LPS-induced IL-8 activation in human intestinal epithelial cells is accompanied by specific histone H3 acetylation and methylation changes

<p>Abstract</p> <p>Background</p> <p>The release of LPS by bacteria stimulates both immune and specific epithelial cell types to release inflammatory mediators. It is known that LPS induces the release of IL-8 by intestinal mucosal cells. Because it is now emerging that bacteria may induce alteration of epigenetic patterns in host cells, we have investigated whether LPS-induced IL-8 activation in human intestinal epithelial cells involves changes of histone modifications and/or DNA methylation at IL-8 gene regulatory region.</p> <p>Results</p> <p>RT-PCR analysis showed that IL-8 mRNA levels rapidly increase after exposure of HT-29 cells to LPS. DNA demethylating agents had no effects on IL-8 expression, suggesting that DNA methylation was not involved in IL-8 gene regulation. Consistently we found that 5 CpG sites located around IL-8 transcription start site (-83, -7, +73, +119, +191) were unmethylated on both lower and upper strand either in LPS treated or in untreated HT-29 cells, as well as in normal intestinal mucosa.</p> <p>Conversely, pretreatment of HT-29 cells with deacetylase inhibitors strengthened the LPS-mediated IL-8 activation. Inhibitors of histone deacetylases could induce IL-8 mRNA expression also in the absence of LPS, suggesting that chromatin modifications could be involved in IL-8 gene regulation. Chromatin immunoprecipitation analyses showed that, concurrently with IL-8 activation, transient specific changes in H3 acetylation and H3K4, H3K9 and H3K27 methylation occurred at IL-8 gene promoter during LPS stimulation. Changes of H3-acetyl, H3K4me2 and H3K9me2 levels occurred early, transiently and corresponded to transcriptional activity, while changes of H3K27me3 levels at IL-8 gene occurred later and were long lasting.</p> <p>Conclusion</p> <p>The results showed that specific chromatin modifications occurring at IL-8 gene, including histone H3 acetylation and methylation, mark LPS-mediated IL-8 activation in intestinal epithelial cells while it is unlikely that DNA methylation of IL-8 promoter is directly involved in IL-8 gene regulation in these cells.</p

Epigenetic Regulation of miR-212 Expression in Lung Cancer

Many studies have shown that microRNA expression in cancer may be regulated by epigenetic events. Recently, we found that in lung cancer miR-212 was strongly down-regulated. However, mechanisms involved in the regulation of miR-212 expression are unknown. Therefore, we addressed this point by investigating the molecular mechanisms of miR-212 silencing in lung cancer. We identified histone modifications rather than DNA hypermethylation as epigenetic events that regulate miR-212 levels in NSCLC. Moreover, we found that miR-212 silencing in vivo is closely associated with the severity of the disease

Epigenetic Regulation of miR-212 Expression in Lung Cancer

Many studies have shown that microRNA expression in cancer may be regulated by epigenetic events. Recently, we found that in lung cancer miR-212 was strongly down-regulated. However, mechanisms involved in the regulation of miR-212 expression are unknown. Therefore, we addressed this point by investigating the molecular mechanisms of miR-212 silencing in lung cancer. We identified histone modifications rather than DNA hypermethylation as epigenetic events that regulate miR-212 levels in NSCLC. Moreover, we found that miR-212 silencing in vivo is closely associated with the severity of the disease

Characterization of micro-RNA in women with different ovarian reserve

Women undergoing infertility treatment are routinely subjected to one or more tests of ovarian reserve. Therefore, an adequate assessment of the ovarian reserve is necessary for the treatment. In this study, we aimed to characterize the potential role of microRNAs (miRNAs) as biomarkers for women with different ovarian reserves. A total of 159 women were recruited in the study and classified according to their anti-Müllerian hormone (AMH) level into three groups: (1) low ovarian reserve (LAMH, n = 39), (2) normal ovarian reserve (NAMH, n = 80), and (3) high ovarian reserve (HAMH, n = 40). SurePrint Human miRNA array screening and reverse transcription-quantitative PCR (RT-qPCR) were respectively employed to screen and validate the miRNA abundance level in the three tested groups. Compared with NAMH, the abundance level of 34 and 98 miRNAs was found to be significantly altered in LAMH and HAMH, respectively. The abundance level of miRNAs was further validated by RT-qPCR in both, the screening samples as well as in an independent set of validation samples. The abundance levels of the validated miRNAs were significantly correlated with the AMH level. The best AUC value for the prediction of the increase and decrease in the AMH level was obtained for the miR-100-5p and miR-21-5p, respectively. The level of miRNAs abundance correlates with the level of AMH, which may serve as a tool for identifying women with a different ovarian reserve and may help to lay the ground for the development of novel diagnostic approaches

Epigenetic Switch at Atp2a2 and Myh7 Gene Promoters in Pressure Overload-Induced Heart Failure

