This study was carried out to determine whether interactions of cell activation, shear stress and platelets at sites of endothelial injury explain the paradoxical maldistribution of activated leukocytes during sepsis away from local sites of infection towards disseminated leukocyte accumulation at remote sites.2). Leukocyte-endothelial interactions were analyzed by videomicroscopy and analysis of covariance.Human umbilical venous endothelial cells (HUVEC) and polymorphonuclear neutrophils (PMN) were activated with lipopolysaccharide at 100 and 10 ng/ml to achieve adhesion molecule patterns as have been reported from the hyper- and hypo-inflammatory stage of sepsis. To examine effects of leukocyte activation on leukocyte-endothelial interactions, activated HUVEC were perfused with activated and non-activated neutrophils in a parallel plate flow chamber. Adhesion molecule expression and function were assessed by flow cytometry and blocking antibodies. In a subset of experiments the sub-endothelial matrix was exposed and covered with platelets to account for the effects of endothelial injury. To investigate interactions of these effects with flow, all experiments were done at various shear stress levels .Activation of neutrophils rendered adhesion increasingly dependent on shear stress reduction. At normal shear stress, shedding of L-selectin decreased adhesion by 56%. Increased rolling fractions of activated PMN at low shear stress revealed impaired integrin affinity despite numerical up-regulation of CD11b. On sub-maximally activated, intact HUVEC shear stress became the prevailing determinant of adhesion. Presence of a platelet-covered injury with high surface density of P-selectin was the strongest variable for adhesion. When compared to maximally activated HUVEC, platelets increased neutrophil adhesion by 2.7-fold. At sub-maximal activation a 10-fold increase was observed (L-selectin shedding and integrin dysfunction render leukocyte adhesion increasingly susceptible to shear stress and alternative adhesion receptors. In combination, these effects inhibit recruitment to normally perfused sites with intact endothelium and favor maldistribution towards sites with compromised perfusion or endothelial injury. Directing leukocytes to local sites of infection is a crucial part of the innate immune response. While intravascular shear forces prevent relevant leukocyte adhesion in a healthy individual, increased concentrations of microbial toxins and pro-inflammatory mediators induce upregulation of endothelial adhesion molecules in inflamed tissue, resulting in a targeted accumulation of leukocytes at the site of infection . InitialIn contrast to local inflammation, systemic sepsis is characterized by profound leukocyte activation throughout the circulation ,3. BecauFirst, does systemic leukocyte activation increase or impair leukocyte recruitment to activated endothelium and what are the mechanisms during the different stages of inflammation? Second, if targeted leukocyte recruitment to locally activated endothelium is impaired, are there mechanisms that favour disseminated leukocyte accumulation at the same time? Third, given that later sepsis is characterized by immunosuppression, endothelial cell damage and organ dysfunction, are there mechanisms, independent of the physiological immune response, that gain a leading role for the distribution of leukocyte accumulation?In compliance with the Helsinki Declaration on experimental research on humans and after obtaining ethical committee approval  and informed consent, human umbilical venous endothelial cells (HUVEC) and polymorphonuclear neutrophils (PMN) were derived from human umbilical veins and citrated blood samples from healthy volunteers as previously described . HUVEC w4Cl on ice. After washing, the PMN pellet was resuspended in cold Medium 199  supplemented with 50% fetal calf serum  at 5 × 107/ml. To avoid assay related activation of PMN during rewarming, we reconstituted the PMN pellet to 106 PMN/ml just before the adhesion assay in normoxic, room temperature Medium 199 only. Final rewarming to 37°C was achieved in the heatable flow chamber.PMN were isolated by density gradient centrifugation at 1,700 rpm on a discontinuous Percoll gradient with 63% and 72% Percoll in buffer . The bottom layer was collected and contaminating erythrocytes were removed by hypotonic lysis in 10% NH2 [PMN adhesion to HUVEC was quantified in a parallel plate flow chamber with a laminar flow profile  × 100)/(No. of rolling cells + No. of firmly adherent cells). Mean rolling velocities were determined from more than 25 individual velocity profiles for each experimental condition as derived from customized software for image recognition .PMN were perfused over HUVEC-containing coverslips for 10 minutes under different conditions of LPS-activation. Thereafter, PMN-adhesion was determined from 10 s video recordings of five different fields of view by phase contrast microscopy . PMN were defined as rolling when traveling below 50% of the velocity of free flowing PMN in close proximity to the endothelium at the given shear stress . A PMN, ficiency ,28, the 2 in presence of functional blocking monoclonal antibodies (mAb). PMN and HUVEC were incubated for 30 minutes prior to the adhesion assay with mAb against endothelial (E)-selectin , leukocyte (L)-selectin , platelet (P)-selectin  or a nonspecific antibody .Selectin function was determined at 2 dyne/cmEscherichia coli, Sigma), dissolved in Medium 199 supplemented with 20% fetal calf serum.By combining different conditions of neutrophil and endothelial activation, we intended to mimic patterns of adhesion molecule expression as they have been observed during local inflammation and different stages of sepsis-associated systemic hyper- or hypo-inflammation -10,29-31The changes in adhesion molecule expression were determined by flow cytometry . Cells were gated using forward and side scatter properties and staining with saturating amounts of fluorochrome conjugated mAb against E-selectin, L-selectin (both from BD Biosciences), ICAM-1  and CD11b . Matching isotype controls were used to define the setup of the instrument. Unintended PMN-activation during cell separation was ruled out by comparison of isolated PMN to leukocytes from whole blood.2 for five minutes prior to the PMN adhesion assay. Since platelet-matrix interactions are much more shear-resistant than leukocyte-endothelial interactions, this resulted in dense platelet accumulation at the site of injury without premature leukocyte adhesion. Before starting the PMN adhesion assay, the chamber was cleared from remaining blood by a thorough rinse with cell free medium. Then, the platelet-covered HUVEC were perfused with the PMN suspension at 2 to 0.25 dyne/cm2.Distinct from true endothelial activation, severe sepsis leads to endothelial cell injury which is likely to persist even in the hypo-inflammatory stage ,32 and rpost hoc t-tests, we examined whether PMN activation , shear stress (continuous effect) or a combination thereof influenced PMN adhesion. Effects of platelets were analyzed accordingly (replacing PMN-activation by PLT-treatment). Effects of antibody blockade were examined by paired t-tests. Results of the adhesion assays are presented as means ± SEM. A P-value <0.05 after Bonferroni-Holm correction was considered significant. All analyses were performed using the statistical software JMP .All experiments were carried out in quadruplicate. The medians of fluorescence intensity (MFI) were calculated from 5,000 single events by flow cytometry. An analysis of variance (ANOVA) was performed to determine whether adhesion molecule expression was influenced by LPS activation. Using an analysis of covariance (ANCOVA) and When compared to non-activated controls (HUVEC-/PMN-), maximal LPS-activation with 100 ng/ml (HUVEC++/PMN++) resulted in maximal upregulation of E-selectin, ICAM-1, CD11b and complete shedding of L-selectin, comparable to systemic hyper-inflammation -31. Simi2 prevented relevant adhesion in non-activated HUVEC-/PMN-. As expected in the model for local inflammation, maximal LPS-activation of HUVEC largely increased adhesion of non-activated PMN at 3 dyne/cm2 from 42 ± 17 (HUVEC-/PMN-) to 894 ± 93 cells/mm2 in HUVEC++/PMN-  down to values obtained with activated PMN whereas no effect was observed on activated PMN. Blockade of P-selectin had no significant effect in both groups, suggesting that P-selectin played no role on intact HUVEC after four hours LPS-activation. Consequently, only E-selectin remained functional under the condition of systemic hyper-inflammation and blocking the molecule in HUVEC++/PMN++ reduced adhesion down to background values observed in HUVEC-/PMN-.Addition of selectin-blocking mAbs at 2 dyne/cmTo determine whether a dissociation of quantitative and qualitative integrin upregulation contributed to the decreased adhesion of LPS-activated PMN, rolling fractions were calculated from the number of rolling PMN in relation to total adhesion as a measure for adhesion efficiency Figure . For simP = 0.59, Figure 2 whereas markedly higher velocities were observed on HUVEC lacking E-selectin (Figure On maximally activated HUVEC with upregulated E-selectin, PMN-activation had no influence on rolling fraction (P < 0.05, Figure 2-integrins [On sub-maximally activated HUVEC without E-selectin, co-activated PMN showed significantly increased rolling fractions at all levels of shear stress, indicating decreased adhesion efficiency (ntegrins .To differentiate effects of endothelial activation from effects of endothelial injury on PMN recruitment -32,38 weP < 0.01, Figure 2 PMN adhesion increased 2.7-fold in maximally activated HUVEC++/PMN++/PLT (Figure P < 0.01, Figure The presence of platelets was the strongest variable for adhesion of activated PMN. At all levels of shear stress PMN adhesion on platelet-covered, injured HUVEC increased significantly when compared to intact HUVEC (T Figure . In sub-T Figure . AdditioP < 0.01, Table Blockade of P-selectin revealed that the increased adhesion was largely due to platelet P-selectin. In contrast to its lacking effect in intact HUVEC++/PMN++, P-selectin blocking WASP12.2 decreased PMN adhesion in injured HUVEC++/PMN++/PLT by 70% (To provide more insight into the mechanisms that might explain the occurrence of disseminated leukocyte-related tissue damage in spite of an impaired leukocyte recruitment to local sites of inflammation during severe sepsis, we investigated the interdependent effects of cell activation, adhesion molecule expression, shear stress and a platelet-covered endothelial injury on PMN-adhesion.In order to mimic different stages of inflammation, as they are frequently observed during the time course of severe sepsis, various constellations of PMN and endothelial activation were combined. Maximal activation of both PMN and HUVEC was considered to reflect maximal systemical inflammation in the hyper-inflammatory stage of sepsis where high concentrations of circulating mediators induce activation of leukocyte and endothelial cell adhesion molecule expression systemically throughout the circulation ,3,10. SuApart from the