ADAMTS9, BDNF, CASS4, COMT, CR1, DNMT3A, DTNBP1, REST, SRR, TOMM40, circadian clock, and Alzheimer’s diseases-associated genes may contribute to susceptibility to cognitive aging. In this review, we first illustrated evidence of the genetic contribution to disease susceptibility to age-related cognitive decline in recent studies ranging from approaches of candidate genes to genome-wide association studies. We then surveyed a variety of association studies regarding age-related cognitive decline with consideration of gene–gene and gene–environment interactions. Finally, we highlighted their limitations and future directions. In light of advances in precision medicine and multi-omics technologies, future research in genomic medicine promises to lead to innovative ideas that are relevant to disease prevention and novel drugs for cognitive aging.With ever-increasing elder populations, age-related cognitive decline, which is characterized as a gradual decline in cognitive capacity in the aging process, has turned out to be a mammoth public health concern. Since genetic information has become increasingly important to explore the biological mechanisms of cognitive decline, the search for genetic biomarkers of cognitive aging has received much attention. There is growing evidence that single-nucleotide polymorphisms (SNPs) within the  Generally speaking, age-related cognitive decline, or cognitive aging, is recognized as a gradual and enduring process of transition in cognitive capacity with increasing age . AlthougPICALM, MS4A6E, MS4A4E, MS4A4A, EPHA1, CR1, CLU, CD33, CD2AP, BIN1, APOE, and ABCA7 gene  identified 14 risk genes in AD, encompassing the ZCWPW1, SORL1, SLC24A4, RIN3, PTK2B, NME8, MEF2C, INPP5D, HLA-DRB4, HLA-DRB1, FERMT2, DSG2, CELF1, and CASS4 genes (PLD3 gene to likely be a risk gene for AD (CR1 rs6656401 SNP by using the established AD-associated genes (CLU-MS4A4E (CD33-MS4A4E (ABCA7 rs3764650 and CD33 rs3865444 SNPs in the elderly Caucasian women (More recent research in genome-wide association studies (GWAS) has implicated that single-nucleotide polymorphisms (SNPs) among 11 genes appear to affect the risk of AD, encompassing the S4 genes . The suce for AD . MoreoveU-MS4A4E , 16 and an women .In this review, we first surveyed some genetic biomarkers that were linked with age-related cognitive decline in several recent association studies Table . FurtherZCWPW1, SORL1, SLC24A4, RIN3, PTK2B, PLD3, PICALM, NME8, MS4A6E, MS4A4E, MS4A4A, MEF2C, INPP5D, HLA-DRB4, HLA-DRB1, FERMT2, EPHA1, DSG2, CR1, CLU, CELF1, CD33, CD2AP, CASS4, BIN1, APOE, and ABCA7 in GWAS and meta-analyses (n = 634) aged over 60 years (mean age: 64.2 years) from the Taiwan Biobank  approach  and Tau protein   also suggested that carriers of the APOE ɛ4 allele, which were linked with late-onset AD, performed worse on several domains of cognitive functions including overall global cognitive ability, episodic memory, and executive functioning  . A meta-ctioning . Furthere adults .BDNF was reported to be implicated in age-related cognitive decline among the elderly  for normal aging by using the mean Z-scores based on three cognitive domains including motor function, memory, and perceptual speed  and German (mean age: 64.9~65.3 years) populations using MMSE scores  for normal aging  as well as for pathological aging  using the cognitive testing data such as memory and global cognition   after performing Bonferroni correction  . The resrrection . In addiapproach .ADAMTS9 is demonstrated in the regulation of a wide variety of processes, such as the control of organ shape during growth, the inhibition of angiogenesis, and the cleavage of proteoglycans   . Their aE scores . This prrrection . On the 4 allele . In addipal loss –67. Addiogenesis –67. Althr adults , 69, theve aging . Consistve aging , several2   . After fve aging . Furtherve aging . The TOMchondria .DTNBP1 gene encodes a protein that plays a role in the biogenesis of organelle linked with lysosomes, platelet dense granules, and melanosomes  and German (mean age: 24.8) populations (DTNBP1 gene was associated with general cognitive ability and cognitive decline in schizophrenia patients (The anosomes . It has ulations –87. In apatients .Hp1bp3 gene to be a novel modulator of cognitive aging (Hp1bp3 may contribute to cognitive aging in both mice and humans (HP1BP3 gene is located on chromosome 1p36.12 and encodes a histone H1 related protein with non-redundant and specific roles vital for viability and gain in humans (In a recent systems genetics study applying a genetically diverse population of mice, Neuner et al. pinpointed ve aging . Their fd humans . The HP1n humans .SRR although, to our knowledge, there are only animal studies but no population studies for this gene in cognitive aging. SRR encodes the serine racemase enzyme which converts L-serine to d-serine. d-serine is an endogenous co-agonist for N-methyl-D-aspartate receptors (SRR rs408067 SNP, located in the promoter region, may affect the transcription activity of the SRR gene (SRR expression impaired hippocampal age-related cognitive function in an animal study, suggesting that the SRR-dependent pathway might be one possible target of the hippocampus-related cognitive decline in aging (SRR mutation (It is worth mentioning a potential gene called eceptors . The SRRSRR gene . Reducedin aging . Anothermutation .Notwithstanding, there were several limitations with respect to the aforementioned studies. First, there is certainly room for development of much further research and comprehensive evaluation to reassess whether the current results remain in other ethnic populations for the investigated genetic variants with cognitive aging in terms of the association and interactions .Second, given the relatively young mean age of the sample in several studies mentioned previously, the current results are unable to be extrapolated to much older populations that have higher risk for developing age-associated diseases and neurodegenerative disorders, such as MCI, AD, and other dementias .Because of logistical and ethical matters, it is challenging to assess homogeneous genetic backgrounds and recruit a large enough cohort of participants at the same time . FurtherIn order to reinforce the statistical findings, it is appealing to seek more supplementary biologically relevant evidences owing to the fact that the investigated SNPs might be greatly enhanced in association studies .Besides, the aforementioned studies utilized various methods to assess cognitive function. A major challenge is to ensure a proper approach for evaluating cognitive function. The well-established MMSE approach is chosen to evaluate cognitive function in several aforementioned studies because it is the most widely used screening test of cognition . NeverthCross-sectional design for cognitive aging studies has been adopted by many researchers because it is less feasible to examine aging trajectories for individual participants with longitudinal studies due to the high cost and long follow-up time . NeverthOver the past decade, advances in genome science have spawned numerous lines of research into precision medicine and multi-omics . In spitBuilding up a set of genetic biomarkers which are immensely dependable as a benchmark of disease status or drug response for cognitive aging will be considerably indispensable in the future . At thisMoreover, machine learning and predictive techniques such as Bayesian networks may present a conceivable approach to forecast novel drug efficacy and establish statistical models for predicting disease status . In futuEssentially, evidence shows that multi-omics data and biomarkers such as genetic, epigenetic, metabolomic, transcriptomic, and proteomic profiles are important in assorted pathophysiology for a certain disease and novel drug treatment , 105. SuIn this review, we have focused primarily on recent findings and relevant studies for age-related cognitive decline. The current review also highlighted the merit of association studies with relatively large sample size to incorporate a wide variety of populations for cognitive aging. In order to advance personalized treatment and prevention strategies worldwide, a main challenge is how best to integrate these findings with other pieces until the picture of cognitive aging is adequately apparent. Similarly, these findings have indicated that machine learning and predictive tools might be beneficial for clinical decision making by integrating multi-omics data and biomarkers.In light of recent developments, novel machine learning and predictive algorithms will be the new frontier in the decades to come for establishing prognostic and diagnostic assessments by using huge data technologies for precision medicine , 105. FuEL, H-YL, and C-HL involved in conception and design; EL and C-HL involved in literature review and interpretation, and manuscript writing; EL, H-YL, and C-HL involved in financial support and final approval of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The objective of this study was to assess the safety of Xemys in the treatment of patients with relapsing-remitting multiple sclerosis (MS) and secondary progressive MS, who failed to achieve a sustained response to first-line disease-modifying therapies. In this phase I, open-label, dose-escalating, proof-of-concept study, 20 patients with relapsing-remitting or secondary progressive MS received weekly subcutaneously injections with ascending doses of Xemys up to a total dose of 2.675 mg. Clinical examinations, including Expanded Disability Status Scale score, magnetic resonance imaging results, and serum cytokine concentrations, were assessed before the first injection and for up to 17 weeks after the final injection. Xemys was safe and well tolerated when administered for 6 weeks to a maximum single dose of 900 μg. Expanded Disability Status Scale scores and numbers of T2-weighted and new gadolinium-enhancing lesions on magnetic resonance imaging were statistically unchanged at study exit compared with baseline; nonetheless, the increase of number of active gadolinium-enhancing lesions on weeks 7 and 10 in comparison with baseline was statistically significant. During treatment, the serum concentrations of the cytokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and interleukin-7 decreased, whereas the level of tumor necrosis factor-α increased. These results provide evidence for the further development of Xemys as an antigen-specific, disease-modifying therapy for patients with MS.Previously, we showed that CD206-targeted liposomal delivery of co-encapsulated immunodominant myelin basic protein (MBP) sequences MBPThe online version of this article (doi:10.1007/s13311-016-0448-0) contains supplementary material, which is available to authorized users. Multiple sclerosis (MS) is a chronic neurodegenerative disease with an evident autoimmune background resulting in inflammatory demyelination and axonal and neuronal injury . MS, whiSeveral treatment strategies for MS have been found to be moderately successful . For exaHowever, some patients remain refractory to these