , young sweet corn , broad bean , japanese radish , and white radish sprout have esrage production potential on induced endothelial cells . therefore , the mechanism by which white radish sprout and the seven other kinds of plants increased esrage production is unclear . indicated that atorvastatin exerted a beneficial effect on diabetic nephropathy with reduced age accumulation , down - regulating rage expression and up - regulating srage in the kidney . from the above - mentioned report , the increase in esrage production may be considered to result from the down - regulation of rage by the polyphenolic extract . however , although lee and lee show that 5 m egcg down - regulates the rage expression , this may not necessarily indicate an inverse correlation between rage and esrage because in our study 10 m egcg does not increase esrage production . effect of methanolic extracts from eight plants on endogenous secretory receptor for advanced glycation end products ( esrage ) production in human umbilical vein endothelial cells cultured in high glucose . the esrage productions of control ( dimethyl sulfoxide ) and samples in human umbilical vein endothelial cells cultured in high ( 4.5 two groups of experiments were formed , one receiving continuous 10 l methanolic extracts from edible plants ( meep ) ( a ) and the other receiving continuous 100 l meep ( b ) values are the mean standard deviation of three measurements . * * p < 0.01 , * p < 0.05 compared with the controls receptor for advanced glycation end products plays the essential role in the pathogenesis of diabetic vascular complications . performed the study to compare concentration of srage and its ligands ( en - rage and hmgb1 ) in type 1 ( t1 dm ) and t2 dm . similarly , en - rage was significantly higher in both diabetic groups and hmgb1 concentrations were elevated in t2 dm patients . on other word , showed that mutagenesis of the g - rich cis - elements caused an increase in the esrage / membrane - bound rage ratio in the minigene - transfected cells . therefore , we examined the correlation between esrage production , and the two indicators mentioned above ( peroxynitrite and 8-ohdg levels ) to determine the mechanism for increasing esrage production in huvecs treated with a eight plant extracts . showed that the amount of 8-ohdg in the medium increased to a significantly greater extent in high glucose - incubated huvecs than in low glucose - incubated huvecs . in our experiment , the level of 8-ohdg was in the range of 0.28 - 0.52 ng / ml ( data not shown ) . the calculated 8-ohdg recovery rates for white radish sprout , broad bean , young sweet corn , japanese radish , eggplant , jew 's marrow , cauliflower , and carrot peel were 61.9% , 47.7% , 68.9% , 55.5% , 43.8% , 62.0% , 60.2% , and 77.1% , respectively in comparison to the normal glucose medium [ figure 2 ] indicating that the samples are potent inhibitors of 8-ohdg . in particular , carrot peel and young sweet corn which had higher 8-ohdg recovery rate potential than genistein , significantly ( p < 0.05 ) decreased 8-ohdg level . the correlation between endogenous secretory receptor for advanced glycation end products production and 8-hydroxydeoxyguanosine level of methanolic extracts from eight plants because peroxynitrite has a short half - life , the measurement of the intracellular scavenging activity is difficult . amongst the eight extracts , white radish sprout revealed the highest scavenging activity ( 44.8% ) figure 5 shows the correlation between esrage production and peroxynitrite level of the eight plant extracts . the peroxynitrite level significantly correlated with esrage production ( r = -0.706 , p < 0.10 ) . this was marked by reduction in the formation of peroxynitrite indicating that there may be an association between peroxynitrite and esrage . * * p < 0.01 , * p < 0.05 compared with the controls the correlation between endogenous secretory receptor for advanced glycation end products production and peroxynitrite level of methanolic extracts from eight plants the tpc of the eight meep significantly correlated with esrage production . a positive correlation between esrage production and tpc this result proved that the tpc of these plants could be clearly attributed to their esrage production . the correlation between endogenous secretory receptor for advanced glycation end products production and total phenolic content of methanolic extracts from eight plants the molecular mechanisms underlying these effects are unknown . in their studies , quiescent ecv304 ( human endothelial - like ) cells were treated in vitro with advanced glycation end products ( ages ) in the presence or absence of hyperoside . furthermore , from the above - mentioned report , the increase in esrage production may be considered a result of the down - regulation of rage by the polyphenolic extract . our findings suggest a novel role for eight edible plants in the treatment and prevention of diabetes .

the mental rotation task is a well - established paradigm to study the cognitive process of mentally rotating objects . typically , observed reaction time ( rt ) profiles show an increase in rt for increased angle of rotation indicating that participants mentally rotate stimuli to the upward position ( shepard and metzler 1971 ; parsons 1994 ; jeannerod and decety 1995 ) . they used a task in which participants had to judge whether differently rotated 3d cube figures were identical or mirror reversed images of that figure ( shepard and metzler 1971 ) . it was found that rts increased linearly with increasing angle of rotation . other mental rotation studies used different paradigms in which the participants had to judge object or body part laterality , referred to as a laterality judgment task , first introduced by sekiyama ( 1982 ) . he found that rts for judging hand laterality also increased with increased angles of rotation . however , rt did not increase linearly but quadratic and was not symmetrical about 180 as is the case for 3d cube figures . this asymmetric quadratic rt profile was interpreted as evidencing kinesthetic influence of the laterality judgment of hands ( sekiyama 1982 ) . it was postulated that participants engage in an embodied mental process in which biomechanical constraints influence the duration of the mental rotation process ( sekiyama 1982 ; parsons 1987 ; jeannerod 1994 ; decety 1996a ; shenton et al . the influence of kinesthetic aspects or biomechanical constraints was further postulated by parsons ( 1994 ) . he showed that the time needed to actually rotate the own hand to an identical position as the displayed rotated hand corresponds to the time needed for a hand laterality judgment ( parsons 1987 ; parsons 1994 ) . more specifically , he showed that hand rotations away of the mid - sagittal plane ( i.e. , lateral rotation ) resulted in larger rts than hand rotation toward the mid - sagittal plane ( i.e. , medial rotation ) for both overt as imagined movement . thus , the rt profiles of imagined hand rotations were subject to the same biomechanical constraints experienced for overt movement . laterality judgments of hands that are subject to biomechanical constraints make it conceivable that participants engage in embodied cognitive processing . this embodied processing is referred to as motor imagery ( mi ) ( de lange et al . , rt differences between lateral- and medial - rotated stimuli can be used as excellent measure to test for engagement in mi . when engaged in mi , participants mentally plan and perform a movement from a first person perspective without overtly performing the movement and without sensory feedback ( decety 1996a , b ) . the embodied nature of mi in a hand laterality judgment task was further evidenced by showing that rt profiles change as a consequence of changing the participant s posture ( parsons 1994 ; sirigu and duhamel 2001 ; de lange et al . 2007 ; ionta et al . 2007 ; ionta and blanke 2009 ) . extending these findings , children at 57 years of age were also found to engage in mi in a hand laterality judgment task , possibly even more than adults ( funk et al . 2005 ) . visual imagery ( vi ) , on the other hand , encompasses simulating executing a movement from a third person perspective ( e.g. , steenbergen et al . 2007 ) . thus , vi is not subject to biomechanical constrains and as such not an embodied process . studies using hand laterality judgment tasks have not unequivocally shown to induce an mi strategy . as an example , we showed that participants with congenital alterations of posture on one side of the body , i.e. , participants with hemiparetic cerebral palsy , lacked the presumed influence of posture on laterality judgments ( steenbergen et al . , it was hypothesized that these participants would show a different rt profile for laterality judgments of their affected versus their non - affected hand , but this was not evidenced by the data . in the study by lust et al . ( 2006 ) , groups of adults , healthy children and children suffering from developmental disorder were tested on their engagement in mi during a hand laterality judgment task with only hand stimuli shown from the back . their results showed that rts for both groups of children were subject to biomechanical constraints , as evident by the increased rt for difficult hand postures when compared to hand postures that are easier to adopt . however , the adult group showed no effect of biomechanical constraints and hence no engagement in mi ( lust et al . we propose that the differences observed in literature on the effects of posture or , more general , biomechanical constraints on laterality judgments are due to the particulars of the stimulus set . the argument for this is following . ( 2006 ) and our recent study , the stimulus set was largely the same . that is , all displayed pictures of hands were presented from a back view perspective in different angles of rotation . in contrast , studies showing strong effects of posture on rt used stimulus sets in which hands were presented from different viewpoints . see table 1 for a short overview of literature on the hand laterality judgment task . obviously , the use of these different perspectives from which the hand stimuli are presented yielded an increase in number of rotational axes and , as a consequence , an increase in overall task difficulty . another factor that differed among studies and which may potentially cause the differential results is the number of rotational steps of the displayed stimuli , see table 1 . however , the number of rotational steps appears to have only a marginal influence because in the study by steenbergen et al . ( 2007 ) eighteen different rotational angles were used but no engagement in mi was found . other studies , however , do show engagement in mi with as little as four angles of rotation ( de lange et al . 2006).table 1studies on hand laterality judgment taskauthoryearviewsteps of rotationmi engagementionta and blanke2009hands and feet , back , palm , thumb , little finger6yesde lange et al.2008back , palm7yesionta et al.2007as ionta and blanke ( 2009)6yessteenbergen et al.2007back18nohelmich et al.2007back , palm8yesde lange et al.2006back , palm4yeslust et al.2006back8yes ( children)no ( adults)thayer and johnson2006back , palm6yessauner et al.2006back , palm8yesfunk et al.2005back , palm4yesshenton et al.2004back , palm6yesparsons1994back , palm , thumb , little finger , front finger , back palm12yesparsons1987back , palm , thumb , little finger , front finger , back palm12yessekiyama1982thumb , little finger , palm8yesnote studies on hand laterality judgment task and results on mi engagement . both studies using only back view hand stimuli show no mi engagement ( for adults ) . in contrast , studies using multiple viewpoints of hands within their stimulus set do show engagement in mi studies on hand laterality judgment task note studies on hand laterality judgment task and results on mi engagement . both studies using only back view hand stimuli show no mi engagement ( for adults ) . in contrast , studies using multiple viewpoints of hands within their stimulus set do show engagement in mi given such diverging results , we hypothesize that the use of more rotational axes within a stimulus set facilitates mi engagement . the argument for this is that simple tasks , in which only in - plane - rotated back view hand stimuli are used ( i.e. , one axis of rotation ) , may promote the use of an alternative strategy based on the combination of finger and thumb orientation to solve the task ( lust et al . 2006 ) . for more complex tasks , including for instance the use of both palm and back view hand stimuli , such a strategy will not suffice because one additional judgment on hand view is necessary next to judging hand laterality from the finger and thumb orientation . the inclusion of multiple rotational axes , and thus judgments , may therefore promote engagement in mi . that there is a neurophysiological difference between two - dimensional ( 2d ) and three - dimensional 2007 ) showed that task difficulty enhanced by rotation dimensionality plays an important role in the selection of a motor strategy . they used three - dimensional cube figures , similar to shepard and metzler ( 1971 ) . participants were presented a set of these figures rotated in 2d ( i.e. , in - plane ) or 3d ( i.e. , in - plane and in - depth ) . the results showed that the right superior parietal lobule was activated for 2d - rotated stimuli . in the case of 3d - rotated stimuli , the right dorsal premotor cortex ( pmd ) these findings indicate that 2d and 3d rotation of cube figures activate different brain regions and may therefore be ( partly ) different neuronal or cognitive processes . in the present study , we used three different conditions in which the number of axes of rotation increased from , respectively , only 1 axis to 3 axes of rotation . we expected an increasing difference in rts between lateral- and medial - rotated pictures of hands as a function of the number of axes of rotation . more specifically , for 1 axis of rotation , we expected to find no or marginal engagement in mi which will be evidenced by a marginal difference in rt between medial and lateral rotation , whereas increasing engagement in mi ( i.e. , increasing difference in rt between lateral- and medial - rotated hand pictures ) is expected in the conditions with 2- and 3 axes of rotation . twelve healthy right - handed participants were included in the present study ( 8 women , age 22.5 3.7 years , mean sd ) . the study was approved by the local ethics committee , and all participants gave written informed consent prior to the experiment , in accordance with the helsinki declaration . we used a custom made 3d hand model designed in a 3d image software package ( autodesk maya 2009 , usa ) . from this realistic model , we constructed all stimuli that were used in the experiments . all stimuli were displayed on a 19 lcd computer screen , at a distance of approximately 70 cm from the participants eyes , resulting in a visual angle of approximately 6. the hand stimuli were rotated over three axes , resulting in three different rotational directions , namely : in - plane , longitudinal ( referred to as 1 . three different sets of stimuli were used , namely : set-1 containing only in - plane - rotated stimuli , set-2 with both in - plane- and longitudinal - rotated stimuli , and set-3 containing in - plane- , longitudinal- , and in - depth - rotated stimuli . with 0 of in - plane angular disparity as the upright position with the fingers pointing upward , see fig . 1 . left and right hands were mirror images of each other but otherwise identical.fig . 1shown are all used hand stimuli for set-1 : upper row , left column ; set-2 : upper row ; set-3 : all stimuli . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity shown are all used hand stimuli for set-1 : upper row , left column ; set-2 : upper row ; set-3 : all stimuli . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity in set-1 , we used back view left and right hand stimuli in six different angles of in - plane rotation ( i.e. , 0 , 60 , 120 , 180 , 240 , and 300 ) , resulting in 12 different stimuli , see upper left cell of fig . 1 . for set-2 , we used both back and palm view ( i.e. , two longitudinal rotational steps ) stimuli of hands . all other aspects were identical to set-1 , resulting in 24 different stimuli , see upper row of fig . 1 . in set-3 , our set of stimuli were identical to the stimuli used in set-2 but with three angles of in - depth rotations ( i.e. , 0 , 60 , 300 ) with 0 parallel to the vertical plane , resulting in 72 different stimuli , see fig . 1 . all stimuli were repeated three times resulting in 36 ( 12 * 3 ) , 72 ( 24 * 3 ) and 216 ( 72 * 3 ) stimuli for set-1 , -2 , and -3 , respectively . all three conditions were preceded by a test of 24 stimuli to familiarize the participants with the task . stimuli were presented using custom - developed software in presentation ( neurobehavioral systems , albany , usa ) . participants had to respond by pressing the left button with their left hand for left - hand stimuli and the right button with their right hand for right - hand stimuli . participants were instructed to judge the laterality of the hand as fast and as accurate as possible , without explicit instructions on how to solve the task . these seven blocks comprise : 1 block of set-1 stimuli , 3 blocks of set-2 stimuli , and 3 blocks of set-3 stimuli . all sets were presented blockwise and sequential , preventing mixing of blocks of the difficulty conditions over the experiment . reaction times smaller than 300 ms and larger than 3500 ms were excluded from analysis ( total loss 3.2% of trials ) ; these upper and lower boundaries are based on similar studies using a hand laterality judgment task ( sekiyama 1987 ; parsons 1994 ; ionta et al . right hand and vice versa and amounted to a total 6.4% of all trials . for analyses purposes , the different in - plane rotations were divided into medial- and lateral - rotated stimuli referred to as direction of rotation ( dor ) in order to measure engagement in mi . medial - rotated stimuli consisted of right hand 240 and 300 and left hand 60 and 120 in - plane - rotated stimuli . lateral - rotated stimuli consisted of right hand 60 and 120 and left hand 240 and 300 in - plane - rotated stimuli . first , we analyzed the influence of increasing angle of rotation in order to test for the use of mental rotation for all sets . to this aim , we used an anova with the following design : 2 within - subject factors ( set , angle ) , with 3 levels for set ( set-1 , -2 , and -3 ) and 4 levels for angle ( 0 , 60 , 120 , and 180 ) . with 60 being the averaged value of both 60 and 300 in - plane - rotated stimuli and 120 being the averaged value of both 120 and 240 in - plane - rotated stimuli . a significant effect for angle with increasing rt with increased angle of rotation would indicate the use of mental rotation ( shepard and metzler 1971 ) . then , we analyzed the effect of biomechanical constraints on the rts by means of an anova with the following design : 2 within - subject factors ( set , dor ) , with 3 levels for set ( set-1 , -2 and -3 ) and 2 levels for dor ( lateral rotation , medial rotation ) . a significant set by dor interaction would indicate that the influence of biomechanical constraints ( as measured with dor ) differs between sets . ad hoc analysis was bonferroni corrected , and alpha level was set at p = 0.05 . twelve healthy right - handed participants were included in the present study ( 8 women , age 22.5 3.7 years , mean sd ) . the study was approved by the local ethics committee , and all participants gave written informed consent prior to the experiment , in accordance with the helsinki declaration . we used a custom made 3d hand model designed in a 3d image software package ( autodesk maya 2009 , usa ) . from this realistic model , we constructed all stimuli that were used in the experiments . all stimuli were displayed on a 19 lcd computer screen , at a distance of approximately 70 cm from the participants eyes , resulting in a visual angle of approximately 6. the hand stimuli were rotated over three axes , resulting in three different rotational directions , namely : in - plane , longitudinal ( referred to as 1 . three different sets of stimuli were used , namely : set-1 containing only in - plane - rotated stimuli , set-2 with both in - plane- and longitudinal - rotated stimuli , and set-3 containing in - plane- , longitudinal- , and in - depth - rotated stimuli . with 0 of in - plane angular disparity as the upright position with the fingers pointing upward , see fig . 1 . left and right hands were mirror images of each other but otherwise identical.fig . 1shown are all used hand stimuli for set-1 : upper row , left column ; set-2 : upper row ; set-3 : all stimuli . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity shown are all used hand stimuli for set-1 : upper row , left column ; set-2 : upper row ; set-3 : all stimuli . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity in set-1 , we used back view left and right hand stimuli in six different angles of in - plane rotation ( i.e. , 0 , 60 , 120 , 180 , 240 , and 300 ) , resulting in 12 different stimuli , see upper left cell of fig . 1 . for set-2 , we used both back and palm view ( i.e. , two longitudinal rotational steps ) stimuli of hands . all other aspects were identical to set-1 , resulting in 24 different stimuli , see upper row of fig . 1 . in set-3 , our set of stimuli were identical to the stimuli used in set-2 but with three angles of in - depth rotations ( i.e. , 0 , 60 , 300 ) with 0 parallel to the vertical plane , resulting in 72 different stimuli , see fig . 1 . all stimuli were repeated three times resulting in 36 ( 12 * 3 ) , 72 ( 24 * 3 ) and 216 ( 72 * 3 ) stimuli for set-1 , -2 , and -3 , respectively . all three conditions were preceded by a test of 24 stimuli to familiarize the participants with the task . stimuli were presented using custom - developed software in presentation ( neurobehavioral systems , albany , usa ) . participants had to respond by pressing the left button with their left hand for left - hand stimuli and the right button with their right hand for right - hand stimuli . participants were instructed to judge the laterality of the hand as fast and as accurate as possible , without explicit instructions on how to solve the task . these seven blocks comprise : 1 block of set-1 stimuli , 3 blocks of set-2 stimuli , and 3 blocks of set-3 stimuli . all sets were presented blockwise and sequential , preventing mixing of blocks of the difficulty conditions over the experiment . reaction times smaller than 300 ms and larger than 3500 ms were excluded from analysis ( total loss 3.2% of trials ) ; these upper and lower boundaries are based on similar studies using a hand laterality judgment task ( sekiyama 1987 ; parsons 1994 ; ionta et al . right hand and vice versa and amounted to a total 6.4% of all trials . for analyses purposes , the different in - plane rotations were divided into medial- and lateral - rotated stimuli referred to as direction of rotation ( dor ) in order to measure engagement in mi . medial - rotated stimuli consisted of right hand 240 and 300 and left hand 60 and 120 in - plane - rotated stimuli . lateral - rotated stimuli consisted of right hand 60 and 120 and left hand 240 and 300 in - plane - rotated stimuli . first , we analyzed the influence of increasing angle of rotation in order to test for the use of mental rotation for all sets . to this aim , we used an anova with the following design : 2 within - subject factors ( set , angle ) , with 3 levels for set ( set-1 , -2 , and -3 ) and 4 levels for angle ( 0 , 60 , 120 , and 180 ) . with 60 being the averaged value of both 60 and 300 in - plane - rotated stimuli and 120 being the averaged value of both 120 and 240 in - plane - rotated stimuli . a significant effect for angle with increasing rt with increased angle of rotation would indicate the use of mental rotation ( shepard and metzler 1971 ) then , we analyzed the effect of biomechanical constraints on the rts by means of an anova with the following design : 2 within - subject factors ( set , dor ) , with 3 levels for set ( set-1 , -2 and -3 ) and 2 levels for dor ( lateral rotation , medial rotation ) . a significant set by dor interaction would indicate that the influence of biomechanical constraints ( as measured with dor ) differs between sets . ad hoc analysis was bonferroni corrected , and alpha level was set at p = 0.05 . the total number of incorrect responses ( i.e. , 6.4% of all trials ) corresponds to former studies ( de lange et al . 2006 ; ionta et al . the amount of erroneous responses differed significantly [ f(2,20 ) = 4.692 , p < 0.05 , = 0.319 ] . within set-1 , set-2 , and set-3 , the amount of errors was 3.3 , 4.6 , and 7.3% of the number of trials per set , respectively . the mental rotation analysis revealed a significant main effect for both set [ f(2,22 ) = 43.380 , p < 0.001 , = 0.798 ] and angle [ f(3,33 ) = 38.772 , p < 0.001 , = 0.779 ] . additionally , a significant interaction of set by angle [ f(6,66 ) = 7.716 , p < 0.001 , = 0.412 ] was found . ad hoc analysis revealed a significant increase in rt with increasing angle of rotation for all three sets . set-1 [ f(3,33 ) = 37.732 , p < 0.001 , = 0.774 ] , set-2 [ f(3,33 ) = 20.941 , p < 0.001 , = 0.656 ] , and set-3 [ f(3,33 ) = 28.789 , p < 0.001 , = 0.724 ] . however , the mean rt for 0 rotated stimuli in set-3 was larger than the mean rt for 60 rotated stimuli . this larger rt for 0 was caused by the in - depth - rotated hand stimuli . overall , rts increased significantly with increasing angle of rotation for all three sets , see fig . 2reaction times for all three sets , mirrored at 180 ( i.e. , 60 represents average rt for 60 and 300 rotated hand stimuli ) . error - bars indicate standard error of the mean ( sem ) reaction times for all three sets , mirrored at 180 ( i.e. , 60 represents average rt for 60 and 300 rotated hand stimuli ) . error - bars indicate standard error of the mean ( sem ) the analysis on the influence of biomechanical constraints showed a significant main effect for set [ f(2,22 ) = 33.422 , p < 0.001 , = 0.75 ] and dor [ f(1,11 ) = 9.890 , p < 0.01 , = 0.47 ] . the set effect was accounted for by the increase in rt for increasing number of axes of rotation , 848 ms , 1,123 ms , and 1,214 ms for set-1 , set-2 , and set-3 , respectively . set-1 differed significantly from both set-2 and set-3 ( p < 0.001 and p < 0.001 , respectively ) . set-2 and set-3 did not differ significantly from each other ( p = 0.168 ) . the dor effect was accounted for by the increased rt for laterally rotated stimuli ( 1,117 ms ) compared to medially rotated stimuli ( 1,006 ms ) . in order to support our hypothesis about increasing difference in rt between laterally and medially rotated stimuli , we expected a significant interaction between set and direction of rotation ( dor ) . the anova resulted in a significant interaction of set by dor [ f(2,22 ) = 8.196 , p < 0.01 , = 0.43 ] , which was accounted for by the increasing difference in rts between laterally and medially rotated stimuli over the stimulus sets , resulting in significant simple dor effects in set-2 [ f(1,11 ) = 5.964 , p < 0.05 , = 0.35 ] and set-3 [ f(1,11 ) = 18.005 , p < 0.01 , = 0.62 ] . crucially , the simple dor effect of set-1 was not significant ( p = 0.578 ) , see fig . 3 . the simple dor effects differed significantly between set-1 and set-2 [ f(1,11 ) = 6.276 , p < 0.05 , = 0.363 ] , and set-1 and set-3 [ f(1,11 ) = 13.344 , p < 0.01 , = 0.548 ] but not between set-2 and set-3 ( p = 0.198).fig . lateral rotation indicates rotations away from the mid - sagittal plane , and medial rotation indicates rotations toward the mid - sagittal plane . as can be seen , the significant interaction of set by dor ( p < 0.01 ) as represented by the differences in rts between lateral and medial rotation ( i.e. , dor ) increases with increasing number of axes of rotation . * indicate significance at the p < 0.05 level , * * indicate significance at the p < 0.01 level . error - bars indicate standard error of the mean ( sem ) reaction times for all 3 sets divided into lateral rotation and medial rotation . lateral rotation indicates rotations away from the mid - sagittal plane , and medial rotation indicates rotations toward the mid - sagittal plane . as can be seen , the significant interaction of set by dor ( p < 0.01 ) as represented by the differences in rts between lateral and medial rotation ( i.e. , dor ) increases with increasing number of axes of rotation . * indicate significance at the p < 0.05 level , * * indicate significance at the p < 0.01 level . error - bars indicate standard error of the mean ( sem ) to control for the differences in mean rts between the sets and its possible influence on the obtained dor effects of the sets , we conducted an additional anova with the data being normalized into the range of [ 01 ] . the anova contained the following design : 2 within - subject factors ( set and dor ) with 3 levels for set ( set-1 , set-2 and set-3 ) and 2 levels for dor ( lateral rotation , medial rotation ) . the results replicate the results with the non - normalized data except for an insignificant set effect ( p = 0.074 ) . the lack of significant set effect indicates that the normalization was effective as it reduced the differences in absolute value of the mean rts between the sets . as we only obtained a significant dor effect in the sets including palm view stimuli , one might argue that only the palm view stimuli accounted for the obtained dor effect in set-2 and set-3 . to test whether a dor effect is also present for in - plane - rotated back view stimuli ( i.e. , set-1 stimuli ) within set-2 and set-3 , we performed an anova identical to the anova testing the influence of biomechanical constraints , described in the data analysis section . however , we now only included rt values of the set-1 stimuli in all sets . this test enables us to see whether a dor effect is also present for back view stimuli in set-2 and set-3 and as a consequence enables us to examine whether only palm view or also back view stimuli induce the use of mi , depending on the context in which they are embedded . additionally , this test shows whether participants used the same strategy for in - plane - rotated back view hand ( i.e. , set-1 ) stimuli for all three sets or not . the anova revealed a significant main effect of set [ f(2,22 ) = 21.694 , p < 0.001 , = 0.664 ] and dor [ f(1,10 ) = 4.999 , p < 0.05 , = 0.312 ] and an interaction of set by dor [ f(2,22 ) = 3.429 , p 0.05 , = 0.238 ] . the interaction was accounted for by a lack of significant dor effect for set-1 stimuli in set-1 ( p = 0.578 ) and set-2 ( p = 0.237 ) but a significant dor effect for set-1 stimuli within set-3 [ f(1,11 ) = 8.620 , p < 0.05 , = 0.439 ] . in this study , we investigated the influence of multiple angles of rotation in the stimulus set in a hand laterality judgment task on participants engagement in mi . we hypothesized that an increase in the number of axes of rotation facilitates an mi strategy . the number of axes of rotation cumulated from 1 ( i.e. , in - plane ) to 2 and 3 axes over 3 separate stimulus sets . according to the low error rates in all sets , which are well below chance level and the increasing rt in all sets for increasing angle of rotation ( see fig . 2 ) , we may assume that participants did indeed mentally rotate the stimuli and were able to solve the mental rotation task accurately ( shepard and metzler 1971 ; sekiyama 1987 ; parsons 1994 ; helmich et al . we showed increased rt differences between laterally and medially rotated hand stimuli ( e.g. , direction of rotation ( dor ) effect ) with increasing number of axes of rotation ( see fig . thus , there is an increasing influence of biomechanical constraints on rt with increasing number of axes of rotation , which exemplifies increased engagement in mi ( parsons 1994 ; lust et al . ( containing only in - plane - rotated back view stimuli ) , no effect of dor was found . in contrast , both set-2 ( in - plane - rotated , back- and palm view stimuli ) and set-3 ( in - plane- and in - depth - rotated , back- and palm view stimuli ) revealed a significant dor effect . the dor effects in both the set-2 and set-3 conditions were accounted for by a smaller rt for medial compared to lateral rotations , which is in correspondence with literature ( parsons 1987 ; parsons 1994 ) . additionally , the difference in obtained dor effects between the sets was not accounted for by the differences in absolute mean rt between the sets as the analysis on the normalized data shows the same results . the process of mi is dependent on embodied cognitive processing and therefore affected by both the desired end - state and the current position of one s body ( jeannerod 1994 ; de lange et al . the desired end - state is the presented hand posture . because mi is subject to biomechanical constraints , rts for lateral rotations of hands are prolonged compared to medial rotations if the participant engages in mi ( parsons 1987 ; parsons 1994 ; decety 1996a ) . however , one might argue that participants memorized the set-1 stimuli together with their laterality , leading to fast responses and no engagement in mi for set-1 . this explanation of the results is plausible because the set-1 stimuli are seen in all three sets , therefore facilitating the memorizing of the stimuli . however , if participants indeed memorized the set-1 stimuli , this would result in a lack of use of mental rotation at all . 2 ) , which was evident from the increase in rt with increasing angle of rotation . hence another possible explanation might be that the simple fact of adding palm view stimuli to a stimulus set results in the obtained difference in rt between medially and laterally rotated stimuli irrespective of the use of back view stimuli . the results indicate that we obtained this influence of biomechanical constraints only for the sets in which palm view stimuli were used . as a consequence , we sought to see whether the strategy used for the set-1 stimuli ( i.e. , only in - plane - rotated back view stimuli ) changes when they are embedded within a set of stimuli rotated around other rotational axes . the results indicate that the context in which the stimuli ( i.e. , set-1 stimuli ) are embedded is of influence on the strategy use , as the interaction of set by dor is significant . the changing effect of dor over the sets indicates that the particulars of a stimulus set ( i.e. , number of rotational axes ) influences the strategy used to solve the mental rotation task for the complete set and that strategies are not linked to a particular type of stimuli . we expected an increase in rt from set-1 till set-3 because of the systematically adding of an extra rotational axis , thereby increasing the task difficulty . however , the general rt increase over all angles does not linearly increase from set-1 to set-3 , as can be seen in both figs . 2 and 3 . , the set-1 stimuli were presented in a two - dimensional framework and the stimuli in both set-2 and set-3 were presented in a three - dimensional framework . it might be possible that , because of the dimensionality differences between the frameworks , the complexity increase is largest from set-1 to set-2 , thereby introducing a larger overall increase in rt . second , the non - linear increase may well be accounted for by the use of mi in both set-2 and set-3 and no mi in set-1 . this converges with findings of de lange and colleagues , who showed that mi is a much slower strategy than the use of a third person s perspective ( i.e. , vi ) ( de lange et al . 2005 ) . these findings beg the question as to what is the underlying cause of the observed differences in engagement in mi between the sets ? this question may be answered by examining the differences in dor effects and the particulars of the stimuli between the three stimulus sets . for the set-1 condition , it was sufficient for participants to judge the hand laterality by focusing on the combined finger and thumb orientation by , for example , determining whether an l-shape can be observed . if this is possible , then the presented hand must be a left hand or otherwise a right hand . this one possible strategy can be used for all angles of in - plane - rotated stimuli . however , when palm view stimuli are added , the use of that same strategy is not sufficient because , for an upright hand orientation , the same thumb orientation can lead to both left and right judgments due to the inverted view ( i.e. , thumb oriented to the left denotes a right back view hand or a left palm view hand ) . therefore , an additional judgment needs to be incorporated into the strategy , namely a view judgment . it might be that the incorporation of multiple judgments into a strategy facilitates the use of the own body representation in order to judge the hand laterality . according to this suggested difference in strategy use , the inclusion of palm view stimuli within a stimulus set is crucial to induce mi engagement . it has already been shown that humans are able to choose between two strategies in order to solve a mental rotation task ( kosslyn et al . studies on the hand laterality judgment task with children also show that there are two different strategies . at the same time , these studies showed that the implicit use of these different strategies differs between adults and children ( lust et al . collectively , it is likely that participants in our study use two different strategies to solve the mental rotation task , which encompasses a visually based strategy for set-1 ( as no influence of biomechanical constraints is evident ) and a motor - guided strategy for set-2 and set-3 . in summary , this study shows that the number of axes of rotation of a stimulus set does critically influence the engagement in mi during a hand laterality judgment task . more specifically , combined use of palm and back view stimuli increases differences in rt between lateral and medial rotation compared to only presenting back view stimuli , implying a facilitated engagement in mi . our results therefore show that participants do not automatically engage in mi in a hand laterality judgment task , but that engagement is critically dependent on the set of stimuli used . a simple set of stimuli might result in developing and using a strategy that lacks mi engagement , whereas a more difficult set of stimuli promotes engagement in mi . these results have implications for generalization of results of different studies on the hand laterality judgment task and may explain some of the differences observed in mi engagement between studies . motor imagery is used for rehabilitation purposes by , for instance , patients with stroke ( braun et al . 2006 ; sharma et al . 2006 ) , patients with parkinson s disease and patients with complex regional pain syndrome ( see dickstein and deutsch 2007 for a review ) . a major challenge in these studies is to engage participants into the use of motor imagery . only then similar brain networks are active as in actual motor control , which enhances motor recovery . one way to establish this is by only including participants that are able to use motor imagery . validated questionnaires such as the movement imagery questionnaire ( miq ) in addition , the use of a task that implicitly induces the use of a mental movement , such as the hand laterality judgment task , would probably facilitate the patient s ability to engage in a mental movement as this engagement does not depend on the conscious effort of the patient in imagining the movement . finally , our results show that a hand laterality judgment task must be designed such that participants do not develop a strategy based on the visual characteristics of the stimuli .

various studies on the hand laterality judgment task , using complex sets of stimuli , have shown that the judgments during this task are dependent on bodily constraints . more specific , these studies showed that reaction times are dependent on the participant s posture or differ for hand pictures rotated away or toward the mid - sagittal plane ( i.e. , lateral or medial rotation , respectively ) . these findings point to the use of a cognitive embodied process referred to as motor imagery . we hypothesize that the number of axes of rotation of the displayed stimuli during the task is a critical factor for showing engagement in a mental rotation task , with an increased number of rotational axes leading to a facilitation of motor imagery . to test this hypothesis , we used a hand laterality judgment paradigm in which we manipulated the difficulty of the task via the manipulation of the number of rotational axes of the shown stimuli . our results showed increased influence of bodily constraints for increasing number of axes of rotation . more specifically , for the stimulus set containing stimuli rotated over a single axis , no influence of biomechanical constraints was present . the stimulus sets containing stimuli rotated over more than one axes of rotation did induce the use of motor imagery , as a clear influence of bodily constraints on the reaction times was found . these findings extend and refine previous findings on motor imagery as our results show that engagement in motor imagery critically depends on the used number of axes of rotation of the stimulus set .

Introduction Materials and methods Participants Stimuli Experimental procedure Data analysis Results Discussion

the mental rotation task is a well - established paradigm to study the cognitive process of mentally rotating objects . other mental rotation studies used different paradigms in which the participants had to judge object or body part laterality , referred to as a laterality judgment task , first introduced by sekiyama ( 1982 ) . this asymmetric quadratic rt profile was interpreted as evidencing kinesthetic influence of the laterality judgment of hands ( sekiyama 1982 ) . it was postulated that participants engage in an embodied mental process in which biomechanical constraints influence the duration of the mental rotation process ( sekiyama 1982 ; parsons 1987 ; jeannerod 1994 ; decety 1996a ; shenton et al . the influence of kinesthetic aspects or biomechanical constraints was further postulated by parsons ( 1994 ) . he showed that the time needed to actually rotate the own hand to an identical position as the displayed rotated hand corresponds to the time needed for a hand laterality judgment ( parsons 1987 ; parsons 1994 ) . more specifically , he showed that hand rotations away of the mid - sagittal plane ( i.e. , lateral rotation ) resulted in larger rts than hand rotation toward the mid - sagittal plane ( i.e. this embodied processing is referred to as motor imagery ( mi ) ( de lange et al . the embodied nature of mi in a hand laterality judgment task was further evidenced by showing that rt profiles change as a consequence of changing the participant s posture ( parsons 1994 ; sirigu and duhamel 2001 ; de lange et al . extending these findings , children at 57 years of age were also found to engage in mi in a hand laterality judgment task , possibly even more than adults ( funk et al . as an example , we showed that participants with congenital alterations of posture on one side of the body , i.e. ( 2006 ) , groups of adults , healthy children and children suffering from developmental disorder were tested on their engagement in mi during a hand laterality judgment task with only hand stimuli shown from the back . their results showed that rts for both groups of children were subject to biomechanical constraints , as evident by the increased rt for difficult hand postures when compared to hand postures that are easier to adopt . however , the adult group showed no effect of biomechanical constraints and hence no engagement in mi ( lust et al . we propose that the differences observed in literature on the effects of posture or , more general , biomechanical constraints on laterality judgments are due to the particulars of the stimulus set . see table 1 for a short overview of literature on the hand laterality judgment task . obviously , the use of these different perspectives from which the hand stimuli are presented yielded an increase in number of rotational axes and , as a consequence , an increase in overall task difficulty . another factor that differed among studies and which may potentially cause the differential results is the number of rotational steps of the displayed stimuli , see table 1 . however , the number of rotational steps appears to have only a marginal influence because in the study by steenbergen et al . 2006).table 1studies on hand laterality judgment taskauthoryearviewsteps of rotationmi engagementionta and blanke2009hands and feet , back , palm , thumb , little finger6yesde lange et al.2008back , palm7yesionta et al.2007as ionta and blanke ( 2009)6yessteenbergen et al.2007back18nohelmich et al.2007back , palm8yesde lange et al.2006back , palm4yeslust et al.2006back8yes ( children)no ( adults)thayer and johnson2006back , palm6yessauner et al.2006back , palm8yesfunk et al.2005back , palm4yesshenton et al.2004back , palm6yesparsons1994back , palm , thumb , little finger , front finger , back palm12yesparsons1987back , palm , thumb , little finger , front finger , back palm12yessekiyama1982thumb , little finger , palm8yesnote studies on hand laterality judgment task and results on mi engagement . in contrast , studies using multiple viewpoints of hands within their stimulus set do show engagement in mi studies on hand laterality judgment task note studies on hand laterality judgment task and results on mi engagement . in contrast , studies using multiple viewpoints of hands within their stimulus set do show engagement in mi given such diverging results , we hypothesize that the use of more rotational axes within a stimulus set facilitates mi engagement . the argument for this is that simple tasks , in which only in - plane - rotated back view hand stimuli are used ( i.e. , one axis of rotation ) , may promote the use of an alternative strategy based on the combination of finger and thumb orientation to solve the task ( lust et al . for more complex tasks , including for instance the use of both palm and back view hand stimuli , such a strategy will not suffice because one additional judgment on hand view is necessary next to judging hand laterality from the finger and thumb orientation . that there is a neurophysiological difference between two - dimensional ( 2d ) and three - dimensional 2007 ) showed that task difficulty enhanced by rotation dimensionality plays an important role in the selection of a motor strategy . the results showed that the right superior parietal lobule was activated for 2d - rotated stimuli . in the case of 3d - rotated stimuli , the right dorsal premotor cortex ( pmd ) these findings indicate that 2d and 3d rotation of cube figures activate different brain regions and may therefore be ( partly ) different neuronal or cognitive processes . in the present study , we used three different conditions in which the number of axes of rotation increased from , respectively , only 1 axis to 3 axes of rotation . we expected an increasing difference in rts between lateral- and medial - rotated pictures of hands as a function of the number of axes of rotation . more specifically , for 1 axis of rotation , we expected to find no or marginal engagement in mi which will be evidenced by a marginal difference in rt between medial and lateral rotation , whereas increasing engagement in mi ( i.e. , increasing difference in rt between lateral- and medial - rotated hand pictures ) is expected in the conditions with 2- and 3 axes of rotation . we used a custom made 3d hand model designed in a 3d image software package ( autodesk maya 2009 , usa ) . all stimuli were displayed on a 19 lcd computer screen , at a distance of approximately 70 cm from the participants eyes , resulting in a visual angle of approximately 6. the hand stimuli were rotated over three axes , resulting in three different rotational directions , namely : in - plane , longitudinal ( referred to as 1 . three different sets of stimuli were used , namely : set-1 containing only in - plane - rotated stimuli , set-2 with both in - plane- and longitudinal - rotated stimuli , and set-3 containing in - plane- , longitudinal- , and in - depth - rotated stimuli . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity in set-1 , we used back view left and right hand stimuli in six different angles of in - plane rotation ( i.e. for set-2 , we used both back and palm view ( i.e. in set-3 , our set of stimuli were identical to the stimuli used in set-2 but with three angles of in - depth rotations ( i.e. participants were instructed to judge the laterality of the hand as fast and as accurate as possible , without explicit instructions on how to solve the task . reaction times smaller than 300 ms and larger than 3500 ms were excluded from analysis ( total loss 3.2% of trials ) ; these upper and lower boundaries are based on similar studies using a hand laterality judgment task ( sekiyama 1987 ; parsons 1994 ; ionta et al . for analyses purposes , the different in - plane rotations were divided into medial- and lateral - rotated stimuli referred to as direction of rotation ( dor ) in order to measure engagement in mi . first , we analyzed the influence of increasing angle of rotation in order to test for the use of mental rotation for all sets . to this aim , we used an anova with the following design : 2 within - subject factors ( set , angle ) , with 3 levels for set ( set-1 , -2 , and -3 ) and 4 levels for angle ( 0 , 60 , 120 , and 180 ) . a significant effect for angle with increasing rt with increased angle of rotation would indicate the use of mental rotation ( shepard and metzler 1971 ) . then , we analyzed the effect of biomechanical constraints on the rts by means of an anova with the following design : 2 within - subject factors ( set , dor ) , with 3 levels for set ( set-1 , -2 and -3 ) and 2 levels for dor ( lateral rotation , medial rotation ) . a significant set by dor interaction would indicate that the influence of biomechanical constraints ( as measured with dor ) differs between sets . we used a custom made 3d hand model designed in a 3d image software package ( autodesk maya 2009 , usa ) . all stimuli were displayed on a 19 lcd computer screen , at a distance of approximately 70 cm from the participants eyes , resulting in a visual angle of approximately 6. the hand stimuli were rotated over three axes , resulting in three different rotational directions , namely : in - plane , longitudinal ( referred to as 1 . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity in set-1 , we used back view left and right hand stimuli in six different angles of in - plane rotation ( i.e. for set-2 , we used both back and palm view ( i.e. in set-3 , our set of stimuli were identical to the stimuli used in set-2 but with three angles of in - depth rotations ( i.e. participants were instructed to judge the laterality of the hand as fast and as accurate as possible , without explicit instructions on how to solve the task . reaction times smaller than 300 ms and larger than 3500 ms were excluded from analysis ( total loss 3.2% of trials ) ; these upper and lower boundaries are based on similar studies using a hand laterality judgment task ( sekiyama 1987 ; parsons 1994 ; ionta et al . for analyses purposes , the different in - plane rotations were divided into medial- and lateral - rotated stimuli referred to as direction of rotation ( dor ) in order to measure engagement in mi . first , we analyzed the influence of increasing angle of rotation in order to test for the use of mental rotation for all sets . to this aim , we used an anova with the following design : 2 within - subject factors ( set , angle ) , with 3 levels for set ( set-1 , -2 , and -3 ) and 4 levels for angle ( 0 , 60 , 120 , and 180 ) . a significant effect for angle with increasing rt with increased angle of rotation would indicate the use of mental rotation ( shepard and metzler 1971 ) then , we analyzed the effect of biomechanical constraints on the rts by means of an anova with the following design : 2 within - subject factors ( set , dor ) , with 3 levels for set ( set-1 , -2 and -3 ) and 2 levels for dor ( lateral rotation , medial rotation ) . a significant set by dor interaction would indicate that the influence of biomechanical constraints ( as measured with dor ) differs between sets . the total number of incorrect responses ( i.e. within set-1 , set-2 , and set-3 , the amount of errors was 3.3 , 4.6 , and 7.3% of the number of trials per set , respectively . error - bars indicate standard error of the mean ( sem ) reaction times for all three sets , mirrored at 180 ( i.e. error - bars indicate standard error of the mean ( sem ) the analysis on the influence of biomechanical constraints showed a significant main effect for set [ f(2,22 ) = 33.422 , p < 0.001 , = 0.75 ] and dor [ f(1,11 ) = 9.890 , p < 0.01 , = 0.47 ] . the set effect was accounted for by the increase in rt for increasing number of axes of rotation , 848 ms , 1,123 ms , and 1,214 ms for set-1 , set-2 , and set-3 , respectively . in order to support our hypothesis about increasing difference in rt between laterally and medially rotated stimuli , we expected a significant interaction between set and direction of rotation ( dor ) . lateral rotation indicates rotations away from the mid - sagittal plane , and medial rotation indicates rotations toward the mid - sagittal plane . as can be seen , the significant interaction of set by dor ( p < 0.01 ) as represented by the differences in rts between lateral and medial rotation ( i.e. , dor ) increases with increasing number of axes of rotation . error - bars indicate standard error of the mean ( sem ) reaction times for all 3 sets divided into lateral rotation and medial rotation . lateral rotation indicates rotations away from the mid - sagittal plane , and medial rotation indicates rotations toward the mid - sagittal plane . as can be seen , the significant interaction of set by dor ( p < 0.01 ) as represented by the differences in rts between lateral and medial rotation ( i.e. , dor ) increases with increasing number of axes of rotation . error - bars indicate standard error of the mean ( sem ) to control for the differences in mean rts between the sets and its possible influence on the obtained dor effects of the sets , we conducted an additional anova with the data being normalized into the range of [ 01 ] . to test whether a dor effect is also present for in - plane - rotated back view stimuli ( i.e. , set-1 stimuli ) within set-2 and set-3 , we performed an anova identical to the anova testing the influence of biomechanical constraints , described in the data analysis section . this test enables us to see whether a dor effect is also present for back view stimuli in set-2 and set-3 and as a consequence enables us to examine whether only palm view or also back view stimuli induce the use of mi , depending on the context in which they are embedded . in this study , we investigated the influence of multiple angles of rotation in the stimulus set in a hand laterality judgment task on participants engagement in mi . we hypothesized that an increase in the number of axes of rotation facilitates an mi strategy . the number of axes of rotation cumulated from 1 ( i.e. 2 ) , we may assume that participants did indeed mentally rotate the stimuli and were able to solve the mental rotation task accurately ( shepard and metzler 1971 ; sekiyama 1987 ; parsons 1994 ; helmich et al . , direction of rotation ( dor ) effect ) with increasing number of axes of rotation ( see fig . thus , there is an increasing influence of biomechanical constraints on rt with increasing number of axes of rotation , which exemplifies increased engagement in mi ( parsons 1994 ; lust et al . however , if participants indeed memorized the set-1 stimuli , this would result in a lack of use of mental rotation at all . hence another possible explanation might be that the simple fact of adding palm view stimuli to a stimulus set results in the obtained difference in rt between medially and laterally rotated stimuli irrespective of the use of back view stimuli . the results indicate that we obtained this influence of biomechanical constraints only for the sets in which palm view stimuli were used . as a consequence , we sought to see whether the strategy used for the set-1 stimuli ( i.e. , only in - plane - rotated back view stimuli ) changes when they are embedded within a set of stimuli rotated around other rotational axes . the results indicate that the context in which the stimuli ( i.e. the changing effect of dor over the sets indicates that the particulars of a stimulus set ( i.e. , number of rotational axes ) influences the strategy used to solve the mental rotation task for the complete set and that strategies are not linked to a particular type of stimuli . we expected an increase in rt from set-1 till set-3 because of the systematically adding of an extra rotational axis , thereby increasing the task difficulty . this converges with findings of de lange and colleagues , who showed that mi is a much slower strategy than the use of a third person s perspective ( i.e. these findings beg the question as to what is the underlying cause of the observed differences in engagement in mi between the sets ? for the set-1 condition , it was sufficient for participants to judge the hand laterality by focusing on the combined finger and thumb orientation by , for example , determining whether an l-shape can be observed . however , when palm view stimuli are added , the use of that same strategy is not sufficient because , for an upright hand orientation , the same thumb orientation can lead to both left and right judgments due to the inverted view ( i.e. it might be that the incorporation of multiple judgments into a strategy facilitates the use of the own body representation in order to judge the hand laterality . it has already been shown that humans are able to choose between two strategies in order to solve a mental rotation task ( kosslyn et al . studies on the hand laterality judgment task with children also show that there are two different strategies . at the same time , these studies showed that the implicit use of these different strategies differs between adults and children ( lust et al . collectively , it is likely that participants in our study use two different strategies to solve the mental rotation task , which encompasses a visually based strategy for set-1 ( as no influence of biomechanical constraints is evident ) and a motor - guided strategy for set-2 and set-3 . in summary , this study shows that the number of axes of rotation of a stimulus set does critically influence the engagement in mi during a hand laterality judgment task . more specifically , combined use of palm and back view stimuli increases differences in rt between lateral and medial rotation compared to only presenting back view stimuli , implying a facilitated engagement in mi . our results therefore show that participants do not automatically engage in mi in a hand laterality judgment task , but that engagement is critically dependent on the set of stimuli used . these results have implications for generalization of results of different studies on the hand laterality judgment task and may explain some of the differences observed in mi engagement between studies . a major challenge in these studies is to engage participants into the use of motor imagery . validated questionnaires such as the movement imagery questionnaire ( miq ) in addition , the use of a task that implicitly induces the use of a mental movement , such as the hand laterality judgment task , would probably facilitate the patient s ability to engage in a mental movement as this engagement does not depend on the conscious effort of the patient in imagining the movement . finally , our results show that a hand laterality judgment task must be designed such that participants do not develop a strategy based on the visual characteristics of the stimuli .

typically , observed reaction time ( rt ) profiles show an increase in rt for increased angle of rotation indicating that participants mentally rotate stimuli to the upward position ( shepard and metzler 1971 ; parsons 1994 ; jeannerod and decety 1995 ) . it was postulated that participants engage in an embodied mental process in which biomechanical constraints influence the duration of the mental rotation process ( sekiyama 1982 ; parsons 1987 ; jeannerod 1994 ; decety 1996a ; shenton et al . he showed that the time needed to actually rotate the own hand to an identical position as the displayed rotated hand corresponds to the time needed for a hand laterality judgment ( parsons 1987 ; parsons 1994 ) . thus , the rt profiles of imagined hand rotations were subject to the same biomechanical constraints experienced for overt movement . , rt differences between lateral- and medial - rotated stimuli can be used as excellent measure to test for engagement in mi . when engaged in mi , participants mentally plan and perform a movement from a first person perspective without overtly performing the movement and without sensory feedback ( decety 1996a , b ) . the embodied nature of mi in a hand laterality judgment task was further evidenced by showing that rt profiles change as a consequence of changing the participant s posture ( parsons 1994 ; sirigu and duhamel 2001 ; de lange et al . extending these findings , children at 57 years of age were also found to engage in mi in a hand laterality judgment task , possibly even more than adults ( funk et al . as an example , we showed that participants with congenital alterations of posture on one side of the body , i.e. , it was hypothesized that these participants would show a different rt profile for laterality judgments of their affected versus their non - affected hand , but this was not evidenced by the data . ( 2006 ) , groups of adults , healthy children and children suffering from developmental disorder were tested on their engagement in mi during a hand laterality judgment task with only hand stimuli shown from the back . their results showed that rts for both groups of children were subject to biomechanical constraints , as evident by the increased rt for difficult hand postures when compared to hand postures that are easier to adopt . however , the adult group showed no effect of biomechanical constraints and hence no engagement in mi ( lust et al . we propose that the differences observed in literature on the effects of posture or , more general , biomechanical constraints on laterality judgments are due to the particulars of the stimulus set . obviously , the use of these different perspectives from which the hand stimuli are presented yielded an increase in number of rotational axes and , as a consequence , an increase in overall task difficulty . another factor that differed among studies and which may potentially cause the differential results is the number of rotational steps of the displayed stimuli , see table 1 . other studies , however , do show engagement in mi with as little as four angles of rotation ( de lange et al . 2006).table 1studies on hand laterality judgment taskauthoryearviewsteps of rotationmi engagementionta and blanke2009hands and feet , back , palm , thumb , little finger6yesde lange et al.2008back , palm7yesionta et al.2007as ionta and blanke ( 2009)6yessteenbergen et al.2007back18nohelmich et al.2007back , palm8yesde lange et al.2006back , palm4yeslust et al.2006back8yes ( children)no ( adults)thayer and johnson2006back , palm6yessauner et al.2006back , palm8yesfunk et al.2005back , palm4yesshenton et al.2004back , palm6yesparsons1994back , palm , thumb , little finger , front finger , back palm12yesparsons1987back , palm , thumb , little finger , front finger , back palm12yessekiyama1982thumb , little finger , palm8yesnote studies on hand laterality judgment task and results on mi engagement . in contrast , studies using multiple viewpoints of hands within their stimulus set do show engagement in mi given such diverging results , we hypothesize that the use of more rotational axes within a stimulus set facilitates mi engagement . , one axis of rotation ) , may promote the use of an alternative strategy based on the combination of finger and thumb orientation to solve the task ( lust et al . for more complex tasks , including for instance the use of both palm and back view hand stimuli , such a strategy will not suffice because one additional judgment on hand view is necessary next to judging hand laterality from the finger and thumb orientation . that there is a neurophysiological difference between two - dimensional ( 2d ) and three - dimensional 2007 ) showed that task difficulty enhanced by rotation dimensionality plays an important role in the selection of a motor strategy . in the case of 3d - rotated stimuli , the right dorsal premotor cortex ( pmd ) these findings indicate that 2d and 3d rotation of cube figures activate different brain regions and may therefore be ( partly ) different neuronal or cognitive processes . in the present study , we used three different conditions in which the number of axes of rotation increased from , respectively , only 1 axis to 3 axes of rotation . we expected an increasing difference in rts between lateral- and medial - rotated pictures of hands as a function of the number of axes of rotation . more specifically , for 1 axis of rotation , we expected to find no or marginal engagement in mi which will be evidenced by a marginal difference in rt between medial and lateral rotation , whereas increasing engagement in mi ( i.e. , increasing difference in rt between lateral- and medial - rotated hand pictures ) is expected in the conditions with 2- and 3 axes of rotation . twelve healthy right - handed participants were included in the present study ( 8 women , age 22.5 3.7 years , mean sd ) . all stimuli were displayed on a 19 lcd computer screen , at a distance of approximately 70 cm from the participants eyes , resulting in a visual angle of approximately 6. the hand stimuli were rotated over three axes , resulting in three different rotational directions , namely : in - plane , longitudinal ( referred to as 1 . three different sets of stimuli were used , namely : set-1 containing only in - plane - rotated stimuli , set-2 with both in - plane- and longitudinal - rotated stimuli , and set-3 containing in - plane- , longitudinal- , and in - depth - rotated stimuli . with 0 of in - plane angular disparity as the upright position with the fingers pointing upward , see fig . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity shown are all used hand stimuli for set-1 : upper row , left column ; set-2 : upper row ; set-3 : all stimuli . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity in set-1 , we used back view left and right hand stimuli in six different angles of in - plane rotation ( i.e. in set-3 , our set of stimuli were identical to the stimuli used in set-2 but with three angles of in - depth rotations ( i.e. for analyses purposes , the different in - plane rotations were divided into medial- and lateral - rotated stimuli referred to as direction of rotation ( dor ) in order to measure engagement in mi . first , we analyzed the influence of increasing angle of rotation in order to test for the use of mental rotation for all sets . to this aim , we used an anova with the following design : 2 within - subject factors ( set , angle ) , with 3 levels for set ( set-1 , -2 , and -3 ) and 4 levels for angle ( 0 , 60 , 120 , and 180 ) . with 60 being the averaged value of both 60 and 300 in - plane - rotated stimuli and 120 being the averaged value of both 120 and 240 in - plane - rotated stimuli . a significant effect for angle with increasing rt with increased angle of rotation would indicate the use of mental rotation ( shepard and metzler 1971 ) . then , we analyzed the effect of biomechanical constraints on the rts by means of an anova with the following design : 2 within - subject factors ( set , dor ) , with 3 levels for set ( set-1 , -2 and -3 ) and 2 levels for dor ( lateral rotation , medial rotation ) . twelve healthy right - handed participants were included in the present study ( 8 women , age 22.5 3.7 years , mean sd ) . all stimuli were displayed on a 19 lcd computer screen , at a distance of approximately 70 cm from the participants eyes , resulting in a visual angle of approximately 6. the hand stimuli were rotated over three axes , resulting in three different rotational directions , namely : in - plane , longitudinal ( referred to as 1 . three different sets of stimuli were used , namely : set-1 containing only in - plane - rotated stimuli , set-2 with both in - plane- and longitudinal - rotated stimuli , and set-3 containing in - plane- , longitudinal- , and in - depth - rotated stimuli . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity shown are all used hand stimuli for set-1 : upper row , left column ; set-2 : upper row ; set-3 : all stimuli . angles within stimuli represent in - plane angular disparity and angles displayed in the in - depth column represent in - depth angular disparity in set-1 , we used back view left and right hand stimuli in six different angles of in - plane rotation ( i.e. in set-3 , our set of stimuli were identical to the stimuli used in set-2 but with three angles of in - depth rotations ( i.e. for analyses purposes , the different in - plane rotations were divided into medial- and lateral - rotated stimuli referred to as direction of rotation ( dor ) in order to measure engagement in mi . first , we analyzed the influence of increasing angle of rotation in order to test for the use of mental rotation for all sets . to this aim , we used an anova with the following design : 2 within - subject factors ( set , angle ) , with 3 levels for set ( set-1 , -2 , and -3 ) and 4 levels for angle ( 0 , 60 , 120 , and 180 ) . with 60 being the averaged value of both 60 and 300 in - plane - rotated stimuli and 120 being the averaged value of both 120 and 240 in - plane - rotated stimuli . a significant effect for angle with increasing rt with increased angle of rotation would indicate the use of mental rotation ( shepard and metzler 1971 ) then , we analyzed the effect of biomechanical constraints on the rts by means of an anova with the following design : 2 within - subject factors ( set , dor ) , with 3 levels for set ( set-1 , -2 and -3 ) and 2 levels for dor ( lateral rotation , medial rotation ) . the mental rotation analysis revealed a significant main effect for both set [ f(2,22 ) = 43.380 , p < 0.001 , = 0.798 ] and angle [ f(3,33 ) = 38.772 , p < 0.001 , = 0.779 ] . set-1 [ f(3,33 ) = 37.732 , p < 0.001 , = 0.774 ] , set-2 [ f(3,33 ) = 20.941 , p < 0.001 , = 0.656 ] , and set-3 [ f(3,33 ) = 28.789 , p < 0.001 , = 0.724 ] . error - bars indicate standard error of the mean ( sem ) the analysis on the influence of biomechanical constraints showed a significant main effect for set [ f(2,22 ) = 33.422 , p < 0.001 , = 0.75 ] and dor [ f(1,11 ) = 9.890 , p < 0.01 , = 0.47 ] . the set effect was accounted for by the increase in rt for increasing number of axes of rotation , 848 ms , 1,123 ms , and 1,214 ms for set-1 , set-2 , and set-3 , respectively . set-1 differed significantly from both set-2 and set-3 ( p < 0.001 and p < 0.001 , respectively ) . in order to support our hypothesis about increasing difference in rt between laterally and medially rotated stimuli , we expected a significant interaction between set and direction of rotation ( dor ) . the anova resulted in a significant interaction of set by dor [ f(2,22 ) = 8.196 , p < 0.01 , = 0.43 ] , which was accounted for by the increasing difference in rts between laterally and medially rotated stimuli over the stimulus sets , resulting in significant simple dor effects in set-2 [ f(1,11 ) = 5.964 , p < 0.05 , = 0.35 ] and set-3 [ f(1,11 ) = 18.005 , p < 0.01 , = 0.62 ] . the simple dor effects differed significantly between set-1 and set-2 [ f(1,11 ) = 6.276 , p < 0.05 , = 0.363 ] , and set-1 and set-3 [ f(1,11 ) = 13.344 , p < 0.01 , = 0.548 ] but not between set-2 and set-3 ( p = 0.198).fig . as can be seen , the significant interaction of set by dor ( p < 0.01 ) as represented by the differences in rts between lateral and medial rotation ( i.e. as can be seen , the significant interaction of set by dor ( p < 0.01 ) as represented by the differences in rts between lateral and medial rotation ( i.e. error - bars indicate standard error of the mean ( sem ) to control for the differences in mean rts between the sets and its possible influence on the obtained dor effects of the sets , we conducted an additional anova with the data being normalized into the range of [ 01 ] . the lack of significant set effect indicates that the normalization was effective as it reduced the differences in absolute value of the mean rts between the sets . as we only obtained a significant dor effect in the sets including palm view stimuli , one might argue that only the palm view stimuli accounted for the obtained dor effect in set-2 and set-3 . this test enables us to see whether a dor effect is also present for back view stimuli in set-2 and set-3 and as a consequence enables us to examine whether only palm view or also back view stimuli induce the use of mi , depending on the context in which they are embedded . the anova revealed a significant main effect of set [ f(2,22 ) = 21.694 , p < 0.001 , = 0.664 ] and dor [ f(1,10 ) = 4.999 , p < 0.05 , = 0.312 ] and an interaction of set by dor [ f(2,22 ) = 3.429 , p 0.05 , = 0.238 ] . the interaction was accounted for by a lack of significant dor effect for set-1 stimuli in set-1 ( p = 0.578 ) and set-2 ( p = 0.237 ) but a significant dor effect for set-1 stimuli within set-3 [ f(1,11 ) = 8.620 , p < 0.05 , = 0.439 ] . in this study , we investigated the influence of multiple angles of rotation in the stimulus set in a hand laterality judgment task on participants engagement in mi . thus , there is an increasing influence of biomechanical constraints on rt with increasing number of axes of rotation , which exemplifies increased engagement in mi ( parsons 1994 ; lust et al . in contrast , both set-2 ( in - plane - rotated , back- and palm view stimuli ) and set-3 ( in - plane- and in - depth - rotated , back- and palm view stimuli ) revealed a significant dor effect . the dor effects in both the set-2 and set-3 conditions were accounted for by a smaller rt for medial compared to lateral rotations , which is in correspondence with literature ( parsons 1987 ; parsons 1994 ) . additionally , the difference in obtained dor effects between the sets was not accounted for by the differences in absolute mean rt between the sets as the analysis on the normalized data shows the same results . the process of mi is dependent on embodied cognitive processing and therefore affected by both the desired end - state and the current position of one s body ( jeannerod 1994 ; de lange et al . hence another possible explanation might be that the simple fact of adding palm view stimuli to a stimulus set results in the obtained difference in rt between medially and laterally rotated stimuli irrespective of the use of back view stimuli . , number of rotational axes ) influences the strategy used to solve the mental rotation task for the complete set and that strategies are not linked to a particular type of stimuli . , the set-1 stimuli were presented in a two - dimensional framework and the stimuli in both set-2 and set-3 were presented in a three - dimensional framework . second , the non - linear increase may well be accounted for by the use of mi in both set-2 and set-3 and no mi in set-1 . these findings beg the question as to what is the underlying cause of the observed differences in engagement in mi between the sets ? collectively , it is likely that participants in our study use two different strategies to solve the mental rotation task , which encompasses a visually based strategy for set-1 ( as no influence of biomechanical constraints is evident ) and a motor - guided strategy for set-2 and set-3 . in summary , this study shows that the number of axes of rotation of a stimulus set does critically influence the engagement in mi during a hand laterality judgment task . more specifically , combined use of palm and back view stimuli increases differences in rt between lateral and medial rotation compared to only presenting back view stimuli , implying a facilitated engagement in mi . validated questionnaires such as the movement imagery questionnaire ( miq ) in addition , the use of a task that implicitly induces the use of a mental movement , such as the hand laterality judgment task , would probably facilitate the patient s ability to engage in a mental movement as this engagement does not depend on the conscious effort of the patient in imagining the movement .