different stages of inflammation, adhesion molecule expression during systemic sepsis differs from local inflammation in another important aspect. In local inflammation upregulation of leukocyte integrins and shedding of L-selectin does not occur before entering the inflamed tissue . To accoThe results demonstrate that impaired recruitment of systemically activated PMN to local sites of inflammation during severe sepsis ,3,8-10 cReflecting the well-known inverse correlation of shear stress and adhesion in general -22 PMN-aFar exceeding the effects of shear stress is the platelet-covered endothelial lesion, which proved to be the strongest determinant of PMN-adhesion at all levels of shear stress. In maximally activated cultures, PLT-PMN interactions increased PMN adhesion by two-fold. At the sub-maximal LPS dose, an even more dramatic 10-fold increase was observed. Both findings indicate that endothelial cell damage gains a leading role for the spatial distribution of leukocyte accumulation through PLT-PMN interactions under conditions of systemic leukocyte activation and becomes exceedingly pronounced when true endothelial cell activation is outweighed by endothelial cell damage, as might occur in the hypo-inflammatory stage of severe sepsis -17,30,32Independent from endothelial cell activation platelet-covered lesions provide a rich source of platelet-derived P-selectin ,34. In oin vivo and, in part, are species-related, we decided to use a flow chamber to examine their interplay in a human setting. Clearly, this simplified in vitro model has other inherent limitations since it neither includes true infection nor simulates all aspects of sepsis in an intact organism. For instance, we had to abstain from inducing true endotoxin tolerance since this would have required prolonged cell culture with inevitable confounding effects on adhesion molecule expression in an otherwise comparative experimental setting. Additionally, the use of cell suspensions instead of whole blood influences rheological properties and the fixed diameter of the flow channel precludes effects of luminal narrowing that may arise in small vessels during leukocyte adhesion. Apart from directly favouring further adhesion, these effects may also influence cell interactions in vivo by decreasing blood flow and oxygen transport.Since the effects of shear stress, tissue hypoxia, cell activation and cell injury are hardly distinguishable from each other during sepsis in vivo [As a necessary simplification instead, we used different LPS-concentrations and standardized reproducible hydrodynamic conditions in an otherwise unchanged comparative model to investigate the mechanisms of leukocyte accumulation during different stages of systemic inflammation. Although this model is artificial in many aspects, flow chamber experiments have proven valid for studying cell interactions in a number of studies including direct comparison with leukocyte adhesion in animals ,46. Addiin vivo ,10,12-17In summary, our findings indicate a maldistribution of systemically activated leukocytes away from sites of local inflammation with intact endothelium and normal blood flow towards sites with compromised perfusion or endothelial cell injury. Because of L-selectin shedding and altered integrin function, this maldistribution might occur even during the early hyper-inflammatory stage. It seems to become exceedingly pronounced, however, when endothelial LPS sensitivity is decreased, as might occur in patients with hypo-inflammatory cytokine profiles -16. From• Activation of leukocytes renders adhesion increasingly susceptible to shear stress.• Presence of a platelet-covered endothelial injury overcomes this effect and seems to become the prevailing factor for leukocyte accumulation under the condition of systemic inflammation.• Together these mechanisms favor the maldistribution of leukocytes away from local sources of inflammation and towards areas with compromised flow and/or endothelial damage.ANOVA: analysis of variance; ANCOVA: analysis of covariance; E-selectin: endothelial selectin; HUVEC: human umbilical venous endothelial cells; ICAM-1: intercellular adhesion molecule-1; IL-10: interleukin-10; LFA-1: lymphocyte function antigen-1; LPS: lipopolysaccharide; L-selectin: leukocyte selectin; mAb: monoclonal antibody; MAC-1: macrophage antigen-1; MFI: median of fluorescence intensity; PLT: platelets; PMN: polymorhponuclear neutrophils; P-selectin: platelet selectin; SEM: standard error of the mean; TNF-α: tumor necrosis factor-α.The authors declare that they have no competing interests.AP and BN conceived of the study, participated in its design and coordination, and drafted the manuscript. VS and AH carried out the adhesion assays. JR and HAH participated in the design of the study and helped to draft the manuscript.


Drosophila melanogaster, genes of the sex-determination hierarchy orchestrate the development and differentiation of sex-specific tissues, establishing sex-specific physiology and neural circuitry. One of these sex-determination genes, fruitless (fru), plays a key role in the formation of neural circuits underlying Drosophila male courtship behavior. Conservation of fru gene structure and sex-specific expression has been found in several insect orders, though it is still to be determined whether a male courtship role for the gene is employed in these species due to the lack of mutants and homologous experimental evidence. We have isolated the fru ortholog (Md-fru) from the common housefly, Musca domestica, and show the gene’s conserved genomic structure. We demonstrate that male-specific Md-fru transcripts arise by conserved mechanisms of sex-specific splicing. Here we show that Md-fru, is similarly involved in controlling male courtship behavior. A male courtship behavioral function for Md-fru was revealed by the behavioral and neuroanatomical analyses of a hypomorphic allele, manMd-tra, which specifically disrupted the expression of Md-fru in males, leading to severely impaired male courtship behavior. In line with a role in nervous system development, we found that expression of Md-fru was confined to neural tissues in the brain, most prominently in optic neuropil and in peripheral sensory organs. We propose that, like in Drosophila, overt sexual differentiation of the housefly depends on a sex-determining pathway that bifurcates downstream of the Md-tra gene to coordinate dimorphic development of non-neuronal tissues mediated by Md-dsx with that of neuronal tissues largely mediated by Md-fru.In  Drosophila melanogaster has been elucidated in great detail Drosophila melanogaster is largely governed by the transcription factor fruitless (fru) fru alleles have been identified which impairs courtship performance of males, but leaves females unaffected fru functions in genotypically female flies elicits some aspects of male courtship behavior fru is not only necessary but also sufficient for implementing male mating behavior in Drosophila has been challenged in recent studies doublesex (dsx) also contributes to the regulation of male mating behavior.The genetic pathway controlling sex-specific behaviors in the invertebrate model system fru gene in Drosophila melanogaster is very large in size spanning approximately 150 kb of genomic sequences. Transcription initiates from four independent promoters (P1–P4) and the resulting transcripts undergo a complex pattern of alternative splicing events at both the 5′ and the 3′ ends, generating 18 different transcript variants which code for a family of transcription factors containing a common BTB domain and one of four alternative zinc-finger DNA binding domains transformer (tra), a key component of the sex-determining pathway. In females, TRA protein forms a splice enhancer complex with TRA2 and RBP1 (RNA Binding Protein 1) binding to nascent fru RNAs where they act to alter splice site choice, resulting in the inclusion of a pre-mature stop codon signal and thereby generating female-specific transcripts (Ffru) with a severely truncated ORF tra gene is functionally OFF, the default splice-site is used and the resulting male-specific transcripts (Mfru) encode functional full-length BTB-Zn-finger type transcription factors. These sex-specific transcripts incorporate alternate 3′ exons A, B, or C to encode the FruM isoforms: FruMA, FruMB and FruMC. FruM isoforms are translated only in males and are expressed in all regions of the nervous system implicated in male courtship behavior M positive neurons located in widely distributed clusters throughout the male brain and nerve chord are key parts of the neuronal circuitry required for behavioral responses to sex pheromones and the display of proper male courtship behavior.The fru in controlling sex-specific behaviors the question arises whether this function is conserved in other insects. The existence of sex-specifically expressed fru homologs in various dipteran and in hymenopteran species  to control various aspects of sexually dimorphic CNS development Md-tra also regulates housefly courtship through a fru-like downstream effector we set out to isolate the fru ortholog in Musca. PCR amplification with degenerate primers flanking the conserved BTB domain of fru yielded a genomic fragment of 190 bp in length. Subsequent extension by 3′ RACE of cDNAs prepared from adult houseflies revealed the existence of four alternative mRNA transcripts  upstream of the BTB coding exons (C1–C3) extending the ORF by an additional 72 amino acids at the amino terminal end (f). In addition, we identified a female-specific exon (f) downstream of exon Sf (cis-regulatory elements (TRA/TRA2 binding sites) known to play an important role in sex-specific splicing of Drosophila fru . The female-specific transcripts, on the other hand, have a severely truncated ORF and thus are likely to represent non-functional splice variants of the same locus. Because of their structural correspondence to the non-functional products of P1-derived female transcripts in Drosophila they are collectively named FMd-fru . Taken together we conclude that the Musca genome harbors an ortholog of fru, Md-fru, which expresses a set of male-specific products and hence is likely to be a target of the sex determining pathway.In inal end , S1. In  exon Sf . These a exon Sf . Four rehila fru . The malMd-fru, suggested that it is sex-specifically spliced and directly targeted like Md-dsx, by Md-tra. To explore this possibility, we tested expression of Md-fru in different genetic backgrounds in which Md-tra activity was experimentally altered . We conclude that female splicing of Md-fru depends on the presence of Md-tra and Md-tra2 activity supporting our notion that Md-fru is a downstream target of Md-tra in the sex determination pathway. To extend this type of epistatic analysis we investigated whether ectopic expression of the female activity of Md-tra is sufficient to direct female-specific splicing of Md-fru in genotypic males. To this end, we used a transgenic line in which FMd-tra is constitutively expressed in flies carrying the male determining factor IIIM on the third chromosome FMd-tra expression overrides repression by IIIM and is sufficient