agents. This may be due to as yet unknown triggers of MS, together with high heterogeneity of this disease. Therefore, searching for novel, antigen-specific immunotherapeutic treatment options for MS is highly feasible . For exa46–62, MBP 124–139, and MBP 147–170 coencapsulated in CD206-targeted small monolammelar liposomes  in patiThe primary endpoint was the safety of Xemys, as determined by the frequency and severity of adverse events (AEs) and SAEs. To ensure patient safety, the patients were divided into 2 cohorts. Dose-limiting toxicities (DLT) and dose adjustments were assessed in the first cohort; doses in the second cohort were limited to the highest first-cohort dose not associated with DLT. If there were no DLTs in the first cohort, the dosing regimen would remain unaltered in the second cohort.Secondary clinical endpoints included the number of relapses during the study period and Expanded Disability Status Scale (EDSS) score at the end of the trial. Secondary magnetic resonance imaging (MRI) endpoints included the number of gadolinium-enhancing T1 lesions and the total number of lesions in T2 and fluid-attenuated inversion recovery (FLAIR) sequences. Laboratory endpoints included the concentrations of pro- and anti-inflammatory cytokines. The study was approved by the Russian Ministry of Health, the Central Ethics Commission, and all local ethic committees, and was conducted in full compliance with the Declaration of Helsinki, International Conference on Harmonisation’s Good Clinical Practice, and appropriate local legislation.Subjects were screened 2 weeks before enrollment into the treatment phase of the study Fig. . The tri2; or hypersensitivity toward components of a test item  or GA. Subjects were also excluded if they had liver decompensation; heart diseases; tuberculosis in anamnesis; significantly abnormal hematological or biochemical parameters; oncological diseases; previous therapy with cladribine, alemtuzumab, rituximab, natalizumab, daclizumab, or intravenous immunoglobulin; treatment with any disease-modifying therapy during the previous 6 months, including cyclophosphamide, mitoxantrone, ciclosporin, mycophenolate mofetil, azathioprine, methotrexate, or plasmapheresis; or had been administered glucocorticoids within the last month at a daily dosage equivalent to > 60 mg of prednisolone.Subjects were excluded if they had primary progressive MS; clinically relevant infection or surgical intervention < 30 days before the screening visit; contraindications to MRI scanning, including hypersensitivity toward gadolinium; a body mass index > 40 kg/mThe study was authorized by the Russian Public Health Ministry #930 (FASEMS-01/01) issued on 28 April 2012. All patients provided written informed consent at enrollment after discussion of the study with investigators including possible alternative treatment options. Details of full medical history and MS pathogenesis were collected.Lyophilized Xemys consists of equimolar amounts of lyophilized, chemically synthesized MBP peptides 46–62 (GGDRGAPKRGSGKDSHH), 124–139 (GFGYGGRASDYKSAHK), and 147–170 (QGTLSKIFKLGGRDSRSGSPMARR)  encapsulated in small unilamellar liposomes prepared from egg phosphatidylcholine and monomannosyl dioleyl glycerol with the addition of α-tocopherol and lactose . Each dose was rehydrated in 1.0 ml sterile water immediately before administration. The starting dose, consisting of 0.05-mg peptides, was chosen as the minimum to detect any unpredictable AEs without significant risk to the patients. Patients received weekly subcutaneous injections of Xemys at escalating doses over 6 weeks of 50 μg, 150 μg, 225 μg, 450 μg, 900 μg, and 900 μg, yielding a total dose of 2.675 mg.exacerbation was defined as a new worsening of neurological function lasting for > 24 h that was unrelated to other comorbidities.Patients were followed-up 1, 4, and 12 weeks after administration of the last dose, corresponding to study weeks 7, 10, and 18, respectively. AEs were monitored by 12-lead electrocardiography, hematology, and laboratory tests, and scored according to the Common Terminology Criteria for Adverse Events version 4.0. Any SAEs deemed “certainly” or “likely” due to the study drug were considered DLTs, with the next lowest dose level considered the maximum tolerated dose. Patients also underwent complete neurological examinations during each study visit. An MS EDSS was determined at baseline (week 2) and at all follow-up visits. Patients underwent MRI scans, including T1-weighted axial scans with and without gadolinium, proton density axial, T2-weighted axial, T2-weighted sagittal, and FLAIR sequence axial images, at baseline and at follow-up visits at weeks 7, 10, and 18. Scans were performed with Philips, Amsterdam, Netherlands Integra 1.5T, Magnetom Avanto 1.5T, and GE Medical Systems, Milwaukee, WI, USA Signa 1.5T scanners.Serum samples for cytokine analysis were collected at baseline and during all follow-up visits. The profiles of 17 cytokines and chemokines were determined using a multiplexed fluorescent magnetic bead-based immunoassay , according to the manufacturer’s instructions. These 17 cytokines and chemokines included interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, interferon-γ, monocyte chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein (MIP-1b/CCL4), and tumor necrosis factor (TNF)-α.t tests were used to compare between-group variables and the Wilcoxon signed rank test for within-group variables. All tests were two sided, and p-values < 0.05 were considered significant. All statistical analyses were performed with SPSS  and GraphPad Prism 6.0 .Demographic data, baseline characteristics, safety and tolerability variables, and other parameters under investigation were calculated using descriptive statistics. Safety and tolerability were assessed in patients who received at least 1 dose of the studied substance. AEs were grouped by dose and classified by MedDRA system organ classes and preferred terms, with severity classified by Common Terminology Criteria for Adverse Events version 4.0. Secondary endpoints were analyzed in patients who received at least 1 dose of the studied substance and underwent at least 1 assessment. The normality of the data was determined using Kolmogorov–Smirnov tests; all datasets were non-normally distributed. Changes from baseline in the number of MRI lesions were assessed by analysis of variance. Mann–Whitney Between April 2013 and July 2014, 20 patients with RRMS or SPMS matching all criteria were directly recruited into the trial at 4 clinical centers in the Russian Federation. Baseline characteristics of patients with MS are listed in Table As no patient experienced a DLT during treatment, an maximum-tolerated dose was not reached, making it likely to be > 900 μg per week. Eight patients 40%) experienced 16 AEs Table , with 110% experiThe most common AE was local reaction at the site of injection, which was observed 8 times in 4 (20%) patients. Most injection site reactions occurred at administration of submaximal (0.45 mg) and maximal (0.9 mg) doses of Xemys; all resolved within 24 h without treatment. Rhinitis occurred twice in 2 patients (10%) each and general weakness twice in 1 (5%) patient. Other AEs occurred only once in single patients. Xemys did not have any effect on laboratory safety tests, vital signs , results of physical examination, or electrocardiography parameters . In general, the administered doses of Xemys were considered safe and tolerable.F = 3.015, p = 0.038), but not at week 18 (p > 0.05). At last follow-up, 16/19 (85%) patients were relapse-free, and EDSS worsened in 21% and improved in 10% of patients. The disease histories, and clinical and MRI outcomes of individual patients are summarized in Table Patients underwent MRI scans at baseline (week –2) and after treatment, at weeks 7, 10, and 18, using T1-weighted (with and without contrast), T2, and FLAIR regimens. Nineteen patients were evaluated at baseline and week 10, and 18 patients at weeks 7 and 18 Table , Fig. 2ATo analyze the immunological consequences of Xemys administration, the concentrations of 17 serum cytokines and chemokines were analyzed at follow-up time points Fig. . CompareThis study showed that subcutaneous administration of Xemys was safe and well tolerated in patients with MS who had previously failed to achieve sustained disease control following treatment with GA or β-interferons. The AEs related to Xemys were of mild or moderate severity and occurred mainly after treatment with submaximal and maximal doses of Xemys. These AEs were self-limiting, required no concomitant medication, and did not cause abnormalities in blood tests or other safety measures. Taken together, these findings showed that once-weekly Xemys has a relatively good safety profile.Patients included in the FASEMS trial have experienced significant disease activity, despite previous first-line disease-modifying treatment. Importantly, 12 weeks after the end of Xemys administration, 7 patients (37%) showed no evidence of disease activity, and 16 (85%) were relapse-free. Although EDSS levels and the numbers of T2-weighted lesions and new gadolinium-enhancing lesions on MRI in comparison with baseline were statistically unchanged at study exit, a statistically significant increase in the number of active gadolinium-enhancing lesions on weeks 7 and 10 in comparison with baseline was detected. Here we emphasize that all patients included in this study had experienced > 1 relapse during the previous year, had progressing EDSS, and at the same time had disease stabilization for at least 30 days before screening. Therefore, the number of contrast-enhancing lesions may have had a natural tendency to increase during the course of the study. The lack of a placebo group reasoned by approved study design did not allow comparative monitoring of disease progression, including the number of gadolinium-enhancing lesions, in the absence of therapeutic intervention. Finally, the statistically significant increase in the number of gadolinium-enhancing lesions was assessed only in a small group of patients who experienced relapse. Thus, the appearance of these lesions was not likely associated with treatment, although this possibility cannot be completely excluded.Our preliminary data also suggest that there were no consistent trends in serum cytokine profiles. However, the concentrations of MCP-1, MIP-1β, and IL-7 were significantly lower after treatment that at baseline. Immunologically, Xemys administration is thought to restrict monocyte cell trafficking. Although a classical proinflammatory cytokine TNF-α has been previously shown to have potent beneficial effects in autoimmune neurodegeneration , reporteIn conclusion, this phase I trial showed that once-weekly subcutaneous Xemys for 6 weeks, at a cumulative dose of 2.675 mg, was safe and well tolerated by patients with RRMS and SPMS. Preclinical and clinical data warrant the further development of Xemys as an antigen-specific disease-modifying therapy for patients with MS.ESM 1(PDF 1225 kb)Below is the link to the electronic supplementary material.


