Previous studies showed that cyclooxygenase(COX) was involved in ischemia/reperfusion (I/R) injuries. Parecoxib, a selective inhibitor for COX −2, has been shown to have protective properties in reducing I/R injury in the heart, kidney and brain. The aim of this study was to investigate the effects of parecoxib on hepatic I/R and to explore the underlying mechanisms.Fifty-two Sprague–Dawley rats were randomly divided into three groups: the sham-operation (Sham) group, the hepatic ischemia/reperfusion (I/R) group, and the parecoxib pretreated I/R (I/R + Pare) group. Partial warm ischemia was produced in the left and middle hepatic lobes of Sprague–Dawley rats for 60 min, followed by 6 h of reperfusion. Rats in the I/R + Pare group received parecoxib (10 mg/kg) intraperitoneally twice a day for three consecutive days prior to ischemia. Blood and tissue samples from the groups were collected 6 h after reperfusion, and a survival study was performed.Pretreatment with parecoxib prior to I/R insult significantly reduced I/R-induced elevations of aminotransferases, and significantly improved the histological status of the liver. Parecoxib significantly suppressed inflammatory cascades, as demonstrated by attenuations in TNF-α and IL-6. Parecoxib significantly inhibited iNOS and nitrotyrosine expression after I/R and significantly attenuated I/R-induced apoptosis. The 7-day survival rate was increased by pre-administration of parecoxib.Administration of parecoxib prior to hepatic I/R attenuates hepatic injury through inhibition of inflammatory response and nitrosative stress. Hepatic ischemia/reperfusion (I/R) is an important factor for the morbidity associated with several clinical conditions and interventions including orthotopic liver transplantation, hepatectomy, and shock. In these conditions, reperfusion induces further hepatocellular damage due to the accumulation of inflammatory cells and mediators, reactive oxygen species, and the subsequent biochemical derangements in intracellular homeostasis . HepaticHepatic injuries induced by I/R are the result of complex interactions between various inflammatory mediators, among which, cyclooxygenase (COX)-derived prostanoids such as prostacyclin (PGI) and prostaglandin E (PGE) have been shown to play a critical role , 4. COX This study was approved by the Animal Care Committee of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, and was performed in accordance with the committee’s guidelines for use of experimental animals. Fifty-two adult specific pathogen-free male Sprague–Dawley rats weighing between 250 and 300 g were obtained from the Animal Center of Sun Yat-sen University, Guangzhou, China. Animals were housed in individual cages in a temperature-controlled room with 12 h light–dark cycles. Food was removed 8 h before the surgery, although all animals had free access to water. The rats were randomly divided into three groups: the sham-operation (Sham) group, the hepatic ischemia/reperfusion (I/R) group, and the parecoxib pretreated I/R (I/R + Pare) group.Parecoxib  was diluted in isotonic saline. The parecoxib treatment group was injected with parecoxib (10 mg/kg) intraperitoneally twice a day for three consecutive days prior to ischemia. The rats in sham and I/R groups were injected intraperitoneally with the same volume of isotonic saline at the same time. The parecoxib dosage was determined based on previous studies , 12.On the day of surgery, 1 h after the last injection of parecoxib or saline, all animals were anesthetized with pentobarbital . A 3-cm midline incision was performed and the hilum of the liver was exposed. All structures in the portal triad  to the left and median liver lobes were occluded by a clip in order to produce 70 % hepatic ischemia. The clip was removed after 60 min, and the abdomen closed. Sham-operated animals underwent midline laparotomy only, without hepatic ischemia. Core body temperature was maintained between 35.5-36.5 °C with the aid of a heating pad. Blood and liver samples of 6 rats of each group were collected 6 h after reperfusion and stored at −80 °C prior to use. The remaining rats in the I/R group and the I/R + Pare group (n = 16-18/group) were monitored for 7 days to record survival.Blood samples were centrifuged at 4,000 rpm for 12 min to collect serum, which was then stored at −80 °C before use. Serum levels for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by a serum autoanalyzer . Liver tissue were taken from the median lobe 6 h after reperfusion and stored in 10 % formalin before being fixed in paraffin. Biopsies were then sectioned and stained with hematoxylin-eosin. Liver histologic injury was assessed using a semi-quantitative light microscopy evaluation. The histologic injury score for each sample was expressed as the sum of the individual scores for 6 different parameters: cytoplasmic color fading, vacuolization, nuclear condensation, nuclear fragmentation, nuclear fading, and erythrocyte stasis . Scores For the survival study, the non-ischematized 30 % of the liver was resected at the onset of reperfusion. The animals in the I/R group and the I/R + Pare group were monitored for 7 days to record survival.Total RNA was isolated from the liver tissue samples using the Trizol reagent  as described in the manufacturer's instructions. Quantitative PCR was performed using a Light-Cycler® 480 Real-Time PCR System  and the SYBR Green qPCR Master Mix (2X) . The primer sequences: TNF-α: Forward: 5’-GTCTGTGCCTCAGCCTCTTC-3’; Reverse: 5’-CCCATTTGGGAACTTCTCCT-3’. IL-6: Forward: 5’-GCCCTTCAGGAACAGCTATG-3’; Reverse: 5’-GTCTCCTCTCCGGACTTGTG-3’. Each expression gene was normalized with GAPDH mRNA using a Delta-Delta CT method. The gene activity in the shame group was assigned as 1 as a reference. Serum TNFα and IL-6 levels were determined using an ELISA kit .2O2. Light absorbance was read at 460 nm over a period of 5 minutes. MPO activity  was expressed as units per gram of tissue.100 mg liver tissue was homogenized in 1 ml of KPO4 buffer containing 0.5 % hexadecyltrimethyl-ammonium bromide by sonication and cultivated at 60 °C for 2 h. Samples were centrifuged to collect the supernatant, and then measured for protein concentration in a 96-well plate by adding samples into phosphate buffer containing o-dianisidine hydrochloride and HFluorescence staining was conducted using a commercially available In Situ Cell Death Detection Kit (Roche). The assay was performed according to the manufacturer’s instructions. The nucleus was stained with propidium iodide. Results were expressed as the average number of TUNEL positive cells per 10 microscopic fields.g for 10 min to collect supernatants, and then centrifuged at 10,000× g for an additional 10 min. The supernatants were isolated for western blot analysis. Protein concentration was determined using the BCA protein assay . Equal amounts of protein were separated on an SDS polyacrylamide gel, and then transferred onto a nitrocellulose membrane . Membranes were incubated with primary antibodies against iNOS  or nitrotyrosine, a marker for peroxynitrite (ONOO−) . All protein bands were detected by species specific infrared fluorescent secondary antibodies . The relative amount of each protein was normalized by the ratio to GAPDH and analyzed using Image J .Liver tissue samples were homogenized in lysis buffer  for protein extraction. The homogenates were centrifuged at 850× P <0.05 in two-tailed testing was considered to be statistically significant.SPSS 16.0 was used for the statistical analysis. All data are expressed as a mean ± SE and compared by one-way analysis of variance (ANOVA) and the Student-Newman-Keuls (SNK) test. Survival rates were analyzed by the Kaplan-Meier method using a log-rank test. Hepatocyte damage was assessed by measuring serum AST and ALT levels, which increased by 46- and 28-fold, respectively, 6 h after hepatic I/R compared with the Sham group Fig. . ContrasTNF-α and IL-6 levels in the liver and change of hepatic neutrophil infiltration were measured to ascertain the inflammatory responses after hepatic I/R. At 6 h after reperfusion, hepatic I/R resulted in a 15-fold increase of TNF-α and 18-fold increase of IL-6 mRNA expression in comparison to sham, which was decreased by 46 % and 65 % when parecoxib was administered Fig.  and b. SThe result of TUNEL staining showed that parecoxib decreased the frequency of apoptotic TUNEL positive cells in livers compared with IR group Fig. .Fig. 4EfIn order to determine the effect of parecoxib on nitrosative burden following I/R, hepatic tissue levels of iNOS and nitrotyrosine were evaulated. Compared with the sham group, we observed a significant increase in iNOS and nitrotyrosine protein expression in the control I/R group, which was reduced by administration of parecoxib Fig. .Fig. 5EfP = 0.04).Only 22.2 % of I/R animals survived 7 days after hepatic I/R. Contrastingly, parecoxib significantly improved survival rate to 56.2 % after hepatic I/R , creating peroxynitrite (ONOO−). Peroxynitrite then aggravates the injury through lipid peroxidation, apoptosis, and necrosis by nitration of tyrosine residues on tissue proteins [−), and iNOS are often used as markers of nitrosative stress. In the present study, we observed that parecoxib administration lead to decreased iNOS and nitrotyrosine levels in hepatic I/R, which suggested that parecoxib decreased the nitrosative stress caused by I/R. This result is consistent with the decrease in number of apoptotic cells.Nitrosative stress also has been recognized to contribute to the cellular damage associated with I/R injury . When iNproteins . It has proteins . NitrotyIn summary, the study showed that pretreatment of parecoxib reduced I/R-induced liver injury, inflammatory response, and nitrosative stress, and improved survival rate after hepatic I/R. These results provides evidence that parecoxib, an analgesic widely used perioperatively, could be a protective pharmacological strategy to prevent hepatic I/R injury.