to direct all aspects of female development. RNA samples analyzed from the heads of these sex-reverted III/+M; p[FMd-tra] individuals showed the presence of only female-specific splice variants of Md-fru  allele of Md-tra to be a hypomorphic mutation with residual Md-tra activity manMd-tra develop into phenotypically normal looking males . However, they are reduced in fertility, as only a fraction (about 10%) produces progeny in single mating tests. Sterility is 100% in homozygous manMd-tra males when their mother carried the dominant gain-of-function allele DMd-tra in trans man2Md-tra males. The genital apparatus of man1Md-tra and man2Md-tra males looked indistinguishable in morphology from those of wildtype males, and normal amounts of motile sperm were present in squashed preparations of mutant testes. To test for gonad and sperm functionality, the larval anlagen of man2Md-tra testes were transplanted into wildtype male hosts   . In singn period . Of the n period . To assele rates . In partmanMd-tra allele caused male courtship failure. We tested this hypothesis by injecting dsRNA of Md-tra into syncytial man2Md-tra embryos. Silencing of Md-tra at this early stage leads to a complete and lasting loss of Md-tra activity man2Md-tra males, 41 were fertile in single mating tests and, due to the absence of an M factor, produced only female offspring when outcrossed with wild-type females. The percentage of successful matings (about 80%) was comparable to those of wild-type control males in single crosses. In contrast, all 20 man2Md-tra males injected with Ringer’s buffer remained infertile. Hence, the sterility of man2Md-tra homozygous males can be rescued by targeted silencing of Md-tra. In a previous study we showed that silencing of Md-tra2, a required co-regulator for female expression of Md-tra, could also completely restore fertility of man2Md-tra mutant males manMd-tra is sufficient to cause male courtship failure.We hypothesized that residual feminizing activity of the Md-tra target Md-fru. The sex-specific splicing of Md-fru was investigated in man2Md-tra males. RT-PCR experiments on RNA extracted from man2Md-tra male heads detected only non-functional FMd-fru transcripts . If Md-fru were indeed part of a developmental program involved in establishing the neuronal circuitry involved in courtship behavior, we would expect a locally restricted expression pattern in the adult CNS similar to that reported in Drosophila. RNA in situ hybridizations were performed on cryosections of adult female and male housefly heads with an antisense probe against 5′ sequences (P1-derived) of Md-fru . The Md-fru probe also labeled neurons located in the base of taste sensilla of the labellum  antibody staining on whole mount preparations of adult male and female CNS ventral ganglions , compared with 100% immunoreactivity in wild-type controls and in man1Md-tra (n = 10 each). The loss of serotonin staining of this cluster correlates with the lack of MMd-fru expression suggesting that, just as Drosophila, Md-FRUM plays a key role in the specification of these male serotonergic cells Within the sexually dimorphic neural circuitry identified in the CNS of anglions . As in Dganglion . Also, pganglion . Signifint males . There wMd-tra acts as the key switch in the sex determining hierarchy of the house fly governing every aspect of somatic dimorphic development Md-tra. Our analysis of manMdtra males revealed that infertility is primarily a result of anomalous behavior rather than of aberrant differentiation of reproductive tissues. We propose that the primary cause for anomalous courtship behavior in mutant males is the presence of female Md-tra activity. When Md-tra is knock downed by RNAi, fertility of these males is restored suggesting the absence of Md-tra activity is a pre-requisite for proper implementation of male courtship behavior. In females, on the other hand, Md-tra is activated in order to prevent the establishment of these behaviors. We propose that Md-tra acts as an upstream switch, coordinating dimorphic differentiation of both morphological and behavioral traits. The central role of tra in coordinating these two facets of dimorphic development has been extensively investigated in Drosophila. For instance, genotypically female XX animals which are mutant for tra develop into morphologically normal males which behave and court like males tra results in the development of pseudofemales, which do not display male behaviors. Morphological and behavioral dimorphic development, however, differ with regard to the spatial and temporal requirements of tra. While tra’s functions in somatic differentiation are needed as early as during embryonic development , the critical developmental window for the control of sex-specific behaviors was mapped to much later stages of development around larval-pupal transition tra is activated at these stages in genotypically male individuals, they will develop into normal looking males however courtship behavior is severely impaired. It suffices to target expression of tra to specific areas of the brain to accomplish a complete loss of courtship behavior in otherwise normally developed males manMd-tra males suggesting that residual Md-tra activity in these males is primarily affecting late events in dimorphic development in particular those during the critical stages when male courtship behavior is established. As feminization is only observed at the behavior level this suggests that the female activity provided by the manMd-tra alleles is either below a threshold needed to differentiate female structures or is confined to neural tissues where it specifically affects CNS related aspects of dimorphic development.We previously reported that the gene Drosophila sex-determination gene fru, Md-fru. We have shown that, in an analogous manner to Drosophila, Md-fru RNAs are alternatively spliced leading to male-specific expression of Md-FRUM proteins from the P1 promoter. The presence of four putative TRA/TRA2 bindings sites located close to the regulated splice sites of the female-specific exons of Md-fru suggests that this regulation is due to its targeting by the female-specific splicing factor Md-tra. This is similar to the regulation of the previously proposed targets of Md-tra, Md-dsx and Md-tra itself Md-tra activity lead to corresponding changes in Md-fru expression. For instance, ablating Md-tra function by RNAi in karyotypically female individuals shifts the mode of Md-fru splicing from female to male. Reciprocally, when Md-tra functions are ectopically expressed in karyotypic males, Md-fru expression switches to the female nonfunctional mode. We additionally show that Md-tra2, a functional splicing co-factor with tra in Drosophila, is involved in Md-fru splicing regulation, by silencing Md-tra2 in man2Md-tra males and showing a shift of Md-fru splicing from the female to the male mode (data not shown) In this study we have identified the house fly homologue of the key Md-dsx and Md-tra, Md-fru is another downstream and possibly direct target of Md-tra in the house fly sex determination pathway. In D. melanogaster, male-specific FRUM products are required for the proper display of male courtship behavior. When tra is ectopically activated in the developing male CNS, it prevents the production of FRUM and, as a consequence, these males exhibit impaired and anomalous courtship Md-fru is a target of Md-tra and that misregulation of Md-fru by residual Md-tra activity is the primary cause for courtship failure of manMd-tra males. Interestingly, the sex-specific splicing of Md-fru, and not Md-dsx, seems to be affected by the hypomorphic activity produced by the manMd-tra alleles. In manMd-tra mutant males the splicing of Md-fru shifts from the male to female mode, resulting in the loss of ability to produce male-specific FRU proteins.Taken together we propose that, like Md-dsx and Md-fru in the hypomorphic manMd-tra background can be explained by either different thresholds requirements or by differences in temporal and spatial requirements for activating the female splicing mode. For instance, Md-fru pre-mRNAs may have a higher affinity for Md-TRA binding than Md-dsx pre-mRNAs or may require lower levels of bound complexes to activate the female-specific splice sites. On the other hand, the differential effects on Md-dsx and Md-fru may result from discontinuous and/or tissue-specific manMd-tra expression during development. In either case, its prominent impact on behavioral dimorphic development suggests that manMd-tra must be expressed in the nervous system around the time when the CNS shapes the neuronal circuits required for proper male behavior fru transcripts (FMd-fru) in the heads of male manMd-tra adults. We propose that Md-fru is a direct target of Md-tra thereby defining an Md-dsx independent branch in the sex-determining pathway, which specifies the fates or activities of neurons that carry out higher order control functions to elicit and coordinate the activities comprising male reproductive behavior.The disparity in splicing behavior of MMd-fru expression is required for the correct display of male courtship in the housefly. This conclusion is based on the strict correlation between proper courtship performance and male expression of Md-fru. In our courtship assays we found that only males that expressed the MMd-fru variants performed as well as normal males, while those that expressed non-functional FMd-fru variants either performed poorly or did not engage in courtship at all. The most compelling argument comes from the observation that silencing of Md-tra in man2Md-tra males not only shifts Md-fru expression from the female to male mode but also completely restores fertility. A more direct test to confirm the role of Md-fru in courtship behavior would require a targeted disruption of MMd-fru function . To overcome this problem we introduced a construct into the housefly genome which contains inverted repeats against P1 derived Md-fru transcripts driven by a constitutively expressed promoter. Unfortunately, none of the tested transgenic lines effectively suppressed Md-fru (data not shown). On the other hand, downregulation of the respective fru homolog in the hemimetabolous species, Blatetella germanica and Schistocerca gregaria, both of which are responsive to systemic effects of dsRNA silencing, have been shown to disrupt proper male behavior suggesting that fru plays an important and widely conserved role in male behavior fru homologs in these species are direct targets of the sex-determining cascade and whether of not these homologs are specifically expressed in the CNS.We propose that male-specific tion cf. . We expefru in male courtship have been studied in great detail at the cellular level in Drosophila. FRUM is expressed in about 1700 neurons which appear to be involved in various aspects of male courtship including olfactory and gustatory neurons required for behavioral responses to sex pheromones. Likewise, we observe that expression of Md-fru is confined to specific areas of the CNS and to neurons of the peripheral nervous system. Though expression appears much broader than in Drosophila and