Epigenetically-regulated RPN2 gene influences lymphocyte activation and is involved in pathogenesis of rheumatoid arthritis.

BACKGROUND: To identify RA-associated genes and to ascertain epigenetic factors and functional mechanisms underlying RA pathogenesis. METHODS: Peripheral blood mononuclear cells (PBMC) transcriptome- and proteome- wide gene expressions were profiled in a case-control study sample. Differentially expressed genes (DEGs) were discovered and validated independently. In-house PBMC genome-wide SNP genotyping data, miRNA expression data and DNA methylation data in the same sample were utilized to identify SNPs [expression quantitative trait locus (eQTLs) and protein quantitative trait locus (pQTLs)], miRNAs, and DNA methylation positions (DMPs) regulating key DEG of interest. Lentivirus transfection was conducted to study the effects of RPN2 on T lymphocyte activation, proliferation, apoptosis, and inflammatory cytokine expression. Rpn2 protein level in plasma was quantitated by ELISA to assess its performance in discriminating RA cases and controls. RESULTS: Twenty-two DEGs were discovered in PBMCs. The most significant DEG, i.e., RPN2, was validated to be up-regulated with RA in PBMCs. A complex regulatory network for RPN2 gene expression in PBMCs was constructed, which consists of 38 eQTL and 53 pQTL SNPs, 3 miRNAs and 2 DMPs. Besides, RPN2 expression was significantly up-regulated with RA in primary T lymphocytes, as well as in PHA-activated T lymphocytes. RPN2 over-expression in T lymphocytes significantly inhibited apoptosis and IL-4 expression and promoted proliferation and activation. PBMCs-expressed RPN2 mRNA and plasma Rpn2 protein demonstrated superior and modest performances in discriminating RA cases and controls, respectively. CONCLUSIONS: RPN2 gene influences T lymphocyte growth and activation and is involved in the pathogenesis of RA. Rpn2 may serve as a novel protein biomarker for RA diagnosis.

ITGA2 protein is associated with rheumatoid arthritis in Chinese and affects cellular function of T cells.

BACKGROUND: We identified proteins significant for rheumatoid arthritis (RA) in peripheral blood mononuclear cells (PBMCs), and to clarify mechanisms mediated by underlying proteins that may involve in the pathogenesis of RA. METHODS: Proteome-wide protein expressions were profiled by employing label-free quantitative proteomics methodology (Easy-nLC1000 and Q-exactive). The t-test was applied to identify differentially expressed proteins (DEP, p ≤ 0.05) between RA case and control samples. Gene Ontology enrichment analyses and Protein-Protein Interaction analyses were performed to annotate functions of DEPs. The selected DEP was validated in independent samples using Simple Western assay. Plasma protein level of α2 component of integrin (ITGA2) was measured by using ELISA. The DEP, ITGA2, was assessed for its effects on T cell proliferation, cell cycle, apoptosis, and inflammatory cytokine expression. RESULTS: Sixty-four DEPs (p < 0.05) were identified in PBMCs. The selected ITGA2 (Fold Change, FC = 2.20, p = 1.49E-02) was validated to be up-regulated (FC = 12.33, p = 4.90E-2) with RA, and plasma ITGA2 protein level significantly elevated in RA patients vs. controls. Over-expression of ITGA2 could promote proliferation and inhibit apoptosis of Jurkat T cell, and induce IL-8, IFN-γ and TNF-α expression in Jurkat T cells. CONCLUSIONS: ITGA2 protein was significantly over-expressed in PBMCs in RA patients, and affects T cell growth and pro-inflammatory cytokine expression in T cells.

PFOS-induced placental cell growth inhibition is partially mediated by lncRNA H19 through interacting with miR-19a and miR-19b.

