Host derived markers on virally infected cells or virions may provide targets for the generation of antiviral agents. Recently, we identified phosphatidylserine (PS) as a host marker of virions and virally-infected cells.Under normal physiological conditions, PS is maintained on the inner leaflet of the plasma membrane facing the cytosol. Following viral infection, activation or pre-apoptotic changes cause PS to become externalized. We have previously shown that bavituximab, a chimeric human-mouse antibody that binds PS complexed with β2-glycoprotein I (β2GP1), protected rodents against lethal Pichinde virus and cytomegalovirus infections.Here, we determined the antiviral activity of a fully human monoclonal antibody, PGN632, that directly binds to PS. Treatment with PGN632 protected 20% of guinea pigs with advanced infections of the hemorrhagic arenavirus, Pichinde, from death. Combining PGN632 with ribavirin improved the antiviral activity of both agents, such that the combination rescued 50% of animals from death.The major mechanisms of action of PGN632 appear to be opsonization of virus and antibody-dependent cellular cytotoxicity of virally-infected cells. PS-targeting agents may have utility in the treatment of viral diseases. Due H and Vκ chains linked to human IgG1 constant domains. It targets a lipid, phosphatidylserine (PS), which is normally intracellular -thymidine for eight hours . Stimulation index was calculated.Antigen-specific immune responses were analyzed using a on assay . Briefly2.14p values are reported. A value was considered significant if p ≤ 0.05.The significance between two means (histograms) was calculated by student’s unpaired t-test. Log-rank analysis for non-normally distributed data  was used to compare the survival of the animals exposed to different treatments, and pairwise comparisons of treatment means were made between groups to determine statistical significance. Chi-square, degrees of freedom, and 2.15Male Hartley guinea pigs , aged 4-6 weeks, and approximately 300-350g in weight, were used for all animal studies. The animals were maintained in a specific pathogen free (SPF) BSL3 facility, and fed a normal diet of pig chow and water (unrestricted access to both). Prior to each animal experiment, guinea pigs were randomized and grouped, weighed, and sub-cutaneously implanted with biometric transponder chips that recorded identification number and temperature . Infected and/or antibody-treated animals were monitored twice daily for the entire duration of the experiment. Animals were euthanatized when their body weight decreased by 20% of their total body weight, or when they were scored as ‘severe’ based upon appearance, clinical signs, or unprovoked behavior in accordance with UT Southwestern Institutional Animal Care and Use Committee guidelines.33.11). Binding was not increased in the presence of human β2GP1 (not shown). Binding was also not due to carryover of trace amounts of contaminating β2GP1, which has confounded other studies [1). PGN632 did not recognize any of the neutral phospholipids, sphingomyelin (SM), phosphatidylcholine (PC), or phosphatidylethanolamine (PE).PGN632 bound directly to PS-coated microplates. 50% maximal binding was observed with PGN632 at a concentration of 0.2 µg/ml (1.3nM) .Immunofluorescence microscopy showed that PGN632 bound specifically to cells infected with Pichinde virus. Uninfected Vero cells were not visibly stained. After infection, the cells developed the same punctate staining pattern that we have previously observed with bavituximab on virally infected cells . By 24-43.33a). To confirm that the PGN632 could bind to live infectious virions, bead depletion assays were performed in which magnetic beads coated with PGN632 were used to pull down infectious virus, and the number of infectious virus particles remaining in suspension were quantified by plaque assay. PGN632-coated beads removed 80% of the infectious virons, the same as for bavituximab-coated beads . Erbituximab-coated beads did not remove the virions, showing that the depletion caused by the PGN632- and bavituximab-coated beads was antigen specific. A further control compared the depletion with that of beads coated with polyclonal guinea pig IgG purified from guinea pigs that had been immunized with Pichinde virus. The polyclonal anti-Pichinde virus-coated beads removed the same amount of virus from the suspensions as did the PGN632-coated beads . Thus, PGN632 is capable of binding to PS on the external surface of the Pichinde virus envelope, at levels similar to those seen with bavituximab.PGN632 bound specifically to Pichinde virions adsorbed onto ELISA plates. The binding of the PGN632 was antigen-specific, since the erbituximab control antibody did not bind to the virions. PGN632 and bavituximab showed the same ability to bind to the immobilized virions .Uninfected guinea pigs treated with PGN632  gained body weight at the same rate as untreated animals. Their physical activity and appearance were indistinguishable from untreated animals. Because PGN632 binds to PS, it has the potential to be anti-coagulating through interference with coagulation factor binding to PS surfaces. We therefore measured PT and aPTT of blood drawn from uninfected guinea pigs 6 hours and 24 hours after treatment with PGN632 . Neither PT nor aPTT in PGN632-treated animals was prolonged relative to erbituximab-treated or bavituximab-treated animals.  of Pichinde virus, and treatment was begun when they showed signs of advanced infection . When administered as a single agent, PGN632 protected 20% of guinea pigs from dying from their infections, the same percentage as for bavituximab . While notable, the difference in survival between groups treated with anti-PS antibody and the PBS control animals was not significant (p = 0.1189). The guinea pigs started to regain body weight and gradually returned to having normal physical signs over the following 10-12 days. As expected, animals treated with erbituximab were not protected from challenge and did not survive past day 15.Guinea pigs were infected with 104b). This sub-optimal dose of ribavirin offered no protection by itself. However, when combined with PGN632, 50% of guinea pigs recovered from their infections and survived, as compared to 20% in the group treated with PGN632 alone . This difference trended towards, but did not reach, statistical significance (P=0.15). However, the difference in survival between the group treated with PGN632 and ribavirin, and the group treated with ribavirin alone was statistically-significant (p ≤ 0.0055). Thus, PGN632 appears to have additive anti-viral activity when combined with ribavirin.We next determined if PGN632 offered better protection when given in combination with ribavirin, the primary drug for treating Lassa virus infection. Animals with advanced disease (as above) were treated with PGN632 three times a week in combination with daily ribavirin  . In contrast, in the group of animals sacrificed on day 14, after 3 treatments, there was a 500-fold reduction in virus load in the blood, and a 10-fold reduction in virus load in the liver, lung, kidney, and heart relative to erbituximab-treated animals (P<0.0001). Bavituximab also reduced the virus load in blood and tissues, but to a somewhat lesser extent than did PGN632 . Virus load in the spleen was not reduced by either PGN632 or bavituximab treatment, probably because the spleen is a major site of clearance of opsonized virus from the blood.Guinea pigs were treated with PGN632 on day 4 after infection, when they showed signs of advanced disease, and were treated again on day 7. Animals were sacrificed ten days post-infection, and the virus load in blood and various tissues was determined. The virus load in the blood was reduced by 50-fold relative to the group of animals that received erbituximab. However, there were no significant reductions of viral load in the liver, lungs, kidneys, or hearts of animals treated with PGN632 as compared to erbituximab recipients and the virus load in the spleen was elevated. Bavituximab recipients also showed no significant reductions in virus load in these tissues . Bavituximab also reduced virus yield by 3-fold. Thus, neither PGN632 nor bavituximab are neutralizing antibodies.We examined the mechanisms by which PGN632 rescued animals dying from Pichinde virus infections. First, we determined whether PGN632 was able to induce direct neutralization of Pichinde virus. Vero cells were infected with Pichinde virus (MOI = 4) in the presence of PGN632, bavituximab or erbituximab. PGN632 reduced the yield of virus only slightly (3-fold) and not significantly  Thus, induction of humoral or cellular immunity does not appear to explain the survival of PGN632-treated animals.Next, virus-specific humoral immune responses to Pichinde virus were assessed in guinea pigs that had been infected with virus 14 days earlier, and then treated with PGN632, bavituximab or erbituximab as above. The day 14 time point was selected because this is the time at which differences in survival became apparent. There were no significant differences in levels of anti-Pichinde virus antibodies in the PGN632 or bavituximab-treated animals compared to erbituximab treated animals (results not shown). Similarly, splenocytes taken from guinea pigs that had been sacrificed 14 days after infection and treated with PGN632 or bavituximab showed no significant proliferative responses to Pichinde antigens compared with splenocytes from erbituximab-treated guinea pigs . Thus, opsonization of virus may contribute to the better survival of PGN632-treated guinea pigs.We determined whether antiviral activity could be due to opsonization of virus by PGN632 