Hmox1, Sepp1, and Srxn1), reactive oxygen species metabolism  and oxygen transport (Ift172 and Slc38a1). Furthermore, multiple genes relevant to mitochondrial respiration  and neuroinflammation  were up-regulated, alongside 73–88% reduction in the expression of anti-inflammatory genes IL4 and IL10, and nuclear translocation and increased expression of Nrf2 protein. These hippocampal changes were associated with elevated levels of pro-inflammatory cytokines and chemokines  and lipid peroxidation byproduct malondialdehyde in the serum, suggesting the presence of an incessant systemic inflammation and elevated oxidative stress. These results imply that chronic oxidative stress, inflammation, and mitochondrial dysfunction in the hippocampus, and heightened systemic inflammation and oxidative stress likely underlie the persistent memory and mood dysfunction observed in GWI.Memory and mood dysfunction are the key symptoms of Gulf war illness (GWI), a lingering multi-symptom ailment afflicting >200,000 veterans who served in the Persian Gulf War-1. Research probing the source of the disease has demonstrated that concomitant exposures to anti-nerve gas agent pyridostigmine bromide (PB), pesticides, and war-related stress are among the chief causes of GWI. Indeed, exposures to GWI-related chemicals (GWIR-Cs) and mild stress in animal models cause memory and mood impairments alongside reduced neurogenesis and chronic low-level inflammation in the hippocampus. In the current study, we examined whether exposure to GWIR-Cs and stress causes chronic changes in the expression of genes related to increased oxidative stress, mitochondrial dysfunction, and inflammation in the hippocampus. We also investigated whether GWI is linked with chronically increased activation of Nrf2 (a master regulator of antioxidant response) in the hippocampus, and inflammation and enhanced oxidative stress at the systemic level. Adult male rats were exposed daily to low-doses of PB and pesticides (DEET and permethrin), in combination with 5 min of restraint stress for 4 weeks. Analysis of the hippocampus performed 6 months after the exposure revealed increased expression of many genes related to oxidative stress response and/or antioxidant activity ( Central nervous system (CNS) impairments, characterized by memory and mood dysfunction, are among the persistent symptoms in Gulf war illness (GWI) . GWI is The multiple chemical exposure theory has been supported by various animal studies. For example, we previously demonstrated that rats exposed to low-doses of GWI-related chemicals (GWIR-Cs), such as PB, DEET, and PM, and mild stress for 4 weeks consistently developed memory and mood impairments, which were akin to those observed in patients with GWI . FurtherNonetheless, it is unknown whether the various morphological alterations observed in the hippocampus at extended time-points after the exposure to GWIR-Cs and stress are accompanied by molecular changes, such as alterations in genes encoding proteins that are relevant to increased oxidative stress, mitochondrial dysfunction, and enhanced inflammation. Therefore, in the current study, using quantitative real-time polymerase chain reaction (qRT-PCR) arrays, we first investigated whether exposure to GWIR-Cs and stress caused lasting modifications in the expression of genes related to oxidative stress response, metabolism of reactive oxygen species (ROS), oxygen transport, antioxidant activity, and inflammatory proteins in the hippocampus. Analyses was performed at a delayed time-point (6 months) after exposure to GWIR-Cs and stress in this study to simulate the scenario in GWI patients in whom over 25 years have passed since the exposure during PGW-1. A delay of 6 months after exposure in a rat model is equivalent to ∼17 years survival period after exposure in humans . Next, sFinally, to determine whether the chronic increase in oxidative stress and inflammation in the hippocampus occurred in parallel to systemic inflammation and oxidative stress, we evaluated multiple pro-inflammatory proteins and malondialdehyde (MDA) in the serum. This analysis is particularly important because GWI patients exhibit increased concentration of multiple inflammatory biomarkers such as C-reactive protein, leptin, brain-derived neurotrophic factor and matrix metalloproteinase-9 in the blood  and multad libitum access to commercial rat chow and water. Following 12–15 days of acclimatization in the vivarium, animals were randomly assigned to the naïve control, vehicle (VEH) control, or GWI groups, which here onward will be referred to as “naïve controls,” “VEH-rats,” and “GWI-rats,” respectively. GWI-rats received daily exposure to GWIR-Cs, namely, PB, DEET, and PM, and mild stress (5 min of restraint stress) for 4 weeks. In contrast, VEH-rats received sterile water (the vehicle solution used to dissolve PB), and 70% alcohol (the vehicle solution used to dissolve PB).Young male Sprague-Dawley rats, aged 3–4 months, were obtained from Harlan Sprague-Dawley Inc.  for this study. A combined institutional animal care and use committee of the Central Texas Veterans Health Care System and the Texas A&M Health Science Center College of Medicine approved all the experiments performed in the current study. Upon arrival, animals were housed with The dosages of PB, DEET, and PM, and duration of stress were based on prior studies of rat models of GWI . PB Sig. Becausen = 8/group) was deeply anesthetized with an overdose of isoflurane and decapitated, 6 months after the exposure to the GWIR-Cs and restraint stress. Following this, the brain was rapidly removed from the skull, snap frozen in dry ice, and stored at -80°C until further analysis. Following quick thawing, the entire hippocampus from both hemispheres of each brain was micro-dissected and processed for molecular biology studies, which comprised analyses of the expression of genes encoding proteins that are relevant for regulating oxidative stress, antioxidant activity, inflammation, and mitochondrial respiration, using qRT-PCR arrays.Each animal . We employed the manufacturer’s instructions, as previously detailed . The con2 qPCR master mix  and 1,248 μl of dH2O to obtain a cocktail of 2,700 μl. This solution was then transferred to wells in a rat oxidative stress RT2 ProfilerTM PCR array plate (25 μl/well), which were comprised of pre-dispensed gene specific primer sets. In addition to primers for the 84 genes related to oxidative stress, each array also included primers for detecting five housekeeping genes, namely, beta-actin, beta-2-microglubulin, hypoxanthine phosphoribosyl transferase 1, lactate dehydrogenase A, and ribosomal protein lateral stalk subunit P1, and three RNA and three PCR quality controls. The reactions were performed according to the manufacturer’s protocol using a CFX96 Real-Time system . The PCR amplification was done using the following two-step cycling program: 10 min denaturizing at 95°C, 40 cycles of 95°C for 15 s, and 60°C for 1 min. At the end of the amplification assay, a melt curve analysis was conducted to evaluate the specificity of the reaction. The conditions for the melt curve were 95°C for 1 min, followed by incremental increases of 0.5°C per 5 min, from 65 to 95°C.The Rat Oxidative Stress PCR Array used in this study  detected the expression of 84 key genes related to oxidative stress response, ROS metabolism, oxygen transport, and antioxidant activity in the hippocampus. A previously described  qRT-PCR ΔCt values for each gene were compared between VEH-rats and GWI-rats (n = 4–5/group).The Rat Mitochondrial Energy Metabolism PCR Array  used in this study profiled the expression of 84 key genes in the hippocampus, which encoded proteins that are relevant for mitochondrial respiration, such as genes encoding components of the electron transport chain and oxidative phosphorylation complexes. The reactions were performed as described above. For data analyses, the Ct (threshold cycle) values of all wells were exported to an Excel spreadsheet and analyzed using web-based SABiosciences PCR array data analysis software. The 22 qPCR master mix, 1 μl of RT2 primer , and 10.5 μl of dH2O to obtain a total volume of 25 μl. Primers relevant to selected genes (n = 35) encoding proteins involved in neurogenesis, learning and memory, and inflammation were used. Genes related to inflammation comprised the following: B-cell lymphoma 6 (Bcl6), complement component 3a receptor 1, chemokine (C-C-motif) ligand 5 , Ccl17, Ccl22, colony stimulating factor 2 (Csf2), interferon gamma (Ifng), insulin-like growth factor-binding protein 1 (Igf1), interleukin 1 alpha (IL1a), IL1b, IL2, IL4, IL6, IL10, IL12b, nuclear factor kappa B subunit 1 (Nfkb1), and tumor necrosis factor alpha (Tnfa). Genes that are relevant to neurodegeneration and/or cognitive and memory function included the following: mitogen-activated protein kinase 1 (Mapk1), Mapk3, protein kinase C alpha (Prkaca), glyoxylate reductase 1 homolog (N-pac), NMDA receptor regulated 2 (Narg2), nerve growth factor (Ngf), fibroblast growth factor-2 (Fgfb), vascular endothelial growth factor A (Vegfa), synaptotagmin-5 (Syt5), and forkhead box O3. Each array also contained two of three housekeeping genes glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase 1, and ribosomal protein lateral stalk subunit P1. The qRT-PCR reactions and data analyses were carried out as described above; 2ΔCt values for each gene were compared between VEH-rats and GWI-rats(n = 6–8/group).The template cDNA (1 μl) from each hippocampal sample was mixed with 12.5 μl of 2X RTn = 8/group) were subjected to terminal anesthesia with isoflurane and intracardiac perfusion with 4% paraformaldehyde solution in phosphate buffer. Following this, the brain was carefully removed from the skull of each animal and post-fixed in 4% paraformaldehyde for approximately 16 h at 4°C. Next, brain tissues were treated with 30% sucrose solution in phosphate buffer until it sank to the bottom, and 30-μm-thick cryostat sections were cut coronally through the entire septo-temporal axis of the hippocampus. Sections were collected serially in 24-well plates filled with phosphate buffer. Every 20th section through the hippocampus was then processed from randomly chosen GWI-rats and naïve controls (n = 5/group) for Nrf2 and NeuN dual immunofluorescence. In brief, sections were first processed for Nrf2 immunofluorescence, which comprised sequential incubation of sections in 10% donkey serum for 30 min and rabbit anti-Nrf2 solution  overnight. Following a wash in phosphate buffer, the sections were incubated in donkey anti-rabbit AF594 solution  for 1 h and thoroughly washed with phosphate buffer thereafter. Next, sections were incubated in mouse anti-NeuN solution  overnight, washed in phosphate buffer, and incubated in donkey anti-mouse AF488 solution  for 1 h. The sections were then thoroughly washed in phosphate buffer, and mounted on slides using SlowFade antifade mounting medium (ThermoFisher Scientific). Thereafter, 2-μm-thick Z-sectioning analysis was performed using confocal microscopy to visualize dense NeuN and speckled Nrf2 expression. Since Nrf2 expression was prominently seen in the CA3 subfield of the hippocampus in both