there is some evidence to suggest that working memory might be impaired in patients who show jtc compared to those who do not . a small study by ormrod et al69 found that visual working memory performance was affected in first - episode psychosis patients who showed a jtc response style . in addition , in sz patients with strong current delusions , working memory ( but not premorbid iq ) was worse in those who demonstrated jtc.70 as noted above , broome et al67 found that jtc in the prodrome was linked to working memory impairments . nevertheless , the presence of a memory aid during the urn task does not affect jtc in patient groups.51,53 this would appear to undermine any suggestion that jtc stems from a relative inability to maintain the task items in memory . working memory impairments might correlate with a tendency to jtc , but the relationship is unlikely to be causal . one explanation for the jtc response style observed in sz is that patients simply make decisions based on less evidence . this liberal acceptance account53,71 was founded on evidence that sz patients tend not to converge on one particular interpretation of a situation ( eg , when asked to judge the plausibility of multiple interpretations of a picture ) . healthy controls ruled out interpretations that patients continued to liberally entertain , giving higher ratings to a wide range of interpretations . sz patients are proposed to more readily accept a response option , while healthy participants are more cautious in doing so . in situations where only two ( mutually exclusive ) options moritz et al71 increased the number of jars to four to provide additional ambiguity : this was found to abolish the jtc in patients . although sz patients made a less systematic information search and were more likely to consider less valid information , they did not inspect fewer pieces of information compared to healthy controls and thus did not demonstrate jtc . effects were found on confidence ratings however , with patients more likely to be overconfident , using extreme confidence ratings under inappropriate circumstances . this is consistent with work showing that sz patients are less confident of their correct answers and overconfident when they make errors , during word recall tasks.72,73 although there were no overall differences between patients and controls , symptomatology in the patient group was linked to information search . a correlation was observed between symptom severity scores ( positive and negative syndrome scale [ panss ] positive , panss delusion ) and the degree of information search , with higher - scoring participants tending to gather less pieces of information . panss score did not predict use of extreme confidence ratings , but nevertheless this study would suggest that jtc can be largely abolished and can manifest as overconfidence only when information is presented in the right manner . impulsiveness is unlikely to be a factor because patients with sz show similar reaction times as healthy controls51,53 and draw more beads when the task is made harder.51,55,74 motivational factors are also unlikely to play a part : the possibility that the patients overestimate the cost of gathering more information ( possibly due to a greater need for closure ) has been discounted because patients do not seem to experience a greater cost for gathering more information.75 an alternative explanation is that jtc manifests not through a lowered threshold for making a decision but through each piece of evidence being relatively overvalued . when asked to report belief estimates after each draw , it has been shown that patients make more drastic updates after each piece of evidence.46,56,77 speechley et al78 found that delusional patients , when asked to give likelihood ratings for each urn on each trial , gave higher ratings for whichever urn matched the current evidence , while ratings for the nonmatching urn did not differ from those of the control groups . the authors argue that this provides evidence of a reasoning bias characterized by hypersalience of evidence that matches a hypothesis , but with reasoning that appears intact for nonmatches . the literature is inconsistent regarding patients responses to nonmatches ( disconfirmatory evidence ) . because delusions are maintained in the face of contradictory evidence , it is unsurprising that patients tend to show a bias against disconfirmatory evidence.79 it has been argued that hypersalience could underlie this effect : hypersalience of evidence hypothesis matches may lead to an enhancement of weak matches , leading to difficulties in integrating disambiguating information.80 some studies point to hypersalience of disconfirmatory , as well as confirmatory , evidence : deluded patients show a tendency to overadjust when presented with potentially disconfirmatory evidence,53,56 although this effect might be linked to miscomprehension of the task,81 especially because such a tendency would contradict the evidence of reversal - learning deficits discussed in the previous section . these findings are consistent with an aberrant salience account of sz,82 whereby dysregulated dopamine transmission generates context - inappropriate salience attributions , potentially due to aberrant signaling in the ventral striatal dopaminergic pathway , which is thought to regulate stimulus response pairings.83 moore and sellen84 built a simple network model in which the gain , or signal - to - noise , parameter ( which describes the likelihood of a node firing when presented with some input ) was varied . the gain parameter was assumed to represent striatal dopaminergic activity , and increasing this parameter meant that the model successfully mimicked the jtc response style seen in research data from delusional patients . this might be overly simplistic however , because imaging studies have shown that the striatum is downregulated in arms85 and sz patients.86 nevertheless , it is important to note that evidence for hypersalience has been observed across various other cognitive biases present in sz8789 and as such represents a convincing account of jtc . as mentioned previously , another suggestion concerning the development of delusions in sz focuses on impairments in theory of mind.42 langdon et al77 compared 35 sz patients with a history of delusions to healthy controls , on a battery of tasks that included two versions of the urn task , three theory - of - mind tasks , and a questionnaire on attributional biases . a jtc response style was found in the patient group , as well as impairments on the theory of mind tasks and evidence of an externalizing attributional bias . performance on the urn tasks correlated with that of the theory - of - mind tasks , while attributional bias scores did not correlate with other task measures . delusion proneness ( measured by the questionnaire ) correlated with probabilistic reasoning and theory - of - mind measures , while externalizing bias did not ; iq and memory ability were accounted for . although it was draws - to - decision that correlated most robustly with delusion proneness , these data prompted the authors to suggest that a common underlying mechanism might operate in sz to drive probabilistic reasoning and theory of mind deficits . they speculate that this could be a difficulty in inhibiting sensory input reflecting the immediate perceived reality , thus making patients more likely to be influenced by current data when making decisions on probabilistic reasoning tasks and making it harder to maintain an abstract viewpoint as required by theory - of - mind tasks . however , it should be noted that a large meta - analysis found no evidence for a link between theory of mind deficits and positive symptoms ; instead , deficits in theory of mind were correlated with negative symptoms , disorganization , and cognitive impairment.90 there is also some suggestion that poor emotion regulation might have a role to play . in one study , 90 healthy individuals with varying levels of psychosis vulnerability ( assessed by the community assessment of psychic experiences ) were recruited.74 half the sample received an anxiety induction procedure , the other half did not . paranoid delusions and jtc were then assessed during the session . the anxiety induction procedure promoted delusions and jtc , and participants with higher psychosis vulnerability showed greater increases in paranoid delusional ideation . furthermore , jtc appeared to mediate the association between anxiety and delusions , prompting the authors to suggest that paranoid delusions result from an interaction of anxiety and reasoning biases . in the glckner and moritz91 study , it was found that increasing stress ( through time pressure and the addition of affective valence ) led to diminished performance in patients . thus , improved self - monitoring in terms of better emotion regulation , combined with metacognitive training targeted at reasoning biases , could be beneficial in reducing delusion formation in sz . on balance , the explanations best supported by evidence are those of liberal acceptance and hypersalience . at present , it is hard to say which is best supported by the evidence available : this is possibly because these two explanations are by no means mutually exclusive . one difficulty in differentiating these explanations is that the urn task offers limited information regarding learning and decision - making processes . in one of our studies,92 we investigated performance in patients on a sequence - learning task ( sequences of four button presses were learnt using two buttons , feedback after each button press ) and the typical urn task . patients were able to learn the correct sequence , but learning was slower compared to that in healthy controls . interestingly , learning from positive ( but not negative ) feedback in the sequence task correlated with draws - to - decision in the urn task : patients who showed a jtc response style were impaired in learning from positive feedback . thus , these findings clearly favor a liberal acceptance account over hypersalience , but further work is needed to distinguish liberal acceptance and hypersalience , as well as to explore potential interactions with emotion regulation and other self - monitoring activities . in the next section , we discuss work aimed at elucidating the neural basis of jtc . various studies have attempted to induce the jtc bias in healthy controls using pharmacological manipulations ; others have used fmri . a drug model of jtc in healthy controls would be informative regarding the neurobiological underpinnings of jtc . there is some evidence that the noncompetitive n - methyl - d - aspartate ( nmda ) receptor antagonist ketamine could serve as such a model . because ketamine can be safely administered under clinical supervision and has relatively short - lived effects , the drug could represent a useful tool for studying processes underlying jtc . ketamine infusions in healthy controls can induce behavioral and cognitive disturbances that are somewhat similar to the symptoms of sz,9395 infusions induce delusional thinking,96,97 and ketamine use has been linked to increased delusional symptoms in recreational users.98 importantly , ketamine infusions in patients with sz cause a worsening of symptoms.99101 ketamine has been shown to affect both glutamatergic and dopaminergic systems,102,103 potentially mimicking the aberrant dopaminergic transmission posited to underlie the jtc effect seen in sz ; ketamine also increases basal ganglia and thalamic activation in a manner similar to that observed in sz patients.104 a recent study by corlett et al105 administered ketamine to healthy controls to investigate whether faulty prediction error signals could underlie delusion formation in sz . prediction error is defined by the mismatch between expectations and experience , and it is probably represented by activity in the mesostriatal dopamine system.106 the prediction error signal in frontostriatal regions has been shown to correlate with delusion - like beliefs in healthy people107 and to be predictive of the severity of delusions in sz patients.108 disrupted prediction error signals could cause individuals to attend to and make associations with inappropriate stimuli ( both internal and external ) , consequently developing beliefs that do not reflect real - world contingencies,108 causing a jtc style of responding . corlett et al105 found that ketamine could strengthen the memory trace of a previously conditioned stimulus when it was presented again , without reinforcement , offering tentative support for this hypothesis . however , another study failed to find a jtc response style during the urn task in healthy controls receiving ketamine . this suggests that although ketamine can induce delusions in healthy controls , delusion formation under ketamine might not be so closely linked to jtc as in sz,109 although possibly higher doses of ketamine might be required for jtc to manifest . interestingly , work investigating the effects of dopamine agonists and antagonists in healthy controls has also failed to induce jtc , suggesting that straightforward modulation of the dopaminergic system might not be sufficient to reproduce the jtc response style seen in sz . l - dopa , had no effect on draws - to - decision , or confidence , whereas a dopamine antagonist ( haloperidol ) was found to reduce overconfidence but had no effect on draws - to - decision.110 likewise , dopaminergic modulation with methamphetamine does not affect draws - to - decision.111 in sum , attempts to promote jtc in controls using pharmacological interventions have been largely unsuccessful . thus , it would seem that jtc can not be attributed solely to general dopaminergic overactivity , at least when induced acutely : the relationship might not be linear , or perhaps chronic aberrations in dopamine firing is required for jtc to manifest . alternatively , the disrupted interactions between several neurotransmitter systems might be critical . fmri can potentially indicate the neural mechanisms contributing to probabilistic reasoning in the urn task . studies in healthy controls implicate a distributed network of brain areas , including parietal cortex ( typically around intraparietal sulcus ) , prefrontal cortex ( typically dorsolateral ) , anterior insula , and striatum . these fmri studies use paradigms , including draw events ( stimuli for which participants choose to gather more information ) and urn events ( in which participants decide they have enough evidence and so select an urn ) . some of these studies compared urn task blocks ( which collapse over draw and urn decision events ) with blocks where participants performed a control task on the same stimuli . the most consistent finding across these block design studies are parietal responses near the intraparietal sulcus , which are larger for urn task blocks,112114 although some of these studies also report enhanced responses in the right dorsolateral prefrontal cortex113115 and anterior insula.113,115 one of these studies included a sz patient group and showed that the enhanced responses for the urn task blocks in the parietal and prefrontal cortices was reduced in these individuals , compared to the responses in healthy controls.113 this conclusion is tentative , however , as this study failed to replicate the classic jtc behavioral effect in sz . these block - design studies are limited , as they can not distinguish fmri responses to draw choice events ( decisions to gather more information ) from fmri responses associated with final choices of an urn . jtc occurs when data gathering is discontinued in favor of choosing an urn , so brain areas contributing to urn choice events are likely to be involved in jtc . studies that contrast fmri responses to urn choices versus draw choices yield similar results as the block - design contrasts . urn choices , compared to draw choices , activate anterior insula , striatum,115,116 acc , and parietal cortex , including the intraparietal sulcus.116 a near - identical network of brain areas has also been observed in the analogous contrast in the closely related best choice task.117 here , this network of areas was associated with deciding on an option currently available ( eg , a used car ) , compared to deciding to sample more possible options ( eg , viewing more cars ) . furl and averbeck116 found further roles for this parietal cortex area in the urn task . parietal cortex was more responsive during urn decisions for participants who tended to draw more and for participants who made greater adjustments to their draws - to - decision depending on prevalence of the minority bead color . these latter findings link parietal responses to individual differences in information - gathering behavior . however , this between - participant variability was within a nonclinical sample , and it remains to be confirmed whether it also extends to clinical cases such as sz . involvement of areas such as striatum , dorsolateral prefrontal cortex , and intraparietal sulcus in deciding on a probabilistic inference rather than continued evidence seeking ( as in jtc ) is perhaps not surprising . for example , measures of evidence accumulation for perceptual decisions have been associated with fmri responses in the prefrontal cortex of psychiatrically healthy participants118 and in neural recordings from the striatum119 and intraparietal sulcus in monkeys.120 measures of evidence accumulation for economic decisions in healthy participants are also associated with fmri responses in dorsolateral prefrontal cortex , striatum , and intraparietal sulcus.121 prefrontal cortex lesions are also associated with jtc.122 we hypothesize that this network of areas , which contributes to the urn task and other decision - making tasks in healthy participants , may be compromised in sz patients who show jtc behavior . the striatum , for instance , signals errors in reward prediction and predicts successful reward learning in healthy participants123 but shows aberrant responses during reward prediction tasks in schizophrenic individuals.124,125 aberrant prediction - related signaling in the dopaminergic striatum might also play a role in jtc behavior , as dopaminergic antipsychotic treatment abolishes jtc on an emotionally salient version of the urn task.64 in addition to the striatum , responses in the dorsolateral prefrontal cortex are also reduced in schizophrenics , compared to those in healthy individuals , when performing tasks requiring reward prediction.126 in sum , schizophrenic individuals appear to show reward - related response reductions in many of the brain areas activated by the urn task in healthy participants . dysfunctional interactions between the dopaminergic striatum and cortical areas , such as intraparietal sulcus and dorsolateral prefrontal cortex , might explain jtc behavior in sz patients . more brain - imaging studies using sz patients and the urn task are required to test this hypothesis . a drug model of jtc in healthy controls would be informative regarding the neurobiological underpinnings of jtc . there is some evidence that the noncompetitive n - methyl - d - aspartate ( nmda ) receptor antagonist ketamine could serve as such a model . because ketamine can be safely administered under clinical supervision and has relatively short - lived effects , the drug could represent a useful tool for studying processes underlying jtc . ketamine infusions in healthy controls can induce behavioral and cognitive disturbances that are somewhat similar to the symptoms of sz,9395 infusions induce delusional thinking,96,97 and ketamine use has been linked to increased delusional symptoms in recreational users.98 importantly , ketamine infusions in patients with sz cause a worsening of symptoms.99101 ketamine has been shown to affect both glutamatergic and dopaminergic systems,102,103 potentially mimicking the aberrant dopaminergic transmission posited to underlie the jtc effect seen in sz ; ketamine also increases basal ganglia and thalamic activation in a manner similar to that observed in sz patients.104 a recent study by corlett et al105 administered ketamine to healthy controls to investigate whether faulty prediction error signals could underlie delusion formation in sz . prediction error is defined by the mismatch between expectations and experience , and it is probably represented by activity in the mesostriatal dopamine system.106 the prediction error signal in frontostriatal regions has been shown to correlate with delusion - like beliefs in healthy people107 and to be predictive of the severity of delusions in sz patients.108 disrupted prediction error signals could cause individuals to attend to and make associations with inappropriate stimuli ( both internal and external ) , consequently developing beliefs that do not reflect real - world contingencies,108 causing a jtc style of responding . corlett et al105 found that ketamine could strengthen the memory trace of a previously conditioned stimulus when it was presented again , without reinforcement , offering tentative support for this hypothesis . however , another study failed to find a jtc response style during the urn task in healthy controls receiving ketamine . this suggests that although ketamine can induce delusions in healthy controls , delusion formation under ketamine might not be so closely linked to jtc as in sz,109 although possibly higher doses of ketamine might be required for jtc to manifest . interestingly , work investigating the effects of dopamine agonists and antagonists in healthy controls has also failed to induce jtc , suggesting that straightforward modulation of the dopaminergic system might not be sufficient to reproduce the jtc response style seen in sz . l - dopa , had no effect on draws - to - decision , or confidence , whereas a dopamine antagonist ( haloperidol ) was found to reduce overconfidence but had no effect on draws - to - decision.110 likewise , dopaminergic modulation with methamphetamine does not affect draws - to - decision.111 in sum , attempts to promote jtc in controls using pharmacological interventions have been largely unsuccessful . thus , it would seem that jtc can not be attributed solely to general dopaminergic overactivity , at least when induced acutely : the relationship might not be linear , or perhaps chronic aberrations in dopamine firing is required for jtc to manifest . alternatively , the disrupted interactions between several neurotransmitter systems might be critical . fmri can potentially indicate the neural mechanisms contributing to probabilistic reasoning in the urn task . studies in healthy controls implicate a distributed network of brain areas , including parietal cortex ( typically around intraparietal sulcus ) , prefrontal cortex ( typically dorsolateral ) , anterior insula , and striatum . these fmri studies use paradigms , including draw events ( stimuli for which participants choose to gather more information ) and urn events ( in which participants decide they have enough evidence and so select an urn ) . some of these studies compared urn task blocks ( which collapse over draw and urn decision events ) with blocks where participants performed a control task on the same stimuli . the most consistent finding across these block design studies are parietal responses near the intraparietal sulcus , which are larger for urn task blocks,112114 although some of these studies also report enhanced responses in the right dorsolateral prefrontal cortex113115 and anterior insula.113,115 one of these studies included a sz patient group and showed that the enhanced responses for the urn task blocks in the parietal and prefrontal cortices was reduced in these individuals , compared to the responses in healthy controls.113 this conclusion is tentative , however , as this study failed to replicate the classic jtc behavioral effect in sz . these block - design studies are limited , as they can not distinguish fmri responses to draw choice events ( decisions to gather more information ) from fmri responses associated with final choices of an urn . jtc occurs when data gathering is discontinued in favor of choosing an urn , so brain areas contributing to urn choice events are likely to be involved in jtc . studies that contrast fmri responses to urn choices versus draw choices yield similar results as the block - design contrasts . urn choices , compared to draw choices , activate anterior insula , striatum,115,116 acc , and parietal cortex , including the intraparietal sulcus.116 a near - identical network of brain areas has also been observed in the analogous contrast in the closely related best choice task.117 here , this network of areas was associated with deciding on an option currently available ( eg , a used car ) , compared to deciding to sample more possible options ( eg , viewing more cars ) . furl and averbeck116 found further roles for this parietal cortex area in the urn task . parietal cortex was more responsive during urn decisions for participants who tended to draw more and for participants who made greater adjustments to their draws - to - decision depending on prevalence of the minority bead color . however , this between - participant variability was within a nonclinical sample , and it remains to be confirmed whether it also extends to clinical cases such as sz . involvement of areas such as striatum , dorsolateral prefrontal cortex , and intraparietal sulcus in deciding on a probabilistic inference rather than continued evidence seeking ( as in jtc ) is perhaps not surprising . for example , measures of evidence accumulation for perceptual decisions have been associated with fmri responses in the prefrontal cortex of psychiatrically healthy participants118 and in neural recordings from the striatum119 and intraparietal sulcus in monkeys.120 measures of evidence accumulation for economic decisions in healthy participants are also associated with fmri responses in dorsolateral prefrontal cortex , striatum , and intraparietal sulcus.121 prefrontal cortex lesions are also associated with jtc.122 we hypothesize that this network of areas , which contributes to the urn task and other decision - making tasks in healthy participants , may be compromised in sz patients who show jtc behavior . the striatum , for instance , signals errors in reward prediction and predicts successful reward learning in healthy participants123 but shows aberrant responses during reward prediction tasks in schizophrenic individuals.124,125 aberrant prediction - related signaling in the dopaminergic striatum might also play a role in jtc behavior , as dopaminergic antipsychotic treatment abolishes jtc on an emotionally salient version of the urn task.64 in addition to the striatum , responses in the dorsolateral prefrontal cortex are also reduced in schizophrenics , compared to those in healthy individuals , when performing tasks requiring reward prediction.126 in sum , schizophrenic individuals appear to show reward - related response reductions in many of the brain areas activated by the urn task in healthy participants . dysfunctional interactions between the dopaminergic striatum and cortical areas , such as intraparietal sulcus and dorsolateral prefrontal cortex , might explain jtc behavior in sz patients . more brain - imaging studies using sz patients and the urn task are required to test this hypothesis . some studies have suggested that the jtc bias could represent an important therapeutic target . using an emotionally salient version of the urn task , menon et al64 found that within 2 weeks of initiating treatment with antipsychotics , patients demonstrated an increase in the number of trials - to - decision alongside attenuation of psychotic symptoms and delusions . although these measures were not correlated , baseline performance on the task had some predictive power over the individuals who would show improvements in symptomology in response to medication : jtc performance at baseline could therefore be useful in guiding treatment . moreover , treatment - related improvements in jtc can predict probability of regaining full employment , measured over a 6-month window . this was not the case for positive symptoms or neuropsychological performance , suggesting that jtc might act independently to influence real - world functioning.127 it is important to note that most studies have failed to show improvements in jtc on the standard urn task following antipsychotic treatment,64,128 although there is some evidence to dispute this.63,65 if jtc does indeed fluctuate with delusional symptoms , it would provide strong evidence of its importance as a treatment target : interventions that specifically target cognitive biases ( known as metacognitive training programs ) have already been shown to improve delusions and other positive symptoms.129,130 it is also important to note that most studies typically detect jtc in only approximately 50% of their patient samples . this heterogeneity needs to be explored further , to determine how it might relate to heterogeneity of symptomatology or whether it could be an issue of task sensitivity . again , individual differences in jtc performance could be useful in determining the best course of treatment . training programs that aim to ameliorate the jtc response style might prove to be an important adjunct to established therapies . in sum , jtc in sz seems to be a consistent finding and there is strong evidence linking jtc to delusion formation . both liberal acceptance and hypersalience accounts of jtc are well supported by the literature , but attempts to replicate jtc in healthy controls using pharmacological manipulations have largely failed , undermining attempts to develop a neurobiological account of jtc . fmri studies have implicated a network involved in making urn choices , which includes striatal , insula , parietal , and prefrontal areas ; further patient work is required , particularly in the context of evidence suggesting that jtc could represent a valuable therapeutic target .

schizophrenia is a mental disorder associated with a variety of symptoms , including hallucinations , delusions , social withdrawal , and cognitive dysfunction . impairments on decision - making tasks are routinely reported : evidence points to a particular deficit in learning from and revising behavior following feedback . in addition , patients tend to make hasty decisions when probabilistic judgments are required . this is known as jumping to conclusions ( jtc ) and has typically been demonstrated by presenting participants with colored beads drawn from one of two urns until they claim to be sure which urn the beads are being drawn from ( the proportions of colors vary in each urn ) . patients tend to make early decisions on this task , and there is evidence to suggest that a hasty decision - making style might be linked to delusion formation and thus be of clinical relevance . various accounts have been proposed regarding what underlies this behavior . in this review , we briefly introduce the disorder and the decision - making deficits associated with it . we then explore the evidence for each account of jtc in the context of a wider decision - making deficit and then go on to summarize work exploring jtc in healthy controls using pharmacological manipulations and functional imaging . finally , we assess whether jtc might have a role in therapy .

Working memory impairment Liberal acceptance Hypersalience of evidence Self-monitoring difficulties Remarks Methods of investigation Pharmacological interventions fMRI studies Role in therapy Conclusion

there is some evidence to suggest that working memory might be impaired in patients who show jtc compared to those who do not . in addition , in sz patients with strong current delusions , working memory ( but not premorbid iq ) was worse in those who demonstrated jtc.70 as noted above , broome et al67 found that jtc in the prodrome was linked to working memory impairments . nevertheless , the presence of a memory aid during the urn task does not affect jtc in patient groups.51,53 this would appear to undermine any suggestion that jtc stems from a relative inability to maintain the task items in memory . working memory impairments might correlate with a tendency to jtc , but the relationship is unlikely to be causal . this liberal acceptance account53,71 was founded on evidence that sz patients tend not to converge on one particular interpretation of a situation ( eg , when asked to judge the plausibility of multiple interpretations of a picture ) . healthy controls ruled out interpretations that patients continued to liberally entertain , giving higher ratings to a wide range of interpretations . this is consistent with work showing that sz patients are less confident of their correct answers and overconfident when they make errors , during word recall tasks.72,73 although there were no overall differences between patients and controls , symptomatology in the patient group was linked to information search . a correlation was observed between symptom severity scores ( positive and negative syndrome scale [ panss ] positive , panss delusion ) and the degree of information search , with higher - scoring participants tending to gather less pieces of information . panss score did not predict use of extreme confidence ratings , but nevertheless this study would suggest that jtc can be largely abolished and can manifest as overconfidence only when information is presented in the right manner . impulsiveness is unlikely to be a factor because patients with sz show similar reaction times as healthy controls51,53 and draw more beads when the task is made harder.51,55,74 motivational factors are also unlikely to play a part : the possibility that the patients overestimate the cost of gathering more information ( possibly due to a greater need for closure ) has been discounted because patients do not seem to experience a greater cost for gathering more information.75 an alternative explanation is that jtc manifests not through a lowered threshold for making a decision but through each piece of evidence being relatively overvalued . when asked to report belief estimates after each draw , it has been shown that patients make more drastic updates after each piece of evidence.46,56,77 speechley et al78 found that delusional patients , when asked to give likelihood ratings for each urn on each trial , gave higher ratings for whichever urn matched the current evidence , while ratings for the nonmatching urn did not differ from those of the control groups . because delusions are maintained in the face of contradictory evidence , it is unsurprising that patients tend to show a bias against disconfirmatory evidence.79 it has been argued that hypersalience could underlie this effect : hypersalience of evidence hypothesis matches may lead to an enhancement of weak matches , leading to difficulties in integrating disambiguating information.80 some studies point to hypersalience of disconfirmatory , as well as confirmatory , evidence : deluded patients show a tendency to overadjust when presented with potentially disconfirmatory evidence,53,56 although this effect might be linked to miscomprehension of the task,81 especially because such a tendency would contradict the evidence of reversal - learning deficits discussed in the previous section . these findings are consistent with an aberrant salience account of sz,82 whereby dysregulated dopamine transmission generates context - inappropriate salience attributions , potentially due to aberrant signaling in the ventral striatal dopaminergic pathway , which is thought to regulate stimulus response pairings.83 moore and sellen84 built a simple network model in which the gain , or signal - to - noise , parameter ( which describes the likelihood of a node firing when presented with some input ) was varied . this might be overly simplistic however , because imaging studies have shown that the striatum is downregulated in arms85 and sz patients.86 nevertheless , it is important to note that evidence for hypersalience has been observed across various other cognitive biases present in sz8789 and as such represents a convincing account of jtc . as mentioned previously , another suggestion concerning the development of delusions in sz focuses on impairments in theory of mind.42 langdon et al77 compared 35 sz patients with a history of delusions to healthy controls , on a battery of tasks that included two versions of the urn task , three theory - of - mind tasks , and a questionnaire on attributional biases . a jtc response style was found in the patient group , as well as impairments on the theory of mind tasks and evidence of an externalizing attributional bias . although it was draws - to - decision that correlated most robustly with delusion proneness , these data prompted the authors to suggest that a common underlying mechanism might operate in sz to drive probabilistic reasoning and theory of mind deficits . they speculate that this could be a difficulty in inhibiting sensory input reflecting the immediate perceived reality , thus making patients more likely to be influenced by current data when making decisions on probabilistic reasoning tasks and making it harder to maintain an abstract viewpoint as required by theory - of - mind tasks . however , it should be noted that a large meta - analysis found no evidence for a link between theory of mind deficits and positive symptoms ; instead , deficits in theory of mind were correlated with negative symptoms , disorganization , and cognitive impairment.90 there is also some suggestion that poor emotion regulation might have a role to play . the anxiety induction procedure promoted delusions and jtc , and participants with higher psychosis vulnerability showed greater increases in paranoid delusional ideation . furthermore , jtc appeared to mediate the association between anxiety and delusions , prompting the authors to suggest that paranoid delusions result from an interaction of anxiety and reasoning biases . at present , it is hard to say which is best supported by the evidence available : this is possibly because these two explanations are by no means mutually exclusive . one difficulty in differentiating these explanations is that the urn task offers limited information regarding learning and decision - making processes . in one of our studies,92 we investigated performance in patients on a sequence - learning task ( sequences of four button presses were learnt using two buttons , feedback after each button press ) and the typical urn task . interestingly , learning from positive ( but not negative ) feedback in the sequence task correlated with draws - to - decision in the urn task : patients who showed a jtc response style were impaired in learning from positive feedback . in the next section , we discuss work aimed at elucidating the neural basis of jtc . various studies have attempted to induce the jtc bias in healthy controls using pharmacological manipulations ; others have used fmri . a drug model of jtc in healthy controls would be informative regarding the neurobiological underpinnings of jtc . ketamine infusions in healthy controls can induce behavioral and cognitive disturbances that are somewhat similar to the symptoms of sz,9395 infusions induce delusional thinking,96,97 and ketamine use has been linked to increased delusional symptoms in recreational users.98 importantly , ketamine infusions in patients with sz cause a worsening of symptoms.99101 ketamine has been shown to affect both glutamatergic and dopaminergic systems,102,103 potentially mimicking the aberrant dopaminergic transmission posited to underlie the jtc effect seen in sz ; ketamine also increases basal ganglia and thalamic activation in a manner similar to that observed in sz patients.104 a recent study by corlett et al105 administered ketamine to healthy controls to investigate whether faulty prediction error signals could underlie delusion formation in sz . prediction error is defined by the mismatch between expectations and experience , and it is probably represented by activity in the mesostriatal dopamine system.106 the prediction error signal in frontostriatal regions has been shown to correlate with delusion - like beliefs in healthy people107 and to be predictive of the severity of delusions in sz patients.108 disrupted prediction error signals could cause individuals to attend to and make associations with inappropriate stimuli ( both internal and external ) , consequently developing beliefs that do not reflect real - world contingencies,108 causing a jtc style of responding . however , another study failed to find a jtc response style during the urn task in healthy controls receiving ketamine . this suggests that although ketamine can induce delusions in healthy controls , delusion formation under ketamine might not be so closely linked to jtc as in sz,109 although possibly higher doses of ketamine might be required for jtc to manifest . l - dopa , had no effect on draws - to - decision , or confidence , whereas a dopamine antagonist ( haloperidol ) was found to reduce overconfidence but had no effect on draws - to - decision.110 likewise , dopaminergic modulation with methamphetamine does not affect draws - to - decision.111 in sum , attempts to promote jtc in controls using pharmacological interventions have been largely unsuccessful . studies in healthy controls implicate a distributed network of brain areas , including parietal cortex ( typically around intraparietal sulcus ) , prefrontal cortex ( typically dorsolateral ) , anterior insula , and striatum . these fmri studies use paradigms , including draw events ( stimuli for which participants choose to gather more information ) and urn events ( in which participants decide they have enough evidence and so select an urn ) . the most consistent finding across these block design studies are parietal responses near the intraparietal sulcus , which are larger for urn task blocks,112114 although some of these studies also report enhanced responses in the right dorsolateral prefrontal cortex113115 and anterior insula.113,115 one of these studies included a sz patient group and showed that the enhanced responses for the urn task blocks in the parietal and prefrontal cortices was reduced in these individuals , compared to the responses in healthy controls.113 this conclusion is tentative , however , as this study failed to replicate the classic jtc behavioral effect in sz . urn choices , compared to draw choices , activate anterior insula , striatum,115,116 acc , and parietal cortex , including the intraparietal sulcus.116 a near - identical network of brain areas has also been observed in the analogous contrast in the closely related best choice task.117 here , this network of areas was associated with deciding on an option currently available ( eg , a used car ) , compared to deciding to sample more possible options ( eg , viewing more cars ) . however , this between - participant variability was within a nonclinical sample , and it remains to be confirmed whether it also extends to clinical cases such as sz . involvement of areas such as striatum , dorsolateral prefrontal cortex , and intraparietal sulcus in deciding on a probabilistic inference rather than continued evidence seeking ( as in jtc ) is perhaps not surprising . for example , measures of evidence accumulation for perceptual decisions have been associated with fmri responses in the prefrontal cortex of psychiatrically healthy participants118 and in neural recordings from the striatum119 and intraparietal sulcus in monkeys.120 measures of evidence accumulation for economic decisions in healthy participants are also associated with fmri responses in dorsolateral prefrontal cortex , striatum , and intraparietal sulcus.121 prefrontal cortex lesions are also associated with jtc.122 we hypothesize that this network of areas , which contributes to the urn task and other decision - making tasks in healthy participants , may be compromised in sz patients who show jtc behavior . the striatum , for instance , signals errors in reward prediction and predicts successful reward learning in healthy participants123 but shows aberrant responses during reward prediction tasks in schizophrenic individuals.124,125 aberrant prediction - related signaling in the dopaminergic striatum might also play a role in jtc behavior , as dopaminergic antipsychotic treatment abolishes jtc on an emotionally salient version of the urn task.64 in addition to the striatum , responses in the dorsolateral prefrontal cortex are also reduced in schizophrenics , compared to those in healthy individuals , when performing tasks requiring reward prediction.126 in sum , schizophrenic individuals appear to show reward - related response reductions in many of the brain areas activated by the urn task in healthy participants . more brain - imaging studies using sz patients and the urn task are required to test this hypothesis . a drug model of jtc in healthy controls would be informative regarding the neurobiological underpinnings of jtc . ketamine infusions in healthy controls can induce behavioral and cognitive disturbances that are somewhat similar to the symptoms of sz,9395 infusions induce delusional thinking,96,97 and ketamine use has been linked to increased delusional symptoms in recreational users.98 importantly , ketamine infusions in patients with sz cause a worsening of symptoms.99101 ketamine has been shown to affect both glutamatergic and dopaminergic systems,102,103 potentially mimicking the aberrant dopaminergic transmission posited to underlie the jtc effect seen in sz ; ketamine also increases basal ganglia and thalamic activation in a manner similar to that observed in sz patients.104 a recent study by corlett et al105 administered ketamine to healthy controls to investigate whether faulty prediction error signals could underlie delusion formation in sz . prediction error is defined by the mismatch between expectations and experience , and it is probably represented by activity in the mesostriatal dopamine system.106 the prediction error signal in frontostriatal regions has been shown to correlate with delusion - like beliefs in healthy people107 and to be predictive of the severity of delusions in sz patients.108 disrupted prediction error signals could cause individuals to attend to and make associations with inappropriate stimuli ( both internal and external ) , consequently developing beliefs that do not reflect real - world contingencies,108 causing a jtc style of responding . this suggests that although ketamine can induce delusions in healthy controls , delusion formation under ketamine might not be so closely linked to jtc as in sz,109 although possibly higher doses of ketamine might be required for jtc to manifest . l - dopa , had no effect on draws - to - decision , or confidence , whereas a dopamine antagonist ( haloperidol ) was found to reduce overconfidence but had no effect on draws - to - decision.110 likewise , dopaminergic modulation with methamphetamine does not affect draws - to - decision.111 in sum , attempts to promote jtc in controls using pharmacological interventions have been largely unsuccessful . studies in healthy controls implicate a distributed network of brain areas , including parietal cortex ( typically around intraparietal sulcus ) , prefrontal cortex ( typically dorsolateral ) , anterior insula , and striatum . these fmri studies use paradigms , including draw events ( stimuli for which participants choose to gather more information ) and urn events ( in which participants decide they have enough evidence and so select an urn ) . the most consistent finding across these block design studies are parietal responses near the intraparietal sulcus , which are larger for urn task blocks,112114 although some of these studies also report enhanced responses in the right dorsolateral prefrontal cortex113115 and anterior insula.113,115 one of these studies included a sz patient group and showed that the enhanced responses for the urn task blocks in the parietal and prefrontal cortices was reduced in these individuals , compared to the responses in healthy controls.113 this conclusion is tentative , however , as this study failed to replicate the classic jtc behavioral effect in sz . urn choices , compared to draw choices , activate anterior insula , striatum,115,116 acc , and parietal cortex , including the intraparietal sulcus.116 a near - identical network of brain areas has also been observed in the analogous contrast in the closely related best choice task.117 here , this network of areas was associated with deciding on an option currently available ( eg , a used car ) , compared to deciding to sample more possible options ( eg , viewing more cars ) . however , this between - participant variability was within a nonclinical sample , and it remains to be confirmed whether it also extends to clinical cases such as sz . involvement of areas such as striatum , dorsolateral prefrontal cortex , and intraparietal sulcus in deciding on a probabilistic inference rather than continued evidence seeking ( as in jtc ) is perhaps not surprising . for example , measures of evidence accumulation for perceptual decisions have been associated with fmri responses in the prefrontal cortex of psychiatrically healthy participants118 and in neural recordings from the striatum119 and intraparietal sulcus in monkeys.120 measures of evidence accumulation for economic decisions in healthy participants are also associated with fmri responses in dorsolateral prefrontal cortex , striatum , and intraparietal sulcus.121 prefrontal cortex lesions are also associated with jtc.122 we hypothesize that this network of areas , which contributes to the urn task and other decision - making tasks in healthy participants , may be compromised in sz patients who show jtc behavior . the striatum , for instance , signals errors in reward prediction and predicts successful reward learning in healthy participants123 but shows aberrant responses during reward prediction tasks in schizophrenic individuals.124,125 aberrant prediction - related signaling in the dopaminergic striatum might also play a role in jtc behavior , as dopaminergic antipsychotic treatment abolishes jtc on an emotionally salient version of the urn task.64 in addition to the striatum , responses in the dorsolateral prefrontal cortex are also reduced in schizophrenics , compared to those in healthy individuals , when performing tasks requiring reward prediction.126 in sum , schizophrenic individuals appear to show reward - related response reductions in many of the brain areas activated by the urn task in healthy participants . more brain - imaging studies using sz patients and the urn task are required to test this hypothesis . using an emotionally salient version of the urn task , menon et al64 found that within 2 weeks of initiating treatment with antipsychotics , patients demonstrated an increase in the number of trials - to - decision alongside attenuation of psychotic symptoms and delusions . this was not the case for positive symptoms or neuropsychological performance , suggesting that jtc might act independently to influence real - world functioning.127 it is important to note that most studies have failed to show improvements in jtc on the standard urn task following antipsychotic treatment,64,128 although there is some evidence to dispute this.63,65 if jtc does indeed fluctuate with delusional symptoms , it would provide strong evidence of its importance as a treatment target : interventions that specifically target cognitive biases ( known as metacognitive training programs ) have already been shown to improve delusions and other positive symptoms.129,130 it is also important to note that most studies typically detect jtc in only approximately 50% of their patient samples . training programs that aim to ameliorate the jtc response style might prove to be an important adjunct to established therapies . in sum , jtc in sz seems to be a consistent finding and there is strong evidence linking jtc to delusion formation . both liberal acceptance and hypersalience accounts of jtc are well supported by the literature , but attempts to replicate jtc in healthy controls using pharmacological manipulations have largely failed , undermining attempts to develop a neurobiological account of jtc . fmri studies have implicated a network involved in making urn choices , which includes striatal , insula , parietal , and prefrontal areas ; further patient work is required , particularly in the context of evidence suggesting that jtc could represent a valuable therapeutic target .

there is some evidence to suggest that working memory might be impaired in patients who show jtc compared to those who do not . in addition , in sz patients with strong current delusions , working memory ( but not premorbid iq ) was worse in those who demonstrated jtc.70 as noted above , broome et al67 found that jtc in the prodrome was linked to working memory impairments . nevertheless , the presence of a memory aid during the urn task does not affect jtc in patient groups.51,53 this would appear to undermine any suggestion that jtc stems from a relative inability to maintain the task items in memory . although sz patients made a less systematic information search and were more likely to consider less valid information , they did not inspect fewer pieces of information compared to healthy controls and thus did not demonstrate jtc . effects were found on confidence ratings however , with patients more likely to be overconfident , using extreme confidence ratings under inappropriate circumstances . this is consistent with work showing that sz patients are less confident of their correct answers and overconfident when they make errors , during word recall tasks.72,73 although there were no overall differences between patients and controls , symptomatology in the patient group was linked to information search . a correlation was observed between symptom severity scores ( positive and negative syndrome scale [ panss ] positive , panss delusion ) and the degree of information search , with higher - scoring participants tending to gather less pieces of information . panss score did not predict use of extreme confidence ratings , but nevertheless this study would suggest that jtc can be largely abolished and can manifest as overconfidence only when information is presented in the right manner . impulsiveness is unlikely to be a factor because patients with sz show similar reaction times as healthy controls51,53 and draw more beads when the task is made harder.51,55,74 motivational factors are also unlikely to play a part : the possibility that the patients overestimate the cost of gathering more information ( possibly due to a greater need for closure ) has been discounted because patients do not seem to experience a greater cost for gathering more information.75 an alternative explanation is that jtc manifests not through a lowered threshold for making a decision but through each piece of evidence being relatively overvalued . when asked to report belief estimates after each draw , it has been shown that patients make more drastic updates after each piece of evidence.46,56,77 speechley et al78 found that delusional patients , when asked to give likelihood ratings for each urn on each trial , gave higher ratings for whichever urn matched the current evidence , while ratings for the nonmatching urn did not differ from those of the control groups . the authors argue that this provides evidence of a reasoning bias characterized by hypersalience of evidence that matches a hypothesis , but with reasoning that appears intact for nonmatches . because delusions are maintained in the face of contradictory evidence , it is unsurprising that patients tend to show a bias against disconfirmatory evidence.79 it has been argued that hypersalience could underlie this effect : hypersalience of evidence hypothesis matches may lead to an enhancement of weak matches , leading to difficulties in integrating disambiguating information.80 some studies point to hypersalience of disconfirmatory , as well as confirmatory , evidence : deluded patients show a tendency to overadjust when presented with potentially disconfirmatory evidence,53,56 although this effect might be linked to miscomprehension of the task,81 especially because such a tendency would contradict the evidence of reversal - learning deficits discussed in the previous section . these findings are consistent with an aberrant salience account of sz,82 whereby dysregulated dopamine transmission generates context - inappropriate salience attributions , potentially due to aberrant signaling in the ventral striatal dopaminergic pathway , which is thought to regulate stimulus response pairings.83 moore and sellen84 built a simple network model in which the gain , or signal - to - noise , parameter ( which describes the likelihood of a node firing when presented with some input ) was varied . the gain parameter was assumed to represent striatal dopaminergic activity , and increasing this parameter meant that the model successfully mimicked the jtc response style seen in research data from delusional patients . this might be overly simplistic however , because imaging studies have shown that the striatum is downregulated in arms85 and sz patients.86 nevertheless , it is important to note that evidence for hypersalience has been observed across various other cognitive biases present in sz8789 and as such represents a convincing account of jtc . as mentioned previously , another suggestion concerning the development of delusions in sz focuses on impairments in theory of mind.42 langdon et al77 compared 35 sz patients with a history of delusions to healthy controls , on a battery of tasks that included two versions of the urn task , three theory - of - mind tasks , and a questionnaire on attributional biases . a jtc response style was found in the patient group , as well as impairments on the theory of mind tasks and evidence of an externalizing attributional bias . performance on the urn tasks correlated with that of the theory - of - mind tasks , while attributional bias scores did not correlate with other task measures . delusion proneness ( measured by the questionnaire ) correlated with probabilistic reasoning and theory - of - mind measures , while externalizing bias did not ; iq and memory ability were accounted for . although it was draws - to - decision that correlated most robustly with delusion proneness , these data prompted the authors to suggest that a common underlying mechanism might operate in sz to drive probabilistic reasoning and theory of mind deficits . they speculate that this could be a difficulty in inhibiting sensory input reflecting the immediate perceived reality , thus making patients more likely to be influenced by current data when making decisions on probabilistic reasoning tasks and making it harder to maintain an abstract viewpoint as required by theory - of - mind tasks . however , it should be noted that a large meta - analysis found no evidence for a link between theory of mind deficits and positive symptoms ; instead , deficits in theory of mind were correlated with negative symptoms , disorganization , and cognitive impairment.90 there is also some suggestion that poor emotion regulation might have a role to play . in one study , 90 healthy individuals with varying levels of psychosis vulnerability ( assessed by the community assessment of psychic experiences ) were recruited.74 half the sample received an anxiety induction procedure , the other half did not . furthermore , jtc appeared to mediate the association between anxiety and delusions , prompting the authors to suggest that paranoid delusions result from an interaction of anxiety and reasoning biases . in the glckner and moritz91 study , it was found that increasing stress ( through time pressure and the addition of affective valence ) led to diminished performance in patients . thus , improved self - monitoring in terms of better emotion regulation , combined with metacognitive training targeted at reasoning biases , could be beneficial in reducing delusion formation in sz . in one of our studies,92 we investigated performance in patients on a sequence - learning task ( sequences of four button presses were learnt using two buttons , feedback after each button press ) and the typical urn task . interestingly , learning from positive ( but not negative ) feedback in the sequence task correlated with draws - to - decision in the urn task : patients who showed a jtc response style were impaired in learning from positive feedback . thus , these findings clearly favor a liberal acceptance account over hypersalience , but further work is needed to distinguish liberal acceptance and hypersalience , as well as to explore potential interactions with emotion regulation and other self - monitoring activities . in the next section , we discuss work aimed at elucidating the neural basis of jtc . there is some evidence that the noncompetitive n - methyl - d - aspartate ( nmda ) receptor antagonist ketamine could serve as such a model . ketamine infusions in healthy controls can induce behavioral and cognitive disturbances that are somewhat similar to the symptoms of sz,9395 infusions induce delusional thinking,96,97 and ketamine use has been linked to increased delusional symptoms in recreational users.98 importantly , ketamine infusions in patients with sz cause a worsening of symptoms.99101 ketamine has been shown to affect both glutamatergic and dopaminergic systems,102,103 potentially mimicking the aberrant dopaminergic transmission posited to underlie the jtc effect seen in sz ; ketamine also increases basal ganglia and thalamic activation in a manner similar to that observed in sz patients.104 a recent study by corlett et al105 administered ketamine to healthy controls to investigate whether faulty prediction error signals could underlie delusion formation in sz . prediction error is defined by the mismatch between expectations and experience , and it is probably represented by activity in the mesostriatal dopamine system.106 the prediction error signal in frontostriatal regions has been shown to correlate with delusion - like beliefs in healthy people107 and to be predictive of the severity of delusions in sz patients.108 disrupted prediction error signals could cause individuals to attend to and make associations with inappropriate stimuli ( both internal and external ) , consequently developing beliefs that do not reflect real - world contingencies,108 causing a jtc style of responding . interestingly , work investigating the effects of dopamine agonists and antagonists in healthy controls has also failed to induce jtc , suggesting that straightforward modulation of the dopaminergic system might not be sufficient to reproduce the jtc response style seen in sz . l - dopa , had no effect on draws - to - decision , or confidence , whereas a dopamine antagonist ( haloperidol ) was found to reduce overconfidence but had no effect on draws - to - decision.110 likewise , dopaminergic modulation with methamphetamine does not affect draws - to - decision.111 in sum , attempts to promote jtc in controls using pharmacological interventions have been largely unsuccessful . thus , it would seem that jtc can not be attributed solely to general dopaminergic overactivity , at least when induced acutely : the relationship might not be linear , or perhaps chronic aberrations in dopamine firing is required for jtc to manifest . fmri can potentially indicate the neural mechanisms contributing to probabilistic reasoning in the urn task . studies in healthy controls implicate a distributed network of brain areas , including parietal cortex ( typically around intraparietal sulcus ) , prefrontal cortex ( typically dorsolateral ) , anterior insula , and striatum . the most consistent finding across these block design studies are parietal responses near the intraparietal sulcus , which are larger for urn task blocks,112114 although some of these studies also report enhanced responses in the right dorsolateral prefrontal cortex113115 and anterior insula.113,115 one of these studies included a sz patient group and showed that the enhanced responses for the urn task blocks in the parietal and prefrontal cortices was reduced in these individuals , compared to the responses in healthy controls.113 this conclusion is tentative , however , as this study failed to replicate the classic jtc behavioral effect in sz . urn choices , compared to draw choices , activate anterior insula , striatum,115,116 acc , and parietal cortex , including the intraparietal sulcus.116 a near - identical network of brain areas has also been observed in the analogous contrast in the closely related best choice task.117 here , this network of areas was associated with deciding on an option currently available ( eg , a used car ) , compared to deciding to sample more possible options ( eg , viewing more cars ) . parietal cortex was more responsive during urn decisions for participants who tended to draw more and for participants who made greater adjustments to their draws - to - decision depending on prevalence of the minority bead color . involvement of areas such as striatum , dorsolateral prefrontal cortex , and intraparietal sulcus in deciding on a probabilistic inference rather than continued evidence seeking ( as in jtc ) is perhaps not surprising . for example , measures of evidence accumulation for perceptual decisions have been associated with fmri responses in the prefrontal cortex of psychiatrically healthy participants118 and in neural recordings from the striatum119 and intraparietal sulcus in monkeys.120 measures of evidence accumulation for economic decisions in healthy participants are also associated with fmri responses in dorsolateral prefrontal cortex , striatum , and intraparietal sulcus.121 prefrontal cortex lesions are also associated with jtc.122 we hypothesize that this network of areas , which contributes to the urn task and other decision - making tasks in healthy participants , may be compromised in sz patients who show jtc behavior . the striatum , for instance , signals errors in reward prediction and predicts successful reward learning in healthy participants123 but shows aberrant responses during reward prediction tasks in schizophrenic individuals.124,125 aberrant prediction - related signaling in the dopaminergic striatum might also play a role in jtc behavior , as dopaminergic antipsychotic treatment abolishes jtc on an emotionally salient version of the urn task.64 in addition to the striatum , responses in the dorsolateral prefrontal cortex are also reduced in schizophrenics , compared to those in healthy individuals , when performing tasks requiring reward prediction.126 in sum , schizophrenic individuals appear to show reward - related response reductions in many of the brain areas activated by the urn task in healthy participants . there is some evidence that the noncompetitive n - methyl - d - aspartate ( nmda ) receptor antagonist ketamine could serve as such a model . ketamine infusions in healthy controls can induce behavioral and cognitive disturbances that are somewhat similar to the symptoms of sz,9395 infusions induce delusional thinking,96,97 and ketamine use has been linked to increased delusional symptoms in recreational users.98 importantly , ketamine infusions in patients with sz cause a worsening of symptoms.99101 ketamine has been shown to affect both glutamatergic and dopaminergic systems,102,103 potentially mimicking the aberrant dopaminergic transmission posited to underlie the jtc effect seen in sz ; ketamine also increases basal ganglia and thalamic activation in a manner similar to that observed in sz patients.104 a recent study by corlett et al105 administered ketamine to healthy controls to investigate whether faulty prediction error signals could underlie delusion formation in sz . prediction error is defined by the mismatch between expectations and experience , and it is probably represented by activity in the mesostriatal dopamine system.106 the prediction error signal in frontostriatal regions has been shown to correlate with delusion - like beliefs in healthy people107 and to be predictive of the severity of delusions in sz patients.108 disrupted prediction error signals could cause individuals to attend to and make associations with inappropriate stimuli ( both internal and external ) , consequently developing beliefs that do not reflect real - world contingencies,108 causing a jtc style of responding . interestingly , work investigating the effects of dopamine agonists and antagonists in healthy controls has also failed to induce jtc , suggesting that straightforward modulation of the dopaminergic system might not be sufficient to reproduce the jtc response style seen in sz . l - dopa , had no effect on draws - to - decision , or confidence , whereas a dopamine antagonist ( haloperidol ) was found to reduce overconfidence but had no effect on draws - to - decision.110 likewise , dopaminergic modulation with methamphetamine does not affect draws - to - decision.111 in sum , attempts to promote jtc in controls using pharmacological interventions have been largely unsuccessful . thus , it would seem that jtc can not be attributed solely to general dopaminergic overactivity , at least when induced acutely : the relationship might not be linear , or perhaps chronic aberrations in dopamine firing is required for jtc to manifest . the most consistent finding across these block design studies are parietal responses near the intraparietal sulcus , which are larger for urn task blocks,112114 although some of these studies also report enhanced responses in the right dorsolateral prefrontal cortex113115 and anterior insula.113,115 one of these studies included a sz patient group and showed that the enhanced responses for the urn task blocks in the parietal and prefrontal cortices was reduced in these individuals , compared to the responses in healthy controls.113 this conclusion is tentative , however , as this study failed to replicate the classic jtc behavioral effect in sz . urn choices , compared to draw choices , activate anterior insula , striatum,115,116 acc , and parietal cortex , including the intraparietal sulcus.116 a near - identical network of brain areas has also been observed in the analogous contrast in the closely related best choice task.117 here , this network of areas was associated with deciding on an option currently available ( eg , a used car ) , compared to deciding to sample more possible options ( eg , viewing more cars ) . parietal cortex was more responsive during urn decisions for participants who tended to draw more and for participants who made greater adjustments to their draws - to - decision depending on prevalence of the minority bead color . for example , measures of evidence accumulation for perceptual decisions have been associated with fmri responses in the prefrontal cortex of psychiatrically healthy participants118 and in neural recordings from the striatum119 and intraparietal sulcus in monkeys.120 measures of evidence accumulation for economic decisions in healthy participants are also associated with fmri responses in dorsolateral prefrontal cortex , striatum , and intraparietal sulcus.121 prefrontal cortex lesions are also associated with jtc.122 we hypothesize that this network of areas , which contributes to the urn task and other decision - making tasks in healthy participants , may be compromised in sz patients who show jtc behavior . the striatum , for instance , signals errors in reward prediction and predicts successful reward learning in healthy participants123 but shows aberrant responses during reward prediction tasks in schizophrenic individuals.124,125 aberrant prediction - related signaling in the dopaminergic striatum might also play a role in jtc behavior , as dopaminergic antipsychotic treatment abolishes jtc on an emotionally salient version of the urn task.64 in addition to the striatum , responses in the dorsolateral prefrontal cortex are also reduced in schizophrenics , compared to those in healthy individuals , when performing tasks requiring reward prediction.126 in sum , schizophrenic individuals appear to show reward - related response reductions in many of the brain areas activated by the urn task in healthy participants . using an emotionally salient version of the urn task , menon et al64 found that within 2 weeks of initiating treatment with antipsychotics , patients demonstrated an increase in the number of trials - to - decision alongside attenuation of psychotic symptoms and delusions . although these measures were not correlated , baseline performance on the task had some predictive power over the individuals who would show improvements in symptomology in response to medication : jtc performance at baseline could therefore be useful in guiding treatment . this was not the case for positive symptoms or neuropsychological performance , suggesting that jtc might act independently to influence real - world functioning.127 it is important to note that most studies have failed to show improvements in jtc on the standard urn task following antipsychotic treatment,64,128 although there is some evidence to dispute this.63,65 if jtc does indeed fluctuate with delusional symptoms , it would provide strong evidence of its importance as a treatment target : interventions that specifically target cognitive biases ( known as metacognitive training programs ) have already been shown to improve delusions and other positive symptoms.129,130 it is also important to note that most studies typically detect jtc in only approximately 50% of their patient samples . fmri studies have implicated a network involved in making urn choices , which includes striatal , insula , parietal , and prefrontal areas ; further patient work is required , particularly in the context of evidence suggesting that jtc could represent a valuable therapeutic target .