and subsequent clearance by the reticuloendothelial system. Guinea pigs were infected with Pichinde virus and treated i.p. with PGN632, bavituximab or erbituximab (6 mg/kg). Treatment with either PGN632 or bavituximab reduced the levels of circulating virus in the blood by 10 to 15-fold (P<0.01) relative to animals treated with erbituximab . PGN632 and bavituximab were equally effective at mediating ADCC  Thus, ADCC by innate immune cells likely contributed to the anti-viral action of PGN632.Lastly, we determined whether the antiviral activity of PGN632 could be due to ADCC of virus-infected cells. PGN632 and bavituximab efficiently mediated lysis of infected primary guinea pig fibroblasts by primary guinea pig macrophages 4in vitro [The use of antibodies as novel therapeutics against infectious diseases has gained traction in recent years as an effective way to combat invasive pathogens. Synagis  is a humanized monoclonal antibody that has been approved for the treatment of respiratory syncytial virus . Numerouin vitro . During 2+ that activate PS exporters while simultaneously inhibiting the major PS importers, the aminophospholipid translocases [We recently identified PS as a generic marker of virus-infected cells and enveloped virions . In uninslocases , 15. Altslocases . Importaslocases , cytomegslocases , 17, infslocases , HIV-1 [slocases , vaccinislocases , and HSVslocases . Envelopslocases . Virusesslocases .The PGN632 antibody has very similar phospholipid specificity to bavituximab, in that both antibodies recognize anionic phospholipids, including PS. Both anti-PS antibodies produced indistinguishable staining patterns on Pichinde virus-infected cells. PS is likely to be the major anionic phospholipid to become exposed on virus-infected cells since it is the most abundant, and it is the lipid whose asymmetry is most tightly regulated. However, it is possible that there is also exposure of the more-minor anionic phospholipids, including PI, PA and PG, which would also be detected by the antibodies. The major differences between PGN632 and bavituximab are: 1) PGN632 is human whereas bavituximab is chimeric and theoretically could induce antibodies against mouse determinants in patients and; 2) PGN632 binds to PS directly, without the need for the β2GP1 cofactor protein, which avoids possible depletion of β2GP1 in patients.6 pfu/ml generally survive [3-108 TCID50/ml [PGN632 treatment of guinea pigs with advanced Pichinde virus infections protected 20% of animals from death and reduced virus load in tissues. When combined with low-dose ribavirin, 50% of animals were protected, which would be expected for drugs with non-overlapping mechanisms of protection. We previously reported that 50% of guinea pigs could be protected from death by treatment with bavituximab alone, a higher percentage than has observed in the present studies. It is possible that these differences relate to the much higher virus inoculum used to infect the animals in the present study. It could also relate to the outbred nature of the guinea pig and genetic non-identity in the two studies. We chose to use the i.p. route for administration of the PGN632 antibody, as we had previously used this same route in our initial studies with bavituximab as an antiviral agent . It is p survive . Indeed,CID50/ml . In the CID50/ml . Combinain vitro. The mechanism was that PGN632 induced β-chemokine production which blocked the R5 coreceptor [Two mechanisms appear to explain the antiviral activity of PGN632: (1) opsonization of virus by the antibody; and (2) the induction of antibody dependent cellular cytotoxicity (ADCC) of virally-infected cells . PGN632 receptor . Viral Preceptor . Thus, iIn conclusion, using antibodies to target exposed PS on virus-infected cells and virions shows promise as an antiviral strategy. Because the antibodies act by different mechanisms, they should be used in combination with conventional antiviral agents. Other strategies for exploiting the exposure of PS on virus-infected cells and/or virus envelopes can be envisioned: antibodies, peptides or small molecules that target exposed anionic phospholipids might be used to deliver or direct agents that interfere with host cell or viral metabolism, or recruit immune effector cells.


To elucidate the mechanisms driving metastasis, we characterized the gene expression differences between LuM cells and parent KYSE150 cells. IL-6, IL-1β, and LCN2, previously associated with tumor growth and metastasis, were up-regulated in LuM cells. Recent studies on cancer have increasingly focused on the tumor microenvironment, from which these cytokines are released. The fact that these three cytokines  were up-regulated in LuM cells indicates that these highly metastatic cells obtained through in vivo selection will be a useful resource for further studies on elucidating the mechanisms underlying the tumor microenvironment which is associated with cytokine-related tumor growth and metastasis. Moreover, LuM cells could disseminate to the lung in shorter period of time in vivo, indicating their utility for in vivo experiments of metastasis and new therapeutic targets in a shorter period of time than currently possible.Esophageal cancer is the eighth most common cancer and the sixth most common cause of cancer-related deaths worldwide. Despite the research progress in understanding the disease, the mechanism underlying the metastasis is still unclear. Here, we successfully generated a highly metastatic cell subline, designated as KYSE150-LuM, derived from an esophageal squamous cell carcinoma cell line (KYSE150) by  ADH1B, ALDH2 [YAP [Esophageal cancer is the eighth most common cancer and the sixth most common cause of cancer-related deaths worldwide . There aB, ALDH2 , and YAPDH2 [YAP  have beeDH2 [YAP . Rapid pin vivo selection [in vivo selection could reflect the ability of tumor cells to survive in the circulation and grow in a distant organ. Therefore, the characterization of highly metastatic variants that were selected by in vivo selection may improve our understanding of the mechanisms driving cancer metastasis.The process of metastasis consists of multiple and sequential steps, including proliferation, angiogenesis, motility, and invasion . The outelection , which ielection , 9. We cin vivo selection. To help elucidate the mechanisms driving metastasis, we characterized the gene expression differences between the highly metastatic cells and parent cells. We particularly focused on the expression and secretion of cytokines known to be involved in tumor development and metastasis between LuM cells and parent KYSE150 cells. This work is expected to provide a new resource for more detailed studies on metastasis in ESCC, facilitating the development of new therapeutic targets for pre-clinical and clinical trials.In the present study, we selected a highly metastatic ESCC subline, designated as KYSE150-LuM , derived from parent ESCC cell line, KYSE150 , by in vin vivo selection [To identify candidate metastasis-related genes in ESCC, we first established the highly metastatic cell subline by election . LuciferTo confirm whether LuM cells had increased metastatic ability, we injected the selected cells into the tail veins of the mice. LuM cells disseminated to the lung approximately 30 days earlier than observed for parent KYSE150 cells, which usually take approximately 90 days to disseminate to the lung Figure . Moreovein vitro experiments. Although the metastatic ability was clearly higher in LuM cells than parent KYSE150 cells in vivo, cell growth was slower in LuM cells in vitro  . TherefoTo examine the specific cytokines associated with LuM cells, the differential expression levels of a subset of the up-regulated genes were evaluated with qRT-PCR, which confirmed increased expression levels of several of the genes, including cytokines, in LuM cells Figure . CytokinFurthermore, the cytokines IL-6 and IL-1β, previously associated with tumor growth and metastasis –16, wereTaken together, these results suggest that the highly metastatic ability of LuM cells may be explained by the interaction of these cytokines.in vivo selection. The selection of highly metastatic populations points to substantial tumor heterogeneity [in vivo experiments of metastasis and new therapeutic targets in a shorter period of time than currently possible.In the present study, we successfully generated a highly metastatic cell line, designated as LuM, derived from parent ESCC cell line, KYSE150 , by in vogeneity . LuM celThe characteristics of these highly metastatic cells suggest the involvement of several cytokines in metastasis. The up-regulated cytokines are mainly associated with inflammation. Indeed, the constitutive activation of inflammatory pathways is known to induce the development of esophageal cancer . Inflammin vitro and in ESCC patients [In particular, the IL-6/signal transducer and activator of transcription 3 (STAT3) pathway and IL-1β are well known to be involved in inflammation-related pathways that play a key role in carcinogenesis , 15, 17.patients , 18, 19.patients . Normallpatients . Moreovepatients . In addipatients , is up-rpatients .in vivo selection will be a useful resource for further studies on elucidating the mechanisms underlying cytokine-related tumor growth and metastasis. In the present study, in order to reveal the mechanism of over-expression of LCN2 and other genes up-regulated in LuM cells compared with parent KYSE150 cells, we performed a subtractive array-CGH analysis between LuM cells and parent KYSE150 cells using array-CGH system. However, any specific copy number aberrations between LuM cells and parent KYSE150 cells could not be detected (data not shown).The