Here, we investigated whether HO-1 induction could attenuate TNF-α-induced hypertension in pregnant rats. In response to TNF-α infusion (100 ng/day i.p.), maternal mean arterial pressure (MAP) increased vs. control animals (104 ± 3 vs. 119 ± 3 mmHg). HO-1 induction had no effect in control animals, but significantly decreased MAP in TNF-α-infused animals (108 ± 2 mmHg). Placental vascular endothelial growth factor (VEGF) was decreased in response to TNF-α infusion (92 ± 4 vs. 76 ± 2 pg/mg). Placental sFlt-1 was increased by TNF-α infusion , which trended to normalization by HO-1 induction (779 ± 98 pg/mg). In contrast, HO-1 induction had no significant effect on placental VEGF in TNF-α-infused animals. Taken together, these data suggest that one of the key mechanisms by which HO exerts cytoprotective actions in the placenta during inflammation due to chronic ischemia is through suppression of sFlt-1. Further work elucidating the bioactive metabolites of HO-1 in innate inflammatory responses to placental ischemia is warranted.Pre-eclampsia is a hypertensive disorder of pregnancy initiated by placental insufficiency and chronic ischemia. In response, several pathways activated in the placenta are responsible for the maternal syndrome, including increased production of the anti-angiogenic protein, sFlt-1, and inflammatory cytokines, especially tumor necrosis factor-alpha (TNF-α). Previous studies have demonstrated that heme oxygenase (HO) induction can block TNF-α pathways  Perhaps the most persistent and pervasive of obstetrical disorders is pre-eclampsia, which occurs in approximately 5% of all births in the United States, is a contributor to up to 15% of all preterm births, and is a leading cause of maternal/fetal morbidity . The claThe identities of these soluble factors have been a major area of investigation, and several have been repeatedly and strongly implicated in both patients and animal models of placental ischemia, including the anti-angiogenic protein, sFlt-1, and circulating autoantibodies to the angiotensin type 1 receptor . One of Several lines of evidence suggest that the detrimental cardiovascular effects of TNF-α elevation could be attenuated by induction of heme oxygenase-1 (HO-1). The predominant function of HO is in the conversion of free heme from heme-containing proteins to biliverdin, which is quickly converted by biliverdin reductase to bilirubin, which can then be conjugated for excretion in the bile. In the process, the biologically-active byproducts, carbon monoxide (CO) and bilirubin, are produced. These in turn are believed to have important roles in blood pressure regulation through anti-inflammatory, antioxidant, and vasorelaxation mechanisms . HO-1 inad libitum. All protocols were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee and followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.Timed pregnant Sprague Dawley rats  were received on gestational day 11, held at a constant 23°C, and put on a 12:12-h light-dark cycle with food and water On gestational day 14, animals in the experimental groups were implanted i.p. with mini-osmotic pumps containing recombinantly-expressed TNF-α  or vehicle alone. TNF-α was received lyophilized and reconstituted in sterile PBS. Mini-pumps  delivered TNF-α at a constant dose of 100 ng/day. Briefly, rats were anesthetized and maintained with 3% isoflurane. A ventral midline incision ∼3 cm in length was made, and pumps were placed i.p. The incision was closed with non-absorbable continuous sutures. Animals were euthanized on gestational day 19 and tissues harvested post-mortem. For HO-1 induction, on gestational day 14, cobalt protoporphyrin IX chloride  was injected i.p. at a dose of 5 mg/kg, which we have previously shown to cause persistent elevation in HO-1 production in the placenta ,b. Each On gestational day 18, animals were anesthetized as above, and implanted with indwelling carotid catheters. The catheter was externalized at the nape of the neck s.c.. The following day, animals were placed in restraint cages and acclimatized. Mean pressure was determined over the course of 30 min via direct pressure transducers .Rats were anesthetized as above. The uterus was externalized thorough a ventral midline incision, and blood was collected by cannulation of the abdominal aorta for isolation of plasma and serum. Records were made of the viable and resorbed pups present in each animal, and individual pups and placentas were weighed and recorded. Representative placental samples from each horn, thoracic aortae, and livers were flash frozen in liquid nitrogen and stored at –80°C for later analysis.Placental protein was prepared from a representative placenta from individual rats. Briefly, the frozen tissue was mechanical ground by mortar and pestle in liquid nitrogen. Tissue fragments were resuspended in radioimmunoassay buffer with protease inhibitor cocktail, phenylmethanesulfonyl fluoride (PMSF), and sodium orthovanadate . Homogenization was performed via automated tissue processor  and the solution was cleared by centrifugation at 12000 × g for 20 min. Resulting protein concentrations were measured by the bicinchoninic acid method . Vascular endothelial growth factor (VEGF) and sFlt-1 were both measured using sandwich ELISA  in duplicate according to manufacturer’s instructions. Measurements of free plasma VEGF were performed with the same kit. Plasma TNF-α levels were measured using a commercial ELISA per manufacturer’s instructions .p < 0.05. All statistical comparisons and graphs were generated with Prism 6 .All date are displayed as means ± SEM. Comparisons between groups were performed by one-way ANOVA with Tukey’s multiple comparisons test at a significance threshold value of n = 5 in each group) in liver and placenta was performed as described previously . Administration of CoPP had no significant effect on the circulating levels of TNF-α in either control (2.8 ± 0.4 pg/mL) or TNF-α-infused rats (8.4 ± 2.3 pg/mL). Taken together, these data demonstrate that TNF-α infusion increases circulating levels of TNF-α, while CoPP administration has no direct effect. The efficacy of CoPP for induction of HO activity was ascertained by examining HO activity in the livers and placentas of all groups. As seen in Figures To determine whether the administered dose of TNF-α was sufficient to exert a biological effect, plasma levels of the protein were determined via sandwich ELISA. Control animals exhibited low circulating levels of TNF-α  in mean arterial pressure (MAP). While administration of the HO-1 inducer CoPP had no effect on control animals (104 ± 3 vs. 105 ± 3 mmHg), it significantly attenuated the rise in blood pressure caused by infusion of TNF-α . These data suggest that induction of HO-1 has no effect on blood pressure under normal conditions in pregnant rats, but blocks the hypertensive effects of TNF-α during pregnancy. In line with other published data in pregnant rodents, there was no effect of HO-1 induction on either fetal or placental mass .To determine the effects of TNF-α infusion and HO-1 induction on the pro-angiogenic protein VEGF and its antagonist sFlt-1, placental levels of both were determined by sandwich ELISA. Interestingly TNF-α infusion resulted in a decrease in placental VEGF  is the induction of inflammatory cytokines—TNF-α in particular.Despite intensive research into the central causes and molecular mechanisms responsible for the maternal pathology of pre-eclampsia, little in the way of new therapeutic approaches has been translated into the clinical setting. It is now widely believed that defects in placental development, specifically failure to adequately remodel the maternal vasculature feeding the placenta, are a key central mechanism in the development of the disorder . In respin vitro , 2012. Bin vitro . Recentlin vitro . In the in vitro . One patElevation of TNF-α has been one of the most consistent findings in both pre-eclamptic patients and experimental forms of placental ischemia-induced pre-eclampsia models . PreviouSeveral previous reports have supported a potential cytoprotective effect of HO-1 in response to TNF-α exposure. Using placental tissue explants, Here, we have attempted to examine directly the effects of HO-1 induction in a TNF-α infusion model of hypertension in rodents during late gestation. As a consequence of TNF-α infusion, circulating levels of the cytokine were elevated approximately 50% compared to controls. These levels were not affected by the induction of HO-1 by CoPP in either control or TNF-α groups. Consistent with previously published reports in pregnant rodent models, infusion of TNF-α resulted in a significant increase in blood pressure of 15 mmHg. Induction of HO by CoPP was measured by enzymatic activity in both liver and placenta, and was found to be elevated in both tissues in both control and TNF-α infused animals at similar levels. HO induction, which had no effect on blood pressure in control animals, returned blood pressure to levels undistinguishable from controls in TNF-α animals. Promisingly, this had no significant detrimental effects on either placental or fetal weight, suggesting that HO-1 induction had no negative effect on placental efficiency. Interestingly, TNF-α infusion was associated with increased placental levels of sFlt-1 and decreased levels of VEGF. In both cases, induction of HO-1 caused trends to normalization of both proteins, though neither reached statistical significance. Together, these data suggest that, among other potential pathways, one of the mechanisms by which HO-1 induction could be attenuating the effects of TNF-α in response to placental ischemia is through modulation of sFlt-1 and/or VEGF.Still unknown is the underlying molecular mechanism by which HO-1 is eliciting this effect. As it has been previously reported that CO suppresses sFlt-1, it is possible that this is the bioactive component at work here . WhetherThe 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.


Inaccurate data in scientific papers can result from honest error or intentional falsification. This study attempted to determine the percentage of published papers that contain inappropriate image duplication, a specific type of inaccurate data. The images from a total of 20,621 papers published in 40 scientific journals from 1995 to 2014 were visually screened. Overall, 3.8% of published papers contained problematic figures, with at least half exhibiting features suggestive of deliberate manipulation. The prevalence of papers with problematic images has risen markedly during the past decade. Additional papers written by authors of papers with problematic images had an increased likelihood of containing problematic images as well. As this analysis focused only on one type of data, it is likely that the actual prevalence of inaccurate data in the published literature is higher. The marked variation in the frequency of problematic images among journals suggests that journal practices, such as prepublication image screening, influence the quality of the scientific literature. Inaccuracies in scientific papers have many causes. Some result from honest mistakes, such as incorrect calculations, use of the wrong reagent, or improper methodology . Others There is also little firm information on temporal trends regarding the prevalence of errors and misconduct. Research errors and misconduct have probably always existed. Even scientific luminaries such as Darwin, Mendel, and Pasteur were accused of manipulating or misreporting their data , 5. HoweConcerns about misconduct have been accompanied by increasing concerns about the reproducibility of the scientific literature. An analysis of 53 landmark papers in oncology reported that only 6 could be reproduced , and othIn this study, we sought to estimate the prevalence of a specific type of inaccurate data that can be readily observed in the published literature, namely, inappropriate image duplication. The results demonstrate that problematic images are disturbingly common in the biomedical literature and may be found in approximately 1 out of every 25 published articles containing photographic image data, in particular Western blotting images.PLoS One) in 2013 and 2014; the other 12,483 (60.5%) papers were published in 39 journals spanning the years 1995 to 2014  “stamping,” in which a defined area was duplicated multiple times within the same image, (ii) “patching,” in which part of an image was obscured by a rectangular area with a different background, and (iii) fluorescence-activated cell sorting (FACS) images sharing conserved regions