Unfaithful Maintenance of Methylation Imprints Due to Loss of Maternal Nuclear Dnmt1 during Somatic Cell Nuclear Transfer

The low success rate of somatic cell nuclear transfer (SCNT) in mammalian cloning is largely due to imprinting problems. However, little is known about the mechanisms of reprogramming imprinted genes during SCNT. Parental origin-specific DNA methylation regulates the monoallelic expression of imprinted genes. In natural fertilization, methylation imprints are established in the parental germline and maintained throughout embryonic development. However, it is unclear whether methylation imprints are protected from global changes of DNA methylation in cloned preimplantation embryos. Here, we demonstrate that cloned porcine preimplantation embryos exhibit demethylation at differentially methylated regions (DMRs) of imprinted genes; in particular, demethylation occurs during the first two cell cycles. By RNAi-mediated knockdown, we found that Dnmt1 is required for the maintenance of methylation imprints in porcine preimplantation embryos. However, no clear signals were detected in the nuclei of oocytes and preimplantation embryos by immunofluorescence. Thus, Dnmt1 is present at very low levels in the nuclei of porcine oocytes and preimplantation embryos and maintains methylation imprints. We further showed that methylation imprints were rescued in nonenucleated metaphase II (MII) oocytes. Our results indicate that loss of Dnmt1 in the maternal nucleus during SCNT significantly contributes to the unfaithful maintenance of methylation imprints in cloned embryos

Gender differences in plaque characteristics of nonculprit lesions in patients with coronary artery disease

Abstract Background Although numerous reports suggest sex-related differences in atherosclerosis, limited data describing gender-associated differences in plaque morphology and composition are currently available. The aim of the present study was to compare coronary nonculprit plaque characteristics in women and men with coronary artery disease (CAD) by optical coherence tomography (OCT). Methods This was a retrospective study. A total of 187 nonculprit plaques were identified in 103 patients with CAD who underwent OCT imaging of all 3 coronary arteries. These patients included 77 (74.8%) men and 26 (25.2%) women. Results Female patients were significantly older than males (mean age, 70.8 ± 7.3 vs 60.8 ± 9.8 years; P < 0.001) and less likely to be current smokers (P = 0.007). OCT analysis included the presence of lipid-rich plaque, maximum lipid arc, lipid-core length, lipid index (LI), fibrous cap thickness, and the incidence of thin-cap fibroatheroma (TCFA). Nonculprit plaques in men exhibited greater lipid-core length and LI compared with those of women (9.4 ± 4.5 vs. 7.3 ± 4.3 mm, P = 0.024; 1615.1 ± 893.8 vs. 1237.8 ± 859.8, P = 0.035, respectively). In the univariate linear regression model, sex and current smoker were all associated with a larger LI, whereas only use of statin was independent risk factor for a larger LI in multivariate analysis. Conclusions Coronary nonculprit plaques in male patients with CAD contain larger lipid cores than those of female patients

Identification of Subpathway Signatures For Ovarian Cancer Prognosis by Integrated Analyses of High-Throughput miRNA and mRNA Expression

Background/Aims: Ovarian cancer (OC) causes more death and serious conditions than any other female reproductive cancers, and many expression signatures have been identified for OC prognoses. However, no significant overlap is found among signatures from different studies, indicating the necessity of signature identifications at the functional level. Methods: We performed an integrated analyses of miRNA and gene expressions to identify OC prognostic subpathways (pathway regions). Using The Cancer Genome Atlas data set, we identified core prognostic subpathways, and calculated subpathway risk scores using both miRNA and gene components. Finally, we performed global risk impact analyses to optimize core subpathways using the random walk algorithm. Results: Subpathway-level analyses displayed more robust results than the gene- and miRNA-level analyses. Moreover, we verified the advantage of core subpathways over the entire pathway-based results and their prognostic performance in two independent validation data sets. Based on the global impact score, 13 subpathway signatures were selected and a combined subpathway-based risk score was further calculated for OC patient prognoses. Conclusions: Overall, it was possible to systematically perform integrated analyses of the expression levels of miRNAs and genes to identify prognostic subpathways and infer subpathway risk scores for use in OC clinical applications

The immunosuppressant Protosappanin A diminished recipient T cell migration into allograft via inhibition of IP-10 in rat heart transplant.