fact that these three cytokines  were up-regulated in LuM cells indicates that these highly metastatic cells obtained through in vivo selection may help to elucidate the characteristics and functions of the TME. It is expected that further studies of the TME may lead to the development of novel therapeutic targets.We consider that this highly metastatic subline is useful resource for further study including comparative expression analysis with clinical samples due to understand a possible mechanism driving metastatic property and to develop therapeutic strategy against metastatic ESCC cells. Furthermore, recent studies on cancer have increasingly focused on the tumor microenvironment (TME), from which these cytokines are released. Several other components of the TME have also been associated with tumor growth and metastasis, such as VEGF, PD-1, and CTLA-4, and treatments targeting these TME factors are currently being explored in clinical trials . However2.The KYSE150 cell line was kindly provided by Dr. Y. Shimada  and was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS). KYSE150 cells were maintained at 37°C with 5% COin vivo selection, luciferase-labeled KYSE150 cells  were injected into the tail vein of the mice. For bioluminescent observation of lung metastasis, the mice were injected intraperitoneally with 150 mg/kg d-luciferin potassium salt  and observed with the luminometer . Upon detection of lung metastasis, the mice were sacrificed and the metastatic nodules were collected, cut into small pieces, and incubated in dissociation solution  with the cells. The cells obtained from the metastasized lung tissues were cultured in a culture dish as described above. After repeating this process three times, the highly metastatic subline, designated as LuM, was established. Short tandem repeat analysis was conducted to confirm that the LuM cells were indeed derived from KYSE150 cells.NOD/SCID mice were purchased from the Charles River Laboratories Japan, and animal experiments were performed according to the institutional animal ethics guidelines. To establish the highly metastatic subline by The Agilent 8 × 60 K array  was used for differential gene expression analysis between parent KYSE150 cells and LuM cells according to the manufacturer's instructions. The raw data were analyzed with GeneSpring GX10 software (Agilent Technologies). After selection, candidate genes associated with metastasis were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to determine the pathways that were enriched.Cell survival was assessed by the crystal violet (CV) staining assay. Cells were washed in PBS and fixed with 0.1% CV in 10% formaldehyde in PBS for 10 min. After excess CV solution was discarded, the stained cells were completely air-dried, and then lysed with a 2% sodium dodecyl sulfate (SDS) solution with shaking for 2 h. Optical density (OD) values of the absorbance were measured at 560 nm using a microplate reader , and the percent absorbance of each well was determined. The OD absorbance values of cells in control wells were arbitrarily set at 100% to determine the percentage of viable cells.Transwell migration and invasion assays were carried out in 24-well modified chambers that were not pre-coated (migration assay) or pre-coated (invasion assay) with Matrigel . Cells in serum-free medium were transferred into the upper chamber. After 48-h incubation, the migrated cells in the lower chamber with 10% FBS as a chemoattractant were fixed and stained with 0.1% CV in 10% formaldehyde and counted with ImageJ software . Each assay was performed in triplicate.Whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidene fluoride membranes . After blocking with Tris-buffered saline containing 0.05% Tween-20 and 5% non-fat dry milk for 1 h, the membrane was reacted with the primary antibody for interleukin (IL)-6 , beta-actin , IL-1β , and lipocalin-2/NGAL . The membrane was washed and exposed to horseradish peroxidase-conjugated anti-mouse or -rabbit IgG antibodies  for 2 h. The bound antibodies were visualized on the LAS3000 imaging system  using a Pierce ECL Western detection kit according to the manufacturer's instructions .qRT-PCR was performed using an ABI PRISM 7500 Fast Real Time PCR System with the KAPA SYBR Fast qPCR kit  according to the manufacturer's instructions. Primers were indicated in ® ELISA kit (R&D Systems) according to the manufacturer›s instructions.Cell culture supernatants were collected and analyzed by ELISA using a QuantikineThe expression levels of human cytokines in the cell culture supernatants, including IL-1β and LCN2, were measured by antibody array (Label-Based (L-Series) Human Antibody Array 507 (L-507) Membrane Kit; RayBiotech, GA, USA) according to the manufacturer's instructions. Information of each position on array were shown in manufacturer's instructions.The siRNA for LCN2  and control non-specific siRNA  were obtained from Thermo Scientific. The siRNA was transfected individually into cells at the indicated concentration using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.t-test. A P-value of < 0.05 was considered statistically significant.Differences between subgroups (LuM cells and parent KYSE150 cells) were tested by the Student


Novel treatments for MDD are certainly desired. However, in our view the evidence in this study does not solve the academic debate on the efficacy of omega-3 fatty acids for MDD. Some food for thought.A recent meta-analysis and meta-regression of 13 randomized clinical trials by Mocking n=26) as very low,2 and the body of evidence as composed of a limited number of predominantly small studies at high risk of selection, performance or attrition bias. Poor evidence quality downgrades the credibility of overall effect size estimates, particularly when the evidence for an effect appears to be driven by poorer quality studies.On the basis of the widely accepted GRADE system, a recent Cochrane review evaluated the overall quality of the evidence of studies on omega-3 fatty acids and depressive symptomatology . Analyses conducted on studies with low risk of bias have consistently produced nonsignificant effect estimates.2 Moreover, based on the mean standardized differences and s.e.'s reported in their et al.1 show an inverse association between study effect size and study precision (r=−0.344): less precise trials produced larger effect sizes. To illustrate the impact that less precise studies can have on meta-analytic results, we repeated the meta-analysis  but without the least precise study3 (N=20), which reduced the overall effect size from a standardized mean difference (SMD) of 0.398 : 0.114, 0.681, P=0.006) to 0.317 . In addition, excluding the second-least precise study4 (N=22) further reduces the effect size to 0.227 . Thus, the observed effect of omega-3 fatty-acid supplementation on depression seems largely driven by the most imprecise studies.Study selection concerns aside, Mocking et al.1 concluded that omega-3 fatty-acid supplementation in MDD patients is especially beneficial in patients using antidepressants and for higher doses of EPA. A low number of trials reduces the probability of a true-negative finding. Whereas the number of trials here may not be exceptionally low compared with other meta-regressions, detecting moderator effects requires more powerful analyses than are employed in most published studies.5 Especially when high heterogeneity is present across studies, as is the case in Mocking et al.1 , power of 80% to detect even the largest of the modest moderator effects reported in Mocking et al.1 may not be achieved except with a much larger number of trials.5 Perhaps counterintuitively, low statistical power also decreases the probability that an observed effect that reaches nominal statistical significance actually reflects a true effect.7 The risk of false-positive findings is further increased by the substantial number of statistical tests conducted in this study.8 Indeed, neither of the results  survives correction for multiple comparisons , and the EPA dose–response relationship is mainly attributable to the two least precise studies4 (Based on nine univariate meta-regressions (one for each study characteristic) across 13 trials, Mocking studies4 .8 Hence, the associations found with EPA dose and antidepressant use could be due to other, known or unknown, trial characteristics. That findings from this meta-regression do not necessarily align with results from intervention studies is illustrated by the largest clinical trial available to date (N=432),9 which stratified randomization by antidepressant use and found evidence for neither an interaction between treatment group and antidepressant use, nor benefit from EPA supplementation among the subgroup of patients also taking antidepressants (n=174).The conclusions on EPA dose and antidepressant use were not based on randomization of these characteristics. Meta-regression is observational and, therefore, susceptible to confounding; it does not allow causal inference.Meta-analyses are critical to evidence-based medicine, but may lead to biased conclusions if the quality of available evidence is not adequately considered. Findings from meta-regression should be interpreted with particular caution, especially when suggesting clinical implications. Even if unbiased, a statistically significant result is not necessarily clinically relevant, and one may wonder whether, for instance, a decrease of 0.04 on the 17-item Hamilton Depression Rating Scale with every 100 mg increase in EPA dose is meaningful. In our view, the current evidence supporting the use of omega-3 fatty-acid supplementation in depression remains weak and clinical implications should be tempered.