and other regions in which some data points had been added or removed. Examples of duplicated images with alteration are shown in Problematic images were classified into three major categories: simple duplications, duplications with repositioning, and duplications with alteration.Journal of Cell Biology):Cuts. Abrupt vertical changes in the background signal between adjacent lanes in a blot or gel suggest that the lanes were not next to each other in the original gel. These splices are of potential concern but do not necessarily indicate inaccurate data representation.Beautification. Part of the background of a blot or gel where no band of interest is expected may show signs of patching, perhaps to remove a smudge or stain. This is not considered to represent best practice according to contemporary guidelines for data presentation  but doesTwo additional types of image modification were not scored as problematic, although they may represent questionable research practices by current standards and would not be accepted by certain journals  images are most likely to result from honest errors, in which an author(s) intended to insert two similar images but mistakenly inserted the same image twice. Alternatively, simple duplications may result from misconduct, for example, if an author(s) intentionally recycled a control panel from a different experiment because the actual control was not performed. Category II (duplication with repositioning) and category III  may be somewhat more likely to result from misconduct, as conscious effort would be required for these actions.In our study, a paper was classified as containing an inappropriate duplication when at least one category I, II, or III problem was identified. Papers were classified according to the highest category of duplicated image, e.g., a paper containing both category I and category II images was classified as a category II paper.Among the 782 problematic papers found in this study, 230 (29.4%) contained simple duplications and 356 (45.5%) contained duplicated images with repositioning, while the remaining 196 (25.1%) contained duplicated figures with alteration.To investigate the prevalence of image duplications and alterations over time, we plotted the percentage of papers containing inappropriate image duplication as a function of publication year . The perPLoS One, from which the largest number of papers was screened, 4.3% of the papers were found to contain inappropriately duplicated images, whereas the percentage of papers with image duplication ranged from 0.3%  to 12.4%  among the other journals, with a mean of 4.4% in the last decade. Hence, even though PLoS One was the journal from which the largest set of papers were evaluated in this study, it is not an outlier with regard to inappropriately duplicated images relative to the other journals examined. To assess the possibility that journals with higher impact factors might be better at detecting problematic images and/or authors might be more careful in preparing images for publication in such journals, the relationship between the prevalence of image duplication and journal impact factor was examined (P = 0.019), with the lowest percentage of problematic images found in journals with high impact factors. The prevalence of image duplication in 12 open-access journals was not significantly different from that in 28 non-open-access journals .Substantial variation in the prevalence of papers with image duplication was observed among the 40 journals investigated. In examined . For thiPLoS One containing duplicated images was compared to the country of origin for all papers published by that journal during the same time interval that were included in our search. In cases where the authors of a paper were affiliated with institutions in multiple countries, all countries were taken into account. A majority of the 8,138 screened papers published in PLoS One during the 16-month study period from 2013 to 2014 were affiliated with China (26.2%) and the United States (40.9%) (P < 0.001), while papers from the United States had a lower probability . Other countries with a higher-than-predicted ratio of papers containing image duplication were India (1.93) and Taiwan (1.20), whereas the prevalence rates of image duplication were lower than predicted in papers from the United Kingdom (0.47), Japan (0.26), and Germany (0.34).To determine whether inappropriate image duplication was more frequent in papers from some countries than others, the country of origin for each of the 348 papers from  (40.9%) . HoweverP > 0.1).Errors or misconduct might be predicted to be more frequent in papers with fewer authors, due to reduced scrutiny. The mean number of authors per paper for the 781 papers containing inappropriate image duplication was 7.28. No significant difference between the mean number of authors for papers with versus without image duplication was found (P < 0.001), indicating that other papers by first or last authors of papers with duplicated images have an increased probability of also containing duplicated images.Our data set of 782 problematic papers contained 28 papers  of papers with a common first author. To determine whether authors of papers containing inappropriate image duplication were more likely to have published additional papers containing image duplication, we screened other papers written by the first and last authors of 559 papers  identified during our initial screening. This analysis encompassed 2,425 papers, or a mean of 4.3 additional papers for each primary paper. In 217 cases (38.8%), at least one additional paper containing duplicated images was identified. In total, 269 additional papers containing duplicated images (11.1%) were found among the 2,425 papers in the secondary data set. The percentage of papers with duplicated images in the secondary data set was significantly higher than that of the first data set  to the International Journal of Oncology (12.4%), a difference of more than 40-fold. The differences among journals are important because, as noted above, these findings suggest that journal editorial policies can have a substantial impact on this problem. Alternatively, the variable prevalence of duplication could be partly accounted for by variations in the average number of figures and the number of panels per figure, which is likely to differ per journal but was not determined in our study. The inverse correlation between the prevalence of problematic papers and journal impact factor contrasts with the positive correlation observed for research misconduct resulting in retraction  would not have been detected. Third, since problematic images were detected by visual inspection, the false-negative rate could not be determined; we readily acknowledge that our screen may have missed problematic papers. It should be noted that our findings contrast with a recent small study by Oksvold that examined 120 papers from three different cancer research journals, which reported duplicated images in 24.2% of the papers examined . Many ofThe high prevalence of inaccurate data in the literature should be a finding of tremendous concern to the scientific community, since the literature is the record of scientific output upon which future research progress depends. Papers containing inaccurate data can reduce the efficiency of the scientific enterprise by directing investigators to pursue false leads or construct unsupportable hypotheses. Although our findings are disturbing, they also suggest specific actions that can be taken to improve the literature. Increased awareness of recurring problems with figure preparation, such as control band duplication, can lead to the reform of laboratory procedures to detect and correct such issues prior to manuscript submission. The variation among journals in the prevalence of problematic papers suggests that individual journal practices, such as image screening, can reduce the prevalence of problematic images , 18. ThePLoS One), 8,138 papers published in 2013 and 2014 were included in the study, comprising 39.5% of the data set. From the remaining journals, a mean of 320.1  papers per journal were included. For most of these journals, if more than 50 papers were found in a given year, screening was limited to the first 40 to 50 papers that were shown in the search field. The selected papers spanned the years 1995 to 2014, with most papers published in 2013 and 2014, primarily as a result of the large contribution of papers from PLoS One .A total of 20,621 papers were selected from 40 different scientific journals in the fields of microbiology and immunology, cancer biology, and general biology. These journals were published by 14 organizations  . All papers were initially screened by one of the authors (E.M.B.). If a possible problematic image or set of images was detected, figures were further examined for evidence of image duplication or manipulation by using the Adjust Color tool in Preview software on an Apple iMac computer. No additional special imaging software was used. Supplementary figures were not part of the initial search but were examined in papers in which problems were found in images in the primary manuscript. All figures found by the screening author (E.M.B.) to contain possible duplications were independently reviewed by the two coauthors (F.C.F. and A.C.). Consensus had to be reached among all three authors for a paper to be considered to contain unequivocal evidence of inappropriate figure duplication. Consensus among all three authors was reached in 90.4% of the papers selected during primary screening.The R software package was used to plot and analyze data. The stat_smooth method implemented from the R ggplot2 library was applied to find the best fit for a linear regression model on semi-log-transformed data examining the relationship between the 2013 Thomson Reuters impact factor and percentage of papers with inappropriately duplicated images for the 40 journals included in this study. Pearson’s correlation and Pearson’s chi-square tests with Yates’ continuity correction were performed in basic R. Details on the R code are available in Data Set S1 in the supplemental material.TABLE S1 The 40 journals screened in this study. Table includes publisher, impact factor (Thomson Reuters 2013), number of papers containing the term “western blot” (WB) screened per year, and number of papers with inappropriate image duplication (IDs) found in that year.Table S1, XLSX file, 0.1 MBData Set S1  Data Set S1, DOCX file, 0.1MB


Virus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast. Due to their repetitive structure, VLPs are extensively used for protein engineering and generation of chimeric VLPs with inserted foreign epitopes. Hamster polyomavirus VP1 represents a promising epitope carrier. However, insertion of large sized protein sequences may interfere with its self-assembly competence. The co-expression of polyomavirus capsid protein VP1 with minor capsid protein VP2 or its fusion protein may result in pseudotype VLPs where an intact VP1 protein mediates VLP formation. In the current study, the capacity of VP1 protein to self-assemble to VLPs and interact with the modified VP2 protein has been exploited to generate pseudotype VLPs displaying large-sized antibody molecules.Saccharomyces cerevisiae and purified by density-gradient centrifugation. Formation of VLPs was confirmed by electron microscopy. Two types of pseudotype VLPs were generated harbouring either the single-chain fragment variable (scFv) or Fc-engineered scFv on the VLP surface. The antigen-binding activity of the purified pseudotype VLPs was evaluated by ELISA and virus-neutralization assay on HBV-susceptible primary hepatocytes from Tupaia belangeri. Both types of the pseudotype VLPs were functionally active and showed a potent HBV-neutralizing activity comparable to that of the parental monoclonal antibody. The VP2-fused scFv molecules were incorporated into the VLPs with higher efficiency as compared to the VP2-fused Fc-scFv. However, the pseudotype VLPs with displayed VP2-fused Fc-scFv molecule showed higher antigen-binding activity and HBV-neutralizing capacity that might be explained by a better accessibility of the Fc-engineered scFv of the VLP surface.Polyomavirus-derived pseudotype VLPs harbouring a surface-exposed functionally active neutralizing antibody specific to hepatitis B virus (HBV) surface antigen (HBsAg) have been generated. The pseudotype VLPs consisting of an intact hamster polyomavirus (HaPyV) major capsid protein VP1 and minor capsid protein VP2 fused with the