GWI-rats and naïve controls, the percentage of CA3 pyramidal neurons displaying neuronal translocation of Nrf2 was quantified using multiple sections in each animal. In addition, since the number of Nrf2 speckles within nuclei varied in different groups, we also quantified the percentage of neurons expressing robust Nrf2 (having more than two speckles) among those expressing neuronal Nrf2. The values were compared between naïve control rats and GWI-rats (n = 5/group).Six months after the exposure to GWIR-Cs and stress, GWI-rats and naïve controls (n = 8/group) were deeply anesthetized with an overdose of isoflurane. The heart was exposed and blood was quickly collected from the right atrium. Following this, the brain was rapidly removed from the skull, snap frozen in dry ice, and stored at -80°C. The serum was extracted from the collected blood using standard procedures and stored at -80°C. The entire hippocampi from both hemispheres were micro-dissected following quick thawing, and the samples were processed to measure Nrf2 levels through an ELISA. Serum samples were processed for measuring the relative concentration of various pro-inflammatory cytokines and chemokines, and MDA.Six months after the exposure to GWIR-Cs and stress, additional GWI-rats and naïve controls (g at 4°C for 15 min) to avoid contamination with cellular debris. Aliquots of the supernatant solution were stored at -80°C until further use. The protein concentration of different samples was measured using a Pierce BCA reagent kit (ThermoFisher Scientific). We measured Nrf2 concentration in hippocampal lysates using the ELISA method, according to manufacturer instructions . Briefly, hippocampal lysates were diluted to 10 μg/100 μl and added to each well of a 96-well plate that was pre-immobilized with the Nrf2 consensus sequencing oligonucleotide, which resulted in binding of activated Nrf2 in the whole cell lysate to the oligonucleotide. Samples were incubated with 50 μl of a specific antibody against an Nrf2 subunit for 1 h at room temperature (RT), washed, incubated with 50 μl of horseradish peroxidase-conjugated secondary antibody for 45 min at RT, washed again, and incubated with 50 μl of substrate solution for 10–30 min or until positive wells turned blue. Thereafter, 25 μl of stop solution was added, which transformed the color of the solution from blue to yellow. The yellow colored reaction product was measured using a Micro plate reader at 450 nm. The amount of Nrf2 in the lysate was directly proportional to the intensity of color. The values were compared between naïve control rats and GWI-rats (n = 6/group).Each hippocampal sample was weighed and lysed in 750 μl of ice-cold tissue extraction buffer  with protease inhibitors , using a sonic dismembrator for 10 s. The lysed solution was centrifuged twice .We used a rapid and sensitive rat cytokine array to measure16 cytokines/chemokines in the serum in a high-throughput manner, as per manufacturer instructions . Briefly, serum samples (1:5 diluted) were dispensed to wells (100 μl per well) of 96-well plates that were coated with specific cytokine capture primary antibodies and incubated for 2 h at RT with gentle shaking. Following three washes with wash buffer, 100 μl of diluted biotin-labeled detection antibody mixture was added to each well and incubated for 1 h at RT with gentle shaking. Then, samples were washed and 100 μl of diluted streptavidin-HRP conjugate was added to each well and incubated for 45 min at RT. After three washes, 100 μl substrate was added to each well and incubated at RT. One hour later, 50 μl of stop solution was added to each well, which transformed the color of the solution from blue to yellow. The yellow reaction product was measured using a Micro plate reader at 450 nm. The concentration of the various cytokines and chemokines in the serum was directly proportional to the intensity of color in this procedure. The values for each cytokine or chemokine were compared between naïve control rats and GWI-rats (n = 6/group).The level of MDA, which is a by-product of lipid peroxidation (a mechanism of cellular injury), in the serum was detected using an ELISA kit . Briefly, each well of the 96-well plate was pre-coated with an antigen-specific capture antibody. Diluted serum and known standards were added to each well followed by a fixed quantity of HRP-conjugated target antigen. The free antigen in the sample competed with the HRP conjugated antigen for binding to the capture antibody. Unbound antigen was washed away. A 3,3′,5,5′-tetramethylbenzidine substrate was then added which reacted with the HRP enzyme resulting in color development. Sulfuric acid stop solution was added to terminate the color development reaction. Then, the absorbance of the well was measured at a wavelength of 450 nm using a plate reader. A standard graph was plotted and the MDA concentration in unknown samples was computed from the best-fit curve. The values were expressed as ng/ml and compared between naïve control rats and GWI-rats  was employed. Numerical data were presented as mean ± SEM and a p-value less than 0.05 was considered as statistically significant.Prism software was used for all statistical analyses. Unpaired, two-tailed, Student’s n = 27) that typically respond to increased oxidative stress (referred to as oxidative stress-response genes) was significantly increased in the hippocampus, at 6 months after exposure to GWIR-Cs and mild stress , which implied long-term changes in the expression of these genes. The genes were as follows: alsin rho guanine nucleotide exchange factor 2 , apolipoprotein E , catalase , cathepsin B , 24-dehydrocholesterol reductase , dual oxidase 2 , excision repair cross-complementation group 6 , glutamate-cysteine ligase catalytic subunit , glutathione peroxidase 3 , Gpx4 (B10), Gpx6 (B11), Gpx7 (B12), heme oxygenase 1 , isocitrate dehydrogenase , myeloperoxidase , NAD(P)H quinone dehydrogenase 1 , nudix hydrolase 1 , parkinsonism associated deglycase , peroxiredoxin1 , Prdx2 (B20), Prdx6 (B21), prion protein , proteasome subunit beta 5 , sequestosome 1 , thioredoxin reductase 1 , Txnrd2 (B26), and Selenoprotein P Plasma 1 .The expression of a multitude of genes , suggesting the chronic presence of enhanced ROS in the hippocampus. This comprised the following genes: flavin containing monooxygenase 2 , superoxide dismutase 2 , Sod3 (B3), copper chaperone for superoxide dismutase , stearoyl-CoA desaturase 1 , and uncoupling protein 2 . There were also increased expression of several genes involved in oxygen transport in the hippocampus at 6 months post-exposure to GWIR-Cs and stress . These genes were the following: cytoglobin , dynamin 2 , intraflagellar transport 172 , and solute carrier family 38 member 1 .Six different genes encoding proteins that are involved in ROS metabolism were upregulated in the hippocampus at 6 months post-exposure to GWIR-Cs and mild stress (n = 20) encoding proteins that are involved in antioxidant activity were upregulated in the hippocampus at 6 months post-exposure to GWIR chemicals and mild stress, further supporting the chronic presence of enhanced oxidative stress in the hippocampus . The up-regulated expression of 12 of these genes are illustrated in Figure 1, which included Cat (B3), Ctsb (B4), Gpx3, Gpx4, Gpx6, and Gpx7 (B9-B12), Mpo (B15), Prdx1, Prdx2 and Prdx6 (B19-B21), and Txnrd1 and Txnrd2 (B25-B26). The enhanced expression of the antioxidant gene Sod3 is illustrated in Figure 2B3. Finally, there were seven additional genes with increased expression , namely, adenomatous polyposis coli , glutathione reductase , glutathione S-transferase kappa 1 , glutathione S-transferase pi 1 , Prdx5 (B5), prostaglandin-endoperoxide synthase 1 , and sulfiredoxin 1 .Many genes (n = 26) involved in mitochondrial respiration were up-regulated in the hippocampus, 6 months post-exposure to GWIR-Cs and mild stress . These comprised genes encoding proteins that are important for the function of complexes I, II, IV, and V of the mitochondrial respiratory chain . The nine genes with altered expression pertaining to complex I were NADH dehydrogenase [ubiquinone] 1 alpha subcomplex 8 , Ndufa9 [B2], Ndufa11 [B3], NADH dehydrogenase [ubiquinone] 1 beta subcomplex 3 , Ndufb5 [B5], NADH dehydrogenase [ubiquinone] Fe-S protein 6 , Ndufs7 [B7], Ndufs8 [B8], and NADH dehydrogenase [ubiquinone] flavoprotein 1 . The only gene that was modified in relation to complex II function was succinate dehydrogenase complex, subunit B, iron sulfur . The five genes associated with complex IV  were cytochrome C oxidase subunit Vb , cytochrome C oxidase subunit V1a polypeptide 1 , cytochrome C oxidase subunit VIIa polypeptide 2 like , surfeit 1 , and cytochrome C-1 . The two genes relevant to complex V , which had an altered expression, were ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1  and Atp5b (E2).Many genes . These included the following: ATPase, H+ transporting, lysosomal accessory protein 1 , ATPase, H+ transporting, lysosomal V0 subunit A2, involved in acidification of cell organelles , phospholysine phosphohistidine inorganic pyrophosphate phosphatase, involved in respiratory electron transport , pyrophosphatase [inorganic] 1, important for phosphate metabolism of cells , solute carrier family 25 member 10, one of mitochondrial membrane carrier proteins , solute carrier family 25 member 20, another mitochondrial membrane carrier protein , uncoupling protein 1, one of mitochondrial anion carrier proteins , ubiquinol-cytochrome C reductase binding protein, participates in the transfer of electrons when ubiquinone is bound , and ubiquinol-cytochrome C reductase core protein I, involved in metabolism .In addition to the above genes, nine other genes relevant to mitochondrial/other organelle function displayed enhanced expression . The increased expression of these pro-inflammatory genes was associated with the decreased expression of anti-inflammatory genes IL4 and IL10 . Further, several genes associated with neurodegenerative disorders and/or cognitive and memory function also had an altered expression. These included the increased expression of Ngf, Fgfb, Mapk1, Mapk3, Prkaca, N-pac, and Narg2 , and the decreased expression of Vegfa .Many genes linked to inflammation, namely, Figure 7). Since the frequency of neurons with nuclear Nrf2 was greatest in the CA3 hippocampal in GWI-rats, we chose this subfield for quantification. In naïve controls, neurons expressed nuclear Nrf2 infrequently. Further, neurons positive for Nrf2 displayed minimal Nrf2 expression (one or two speckles) in the nucleus . In comparison, more CA3 pyramidal neurons expressed nuclear Nrf2 in GWI-rats . The overall amount of nuclear Nrf2 in these neurons was also greater, which was typically evidenced by three or more speckles of positive staining . GWI-rats had significantly more neurons with nuclear Nrf2  and a greater fraction of neurons with robust nuclear Nrf2 . To validate the activation of Nrf2 in the hippocampus, we performed an ELISA for activated Nrf2 using hippocampal tissue lysate. The overall amount of activated Nrf2 was greater in the hippocampus of GWI-rats, in comparison to naïve controls . Thus, we confirmed that chronic