Perfluorooctane sulfonic acid (PFOS), a persistent environmental pollutant, has been associated with decreased birth weight. The dysregulation of long non-coding RNA (lncRNA) H19 has been implicated in pregnancy complications such as intra-uterine growth retardation (IUGR), preeclampsia (PE), however, the expression and function of H19 in PFOS-exerted detrimental effects in the placenta remains to be unveiled. Here, we explored the role of H19 in PFOS-induced placental toxicity. Results showed that PFOS caused decreased cell growth in human HTR-8/SVneo cells. Expression of H19 was increased, while miR-19a and miR-19b expression were decreased in mice placenta tissues and in HTR-8/SVneo cells exposed to PFOS. A significant hypomethylation was observed at the H19 promoter in the placentas of mice that were gestational exposed to high dose of PFOS. H19 was confirmed to bind with miR-19a and miR-19b, targeting SMAD4. Furthermore, H19 appeared to partially improve the cell growth of HTR-8/SVneo cells exposed to PFOS via upregulation of miR-19a and miR-19b. In summary, our findings revealed that H19/miR-19a and miR-19b/SMAD4 axis exerted important functions in PFOS-induced placenta cell toxicity.

Petunidin-Based Anthocyanin Relieves Oxygen Stress in Lactobacillus plantarum ST-III.

Application of probiotics in the food industry has been hampered by their sensitivity to challenging conditions that reduce their vitality in food matrices. A lot of attempts have been made to promote the growth of these probiotics in the aspect of nutrition demands. Among the other adverse conditions, oxygen stress can restrict the growth of probiotics and has not yet been paid enough attention to. In this study, the effect of a petunidin-based anthocyanin (ACN) on the growth of probiotic Lactobacillus plantarum ST-III was investigated under oxygen stress. The growth of ST-III was analyzed through spot assay on agar plates as well as plating-based enumeration of the viable cells in the liquid culture. Results indicated that ACN could efficiently improve the growth of ST-III under oxygen stress, whereas no effect was observed in the absence of oxygen stress. Further investigations indicated that ACN reduced the oxido-reduction potential of the culture; meanwhile, it exerted a positive transcriptional regulation on the thioredoxin system of ST-III, leading to a decrease in reactive oxygen species accumulation within the cells. Moreover, ACN enabled the growth of ST-III in reconstituted skim milk and promoted the formation of milk clots. These results revealed the role of a petunidin-based ACN in oxygen stress relief and highlighted its potential in manufacture and preservation of L. plantarum-based dairy products.

Evaluation of plasma cytokine protein array profile: the highlighted PDGF-BB in rheumatoid arthritis.

OBJECTIVES: The cytokines play critical roles in the complex pathogenesis of rheumatoid arthritis (RA), but the specific cytokines are still in need of being discovered. This multi-stage study was performed to identify novel RA cytokines in plasma and further understand the pathological mechanism of the identified cytokines. METHOD: The plasma cytokine protein profile was evaluated by using Human Cytokine Antibody Array 440 in 18 subjects (RA: healthy control = 9:9). Then, enzyme-linked immunosorbent assay (ELISA) was used to validate the highlighted cytokines in 80 subjects (RA: healthy control = 40:40). Further functional experiments on fibroblast-like synoviocytes were performed to identify the pathological mechanisms of the highlighted cytokines for RA. RESULTS: A total of seven significant cytokines have differential expressions between RA patients and controls (fold change (FC) > 2, P value < 0.05). The difference in plasma for the highlighted platelet-derived growth factor (PDGF)-BB was validated in an independent validation sample (P = 0.005). Further, the PDGF-BB obviously promotes cell proliferation of MH7A cell, probably by inhibiting cell apoptosis and accelerating the cell cycle. The PDGF-BB can also promote MH7A cell migration. CONCLUSIONS: This study evaluated the plasma cytokine protein array profile associated with RA and highlighted the importance of PDGF-BB. PDGF-BB has an important role in RA-FLS proliferation and migration. These results suggest that PDGF-BB might contribute to occurrence and development of RA. Key Points • The levels of plasma cytokines were systemically tested using Human Cytokine Antibody Arrays. • The expression difference of PDGF-BB was validated in an independent sample. • PDGF-BB obviously promotes cell proliferation and migration in RA-FLS.

Multi-omics integrative analysis identified SNP-methylation-mRNA: Interaction in peripheral blood mononuclear cells.