anti-HBsAg molecule were efficiently produced in yeast Polyomavirus-derived pseudotype VLPs harbouring multiple functionally active antibody molecules with virus-neutralizing capability may represent a novel platform for developing therapeutic tools with a potential application for post-exposure or therapeutic treatment of viral infections. INK4A [Viral structural proteins produced by heterologous expression may have the intrinsic ability to spontaneously self-assemble into highly organized particles – virus-like particles (VLPs) – that morphologically resemble viral capsid and represent a promising tool for protein engineering. The simplest VLPs are composed of a single viral structural protein, such as the assembly-competent envelope or capsid protein –4. More INK4A  and the INK4A . The firINK4A  was fuseINK4A . To joinINK4A , 22–24. In the current study, we describe generation of polyomavirus-derived pseudotype VLPs harbouring surface-exposed scFv and scFv-Fc molecules specific to hepatitis B virus surface antigen (HBsAg). To our knowledge, this is the first example of pseudotype VLPs with an anti-viral activity.4S)4-VH was derived from the in-house produced hybridoma HB1 secreting neutralizing MAb against HBsAg (unpublished data). The cDNA sequences encoding VH and VL regions of anti-HBsAg MAb were deposited in GenBank under accession numbers KP972453 and KP972454. Two recombinant proteins harbouring HBsAg-specific antibody molecule were produced in yeast S. cerevisiae strain AH22-214p: one carrying the scFv-Fc molecule and one carrying the scFv without the Fc part  residues. The production of both HaPyV VP1 protein and the modified VP2 protein was induced by adding galactose into yeast growth medium. Anti-HbsAg scFv comprising variable regions of mouse IgG in the orientation of VL- by SDS-PAGE revealed a protein band of approximately 42 kDa that was identified as an intact VP1 protein  and VP1/scFv-VP2 (construct #2) were successfully purified by the method adapted for the purification of VLPs using CsCl density centrifugation. The analysis of purified recombinant proteins by SDS-PAGE revealed the expected homogenous protein bands of 78.4 kDa and 42 kDa in case of construct #1 Fig. , lane 1 As calculated from the SDS-PAGE with subsequent densitometry analysis, the VP1: scFv-Fc-VP2 ratio (w/w) in the VP1/scFv-Fc-VP2 (construct #1) is approximately 13:1, while the ratio the VP1: scFv-VP2 (w/w) in the VP1/scFv-VP2 (construct #2) is approximately 6:1. Based on the molecular weight (MW) of the VP2-fused proteins , the calculated average number of the scFv-Fc-VP2 fused protein per one pseudotype VLP was 14 (construct #1), while the calculated number of the scFv-VP2 fused protein per one pseudotype VLP was 46 (construct #2).The purified recombinant proteins were negatively stained and subjected to electron microscopy (EM) analysis that confirmed an efficient self-assembly of both recombinant proteins to VLPs Fig. . The pse−12 M) as the parental MAb HB1 used as a positive control (starting from 0.7 × 10−12 M). Meanwhile, the scFv-VP2 fused protein (construct #2) was reactive with the plate-bound HBsAg when added at concentrations exceeding 24 × 10−12 M. The VP1/GFP-VP2 pseudotype VLPs used as a negative control did not show any reactivity with the immobilized antigen, which confirms the specificity of the ELISA  [−10 M) of scFv-Fc-VP2 fused protein displayed on the pseudotype VLPs (construct #1) were sufficient to induce complete HBV neutralization while 1 μg/mL (1.9 × 10−10 M) of the scFv-VP2 fused protein displayed on the pseudotype VLPs (construct #2) induced only partial HBV neutralization  of scFv-Fc-VP2 fused protein was comparable to that obtained with 1 μg /mL (0.67 × 10−10 M) of the full-length parental MAb HB1. The specificity of the neutralization test was confirmed by the use of HaPyV-derived VP1/GFP-VP2 pseudotype VLPs (negative control) that did not induce HBV neutralization. The incomplete HBV neutralization by the construct #2 harbouring the scFv without the Fc part might indicate that the scFv molecule is less accessible on the surface of pseudotype VLPs as compared to the Fc-engineered scFv. The results of the HBV-neutralization test are consistent with the results of an indirect ELISA  coated on the microtiter plate and the HaPyV VP1-specific MAb 3D10 as a detection antibody. Both anti-HBsAg fused proteins displayed on the respective pseudotype VLPs were reactive with the immobilized HBsAg Fig. . The scFISA Fig. . Furtheres (PTH) . The neuion Fig. . The obsISA Fig.  where thin vivo studies have demonstrated the efficiency of neutralizing antibodies in established infections of influenza A, HIV, polioviruses, filoviruses and other viral infections [Recombinant VLPs are extensively used for various applications, from basic virus assembly and structure studies to the production of human and animal vaccines –5. Due tfections –30. The fections , 32 may in vitro HBV infection assay as compared to construct #1 harbouring Fc-engineered scFv molecules. The observed difference might be explained by a better accessibility of the Fc-engineered scFvs on the surface of VLPs. The quantity of anti-HBsAg molecules incorporated into the pseudotype VLPs of our study (14 anti-HBsAg Fc-scFv-VP2 molecules per one VLP) is consistent with a previous report where approx. 19 molecules of anti-VLY Fc-scFv-VP2 molecules were found to be displayed the pseudotype VLP [The current study demonstrates that antigen-binding capability of the pseudotype VLPs depends on the structure of the antibody molecule displayed on the VLPs. The anti-HBsAg molecule was presented on the VP2 protein in two different formats – either scFv alone or Fc-engineered scFv comprising CH2 and CH3 domains and the hinge region of human IgG1 . The caltype VLP . Based otype VLP , 13, 14.type VLP , 34 mighThe current study demonstrates that HaPyV-derived pseudotype VLPs are suitable carriers for antibody molecules, both in the format of scFv and Fc-scFv. The pseudotype VLPs harbouring the Fc-scFv-VP2 molecule show higher antigen-binding activity and virus-neutralizing potency as compared to those harbouring the scFv-VP2 molecule. This difference might be explained by a more efficient exposure of the Fc-engineered scFv molecule on the surface of the pseudotype VLPs as compared to the scFv molecule.The produced pseudotype VLPs harbouring surface-exposed anti-HBsAg molecules is the first example of anti-viral VLPs with virus-neutralizing capability.The pseudotype VLPs harbouring multiple functionally active antibody molecules with virus-neutralizing capability represents a novel platform for the construction of therapeutic vaccines with a potential application for post-exposure or therapeutic treatment of viral infections. The VLP approach combining the advantages of virus targeting by the surface-exposed antibody molecule and the capability of VLPs to encapsidate and deliver cargo, such as therapeutic nucleic acids or proteins, might be further exploited for the development of novel efficient anti-viral strategies.6 hybridoma cells (clone HB1) using GeneJET RNA Purification Kit (Thermo Fisher Scientific) according to manufacturer’s recommendations. Hybridoma clone HB1 was produced in-house using spleen cells of BALB/c mouse immunized with recombinant HBsAg (unpublished data). The first strand of cDNA was prepared using RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. The cDNA corresponding to the variable regions of mouse IgG VH and VL was obtained by polymerase chain reaction (PCR) using sets of specific primers described previously [4S)4-VH.All DNA manipulations were carried out according to standard procedures . Enzymeseviously , 36. In E. coli DH10B cells.The anti-HBsAg scFv was displayed on the pseudotype VLPs either alone or fused with the Fc region of human immunoglobulin G1 encoded in pFUSE-hIgG1-Fc2 vector . The resThe plasmid pFGG3-VP1/ VP2-Fc-scFv used for the co-expression of chimeric HaPyV-derived protein scFv-Fc-VP2 and the intact HaPyV VP1 protein (construct #1) were generated as described previously . The conS. cerevisiae strain AH22-214p . Transformed yeast cells were cultivated as described previously [S. cerevisiae transformed with pFGG3 vector without any insert was used as a negative control.The constructed plasmids pFGG3-VP1/VP2-Fc-scFv and pFGG3-VP1/VP2-scFv (Table Recombinant proteins were fractionated by electrophoresis on 12.5 % sodium dodecylsulfate-polyacrylamide gels (SDS-PAGE) and then either visualized by staining with Coomassie brilliant blue . The quantities of loaded recombinant proteins were determined by densitometry using the ImageScanner III  device supplied with the ImageQuantTL software. The determination of the quantities of VP1 and VP2-fused antibodies in the preparations of pseudotype VLPs was based on the comparison with the known quantities of VP1 and BSA proteins run in the same SDS-PAGE gel and scanned by the ImageScanner III. The molar quantities of VP1 and VP2-fused proteins in the samples of purified VP1/scFv-Fc-VP2 and VP1/scFv-VP2 constructs were calculated in accordance with their molecular weight (MW). The average number of VP2-fused proteins presented on one VLP was calculated as described previously  and the Recombinant proteins fractionated by SDS-PAGE were electrotransferred to Immobilon P membrane . The membranes were blocked with 5 % milk powder in phosphate-buffered saline (PBS) for 2 h at room temperature (RT) and rinsed in PBS with 1 % Tween-20 (PBST). For the detection of HaPyV VP1 protein, the membranes were incubated for 1 h at RT with monoclonal antibody (MAb) 3D10 [S. cerevisiae yeast biomass containing recombinant proteins was suspended and homogenized in DB450 buffer  containing 2 mM PMSF, EDTA-free Complete Protease Inhibitor Cocktail (Thermo Fisher Scientific) and mechanically disrupted using French press. After centrifugation of yeast lysate, the supernatant containing recombinant proteins was collected and subjected to the ultracentrifugation on 30–65 % sucrose gradient and subsequently on CsCl density gradient ranging to buoyant densities from 1.23 to 1.38 g/mL as described previously [eviously . The purThe samples of purified recombinant proteins were placed on 400-mesh carbon coated palladium grids, negatively stained with 2 % aqueous uranyl acetate solution and examined by Morgagni 268 electron microscope .Polystyrene microtiter plates  were coated with 100 μL per well of the recombinant HBsAg ayw  diluted in coating buffer  to a concentration of 5 μg/mL and incubated overnight at 4 °C. The coated plates were blocked with 2 % bovine serum albumin (BSA) for 1 h at RT and rinsed twice with PBST. Recombinant pseudotype VLPs were serially diluted in PBST, added to the wells and incubated for 1 h at RT. As a negative control, non-modified HaPyV VP1/VP2 VLPs were used. The plates were rinsed with PBST and incubated for 1 h with the MAb 3D10 diluted 1:1000 in PBST. The plates were rinsed with PBST and then incubated for 1 h with HRP-conjugated rabbit anti-human IgG (Bio-Rad) diluted 1:1000 in PBST. The plates were rinsed 5–7 times with PBST. Enzymatic reaction was visualized by the addition of 100 μL of ready-to-use TMB substrate (Sigma–Aldrich) to each well. After 10 min of incubation at RT, the reaction was stopped by adding 50 μL/well of 10 % sulphuric acid. The optical density (OD) was measured at 450 nm (reference filter 620 nm) in a microplate reader .Tupaia belangeri hepatocyte cultures (PTHs) for 16 h at 37 °C. As a positive control, the full-length MAb HB1 previously shown to exhibit the HBV-neutralizing activity in the in vitro assay was used. As a negative control, irrelevant pseudotype VLPs harbouring green-fluorescent protein (VP1/GFP-VP2) were used. The HBV-neutralization potency of the pseudotype VLPs was evaluated by measuring the concentration of the newly produced HBsAg in the culture supernatant on day 11th after infection. Animals were handled in a full accordance with ethical requirements. Permission to use primary hepatocytes from Tupaia belangeri was obtained from the Institutional Review Board of Justus-Liebig University of Giessen.Neutralization test was performed as described previously . Briefly


