The potential DNA methylation markers of cardiovascular disease in patients with type 2 diabetes.

BACKGROUND: DNA methylation is associated with cardiovascular (CV) disease. However, in type 2 diabetes (T2D) patients, the role of gene methylation in the development of CV disease is under-studied. We aimed to identify the CV disease-related DNA methylation loci in patients with T2D and to explore the potential pathways underlying the development of CV disease using a two-stage design. METHODS: The participants were from the Jinan Diabetes Cohort Study (JNDCS), an ongoing longitudinal study designed to evaluate the development of CV risk in patients with T2D. In the discovery cohort, 10 diabetic patients with CV events at baseline were randomly selected as the case group, and another 10 diabetic patients without CV events were matched for sex, age, smoking status, and body mass index as the control group. In 1438 T2D patients without CV disease at baseline, 210 patients with CV events were identified after a mean 6.5-year follow-up. Of whom, 100 patients who experienced CV events during the follow-up were randomly selected as cases, and 100 patients who did not have CV events were randomly selected as the control group in the validation cohort. Reduced representation bisulfite sequencing and Targeted Bisulfite Sequencing were used to measure the methylation profiles in the discovery and validation cohort, respectively. RESULTS: In the discover cohort, 127 DMRs related to CV disease were identified in T2D patients. Further, we validated 23 DMRs mapped to 25 genes, of them, 4 genes (ARSG, PNPLA6, NEFL, and CRYGEP) for the first time were reported. There was evidence that the addition of DNA methylation data improved the prediction performance of CV disease in T2D patients. Pathway analysis identified some significant signaling pathways involved in CV comorbidities, T2D, and inflammation. CONCLUSIONS: In this study, we identified 23 DMRs mapped to 25 genes associated with CV disease in T2D patients, of them, 4 DMRs for the first time were reported. DNA methylation testing may help identify a high CV-risk population in T2D patients.

SIRT1 upregulation promotes epithelial-mesenchymal transition by inducing senescence escape in endometriosis.

Endometrial epithelial cells carry distinct cancer-associated alterations that may be more susceptible to endometriosis. Mouse models have shown that overexpression of SIRT1 associated with oncogene activation contributes to the pathogenesis of endometriosis, but the underlying reason remains elusive. Here, we used integrated systems biology analysis and found that enrichment of endometrial stromal fibroblasts in endometriosis and their cellular abundance correlated negatively with epithelial cells in clinical specimens. Furthermore, endometrial epithelial cells were characterized by significant overexpression of SIRT1, which is involved in triggering the EMT switch by escaping damage or oncogene-induced induced senescence in clinical specimens and in vitro human cell line models. This observation supports that genetic and epigenetic incident favors endometrial epithelia cells escape from senescence and fuel EMT process in endometriosis, what could be overcome by downregulation of SIRT1.

Role of Slit2 upregulation in recurrent miscarriage through regulation of stromal decidualization.

INTRODUCTION: Knockout mouse model has shown a relationship between Slit2/Robo1 signalling and altered fertility. Altered expression by endometrial epithelium and trophoblast and is associated with the pathogenesis of pregnancy complications but few studies have investigated the expression of decidual Slit2 in miscarriage. METHODS: Expression profiles of Slit2 and Robo1 were measured in human endometrial tissues during the menstrual cycle phases (n = 30), in decidua tissues from recurrent miscarriage (n = 20) and healthy control (n = 20) at 6-8 weeks of gestation. The hormonal regulation of Slit2/Robo1 expression and the role of Slit2/Robo1 signalling in decidualization was investigated in vitro, along with its effects on ß-catenin and MET expression. RESULTS: In human endometrium, Slit2 and Robo1 protein expression in stromal cells were decreased between the late-proliferative and early-secretory phase. In recurrent miscarriage patients, decidual expression Slit2 was increased and associated with lower expression of E-cadherin and higher level vimentin compared to controls. In vitro, the expression of Slit2 was downregulated by cAMP and progesterone in hESCs. Upregulation of Slit2 resulted in inhibition of cell decidualization and ß-catenin translocation to nucleus. DISCUSSION: This study indicates a functional role for Slit2 in endometrial stromal cell decidualization and the pathogenesis of recurrent miscarriage. Aberrant Increase in Slit2 expression may impairs decidualization of endometrial stromal cells leading to recurrent in recurrent miscarriage.

Baicalin rescues hyperglycemia-induced neural tube defects via targeting on retinoic acid signaling.