apparent in the optic neuropils, its neural-specific expression suggests that Md-fru is also involved in building and specifying neuronal circuits required for male behavior. High levels of expression in the neuropils which process visual cues hint at a prevalent role of the visual system in male behavior of the housefly. Previous studies showed that visually guided behavior patterns in houseflies differ between the sexes. Male houseflies chase females in the air using visual cues, but are not chased by females Md-fru in the optic neuropils is involved in establishing the sexual difference of neural architecture involved in motion detection.The functions of fru mutant males this cluster is substantially reduced in size and projection patterns are aberrant man2Md-tra housefly males where the cluster is substantially diminished in size and projections to the internal reproductive organs are lost. In Drosophila the formation and/or specification of the male-specific serotonergic cluster depends on presence of FRUM and DSXM activity M is the cause for their absence in man2Md-tra males, suggesting a conserved mechanism in the specification of these male-specific neurons.One striking dimorphic neural trait common to houseflies and fruit flies is the presence of a male-specific cluster of serotonergic neurons in the dorsal posterior part of the abdominal ganglion. In both types of dipterans these neurons send projections to the male internal reproductive organs where they innervate contractile muscles. It has been proposed that these muscles are required for transferring sperm and seminal fluids during copulation fru and dsx, at the level of tra is a widely used strategy in holometabolous insects  autosomal M strain: females XX; pw, bwb/pw, bwb, males III, pw+, bwb+/pw, bwbXX; M; (3) man1Md-tra strain: females man/Md-tra+XX, Md-tra, males man/Md-tramanXX, Md-traman2Md-tra strain: females D, Ba/Mdtraman, Ba+XX; Mdtra, males man, Ba+/Mdtraman, Ba+XX; Mdtra; (5) multimarked strain: females XX; ac/ac; ar/ar; bwb/bwb; ye/ye; snp/snp, males XY; ac/ac; ar/ar; bwb/bwb; ye/ye; snp/snp.(1) Wild-type strain: females man2, bwbMdtra+ testes were dissected and transplanted into the posterior region of bwb male host larvae in Ringer’s solution. These larvae were then reared on porcine manure and surviving adults were outcrossed with bwb females of the host strain. In the progeny we screened for presence of donor derived bwb+ individuals.Crossing schemes and used genotypes are shown in Testes of 5-days old adult males were dissected in Ringer’s solution and transferred to a coverslip where they were gently squeezed to release sperm which was then dispersed with a needle. Morphology and mobility of sperm were examined by phase-contrast microscopy.Male and female flies collected for courtship assays were separated shortly after hatching and kept at 25°C for 6 days in plastic beakers with sugar water and milk powder. The air humidity was kept between 65% and 75% and flies were exposed to a 12 hours light/dark cycle. The courtship assays were performed at the same time of the day. The courtship arena consisted of a see-through plastic cylinder with a height of 40 mm and a length of 160 mm. For each couple courtship activities were examined during a period of at least 20 minutes.Md-tra dsRNA as described in Md-tra forward primer located in the 5′UTR of exon 1 linked to T7 promoter sequences : (5′ gtaatacgactcactatagggTGGTGTAATATGGCTCTATCG 3′) and Md-tra reverse primer located in exon 5 linked to T7 promoter sequences : (5′ gtaatacgactcactatagggGCTGCCATACAAACGTGTC 3′).The same primers were used for synthesis of Total RNA from adult females was reverse transcribed and the resulting cDNA was amplified with this primer pair to generate a female-specific template of 620 bp in length. In vitro transcription of this template with T7 RNA polymerase yielded dsRNA that was ethanol precipitated and resuspended in injection buffer to a final concentration of 1 µg/µl. Embryos were collected within 1 h after egg laying and dechorionated. Then 1–2 nl of the dsRNA solution was injected into the posterior pole of syncytial blastoderm embryos Md-fru fragment spanning the conserved BTB domain was previously recovered by David Dolezel  using degenerated primers and made available to us for further investigation. Based on sequence alignments between fru sequences of Drosophila and Anopheles gambiaeA small domain A: Md-fru-6 (5′ RCAATGNGCYTTCATRTTATC 3′),domain B: Md-fru-7 (5′ ATGNSWATGRAAATGATGCCA 3′),domain C: Md-fru-8 (5′ CATNGGATGYTTRAAYTTRCA 3′).These reverse primers were used in combination with the forward primer MdFRU-3E (5′CAGTTAAGGCTCATCAAGCC3′) located in the BTB domain to amplify cDNA variants containing zincfinger domains B and C. Forward primer MdFRU-15s (5′CATGGGCCTCAATCAGTCG3′) was used to amplify the variant which contains zinc finger domain A. The zinc finger domain D was identified by bioinformatics in the genomic region between exon C4 and exon C5.Female and male heads were collected from freshly hatched adults and total RNA was prepared according to the protocol of the manufacturer TRI REAGENT  or to the protocol of the “NucleoSpin RNAII” kit . For long term storage, total RNA was dissolved in 70% ethanol and stored at −80°C.nd Generation” . For 3′ RACE, the supplied oligo-dT primers were used for reverse strand cDNA synthesis. Forward primers located in the zincfinger encoding exons A, B, and C, were used for second strand synthesis and subsequent amplification: Md-fru-70B in domain A (5′ GCGATGACTTGAGTCTGACA 3′), Md-fru-35 in domain B (5′ CAACCTGAGGACACATTGCA 3′), Md-fru-36 in domain C (5′ GGACGTATGTCAAGAACCTC 3′) in combination with the shortened PCR-anchor primer A-3 (5′ ACGCGTATCGATGTCGAC 3′). For 5′ RACEs, gene-specific primer Md-fru-1 (5′ TCTGAGTATCTGACATCCTTC 3′) was used for reverse transcription. First round PCR amplification used gene-specific primer Md-fru-4E (5′ GCAGGCTAGAGTAACATCAC 3′) and the oligo-dT primer supplied with the Kit. Second round PCR amplification used gene-specific primer Md-fru-23 (5′ GACACCGGTTAAATTTGTCGG 3′) and a shortened PCR-anchor primer A-3 provided by the manufacturer Boehringer. In the first round PCR, the polymerase and Buffer 3 of the “Expand Long Template PCR System” kit by the manufacturer Roche Diagnostics  was used. For the second round PCR, the “Taq DNA Polymerase in Storage Buffer B” by the manufacturer Promega  was used.3′ RACE was performed according to the protocol “5′/3′ RACE Kit, 2Md-fru transcripts. Forward primers: Md-fru-18 (5′ TCCATCATGAGCGAACTCTGT 3′) is located in the female-specific sequences of the P1 transcripts, Md-fru-27 (5′ GTGAAGTGAACTTAGTACATGG 3′) is located in the common sequences of the P1 transcripts; reverse primer: Md-fru-1 (5′ TCTGAGTATCTGACATCCTTC 3′), located in the C1 exon. As an internal control the cytochromatic gene CYP6D3 was amplified with the following primers: CYP6D3-1 (5′ GTTCGGTAATATTTGGCTTGG 3′) and CYP6D3-2 (5′ CCCGTATTCCGTAGTTGAATT 3′). Primer pair Md-fru-18 and Md-fru-1 yields a fragment of 289 bp in females, and primer pair Md-fru-27 and Md-fru-1 generates a 602 bp long PCR product in males.The following primers were used to monitor amplify the sex-specific splice variants of Md-fru in Mdtra RNAi treated XX males, the following primer pair was used for reamplification: Md-fru-2  and Mdfru-28 .When testing the splicing mode of http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used.For multiple sequence alignments the ClustalW algorithm provided by EMBL (GTGAAGTGAACTTAGTACATGG 3′) and Md-fru-29 : A T7 promoter sequence (GTAATACGACTCACTATAGGG) was added to the Md-fru-27 primer for preparing sense probes (control) and to the Md-fru-29 primer for preparing antisense probes. Both amplicons were transcribed with T7 polymerase to generate labelled single stranded RNA probes . Heads were dissected from adult flies, mounted in O.C.T medium and frozen at −20°C prior cryosectioning with a Leica rotary microtome. In situ hybridizations were carried out on adult head sections according to the protocol of Probes were derived from templates generated by PCR with primer Md-fru-27  normal goat serum), followed by the incubation with the primary antibody for 24–36 hours at 4°C. The rabbit anti-serotonin (5-HT) antibody was purchased from SIGMA-ALDRICH (cat # S5545). Dilution of the antibody was 1/400 in PTN. After the incubation of the primary antibody the ganglions were washed four times in PBT for 30 minutes each, followed by the incubation of the secondary antibody at 4°C overnight or at room temperature for 3 to 4 hours. Dilution of the secondary antibody  was 1/600 in PTN. To remove unbound secondary antibody the samples were washed four times in PBT for 1 hour each. Thereafter, the samples were washed several times in PBS for several hours each . Finally, the samples were mounted in VectaShield medium  on glass slides and covered with cover slips. The mounted samples were examined and analysed by confocal laser scanning microscopy (Zeiss LSM 510). The genotypes of all animals were coded before dissections to avoid bias in either preparation or scoring of the samples.Figure S1Amino acid sequence alignments of fru homologs in different dipterans and in Tribolium and Nasonia. (A) Alignments of the male-specific N-terminal extension of fru in M.domestica, D. melanogaster, A. gambiae, N. vitripennis. The putative start codon is labelled in bold. (B) Alignment of the BTB domain of fru homologs from the same species as in (A) with the addition of in silico identified fru sequences of T. castaneum. The BTB domain overall shows a high degree of sequence similarity. (C) Alignment of the zinc-finger A encoding exon. (D) Alignment of the zinc-finger B encoding exon. (E) Alignment of the zinc-finger C encoding exon. (F) Alignment of the zinc-finger D encoding exons. Identical amino acids are indicated with an asterisk (*). Highly similar amino acids are labelled with a colon (:) while amino acids with weak similarity are indicated with a dot (.). Similarity scores between the different domains of Musca and Drosophila fru are: 54% (A), 98% (B), 90% (C), 74% (D), 54% (E) and 54% (F).(TIF)Click here for additional data file.Figure S2Larval testes transplantations. Male man2Md-tra progeny (donor) was generated by crossing bwb+; manMd-tra males with bwb+; manMd-tra/DMd-tra females. Gonads were dissected from these male larvae and transplanted into male bwb, IIIM; +Md-tra larvae (host). These larvae were reared to adulthood and outcrossed with females of the host strain. Three different phenotypes of progeny were expected, bwb males and females (light brown) from host derived sperm and bwb+ females (dark brown) from donor derived sperm. We recovered 7 successfully transplanted males which sired in total 728 individuals with a host genotype (bwb) and 154 individuals with a donor genotype (bwb+).(TIF)Click here for additional data file.Table S1Courtship latency and duration of wild-type males and mutant manMd-tra males.(DOCX)Click here for additional data file.Table S2Frequencies of discrete courtship elements observed in courting wild-type males and manMd-tra males.(DOCX)Click here for additional data file.