TNF-α gene can influence its production. The aim of the present study was to determine the association of functional TNF-α SNPs and its associated parameters related to apoptosis that may influence the rate of HIV-1 disease progression.The natural history of HIV-1 infection and its progression towards AIDS vary considerably among individuals. Host genetic factors may be one of the possible reasons for variable HIV-1 disease progression. Single nucleotide polymorphisms (SNPs) in the promoter region of TNF-α variants  was done using PCR-RFLP. CD4 counts were determined by flow cytometry. Plasma viral load was estimated by COBAS AMPLICOR HIV-1 monitor test. Plasma TNF-α concentration was estimated by Human CBA Th1/Th2 cytokine kit. The lymphocyte mitochondrial membrane potential was measured by JC-1 dye by flow cytometry.Therapy naive, 100 HIV slow progressors (SPs), 100 HIV fast progressors (FPs), 50 HIV exposed but seronegative individuals (ESNs) and 260 healthy controls from same ethnic origin were recruited. Genotyping of TNF-α -238G/A and -863C/A was not significantly different in HIV-1-infected patients when compared to controls, while that of TNF-α -308G/A variant (high TNF-α producer) was significantly higher in FPs compared to SPs . Haplotype analyses also showed that carriers of high TNF-α producing haplotype CAG was significantly more common among FPs compared to SPs . The circulating TNF-α levels in blood also correlated well with genotypes. The lymphocyte mitochondrial membrane potential of FPs having CAG haplotype was significantly low as compared to wild type (CGG) haplotype .Genotype and allele frequency of TNF-α gene associates with enhanced apoptosis of lymphocytes in HIV-1 infected individuals, hence faster progression to AIDS. However, further functional studies are needed to confirm this association and this knowledge may help clinicians to better understand the disease outcome.High producer haplotype, CAG of  Polymorphism of the ‘immune response genes’ including chemokine receptor/ligand system may be one of the possible reasons for this variable rate of disease progression TNF-α gene SNPs, both individually and at the haplotype level, with the rate of HIV disease progression or risk of HIV infection in individuals from North India. In addition, we have attempted to explore whether any of these polymorphisms are associated with parameters related to apoptosis that may influence the rate of decline of CD4 cells.The aim of the present study was to determine the associations, if any, of potentially functional The study was approved by the Institutional Ethics Committee (IEC) of PGIMER, Chandigarh, India and an informed consent was obtained from all the patients and healthy controls before obtaining the blood specimen.Patients in different study groups were recruited from ICTC (Intergraded Counseling and Testing Center) in the Department of Immunopathology, PGIMER, Chandigarh, India. The study was carried out on HIV seropositive cohorts from North India that included: Group-I : therapy naïve individuals who maintained CD4 count of >400 cells/µL for past 3 years or more (slope of decline<-32 cells/µL/Year); Group II : individuals with fast depletion of CD4 count to <200 cells/µL within one year (slope of decline>-200 cells/µL/Year). Group III : individuals who were exposed to HIV but remained seronegative. Inclusion criteria in this group was: the HIV seronegative partners of discordant couples, having unprotected sexual intercourse for past one year with a minimum of 4 to 5 exposures every month. The recruitment was based on a detailed history of each couple by the counselor to ascertain the unprotected exposure for a specified period of time and the HIV negative status based on serology as well as HIV-1 DNA PCR. Group IV : HIV seronegative healthy volunteers from the same geographical region. HIV patients on anti-retroviral therapy (ART) and HIV patients with TB and other chronic co-infections  were excluded from the study.Fresh peripheral blood samples (5 mL) were obtained from antecubital vein in evacuated EDTA vials . DNA extraction was carried out using DNA extraction kit  from the whole blood. The quality and quantity of DNA were checked spectrophotometerically .Absence of pro-viral DNA of HIV-1 was confirmed in each ESN individual using an in-house developed nested-PCR, as described previously CCR5 gene (CCR5Δ32), which confers nearly complete protection from HIV-1 infection in homozygous individuals was ruled out in ESNs by PCR as described previously The most widely known genetic association with 32 bp deletion in the TNF-α gene. For genotyping nested PCR was carried out resulting in amplification of 266 bp product (spanning -372 bp to -106 bp). The reaction mixture (20 µL) comprised of 50–100 ng of template DNA, 10 pM of each primer , 200 µM dNTP mix  and 1 unit of Taq DNA polymerase  along with 2 µL of 10X buffer . The primers 5′-AGG CAA TAG GTT TTC AGG TCC ATG-3′ and TNF-Bg R: 5′–CAC ACT CCC CAT CCT CCC AGA TC-3′. The PCR conditions were same as mentioned above. Four micrograms of nested PCR product (118 bp) was digested at 37°C for 8 hours in a 10 µL reaction volume containing 2.5 units of restriction enzyme NcoI  to detect -308G/A polymorphism BglII  to detect -238G/A polymorphism, and then analyzed on a 15% non-denaturing polyacrylamide gel (PAGE). Restriction fragment length polymorphism (RFLP) analysis for -308G/A SNP resulted in two bands (94 and 24 bp) for GG genotype, three bands  for GA heterozygous and single uncut band (118 bp) corresponding to AA genotype. Similarly RFLP analysis for -238G/A SNP resulted in two bands (95 and 23 bp) for AA genotype, three bands  for GA heterozygous and single uncut band (118 bp) corresponding to GG genotype.This polymorphism consists of a G to A substitution at positions -238 and -308 in the proximal promoter of the TNF-α gene. The subjects were genotyped for this polymorphism by PCR–RFLP as described previously 2, 0.2 mM of each nucleotide , and 1 U of Taq DNA polymerase . PCR conditions included initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 Sec, annealing at 62°C for 1 min and extension at 72°C for 30 Sec, the final extension being at 72°C for 5 min. Four micrograms of amplified product (126 bp) was then digested with BsaAI restriction enzyme  at 37°C for 8 hours, electrophoresed on 15% PAGE at 60 V and stained with ethidium bromide. The 126 bp band corresponded to the C wild-type C allele and a set of 103 bp and 23 bp bands corresponded to the variant A allele.This polymorphism consists of a C to A substitution at position -863 in the proximal promoter of the Plasma TNF-α concentration was estimated by Human CBA Th1/Th2 cytokine kit  according to the manufacturer’s instructions. The lower limit of TNF-α detection using this kit was 2.8 pg/mL. Standard curves generated and plasma TNF-α concentrations were calculated using the BD CBA Software .Absolute CD4 T-lymphocyte counts were estimated in the whole blood by flow cytometry using BD Tritest CD3 FITC/CD4 PE/CD45 PerCP with BD Trucount tubes  and plasma viral load (pVL) was estimated using COBAS Amplicor HIV-1 monitor test, version 1.5  as per the manufacturer’s guidelines.6 cells/mL before staining with JC-1 dye  by incubation at 37°C (5% CO2 humidified atmosphere) for 15 minutes in dark. Stained cells were washed with PBS and acquired immediately on flow cytometer (FACSCanto II). The data was analysed using software FACSDiva Version 6.1.3 . As a positive control for maximum depolarization of mitochondrial membranes, the mitochondrial uncoupling agent protonophore carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)  was used at final concentration of 2 µM. A total of 50,000 events were acquired for each assayed sample. Lymphocytes were gated on forward and side scatter to exclude debris and non-lymphoid cells. JC-1 fluorescence was analysed on FL1 and FL2 channels for the detection of dye monomers (Green) and J-aggregates (Red), respectively. The ratio of red/green fluorescence reflected mitochondrial trans-membrane potential (Δψm). The treatment with the protonophore FCCP resulted in decreased JC-1 fluorescence ratio and served as a positive control for disruption of mitochondrial membrane causing change in membrane potential. The Δψm is expressed as percent median fluorescence intensity (FL2:FL1/FL2 FCCP: FL1 FCCP)×100.Mitochondrial membrane potential (Δψm) was measured flow cytometrically using 5, 5′, 6, 6′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) dye  http://hydra.usc.edu/gxe). Hardy-Weinberg equilibrium (HWE) was examined for each SNP by online HWE calculator . The frequencies of genotypes and alleles were compared by Fisher’s exact test. Fisher’s exact test was also used to analyze association between HIV disease progression and TNF-α variants. Odds ratio (OR) and its 99% confidence interval (C.I.) were calculated to assess the risk conferred by a particular allele and genotype. Haplotype analysis was performed using PHASE (http://www.stat.washington.edu/stephens/software.html) v2.1. Slope of CD4 T cell decline was calculated by linear regression model. Discrete and continuous variables were compared between cases and control subjects using unpaired Student’s t-test, Two way ANOVA and Mann–Whitney test as appropriate. The Bonferroni correction for multiple testing was applied when necessary. A two sided p-value of <0.05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism version 5.0 . Power analysis was performed using Quanto software , 200 HIV-1 seropositive patients and 50 ESNs were genotyped for -863C/A, -308G/A, and 238G/A polymorphisms in the promoter region of CCR5Δ32 mutation (data not shown). For the HIV-1 seropositive individuals included in this study, we had the baseline values of viral load and CD4 count both at the time of registration in the ART clinic as well as at the time of recruitment in this study. At the time of registration the FPs had initially higher median CD4 count than SPs . The HIV plasma mean log viral load was also significantly  higher in FPs (5.4 Log10 RNA copies/mL) as compared to SPs  included in this study were ethnically from the North Indian states. The demographic and clinical profile of individuals in different study groups is summarized in TNF-α gene we found genotype distribution of GG (91%) and GA (9%) in the healthy controls with allele frequency of G allele at 95.38% and A allele at 4.62%, which was considered as wild type distribution . Further comparison between allele frequencies between FPs vs SPs also showed A allele to be significantly over-represented in FPs  (TNF-α -308G/A is shown in The genotype distribution of GG (90%), GA (9%) and AA (1%) was observed in healthy controls with allele frequency for G allele as 92.42% and for A allele 5.58%. No significant difference was found in genotype and allele frequency when compared with overall HIV-1 seropositive group . However = 3.39) . RFLP anTNF-α -863C/A SNP revealed, CC (50%), CA (43.85%) and AA (6.15%) in healthy controls with frequency of C allele as 71.92% and of A allele as 28.08%. No significant difference was observed in the frequency distribution of these alleles among HIV positive groups when compared to HC  was not significantly different from that in SPs (68%). However, the CAG haplotype, which is high producer of TNF-α, was significantly overrepresented in FPs  as compared to HCs and SPs  ] . The frep<0.001) higher in FPs (20.9±1.4 pg/mL) as compared to SPs (7.9±1.1 pg/mL) and ESNs . We further investigated the effect of haplotypes on TNF-α levels within FP group. Individuals having CAG (high producer) haplotype had significantly (p<0.01) higher levels  of TNF-α as compared to carriers of low producer AGG  & wild type CGG haplotype  (p<0.01). Further, in FPs, positive correlation was also observed between plasma TNF-α concentration and plasma viral load .TNF-α levels were measured in the plasma samples of 18 subjects in each group. The plasma TNF-α levels were significantly ( p<0.01) . HoweverTNF-α haplotypes and baseline viral load and CD4 T-cell counts within FP group. High TNF-α producing haplotype CAG within FP group had significantly higher mean log viral load as compared to carriers of wild type haplotype (CGG)  as well as low producer AGG and faster decline of CD4 counts as observed by steeper decline of CD4 slope (mean slope of decline for CD4 count -484 cells/µL/Year vs -464 cells/µL/Year)  (We tried to investigate the distribution of  p<0.01) .p<0.003). Also, Δψm was significantly lower in FP group as compared to SP group . Further, when we compared Δψm of different haplotypes within each group or between groups (p<0.01), indicating higher cell death among high TNF-α producing haplotype carriers as compared to wild type. Similarly in case of SP group also, individuals having CAG haplotype had lower Δψm as compared to carriers of wild type (CGG) .Since, the high producer genotypes and haplotypes combined with high plasma TNF-α concentration