we identified key actors by applying a snowball method starting with key actors in our own networks . because the netherlands is a relatively small country , this was a feasible method of reaching sufficient people . the interviews were conducted by two researchers who had not published any research on threatening communication ( gjp and kees de jong ) to minimize socially desirable answers based on familiarity with an interviewer 's work . specifically , we interviewed 18 intervention developers and two administrators at health promoting service providers , four scientists , three policymakers , three politicians , and three advertising professionals . five participants worked at regional hsps ( e.g. , municipal health services ) ; the remaining 14 worked at national hsps , each addressing one or several behavioral themes such as smoking , safe sex , exercise , substance use , and diet patterns . all four scientists worked at dutch universities , specifically in the fields of medicine , business communication , epidemiology , and risk communication . of the policymakers , one worked at a dutch ministry ; one at a national funding organization ; one at a national hsp ; and one at a regional hsp . of the politicians , one was active in national politics as a member of parliament , and the other two in regional politics as municipal counselors . the advertising agency employees all worked at advertising agencies that had developed health promotion campaigns for national hsps . the interview protocol ( see http://sciencerep.org/5 ) started with gathering information on the background of each participant . after that , questions addressed participants ' experience with intervention development in general ; what information participants gathered before making decisions about interventions ; how certain choices were made ; and what were particularly good or bad interventions in participants ' opinion . specifically , the interviews focused on interventions with behavior change as a goal in general ( no specific interventions participants had developed ) . during these conversations , when threatening communication would come up , this issue would be probed in more depth . to ensure that threatening communication would be discussed in all interviews , a number of examples of interventions were included for the interviewee to judge , two of which used threatening communication ( a picture of blackened lungs on a pack of cigarettes and a speeding intervention ; see http://sciencerep.org/5 for these pictures ) . we chose this approach to let threatening communication come up in the interview naturally , which decreased the possibility of defensive reactions . we anticipated defensive reactions when intervention designers would be confronted directly with the question of why they use threatening communication . the interviews were recorded , transcribed , and imported into qsr nvivo , where they were coded by gjp . first , all interview fragments where threatening communication was discussed were coded in the same fear appeal category . then these fragments were reread and coded with more specific categories ( see results section ) . the second coding sweep yielded a number of systematically occurring beliefs , which were categorized in a tree structure with the following main categories : reasons for fear appeal use ; reasons against fear appeal use ; the process of intervention development ; related beliefs ( not otherwise categorized ) ; and environmental constraints . within some categories , subcategories were formed , such as within the process of intervention development category , the subcategories goals of intervention development and target group involvement . the beliefs identified in these categories and subcategories were then organized in a narrative in four sections , each describing largely coherent sets of beliefs . all participants knew a lot about how to develop effective interventions . in this paper , however , we focus on beliefs that were inconsistent with the scientific state of the art . that narrow focus may cause these results to paint a bleak picture , but this does not mean that the interviewed participants were ignorant or incompetent in terms of intervention development . yet there is always room for improvement , and this paper aims to facilitate such improvement . inducing fear was cited relatively rarely as a reason to use threatening communication . more often , the goal was to confront people with the negative consequences of a given risky behavior . the most common argument for this need for confrontation was rooted in the fact that attention is a first necessary condition for further processing , and attention is a limited resource . given that target population members are exposed simultaneously to multiple stimuli , behavior change interventions need to compete for attention . evoking strong emotions the need to break through and draw attention was often reported : of course , we do n't have as much media budget as a commercial organization . you want to draw attention , you do n't want to shock , i do n't think that 's the right word , but you do want to do something that reaches people . so you want to hit a certain emotion , and i think that we managed with our campaign . because many people saw it . ( intervention developer , 191110 ) of course , we do n't have as much media budget as a commercial organization . you want to draw attention , you do n't want to shock , i do n't think that 's the right word , but you do want to do something that reaches people . so you want to hit a certain emotion , and i think that we managed with our campaign . because many people saw it . ( intervention developer , 191110 ) emotion was also assumed to render interventions memorable , which , combined with the memory of the evoked emotion itself , was assumed to prompt desirable behavior : and why do you have to strike an emotion ? yes , again , people get to see so many communications in a day , that if you want to strike somebody at all , then especially that emotion is important . because only by letting people really feel something , you can prompt people to act . so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . or maybe even extra this year . oh , yes , i saw that commercial , that struck me personally . then that works . ( intervention developer , 191110 ) and why do you have to strike an emotion ? yes , again , people get to see so many communications in a day , that if you want to strike somebody at all , then especially that emotion is important . because only by letting people really feel something , you can prompt people to act . so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . or maybe even extra this year . oh , yes , i saw that commercial , that struck me personally . then that works . ( intervention developer , 191110 ) a second example of this reasoning was provided by a participant from the advertising world : ( discussing the first prompting intervention , see http://sciencerep.org/5 ) what do you think ? is this a good intervention , or not ? i think that in this case , you want to address a specific target group , and for that , i think it 's good . i think that in that case you do n't have to bother with a boring communication indeed , i think that if you want to address a certain target group , with a lot of sexual contacts , it 's fine . before you know it , you have it , so be wise . becoming aware of easily contracting an sti and continuing your old behavior does n't seem useful . ( advertising professional , 51183 ) ( discussing the first prompting intervention , see http://sciencerep.org/5 ) what do you think ? i think that in this case , you want to address a specific target group , and for that , i think it 's good . i think that in that case you do n't have to bother with a boring communication indeed , i think that if you want to address a certain target group , with a lot of sexual contacts , it 's fine . before you know it , you have it , so be wise . becoming aware of easily contracting an sti and continuing your old behavior does n't seem useful . ( advertising professional , 51183 ) another common belief was that when a communication manages to evoke emotions in target population members , this would cause reflection about the relevant behavior : for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . so one picture of a child who had not consumed alcohol , and another scan of a child who had consumed alcohol . and showed what it does to your brains . well , they showed that at the meeting , and then people could ask questions . and then i thought , i think that works , because people then start thinking . ( politician , 191115 ) for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . so one picture of a child who had not consumed alcohol , and another scan of a child who had consumed alcohol . and showed what it does to your brains . well , they showed that at the meeting , and then people could ask questions . and then i thought , i think that works , because people then start thinking . ( politician , 191115 ) this belief was based on the assumption that target population members act and reason rationally ( i.e. consistent with a subjective expected utility model ; see e.g. tversky & kahneman , 1981 ) . a related and possibly derived belief was participants ' presumption that their own common - sense reasoning about how communications would be received and processed further would resemble reality . for example , one scientist explained : i would , if i would read , like , it gives you lung cancer ( scientist , 4119 ) i would , if i would read , like , it gives you lung cancer ( scientist , 4119 ) also related , achieving awareness ( or increasing risk perception to a certain threshold ) was often considered a sufficient condition for behavior change : you try to increase this motivation to quit . so the more you make them aware of the fact that it 's bad , the stronger your motivation becomes with respect to your addiction . so the more you make them aware of the fact that it 's bad , the stronger your motivation becomes with respect to your addiction . ( advertising professional , 22114 ) to participants who assumed that target population members who perform the undesirable behavior are ignorant as to the negative consequences , showing these negative consequences was considered necessary education . similarly , target population members were generally assumed to underestimate the negative consequences of the behavior . thus , participants assumed that generally even target population members who are aware of a causal link between a behavior and a negative consequence would under - estimate the severity of the negative consequence . intervention developers had often heard negative messages about threatening communication , but did not usually understand the dynamics at play . in general , it could be said that interviewees seemed to have some pieces of the puzzle , but lacked a coherent framework of the working mechanisms of threatening communication . this sometimes manifested as ambivalence regarding the effectiveness of threatening communication : yes , but risk communication does not work with this population ? yes , i think that this [ pictures on cigarette packs ] is a good method , but i also think that there 's no other way than this [ to change behavior ] . ( scientist , 4119 ) yes , but risk communication does not work with this population ? yes , i think that this [ pictures on cigarette packs ] is a good method , but i also think that there 's no other way than this [ to change behavior ] . ( scientist , 4119 ) when logical arguments against the use of threatening communication were acknowledged , this ineffectiveness was sometimes attributed to specific characteristics of the situation at hand , often the nature of the behavior at hand ( e.g. , smoking is a difficult behavior ) . it was not uncommon for participants to express beliefs that in terms of the eppm were logically inconsistent . for example , participants could state that they thought that low susceptibility , rather than low severity , was the main cause of unhealthy behavior ; and several minutes later explain the importance of emphasizing the grave consequences of unhealthy behavior . one participant explained that they tried to solve the problem of low susceptibility in their target population by tailoring an intervention , enabling them to communicate that target population members belonged to a subpopulation that was at a relatively high risk . while this reasoning is partly in line with eppm , for example , one participant remarked : yes , this [ picture of blackened lungs ] is ok , because you can do something about it by quitting smoking , so , um , i mean , if you would say , ( advertising professional , 22114 ) yes , this [ picture of blackened lungs ] is ok , because you can do something about it by quitting smoking , so , um , i mean , if you would say , breathing causes lung cancer [ that would be a different story ] . ( advertising professional , 22114 ) related to this , participants sometimes were aware of the theoretical constraint that threatening information would not work unless efficacy was high , but then often underestimated the required intensity of an intervention to enhance efficacy . in fact , this was sometimes reported as a necessary ingredient of interventions using threatening communication . scientists were often cognizant of the eppm but , like other participants , did not always realize that the threatening stimulus in this model can be any stimulus . some classes of intervention types were considered to not be fear - inducing ( i.e. , carry the risk of fear control responses ) , such as scenario messages or personalized risk assessments . similarly , often participants indicated that they did not use threatening communication , while from their explanation it became clear that they did : [ discussing a campaign against smoking that would focus on cancer ] some people may call this a fear inducing message i do n't call this fear inducing , because question one is , what is fear exactly and what is fear inducing ? i consider it more of an awareness campaign . because people often say , well , the risk is not so bad , and well , cancer it is more of a confronting campaign , where the health effects you can expect from smoking regarding cancer are made clear , visually . ( policymaker , 26114 ) [ discussing a campaign against smoking that would focus on cancer ] some people may call this a fear inducing message i do n't call this fear inducing , because question one is , what is fear exactly and what is fear inducing ? i consider it more of an awareness campaign . because people often say , well , the risk is not so bad , and well , cancer it is more of a confronting campaign , where the health effects you can expect from smoking regarding cancer are made clear , visually . in fact , few participants were aware of the conceptual distinction between personal determinants ( psychological variables that can be influenced to eventually engender behavior change , such as risk perception or self - efficacy ) , behavior change methods or techniques ( theoretical methods to influence a given determinant , such as fear appeals or modeling ) , and applications of these methods ( an incarnation of a theoretical method , such as a picture of blackened lungs or a movie with a role model ) . one participant who was partially aware of this distinction explained : but then the step from those modifiable determinants to good methods to change that , kind of , that is often difficult . what are effective methods to achieve those goals and how do you utilize these ? often , the time and knowledge to do this thoroughly is lacking . ( intervention developer , 22612 ) but then the step from those modifiable determinants to good methods to change that , kind of , that is often difficult . what are effective methods to achieve those goals and how do you utilize these ? often , the time and knowledge to do this thoroughly is lacking . ( intervention developer , 22612 ) this lack of knowledge about methods for behavior change was one of the reasons for the use of threatening communication : there were no known good alternatives . intervention developers often did not have an array of behavior change methods at their disposal , and sometimes they applied the same method in all interventions . some other participants had naive ideas of behavior change methods that carried a high risk of yielding non evidence - based results . specifically , some participants indicated that it was important that an intervention originated from within the target population . however , these target population members generally advocated threatening communication approaches : yes , or , they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign . , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . ( intervention developer , 21210 ) yes , or , they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign yes , no , it has to be more serious . for example , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . ( intervention developer , 21210 ) because participants often assumed that target population members knew how they themselves could be influenced , this reliance on target population members ' preferences for threatening communication could easily result in the development of threatening interventions . among employees of advertising agencies and policymakers , the belief existed that there are no ways to predict in advance whether a campaign would be successful : and can you estimate a bit , when you 've made many campaigns , whether campaigns work or not ? regardless of indicators like market share , on the basis of your experience ? yes , that is very hard to say . because sometimes you 're surprised by campaigns that turn out to work very well , while you think , this has to be great , but it does n't work , or a customer gives it insufficient chance . that is , um , when you would be able to determine this in advance , you would have found the holy grail . ( advertising professional , 9416 ) and can you estimate a bit , when you 've made many campaigns , whether campaigns work or not ? regardless of indicators like market share , on the basis of your experience ? yes , that is very hard to say . because sometimes you 're surprised by campaigns that turn out to work very well , while you think , or campaigns of which you think , this has to be great , but it does n't work , or a customer gives it insufficient chance . that is , um , when you would be able to determine this in advance , you would have found the holy grail . ( advertising professional , 9416 ) intervention development was considered largely a trial - and - error endeavor , with the development process often being seen as a black box ( in advertising , a creative process engaged in by the creatives ) . yet , at the same time , in advertising , there were some basic guidelines on campaign development . for example , one interviewee explained that a commercial for a product such as soup should always visually show both the soup and the packaging . another such heuristic was the merit of creativity or originality : you can do something very strange and very new , but if you do it a second or a third time , then it 's already not surprising any more . i think that in the end clients choose to be creative within a given campaign , with a given idea . and ( advertising professional , 9416 ) you can do something very strange and very new , but if you do it a second or a third time , then it 's already not surprising any more . i think that in the end clients choose to be creative within a given campaign , with a given idea . and ( advertising professional , 9416 ) among participants from advertising agencies , sometimes confrontation and humor were two options to choose from when trying to reach people . when confrontation was considered inappropriate , humor would be the alternative approach : well , i think that you choose humor at a time you actually want to point something out to people , but you do n't want to pedantically wag your finger . and if you do it in a humorous way then it does come across differently to people than when you say , eh , you 're not allowed to do this , or you have to do that . yes . that you then mainly choose humor . so that it does come across , but that people do n't think , ( advertising professional , 22111 ) well , i think that you choose humor at a time you actually want to point something out to people , but you do n't want to pedantically wag your finger . and if you do it in a humorous way then it does come across differently to people than when you say , eh , you 're not allowed to do this , or you have to do that . yes . that you then mainly choose humor . so that it does come across , but that people do n't think , ( advertising professional , 22111 ) beliefs in advertising agencies about behavior change methods proved relevant because intervention developers sometimes trusted advertising agencies to know how to influence target population members . at the same time , when advertising agencies delivered a product or presented an intermediate step that intervention developers judged to be unacceptable ( i.e. , not in line with known goals or constraints ) , intervention developers would generally communicate this and demand adjustments . participants generally conceded that evaluating whether an intervention had successfully induced behavior change was complicated . understandably , besides the scientists , very few participants mentioned the possibility of examining such evaluations in a laboratory setting . instead one such indicator was the degree to which an intervention was recognized or remembered , or the perceived impact as reported by introspection of target population members : so how do you want to verify whether goals you set for an intervention , whether those are met after some time ? well , for now that is mainly in narrative form , it 's mainly about statements of people , to go start and maintain a dialogue with young people and teachers and other involved people , like , well , did it touch you , did it do something for you ? ( intervention developer , 18314 ) so how do you want to verify whether goals you set for an intervention , whether those are met after some time ? well , for now that is mainly in narrative form , it 's mainly about statements of people , to go start and maintain a dialogue with young people and teachers and other involved people , like , well , did it touch you , did it do something for you ? ( intervention developer , 18314 ) some participants reported favoring pictures on packs of cigarettes , not to induce fear , but simply to make the packaging less attractive : i also do n't believe that a picture in itself necessarily causes somebody to quit , if you say that i do n't think you can deliver . now the tobacco industry still tries , very glamorizing , to make it attractive , make it pretty . in america , they have special pink packages , to target women . in other countries they have packages with only five cigarettes , to make it more attractive for young people . ( policymaker , 26114 ) i also do n't believe that a picture in itself necessarily causes somebody to quit , if you say that i do n't think you can deliver . . now the tobacco industry still tries , very glamorizing , to make it attractive , make it pretty . in america , they have special pink packages , to target women . in other countries they have packages with only five cigarettes , to make it more attractive for young people . participants indicated that when politicians or policymakers pressed for interventions that addressed a given problem , they would often prioritize quick and clearly visible results over effective evidence - based interventions ( note that this fits well with advertising agencies ' drive for originality ) : and in practice people then start to do something , eh , because the municipality also has to show that something is happening , and politics also asks for it , and also asks for visible activities and results preferably . ( intervention developer , 31317 ) and in practice people then start to do something , eh , because the municipality also has to show that something is happening , and politics also asks for it , and also asks for visible activities and results preferably . ( intervention developer , 31317 ) in addition , intervention developers sometimes operated within intermediary organizations , such as schools , that were in favor of threatening approaches , and were sometimes hard to convince otherwise . when participants were not in favor of threatening communication , their reasons were not always based on the understanding of the inefficacy of threatening communications . other reasons included that fear was not considered an intrinsic motivator ( reflecting the implicit assumption that intrinsic motivation is superior to extrinsic motivation ) and that their organization just did not believe in motivating through fear . one participant stated that there was a risk that the negative affect would be associated with the health - promoting organization where the intervention originated , which might decrease donations or susceptibility to future messages . finally , one additional overarching observation was that although most beliefs did not seem specific to one or a few categories of key actors , one clear pattern emerged . participants who were closer to actual intervention development often had a generic idea that there was something wrong with inducing fear ( though they rarely grasped the underlying dynamics ) . participants who were further removed from the intervention development activities often did not have this basic heuristic belief . inducing fear was cited relatively rarely as a reason to use threatening communication . more often , the goal was to confront people with the negative consequences of a given risky behavior . the most common argument for this need for confrontation was rooted in the fact that attention is a first necessary condition for further processing , and attention is a limited resource . given that target population members are exposed simultaneously to multiple stimuli , behavior change interventions need to compete for attention . evoking strong emotions the need to break through and draw attention was often reported : of course , we do n't have as much media budget as a commercial organization . you want to draw attention , you do n't want to shock , i do n't think that 's the right word , but you do want to do something that reaches people . so you want to hit a certain emotion , and i think that we managed with our campaign . because many people saw it . ( intervention developer , 191110 ) of course , we do n't have as much media budget as a commercial organization . you want to draw attention , you do n't want to shock , i do n't think that 's the right word , but you do want to do something that reaches people . so you want to hit a certain emotion , and i think that we managed with our campaign . because many people saw it . ( intervention developer , 191110 ) emotion was also assumed to render interventions memorable , which , combined with the memory of the evoked emotion itself , was assumed to prompt desirable behavior : and why do you have to strike an emotion ? yes , again , people get to see so many communications in a day , that if you want to strike somebody at all , then especially that emotion is important . because only by letting people really feel something , you can prompt people to act . so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . or maybe even extra this year . oh , yes , i saw that commercial , that struck me personally . then that works . ( intervention developer , 191110 ) and why do you have to strike an emotion ? yes , again , people get to see so many communications in a day , that if you want to strike somebody at all , then especially that emotion is important . because only by letting people really feel something , you can prompt people to act . so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . or maybe even extra this year . oh , yes , i saw that commercial , that struck me personally . then that works . ( intervention developer , 191110 ) a second example of this reasoning was provided by a participant from the advertising world : ( discussing the first prompting intervention , see http://sciencerep.org/5 ) what do you think ? i think that in this case , you want to address a specific target group , and for that , i think it 's good . i think that in that case you do n't have to bother with a boring communication indeed , i think that if you want to address a certain target group , with a lot of sexual contacts , it 's fine . before you know it , you have it , so be wise . is this of course , you have behavior change and awareness . what do you think of it in those respects ? becoming aware of easily contracting an sti and continuing your old behavior does n't seem useful . ( advertising professional , 51183 ) ( discussing the first prompting intervention , see http://sciencerep.org/5 ) what do you think ? is this a good intervention , or not ? do you think this would work , or not ? i think that in this case , you want to address a specific target group , and for that , i think it 's good . i think that in that case you do n't have to bother with a boring communication indeed , i think that if you want to address a certain target group , with a lot of sexual contacts , it 's fine . before you know it , you have it , so be wise . becoming aware of easily contracting an sti and continuing your old behavior does n't seem useful . ( advertising professional , 51183 ) another common belief was that when a communication manages to evoke emotions in target population members , this would cause reflection about the relevant behavior : for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . so one picture of a child who had not consumed alcohol , and another scan of a child who had consumed alcohol . and showed what it does to your brains . well , they showed that at the meeting , and then people could ask questions . and then i thought , i think that works , because people then start thinking . ( politician , 191115 ) for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . so one picture of a child who had not consumed alcohol , and another scan of a child who had consumed alcohol . and showed what it does to your brains . well , they showed that at the meeting , and then people could ask questions . and then i thought , i think that works , because people then start thinking . ( politician , 191115 ) this belief was based on the assumption that target population members act and reason rationally ( i.e. consistent with a subjective expected utility model ; see e.g. tversky & kahneman , 1981 ) . a related and possibly derived belief was participants ' presumption that their own common - sense reasoning about how communications would be received and processed further would resemble reality . for example , one scientist explained : i would , if i would read , like , it gives you lung cancer ( scientist , 4119 ) i would , if i would read , like , it gives you lung cancer ( scientist , 4119 ) also related , achieving awareness ( or increasing risk perception to a certain threshold ) was often considered a sufficient condition for behavior change : you try to increase this motivation to quit . so the more you make them aware of the fact that it 's bad , the stronger your motivation becomes with respect to your addiction . so the more you make them aware of the fact that it 's bad , the stronger your motivation becomes with respect to your addiction . ( advertising professional , 22114 ) to participants who assumed that target population members who perform the undesirable behavior are ignorant as to the negative consequences , showing these negative consequences was considered necessary education . similarly , target population members were generally assumed to underestimate the negative consequences of the behavior . thus , participants assumed that generally even target population members who are aware of a causal link between a behavior and a negative consequence would under - estimate the severity of the negative consequence . intervention developers had often heard negative messages about threatening communication , but did not usually understand the dynamics at play . in general , it could be said that interviewees seemed to have some pieces of the puzzle , but lacked a coherent framework of the working mechanisms of threatening communication . this sometimes manifested as ambivalence regarding the effectiveness of threatening communication : yes , but risk communication does not work with this population ? yes , i think that this [ pictures on cigarette packs ] is a good method , but i also think that there 's no other way than this [ to change behavior ] . ( scientist , 4119 ) yes , but risk communication does not work with this population ? yes , i think that this [ pictures on cigarette packs ] is a good method , but i also think that there 's no other way than this [ to change behavior ] . ( scientist , 4119 ) when logical arguments against the use of threatening communication were acknowledged , this ineffectiveness was sometimes attributed to specific characteristics of the situation at hand , often the nature of the behavior at hand ( e.g. , smoking is a difficult behavior ) . it was not uncommon for participants to express beliefs that in terms of the eppm were logically inconsistent . for example , participants could state that they thought that low susceptibility , rather than low severity , was the main cause of unhealthy behavior ; and several minutes later explain the importance of emphasizing the grave consequences of unhealthy behavior . one participant explained that they tried to solve the problem of low susceptibility in their target population by tailoring an intervention , enabling them to communicate that target population members belonged to a subpopulation that was at a relatively high risk . while this reasoning is partly in line with eppm , for example , one participant remarked : yes , this [ picture of blackened lungs ] is ok , because you can do something about it by quitting smoking , so , um , i mean , if you would say , breathing causes lung cancer ( advertising professional , 22114 ) yes , this [ picture of blackened lungs ] is ok , because you can do something about it by quitting smoking , so , um , i mean , if you would say , breathing causes lung cancer [ that would be a different story ] . ( advertising professional , 22114 ) related to this , participants sometimes were aware of the theoretical constraint that threatening information would not work unless efficacy was high , but then often underestimated the required intensity of an intervention to enhance efficacy . in fact , this was sometimes reported as a necessary ingredient of interventions using threatening communication . scientists were often cognizant of the eppm but , like other participants , did not always realize that the threatening stimulus in this model can be any stimulus . some classes of intervention types were considered to not be fear - inducing ( i.e. , carry the risk of fear control responses ) , such as scenario messages or personalized risk assessments . similarly , often participants indicated that they did not use threatening communication , while from their explanation it became clear that they did : [ discussing a campaign against smoking that would focus on cancer ] some people may call this a fear inducing message i do n't call this fear inducing , because question one is , what is fear exactly and what is fear inducing ? i consider it more of an awareness campaign . because people often say , well , the risk is not so bad , and well , cancer , my grandfather lived to 92 with two packs of cigarettes a day . it is more of a confronting campaign , where the health effects you can expect from smoking regarding cancer are made clear , visually . ( policymaker , 26114 ) [ discussing a campaign against smoking that would focus on cancer ] some people may call this a fear inducing message i do n't call this fear inducing , because question one is , what is fear exactly and what is fear inducing ? i consider it more of an awareness campaign . because people often say , well , the risk is not so bad , and well , cancer it is more of a confronting campaign , where the health effects you can expect from smoking regarding cancer are made clear , visually . in fact , few participants were aware of the conceptual distinction between personal determinants ( psychological variables that can be influenced to eventually engender behavior change , such as risk perception or self - efficacy ) , behavior change methods or techniques ( theoretical methods to influence a given determinant , such as fear appeals or modeling ) , and applications of these methods ( an incarnation of a theoretical method , such as a picture of blackened lungs or a movie with a role model ) . one participant who was partially aware of this distinction explained : but then the step from those modifiable determinants to good methods to change that , kind of , that is often difficult . what are effective methods to achieve those goals and how do you utilize these ? often , the time and knowledge to do this thoroughly is lacking . ( intervention developer , 22612 ) but then the step from those modifiable determinants to good methods to change that , kind of , that is often difficult . what are effective methods to achieve those goals and how do you utilize these ? often , the time and knowledge to do this thoroughly is lacking . ( intervention developer , 22612 ) this lack of knowledge about methods for behavior change was one of the reasons for the use of threatening communication : there were no known good alternatives . intervention developers often did not have an array of behavior change methods at their disposal , and sometimes they applied the same method in all interventions . some other participants had naive ideas of behavior change methods that carried a high risk of yielding non evidence - based results . specifically , some participants indicated that it was important that an intervention originated from within the target population . however , these target population members generally advocated threatening communication approaches : yes , or , they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign . , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . ( intervention developer , 21210 ) yes , or , they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign . , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . ( intervention developer , 21210 ) because participants often assumed that target population members knew how they themselves could be influenced , this reliance on target population members ' preferences for threatening communication could easily result in the development of threatening interventions . among employees of advertising agencies and policymakers , the belief existed that there are no ways to predict in advance whether a campaign would be successful : and can you estimate a bit , when you 've made many campaigns , whether campaigns work or not ? regardless of indicators like market share , on the basis of your experience ? yes , that is very hard to say . because sometimes you 're surprised by campaigns that turn out to work very well , while you think , this has to be great , but it does n't work , or a customer gives it insufficient chance . that is , um , when you would be able to determine this in advance , you would have found the holy grail . ( advertising professional , 9416 ) and can you estimate a bit , when you 've made many campaigns , whether campaigns work or not ? regardless of indicators like market share , on the basis of your experience ? yes , that is very hard to say . because sometimes you 're surprised by campaigns that turn out to work very well , while you think , or campaigns of which you think , this has to be great , but it does n't work , or a customer gives it insufficient chance . that is , um , when you would be able to determine this in advance , you would have found the holy grail . ( advertising professional , 9416 ) intervention development was considered largely a trial - and - error endeavor , with the development process often being seen as a black box ( in advertising , a creative process engaged in by the creatives ) . yet , at the same time , in advertising , there were some basic guidelines on campaign development . for example , one interviewee explained that a commercial for a product such as soup should always visually show both the soup and the packaging . another such heuristic was the merit of creativity or originality : you can do something very strange and very new , but if you do it a second or a third time , then it 's already not surprising any more . i think that in the end clients choose to be creative within a given campaign , with a given idea . and ( advertising professional , 9416 ) you can do something very strange and very new , but if you do it a second or a third time , then it 's already not surprising any more . i think that in the end clients choose to be creative within a given campaign , with a given idea . and ( advertising professional , 9416 ) among participants from advertising agencies , sometimes confrontation and humor were two options to choose from when trying to reach people . when confrontation was considered inappropriate , humor would be the alternative approach : well , i think that you choose humor at a time you actually want to point something out to people , but you do n't want to pedantically wag your finger . and if you do it in a humorous way then it does come across differently to people than when you say , eh , you 're not allowed to do this , or you have to do that . yes . that you then mainly choose humor . so that it does come across , but that people do n't think , ( advertising professional , 22111 ) well , i think that you choose humor at a time you actually want to point something out to people , but you do n't want to pedantically wag your finger . and if you do it in a humorous way then it does come across differently to people than when you say , eh , you 're not allowed to do this , or you have to do that . yes . that you then mainly choose humor . so that it does come across , but that people do n't think , ( advertising professional , 22111 ) beliefs in advertising agencies about behavior change methods proved relevant because intervention developers sometimes trusted advertising agencies to know how to influence target population members . at the same time , when advertising agencies delivered a product or presented an intermediate step that intervention developers judged to be unacceptable ( i.e. , not in line with known goals or constraints ) , intervention developers would generally communicate this and demand adjustments . participants generally conceded that evaluating whether an intervention had successfully induced behavior change was complicated . understandably , besides the scientists , very few participants mentioned the possibility of examining such evaluations in a laboratory setting . instead one such indicator was the degree to which an intervention was recognized or remembered , or the perceived impact as reported by introspection of target population members : so how do you want to verify whether goals you set for an intervention , whether those are met after some time ? well , for now that is mainly in narrative form , it 's mainly about statements of people , to go start and maintain a dialogue with young people and teachers and other involved people , like , well , did it touch you , did it do something for you ? ( intervention developer , 18314 ) so how do you want to verify whether goals you set for an intervention , whether those are met after some time ? well , for now that is mainly in narrative form , it 's mainly about statements of people , to go start and maintain a dialogue with young people and teachers and other involved people , like , well , did it touch you , did it do something for you ? some participants reported favoring pictures on packs of cigarettes , not to induce fear , but simply to make the packaging less attractive : i also do n't believe that a picture in itself necessarily causes somebody to quit , if you say that i do n't think you can deliver . now the tobacco industry still tries , very glamorizing , to make it attractive , make it pretty . in america , they have special pink packages , to target women . in other countries they have packages with only five cigarettes , to make it more attractive for young people . ( policymaker , 26114 ) i also do n't believe that a picture in itself necessarily causes somebody to quit , if you say that i do n't think you can deliver . now the tobacco industry still tries , very glamorizing , to make it attractive , make it pretty . in america , they have special pink packages , to target women . in other countries they have packages with only five cigarettes , to make it more attractive for young people . participants indicated that when politicians or policymakers pressed for interventions that addressed a given problem , they would often prioritize quick and clearly visible results over effective evidence - based interventions ( note that this fits well with advertising agencies ' drive for originality ) : and in practice people then start to do something , eh , because the municipality also has to show that something is happening , and politics also asks for it , and also asks for visible activities and results preferably . ( intervention developer , 31317 ) and in practice people then start to do something , eh , because the municipality also has to show that something is happening , and politics also asks for it , and also asks for visible activities and results preferably . ( intervention developer , 31317 ) in addition , intervention developers sometimes operated within intermediary organizations , such as schools , that were in favor of threatening approaches , and were sometimes hard to convince otherwise . when participants were not in favor of threatening communication , their reasons were not always based on the understanding of the inefficacy of threatening communications . other reasons included that fear was not considered an intrinsic motivator ( reflecting the implicit assumption that intrinsic motivation is superior to extrinsic motivation ) and that their organization just did not believe in motivating through fear . one participant stated that there was a risk that the negative affect would be associated with the health - promoting organization where the intervention originated , which might decrease donations or susceptibility to future messages . finally , one additional overarching observation was that although most beliefs did not seem specific to one or a few categories of key actors , one clear pattern emerged . participants who were closer to actual intervention development often had a generic idea that there was something wrong with inducing fear ( though they rarely grasped the underlying dynamics ) . participants who were further removed from the intervention development activities often did not have this basic heuristic belief . one of the main reasons for using threatening communication was to confront people with the consequences of a behavior . the first perceived goal of emotions was to draw attention to the intervention and prompt self - reflection , which would then lead to the desirable behavior , because target population members were assumed to act rationally on hypothesized ( successful ) increments in risk perceptions . this assumption of rationality also explained the presupposition that achieving awareness or raising risk perception would cause behavior change . this enhanced awareness was the second perceived goal emotions could serve : explicitly emotionally defined risk was often assumed to directly enhance awareness . this resulted in , for example , threatening interventions not being considered fear - inducing ; underestimation of what is required to enhance self - efficacy ; and overestimation of efficacy levels in the target population . most participants did not know many behavior change methods , threatening communication sometimes being the best - known solution . participants often believed the target population could help in identifying useful methods , and some relied on advertising agencies , which mainly advocated originality , confrontation , and humor . the complexity of the evaluation of behavior change interventions often led participants to adopt proxies for effectiveness , such as how well known an intervention was . finally , working with external organizations sometimes facilitated the choice for threatening communications : funders or intermediary organizations such as schools sometimes preferred threatening communication , and politicians often desired quick and salient , rather than thoroughly researched , interventions . this last finding was already hypothesized decades ago ( soames job , 1988 ) , but has not been empirically investigated until now . it is clear that the decades of communication by behavior change scientists regarding the ineffectiveness of health threats has had results , as most intervention developers got the gist of it , albeit usually not more than the gist which still leaves many opportunities for threatening communications . this makes sense , as most intervention developers were not behavior change scientists , or even psychologists . this also explains a second erroneous assumption , namely that humans act rationally , being driven by something similar to expected utility theory ( which is known to be incorrect ; see kahneman & tversky , 1979 ) . at the same time , the choice for threatening communication was also based on some characteristics of behavior change communications that were consistent with the scientific evidence : it is necessary to draw attention to an intervention , and the interventions do have to inspire self - reflection to a degree . however , people generally direct their attention away from threatening information ( bar - haim , lamy , pergamin , bakermans - kranenburg , & van ijzendoorn , 2007 ) , especially if they are not confident they can deflect the danger ( kessels et al . , 2010 ; nielsen & shapiro , 2009 ) . it became clear that intervention developers often did not know many behavior change methods , sometimes knowing no alternatives to threatening communication at all . sometimes they relied on target population members to tell them which methods to use ; but target population members often advocated fear ( biener , ji , gilpin , & albers , 2004 ; goodall & appiah , 2008 ; lennon & rentfro , 2010 ) . some intervention developers also relied on advertising agency employees , who lacked knowledge on behavior change methods ( and those advertising agency employees following the literature in their field may in fact be in favor of threatening communication ; see for example latour , snipes , & bliss , 1996 ) . at the same time , intervention developers would also correct advertising agencies when they noticed errors , although time constraints often restrained possibilities for adjustments . integrating these various aspects of the decision - making process , some possible actions emerge that may be taken to decrease the use of threatening communication . first , of course , it is important to provide intervention developers with a toolbox of different behavior change methods . such a toolbox is available , for example , in intervention development protocols such as intervention mapping ( kok , bartholomew , parcel , gottlieb , & fernandez , 2014 , this issue ; see also bartholomew et al . , 2011 ) and the methodology being developed based on the behavior change techniques ( bct ) taxonomy ( abraham & michie , 2008 ) and the behavior change wheel ( michie et al . , 2011 ) second , it seems useful to educate intervention developers on two psychological knowledge elements : firstly , it is unwise to consider humans rational ( kahneman & tversky , 1979 ) ; and secondly , threatening information averts attention ( bar - haim et al . , 2007 ) and prompts defensive reactions ( erceg - hurn & steed , 2011 ; kessels et al . , 2014 , this issue ) , use of negative emotions is very dangerous , and one should exercise caution when labeling something as not fear - inducing . this is connected to the third point : intervention developers should be urged to apply the framework for ethical justification provided by brown and whiting ( 2014 , this issue ) . fourth , the use of determinant studies as a method of establishing intervention targets can be promoted . comparison of the relevance of several cognitive determinants shows that often threat ( i.e. , severity and susceptibility ) is not the most expedient intervention target ( but note that when the target of the threat is somebody else , such as one 's children , threat does seem effective ) . sixth , and this is a very important point indeed : it would be beneficial if intervention developers were convinced of their expertise in this matter . intervention developers need to be empowered to act as professionals when dealing with intermediary organizations , advertising agencies , and potentially even funders and politicians , who as well - meaning lay people are susceptible to the intuitive appeal of threatening communication . to this end , providing intervention developers with an overview of the evidence regarding the behavior change potential of threatening information can be helpful . finally , alternative tools should be provided to attract attention , as this was one of the reasons for using threatening communication . somewhat ironically , such tools may be found in the marketing and advertising literature ( maughan , gutnikov , & stevens , 2007 ; pieters & wedel , 2012 ) . a concrete recommendation is simply the opposite of the belief that threatening communication attracts attention : people look more at advertisements they like ( maughan et al . , 2007 ) . note that in compiling guidelines for attracting attention , it is crucial to respect the parameters of effectiveness of behavior change methods ( bartholomew et al . , 2011 ) . an intervention implementing these elements may succeed in eradicating the widespread reliance on threatening interventions , paving the way to the use of more effective behavior change methods . in addition , the findings from the current study should be corroborated by quantitative studies , so that the pertinent beliefs of these groups of key actors can be mapped in terms of their relative importance .

threatening communication is a widely applied method in behavior change interventions , which at the same time has been heavily criticized in the psychological literature . the current paper describes a study of the reasons for this persistent wide application of threatening communication . we conducted qualitative interviews with 33 key actors in behavior change intervention development in the netherlands . specifically , we interviewed intervention developers , policymakers , politicians , scientists , and advertising professionals . the interviews were transcribed and subsequently coded using nvivo . we found that participants most closely involved with the actual intervention development were generally convinced that threatening information was to be prevented , but often did not understand the exact processes involved . they were often under the impression that rather than a potent efficacy enhancing element , a behavioral suggestion would suffice to prevent threatening communication from backfiring . as participants were further removed from the actual intervention development , they generally tended to be more in favor of threatening communication . the main reasons for use of threatening information were to attract attention or prompt self - reflection through confrontation , because target population members were assumed to like threatening information and respond rationally to increased risk perceptions by changing their behavior , or simply because no alternatives were available . in addition , intervention developers frequently had to deal with supervisors or funders who preferred threatening communication . thus , when communicating with practitioners , it seems fruitful to provide them with a toolbox of evidence - based behavior change methods that promote adaptive , rather than maladaptive , behavior ; to promote basing interventions on the most relevant behavioral determinants as identified by determinant analyses ; and to equip intervention developers with the tools to persuade other key stakeholders that fear is a bad counselor .

METHOD RESULTS Confrontation and emotion through fear Partial knowledge of the theory More, or less, systematic approaches to intervention development Other reasons to use or not use threatening communication and differences between key actors DISCUSSION

the interviews were conducted by two researchers who had not published any research on threatening communication ( gjp and kees de jong ) to minimize socially desirable answers based on familiarity with an interviewer 's work . specifically , we interviewed 18 intervention developers and two administrators at health promoting service providers , four scientists , three policymakers , three politicians , and three advertising professionals . of the policymakers , one worked at a dutch ministry ; one at a national funding organization ; one at a national hsp ; and one at a regional hsp . of the politicians , one was active in national politics as a member of parliament , and the other two in regional politics as municipal counselors . after that , questions addressed participants ' experience with intervention development in general ; what information participants gathered before making decisions about interventions ; how certain choices were made ; and what were particularly good or bad interventions in participants ' opinion . the interviews were recorded , transcribed , and imported into qsr nvivo , where they were coded by gjp . first , all interview fragments where threatening communication was discussed were coded in the same fear appeal category . the second coding sweep yielded a number of systematically occurring beliefs , which were categorized in a tree structure with the following main categories : reasons for fear appeal use ; reasons against fear appeal use ; the process of intervention development ; related beliefs ( not otherwise categorized ) ; and environmental constraints . in this paper , however , we focus on beliefs that were inconsistent with the scientific state of the art . that narrow focus may cause these results to paint a bleak picture , but this does not mean that the interviewed participants were ignorant or incompetent in terms of intervention development . the most common argument for this need for confrontation was rooted in the fact that attention is a first necessary condition for further processing , and attention is a limited resource . given that target population members are exposed simultaneously to multiple stimuli , behavior change interventions need to compete for attention . ( intervention developer , 191110 ) emotion was also assumed to render interventions memorable , which , combined with the memory of the evoked emotion itself , was assumed to prompt desirable behavior : and why do you have to strike an emotion ? so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . ( advertising professional , 51183 ) another common belief was that when a communication manages to evoke emotions in target population members , this would cause reflection about the relevant behavior : for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . well , they showed that at the meeting , and then people could ask questions . ( politician , 191115 ) for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . well , they showed that at the meeting , and then people could ask questions . ( politician , 191115 ) this belief was based on the assumption that target population members act and reason rationally ( i.e. similarly , target population members were generally assumed to underestimate the negative consequences of the behavior . thus , participants assumed that generally even target population members who are aware of a causal link between a behavior and a negative consequence would under - estimate the severity of the negative consequence . intervention developers had often heard negative messages about threatening communication , but did not usually understand the dynamics at play . in general , it could be said that interviewees seemed to have some pieces of the puzzle , but lacked a coherent framework of the working mechanisms of threatening communication . this sometimes manifested as ambivalence regarding the effectiveness of threatening communication : yes , but risk communication does not work with this population ? ( scientist , 4119 ) when logical arguments against the use of threatening communication were acknowledged , this ineffectiveness was sometimes attributed to specific characteristics of the situation at hand , often the nature of the behavior at hand ( e.g. for example , participants could state that they thought that low susceptibility , rather than low severity , was the main cause of unhealthy behavior ; and several minutes later explain the importance of emphasizing the grave consequences of unhealthy behavior . ( advertising professional , 22114 ) related to this , participants sometimes were aware of the theoretical constraint that threatening information would not work unless efficacy was high , but then often underestimated the required intensity of an intervention to enhance efficacy . in fact , few participants were aware of the conceptual distinction between personal determinants ( psychological variables that can be influenced to eventually engender behavior change , such as risk perception or self - efficacy ) , behavior change methods or techniques ( theoretical methods to influence a given determinant , such as fear appeals or modeling ) , and applications of these methods ( an incarnation of a theoretical method , such as a picture of blackened lungs or a movie with a role model ) . ( intervention developer , 22612 ) this lack of knowledge about methods for behavior change was one of the reasons for the use of threatening communication : there were no known good alternatives . intervention developers often did not have an array of behavior change methods at their disposal , and sometimes they applied the same method in all interventions . some other participants had naive ideas of behavior change methods that carried a high risk of yielding non evidence - based results . however , these target population members generally advocated threatening communication approaches : yes , or , they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign . , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . ( intervention developer , 21210 ) yes , or , they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign yes , no , it has to be more serious . for example , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . ( intervention developer , 21210 ) because participants often assumed that target population members knew how they themselves could be influenced , this reliance on target population members ' preferences for threatening communication could easily result in the development of threatening interventions . because sometimes you 're surprised by campaigns that turn out to work very well , while you think , or campaigns of which you think , this has to be great , but it does n't work , or a customer gives it insufficient chance . yet , at the same time , in advertising , there were some basic guidelines on campaign development . i think that in the end clients choose to be creative within a given campaign , with a given idea . so that it does come across , but that people do n't think , ( advertising professional , 22111 ) beliefs in advertising agencies about behavior change methods proved relevant because intervention developers sometimes trusted advertising agencies to know how to influence target population members . at the same time , when advertising agencies delivered a product or presented an intermediate step that intervention developers judged to be unacceptable ( i.e. instead one such indicator was the degree to which an intervention was recognized or remembered , or the perceived impact as reported by introspection of target population members : so how do you want to verify whether goals you set for an intervention , whether those are met after some time ? participants indicated that when politicians or policymakers pressed for interventions that addressed a given problem , they would often prioritize quick and clearly visible results over effective evidence - based interventions ( note that this fits well with advertising agencies ' drive for originality ) : and in practice people then start to do something , eh , because the municipality also has to show that something is happening , and politics also asks for it , and also asks for visible activities and results preferably . ( intervention developer , 31317 ) in addition , intervention developers sometimes operated within intermediary organizations , such as schools , that were in favor of threatening approaches , and were sometimes hard to convince otherwise . when participants were not in favor of threatening communication , their reasons were not always based on the understanding of the inefficacy of threatening communications . participants who were further removed from the intervention development activities often did not have this basic heuristic belief . the most common argument for this need for confrontation was rooted in the fact that attention is a first necessary condition for further processing , and attention is a limited resource . given that target population members are exposed simultaneously to multiple stimuli , behavior change interventions need to compete for attention . ( intervention developer , 191110 ) emotion was also assumed to render interventions memorable , which , combined with the memory of the evoked emotion itself , was assumed to prompt desirable behavior : and why do you have to strike an emotion ? so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . ( advertising professional , 51183 ) another common belief was that when a communication manages to evoke emotions in target population members , this would cause reflection about the relevant behavior : for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . well , they showed that at the meeting , and then people could ask questions . ( politician , 191115 ) this belief was based on the assumption that target population members act and reason rationally ( i.e. similarly , target population members were generally assumed to underestimate the negative consequences of the behavior . thus , participants assumed that generally even target population members who are aware of a causal link between a behavior and a negative consequence would under - estimate the severity of the negative consequence . intervention developers had often heard negative messages about threatening communication , but did not usually understand the dynamics at play . in general , it could be said that interviewees seemed to have some pieces of the puzzle , but lacked a coherent framework of the working mechanisms of threatening communication . ( scientist , 4119 ) when logical arguments against the use of threatening communication were acknowledged , this ineffectiveness was sometimes attributed to specific characteristics of the situation at hand , often the nature of the behavior at hand ( e.g. for example , participants could state that they thought that low susceptibility , rather than low severity , was the main cause of unhealthy behavior ; and several minutes later explain the importance of emphasizing the grave consequences of unhealthy behavior . ( advertising professional , 22114 ) related to this , participants sometimes were aware of the theoretical constraint that threatening information would not work unless efficacy was high , but then often underestimated the required intensity of an intervention to enhance efficacy . scientists were often cognizant of the eppm but , like other participants , did not always realize that the threatening stimulus in this model can be any stimulus . similarly , often participants indicated that they did not use threatening communication , while from their explanation it became clear that they did : [ discussing a campaign against smoking that would focus on cancer ] some people may call this a fear inducing message i do n't call this fear inducing , because question one is , what is fear exactly and what is fear inducing ? in fact , few participants were aware of the conceptual distinction between personal determinants ( psychological variables that can be influenced to eventually engender behavior change , such as risk perception or self - efficacy ) , behavior change methods or techniques ( theoretical methods to influence a given determinant , such as fear appeals or modeling ) , and applications of these methods ( an incarnation of a theoretical method , such as a picture of blackened lungs or a movie with a role model ) . ( intervention developer , 22612 ) this lack of knowledge about methods for behavior change was one of the reasons for the use of threatening communication : there were no known good alternatives . intervention developers often did not have an array of behavior change methods at their disposal , and sometimes they applied the same method in all interventions . some other participants had naive ideas of behavior change methods that carried a high risk of yielding non evidence - based results . however , these target population members generally advocated threatening communication approaches : yes , or , they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign . , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . ( intervention developer , 21210 ) because participants often assumed that target population members knew how they themselves could be influenced , this reliance on target population members ' preferences for threatening communication could easily result in the development of threatening interventions . because sometimes you 're surprised by campaigns that turn out to work very well , while you think , this has to be great , but it does n't work , or a customer gives it insufficient chance . because sometimes you 're surprised by campaigns that turn out to work very well , while you think , or campaigns of which you think , this has to be great , but it does n't work , or a customer gives it insufficient chance . ( advertising professional , 9416 ) intervention development was considered largely a trial - and - error endeavor , with the development process often being seen as a black box ( in advertising , a creative process engaged in by the creatives ) . i think that in the end clients choose to be creative within a given campaign , with a given idea . so that it does come across , but that people do n't think , ( advertising professional , 22111 ) beliefs in advertising agencies about behavior change methods proved relevant because intervention developers sometimes trusted advertising agencies to know how to influence target population members . at the same time , when advertising agencies delivered a product or presented an intermediate step that intervention developers judged to be unacceptable ( i.e. instead one such indicator was the degree to which an intervention was recognized or remembered , or the perceived impact as reported by introspection of target population members : so how do you want to verify whether goals you set for an intervention , whether those are met after some time ? participants indicated that when politicians or policymakers pressed for interventions that addressed a given problem , they would often prioritize quick and clearly visible results over effective evidence - based interventions ( note that this fits well with advertising agencies ' drive for originality ) : and in practice people then start to do something , eh , because the municipality also has to show that something is happening , and politics also asks for it , and also asks for visible activities and results preferably . ( intervention developer , 31317 ) in addition , intervention developers sometimes operated within intermediary organizations , such as schools , that were in favor of threatening approaches , and were sometimes hard to convince otherwise . when participants were not in favor of threatening communication , their reasons were not always based on the understanding of the inefficacy of threatening communications . participants who were further removed from the intervention development activities often did not have this basic heuristic belief . one of the main reasons for using threatening communication was to confront people with the consequences of a behavior . the first perceived goal of emotions was to draw attention to the intervention and prompt self - reflection , which would then lead to the desirable behavior , because target population members were assumed to act rationally on hypothesized ( successful ) increments in risk perceptions . this resulted in , for example , threatening interventions not being considered fear - inducing ; underestimation of what is required to enhance self - efficacy ; and overestimation of efficacy levels in the target population . most participants did not know many behavior change methods , threatening communication sometimes being the best - known solution . participants often believed the target population could help in identifying useful methods , and some relied on advertising agencies , which mainly advocated originality , confrontation , and humor . the complexity of the evaluation of behavior change interventions often led participants to adopt proxies for effectiveness , such as how well known an intervention was . finally , working with external organizations sometimes facilitated the choice for threatening communications : funders or intermediary organizations such as schools sometimes preferred threatening communication , and politicians often desired quick and salient , rather than thoroughly researched , interventions . this makes sense , as most intervention developers were not behavior change scientists , or even psychologists . at the same time , the choice for threatening communication was also based on some characteristics of behavior change communications that were consistent with the scientific evidence : it is necessary to draw attention to an intervention , and the interventions do have to inspire self - reflection to a degree . it became clear that intervention developers often did not know many behavior change methods , sometimes knowing no alternatives to threatening communication at all . some intervention developers also relied on advertising agency employees , who lacked knowledge on behavior change methods ( and those advertising agency employees following the literature in their field may in fact be in favor of threatening communication ; see for example latour , snipes , & bliss , 1996 ) . at the same time , intervention developers would also correct advertising agencies when they noticed errors , although time constraints often restrained possibilities for adjustments . integrating these various aspects of the decision - making process , some possible actions emerge that may be taken to decrease the use of threatening communication . first , of course , it is important to provide intervention developers with a toolbox of different behavior change methods . , 2011 ) second , it seems useful to educate intervention developers on two psychological knowledge elements : firstly , it is unwise to consider humans rational ( kahneman & tversky , 1979 ) ; and secondly , threatening information averts attention ( bar - haim et al . sixth , and this is a very important point indeed : it would be beneficial if intervention developers were convinced of their expertise in this matter . intervention developers need to be empowered to act as professionals when dealing with intermediary organizations , advertising agencies , and potentially even funders and politicians , who as well - meaning lay people are susceptible to the intuitive appeal of threatening communication . to this end , providing intervention developers with an overview of the evidence regarding the behavior change potential of threatening information can be helpful . finally , alternative tools should be provided to attract attention , as this was one of the reasons for using threatening communication . a concrete recommendation is simply the opposite of the belief that threatening communication attracts attention : people look more at advertisements they like ( maughan et al . note that in compiling guidelines for attracting attention , it is crucial to respect the parameters of effectiveness of behavior change methods ( bartholomew et al . an intervention implementing these elements may succeed in eradicating the widespread reliance on threatening interventions , paving the way to the use of more effective behavior change methods . in addition , the findings from the current study should be corroborated by quantitative studies , so that the pertinent beliefs of these groups of key actors can be mapped in terms of their relative importance .