However, known disease-related lncRNAs are still comparatively small in number, and experimental identification is time-consuming and labor-intensive. Therefore, developing a useful computational method for inferring potential associations between lncRNAs and diseases has become a hot topic, which can significantly help people to explore complex human diseases at the molecular level and effectively advance the quality of disease diagnostics, therapy, prognosis and prevention. In this paper, we propose a novel prediction of lncRNA-disease associations via lncRNA-disease-gene tripartite graph (TPGLDA), which integrates gene-disease associations with lncRNA-disease associations. Compared to previous studies, TPGLDA can be used to better delineate the heterogeneity of coding-non-coding genes-disease association and can effectively identify potential lncRNA-disease associations. After implementing the leave-one-out cross validation, TPGLDA achieves an AUC value of 93.9% which demonstrates its good predictive performance. Moreover, the top 5 predicted rankings of lung cancer, hepatocellular carcinoma and ovarian cancer are manually confirmed by different relevant databases and literatures, affording convincing evidence of the good performance as well as potential value of TPGLDA in identifying potential lncRNA-disease associations. Matlab and R codes of TPGLDA can be found at following:  Accumulating studies have indicated that a large quantity of lncRNAs are critical in many important biological processes such as chromatin modification, transcriptional and post-transcriptional regulation, genomic splicing, differentiation, immune responses, cell cycle control and so on6. Especially, it has been demonstrated that a large number of lncRNAs are involved in numerous complex human diseases4, such as neurological disorders7, coronary artery diseases8, cardiovascular diseases9, and various cancers10. Accordingly, inferring potential associations between lncRNAs and diseases can help us understand the pathogenesis of complex diseases at the molecular level and benefit biomarker identification for disease diagnosis, therapy, prognosis and monitoring5.Long non-coding RNAs (lncRNAs) are a new class of transcripts, with the length longer than 200nt13 and only few associations between lncRNAs and diseases have been reported. Collecting and integrating these data from a great number of literatures and databases is costly. Moreover, many biological experiments are time-consuming and expensive. Accordingly, researchers have started to focus on developing computational prediction approaches based on the existing datasets, which can quantify the associations’ probability between lncRNAs and diseases, and the most promising lncRNA-disease associations are used for further biological experimental validation. In this case, the time and cost of biological experiments can be effectively reduced14.Up to now, a large amount of lncRNA-related biological data has been distributed in different public databases5, Chen and Yan propose a computational approach of LRLSLDA to identify potential disease–related lncRNAs. LRLSLDA is a novel semi-supervised learning method in Laplacian regularized least squares framework. Moreover, this method does not require negative samples and can produce reliable results based on lncRNA expression profile and known lncRNA-disease associations. Subsequently, based on the finding that functionally related genes are often associated with phenotypically similar diseases16, Sun et al.17. Construct an lncRNA-lncRNA functional similarity network. Then, they develop a global network-based computational approach, RWRlncD, to identify potential lncRNA–disease associations by integrating disease similarity network, lncRNA functional similarity network and experimentally verified lncRNA-disease associations. Based on the same assumption, Ganegoda et al.18. Further propose a kernel based random walk with restart in heterogeneous network model (KRWRH), which incorporates with lincRNA tissue specific information, disease phenotype information and experimentally validated disease-lincRNA associations. KRWRH uses Gaussian interaction profile kernel to calculate the similarities of diseases and lincRNAs, and random walk with restart method is utilized for final prediction. The good experimental results highlight the importance and effectiveness of inferring potential disease-lincRNA associations using different biological information18.In recent years, some computational models have been proposed to identify potential associations between lncRNAs and diseases. For example, based on important assumption that similar diseases are often associated with functionally similar lncRNAs22 For example, Sahu et al.23 demonstrate that coding gene-TAF1D and lncRNA-SNHG1 are highly co-expressed in neuroblastoma. At the same time, rich information about gene-disease associations is available in database such as DisGeNET24 and PsyGeNET25. Therefore, if effectively used, such information may be of great help to infer potential associations between lncRNAs and diseases. Recently, Yang et al.22 conduct a pioneer study in which the authors integrate coding gene-disease associations and propose a propagation algorithm to infer potential lncRNA-disease associations based on a bipartite graph of coding-non-coding genes-disease. The authors show integrating coding gene-disease associations with lncRNA-disease associations can significantly enhance its prediction performance. Despite its low complexity and effectiveness, the bipartite graph model used in this study treats coding and non-coding genes without distinction and therefore cannot fully account for the heterogeneity of coding-non-coding genes-disease association. In addition, it cannot work for the lncRNAs and diseases without any known associations (hereafter isolated nodes), due to the fact that this method depends on the topological structure of bipartite graph and in consequence isolated nodes cannot get any information22. Another pioneer study called ncPred uses a tripartite network to infer potential ncRNA-disease association by integrating ncRNA-target associations and disease-target associations26. In this excellent work, the target information gives rise to a bridge connecting the ncRNA and diseases which significantly improve its predictive performance.Despite the success achieved by aforementioned methods, another important factor contributing to infer potential lncRNA-disease associations lies in the fact that coding and non-coding genes are often cooperated in human diseases, which has been demonstrated in many previous studiesTripartite Graph for potential LncRNA-Disease Association identification (TPGLDA) by integrating gene-disease associations and lncRNA-disease associations. The method begins with an lncRNA-disease-gene tripartite graph to delineate the heterogeneity of coding-non-coding genes-disease association. Subsequently, an effective resource allocation algorithm is proposed to accurately identify potential lncRNA-disease associations. Furthermore, lncRNA expression similarity and disease semantic similarity are introduced into TPGLDA to make inference for isolated nodes27. To perform a proper evaluation of our proposed method, we utilize leave-one-out cross validation experiment to demonstrate its superior performance compared with existing approaches. Besides, the analyses of several important cancers  effectively support the practical application of our method. We then use TPGLDA to infer potential lncRNA–disease associations and some high-ranking results are successfully verified by related literatures and databases, such as Lnc2Cancer28, LncRNA2Target29 and MNDR30. These results afford convincing evidence of the good performance of TPGLDA as well as potential value in supporting further biological experiments and promoting research productivity.Inspired by the above methods, in this paper we present a novel computational approach of a The overview of TPGLDA in identifying potential lncRNA-disease associations can be simple summarized as following four steps Fig. . First, 22. At each step of the LOOCV experiment, each known lncRNA-disease association is removed from the training samples in turn as test sample, whereas other known associations are taken as training samples for model learning22. Then, the receiver operating characteristics (ROC) curve is utilized to demonstrate the predictive performance of our proposed method and other methods mentioned in this paper by plotting the true positive rate  and the false positive rate  at different cutoff points. Here, sensitivity refers to the ratio of positive cases which can be correctly identified, and specificity represents the percentage of negative cases which can be correctly predicted. The value of AUC is calculated from the corresponding area under ROC curve, and the perfect performance appears in AUC = 1 while the random performance emerges in AUC = 0.532. Besides, we also adopt other evaluation measures such as accuracy (Acc), precision (Pre), and Matthews’s correlation coefficient (MCC)33. The definitions can be obtained as follows:Leave-one-out cross validation (LOOCV) is implemented on our gold standard dataset to evaluate the performance of TPGLDA in inferring potential associations between lncRNAs and diseases5 and KRWRH18, and the corresponding ROC curves of different methods are shown in Fig. 22 is not assessed here as it requires the node degree of each candidate ≥2. As a result, both LRLSLDA and KRWRH achieve reliable performance with AUC values of 82.2% and 83.8%, respectively, and TPGLDA has improved with an AUC value of 93.9%. Besides, other common performance evaluation measures, including Sn, Sp, Pre, Acc, and MCC, are also used to measure the predictive performance of these methods, and the results are shown in Table 34. At medium stringency level of specificity (Sp = 95.0%), KRWRH achieves the values of Sn, Pre, Acc, and MCC are 42.6%, 18.7%, 93.6% and 25.4%, respectively, which performs slightly better than LRLSLDA. By contrast, the corresponding values of TPGLDA are 76.8%, 29.4%, 94.5%, 45.4%, respectively. When the stringency level of specificity enlarges to 99.0%, the performance of our proposed method and the other two methods are consistently improved across all measures. Overall, these assessments generally confirm the good performance of TPGLDA in recovering experimentally verified lncRNA-disease associations.In order to comprehensively assess the predictive ability of TPGLDA to predict lncRNA–disease associations, we compare our method with two state-of-the-art methods: LRLSLDA32, the corresponding AUC