We, in this study, studied whether or not antioxidant activities of Baicalin could reduce the incidence of neural tube defects (NTDs) in the presence of hyperglycemia. Using early chick embryos, we demonstrated that Baicalin at 6 µM dramatically reduced NTDs rate and impaired neurogenesis in E4.5-day and HH10 chick embryo neural tubes induced by high glucose (HG). Likewise, immunofluorescent staining showed that Baicalin mitigated the HG-induced regression of Pax7 expression in neural tubes of both HH10 and E4.5-day chick embryos. Additionally, PHIS3 immunofluorescent staining in neural tubes of both HH10 and E4.5-day chick embryos manifested that cell proliferation inhibited by HG was significantly reversed by the administration of Baicalin, and similar result could also be observed in neurosphere assay in vitro. c-Caspase3 or γH2AX immunofluorescent staining and quantitative PCR showed that Baicalin administration alleviated HG-induced cell apoptosis and DNA damage. Bioinformatics results indicated that retinoic acid (RA) was likely to be the signaling pathway that Baicalin targeted on, and this was confirmed by whole-mount RALDH2 in situ hybridization and quantitative PCR of HH10 chick embryos in the absence/presence of Baicalin. In addition, blocking RA with an inhibitor abolished Baicalin's protective role in HG-induced NTDs, suppression of neurogenesis and cell proliferation, and induction of apoptosis, which further verified the centrality of RA in the process of Baicalin confronting HG-induced abnormal neurodevelopment.

Toxoplasma gondii Cathepsin C1 inhibits NF-κB signalling through the positive regulation of the HIF-1α/EPO axis.

Toxoplasma gondii has evolved many successful strategies for immune evasion. However, the parasite-derived effectors involved in modulating NF-κB signalling pathway are largely unknown. T. gondii Cathepsin C1 (CPC1) is widely conserved among T. gondii strains and is important for T. gondii intracellular growth and proliferation. Our study showed that CPC1 protein could abrogate NF-κB activation after screening dense granule proteins. CPC1 suppressed NF-κB activation at or downstream of p65 and decreased the production of IL-1, IL-8, IL-6, IL-12, and TNF-α. Western blot analysis revealed that CPC1 inhibited phospho-p65 and CPC1 proteins primarily settled in cytoplasm. RNA sequencing analysis revealed that overexpression of CPC1 significantly upregulated erythropoietin (EPO), which can be induced by the hypoxia-inducible factor -1α (HIF-1α) during hypoxia. Furthermore, dual-luciferase reporter assays confirmed that CPC1 upregulated HIF-1α. Finally, both the knockdown of EPO and restriction of HIF-1α partially eliminated the suppression impact of CPC1 on the NF-κB signalling pathway. Our study identified a previously unrecognized role of CPC1 in the negative regulation of NF-κB activation through positive regulation of the HIF-1α/EPO axis. For the first time, CPC1 was shown to play an important role in immune evasion during T. gondii infection.

MiR-30c regulates cisplatin-induced apoptosis of renal tubular epithelial cells by targeting Bnip3L and Hspa5.

As a common anticancer drug, cisplatin has been widely used for treating tumors in the clinic. However, its side effects, especially its nephrotoxicity, noticeably restrict the application of cisplatin. Therefore, it is imperative to investigate the mechanism of renal injury and explore the corresponding remedies. In this study, we showed the phenotypes of the renal tubules and epithelial cell death as well as elevated cleaved-caspase3- and TUNEL-positive cells in rats intraperitoneally injected with cisplatin. Similar cisplatin-induced cell apoptosis was found in HK-2 and NRK-52E cells exposed to cisplatin as well. In both models of cisplatin-induced apoptosis in vivo and in vitro, quantitative PCR data displayed reductions in miR-30a-e expression levels, indicating that miR-30 might be involved in regulating cisplatin-induced cell apoptosis. This was further confirmed when the effects of cisplatin-induced cell apoptosis were found to be closely correlated with alterations in miR-30c expression, which were manipulated by transfection of either the miR-30c mimic or miR-30c inhibitor in HK-2 and NRK-52E cells. Using bioinformatics tools, including TargetScan and a gene expression database (Gene Expression Omnibus), Adrb1, Bnip3L, Hspa5 and MAP3K12 were predicted to be putative target genes of miR-30c in cisplatin-induced apoptosis. Subsequently, Bnip3L and Hspa5 were confirmed to be the target genes after determining the expression of these putative genes following manipulation of miR-30c expression levels in HK-2 cells. Taken together, our current experiments reveal that miR-30c is certainly involved in regulating the renal tubular cell apoptosis induced by cisplatin, which might supply a new strategy to minimize cisplatin-induced nephrotoxicity.