The hematopoietic protein Granulocyte-colony stimulating factor (G-CSF) hasneuroprotective and -regenerative properties. The G-CSF receptor isexpressed by motoneurons, and G-CSF protects cultured motoneuronal cellsfrom apoptosis. It therefore appears as an attractive and feasible drugcandidate for the treatment of amyotrophic lateral sclerosis (ALS). Thecurrent pilot study was performed to determine whether treatment with G-CSFin ALS patients is feasible.Ten patients with definite ALS were entered into a double-blind,placebo-controlled, randomized trial. Patients received either 10µg/kg BW G-CSF or placebo subcutaneously for the first 10 days andfrom day 20 to 25 of the study. Clinical outcome was assessed by changes inthe ALS functional rating scale (ALSFRS), a comprehensive neuropsychologicaltest battery, and by examining hand activities of daily living over thecourse of the study (100 days). The total number of adverse events (AE) andtreatment-related AEs, discontinuation due to treatment-related AEs,laboratory parameters including leukocyte, erythrocyte, and platelet count,as well as vital signs were examined as safety endpoints.Furthermore, we explored potential effects of G-CSF on structural cerebralabnormalities on the basis of voxel-wise statistics of Diffusion TensorImaging (DTI), brain volumetry, and voxel-based morphometry.Treatment was well-tolerated. No significant differences were found betweengroups in clinical tests and brain volumetry from baseline to day 100.However, DTI analysis revealed significant reductions of fractionalanisotropy (FA) encompassing diffuse areas of the brain when patients werecompared to controls. On longitudinal analysis, the placebo group showedsignificant greater and more widespread decline in FA than the ALS patientstreated with G-CSF.Subcutaneous G-CSF treatment in ALS patients appears as feasible approach.Although exploratory analysis of clinical data showed no significant effect,DTI measurements suggest that the widespread and progressive microstructuralneural damage in ALS can be modulated by G-CSF treatment. These findings maycarry significant implications for further clinical trials on ALS usinggrowth factors.NCT00298597ClinicalTrials.gov  Amyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerativedisease with progressive loss of motor neurons. It is characterized by motorweakness and muscle wasting finally leading to death due to respiratory failurewithin 2–5 years after diagnosis Recent studies have uncovered the neuroprotective and regenerative properties of thehaematopoietic protein granulocyte-colony stimulating factor (G-CSF) The purpose of this pilot study was to assess whether G-CSF treatment in ALS patientsis feasible, and to explore potential subclinical effects of G-CSF on structuralcerebral abnormalities by using a combination of voxel-based morphometry (VBM) andwhole-brain, voxel-based diffusion tensor imaging (DTI) analysis.Ten patients  withdefinite ALS based on revised El Escorial criteria were recruited for thisdouble-blind, placebo-controlled and randomized study Each patient was physically examined and a questionnaire for the ALS functionalrating scale (ALSFRS) was used to assess disease severity Image data were obtained on a 3.0 T system with a high resolution structuralT1-weighted 3D turbo-field-echo sequence , as well asT2-weighted, and FLAIR imaging. For DTI we employed echo planar imaging (EPI)with 20 diffusion directions .MRI examinations of the ALS patients were performed at Day 0 and Day 100 of thestudy.Diffusion tensor and FA  field maps were calculated fromspatially normalised images. The method was described in detail previously Patterns of cerebral atrophy were assessed using the automated and unbiasedtechnique of VBM. An optimized method of VBM was applied using both customizedtemplates and prior probability maps, implemented using SPM5. The processingsteps were performed as previously described Total brain tissue volumes, normalized for subject head size, were calculatedfrom the high-resolution T1-weighted images, using the well-establishedcross-sectional version of the Structural Imaging Evaluation of NormalizedAtrophy (SIENA) software (SIENAx) Data were testes for normal distribution with the Wilk-Shapiro test. Changes ofALSFRS, JTT and cognitive performance from baseline to Day 30 and 100 wereexamined between groups using a two sample t-test. Fisher's Exact Test wasused to assess differences in the number of AEs between groups. Differences inlaboratory parameters and vital signs were assessed with a two samplet-test.U testas appropriate. All data are given as means ± SD, unless statedotherwise. A two-tailed P value <0.05 was considered significant. Allstatistical analyses were performed using SPSS 16.Single neurocognitive test results were Z-transformed with a mean score of 0 andstandard deviation of 1; mean Z-scores of cognitive domains were then calculatedby taking the mean of the individual Z-scores. For timed tests, the sign of theZ-score was reversed so that improved performance resulted in a higher score inall tests. Differences in neurocognitive test results between groups wereassessed with either two sample t- test or Mann-Whitney http://www.fil.ion.ucl.ac.uk/spm/). We hypothesized that all ALSpatients (at Visit 1) would show damage to white matter tracts reflected asdecreased FA when compared to the 32 controls. This hypothesis was tested usingcross-sectional comparisons, i.e. two-sample t-tests performed on Visit 1 data(pre-medication). Differences between both ALS-groups vs. healthy volunteers aswell as each single ALS-group vs. controls were calculated. Secondly,differences in FA changes between ALS patients treated with G-CSF and theALS-Placebo group in the 100 days following Visit 1 were assessed. This wastested using a factorial design, with VISIT  as the firstfactor, and GROUP , as the second factor. We hypothesizedthat there would be an overall group difference in FA change between thesegroups, manifesting as a significant interaction between GROUP and VISIT. Agewas included as covariate because of the known or potential effects on FA.Additionally, follow-up paired comparisons (t-tests) were used to investigatewithin group longitudinal changes in FA values.Voxel-wise statistical tests were performed on FA and gray matter values usingFactorial ANOVA in SPM5  and longitudinal GM changes within both ALS groups were tested .Differences in total brain volumes between both ALS groups  and to the group of healthy controls were assessed byanalysis of covariance, modeling the factor age as co-variable.The protocol for this trial and supporting CONSORT checklist are available assupporting information; see Five placebo-treated patients and 4 ALS patients treated with G-CSF completed thestudy, including both MRI scans. One patient died of intracranial hemorrhage due toan accidental fall shortly after enrolment and randomization, but before beingtreated with G-CSF. All individuals who received at least one dose of studymedication tolerated the medication well and were able to complete the study.Clinical tests  did not show a significant difference between both groups in change frombaseline to day 30 and day 100 . All cliOn cross-sectional comparison, voxel-based analysis revealed a widespread decline inFA in ALS patients when compared to the healthy controls  compared to the ALSpatients treated with G-CSF. As shown in Post hoc t-test of SPM-ANOVA indicated that FA decreased from Visit 1 to Visit 2mainly bilateral in the subcortical WM of the precentral gyrus in the ALS-G-CSFgroup  was 75% in the G-CSF, 80% in the placebo group (notsignificant); no severe AE that led to discontinuation of the study was reported.72% of AEs in the G-CSF group and 53% in the placebo group wereclassified as possibly or probably related to the treatment. This effect was alsonot significant . Surprisingly,we did detect signals of efficacy using MR imaging: We discovered a reduction instructural disintegration of white matter tracts in ALS patients treated withG-CSF.So far, only data from one randomized controlled trial with G-CSF in ALS patients areavailable The FA changes were most prominent in the corticospinal tracts, in subcortical WM ofthe precentral gyrus, and its connecting fibres in the corpus callosum. Thisneuroanatomical pattern was in considerable accordance to former studies usingvoxel-based DTI analysis in ALS patients and reflects the presence ofmicrostructural damage along motor fibres, which was correlated with the degree ofmotor disability The extra-motor and widespread FA decline in the untreated patient group during thestudy period supported the notion, that ALS is a multisystem disorder. These changesin subcortical regions beyond the limits of the primary motor areas are well-knownand also clinically relevant In previous experimental studies, G-CSF was shown to protect cultured motoneuronsfrom apoptosis, led to a significant improvement in motor performance, and prolongedoverall survival of ALS-mice The commonly used ALSFRS score is a rather insensitive, non-parametric tool tomeasure activities of daily living in ALS patients, which could explain some of thenegative results In conclusion, our results are paving the way for properly powered trials withoptimized regimes and escalated G-CSF dosages, combined with voxel-wise DTI analysisas a sensitive tool to quantify subtle brain tissue alterations.Checklist S1(DOC)Click here for additional data file.Protocol S1(DOC)Click here for additional data file.