values within the top k candidates of ranking lists are measured and the detailed results are shown in Fig. k candidates of ranking lists32. In the top 20 candidates, our method can successfully rank about 76% of known lncRNA-disease association pairs. When the rank threshold reaches to 100, the recall values of LRLSLDA and KRWRH are improved to 72.6% and 78.7%, respectively, and the corresponding value obtained by TPGLDA is 93.6%. Taken together, TPGLDA achieves decent recall in above different top k ranking lists, suggesting that our method can infer the largest number of positive samples based on different cutoffs.Furthermore, considering the importance of top portion of the prediction results32, we apply the LOOCV experiment on 15 diseases for demonstrating the practical predictive ability of different methods, and the corresponding AUC values are shown in Table Similar to previous studieset al. (2013)35 dataset used in ncPred26 is applied to make comparison with ncPred  and O(nrm), respectively. Considering the fact that the number of disease-related genes is an order of larger than the number of lncRNAs and diseases, the overall time complexity of resource allocation in tripartite graph is O(nrm), which is more efficient to that of ncPred (O(r2m))26. However, the parallelization and optimization techniques can greatly reduce the differences in computational speed. Also, we compare the running time of different methods and the results show the running time of TPGLDA is 0.6 second in average, which is comparable with other methods  information and lncRNAs without expression profiles in ArrayExpress59 (http://www.ebi.ac.uk/arrayexpress/), and eventually obtain 540 experimentally verified lncRNA-disease associations. This dataset is utilized as the gold standard dataset in the leave-one-out cross validation experiment and as the training dataset for inference of lncRNA–disease association5. Using the information of lncRNA-related diseases, we further collect 5212 gene–disease associations from DisGeNET database24 (http://www.disgenet.org/web/DisGeNET/) and construct a lncRNA-disease-gene tripartite graph, which includes 115 lncRNAs, 178 diseases and 1415 genes.The recent version of lncRNA-disease associations are downloaded from the LncRNADisease59 which contains more than 60000 expression profiles across 16 human tissues generated through RNA-Seq technology. Following previous approaches60, we calculate the lncRNA expression similarity  including all related annotation terms which can be obtained from the U.S. National Library of Medicine . Based on their DAGs, the similarities between diseases are measured and the detailed calculations are illustrated in the DOSE package63. We eventually calculate the semantic similarities , where L = {D = {G = {n long non-coding RNAs, the nodes set of m diseases and the node set of r genes, respectively. E denotes the interactions (edges) set between nodes in L with D and D with G. The tripartite graph can also be represented by two adjacency matrices Inspired by previous study using tripartite graph of users, items and tags for recommendation56 on the lncRNA-disease-gene tripartite graph. In order to help readers better understand the procedure of our model, a simple example of resource allocation in the tripartite graph is shown in Fig. We model the prediction process of lncRNA–disease associations as resource allocationD to those in L and G, respectively. In the second step, the resource is transferred back from nodes in L and G to the D nodes. We use the corresponding weight matrix j-th node to i-th node in L, and can be described as the similarity58 between lncRNA D. We further modify the resource allocation algorithm by considering the level of consistency between the contribution of resource moved in both directions57, which reflects the impact of co-selection (58. Accordingly, we define a consistence-based resource allocation to represent lncRNA-disease association as follows:i-th node and j-th node in L. The corresponding weight matrix is then rewritten as D nodes is defined as:The initial resource vector is then denoted as RNA Fig. . The resD nodes56 are allocated from nodes in D to nodes in G and then transferred back, and the final resource vector D nodes can be calculated as:G and With respect to resource allocation between genes and diseases, the same initial resource located on 27, which can be summarized as follows. First, we calculate the similarity 27, which is then integrated into tripartite graph for further resource allocation.The inference of the isolated node is implemented by following procedure proposed in previous studySupplementary InformationSupplementary Table S1Supplementary Table S2Supplementary Table S3


Anopheles gambiae and An. coluzzii, the two most important malaria vectors in sub-Saharan Africa, are recently radiated sibling species that are reproductively isolated even in areas of sympatry. In females from these species, sexual transfer of male accessory gland products, including the steroid hormone 20-hydroxyecdysone (20E), induces vast behavioral, physiological, and transcriptional changes that profoundly shape their post-mating ecology, and that may have contributed to the insurgence of post-mating, prezygotic reproductive barriers. As these barriers can be detected by studying transcriptional changes induced by mating, we set out to analyze the post-mating response of An. gambiae and An. coluzzii females captured in natural mating swarms in Burkina Faso. While the molecular pathways shaping short- and long-term mating-induced changes are largely conserved in females from the two species, we unravel significant inter-specific differences that suggest divergent regulation of key reproductive processes such as egg development, processing of seminal secretion, and mating behavior, that may have played a role in reproductive isolation. Interestingly, a number of these changes occur in genes previously shown to be regulated by the sexual transfer of 20E and may be due to divergent utilization of this steroid hormone in the two species. Plasmodium parasites1. One of the main reasons for this higher burden in the African continent is the presence of a very efficient mosquito vectorial system, principally represented by the two most recently radiated species of the Anopheles gambiae complex, i.e. Anopheles gambiae and An. coluzzii3. These species are sympatric in West and Central Africa5, but differ in their larval ecology, with An. gambiae being more adapted to temporary rain-dependent and An. coluzzii to permanent anthropogenic breeding sites9. Due to their major role as malaria vectors, the two species are the target of several studies aimed at developing novel approaches for the control of disease transmission in sub-Saharan Africa, with the view to complement or strengthen current insecticide-based control methods12.Although overall malaria mortality rates have significantly declined since 2010 due to increased prevention and control measures, Sub-Saharan Africa continues to carry a disproportionately high share of the global malaria burden, bearing more than 90% of the 212 million cases and of the estimated 429,000 deaths caused by An. gambiae virgin females virtually sterilizes them by preventing their insemination and reducing egg development10. This hormone is a potent regulator of gene transcription during both juvenile development and oogenesis in adults14, and in some anopheline species is synthetized in the male accessory glands (MAGs) and transferred during mating to the female atrium together with other seminal secretions embedded in a gelatinous structure named the mating plug20. Several studies have shown that in species of the Afrotropical An. gambiae complex, sexual transfer of 20E is essential for proper induction of female post-mating behaviors, such as refractoriness to further mating, enhanced egg production, triggered egg laying and increased fertility21. In addition, 20E injections in the G3 strain, which is a mixture of An. gambiae and An. coluzzii, induce a broad transcriptional response in the female reproductive tract, closely overlapping with the vast response induced by mating in the same strain, where hundreds of genes are up-and down-regulated at different time points after copulation22. However, the extent to which the response to mating is conserved between An. gambiae and An. coluzzii females is currently unknown.One of these novel approaches consists in manipulating the mosquito reproductive success. A recent study showed that application of non-steroidal agonists of the steroid hormone 20-hydroxyecdysone (20E) on Drosophila, multiple lines of evidence point to a role of female post-mating biology in the insurgence of post-mating, prezygotic reproductive barriers. For instance, in crosses between recently diverged species, failure in sperm transfer and/or storage in hetero-specific crosses was attributed to mating-induced changes24. Additionally, processing of the insemination “plug” that forms in Drosophila females immediately after mating takes longer in hetero- than in homo-specific crosses26. It has also been postulated that fast evolving male-female molecular interactions or post-mating changes in transcript abundance may represent signatures of natural selection shaping the evolutionary arms race between the sexes30.In An. gambiae and An. coluzzii. While hybrid males from most crosses between species of the An. gambiae complex are sterile, males from crosses between An. gambiae and An. coluzzii do not show signatures of genetic incompatibilities and are fully fertile, with no obvious loss in fitness under laboratory conditions31. Nevertheless, hybrids between these two species are rarely observed in most areas of sympatry32. Where the two species are sympatric, e.g. in Burkina Faso, spatial and temporal segregation of the swarms is significantly contributing to assortative mating34, while close-range mate recognition cues, such as species-specific flight tones and/or contact cuticular pheromones, are believed to reinforce pre-mating isolation41. Inter-specific mating couples have, however, been repeatedly collected in the field43, suggesting the co-occurrence of intrinsic and/or extrinsic post-mating isolation mechanisms. While the latter have been shown to play a role5, intrinsic post-mating isolation mechanisms have never been investigated.Post-mating events may have also played a role in the recent divergence between An. gambiae and An. coluzzii females captured in natural mating swarms from Burkina Faso. Our results corroborate previous data obtained under laboratory conditions22, allow the identification of factors