From the Cover: Exposing Imidacloprid Interferes With Neurogenesis Through Impacting on Chick Neural Tube Cell Survival.

As a neonicotinoid pesticide, imidacloprid is widely used to control insects in agriculture and fleas on domestic animals. However, it is not known whether imidacloprid exposure negatively affects neurogenesis during embryonic development. In this study, using a chick embryo model, we investigated the effects of imidacloprid exposure on neurogenesis at the earliest stage and during late-stage embryo development. Exposing HH0 chick embryos to imidacloprid in EC culture caused neural tube defects (NTDs) and neuronal differentiation dysplasia as determined by NF/Tuj1 labeling. Furthermore, we found that F-actin accumulation on the apical side of the neural tube was suppressed by exposure to imidacloprid, and the expression of BMP4 and Shh on the dorsal and ventral sides of the neural tubes, respectively, were also reduced, which in turn affects the dorsolateral hinge points during bending of the neural plate. In addition, exposure to imidacloprid reduced cell proliferation and increased cell apoptosis, as determined by pHIS3 labeling and TUNEL staining, respectively, also contributing to the malformation. We obtained similar results in late-stage embryos exposed to imidacloprid. Finally, a bioinformatics analysis was employed to determine which genes identified in this study were involved in NTDs. The experimental evidence and bioinformatics analysis suggested that imidacloprid exposure during chick embryo development could increase the risk of NTDs and neural dysplasia.

A new gene ontology-based measure for the functional similarity of gene products.

BACKGROUND: Although biomedical ontologies have standardized the representation of gene products across species and databases, a method for determining the functional similarities of gene products has not yet been developed. METHODS: We proposed a new semantic similarity measure based on Gene Ontology that considers the semantic influences from all of the ancestor terms in a graph. Our measure was compared with Resnik's measure in two applications, which were based on the association of the measure used with the gene co-expression and the protein-protein interactions. RESULTS: The results showed a considerable association between the semantic similarity and the expression correlation and between the semantic similarity and the protein-protein interactions, and our measure performed the best overall. CONCLUSION: These results revealed the potential value of our newly proposed semantic similarity measure in studying the functional relevance of gene products.

Heterogeneity of the biological properties and gene expression profiles of murine bone marrow stromal cells.

Although mesenchymal stromal cells (MSCs) have demonstrated great therapeutic potential, the heterogeneity of MSCs may be responsible for the incongruent data obtained in MSC-based preclinical studies and clinical trials. Here, four mouse clonal MSC lines, termed MSC1, MSC2, MSC3, and MSC4, were isolated and extensively characterized. MSC4 cells grew most rapidly and formed colonies of the largest size, whereas MSC3 cells exhibited the slowest growth and formed only a few tiny clusters. MSC4 cells could differentiate into adipocytes, osteoblasts, and chondrocytes in vitro, and more importantly, establish hematopoietic microenvironment in vivo; whereas the other lines displayed uni-adipogenic, osteo-chondrogenic, or non-differentiation potential. All lines were positive for Sca-1, CD106, and CD44; MSC4 was also positive for CD90.2. In terms of immunosuppressive capacity, MSC2, MSC3, and MSC4 cells exerted clear inhibitory effects on lymphocyte proliferation, whereas MSC1 did not. Further investigation revealed that the NO and not the PGE2 pathway may play a role in the different immunomodulatory effects of the cell lines. To clarify the molecular basis of this heterogeneity, we employed RNA sequencing to compare the gene expression profiles of the four subtypes, revealing a relationship between gene expression and variability in subtype function. This study provides novel information about the heterogeneity of MSCs and insight into the selection of optimal cell sources for therapeutic applications.

Coseeded Schwann cells myelinate neurites from differentiated neural stem cells in neurotrophin-3-loaded PLGA carriers.

Biomaterials and neurotrophic factors represent promising guidance for neural repair. In this study, we combined poly-(lactic acid-co-glycolic acid) (PLGA) conduits and neurotrophin-3 (NT-3) to generate NT-3-loaded PLGA carriers in vitro. Bioactive NT-3 was released stably and constantly from PLGA conduits for up to 4 weeks. Neural stem cells (NSCs) and Schwann cells (SCs) were coseeded into an NT-releasing scaffold system and cultured for 14 days. Immunoreactivity against Map2 showed that most of the grafted cells (>80%) were differentiated toward neurons. Double-immunostaining for synaptogenesis and myelination revealed the formation of synaptic structures and myelin sheaths in the coculture, which was also observed under electron microscope. Furthermore, under depolarizing conditions, these synapses were excitable and capable of releasing synaptic vesicles labeled with FM1-43 or FM4-64. Taken together, coseeding NSCs and SCs into NT-3-loaded PLGA carriers increased the differentiation of NSCs into neurons, developed synaptic connections, exhibited synaptic activities, and myelination of neurites by the accompanying SCs. These results provide an experimental basis that supports transplantation of functional neural construction in spinal cord injury.