GWI was associated with considerable activation of Nrf2 in the hippocampus.Using confocal microscopy and dual immunofluorescence, we examined if NeuN + neurons in the hippocampus expressed nuclear Nrf2 (activated Nrf2) . These were the following: Tnfa, IL1b, IL1a, transforming growth factor beta (Tgfb), macrophage inflammatory protein 1 alpha , IL6, interferon gamma-induced protein 10 , stem cell factor, a cytokine that binds to c-KIT receptor (Scf), Vegf, Fgfb, IL5, and IL15 . Cytokines/chemokines that were unchanged included macrophage chemoattractant protein 1 (Mcp1), Ccl5 (or Rantes), Ifng, and leptin. The concentration of MDA (a by-product of lipid peroxidation and increased oxidative stress) was also greatly enhanced in the serum of GWI-rats . Thus, multiple cytokines and chemokines were upregulated, at a systemic level, in association with the increased oxidative stress in a rat model of chronic GWI.Many cytokines and chemokines were elevated in the serum, 6 months after exposure to GWIR-Cs and stress , 6 genes encoding proteins involved in ROS metabolism , 4 genes coding proteins involved in oxygen transport , and 20 antioxidant genes . The proteins encoded by these genes serve multiple functions, some of which are briefly outlined in Table 1. Among genes that were statistically up-regulated (Table 1), 8 genes displayed the highest levels of expression (1.5-fold or greater increase). These include Fmo2, Hmox1, Ift172, Sepp1, Slc38a1, Sod3, Srxn1, and Ucp2. Greatly enhanced expression of these genes implied a compensatory reaction against oxidative stress that prevailed in the hippocampus of GWI-rats because, proteins coding some of these genes are directly involved in the regulation of ROS production , inflammation (Mpo), neuroprotection , neurocognitive disorders (Apc and Ctsb), Alzheimer’s disease , amyotrophic lateral sclerosis (Als2), hippocampal resting state functional connectivity (Apoe), reduced neurogenesis (Mpo), anti-apoptotic activity (Dhcr24), axon growth and vesicular trafficking (Dnm2), cellular detoxification and stress response (Gstk1 and Gstp1), DNA repair (Ercc6), and altered presynaptic glutamatergic function (Slc38a1). From these, it appears that persistent oxidative stress and inflammation in the hippocampus caused multiple adverse effects on hippocampus function. Persistently reduced neurogenesis, and hippocampus-dependent cognitive and memory impairments are among the conspicuous symptoms observed in GWI-rats , complex II (Sdhb), complex IV , and complex V (Atp5a1 and Atp5b) of the mitochondrial respiratory chain. Furthermore, nine genes, namely, Atp6ap1, Atp6voa2, Lhpp, Ppa1, Slc25a10, Slc25a20, Ucp1, Uqcrb, and Uqcrc1, which are relevant to mitochondrial/other organelle function also showed elevated expression. Among genes that were statistically up-regulated, eight genes displayed higher levels of expression (1.3-fold or greater increase). These include Atp6a1, Cox6a1, Cox7a2L, Ndufs7, Ndufv1, Slc25a10, and Ucp1. Mitochondrial electron transport generates ATP that is essential for the excitability and survival of neurons (Atp5a1 and Atp5b) could also be attributed to the altered energy requirement due to augmented levels of protein degradation , is typically upregulated in glioblastoma patients and believed to promote alternative activation of microglia , and are elevated during increased oxidative stress and in neurodegenerative conditions such as Alzheimer’s disease  quinone oxidoreductase, and genes coding enzymes for glutathione metabolism and protein degradation via the proteasome and autophagy routes (Alterations in the expression of genes related to oxidative stress, inflammation, neurodegeneration, cognition, and memory were also associated with increased Nrf2 immunoreactivity in hippocampal neuronal nuclei and activated Nrf2 in the hippocampus in general, in GWI-rats. The transcription factor Nrf2 is a dominant regulator of redox homeostasis that curbs inflammation. Nrf2 generates antioxidant and phase II detoxification enzymes under conditions of augmented oxidative stress, which inherently decreases oxidative stress and buildup of toxic metabolites. Nrf2 regulates the expression of multiple cytoprotective genes that share a y routes , 2014. Hy routes . Nrf2 acy routes . Thus, NInterestingly, changes in the hippocampus of GWI-rats were accompanied with increased oxidative stress and inflammation at the systemic level. The concentration of MDA was enhanced in the serum, 6 months after exposure to GWIR chemicals and stress. Furthermore, elevated levels of multiple cytokines and chemokines, which include Tnfa, IL1b, IL1a, Tgfb, Mip1a, IL6, IP10, Scf, Vegf, Fgfb, IL5, and IL15, were observed in the serum. How do these inflammatory markers relate to biomarkers identified in GWI patients? Systemic inflammation has also been reported in GWI patients . StudiesThis study validated that unrelenting oxidative stress, mitochondrial impairment, Nrf2 activation, and inflammation can occur in the hippocampus with exposure to relatively low-doses of GWIR-Cs and mild stress in a rat model. Previous studies have demonstrated hippocampus-dependent cognitive, memory and mood impairments in the same animal prototype. Since the various enduring modifications in the hippocampus elucidated in this study can impede cognitive, memory and mood function, potential therapies for GWI would need to alleviate these adverse changes in the hippocampus for improving function. In this context, effective antioxidant and/or anti-inflammatory drug therapies or compounds and small molecules that can directly or indirectly modulate oxidative stress and inflammation signaling pathways or repair mitochondrial dysfunction in the hippocampus have promise to improve the quality of life in GWI patients. Nonetheless, this study also revealed that persistently increased oxidative stress and inflammation are not restricted to the hippocampus but global in nature. This was evidenced by considerable systemic inflammation typified by enhanced levels of multiple pro-inflammatory cytokines in the serum, consistent with results of multiple blood biomarker studies performed in GWI patients. This finding raises the possibility that hippocampal dysfunction in GWI is one of the adverse outcomes of persistently elevated oxidative stress and inflammation at the systemic level. It is plausible that incessantly higher levels of pro-inflammatory cytokines and chemokines in the circulating blood cause brain dysfunction. Therefore, a larger question to address in the future is whether long-term suppression/modulation of systemic inflammation would be efficacious for improving brain function in GWI patients, in addition to relieving other symptoms such as sickness-like behavior and chronic pain.AS, GS, and BH designed research; GS, BH, DU, AB, SA, BS, and MK performed research and collected data. AS, GS, BH, DU, and AB analyzed and interpreted data; GS and AS wrote the paper. All authors gave inputs to the manuscript text and approved the final version of the manuscript.The contents of this article suggest the views of authors and do not represent the views of the United States Department of Veterans Affairs, Department of Defense or the United States Government.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cysteinyl leukotrienes (CysLTs) play a key role in inflammatory diseases such as asthma and their receptors’ antagonists are currently used as anti-asthmatic drugs. CysLTs have also been found to participate in other inflammatory reactions. Here, we reported that in rheumatoid arthritis (RA) animals model, collagen-induced arthritis, (CIA), CysLT1, a receptor for CysLTs, was up-regulated in hind paw and lymph node, while CysLTs levels in the blood were also higher than normal mice. Montelukast, a drug targeting CysLT1, has been shown to effectively reduce the CIA incidence, peak severity, and cumulative disease scores. Further study indicated that CysLT1 signaling did not affect the differentiation of pathogenic T helper cells. We conclude that montelukast may play important roles in the pathogenesis of CIA, mainly by inducing infiltration of pathogenic T cells, increasing IL-17A secretion and expression of IL-17A, while these effects can be blocked by CysLT1 antagonists. Our findings indicate that antagonist of CysLT1 receptor may be used to treat rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by immune-mediated synovitis, local inflammatory cells infiltration and neoangiogenesis , 2. AlthG protein-coupled receptors (GPCRs) mediate many important biological pathways and are targeted by the largest proportion in the current medicine market , 11. ManLeukotrienes (LTs), an endogenous signaling molecule, are potent pro-inflammatory mediators which involve in host defense and some inflammatory diseases -18 via dcPLA2α, 5-LO and CysLT1 mRNA levels were up-regulated, and CysLTs levels in serum were also elevated. Blocking of CysLT1 with its antagonist, montelukast, relieved the CIA’s clinical symptoms. We demonstrated that CysLTs signaling pathway play a vital role in the pathogenesis of CIA, primarily by up-regulating the expression of IL-17A gene, then inducing production of IL-17A, and infiltration of pathogenic T cells which can be relieved by blocked CysLT1 signaling pathway. Our results demonstrate that the CysLT1 antagonists may be used to treat RA.The role of CysLT1 signaling pathway in the pathogenesis of RA is unclear. In our study, we discovered in CIA mouse, a RA animal model, cPLA2α mRNA was increased at week 3 and week 6 post booster immunization  in CIA mice. Compare to control mice, the CysLTs levels in serum , CD4+ T cells, CD8+ T cells and B cells in blood  or montelukast (100μm). Cells were harvested 3 days later and performed intracellular staining for IFN-γ, IL-17, or Foxp3 , 150 mm NaCl, 0.1% CHAPS, 1 mm EDTA, 1 mm NaF, 1 mm Nap < 0.05 was considered statistically significant.Data are presented as mean±SEM. The statistical significance of the CIA clinical scores between treatments was analyzed with two-way ANOVA test. And the CIA scores at a given date were analyzed by Mann-Whitney test. Other analyses, including gene expression, cytokine production, were assessed by Student’s t-test or two-way ANOVA test.


Hemophagocytic lymphohistiocytosis (HLH) is a histiocytic disorder characterized by a highly stimulated, but ineffective, immune response to antigens, which results in life-threatening cytokine storm and inflammatory reaction. Considerable progress has been made during the past 2 decades. Detection of molecular genetic abnormalities in genes involved in immune response pathways, such as PRF1, STX11, UNC13D, STXBP2, RAB27A, LYST, AP3B1, SH2D1A, and BIRC4, is confirmatory for the diagnosis. Clinical diagnosis is largely made according to HLH-2004 criteria. However, a new finding of the Th1/Th2 cytokine pattern  is a useful biomarker for the early diagnosis, differential diagnosis, and the monitoring of the disease. Intensive immunosuppressive therapy is generally accepted as treatment for the relief of clinical symptoms/signs, while allogeneic hematopoietic stem cell transplantation is currently the only potentially curative therapy option for severe familial forms of HLH.