potentially important for mating, fertility and reproductive success in each species, and provide novel insights on inter-specific differences that shape their reproductive ecology and may help unravel the mechanisms of their reproductive isolation.Here we report the first data on the transcriptional changes induced by mating in An. gambiae and 75 An. coluzzii mating couples from different swarms in the villages of Soumousso and Vallèe du Kou (Burkina Faso) , three serine proteases , one amino protease (AGAP000885), two metallopeptidases (AGAP001791 and AGAP009791), a protease inhibitor (AGAP009766), and a putative anti-microbial Andropin-like gene (AGAP009429)45. The last gene was the mating induced stimulator of oogenesis , which is induced by the sexual transfer of the steroid hormone 20E and is implicated in the increase in egg development experienced by mated An. gambiae females after blood feeding17.In our analysis of the post-mating response in the LRT, we focused on ten genes shown to be strongly up- or down-regulated after mating in laboratory experiments and thus likely to be involved in the reproductive processes triggered by copulationAn. gambiae females, changes in gene expression were detected in five proteases , all downregulated at 1 day PM . Moreover, four of these genes showed reduced expression levels also at 4 days PM  , AGAP005194 at 1 day PM , and AGAP009791 at both time points analyzed . Furthermore, MISO was strongly up-regulated at 1 day PM , while the ABC transporter AGAP011518 was downregulated at 4 days PM  , which encodes a yolk protein that is needed for egg development46 and which in Aedes aegypti mosquitoes is strongly upregulated by 20E synthetized after blood feeding47, and other six genes shown to be differentially expressed in An. gambiae and An. coluzzii virgin females48 that we reasoned might be associated with assortative mating behavior. These included: the sex determining gene doublesex , the antennal carrier protein AP-1, (AGAP004799), the odorant binding protein 25 , the cuticular protein CPF3 (AGAP004690), the glutathione S transferases – epsilon class 2 ; and lingerer (AGAP004817). dsx regulates the terminal sexual differentiation in most insects49, and specifically it determines the differentiation of neurons that control male courtship behavior50 and female sexual receptivity53. In Drosophila, lingerer is involved in the control of male copulatory organs during courtship54. CPF3 is a non-canonical cuticular protein with no chitin-binding capacity, which may be part of the epicuticle55 where it could bind to sex pheromones such as cuticular hydrocarbons (CHCs)48. Odorant binding proteins such as AP-1 and OBP25 transfer odorants to specific receptors56 and might play a role in female mate choice by helping the identification of co-specific males. GST-E2 might instead be involved in the metabolism of chemical stimuli from antennae and other sensory organs57, thus regulating the availability of stimulants such as CHCs.We next analyzed the expression of genes in the female carcass, initially focusing on seven factors that may be related to reproductive success or mating behavior Table ; Fig. 3.An. coluzzii . dsx, CPF3 and AP-1 were downregulated at 1 day PM  and AP-1 was down-regulated also at 4 days PM (P = 0.0342). OBP25 and Vg were instead upregulated at 1 day PM  , which is a complement-like factor, homologous to the human C3, that binds and mediates killing of pathogens including Plasmodium parasites60; the leucine-rich immune protein 1 (LRIM1), which circulates in the hemolymph as a disulphide-bounded complex with the leucine-rich protein APL1C and interacts with TEP1 controlling its activity62; and the antimicrobial peptides cecropin 1 (CEC1), CEC3, and gambicin (GAMB)64. Only TEP1 showed to be upregulated in An. gambiae females at 1 day PM .We finally tested whether mating induces a differential immune response in the two species, possibly driven by diverging sexually transmitted pathogensAn. gambiae and An. coluzzii females collected from natural mating swarms largely corroborate previous data obtained under laboratory conditions22, demonstrating the opportunity of studying complex phenomena such as mating and post-mating behavior in laboratory colonies. This result is remarkable when considering that gene expression is age-dependent67 and that in our study it was not possible to precisely age-match mated females to virgin ones. For this reason - as well as for the limited number of samples we analyzed due to intrinsic difficulties in collecting couples from natural mating swarms - we observed some variability in our results that probably limited our power to detect subtler, age-dependent changes.Our results on the transcriptional response to mating in An. gambiae and An. coluzzii males and females from the same geographic areas studied here share largely overlapping reproductive microbiomes42, we detected a mating-induced regulation of TEP1, a key immune gene, in the carcass of An. gambiae females. This species-specific upregulation may be due to sexual transfer of microorganisms populating the An. gambiae male reproductive tract, similarly to what observed in D. melanogaster where mating anticipates immune reactions to sexually transmitted pathogens possibly as a mechanism to enhance fecundity68.Despite these limitations, some interesting differences were detected in the post-mating responses of the two species samples. Although field MISO – an atrial gene strongly induced by sexual transfer of 20E that regulates the number of eggs developed by females after mating and blood feeding19 – was significantly upregulated only in An. coluzzii at 1 day PM, although a trend towards an increase was also observed in An. gambiae at the same time point  was regulated by mating in An. gambiae females only. dsx is a key gene in the sexual differentiation cascade, and is produced as sex-specific isoforms78 that in Drosophila govern multiple aspects of reproductive biology, including the female receptivity to mating and the development and the activity of neural circuit that regulate sex-specific sexual behavior53. Furthermore, in the fruit fly dsx controls the expression of genes that synthetize female-specific long-chain cuticular hydrocarbons (CHC), notably the desaturase DESAT-F, that are potent pheromones for male courtship behavior79. It is therefore possible that dsx may affect the synthesis of CHC pheromones also in An. gambiae females, consistent with the observation that An. gambiae and An. coluzzii have, indeed, slightly different CHC profiles that are altered after mating38. Interestingly, CPF3, another gene related to CHC, is also downregulated in An. gambiae females. This cuticular protein is likely expressed in the epicuticle, where it is postulated to bind to cuticular pheromones such as CHCs48. Because post-mating changes in the CHCs profiles affect female attractiveness in many monandrous insect species81, the An. gambiae-specific downregulation of genes related to chemical contact cues might reflect the occurrence of different post-mating signals in the two species.Finally, the expression of four genes encoding for factors possibly involved in mating behavior  and CHCs have been linked to 20E function, as this ecdysteroid reduces CP expression levels during developmentAnopheles gambiae and An. coluzzii were collected in September 2009 in the Western part of Burkina Faso, i.e. in the village of Soumousso  and in Vallèe du Kou , located 55 km east and 30 km north-west of Bobo-Dioulasso, respectively. While in Soumousso larval breeding sites are mostly temporary, rain-dependent puddles more favorable to An. gambiae, the irrigation scheme in Vallèe du Kou largely favors An. coluzzii.Virgin females were obtained from larvae collected in natural breeding sites  and reared to the adult stage in natural climatic conditions and photoperiod using cages placed in the outdoor space available at the IRSS laboratory in Bobo-Dioulasso. In order to ensure females would not mate, pupae were individually transferred in single cups and their sex determined at emergence. Using this method, adult females and males were never in contact with each other.87, allowed to complete copulation, transferred to single cups using mouth aspirators, and brought to the laboratory in sealed containers avoiding shifts in temperature and humidity.Mating couples were collected from naturally occurring swarms as previously described88 prior to dissections of reproductive organs. These were carried out under a dissecting stereo-microscope (5x magnification lens) at different time intervals, i.e. 2 and 5 days post emergence in the case of virgin females, and 24 hours and 4 days post-mating in the case of mated ones . The time points for virgin females were selected to match as much as possible the age of mated females based on data showing that most females mate on the second night after emergence44. The lower reproductive tract  and the rest of the body (carcass) of single females were stored separately in RNAlater solution (Ambion) and pools of five individual tissues/species/time interval were obtained for each time point (Table S1).Virgin and mated females were maintained in individual cups and DNA was extracted from single legs removed from live specimens for genotypingFor tissue-specific analysis, total RNA was extracted using TRI Reagent (Helena Biosciences). The amount of RNA for female carcasses was limited to 1 μg. All samples were treated with DNase I (Invitrogen), according to manufacturer’s guidelines. cDNAs were synthesized in 100 μl reactions using 1x First Strand buffer, 5 mM DDT, 0.5 mM dNTPs, 2.5 μM random hexamers, 40 units RNaseOut recombinant ribonuclease inhibitor, and 125 units of M-MLV reverse transcriptase .Samples were run in 15 μl reaction volume using 1x Fast SYBR Green Master Mix (Applied Biosystems). Gene expression was quantified in duplicates on a StepOnePlus Real-Time thermocycler (Applied Biosystems) using the following program: 95 °C for 15 min, then 40 cycles  followed by a dissociation curve analysis. Primers used for qRT-PCR are listed in Table S2. Three technical replicates were used for each biological replicate for each gene. A standard curve against serial dilutions of cDNA templates (mated and virgin) was used for each gene to determine the linear range of the assay.RpL19 (AGAP004422), which is expressed at high levels and does not respond to mating22. To test for mating-induced changes in gene expression, the two species were studied separately. As we do not know if the primers anneal with the same efficiency or if the reference gene is expressed at the same levels in the two species, we did not perform cross-species comparisons. An ANOVA was first used to test whether each gene showed significant changes in the two time points analyzed, including in the analysis both mated and virgin samples. If the global F test gave positive results, pairwise posthoc contrast tests (Tukey-Kramer procedure) have been used to determine differences between mated and virgin females at each time point analyzed. To control for possible Type I error arising through use of multiple ANOVA tests, the P values were corrected by applying a False Discovery Rate procedure (FDR).Gene expression levels were normalized using deltaCt method against the ribosomal gene