were found to be associated with faster decline in CD4 count, we investigated further, whether these genotypic differences could influence the apoptosis of lymphocytes in terms of decrease in mitochondrial membrane potential (Δψm) in HIV infected individuals . In orden groups , we founTNF-α promoter polymorphisms and their association with HIV-1 disease progression or risk of HIV infection, plasma levels of TNF-α and mitochondrial membrane potential in ethnically defined North Indian population. The TNF-α gene is located within the class III region of the major histocompatibility complex (MHC) between HLA-B and DR, and its expression in the host is controlled in a regulated manner at the transcriptional as well as post transcriptional level. Polymorphism in the TNF-α promoter region has been shown to play an important role in the development of autoimmune diseases and several infections like HBV, HCV, leishmaniasis, etc. TNF-α promoter is a G/A transition and is the most studied SNP in this gene. Our data indicate that TNF-α -308 G/A polymorphism is associated with faster progression of HIV disease both at genotype and allele frequency level. Although, when Bonferroni’s corrections are applied for multiple comparisons, the significance level comes at border line only. Haplotype analyses also suggest that the combination of some genetic variants within the TNF-α gene (e.g. CAG haplotype), conforming to higher production of the cytokine was represented in significantly higher number of FPs as compared to SPs, suggesting that the high producers of TNF-α would show faster disease progression. These results do agree with some previous reports, which have shown an association between TNF-α SNPs -238G/A and -308G/A and the risk of HIV-1 infection The present study evaluated the frequency of Furthermore, we observed a significantly lower mean lymphocyte mitochondrial membrane potential  in FPs as compared to SPs and even within group (FPs) carriers of CAG haplotype as compared to carriers of wild type haplotype (CGG), indicating that the individuals having high TNF-α producing genetic background will have faster disease progression due to predisposition to higher rate of cell death.TNF-α -238G/A and -308G/A allele frequencies as reported earlier from North Indian population in studies related to Mycobacterium tuberculosis infection and Type-1 Diabetes TNF-α -308A in the Caucasian population is around 17–22%, which is higher than that observed in the Asian populations (5–6%), which is rare as compared to -308G allele (found in 93%), suggesting large variation in the allele frequencies among different ethnic groups TNF-α alleles or haplotypes in the ESNs and HIV infected groups, indicating no association of these polymorphisms with risk of HIV infection in North Indian cohorts. In contrast to our results, a recent study from Western India (Mumbai) showed a higher frequency of -238GG genotype in exposed uninfected (EU) individuals as compared to those who acquired infection, indicating a possible association with HIV transmission TNF-α gene may positively modulate the risk of HIV infection in an individual TNF-α polymorphism with risk of infection.In the current study, we have found almost similar TNF-α -308A allele with HIV-1-associated dementia and other HIV therapy related complications like Lipodystrophy Syndrome in infected patients Similar observations have been made in relation to different infections, like in case of HBV infection, the presence of -308A and -863C was found to be associated with viral clearance The immuno-regulatory response of the host influences the pathogenesis of HIV-1 infection, triggering monocytes, macrophages, and natural killer cells to produce TNF-α TNF-α promoter gene polymorphism in HIV-1 infection having different clinical forms. Also, we have tried to find association at the haplotype level as haplotype-based approaches are more powerful than single SNP approaches in the setting of multiple susceptibility loci. However, additional multi-centric studies with larger number of patients from similar cohorts are warranted to establish the detailed mechanisms underlying the functional effects of these genetic polymorphisms. Also, HLA typing of these HIV-1 infected patients is required as recently in genome wide association studies (GWAS) it has been shown that the TNF-α SNPs are in linkage disequilibrium with MHC class I gene. It becomes more important in the light of well documented association of some HLA types with the rate of HIV disease progression The current study is novel in terms of our study population as the three highly unique HIV-1 subtype-C infected cohorts from a previously not seriously investigated ethnic population, were studied for the expression of polymorphic alleles that could serve as clinical biomarkers for the progression of HIV-1 infection. To the best of our knowledge this is the first study from North Indian population on


Incidence and prevalence of heart failure (HF) with preserved ejection fraction (HFpEF) is rising, with half of all HF patients having a preserved left ventricular ejection fraction (LVEF). These patients have similar mortality rates as patients with HF and reduced EF (HFrEF). HFpEF is observed predominantly in the elderly with a high burden of comorbidities that may contribute to the disease process itself as well as to noncardiac morbidity and mortality.A unifying, but untested, theory of the pathophysiology of HFpEF suggests that comorbidities lead to a systemic inflammation, which triggers endothelial and microvascular dysfunction. This results in diastolic stiffness as well as concentric LV remodeling and fibrosis. Presence of diastolic and vascular dysfunction out of proportion to comorbidities and the lack of neurohormonal antagonism benefitting HFpEF patients might suggest a different pathophysiology of HFpEF compared to those with HFrEF. Alternatively, greater etiological heterogeneity in the less‐specific definition of HFpEF or LVEF‐fibrosis interactions might explain the lack of apparent benefit to neurohormonal antagonism in HFpEF.Analysis of hypertensive patients with and without HFpEF reveals elevated serum markers of collagen production and turnover.NO‐dependent microvascular dysfunction may be measured using coronary flow reserve (CFR) and possibly index of microcirculatory resistance (IMR) after administration of acetylcholine, whereas NO‐independent microvascular dysfunction may be measured using CFR and IMR after administration of adenosine. Assessment of coronary vascular endothelial function lacks consistency, whereas coronary microvascular function has been only partly assessed in HFpEF. There is therefore an unmet need to determine the presence and severity of endothelial and microvascular dysfunction in patients at risk for, and those with, overt HFpEF. In contrast, vascular endothelial function in peripheral arteries and aortic stiffness has received more attention in HFpEF. Aortic stiffening is greater in HFpEF and vascular endothelial function has been found to be impaired in the digital microvasculature and preserved in large conduit vessels in HFpEF.A recent study defined structural changes in full‐thickness myocardial autopsy specimens from 124 subjects with HFpEF and 104 controls dying of noncardiac causes.In this respect, the study by Kato et al. sheds further light to this important concept. The investigators used phase‐contrast cine‐magnetic resonance imaging (cMRI) of the coronary sinus to assess blood flow at rest and during adenosine triphosphate (ATP) infusion for noninvasive evaluation of CFR in patients with HFpEF, those with hypertensive left ventricular hypertrophy (LVH), and controls.1 mapping by cMRI compared to patients with NO‐dependent endothelial dysfunction? And, most important, does this HFpEF subpopulation experience worse outcome compared to the HFpEF subpopulation with NO‐dependent endothelial dysfunction?Endothelial and microvascular dysfunction undoubtedly play an important role in the diastolic abnormalities in HFpEF. There are several questions that remain unanswered, however, the most important being: (1) How important are myocardial endothelial and microvascular dysfunction in the pathogenesis of HFpEF? and (2) What role does myocardial fibrosis play? Are NO‐dependent endothelial dysfunction and inflammation‐induced fibrosis part of the same continuum of disease in HFpEF or separate entities that independently contribute to abnormalities in diastolic function? What are their prognostic implications alone and in combination in patients with HFpEF? Does comorbidity burden increase the likelihood of abnormal CFR in the absence of HFpEF? Is the severity of this abnormal CFR per certain comorbidity burden amplified by the presence of HFpEF? What are the physiological and clinical consequences of myocardial endothelial and microvascular dysfunction in subjects with HFpEF? Do HFpEF patients with NO‐independent microvascular dysfunction have higher levels of biomarkers for fibrosis and a larger amount of diffuse myocardial fibrosis on extracellular volume fraction and TThere is an unmet need to classify HFpEF patients based on their myocardial vasodilator response as well as characterize their ventricular mechanics, inflammatory and neurohormonal milieu, myocardial substrate, and overall outcomes. To date, no study has evaluated and phenotyped the myocardial substrate in patients with HFpEF and myocardial microvascular dysfunction. Accurate phenotyping may lead to better trial design for future therapeutic advances for treatment of HFpEF. Endothelial and microvascular dysfunction may be early steps in the pathogenesis of HFpEF, and identification of patients with either endothelial or microvascular dysfunction may help identify those without HFpEF at risk for progression to overt HF and other clinical events. Identification and classification of HFpEF based on the presence of endothelial or microvascular dysfunction may identify high‐risk subgroups that may benefit from therapy targeted to the endothelium and/or microvasculature. In this respect, Kato et al. should be congratulated for their important findings that certainly will move this field forward.None.


CXCR4 is a crucial chemokine receptor that plays key roles in primordial germ cell (PGC) homing. To further characterize the CXCR4-mediated migration of PGCs, we screened CXCR4-interacting proteins using yeast two-hybrid screening. We identified reticulon3 (RTN3), a member of the reticulon family, and considered an apoptotic signal transducer, as able to interact directly with CXCR4. Furthermore, we discovered that the mRNA and protein expression levels of CXCR4 could be regulated by RTN3. We also found that RTN3 altered CXCR4 translocation and localization. Moreover, increasing the signaling of either CXCR4b or RTN3 produced similar PGC mislocalization phenotypes in zebrafish. These results suggested that RTN3 modulates PGC migration through interaction with, and regulation of, CXCR4. Directional cell migration is crucial for embryonic development, as well as during early development and adult life. Identification of the molecular cues regulating cell migration is important for understanding the mechanism of cell movement and for developing therapies to treat diseases resulting from aberrant cell movement. An excellent model system for studying directional cell migration is that of primordial germ cells (PGCs). In vertebrates, PGCs actively migrate during embryonic development from the position where they are specified toward the site of the genital ridge, where they will differentiate into gametes with the unique role of transmitting genetic information between generations ,2.in vitro, and selectively influence seminoma migration and metastasis [Studies on mouse and zebrafish germ cells show that during migration, chemokine receptor-4 (CXCR4), a seven-transmembrane G protein-coupled receptor, and its ligand, stromal cell-derived factor-1 (SDF1), are involved in the proliferation, motility, and survival of PGCs, gonocytes, and spermatogonial stem cells ,4. CXCR4tastasis ,9,10. Intastasis , and distastasis .Reticulons (RTNs) are a group of evolutionarily-conserved proteins present in plants, yeast, and vertebrates ,14,15. AIn this study, we identified RTN3 as an important component guiding PGC migration in zebrafish. We showed that RTN3 could directly interact with CXCR4, regulate the expression level of CXCR4 and alter the localization of CXCR4 at the plasma membrane in HEK293 and HeLa cells, suggests a mechanism how RTN3 affects PGC migration in zebrafish.