The immunosuppressant Protosappanin A (PrA), isolated from the medicinal herb, promotes cardiac allograft survival, diminishes inflammatory cell infiltration, and inhibits interferon γ-induced protein 10 kDa (IP-10) mRNA expression in rats cardiac grafts. Binding of the chemokine IP-10 to its cognate receptor, CXCR3, plays crucial roles in allograft immunity, especially by mediating the recruitment of effector T cells to allografted tissues. In this study, we attempted to determine whether PrA-mediated inhibition of IP-10 contributes to the effect of reduced T cell infiltration into cardiac allograft within a rat model. Administration of PrA (25 mg/kg daily) via oral gavage following heart transplantation significantly reduced the increase of IP-10 mRNA level in allograft and prevented IP-10 secretion by peripheral blood mononuclear cells (PBMC) isolated from recipient rats seven days posttransplantation. Furthermore, in vitro experiments demonstrated that PrA addition to control PBMC prevented IP-10 secretion. Chemotactic migration assays were utilized to evaluate recipient T cell migration towards PBMC supernatant. PrA administration impaired PBMC supernatant-induced T cell migration. Additional in vitro experiments revealed that PrA slightly reduced naïve T cell migration towards chemokines. The presence of IP-10 in PBMC supernatant prevented PrA from reducing T cell migration in PrA-treated recipients. Neither CXCR3 chemokine ligand Mig nor non-CXCR3 chemokine ligand SDF-1 had any effect on T cell migration in PrA-treated recipients. The addition of anti-CXCR3 antibody restored PrA-mediated inhibition of T cell migration. Immunofluorescence microscopy showed that IP-10 was expressed mainly in CD68 positive infiltrating monocytes. Furthermore, PrA consistently reduced CXCR3+T cell infiltration into cardiac allografts. The reduced intensity of CXCR3 staining in PrA-treated allografts contributed to the previously depressed naïve T cell migrating activity induced by PrA. Collectively, these data indicate that PrA inhibition of IP-10 activity reduced recipient T cell migration and infiltration of cardiac allografts, thus partially explaining the immunosuppressive effect of PrA

IRGM/Irgm1 deficiency inhibits neutrophil-platelet interactions and thrombosis in experimental atherosclerosis and arterial injury

Background: Neutrophil extracellular traps (NETs) closely link inflammation and thrombosis. The immune-related GTPase family M protein (IRGM) and its ortholog of mouse IRGM1 are positively correlated with plaque rupture during atherosclerosis process. However, whether and how IRGM/IRGM1 affects NETs formation and atherosclerotic thrombosis remains unknown, which will further promote the development of antithrombotic treatment tools. Methods: The thrombi images, platelet activation makers and NETs makers were detected in the serum of STEMI patients and controls. To futher investigate IRGM/IRGM1 affects NETs formation and atherothrombosis in vivo, ApoE-/-Irgm1+/- and ApoE-/- mice received diets rich in fat and 2.5% FeCl3 was then used to induce experimental arterial thrombosis in an atherosclerosis background. In vitro, PMA and thrombin were used to stimulate neutrophils and platelets, respectively, and the expression of IRGM/IRGM1 were modified. To reveal the molecular mechanisms, MAPK-cPLA2 signals inhibitors were used. Results: Serum IRGM was positively correlated with PF4 and neutrophil elastase. Subsequently, Irgm1 deficient mice have a longer occlusion time and lower growth rate. In vitro, as expected, IRGM/Irgm1 deficiency inhibits platelet activation and platelet-neutrophil interaction. More importantly, IRGM promoted NETs production through activating MAPK-cPLA2 signals in PMA stimulated neuropils, whereas inhibiting the production of NETs eliminated the difference in platelet activation and thrombosis caused by IRGM/Irgm1 modification in vivo and vitro. Similarly, inhibition of platelet activation also eliminated the influence of IRGM/Irgm1 modification on NETs production. Conclusions: Overall, our data indicate that IRGM/Irgm1 deficiency in neuropils inhibits the intense interaction between neutrophils and platelets, and ultimately inhibits thrombosis

PrA reduced T cell migration to PBMC supernatant through inhibition of IP-10-CXCR3 receptor interaction.