Follow-up observation is required for mild-to-moderate hydrocephalic patients because of the potential damage to brain. However, effects of mild-to-moderate hydrocephalus on gray and white matter remain unclear in vivo. Using structural MRI and diffusion tensor imaging (DTI), current study compared the cortical thickness and white matter integrity between children with mild-to-moderate communicating hydrocephalus and healthy controls. The relationships between cortical changes and intelligence quota were also examined in patients. We found that cortical thickness in the left middle temporal and left rostral middle frontal gyrus was significantly lower in the hydrocephalus group compared with that of controls. Fractional anisotropy in the right corpus callosum body was significantly lower in the hydrocephalus group compared with that of controls. In addition, there was no association of cortical thinning or white matter fractional anisotropy with intelligence quota in either group. Thus, our findings provide clues to that mild-to-moderate hydrocephalus could lead to structural brain deficits especially in the middle temporal and middle frontal gyrus prior to the behavior changes. Hydrocephalus is a pathologic condition in which excessive cerebrospinal fluid (CSF) accumulates in the ventricular system because of obstruction along CSF pathways or an imbalance between CSF production and reabsorption , 2. The Although conventional MRI techniques, including T1 weighted image (T1WI) or T2 weighted image (T2WI), can detect major structural abnormalities, they cannot detect more subtle GM and WM abnormalities, such as cortical thickness and ultrastructural changes, in hydrocephalic patients during follow-up observation. By contrast, 3-dimensional fast spoiled gradient-recalled sequence can provide high-resolution T1WI data that allowed us to observe and measure cortical thickness. DTI provides quantitative information on anisotropic diffusion properties in WM and has been used to investigate WM damage and recovery in various neurologic and pathologic disorders –8. ChangIn the present study, we performed MRI studies on school-aged children with mild-to-moderate communicating hydrocephalus to identify regional changes of cortical thickness and WM fractional anisotropy (FA) and assessed the correlation between cortical changes and intelligence quota (IQ).This study included 15 treatment-naive patients with communicating hydrocephalic . All hydrocephalus subjects were recruited from patients who visited The Second Affiliated Hospital of Wenzhou Medical University from December 2013 to June 2015. The inclusion criteria were (1) age between 6 and14 years, (2) IQ within the normal range, (3) no evidence of obstruction in CSF flow and no GRASS MRI finding of other diseases, (4) Evan's index between 0.33 and 0.50, (5) duration of illness more than 1 year, (6) no history of other intracranial or other related diseases , (7) right-handed, and (8) no claustrophobia [The Wechsler Intelligence Scale for Children-Chinese Revised, which demonstrates high reliability and validity, was used to assess general intellectual ability of all subjects and the linguistic IQ and performance IQ, and senior medical staff that majored on intelligence assessment calculated full scale IQ.2, matrix = 256 × 256, and in-plane resolution = 0.94 × 0.94 mm2). DTI images were acquired with a diffusion-weighted spin-echo sequence .MRI data were obtained on a 3.0 Tesla system (GE Signa HDxt) with an 8-channel phase array head coil. High-resolution T1WI was acquired with a volumetric 3-dimensional fast spoiled gradient-recalled sequence . Cortical thickness was measured as the difference between the position of equivalent vertices in the pial and GM/WM surfaces, by utilizing both intensity and continuity information from the entire 3-dimensional MR volume in the segmentation and deformation procedures. The reliability of the method was previously validated against histological analysis on postmortem brains and manual measurements, and the test-retest reliability is high [High-resolution T1WI analysis was performed using the FreeSurfer package . Data were inspected for movement artifacts using FSL-MCFLIRT (<1° rotation and <1 mm translation) and then corrected for eddy current-induced distortions. Brain extraction and calculation of diffusion parameter maps were performed using FSL. FA maps for each participant were registered into a standard brain template  using the nonlinear spatial transformation tool FNIRT. A mean FA image was then compiled by averaging aligned FA maps from each participant. To generate a mean FA skeleton representing the centers of all tracts common to the group, the map threshold was then set for voxels showing FA values ≥ 0.2. Aligned FA maps for each participant were projected onto the standard skeletonized FA image  by searching the area around the skeleton in the direction perpendicular to each tract, finding the highest local FA value, and assigning this value to the skeleton [DTI image processing was performed using the FSL package (skeleton .P < 0.05), to find regions with significant group differences.In the graphical interface of FreeSurfer , structural parameters were smoothed with full width at half maximum at 10 mm, mesh surface-base, and concomitant variable of age. Cortical thickness was compared between the two groups, corrected using the Monte Carlo simulation .Using tract based spatial statistics tools, voxelwise spatial statistical analysis comparing the hydrocephalus and control groups was performed using the “randomize” program within FSL, which involves permutation testing . The mea t-test was performed to compare age and IQ of the hydrocephalus and control groups, while Chi-square test was performed to compare gender ratio (SPSS v19.0 software). Bivariate correlations were performed between cortical thickness, FA changes, and IQ for each group.To evaluate the relationship between cortical changes and IQ, we extracted the mean value of cortical thickness of regions with significant intergroup differences. A 2-sampleP = 0.80) or gender composition (P = 0.73) between the hydrocephalus and control groups  and in the left rostral middle frontal gyrus . There were no brain regions with increased cortical thickness in hydrocephalus patients . There were no brain regions with increased FA in hydrocephalus patients (Compared with the control group, hydrocephalus patients had a lower FA in the right corpus callosum body (region size = 836 voxels; MNI coordinates = 11, −11, 31; patients , Table 3In the control group, there were a trend towards a decrease in cortical thickness in the left middle temporal gyrus with increasing linguistic IQ and full scale IQ and a trend towards an increase in cortical thickness in other regions with increasing linguistic IQ, performance IQ, and full scale IQ. In both the control and the hydrocephalus groups, there were a trend towards an increase in cortical thickness in the left rostral middle frontal gyrus with increasing linguistic IQ, performance IQ, and full scale IQ and a trend towards a decrease in FA in the right corpus callosum body with increasing linguistic IQ. Further, in the hydrocephalus group, there was a trend towards a decrease in full scale IQ. By contrast, there was no association of cortical thinning or decreased FA with IQ in either group Tables –6.Unlike the widespread cortical deficits of the whole brain in patients with severe hydrocephalus, school-aged children with mild-to-moderate communicating hydrocephalus showed significant cortical thinning in the left middle temporal gyrus and left rostral middle frontal gyrus and decreased FA in the right corpus callosum body, though the two groups showed no difference of IQ. Furthermore, there was no correlation between the mean cortical thickness nor the FA and IQ in either group. These findings suggest that the gray matter of left temporal and frontal lobe and white matter of corpus callosum are the most vulnerable regions in children with mild-to-moderate communicating hydrocephalus, which may happen before the behavior changes.Widespread cortical thinning was previously reported in animal models and children with severe hydrocephalus , 28, 29.The decreased FA in the right corpus callosum body of infants and children with severe hydrocephalus was also reported in previous studies –37. DTI We acknowledge several potential limitations of our study. First, our results were derived from a limited number of participants, which may have had an adverse effect on the power of statistical analysis. Second, a confounding factor is the relationship between age of onset and disease duration, which is difficult to resolve in a cross-sectional design. Thus, further longitudinal studies with more patients are required to confirm the changes in cortical thickness and WM ultrastructure in children with mild-to-moderate communicating hydrocephalus. A third limitation is that the Wechsler Intelligence Scale for Children-Chinese Revised is a basic and very ordinary intelligence scale for children, which may be the reason why no association of cortical thinning and decreased FA with IQ was found. Therefore, more detailed intelligence scales may be required to detect specific differences. In addition, we use Evan's index instead of cerebrospinal fluid pressure as crucial indicator for noninvasiveness. However, the accuracy of Evan's index is still not very ideal and more reference indexes will be helpful for noninvasive indicators . MeanwhiBy studying the school-aged children with mild-to-moderate communicating hydrocephalus, our findings provide evidences that the gray matter of left temporal and frontal lobe and white matter of corpus callosum are the most vulnerable regions in mild-to-moderate hydrocephalus, which happens even before the behavior changes. Thus, structural analysis by MRI can be used to monitor long-term outcomes and follow-up observation in mild-to-moderate hydrocephalic patients.