specifically , we interviewed 18 intervention developers and two administrators at health promoting service providers , four scientists , three policymakers , three politicians , and three advertising professionals . , municipal health services ) ; the remaining 14 worked at national hsps , each addressing one or several behavioral themes such as smoking , safe sex , exercise , substance use , and diet patterns . all four scientists worked at dutch universities , specifically in the fields of medicine , business communication , epidemiology , and risk communication . of the politicians , one was active in national politics as a member of parliament , and the other two in regional politics as municipal counselors . specifically , the interviews focused on interventions with behavior change as a goal in general ( no specific interventions participants had developed ) . to ensure that threatening communication would be discussed in all interviews , a number of examples of interventions were included for the interviewee to judge , two of which used threatening communication ( a picture of blackened lungs on a pack of cigarettes and a speeding intervention ; see http://sciencerep.org/5 for these pictures ) . we chose this approach to let threatening communication come up in the interview naturally , which decreased the possibility of defensive reactions . we anticipated defensive reactions when intervention designers would be confronted directly with the question of why they use threatening communication . the second coding sweep yielded a number of systematically occurring beliefs , which were categorized in a tree structure with the following main categories : reasons for fear appeal use ; reasons against fear appeal use ; the process of intervention development ; related beliefs ( not otherwise categorized ) ; and environmental constraints . within some categories , subcategories were formed , such as within the process of intervention development category , the subcategories goals of intervention development and target group involvement . the beliefs identified in these categories and subcategories were then organized in a narrative in four sections , each describing largely coherent sets of beliefs . in this paper , however , we focus on beliefs that were inconsistent with the scientific state of the art . that narrow focus may cause these results to paint a bleak picture , but this does not mean that the interviewed participants were ignorant or incompetent in terms of intervention development . yet there is always room for improvement , and this paper aims to facilitate such improvement . more often , the goal was to confront people with the negative consequences of a given risky behavior . the most common argument for this need for confrontation was rooted in the fact that attention is a first necessary condition for further processing , and attention is a limited resource . ( intervention developer , 191110 ) emotion was also assumed to render interventions memorable , which , combined with the memory of the evoked emotion itself , was assumed to prompt desirable behavior : and why do you have to strike an emotion ? so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . so at the moment you , for example , strike someone , in the sense of , we have a collection campaign , and you manage to strike someone , then such a person will be willing to donate money in the collection box . ( intervention developer , 191110 ) a second example of this reasoning was provided by a participant from the advertising world : ( discussing the first prompting intervention , see http://sciencerep.org/5 ) what do you think ? i think that in this case , you want to address a specific target group , and for that , i think it 's good . i think that in this case , you want to address a specific target group , and for that , i think it 's good . ( advertising professional , 51183 ) another common belief was that when a communication manages to evoke emotions in target population members , this would cause reflection about the relevant behavior : for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . ( politician , 191115 ) for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . for example , one scientist explained : i would , if i would read , like , it gives you lung cancer ( scientist , 4119 ) i would , if i would read , like , it gives you lung cancer ( scientist , 4119 ) also related , achieving awareness ( or increasing risk perception to a certain threshold ) was often considered a sufficient condition for behavior change : you try to increase this motivation to quit . ( advertising professional , 22114 ) to participants who assumed that target population members who perform the undesirable behavior are ignorant as to the negative consequences , showing these negative consequences was considered necessary education . thus , participants assumed that generally even target population members who are aware of a causal link between a behavior and a negative consequence would under - estimate the severity of the negative consequence . in general , it could be said that interviewees seemed to have some pieces of the puzzle , but lacked a coherent framework of the working mechanisms of threatening communication . ( scientist , 4119 ) when logical arguments against the use of threatening communication were acknowledged , this ineffectiveness was sometimes attributed to specific characteristics of the situation at hand , often the nature of the behavior at hand ( e.g. for example , participants could state that they thought that low susceptibility , rather than low severity , was the main cause of unhealthy behavior ; and several minutes later explain the importance of emphasizing the grave consequences of unhealthy behavior . while this reasoning is partly in line with eppm , for example , one participant remarked : yes , this [ picture of blackened lungs ] is ok , because you can do something about it by quitting smoking , so , um , i mean , if you would say , ( advertising professional , 22114 ) yes , this [ picture of blackened lungs ] is ok , because you can do something about it by quitting smoking , so , um , i mean , if you would say , breathing causes lung cancer [ that would be a different story ] . scientists were often cognizant of the eppm but , like other participants , did not always realize that the threatening stimulus in this model can be any stimulus . , carry the risk of fear control responses ) , such as scenario messages or personalized risk assessments . similarly , often participants indicated that they did not use threatening communication , while from their explanation it became clear that they did : [ discussing a campaign against smoking that would focus on cancer ] some people may call this a fear inducing message i do n't call this fear inducing , because question one is , what is fear exactly and what is fear inducing ? because people often say , well , the risk is not so bad , and well , cancer it is more of a confronting campaign , where the health effects you can expect from smoking regarding cancer are made clear , visually . ( policymaker , 26114 ) [ discussing a campaign against smoking that would focus on cancer ] some people may call this a fear inducing message i do n't call this fear inducing , because question one is , what is fear exactly and what is fear inducing ? because people often say , well , the risk is not so bad , and well , cancer it is more of a confronting campaign , where the health effects you can expect from smoking regarding cancer are made clear , visually . in fact , few participants were aware of the conceptual distinction between personal determinants ( psychological variables that can be influenced to eventually engender behavior change , such as risk perception or self - efficacy ) , behavior change methods or techniques ( theoretical methods to influence a given determinant , such as fear appeals or modeling ) , and applications of these methods ( an incarnation of a theoretical method , such as a picture of blackened lungs or a movie with a role model ) . one participant who was partially aware of this distinction explained : but then the step from those modifiable determinants to good methods to change that , kind of , that is often difficult . ( intervention developer , 22612 ) this lack of knowledge about methods for behavior change was one of the reasons for the use of threatening communication : there were no known good alternatives . some other participants had naive ideas of behavior change methods that carried a high risk of yielding non evidence - based results . however , these target population members generally advocated threatening communication approaches : yes , or , they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign . , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . ( intervention developer , 21210 ) yes , or , they had experience with these kind of campaigns where young people are in a panel that we use for developing such a campaign yes , no , it has to be more serious . among employees of advertising agencies and policymakers , the belief existed that there are no ways to predict in advance whether a campaign would be successful : and can you estimate a bit , when you 've made many campaigns , whether campaigns work or not ? ( advertising professional , 9416 ) intervention development was considered largely a trial - and - error endeavor , with the development process often being seen as a black box ( in advertising , a creative process engaged in by the creatives ) . well , for now that is mainly in narrative form , it 's mainly about statements of people , to go start and maintain a dialogue with young people and teachers and other involved people , like , well , did it touch you , did it do something for you ? well , for now that is mainly in narrative form , it 's mainly about statements of people , to go start and maintain a dialogue with young people and teachers and other involved people , like , well , did it touch you , did it do something for you ? participants indicated that when politicians or policymakers pressed for interventions that addressed a given problem , they would often prioritize quick and clearly visible results over effective evidence - based interventions ( note that this fits well with advertising agencies ' drive for originality ) : and in practice people then start to do something , eh , because the municipality also has to show that something is happening , and politics also asks for it , and also asks for visible activities and results preferably . when participants were not in favor of threatening communication , their reasons were not always based on the understanding of the inefficacy of threatening communications . one participant stated that there was a risk that the negative affect would be associated with the health - promoting organization where the intervention originated , which might decrease donations or susceptibility to future messages . the most common argument for this need for confrontation was rooted in the fact that attention is a first necessary condition for further processing , and attention is a limited resource . ( intervention developer , 191110 ) emotion was also assumed to render interventions memorable , which , combined with the memory of the evoked emotion itself , was assumed to prompt desirable behavior : and why do you have to strike an emotion ? ( advertising professional , 51183 ) another common belief was that when a communication manages to evoke emotions in target population members , this would cause reflection about the relevant behavior : for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . ( politician , 191115 ) for example , i spoke to a doctor , a doctor for young people from the municipal health centre , and he had a parent meeting at school , in group 7/8 [ ages 1012 ] , and there they showed a brain scan with pictures . for example , one scientist explained : i would , if i would read , like , it gives you lung cancer ( scientist , 4119 ) i would , if i would read , like , it gives you lung cancer ( scientist , 4119 ) also related , achieving awareness ( or increasing risk perception to a certain threshold ) was often considered a sufficient condition for behavior change : you try to increase this motivation to quit . thus , participants assumed that generally even target population members who are aware of a causal link between a behavior and a negative consequence would under - estimate the severity of the negative consequence . in general , it could be said that interviewees seemed to have some pieces of the puzzle , but lacked a coherent framework of the working mechanisms of threatening communication . ( scientist , 4119 ) when logical arguments against the use of threatening communication were acknowledged , this ineffectiveness was sometimes attributed to specific characteristics of the situation at hand , often the nature of the behavior at hand ( e.g. while this reasoning is partly in line with eppm , for example , one participant remarked : yes , this [ picture of blackened lungs ] is ok , because you can do something about it by quitting smoking , so , um , i mean , if you would say , breathing causes lung cancer ( advertising professional , 22114 ) yes , this [ picture of blackened lungs ] is ok , because you can do something about it by quitting smoking , so , um , i mean , if you would say , breathing causes lung cancer [ that would be a different story ] . similarly , often participants indicated that they did not use threatening communication , while from their explanation it became clear that they did : [ discussing a campaign against smoking that would focus on cancer ] some people may call this a fear inducing message i do n't call this fear inducing , because question one is , what is fear exactly and what is fear inducing ? because people often say , well , the risk is not so bad , and well , cancer it is more of a confronting campaign , where the health effects you can expect from smoking regarding cancer are made clear , visually . in fact , few participants were aware of the conceptual distinction between personal determinants ( psychological variables that can be influenced to eventually engender behavior change , such as risk perception or self - efficacy ) , behavior change methods or techniques ( theoretical methods to influence a given determinant , such as fear appeals or modeling ) , and applications of these methods ( an incarnation of a theoretical method , such as a picture of blackened lungs or a movie with a role model ) . one participant who was partially aware of this distinction explained : but then the step from those modifiable determinants to good methods to change that , kind of , that is often difficult . ( intervention developer , 22612 ) this lack of knowledge about methods for behavior change was one of the reasons for the use of threatening communication : there were no known good alternatives . , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . , last year , we had a study done into interventions targeting young immigrants , who were also like yes , we actually do n't want these things addressed with humor , but actually it should just be , not too hard , but confronting , because bad things can happen if you have unsafe sex . among employees of advertising agencies and policymakers , the belief existed that there are no ways to predict in advance whether a campaign would be successful : and can you estimate a bit , when you 've made many campaigns , whether campaigns work or not ? ( advertising professional , 9416 ) intervention development was considered largely a trial - and - error endeavor , with the development process often being seen as a black box ( in advertising , a creative process engaged in by the creatives ) . well , for now that is mainly in narrative form , it 's mainly about statements of people , to go start and maintain a dialogue with young people and teachers and other involved people , like , well , did it touch you , did it do something for you ? well , for now that is mainly in narrative form , it 's mainly about statements of people , to go start and maintain a dialogue with young people and teachers and other involved people , like , well , did it touch you , did it do something for you ? participants indicated that when politicians or policymakers pressed for interventions that addressed a given problem , they would often prioritize quick and clearly visible results over effective evidence - based interventions ( note that this fits well with advertising agencies ' drive for originality ) : and in practice people then start to do something , eh , because the municipality also has to show that something is happening , and politics also asks for it , and also asks for visible activities and results preferably . when participants were not in favor of threatening communication , their reasons were not always based on the understanding of the inefficacy of threatening communications . one participant stated that there was a risk that the negative affect would be associated with the health - promoting organization where the intervention originated , which might decrease donations or susceptibility to future messages . the first perceived goal of emotions was to draw attention to the intervention and prompt self - reflection , which would then lead to the desirable behavior , because target population members were assumed to act rationally on hypothesized ( successful ) increments in risk perceptions . it is clear that the decades of communication by behavior change scientists regarding the ineffectiveness of health threats has had results , as most intervention developers got the gist of it , albeit usually not more than the gist which still leaves many opportunities for threatening communications . at the same time , the choice for threatening communication was also based on some characteristics of behavior change communications that were consistent with the scientific evidence : it is necessary to draw attention to an intervention , and the interventions do have to inspire self - reflection to a degree . some intervention developers also relied on advertising agency employees , who lacked knowledge on behavior change methods ( and those advertising agency employees following the literature in their field may in fact be in favor of threatening communication ; see for example latour , snipes , & bliss , 1996 ) . integrating these various aspects of the decision - making process , some possible actions emerge that may be taken to decrease the use of threatening communication . such a toolbox is available , for example , in intervention development protocols such as intervention mapping ( kok , bartholomew , parcel , gottlieb , & fernandez , 2014 , this issue ; see also bartholomew et al . , 2011 ) and the methodology being developed based on the behavior change techniques ( bct ) taxonomy ( abraham & michie , 2008 ) and the behavior change wheel ( michie et al . , 2011 ) second , it seems useful to educate intervention developers on two psychological knowledge elements : firstly , it is unwise to consider humans rational ( kahneman & tversky , 1979 ) ; and secondly , threatening information averts attention ( bar - haim et al . , severity and susceptibility ) is not the most expedient intervention target ( but note that when the target of the threat is somebody else , such as one 's children , threat does seem effective ) . to this end , providing intervention developers with an overview of the evidence regarding the behavior change potential of threatening information can be helpful . note that in compiling guidelines for attracting attention , it is crucial to respect the parameters of effectiveness of behavior change methods ( bartholomew et al . an intervention implementing these elements may succeed in eradicating the widespread reliance on threatening interventions , paving the way to the use of more effective behavior change methods . in addition , the findings from the current study should be corroborated by quantitative studies , so that the pertinent beliefs of these groups of key actors can be mapped in terms of their relative importance .

in voltage - dependent sodium , potassium , and calcium channels , the voltage dependence of gating has usually been attributed to that intrinsic part of the protein molecule known as the voltage sensor ( s4 region ) , although a number of very recent studies have reported some dependence on the presence and nature of the permeating cation ( yellen , 1997 ) . for example , lower than normal concentrations of external potassium substantially reduce the open probability of some k channels . by comparison , cloning and sequencing of the voltage - dependent clc family of cl channels has failed to reveal an s4 type of voltage sensor and experiments on the torpedo cl channel , clc-0 , indicate that there is little or no contribution to gating from intrinsic protein charge movement ( chen and miller , 1996 ) . indeed , for two family members , clc-0 , and the skeletal muscle cl channel , clc-1 , a number of experiments in which cl has been replaced by glucose or by other anions suggest that activation of these channels is mainly controlled by the presence and concentration of external cl ( pusch et al . these authors submit that the permeant anion also serves as a ligand that regulates channel opening when it binds to a specific intra - pore site . for a constant external cl concentration , the availability of extracellularly derived cl at the regulatory site depends on the transmembrane electric field and this confers the experimentally observed voltage dependence upon gating , no intrinsic voltage sensing component of the channel protein being required . similar experiments using methanesulfonate as the impermeant cl substitute along with the effect of a mutation in clc-1 ( d136 g ) , meanwhile , led fahlke et al . ( 1995 , 1996 ) to conclude that d136 is part of a voltage sensor that controls gating , this voltage sensor possibly being subject to electrostatic perturbation by anion binding within the channel . mutations in clc-0 ( ludewig et al . , 1997 ) and clc-1 ( fahlke et al . , 1997c ) at very different positions , however , cause the same inwardly rectifying gating as d136 g , making it unlikely that this residue serves as a voltage sensor . in their latest interpretation of gating , fahlke et al . ( 1997b ) endorse the functional linkage between ion permeation and gating , but maintain their different viewpoint with respect to the involvement of a voltage sensor . they argue that the voltage responsiveness resulting from voltage - dependent conformational changes can be modulated by permeant ions , and hence be modified by the presence of foreign anions . methanesulfonate , which has often been used as the preferred impermeant cl substitute because of its negligible permeability and apparent lack of interaction with cl in mole fraction substitution studies of membrane conductance in the rat diaphragm muscle ( palade and barchi , 1977 ) . other anions ( br , i , so4 , and methylsulphate ) tested by these authors , appeared to interact to varying degrees with cl , displaying nonlinear and anomalous mole fraction effects on membrane conductance when substituted for cl . similar effects of foreign anion substitution on fish skeletal muscle had been demonstrated previously ( hagiwara and takahashi , 1974 ) . early work on frog muscle with halide and no3 substitution defined the substantial specificity of skeletal muscle anion channels for cl over other anions ( for review see bretag , 1987 ) . woodbury and miles ( 1973 ) tested an extensive range of foreign anions on frog muscle and found that they could be separated into two groups according to how they affected the ph dependence of membrane conductance . for cl itself and chloride - like anions , membrane conductance decreased at low ph , while , for benzoate - like anions , it increased . more recently , the effects of foreign anions on cl channels of tissues other than muscle have been analyzed at the single channel level ( bormann et al . , 1987 ; halm and frizzell , 1992 ; linsdell et al . , 1997a ; tabcharani et al . , 1997 ) and , based on the extensive kinetic information obtained , substantial models of multi - ion pores have been developed ( bormann et al . , 1987 ; halm and frizzell , 1992 ; linsdell et al . , 1997b ) . the skeletal muscle cl channel , clc-1 , has a conductance that is too small to allow observation of single channel currents ( pusch et al . . nevertheless , investigation of ensemble currents can provide valuable insight into the gating and permeation characteristics of this channel ( steinmeyer et al . ; astill et al . , 1996 ; fahlke et al . , 1996 ; rychkov et al . , 1996 ; fahlke et al we have investigated the influence of foreign anions on current kinetics , apparent popen permeability , and conductance of clc-1 channels that allow a number of predictions to be made about the nature of the channel pore . rat clc-1 ( rclc-1 ) was expressed in sf9 ( a spondoptera frugiperda insect cell line ) cells as described in detail previously ( astill et al . , 1996 ) . cultured sf9 cells were infected with baculovirus bvda6.3 containing rclc-1 cdna and incubated for 2830 h at 28c in air . after incubation , infected cells were seeded onto glass coverslips and maintained at room temperature until required . whole - cell patch - clamp experiments were performed on sf9 cells between 28 and 34 h postinfection using an epc7 patch - clamp amplifier and associated standard equipment ( list electronic , darmstadt , germany ) . the usual bath solution contained ( mm ) : 170 nacl , 2 mgso4 , 2 ca - gluconate , and 10 hepes , adjusted to ph 7.5 with naoh . lower concentrations of cl in the bath solution were achieved by equimolar substitution of 5 , 10 , 25 , 50 , 75 , 90 , or 100% of external cl by a particular anion ; for impermeant anions the highest concentration in the bath solution was 95% . electrodes were made of borosilicate glass and had a resistance of 13 m when filled with a normal internal solution containing ( mm ) : 40 kcl , 120 k - glutamate , 10 egta - na , and 10 hepes , adjusted to ph 7.2 with naoh . in some experiments , the concentration of nacl in the bath solution was raised to 340 mm , in which case the concentrations of kcl and k - glutamate in the internal solution were doubled . when hco3 was used to substitute for cl , bath hepes concentration was increased to 20 mm and the solution was used within 20 min . to reduce possible acidification of the internal medium due to co2 diffusion into the cell , 120 mm k - glutamate in the internal solution was replaced with equimolar tris - glutamate , a substitution that , of itself , had no effect on clc-1 channel properties . when necessary , pentobarbitone ( 0.5 mm ) , added to the bath solution , was used to block native anion channels in sf9 cells ( birnir et al . , 1992 ) . data were collected , filtered at 3 or 10 khz , and analyzed on an ibm - compatible pc using pclamp v6.0 software ( axon instruments , foster city , ca ) . liquid junction potentials between the bath and electrode solutions were estimated by using jpcalc ( barry , 1994 ) and corrected where specified . experiments were conducted at a room temperature of 24 1c . to obtain permeability ratios , membrane potentials for at least seven different concentrations of each foreign anion were measured in current clamp mode . shifts in potential ( relative to control cl solution ) were plotted against mole fraction of the anion and fitted with a goldman - hodgkin - katz equation of the form : 1\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*}y=58.4\;log\;\;\{1/[(1-m_{f})+am_{f}]\},\end{equation*}\end{document } where y is the shift of the membrane potential , mf ( mole fraction ) is the concentration of foreign anion , x , relative to the control cl concentration ( mf = [ x]/[x ] + [ cl ] ) , and a = ( px/ pcl ) is the permeability ratio for this anion relative to cl . to determine relative conductances in the presence of external foreign anions , outward conductance was estimated as a chord conductance ( approximating the slope conductance ) from the outward current interpolated at a point 20 mv to the right of the reversal potential on the current voltage ( i - v ) plot . we constructed these i - v plots from steady state currents obtained at the end of 100-ms test voltage pulses . tail currents for voltage steps to 100 mv after 150-ms test pulses in the range from 160 to + 100 mv were extrapolated back to the beginning of the pulse by fitting with two exponentials plus a constant ( rychkov et al . , 1996 ) . instantaneous currents so obtained were used to produce apparent popen curves by fitting with a boltzmann distribution of the form : 2\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*}x(v)=x_{o}+(1-x_{o})/\{1+\;exp[(v_{1/2}-v)/k]\},\end{equation*}\end{document } where xo is an offset , v is the membrane potential , v1/2 is the potential at which x = ( 1 + xo)/2 , and k is the slope factor . the slope factor k was used to calculate the apparent gating valence z , ( z = 25.4/k ) . in these experiments , the bath solution contained ( mm ) : 100 nax ( x = cl , br , etc . , or 50 cl + 50 i ) , 2 mgcl2 , 5 hepes , adjusted to ph 7.3 with naoh , and the pipette solution contained ( mm ) : 100 nacl , 2 mgcl2 , 5 egta , 5 hepes , adjusted to ph 7.3 with naoh . inside - out patches containing hclc-1 were also produced from xenopus oocytes . in this case , the pipettes were filled with control bath solution containing ( mm ) : 100 nacl , 2 mgcl2 , 5 hepes , adjusted to ph 7.3 with naoh , and the patch was exposed to ( mm ) : 100 nacl , 2 mgcl2 , 5 egta , 5 hepes , adjusted to ph 7.3 with naoh . where specified , various fractions of the 100 mm nacl were replaced by equimolar amounts of nabr or nai . various foreign anion substitutions were first tested against the effects of nacl replacement by glucose . our presumptions were that permeation through clc-1 would not be influenced by glucose , that glucose would not interact with any regulatory site or any process modulating clc-1 , and that anion permeation through clc-1 could be satisfactorily monitored without interference from any other permeation pathways through the sf9 cell membrane . when nacl in the external solution was replaced by glucose , the slope conductance of clc-1 , estimated near the equilibrium potential , showed saturation at physiological external cl concentrations , only becoming dependent on external cl at low concentrations ( fig . almost identical results were obtained when glutamate , gluconate , glucuronate , and diatrizoate were substituted for cl ( fig . 1 , relative permeabilities for these anions , obtained from the fit of the goldman - hodgkin - katz equation to shifts in membrane potential , were < 1% and could not be considered different from zero . substitution of cl by glutamate or glucose influenced current kinetics , increasing the rate of deactivation of inward currents , and shifted the apparent popen curve in the depolarizing direction , as we reported previously ( rychkov et al . , 1996 ) . both of these were dependent on the extent to which cl had been replaced . when other anions in this group were substituted for cl , they also made current deactivation faster and shifted the apparent popen curve to more positive potentials to the same extent as glutamate ( fig . two much smaller anions , hco3 and bro3 , although having a measurable permeability relative to cl ( table i ) had the same ( fig . 2 c ) or a similar ( fig . 2 d ) effect on current kinetics as the large impermeant organic anions from the previous group . they also shifted the apparent popen curves to more positive potentials without changing the gating charge ( table i ) , but unlike glutamate and other anions from that group they appeared to interact with cl inside the channel to some extent , since the relationship between conductance and concentration was different from that for glucose ( fig . bicarbonate was less permeant than bro3 ( table i ) and its effects were closer to those of glutamate : at 95% concentration , hco3 shifted v1/2 by +60 mv , while bro3 , at the same concentration , shifted v1/2 rather less , by +50 mv ( compare with the shift of +70 mv for glutamate ; table i ) . bromate showed stronger interaction with cl inside the channel than hco3 since conductance of the channel in the outward direction in the presence of bro3 was smaller than in the presence of hco3 ( figs . 1 and 2 , c and d ) . at the same time , bro3 weakly blocked inward current , while hco3 did not show any block ( compare the relative amplitudes of inward current in the presence of bro3 and hco3 ; fig . 2 ) . cyclamate and methanesulfonate were as impermeant as the other large organic anions , but they changed currents through clc-1 very differently from glutamate ( fig . 3 ) . cyclamate ( when substituting 95% of cl ) blocked 35% of inward current , shifted v1/2 by + 28 mv ( compared with +70 mv for glutamate ; table i ) , and increased the slope coefficient , k , of the popen curve , thus reducing the apparent gating charge , z , to 0.63 . methanesulfonate , at the same concentration , blocked up to 70% of inward current and substantially modified the kinetics of current deactivation . furthermore , the apparent popen curve was shifted in the opposite direction , to more negative potentials , by 45 mv , and the apparent gating charge was decreased ( fig . bromide , no3 , clo3 , i , clo4 , and scn fall into a separate category of foreign anions in regard to their action on clc-1 . with increasing amounts of br replacing cl in the external solution , the slope conductance , calculated for outward current near the reversal potential , decreased until just 15% of its initial value remained when 100% br substituted for cl ( fig . inward current amplitude ( at 120 mv ) was reduced by 50% , indicating that br blocks the clc-1 channel ( fig . 5 ) . the relative permeability for br , again obtained using the goldman - hodgkin - katz equation and data for different mole fractions of br , was 0.37 0.01 . there was no evidence of an anomalous mole - fraction effect on permeability for br . kinetics of current deactivation were not substantially altered , nor was there any significant shift in the voltage dependence of the apparent popen , as indicated by v1/2 ( table i ) . the main difference was an increased proportion of steady state current at negative potentials ( compare figs . 2 and 5 ) . 4 ) and , with relative permeabilities of 0.25 , were somewhat less permeant than br . in addition , they blocked up to 80% of the inward current at 120 mv when substituting 100% of the cl ( fig . apparent popen in the presence of either of these anions at 50% concentration was shifted to more negative potentials without significant change in the slope of the curve , but if the concentration of no3 or clo3 was increased up to 100% , the slope factor of the apparent popen curve was increased ( table i ) , implying a reduction in gating charge to 0.6 ( normally 1.2 ) . time constants of the deactivating components of the inward current were not appreciably changed by these anions , but the relative proportion of steady state current at 120 mv increased from 7 to 50% ( compare figs . 2 and 5 ) . although i was quite permeant , with a relative permeability of 0.22 0.02 , conductance of i ions through clc-1 was negligibly small and not readily distinguishable from leakage . inward current was blocked by 90% at 120 mv when all cl was replaced with i and current showed little deactivation at negative potentials ( fig . apparent popen curves obtained at lower mole fractions of i , where current deactivation at negative potentials was more obvious , showed that the voltage dependence of channel gating was shifted to more negative potentials by i and the apparent gating charge decreased ( table i ) . thiocyanate and clo4 at low concentrations ( up to 10% ) had effects very similar to i , they blocked inward current ( by 90% in the case of 10% scn ) , reduced current deactivation at negative potentials , shifted v1/2 to more negative potentials , decreased the slope of apparent popen curves ( table i ) , and shifted membrane potentials towards more positive values . when the concentration of scn ( or clo4 ) was raised above 10% , inward current was blocked almost completely , but the amplitude of the outward current started to increase and reached its maximum when all cl in the external solution was replaced with scn ( fig . 5 ) , and scn was more permeant than cl since membrane potentials were shifted to values more negative than the cl equilibrium potential ( fig . endogenous volume - regulated cl channels in sf9 cells were more permeable to scn than to cl and , furthermore , control cells showed outwardly rectifying currents in the presence of scn qualitatively similar to the currents recorded under the same conditions from the cells expressing clc-1 ( not shown ) . to make sure that the anomalous mole - fraction effect and outward rectification in solutions containing scn were not the result of increased activity of the native cl channels , concomitant with complete block of clc-1 channels , experiments with scn substitution for cl if currents recorded in the presence of scn were passing only through native channels , their amplitudes should not be much different from cell to cell and should not depend on the cl current amplitude through clc-1 . in reality , there was a high level of correlation between the amplitudes of outward currents , measured in cells expressing clc-1 , bathed in control cl solution , and when scn replaced cl ( fig . we can , therefore , have confidence that clc-1 is permeable to scn ( and , likewise , to clo4 ) and that outward currents recorded in the presence of scn are mainly due to conductance through clc-1 . external substitution of br , no3 , and i on hclc-1 expressed in xenopus oocytes had very similar effects to those described above for rclc-1 , although block by br , i , and methanesulfonate may have been slightly weaker ( table ii ) . 7 ) , performed on outside - out patches containing hclc-1 at 140 mv to gain information about the mechanism of this block , show ( table ii ) that single channel current amplitude is reduced by foreign anion substitution in the same proportion as whole - cell current . as illustrated in figs . 1 and 8 , conductance to outward currents is saturated at physiological cl concentrations with a kd of 6 mm . since it was not possible to change internal cl concentration during whole - cell patch - clamp experiments in sf9 cells , results from separate whole - cell experiments were combined after normalizing in the following way . for different cells , the ratio between chord conductance at 120 mv and current at + 80 mv was found to be constant for external cl concentrations from 170 to 340 mm and a fixed internal cl concentration , presumably reflecting the ratio between single channel currents at 120 and + 80 mv . similarly , ratios were calculated from results obtained using a variety of cl concentrations in the pipette , plotted against internal cl concentrations and fitted with a one - binding site hyperbola . the maximal ratio obtained from the fit was used to normalize the data points , making it possible to present the saturation curves for internal and external cl on the one graph ( fig . 8) . similar to its external application , br applied internally had little effect on apparent popen curves ( table iii ) . but unlike external br , which did not affect inward current kinetics , internal br slowed down current deactivation and appeared to have a stronger blocking action ( table ii indicates 20% block of hclc-1 and 40% block of rclc-1 from the outside , while fig . these estimates of block from attenuation of inward currents are not strictly comparable as br applied from inside blocks inward current to a greater extent than outward current , whereas the opposite is true for block by externally applied br . in each case , the block is voltage dependent . 9 ) than br , just as occurs when they are applied from the outside . in 100 mm intracellular i ( leaving 4 mm internal cl ) , inward currents through hclc-1 were almost completely blocked and it was impossible to extract parameters describing the voltage dependence . partial replacement of cl by i led to a slight shift of v1/2 to more positive values and to a decrease in the apparent gating charge , z. the shift of v1/2 is opposite to the effect of external i , whereas both external and internal i led to a reduction in gating charge ( table iii ) . in our receptor - operated model of gating in the clc-1 channel , open probability is controlled by a binding site for cl , or certain other anions , which is accessible only from outside and which must be correctly occupied for opening to occur ( rychkov et al . , 1996 ) . consistent with this model , anions we have studied can be divided into three groups according to their ability to open the channel and to permeate it : impermeant anions that are unable to open the channel , anions that can open the channel but can not permeate it , and anions that can both open the channel and permeate it . large anions such as glutamate , gluconate , and glucuronate belong to the first group . all of these cause a shift of the deactivation curve to positive potentials similar to that caused by glucose , which provides evidence that the shift in voltage dependence of the channel is due to the absence of cl rather than to the presence of the foreign anion . since glucose , presumably , does not interact with binding sites regulating the behavior of clc-1 , any difference between those relative conductances obtained when glucose substitutes for nacl and those where a foreign anion substitutes for cl , reflects interaction between cl and that anion in the pore . impermeant anions that do not interfere with the gating , such as glutamate and glucuronate , also do not change relative cl conductance . by contrast , hco3 , which has just 3% of cl permeability , clearly reduces cl conductance ( fig . 1 ) and shifts the apparent popen curve to less positive potentials than the impermeant glutamate . the slightly more permeant bro3 has more pronounced effects on channel conductance and gating . that gating is intrinsically linked to permeation and appears to be supported by the behavior of these anions . in the present work , however , we have found other anions , such as cyclamate and methanesulfonate , that can have a substantial effect on gating without being permeant . for example , cyclamate and bro3 have much the same effect on conductance and current kinetics and the amount of block of the inward current is also very similar , but while bro3 is permeant , cyclamate is impermeant and , in addition , it reduces the gating charge to 0.62 . these results suggest that cyclamate can bind ( more readily than bicarbonate or bro3 ) to the regulatory binding site that opens the channel and that this site lies external to the selectivity filter , which cyclamate can not pass . thus , gating is not always coupled to permeation , probably because specificity of the pore selectivity filter differs from that of the site within the pore that regulates channel opening . it could be that when blocking anions , such as methanesulfonate , are bound to the latter site , they hinder cl passage through the pore . alternatively , although less likely ( see below ) , block could be due to binding at some second site ( perhaps the selectivity filter ) , which must also be deep inside the pore , as indicated by diminished inward current block at negative potentials . in these ways , clc-1 appears to differ from clc-0 , not because of any profound difference in their mechanisms of fast gating , but , probably , because the selectivity filter and regulatory binding site have very similar specificity in clc-0 , which allows channel activation to be closely linked to permeation , whereas these specificities are different in clc-1 with the consequences that this entails . small inorganic anions all showed potential - dependent block at low concentrations in the sequence : br < no3 < clo3 < i < clo4 < scn , which coincides with the ability to shift the voltage dependence of gating , to modify current kinetics , and also coincides with the lyotropic series . for br , no3 , i , and methanesulfonate , we have good evidence from noise analysis that all reduction in current amplitude is due to a reduction in single channel current . in the lyotropic series , anions are arranged in order according to their ability to bind or adsorb to proteins , to potentiate the strength of a muscle twitch , and to unwind macromolecules . it is believed that , for binding to follow the lyotropic series , a site requires the combination of two molecular attributes : appropriate anion - attracting groups and neighboring hydrophobic groups ( dani et al . , 1983 ) . if conductance is limited by binding inside the pore , then , the stronger the binding , the smaller the conductance . this seems to be the case when scn concentration is low , where strong binding to only one of several intrapore binding sites is sufficient to block the channel . possibly , as concentration is increased , all intrapore binding sites are occupied by scn , and electrostatic repulsion between these ions accelerates the rate - limiting exit steps . when all cl outside is replaced by scn , positive potentials applied from the inside reduce the probability of internal cl entering the channel , so the channel starts to rectify outward just as it also rectifies inward at strongly negative potentials when all scn is likely to be displaced from the channels . as is typical of the anomalous mole fraction effects seen in pores with multiple intrapore binding sites , the lowest conductance occurs when a mixture of ions is present in the channel . an anomalous mole - fraction effect for mixtures of cl and scn is not unique for clc-1 . some other cl channels , such as the -aminobutyric acid ( gaba ) and glycine receptor channels ( bormann et al . , 1987 ) and the cystic fibrosis transmembrane conductance regulator ( tabcharani et al . , 1993 ) , have similar properties . with a kd of 6 mm , outward cl conductance for outward currents through clc-1 is saturated at physiological external cl concentrations . saturation is presumed to arise when the binding unbinding steps of permeation become rate limiting ( rate of ion entry approaches the maximum rate for the unbinding steps ) . for inward currents , the kd is 33 mm , which suggests that there is a deeper energy well for cl ions passing through the channel from the external side than for those passing from the inside . this can be interpreted in terms of the rate limiting binding site being closer to the external side . when positive potential is applied from inside , this binding site is occupied by cl , but other intrapore sites will be free . conductance is then very low due to strong binding of cl to this site . when cl is moving out under the influence of a positive potential on the outside , all binding sites in the channel will be occupied . electrostatic repulsion between cl ions can then accelerate the rate - limiting exit step and increase conductance ( hille , 1992 ) . in this way , open channel rectification and the different kds on either side of the membrane may be explained . the effects of foreign anions applied to the inside are also different from their effects on the outside . from inside , br and i block inward current without affecting outward current , whereas , from outside , both currents are blocked . also , from the inside , i shifts the apparent popen curve in the opposite direction , to more positive potentials . these results are all consistent with and extend the view of clc-1 as a channel with multiple internal binding sites having different characteristics and , especially , they support the proposal that there is at least one inner and one outer binding site inside the channel that are quite distinct from each other ( see fahlke et al . , 1997b ) . > hco3 ( f ) , while just for halides it was cl > br > i > f. this sequence corresponds to a cationic site of moderately strong field strength in the membrane ( eisenman sequence 4 ; eisenman , 1965 ) . however , not only electrostatic but also hydrophobic interactions are involved in anion permeation through clc-1 , and so the permeability sequence need not be directly related to the electrostatic field strength of the relevant binding site . from blocking ability when applied externally , the binding sequence is scn > br > bro3 > hco3 ( f ) , which corresponds to the low field strength eisenman sequence 1 ( eisenman , 1965 ) . these results agree with early studies of foreign anion permeability ( woodbury and miles , 1973 ) and of block of cl efflux ( harris , 1958 ) by foreign anions in frog muscle where the same sequence difference occurred . this would indicate that the site accessible from outside , at which block occurs , is different from the site governing selectivity and because blocking ability corresponds to opening ability , that block probably occurs at the regulatory binding site . the effects of some of the anions studied in this work on rat clc-1 have been investigated in human clc-1 by fahlke et al . ( 1997a ) , who obtained a similar sequence for relative permeability : cl scn > br > ( 1997a ) were somewhat higher than in our work , which can be explained by differences in internal cl concentration . in a multi - ion pore although for halides the differences were relatively small , methanesulfonate was found to be impermeant in rat clc-1 , while in human clc-1 , pmetsulf / pcl was 0.2 . with impermeant anion substitution , we have found that the sf9 cells can not for long sustain membrane potentials as highly positive as +80 mv with 140 mm of cl inside and only 8 mm of cl remaining outside . as external cl was replaced with impermeant anion to achieve this condition , membrane potential increased to approximately the predicted + 80 mv , but soon dropped to less positive values due to increased leakage . we therefore generally used a lower concentration of cl ( 40 mm ) in the internal solution . the difference in results might also be accounted for by cl accumulation near the external mouth of the channel , which could be higher at higher internal cl concentration . it is also possible that the inconsistencies could be due to a real difference between rat and human clc-1 . some results , such as nondeactivating currents at negative potentials in the presence of i , are similar in both studies but explained differently . ( 1997b ) sug - gest that inherently voltage - dependent conformational changes are modulated by i , which locks the channel in a nondeactivating state . while we do not dispute that i might lock the channel in the open state , evidence is lacking for the existence of conformational changes dependent upon an intrinsic voltage sensor in clc channels ( see introduction ) . our interpretation is , instead , based on a mechanism ( pusch et al . 1995a ; rychkov et al . , 1996 ) that is more akin to gating in receptor - operated channels . hence , a binding site that has a higher affinity for i than for cl will maintain channel opening in the presence of i , achievable negative potentials being unable to displace i from the site . estimates of the dimensions of a number of cation and anion channels have been made using a simple cylindrical pore model ( hille , 1975 ; bormann et al . , 1987 ) relative permeabilities can be described by the equation : 3\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*}p_{x}/p_{cl}=c / d(1-d / a)^{2},\end{equation*}\end{document } where c is a constant , a is the pore diameter , and d is the stokes diameter for the particular ion . although for the clc-1 channel this equation could not reliably be fitted to the experimental points , the relative permeability of anions included in the present study clearly decreased with increasing size of the anion ( fig . is still measurably permeant and methanesulfonate ( 5 ) is not , this might place an upper limit on the pore diameter . for comparison , stokes diameters of the permeant hydrophobic anions is significantly bigger , up to 6 for hexanoate . however , if those anions are approximated by a cylinder , their cylindrical diameter is 4.3 ( halm and frizzell , 1992 ) . still , they are much more permeant than hco3 and their relative permeability increases along with the size of the hydrophobic part of the molecule , suggesting the likelihood of significant hydrophobic interactions with the wall of the pore ( see woodbury and miles , 1973 , for similar results on frog muscle ) . as well as having an anomalous relationship between permeability and size , these hydrophobic anions have complicated effects on clc-1 channel gating , which will be described in detail elsewhere . it may well be that the aromatic monocarboxylate blockers ( bretag , 1987 ; astill et al . 1997 ) have their effect due to their ability to enter the pore because of their charge but interact very strongly with the pore wall . for several anion channels that were studied at the single channel level , multiple binding site models have been developed based on extensive kinetic data ( bormann et al . , 1987 ; halm and frizzell , 1992 ; linsdell et al . , 1997b ) . it is apparent that very similar models of anion permeation could account for the characteristics of clc-1 , but that a detailed quantitative description of such a model must await more information at the single channel level . in conclusion , we have shown ( a ) the multi - ion nature of clc-1 with demonstrable anomalous mole fraction effects of scn and clo4 ; ( b ) two of the anion binding sites , an inner and an outer site , are accessible from the cytoplasmic solution and from the external solution , respectively , and have different binding properties even for cl ; ( c ) the outer binding site appears to have characteristics that make it the prime candidate for regulation of channel opening and for channel block by external anions , but it is not simultaneously the site that determines channel selectivity ; ( d ) block of macroscopic currents by foreign anions can be accounted for entirely by a reduction in single channel conductance without any requirement for an effect on popen ; ( e ) minimum pore diameter is 4.5 ; and ( f ) foreign anions can be clearly categorized into three different groups according to their interaction with clc-1 . membrane conductance of sf9 cells expressing rclc-1 when impermeant or poorly permeant anions replace cl in the external solution . relative conductance , gx + cl / gcl , is plotted against mole fraction , [ x]/([x]+[cl ] ) , of the foreign anion . kinetics of whole - cell currents in sf9 cells expressing rclc-1 when impermeant or poorly permeant anions replace 95% of cl in the external solution . currents were elicited by the standard activation protocol from a holding potential of 30 mv with a prepulse to + 40 mv followed by steps from 120 to + 80 mv in 20-mv increments . since experiments were performed on different cells , currents have been normalized to the peak inward current in cl solution at 120 mv . peak inward currents from cell to cell ranged between 5 and 10 na . relative permeabilities for different foreign anions and their effect on the parameters of the popen curves voltages have been corrected for liquid junction potentials ( see methods ) . the same number of experiments was performed with a particular anion for each different percent chloride replacement . 2 ) and 95% of external cl was replaced by ( a ) cyclamate or ( b ) methanesulfonate . apparent popen curves ( c ) are plotted for control ( ) , methanesulfonate ( ) and cyclamate ( ) . membrane conductance of sf9 cells expressing rclc-1 when various permeant anions replace cl in the external solution . relative conductance has been plotted against mole fraction of foreign anion as for fig . kinetics of whole - cell currents in sf9 cells expressing rclc-1 when various permeant anions replace 100% of cl in the external solution . amplitudes of outward current in cl solution and in scn solution were found to be closely correlated ( a ) . anomalous mole fraction effects were apparent for both permeability and conductance ( b ) . relative conductance ( ) in this case was determined from the chord conductance measured at + 80 mv . foreign anion block of macroscopic currents , i , from whole - cell studies and of single channel currents , i , estimated by noise analysis for ease of comparison , peak inward currents for a step to 140 mv in the presence of foreign anions have been normalized to the current in external chloride solution for rc1c-1 and hc1c-1 as appropriate . currents , ix and ix , are inward currents carried by internal chloride in the presence of external anion x. these anions were substituted in equimolar amounts for 127.5 mm cl ( 75% of external cl substituted ) in experiments on rclc-1 expressed in sf9 cells and for 100 mm cl ( 98% of external cl substituted ) in experiments on hclc-1 expressed in xenopus oocytes . nonstationary noise analysis performed on outside - out patches from xenopus oocytes expressing hclc-1 in the presence of cl , no3 , i , or br . variances ( top ) and means ( middle ) of current records ( n = 150 ) are shown for standard voltage protocols incorporating a prepulse to + 60 mv followed by a 70-ms test pulse to 140 mv . ( bottom ) variance mean current plots are fitted with parabolas according to the equation : = 0 + ii i /n , where 0 is baseline noise variance , i is single channel current , i is mean current , and n is number of channels . fitted values for the parabolas shown are : ( a ) for chloride , i = 0.26 pa ; ( b ) for nitrate , i = 0.1 pa ; ( c ) for 50% iodide/50% chloride , i = 0.077 pa ; ( d ) for bromide , i = 0.21 pa . relationship between cl conductance and concentration in rclc-1 . in this case , cl was replaced by glucose in the external solution but by glutamate in the internal solution . from fig . 1 , it can be seen that the effects of glucose or glutamate replacement in the external solution are almost identical . parameters of apparent popen curves from inside - out patches of xenopus oocytes after internal application of foreign anions kinetics of currents in inside - out patches from xenopus oocytes expressing hclc-1 when br and i are substituted for cl in the internal solution . a shows control currents in the patch to which 100% br was then applied ( b ) , while c shows control currents in the patch to which 50% i was later applied ( d ) . voltage protocol : after a 50-ms prepulse to + 60 mv , the membrane potential was stepped for 150 ms from 140 to + 80 mv in 20-mv increments , followed by a step to 100 mv for 50 ms . dependence of relative permeability of rclc-1 expressed in sf9 cells on the apparent ionic diameter of various anions . apparent ionic diameters , d , were calculated from the einstein - stokes relation , d = 183.6/ , where is the limiting conductance for the ion ( robinson and stokes , 1959 ) . relative permeability for f was determined from the biionic potential with 160 mm of f in the internal solution and 170 mm of cl in the external solution .

a distinctive feature of the voltage - dependent chloride channels clc-0 ( the torpedo electroplaque chloride channel ) and clc-1 ( the major skeletal muscle chloride channel ) is that chloride acts as a ligand to its own channel , regulating channel opening and so controlling the permeation of its own species . we have now studied the permeation of a number of foreign anions through clc-1 using voltage - clamp techniques on xenopus oocytes and sf9 cells expressing human ( hclc-1 ) or rat ( rclc-1 ) isoforms , respectively . from their effect on channel gating , the anions presented in this paper can be divided into three groups : impermeant or poorly permeant anions that can not replace cl as a channel opener and do not block the channel appreciably ( glutamate , gluconate , hco3 , bro3 ) ; impermeant anions that can open the channel and show significant block ( methanesulfonate , cyclamate ) ; and permeant anions that replace cl at the regulatory binding site but impair cl passage through the channel pore ( br , no3 , clo3 , i , clo4 , scn ) . the permeability sequence for rclc-1 , scn clo4 > cl > br > no3 clo3 > i > > bro3 > hco3 > > methanesulfonate cyclamate glutamate , was different from the sequence determined for blocking potency and ability to shift the popen curve , scn clo4 > i > no3 clo3 methanesulfonate > br > cyclamate > bro3 > hco3 > glutamate , implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side . channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance . minimum pore diameter was estimated to be 4.5 . anomalous mole - fraction effects found for permeability ratios and conductance in mixtures of cl and scn or clo4 suggest a multi - ion pore . hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size .

introduction methods results discussion Figures and Tables

in voltage - dependent sodium , potassium , and calcium channels , the voltage dependence of gating has usually been attributed to that intrinsic part of the protein molecule known as the voltage sensor ( s4 region ) , although a number of very recent studies have reported some dependence on the presence and nature of the permeating cation ( yellen , 1997 ) . by comparison , cloning and sequencing of the voltage - dependent clc family of cl channels has failed to reveal an s4 type of voltage sensor and experiments on the torpedo cl channel , clc-0 , indicate that there is little or no contribution to gating from intrinsic protein charge movement ( chen and miller , 1996 ) . indeed , for two family members , clc-0 , and the skeletal muscle cl channel , clc-1 , a number of experiments in which cl has been replaced by glucose or by other anions suggest that activation of these channels is mainly controlled by the presence and concentration of external cl ( pusch et al . these authors submit that the permeant anion also serves as a ligand that regulates channel opening when it binds to a specific intra - pore site . for a constant external cl concentration , the availability of extracellularly derived cl at the regulatory site depends on the transmembrane electric field and this confers the experimentally observed voltage dependence upon gating , no intrinsic voltage sensing component of the channel protein being required . , 1997 ) and clc-1 ( fahlke et al . ( 1997b ) endorse the functional linkage between ion permeation and gating , but maintain their different viewpoint with respect to the involvement of a voltage sensor . they argue that the voltage responsiveness resulting from voltage - dependent conformational changes can be modulated by permeant ions , and hence be modified by the presence of foreign anions . other anions ( br , i , so4 , and methylsulphate ) tested by these authors , appeared to interact to varying degrees with cl , displaying nonlinear and anomalous mole fraction effects on membrane conductance when substituted for cl . more recently , the effects of foreign anions on cl channels of tissues other than muscle have been analyzed at the single channel level ( bormann et al . , 1996 ; fahlke et al we have investigated the influence of foreign anions on current kinetics , apparent popen permeability , and conductance of clc-1 channels that allow a number of predictions to be made about the nature of the channel pore . to reduce possible acidification of the internal medium due to co2 diffusion into the cell , 120 mm k - glutamate in the internal solution was replaced with equimolar tris - glutamate , a substitution that , of itself , had no effect on clc-1 channel properties . shifts in potential ( relative to control cl solution ) were plotted against mole fraction of the anion and fitted with a goldman - hodgkin - katz equation of the form : 1\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*}y=58.4\;log\;\;\{1/[(1-m_{f})+am_{f}]\},\end{equation*}\end{document } where y is the shift of the membrane potential , mf ( mole fraction ) is the concentration of foreign anion , x , relative to the control cl concentration ( mf = [ x]/[x ] + [ cl ] ) , and a = ( px/ pcl ) is the permeability ratio for this anion relative to cl . to determine relative conductances in the presence of external foreign anions , outward conductance was estimated as a chord conductance ( approximating the slope conductance ) from the outward current interpolated at a point 20 mv to the right of the reversal potential on the current voltage ( i - v ) plot . almost identical results were obtained when glutamate , gluconate , glucuronate , and diatrizoate were substituted for cl ( fig . they also shifted the apparent popen curves to more positive potentials without changing the gating charge ( table i ) , but unlike glutamate and other anions from that group they appeared to interact with cl inside the channel to some extent , since the relationship between conductance and concentration was different from that for glucose ( fig . bicarbonate was less permeant than bro3 ( table i ) and its effects were closer to those of glutamate : at 95% concentration , hco3 shifted v1/2 by +60 mv , while bro3 , at the same concentration , shifted v1/2 rather less , by +50 mv ( compare with the shift of +70 mv for glutamate ; table i ) . at the same time , bro3 weakly blocked inward current , while hco3 did not show any block ( compare the relative amplitudes of inward current in the presence of bro3 and hco3 ; fig . cyclamate ( when substituting 95% of cl ) blocked 35% of inward current , shifted v1/2 by + 28 mv ( compared with +70 mv for glutamate ; table i ) , and increased the slope coefficient , k , of the popen curve , thus reducing the apparent gating charge , z , to 0.63 . bromide , no3 , clo3 , i , clo4 , and scn fall into a separate category of foreign anions in regard to their action on clc-1 . with increasing amounts of br replacing cl in the external solution , the slope conductance , calculated for outward current near the reversal potential , decreased until just 15% of its initial value remained when 100% br substituted for cl ( fig . there was no evidence of an anomalous mole - fraction effect on permeability for br . apparent popen in the presence of either of these anions at 50% concentration was shifted to more negative potentials without significant change in the slope of the curve , but if the concentration of no3 or clo3 was increased up to 100% , the slope factor of the apparent popen curve was increased ( table i ) , implying a reduction in gating charge to 0.6 ( normally 1.2 ) . apparent popen curves obtained at lower mole fractions of i , where current deactivation at negative potentials was more obvious , showed that the voltage dependence of channel gating was shifted to more negative potentials by i and the apparent gating charge decreased ( table i ) . endogenous volume - regulated cl channels in sf9 cells were more permeable to scn than to cl and , furthermore , control cells showed outwardly rectifying currents in the presence of scn qualitatively similar to the currents recorded under the same conditions from the cells expressing clc-1 ( not shown ) . to make sure that the anomalous mole - fraction effect and outward rectification in solutions containing scn were not the result of increased activity of the native cl channels , concomitant with complete block of clc-1 channels , experiments with scn substitution for cl if currents recorded in the presence of scn were passing only through native channels , their amplitudes should not be much different from cell to cell and should not depend on the cl current amplitude through clc-1 . external substitution of br , no3 , and i on hclc-1 expressed in xenopus oocytes had very similar effects to those described above for rclc-1 , although block by br , i , and methanesulfonate may have been slightly weaker ( table ii ) . partial replacement of cl by i led to a slight shift of v1/2 to more positive values and to a decrease in the apparent gating charge , z. the shift of v1/2 is opposite to the effect of external i , whereas both external and internal i led to a reduction in gating charge ( table iii ) . consistent with this model , anions we have studied can be divided into three groups according to their ability to open the channel and to permeate it : impermeant anions that are unable to open the channel , anions that can open the channel but can not permeate it , and anions that can both open the channel and permeate it . large anions such as glutamate , gluconate , and glucuronate belong to the first group . all of these cause a shift of the deactivation curve to positive potentials similar to that caused by glucose , which provides evidence that the shift in voltage dependence of the channel is due to the absence of cl rather than to the presence of the foreign anion . impermeant anions that do not interfere with the gating , such as glutamate and glucuronate , also do not change relative cl conductance . in the present work , however , we have found other anions , such as cyclamate and methanesulfonate , that can have a substantial effect on gating without being permeant . these results suggest that cyclamate can bind ( more readily than bicarbonate or bro3 ) to the regulatory binding site that opens the channel and that this site lies external to the selectivity filter , which cyclamate can not pass . it could be that when blocking anions , such as methanesulfonate , are bound to the latter site , they hinder cl passage through the pore . in these ways , clc-1 appears to differ from clc-0 , not because of any profound difference in their mechanisms of fast gating , but , probably , because the selectivity filter and regulatory binding site have very similar specificity in clc-0 , which allows channel activation to be closely linked to permeation , whereas these specificities are different in clc-1 with the consequences that this entails . small inorganic anions all showed potential - dependent block at low concentrations in the sequence : br < no3 < clo3 < i < clo4 < scn , which coincides with the ability to shift the voltage dependence of gating , to modify current kinetics , and also coincides with the lyotropic series . for br , no3 , i , and methanesulfonate , we have good evidence from noise analysis that all reduction in current amplitude is due to a reduction in single channel current . when all cl outside is replaced by scn , positive potentials applied from the inside reduce the probability of internal cl entering the channel , so the channel starts to rectify outward just as it also rectifies inward at strongly negative potentials when all scn is likely to be displaced from the channels . as is typical of the anomalous mole fraction effects seen in pores with multiple intrapore binding sites , the lowest conductance occurs when a mixture of ions is present in the channel . an anomalous mole - fraction effect for mixtures of cl and scn is not unique for clc-1 . for inward currents , the kd is 33 mm , which suggests that there is a deeper energy well for cl ions passing through the channel from the external side than for those passing from the inside . this can be interpreted in terms of the rate limiting binding site being closer to the external side . the effects of foreign anions applied to the inside are also different from their effects on the outside . also , from the inside , i shifts the apparent popen curve in the opposite direction , to more positive potentials . these results are all consistent with and extend the view of clc-1 as a channel with multiple internal binding sites having different characteristics and , especially , they support the proposal that there is at least one inner and one outer binding site inside the channel that are quite distinct from each other ( see fahlke et al . > hco3 ( f ) , while just for halides it was cl > br > i > f. this sequence corresponds to a cationic site of moderately strong field strength in the membrane ( eisenman sequence 4 ; eisenman , 1965 ) . however , not only electrostatic but also hydrophobic interactions are involved in anion permeation through clc-1 , and so the permeability sequence need not be directly related to the electrostatic field strength of the relevant binding site . from blocking ability when applied externally , the binding sequence is scn > br > bro3 > hco3 ( f ) , which corresponds to the low field strength eisenman sequence 1 ( eisenman , 1965 ) . these results agree with early studies of foreign anion permeability ( woodbury and miles , 1973 ) and of block of cl efflux ( harris , 1958 ) by foreign anions in frog muscle where the same sequence difference occurred . this would indicate that the site accessible from outside , at which block occurs , is different from the site governing selectivity and because blocking ability corresponds to opening ability , that block probably occurs at the regulatory binding site . the effects of some of the anions studied in this work on rat clc-1 have been investigated in human clc-1 by fahlke et al . ( 1997a ) , who obtained a similar sequence for relative permeability : cl scn > br > ( 1997a ) were somewhat higher than in our work , which can be explained by differences in internal cl concentration . in a multi - ion pore although for halides the differences were relatively small , methanesulfonate was found to be impermeant in rat clc-1 , while in human clc-1 , pmetsulf / pcl was 0.2 . with impermeant anion substitution , we have found that the sf9 cells can not for long sustain membrane potentials as highly positive as +80 mv with 140 mm of cl inside and only 8 mm of cl remaining outside . the difference in results might also be accounted for by cl accumulation near the external mouth of the channel , which could be higher at higher internal cl concentration . ( 1997b ) sug - gest that inherently voltage - dependent conformational changes are modulated by i , which locks the channel in a nondeactivating state . hence , a binding site that has a higher affinity for i than for cl will maintain channel opening in the presence of i , achievable negative potentials being unable to displace i from the site . estimates of the dimensions of a number of cation and anion channels have been made using a simple cylindrical pore model ( hille , 1975 ; bormann et al . still , they are much more permeant than hco3 and their relative permeability increases along with the size of the hydrophobic part of the molecule , suggesting the likelihood of significant hydrophobic interactions with the wall of the pore ( see woodbury and miles , 1973 , for similar results on frog muscle ) . for several anion channels that were studied at the single channel level , multiple binding site models have been developed based on extensive kinetic data ( bormann et al . in conclusion , we have shown ( a ) the multi - ion nature of clc-1 with demonstrable anomalous mole fraction effects of scn and clo4 ; ( b ) two of the anion binding sites , an inner and an outer site , are accessible from the cytoplasmic solution and from the external solution , respectively , and have different binding properties even for cl ; ( c ) the outer binding site appears to have characteristics that make it the prime candidate for regulation of channel opening and for channel block by external anions , but it is not simultaneously the site that determines channel selectivity ; ( d ) block of macroscopic currents by foreign anions can be accounted for entirely by a reduction in single channel conductance without any requirement for an effect on popen ; ( e ) minimum pore diameter is 4.5 ; and ( f ) foreign anions can be clearly categorized into three different groups according to their interaction with clc-1 . membrane conductance of sf9 cells expressing rclc-1 when impermeant or poorly permeant anions replace cl in the external solution . kinetics of whole - cell currents in sf9 cells expressing rclc-1 when impermeant or poorly permeant anions replace 95% of cl in the external solution . relative permeabilities for different foreign anions and their effect on the parameters of the popen curves voltages have been corrected for liquid junction potentials ( see methods ) . membrane conductance of sf9 cells expressing rclc-1 when various permeant anions replace cl in the external solution . kinetics of whole - cell currents in sf9 cells expressing rclc-1 when various permeant anions replace 100% of cl in the external solution . foreign anion block of macroscopic currents , i , from whole - cell studies and of single channel currents , i , estimated by noise analysis for ease of comparison , peak inward currents for a step to 140 mv in the presence of foreign anions have been normalized to the current in external chloride solution for rc1c-1 and hc1c-1 as appropriate . nonstationary noise analysis performed on outside - out patches from xenopus oocytes expressing hclc-1 in the presence of cl , no3 , i , or br . ( bottom ) variance mean current plots are fitted with parabolas according to the equation : = 0 + ii i /n , where 0 is baseline noise variance , i is single channel current , i is mean current , and n is number of channels .