<p>IP-10 (50 ng/ml), Mig (100 ng/ml), or SDF-1 (100 ng/ml) was added to the PBMC supernatant. T cells were incubated with the isotype control Ab (20 µg/ml) or anti-CXCR3 Ab (20 µg/ml) prior to chemotactic migration assays (n = 3 in each group). (A) IP-10 addition to the supernatant of PrA-exposed PBMCs improved the migration of PrA-treated recipients T cells. Furthermore, addition of anti-CXCR3 Ab restored PrA inhibition of IP-10-induced T cell migration, but isotype Ab did not have the same effect. (B) The addition of Mig and SDF-1 did not significantly increase the migration of PrA-treated recipients T cells. However the addition of Mig, but not SDF-1 addition increased the migration of non PrA-treated recipients T cells. *indicates <i>p</i><0.05, and bars indicate comparators.</p

PrA inhibited IP-10 expression in infiltrating monocytes of cardiac allografts.

<p>Immunofluorescence staining of heart allograft sections stained with Abs to CD68 (FITC, green) and IP-10 (TMRITC, red). Nuclei were counterstained with DAPI (blue). The IP-10 expression in CD68 positive infiltrating monocytes was reduced after PrA treatment. The results are representative of three independent experiments (n = 3).</p

The migrating capacity of naïve T cells was slightly affected by PrA.

<p>The naïve T cells isolated from rat spleen were cultured with or without PrA (20 nM) for 72 h. A chemotactic migration assay was used to assess the migration of naïve T cells in the presence or absence of PrA towards different doses of either recombinant IP-10 or Mig. (A) PrA-conditioned T cells exhibited decreased migration in presence of 25 ng/mL of IP-10, but not at 50 ng/mL or 100 ng/mL, compared with those of non-condition T cells. (B) The migration of PrA-conditioned T cells was decreased at 50 ng/mL and 100 ng/mL of Mig, but not at 200 ng/mL, as compared with those of non-conditioned T cells. The result suggests that the capacity of T cell migration was partially affected by PrA <i>in vitro</i>. * indicates <i>p</i><0.05, and bars indicate comparators.</p

PrA conditioning reduced PBMC IP-10 secretion <i>in vitro</i>.

<p>PBMCs isolated from control recipient rats on day four were cultured with different doses of PrA for 72(n = 3 per group). (A) PBMC viability was measured by MTT assay. The highest dose induced a significant decrease in viable cell count, therefore, only 5 nM and 20 nM concentrations were used in subsequent experiments. (B) Culture supernatants of PBMC with or without PrA condition were harvested and analyzed for the production of IP-10 by ELISA. * indicates <i>p</i><0.05 when comparing treatment condition to control.</p

PrA prevented CXCR3⁺T cell infiltration into allografts.

<p>(A) The heart allograft was harvested on day seven post heart transplantation, sectioned, and stained for TCR (TMRITC, red) and CXCR3 (FITC, green). The extent of colocalization (yellow) of TCR (T cell marker) and CXCR3 indicating CXCR3⁺ T cell infiltration into allografts was significantly reduced after PrA administration. The results are representative of three independent experiments. (B) The relative quantitative analysis to determine the percentage of CXCR3⁺TCR⁺ cells within infiltrating cells of allografts further showed that PrA treatment inhibited the CXCR3⁺ T cell infiltration into allografts. *indicates <i>p</i><0.05 when comparing treatment to control.</p