Epstein–Barr virus (EBV) was first discovered in cells from a patient with Burkitt lymphoma (BL), and is now known to be a contributory factor in 1–2% of all cancers, for which there are as yet, no EBV-targeted therapies available. Like other herpesviruses, EBV adopts a persistent latent infection in vivo and only rarely reactivates into replicative lytic cycle. Although latency is associated with restricted patterns of gene expression, genes are never expressed in isolation; always in groups. Here, we discuss (1) the ways in which the latent genes of EBV are known to modulate cell death, (2) how these mechanisms relate to growth transformation and lymphomagenesis, and (3) how EBV genes cooperate to coordinately regulate key cell death pathways in BL and lymphoblastoid cell lines (LCLs). Since manipulation of the cell death machinery is critical in EBV pathogenesis, understanding the mechanisms that underpin EBV regulation of apoptosis therefore provides opportunities for novel therapeutic interventions. All viruses possess methods to negotiate and subvert the cell death pathways of their hosts, but for viruses such as influenza that lead to acute infections, the battle for host cell survival is ultimately lost when the host clears the virus. Persistent viruses however, such as those of the herpesvirus family that are carried by the host for life, must avoid elimination by immune cells, whilst continuing to disseminate within the host and avoid being lost through normal cell turnover. Human herpesvirus 4, also known as Epstein–Barr virus (EBV), is extremely efficient at establishing a persistent life-long infection in human B cells. Most primary infections occur asymptomatically in early childhood and by adulthood the majority (95% world-wide) of the adult population are infected with EBV . SomewhaThe existence of EBV was first suspected by Denis Parsons Burkitt, a surgeon working in post-war Uganda. In 1958, he published the first detailed clinical study of a strange lymphoma affecting the jaws and abdomens of children across sub-Saharan Africa . BurkittBamHI A rightward transcripts (BARTs) and the BHRF1 locus (BHRF1 miRNAs), respectively  ,14. Thisectively  [18,19,2Upon infection of resting B cells, EBV gene expression, driven by host cell RNA polymerase II, begins almost immediately; the Wp promoter that drives early latent gene expression reaches maximal activity around 8–12 h post-infection (PI). These long and differentially spliced Wp-transcripts preferentially encode EBNA-LP, EBNA-2 and BHRF1 ,26. The MYC, that can both increase proliferation and sensitise cells to apoptosis, through long-range interactions [ARF in a yeast 2-hybrid system and colocalised with p14ARF and p53 transcripts in LCLs [EBNA-2 and EBNA-LP are the first proteins to be expressed following infection of B cells. EBNA2 is a functional mimic of cellular Notch ,35,36 anractions . EBNA-LPractions , is esseractions ,50,51, bractions . Another in LCLs . TherefoINK4a and p14ARF [ARF by EBNA-3A and -3C occurs through epigenetic silencing at their transcriptional start sites (TSS) [The EBNA-3s are a family of three large proteins , which likely arose by gene duplication, that predominantly function as regulators of host cell and virus transcription. Like EBNA-2, the EBNA-3 proteins do not bind DNA directly, but instead transactivate or repress gene expression via interactions with transcription factors, such as RBP-Jκ, for which all four EBNAs compete (reviewed in ). Althoud p14ARF ,67,68,69d p14ARF ,71,72. Aes (TSS) ,64,66,68es (TSS) . It is eThe primary function of EBNA-1 protein is to tether EBV episomes onto host cell chromatin to ensure the viral DNA is replicated during cell mitosis. Therefore, EBNA1 expression is essential for the maintenance of latent infection ,75,76. TLMP1 promotes cell growth and survival and is essential for B cell transformation ,87. FuncLMP2A and 2B are expressed from a series of complex, overlapping transcripts, each driven by its own unique promoter that, like the LMP1p, are transactivated by EBNA-2 during transformation ,109,110.A number of viruses, including EBV, encode homologues of cellular pro-survival BCL-2 family proteins in order to evade apoptosis triggered as part of the anti-viral response. These viral BCL-2 proteins (vBCL-2s) vary in the degree of similarity they display to their cellular counterparts in terms of sequence, structure and function (reviewed in ). AlthouEBV encodes two BCL-2 homologues, called BHRF1 and BALF1. These viral proteins have long been known to be expressed at high levels during lytic cycle, presumably to keep host cells alive to ensure efficient virus replication. They have more recently been shown to also be expressed during the early stages of primary B cell infection in vitro: BHRF1 and BALF1 cDNAs can be detected by day 1 PI ,129. RecL. Like cellular BCL-2 pro-survival proteins, BHRF1 protein has a hydrophobic groove formed by α-helices 2–5 of its 7-helix bundle [Eμ-Myc mouse lymphoma cells in vivo can confer protection from DNA damaging chemotherapeutics [Both BHRF1 and BALF1 contain all four of the functionally important BCL-2 homology domains, BH1-4 ,131. BHRx bundle , into whx bundle ,141,142.apeutics .The mechanism by which BHRF1 confers apoptosis protection is through binding and sequestration of cellular pro-apoptotic proteins. BHRF1 can bind to the cellular BH3-only proteins BIM, PUMA and BID and the apoptosis effector (or executioner) protein, BAK  see Fig. One repBamHI A rightward transcripts (BARTs). Despite abundant BHRF1 and BART primary transcript expression at early time points during transformation, processing of their derivative miRNAs is delayed, peaking at around 72–120 h PI [A number of viral non-coding RNAs are expressed during transformation. Some of these transcripts are reported to regulate cell death during transformation and latency; however, their functions are less well understood than those of the latent proteins. The Epstein–Barr virus encoded RNAs (EBERs) are two highly transcribed small (~170 nt) nuclear RNAs (EBER1 and EBER2) ,149 whos120 h PI .The EBERs are ubiquitously expressed from their own viral promoters in all forms of latent infection. There have been conflicting reports regarding the ability of EBER-KO recombinant viruses to transform B cells, although it should be noted that no previous studies have found the EBERs to be essential for transformation. Interestingly, different groups have used different strains of EBV in their experiments, suggesting that the role of the EBERs in transformation may be context dependent and influenced by the other co-expressed viral proteins. Swaminathan et al. used the transformation incompetent P3HR1 virus strain, which harbours a large genomic deletion spanning the EBNA2 locus, and recombined it with a large fragment of the prototypic B95.8 EBV that was either wt for or lacked the EBER locus (lacking both EBER1 and EBER2). The resulting recombinant wt or EBER-KO EBVs were both transformation competent  and wereThe BHRF1 family of miRNAs consists of one miRNA encoded upstream (miR-BHRF1-1) and two miRNAs encoded downstream (miR-BHRF1-2 and miR-BHRF1-3) of the BHRF1 ORF. During viral transformation of B cells, the BHRF1 miRNAs are processed from the polycistronic Wp and Cp-driven latent transcripts that encode the EBNA proteins and latent BHRF1 protein ,165,166.Other than regulating the expression of the pro-survival, BHRF1 protein, it is also somewhat unclear how the BHRF1 miRNAs might further regulate apoptosis. This is, in part, because it can be difficult to experimentally confirm bona fide miRNA targets and because viral miRNAs sometimes display unique functional features compared to their cellular counterparts, such as tolerating ‘bulges’ in their mRNA seed region binding sites ,171  [BamHI A-derived proteins in EBV-positive cell lines [The  and P2) ,178,179. and P2) . It had  and P2) ,181,182,ll lines ,184. Thell lines . Interesll lines ,165.The prototypic EBV strain, B95.8, from which many of the widely used recombinant EBVs are derived, harbours a large (12 kbp) deletion compared to other EBV isolates ,186, andIn order to identify targets of EBV miRNAs, Dolken et al. carried out immunoprecipitation of RISC complexes (containing miRNAs and their targets) in two EBV-infected BL cell lines and then identified the target mRNA using microarray analyses. They showed that miR-BART3 and miR-BART16 downregulated IPO7 and TOM22 transcripts, respectively , both ofThe use of RNA sequencing technologies has also led to the identification of a large number of novel EBV transcripts in latently infected cells , some ofAlthough EBV is usually found to be latent in infected cells both in vivo and in vitro, the virus must periodically enter into the lytic cycle to generate infectious viral progeny to infect new cells within the host and thereby guarantee persistence and dissemination into new hosts. In vivo, the lytic cycle is thought to occur in terminally differentiated plasma cells and epithelial cells (reviewed in ). In vitThe lytic cycle is also closely linked with cell death since the thousands of EBV particles that are produced eventually cause any cell undergoing lytic replication to lyse, releasing infectious virus ,205. ConIn primary B cells, a number of lytic-cycle-associated EBV genes have been shown to be transcribed early during transformation (1–4 days PI.) including BZLF1, BRLF1, BCRF1 , BALF1, BHRF1 and BMRF1 in the absence of any production of infectious virions ,211,212.ARF [A number of studies have provided insight into the interplay between latent proteins and host cell survival pathways through analyses of early infection (the first 7–10 days PI.) during the establishment of latency . EBV infARF . InteresARF .Following activation of p53, cell death proceeds through the BCL-2 family/intrinsic pathway to apoptosis, via p53-induced upregulation of the BH3-only proteins PUMA and NOXA. These BH3-only proteins initiate cell death by binding to the BCL-2 pro-survival proteins, thereby releasing the executioners of apoptosis BAX and BAK. These proteins form pores in the mitochondrial outer membrane (MOMP) which commits the cell to apoptosis. BAK and BAX can also be directly activated by the BH3-only proteins, BIM, tBID and PUMA, though only PUMA is upregulated by p53 activation (reviewed in ) see Fi. Many huThe in vitro growth transformation of B cells by EBV requires that the virus efficiently activates the cell cycle such that the infected cells will proliferate continually, but also necessitates robust and comprehensive inhibition of cell death . StudiesEBV is known to contribute to various forms of malignancy of lymphoid and epithelial cell origin, together accounting for