-hCXCR4 was transiently co-transfected into HEK293 cells with pCMV-Flag-hRTN3 or pEGFP-N1. The results of this assay showed that Flag-RTN3, but not the GFP control, formed a stable complex with Myc-CXCR4 and allowed it to be precipitated with an antibody recognizing Myc-CXCR4 but not with a control IgG. Furthermore, the reverse co-IP assay verified that Flag-RTN3 interacted with Myc-CXCR4, but not the GFP control and was recognized by the anti-Flag antibody but not the control IgG  was used to inhibit endogenous RTN3 expression. Small interfering RNAs (siRNAs) for RTN3 were transfected into HEK293 cells to inhibit endogenous RTN3 expression, and the non-silencing control siRNA was not complementary to RTN3 D. SpecifTo investigate the subcellular localization of CXCR4 and RTN3, an immunofluorescence assay was performed in HeLa cells. The eukaryotic expression plasmids pcDNA3.1-Myc-hCXCR4 encoding Myc-tagged human CXCR4 and pCMV-Flag-hRTN3 encoding Flag-tagged human RTN3 were overexpressed in HeLa cells, and the localization patterns were screened using confocal microscopy . When HeLa cells were separately transfected with CXCR4 or RTN3, CXCR4 protein expression was mainly localized on the plasma membrane in HeLa cells, and the predominant subcellular localization of RTN3 was diffusely distributed in the cytoplasm A,B. HoweTo investigate whether overexpressing the zebrafish CXCR4 and RTN3 produces a similar effect, pcDNA3.1-Myc-zCXCR4 and pCMV-Flag-zRTN3, encoding the zebrafish CXCR4b and RTN3 genes, which are homologous to the human genes, were co-transfected into HeLa cells. Similar levels of colocalization and translocation of CXCR4 and RTN3 were observed D, indicaCXCR4b activation is essential for PGC directional migration, and based on the data shown above, we investigated whether RTN3 could impact the biological function of CXCR4 and lead to mislocalized PGCs. For this purpose, we used zebrafish from the kop-EGFP-nos1-3′UTR transgenic fish line, with specific expression of EGFP in the PGCs, to investigate the function of RTN3 in PGC migration. To confirm the specificity of the EGFP labeling of PGCs, fluorescence immunohistochemistry was performed with an antibody against zebrafish Vasa protein, which is a specific gene marker of germ cells. These results showed that the EGFP-labeled cells were PGCs A. Next, To ensure that all PGCs reached their destination, we observed PGC distribution at approximately 30 h post-fertilization (hpf) instead of 24 hpf, when PGCs had already migrated to the gonad in wild-type embryos B. These In this report, we investigated the interaction between RTN3 and CXCR4 as well as the potential function of RTN3 in PGC migration in zebrafish. We demonstrated that CXCR4 directly interacts with the RHD domain of RTN3 and that RTN3 mediates endogenous CXCR4 expression, changes the subcellular localization of CXCR4, and co-localizes with CXCR4. We also observed that increased RTN3 signaling could disturb PGC migration and could increase localization at ectopic positions. This phenotype of PGC migration defects is similar to the effect of aberrantly expressed CXCR4b in zebrafish. Taken together, our data suggest that RTN3 can modulate CXCR4 signaling and mediates PGC movement to the region of the developed gonad.Members of the RTN family were previously considered as candidate modulators that play important roles in apoptotic signal transduction and cell death. The RTN family may represent the only molecules to participate in all three apoptosis signaling pathways, the extrinsic pathway, intrinsic pathway and endoplasmic reticulum (ER)-stress pathway, and RTN proteins have demonstrated a strong tendency for aggregation. RTN3 can form homodimers or heterodimers with RTN4 and can promote or suppress apoptosis . In addiAlmost all RTN family members contain variable amino termini that do not display significant sequence similarity and a highly conserved carboxy-terminus named the RHD. The divergent amino terminus plays specific roles in mammals, showing species and tissue specificity, whereas the RHD may execute more basic cellular functions . Among mCXCR4 plays important roles in immune trafficking and in the migration of cells including neurons, germ cells, cancer cells, and other cell types. Dysregulated expression of CXCR4 directly results in disruptions of stem cell homing and neuronal migration . We inveN-glycosylation, disulfide formation, oligomerization, and proteolysis. However, different MW forms of CXCR4 might perform different biological functions. For instance, Sloane et al. [et al. [Moreover, multiple forms of CXCR4 with different molecular weight (MW) exist in mammalian cells; there are numerous post-translational modifications that can generate this MW heterogeneity, including e et al.  found 11 [et al.  showed tPrevious studies have suggested that the level of CXCR4 signaling is important for proper migration of PGCs and that CXCR4 subcellular localization is also necessary for PGC homing . We examTo confirm the functional significance of RTN3 in guided PGC migration in zebrafish, we evaluated the capacity of PGCs guided by RTN3 to arrive at the gonad. The PGCs of the embryos overexpressing RTN3 were found at random sites throughout the embryos and formed distinctly-dispersed cell clusters rather than the compact clusters found at the gonad in wild-type embryos. Similar results were obtained when CXCR4b was overexpressed in embryos.Our study indicates that RTN3 interacts with CXCR4 directly and regulates CXCR4 expression and localization in HEK293 cells. Furthermore, the expression of particularly high levels of RTN3 results in imprecise cell migration in zebrafish, which indicates that RTN3 participates in the process of PGC homing based on CXCR4b-induced migration in zebrafish.The primary role of RTN3 maybe to contribute to PGC migration. Considering that we did not observe any obvious change in PGC number, the overexpression of RTN3 may not affect PGC survival. One possible mechanism by which RTN3 may regulate PGC migration is by changing the subcellular localization of CXCR4 to affect cell polarity and motility. This hypothesis requires confirmation through further experiments, such as measuring the distance and speed of PGC migration during zebrafish embryonic development using a time-lapse camera ,34. The The open reading frames (ORFs) of hCXCR4 and hRTN3 were separately cloned from HEK293 cell cDNA using the primer pairs hCXCR4-F, hCXCR4-R, hRTN3-F, hRTN3-R, hRTN2-F, and hRTN2-R based on the human CXCR4 (GenBank accession no. BC020968.2), RTN3 (NM_006054.3), and RTN2 (NM_206901.2) sequences. The zCXCR4b and zRTN3 ORFs were cloned from cDNA isolated from zebrafish embryo cDNA using the primer pairs zCXCR4b-F, zCXCR4b-R, zRTN3-F, and zRTN3-R based on the zebrafish CXCR4b (AF201451.1) and RTN3 (NM_201072.1) sequences. Detailed information about the primer pairs is provided in pGBKT7-hCXCR4 was constructed by cloning hCXCR4 into the expression vector pGBKT7 , which encoded the full-length CXCR4 fused to the GAL4 DNA-binding domain for yeast two-hybrid screening. pcDNA3.1-Myc-hCXCR4 and pcDNA3.1-Myc-zCXCR4b were obtained by the respective cloning of human and zebrafish CXCR4b genes into the mammalian expression vector pcDNA3.1-Myc  to express human CXCR4 or zebrafish CXCR4b fused to a N-terminal Myc epitope tag. pCMV-Flag-hRTN3, pCMV-Flag-hRTN2, and pCMV-Flag-zRTN3 were created by the respective cloning of human RTN3, RTN2, and zebrafish RTN3 genes into the mammalian expression vector pCMV-N-Flag  encoding human RTN3, RTN2, or zebrafish RTN3 fused to a N-terminal Flag epitope tag. pCMV-Flag-hRTN3-RHD was constructed using standard PCR-subcloning techniques; subcloned PCR products were amplified from pCMV-Flag-hRTN3 using the primer pairs hRTN3-RHD-F and hRTN3-R and were subsequently inserted into the pCMV-N-Flag vector. pEGFP-N1 empty vector (Clontech) encoding enhanced green fluorescent protein (EGFP), and was used as a control. pGEM-zCXCR4 and pGEM-zRTN3 were constructed by cloning zCXCR4 and zRTN3 into the transcription vector pGEM-4Z .For knockdown of human RTN3 in HEK293 cells, three small interfering RNAs (siRNAs) were designed by Suzhou GenePharma Co., LTD . The specific siRNAs sequence is 5′-CCACUCAGUCCCAUUCCAUTT-3′, 5′-GGAUCUACAAGUCCGUCAUTT-3′, and 5′-CCUUCUAAUUCUUGCUGAATT-3′. Non-silencing control siRNA was designed with a 21-nt random sequence, 5′-UUCUCCGAACGUGUCACGUTT-3′, which does not match within the human genome.2 at 37 °C. The cells were plated in 12-well, six-well, or 10 cm plates, transfected with the expression vectors using Lipofectamine 2000 (Invitrogen) reagent and maintained in the complete medium.HEK293 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) basic supplemented with 10% FBS and antibiotics in 5% COThe AH109 yeast strain transfected with pGBKT7-hCXCR4 was used to screen a human fetal liver cDNA library cloned into the GAL4 activation domain vector . Details of the methods have been described previously .HEK293 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing 25 mM Tris base-HCl (pH 7.5), 150 mM NaCl, 1.0 mM EDTA (pH 8.0), 1.0 mM EGTA (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, protease inhibitor cocktail , and phosphatase inhibitor cocktail (Roche). After removing the insoluble debris by centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatants were subjected to immunoprecipitation with anti-Flag M2 Affinity Gel  or Anti-Myc Mouse Monoclonal Antibody Agarose Resin  overnight at 4 °C. After five washes with RIPA buffer, the bound protein was eluted with either sodium dodecyl sulfate (SDS) sample buffer or elution buffer . Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto nitrocellulose (NC) membranes . The immunoblot analyses were conducted with relative antibodies: mouse monoclonal against Flag (Sigma), Myc , GFP (CWBIO), or GAPDH (CWBIO), goat polyclonal against CXCR4 , and horseradish peroxidase (HRP)-labeled secondary antibodies.Ct−ΔΔ method with GAPDH or β-actin as the reference and plotted as the fold change compared with the control or mock samples.Total RNA was extracted using TRIzol (Invitrogen) reagent. Reverse transcription was performed using the GoScript reverse transcription system (Promega). The cDNA was amplified with specific primers in a LightCycler 480 (Roche) with SYBR Green. The specific primers hRTN3-RT-F and hRTN3-RT-R for human RTN3 were designed using Primer Express 3.0 , and thein vitro using a mMessage Machine Kit . Zebrafish embryos were injected at the one-cell stage with 1 nL per injection. Synthetic mRNA was microinjected at a nominal concentration of 300 pg per embryo (CXCR4b) or 250 pg per embryo (RTN3). For controls, 1 nL of Rhodamine B (Sigma) was injected per embryo.Capped sense RNAs were synthesized from linearized pGEM-zCXCR4 and pGEM-zRTN3 Danio rerio) of the AB genetic background were maintained and raised as previously described [in vivo was a gift from Chen at the Chinese Academy of Sciences. The animal welfare and experimental procedures adhered to the Institutional Guidelines of the Care and Use of Laboratory Animals at China Agricultural University . All efforts were made to minimize suffering.Zebrafish  Triton X-100 in PBS for 10 min, and blocked with 0.5% bovine serum albumin (BSA) in PBS. Zebrafish embryos were collected and fixed with 4% paraformaldehyde for 4 h at room temperature, permeabilized with 1% (v/v) Triton X-100 in PBS for 30 min, and blocked with PBTA (1.5% BSA and 0.3% Tween-20 in PBS) for 1 h. The cells or embryos were then successively stained with anti-Flag or anti-Myc antibodies, fluorescein isothiocyanate (FITC) or Cy3-conjugated secondary antibodies (CWBIO), and 4′,6-diamidino-2-phenylindole . Then, images were captured using a Zeiss microscope . The embryos were oriented in 1.5% low-melting-pointagarose on a confocal dish (coverglass bottom dish) and were visualized on a Zeiss microscope.HeLa cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% . Data are presented as the means ± SEM and were obtained from at least three independent experiments. The mean relative changes in the mRNA levels were compared among treatment groups and control or mock groups using the paired Student’s By screening CXCR4-interacting proteins using a yeast two-hybrid approach, we have identified a new CXCR4-interacting proteins, RTN3, which can regulate CXCR4 expression and translocation. Moreover, increased RTN3 signaling in zebrafish can disrupt normal PGC migration. These results indicate that the level of RTN3 expression may play a role in PGC migration through interaction with CXCR4.