Data on the prognostic value of cardiac magnetic resonance imaging (CMR) parameters in patients with ST-elevation myocardial infarction (STEMI) are limited due to analysis of single-center cohorts and small study sample sizes. Aim of the current study was thus to investigate the impact of infarct size, microvascular obstruction (MO) and myocardial salvage index (MSI) on clinical outcome in a large cohort of STEMI patients derived from a multi-center study.STEMI patients (n=795) reperfused by primary percutaneous coronary intervention (PCI) within 12 hours after symptom onset underwent CMR in 8 centers in Germany. CMR was performed at day 1 to 4 after the index event. Infarct size and microvascular obstruction (MO) were measured 15 minutes after gadolinium injection. T2-weighted and contrast-enhanced CMR were used to calculate the MSI. The primary endpoint was defined as a composite of death, non-fatal myocardial reinfarction and congestive heart failure (MACE). Clinical follow-up was conducted after 12 months.Infarct size, MO and MSI were significantly associated with MACE in univariable Cox regression analysis . In multivariable Cox regression analysis including TIMI-risk score, TIMI-flow pre- and post-PCI, ST-segment resolution, left ventricular ejection fraction, as well as infarct size, MO and MSI, infarct size was independently associated with the occurrence of MACE .In this largest multi-center cohort of patients with STEMI undergoing CMR reported so far, infarct size as well as MO and MSI were significantly associated with the occurrence of death, non-fatal myocardial reinfarction and congestive heart failure. Infarct size was identified as an independent predictor for adverse clinical outcome after STEMI even after adjustment for traditional outcome markers.None.


Background. Endothelin-1 (ET-1) is implicated in left ventricular dysfunction after ischaemia-reperfusion. ETA and ETB receptors mediate diverse actions, but it is unknown whether these actions depend on ischaemia type and duration. We investigated the role of ETB receptors after four ischaemia-reperfusion protocols in isolated rat hearts. Methods. Left ventricular haemodynamic variables were measured in the Langendorff-perfused model after 40- and 20-minute regional or global ischaemia, followed by 30-minute reperfusion. Wild-type (n = 39) and ETB-deficient (n = 41) rats were compared. Infarct size was measured using fluorescent microspheres after regional ischaemia-reperfusion. Results. Left ventricular dysfunction was more prominent in ETB-deficient rats, particularly after regional ischaemia. Infarct size was smaller (P = 0.006) in wild-type (31.5 ± 4.4%) than ETB-deficient (45.0 ± 7.3%) rats after 40 minutes of regional ischaemia-reperfusion. Although the recovery of left ventricular function was poorer after 40-minute ischaemia-reperfusion, end-diastolic pressure in ETB-deficient rats was higher after 20 than after 40 minutes of regional ischaemia-reperfusion. Conclusion. ETB receptors exert cytoprotective effects in the rat heart, mainly after regional ischaemia-reperfusion. Longer periods of ischaemia suppress the recovery of left ventricular function after reperfusion, but the role of ETB receptors may be more important during the early phases. Myocardial infarction is the most common clinical manifestation of cardiovascular disease, which remains a leading cause of death worldwide . Prompt Endothelin-1 (ET-1) is a 21-amino-acid peptide, first identified by Yanagisawa and coworkers in 1988 ; in addiPrevious reports have demonstrated elevated circulating ET-1 levels –8, as weBased on the pathophysiologic role of ET-1 during ischaemia-reperfusion, a large number of experimental studies (reviewed in references –14) haveFirst, prolonged ischaemic periods in the Langendorff-perfused rat-heart model result in pronounced and sustained LV dysfunction . No significant differences were observed in maximum +dp/dt and in maximum–dp/dt. Surprisingly, EDP was higher after 20 than after 40 minutes of regional ischaemia; specifically, there was a trend (P = 0.06329) towards higher values at the 10th minute, but significant differences were present at the 20th (P = 0.02227) and 30th (P = 0.00427) minutes of reperfusion.These differences were less pronounced in regional ischaemia protocols and were observed in DP [at the 10th minute  compared to that reported by Yamamoto et al. . Since hWe evaluated the duration of the ischaemic period, by comparing two commonly used protocols –22. In aThe correlation between ischaemia duration (in the ischaemia-reperfusion isolated rat-heart model) and the time to treatment onset (thrombolysis or primary percutaneous coronary intervention) in patients with myocardial infarction is unknown. A recent meta-analysis  indicateThe mechanisms of ventricular cytoprotective effects of ETB receptors after ischaemia-reperfusion remain under investigation. ETB receptors may be involved in the local sequestration and clearance of ET-1, not only under normal, but also during ischaemic conditions . Increasin vivo model of coronary artery ligation in the same ETB-deficient rat strain; in this study, we found markedly higher sympathetic activation in ETB-deficient rats, assessed by fast Fourier analysis of heart rate variability, as well as by measurement of serum epinephrine and norepinephrine levels [Yamamoto et al.  reportede levels . The wele levels  mechanisIn our experiments, the differences in haemodynamic variables between wild-type and ETB-deficient rats were less evident after global ischaemia, providing another potential explanation for the discrepant conclusions of previous studies , 18 repoA paradoxical finding in our experiments was the higher EDP observed in ETB- deficient rats after 20 than after 40 minutes of regional ischaemia. Although difficult to explain, this is in line with our previous observations , indicatThe time course of norepinephrine release from the sympathetic nerve terminals in the ventricular myocardium in response to prolonged ischaemia has been subject of intense research . During First, coronary flow was held constant throughout the experiments, enabling the assessment of postischaemic LV dysfunction without the confounding factor of coronary autoregulation; nonetheless, the addition of protocols using constant perfusion pressure would have permitted the assessment of the effects of ETB receptors on coronary vascular resistance. Second, our study did not address the issue of the pathophysiologic mediators of ETB receptor activation. More specifically, we did not include measurements of norepinephrine levels in the perfusate, that could have expanded previous findings [Third, our protocol did not include the calculation of infarct size after global ischaemia-reperfusion; although measurement of diffuse necrosis is more difficult, its addition in the protocol could have increased the value of our results.We examined the role of ETB receptors using four ischaemia-reperfusion protocols in the isolated Langendorff-perfused rat heart. The evaluation of ischaemia duration and the induction of clinically relevant ischaemia represent major strengths of our work. However, three limitations should be acknowledged. findings , 44. ThiETB receptors exert cytoprotective effects on the ventricular myocardium after ischaemia-reperfusion in the isolated, Langendorff-perfused working rat heart. These effects are more prominent after regional than after global ischaemia (followed by reperfusion). Longer periods of ischaemia suppress the recovery of LV function after reperfusion, but the role of ETB receptors may be more important during the early phases.


Heart failure with preserved ejection fraction accounts for approximately half of all heart failure cases and is associated with similar morbidity and mortality. Although abnormalities of diastolic function are felt to play an important role, no specific treatments have been identified for this common condition, primarily because of poor understanding of its pathophysiology. Despite development of several murine models of this disease, accurate non-invasive assessment of diastolic function has been challenging. Echocardiographic measurements have been limited by small heart sizes, rapid ventricular rates and high inter-observer variability. The aim of this study was to assess the ability of ultra-high field, high temporal resolution CMR tagging to assess diastolic function in mice, compared to the gold-standard technique of invasive pressure-volume loop analysis.High fat diet-induced diabetic mice (n=7) and age-matched control mice (n=4) underwent tissue tagged CMR using a 14.1-Tesla, 89 mm vertical bore magnet with a 600 MHz birdcage resonator. Myocardial tagged images were obtained at the mid-ventricular short axis from end-systole to end-diastole using a spatial modulation of magnetization (SPAMM) sequence Figure . Tagged LV ejection fraction was similar in both groups . However, Ecc rate was significantly reduced in diabetic mice compared with controls , indicating significant relaxation impairment during the rapid filling phase in diabetic mice Figure . PressurThis study provides preliminary validation of ultra-high field, high temporal resolution CMR tagging for non-invasive assessment of diastolic function in mice compared to the gold standard of invasive pressure-volume loop analysis. This technique has potential for assessing the effects of putative therapeutic compounds on mice models of diastolic dysfunction.This work was supported by NIH R01 and T32.


It is well recognized that highly active antiretroviral therapy (HAART) can control HIV/AIDS and prolong patient’s life. However, HAART is associated with drug toxicity, drug resistant, and patient's affordability. Therefore, developing an effective therapeutic vaccine that could induce HIV-specific immune responses may provide a solution to reduce the need of antiretroviral therapy. Adenovirus based vaccines have been extensively evaluated as a vaccine vehicle for HIV/AIDS and a variety infectious diseases, however, it has been a major concern that anti-Ad5 neutralizing antibodies in general population or after the first dose of immunization can hinder the its practical application. The idea of using less prevalent adenovirus serotypes lack of long-term safety record including oncogeneicity. Moreover, any adenovirus serotype will inevitablely induce neutralizing antibodies after one single use which render the strategy of using more serotypes infeasible.In this study, we explored a novel “one-size-fits-all” strategy, namely AVIP , to circumvent the attenuated efficacy of Ad-based vaccines due to anti-Ad immunity.We demonstrated that this AVIP strategy can elicit SIV-specific responses in both Ad5-seropositive and Ad5-seronegative macaques. Interestingly, comparable SIV-specific but weaker Ad5-specific responses were elicited in Ad5-seronegative monkeys received AVIP immunization as compared to direct injection of Ad5-SIV vaccines. Then a cohort of Ad5-seropositive SIVmac239-infected monkeys received AVIP immunization with Ad5 vectors expressing SIVmac239 Gag, Pol and Env with HAART. After therapeutic immunization, SIV-specific response was induced in vaccinated monkeys whereas a slower responses were observed in non-vaccinated monkeys. The functional immunological profiles were also being detected, and we are also monitoring whether this immunological benefit would afford a virological benefit.Our goal is to further develop the AVIP strategy as a simple but practically effective method for repeated delivery of Ad5-based vaccines in humans for HIV and other diseases.