Genetic variants have potential influence on DNA methylation and thereby regulate mRNA expression. This study aimed to comprehensively reveal the relationships among SNP, methylation and mRNA, and identify methylation-mediated regulation patterns in human peripheral blood mononuclear cells (PBMCs). Based on in-house multi-omics datasets from 43 Chinese Han female subjects, genome-wide association trios were constructed by simultaneously testing the following three association pairs: SNP-methylation, methylation-mRNA and SNP-mRNA. Causal inference test (CIT) was used to identify methylation-mediated genetic effects on mRNA. A total of 64,184 significant cis-methylation quantitative trait loci (meQTLs) were identified (FDR < 0.05). Among the 745 constructed trios, 464 trios formed SNP-methylation-mRNA regulation chains (CIT). Network analysis (Cytoscape 3.3.0) constructed multiple complex regulation networks among SNP, methylation and mRNA (eg a total of 43 SNPs simultaneously connected to cg22517527 and further to PRMT2, DIP2A and YBEY). The regulation chains were supported by the evidence from 4DGenome database, relevant to immune or inflammatory related diseases/traits, and overlapped with previous eQTLs from dbGaP and GTEx. The results provide new insights into the regulation patterns among SNP, DNA methylation and mRNA expression, especially for the methylation-mediated effects, and also increase our understanding of functional mechanisms underlying the established associations.

Synthesis and biological evaluation of novel H6 analogues as drug resistance reversal agents.

Hederagenin is a naturally occurring pentacyclic triterpenoids compound with multiple pharmacological activities. We recently showed that H6, a synthetic derivative of hederagenin, could enhance the anticancer activity of paclitaxel in drug-resistant cells in vitro and in vivo, but showed poor solubility. With the aim of improving the drug resistant reversal activity of H6, here we designed and synthesized a series of novel H6 analogues. Our results showed that compound 10 at the concentration of 5 µM significantly enhanced the cytotoxicity of paclitaxel to drug-resistant KBV cells and sensitized cells to paclitaxel in arresting cells in G2/M phase and inducing apoptosis. We found that compound 10 might block the drug efflux of P-gp via stimulating P-gp ATPase activity. Importantly, compound 10 enhanced the efficacy of paclitaxel against KBV cancer cell-derived xenograft tumors. Finally, we summarized a preliminary structure-activity relationship of hederagenin by the drug resistant reversal activity of H6 analogues in vitro and compound 10 and H6in vivo. This study highlights the importance of nitrogen-containing derivatives of hederagenin C-28 in the development of novel drug resistance reversal agents.

Extrusion of Dissolved Oxygen by Exopolysaccharide From Leuconostoc mesenteroides and Its Implications in Relief of the Oxygen Stress.

Strains of Leuconostoc are generally facultatively anaerobic and exposure to oxygen might be detrimental; therefore, strategies to combat the oxygen stress are essential for these bacteria to survive and flourish in the oxygenic atmosphere. Despite the extensive applications in industry, the fundamental issues concerning the aerobic life of Leuconostocs remain to be addressed. In this study, we have demonstrated that Leuconostoc mesenteroides CGMCC10064 cultivated in sucrose medium would acquire a growth advantage over that in glucose medium under oxygenic conditions, as reflected by more viable cells and less accumulation of reactive oxygen species. Further analysis showed that the growth advantage was dependent on exopolysaccharide (EPS) synthesized by a secreted glucansucrase. Determination of the dissolved oxygen in the culture suggested that the growth improvement was mediated by extrusion of dissolved oxygen from the aqueous circumstances. Growth experiments performed with the purified EPS showed that supplementation of 5 g/L EPS in the medium could improve the aerobic growth of L. mesenteroides by ∼10-fold. Moreover, the purified EPS was also effective in promoting the aerobic growth of oxygen-sensitive Lactobacillus and Bifidobacterium. These results demonstrate that EPS of L. mesenteroides plays a critical role in relief of the oxygen stress, and suggest the potential of the EPS in manufacture as well as preservation of oxygen-sensitive probiotics.