around 200,000 new cancer diagnoses annually . AlthougThe frequency and geographical incidence of BL in malarial endemic regions of sub-Saharan Africa is consistent with an infectious aetiology and this led to the identification of BL as the first cancer to be associated with EBV infection. Accordingly, EBV was found to reside in more than 95% of BLs in high incidence or endemic BL (eBL) areas. BL also occurs worldwide at a lower incidence, and in these cases, known as sporadic BLs (spBL), EBV is found in 15–85% of tumours, varying by geographical region ,238,239.c-MYC oncogene and a constitutively active immunoglobulin (Ig) gene promoter/enhancer [c-MYC also sensitises cells to apoptosis under conditions of stress [c-MYC is the ARF-MDM2-p53-axis. Accordingly, around 30% of BL biopsies [ARF or MDM2 is frequently altered in their expression in order to circumvent p53-dependent cell death [Eμ-Myc mouse model of BL, which carries a c-MYC; IgM heavy chain enhancer transgene. Blocking the intrinsic apoptotic pathway through genetic deletion of pro-apoptotic BCL-2 family genes or constitutive overexpression of BCL-2 pro-survival proteins can all accelerate lymphoma onset in this mouse model [Common to all cases of BL is the hallmark reciprocal translocation between the enhancer ,241,242.enhancer ,244,245.f stress . Therefobiopsies  and 70% biopsies ,249,250.ll death ,252. Addse model ,258,259.se model ,261.c-MYC itself is frequently mutated in BL, and this is hypothesised to reflect a need to avoid apoptosis activation whilst retaining or enhancing proliferative capability [c-MYC mutant that commonly occurs in BL has been reported to be incapable of inducing BIM and consequently cannot trigger cells into apoptosis [Interestingly, pability ,264,265.poptosis . Outsidepoptosis ,268,269.poptosis ).c-MYC translocations [MYC-driven lymphomagenesis [In EBV-positive BL, the virus is present as multiple viral episomes in every tumour cell and is maintained indefinitely, suggesting a selective advantage associated with virus retention. Treating BL cells with a dominant negative derivative of the viral episome maintenance protein, EBNA1 (dnEBNA1), enforces loss of viral genomes and induces apoptosis, suggesting that EBV is essential for the continued survival of these BL cells ,272. Incocations ,276,277.agenesis , it has In 85% of eBLs, the virus expresses only EBNA1, the EBER transcripts and the miR-BARTs, collectively termed Latency I . EBNA1, The mechanism of apoptosis protection by EBV in Latency I BL appears to be cooperative and to exhibit redundancy. Whilst EBERs are reported to promote survival by upregulating IL10 in some clonal EBV-loss variants of Akata-BL , we and INK4a through epigenetic silencing leading to reduced sensitivity to cell death [In the remaining ~15% of eBLs, EBV adopts a more extensive pattern of gene expression involving EBNA-3A, -3B and -3C, a truncated form of EBNA-LP and the BHRF1 protein, in addition to EBNA1, EBERs and miR-BARTs ,302,303.ll death ,65,304. ll death . Wp-BLs ll death . BHRF1, ll death ,133. Thell death .INK4A, and frequent characteristic mutations in PIK3CA, ARID1A and BCOR genes [INK4A and the 80% incidence of mutations in the PI3K family gene, PIK3CA, suggest that the same key signalling pathways identified in BL and LCLs are also important in GC pathogenesis. Similarly, in the setting of NPC, where EBV establishes a Latency II infection, the INK4A locus (encoding p16INK4A and p14ARF) is commonly mutated or silenced. Therefore, possible cooperation between EBV genes to regulate key survival signalling pathways, in particular BCL-2 family signalling, PI3K signalling and the ARF-MDM2-p53-axis, in EBV-associated cancers warrants further investigation.Cooperation among EBV genes to inhibit cell death is clearly important for both growth transformation and the pathogenesis of BL; however, relatively little is known about how EBV genes cooperate outside of these models. There is evidence in the literature to suggest that EBV targets common pathways in different disease settings. For example, EBV is now known to be an etiologic factor in around 9% of gastric carcinomas (GC). As in other EBV-associated cancers, the virus is present as a latent infection in these tumours, although there are conflicting reports as to whether it establishes Latency I or II infection (the latter involves expression of LMP1 and LMP2 proteins in addition to the Latency I genes) . Like BLOR genes ,307. TheWhilst it is clear that the known latent genes of EBV make a considerable contribution to the apoptosis phenotype of EBV-associated cancers, the roles of other EBV-encoded genes in this regard require further attention. Lytic-cycle-associated transcripts can be readily detected in EBV-positive cancers ,306,308 Whilst the evidence that EBV can regulate apoptosis is compelling, future investigations may also uncover mechanisms through which EBV can modulate additional forms of cell death. For example, the LMP1 protein has been reported to modulate autophagy by both NF-κB-dependent and -independent mechanisms ,319 and


However, simultaneous substitutions of these amino acids did not significantly increase the virulence of JaOArS982, suggesting that high mortality due to JaTH160 viral infection cannot be simply attributed to the specific amino acids. Multiple and complex, but not simple, mechanisms may induce the high mortality of JaTH160 infection in mice.For the development of effective treatment strategies for Japanese encephalitis (JE), it is important to identify the viral factors causing severe disease during JE virus (JEV) infection. In this study, we assessed whether amino acid substitutions are critical factors for higher mortality of JaTH160 compared with JaOArS982 in mice using the technique of infectious cDNA clones. We raised the possibility that two amino acids of C Flaviviridae, genus Flavivirus [Japanese encephalitis (JE) virus (JEV) causes approximately 30,000 to 50,000 cases and 10,000 to 15,000 deaths in Asian countries annually . JEV belavivirus , whose gavivirus ,7. HowevTo evaluate the disease pathogenesis in JEV infection, mice have been employed as a useful infection model ,9,10. Mo49, E123, E138, E176, E306, and E389/390 affect the virulence of flaviviruses in the CNS [42,43, prM15,17, NS2A23, and NS5879–881,891, contribute to the flavivirus pathogenicity in mice [Previous studies have demonstrated that amino acid substitutions are major factors affecting the virulence in flavivirus infections. For example, it has been shown that amino acid substitutions in the E protein, such as E the CNS ,12,13,14 in mice ,17,18. WJaTH160, but not JaOArS982, expresses the NS1’ protein and that NS1’ enhances JEV production in avian cells and embryonated chicken eggs . Previou113 phenylalanine that affects the efficient propagation of JaTH160 in neuroblastoma Neuro-2a cells, but not extraneural origin cells compared with the JaOArS982 strain [113 phenylalanine is responsible for the high pathogenicity of JaTH160. However, this amino acid did not affect viral loads in the brain nor survival curves in mice, suggesting that virus propagation in vitro may not reflect the level of virus neurovirulence in vivo [We also identified a unique amino acid of NS2A2 strain . Therefo in vivo .There are 19 amino acid differences between JaTH160 and JaOArS982. Therefore, in the present study, we aimed to identify the amino acids affecting the virulence and to assess whether those amino acid substitutions are critical to determine the high mortality due to JaTH160 infection by a reductionist approach using the infectious cDNA clone techniques of JaTH160 and JaOArS982 strains.The animal experiment protocols were approved by the Animal Care and Use Committee of Nagasaki University . The animal experiments were performed in accordance with the recommendations in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology.The full-length cDNA clones S982-IC and JaTH-IC were developed from the genome sequences of JaOArS982 and JaTH160 strains, respectively, as described previously . Based o4 PFU of JEV diluted in EMEM containing 2% FCS. Mock infected mice were inoculated with supernatant of cultured BHK cells. Mice were weighed daily, and survivals were recorded for 21 days.C57BL/6j (B6) mice were purchased from Japan SLC, Inc, or Japan CLEA, Inc. Five- to seven-week-old mice were subcutaneously inoculated with 10p value of <0.05 was considered statistically significant.The log-rank (Mantel-Cox) test was used for statistical analysis to assess the significant differences of survival curves between each recombinant virus and S982-infectious clone (IC) virus. A Amino acid differences between JaOArS982 and JaTH160 are shown in We first constructed four recombinant viruses  based on S982-IC by introducing four regions of JaTH-IC C. MortalWe next constructed recombinant viruses of J-S1, J-S2, J-S3, and J-S4 based on JaTH-IC by replacing the four regions of S982-IC A. Infect323 and NS336-NS5567 may contribute to the high mortality due to JaTH-IC virus infection. Thus, we examined the mortality due to S-J13 virus infection in mice, whose regions of 5’UTR-NS1323 and NS336-NS5567 were of JaTH-IC, and other regions were of S982-IC  and examined the mortality in virus-infected mice , interferon gamma (IFN-γ), interleukin (IL)-2, and IL-10 in the brain, and the decrease of TNF-α in the spleen in JaTH160 infected-mice . From th


Pseudoalteromonas sp. strain SAO4-4, a protease-producing bacterium from seawater, is composed of two circular chromosomes and one plasmid. This genome sequence will provide a better understanding of the ecological roles of protease-producing bacteria in the degradation of organic matter in marine aquatic environments.The complete genome of  Pseudoalteromonas sp. strain SAO4-4, was isolated from seawater of the Atlantic Ocean. The total genomic DNA was extracted using a PowerMax soil DNA isolation kit . The complete genome sequence of strain SAO4-4 was determined using a combination of PacBio RS II single-molecule real-time (SMRT) sequencing technology  and Solexa sequencing . A total of 808 Mb of clean data  from the Solexa sequencing were obtained, representing 162-fold coverage. De novo assembly was performed using the Hierarchical Genome Assembly Process (HGAP) pipeline of the SMRT Analysis version 2.3.0. A total of 3 contigs greater than 500 bp were obtained. Gene calling and annotation were performed using Rapid Annotations using Subsystems Technology (RAST) version 2.0  and one plasmid, having a total size of 5,099,119 bp, with a mean G+C content of 40.7 mol%. Chromosomes 1 and 2 contain 3,282,406 bp and 1,667,761 bp, respectively, and have similar G+C contents  and percentages of coding regions . The genome in total has 4,556 protein-coding genes and 122 RNA genes.MEROPS peptidase database  are serine peptidases and metallopeptidases. These 83 serine peptidases and metallopeptidases can be assigned into 19 metallopeptidase families and 10 serine peptidase families listed in the database  as nutriIn summary, the genome analysis for strain SAO4-4 identifies many genes coding for extracellular enzymes able to decompose protein and other polymeric organic substances, including polysaccharides, lipids, and DNA, providing a better understanding of the ecological roles of the strain and its extracellular enzymes, especially proteases, in the degradation of organic matter and elemental cycles  in seawater.Pseudoalteromonas sp. SAO4-4 has been deposited in DDBJ/EMBL/GenBank under the accession numbers CP023398 for chromosome 1, CP023399 for chromosome 2, and CP023400 for plasmid pl.The complete genome sequence of