Adenoviral infections associated with respiratory illness in military trainees involve multiple co-infecting species and serotypes. Despite the success of the adenovirus vaccine administered to US military trainees, acute respiratory disease (ARD) surveillance still detected breakthrough infections . To explore the role of adenoviral co-infection (simultaneous infection by multiple pathogenic adenovirus species) in breakthrough disease, we examined specimens from patients with ARD by using 3 methods to detect multiple adenoviral species: a DNA microarray, a polymerase chain reaction (PCR)–enzyme-linked immunosorbent assay, and a multiplex PCR assay. Analysis of 52 samples  collected from 1996 to 2000 showed that all vaccinated samples had co-infections. Most of these co-infections were community-acquired serotypes of species B1 and E. Unvaccinated samples primarily contained only 1 species (species E) associated with adult respiratory illness. This study highlights the rarely reported phenomenon of adenoviral co-infections in a clinically relevant environment suitable for the generation of new recombinational variants. Adenoviruses cause an estimated 8% of clinically relevant viral disease globally . Human aHAdV seems to have found a particularly destructive niche in military training camps. HAdV-B1 serotypes 3, 7, and 21; HAdV-E serotype 4; and HAdV-B2 serotype 14 have caused severe outbreaks of acute respiratory disease (ARD) among military recruits in training centers ,10. BefoTo explore the possibility that unique HAdV strains were causing ARD in vaccinated persons, throat swab samples were selected from the Naval Health Research Center population-based febrile respiratory illness surveillance collection from vaccinated (n = 21) and unvaccinated (n = 31) recruits who reported ARD from 1996 to 2000. Samples were chosen that had tested positive for serotypes 4 or 7 by culture and serotypic antibody neutralization. The gene coding for the primary adenoviral antigen, the hexon coat protein, was sequenced from these isolates. The sequence data suggested that the detectable serotype 4 and 7 strains apparently responsible for breakthrough infection were the same as those circulating in unvaccinated military and civilian populations . In thisSamples were collected as throat swabs into viral transport medium from military recruits with ARD at a variety of training camps as previously described . The thrE1A, hexon, and fiber genes. The primers, degenerate polymerase chain reaction (PCR) amplification protocol, probes, and microarray fabrication techniques have been previously described  uses a PCR–enzyme-linked immunosorbent assay that amplifies a fragment from the adenovirus virus-associated (VA) RNA gene and subsequently detects and types the amplicon with species-specific biotinylated oligonucleotide probes in a colorimetric microwell format . ResultsThe species-specific PCR amplification was performed with previously published primers  and a MuLimiting dilutions of ITCF sample 7151 were plated on A549 cells and allowed to adsorb for 16 hours, after which agarose overlays  were added to each infected monolayer. Well-separated virus plaques were picked 5 days postinfection, placed into viral transport medium, and tested by PCR for HAdV-B and HAdV-C. A second round of plaque purification was performed on several plaque isolates that were treated with 0.05% Triton-X 100 to potentially disrupt virus clumps before their dilution and plating. After 6 hours of adsorption, the original inoculum was removed, and the monolayers were overlayed with agarose solution. The newly formed plaques were tested as described above.By using a new 70-mer spotted microarray , a PCR–eIn contrast, the microarray profile of unvaccinated sample 10756 detected a single serotype, HAdV-4 , panel EPreviously vaccinated persons showed a high rate of co-infection with both species commonly associated with ARD (HAdV-B1 and HAdV-E), whereas unvaccinated persons were primarily infected with HAdV-E. Since HAdV-4 and HAdV-7 are the 2 most common ARD-associated serotypes, that they were also the most commonly paired respiratory pathogenic co-infectants detected in vaccinated persons is not surprising. When the vaccine was used, the rates of other respiratory adenoviruses were much higher than when the vaccine was not used . HoweverThe ability of the microarray to identify to the serotype level resulted in the detection of the greatest number of co-infections, despite its inability to detect members of species B2 when a co-infecting HAdV-7 was present (hybridization pattern interference) and members of species F that were not targeted . MicroarAlthough most apparent co-infections could be verified by each of the primary methods tested and by serotype-specific PCR , some could not be verified . Those co-infectant signals that could not be verified were usually weak positives. The strains responsible for these signals appeared to be subordinate co-infectants because the predominant serotype or species signals generated for the associated samples by the microarray, Adenovirus Consensus kit, and serotype-specific PCR were corroborated in every case and matched the results obtained from the sequencing experiments previously reported .The microarray and Adenovirus Consensus kit use detection and signal amplification techniques that enhance assay sensitivity and thus render them more sensitive than traditional PCR/agarose gel visualization techniques, as shown by the number of triple and quadruple co-infections detected with these techniques . Thus, aTo determine whether >1 replication-competent serotype or strain was present in the samples with evidence of co-infection, we attempted to physically separate the paired co-infectants in sample 7151 by plaque purification. Of 92 plaques picked from the initial plate, all tested positive for HAdV-C by PCR and 12 of 92 also tested positive for HAdV-B. Several of the plaques that retained both HAdV-B and HAdV-C signals were replaqued, and PCR testing of these plaques yielded only HAdV-C isolates. Further efforts that used a detergent to increase separation within the original ITCF sample 7151 and applied the agarose overlay more quickly (6 hours) to prevent interplaque contamination also yielded only HAdV-C plaques (data not shown).We demonstrate the rarely reported phenomenon of co-infections with multiple adenoviral species. Two previous studies have noted rare instances of HAdV-C dual infections in small numbers ,28. HAdVMany of the identified co-infectants in this study were species not generally associated with ARD in the military . Although these species were not likely the cause of ARD observed in these patients, since they are not believed to cause ARD in adults and because they have a high potential for latent carriage ,7,8,29, Recombination can generate new strains with unique and stable phenotypes. Intraspecies adenovirus recombination has been demonstrated in laboratory cell-culture co-infection studies –32. ThesThe HAdV vaccine, an enteric-coated live-virus tablet designed to transiently and selectively infect the gastrointestinal tract with normal respiratory HAdV strains, contains viable HAdV-4 and HAdV-7. Thus, we cannot assume whether the detected co-infectants arose from the vaccine itself or from community acquisition of circulating strains. Most HAdV-4 strains in this study are not the vaccine strain but rather a highly divergent variant that has recently been dominant in military training centers (GenBank strain Z-G 95-873). This identity was shown by sequence analysis of 1,500 bp of the hexon gene from many primary infectants identified in the same sample set that was analyzed here . The varFour lines of evidence support the idea that most of the apparent genetic complexity in the throat swab samples comes from multiple strains, as opposed to recombinants with mixed genetic characteristics. The first comes from the microarray data. The microarray tests for hybridization of 6 independent probes designed to match serotype-specific sequences in 3 genes . Since dThe second line of evidence supporting co-infection with independent genomes comes from comparisons of relative co-infectant titers before and after potentially selective events, such as growth in tissue culture. PCR amplification of fiber gene sequences using species B- and E-specific primers was performed on serial dilutions of vaccinated sample 7274 before and after passage of the original ITCF through 2 additional cycles of growth in A549 cells. In this instance, the relative titers of HAdV-4 and HAdV-7, as measured by serial-dilution PCR, changed by 2 orders of magnitude (data not shown). The rapid drift in relative concentrations of PCR targets from paired co-infecting strains strongly suggests that the co-infectants' genomes are replicating independently and thus likely to be physically separate entities.The third line of evidence supporting co-infectant independence comes from whole-genome sequencing efforts. Several molecular methods indicated that vaccinated sample 7151 harbored an HAdV-5/HAdV-21 co-infection . The gen4 in favor of HAdV-7) and, as expected, yielded only HAdV-7 in >300 plaques tested. The data demonstrated the functional independence of 1 co-infectant (HAdV-7) and physical independence of the co-infecting entities but could not conclusively demonstrate functional independence of the minor co-infectant.The fourth line of evidence comes from our attempts to physically separate paired co-infectants by plaque purification. Sample 7151, which contained the HAdV-5/HAdV-21 co-infection, was used initially because it contained relatively equal titers of both co-infectants. Although most of the plaques tested contained HAdV-5, some contained both HAdV-5 and HAdV-21. Although we were unable to identify plaques that contained only HAdV-21, our results demonstrate the physical independence of the co-infecting entities and the functional independence of HAdV-5. Our results also suggest that either the HAdV-21 co-infectant is functionally dependent on HAdV-5 or is effectively outgrown by HAdV-5 to a degree that prevents independent isolation. Similar attempts were made with a few samples that had HAdV-4/HAdV-7 co-infections, but these were generally biased in titer  in a sample. Secondary infections are masked in these methods by the tests . Likewise, direct sequencing  may restFinally, each of the methods designed to test for multiple species or serotypes showed a higher number of HAdV (and accepted virulent HAdV species and serotypes) in vaccinated persons than in unvaccinated persons. HAdV vaccine was administered routinely to all trainees until supplies were exhausted, at which point adenovirus vaccination was stopped. Since trainees were vaccinated systematically, persons tended to be sampled at times when either all or no recruits were being vaccinated. Therefore, vaccinated samples collected and tested (from 1996 to 1998) are not concurrent with unvaccinated samples (collected from 1998 to 2000). Because of this sampling limitation, we could not confidently correlate HAdV co-infection with breakthrough infections in previously vaccinated persons. Thus, although this study highlights the previously underappreciated phenomenon of adenoviral co-infection, the conclusive examination of its relationship to vaccination must await reintroduction of HAdV vaccine .