Association of Plasma Irisin with Bone Mineral Density in a Large Chinese Population Using an Extreme Sampling Design.

Irisin, a myokine produced by skeletal muscle in response to physical exercise, promotes trans-differentiation of white adipose tissue into brown adipose tissue. Recent evidences suggested that irisin also plays an important role in the control of bone metabolism. This study aimed to ascertain the relationship between plasma irisin and bone mineral density (BMD) in Chinese population by adoption of an extreme sampling method. Based on a large and screened Chinese elderly population (N = 6308), two subgroups with extremely high and low hip BMD were selected for discovery (N = 80, high vs. low BMD = 44:36) and validation (N = 60, high vs. low BMD = 30:30), respectively. Plasma irisin, P1NP, and ß-CTx were measured using commercially available ELISA kits. Other metabolic parameters (e.g., blood glucose, total cholesterol and triglycerides) were collected. Student's t test and Spearman correlation analyses were conducted in SPSS. Significant difference was discovered for plasma irisin between females and age-matched males (N = 80, male vs. female = 42:38, P = 0.002). The plasma irisin levels were significantly higher in high BMD subjects than in low BMD subjects, which was observed in both discovery (P = 0.012) and validation samples (P = 0.022). However, such observation was limited to males only. Further correlation analyses in males showed that plasma irisin was correlated with BMD (r = 0.362, P = 0.025) and triglyceride (r = - 0.354, P = 0.032). Plasma irisin levels were associated with hip BMD in Chinese elderly men. This study represented the first effort of investigating the relationship of plasma irisin and BMD in elderly population. The positive correlation between plasma irisin and BMD hints intrinsic communication between muscle and bone.

Molecular and Functional Study of a Branching Sucrase-Like Glucansucrase Reveals an Evolutionary Intermediate between Two Subfamilies of the GH70 Enzymes.

Glucansucrases (GSs) in glycoside hydrolase family 70 (GH70) catalyze the synthesis of α-glucans from sucrose, a reaction that is widely seen in lactic acid bacteria (LAB). These enzymes have been implicated in many aspects of microbial life. Products of GSs have great commercial value as food supplements and medical materials; therefore, these enzymes have attracted much attention from both science and industry. Certain issues concerning the origin and evolution of GSs are still to be addressed, although an increasing number of GH70 enzymes have been characterized. This study describes a GS enzyme with the appearance of a branching sucrase (BrS). Structural analysis indicated that this GS enzyme produced a type of glucan composed of an α-(1→6) glucosidic backbone and α-(1→4) branches, as well as a considerable amount of α-(1→3) branches, distinguishing it from the GSs identified so far. Moreover, sequence-based analysis of the catalytic core of this enzyme suggested that it might be an evolutionary intermediate between the BrS and GS subgroups. These results provide an evolutionary link between these subgroups of GH70 enzymes and shed new light on the origination of GSs.IMPORTANCE GH70 GSs catalyze the synthesis of α-glucans from sucrose, a reaction that is widely seen in LAB. Products of these enzymes have great commercial value as food supplements and medical materials. Moreover, these enzymes have attracted much attention from scientists because they have potential in tailored synthesis of α-glucans with desired structures and properties. Although more and more GSs have been characterized, the origin and evolution of these enzymes have not been well addressed. This study describes a GS with the appearance of a BrS (i.e., high levels of similarity to BrSs in sequence analysis). Further analysis indicated that this enzyme synthesized a type of insoluble glucan composed of an α-(1→6) glucosidic backbone and many α-(1→4)- and α-(1→3)-linked branches, the linkage composition of which has rarely been reported in the literature. This BrS-like GS enzyme might be an evolutionary intermediate between BrS and GS enzymes.

The distribution and functional relevance analysis of runs of homozygosity (ROHs) in Chinese Han female population.