CaAGO, 4 CaDCL and 6 CaRDR genes in pepper and compared them with those of Arabidopsis, tobacco, potato and tomato. Detailed phylogenetic analyses revealed that each CaAGO, CaDCL and CaRDR protein family were classified into four clades. The tissue specific expression and respond to abiotic or biotic stress were studied. The real-time quantitative polymerase chain reaction (PCR) results demonstrated that CaAGO2, CaAGO10b, CaDCL2 and CaDCL4 were upregulated with cucumber mosaic virus (CMV), potato virus Y (PVY) and tobacco mosaic virus (TMV) infections, whereas they showed difference expression patterns in response to abiotic stress. In addition, we found that many of the candidate genes were induced by phytohormones and H2O2 treatment. Our results provide useful information for further elucidation of gene silencing pathways and RNAi-mediated host immunity in pepper.RNA silencing is an evolutionarily conserved mechanism that regulates variety of cellular processes in plants. Argonaute protein (AGO), Dicer-like protein (DCL) and RNA-dependent RNA polymerase (RDR) are critical components of RNA silencing. These efficient and indispensable components of the RNAi pathway have not been identified and characterized in pepper. In this study, we identified 12  Plants have evolved some control mechanisms that efficiently prevent pathogen invasion to protect themselves from pathogen attack throughout their life-cycles . RNA silAGO, DCL and RDR gene families in Arabidopsis, rice, tomato and maize have advanced our understanding of RNA silencing [AtAGOs, four AtDCLs and six AtRDRs in Arabidopsis thaliana [OsDCLs, 19 OsAGOs and five OsRDRs genes were identified, in which OsAGO2 showed specific upregulation in response to cold, salt and dehydration stress [SlDCLs, 15 SlAGOs and six SlRDRs were identified in tomato. The expression models of tandem gene duplications among SlDCL2s indicate that the DCL2 family plays an important role in the evolution of tomato [CsAGOs, CsDCLs, and CsRDR genes, respectively, have been identified in cucumber. All CsAGOs, especially CsAGO1c, CsAGO1d, and CsAGO7, were profusely upregulated in leaves and tendrils compared to that in other organs, whereas all CsDCL genes showed a higher up regulation in tendrils, with almost no expression of CsDCL1, CsDCL4a, or CsDCL4b in other organs. In addition, CsRDR1a, CsRDR2, CsRDR3, and CsRDR6 were relatively upregulated in tendrils, but almost all CsRDRs are downregulated in other organs [Brassica napus possessed eight BnDCLs, 27 BnAGOs, and 16 BnRDRs [VvDCLs, 13 VvAGOs, and five VvRDRs were identified. It was worth mentioning that one gene, VvAGO10a, was only expressed in the stem, suggesting that VvAGO10a might function in the regulation of siRNAs in the grapevine stem [In recent years, studies of the ilencing ,10,11. Tr organs . Genome 6 BnRDRs ,14. In gCaRDR1 from pepper, which was induced by salicylic acid (SA) and tobacco mosaic virus (TMV). CaRDR1 played a positive role in pepper TMV resistance by regulating antioxidant enzymes’ activities and the expression of RNA silencing-related genes [AGO, DCL and RDR gene families were examined in response to biotic/abiotic stress. These results provide useful information for further elucidation of RNA silencing pathways and RNAi-mediated host immunity in pepper.Pepper is one of the most important vegetable crops in the world. However, its productivity is severely affected by viral disease ,17. In ped genes . In thisCaPR1 was induced by cucumber mosaic virus (CMV), potato virus Y (PVY) and TMV infections. The expression level of CaDEF1 upregulated after abscisic acid (ABA), H2O2, MeJA, SA, NaCl and PEG treatments, and CaEREBP-C1 was induced by cold treatment  profiles of the conserved PIWI, DCL (RNase III) and RdRP, and then used BLAST-p to search a draft pepper genome sequence on the genome database  in flower, especially CaAGO1b, CaAGO5 and CaAGO10b  was performed to analyze the transcript level of CaAGO10b . CaAGO2 s organs . All of to roots . CaRDR1  tissues .CaAGOs, CaDCLs and CaRDRs in response to biotic stressors, we inoculated pepper leaves with TMV, CMV and PVY, and measured the expression of CaAGOs, CaDCLs and CaRDRs. At 7 day-post inoculation (dpi) with the viruses, the expression of CaAGO genes was differentially expressed in pepper leaves  and SA treatment. Gene expression analysis at 24 h after phytohormones and H2O2 treatment showed that CaAGO10a and CaAGO10b were significantly induced by ABA, especially CaAGO10b (20 folds). Similarly, H2O2 increased the transcripts of CaAGO1a, CaAGO2, CaAGO5, CaAGO6, CaAGO10a and CaAGO10b  in wild-type, ago2 mutants displayed an enhanced susceptibility to Pst [Nicotiana benthamiana, NbAGO2 contributed to anti-viral defense, and suppression of the NbAGO2 expression enhanced susceptibility to TBSV [ago1 and ago2 mutants were hypersensitive to viral infection in plant [AGO2 mRNA is targeted by the miR403 [AGO1 but not AGO2 were repressed after SMV infection, which were regulated by the upregulation of miR168a and miR403a [CaAGO2 contained an miR403 binding site  [CaAGO1a/b and CaAGO10b increased by CMV, PVY and TMV infection. It implied that CaAGO1 might have played a positive role in pepper through other regulatory mechanisms. In line with previous reports, our findings suggested that the AGO family genes might function collaboratively in RNA silencing-mediated viral defense in plants. Importantly, CaAGOs were also induced by aboitic stress  . AGO1 fug (PTGS) . The expc stress . Here, co stress . CaAGO2 d by ABA . ABA is CaDCL1 and CaDCL3 exhibited a higher expression level in flowers than other organs. In Arabidopsis, DCL1 and DCL3 promote flowering, whereas double mutants of dcl1 and dcl3 exhibited a delay in flowering [CaDCL1 and CaDCL3 might also be involved in pepper flower development. Moreover, the transcripts of CaDCL2 and CaDCL4 were significantly induced upon virus inoculation , DCL2 generated 22-nt siRNAs, which share functional overlap in antiviral defense with DCL4-generated 21-nt siRNAs, and DCL3-produced 24-nt heterochromatic siRNA (hc-siRNA) mediated DNA methylation, gene silencing and chromatin modification ,60,61,62lowering . It implculation B. In tom results . The expreatment . These fs stress .RDRs had been identified in Arabidopsis [CaRDR genes in pepper, which was expressed in all five tissues. Several lines of evidence suggested that plant RDR1 was involved in antiviral defense [NgRDR6 could be induced by ABA, GA, MeJA, CMV, but not by PVY, TMV, H2O2 and SA in Nicotiana glutinosa [CaRDR6 was induced by CMV, PVY, TMV, ABA and H2O2, indicating an important role of RDR6 in different signal pathways in a range of plant species  and SGN (https://solgenomics.net/). Protein sequence was analyzed for domain structure using Pfam (http://pfam.xfam.org/). DCLs, AGOs, and RDRs in pepper were identified by using Hidden Markov Model (HMM) profiles and BLAST-P to search the Pepper Genome database (http://peppersequence.genomics.cn/page/species/index.jsp). All identified genes in this study were named after the homologies sequence in the same gene family of tomato. The conserved domains of the gene sequences were searched using the Simple Modular Architecture Research Tool . The molecular weight (MW) of CaDCL, CaAGO, and CaRDR proteins were predicted using ExPASy ComputepI/Mwtool (http://au.expasy.org/tools/pi_tool.html). The gene information, including accession number, chromosome location, coding sequence (CDS) length and encoded protein length were downloaded from the pepper genome database.Protein sequences of tomato DCLs, AGOs, and RDRs were downloaded from NCBI (The phylogenetic trees were constructed by the Neighbor-Joining (NJ) method following the Poisson model using MEGA 5.0 ,76. The Capsicum annuum L.) cultivars P79 was used in the present study. Pepper seedlings were grown in a plant growth chamber under a 16 h/8 h light/dark period at 25 °C/20 °C. Tissue samples were collected from roots, leaves, stems, flowers and fruit.Pepper  polyethylene glycol (PEG), 200 mM sodium chloride (NaCl) and placed at 4 °C, respectively. Leaves were collected at 24 h after treatment. Samples were frozen in liquid nitrogen and stored at −80 °C.For abiotic stress, such as drought, salinity and cold, 8-week-old seedlings of pepper were treated with 20%  mechanically. Leaves were collected at 7 d after treatment. Samples were frozen in liquid nitrogen and stored at −80 °C.® Premix Ex Taq . Pepper ubiquitin-conjugating protein (CaUbi3) was used as an internal reference gene [Ct−ΔΔ method [An Omega plant RNA kit  was used for RNA extracted. qRT-PCR performed using iQ5 Real-Time PCR Detection System  with SYBRnce gene . Three bt method . Primersp < 0.05.SPSS software was used for statistical analysis. The treatments were compared with the control using Tukey’s test at CaAGO, four CaDCL and six CaRDR genes were identified in pepper plants. We discussed the structures and conserved domains of these genes and performed a detailed phylogenetic analysis that classified CaAGO, CaDCL and CaRDR gene families into four clades. Tissue specific expression analysis revealed that CaAGO, CaDCL and CaRDR genes showed multiple patterns of expression in different organs. We also analyzed expression of CaAGOs, CaDCLs and CaRDRs in response to abiotic and biotic stressors. CMV, PVY and TMV infections upregulated CaAGO2, CaAGO10b, CaDCL2, CaDCL4 and CaRDR6 expression, whereas cold, drought and salinity treatments induced various CaAGOs, CaDCLs and CaRDRs expression in pepper. Additionally, a potential involvement of phytohormones in regulating many of the candidate genes was speculated. RNA silencing components showed distinct role in stress responses of pepper. The results play a basis role in further functional characterization of these genes.In this study, a total of 12