Metastasis-associated protein 1 induces VEGF-C and facilitates lymphangiogenesis in colorectal cancer.

AIM: To study the correlation between high metastasis-associated protein 1 (MTA1) expression and lymphangiogenesis in colorectal cancer (CRC) and its role in production of vascular endothelial growth factor-C(VEGF-C). METHODS: Impact of high MTA1 and VEGF-C expression levels on disease progression and lymphovascular density (LVD, D2-40-immunolabeled) in 81 cases of human CRC was evaluated by immunohistochemistry. VEGF-C mRNA and protein expressions in human LoVo and HCT116 cell lines were detected by real-time polymerase chain reaction and Western blotting, respectively, with a stable expression vector or siRNA. RESULTS: The elevated MTA1 and VEGF-C expression levels were correlated with lymph node metastasis and Dukes stages (P < 0.05). Additionally, high MTA1 expression level was correlated with a large tumor size (P < 0.05). A significant correlation was found between MTA1 and VEGF-C protein expressions in tumor cells (r = 0.371, P < 0.05). Similar to the VEGF-C expression level, high MTA1 expression level was correlated with high LVD in CRC (P < 0.05). Furthermore, over-expression of MTA1 significantly enhanced the VEGF-C mRNA and protein expression levels, whereas siRNAs - knocked down MTA1 decreased the VEGF-C expression level. CONCLUSION: MTA1, as a regulator of tumor-associated lymphangiogenesis, promotes lymphangiogenesis in CRC by mediating the VEGF-C expression.

Copy number increase of HER-2 in colorectal cancers.

HER-2 is involved in genetic instability and is overexpressed in a number of human carcinomas, including colorectal cancer (CRC). The choromosomal locus of HER-2, 17q21, is frequently amplified in breast cancer, but the correlation between copy-number variations and HER-2 overexpression in CRC has yet to be elucidated. The functional impact of such regions requires extensive investigation in large numbers of CRC samples. Case-matched tissues of colorectal adenocarcinomas and adjacent normal epithelia (n=134) were included in this study. Quantitative PCR was performed to examine the copy number and mRNA expression of HER-2 in CRC. The results showed that copy number gains of HER-2 were detected in a relatively high percentage of CRC samples (35.1%, 47 out of 134). A positive correlation was noted between the copy number increase of HER-2 and tumor progression. Furthermore, copy number gains of HER-2 showed a positive correlation with mRNA overexpression in CRC. However, the expression levels of HER-2 mRNA were also enhanced in the group of CRC samples with unaltered copy numbers. In conclusion, the findings suggest that a copy number increase of HER-2 is a potential diagnostic indicator for CRC; whether alone or in combination with other markers.

[Concurrent chemoradiotherapy versus radiotherapy alone for locoregionally advanced nasopharyngeal carcinoma: a meta-analysis].

OBJECTIVE: To determine the value of concurrent chemoradiotherapy in patients with locoregionally advanced nasopharyngeal carcinoma from the mainland of China. METHODS: Data were extracted from randomized trials comparing chemotherapy plus radiotherapy with radiotherapy alone in locally advanced nasopharyngeal carcinoma. Actuarial rates of survival and distant metastases were calculated. The followed electronic databases were searched the Chinese Biomedicine database, Pubmed, Medline, Embase and Cochrane library; Data were extracted by tow reviewers and Review manager 4.1 software was applied for statistical analysis. RESULTS: Eighteen trials with 1993 patients were include according to the including criterion. The 3-year overall survival rate of the chemoradiotherapy group and the radiotherapy group were 68.47% and 56.38% respectively, and the 5-year overall survival rate of the two groups above were 51.91% and 41.09% respectively, while the distant metastases rate of the chemoradiotherapy group and the radiotherapy group were 26.19% and 38.71% respectively. The result demonstrated that chemoradiotherapy increased overall survival by 12% at 3 years, and 11% at 5 years after treatment. After chemoradiotherapy, the rate of distant metastasis was reduce by 12%. CONCLUSIONS: In patients with locoregionally advanced nasopharyngeal carcinoma, chemoradiotherapy significantly improves overall survival at 3 years, and 5 years compared with radiotherapy alone.