Mycobacterium tuberculosis. In addition, Pneumocystis jirovecii and cytomegalovirus are important opportunistic pathogens. As the vertical transmission risk of HIV decreases and access to antiretroviral therapy increases, the epidemiology of these infections is changing, but HIV-infected infants and children still carry a disproportionate burden of these infections. There is also increasing recognition of the impact of in utero exposure to HIV on the general health of exposed but uninfected infants. The reasons for this increased risk are not limited to socioeconomic status or adverse environmental conditions—there is emerging evidence that these HIV-exposed but uninfected infants may have particular immune deficits that could increase their vulnerability to respiratory pathogens. We discuss the impact of tuberculosis and other lower respiratory tract infections on the health of HIV-infected infants and children.Pneumonia remains the most common cause of hospitalization and the most important cause of death in young children. In high human immunodeficiency virus (HIV)-burden settings, HIV-infected children carry a high burden of lower respiratory tract infection from common respiratory viruses, bacteria and  To prevent vertical transmission of human immunodeficiency virus (HIV) from mother to child and to improve long-term outcomes for HIV-infected pregnant women, the current standard of care is to initiate lifelong suppressive antiretroviral therapy (ART) . With thPneumocystis jirovecii, cytomegalovirus (CMV) and Mycobacterium tuberculosis [Despite major reductions in vertical HIV transmission among adequately treated mothers, large numbers of HIV-infected children continue to be born, especially in settings where HIV service delivery or maternal uptake of available services is sub-optimal. In 2013, it was estimated that globally there were 240,000 new pediatric HIV infections . Despiterculosis –11.The overlap in the epidemiology of TB and HIV is well known, with its epicenter in sub-Saharan Africa. All of the countries with HIV prevalence of more than 10% in persons aged 15–49 years are in sub-Saharan Africa, and more than 90% of all HIV-infected children live in this region , 12, 13.Major differences in the number of children diagnosed in TB endemic settings with comparable adult TB incidence rates probably reflect varying ability to diagnose TB in young children . IrrespeReasons for the high rates of TB in HIV-infected infants are multifactorial. HIV-infected and HEU infants and children are highly exposed to adult TB source cases. HIV-infected adults are 21–34 times more likely to develop TB in high burden settings, with TB accounting for up to a quarter of HIV-associated mortality . Woman oAlongside exposure and infection, young age is one of the most important risk factors for developing severe disease . HoweverThe most important tools we have to mitigate the HIV-associated risk of developing TB infection and disease are (i) access to ART for adults and children with HIV; and (ii) use of IPT. In the Children with HIV Early Antiretroviral Therapy (CHER) trial, conducted in Cape Town and Johannesburg, South Africa, TB occurred in 8.3% of children receiving early ART (initiated before12 weeks of age) and in 20% of children receiving delayed ART  . When ARThough there is a decline in the number of children commencing ART only when they are severely immunocompromised, this situation is still common. In 2010 in Asia, Africa and South America, 63% of HIV-infected children in low-income countries, 66% in low–middle-income and 58% in upper–middle-income countries were already severely immunocompromised when commencing ART, compared with 19% of HIV-infected children in the United States . Access In 2014, The Joint United Nations Programme on HIV/AIDS (UNAIDS) defined its ambitious 90–90–90 targets. The three targets to be achieved by 2020 are: 90% of HIV-infected persons must be diagnosed, 90% of those diagnosed should be on sustained therapy, and 90% of those on therapy should have an undetectable viral load . If ART IPT is a well known prevention strategy in HIV-uninfected children and adults with known exposure to TB. In HIV-infected children there are no studies assessing post-exposure prevention; however, in a cohort of 494 children beginning ART when younger than 24 months of age in Cape Town, 127 courses of post-exposure IPT were provided, with only two children developing confirmed TB. Both had fully drug-susceptible isolates . In a prThe temporal associations between TB, influenza, and pneumococcal disease in children suggest a complex interaction between these infections. There is an increase in TB diagnosis approximately 3 months after the annual influenza season and a reduction in culture-confirmed TB in HIV-infected children following receipt of pneumococcal conjugate vaccines , 47. ThePulmonary TB is the most common disease manifestation in HIV-infected children, with 85% diagnosed in an early series of hospitalized South African children . HoweverM. tuberculosis and which drugs should be used?What is the suspected or confirmed susceptibility pattern of Are alternative rifamycins to rifampicin available?Which ART regimen is preferred AND can a suppressive regimen be given safely?What are the potential drug interactions AND can adjustments be made if rifampicin is the only rifamycin available?The potential drug interactions between the TB therapy and ART are well known and, in general, TB therapy should not change. A dual treatment plan should consider the following questions:In the absence of ART, high mortality in HIV-infected children with TB was reported , 54 and Pharmacokinetic studies illustrate that both efavirenz and lopinavir-ritonavir 1:1 in their usual doses provide appropriate drug levels when co-administered with rifampicin , 56. ThePneumonia remains the leading cause of pediatric mortality and morbidity. Globally, there are approximately 12–14 million hospitalizations and 0.68–0.92 million deaths annually from pneumonia in children –72. HIV-P. jirovecii and mycobacterial infections—is well recognized. Mortality is increased significantly with increasing numbers of pathogens. Children with polymicrobial pneumonia have a 10-fold greater risk of dying than children in whom only a single organism is identified [Even with early access to ART, pneumonia remains common among HIV-infected infants and children. In the CHER study, pneumonia occurred at a rate of 12.2 infections/100 person-years in children accessing ART before 12 weeks of age and 26.5 infections/100 person-years in children accessing ART only after laboratory evidence of immune suppression and/or clinical features of HIV infection became apparent . Poor geentified .Streptococcus pneumoniae, but Staphylococcus aureus is increasingly important. Amongst Gram-negative bacteria associated with pneumonia in HIV-infected children, Klebsiella species, Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa and non-typhoidal Salmonella species are also important [HIV-infected children have a higher risk of developing bacterial pneumonia than uninfected children. This remains a significant problem, even after the introduction of ART. HIV-infected children with pneumonia are more likely to have severe disease (including bacteremia), more complications, higher rates of treatment failure and death, and possibly more antimicrobial resistance than HIV-uninfected children . The mosmportant .S. pneumoniae, and although in the authors’ experience it also occurs in HIV-infected children, this has not been reported to date in the literature. The chest radiographs in necrotizing pneumonia frequently demonstrate both pleural and parenchymal involvement. The so-called ‘atypical pneumonias’ can present with a variable radiographic appearance, which includes reticulonodular infiltration, patchy or confluent air-space disease or a combination of all three patterns [HIV does not affect the radiographic appearance of bacterial pneumonia. The two classic patterns of either diffuse patchy air-space disease of bronchopneumonia, or confluent air-space disease with air bronchograms of lobar or segmental pneumonia can be found . Necrotipatterns . It is, patterns .Without appropriate vaccination or ART, HIV-infected children experience a 40-fold increase in the risk of pneumococcal bacteremia, pneumonia and meningitis compared to vaccinated HIV-uninfected children, and they also often experience multiple episodes of invasive disease . In SoutViral infections play a major role in LRTIs and viruses are commonly identified in HIV-infected children hospitalized with these infections. Viral LRTIs may also play an important role in the pathogenesis of bacterial pneumonia and in the epidemiology of TB. In a large study from South Africa at least one virus was identified in 68% of HIV-infected children younger than 5 years of age and in 58% of those aged 5–14 years presenting with symptoms of a LRTI. More than one respiratory virus was found in 28 and 19% of children within these age groups, respectively. In children younger than 5 years the most common viruses detected were human rhinovirus (36%), adenovirus (32%), respiratory syncytial virus (RSV) (13%), enterovirus (8%), parainfluenza (9%) and influenza viruses (7%) , 84. In RSV is a very important pathogen in infants and young children and a common cause of LRTIs that require healthcare visits and hospitalization. HIV is an important risk factor for severe RSV infections, especially in high-burden settings . HIV-infp = 0.01). Interstitial pneumonia was more frequent in HIV-positive children and consolidation was more frequent in HIV-negative children. Overall, 91% of HIV-positive children received oseltamivir. The H1N1 influenza attack rate was very high (20%) in HIV-infected children, but it consistently ran a mild course [Influenza is an important cause of pneumonia hospitalization and death. ART naïve HIV-infected children are up to 8 times more likely to be hospitalized and have a higher case fatality rate (8% in HIV-infected vs. 2% in uninfected children) , 89. In d course .P. jiroveci pneumonia (PJP), is poorly understood, but CMV itself can cause fatal pneumonia [P. jirovecci (51.3%) [CMV is a common co-infection in HIV-infected children and is frequently isolated from urine and respiratory secretions , 94. Conneumonia . Postmor (51.3%) , 98. CMV (51.3%) . In HIV-Despite access to effective vaccination, measles remains an important pathogen in children . In settP. jirovecii is a common and important pathogen in HIV-infected children and causes a severe, rapidly progressive pneumonia with a high mortality rate despite access to antibiotic therapy [ therapy , 94. Thi therapy , 106. Th therapy . Clinica therapy . When PJThe most important strategy to prevent PJP is by using co-trimoxazole prophylactic therapy from 6 weeks of age . StoppinM. tuberculosis and other lung pathogens is a major cause of disease and death in HIV-infected children. Furthermore, many also develop chronic lung disease with bronchiectasis due to recurrent lung infections [Acute respiratory compromise caused by fections , 111. Infections . This hi