We report a case of HER-2-positive recurrent breast cancer showing a clinically complete response to trastuzumab-containing chemotherapy 6 years after primary treatment of triple-negative breast cancer. The primary tumor was negative for HER-2 as determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) , but examination of the recurrent lymph node metastasis showed positivity for HER-2 by FISH . No lesions were detected in either her left breast or in other organs, and the patient was diagnosed as having HER-2-positive recurrent disease. Combination chemotherapy using weekly paclitaxel and trastuzumab was initiated, and a clinically complete response was achieved. This report suggests the benefit of routine evaluation of HER-2 status in recurrent breast cancer with the introduction of HER-2-targeting agents. Recently, several reports have demonstrated that there are substantial discordances in hormone receptor expression and HER-2 status between primary tumors and metastases, which could alter the treatment and prognosis of recurrent breast cancer -5. The d2 and cyclophosphamide 600 mg/m2 every 3 weeks. After the completion of adjuvant chemotherapy, she became postmenopausal and was followed without any treatment.A 49-year-old premenopausal woman had undergone total mastectomy and sentinel lymph node biopsy for stage I right breast cancer in April 2003. The histological diagnosis was invasive ductal carcinoma of the right breast with no metastasis in one sentinel lymph node. Immunohistochemical (IHC) examinations of the tumor cells showed negative results for both estrogen receptor (ER) and progesterone receptor (PgR) and showed weak membrane staining of HER-2 1+ score)   scan confirmed left axillary and infraclavicular lymph node swellings. FDG-PET (2-[18F]Fluoro-2-deoxyglucose positron emission tomography) showed an accumulation of FDG in the left axilla and infraclavicular lymph nodes Figure . The paton ratio .7  in combination with weekly trastuzumab . After 3 courses of administrations, a PET-CT scan showed complete remission of swelling lymph nodes and no accumulation of FDG was detected (Figure She was treated with weekly paclitaxel (80 mg/md Figure . The eleWith the introduction of molecular targeting therapies, such as those utilizing monoclonal antibodies or small-molecule tyrosine kinase inhibitors directed to HER2 signaling and hormonal agents, hormone receptor expression and HER-2 status have become the most important prognostic and predictive factors in breast cancer -10. It iCompared with hormone receptor status, discordance in the HER-2 status between primary and metastatic lesions is less common, and the impact of this discordance on the prognosis of recurrent breast cancer is less clear ,7. HowevHER-2 gene amplification of recurrent disease was detected in samples of fine needle aspirates by HER-2 FISH. In a daily setting, HER-2 FISH is performed on tissue sections according to published guidelines Fluoro-2-deoxyglucose positron emission tomography: FDG-PET; fine needle aspiration: FNA.The authors declare that they have no competing interests.HS, YK, TS and NM tracked the clinical data and drafted the manuscript. TK, KK, TS, AE, RH and TK participated in the design of the study and revised the manuscript. Pathologists MO and KA evaluated the results of the immunohistochemical analysis and fluorescence in situ hybridization. MO conceived of the study, participated in its design and coordination, and revised the manuscript for important intellectual content. All authors read and approved the final manuscript.


Torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor which raises high-density lipoprotein (HDL) cholesterol and reduces low-density lipoprotein (LDL) cholesterol level, has been documented to increase mortality and cardiac events associated with adverse effects. However, it is still unclear the underlying mechanisms of the off-target effects of torcetrapib.in silico between torcetrapib and transmembrane receptors was conducted to identify the potential off-targets. This screening was carried out based on the enriched signaling network analysis.In the present study, we developed a systems biology approach by combining a human reassembled signaling network with the publicly available microarray gene expression data to provide unique insights into the off-target adverse effects for torcetrapib. Cytoscape with three plugins including BisoGenet, NetworkAnalyzer and ClusterONE was utilized to establish a context-specific drug-gene interaction network. The DAVID functional annotation tool was applied for gene ontology (GO) analysis, while pathway enrichment analysis was clustered by ToppFun. Furthermore, potential off-targets of torcetrapib were predicted by a reverse docking approach. In general, 10503 nodes were retrieved from the integrative signaling network and 47660 inter-connected relations were obtained from the BisoGenet plugin. In addition, 388 significantly up-regulated genes were detected by Significance Analysis of Microarray (SAM) in adrenal carcinoma cells treated with torcetrapib. After constructing the human signaling network, the over-expressed microarray genes were mapped to illustrate the context-specific network. Subsequently, three conspicuous gene regulatory networks (GRNs) modules were unearthed, which contributed to the off-target effects of torcetrapib. GO analysis reflected dramatically over-represented biological processes associated with torcetrapib including activation of cell death, apoptosis and regulation of RNA metabolic process. Enriched signaling pathways uncovered that IL-2 Receptor Beta Chain in T cell Activation, Platelet-Derived Growth Factor Receptor (PDGFR) beta signaling pathway, IL2-mediated signaling events, ErbB signaling pathway and signaling events mediated by Hepatocyte Growth Factor Receptor  might play decisive characters in the adverse cardiovascular effects associated with torcetrapib. Finally, a reverse docking algorithm Our study provided unique insights into the biological processes of torcetrapib-associated off-target adverse effects in a systems biology visual angle. In particular, we highlighted the importance of PDGFR, HGFR, IL-2 Receptor and ErbB1tyrosine kinase might be direct off-targets, which were highly related to the unfavorable adverse effects of torcetrapib and worthy of further experimental validation. Cardiovascular disease remains to be the most unexceptional cause of morbidity over the past few years in spite of the usage of hydroxymethylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) that lower low-density lipoprotein (LDL) cholesterol . ElevateTorcetrapib Figure , a CETP With the rapid development of high-throughput screen (HTS) technology such as microarray, the superiority of systems biology and network pharmacology gradually embodied ,8. ReconTo better expound the unfavorable adverse reactions of torcetrapib, a novel network systems approach was proposed by integrating high quality manually curated data with microarray gene expression profiling into a context-specific network, which allowed us to explicate the off-target adverse effects of torcetrapib in a different angle. Detailed illustrations are as follows.Although statins had been well characterized as the best studied contemporary cardiovascular therapies over the past few years, the optimal approach to LDL reduction remained to be controversial. Meanwhile, the prejudice of low levels of HDL cholesterol in cardiovascular system became increasingly prominent, which had a tight consanguinity with myocardial infarction and death from coronary heart disease (CHD). Thus, strategies targeting HDL had been a therapeutic tactic for anti-atherosclerosis. As a novel CETP inhibitor, torcetrapib had been recognized as one of the auspicious foremost candidates for elevating HDL. However, owing to its high risk of mortality, torcetrapib experienced the battle of “Waterloo”, which overshadowed the entire prospect of anti-cholesterol drugs.With the speedy development of bioinformatics, organization of knowledge on drug, disease and target inaugurated a brand-new era in drug target identification and discovery. Network pharmacology comprehended the complexity of biological processes by integrating network biology and poly-pharmacological perspective to create predictive models . Networkhttp://www.hprd.org/) with a manually curated human signaling network acquired from Cui et al. -1H-pyrazolopyrimidin-3-yl}-N-[4- (propan-2-yl)phenyl]benzamide) was downloaded from PDB (PDB code 4HCV). Compound 13 J Figure , a nonrein vitro, which indicated that PDGF could facilitate the formation of atherosclerosis via accelerating the migration and proliferation of plaque.Platelet derived growth factor (PDGF), a 24ku cationic glycoprotein, mainly indwelt in platelet alpha granule, impaired endothelial cell, macrophages, smooth muscle cells, fibroblasts and mesangia cells, which mediated multiple interactions between tissues and endothelial cells through releasing PDGF in an autocrine and paracrine chain amplificated reaction forms ,34. A vaDespite the fact that percutaneous coronary intervention (PCI) was one of the most effective therapeutic approaches for CHD by far, restenosis after stenting was still unavoidable, which affected the long term efficacy. Li and colleagues  discloseOwing to the availability of synthetical PDGF tyrosine kinase inhibitors, it might be conceivable to use crenolanib to exploit the binding pocket region of PDGF protein. Figure Except for cancer, the ErbB family of four receptor tyrosine kinases  also engaged in certain non-neoplastic pathologies, such as hypertension , infectiThe three dimensional structure of ErbB1 tyrosine kinase with compound POX  was downloaded from PDB (code number 3BEL). As shown in Figure As a heparin binding glycoprotein originated from mesenchymal cells, hepatocyte growth factor (HGF) possessed various biological activities including regulating mitosis, morphogenesis, hematopoiesis, myocardial hypertrophy, angiogenesis, fibrosis and tissue regeneration, which were took effect via binding HGF specific receptor kinase (c-Met) . HGF proThe three dimensional crystal structure of HGF receptor and compound 03X oxy]-3-fluorophenyl}-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide) showed that the 6-dimethoxyquinoline and the carbonyl group in 1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide could interact with the hinge region of the active site via directly coalescing with ARG1086 and ASN1171  and revealed that PDGFR, HGFR, IL-2 Receptor and ErbB1tyrosine kinase were direct off-targets for torcetrapib.in silico were profitable for illustrating the relationship between drug and disease related off-targets for interventions. However, due to the false positive connection and noises in the reassembled network, the predictive model in this study was still far more completed. We proposed that our study on the off-target effects of torcetrapib based on network pharmacology will provide beneficial insights for further experimental validations.Taken together, these findings suggested that the network off-target effects prediction methods http://www.ncbi.nlm.nih.gov/geo/) database under the accession number GDS3556 [The microarray gene expression profiling associated with torcetrapib was acquired from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus  docking method through CHARMm force field docking tool . DockingThe authors declare that they have no competing interests.XJL and SJF conceived this study. XJL, SJF, QG and ZYP carried out the data analysis, simulations, drafted the manuscript and analyzed the results. By carried out extensive revisions to the manuscript. All authors read and approved the final manuscript.List of significant over-expressed genes by Significance Analysis of Microarray (SAM).Click here for fileList of raw genes obtained from Human Protein Reference Database (HPRD).Click here for fileList of the core gene regulatory networks (GRNs) up-regulated by torcetrapib.Click here for file