in voltage - dependent sodium , potassium , and calcium channels , the voltage dependence of gating has usually been attributed to that intrinsic part of the protein molecule known as the voltage sensor ( s4 region ) , although a number of very recent studies have reported some dependence on the presence and nature of the permeating cation ( yellen , 1997 ) . by comparison , cloning and sequencing of the voltage - dependent clc family of cl channels has failed to reveal an s4 type of voltage sensor and experiments on the torpedo cl channel , clc-0 , indicate that there is little or no contribution to gating from intrinsic protein charge movement ( chen and miller , 1996 ) . indeed , for two family members , clc-0 , and the skeletal muscle cl channel , clc-1 , a number of experiments in which cl has been replaced by glucose or by other anions suggest that activation of these channels is mainly controlled by the presence and concentration of external cl ( pusch et al . for a constant external cl concentration , the availability of extracellularly derived cl at the regulatory site depends on the transmembrane electric field and this confers the experimentally observed voltage dependence upon gating , no intrinsic voltage sensing component of the channel protein being required . methanesulfonate , which has often been used as the preferred impermeant cl substitute because of its negligible permeability and apparent lack of interaction with cl in mole fraction substitution studies of membrane conductance in the rat diaphragm muscle ( palade and barchi , 1977 ) . , 1996 ; fahlke et al we have investigated the influence of foreign anions on current kinetics , apparent popen permeability , and conductance of clc-1 channels that allow a number of predictions to be made about the nature of the channel pore . lower concentrations of cl in the bath solution were achieved by equimolar substitution of 5 , 10 , 25 , 50 , 75 , 90 , or 100% of external cl by a particular anion ; for impermeant anions the highest concentration in the bath solution was 95% . to reduce possible acidification of the internal medium due to co2 diffusion into the cell , 120 mm k - glutamate in the internal solution was replaced with equimolar tris - glutamate , a substitution that , of itself , had no effect on clc-1 channel properties . shifts in potential ( relative to control cl solution ) were plotted against mole fraction of the anion and fitted with a goldman - hodgkin - katz equation of the form : 1\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*}y=58.4\;log\;\;\{1/[(1-m_{f})+am_{f}]\},\end{equation*}\end{document } where y is the shift of the membrane potential , mf ( mole fraction ) is the concentration of foreign anion , x , relative to the control cl concentration ( mf = [ x]/[x ] + [ cl ] ) , and a = ( px/ pcl ) is the permeability ratio for this anion relative to cl . to determine relative conductances in the presence of external foreign anions , outward conductance was estimated as a chord conductance ( approximating the slope conductance ) from the outward current interpolated at a point 20 mv to the right of the reversal potential on the current voltage ( i - v ) plot . when nacl in the external solution was replaced by glucose , the slope conductance of clc-1 , estimated near the equilibrium potential , showed saturation at physiological external cl concentrations , only becoming dependent on external cl at low concentrations ( fig . substitution of cl by glutamate or glucose influenced current kinetics , increasing the rate of deactivation of inward currents , and shifted the apparent popen curve in the depolarizing direction , as we reported previously ( rychkov et al . they also shifted the apparent popen curves to more positive potentials without changing the gating charge ( table i ) , but unlike glutamate and other anions from that group they appeared to interact with cl inside the channel to some extent , since the relationship between conductance and concentration was different from that for glucose ( fig . bicarbonate was less permeant than bro3 ( table i ) and its effects were closer to those of glutamate : at 95% concentration , hco3 shifted v1/2 by +60 mv , while bro3 , at the same concentration , shifted v1/2 rather less , by +50 mv ( compare with the shift of +70 mv for glutamate ; table i ) . bromate showed stronger interaction with cl inside the channel than hco3 since conductance of the channel in the outward direction in the presence of bro3 was smaller than in the presence of hco3 ( figs . cyclamate ( when substituting 95% of cl ) blocked 35% of inward current , shifted v1/2 by + 28 mv ( compared with +70 mv for glutamate ; table i ) , and increased the slope coefficient , k , of the popen curve , thus reducing the apparent gating charge , z , to 0.63 . furthermore , the apparent popen curve was shifted in the opposite direction , to more negative potentials , by 45 mv , and the apparent gating charge was decreased ( fig . with increasing amounts of br replacing cl in the external solution , the slope conductance , calculated for outward current near the reversal potential , decreased until just 15% of its initial value remained when 100% br substituted for cl ( fig . kinetics of current deactivation were not substantially altered , nor was there any significant shift in the voltage dependence of the apparent popen , as indicated by v1/2 ( table i ) . apparent popen in the presence of either of these anions at 50% concentration was shifted to more negative potentials without significant change in the slope of the curve , but if the concentration of no3 or clo3 was increased up to 100% , the slope factor of the apparent popen curve was increased ( table i ) , implying a reduction in gating charge to 0.6 ( normally 1.2 ) . time constants of the deactivating components of the inward current were not appreciably changed by these anions , but the relative proportion of steady state current at 120 mv increased from 7 to 50% ( compare figs . apparent popen curves obtained at lower mole fractions of i , where current deactivation at negative potentials was more obvious , showed that the voltage dependence of channel gating was shifted to more negative potentials by i and the apparent gating charge decreased ( table i ) . thiocyanate and clo4 at low concentrations ( up to 10% ) had effects very similar to i , they blocked inward current ( by 90% in the case of 10% scn ) , reduced current deactivation at negative potentials , shifted v1/2 to more negative potentials , decreased the slope of apparent popen curves ( table i ) , and shifted membrane potentials towards more positive values . when the concentration of scn ( or clo4 ) was raised above 10% , inward current was blocked almost completely , but the amplitude of the outward current started to increase and reached its maximum when all cl in the external solution was replaced with scn ( fig . endogenous volume - regulated cl channels in sf9 cells were more permeable to scn than to cl and , furthermore , control cells showed outwardly rectifying currents in the presence of scn qualitatively similar to the currents recorded under the same conditions from the cells expressing clc-1 ( not shown ) . to make sure that the anomalous mole - fraction effect and outward rectification in solutions containing scn were not the result of increased activity of the native cl channels , concomitant with complete block of clc-1 channels , experiments with scn substitution for cl if currents recorded in the presence of scn were passing only through native channels , their amplitudes should not be much different from cell to cell and should not depend on the cl current amplitude through clc-1 . in reality , there was a high level of correlation between the amplitudes of outward currents , measured in cells expressing clc-1 , bathed in control cl solution , and when scn replaced cl ( fig . we can , therefore , have confidence that clc-1 is permeable to scn ( and , likewise , to clo4 ) and that outward currents recorded in the presence of scn are mainly due to conductance through clc-1 . external substitution of br , no3 , and i on hclc-1 expressed in xenopus oocytes had very similar effects to those described above for rclc-1 , although block by br , i , and methanesulfonate may have been slightly weaker ( table ii ) . 7 ) , performed on outside - out patches containing hclc-1 at 140 mv to gain information about the mechanism of this block , show ( table ii ) that single channel current amplitude is reduced by foreign anion substitution in the same proportion as whole - cell current . since it was not possible to change internal cl concentration during whole - cell patch - clamp experiments in sf9 cells , results from separate whole - cell experiments were combined after normalizing in the following way . for different cells , the ratio between chord conductance at 120 mv and current at + 80 mv was found to be constant for external cl concentrations from 170 to 340 mm and a fixed internal cl concentration , presumably reflecting the ratio between single channel currents at 120 and + 80 mv . but unlike external br , which did not affect inward current kinetics , internal br slowed down current deactivation and appeared to have a stronger blocking action ( table ii indicates 20% block of hclc-1 and 40% block of rclc-1 from the outside , while fig . partial replacement of cl by i led to a slight shift of v1/2 to more positive values and to a decrease in the apparent gating charge , z. the shift of v1/2 is opposite to the effect of external i , whereas both external and internal i led to a reduction in gating charge ( table iii ) . in our receptor - operated model of gating in the clc-1 channel , open probability is controlled by a binding site for cl , or certain other anions , which is accessible only from outside and which must be correctly occupied for opening to occur ( rychkov et al . consistent with this model , anions we have studied can be divided into three groups according to their ability to open the channel and to permeate it : impermeant anions that are unable to open the channel , anions that can open the channel but can not permeate it , and anions that can both open the channel and permeate it . all of these cause a shift of the deactivation curve to positive potentials similar to that caused by glucose , which provides evidence that the shift in voltage dependence of the channel is due to the absence of cl rather than to the presence of the foreign anion . since glucose , presumably , does not interact with binding sites regulating the behavior of clc-1 , any difference between those relative conductances obtained when glucose substitutes for nacl and those where a foreign anion substitutes for cl , reflects interaction between cl and that anion in the pore . in the present work , however , we have found other anions , such as cyclamate and methanesulfonate , that can have a substantial effect on gating without being permeant . for example , cyclamate and bro3 have much the same effect on conductance and current kinetics and the amount of block of the inward current is also very similar , but while bro3 is permeant , cyclamate is impermeant and , in addition , it reduces the gating charge to 0.62 . these results suggest that cyclamate can bind ( more readily than bicarbonate or bro3 ) to the regulatory binding site that opens the channel and that this site lies external to the selectivity filter , which cyclamate can not pass . in these ways , clc-1 appears to differ from clc-0 , not because of any profound difference in their mechanisms of fast gating , but , probably , because the selectivity filter and regulatory binding site have very similar specificity in clc-0 , which allows channel activation to be closely linked to permeation , whereas these specificities are different in clc-1 with the consequences that this entails . small inorganic anions all showed potential - dependent block at low concentrations in the sequence : br < no3 < clo3 < i < clo4 < scn , which coincides with the ability to shift the voltage dependence of gating , to modify current kinetics , and also coincides with the lyotropic series . in the lyotropic series , anions are arranged in order according to their ability to bind or adsorb to proteins , to potentiate the strength of a muscle twitch , and to unwind macromolecules . it is believed that , for binding to follow the lyotropic series , a site requires the combination of two molecular attributes : appropriate anion - attracting groups and neighboring hydrophobic groups ( dani et al . when all cl outside is replaced by scn , positive potentials applied from the inside reduce the probability of internal cl entering the channel , so the channel starts to rectify outward just as it also rectifies inward at strongly negative potentials when all scn is likely to be displaced from the channels . these results are all consistent with and extend the view of clc-1 as a channel with multiple internal binding sites having different characteristics and , especially , they support the proposal that there is at least one inner and one outer binding site inside the channel that are quite distinct from each other ( see fahlke et al . in a multi - ion pore although for halides the differences were relatively small , methanesulfonate was found to be impermeant in rat clc-1 , while in human clc-1 , pmetsulf / pcl was 0.2 . with impermeant anion substitution , we have found that the sf9 cells can not for long sustain membrane potentials as highly positive as +80 mv with 140 mm of cl inside and only 8 mm of cl remaining outside . while we do not dispute that i might lock the channel in the open state , evidence is lacking for the existence of conformational changes dependent upon an intrinsic voltage sensor in clc channels ( see introduction ) . still , they are much more permeant than hco3 and their relative permeability increases along with the size of the hydrophobic part of the molecule , suggesting the likelihood of significant hydrophobic interactions with the wall of the pore ( see woodbury and miles , 1973 , for similar results on frog muscle ) . in conclusion , we have shown ( a ) the multi - ion nature of clc-1 with demonstrable anomalous mole fraction effects of scn and clo4 ; ( b ) two of the anion binding sites , an inner and an outer site , are accessible from the cytoplasmic solution and from the external solution , respectively , and have different binding properties even for cl ; ( c ) the outer binding site appears to have characteristics that make it the prime candidate for regulation of channel opening and for channel block by external anions , but it is not simultaneously the site that determines channel selectivity ; ( d ) block of macroscopic currents by foreign anions can be accounted for entirely by a reduction in single channel conductance without any requirement for an effect on popen ; ( e ) minimum pore diameter is 4.5 ; and ( f ) foreign anions can be clearly categorized into three different groups according to their interaction with clc-1 . foreign anion block of macroscopic currents , i , from whole - cell studies and of single channel currents , i , estimated by noise analysis for ease of comparison , peak inward currents for a step to 140 mv in the presence of foreign anions have been normalized to the current in external chloride solution for rc1c-1 and hc1c-1 as appropriate . currents , ix and ix , are inward currents carried by internal chloride in the presence of external anion x. these anions were substituted in equimolar amounts for 127.5 mm cl ( 75% of external cl substituted ) in experiments on rclc-1 expressed in sf9 cells and for 100 mm cl ( 98% of external cl substituted ) in experiments on hclc-1 expressed in xenopus oocytes . fitted values for the parabolas shown are : ( a ) for chloride , i = 0.26 pa ; ( b ) for nitrate , i = 0.1 pa ; ( c ) for 50% iodide/50% chloride , i = 0.077 pa ; ( d ) for bromide , i = 0.21 pa . parameters of apparent popen curves from inside - out patches of xenopus oocytes after internal application of foreign anions kinetics of currents in inside - out patches from xenopus oocytes expressing hclc-1 when br and i are substituted for cl in the internal solution .

the utilization of radial ( ra ) over femoral arterial access ( fa ) for acute coronary intervention in stelevation myocardial infarction ( stemi ) is supported by a reduction in mortality , ischemic , and bleeding endpoints.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 this has led various interventional societies to encourage its utilization over femoral access.9 , 10 , 11 although the basis of this evidence includes patients undergoing both primary and rescue percutaneous coronary intervention ( pci ) , the majority of randomized data are confined to patients treated with primary pci ( ppci).2 , 7 ra in patients treated with a fibrinolytic pharmacoinvasive strategy ( pi ) has had limited exploration,12 , 13 , 14 as has any direct comparison with patients treated with ppci . moreover , the application of a pi strategy creates at least 2 distinct patient subsets at the time of angiography , each with a different risk profile with respect to ischemic outcomes and major bleeding / accesssite complications.15 the first is the nonreperfused patient requiring urgent rescue pci who has received recent fibrinolysis with adjunctive antithrombotic and antiplatelet therapy sometimes associated with hemodynamic or electrical instability . the second is the stable reperfused stemi undergoing scheduled pci 6 to 24 hours later . intuitively , a comparable efficacy and safety advantage as observed in ppci in favor of ra is expected in patients treated with a pi strategy , particularly in the subset undergoing rescue pci . the strategic reperfusion early after myocardial infarction ( stream ) trial,16 which randomized patients to a fibrinolytic pi ( rescue and scheduled pci ) versus ppci treatment strategy , provided a unique opportunity to address this issue in an early presenting , rapidly treated contemporary stemi cohort . accordingly , within stream , we evaluated the relationship between arterial access site and the 30day primary composite ( all cause death , shock , congestive heart failure [ chf ] , and reinfarction ) as well as major bleeding events in stemi according to the treatment strategy received . the stream trial protocol and primary results have been published previously.16 , 17 the study protocol was approved by national regulatory authorities and by the local ethics committee at each study center . acute stemi patients presenting within 3 hours of symptom onset and unable to undergo ppci within 1 hour of first medical contact were randomized to either fibrinolysis with a protocoldefined pi strategy or ppci . in the pi strategy , bolus weightbased tenecteplase ( tnk ) , aspirin , clopidogrel , and enoxaparin were administered according to guideline recommendations and followed by either rescue pci or scheduled angiography ( within 624 hours ) . after 21% of the ultimate population had been enrolled , the executive committee amended the protocol on august 24 , 2009 , to reduce the dose of tnk by 50% in patients 75 years of age or older because of an excess of intracranial hemorrhage ( ich ) in that age group . the need for rescue pci was determined by site investigators according to < 50% st segment resolution in the electrocardiogram ( ecg ) lead with the maximal stelevation 90 minutes after tnk bolus , hemodynamic instability , or refractory ventricular arrhythmias as mandated by the study protocol . primary pci was conducted after expeditious transfer from the point of randomization , and early initiation of aspirin , clopidogrel , and antithrombotic therapy , including discretionary glycoprotein iib / iiia ( gp 2b/3a ) antagonists based on best standard practice . ecgs were performed at baseline , 90 minutes posttnk , and 30 minutes postcatheterization in the pharmacoinvasive arm ( including postpci in those undergoing pci ) and baseline and 30 minutes postcatheterization or postpci ( if performed ) in the primary pci arm . interpretation was performed at the canadian vigour center ecg core laboratory ( edmonton , alberta , canada ) , and , for this study , the prespecified ecg metrics included : worst lead stelevation resolution ; worst lead stelevation resolution 50% ; and worst lead residual stelevation . both worst lead stelevation resolution and worst lead residual stelevation have previously been shown to have prognostic utility in stemi patients undergoing primary pci.18 interventional cardiologists in participating sites determined the choice of the arterial access , either ra or fa based upon local practice ( table s1 ) . angiographic assessment detailing coronary anatomy , need for percutaneous intervention after diagnostic angiography and thrombolysis in myocardial infarction ( timi ) flow grade postpci was performed locally by sitespecific investigators using standard definitions.19 management of the arterial sheath postangiography was also determined by sitespecific vascular access best practice protocols . the results of the current report are based on the perprotocol analysis according to the access site utilized to complete the angiographic procedure . the primary outcome of this study was a 30day composite of death , cardiogenic shock , chf , or reinfarction whereas the key secondary outcomes included components of the primary outcome at 30 days , nonintracranial major bleeding or ich , stroke , and 1year allcause mortality . detailed definitions for both the components of the primary composite and bleeding endpoints have been published previously.17 briefly , a stroke review independent panel adjudicated all strokes centrally . the global use of strategies to open occluded arteries ( gusto ) definition of bleeding20 was implemented in this trial . major bleeding was classified as : ( 1 ) intracranial or ( 2 ) nonintracranial severe bleed ( bleed that leads to hemodynamic compromise requiring intervention [ blood or fluid replacement , inotropic support , ventricular assist device , or surgical repair ] or lifethreatening or fatal bleed ) or moderate bleed ( bleeding requiring blood transfusion , but that does not lead to hemodynamic compromise requiring intervention ) . all bleeds , excluding intracranial bleeds , were investigator reported . related major bleeding was defined as major bleeding that occurred less than 48 hours postpci . categorical variables are summarized as percentages and as median ( 25th , 75th percentiles ) for continuous variables . baseline characteristics and concomitant treatment are reported according to access site ( radial vs femoral ) and study treatment received . differences between groups were tested with the chisquare test ( or fisher 's exact test when count was < 5 for at least 1 cell ) for categorical variables and wilcoxon ranksum test for continuous variables , respectively . the association between access site and primary clinical outcome ( composite of death , cardiogenic shock , chf , or reinfarction ) within 30 days was examined using a univariable logistic regression model where a propensity score for access site was used to construct an inverse probability weight.21 given the assignment of access site was not randomized , a propensity score for access site was created using a multivariable logistic regression model . variables considered in the model were based on literature review , expert opinion , and univariate tests with p<0.10 . the covariates in the final model were forced in ; that is , no conventional statistical selection techniques ( eg , stepwise , forward , or backward ) were used . variables included in the final propensity score model were age , sex , weight , history of hypertension , history of diabetes mellitus , heart rate , systolic blood pressure , killip class , inferior myocardial infarction ( mi ) , sum st elevation at baseline , q waves at baseline , time from symptom onset to randomization , and country of enrollment ( table s2 ) . the interaction between access site and study treatment ( ie , pi vs ppci ) on the 30day primary clinical composite outcome was also examined . a further prespecified similar subgroup analysis of access site within the pi ( rescue vs scheduled ) strategy was performed . to test whether gp 2b/3a use and protocol amendment modulate the association between access site and 30day primary outcomes , the interaction between gp 2b/3a use and access site and the interaction between protocol amendment and access site were examined . statistical analyses were performed using sas software ( version 9.4 ; sas institute inc . , cary , nc ) . the stream trial protocol and primary results have been published previously.16 , 17 the study protocol was approved by national regulatory authorities and by the local ethics committee at each study center . acute stemi patients presenting within 3 hours of symptom onset and unable to undergo ppci within 1 hour of first medical contact were randomized to either fibrinolysis with a protocoldefined pi strategy or ppci . in the pi strategy , bolus weightbased tenecteplase ( tnk ) , aspirin , clopidogrel , and enoxaparin were administered according to guideline recommendations and followed by either rescue pci or scheduled angiography ( within 624 hours ) . after 21% of the ultimate population had been enrolled , the executive committee amended the protocol on august 24 , 2009 , to reduce the dose of tnk by 50% in patients 75 years of age or older because of an excess of intracranial hemorrhage ( ich ) in that age group . the need for rescue pci was determined by site investigators according to < 50% st segment resolution in the electrocardiogram ( ecg ) lead with the maximal stelevation 90 minutes after tnk bolus , hemodynamic instability , or refractory ventricular arrhythmias as mandated by the study protocol . primary pci was conducted after expeditious transfer from the point of randomization , and early initiation of aspirin , clopidogrel , and antithrombotic therapy , including discretionary glycoprotein iib / iiia ( gp 2b/3a ) antagonists based on best standard practice . ecgs were performed at baseline , 90 minutes posttnk , and 30 minutes postcatheterization in the pharmacoinvasive arm ( including postpci in those undergoing pci ) and baseline and 30 minutes postcatheterization or postpci ( if performed ) in the primary pci arm . interpretation was performed at the canadian vigour center ecg core laboratory ( edmonton , alberta , canada ) , and , for this study , the prespecified ecg metrics included : worst lead stelevation resolution ; worst lead stelevation resolution 50% ; and worst lead residual stelevation . both worst lead stelevation resolution and worst lead residual stelevation have previously been shown to have prognostic utility in stemi patients undergoing primary pci.18 interventional cardiologists in participating sites determined the choice of the arterial access , either ra or fa based upon local practice ( table s1 ) . angiographic assessment detailing coronary anatomy , need for percutaneous intervention after diagnostic angiography and thrombolysis in myocardial infarction ( timi ) flow grade postpci was performed locally by sitespecific investigators using standard definitions.19 management of the arterial sheath postangiography was also determined by sitespecific vascular access best practice protocols . the results of the current report are based on the perprotocol analysis according to the access site utilized to complete the angiographic procedure . the primary outcome of this study was a 30day composite of death , cardiogenic shock , chf , or reinfarction whereas the key secondary outcomes included components of the primary outcome at 30 days , nonintracranial major bleeding or ich , stroke , and 1year allcause mortality . detailed definitions for both the components of the primary composite and bleeding endpoints have been published previously.17 briefly , a stroke review independent panel adjudicated all strokes centrally . the global use of strategies to open occluded arteries ( gusto ) definition of bleeding20 was implemented in this trial . major bleeding was classified as : ( 1 ) intracranial or ( 2 ) nonintracranial severe bleed ( bleed that leads to hemodynamic compromise requiring intervention [ blood or fluid replacement , inotropic support , ventricular assist device , or surgical repair ] or lifethreatening or fatal bleed ) or moderate bleed ( bleeding requiring blood transfusion , but that does not lead to hemodynamic compromise requiring intervention ) . all bleeds , excluding intracranial bleeds , were investigator reported . related major bleeding was defined as major bleeding that occurred less than 48 hours postpci . categorical variables are summarized as percentages and as median ( 25th , 75th percentiles ) for continuous variables . baseline characteristics and concomitant treatment are reported according to access site ( radial vs femoral ) and study treatment received . differences between groups were tested with the chisquare test ( or fisher 's exact test when count was < 5 for at least 1 cell ) for categorical variables and wilcoxon ranksum test for continuous variables , respectively . the association between access site and primary clinical outcome ( composite of death , cardiogenic shock , chf , or reinfarction ) within 30 days was examined using a univariable logistic regression model where a propensity score for access site was used to construct an inverse probability weight.21 given the assignment of access site was not randomized , a propensity score for access site was created using a multivariable logistic regression model . variables considered in the model were based on literature review , expert opinion , and univariate tests with p<0.10 . the covariates in the final model were forced in ; that is , no conventional statistical selection techniques ( eg , stepwise , forward , or backward ) were used . variables included in the final propensity score model were age , sex , weight , history of hypertension , history of diabetes mellitus , heart rate , systolic blood pressure , killip class , inferior myocardial infarction ( mi ) , sum st elevation at baseline , q waves at baseline , time from symptom onset to randomization , and country of enrollment ( table s2 ) . the interaction between access site and study treatment ( ie , pi vs ppci ) on the 30day primary clinical composite outcome was also examined . a further prespecified similar subgroup analysis of access site within the pi ( rescue vs scheduled ) strategy was performed . to test whether gp 2b/3a use and protocol amendment modulate the association between access site and 30day primary outcomes , the interaction between gp 2b/3a use and access site and the interaction between protocol amendment and access site were examined . statistical analyses were performed using sas software ( version 9.4 ; sas institute inc . , cary , nc ) . figure 1 illustrates the 2 treatment group cohorts from the 1820 perprotocol treated patients enrolled in stream categorized by access site . as evident , there was comparable utilization of either access site within each treatment strategy ( fa : pi 53.4% [ n=478 ] and ppci 57.6% [ n=533 ] ) . in addition , within the pi strategy , both access sites were comparably distributed in the rescue ( fa : 52.8% ; n=200 ) and scheduled pci ( fa : 53.9% ; n=278 ) subgroups . fa indicates femoral ; pi , pharmacoinvasive ; ppci , primary percutaneous coronary intervention ; ra , radial . overall , stemi patients treated by fa were younger , had more past hypertension , lower systolic bp at presentation , lower timi risk score , and more stelevation on the baseline ecg compared to the ra group ( table 1 ) . selected baseline patient characteristics according to access site and study treatment continuous variables presented as median ( 25th75th percentiles ) . fa indicates femoral ; mi , myocardial infarction ; ppci , primary percutaneous coronary intervention ; ra , radial ; ste , stsegment elevation ; timi , thrombolysis in myocardial infarction . evaluated by ecg core laboratory at the canadian vigour centre . as described in table 2 , patients treated by fa had shorter time from symptom onset to randomization in both treatment strategies . in addition , shorter times from symptom onset to femoral , compared to radial sheath , insertion were observed in the ppci , but not in the pi , strategy . ischemic times , medications , angiographic findings , and posttreatment ecg according to access site and study treatment continuous variables presented as median ( 25th75th percentiles ) . ecg indicates electrocardiogram ; fa , femoral ; gp , glycoprotein ; iabp , intraaortic balloon pump ; ppci , primary percutaneous coronary intervention ; ra , radial ; timi , thrombolysis in myocardial infarction ; tnk , tenecteplase . evaluated by ecg core laboratory at the canadian vigour center . a significantly higher utilization of gp2b/3a inhibitor use was noted in the ra , compared to fa , group ( 40.2% vs 24.1% ; p<0.001 ) , particularly in those undergoing ppci . those patients treated with ppci strategy and ra had higher rates of postpci timi3 flow grade than fa patients . evaluation of the posttreatment ecg revealed consistently better indices of reperfusion for patients with ra in both the pi and ppci cohorts , as evidenced by higher rates of worst lead residual st elevation < 1 mm and lesser rates in patients with 2 mm residual st elevation . irrespective of treatment strategy , the unadjusted primary composite of 30day death , shock , chf , or reinfarction occurred in 8.9% in the ra compared to 15.7% in fa group ( table 3 ) . after adjustment , the benefit favoring ra persisted ( adjusted or , 0.59 ; 95% ci , 0.440.78 ; p<0.001 ) , as seen in figure 2 . efficacy and safety outcomes according to access site and study treatment chf indicates congestive heart failure ; ppci , primary percutaneous coronary intervention . fa indicates femoral ; pi , pharmacoinvasive ; ppci , primary percutaneous coronary intervention ; ra , radial . analysis of access site categorized by study treatment received revealed that the advantage associated with ra was present in both the ppci ( adjusted or , 0.63 ; 95% ci , 0.430.92 ) and pi cohorts ( adjusted or , 0.57 ; 95% ci , 0.370.86 ; p [ interaction]=0.730 ; figure 2 ) . within the pi group , a trend for ra advantage was evident in the highrisk rescue pci ( 13.4% vs 26.3% ; adjusted or , 0.65 ; 95% ci , 0.391.07 ) subgroup with no significant difference in patients undergoing scheduled pci ( 5.5% vs 5.4% ; adjusted or , 0.55 ; 95% ci , 0.241.26 ) . however , no interaction was evident between rescue pci or scheduled pci as it relates to the advantage of ra after adjustment ( p [ interaction]=0.739 ) . the observed increase in gp 2b/3a use within the ra group did not appear to modulate the association with the 30day primary composite outcome ( ra : gp 2b/3a use vs not , 9.3% versus 8.7% ; and fa : 20.2% vs 14.3% ; p [ interaction]=0.988 ) ; neither did the implementation of the amendment ( halfdose lytic in patients 75 years ) of the stream trial protocol ( ra and fa : pre and postamendment , respectively , 9.0% vs 8.9% and 18.4% vs 15.2% ; p [ interaction]=0.920 ) . the increased gp 2b/3a use within the ra group also did not appear to modulate the association with major bleeding ( ra vs fa : adjusted hazard ratio , 0.56 ; 95% ci , 0.281.12 ; p [ interaction]=0.087 ) . radial access was associated with an observed reduction in 30day mortality ( 2.4% vs 4.7% ; p=0.009 ) , cardiogenic shock ( 2.9% vs 6.4% ; p=0.001 ) , and heart failure ( 5.0% vs 8.4% ; p=0.005 ; table 3 ) . no significant differences in ischemic stroke or ich were noted in either vascular access site across the 2 treatment groups . at 1 year , no difference in allcause mortality was noted in either accesssite category across both study treatment groups . overall , a comparable rate of nonintracranial major bleeding was noted in the ra versus fa group ( 5.2% vs 6.0% ; p=0.489 ; table 3 ) . this was also evident within the 2 treatment strategies ( pi and ppci : ra vs fa , 5.5% vs 7.8% [ p=0.179 ] and 4.9% vs 4.3% [ p=0.698 ] , respectively ) ; however , within the pi strategy , a trend to increased nonintracranial major bleeding in patients treated by fa within the rescue , compared to the scheduled , subgroup was observed ( rescue and scheduled : ra vs fa , 6.1% vs 11.6% [ p=0.064 ] and 5.1% vs 5.1% [ p=0.996 ] ) . both major accesssite ( ra vs fa , 2.8% vs 4.1% ; p=0.163 ) and nonaccesssite ( ra vs fa , 2.3% vs 1.9% ; p=0.487 ) bleeding were similarly distributed in the overall study population . major vascular access complication ( pseudoaneurysm or arteriovenous fistula development ) occurred in 0% in the ra and 1.4% in the fa group . figure 1 illustrates the 2 treatment group cohorts from the 1820 perprotocol treated patients enrolled in stream categorized by access site . as evident , there was comparable utilization of either access site within each treatment strategy ( fa : pi 53.4% [ n=478 ] and ppci 57.6% [ n=533 ] ) . in addition , within the pi strategy , both access sites were comparably distributed in the rescue ( fa : 52.8% ; n=200 ) and scheduled pci ( fa : 53.9% ; n=278 ) subgroups . fa indicates femoral ; pi , pharmacoinvasive ; ppci , primary percutaneous coronary intervention ; ra , radial . overall , stemi patients treated by fa were younger , had more past hypertension , lower systolic bp at presentation , lower timi risk score , and more stelevation on the baseline ecg compared to the ra group ( table 1 ) . selected baseline patient characteristics according to access site and study treatment continuous variables presented as median ( 25th75th percentiles ) . fa indicates femoral ; mi , myocardial infarction ; ppci , primary percutaneous coronary intervention ; ra , radial ; ste , stsegment elevation ; timi , thrombolysis in myocardial infarction . evaluated by ecg core laboratory at the canadian vigour centre . as described in table 2 , patients treated by fa had shorter time from symptom onset to randomization in both treatment strategies . in addition , shorter times from symptom onset to femoral , compared to radial sheath , insertion were observed in the ppci , but not in the pi , strategy . ischemic times , medications , angiographic findings , and posttreatment ecg according to access site and study treatment continuous variables presented as median ( 25th75th percentiles ) . ecg indicates electrocardiogram ; fa , femoral ; gp , glycoprotein ; iabp , intraaortic balloon pump ; ppci , primary percutaneous coronary intervention ; ra , radial ; timi , thrombolysis in myocardial infarction ; tnk , tenecteplase . evaluated by ecg core laboratory at the canadian vigour center . a significantly higher utilization of gp2b/3a inhibitor use was noted in the ra , compared to fa , group ( 40.2% vs 24.1% ; p<0.001 ) , particularly in those undergoing ppci . those patients treated with ppci strategy and ra had higher rates of postpci timi3 flow grade than fa patients . evaluation of the posttreatment ecg revealed consistently better indices of reperfusion for patients with ra in both the pi and ppci cohorts , as evidenced by higher rates of worst lead residual st elevation < 1 mm and lesser rates in patients with 2 mm residual st elevation . irrespective of treatment strategy , the unadjusted primary composite of 30day death , shock , chf , or reinfarction occurred in 8.9% in the ra compared to 15.7% in fa group ( table 3 ) . after adjustment , the benefit favoring ra persisted ( adjusted or , 0.59 ; 95% ci , 0.440.78 ; p<0.001 ) , as seen in figure 2 . efficacy and safety outcomes according to access site and study treatment chf indicates congestive heart failure ; ppci , primary percutaneous coronary intervention . fa indicates femoral ; pi , pharmacoinvasive ; ppci , primary percutaneous coronary intervention ; ra , radial . analysis of access site categorized by study treatment received revealed that the advantage associated with ra was present in both the ppci ( adjusted or , 0.63 ; 95% ci , 0.430.92 ) and pi cohorts ( adjusted or , 0.57 ; 95% ci , 0.370.86 ; p [ interaction]=0.730 ; figure 2 ) . within the pi group , a trend for ra advantage was evident in the highrisk rescue pci ( 13.4% vs 26.3% ; adjusted or , 0.65 ; 95% ci , 0.391.07 ) subgroup with no significant difference in patients undergoing scheduled pci ( 5.5% vs 5.4% ; adjusted or , 0.55 ; 95% ci , 0.241.26 ) . however , no interaction was evident between rescue pci or scheduled pci as it relates to the advantage of ra after adjustment ( p [ interaction]=0.739 ) . the observed increase in gp 2b/3a use within the ra group did not appear to modulate the association with the 30day primary composite outcome ( ra : gp 2b/3a use vs not , 9.3% versus 8.7% ; and fa : 20.2% vs 14.3% ; p [ interaction]=0.988 ) ; neither did the implementation of the amendment ( halfdose lytic in patients 75 years ) of the stream trial protocol ( ra and fa : pre and postamendment , respectively , 9.0% vs 8.9% and 18.4% vs 15.2% ; p [ interaction]=0.920 ) . the increased gp 2b/3a use within the ra group also did not appear to modulate the association with major bleeding ( ra vs fa : adjusted hazard ratio , 0.56 ; 95% ci , 0.281.12 ; p [ interaction]=0.087 ) . radial access was associated with an observed reduction in 30day mortality ( 2.4% vs 4.7% ; p=0.009 ) , cardiogenic shock ( 2.9% vs 6.4% ; p=0.001 ) , and heart failure ( 5.0% vs 8.4% ; p=0.005 ; table 3 ) . no significant differences in ischemic stroke or ich were noted in either vascular access site across the 2 treatment groups . at 1 year , no difference in allcause mortality was noted in either accesssite category across both study treatment groups . overall , a comparable rate of nonintracranial major bleeding was noted in the ra versus fa group ( 5.2% vs 6.0% ; p=0.489 ; table 3 ) . this was also evident within the 2 treatment strategies ( pi and ppci : ra vs fa , 5.5% vs 7.8% [ p=0.179 ] and 4.9% vs 4.3% [ p=0.698 ] , respectively ) ; however , within the pi strategy , a trend to increased nonintracranial major bleeding in patients treated by fa within the rescue , compared to the scheduled , subgroup was observed ( rescue and scheduled : ra vs fa , 6.1% vs 11.6% [ p=0.064 ] and 5.1% vs 5.1% [ p=0.996 ] ) . both major accesssite ( ra vs fa , 2.8% vs 4.1% ; p=0.163 ) and nonaccesssite ( ra vs fa , 2.3% vs 1.9% ; p=0.487 ) bleeding were similarly distributed in the overall study population . major vascular access complication ( pseudoaneurysm or arteriovenous fistula development ) occurred in 0% in the ra and 1.4% in the fa group . the results of this study indicate that in early presenting stemi ( <3 hours from symptom onset ) , utilization of ra over fa is associated with a significant reduction in the composite of major adverse cardiovascular events regardless of the application of a fibrinolytic pi or ppci strategy . in addition , within the pi strategy , the prognostic advantage of ra applies particularly to the higher risk rescue pci cohort in whom a doubling of adverse outcomes appeared evident within the femoral access subgroup . within the stream study at baseline , patients treated by fa were more hypotensive and had greater st elevation at presentation , reflective of adverse outcomes . given this clinical profile , it is reasonable to suppose that this may have influenced the choice of fa and subsequently be reflected in worse outcomes . however , even after adjustment , the ra 30day composite clinical outcomes and mortality advantage persisted . the stream study enrolled patients presenting within 3 hours of symptom onset and thus represents a very early presenting stemi population as compared to other trials that randomized access site in stemi patients ( 1224 hours).2 , 3 , 7 hence , this study evaluated a distinct stemi cohort undergoing early cardiac catheterizations ( except for scheduled pci cohort ) in the presence of potent antiantithrombotic and fibrinolytic agents . it would therefore seem that lower major bleeding would be associated with the observed significant reduction in the primary composite and 30day mortality favoring ra across both treatment strategies . although there was nominally less nonintracranial major accesssite and nonaccesssite bleeding in those pharmacoinvasivetreated patients undergoing ra , this was not statistically significant . apart from lower major bleeding , the mechanism by which radial access might relate to allcause mortality currently remains unclear in the existing literature.13 , 22 one plausible explanation for the observed prognostic difference favoring ra relates to selection bias introduced by operator experience and centerspecific differences . greater radial interventional expertise in highvolume centers may have been associated with improved outcomes resulting from reduced vascular complications . to provide some context , it is noteworthy that 50% of the patients analyzed in the current study were treated with a fibrinolytic pi strategy , as compared to a much lower incidence in the radial versus femoral randomized investigation in stelevation acute coronary syndrome ( rifle steacs ; 7.6% failed fibrinolysis)2 and radial versus femoral access for coronary intervention ( rival ; 12%)7 trials . hence , the current results not only support the effectiveness of ra within a pitreated stemi patient subset , but also allow comparison of outcomes between roughly equalsized groups undergoing different reperfusion strategies . compared to randomized trials that enrolled postfibrinolytic patients,2 , 7 a higher proportion of major overall and major accesssite bleeding was noted in the current study ; for instance , radial versus femoral , respectively , in rival,7 non coronary artery bypass graft major bleeding ( rival definition ) was noted in 0.84% versus 0.91% , whereas in riflesteacs ( timi definition ) 1.8% versus 2.8% compared to 5.2% and 6.0% in our study . the reasons for this disparity in major bleeding is unclear , but could relate to : ( 1 ) investigator rather than central adjudication of major bleeding in the current study ; ( 2 ) heterogeneity in definition of major bleeding ; ( 3 ) differences in the proportion of the postfibrinolytic stemi population ; and ( 4 ) variability in operator expertise and sitespecific vascular access protocols . despite no difference in nonintracranial major bleeding between the 2 access sites in this study , our adjusted analysis of 30day mortality showed a persisting benefit associated with the radial approach . it is noteworthy that a recent study from the national cardiovascular data registry 's cathpci registry on bleeding complications in fibrinolytictreated patients undergoing rescue pci reported a major bleeding rate of 6.9% in radial versus 12.0% femoral access patients ( adjusted or , 0.67 ; 95% ci , 0.520.87 ; p=0.003).23 ra was employed in only 16% of these patients , and the authors highlighted the need for further data in this given that the choice of vascular access site was left to investigator discretion and absence of access to detailed procedural elements , we can not exclude the impact of unmeasured confounders . additionally , selection bias introduced by absence of information on center and operatorspecific interventional volumes can not be excluded . although we found no overall difference in vascular access bleeding , the trend toward more fa access bleeding in the pi patients undergoing rescue pci suggests less bleeding hazard in the presence of recent fibrinolytic treatment when radial access is employed . stream excluded patients in cardiogenic shock and advanced kidney disease : hence , our findings do not apply to this population . given that stream was an openlabel trial without central adjudication of bleeding endpoints , investigator bias may have played a role in the disparity in bleeding rates between this study and existing literature . however , to the best of our knowledge , it is the largest data set comparing a randomized , multicenter fibrinolytic pi strategy ( rescue and scheduled pci ) in a very earlytreated stemi population to ppci , demonstrating that the outcomes advantage with ra occurs in both the pi and ppci strategy . irrespective of whether a ppci or pi reperfusion strategy is used , these results support the utilization of radial access as the preferred arterial access site in stemi . funding for this trial was provided by boehringer ingelheim . clinical trials identifier : nct00623623 . welsh r discloses research funding from abbott vascular , alere , astrazeneca , bayer , boehringer ingelheim , canadian institute of health research , csl behring llc , edwards lifesciences , eli lilly , jansen , johnson & johnson , matrizyme pharma , pfizer , population health research institute , and university of alberta hospital foundation and personal funding from astrazeneca , bayer , and bristolmyers squibb / pfizer . steg discloses a research grant ( to inserm u1148 ) from sanofi and servier ; has received speaking or consulting fees from amarin , astrazeneca , bayer , boehringer ingelheim , bristolmyers squibb , cslbehring , daiichisankyo , glaxosmithkline , janssen , lilly , merck , novartis , pfizer , regeneron , roche , sanofi , servier , and the medicines company ; and owns stocks in aterovax . dr armstrong 's financial activities outside the submitted work are posted and routinely updated through http://www.vigour.ualberta.ca/en/about/conflictofinterest.aspx . radial versus femoral access according to country of enrollment table s2 . logistic regression model ( propensity score model ) of access site and baseline characteristics appendix s1 .

backgroundthe effectiveness of radial access ( ra ) in stelevation myocardial infarction ( stemi ) has been predominantly established in primary percutaneous coronary intervention ( ppci ) with limited exploration of this issue in the early postfibrinolytic patient . the purpose of this study was to compare the effectiveness and safety of ra versus femoral ( fa ) access in stemi undergoing either a pharmacoinvasive ( pi ) strategy or ppci.methods and resultswithin strategic reperfusion early after myocardial infarction ( stream ) , we evaluated the relationship between arterial access site and primary outcome ( 30day composite of death , shock , congestive heart failure , or reinfarction ) and major bleeding according to the treatment strategy received . a total of 1820 stemi patients were included : 895 pi ( 49.2% ; rescue pci [ n=379 ; 42.3% ] , scheduled pci [ n=516 ; 57.7% ] ) and 925 ppci ( 50.8% ) . irrespective of treatment strategy , there was comparable utilization of either access site ( fa : pi 53.4% and ppci 57.6% ) . fa stemi patients were younger , had lower presenting systolic blood pressure , lesser thrombolysis in myocardial infarction risk , and more stelevation at baseline . the primary composite endpoint occurred in 8.9% ra versus 15.7% fa patients ( p<0.001 ) . on multivariable analysis , this benefit on the primary composite outcome favoring ra persisted ( adjusted odds ratio [ or ] , 0.59 ; 95% ci , 0.440.78 ; p<0.001 ) and was evident in both ppci ( adjusted or , 0.63 ; 95% ci , 0.430.92 ) and pi cohorts ( adjusted or , 0.57 95% ci , 0.370.86 ; p interaction=0.730 ) . there was no difference in nonintracranial major bleeding with either access group ( ra vs fa , 5.2% vs 6.0% ; p=0.489).conclusionsregardless of the application of a pi or ppci strategy , ra was associated with improved clinical outcomes , supporting current stemi evidence in favor of ra in pci.clinical trial registration url : https://www.clinicaltrials.gov/. unique identifier : nct00623623 .

Clinical Trial Registration Introduction Methods Study Design and Patient Population Statistical Analysis Results Baseline Characteristics Primary Outcome Secondary Outcomes Major Bleeding Discussion Conclusion Sources of Funding Disclosures Supporting information

the utilization of radial ( ra ) over femoral arterial access ( fa ) for acute coronary intervention in stelevation myocardial infarction ( stemi ) is supported by a reduction in mortality , ischemic , and bleeding endpoints.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 this has led various interventional societies to encourage its utilization over femoral access.9 , 10 , 11 although the basis of this evidence includes patients undergoing both primary and rescue percutaneous coronary intervention ( pci ) , the majority of randomized data are confined to patients treated with primary pci ( ppci).2 , 7 ra in patients treated with a fibrinolytic pharmacoinvasive strategy ( pi ) has had limited exploration,12 , 13 , 14 as has any direct comparison with patients treated with ppci . moreover , the application of a pi strategy creates at least 2 distinct patient subsets at the time of angiography , each with a different risk profile with respect to ischemic outcomes and major bleeding / accesssite complications.15 the first is the nonreperfused patient requiring urgent rescue pci who has received recent fibrinolysis with adjunctive antithrombotic and antiplatelet therapy sometimes associated with hemodynamic or electrical instability . intuitively , a comparable efficacy and safety advantage as observed in ppci in favor of ra is expected in patients treated with a pi strategy , particularly in the subset undergoing rescue pci . the strategic reperfusion early after myocardial infarction ( stream ) trial,16 which randomized patients to a fibrinolytic pi ( rescue and scheduled pci ) versus ppci treatment strategy , provided a unique opportunity to address this issue in an early presenting , rapidly treated contemporary stemi cohort . accordingly , within stream , we evaluated the relationship between arterial access site and the 30day primary composite ( all cause death , shock , congestive heart failure [ chf ] , and reinfarction ) as well as major bleeding events in stemi according to the treatment strategy received . in the pi strategy , bolus weightbased tenecteplase ( tnk ) , aspirin , clopidogrel , and enoxaparin were administered according to guideline recommendations and followed by either rescue pci or scheduled angiography ( within 624 hours ) . ecgs were performed at baseline , 90 minutes posttnk , and 30 minutes postcatheterization in the pharmacoinvasive arm ( including postpci in those undergoing pci ) and baseline and 30 minutes postcatheterization or postpci ( if performed ) in the primary pci arm . the primary outcome of this study was a 30day composite of death , cardiogenic shock , chf , or reinfarction whereas the key secondary outcomes included components of the primary outcome at 30 days , nonintracranial major bleeding or ich , stroke , and 1year allcause mortality . the association between access site and primary clinical outcome ( composite of death , cardiogenic shock , chf , or reinfarction ) within 30 days was examined using a univariable logistic regression model where a propensity score for access site was used to construct an inverse probability weight.21 given the assignment of access site was not randomized , a propensity score for access site was created using a multivariable logistic regression model . variables included in the final propensity score model were age , sex , weight , history of hypertension , history of diabetes mellitus , heart rate , systolic blood pressure , killip class , inferior myocardial infarction ( mi ) , sum st elevation at baseline , q waves at baseline , time from symptom onset to randomization , and country of enrollment ( table s2 ) . the interaction between access site and study treatment ( ie , pi vs ppci ) on the 30day primary clinical composite outcome was also examined . in the pi strategy , bolus weightbased tenecteplase ( tnk ) , aspirin , clopidogrel , and enoxaparin were administered according to guideline recommendations and followed by either rescue pci or scheduled angiography ( within 624 hours ) . ecgs were performed at baseline , 90 minutes posttnk , and 30 minutes postcatheterization in the pharmacoinvasive arm ( including postpci in those undergoing pci ) and baseline and 30 minutes postcatheterization or postpci ( if performed ) in the primary pci arm . the primary outcome of this study was a 30day composite of death , cardiogenic shock , chf , or reinfarction whereas the key secondary outcomes included components of the primary outcome at 30 days , nonintracranial major bleeding or ich , stroke , and 1year allcause mortality . the association between access site and primary clinical outcome ( composite of death , cardiogenic shock , chf , or reinfarction ) within 30 days was examined using a univariable logistic regression model where a propensity score for access site was used to construct an inverse probability weight.21 given the assignment of access site was not randomized , a propensity score for access site was created using a multivariable logistic regression model . variables included in the final propensity score model were age , sex , weight , history of hypertension , history of diabetes mellitus , heart rate , systolic blood pressure , killip class , inferior myocardial infarction ( mi ) , sum st elevation at baseline , q waves at baseline , time from symptom onset to randomization , and country of enrollment ( table s2 ) . the interaction between access site and study treatment ( ie , pi vs ppci ) on the 30day primary clinical composite outcome was also examined . as evident , there was comparable utilization of either access site within each treatment strategy ( fa : pi 53.4% [ n=478 ] and ppci 57.6% [ n=533 ] ) . in addition , within the pi strategy , both access sites were comparably distributed in the rescue ( fa : 52.8% ; n=200 ) and scheduled pci ( fa : 53.9% ; n=278 ) subgroups . overall , stemi patients treated by fa were younger , had more past hypertension , lower systolic bp at presentation , lower timi risk score , and more stelevation on the baseline ecg compared to the ra group ( table 1 ) . fa indicates femoral ; mi , myocardial infarction ; ppci , primary percutaneous coronary intervention ; ra , radial ; ste , stsegment elevation ; timi , thrombolysis in myocardial infarction . ecg indicates electrocardiogram ; fa , femoral ; gp , glycoprotein ; iabp , intraaortic balloon pump ; ppci , primary percutaneous coronary intervention ; ra , radial ; timi , thrombolysis in myocardial infarction ; tnk , tenecteplase . a significantly higher utilization of gp2b/3a inhibitor use was noted in the ra , compared to fa , group ( 40.2% vs 24.1% ; p<0.001 ) , particularly in those undergoing ppci . irrespective of treatment strategy , the unadjusted primary composite of 30day death , shock , chf , or reinfarction occurred in 8.9% in the ra compared to 15.7% in fa group ( table 3 ) . after adjustment , the benefit favoring ra persisted ( adjusted or , 0.59 ; 95% ci , 0.440.78 ; p<0.001 ) , as seen in figure 2 . efficacy and safety outcomes according to access site and study treatment chf indicates congestive heart failure ; ppci , primary percutaneous coronary intervention . analysis of access site categorized by study treatment received revealed that the advantage associated with ra was present in both the ppci ( adjusted or , 0.63 ; 95% ci , 0.430.92 ) and pi cohorts ( adjusted or , 0.57 ; 95% ci , 0.370.86 ; p [ interaction]=0.730 ; figure 2 ) . within the pi group , a trend for ra advantage was evident in the highrisk rescue pci ( 13.4% vs 26.3% ; adjusted or , 0.65 ; 95% ci , 0.391.07 ) subgroup with no significant difference in patients undergoing scheduled pci ( 5.5% vs 5.4% ; adjusted or , 0.55 ; 95% ci , 0.241.26 ) . the observed increase in gp 2b/3a use within the ra group did not appear to modulate the association with the 30day primary composite outcome ( ra : gp 2b/3a use vs not , 9.3% versus 8.7% ; and fa : 20.2% vs 14.3% ; p [ interaction]=0.988 ) ; neither did the implementation of the amendment ( halfdose lytic in patients 75 years ) of the stream trial protocol ( ra and fa : pre and postamendment , respectively , 9.0% vs 8.9% and 18.4% vs 15.2% ; p [ interaction]=0.920 ) . the increased gp 2b/3a use within the ra group also did not appear to modulate the association with major bleeding ( ra vs fa : adjusted hazard ratio , 0.56 ; 95% ci , 0.281.12 ; p [ interaction]=0.087 ) . radial access was associated with an observed reduction in 30day mortality ( 2.4% vs 4.7% ; p=0.009 ) , cardiogenic shock ( 2.9% vs 6.4% ; p=0.001 ) , and heart failure ( 5.0% vs 8.4% ; p=0.005 ; table 3 ) . overall , a comparable rate of nonintracranial major bleeding was noted in the ra versus fa group ( 5.2% vs 6.0% ; p=0.489 ; table 3 ) . this was also evident within the 2 treatment strategies ( pi and ppci : ra vs fa , 5.5% vs 7.8% [ p=0.179 ] and 4.9% vs 4.3% [ p=0.698 ] , respectively ) ; however , within the pi strategy , a trend to increased nonintracranial major bleeding in patients treated by fa within the rescue , compared to the scheduled , subgroup was observed ( rescue and scheduled : ra vs fa , 6.1% vs 11.6% [ p=0.064 ] and 5.1% vs 5.1% [ p=0.996 ] ) . both major accesssite ( ra vs fa , 2.8% vs 4.1% ; p=0.163 ) and nonaccesssite ( ra vs fa , 2.3% vs 1.9% ; p=0.487 ) bleeding were similarly distributed in the overall study population . as evident , there was comparable utilization of either access site within each treatment strategy ( fa : pi 53.4% [ n=478 ] and ppci 57.6% [ n=533 ] ) . in addition , within the pi strategy , both access sites were comparably distributed in the rescue ( fa : 52.8% ; n=200 ) and scheduled pci ( fa : 53.9% ; n=278 ) subgroups . overall , stemi patients treated by fa were younger , had more past hypertension , lower systolic bp at presentation , lower timi risk score , and more stelevation on the baseline ecg compared to the ra group ( table 1 ) . fa indicates femoral ; mi , myocardial infarction ; ppci , primary percutaneous coronary intervention ; ra , radial ; ste , stsegment elevation ; timi , thrombolysis in myocardial infarction . ecg indicates electrocardiogram ; fa , femoral ; gp , glycoprotein ; iabp , intraaortic balloon pump ; ppci , primary percutaneous coronary intervention ; ra , radial ; timi , thrombolysis in myocardial infarction ; tnk , tenecteplase . a significantly higher utilization of gp2b/3a inhibitor use was noted in the ra , compared to fa , group ( 40.2% vs 24.1% ; p<0.001 ) , particularly in those undergoing ppci . irrespective of treatment strategy , the unadjusted primary composite of 30day death , shock , chf , or reinfarction occurred in 8.9% in the ra compared to 15.7% in fa group ( table 3 ) . after adjustment , the benefit favoring ra persisted ( adjusted or , 0.59 ; 95% ci , 0.440.78 ; p<0.001 ) , as seen in figure 2 . efficacy and safety outcomes according to access site and study treatment chf indicates congestive heart failure ; ppci , primary percutaneous coronary intervention . analysis of access site categorized by study treatment received revealed that the advantage associated with ra was present in both the ppci ( adjusted or , 0.63 ; 95% ci , 0.430.92 ) and pi cohorts ( adjusted or , 0.57 ; 95% ci , 0.370.86 ; p [ interaction]=0.730 ; figure 2 ) . within the pi group , a trend for ra advantage was evident in the highrisk rescue pci ( 13.4% vs 26.3% ; adjusted or , 0.65 ; 95% ci , 0.391.07 ) subgroup with no significant difference in patients undergoing scheduled pci ( 5.5% vs 5.4% ; adjusted or , 0.55 ; 95% ci , 0.241.26 ) . however , no interaction was evident between rescue pci or scheduled pci as it relates to the advantage of ra after adjustment ( p [ interaction]=0.739 ) . the observed increase in gp 2b/3a use within the ra group did not appear to modulate the association with the 30day primary composite outcome ( ra : gp 2b/3a use vs not , 9.3% versus 8.7% ; and fa : 20.2% vs 14.3% ; p [ interaction]=0.988 ) ; neither did the implementation of the amendment ( halfdose lytic in patients 75 years ) of the stream trial protocol ( ra and fa : pre and postamendment , respectively , 9.0% vs 8.9% and 18.4% vs 15.2% ; p [ interaction]=0.920 ) . the increased gp 2b/3a use within the ra group also did not appear to modulate the association with major bleeding ( ra vs fa : adjusted hazard ratio , 0.56 ; 95% ci , 0.281.12 ; p [ interaction]=0.087 ) . radial access was associated with an observed reduction in 30day mortality ( 2.4% vs 4.7% ; p=0.009 ) , cardiogenic shock ( 2.9% vs 6.4% ; p=0.001 ) , and heart failure ( 5.0% vs 8.4% ; p=0.005 ; table 3 ) . overall , a comparable rate of nonintracranial major bleeding was noted in the ra versus fa group ( 5.2% vs 6.0% ; p=0.489 ; table 3 ) . this was also evident within the 2 treatment strategies ( pi and ppci : ra vs fa , 5.5% vs 7.8% [ p=0.179 ] and 4.9% vs 4.3% [ p=0.698 ] , respectively ) ; however , within the pi strategy , a trend to increased nonintracranial major bleeding in patients treated by fa within the rescue , compared to the scheduled , subgroup was observed ( rescue and scheduled : ra vs fa , 6.1% vs 11.6% [ p=0.064 ] and 5.1% vs 5.1% [ p=0.996 ] ) . both major accesssite ( ra vs fa , 2.8% vs 4.1% ; p=0.163 ) and nonaccesssite ( ra vs fa , 2.3% vs 1.9% ; p=0.487 ) bleeding were similarly distributed in the overall study population . the results of this study indicate that in early presenting stemi ( <3 hours from symptom onset ) , utilization of ra over fa is associated with a significant reduction in the composite of major adverse cardiovascular events regardless of the application of a fibrinolytic pi or ppci strategy . the stream study enrolled patients presenting within 3 hours of symptom onset and thus represents a very early presenting stemi population as compared to other trials that randomized access site in stemi patients ( 1224 hours).2 , 3 , 7 hence , this study evaluated a distinct stemi cohort undergoing early cardiac catheterizations ( except for scheduled pci cohort ) in the presence of potent antiantithrombotic and fibrinolytic agents . it would therefore seem that lower major bleeding would be associated with the observed significant reduction in the primary composite and 30day mortality favoring ra across both treatment strategies . to provide some context , it is noteworthy that 50% of the patients analyzed in the current study were treated with a fibrinolytic pi strategy , as compared to a much lower incidence in the radial versus femoral randomized investigation in stelevation acute coronary syndrome ( rifle steacs ; 7.6% failed fibrinolysis)2 and radial versus femoral access for coronary intervention ( rival ; 12%)7 trials . compared to randomized trials that enrolled postfibrinolytic patients,2 , 7 a higher proportion of major overall and major accesssite bleeding was noted in the current study ; for instance , radial versus femoral , respectively , in rival,7 non coronary artery bypass graft major bleeding ( rival definition ) was noted in 0.84% versus 0.91% , whereas in riflesteacs ( timi definition ) 1.8% versus 2.8% compared to 5.2% and 6.0% in our study . despite no difference in nonintracranial major bleeding between the 2 access sites in this study , our adjusted analysis of 30day mortality showed a persisting benefit associated with the radial approach . it is noteworthy that a recent study from the national cardiovascular data registry 's cathpci registry on bleeding complications in fibrinolytictreated patients undergoing rescue pci reported a major bleeding rate of 6.9% in radial versus 12.0% femoral access patients ( adjusted or , 0.67 ; 95% ci , 0.520.87 ; p=0.003).23 ra was employed in only 16% of these patients , and the authors highlighted the need for further data in this given that the choice of vascular access site was left to investigator discretion and absence of access to detailed procedural elements , we can not exclude the impact of unmeasured confounders . however , to the best of our knowledge , it is the largest data set comparing a randomized , multicenter fibrinolytic pi strategy ( rescue and scheduled pci ) in a very earlytreated stemi population to ppci , demonstrating that the outcomes advantage with ra occurs in both the pi and ppci strategy . irrespective of whether a ppci or pi reperfusion strategy is used , these results support the utilization of radial access as the preferred arterial access site in stemi .