Treatment with 5-LOX inhibitors significantly decreased TNF-α-induced pro-inflammatory mediators including interleukin-6 (IL-6) and monocyte chemo-attractant protein-1 (MCP-1) in human synovial fibroblasts. Knockdown of 5-LOX using shRNA exerted similar inhibitory effects. The abrogation of NF-κB activation was involved in the antagonizing effects of these inhibitors. Furthermore, 5-LOX inhibitor decreased TNF-α-induced up-regulation of serum MCP-1 level and paw edema in mouse model. Our results provide the evidence that the administration of 5-LOX inhibitors is able to ameliorate TNF-α-induced cytokine/chemokine release and paw edema, indicating that 5-LOX inhibitors may be developed for therapeutic treatment of inflammatory arthritis.The lipoxygenase isoform of 5-lipoxygenase (5-LOX) is reported to be overexpressed in human rheumatoid arthritis synovial tissue and involved in the progress of inflammatory arthritis. However, the detailed mechanism of how 5-lipoxygenase regulates the inflammatory response in arthritis synovial tissue is still unclear. The aim of this study was to investigate the involvement of lipoxygenase pathways in TNF-α-induced production of cytokines and chemokines. Human synovial fibroblasts from rheumatoid patients were used in this study. 5-LOX inhibitors and shRNA were used to examine the involvement of 5-LOX in TNF-α-induced cytokines and chemokines expression. The signaling pathways were examined by Western Blotting or immunofluorescence staining. The effect of 5-LOX inhibitor on TNF-α-induced chemokine expression and paw edema was also explored  Rheumatoid arthritis (RA) is a chronic and systemic autoimmune syndrome, which is characterized by massive synovial proliferation and inflammation and leads to the destruction of joint cartilage and bone etal. Arachidonic acid (AA) is a key inflammatory intermediate from the lipid composition. In response to a variety of stimuli, AA is released from membrane phospholipid by phospholipase. Pruzanski etal. The inflammatory synovial fluid in RA patients contains many kinds of cytokines including high levels of leukotrienes etal. The 5-LOX cascades and the role of LTB4 in RA are well documented. 5-LOX is required for the production of leukotrienes  which are reported to be potent broncho-constrictors and proinflammatory mediators PF-4191834, a novel selective 5-LOX inhibitor developed by Pfizer, is found to decrease arthritis-associated pain and inflammation in rat model The study was approved by the Institutional Review Board of Taichung Veterans General Hospital (IRB TCVGH NO: C09248), and informed written consent was obtained from patients. All animal experiments were conducted in accordance with the Guidelines for Animals Research of Agriculture Council, ROC and approved by the Ethical Committee for Animal Research of the National Taiwan University (IACUC: 20120439).Mouse monoclonal antibody for α-tubulin, C23, NF-κB p65 and rabbit polyclonal antibody for IgG, IKKα/β, IκBα, NF-κB p50, NF-κB p65 and goat anti-mouse or anti-rabbit secondary antibody conjugated with horseradish peroxidase were purchased from Santa Cruz Biotechnology . Mouse monoclonal antibody for phosphor-IκBα and rabbit monoclonal antibody for phosphor-IKKα/β were from Cell Signaling Technology . Rabbit anti-5-LOX antibody was from Novus . 5-LOX inhibitors, including MK-886, Nordihydroguaiaretic acid (NDGA); leukotriene B4 and leukotriene B4 receptor antagonist LY29311 were from Cayman Chemical Company . Collagenase and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich . Recombinant human TNF-α and enhanced chemiluminescent HRP substrate (ECL) were from Millipore . We purchased RPMI-1640 medium, trypsin and anti-rabbit secondary antibody conjugated with Alexa Fluor 488 from Invitrogen  and fetal bovine serum (FBS) from Biological Industries . Tri-zol was from MDBIO . MMLV Reverse Transcriptase kit was from Promega . Taqman PCR Master Mix and qPCR probes were from Applied Biosystems/Invitrogen .2 with RPMI-1640, which was supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml fungizone (pH 7.6). Over 90% cultured cells were fibroblasts which were characterized by flow cytometry by using CD90 (Thy-1) antibody Human synovial fibroblasts were isolated by collagenase treatment from synovial tissues obtained from patients with rheumatoid arthritis (RA) undergoing total knee replacement surgeries  Total RNA were extracted from human synovial fibroblasts using a Tri-zol kit. The absorbance was measured in a spectrophotometer, Picodrop  at 260 and 280 nm. RNA was used for RT-PCR by using two-step MMLV RT kit. Gene expression was detected by Real-Time PCR which was executed by using a SYBR Green PCR Master Mix  and an ABI StepOnePlus Real-time PCR system (Applied Biosystems). The cDNA was amplified with gene specific primers as shown below:GAPDH: TGGGTGTGAACCATGAGAAG.Forward primer: GCTAAGCAGTTGGTGGTGC.Reverse primer: IL-6: TCACTGGTCTTTTGGAGTTTGA.Forward primer: AGAGCCCTCAGGCTGGACT.Reverse primer: MCP-1/CCL-2: ATGCAATCAATGCCCCAGTC.Forward primer: TGCAGATTCTTGGGTTGTGG.Reverse primer: −ΔΔCT.Amplification was performed in the following cycling conditions: 50°C for 2 min and 95°C for 10 min and then 40 cycles at 95°C for 15 s followed by 60°C for 1 min. The optimal concentrations of primers and templates used in each reaction were established based on the standard curve created before reaction and corresponding to nearly 100% efficiency of the reaction. The reference household gene used to normalize the amount of mRNA was GAPDH. The fold change in gene expression relative to control was calculated by 2After treatment with TNF-α or test substances, the levels of IL-6 or MCP-1 in the culture medium of human synovial fibroblasts and serum from mice were determined by enzyme-linked immunosorbent assay (ELISA). The conditioned medium or serum from mice was obtained and IL-6 or MCP-1 was detected according to the manufacturer’s protocol . All independent experiments were performed and the absorbance was determined using microplate reader .The 5-LOX-shRNA conjugated on the vector of pLKO.1 with ampicillin-resistant region was purchased from National RNAi Core Facility . 3 µg 5-LOX-shRNA and 6 µl Lipofectamine 2000  were premixed with OPTI-MEM separately for 5 min and then mixed with each other for 25 min and then applied to human synovial fibroblasts. The empty vector of shRNA was used as negative control. After 36 hr, the cells were harvested or the culture medium was replaced with serum-free medium and test substances.After treatment with test substances, synovial fibroblasts were then washed with cold PBS and lysed for 30 min at 4°C with lysis buffer as described previously etal., 2011) overnight and then with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody . The nucleus was counterstained by DAPI and the confocal images were obtained by using excitation wavelength 494 nm and emission wavelength 519 nm, respectively (for Alexa-488)  and the thickness of optical sections was set at 0.8 µm.For immunolabeling studies, synovial fibroblasts were seeded on glass overnight and then treated with test substances. After TNF-α (25 ng/ml) treatment for 30 min with or without inhibitors, 4% paraformaldehyde was used to fix the cells for 15 min. 0.1% triton was used to permeabilize the cells and 4% BSA was used for blocking non-specific binding for 1 hr. Synovial fibroblasts were stained with primary mouse monoclonal antibody against NFκB-p65 (1∶200) (Santa Cruz sc-8008)  in the presence or absence of 5-LOX inhibitor NDGA (10 mg/kg) was injected intravenously from femoral vein of male C57BL/6 mice. Mice were sacrificed 6 hr later and serum was collected to perform ELISA analysis.For the paw swelling experiment, TNF-α (200 ng dissolved in 10 µl PBS) in the presence or absence of NDGA (10 µM) or MK-886 (10 µM) was injected intraplantarly into the right hind paw and the PBS-injected left hind paw was used as control. Paw thickness was measured with digimatic caliper . The change of paw thickness was compared to the paw thickness at 0 hr.p value is less than 0.05.The data given were means ± S.E.M. The significance of difference between the experimental group and control was assessed by one-way analysis of variance (ANOVA) and 2-tailed Student’s t-test. The difference is significant if the In inflammatory arthritis, synovial fibroblasts contribute to synovium inflammation, angiogenesis, matrix degradation by its abilities to produce inflammatory cytokines, matrix degradation enzymes and angiogenic factor To investigate the role of 5-LOX in rheumatoid arthritis, human synovial fibroblasts were pretreated with 5-LOX inhibitors NDGA (5 and 10 µM) and MK-886 (5 µM) for 1 hr and then treated with TNF-α (10 ng/ml) for next 6 hr. mRNA extracted from cell lysates was evaluated by quantitative PCR and the conditioned medium collected was measured for IL-6 protein release by ELISA assay. Real-time PCR analysis showed that TNF-α significantly increased IL-6 mRNA levels and pretreatment with 5-LOX inhibitors NDGA (5 and 10 µM) and MK-886 (5 µM) could inhibit the upregulatory effect of TNF-α by 54.7±6.0%, 91.4±4.3% and 57.3±12.6%, respectively . In addiOne of potential therapy in RA is targeting chemokines and a randomized clinical trial with an anti-CCL2/MCP-1 monoclonal antibody in RA patients has been reported previously Leukotriene B4 (LTB4) is one of 5-Lipoxygenase downstream metabolites and plays an important role in pathogenesis of RA To further examine the role of 5-LOX in TNF-α-induced IL-6 and MCP-1 expression, 5-LOX knockdown was performed by using 5-LOX shRNA and the protein release in the conditioned medium in response to TNF-α administration was measured. Total protein extracts from RASFs transfected with empty vector or 5-LOX shRNA were analyzed by immunoblotting. etal. NF-κB is implicated in the transcriptional regulation of inflammatory response by TNF-α. Bondeson invitro. We then examined the effect of 5-LOX inhibitor in animal model. Male C57BL/6 mice were injected with TNF-α  with or without 5-LOX inhibitor NDGA (10 mg/kg). After 6 hr, mice were sacrificed and serum was collected to perform ELISA analysis. It was found that mice injected with TNF-α significantly increased MCP-1 levels in serum to 4.4±0.6 fold of vehicle-treated mice. However, co-treatment TNF-α with 5-LOX inhibitor NDGA (10 mg/kg) significantly reduced TNF-α-induced MCP-1 protein release in serum by 67.9±4.3%  paws, and combination of COX-2 and 5-LOX inhibitors is able to inhibit the development of CIA 2 and IL-6, and IL-6 can also be elevated by the addition of PGE22 alone does not increase the expression of MCP-1 in RASF Here we found that mice treated with NDGA significantly reduced TNF-α-induced up-regulation of MCP-1 in the serum and TNF-α-induced paw edema, whereas 5-LOX inhibitor could not completely reverse TNF-α-induced expression of IL-6 and MCP-1, indicating that other signals are involved in this action. In addition to LOX, arachidonic acid could also be metabolized by COX-2 to produce the bioactive eicosanoids, which play another pivotal part in the inflammatory arthritis. Developing novel 5-LOX inhibitor or 5-LOX/COX-2 dual inhibitor is a therapeutic strategy to treat inflammatory diseases including arthritis invivo. Our results reveal a novel mechanism in TNF-α-induced inflammation and suggest new therapeutic strategies targeting 5-LOX for the treatment of rheumatoid arthritis.In conclusion, 5-LOX exerts a crucial role in chronic systemic inflammation. We demonstrated that both 5-LOX inhibitors antagonized TNF-α-induced IL-6 and MCP-1 expression via inhibition of NF-κB activation in human RA synovial fibroblasts . MoreoveFigure S1Leukotriene B4 receptor antagonist LY29311 reverses TNF-α-induced IκBα phosphorylation and NF-κB nuclear translocation in human synovial fibroblasts. Human synovial fibroblasts were pre-incubated with leukotriene B4 receptor antagonist LY29311 for 1 hr and then exposed to TNF-α (10 ng/ml) for another 30 min. The whole cell lysate results show that the phosphorylation of IκBα was decreased with the treatment of LY29311 in RASF. Cytosolic and nuclear extracts were separated by NE-PER kit. Note that LY29311 significantly antagonized the nuclear translocation of NF-κB subunits of p65. C23 was used as nucleus marker.(TIF)Click here for additional data file.