Extended homozygosity is a genomic region in which the copies inherited from parents are identical, and has obvious inter-individual differences in length and frequency. Runs of homozygosity (ROHs), regarded as a type of structure variations, may have potential capacity in regulating gene transcription. To learn more about the genome-wide distribution of ROH regions in humans and understand the potential roles, this study applied ROH-based approach to quantify and characterize ROHs in 41 Chinese Han female subjects, and test potential associations between ROHs and mRNA expressions by eQTL analysis to ascertain whether ROHs are relevant to gene transcription in peripheral blood mononuclear cells (PBMCs). 10,884 ROH regions were identified in human genome. The average cumulative length of ROH regions was 217,250 ± 20,241 kb. The number of core segments in each chromosome generally matched the total length of corresponding chromosome, i.e., the longer the chromosome, the more the core segments. Genes located in the core regions of ROH were significantly enriched in multiple basic metabolism pathways. A total of 226 cis-eQTLs and 178 trans-eQTLs were identified. The cis-effect size was mainly concentrated at ± 0.5; and the trans-effect size was mainly concentrated at -1.5 and 1.0. Genes with eQTL effects were significantly enriched in functions related to protein binding, cytosol, nucleoplasm, nuclear membrane, protein binding and citrate metabolic process. This study described comprehensive distributions and characteristics of ROH in Han female Chinese, and recognized the significant role of ROH associated with gene transcription in human PBMC.

Relative abundance of mature myostatin rather than total myostatin is negatively associated with bone mineral density in Chinese.

Myostatin is mainly secreted by skeletal muscle and negatively regulates skeletal muscle growth. However, the roles of myostatin on bone metabolism are still largely unknown. Here, we recruited two large populations containing 6308 elderly Chinese and conducted comprehensive statistical analyses to evaluate the associations among lean body mass (LBM), plasma myostatin, and bone mineral density (BMD). Our data revealed that total myostatin in plasma was mainly determined by LBM. The relative abundance of mature myostatin (mature/total) was significantly lower in high versus low BMD subjects. Moreover, the relative abundance of mature myostatin was positively correlated with bone resorption marker. Finally, we carried out in vitro experiments and found that myostatin has inhibitory effects on the proliferation and differentiation of human osteoprogenitor cells. Taken together, our results have demonstrated that the relative abundance of mature myostatin in plasma is negatively associated with BMD, and the underlying functional mechanism for the association is most likely through inhibiting osteoblastogenesis and promoting osteoclastogenesis.

[Relationship between serum zinc levels and attention deficit hyperactivity disorder in children].

OBJECTIVE: To study the possible relationship between serum zinc levels and attention deficit hyperactivity disorder (ADHD) in Chinese children. METHODS: Following a systematic search for case-control studies on the serum zinc levels in Chinese children with ADHD published between 2000 and 2015, a Meta analysis was conducted using Stata 12.0 software. RESULTS: A total of 17 studies, including 2 177 children with ADHD and 2 900 normal children, were enrolled. The Meta analysis showed that serum zinc levels in children with ADHD were lower than normal children (SMD= -1.33; 95%CI: -2.22, -0.44; P=0.003). The sensitivity analysis indicated that the results were reliable. Eggerγs test did not find the existence of publication bias. CONCLUSIONS: Serum zinc levels may be associated with susceptibility to ADHD in children.

[Pollution Status and Migration of Mercury in the Sediments of Nansi Lake in Shandong Province].

The content of mercury and fractions were determined, in order to discuss the pollution situation and migration ability of mercury in inflow rivers and surface sediments of Nansi Lake. The average content of mercury in the sediments of the Nansi Lake was 0.046 mg · kg(-1), significantly higher than the environmental background values, which showed that Nansi Lake suffered from mercury pollution. Mercury in sediments existed mainly in the residual fraction, accounting for 65. 15%. The proportion of non-residual fractions was relatively small, in the order of organic bound fraction (30.61%), extractable fraction (2.93%), and Fe/Mn oxide (1.31%). The spatial change of non-residual fractions was that Weishan Lake and Nanyang Lake had higher non-residual mercury content than Zhaoyang Lake and Dushan Lake, and the non-residual mercury had a certain potential ecological harm on Weishan Lake and Nanyang Lake. From the horizontal view, the mercury content in the surface sediments of Nansi Lake had visible difference and the pollution degree was different among sub-lakes, so horizontal migration of mercury was obvious. Vertically, the mercury content showed a trend of increasing from the bottom to the top in core sediments, which might be associated with social and economic conditions in different periods.

eNOS-JNK1-AR signaling pathway mediates deltamethrin-induced germ cells apoptosis in testes of adult rats.