the utilization of radial ( ra ) over femoral arterial access ( fa ) for acute coronary intervention in stelevation myocardial infarction ( stemi ) is supported by a reduction in mortality , ischemic , and bleeding endpoints.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 this has led various interventional societies to encourage its utilization over femoral access.9 , 10 , 11 although the basis of this evidence includes patients undergoing both primary and rescue percutaneous coronary intervention ( pci ) , the majority of randomized data are confined to patients treated with primary pci ( ppci).2 , 7 ra in patients treated with a fibrinolytic pharmacoinvasive strategy ( pi ) has had limited exploration,12 , 13 , 14 as has any direct comparison with patients treated with ppci . moreover , the application of a pi strategy creates at least 2 distinct patient subsets at the time of angiography , each with a different risk profile with respect to ischemic outcomes and major bleeding / accesssite complications.15 the first is the nonreperfused patient requiring urgent rescue pci who has received recent fibrinolysis with adjunctive antithrombotic and antiplatelet therapy sometimes associated with hemodynamic or electrical instability . the strategic reperfusion early after myocardial infarction ( stream ) trial,16 which randomized patients to a fibrinolytic pi ( rescue and scheduled pci ) versus ppci treatment strategy , provided a unique opportunity to address this issue in an early presenting , rapidly treated contemporary stemi cohort . accordingly , within stream , we evaluated the relationship between arterial access site and the 30day primary composite ( all cause death , shock , congestive heart failure [ chf ] , and reinfarction ) as well as major bleeding events in stemi according to the treatment strategy received . in the pi strategy , bolus weightbased tenecteplase ( tnk ) , aspirin , clopidogrel , and enoxaparin were administered according to guideline recommendations and followed by either rescue pci or scheduled angiography ( within 624 hours ) . after 21% of the ultimate population had been enrolled , the executive committee amended the protocol on august 24 , 2009 , to reduce the dose of tnk by 50% in patients 75 years of age or older because of an excess of intracranial hemorrhage ( ich ) in that age group . primary pci was conducted after expeditious transfer from the point of randomization , and early initiation of aspirin , clopidogrel , and antithrombotic therapy , including discretionary glycoprotein iib / iiia ( gp 2b/3a ) antagonists based on best standard practice . both worst lead stelevation resolution and worst lead residual stelevation have previously been shown to have prognostic utility in stemi patients undergoing primary pci.18 interventional cardiologists in participating sites determined the choice of the arterial access , either ra or fa based upon local practice ( table s1 ) . angiographic assessment detailing coronary anatomy , need for percutaneous intervention after diagnostic angiography and thrombolysis in myocardial infarction ( timi ) flow grade postpci was performed locally by sitespecific investigators using standard definitions.19 management of the arterial sheath postangiography was also determined by sitespecific vascular access best practice protocols . the primary outcome of this study was a 30day composite of death , cardiogenic shock , chf , or reinfarction whereas the key secondary outcomes included components of the primary outcome at 30 days , nonintracranial major bleeding or ich , stroke , and 1year allcause mortality . the association between access site and primary clinical outcome ( composite of death , cardiogenic shock , chf , or reinfarction ) within 30 days was examined using a univariable logistic regression model where a propensity score for access site was used to construct an inverse probability weight.21 given the assignment of access site was not randomized , a propensity score for access site was created using a multivariable logistic regression model . variables included in the final propensity score model were age , sex , weight , history of hypertension , history of diabetes mellitus , heart rate , systolic blood pressure , killip class , inferior myocardial infarction ( mi ) , sum st elevation at baseline , q waves at baseline , time from symptom onset to randomization , and country of enrollment ( table s2 ) . to test whether gp 2b/3a use and protocol amendment modulate the association between access site and 30day primary outcomes , the interaction between gp 2b/3a use and access site and the interaction between protocol amendment and access site were examined . in the pi strategy , bolus weightbased tenecteplase ( tnk ) , aspirin , clopidogrel , and enoxaparin were administered according to guideline recommendations and followed by either rescue pci or scheduled angiography ( within 624 hours ) . after 21% of the ultimate population had been enrolled , the executive committee amended the protocol on august 24 , 2009 , to reduce the dose of tnk by 50% in patients 75 years of age or older because of an excess of intracranial hemorrhage ( ich ) in that age group . primary pci was conducted after expeditious transfer from the point of randomization , and early initiation of aspirin , clopidogrel , and antithrombotic therapy , including discretionary glycoprotein iib / iiia ( gp 2b/3a ) antagonists based on best standard practice . both worst lead stelevation resolution and worst lead residual stelevation have previously been shown to have prognostic utility in stemi patients undergoing primary pci.18 interventional cardiologists in participating sites determined the choice of the arterial access , either ra or fa based upon local practice ( table s1 ) . angiographic assessment detailing coronary anatomy , need for percutaneous intervention after diagnostic angiography and thrombolysis in myocardial infarction ( timi ) flow grade postpci was performed locally by sitespecific investigators using standard definitions.19 management of the arterial sheath postangiography was also determined by sitespecific vascular access best practice protocols . the primary outcome of this study was a 30day composite of death , cardiogenic shock , chf , or reinfarction whereas the key secondary outcomes included components of the primary outcome at 30 days , nonintracranial major bleeding or ich , stroke , and 1year allcause mortality . the association between access site and primary clinical outcome ( composite of death , cardiogenic shock , chf , or reinfarction ) within 30 days was examined using a univariable logistic regression model where a propensity score for access site was used to construct an inverse probability weight.21 given the assignment of access site was not randomized , a propensity score for access site was created using a multivariable logistic regression model . variables included in the final propensity score model were age , sex , weight , history of hypertension , history of diabetes mellitus , heart rate , systolic blood pressure , killip class , inferior myocardial infarction ( mi ) , sum st elevation at baseline , q waves at baseline , time from symptom onset to randomization , and country of enrollment ( table s2 ) . to test whether gp 2b/3a use and protocol amendment modulate the association between access site and 30day primary outcomes , the interaction between gp 2b/3a use and access site and the interaction between protocol amendment and access site were examined . overall , stemi patients treated by fa were younger , had more past hypertension , lower systolic bp at presentation , lower timi risk score , and more stelevation on the baseline ecg compared to the ra group ( table 1 ) . a significantly higher utilization of gp2b/3a inhibitor use was noted in the ra , compared to fa , group ( 40.2% vs 24.1% ; p<0.001 ) , particularly in those undergoing ppci . evaluation of the posttreatment ecg revealed consistently better indices of reperfusion for patients with ra in both the pi and ppci cohorts , as evidenced by higher rates of worst lead residual st elevation < 1 mm and lesser rates in patients with 2 mm residual st elevation . irrespective of treatment strategy , the unadjusted primary composite of 30day death , shock , chf , or reinfarction occurred in 8.9% in the ra compared to 15.7% in fa group ( table 3 ) . analysis of access site categorized by study treatment received revealed that the advantage associated with ra was present in both the ppci ( adjusted or , 0.63 ; 95% ci , 0.430.92 ) and pi cohorts ( adjusted or , 0.57 ; 95% ci , 0.370.86 ; p [ interaction]=0.730 ; figure 2 ) . within the pi group , a trend for ra advantage was evident in the highrisk rescue pci ( 13.4% vs 26.3% ; adjusted or , 0.65 ; 95% ci , 0.391.07 ) subgroup with no significant difference in patients undergoing scheduled pci ( 5.5% vs 5.4% ; adjusted or , 0.55 ; 95% ci , 0.241.26 ) . the observed increase in gp 2b/3a use within the ra group did not appear to modulate the association with the 30day primary composite outcome ( ra : gp 2b/3a use vs not , 9.3% versus 8.7% ; and fa : 20.2% vs 14.3% ; p [ interaction]=0.988 ) ; neither did the implementation of the amendment ( halfdose lytic in patients 75 years ) of the stream trial protocol ( ra and fa : pre and postamendment , respectively , 9.0% vs 8.9% and 18.4% vs 15.2% ; p [ interaction]=0.920 ) . the increased gp 2b/3a use within the ra group also did not appear to modulate the association with major bleeding ( ra vs fa : adjusted hazard ratio , 0.56 ; 95% ci , 0.281.12 ; p [ interaction]=0.087 ) . radial access was associated with an observed reduction in 30day mortality ( 2.4% vs 4.7% ; p=0.009 ) , cardiogenic shock ( 2.9% vs 6.4% ; p=0.001 ) , and heart failure ( 5.0% vs 8.4% ; p=0.005 ; table 3 ) . this was also evident within the 2 treatment strategies ( pi and ppci : ra vs fa , 5.5% vs 7.8% [ p=0.179 ] and 4.9% vs 4.3% [ p=0.698 ] , respectively ) ; however , within the pi strategy , a trend to increased nonintracranial major bleeding in patients treated by fa within the rescue , compared to the scheduled , subgroup was observed ( rescue and scheduled : ra vs fa , 6.1% vs 11.6% [ p=0.064 ] and 5.1% vs 5.1% [ p=0.996 ] ) . both major accesssite ( ra vs fa , 2.8% vs 4.1% ; p=0.163 ) and nonaccesssite ( ra vs fa , 2.3% vs 1.9% ; p=0.487 ) bleeding were similarly distributed in the overall study population . overall , stemi patients treated by fa were younger , had more past hypertension , lower systolic bp at presentation , lower timi risk score , and more stelevation on the baseline ecg compared to the ra group ( table 1 ) . a significantly higher utilization of gp2b/3a inhibitor use was noted in the ra , compared to fa , group ( 40.2% vs 24.1% ; p<0.001 ) , particularly in those undergoing ppci . evaluation of the posttreatment ecg revealed consistently better indices of reperfusion for patients with ra in both the pi and ppci cohorts , as evidenced by higher rates of worst lead residual st elevation < 1 mm and lesser rates in patients with 2 mm residual st elevation . irrespective of treatment strategy , the unadjusted primary composite of 30day death , shock , chf , or reinfarction occurred in 8.9% in the ra compared to 15.7% in fa group ( table 3 ) . analysis of access site categorized by study treatment received revealed that the advantage associated with ra was present in both the ppci ( adjusted or , 0.63 ; 95% ci , 0.430.92 ) and pi cohorts ( adjusted or , 0.57 ; 95% ci , 0.370.86 ; p [ interaction]=0.730 ; figure 2 ) . within the pi group , a trend for ra advantage was evident in the highrisk rescue pci ( 13.4% vs 26.3% ; adjusted or , 0.65 ; 95% ci , 0.391.07 ) subgroup with no significant difference in patients undergoing scheduled pci ( 5.5% vs 5.4% ; adjusted or , 0.55 ; 95% ci , 0.241.26 ) . the observed increase in gp 2b/3a use within the ra group did not appear to modulate the association with the 30day primary composite outcome ( ra : gp 2b/3a use vs not , 9.3% versus 8.7% ; and fa : 20.2% vs 14.3% ; p [ interaction]=0.988 ) ; neither did the implementation of the amendment ( halfdose lytic in patients 75 years ) of the stream trial protocol ( ra and fa : pre and postamendment , respectively , 9.0% vs 8.9% and 18.4% vs 15.2% ; p [ interaction]=0.920 ) . the increased gp 2b/3a use within the ra group also did not appear to modulate the association with major bleeding ( ra vs fa : adjusted hazard ratio , 0.56 ; 95% ci , 0.281.12 ; p [ interaction]=0.087 ) . radial access was associated with an observed reduction in 30day mortality ( 2.4% vs 4.7% ; p=0.009 ) , cardiogenic shock ( 2.9% vs 6.4% ; p=0.001 ) , and heart failure ( 5.0% vs 8.4% ; p=0.005 ; table 3 ) . this was also evident within the 2 treatment strategies ( pi and ppci : ra vs fa , 5.5% vs 7.8% [ p=0.179 ] and 4.9% vs 4.3% [ p=0.698 ] , respectively ) ; however , within the pi strategy , a trend to increased nonintracranial major bleeding in patients treated by fa within the rescue , compared to the scheduled , subgroup was observed ( rescue and scheduled : ra vs fa , 6.1% vs 11.6% [ p=0.064 ] and 5.1% vs 5.1% [ p=0.996 ] ) . both major accesssite ( ra vs fa , 2.8% vs 4.1% ; p=0.163 ) and nonaccesssite ( ra vs fa , 2.3% vs 1.9% ; p=0.487 ) bleeding were similarly distributed in the overall study population . the results of this study indicate that in early presenting stemi ( <3 hours from symptom onset ) , utilization of ra over fa is associated with a significant reduction in the composite of major adverse cardiovascular events regardless of the application of a fibrinolytic pi or ppci strategy . in addition , within the pi strategy , the prognostic advantage of ra applies particularly to the higher risk rescue pci cohort in whom a doubling of adverse outcomes appeared evident within the femoral access subgroup . the stream study enrolled patients presenting within 3 hours of symptom onset and thus represents a very early presenting stemi population as compared to other trials that randomized access site in stemi patients ( 1224 hours).2 , 3 , 7 hence , this study evaluated a distinct stemi cohort undergoing early cardiac catheterizations ( except for scheduled pci cohort ) in the presence of potent antiantithrombotic and fibrinolytic agents . apart from lower major bleeding , the mechanism by which radial access might relate to allcause mortality currently remains unclear in the existing literature.13 , 22 one plausible explanation for the observed prognostic difference favoring ra relates to selection bias introduced by operator experience and centerspecific differences . to provide some context , it is noteworthy that 50% of the patients analyzed in the current study were treated with a fibrinolytic pi strategy , as compared to a much lower incidence in the radial versus femoral randomized investigation in stelevation acute coronary syndrome ( rifle steacs ; 7.6% failed fibrinolysis)2 and radial versus femoral access for coronary intervention ( rival ; 12%)7 trials . hence , the current results not only support the effectiveness of ra within a pitreated stemi patient subset , but also allow comparison of outcomes between roughly equalsized groups undergoing different reperfusion strategies . compared to randomized trials that enrolled postfibrinolytic patients,2 , 7 a higher proportion of major overall and major accesssite bleeding was noted in the current study ; for instance , radial versus femoral , respectively , in rival,7 non coronary artery bypass graft major bleeding ( rival definition ) was noted in 0.84% versus 0.91% , whereas in riflesteacs ( timi definition ) 1.8% versus 2.8% compared to 5.2% and 6.0% in our study . the reasons for this disparity in major bleeding is unclear , but could relate to : ( 1 ) investigator rather than central adjudication of major bleeding in the current study ; ( 2 ) heterogeneity in definition of major bleeding ; ( 3 ) differences in the proportion of the postfibrinolytic stemi population ; and ( 4 ) variability in operator expertise and sitespecific vascular access protocols . it is noteworthy that a recent study from the national cardiovascular data registry 's cathpci registry on bleeding complications in fibrinolytictreated patients undergoing rescue pci reported a major bleeding rate of 6.9% in radial versus 12.0% femoral access patients ( adjusted or , 0.67 ; 95% ci , 0.520.87 ; p=0.003).23 ra was employed in only 16% of these patients , and the authors highlighted the need for further data in this given that the choice of vascular access site was left to investigator discretion and absence of access to detailed procedural elements , we can not exclude the impact of unmeasured confounders . although we found no overall difference in vascular access bleeding , the trend toward more fa access bleeding in the pi patients undergoing rescue pci suggests less bleeding hazard in the presence of recent fibrinolytic treatment when radial access is employed . however , to the best of our knowledge , it is the largest data set comparing a randomized , multicenter fibrinolytic pi strategy ( rescue and scheduled pci ) in a very earlytreated stemi population to ppci , demonstrating that the outcomes advantage with ra occurs in both the pi and ppci strategy .

spinal subdural hematomas ( ssdhs ) have been reported to occur after minor trauma , lumbar puncture , spinal anesthesia,1 or spinal surgery , especially in the presence of intraoperative dural tears.2 nontraumatic ( spontaneous ) ssdhs are much more rare , with a recent review having identified 106 cases reported in the english literature.3 several predisposing factors have been associated with the occurrence of spontaneous ssdhs , such as coagulation abnormalities , anticoagulation therapy,4 platelet dysfunction,5 polycythemia vera,6 pregnancy,7 arterial wall abnormalities,8 and , more rarely , the presence of spinal arteriovenous malformations.9 most ssdhs occur at the lower thoracic region ( the second most common location being the upper cervical spine ) and typically extend from two to five segments.10 the vast majority of ssdhs are located anteriorly to the spinal cord , while most epidural spinal hematomas are located posteriorly to the spinal cord.11 this difference is related to the fact that the posterior longitudinal ligament closely adheres to the vertebral bodies , thus limiting anterior epidural collections . interestingly , the vast majority of case reports on operated ssdhs identified no intermingling between the subdural blood and the cerebrospinal fluid ( csf ) . because the hematoma is confined to the extra - arachnoid space , some authors have suggested that what is commonly designated as an ssdh is , in fact , a dissection between the two inner layers of the spinal dura.12 this concept is supported by anatomic studies that demonstrated that under physiologic conditions , the spinal subdural space is only a capillary slit , which may occasionally extend into a genuine space under pathologic conditions such as during a subdural bleeding.10 13 percutaneous vertebroplasty is a therapeutic strategy that has gained increasing interest in the neurosurgical community for the treatment of refractory axial mechanical pain in patients with vertebral compression fractures . one of the main therapeutic mechanisms of cement augmentation has been proposed to be the improvement in the spinal stability provided by the injection of polymethyl methacrylate ( pmma ) into the fractured vertebral body.14 several complications of vertebroplasty have been reported in the literature , with the vast majority of them being related to cement extravasation to the epidural canal leading to spinal cord compression ( some series report extravasation rates of up to 20% with approximately one - third of such patients being neurologically symptomatic and requiring surgical intervention)15 or related to cement migration through the epidural veins to the venous system leading to pulmonary embolism ( reported to occur in 0.8 to 2.1% of the patients , with the vast majority of patients being asymptomatic).16 in this context , the severity grade of the fracture and a low viscosity of pmma cement have been identified as strong and independent risk factors for cement leakage.17 in this article , we report a patient who developed an acute ssdh following a percutaneous vertebroplasty without signs of major cement extravasation to the spinal canal . the authors perform a comprehensive literature review on the pathophysiology of ssdh and highlight the specific nuances of the presented case report to support a new hypothetical role of venous congestion as a possible etiologic factor involved in the pathophysiology of ssdh . finally , directions for future experimental and clinical research for further investigation of such hypothesis are also delineated . a 49-year - old woman presented to the outpatient clinic with complaints of axial midthoracic pain and a history of a recent fall down the stairs . at that time , the computed tomography ( ct ) scan of the thoracic spine demonstrated a t8 compression fracture affecting mainly the midportion of the vertebral body , with preservation of the posterior cortex ( fig . she was treated conservatively with a tlso brace and analgesics . however , at the 3-months follow - up , she still presented with episodes of severe , deep axial pain centered on her midthoracic spine . the pain was essentially mechanical in nature , with worsening of the symptoms with activity and partial improvement with bedrest . the 3-months follow - up x - ray demonstrated progression of the compression fracture with further loss of height especially in the anterior third of the vertebral body leading to worsening of the kyphotic deformity ( fig . ( a ) initial sagittal computed tomography scan demonstrating a t8 compression fracture affecting mainly the midportion of the vertebral body , with preservation of the posterior cortex . ( b ) lateral plain x - ray of the thoracic spine performed at the 3-months follow - up after failed conservative treatment demonstrating progression of the fracture with further loss of height especially in the anterior third of the vertebral body , leading to worsening of the kyphotic deformity . after cannulation of the left t8 pedicle and the initial injection of pmma , a small posterior extravasation of cement to the epidural veins was observed ( fig . after awaking , the patient presented diffuse numbness on the left side ( both in the superior and inferior limbs ) as well as diffuse weakness ( strength 3/5 ) in the left leg . an immediate ct scan demonstrated only a very small posterior leakage of pmma toward the epidural space as well as into the adjacent costotransverse joint ( fig . 2 ) . however , it was possible to observe a hyperdense collection anterior to the spinal cord beginning one level above the site of the vertebroplasty and extending up to the cervical spine . the presence of pneumorrhachis at a point distant to the level of the vertebroplasty was also identified . ( a ) intraoperative fluoroscopy demonstrating a small extravasation of cement to the posterior epidural veins in the most superior region of the vertebral body . immediate postprocedure ( b ) sagittal and ( c ) axial computed tomography scans demonstrating only a very small posterior leakage toward the spinal canal , as well as the presence of ( d ) a spinal subdural hematoma ( black arrow ) extending from one level above the vertebroplasty to the lower cervical spine . note also the presence of pneumorrhachis ( white arrow ) . due to the acute motor deficit in the left lower limb , the patient was submitted to an immediate decompressive laminectomy at the level of the vertebroplasty and extending one level above and one level below to encompass those levels in which the cross - sectional area of the spinal canal was significantly compromised . no major compression of the thecal sac or epidural bleeding was identified during the surgical procedure . the postoperative magnetic resonance imaging ( fig . 3 ) confirmed the presence of a collection with imaging characteristics of an ssdh , beginning one level above the vertebroplasty and extending up to the lower cervical spine , but without any residual signs of compression , as demonstrated by the presence of csf posterior to the spinal cord ( fig . the subdural collection is located anterior to the spinal cord and extends up to the cervical spine . the acute spinal subdural hematoma is hyperdense in both t1- ( a ) and t2-weighted imaging ( b ) . the axial images demonstrate no residual compression either at the laminectomy level ( c ) or in the levels above ( d ) as evidenced by the presence of cerebrospinal fluid posterior to the spinal cord . after physical therapy and rehabilitation the patient progressively recovered the motor function in the left leg . at the 3-months follow - up , she presented with almost complete recovery of her strength in the left leg ( grade 4+/5 for hip flexion , hip extension , leg flexion and extension , and dorsiflexion and plantar flexion ) , although the sensory symptoms still persisted in the whole left side , both in the superior and inferior limbs , requiring treatment with gabapentin . the vertebral venous plexus is a complex , large - capacity , plexiform venous system that is believed to play an important role in the regulation of intracranial pressure with posture changes . additionally , due to this plexus ' valveless , bidirectional flow , it also provides a direct route for tumor or infection dissemination from the pelvic and lower lumbar region to the thoracolumbar spine . the fact that the vertebral venous plexus appears to be much larger than what would be expected for the drainage of the spinal cord and meninges has led some authors to suggest that this vascular bed may also function as an alternate route for venous blood drainage between the inferior and superior vena cava.18 19 moreover , the fact that such a large plexus does not contain valves has led some authors to infer a possible secondary role as a pressure - regulating system that can protect the spinal cord from the volume and pressure peaks occurring in the intra - abdominal , intrathoracic , intracranial , and intraspinal spaces.10 11 the anatomy of the vertebral venous plexus has been somewhat ignored by ancient anatomic reports and only received the due attention in recent centuries ; special acknowledgments must be given to the works of batson ( 18941979 ) , after whom this venous plexus is named , and breschet ( 17841845).20 the vertebral venous plexus has been classically divided into an internal ( intradural ) vertebral plexus ( which possesses an anterior and a posterior component ) and an external ( or epidural ) vertebral plexus . recently , it has been demonstrated that the epidural vertebral plexus is closely connected to the intracranial venous sinuses in what could be described as the cerebrospinal venous system,21 or the so - called extradural neural axis compartment.22 in relation to the vertebral bodies , several small subsystems of plexiform veins work synergistically to accomplish their venous drainage ( fig . 4 ) . the basivertebral system , which is arranged horizontally in the middle of the vertebral body , forms a large - scale venous grid into which the vertical veins of the vertebral body flow from above and below . the subarticular collecting system is another important plexus formed by large - caliber vertical tributary veins , which abruptly turn to run horizontally , parallel to the vertebral endplates , ultimately draining posteriorly into the epidural venous plexus.23 ( a ) schematic representation of the vertebral venous system , which can be divided into an internal vertebral plexus ( composed of 1 : anterior internal vertebral venous plexus ; 2 : posterior internal vertebral venous plexus ) , and an external ( or epidural ) vertebral plexus ( composed of 4 : posterior external vertebral venous plexus ; 5 : anterior external vertebral venous plexus ; and 8 : radicular vein ) and the ( 6 ) radiculomedullary vein , which connects both . the drainage of the vertebral body is mainly performed by the ( 3 ) basivertebral plexus and the subarticular collecting plexus ( not shown in this image ) . ( b ) a radiograph of a thin coronal section near the central area of a lumbar vertebra . note the stellate arrangement of tributaries draining into the central vein of the basivertebral plexus . ( c ) radiograph of a thin sagittal section cut laterally near the vertebral pedicle . the horizontal subarticular collecting venous system of the vertebral body can be seen running parallel to the inferior vertebral endplate . this system drains by vertical stems through perforations in the vertebral endplates into the larger horizontal subarticular collecting vein system . ( a ) reproduced with permission from groen rjm , grobbelaar m , muller cjf , et al . morphology of the human internal vertebral venous plexus : a cadaver study after latex injection in the 2125-week fetus . ( b and c ) reproduced with permission and copyright of the british editorial society of bone and joint surgery from crock hv , yoshizawa h , kame sk . j bone joint surg br 1973;55(3):528533.23 similar to its arterial counterparts , multiple radiculomedullary veins provide segmental drainage to the emerging spinal nerve roots . these radiculomedullary veins run in the same oblique course as the radiculomedullary arteries , but typically arise at different spinal levels . it has already been demonstrated that the radiculomedullary veins constitute the weakest link between the intradural venous system ( the internal vertebral venous plexus ) and the epidural space ( the external vertebral venous plexus).24 although presenting a small caliber , such radiculomedullary veins are believed to be of crucial importance for the venous drainage of the nerve roots of the cauda equina . in fact , several studies have suggested that the symptoms of neurogenic claudication and radicular pain in patients with lumbar canal stenosis may be more related to venous congestion than to deficits from an impaired arterial supply of the cauda equina.22 25 although the radiculomedullary veins have already been extensively investigated for their supposed role in the pathophysiology of spinal dural arteriovenous fistulas,21 very few studies have mentioned their possible role in the etiology of spontaneous or traumatic spinal subdural hematomas.26 27 moreover , although several previous studies have investigated the general anatomy of the epidural vertebral venous plexus20 28 29 30 31 and the internal vertebral venous plexus,32 33 no specific study focusing on the anatomy of the radiculomedullary veins exists . additionally , no previous histologic study has investigated the actual vulnerability of such apparently weak connection point between the epidural vertebral venous system and the internal venous plexus , as well as its response to congestive venous hypertension . although several reported cases of ssdh have been associated with minor traumatic events or some predisposing coagulation disorder , the exact pathophysiology of ssdh still remains obscure . intracranial subdural hematomas have been commonly ascribed to the rupture of subdural bridging veins . however , unlike the intracranial subdural space , it has already been shown that the spinal subdural compartment lacks bridging veins.34 interestingly , previous reports have demonstrated the association of ssdh with sudden episodes of increased intra - abdominal or intrathoracic pressure ( such as coughing or straining),26 35 36 suggesting the presence of a so - called locus minoris resistentiae ( a place of lower resistance ) in the vascular venous system between the internal and external vertebral venous plexus , which , when submitted to excessive pressures due to venous congestion , would possibly rupture , ultimately leading to extravasation of blood into the subdural space.35 36 alternatively , some authors have suggested that ssdhs might possibly originate from a few thin and delicate extra - arachnoid vessels that have been identified on the inner dural surface.37 38 although such etiology might explain some specific cases of ssdh in which the subdural hematoma occurs in association with a subarachnoid hemorrhage of traumatic origin , it does not explain the several reported cases of ssdhs in which it has been confirmed intraoperatively that the blood was confined to the extra - arachnoid space.39 in the reported case , another factor suggesting an etiology involving venous congestion ( related to the obstruction by pmma of the vertebral venous plexus responsible for the venous drainage of the vertebral body ) is the occurrence of neurologic deficits in the absence of signs of spinal cord compression . in the same way , the occurrence of secondary neurologic deficits due to venous congestion and hypoxia secondary to venous outflow obstruction has already been described in a previous report of a patient who presented with reversible myelopathy and spinal cord edema after a traumatic mediastinal hematoma leading to compression of the brachiocephalic vein.40 similar neurologic deficits related to intraspinal venous hypertension and hypoxia have already been reported after venous thrombosis and venous outflow obstruction during embolization of spinal dural arteriovenous fistulas.41 additionally , the occurrence of a special form of chronic necrotic myelopathy is a well - known phenomenon related to the chronic venous congestion that occurs in patients with spinal dural arteriovenous fistulas ( the so - called foix - alajouanine syndrome).42 the appearance of pneumorrhachis ( presence of air in the subdural space ) in the presented case also supports the hypothesis of a breakdown in the venous system that generated a differential pressure with the surrounding spaces due to its negative intraluminal pressure , ultimately leading to focal accumulation of air in the subdural space . similarly , previous reports have demonstrated the occurrence of pneumorrhachis in situations associated with an increased intrathoracic or intra - abdominal pressure ( such as cardiopulmonary resuscitation and airway obstruction because of foreign body aspiration ) leading to hypertensive venous obstruction of the epidural venous system.43 44 although the management of ssdh is controversial in the literature ( with some authors proposing surgery in the acute phase for spinal decompression and hematoma drainage ) , it has been demonstrated that patients with incomplete neurologic deficits tend to present a positive recovery without drainage of the hematoma . also , in the absence of radiologic evidence of spinal cord compression ( such as in the reported case , in which it was possible to visualize pouches of csf posterior to the spinal cord in the affected levels),27 surgical attempts to open the dura and drain the hematoma seem to be of questionable value , especially in extensive lesions involving several levels as in the reported case.26 in the long term , it has been demonstrated that the physiologic flow of csf tends to dilute the ssdh , ultimately leading to its spontaneous resolution as observed by follow - up imaging.26 45 the vertebral venous plexus is a complex , large - capacity , plexiform venous system that is believed to play an important role in the regulation of intracranial pressure with posture changes . additionally , due to this plexus ' valveless , bidirectional flow , it also provides a direct route for tumor or infection dissemination from the pelvic and lower lumbar region to the thoracolumbar spine . the fact that the vertebral venous plexus appears to be much larger than what would be expected for the drainage of the spinal cord and meninges has led some authors to suggest that this vascular bed may also function as an alternate route for venous blood drainage between the inferior and superior vena cava.18 19 moreover , the fact that such a large plexus does not contain valves has led some authors to infer a possible secondary role as a pressure - regulating system that can protect the spinal cord from the volume and pressure peaks occurring in the intra - abdominal , intrathoracic , intracranial , and intraspinal spaces.10 11 the anatomy of the vertebral venous plexus has been somewhat ignored by ancient anatomic reports and only received the due attention in recent centuries ; special acknowledgments must be given to the works of batson ( 18941979 ) , after whom this venous plexus is named , and breschet ( 17841845).20 the vertebral venous plexus has been classically divided into an internal ( intradural ) vertebral plexus ( which possesses an anterior and a posterior component ) and an external ( or epidural ) vertebral plexus . recently , it has been demonstrated that the epidural vertebral plexus is closely connected to the intracranial venous sinuses in what could be described as the cerebrospinal venous system,21 or the so - called extradural neural axis compartment.22 in relation to the vertebral bodies , several small subsystems of plexiform veins work synergistically to accomplish their venous drainage ( fig . 4 ) . the basivertebral system , which is arranged horizontally in the middle of the vertebral body , forms a large - scale venous grid into which the vertical veins of the vertebral body flow from above and below . the subarticular collecting system is another important plexus formed by large - caliber vertical tributary veins , which abruptly turn to run horizontally , parallel to the vertebral endplates , ultimately draining posteriorly into the epidural venous plexus.23 ( a ) schematic representation of the vertebral venous system , which can be divided into an internal vertebral plexus ( composed of 1 : anterior internal vertebral venous plexus ; 2 : posterior internal vertebral venous plexus ) , and an external ( or epidural ) vertebral plexus ( composed of 4 : posterior external vertebral venous plexus ; 5 : anterior external vertebral venous plexus ; and 8 : radicular vein ) and the ( 6 ) radiculomedullary vein , which connects both . the drainage of the vertebral body is mainly performed by the ( 3 ) basivertebral plexus and the subarticular collecting plexus ( not shown in this image ) . ( b ) a radiograph of a thin coronal section near the central area of a lumbar vertebra . note the stellate arrangement of tributaries draining into the central vein of the basivertebral plexus . ( c ) radiograph of a thin sagittal section cut laterally near the vertebral pedicle . the horizontal subarticular collecting venous system of the vertebral body can be seen running parallel to the inferior vertebral endplate . this system drains by vertical stems through perforations in the vertebral endplates into the larger horizontal subarticular collecting vein system . ( a ) reproduced with permission from groen rjm , grobbelaar m , muller cjf , et al . morphology of the human internal vertebral venous plexus : a cadaver study after latex injection in the 2125-week fetus . ( b and c ) reproduced with permission and copyright of the british editorial society of bone and joint surgery from crock hv , yoshizawa h , kame sk . j bone joint surg br 1973;55(3):528533.23 similar to its arterial counterparts , multiple radiculomedullary veins provide segmental drainage to the emerging spinal nerve roots . these radiculomedullary veins run in the same oblique course as the radiculomedullary arteries , but typically arise at different spinal levels . it has already been demonstrated that the radiculomedullary veins constitute the weakest link between the intradural venous system ( the internal vertebral venous plexus ) and the epidural space ( the external vertebral venous plexus).24 although presenting a small caliber , such radiculomedullary veins are believed to be of crucial importance for the venous drainage of the nerve roots of the cauda equina . in fact , several studies have suggested that the symptoms of neurogenic claudication and radicular pain in patients with lumbar canal stenosis may be more related to venous congestion than to deficits from an impaired arterial supply of the cauda equina.22 25 although the radiculomedullary veins have already been extensively investigated for their supposed role in the pathophysiology of spinal dural arteriovenous fistulas,21 very few studies have mentioned their possible role in the etiology of spontaneous or traumatic spinal subdural hematomas.26 27 moreover , although several previous studies have investigated the general anatomy of the epidural vertebral venous plexus20 28 29 30 31 and the internal vertebral venous plexus,32 33 no specific study focusing on the anatomy of the radiculomedullary veins exists . additionally , no previous histologic study has investigated the actual vulnerability of such apparently weak connection point between the epidural vertebral venous system and the internal venous plexus , as well as its response to congestive venous hypertension . although several reported cases of ssdh have been associated with minor traumatic events or some predisposing coagulation disorder , the exact pathophysiology of ssdh still remains obscure . intracranial subdural hematomas have been commonly ascribed to the rupture of subdural bridging veins . however , unlike the intracranial subdural space , it has already been shown that the spinal subdural compartment lacks bridging veins.34 interestingly , previous reports have demonstrated the association of ssdh with sudden episodes of increased intra - abdominal or intrathoracic pressure ( such as coughing or straining),26 35 36 suggesting the presence of a so - called locus minoris resistentiae ( a place of lower resistance ) in the vascular venous system between the internal and external vertebral venous plexus , which , when submitted to excessive pressures due to venous congestion , would possibly rupture , ultimately leading to extravasation of blood into the subdural space.35 36 alternatively , some authors have suggested that ssdhs might possibly originate from a few thin and delicate extra - arachnoid vessels that have been identified on the inner dural surface.37 38 although such etiology might explain some specific cases of ssdh in which the subdural hematoma occurs in association with a subarachnoid hemorrhage of traumatic origin , it does not explain the several reported cases of ssdhs in which it has been confirmed intraoperatively that the blood was confined to the extra - arachnoid space.39 in the reported case , another factor suggesting an etiology involving venous congestion ( related to the obstruction by pmma of the vertebral venous plexus responsible for the venous drainage of the vertebral body ) is the occurrence of neurologic deficits in the absence of signs of spinal cord compression . in the same way , the occurrence of secondary neurologic deficits due to venous congestion and hypoxia secondary to venous outflow obstruction has already been described in a previous report of a patient who presented with reversible myelopathy and spinal cord edema after a traumatic mediastinal hematoma leading to compression of the brachiocephalic vein.40 similar neurologic deficits related to intraspinal venous hypertension and hypoxia have already been reported after venous thrombosis and venous outflow obstruction during embolization of spinal dural arteriovenous fistulas.41 additionally , the occurrence of a special form of chronic necrotic myelopathy is a well - known phenomenon related to the chronic venous congestion that occurs in patients with spinal dural arteriovenous fistulas ( the so - called foix - alajouanine syndrome).42 the appearance of pneumorrhachis ( presence of air in the subdural space ) in the presented case also supports the hypothesis of a breakdown in the venous system that generated a differential pressure with the surrounding spaces due to its negative intraluminal pressure , ultimately leading to focal accumulation of air in the subdural space . similarly , previous reports have demonstrated the occurrence of pneumorrhachis in situations associated with an increased intrathoracic or intra - abdominal pressure ( such as cardiopulmonary resuscitation and airway obstruction because of foreign body aspiration ) leading to hypertensive venous obstruction of the epidural venous system.43 44 although the management of ssdh is controversial in the literature ( with some authors proposing surgery in the acute phase for spinal decompression and hematoma drainage ) , it has been demonstrated that patients with incomplete neurologic deficits tend to present a positive recovery without drainage of the hematoma . also , in the absence of radiologic evidence of spinal cord compression ( such as in the reported case , in which it was possible to visualize pouches of csf posterior to the spinal cord in the affected levels),27 surgical attempts to open the dura and drain the hematoma seem to be of questionable value , especially in extensive lesions involving several levels as in the reported case.26 in the long term , it has been demonstrated that the physiologic flow of csf tends to dilute the ssdh , ultimately leading to its spontaneous resolution as observed by follow - up imaging.26 45 the occurrence of such a complication in a level different from that of the intervention and in the absence of a major pmma extravasation into the spinal canal ( as well as in the presence of pneumorrhachis ) strongly suggests a venous congestive mechanism as the most plausible etiologic explanation . in this article , we propose a new pathophysiological scheme that may explain the development of ssdhs , at least in a subset of patients . this newly proposed etiologic pathway emphasizes the role of venous congestion leading to the rupture of the fragile radiculomedullary veins into the subdural space . ultimately , this case report highlights the necessity of further anatomical and histologic studies of the radiculomedullary veins to better investigate their behavior in the face of congestive venous hypertension , as well as the supposed relationship between their possible rupture and the development of ssdhs .

study design case report and literature review . objective spinal subdural hematomas are rare events that often progress with severe neurologic deficits . although there have been several case reports in the literature of spontaneous spinal subdural hematomas in the setting of anticoagulation , antiplatelet therapy , or coagulation disorders , the exact pathophysiology of such phenomena remains obscure . methods we present the first report of a subdural hematoma after a percutaneous vertebroplasty and provide a comprehensive review on the anatomy of venous drainage of the vertebral bodies with emphasis on the possible effects of venous congestion caused by cement obstruction . results because the subdural hematoma occurred in the absence of major cement extravasation to the spinal canal and two levels above the site of the vertebroplasty , we discuss the possible role of venous congestion as the main etiologic factor leading to rupture of the fragile , valveless radiculomedullary veins into the subdural space . conclusions the reported case supports a possible new pathophysiological scheme for the development of spinal subdural hematoma in which venous congestion plays a pivotal etiologic role . the reported findings suggests that future anatomical and histologic studies investigating the response of the radiculomedullary veins to congestive venous hypertension may shed new light into the pathophysiology of spinal subdural hematomas .

Introduction Case Report Discussion Anatomy of Spinal Venous Drainage Pathophysiology of Spinal Subdural Hematomas Conclusions

spinal subdural hematomas ( ssdhs ) have been reported to occur after minor trauma , lumbar puncture , spinal anesthesia,1 or spinal surgery , especially in the presence of intraoperative dural tears.2 nontraumatic ( spontaneous ) ssdhs are much more rare , with a recent review having identified 106 cases reported in the english literature.3 several predisposing factors have been associated with the occurrence of spontaneous ssdhs , such as coagulation abnormalities , anticoagulation therapy,4 platelet dysfunction,5 polycythemia vera,6 pregnancy,7 arterial wall abnormalities,8 and , more rarely , the presence of spinal arteriovenous malformations.9 most ssdhs occur at the lower thoracic region ( the second most common location being the upper cervical spine ) and typically extend from two to five segments.10 the vast majority of ssdhs are located anteriorly to the spinal cord , while most epidural spinal hematomas are located posteriorly to the spinal cord.11 this difference is related to the fact that the posterior longitudinal ligament closely adheres to the vertebral bodies , thus limiting anterior epidural collections . because the hematoma is confined to the extra - arachnoid space , some authors have suggested that what is commonly designated as an ssdh is , in fact , a dissection between the two inner layers of the spinal dura.12 this concept is supported by anatomic studies that demonstrated that under physiologic conditions , the spinal subdural space is only a capillary slit , which may occasionally extend into a genuine space under pathologic conditions such as during a subdural bleeding.10 13 percutaneous vertebroplasty is a therapeutic strategy that has gained increasing interest in the neurosurgical community for the treatment of refractory axial mechanical pain in patients with vertebral compression fractures . one of the main therapeutic mechanisms of cement augmentation has been proposed to be the improvement in the spinal stability provided by the injection of polymethyl methacrylate ( pmma ) into the fractured vertebral body.14 several complications of vertebroplasty have been reported in the literature , with the vast majority of them being related to cement extravasation to the epidural canal leading to spinal cord compression ( some series report extravasation rates of up to 20% with approximately one - third of such patients being neurologically symptomatic and requiring surgical intervention)15 or related to cement migration through the epidural veins to the venous system leading to pulmonary embolism ( reported to occur in 0.8 to 2.1% of the patients , with the vast majority of patients being asymptomatic).16 in this context , the severity grade of the fracture and a low viscosity of pmma cement have been identified as strong and independent risk factors for cement leakage.17 in this article , we report a patient who developed an acute ssdh following a percutaneous vertebroplasty without signs of major cement extravasation to the spinal canal . the authors perform a comprehensive literature review on the pathophysiology of ssdh and highlight the specific nuances of the presented case report to support a new hypothetical role of venous congestion as a possible etiologic factor involved in the pathophysiology of ssdh . however , it was possible to observe a hyperdense collection anterior to the spinal cord beginning one level above the site of the vertebroplasty and extending up to the cervical spine . immediate postprocedure ( b ) sagittal and ( c ) axial computed tomography scans demonstrating only a very small posterior leakage toward the spinal canal , as well as the presence of ( d ) a spinal subdural hematoma ( black arrow ) extending from one level above the vertebroplasty to the lower cervical spine . due to the acute motor deficit in the left lower limb , the patient was submitted to an immediate decompressive laminectomy at the level of the vertebroplasty and extending one level above and one level below to encompass those levels in which the cross - sectional area of the spinal canal was significantly compromised . 3 ) confirmed the presence of a collection with imaging characteristics of an ssdh , beginning one level above the vertebroplasty and extending up to the lower cervical spine , but without any residual signs of compression , as demonstrated by the presence of csf posterior to the spinal cord ( fig . the fact that the vertebral venous plexus appears to be much larger than what would be expected for the drainage of the spinal cord and meninges has led some authors to suggest that this vascular bed may also function as an alternate route for venous blood drainage between the inferior and superior vena cava.18 19 moreover , the fact that such a large plexus does not contain valves has led some authors to infer a possible secondary role as a pressure - regulating system that can protect the spinal cord from the volume and pressure peaks occurring in the intra - abdominal , intrathoracic , intracranial , and intraspinal spaces.10 11 the anatomy of the vertebral venous plexus has been somewhat ignored by ancient anatomic reports and only received the due attention in recent centuries ; special acknowledgments must be given to the works of batson ( 18941979 ) , after whom this venous plexus is named , and breschet ( 17841845).20 the vertebral venous plexus has been classically divided into an internal ( intradural ) vertebral plexus ( which possesses an anterior and a posterior component ) and an external ( or epidural ) vertebral plexus . recently , it has been demonstrated that the epidural vertebral plexus is closely connected to the intracranial venous sinuses in what could be described as the cerebrospinal venous system,21 or the so - called extradural neural axis compartment.22 in relation to the vertebral bodies , several small subsystems of plexiform veins work synergistically to accomplish their venous drainage ( fig . it has already been demonstrated that the radiculomedullary veins constitute the weakest link between the intradural venous system ( the internal vertebral venous plexus ) and the epidural space ( the external vertebral venous plexus).24 although presenting a small caliber , such radiculomedullary veins are believed to be of crucial importance for the venous drainage of the nerve roots of the cauda equina . in fact , several studies have suggested that the symptoms of neurogenic claudication and radicular pain in patients with lumbar canal stenosis may be more related to venous congestion than to deficits from an impaired arterial supply of the cauda equina.22 25 although the radiculomedullary veins have already been extensively investigated for their supposed role in the pathophysiology of spinal dural arteriovenous fistulas,21 very few studies have mentioned their possible role in the etiology of spontaneous or traumatic spinal subdural hematomas.26 27 moreover , although several previous studies have investigated the general anatomy of the epidural vertebral venous plexus20 28 29 30 31 and the internal vertebral venous plexus,32 33 no specific study focusing on the anatomy of the radiculomedullary veins exists . although several reported cases of ssdh have been associated with minor traumatic events or some predisposing coagulation disorder , the exact pathophysiology of ssdh still remains obscure . however , unlike the intracranial subdural space , it has already been shown that the spinal subdural compartment lacks bridging veins.34 interestingly , previous reports have demonstrated the association of ssdh with sudden episodes of increased intra - abdominal or intrathoracic pressure ( such as coughing or straining),26 35 36 suggesting the presence of a so - called locus minoris resistentiae ( a place of lower resistance ) in the vascular venous system between the internal and external vertebral venous plexus , which , when submitted to excessive pressures due to venous congestion , would possibly rupture , ultimately leading to extravasation of blood into the subdural space.35 36 alternatively , some authors have suggested that ssdhs might possibly originate from a few thin and delicate extra - arachnoid vessels that have been identified on the inner dural surface.37 38 although such etiology might explain some specific cases of ssdh in which the subdural hematoma occurs in association with a subarachnoid hemorrhage of traumatic origin , it does not explain the several reported cases of ssdhs in which it has been confirmed intraoperatively that the blood was confined to the extra - arachnoid space.39 in the reported case , another factor suggesting an etiology involving venous congestion ( related to the obstruction by pmma of the vertebral venous plexus responsible for the venous drainage of the vertebral body ) is the occurrence of neurologic deficits in the absence of signs of spinal cord compression . in the same way , the occurrence of secondary neurologic deficits due to venous congestion and hypoxia secondary to venous outflow obstruction has already been described in a previous report of a patient who presented with reversible myelopathy and spinal cord edema after a traumatic mediastinal hematoma leading to compression of the brachiocephalic vein.40 similar neurologic deficits related to intraspinal venous hypertension and hypoxia have already been reported after venous thrombosis and venous outflow obstruction during embolization of spinal dural arteriovenous fistulas.41 additionally , the occurrence of a special form of chronic necrotic myelopathy is a well - known phenomenon related to the chronic venous congestion that occurs in patients with spinal dural arteriovenous fistulas ( the so - called foix - alajouanine syndrome).42 the appearance of pneumorrhachis ( presence of air in the subdural space ) in the presented case also supports the hypothesis of a breakdown in the venous system that generated a differential pressure with the surrounding spaces due to its negative intraluminal pressure , ultimately leading to focal accumulation of air in the subdural space . similarly , previous reports have demonstrated the occurrence of pneumorrhachis in situations associated with an increased intrathoracic or intra - abdominal pressure ( such as cardiopulmonary resuscitation and airway obstruction because of foreign body aspiration ) leading to hypertensive venous obstruction of the epidural venous system.43 44 although the management of ssdh is controversial in the literature ( with some authors proposing surgery in the acute phase for spinal decompression and hematoma drainage ) , it has been demonstrated that patients with incomplete neurologic deficits tend to present a positive recovery without drainage of the hematoma . also , in the absence of radiologic evidence of spinal cord compression ( such as in the reported case , in which it was possible to visualize pouches of csf posterior to the spinal cord in the affected levels),27 surgical attempts to open the dura and drain the hematoma seem to be of questionable value , especially in extensive lesions involving several levels as in the reported case.26 in the long term , it has been demonstrated that the physiologic flow of csf tends to dilute the ssdh , ultimately leading to its spontaneous resolution as observed by follow - up imaging.26 45 the vertebral venous plexus is a complex , large - capacity , plexiform venous system that is believed to play an important role in the regulation of intracranial pressure with posture changes . the fact that the vertebral venous plexus appears to be much larger than what would be expected for the drainage of the spinal cord and meninges has led some authors to suggest that this vascular bed may also function as an alternate route for venous blood drainage between the inferior and superior vena cava.18 19 moreover , the fact that such a large plexus does not contain valves has led some authors to infer a possible secondary role as a pressure - regulating system that can protect the spinal cord from the volume and pressure peaks occurring in the intra - abdominal , intrathoracic , intracranial , and intraspinal spaces.10 11 the anatomy of the vertebral venous plexus has been somewhat ignored by ancient anatomic reports and only received the due attention in recent centuries ; special acknowledgments must be given to the works of batson ( 18941979 ) , after whom this venous plexus is named , and breschet ( 17841845).20 the vertebral venous plexus has been classically divided into an internal ( intradural ) vertebral plexus ( which possesses an anterior and a posterior component ) and an external ( or epidural ) vertebral plexus . recently , it has been demonstrated that the epidural vertebral plexus is closely connected to the intracranial venous sinuses in what could be described as the cerebrospinal venous system,21 or the so - called extradural neural axis compartment.22 in relation to the vertebral bodies , several small subsystems of plexiform veins work synergistically to accomplish their venous drainage ( fig . it has already been demonstrated that the radiculomedullary veins constitute the weakest link between the intradural venous system ( the internal vertebral venous plexus ) and the epidural space ( the external vertebral venous plexus).24 although presenting a small caliber , such radiculomedullary veins are believed to be of crucial importance for the venous drainage of the nerve roots of the cauda equina . in fact , several studies have suggested that the symptoms of neurogenic claudication and radicular pain in patients with lumbar canal stenosis may be more related to venous congestion than to deficits from an impaired arterial supply of the cauda equina.22 25 although the radiculomedullary veins have already been extensively investigated for their supposed role in the pathophysiology of spinal dural arteriovenous fistulas,21 very few studies have mentioned their possible role in the etiology of spontaneous or traumatic spinal subdural hematomas.26 27 moreover , although several previous studies have investigated the general anatomy of the epidural vertebral venous plexus20 28 29 30 31 and the internal vertebral venous plexus,32 33 no specific study focusing on the anatomy of the radiculomedullary veins exists . although several reported cases of ssdh have been associated with minor traumatic events or some predisposing coagulation disorder , the exact pathophysiology of ssdh still remains obscure . however , unlike the intracranial subdural space , it has already been shown that the spinal subdural compartment lacks bridging veins.34 interestingly , previous reports have demonstrated the association of ssdh with sudden episodes of increased intra - abdominal or intrathoracic pressure ( such as coughing or straining),26 35 36 suggesting the presence of a so - called locus minoris resistentiae ( a place of lower resistance ) in the vascular venous system between the internal and external vertebral venous plexus , which , when submitted to excessive pressures due to venous congestion , would possibly rupture , ultimately leading to extravasation of blood into the subdural space.35 36 alternatively , some authors have suggested that ssdhs might possibly originate from a few thin and delicate extra - arachnoid vessels that have been identified on the inner dural surface.37 38 although such etiology might explain some specific cases of ssdh in which the subdural hematoma occurs in association with a subarachnoid hemorrhage of traumatic origin , it does not explain the several reported cases of ssdhs in which it has been confirmed intraoperatively that the blood was confined to the extra - arachnoid space.39 in the reported case , another factor suggesting an etiology involving venous congestion ( related to the obstruction by pmma of the vertebral venous plexus responsible for the venous drainage of the vertebral body ) is the occurrence of neurologic deficits in the absence of signs of spinal cord compression . in the same way , the occurrence of secondary neurologic deficits due to venous congestion and hypoxia secondary to venous outflow obstruction has already been described in a previous report of a patient who presented with reversible myelopathy and spinal cord edema after a traumatic mediastinal hematoma leading to compression of the brachiocephalic vein.40 similar neurologic deficits related to intraspinal venous hypertension and hypoxia have already been reported after venous thrombosis and venous outflow obstruction during embolization of spinal dural arteriovenous fistulas.41 additionally , the occurrence of a special form of chronic necrotic myelopathy is a well - known phenomenon related to the chronic venous congestion that occurs in patients with spinal dural arteriovenous fistulas ( the so - called foix - alajouanine syndrome).42 the appearance of pneumorrhachis ( presence of air in the subdural space ) in the presented case also supports the hypothesis of a breakdown in the venous system that generated a differential pressure with the surrounding spaces due to its negative intraluminal pressure , ultimately leading to focal accumulation of air in the subdural space . similarly , previous reports have demonstrated the occurrence of pneumorrhachis in situations associated with an increased intrathoracic or intra - abdominal pressure ( such as cardiopulmonary resuscitation and airway obstruction because of foreign body aspiration ) leading to hypertensive venous obstruction of the epidural venous system.43 44 although the management of ssdh is controversial in the literature ( with some authors proposing surgery in the acute phase for spinal decompression and hematoma drainage ) , it has been demonstrated that patients with incomplete neurologic deficits tend to present a positive recovery without drainage of the hematoma . also , in the absence of radiologic evidence of spinal cord compression ( such as in the reported case , in which it was possible to visualize pouches of csf posterior to the spinal cord in the affected levels),27 surgical attempts to open the dura and drain the hematoma seem to be of questionable value , especially in extensive lesions involving several levels as in the reported case.26 in the long term , it has been demonstrated that the physiologic flow of csf tends to dilute the ssdh , ultimately leading to its spontaneous resolution as observed by follow - up imaging.26 45 the occurrence of such a complication in a level different from that of the intervention and in the absence of a major pmma extravasation into the spinal canal ( as well as in the presence of pneumorrhachis ) strongly suggests a venous congestive mechanism as the most plausible etiologic explanation . this newly proposed etiologic pathway emphasizes the role of venous congestion leading to the rupture of the fragile radiculomedullary veins into the subdural space . ultimately , this case report highlights the necessity of further anatomical and histologic studies of the radiculomedullary veins to better investigate their behavior in the face of congestive venous hypertension , as well as the supposed relationship between their possible rupture and the development of ssdhs .