Re-induction of fetal genes and/or re-expression of postnatal genes represent hallmarks of pathological cardiac remodeling, and are considered important in the progression of the normal heart towards heart failure (HF). Whether epigenetic modifications are involved in these processes is currently under investigation. Here we hypothesized that histone chromatin modifications may underlie changes in the gene expression program during pressure overload-induced HF. We evaluated chromatin marks at the promoter regions of the sarcoplasmic reticulum Ca2+ATPase (SERCA-2A) and β-myosin-heavy chain (β-MHC) genes (Atp2a2 and Myh7, respectively) in murine hearts after one or eight weeks of pressure overload induced by transverse aortic constriction (TAC). As expected, all TAC hearts displayed a significant reduction in SERCA-2A and a significant induction of β-MHC mRNA levels. Interestingly, opposite histone H3 modifications were identified in the promoter regions of these genes after TAC, including H3 dimethylation (me2) at lysine (K) 4 (H3K4me2) and K9 (H3K9me2), H3 trimethylation (me3) at K27 (H3K27me3) and dimethylation (me2) at K36 (H3K36me2). Consistently, a significant reduction of lysine-specific demethylase KDM2A could be found after eight weeks of TAC at the Atp2a2 promoter. Moreover, opposite changes in the recruitment of DNA methylation machinery components (DNA methyltransferases DNMT1 and DNMT3b, and methyl CpG binding protein 2 MeCp2) were found at the Atp2a2 or Myh7 promoters after TAC. Taken together, these results suggest that epigenetic modifications may underlie gene expression reprogramming in the adult murine heart under conditions of pressure overload, and might be involved in the progression of the normal heart towards HF

Chromatin and DNA methylation dynamics during retinoic acid-induced RET gene transcriptional activation in neuroblastoma cells.

Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci

Ticagrelor, but not clopidogrel, reduces arterial thrombosis via endothelial tissue factor suppression

The P2Y12 antagonist ticagrelor reduces mortality in patients with acute coronary syndrome (ACS), compared with clopidogrel, and the mechanisms underlying this effect are not clearly understood. Arterial thrombosis is the key event in ACS; however, direct vascular effects of either ticagrelor or clopidogrel with focus on arterial thrombosis and its key trigger tissue factor have not been previously investigated.Methods and results: Human aortic endothelial cells were treated with ticagrelor or clopidogrel active metabolite (CAM) and stimulated with tumour necrosis factor-alpha (TNF-α); effects on procoagulant tissue factor (TF) expression and activity, its counter-player TF pathway inhibitor (TFPI) and the underlying mechanisms were determined. Further, arterial thrombosis by photochemical injury of the common carotid artery, and TF expression in the murine endothelium were examined in C57BL/6 mice treated with ticagrelor or clopidogrel. Ticagrelor, but not CAM, reduced TNF-α-induced TF expression via proteasomal degradation and TF activity, independently of the P2Y12 receptor and the equilibrative nucleoside transporter 1 (ENT1), an additional target of ticagrelor. In C57BL/6 mice, ticagrelor prolonged time to arterial occlusion, compared with clopidogrel, despite comparable antiplatelet effects. In line with our in vitro results, ticagrelor, but not clopidogrel, reduced TF expression in the endothelium of murine arteries.Conclusion: Ticagrelor, unlike clopidogrel, exhibits endothelial-specific antithrombotic properties and blunts arterial thrombus formation. The additional antithrombotic properties displayed by ticagrelor may explain its greater efficacy in reducing thrombotic events in clinical trials. These findings may provide the basis for new indications for ticagrelor

Generating bovine embryos through ICSI

Through ICSI, competition between sperms and also sperm-oocyte interaction are avoided thus ICSI proving reliable when sperm is not suitable for IVF. In bovine, the limiting step is represented by low rate of sperm head decondensation subsequent ICSI. Intracytoplasmatic sperm injection allows avoiding many critical moments that may occur during normal or in vitro fertilization. Oocytes were obtained from ovaries from slaughtered cows. These were transported in 0.9% NaCl solution in isothermal bags at a temperature of 25-30 ° C. The ovaries were brought from the slaughterhouse within 2 hours. Harvesting of the oocytes was made through the aspiration method. After maturation, oocytes were fertilized using sperm that was prepared using Percoll method and then treated with TritonX. The volume of the TritonX solution that accompanies the sperma and which remains in the oocyte is extremely important given that by its action, TritonX removes the acrosome, thus releasing a rich enzyme content and facilitating the dehydration of the male pronucleus. Even though the number of 2 nucleus, 2 cells or 4 cells oocytes is inferior to the data found in the literature, compared to the results achieved last year in the assisted reproduction laboratory within CLC-HC Timisoara, it marks significant progress. At the 2 cells stage, there were several oocytes from group 1 (24.39% vs. 12.5%), while at the 4 cells stage there were 14.63% oocytes from group 1 and 25% group 2. The use of TritonX solution for sperm treatment as well as shortening the duration of ICSI execution allowed us to get encouraging results. The results obtained are inferior to those presented in the literature but are far superior to those we obtained last year when the ICSI technique was assembled. Achieving the two- and four-cell embryonic stages justifies us thinking that we are mastering the ICSI technique