The purpose of this study is to examine germ cells apoptosis and reduction of spermatogenesis which might be induced by deltamethrin (DM). Furthermore, the study is performed to determine if the apoptosis is mediated by the signaling proteins: eNOS, JNK1 and androgen receptor (AR). Fifty-four male SD rats were divided into nine groups (six rats each): blank control group; corn oil treated group; DM treated group; saline treated group; DM+saline treated group; DM+histamine (eNOS specific agonist) treated group; 50% ethanol treated group; DM+50% ethanol group and DM+quercetagetin (JNK1 specific inhibitor) treated group. The experiment was conducted for 15 days. Apoptosis was evaluated by TUNEL; S-nitrosylation of JNK1 was examined by the biotin switch assay; eNOS expression and Ser650 phosphorylation of AR were assessed by immunoblotting and immunohistochemical analysis, respectively. DM treated group showed notable apoptotic cells and reduced production of sperm, while DM plus histamine group and DM plus quercetagetin group showed remarkably decreased apoptosis and improved production of sperm. Administration of DM inhibited spermatogenesis, the activity of eNOS and S-nitrosylation of JNK1. Meanwhile, phosphorylation of AR was shown to be elevated. Histamine and quercetagetin were also examined to have a further confirmation. It is suggested DM-induced germ cells apoptosis and reduction of sperm production were mediated by eNOS-JNK1-AR signaling pathway.

Expression of epoxygenases belonging to CYP2 family in rat myocardial ischemia/reperfusion injury in vivo.

OBJECTIVE: To elucidate the expression of epoxygenases belonging to cytochrome P-450 mono-oxygenases (CYP2) family in rat ischemic myocardium at varying reperfusion periods, and the effect of epoxygenase inhibition on the post-ischemic heart. METHODS: The current study was conducted in the Department of Pharmacology, Medical College of Wuhan University, China, between September 2004 and June 2005. Rats were subjected to 40 minutes of myocardial ischemia, followed by 0, 15, 60, and 180 minutes of reperfusion. Superoxide generation was assayed by confocal microscopy, CYP2B1/2, 2C6, 2E1, 2J3 gene expressions were determined by reverse transcriptase polymerase chain reaction. Fourteen, 15-dihydroxyeicosatrienoic acid (DHET) concentration was measured by enzyme-linked immunosorbent assay. The effects of the CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH) on myocardial damage and superoxide generation caused by 60 minutes of reperfusion were also evaluated. RESULTS: During myocardial ischemia/reperfusion, CYP2C6 and 2J3 mRNA expression were up-regulated with the peak level at 15 minutes of reperfusion; CYP2E1 gene expression decreased in a time dependent manner and reached the minimum level at 180 minutes of post-ischemia. Meanwhile, no obvious variations of CYP2B1/2 gene expression were detected during different reperfusion periods. Fourteen, 15-DHET significantly increased during reperfusion in ischemic hearts. The MS-PPOH pretreatment (15 mg/kg) effectively reduced myocardial damage and superoxide production. CONCLUSION: There are changes in gene expression of individual isozymes and an elevation of CYP epoxygenase activity involved in myocardial reperfusion injury in vivo. Epoxygenase inhibition plays a protective role in cardiac post-ischemic damage.

Outbreak of tuberculosis in a 2000-year-old Chinese population.

The molecular identification of Mycobacterium tuberculosis DNA in ancient human remains has been achieved mainly in mummies with macroscopic changes but not in the skeletons without bone tuberculosis. Using polymerase chain reaction studies, we identified mycobacterial DNA in 2000-year-old human skeletons without pathological changes. Our findings suggest that these people suffered from an outbreak of tuberculosis. Molecular examinations for mycobacterial DNA in the bone marrow of skeletons may contribute to the clarification of ancient diseases in old human populations.