spinal subdural hematomas ( ssdhs ) have been reported to occur after minor trauma , lumbar puncture , spinal anesthesia,1 or spinal surgery , especially in the presence of intraoperative dural tears.2 nontraumatic ( spontaneous ) ssdhs are much more rare , with a recent review having identified 106 cases reported in the english literature.3 several predisposing factors have been associated with the occurrence of spontaneous ssdhs , such as coagulation abnormalities , anticoagulation therapy,4 platelet dysfunction,5 polycythemia vera,6 pregnancy,7 arterial wall abnormalities,8 and , more rarely , the presence of spinal arteriovenous malformations.9 most ssdhs occur at the lower thoracic region ( the second most common location being the upper cervical spine ) and typically extend from two to five segments.10 the vast majority of ssdhs are located anteriorly to the spinal cord , while most epidural spinal hematomas are located posteriorly to the spinal cord.11 this difference is related to the fact that the posterior longitudinal ligament closely adheres to the vertebral bodies , thus limiting anterior epidural collections . because the hematoma is confined to the extra - arachnoid space , some authors have suggested that what is commonly designated as an ssdh is , in fact , a dissection between the two inner layers of the spinal dura.12 this concept is supported by anatomic studies that demonstrated that under physiologic conditions , the spinal subdural space is only a capillary slit , which may occasionally extend into a genuine space under pathologic conditions such as during a subdural bleeding.10 13 percutaneous vertebroplasty is a therapeutic strategy that has gained increasing interest in the neurosurgical community for the treatment of refractory axial mechanical pain in patients with vertebral compression fractures . one of the main therapeutic mechanisms of cement augmentation has been proposed to be the improvement in the spinal stability provided by the injection of polymethyl methacrylate ( pmma ) into the fractured vertebral body.14 several complications of vertebroplasty have been reported in the literature , with the vast majority of them being related to cement extravasation to the epidural canal leading to spinal cord compression ( some series report extravasation rates of up to 20% with approximately one - third of such patients being neurologically symptomatic and requiring surgical intervention)15 or related to cement migration through the epidural veins to the venous system leading to pulmonary embolism ( reported to occur in 0.8 to 2.1% of the patients , with the vast majority of patients being asymptomatic).16 in this context , the severity grade of the fracture and a low viscosity of pmma cement have been identified as strong and independent risk factors for cement leakage.17 in this article , we report a patient who developed an acute ssdh following a percutaneous vertebroplasty without signs of major cement extravasation to the spinal canal . ( b ) lateral plain x - ray of the thoracic spine performed at the 3-months follow - up after failed conservative treatment demonstrating progression of the fracture with further loss of height especially in the anterior third of the vertebral body , leading to worsening of the kyphotic deformity . immediate postprocedure ( b ) sagittal and ( c ) axial computed tomography scans demonstrating only a very small posterior leakage toward the spinal canal , as well as the presence of ( d ) a spinal subdural hematoma ( black arrow ) extending from one level above the vertebroplasty to the lower cervical spine . due to the acute motor deficit in the left lower limb , the patient was submitted to an immediate decompressive laminectomy at the level of the vertebroplasty and extending one level above and one level below to encompass those levels in which the cross - sectional area of the spinal canal was significantly compromised . at the 3-months follow - up , she presented with almost complete recovery of her strength in the left leg ( grade 4+/5 for hip flexion , hip extension , leg flexion and extension , and dorsiflexion and plantar flexion ) , although the sensory symptoms still persisted in the whole left side , both in the superior and inferior limbs , requiring treatment with gabapentin . the fact that the vertebral venous plexus appears to be much larger than what would be expected for the drainage of the spinal cord and meninges has led some authors to suggest that this vascular bed may also function as an alternate route for venous blood drainage between the inferior and superior vena cava.18 19 moreover , the fact that such a large plexus does not contain valves has led some authors to infer a possible secondary role as a pressure - regulating system that can protect the spinal cord from the volume and pressure peaks occurring in the intra - abdominal , intrathoracic , intracranial , and intraspinal spaces.10 11 the anatomy of the vertebral venous plexus has been somewhat ignored by ancient anatomic reports and only received the due attention in recent centuries ; special acknowledgments must be given to the works of batson ( 18941979 ) , after whom this venous plexus is named , and breschet ( 17841845).20 the vertebral venous plexus has been classically divided into an internal ( intradural ) vertebral plexus ( which possesses an anterior and a posterior component ) and an external ( or epidural ) vertebral plexus . recently , it has been demonstrated that the epidural vertebral plexus is closely connected to the intracranial venous sinuses in what could be described as the cerebrospinal venous system,21 or the so - called extradural neural axis compartment.22 in relation to the vertebral bodies , several small subsystems of plexiform veins work synergistically to accomplish their venous drainage ( fig . the subarticular collecting system is another important plexus formed by large - caliber vertical tributary veins , which abruptly turn to run horizontally , parallel to the vertebral endplates , ultimately draining posteriorly into the epidural venous plexus.23 ( a ) schematic representation of the vertebral venous system , which can be divided into an internal vertebral plexus ( composed of 1 : anterior internal vertebral venous plexus ; 2 : posterior internal vertebral venous plexus ) , and an external ( or epidural ) vertebral plexus ( composed of 4 : posterior external vertebral venous plexus ; 5 : anterior external vertebral venous plexus ; and 8 : radicular vein ) and the ( 6 ) radiculomedullary vein , which connects both . it has already been demonstrated that the radiculomedullary veins constitute the weakest link between the intradural venous system ( the internal vertebral venous plexus ) and the epidural space ( the external vertebral venous plexus).24 although presenting a small caliber , such radiculomedullary veins are believed to be of crucial importance for the venous drainage of the nerve roots of the cauda equina . in fact , several studies have suggested that the symptoms of neurogenic claudication and radicular pain in patients with lumbar canal stenosis may be more related to venous congestion than to deficits from an impaired arterial supply of the cauda equina.22 25 although the radiculomedullary veins have already been extensively investigated for their supposed role in the pathophysiology of spinal dural arteriovenous fistulas,21 very few studies have mentioned their possible role in the etiology of spontaneous or traumatic spinal subdural hematomas.26 27 moreover , although several previous studies have investigated the general anatomy of the epidural vertebral venous plexus20 28 29 30 31 and the internal vertebral venous plexus,32 33 no specific study focusing on the anatomy of the radiculomedullary veins exists . however , unlike the intracranial subdural space , it has already been shown that the spinal subdural compartment lacks bridging veins.34 interestingly , previous reports have demonstrated the association of ssdh with sudden episodes of increased intra - abdominal or intrathoracic pressure ( such as coughing or straining),26 35 36 suggesting the presence of a so - called locus minoris resistentiae ( a place of lower resistance ) in the vascular venous system between the internal and external vertebral venous plexus , which , when submitted to excessive pressures due to venous congestion , would possibly rupture , ultimately leading to extravasation of blood into the subdural space.35 36 alternatively , some authors have suggested that ssdhs might possibly originate from a few thin and delicate extra - arachnoid vessels that have been identified on the inner dural surface.37 38 although such etiology might explain some specific cases of ssdh in which the subdural hematoma occurs in association with a subarachnoid hemorrhage of traumatic origin , it does not explain the several reported cases of ssdhs in which it has been confirmed intraoperatively that the blood was confined to the extra - arachnoid space.39 in the reported case , another factor suggesting an etiology involving venous congestion ( related to the obstruction by pmma of the vertebral venous plexus responsible for the venous drainage of the vertebral body ) is the occurrence of neurologic deficits in the absence of signs of spinal cord compression . in the same way , the occurrence of secondary neurologic deficits due to venous congestion and hypoxia secondary to venous outflow obstruction has already been described in a previous report of a patient who presented with reversible myelopathy and spinal cord edema after a traumatic mediastinal hematoma leading to compression of the brachiocephalic vein.40 similar neurologic deficits related to intraspinal venous hypertension and hypoxia have already been reported after venous thrombosis and venous outflow obstruction during embolization of spinal dural arteriovenous fistulas.41 additionally , the occurrence of a special form of chronic necrotic myelopathy is a well - known phenomenon related to the chronic venous congestion that occurs in patients with spinal dural arteriovenous fistulas ( the so - called foix - alajouanine syndrome).42 the appearance of pneumorrhachis ( presence of air in the subdural space ) in the presented case also supports the hypothesis of a breakdown in the venous system that generated a differential pressure with the surrounding spaces due to its negative intraluminal pressure , ultimately leading to focal accumulation of air in the subdural space . similarly , previous reports have demonstrated the occurrence of pneumorrhachis in situations associated with an increased intrathoracic or intra - abdominal pressure ( such as cardiopulmonary resuscitation and airway obstruction because of foreign body aspiration ) leading to hypertensive venous obstruction of the epidural venous system.43 44 although the management of ssdh is controversial in the literature ( with some authors proposing surgery in the acute phase for spinal decompression and hematoma drainage ) , it has been demonstrated that patients with incomplete neurologic deficits tend to present a positive recovery without drainage of the hematoma . also , in the absence of radiologic evidence of spinal cord compression ( such as in the reported case , in which it was possible to visualize pouches of csf posterior to the spinal cord in the affected levels),27 surgical attempts to open the dura and drain the hematoma seem to be of questionable value , especially in extensive lesions involving several levels as in the reported case.26 in the long term , it has been demonstrated that the physiologic flow of csf tends to dilute the ssdh , ultimately leading to its spontaneous resolution as observed by follow - up imaging.26 45 the vertebral venous plexus is a complex , large - capacity , plexiform venous system that is believed to play an important role in the regulation of intracranial pressure with posture changes . the fact that the vertebral venous plexus appears to be much larger than what would be expected for the drainage of the spinal cord and meninges has led some authors to suggest that this vascular bed may also function as an alternate route for venous blood drainage between the inferior and superior vena cava.18 19 moreover , the fact that such a large plexus does not contain valves has led some authors to infer a possible secondary role as a pressure - regulating system that can protect the spinal cord from the volume and pressure peaks occurring in the intra - abdominal , intrathoracic , intracranial , and intraspinal spaces.10 11 the anatomy of the vertebral venous plexus has been somewhat ignored by ancient anatomic reports and only received the due attention in recent centuries ; special acknowledgments must be given to the works of batson ( 18941979 ) , after whom this venous plexus is named , and breschet ( 17841845).20 the vertebral venous plexus has been classically divided into an internal ( intradural ) vertebral plexus ( which possesses an anterior and a posterior component ) and an external ( or epidural ) vertebral plexus . the subarticular collecting system is another important plexus formed by large - caliber vertical tributary veins , which abruptly turn to run horizontally , parallel to the vertebral endplates , ultimately draining posteriorly into the epidural venous plexus.23 ( a ) schematic representation of the vertebral venous system , which can be divided into an internal vertebral plexus ( composed of 1 : anterior internal vertebral venous plexus ; 2 : posterior internal vertebral venous plexus ) , and an external ( or epidural ) vertebral plexus ( composed of 4 : posterior external vertebral venous plexus ; 5 : anterior external vertebral venous plexus ; and 8 : radicular vein ) and the ( 6 ) radiculomedullary vein , which connects both . in fact , several studies have suggested that the symptoms of neurogenic claudication and radicular pain in patients with lumbar canal stenosis may be more related to venous congestion than to deficits from an impaired arterial supply of the cauda equina.22 25 although the radiculomedullary veins have already been extensively investigated for their supposed role in the pathophysiology of spinal dural arteriovenous fistulas,21 very few studies have mentioned their possible role in the etiology of spontaneous or traumatic spinal subdural hematomas.26 27 moreover , although several previous studies have investigated the general anatomy of the epidural vertebral venous plexus20 28 29 30 31 and the internal vertebral venous plexus,32 33 no specific study focusing on the anatomy of the radiculomedullary veins exists . however , unlike the intracranial subdural space , it has already been shown that the spinal subdural compartment lacks bridging veins.34 interestingly , previous reports have demonstrated the association of ssdh with sudden episodes of increased intra - abdominal or intrathoracic pressure ( such as coughing or straining),26 35 36 suggesting the presence of a so - called locus minoris resistentiae ( a place of lower resistance ) in the vascular venous system between the internal and external vertebral venous plexus , which , when submitted to excessive pressures due to venous congestion , would possibly rupture , ultimately leading to extravasation of blood into the subdural space.35 36 alternatively , some authors have suggested that ssdhs might possibly originate from a few thin and delicate extra - arachnoid vessels that have been identified on the inner dural surface.37 38 although such etiology might explain some specific cases of ssdh in which the subdural hematoma occurs in association with a subarachnoid hemorrhage of traumatic origin , it does not explain the several reported cases of ssdhs in which it has been confirmed intraoperatively that the blood was confined to the extra - arachnoid space.39 in the reported case , another factor suggesting an etiology involving venous congestion ( related to the obstruction by pmma of the vertebral venous plexus responsible for the venous drainage of the vertebral body ) is the occurrence of neurologic deficits in the absence of signs of spinal cord compression . in the same way , the occurrence of secondary neurologic deficits due to venous congestion and hypoxia secondary to venous outflow obstruction has already been described in a previous report of a patient who presented with reversible myelopathy and spinal cord edema after a traumatic mediastinal hematoma leading to compression of the brachiocephalic vein.40 similar neurologic deficits related to intraspinal venous hypertension and hypoxia have already been reported after venous thrombosis and venous outflow obstruction during embolization of spinal dural arteriovenous fistulas.41 additionally , the occurrence of a special form of chronic necrotic myelopathy is a well - known phenomenon related to the chronic venous congestion that occurs in patients with spinal dural arteriovenous fistulas ( the so - called foix - alajouanine syndrome).42 the appearance of pneumorrhachis ( presence of air in the subdural space ) in the presented case also supports the hypothesis of a breakdown in the venous system that generated a differential pressure with the surrounding spaces due to its negative intraluminal pressure , ultimately leading to focal accumulation of air in the subdural space . similarly , previous reports have demonstrated the occurrence of pneumorrhachis in situations associated with an increased intrathoracic or intra - abdominal pressure ( such as cardiopulmonary resuscitation and airway obstruction because of foreign body aspiration ) leading to hypertensive venous obstruction of the epidural venous system.43 44 although the management of ssdh is controversial in the literature ( with some authors proposing surgery in the acute phase for spinal decompression and hematoma drainage ) , it has been demonstrated that patients with incomplete neurologic deficits tend to present a positive recovery without drainage of the hematoma . also , in the absence of radiologic evidence of spinal cord compression ( such as in the reported case , in which it was possible to visualize pouches of csf posterior to the spinal cord in the affected levels),27 surgical attempts to open the dura and drain the hematoma seem to be of questionable value , especially in extensive lesions involving several levels as in the reported case.26 in the long term , it has been demonstrated that the physiologic flow of csf tends to dilute the ssdh , ultimately leading to its spontaneous resolution as observed by follow - up imaging.26 45 the occurrence of such a complication in a level different from that of the intervention and in the absence of a major pmma extravasation into the spinal canal ( as well as in the presence of pneumorrhachis ) strongly suggests a venous congestive mechanism as the most plausible etiologic explanation .

the methionine cycle provides methyl units for a variety of reactions such as the methylation of proteins , dna , rna and lipids , allowing for the modulation of their biological functions ( bauerle et al . , 2015 , gut and verdin , 2013 ) . s - adenosyl methionine ( sam ) is the primary methyl donor molecule utilized in cellular methylation reactions and is synthesized directly from the essential amino acid methionine ( cantoni , 1952 ) ( figure s1a ) . the function of methylation reactions is context and substrate dependent ; for example , dna and histone methylation are epigenetic modifications that have important influences on chromatin structure and regulation of gene expression ( cedar and bergman , 2009 , gut and verdin , 2013 ) . previously , dna methylation was perceived as a relatively stable epigenetic modification with the propensity to encode heritable epigenetic information ( dolinoy , 2007 , dolinoy et al . however , recent work has highlighted the importance of dynamic control of dna methylation , for example , in embryogenesis ( guo et al . , 2014 , smith et al . , 2014 ) , cardiomyocyte development ( gilsbach et al . , 2014 ) , and actively transcribed and regulatory regions of dna ( kangaspeska et al . , 2008 , mtivier et al . , 2008 , schbeler , 2015 ) . it is therefore increasingly appreciated that existing dna , as well as newly synthesized dna , can be dynamically methylated and demethylated ( bhutani et al . , 2011 , , 2012 , feldmann et al . , 2013 , kohli and zhang , 2013 , yu et al . , 2012 ) . accordingly , it is important to define the cellular processes that control dynamic methylation of nucleic acids . as the fields of epigenetics and cellular metabolism particularly cancer cell metabolism have developed in recent years , so has the appreciation of the fundamental crosstalk between these processes ( gut and verdin , 2013 , hino et al . , 2013 , previous studies have shown that histone modifications are responsive to metabolite levels ; for example , glucose - derived acetyl - coa influences histone acetylation via atp - citrate lyase ( wellen et al . , 2009 ) . in gliomas , idh1 mutation is responsible for the generation of 2hg , which inhibits the dna hydroxylase tet and leads to altered methylation of histones and dna , so driving the phenotype of these tumors ( christensen et al . , 2011 , figueroa et al . , 2010 , turcan et al . , 2012 ) . mouse es cells depend on threonine to maintain sam synthesis , with threonine starvation leading to decreased histone methylation and inhibited proliferation ( shyh - chang et al . , 2013 ) . these findings and others the importance of folate - mediated one - carbon metabolism for cancer cell proliferation has long been appreciated ( locasale , 2013 ) . serine plays a key role in feeding one - carbon units to the tetrahydrofolate ( thf ) cycle and supports both nucleotide synthesis and nadph production ( fan et al . cancer cells have high demand for serine , which they meet by a combination of exogenous serine uptake and de novo synthesis from glucose ( locasale and cantley , 2011 , maddocks et al . , 2013 , interestingly , the serine synthesis pathway enzymes can be epigenetically activated by histone h3 methyltransferase g9a to support cancer cell survival and proliferation ( ding et al . , 2013 ) . in addition to nucleotide production , one - carbon metabolism can also contribute to the methionine cycle by providing one - carbons , in the form of methyl groups , to recycle homocysteine to methionine ( herbig et al . , 2002 , lu and mato , 2012 ) . partitioning of one - carbon units between nucleotide synthesis and homocysteine remethylation can be controlled by cytoplasmic serine hydroxymethyltransferase ( shmt1 ) ( herbig et al . , 2002 , with a decreased flux to thymidine synthesis associated with increased uracil incorporation into dna and enhanced cancer risk in some models ( macfarlane et al . , 2011 ) . by contrast , modulation of mthfd1 has been shown to lower sam levels but decrease uracil incorporation into dna , suggesting a switch toward nucleotide synthesis ( macfarlane et al . , 2009 ) . nevertheless , this change in folate metabolism also increased tumorigenesis in some tumor models ( macfarlane et al . , 2011 ) . dietary and genetic perturbation of folate mediated one - carbon metabolism have been shown to disrupt both the synthesis and methylation of dna ( stover , 2004 ) , and while these two pathways are intimately linked , how they are coordinated remains to be fully elucidated . atp is the major energy carrier used in cellular metabolism ( hardie et al . , 2012 ) . the inter - conversion of amp , adp , and atp is a fundamental reaction in cellular biology allowing for the transfer of energy from energy yielding reactions ( e.g. , glycolysis ) to energy consuming reactions ( e.g. , fatty acid synthesis)(hardie et al . , however , there is a clear but potentially underappreciated biochemical distinction between atp turnover via energetic reactions ( i.e. , regeneration of atp by re - phosphorylation of amp and adp ) and atp synthesis ( i.e. , atp generated by de novo purine synthesis , a process that requires glycine and one - carbon units , which are both commonly derived from serine in cancer cells ) . our previous studies highlighted the importance of serine in supporting purine synthesis , showing that glycine did not provide the one - carbon units necessary for the de novo synthesis of amp , adp , and atp in the cancer cells we studied ( labuschagne et al . while purine synthesis provides vital nucleotides for nucleic acid synthesis , atp generated by this biosynthetic pathway has the potential to be used in other metabolic reactions . in this study , we analyze the transfer of c labeled carbon from methionine and serine directly onto dna or rna in cancer cells . under methionine - fed conditions , serine did not contribute one - carbon units to recycle methionine , although serine did support dna and rna methylation under these conditions . surprisingly , we show that this contribution of serine is through the de novo synthesis of atp , which is required to convert methionine to sam . our data show that non - energetic metabolic stress can cause a dramatic decrease in total atp levels in rapidly proliferating cells , which can lead to changes in methyl group transfer without inducing activation of amp - activated protein kinase ( ampk ) . we have previously seen that serine availability has a major influence on the growth and metabolism of colorectal cancer cells ( labuschagne et al . , 2014 , maddocks et al . , 2013 ) , with a clear impact on one - carbon metabolism and purine synthesis . to analyze the contribution of serine - dependent one - carbon metabolism to the methionine cycle and methylation reactions ( figure s1a ) in these cells , we developed an assay to track one - carbons from extracellular nutrients , through the methionine cycle , into methylated substrates ( figure 1a ) . we chose to focus on the nucleic acids dna and rna as the methyl acceptor substrates , as the most common forms of methylated dna and rna ( methyl - cytidine and methyl - adenosine , respectively ) are well characterized ( fu et al . a number of assays have been developed to analyze relative quantities of modified and unmodified dna bases by mass spectrometry , including global dna methylation ( kok et al . , 2007 ) , , 2015 , herbig et al . , 2002 ) , and incorporation of deuterium - c - labeled methionine into dna ( bachman et al . , 2014 ) . based on an acid hydrolysis technique ( kok et al . , 2007 ) , we developed our assay to use cn - serine or cn - methionine in cell culture medium for 3 hr . using this method , we were able to track c - labeled serine and methionine into methyl groups on dna and rna . this method permitted relative quantification of the one - carbon contribution from extracellular serine and methionine onto dna and rna over a fixed time period ( figure 1a ) . using this method we found as expected that one - carbons from labeled methionine were readily used to methylate dna and rna . however , no significant contribution of one - carbons from serine was detected under the same conditions ( figures 1b and 1c ) . as re - methylated methionine is derived from its downstream metabolite homocysteine ( figure s1a ) , removal of exogenous methionine also deprives cells of the homocysteine they require for re - methylation ( shlomi et al . therefore we supplemented methionine - starved cells with exogenous homocysteine and vitamin b12 ( co - factor for re - methylation reaction ) to ensure these nutrients were not limiting . surprisingly , although serine did not contribute one - carbons under these conditions , serine starvation clearly decreased the contribution of one - carbon units from methionine to dna and rna after 9 hr ( figures 1b and 1c ) . treatment with a low dose of the dna methyltransferase inhibitor azacytidine , which directly blocks dna methyl transfer , caused a similar decrease in the transfer of methyl groups to dna as serine starvation ( figure 1d ) . after 30 hr of serine starvation , cells restored their ability to maintain the transfer of methionine - derived methyl groups to dna ( figure 1e ) . cancer cells adapt to serine starvation both by controlling serine utilization and by upregulating de novo serine synthesis , which allows recovery of serine levels ( maddocks et al . , 2013 ) , potentially explaining the recovery of methyl transfer by this later time point . analysis of total dna and rna methylation showed that serine depletion did not significantly impact total levels of methylated dna or rna , whereas methionine starvation caused decreased methylation ( figure s1b ) . this finding suggests a degree of coordination between serine availability and methyl group transfer , which is lacking for methionine . as expected , azacytidine caused a decrease in total dna methylation , but did not alter total rna methylation ( figure s1b ) . as serine starvation decreases the proliferation rate of cancer cells , we tested whether serum starvation , which also decreases proliferation rate ( figure s1c ) , had a similar effect . serum starvation did not impede transfer of methyl groups to dna to the same extent as serine starvation ( figure s1d ) , suggesting that the changes in methyl transfer that we detected are not simply a reflection of decreased proliferation . to understand why serine availability influenced the transfer of methyl groups to dna and rna , despite not directly contributing one - carbons for methylation , we analyzed metabolite levels in the methionine cycle by liquid chromatography mass spectrometry ( lcms ) . addition of cn - labeled methionine showed that serine - starved cells had increased levels of methionine but lower levels of downstream metabolites sam and sah ( figures 2a and 2b ) , suggesting a decrease in the conversion of methionine into the methyl donor molecule sam . given that conversion of methionine into sam is atp dependent , we hypothesized that methylation could be influenced by cellular atp levels . to test this , we assessed the impact of serine depletion on atp levels and compared this to glucose starvation as a positive control . glucose is a major cellular energy source , and as expected , glucose depletion caused increased levels of amp and lower levels of atp ( figure 3a ) . this corresponded with activation ( by phosphorylation ) of the cellular energy sensor ampk ( figure 3b ) . by comparison , we noted that serine starvation alone decreased both atp and amp levels ( figure 3a ) . this is a reflection of the requirement of serine - derived one - carbon units for de novo purine synthesis , and as reported previously ( labuschagne et al . , 2014 ) , this effect was further exacerbated by both removing serine and increasing glycine , which further inhibits nucleotide synthesis . in previous work , we found that pyruvate partially rescued cells from serine starvation and showed that acute depletion of exogenous pyruvate and serine resulted in activation of ampk ( maddocks et al . , 2013 ) . in the present study , cells were routinely grown without pyruvate , hence were adapted to its absence . in this context , the subsequent removal of serine led to a coordinated decrease in both amp and atp levels ( figure 3a ) , without activation of ampk ( figure 3b ) . while de novo atp synthesis may be slower in comparison to the rate of atp turnover ( as in energy generating reactions ) , these results suggest that without significant de novo atp synthesis , cancer cells do not have an adequate pool of adenine nucleotides for biosynthetic reactions ( such as sam synthesis ) that require atp to contribute adenosine . in order to test whether de novo synthesis of atp could make a direct contribution to the methionine cycle , we analyzed sam cycle metabolites by lcms after feeding cells cn - serine . in support of the dna and rna methylation data ( figures 1b and 1c ) , labeled serine did not contribute one - carbons to methionine re - methylation ( i.e. , the m+1 pool of methionine was at background levels , as seen in the control cells ) . however , after just 3 hr , both sam and sah were labeled by serine - derived carbon and nitrogen ( figures 4 and s2a ) . labeling was not seen in homocysteine , reflecting the fact that the atp - derived adenosine nucleotide is lost on conversion of sah to homocysteine ( figure s2a ) . as a result , atp also contributes adenosine to the synthesis other metabolites ( e.g. , nad(h ) ) . in comparison to sam , the contribution of labeled serine to nad(h ) was smaller over 3 hr ( figure s2a ) , with serine starvation having a relatively modest impact on total nad(h ) levels over 624 hr . excess glycine had a greater impact on nad(h ) , in line with its more dramatic effects on atp levels . these data suggest that sam levels may be more sensitive to variations in de novo atp synthesis than other metabolites . to test whether de novo nucleotide synthesis is required for incorporation of methyl units into dna , we cultured cells under conditions which inhibited de novo nucleotide synthesis ( i.e. , no serine / no serine + high glycine ) . analysis of total atp , methionine , and sam pools ( without using labeled metabolites ) showed that these conditions caused a dramatic decrease in cellular atp levels concurrent with increased methionine , and lower sam levels ( figures 5a and s3a ) . these metabolic changes translated to an increased methionine / sam ratio ( figures 5b and s3b ) and a decrease in the contribution of methyl units from the methionine cycle to dna ( figure 5c ) . exogenous formate directly provides one - carbons to the thf cycle and restores de novo nucleotide synthesis in the presence of excess glycine ( labuschagne et al . , 2014 ) . consistent with the requirement for de novo atp to support methionine to sam conversion , the addition of formate restored transfer of methyl groups from methionine to dna ( figure 5c ) . wider analysis of nucleotide synthesis intermediates showed that serine starvation generally impeded the synthesis of purines ( due to lack of one - carbon units , as previously reported ; labuschagne et al . , 2014 ) . by contrast , pyrimidine levels upstream of dtmp either showed little change or increased in response to serine starvation ( figure s4 ) . as the pyrimidine nucleotides upstream of dtmp do not require one - carbon units for de novo synthesis , they can be made in the absence of serine . furthermore , the lack of purines will inhibit nucleic acid synthesis , allowing unused pyrimidines to accumulate . the synthesis of dtmp from dump requires serine - derived one - carbon , and accordingly , the levels of dtmp were also lower in serine - starved cells . as we were unable to detect the use of serine - derived one - carbons for methionine re - methylation after 9 hr ( figures 1b and 1c ) , we considered that a longer period of methionine starvation could be necessary to promote detectable re - methylation . after 24 hr of methionine starvation ( with supplementary vitamin b12 and homocysteine ) with labeled serine for the final 3 hr , we were able to detect the transfer of serine - derived one - carbons to dna and rna ( figure 6a ) . supplementing cells with homocysteine and vitamin b12 provided varying degrees of proliferative rescue ( figure 6b ) , most likely reflecting the efficiency with which these cells can recycle methionine from homocysteine . the small additional gain in rescue achieved by supplementary vitamin b12 suggests that background vitamin b12 levels are already present in the dialyzed serum in the protein bound form . our data suggest that serine can contribute to the methionine cycle both by providing one - carbon units for re - methylation of methionine and by supporting de novo atp synthesis to allow the conversion of methionine to sam . to assess the full extent of the contribution of serine to nucleotide synthesis and the methionine cycle , we cultured cells with labeled serine for 24 hr , either in the presence of methionine or without methionine plus homocysteine and vitamin b12 . all three cell lines readily took up the exogenous serine , which was converted into glycine . one - carbons from serine and serine - derived glycine molecules entered de novo nucleotide synthesis so that almost the entire cellular pools of amp , adp , atp gmp , gdp , and gtp were labeled after 24 hr ( figures 7a and s5 ) . at this time point most nad(h ) was also labeled , although the proportion of the pool labeled was less than that seen for atp and sam ( figures 7a and s5 ) . the major atp isotopomer contained four labeled carbons and one labeled nitrogen ( m+5 ) , indicating incorporation of serine - derived glycine ( two carbons , one nitrogen ) and two serine - derived one - carbon units ( figures 7a and 7b ) . while total methionine levels were lower in the methionine - starved cells , an increase in the proportion of m+1 labeled methionine was observed . hence the ratio of labeled to unlabeled methionine was dramatically increased in the methionine - starved cells . labeling of sam generally mirrored atp labeling ; however , an additional m+6 isotopomer was also observed in sam , which was increased during methionine starvation . this m+6 isotopomer is a result of sam labeling via re - methylated methionine ( one carbon from serine ) plus serine - labeled atp ( four carbons and one nitrogen ) ( figures 7a and 7b ) . this labeling pattern indicates that serine can contribute simultaneously to two independent pathways that support the sam cycle . for technical clarity , we have provided examples of chromatogram peaks for adenine , methyl - adenine , cytosine , and methyl - cytosine with theoretical and measured masses ( figure s6 ) , as well as a breakdown of our data analysis and presentation for c methyl transfer ( figures s7a s7c ) and total rna / dna methylation analysis ( figures s7d s7f ) . one of the most important cellular uses for serine is to provide one - carbon units to support the thf - cycle ( tibbetts and appling , 2010 ) . while the cleavage of glycine could also provide one - carbons , we showed previously that glycine can not substitute for serine , and increasing glycine in cells starved of serine further depletes the one - carbon pool as cells convert the glycine to serine ( labuschagne et al . , 2014 ) . serine - dependent one - carbon metabolism is necessary for purine synthesis , but it can also contribute to several other metabolic pathways . the biochemical capacity for transfer of one - carbons from the thf cycle to homocysteine re - methylation is well known ( field et al . however , the activity of this pathway in cancer cells is poorly characterized . in the present study , we show that during methionine starvation , serine can provide one - carbon units to recycle homocysteine to methionine and so support the methionine cycle . intriguingly , however , we also found that serine contributes to the sam cycle and subsequent dna and rna methylation , even in methionine - fed cells , although this was not a reflection of methionine recycling . instead , we found that serine - dependent de novo atp synthesis is needed to support the conversion of methionine to sam and that limitation of this atp pool can impact the rate of sam generation and new methylation of dna and rna . the method we have used to track transfer of methyl units from c - labeled methionine and serine has potential utility to evaluate methylation dynamics of dna and rna in other contexts . there is a growing realization that methylation of dna and rna is more dynamic than previously appreciated . the ability to analyze these changes with a temporal dimension ( e.g. , by adapting the method to include multiple time points ) may allow this technique to be used to achieve this goal . in addition , the analysis method we employed for total cytosine / adenine methylation offers a robust method for global methylation analysis . the advantages of this lcms method compared to other measures of global methylation ( e.g. , immuno - detection of 5 mc ) are that it is direct and properly normalized for total cytosine / adenine content . however , the present method is not sensitive enough to detect small changes ( e.g. , less than 2% ) in methylation or to provide information on where in the genome these changes may occur . serine has been shown to play an important role in supporting the proliferation of cancer cells . although not an essential amino acid , serine starvation promotes the induction of a program of metabolic adaptation including the de novo serine synthesis pathway , allowing cells to generate serine from glycolytic intermediates . in some cancers , amplification of the ssp enzymes renders the cells less dependent on exogenous serine . however , most cancer cells respond to serine starvation with an abrupt depletion of intracellular serine . successful adaptation to serine starvation depends on the ability to partition the depleted serine pool into pathways important for cell survival ( such as the generation of glutathione to control ros ) while limiting flow of serine - derived one - carbon units into nucleotide synthesis . while this strategy is consistent with meeting the needs of cells that have undergone a transient cell - cycle arrest ( in response to serine depletion ) and so have lower need for nucleic acid synthesis , our data indicate that a broader view of the contribution of purines especially atp to cell growth and survival needs to be considered . numerous studies demonstrate that cells monitor and sense energetic stress through ampk , which detects elevated amp / adp levels , signifying an increase in the amp - to - atp ratio ( hardie et al . , 2012 , xiao et al . , 2013 ) . ampk activation causes a host of changes that promote atp regeneration and inhibit atp consumption , so allowing the cell to balance atp demand with atp supply ( hardie et al . , 2012 , hochachka and mcclelland , 1997 ) . the intracellular atp level is frequently considered to be a function of changes in atp turnover ( i.e. , the regeneration of atp by phosphorylation of amp / adp ) ( hardie et al . , 2012 , hochachka and mcclelland , 1997 , xiao et al . , 2013 ) , and clearly , the changing ratios of these metabolites in relation to atp are signals for the activation of energetic stress responses like ampk ( xiao et al . , 2013 ) . less explored , however , is the contribution of de novo atp synthesis ( i.e. , atp generated by de novo purine synthesis , a biosynthetic pathway that requires glucose and amino acids ) . cancer cells show enhanced rates of de novo purine synthesis , which has been interpreted as contributing to increased demand for nucleic acid ( primarily rna and dna ) synthesis . however , atp generated by this pathway has the potential to be used in other metabolic reactions , including functions in energy transfer , as a phosphate donor , or as an adenosine donor as in sam and nad(h ) synthesis . it is possible that these alternative functions for the de novo synthesized purines ( both atp and gtp ) could help explain why cancer cells devote so many resources to this pathway . indeed , guanine nucleotides ( gmp , gdp , and gtp ) are critical for supporting the ras pathway and a range of other signaling and metabolic processes important in cancer ( grewal et al . , 2011 , ostrem et al . , 2013 ) . given that we observed almost complete labeling of the atp pool within 24 hr , our data also suggest that just as other pools of cellular components ( such as dna and proteins ) need to be replicated to support cell division , so do pools of free nucleotides such as atp and gtp . since ampk is seemingly insensitive to concurrent decreases in amp and atp , it is tempting to speculate that other mechanisms exist to detect absolute cellular atp ( and gtp ) content . given the dependence of the methionine cycle on atp and its ability to influence gene expression and protein activity by methylation , it is conceivable that the methionine cycle has a role in sensing absolute atp levels . in the present work , we observe the contribution of newly synthesized atp to the sam cycle and a requirement for serine in supporting transfer of methyl groups to dna and rna through atp synthesis . a wealth of literature supports the role of serine - derived one - carbon in methionine re - methylation through the synthesis of 5-methyl thf , and polymorphisms in one - carbon metabolism enzymes can influence chromatin methylation and ultimately disease risk ( stover , 2011 ) . however , we did not observe the expected serine - dependent re - methylation in cancer cell lines when methionine was present . we show that non - energetic metabolic stress can have a dramatic and rapid effect on total atp levels , causing an even greater decrease than glucose starvation . however , this drop in atp is not clearly detected by the classic energetic stress sensor ampk , likely because lower de novo atp synthesis is accompanied by lower levels of amp . we see that despite dramatic decreases in atp levels , cells survive and maintain proliferative potential . these are important observations given the widely held belief based on studies involving energetic stress that cells maintain constant atp levels at all costs and that atp concentration is universally homeostatic the term atp synthesis is very commonly used ( pubmed returns over 600 papers with this term in the title ) to describe the generation of atp by adding phosphate(s ) to amp or adp . however , this form of atp generation can more accurately be referred to as atp turnover or regeneration , as the adenosine nucleotide is conserved during such reactions . as the present study demonstrates , true ( i.e. , de novo ) atp synthesis achieved by the assembly of glucose and amino acids through multi - step de novo purine synthesis is also a major contributor to the functional atp pool in cancer cells . hct116 , rko , and sw480 cells were obtained from atcc and subsequently authenticated using the promega geneprint 10 system according to the manufacturer s instructions . unless otherwise stated , cell culture media were purchased from gibco , product numbers are shown in parenthesis . stock hct116 and rko cells were grown in mccoys 5a medium ( 26600 ) supplemented with 10% fbs and penicillin - streptomycin . sw480 cells were grown in dmem ( 21969 ) supplemented with 2 mm l - glutamine 10% fbs . all stock cell culture and experiments were conducted in 37c , 5% co2 incubators . for experiments where nutrient levels were manipulated , cells received formulated medium containing mem vitamins ( 11120 ) , dialysed fbs ( hyclone , thermo scientific ) , penicillin - streptomycin , 17 mm d - glucose ( sigma ) , sodium bicarbonate ( sigma ) lacking methionine , and serine and glycine ( but containing all other amino acids ) , with varying concentrations of these nutrients added back as specified in each experiment . for re - methylation experiments ( where cells were deprived of methionine ) 0.8 mm homocysteine ( sigma ) and 1 m vitamin b12 ( methylcobalamin , sigma ) were added . usa , via ck gas ltd , uk ) were used at concentrations specified in each experiment . cells were seeded in mccoys / dmem in 6-well plates and left for 1624 hr ; initial seeding density was 2 to 8 10 cells per well and was calculated so that cells were approximately 80% confluent at the time of harvest . cells were washed once with pbs and received formulated assay medium as described above under nutrient deprivation . for each experiment , cells were initially grown with unlabeled nutrients for various time periods ( stated in each experiment ) followed by a fixed period of 3 hr with labeled c3n1-serine or c5n1-methionine . the relative contribution of these labeled metabolites to dna / rna methylation over the 3 hr period was then assessed as follows : cells were removed from wells with trypsin , and each well was split into two cell pellets that were frozen on dry ice/80c . one pellet was used for dna extraction using qiaamp dna mini kit ( qiagen , 51304 ) with rnase treatment , rna was extracted from the second pellet using rneasy mini kit ( qiagen , 74104 ) with dnase treatment according to manufacturer s instructions . the protocol for dna / rna acid hydrolysis 1ug dna or 3ug rna was placed in a 1.5ml tube and dried at 40c under nitrogen gas . 100ul of formic acid ( sigma ) was added to the dry pellets and incubated at 130c for 3.5 hr . after cooling , the acid was dried off at 40c under nitrogen gas , dry pellets were re - suspended in 25ul lcms - grade water for 20 min at room temperature . 100ul of ice - cold lcms - grade methanol ( 62.5% ) acetonitrile ( 37.5% ) solution was added to each sample . samples were vortexed and spun at 4c for 15 min , supernatant were transferred to lcms vials . bases from hydrolyzed dna and rna were analyzed on a dionex ultimate 3000 lc system coupled to a q exactive mass spectrometer ( thermo scientific ) . chromatographic separation was achieved using a sequant zic - philic column ( 2.1 150 mm , 5 m ) ( merck ) with elution buffers ( a ) and ( b ) consisting of 20 mm ( nh4)2co3 , 0.1% nh4oh in h2o ) and acetonitrile , respectively . the lc system was programmed to maintain a flow rate of 200 l / min with the staring condition at 80% ( a ) , which linearly decreased to 20% ( a ) over 10 min followed by washing and re - equilibration steps ( 20%80% [ a ] ) over 7 min . ionization of the analytes occurred in a heated electrospray ionization ( hesi ) probe fitted to the mass spectrometer that operated in negative ion mode over a mass range between 75 and 200 m / z at a resolution of 70,000 . thermo lcquan software was used to identify and analyze the nucleotides . to quantify the contribution of labeled serine / methionine to dna / rna methylation the peak area for m+1,methyl - cytosine / m+1,methyl - adenine for quantification of total dna / rna methylation , only unlabeled metabolites were used , and the peak area for methyl - cytosine / methyl - adenine was divided by peak area for cytosine / adenine , respectively . examples of chromatogram peaks and a graphical representation of the data analysis and presentation are included in figures s6 and s7 . assays were performed as described previously ( labuschagne et al . , 2014 ) . briefly , cells were plated in 6-well plates and cultured in mccoys 5a medium or dmem for 24 hr before replacing the medium with fresh medium containing labeled amino acids and incubated for the indicated times . cells were washed with pbs followed by metabolite extraction with ice - cold extraction buffer consisting of methanol , acetonitrile , and h2o ( 50:30:20 ) . extracts were analyzed by lcms using a dionex ultimate 3000 lc system coupled to a q exactive mass spectrometer ( thermo scientific ) . a sequant zic - philic column ( 2.1 150 mm , 5 m ) ( merck ) was used to separate the metabolites using the same elution buffers ( [ a ] and [ b ] ) as described above . a gradient program starting at 80% ( a ) and linearly decreasing to 20% ( a ) over 17 min was used followed by washing and re - equilibration steps . the q exactive was operated in full scan mode over a mass range of 751,000 m / z at a resolution of 35,000 with polarity switching . whole - cell protein lysates were prepared in ripa - buffer supplemented with complete protease inhibitors ( roche ) , sodium orthovanadate , and sodium fluoride ( both sigma ) . lysates were separated using precast nupage gels ( invitrogen , life technologies ) and transferred to nitrocellulose membranes . proteins were detected and quantified using a li - cor odyssey infrared scanner and software ( li - cor biosciences ) . primary antibodies used were as follows : anti - ampk - a ( 2532 ) and anti - phospho - ampk - a ( 2535 ) both from cell signaling technology . secondary antibodies for the relevant species were irdye680lt and irdye800cw conjugated ( li - cor biosciences ) . hct116 , rko ( 2 to 3 10 cells per well in mccoys medium ) and sw480 ( 5 10 cell per well in dmem ) were seeded in 24-well plates and allowed to adhere overnight . adherent cells were washed with pbs and were fed formulated assay medium supplemented with the stated nutrients ( see nutrient deprivation above ) . a separate time - zero media were changed every 24 hr , and plates were counted after 2 and 4 days . relative cell number was calculated by comparison to cell number at time - zero . for counting cells were trypsinized , re - suspended in pbs - edta , and counted with a casy model tt cell counter ( innovatis , roche applied science ) .

summarycrosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior . changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation . here we analyzed the contribution of methionine and serine metabolism to methylation of dna and rna . serine can contribute to this pathway by providing one - carbon units to regenerate methionine from homocysteine . while we observed this contribution under methionine - depleted conditions , unexpectedly , we found that serine supported the methionine cycle in the presence and absence of methionine through de novo atp synthesis . serine starvation increased the methionine / s - adenosyl methionine ratio , decreasing the transfer of methyl groups to dna and rna . while serine starvation dramatically decreased atp levels , this was accompanied by lower amp and did not activate ampk . this work highlights the difference between atp turnover and new atp synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids .

Introduction Results Discussion Experimental Procedures

the methionine cycle provides methyl units for a variety of reactions such as the methylation of proteins , dna , rna and lipids , allowing for the modulation of their biological functions ( bauerle et al . s - adenosyl methionine ( sam ) is the primary methyl donor molecule utilized in cellular methylation reactions and is synthesized directly from the essential amino acid methionine ( cantoni , 1952 ) ( figure s1a ) . the function of methylation reactions is context and substrate dependent ; for example , dna and histone methylation are epigenetic modifications that have important influences on chromatin structure and regulation of gene expression ( cedar and bergman , 2009 , gut and verdin , 2013 ) . accordingly , it is important to define the cellular processes that control dynamic methylation of nucleic acids . as the fields of epigenetics and cellular metabolism particularly cancer cell metabolism have developed in recent years , so has the appreciation of the fundamental crosstalk between these processes ( gut and verdin , 2013 , hino et al . these findings and others the importance of folate - mediated one - carbon metabolism for cancer cell proliferation has long been appreciated ( locasale , 2013 ) . serine plays a key role in feeding one - carbon units to the tetrahydrofolate ( thf ) cycle and supports both nucleotide synthesis and nadph production ( fan et al . in addition to nucleotide production , one - carbon metabolism can also contribute to the methionine cycle by providing one - carbons , in the form of methyl groups , to recycle homocysteine to methionine ( herbig et al . partitioning of one - carbon units between nucleotide synthesis and homocysteine remethylation can be controlled by cytoplasmic serine hydroxymethyltransferase ( shmt1 ) ( herbig et al . dietary and genetic perturbation of folate mediated one - carbon metabolism have been shown to disrupt both the synthesis and methylation of dna ( stover , 2004 ) , and while these two pathways are intimately linked , how they are coordinated remains to be fully elucidated . the inter - conversion of amp , adp , and atp is a fundamental reaction in cellular biology allowing for the transfer of energy from energy yielding reactions ( e.g. , however , there is a clear but potentially underappreciated biochemical distinction between atp turnover via energetic reactions ( i.e. , regeneration of atp by re - phosphorylation of amp and adp ) and atp synthesis ( i.e. , atp generated by de novo purine synthesis , a process that requires glycine and one - carbon units , which are both commonly derived from serine in cancer cells ) . our previous studies highlighted the importance of serine in supporting purine synthesis , showing that glycine did not provide the one - carbon units necessary for the de novo synthesis of amp , adp , and atp in the cancer cells we studied ( labuschagne et al . in this study , we analyze the transfer of c labeled carbon from methionine and serine directly onto dna or rna in cancer cells . under methionine - fed conditions , serine did not contribute one - carbon units to recycle methionine , although serine did support dna and rna methylation under these conditions . surprisingly , we show that this contribution of serine is through the de novo synthesis of atp , which is required to convert methionine to sam . our data show that non - energetic metabolic stress can cause a dramatic decrease in total atp levels in rapidly proliferating cells , which can lead to changes in methyl group transfer without inducing activation of amp - activated protein kinase ( ampk ) . , 2013 ) , with a clear impact on one - carbon metabolism and purine synthesis . to analyze the contribution of serine - dependent one - carbon metabolism to the methionine cycle and methylation reactions ( figure s1a ) in these cells , we developed an assay to track one - carbons from extracellular nutrients , through the methionine cycle , into methylated substrates ( figure 1a ) . we chose to focus on the nucleic acids dna and rna as the methyl acceptor substrates , as the most common forms of methylated dna and rna ( methyl - cytidine and methyl - adenosine , respectively ) are well characterized ( fu et al . using this method , we were able to track c - labeled serine and methionine into methyl groups on dna and rna . this method permitted relative quantification of the one - carbon contribution from extracellular serine and methionine onto dna and rna over a fixed time period ( figure 1a ) . using this method we found as expected that one - carbons from labeled methionine were readily used to methylate dna and rna . however , no significant contribution of one - carbons from serine was detected under the same conditions ( figures 1b and 1c ) . surprisingly , although serine did not contribute one - carbons under these conditions , serine starvation clearly decreased the contribution of one - carbon units from methionine to dna and rna after 9 hr ( figures 1b and 1c ) . treatment with a low dose of the dna methyltransferase inhibitor azacytidine , which directly blocks dna methyl transfer , caused a similar decrease in the transfer of methyl groups to dna as serine starvation ( figure 1d ) . after 30 hr of serine starvation , cells restored their ability to maintain the transfer of methionine - derived methyl groups to dna ( figure 1e ) . cancer cells adapt to serine starvation both by controlling serine utilization and by upregulating de novo serine synthesis , which allows recovery of serine levels ( maddocks et al . analysis of total dna and rna methylation showed that serine depletion did not significantly impact total levels of methylated dna or rna , whereas methionine starvation caused decreased methylation ( figure s1b ) . as serine starvation decreases the proliferation rate of cancer cells , we tested whether serum starvation , which also decreases proliferation rate ( figure s1c ) , had a similar effect . serum starvation did not impede transfer of methyl groups to dna to the same extent as serine starvation ( figure s1d ) , suggesting that the changes in methyl transfer that we detected are not simply a reflection of decreased proliferation . to understand why serine availability influenced the transfer of methyl groups to dna and rna , despite not directly contributing one - carbons for methylation , we analyzed metabolite levels in the methionine cycle by liquid chromatography mass spectrometry ( lcms ) . addition of cn - labeled methionine showed that serine - starved cells had increased levels of methionine but lower levels of downstream metabolites sam and sah ( figures 2a and 2b ) , suggesting a decrease in the conversion of methionine into the methyl donor molecule sam . given that conversion of methionine into sam is atp dependent , we hypothesized that methylation could be influenced by cellular atp levels . to test this , we assessed the impact of serine depletion on atp levels and compared this to glucose starvation as a positive control . by comparison , we noted that serine starvation alone decreased both atp and amp levels ( figure 3a ) . this is a reflection of the requirement of serine - derived one - carbon units for de novo purine synthesis , and as reported previously ( labuschagne et al . , 2014 ) , this effect was further exacerbated by both removing serine and increasing glycine , which further inhibits nucleotide synthesis . in previous work , we found that pyruvate partially rescued cells from serine starvation and showed that acute depletion of exogenous pyruvate and serine resulted in activation of ampk ( maddocks et al . in this context , the subsequent removal of serine led to a coordinated decrease in both amp and atp levels ( figure 3a ) , without activation of ampk ( figure 3b ) . while de novo atp synthesis may be slower in comparison to the rate of atp turnover ( as in energy generating reactions ) , these results suggest that without significant de novo atp synthesis , cancer cells do not have an adequate pool of adenine nucleotides for biosynthetic reactions ( such as sam synthesis ) that require atp to contribute adenosine . in order to test whether de novo synthesis of atp could make a direct contribution to the methionine cycle , we analyzed sam cycle metabolites by lcms after feeding cells cn - serine . in support of the dna and rna methylation data ( figures 1b and 1c ) , labeled serine did not contribute one - carbons to methionine re - methylation ( i.e. , the m+1 pool of methionine was at background levels , as seen in the control cells ) . in comparison to sam , the contribution of labeled serine to nad(h ) was smaller over 3 hr ( figure s2a ) , with serine starvation having a relatively modest impact on total nad(h ) levels over 624 hr . these data suggest that sam levels may be more sensitive to variations in de novo atp synthesis than other metabolites . to test whether de novo nucleotide synthesis is required for incorporation of methyl units into dna , we cultured cells under conditions which inhibited de novo nucleotide synthesis ( i.e. these metabolic changes translated to an increased methionine / sam ratio ( figures 5b and s3b ) and a decrease in the contribution of methyl units from the methionine cycle to dna ( figure 5c ) . exogenous formate directly provides one - carbons to the thf cycle and restores de novo nucleotide synthesis in the presence of excess glycine ( labuschagne et al . consistent with the requirement for de novo atp to support methionine to sam conversion , the addition of formate restored transfer of methyl groups from methionine to dna ( figure 5c ) . wider analysis of nucleotide synthesis intermediates showed that serine starvation generally impeded the synthesis of purines ( due to lack of one - carbon units , as previously reported ; labuschagne et al . as the pyrimidine nucleotides upstream of dtmp do not require one - carbon units for de novo synthesis , they can be made in the absence of serine . the synthesis of dtmp from dump requires serine - derived one - carbon , and accordingly , the levels of dtmp were also lower in serine - starved cells . as we were unable to detect the use of serine - derived one - carbons for methionine re - methylation after 9 hr ( figures 1b and 1c ) , we considered that a longer period of methionine starvation could be necessary to promote detectable re - methylation . after 24 hr of methionine starvation ( with supplementary vitamin b12 and homocysteine ) with labeled serine for the final 3 hr , we were able to detect the transfer of serine - derived one - carbons to dna and rna ( figure 6a ) . supplementing cells with homocysteine and vitamin b12 provided varying degrees of proliferative rescue ( figure 6b ) , most likely reflecting the efficiency with which these cells can recycle methionine from homocysteine . our data suggest that serine can contribute to the methionine cycle both by providing one - carbon units for re - methylation of methionine and by supporting de novo atp synthesis to allow the conversion of methionine to sam . to assess the full extent of the contribution of serine to nucleotide synthesis and the methionine cycle , we cultured cells with labeled serine for 24 hr , either in the presence of methionine or without methionine plus homocysteine and vitamin b12 . one - carbons from serine and serine - derived glycine molecules entered de novo nucleotide synthesis so that almost the entire cellular pools of amp , adp , atp gmp , gdp , and gtp were labeled after 24 hr ( figures 7a and s5 ) . the major atp isotopomer contained four labeled carbons and one labeled nitrogen ( m+5 ) , indicating incorporation of serine - derived glycine ( two carbons , one nitrogen ) and two serine - derived one - carbon units ( figures 7a and 7b ) . while total methionine levels were lower in the methionine - starved cells , an increase in the proportion of m+1 labeled methionine was observed . hence the ratio of labeled to unlabeled methionine was dramatically increased in the methionine - starved cells . this labeling pattern indicates that serine can contribute simultaneously to two independent pathways that support the sam cycle . one of the most important cellular uses for serine is to provide one - carbon units to support the thf - cycle ( tibbetts and appling , 2010 ) . while the cleavage of glycine could also provide one - carbons , we showed previously that glycine can not substitute for serine , and increasing glycine in cells starved of serine further depletes the one - carbon pool as cells convert the glycine to serine ( labuschagne et al . serine - dependent one - carbon metabolism is necessary for purine synthesis , but it can also contribute to several other metabolic pathways . the biochemical capacity for transfer of one - carbons from the thf cycle to homocysteine re - methylation is well known ( field et al . however , the activity of this pathway in cancer cells is poorly characterized . in the present study , we show that during methionine starvation , serine can provide one - carbon units to recycle homocysteine to methionine and so support the methionine cycle . intriguingly , however , we also found that serine contributes to the sam cycle and subsequent dna and rna methylation , even in methionine - fed cells , although this was not a reflection of methionine recycling . instead , we found that serine - dependent de novo atp synthesis is needed to support the conversion of methionine to sam and that limitation of this atp pool can impact the rate of sam generation and new methylation of dna and rna . the method we have used to track transfer of methyl units from c - labeled methionine and serine has potential utility to evaluate methylation dynamics of dna and rna in other contexts . there is a growing realization that methylation of dna and rna is more dynamic than previously appreciated . successful adaptation to serine starvation depends on the ability to partition the depleted serine pool into pathways important for cell survival ( such as the generation of glutathione to control ros ) while limiting flow of serine - derived one - carbon units into nucleotide synthesis . while this strategy is consistent with meeting the needs of cells that have undergone a transient cell - cycle arrest ( in response to serine depletion ) and so have lower need for nucleic acid synthesis , our data indicate that a broader view of the contribution of purines especially atp to cell growth and survival needs to be considered . numerous studies demonstrate that cells monitor and sense energetic stress through ampk , which detects elevated amp / adp levels , signifying an increase in the amp - to - atp ratio ( hardie et al . the intracellular atp level is frequently considered to be a function of changes in atp turnover ( i.e. less explored , however , is the contribution of de novo atp synthesis ( i.e. it is possible that these alternative functions for the de novo synthesized purines ( both atp and gtp ) could help explain why cancer cells devote so many resources to this pathway . indeed , guanine nucleotides ( gmp , gdp , and gtp ) are critical for supporting the ras pathway and a range of other signaling and metabolic processes important in cancer ( grewal et al . given that we observed almost complete labeling of the atp pool within 24 hr , our data also suggest that just as other pools of cellular components ( such as dna and proteins ) need to be replicated to support cell division , so do pools of free nucleotides such as atp and gtp . given the dependence of the methionine cycle on atp and its ability to influence gene expression and protein activity by methylation , it is conceivable that the methionine cycle has a role in sensing absolute atp levels . in the present work , we observe the contribution of newly synthesized atp to the sam cycle and a requirement for serine in supporting transfer of methyl groups to dna and rna through atp synthesis . a wealth of literature supports the role of serine - derived one - carbon in methionine re - methylation through the synthesis of 5-methyl thf , and polymorphisms in one - carbon metabolism enzymes can influence chromatin methylation and ultimately disease risk ( stover , 2011 ) . however , we did not observe the expected serine - dependent re - methylation in cancer cell lines when methionine was present . we show that non - energetic metabolic stress can have a dramatic and rapid effect on total atp levels , causing an even greater decrease than glucose starvation . however , this drop in atp is not clearly detected by the classic energetic stress sensor ampk , likely because lower de novo atp synthesis is accompanied by lower levels of amp . we see that despite dramatic decreases in atp levels , cells survive and maintain proliferative potential . these are important observations given the widely held belief based on studies involving energetic stress that cells maintain constant atp levels at all costs and that atp concentration is universally homeostatic the term atp synthesis is very commonly used ( pubmed returns over 600 papers with this term in the title ) to describe the generation of atp by adding phosphate(s ) to amp or adp . however , this form of atp generation can more accurately be referred to as atp turnover or regeneration , as the adenosine nucleotide is conserved during such reactions . , de novo ) atp synthesis achieved by the assembly of glucose and amino acids through multi - step de novo purine synthesis is also a major contributor to the functional atp pool in cancer cells . the relative contribution of these labeled metabolites to dna / rna methylation over the 3 hr period was then assessed as follows : cells were removed from wells with trypsin , and each well was split into two cell pellets that were frozen on dry ice/80c . bases from hydrolyzed dna and rna were analyzed on a dionex ultimate 3000 lc system coupled to a q exactive mass spectrometer ( thermo scientific ) . to quantify the contribution of labeled serine / methionine to dna / rna methylation the peak area for m+1,methyl - cytosine / m+1,methyl - adenine for quantification of total dna / rna methylation , only unlabeled metabolites were used , and the peak area for methyl - cytosine / methyl - adenine was divided by peak area for cytosine / adenine , respectively .

the methionine cycle provides methyl units for a variety of reactions such as the methylation of proteins , dna , rna and lipids , allowing for the modulation of their biological functions ( bauerle et al . s - adenosyl methionine ( sam ) is the primary methyl donor molecule utilized in cellular methylation reactions and is synthesized directly from the essential amino acid methionine ( cantoni , 1952 ) ( figure s1a ) . the function of methylation reactions is context and substrate dependent ; for example , dna and histone methylation are epigenetic modifications that have important influences on chromatin structure and regulation of gene expression ( cedar and bergman , 2009 , gut and verdin , 2013 ) . previously , dna methylation was perceived as a relatively stable epigenetic modification with the propensity to encode heritable epigenetic information ( dolinoy , 2007 , dolinoy et al . however , recent work has highlighted the importance of dynamic control of dna methylation , for example , in embryogenesis ( guo et al . accordingly , it is important to define the cellular processes that control dynamic methylation of nucleic acids . as the fields of epigenetics and cellular metabolism particularly cancer cell metabolism have developed in recent years , so has the appreciation of the fundamental crosstalk between these processes ( gut and verdin , 2013 , hino et al . , 2013 , previous studies have shown that histone modifications are responsive to metabolite levels ; for example , glucose - derived acetyl - coa influences histone acetylation via atp - citrate lyase ( wellen et al . in gliomas , idh1 mutation is responsible for the generation of 2hg , which inhibits the dna hydroxylase tet and leads to altered methylation of histones and dna , so driving the phenotype of these tumors ( christensen et al . these findings and others the importance of folate - mediated one - carbon metabolism for cancer cell proliferation has long been appreciated ( locasale , 2013 ) . serine plays a key role in feeding one - carbon units to the tetrahydrofolate ( thf ) cycle and supports both nucleotide synthesis and nadph production ( fan et al . , 2013 , interestingly , the serine synthesis pathway enzymes can be epigenetically activated by histone h3 methyltransferase g9a to support cancer cell survival and proliferation ( ding et al . in addition to nucleotide production , one - carbon metabolism can also contribute to the methionine cycle by providing one - carbons , in the form of methyl groups , to recycle homocysteine to methionine ( herbig et al . , 2002 , with a decreased flux to thymidine synthesis associated with increased uracil incorporation into dna and enhanced cancer risk in some models ( macfarlane et al . by contrast , modulation of mthfd1 has been shown to lower sam levels but decrease uracil incorporation into dna , suggesting a switch toward nucleotide synthesis ( macfarlane et al . dietary and genetic perturbation of folate mediated one - carbon metabolism have been shown to disrupt both the synthesis and methylation of dna ( stover , 2004 ) , and while these two pathways are intimately linked , how they are coordinated remains to be fully elucidated . the inter - conversion of amp , adp , and atp is a fundamental reaction in cellular biology allowing for the transfer of energy from energy yielding reactions ( e.g. , regeneration of atp by re - phosphorylation of amp and adp ) and atp synthesis ( i.e. our previous studies highlighted the importance of serine in supporting purine synthesis , showing that glycine did not provide the one - carbon units necessary for the de novo synthesis of amp , adp , and atp in the cancer cells we studied ( labuschagne et al . while purine synthesis provides vital nucleotides for nucleic acid synthesis , atp generated by this biosynthetic pathway has the potential to be used in other metabolic reactions . in this study , we analyze the transfer of c labeled carbon from methionine and serine directly onto dna or rna in cancer cells . surprisingly , we show that this contribution of serine is through the de novo synthesis of atp , which is required to convert methionine to sam . our data show that non - energetic metabolic stress can cause a dramatic decrease in total atp levels in rapidly proliferating cells , which can lead to changes in methyl group transfer without inducing activation of amp - activated protein kinase ( ampk ) . we have previously seen that serine availability has a major influence on the growth and metabolism of colorectal cancer cells ( labuschagne et al . to analyze the contribution of serine - dependent one - carbon metabolism to the methionine cycle and methylation reactions ( figure s1a ) in these cells , we developed an assay to track one - carbons from extracellular nutrients , through the methionine cycle , into methylated substrates ( figure 1a ) . we chose to focus on the nucleic acids dna and rna as the methyl acceptor substrates , as the most common forms of methylated dna and rna ( methyl - cytidine and methyl - adenosine , respectively ) are well characterized ( fu et al . , 2007 ) , we developed our assay to use cn - serine or cn - methionine in cell culture medium for 3 hr . as re - methylated methionine is derived from its downstream metabolite homocysteine ( figure s1a ) , removal of exogenous methionine also deprives cells of the homocysteine they require for re - methylation ( shlomi et al . therefore we supplemented methionine - starved cells with exogenous homocysteine and vitamin b12 ( co - factor for re - methylation reaction ) to ensure these nutrients were not limiting . surprisingly , although serine did not contribute one - carbons under these conditions , serine starvation clearly decreased the contribution of one - carbon units from methionine to dna and rna after 9 hr ( figures 1b and 1c ) . treatment with a low dose of the dna methyltransferase inhibitor azacytidine , which directly blocks dna methyl transfer , caused a similar decrease in the transfer of methyl groups to dna as serine starvation ( figure 1d ) . after 30 hr of serine starvation , cells restored their ability to maintain the transfer of methionine - derived methyl groups to dna ( figure 1e ) . analysis of total dna and rna methylation showed that serine depletion did not significantly impact total levels of methylated dna or rna , whereas methionine starvation caused decreased methylation ( figure s1b ) . as serine starvation decreases the proliferation rate of cancer cells , we tested whether serum starvation , which also decreases proliferation rate ( figure s1c ) , had a similar effect . serum starvation did not impede transfer of methyl groups to dna to the same extent as serine starvation ( figure s1d ) , suggesting that the changes in methyl transfer that we detected are not simply a reflection of decreased proliferation . to understand why serine availability influenced the transfer of methyl groups to dna and rna , despite not directly contributing one - carbons for methylation , we analyzed metabolite levels in the methionine cycle by liquid chromatography mass spectrometry ( lcms ) . addition of cn - labeled methionine showed that serine - starved cells had increased levels of methionine but lower levels of downstream metabolites sam and sah ( figures 2a and 2b ) , suggesting a decrease in the conversion of methionine into the methyl donor molecule sam . given that conversion of methionine into sam is atp dependent , we hypothesized that methylation could be influenced by cellular atp levels . to test this , we assessed the impact of serine depletion on atp levels and compared this to glucose starvation as a positive control . glucose is a major cellular energy source , and as expected , glucose depletion caused increased levels of amp and lower levels of atp ( figure 3a ) . in previous work , we found that pyruvate partially rescued cells from serine starvation and showed that acute depletion of exogenous pyruvate and serine resulted in activation of ampk ( maddocks et al . in this context , the subsequent removal of serine led to a coordinated decrease in both amp and atp levels ( figure 3a ) , without activation of ampk ( figure 3b ) . while de novo atp synthesis may be slower in comparison to the rate of atp turnover ( as in energy generating reactions ) , these results suggest that without significant de novo atp synthesis , cancer cells do not have an adequate pool of adenine nucleotides for biosynthetic reactions ( such as sam synthesis ) that require atp to contribute adenosine . in order to test whether de novo synthesis of atp could make a direct contribution to the methionine cycle , we analyzed sam cycle metabolites by lcms after feeding cells cn - serine . in support of the dna and rna methylation data ( figures 1b and 1c ) , labeled serine did not contribute one - carbons to methionine re - methylation ( i.e. labeling was not seen in homocysteine , reflecting the fact that the atp - derived adenosine nucleotide is lost on conversion of sah to homocysteine ( figure s2a ) . in comparison to sam , the contribution of labeled serine to nad(h ) was smaller over 3 hr ( figure s2a ) , with serine starvation having a relatively modest impact on total nad(h ) levels over 624 hr . analysis of total atp , methionine , and sam pools ( without using labeled metabolites ) showed that these conditions caused a dramatic decrease in cellular atp levels concurrent with increased methionine , and lower sam levels ( figures 5a and s3a ) . these metabolic changes translated to an increased methionine / sam ratio ( figures 5b and s3b ) and a decrease in the contribution of methyl units from the methionine cycle to dna ( figure 5c ) . exogenous formate directly provides one - carbons to the thf cycle and restores de novo nucleotide synthesis in the presence of excess glycine ( labuschagne et al . consistent with the requirement for de novo atp to support methionine to sam conversion , the addition of formate restored transfer of methyl groups from methionine to dna ( figure 5c ) . the synthesis of dtmp from dump requires serine - derived one - carbon , and accordingly , the levels of dtmp were also lower in serine - starved cells . as we were unable to detect the use of serine - derived one - carbons for methionine re - methylation after 9 hr ( figures 1b and 1c ) , we considered that a longer period of methionine starvation could be necessary to promote detectable re - methylation . after 24 hr of methionine starvation ( with supplementary vitamin b12 and homocysteine ) with labeled serine for the final 3 hr , we were able to detect the transfer of serine - derived one - carbons to dna and rna ( figure 6a ) . supplementing cells with homocysteine and vitamin b12 provided varying degrees of proliferative rescue ( figure 6b ) , most likely reflecting the efficiency with which these cells can recycle methionine from homocysteine . the small additional gain in rescue achieved by supplementary vitamin b12 suggests that background vitamin b12 levels are already present in the dialyzed serum in the protein bound form . our data suggest that serine can contribute to the methionine cycle both by providing one - carbon units for re - methylation of methionine and by supporting de novo atp synthesis to allow the conversion of methionine to sam . to assess the full extent of the contribution of serine to nucleotide synthesis and the methionine cycle , we cultured cells with labeled serine for 24 hr , either in the presence of methionine or without methionine plus homocysteine and vitamin b12 . one - carbons from serine and serine - derived glycine molecules entered de novo nucleotide synthesis so that almost the entire cellular pools of amp , adp , atp gmp , gdp , and gtp were labeled after 24 hr ( figures 7a and s5 ) . the major atp isotopomer contained four labeled carbons and one labeled nitrogen ( m+5 ) , indicating incorporation of serine - derived glycine ( two carbons , one nitrogen ) and two serine - derived one - carbon units ( figures 7a and 7b ) . while total methionine levels were lower in the methionine - starved cells , an increase in the proportion of m+1 labeled methionine was observed . for technical clarity , we have provided examples of chromatogram peaks for adenine , methyl - adenine , cytosine , and methyl - cytosine with theoretical and measured masses ( figure s6 ) , as well as a breakdown of our data analysis and presentation for c methyl transfer ( figures s7a s7c ) and total rna / dna methylation analysis ( figures s7d s7f ) . one of the most important cellular uses for serine is to provide one - carbon units to support the thf - cycle ( tibbetts and appling , 2010 ) . while the cleavage of glycine could also provide one - carbons , we showed previously that glycine can not substitute for serine , and increasing glycine in cells starved of serine further depletes the one - carbon pool as cells convert the glycine to serine ( labuschagne et al . in the present study , we show that during methionine starvation , serine can provide one - carbon units to recycle homocysteine to methionine and so support the methionine cycle . intriguingly , however , we also found that serine contributes to the sam cycle and subsequent dna and rna methylation , even in methionine - fed cells , although this was not a reflection of methionine recycling . instead , we found that serine - dependent de novo atp synthesis is needed to support the conversion of methionine to sam and that limitation of this atp pool can impact the rate of sam generation and new methylation of dna and rna . the method we have used to track transfer of methyl units from c - labeled methionine and serine has potential utility to evaluate methylation dynamics of dna and rna in other contexts . in addition , the analysis method we employed for total cytosine / adenine methylation offers a robust method for global methylation analysis . although not an essential amino acid , serine starvation promotes the induction of a program of metabolic adaptation including the de novo serine synthesis pathway , allowing cells to generate serine from glycolytic intermediates . successful adaptation to serine starvation depends on the ability to partition the depleted serine pool into pathways important for cell survival ( such as the generation of glutathione to control ros ) while limiting flow of serine - derived one - carbon units into nucleotide synthesis . while this strategy is consistent with meeting the needs of cells that have undergone a transient cell - cycle arrest ( in response to serine depletion ) and so have lower need for nucleic acid synthesis , our data indicate that a broader view of the contribution of purines especially atp to cell growth and survival needs to be considered . numerous studies demonstrate that cells monitor and sense energetic stress through ampk , which detects elevated amp / adp levels , signifying an increase in the amp - to - atp ratio ( hardie et al . , 2013 ) , and clearly , the changing ratios of these metabolites in relation to atp are signals for the activation of energetic stress responses like ampk ( xiao et al . however , atp generated by this pathway has the potential to be used in other metabolic reactions , including functions in energy transfer , as a phosphate donor , or as an adenosine donor as in sam and nad(h ) synthesis . indeed , guanine nucleotides ( gmp , gdp , and gtp ) are critical for supporting the ras pathway and a range of other signaling and metabolic processes important in cancer ( grewal et al . given that we observed almost complete labeling of the atp pool within 24 hr , our data also suggest that just as other pools of cellular components ( such as dna and proteins ) need to be replicated to support cell division , so do pools of free nucleotides such as atp and gtp . given the dependence of the methionine cycle on atp and its ability to influence gene expression and protein activity by methylation , it is conceivable that the methionine cycle has a role in sensing absolute atp levels . in the present work , we observe the contribution of newly synthesized atp to the sam cycle and a requirement for serine in supporting transfer of methyl groups to dna and rna through atp synthesis . a wealth of literature supports the role of serine - derived one - carbon in methionine re - methylation through the synthesis of 5-methyl thf , and polymorphisms in one - carbon metabolism enzymes can influence chromatin methylation and ultimately disease risk ( stover , 2011 ) . however , we did not observe the expected serine - dependent re - methylation in cancer cell lines when methionine was present . we show that non - energetic metabolic stress can have a dramatic and rapid effect on total atp levels , causing an even greater decrease than glucose starvation . however , this drop in atp is not clearly detected by the classic energetic stress sensor ampk , likely because lower de novo atp synthesis is accompanied by lower levels of amp . these are important observations given the widely held belief based on studies involving energetic stress that cells maintain constant atp levels at all costs and that atp concentration is universally homeostatic the term atp synthesis is very commonly used ( pubmed returns over 600 papers with this term in the title ) to describe the generation of atp by adding phosphate(s ) to amp or adp . , de novo ) atp synthesis achieved by the assembly of glucose and amino acids through multi - step de novo purine synthesis is also a major contributor to the functional atp pool in cancer cells . the relative contribution of these labeled metabolites to dna / rna methylation over the 3 hr period was then assessed as follows : cells were removed from wells with trypsin , and each well was split into two cell pellets that were frozen on dry ice/80c . to quantify the contribution of labeled serine / methionine to dna / rna methylation the peak area for m+1,methyl - cytosine / m+1,methyl - adenine for quantification of total dna / rna methylation , only unlabeled metabolites were used , and the peak area for methyl - cytosine / methyl - adenine was divided by peak area for cytosine / adenine , respectively .

the reconstruction of defective and degenerative bones has been a significant challenge in oral and maxillofacial surgery [ 1 , 2 ] . autologous tissues derived from intra- or extra - oral sites are considered to be a gold - standard in terms of regenerative potential . however , the limited access and amount of bones as well as the pain and psychological fear that patients tend to suffer due to the additional surgical operation required have been raised as concerns . bone grafts have been introduced as an alternative to the use of autologous bones , and they have been found to be useful for filling bony defects and expanding the quantity and quality of bones available for implantation [ 46 ] . when compared to allogenic or xenogenic materials , which may cause immune and/or disease problems , synthetic bone grafts are considered relatively safe for use in large quantities [ 3 , 4 ] . over the past few decades , several types of synthetic bone grafts have been proved by clinical trials and commercialized , and ceramics or glasses classified as bioactive materials comprise the major category [ 58 ] . bioactive glasses obtained from melt - quenching or sol - gel process are known to form direct bonds with hard human tissues through a specific attachment mechanism , such as the formation of a bone mineral like carbonated hydroxyapatite layer that forms on the surface of the glasses under physiological conditions . one of the benefits of bioactive glasses is the ability to easily incorporate elements tuned to the required tissue responses . for example , the addition of magnesium to melt glass 45s5 has been found to improve bioactivity , while the addition of silver to bioactive glass was reported to elicit antimicrobial activity and the addition of strontium to sol - gel bioactive glass was shown to enhance bone cell responses . in this study , zinc was added to the sol - gel bioactive glass based on the previous reports demonstrating zinc roles in bone formation [ 1315 ] . as one of the trace elements found in natural bone , zinc has been studied in bone biology , particularly in osteoporotic patients [ 13 , 14 ] . furthermore , several recent studies have utilized zinc as an additive to medical materials , including zinc - substituted hydroxyapatite powders , and coatings , tricalcium phosphate powders , and bioactive glass granules [ 1520 ] . small concentrations of zinc within the bioactive glasses have been shown to affect bone - associated cell responses such as proliferation and matrix synthesis in vitro [ 1820 ] . stem cell - based tissue engineering is gaining considerable opportunities for the successful reconstruction of bone tissue . mesenchymal stem cells ( mscs ) derived from adult bone marrow are one of the most popular cell sources available for the regenerative therapy , including osteogenesis . compared to the stem cells of embryonic origin , relatively large quantities of mscs can be obtained without significant ethical concerns , and these cells can be applied clinically using autologous cells [ 21 , 22 ] . in this study , as a first step towards bone tissue engineering using zinc - added bioactive glass , the effects of the addition of small concentrations ( 2 and 5% by mole ) of zinc to the sol - gel bioactive glass on the growth and osteogenic potential of mscs were investigated . the sol - gel - derived bioactive glass was produced using precursors of tetraethyl orthosilicate and calcium nitrate tetrahydrate ( from sigma - aldrich ) in an ethanol - water solvent , with hydrochloric acid as a catalyst . to incorporate the zinc , calcium nitrate tetrahydrate was replaced with zinc nitrate hexahydrate ( from sigma - aldrich ) at 2 and 5 mol% to produce three different compositions of the bioactive glasses : 70sio2 - 30cao ( 0% zn ) , 70sio2 - 28cao-2zno ( 2% zn ) , and 70sio2 - 25cao-5zno ( 5% zn ) . the prepared sol was then left to age for 24 h and transformed into a gel , which was subsequently dried in an oven ( 60c ) and then thermal - treated by heating to 650c at a ramping rate of 2c / min , where it was maintained for 3 h , after which the sol was furnace - cooled in the air . the calcined granules containing diameters ranging from 500 to 1000 micrometers were then gathered using metal sieves . for the cell test samples , the glass granules were sterilized and different quantities ( 3 , 10 , and 30 ) of the granules of each composition ( 0% zn , 2% zn , and 5% zn ) were placed in the individual wells of 24 culture well plates . the surface morphology of the glass granules was observed by scanning electron microscopy ( sem , hitachi ) . for observation of the bone bioactivity , the zinc - added bioactive glass granules were soaked in simulated body fluid ( ionic concentration : 142.0 mm na , 5 mm k , 1.5 mm mg , 2.5 mm ca , 147.8 mm cl , 4.2 mm hco3 , 1.0 mm hpo4 , and 0.5 mm so4 ) for 7 days , after which the surface was examined by sem . the dissolution of ions ( si , ca and zn ) from the glasses was detected using inductively coupled plasma atomic emission spectroscopy ( icp - aes ) . for the dissolution test , glass granules were incubated in an acellular culture medium ( serum free ) for up to 7 days , during which time the medium samples were analyzed . mesenchymal stem cells ( mscs ) were isolated from the bone marrow of tibia and femora of adult rats ( 48 weeks , korea ) using a method described in our previous study . the bone marrow mixture was then centrifuged , after which the supernatant was gathered and maintained on a culture flask containing growth medium ( -minimal essential medium ; mem , 2 mm glutamine , 100 u / ml penicillin , and 100 mg / ml streptomycin ) supplemented with 10% fetal bovine serum ( fbs ) . after cell adhesion for 1 day , the cells were washed with phosphate buffered saline solution ( pbs ) and cultured for up to 7 days until they reached near confluence . glass granules ( 5001000 micrometers in size ) with three different compositions ( 0 , 2 , and 5% zn ) were contained within the individual wells of 24-well plates at three different quantities ( 3 , 10 , and 30 ) . thus , a total of ten sample groups were used , including a glass - free blank control . an aliquot of 100 l of the cell suspension prepared at a density of 5 10 cells / ml was then seeded into each well . the medium for the cell growth was supplemented with 50 g / ml sodium ascorbate , 10 m sodium -glycerol phosphate , and 10 m dexamethasone to induce osteoblastic differentiation . the samples were then incubated for 6 h , after which 0.1 ml of culture medium without cells was added to each well . next , the samples were cultured for up to 21 days , with the medium being refreshed every 2 - 3 days . the cell growth level was measured at days 3 and 7 based on the mitochondrial nadh / nadph - dependant dehydrogenase activity , which was measured using a cell proliferation assay kit ( celltiter 96 aqueous one solution , promega ) . briefly , 100 l of mts regent was added to each sample and incubated for 3 h. the absorbance was then determined at 490 nm using an elisa plate reader . the morphology of cells at each culture time was determined by optical microscopy ( motic ) . the cell growth on the glass granules was examined by sem after fixation of the samples with glutaraldehyde , dehydration in a graded series of ethanol , and treatment with hexamethyldisilazane solution . sem was conducted at an accelerating voltage of 15 kv after gold coating the surface of the samples . the effect of the glass granules on the in vitro osteogenic differentiation of the mscs was assessed based on the alkaline phosphatase activity . cells were seeded onto each sample at 5 10 cells / ml and then cultured for 7 and 14 days . after washing the samples with pbs the cells were gathered and added with cell lysis buffer ( 10% triton x-100 , 1 m tris - hcl , 0.5 m nacl , and 0.5 m edta ) containing protease inhibitor . total protein content was measured using a commercial dc protein assay kit ( biorad , hercules , usa ) . bovine serum albumin ( from sigma ) was used as the reference curve for the protein assay . the quantity of each sample used for an enzymatic reaction was determined when normalized to the total protein content . the enzymatic activity of the alp produced was determined using an alp assay kit ( procedure no . three replicate samples were used for the assay ( n = 3 ) . to observe the osteogenic marker , bone sialoprotein ( bsp ) , next , the samples were washed with pbs and incubated with the primary antibody at a concentration of 1 : 500 ( polyclonal rabbit antibone sialoprotein : bsp ) at 4c , after which they were washed with pbs . subsequently , the alexfluor 555 secondary antibody ( molecular probe ) was added to the samples at 1 : 200 and incubated at ~25c in dark condition . after washing with pbs , the samples were mounted and visualized by fluorescence microscopy . for the mineralization study , the cells were cultured under the influence of the eluted products from glass granules that were present within the transwell insert . after culturing the cells , the samples were rinsed in pbs , fixed with 10% formaldehyde , and then stained with 1% alizarin red s ( ars ) solution ( ph 4.1 ) for 30 min . after washing fully with distilled water the mineralization was quantified by detecting the amount of calcium present on the cellular constructs . after culturing , the cell layer ( including extracellular matrix ) collected from each specimen was homogenized in diluted hcl solution by vigorous shaking . the cell lysate was centrifuged and the supernatant was used to determine the calcium content . the calcium concentration in each sample was quantified with a regent solution using orthocresolphthalein complexone ( ocpc ) as a metallochromic indicator . standard calcium solutions were prepared after suitable dilutions of 1 mg / ml cacl2 solution . an aliquot of the regent was added to each sample to induce ca - ocpc complexometric reaction , and the absorbance was read at 590 nm using a microplate reader . data were presented as mean one standard deviation for three or five different sets of samples in each group . statistical analysis was performed by the student t - test and significant level was considered at p < .05 . figure 1 shows the typical morphology of the glass granules ( 2% zn ) that were prepared by the sol - gel synthesis and subsequent heat treatment ( figure 1(a ) ) . granules with sizes of approximately 500 to 1000 m were chosen for further biological tests . when the glass granules were immersed in a simulated body fluid and incubated for 3 days at 37c , calcium phosphate crystallites were found to precipitate on the surface of the glass granules , and after 7 days of incubation , the surface of the granules was fully covered by a thick mineral layer ( representative image of 2% zn - added glass shown in figures 1(b ) and 1(c ) ) . similar in vitro mineralization behavior in sbf was observed in the other compositions ( data not shown here ) . for the biological tests , the glass granules were evaluated at three different concentrations ( low ( 3 ) , medium ( 10 ) , and high ( 30 ) ) contained within each well of a 24-well culture dish . the ionic elusion ( si , ca , and zn ) from glass samples cultured in an acellular medium was measured using icp - aes for up to 7 days ( table 1 ) . the results revealed that the addition of zinc to the glass led to a slight decrease in the dissolution of silicon and calcium . in addition , a trace amount of zinc was released from the zinc - added glass granules . the reduced release rate of the cations with time may be due to the depletion of cations with time and the resultant slow - down in further diffusion release process . mesenchymal stem cells ( mscs ) extracted from rat bone marrow were cultured in the presence of the bioactive glass granules . three different quantities ( 3 , 10 , and 30 ) of glass granules were placed in each well of a 24-well culture dish , and cellular responses such as growth and osteoblastic differentiation were investigated . figure 2 shows the optical morphology of cells grown in culture wells for 7 days , including wells that contained the glass granules ( 0 , 2 , and 5% zn ) at different concentrations ( 3 , 10 , and 30 granules ) , as well as a glass - free culture well as a control ( blank ) . when low and medium concentrations ( 3 and 10 ) of the glass granules were present , the cells reached near confluence and had an elongated and well - spreading morphology similar to that of the control . however , when the high concentration ( 30 ) of the granules was present , the cell morphology became less elongated and more granular - like . the cell morphology on the contained glass granules was observed with sem ( figure 3 ) . after 14 and 21 days of culture , the cells were found to actively grow on the surface of glass granules . furthermore , the cells had flattened bodies and many cytoskeletal extensions that were in intimate contact with the glass substrate . at day 21 , the cells appeared to be aggregated ( figure 3(e ) ) , and mineral - like products were noticeable around / below the cellular construct ( figures 3(e ) and 3(f ) ) . figure 4 summarizes the cell viability for periods of up to 7 days in the presence of bioactive glass granules with different compositions and concentrations , as measured by an mts method . when cultured in the presence of low and medium concentrations of granules for 3 and 7 days , the cell growth was equal to or higher than that of the control . however , a slight decrease in cell growth was observed with a high quantity ( 30 ) of the glass ( 2% and 5% zn - added ) , and even significant decrease was noticed in the 0% zn - added glass . in particular , zinc - containing glasses induced significantly higher cell growth at day 3 when they were contained at low ( 3 ) and medium ( 10 ) quantity . after prolonged culture periods ( 7 days ) , the cell viability affected by the glass granules became similar to that of the blank control , regardless of the composition of the glass . the alkaline phosphatase ( alp ) activity of the mscs cultured in the presence of various glasses was evaluated ( figure 5 ) . two sets of experiments were conducted using different quantities of glasses ( 10 and 30 granules ) . at day 14 , the addition of medium quantity ( 10 ) of glass granules significantly increased the alp level , and this improvement increased as the zinc content within the glass increased ( figure 5(a ) ) . a similar trend was observed in samples containing a high quantity of glass granules ( 30 granules ) ( figure 5(b ) ) . the expression of the osteogenic marker bone sialoprotein ( bsp ) produced by the cells in the presence of bioactive glass granules was observed by immunofluorescence staining . as shown in figure 6 , the red - colored spots representing the expressed bsp were obvious ( 2% zn shown as a representative sample ) , particularly near the bioactive glass granules . the immunofluorescence stain was more obvious in the samples that contained the glass granules than in the control . when compared to the blank control , the addition of glass granules significantly enhanced the expression of bsp . moreover , the stimulation of bsp was greater in response to a higher concentration of glass granules . the mineralization behavior of the mscs was investigated by alizarin red s ( ars ) staining . specifically , 30 glass granules for each composition ( 0 , 2 , and 5% zn ) were placed in a transwell insert , and the mscs were then cultured under the influence of the ionic eluants from the glass granules . this was conducted to eliminate the possible interference between ca within the glass and ars dye . as shown in figure 8(a ) , all the glass - containing groups showed the cellular constructs stained dark - red in response to the influence of the glass eluants , confirming significant level of cellular mineralization . the mineralized level was assessed by detection of calcium quantity of the cellular constructs . as presented in figure 8(b ) , the mineralization was significantly higher on all the glass - containing samples than on the blank control , and furthermore , the 2% zn - added glass sample showed significantly higher level of mineralization than the pure bioactive glass . the use of bioactive inorganics is considered a promising tool in the reconstruction of oral and maxillofacial defects [ 1 , 2 ] . among the various compositions evaluated , bioactive glasses have been widely studied due to their excellent bioactivity and ability to direct - bonding to bone , which is primarily associated with the bone mineral - like phase created on their surface [ 8 , 9 ] . moreover , the composition of bioactive glasses is believed to be easily controlled by the introduction of biologically relevant elements , such as silicon , magnesium , strontium , silver , and zinc [ 1012 ] . zinc is considered a key element present in bone and has been shown to regulate osteoblastic function and bone formation . osteoporotic patients have often been found to have a lack of zinc ; therefore , it is considered to be useful for the treatment of osteoporosis [ 13 , 14 ] . additionally , several recent studies have been conducted to utilize zinc within the composition of biomaterials . those studies have shown that zinc played an effective role in the stimulation of bone cell functions when it was present in calcium phosphate compounds such as hydroxyapatite and tricalcium phosphate [ 1518 ] . in the present study , zinc was added to the composition of bioactive glass during the sol - gel synthesis , and the effects of zinc addition ( 2 and 5% ) on the responses of rat bone marrow mscs were evaluated . mscs are considered a potential cell source for application in regenerative medicine , including the treatment of bone disorders [ 2123 ] . for stem cell - based bone tissue engineering , the material substrate should guide the initial anchorage of cells and their multiplication and further stimulate the differentiation into an osteogenic lineage . in order to utilize the zinc - bioactive glass as a scaffold for the mscs , this study was carried out as a first step to gain insight into the behavior of mscs in response to the glasses that contain zinc . different quantities of granules ( 3 , 10 , and 30 ) with variable glass compositions ( 0 , 2 , and 5% zn - added ) were used to evaluate the mscs responses . for all compositions , the initial cell adhesion and growth were shown to be favorable in the presence of small quantities of glass granules ( figures 2 and 4 ) . although some downregulation of cell viability was noticed initially , particularly in the case of the high concentration of 0% zn - added glass , the cell growth level became almost equal to that of the blank control after a prolonged culture time ( 7 days ) . conversely , a small quantity of glass granules led to a significant increase in cell viability , suggesting that the existence of glasses and their eluted products should favor the proliferative potential of mscs . based on the ionic release data ( table 1 ) , the addition of zinc to the glass led to a slight decrease in the elution of ions such as si and ca . thus , the initial high release of ions from the 0% zn - added glass might be one of the possible reasons for the decreased cell viability . although the initial ionic release was high , the release rate decreased with time , with cells eventually reaching their normal proliferation level after a prolonged culture period ( over 7 days ) . the images of the cells grown directly on the glass particles at day 14 ( figure 3 ) confirmed the viable cellular status , that is , the presence of extensive cytoskeletal processes and the formation of collagenous fibrillar extracellular matrix by the proliferative cells . the effects of the zinc - added bioactive glass on the osteogenic differentiation of the mscs were evaluated based on the alkaline phosphatase ( alp ) activity of cells . alp is a marker of osteoblastic cells that is evident at relatively early stage of osteogenic differentiation of progenitor or stem cells . in the present study , alp was significantly upregulated when grown in the presence of bioactive glass granules , particularly with larger quantity of granules and at later culture times ( over 14 days ) ( figure 5 ) . moreover , the increase appeared to be greater in the presence of zinc - added glasses . as another marker for osteogenic differentiation , bone sialo - protein ( bsp ) was evident in the presence of glass , particularly near the glass granules ( figures 6 and 7 ) . these results indicate that mscs are triggered to undergo osteogenic development by the eluants of the bioactive glass particles . the ions released from the bioactive glasses further stimulated cellular mineralization , as confirmed by the ars staining and calcium quantification of samples ( figure 8) . the released calcium ion should participate directly in the mineralization of extracellular matrices ( ecms ) , which would be secreted by the differentiated cells . although the mscs differentiate down to an osteogenic lineage within the total culture medium used herein , the presence of bioactive glasses should alter the secretion of proteins , and thus the ecm components . as noticed , the mineralization behavior under the influence of the glasses was noteworthy with respect to that of glass - free control which was conditioned only with the osteogenic medium . these results indicate that the differentiated cells and the synthesized ecm must be properly conditioned to induce mineralization under the influence of the bioactive glasses eluants . as to the effects of zinc , the results demonstrated that the mscs expressed alp and bsp at levels largely similar to those of zinc - free bioactive glass , and sometimes at even higher levels depending on the zinc concentration and the quantity of glass used . the different levels of ions released from the zinc - added bioactive glasses should affect the differentiation responses of stem cells and further cellular mineralization , and the zinc ions released may participate in the regulation of cellular functions to some extent . although no significant difference in bsp levels was noticed , the upregulations of alp and mineralization by the zinc - added glass support the possible role of zinc in the differentiation of mscs to the osteoblastic pathway . taken together this study may provide some useful information regarding the effects of zinc addition to bioactive glasses on the mscs growth and their stimulation into osteogenic differentiation . accordingly , we are currently developing zinc - added bioactive glasses as 3d scaffolds to use them in bone tissue engineering with the mscs . the cell growth and osteogenic differentiation of mscs with respect to zinc - added bioactive glasses ( 0 , 2 , and 5% ) in granular form were investigated . the presence of a small quantity of glass enhanced the initial cell growth , whereas a downregulation was noticed in response to a large quantity of zinc - free glass . the osteogenic development , as determined based on the alkaline phosphatase ( alp ) activity and bone - sialo protein ( bsp ) secretion , was significantly enhanced by the bioactive glass granules . moreover , the zinc - added glass induced cells to differentiate to a level similar to or even greater than that of zinc - free glass . the cellular mineralization was also significantly stimulated by the zinc addition to the bioactive glass . taken together , zinc - added bioactive glasses may be useful in bone regeneration and tissue engineering with adult stem cells because they preserve the active growth of mscs and stimulate further osteogenic differentiation .

responses of mesenchymal stem cells ( mscs ) cultured with zinc - added ( 2 and 5% ) bioactive glass granules were evaluated in terms of cell growth and osteogenic differentiation . mscs were cultured with different quantities ( 3 , 10 and 30 ) of glass granules for up to 21 days in the osteogenic medium . cell growth was stimulated by a small quantity of glasses , particularly those that contained zinc . osteogenic differentiation , as assessed by alkaline phosphatase activity ( alp ) activity , was significantly enhanced by the glasses , particularly with large quantities of glass and for prolonged culturing . expression of bone - sialo protein ( bsp ) was significantly up - regulated around the bioactive glass granules . moreover , the zinc addition significantly altered the alp and bsp depending on the culture time and glass quantity . cellular mineralization was improved in all glass samples , and particularly in the 2% zinc - glass . taken together , the zinc addition to bioactive glass induced the mscs growth and their osteogenic differentiation , at least to the level of zinc - free glass , and with even higher level observed depending on the quantity and culture time . these findings indicate that the zinc addition to bioactive glass may be useful in development of biomaterials for the stimulation of adult stem cell in bone tissue engineering .

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

autologous tissues derived from intra- or extra - oral sites are considered to be a gold - standard in terms of regenerative potential . however , the limited access and amount of bones as well as the pain and psychological fear that patients tend to suffer due to the additional surgical operation required have been raised as concerns . bone grafts have been introduced as an alternative to the use of autologous bones , and they have been found to be useful for filling bony defects and expanding the quantity and quality of bones available for implantation [ 46 ] . when compared to allogenic or xenogenic materials , which may cause immune and/or disease problems , synthetic bone grafts are considered relatively safe for use in large quantities [ 3 , 4 ] . bioactive glasses obtained from melt - quenching or sol - gel process are known to form direct bonds with hard human tissues through a specific attachment mechanism , such as the formation of a bone mineral like carbonated hydroxyapatite layer that forms on the surface of the glasses under physiological conditions . one of the benefits of bioactive glasses is the ability to easily incorporate elements tuned to the required tissue responses . for example , the addition of magnesium to melt glass 45s5 has been found to improve bioactivity , while the addition of silver to bioactive glass was reported to elicit antimicrobial activity and the addition of strontium to sol - gel bioactive glass was shown to enhance bone cell responses . in this study , zinc was added to the sol - gel bioactive glass based on the previous reports demonstrating zinc roles in bone formation [ 1315 ] . as one of the trace elements found in natural bone , zinc has been studied in bone biology , particularly in osteoporotic patients [ 13 , 14 ] . furthermore , several recent studies have utilized zinc as an additive to medical materials , including zinc - substituted hydroxyapatite powders , and coatings , tricalcium phosphate powders , and bioactive glass granules [ 1520 ] . small concentrations of zinc within the bioactive glasses have been shown to affect bone - associated cell responses such as proliferation and matrix synthesis in vitro [ 1820 ] . stem cell - based tissue engineering is gaining considerable opportunities for the successful reconstruction of bone tissue . mesenchymal stem cells ( mscs ) derived from adult bone marrow are one of the most popular cell sources available for the regenerative therapy , including osteogenesis . compared to the stem cells of embryonic origin , relatively large quantities of mscs can be obtained without significant ethical concerns , and these cells can be applied clinically using autologous cells [ 21 , 22 ] . in this study , as a first step towards bone tissue engineering using zinc - added bioactive glass , the effects of the addition of small concentrations ( 2 and 5% by mole ) of zinc to the sol - gel bioactive glass on the growth and osteogenic potential of mscs were investigated . the sol - gel - derived bioactive glass was produced using precursors of tetraethyl orthosilicate and calcium nitrate tetrahydrate ( from sigma - aldrich ) in an ethanol - water solvent , with hydrochloric acid as a catalyst . to incorporate the zinc , calcium nitrate tetrahydrate was replaced with zinc nitrate hexahydrate ( from sigma - aldrich ) at 2 and 5 mol% to produce three different compositions of the bioactive glasses : 70sio2 - 30cao ( 0% zn ) , 70sio2 - 28cao-2zno ( 2% zn ) , and 70sio2 - 25cao-5zno ( 5% zn ) . for the cell test samples , the glass granules were sterilized and different quantities ( 3 , 10 , and 30 ) of the granules of each composition ( 0% zn , 2% zn , and 5% zn ) were placed in the individual wells of 24 culture well plates . for observation of the bone bioactivity , the zinc - added bioactive glass granules were soaked in simulated body fluid ( ionic concentration : 142.0 mm na , 5 mm k , 1.5 mm mg , 2.5 mm ca , 147.8 mm cl , 4.2 mm hco3 , 1.0 mm hpo4 , and 0.5 mm so4 ) for 7 days , after which the surface was examined by sem . for the dissolution test , glass granules were incubated in an acellular culture medium ( serum free ) for up to 7 days , during which time the medium samples were analyzed . mesenchymal stem cells ( mscs ) were isolated from the bone marrow of tibia and femora of adult rats ( 48 weeks , korea ) using a method described in our previous study . after cell adhesion for 1 day , the cells were washed with phosphate buffered saline solution ( pbs ) and cultured for up to 7 days until they reached near confluence . glass granules ( 5001000 micrometers in size ) with three different compositions ( 0 , 2 , and 5% zn ) were contained within the individual wells of 24-well plates at three different quantities ( 3 , 10 , and 30 ) . the medium for the cell growth was supplemented with 50 g / ml sodium ascorbate , 10 m sodium -glycerol phosphate , and 10 m dexamethasone to induce osteoblastic differentiation . next , the samples were cultured for up to 21 days , with the medium being refreshed every 2 - 3 days . the cell growth level was measured at days 3 and 7 based on the mitochondrial nadh / nadph - dependant dehydrogenase activity , which was measured using a cell proliferation assay kit ( celltiter 96 aqueous one solution , promega ) . the cell growth on the glass granules was examined by sem after fixation of the samples with glutaraldehyde , dehydration in a graded series of ethanol , and treatment with hexamethyldisilazane solution . the effect of the glass granules on the in vitro osteogenic differentiation of the mscs was assessed based on the alkaline phosphatase activity . bovine serum albumin ( from sigma ) was used as the reference curve for the protein assay . the quantity of each sample used for an enzymatic reaction was determined when normalized to the total protein content . to observe the osteogenic marker , bone sialoprotein ( bsp ) , next , the samples were washed with pbs and incubated with the primary antibody at a concentration of 1 : 500 ( polyclonal rabbit antibone sialoprotein : bsp ) at 4c , after which they were washed with pbs . subsequently , the alexfluor 555 secondary antibody ( molecular probe ) was added to the samples at 1 : 200 and incubated at ~25c in dark condition . for the mineralization study , the cells were cultured under the influence of the eluted products from glass granules that were present within the transwell insert . after culturing the cells , the samples were rinsed in pbs , fixed with 10% formaldehyde , and then stained with 1% alizarin red s ( ars ) solution ( ph 4.1 ) for 30 min . after washing fully with distilled water the mineralization was quantified by detecting the amount of calcium present on the cellular constructs . figure 1 shows the typical morphology of the glass granules ( 2% zn ) that were prepared by the sol - gel synthesis and subsequent heat treatment ( figure 1(a ) ) . when the glass granules were immersed in a simulated body fluid and incubated for 3 days at 37c , calcium phosphate crystallites were found to precipitate on the surface of the glass granules , and after 7 days of incubation , the surface of the granules was fully covered by a thick mineral layer ( representative image of 2% zn - added glass shown in figures 1(b ) and 1(c ) ) . for the biological tests , the glass granules were evaluated at three different concentrations ( low ( 3 ) , medium ( 10 ) , and high ( 30 ) ) contained within each well of a 24-well culture dish . the ionic elusion ( si , ca , and zn ) from glass samples cultured in an acellular medium was measured using icp - aes for up to 7 days ( table 1 ) . the results revealed that the addition of zinc to the glass led to a slight decrease in the dissolution of silicon and calcium . in addition , a trace amount of zinc was released from the zinc - added glass granules . the reduced release rate of the cations with time may be due to the depletion of cations with time and the resultant slow - down in further diffusion release process . mesenchymal stem cells ( mscs ) extracted from rat bone marrow were cultured in the presence of the bioactive glass granules . three different quantities ( 3 , 10 , and 30 ) of glass granules were placed in each well of a 24-well culture dish , and cellular responses such as growth and osteoblastic differentiation were investigated . figure 2 shows the optical morphology of cells grown in culture wells for 7 days , including wells that contained the glass granules ( 0 , 2 , and 5% zn ) at different concentrations ( 3 , 10 , and 30 granules ) , as well as a glass - free culture well as a control ( blank ) . when low and medium concentrations ( 3 and 10 ) of the glass granules were present , the cells reached near confluence and had an elongated and well - spreading morphology similar to that of the control . however , when the high concentration ( 30 ) of the granules was present , the cell morphology became less elongated and more granular - like . the cell morphology on the contained glass granules was observed with sem ( figure 3 ) . after 14 and 21 days of culture , the cells were found to actively grow on the surface of glass granules . furthermore , the cells had flattened bodies and many cytoskeletal extensions that were in intimate contact with the glass substrate . at day 21 , the cells appeared to be aggregated ( figure 3(e ) ) , and mineral - like products were noticeable around / below the cellular construct ( figures 3(e ) and 3(f ) ) . figure 4 summarizes the cell viability for periods of up to 7 days in the presence of bioactive glass granules with different compositions and concentrations , as measured by an mts method . when cultured in the presence of low and medium concentrations of granules for 3 and 7 days , the cell growth was equal to or higher than that of the control . however , a slight decrease in cell growth was observed with a high quantity ( 30 ) of the glass ( 2% and 5% zn - added ) , and even significant decrease was noticed in the 0% zn - added glass . in particular , zinc - containing glasses induced significantly higher cell growth at day 3 when they were contained at low ( 3 ) and medium ( 10 ) quantity . after prolonged culture periods ( 7 days ) , the cell viability affected by the glass granules became similar to that of the blank control , regardless of the composition of the glass . the alkaline phosphatase ( alp ) activity of the mscs cultured in the presence of various glasses was evaluated ( figure 5 ) . two sets of experiments were conducted using different quantities of glasses ( 10 and 30 granules ) . at day 14 , the addition of medium quantity ( 10 ) of glass granules significantly increased the alp level , and this improvement increased as the zinc content within the glass increased ( figure 5(a ) ) . a similar trend was observed in samples containing a high quantity of glass granules ( 30 granules ) ( figure 5(b ) ) . the expression of the osteogenic marker bone sialoprotein ( bsp ) produced by the cells in the presence of bioactive glass granules was observed by immunofluorescence staining . as shown in figure 6 , the red - colored spots representing the expressed bsp were obvious ( 2% zn shown as a representative sample ) , particularly near the bioactive glass granules . the immunofluorescence stain was more obvious in the samples that contained the glass granules than in the control . when compared to the blank control , the addition of glass granules significantly enhanced the expression of bsp . moreover , the stimulation of bsp was greater in response to a higher concentration of glass granules . specifically , 30 glass granules for each composition ( 0 , 2 , and 5% zn ) were placed in a transwell insert , and the mscs were then cultured under the influence of the ionic eluants from the glass granules . this was conducted to eliminate the possible interference between ca within the glass and ars dye . as shown in figure 8(a ) , all the glass - containing groups showed the cellular constructs stained dark - red in response to the influence of the glass eluants , confirming significant level of cellular mineralization . the mineralized level was assessed by detection of calcium quantity of the cellular constructs . as presented in figure 8(b ) , the mineralization was significantly higher on all the glass - containing samples than on the blank control , and furthermore , the 2% zn - added glass sample showed significantly higher level of mineralization than the pure bioactive glass . moreover , the composition of bioactive glasses is believed to be easily controlled by the introduction of biologically relevant elements , such as silicon , magnesium , strontium , silver , and zinc [ 1012 ] . osteoporotic patients have often been found to have a lack of zinc ; therefore , it is considered to be useful for the treatment of osteoporosis [ 13 , 14 ] . those studies have shown that zinc played an effective role in the stimulation of bone cell functions when it was present in calcium phosphate compounds such as hydroxyapatite and tricalcium phosphate [ 1518 ] . in the present study , zinc was added to the composition of bioactive glass during the sol - gel synthesis , and the effects of zinc addition ( 2 and 5% ) on the responses of rat bone marrow mscs were evaluated . for stem cell - based bone tissue engineering , the material substrate should guide the initial anchorage of cells and their multiplication and further stimulate the differentiation into an osteogenic lineage . in order to utilize the zinc - bioactive glass as a scaffold for the mscs , this study was carried out as a first step to gain insight into the behavior of mscs in response to the glasses that contain zinc . different quantities of granules ( 3 , 10 , and 30 ) with variable glass compositions ( 0 , 2 , and 5% zn - added ) were used to evaluate the mscs responses . for all compositions , the initial cell adhesion and growth were shown to be favorable in the presence of small quantities of glass granules ( figures 2 and 4 ) . although some downregulation of cell viability was noticed initially , particularly in the case of the high concentration of 0% zn - added glass , the cell growth level became almost equal to that of the blank control after a prolonged culture time ( 7 days ) . conversely , a small quantity of glass granules led to a significant increase in cell viability , suggesting that the existence of glasses and their eluted products should favor the proliferative potential of mscs . based on the ionic release data ( table 1 ) , the addition of zinc to the glass led to a slight decrease in the elution of ions such as si and ca . thus , the initial high release of ions from the 0% zn - added glass might be one of the possible reasons for the decreased cell viability . the images of the cells grown directly on the glass particles at day 14 ( figure 3 ) confirmed the viable cellular status , that is , the presence of extensive cytoskeletal processes and the formation of collagenous fibrillar extracellular matrix by the proliferative cells . the effects of the zinc - added bioactive glass on the osteogenic differentiation of the mscs were evaluated based on the alkaline phosphatase ( alp ) activity of cells . alp is a marker of osteoblastic cells that is evident at relatively early stage of osteogenic differentiation of progenitor or stem cells . in the present study , alp was significantly upregulated when grown in the presence of bioactive glass granules , particularly with larger quantity of granules and at later culture times ( over 14 days ) ( figure 5 ) . moreover , the increase appeared to be greater in the presence of zinc - added glasses . as another marker for osteogenic differentiation , bone sialo - protein ( bsp ) was evident in the presence of glass , particularly near the glass granules ( figures 6 and 7 ) . these results indicate that mscs are triggered to undergo osteogenic development by the eluants of the bioactive glass particles . the ions released from the bioactive glasses further stimulated cellular mineralization , as confirmed by the ars staining and calcium quantification of samples ( figure 8) . the released calcium ion should participate directly in the mineralization of extracellular matrices ( ecms ) , which would be secreted by the differentiated cells . although the mscs differentiate down to an osteogenic lineage within the total culture medium used herein , the presence of bioactive glasses should alter the secretion of proteins , and thus the ecm components . as noticed , the mineralization behavior under the influence of the glasses was noteworthy with respect to that of glass - free control which was conditioned only with the osteogenic medium . these results indicate that the differentiated cells and the synthesized ecm must be properly conditioned to induce mineralization under the influence of the bioactive glasses eluants . as to the effects of zinc , the results demonstrated that the mscs expressed alp and bsp at levels largely similar to those of zinc - free bioactive glass , and sometimes at even higher levels depending on the zinc concentration and the quantity of glass used . the different levels of ions released from the zinc - added bioactive glasses should affect the differentiation responses of stem cells and further cellular mineralization , and the zinc ions released may participate in the regulation of cellular functions to some extent . although no significant difference in bsp levels was noticed , the upregulations of alp and mineralization by the zinc - added glass support the possible role of zinc in the differentiation of mscs to the osteoblastic pathway . taken together this study may provide some useful information regarding the effects of zinc addition to bioactive glasses on the mscs growth and their stimulation into osteogenic differentiation . accordingly , we are currently developing zinc - added bioactive glasses as 3d scaffolds to use them in bone tissue engineering with the mscs . the cell growth and osteogenic differentiation of mscs with respect to zinc - added bioactive glasses ( 0 , 2 , and 5% ) in granular form were investigated . the presence of a small quantity of glass enhanced the initial cell growth , whereas a downregulation was noticed in response to a large quantity of zinc - free glass . the osteogenic development , as determined based on the alkaline phosphatase ( alp ) activity and bone - sialo protein ( bsp ) secretion , was significantly enhanced by the bioactive glass granules . moreover , the zinc - added glass induced cells to differentiate to a level similar to or even greater than that of zinc - free glass . the cellular mineralization was also significantly stimulated by the zinc addition to the bioactive glass . taken together , zinc - added bioactive glasses may be useful in bone regeneration and tissue engineering with adult stem cells because they preserve the active growth of mscs and stimulate further osteogenic differentiation .

the reconstruction of defective and degenerative bones has been a significant challenge in oral and maxillofacial surgery [ 1 , 2 ] . however , the limited access and amount of bones as well as the pain and psychological fear that patients tend to suffer due to the additional surgical operation required have been raised as concerns . bone grafts have been introduced as an alternative to the use of autologous bones , and they have been found to be useful for filling bony defects and expanding the quantity and quality of bones available for implantation [ 46 ] . when compared to allogenic or xenogenic materials , which may cause immune and/or disease problems , synthetic bone grafts are considered relatively safe for use in large quantities [ 3 , 4 ] . over the past few decades , several types of synthetic bone grafts have been proved by clinical trials and commercialized , and ceramics or glasses classified as bioactive materials comprise the major category [ 58 ] . bioactive glasses obtained from melt - quenching or sol - gel process are known to form direct bonds with hard human tissues through a specific attachment mechanism , such as the formation of a bone mineral like carbonated hydroxyapatite layer that forms on the surface of the glasses under physiological conditions . one of the benefits of bioactive glasses is the ability to easily incorporate elements tuned to the required tissue responses . for example , the addition of magnesium to melt glass 45s5 has been found to improve bioactivity , while the addition of silver to bioactive glass was reported to elicit antimicrobial activity and the addition of strontium to sol - gel bioactive glass was shown to enhance bone cell responses . in this study , zinc was added to the sol - gel bioactive glass based on the previous reports demonstrating zinc roles in bone formation [ 1315 ] . as one of the trace elements found in natural bone , zinc has been studied in bone biology , particularly in osteoporotic patients [ 13 , 14 ] . furthermore , several recent studies have utilized zinc as an additive to medical materials , including zinc - substituted hydroxyapatite powders , and coatings , tricalcium phosphate powders , and bioactive glass granules [ 1520 ] . small concentrations of zinc within the bioactive glasses have been shown to affect bone - associated cell responses such as proliferation and matrix synthesis in vitro [ 1820 ] . stem cell - based tissue engineering is gaining considerable opportunities for the successful reconstruction of bone tissue . mesenchymal stem cells ( mscs ) derived from adult bone marrow are one of the most popular cell sources available for the regenerative therapy , including osteogenesis . compared to the stem cells of embryonic origin , relatively large quantities of mscs can be obtained without significant ethical concerns , and these cells can be applied clinically using autologous cells [ 21 , 22 ] . in this study , as a first step towards bone tissue engineering using zinc - added bioactive glass , the effects of the addition of small concentrations ( 2 and 5% by mole ) of zinc to the sol - gel bioactive glass on the growth and osteogenic potential of mscs were investigated . the sol - gel - derived bioactive glass was produced using precursors of tetraethyl orthosilicate and calcium nitrate tetrahydrate ( from sigma - aldrich ) in an ethanol - water solvent , with hydrochloric acid as a catalyst . to incorporate the zinc , calcium nitrate tetrahydrate was replaced with zinc nitrate hexahydrate ( from sigma - aldrich ) at 2 and 5 mol% to produce three different compositions of the bioactive glasses : 70sio2 - 30cao ( 0% zn ) , 70sio2 - 28cao-2zno ( 2% zn ) , and 70sio2 - 25cao-5zno ( 5% zn ) . the prepared sol was then left to age for 24 h and transformed into a gel , which was subsequently dried in an oven ( 60c ) and then thermal - treated by heating to 650c at a ramping rate of 2c / min , where it was maintained for 3 h , after which the sol was furnace - cooled in the air . for the cell test samples , the glass granules were sterilized and different quantities ( 3 , 10 , and 30 ) of the granules of each composition ( 0% zn , 2% zn , and 5% zn ) were placed in the individual wells of 24 culture well plates . the surface morphology of the glass granules was observed by scanning electron microscopy ( sem , hitachi ) . for observation of the bone bioactivity , the zinc - added bioactive glass granules were soaked in simulated body fluid ( ionic concentration : 142.0 mm na , 5 mm k , 1.5 mm mg , 2.5 mm ca , 147.8 mm cl , 4.2 mm hco3 , 1.0 mm hpo4 , and 0.5 mm so4 ) for 7 days , after which the surface was examined by sem . the dissolution of ions ( si , ca and zn ) from the glasses was detected using inductively coupled plasma atomic emission spectroscopy ( icp - aes ) . for the dissolution test , glass granules were incubated in an acellular culture medium ( serum free ) for up to 7 days , during which time the medium samples were analyzed . mesenchymal stem cells ( mscs ) were isolated from the bone marrow of tibia and femora of adult rats ( 48 weeks , korea ) using a method described in our previous study . the bone marrow mixture was then centrifuged , after which the supernatant was gathered and maintained on a culture flask containing growth medium ( -minimal essential medium ; mem , 2 mm glutamine , 100 u / ml penicillin , and 100 mg / ml streptomycin ) supplemented with 10% fetal bovine serum ( fbs ) . glass granules ( 5001000 micrometers in size ) with three different compositions ( 0 , 2 , and 5% zn ) were contained within the individual wells of 24-well plates at three different quantities ( 3 , 10 , and 30 ) . thus , a total of ten sample groups were used , including a glass - free blank control . an aliquot of 100 l of the cell suspension prepared at a density of 5 10 cells / ml was then seeded into each well . the medium for the cell growth was supplemented with 50 g / ml sodium ascorbate , 10 m sodium -glycerol phosphate , and 10 m dexamethasone to induce osteoblastic differentiation . next , the samples were cultured for up to 21 days , with the medium being refreshed every 2 - 3 days . the cell growth level was measured at days 3 and 7 based on the mitochondrial nadh / nadph - dependant dehydrogenase activity , which was measured using a cell proliferation assay kit ( celltiter 96 aqueous one solution , promega ) . the cell growth on the glass granules was examined by sem after fixation of the samples with glutaraldehyde , dehydration in a graded series of ethanol , and treatment with hexamethyldisilazane solution . sem was conducted at an accelerating voltage of 15 kv after gold coating the surface of the samples . the effect of the glass granules on the in vitro osteogenic differentiation of the mscs was assessed based on the alkaline phosphatase activity . after washing the samples with pbs the cells were gathered and added with cell lysis buffer ( 10% triton x-100 , 1 m tris - hcl , 0.5 m nacl , and 0.5 m edta ) containing protease inhibitor . bovine serum albumin ( from sigma ) was used as the reference curve for the protein assay . to observe the osteogenic marker , bone sialoprotein ( bsp ) , next , the samples were washed with pbs and incubated with the primary antibody at a concentration of 1 : 500 ( polyclonal rabbit antibone sialoprotein : bsp ) at 4c , after which they were washed with pbs . subsequently , the alexfluor 555 secondary antibody ( molecular probe ) was added to the samples at 1 : 200 and incubated at ~25c in dark condition . for the mineralization study , the cells were cultured under the influence of the eluted products from glass granules that were present within the transwell insert . after culturing the cells , the samples were rinsed in pbs , fixed with 10% formaldehyde , and then stained with 1% alizarin red s ( ars ) solution ( ph 4.1 ) for 30 min . after washing fully with distilled water the mineralization was quantified by detecting the amount of calcium present on the cellular constructs . figure 1 shows the typical morphology of the glass granules ( 2% zn ) that were prepared by the sol - gel synthesis and subsequent heat treatment ( figure 1(a ) ) . when the glass granules were immersed in a simulated body fluid and incubated for 3 days at 37c , calcium phosphate crystallites were found to precipitate on the surface of the glass granules , and after 7 days of incubation , the surface of the granules was fully covered by a thick mineral layer ( representative image of 2% zn - added glass shown in figures 1(b ) and 1(c ) ) . similar in vitro mineralization behavior in sbf was observed in the other compositions ( data not shown here ) . for the biological tests , the glass granules were evaluated at three different concentrations ( low ( 3 ) , medium ( 10 ) , and high ( 30 ) ) contained within each well of a 24-well culture dish . the ionic elusion ( si , ca , and zn ) from glass samples cultured in an acellular medium was measured using icp - aes for up to 7 days ( table 1 ) . the results revealed that the addition of zinc to the glass led to a slight decrease in the dissolution of silicon and calcium . the reduced release rate of the cations with time may be due to the depletion of cations with time and the resultant slow - down in further diffusion release process . mesenchymal stem cells ( mscs ) extracted from rat bone marrow were cultured in the presence of the bioactive glass granules . figure 2 shows the optical morphology of cells grown in culture wells for 7 days , including wells that contained the glass granules ( 0 , 2 , and 5% zn ) at different concentrations ( 3 , 10 , and 30 granules ) , as well as a glass - free culture well as a control ( blank ) . when low and medium concentrations ( 3 and 10 ) of the glass granules were present , the cells reached near confluence and had an elongated and well - spreading morphology similar to that of the control . however , when the high concentration ( 30 ) of the granules was present , the cell morphology became less elongated and more granular - like . after 14 and 21 days of culture , the cells were found to actively grow on the surface of glass granules . at day 21 , the cells appeared to be aggregated ( figure 3(e ) ) , and mineral - like products were noticeable around / below the cellular construct ( figures 3(e ) and 3(f ) ) . figure 4 summarizes the cell viability for periods of up to 7 days in the presence of bioactive glass granules with different compositions and concentrations , as measured by an mts method . when cultured in the presence of low and medium concentrations of granules for 3 and 7 days , the cell growth was equal to or higher than that of the control . however , a slight decrease in cell growth was observed with a high quantity ( 30 ) of the glass ( 2% and 5% zn - added ) , and even significant decrease was noticed in the 0% zn - added glass . in particular , zinc - containing glasses induced significantly higher cell growth at day 3 when they were contained at low ( 3 ) and medium ( 10 ) quantity . after prolonged culture periods ( 7 days ) , the cell viability affected by the glass granules became similar to that of the blank control , regardless of the composition of the glass . the alkaline phosphatase ( alp ) activity of the mscs cultured in the presence of various glasses was evaluated ( figure 5 ) . at day 14 , the addition of medium quantity ( 10 ) of glass granules significantly increased the alp level , and this improvement increased as the zinc content within the glass increased ( figure 5(a ) ) . the expression of the osteogenic marker bone sialoprotein ( bsp ) produced by the cells in the presence of bioactive glass granules was observed by immunofluorescence staining . as shown in figure 6 , the red - colored spots representing the expressed bsp were obvious ( 2% zn shown as a representative sample ) , particularly near the bioactive glass granules . when compared to the blank control , the addition of glass granules significantly enhanced the expression of bsp . moreover , the stimulation of bsp was greater in response to a higher concentration of glass granules . specifically , 30 glass granules for each composition ( 0 , 2 , and 5% zn ) were placed in a transwell insert , and the mscs were then cultured under the influence of the ionic eluants from the glass granules . as shown in figure 8(a ) , all the glass - containing groups showed the cellular constructs stained dark - red in response to the influence of the glass eluants , confirming significant level of cellular mineralization . as presented in figure 8(b ) , the mineralization was significantly higher on all the glass - containing samples than on the blank control , and furthermore , the 2% zn - added glass sample showed significantly higher level of mineralization than the pure bioactive glass . the use of bioactive inorganics is considered a promising tool in the reconstruction of oral and maxillofacial defects [ 1 , 2 ] . among the various compositions evaluated , bioactive glasses have been widely studied due to their excellent bioactivity and ability to direct - bonding to bone , which is primarily associated with the bone mineral - like phase created on their surface [ 8 , 9 ] . moreover , the composition of bioactive glasses is believed to be easily controlled by the introduction of biologically relevant elements , such as silicon , magnesium , strontium , silver , and zinc [ 1012 ] . osteoporotic patients have often been found to have a lack of zinc ; therefore , it is considered to be useful for the treatment of osteoporosis [ 13 , 14 ] . those studies have shown that zinc played an effective role in the stimulation of bone cell functions when it was present in calcium phosphate compounds such as hydroxyapatite and tricalcium phosphate [ 1518 ] . in the present study , zinc was added to the composition of bioactive glass during the sol - gel synthesis , and the effects of zinc addition ( 2 and 5% ) on the responses of rat bone marrow mscs were evaluated . mscs are considered a potential cell source for application in regenerative medicine , including the treatment of bone disorders [ 2123 ] . for stem cell - based bone tissue engineering , the material substrate should guide the initial anchorage of cells and their multiplication and further stimulate the differentiation into an osteogenic lineage . in order to utilize the zinc - bioactive glass as a scaffold for the mscs , this study was carried out as a first step to gain insight into the behavior of mscs in response to the glasses that contain zinc . different quantities of granules ( 3 , 10 , and 30 ) with variable glass compositions ( 0 , 2 , and 5% zn - added ) were used to evaluate the mscs responses . for all compositions , the initial cell adhesion and growth were shown to be favorable in the presence of small quantities of glass granules ( figures 2 and 4 ) . although some downregulation of cell viability was noticed initially , particularly in the case of the high concentration of 0% zn - added glass , the cell growth level became almost equal to that of the blank control after a prolonged culture time ( 7 days ) . conversely , a small quantity of glass granules led to a significant increase in cell viability , suggesting that the existence of glasses and their eluted products should favor the proliferative potential of mscs . based on the ionic release data ( table 1 ) , the addition of zinc to the glass led to a slight decrease in the elution of ions such as si and ca . thus , the initial high release of ions from the 0% zn - added glass might be one of the possible reasons for the decreased cell viability . the images of the cells grown directly on the glass particles at day 14 ( figure 3 ) confirmed the viable cellular status , that is , the presence of extensive cytoskeletal processes and the formation of collagenous fibrillar extracellular matrix by the proliferative cells . the effects of the zinc - added bioactive glass on the osteogenic differentiation of the mscs were evaluated based on the alkaline phosphatase ( alp ) activity of cells . in the present study , alp was significantly upregulated when grown in the presence of bioactive glass granules , particularly with larger quantity of granules and at later culture times ( over 14 days ) ( figure 5 ) . moreover , the increase appeared to be greater in the presence of zinc - added glasses . as another marker for osteogenic differentiation , bone sialo - protein ( bsp ) was evident in the presence of glass , particularly near the glass granules ( figures 6 and 7 ) . although the mscs differentiate down to an osteogenic lineage within the total culture medium used herein , the presence of bioactive glasses should alter the secretion of proteins , and thus the ecm components . as noticed , the mineralization behavior under the influence of the glasses was noteworthy with respect to that of glass - free control which was conditioned only with the osteogenic medium . these results indicate that the differentiated cells and the synthesized ecm must be properly conditioned to induce mineralization under the influence of the bioactive glasses eluants . as to the effects of zinc , the results demonstrated that the mscs expressed alp and bsp at levels largely similar to those of zinc - free bioactive glass , and sometimes at even higher levels depending on the zinc concentration and the quantity of glass used . the different levels of ions released from the zinc - added bioactive glasses should affect the differentiation responses of stem cells and further cellular mineralization , and the zinc ions released may participate in the regulation of cellular functions to some extent . although no significant difference in bsp levels was noticed , the upregulations of alp and mineralization by the zinc - added glass support the possible role of zinc in the differentiation of mscs to the osteoblastic pathway . taken together this study may provide some useful information regarding the effects of zinc addition to bioactive glasses on the mscs growth and their stimulation into osteogenic differentiation . the cell growth and osteogenic differentiation of mscs with respect to zinc - added bioactive glasses ( 0 , 2 , and 5% ) in granular form were investigated . the osteogenic development , as determined based on the alkaline phosphatase ( alp ) activity and bone - sialo protein ( bsp ) secretion , was significantly enhanced by the bioactive glass granules . taken together , zinc - added bioactive glasses may be useful in bone regeneration and tissue engineering with adult stem cells because they preserve the active growth of mscs and stimulate further osteogenic differentiation .