Genome-Wide DNA Methylation Analysis Reveals Phytoestrogen Modification of Promoter Methylation Patterns during Embryonic Stem Cell Differentiation

BACKGROUND: Environmental challenges during development affect the fetal epigenome, but the period(s) vulnerable to epigenetic dysregulation is(are) not clear. By employing a soy phytoestrogen, genistein, that is known to alter the epigenetic states of the A(vy) allele during embryogenesis, we have explored the sensitive period for epigenetic regulation. The post-implantation period, when de novo DNA methylation actively proceeds, is amenable to in vitro analysis using a mouse embryonic stem (ES) cell differentiation system. METHODS AND FINDINGS: Mouse ES cells were differentiated in the presence or absence of genistein, and DNA methylation patterns on day 10 were compared by microarray-based promoter methylation analysis coupled with a methylation-sensitive endonuclease (HpaII/McrBC)-dependent enrichment procedure. Moderate changes in methylation levels were observed in a subset of promoters following genistein treatment. Detailed investigation of the Ucp1 and Sytl1 promoters further revealed that genistein does not affect de novo methylation occurring between day 0 and day 4, but interferes with subsequent regulatory processes and leads to a decrease in methylation level for both promoters. CONCLUSION: Genistein perturbed the methylation pattern of differentiated ES cells after de novo methylation. Our observations suggest that, for a subset of genes, regulation after de novo DNA methylation in the early embryo may be sensitive to genistein

Identification of Dietary Phytochemicals Capable of Enhancing the Autophagy Flux in HeLa and Caco-2 Human Cell Lines

Autophagy is a major degradation system for intracellular macromolecules. Its decline with age or obesity is related to the onset and development of various intractable diseases. Although dietary phytochemicals are expected to enhance autophagy for preventive medicine, few studies have addressed their effects on the autophagy flux, which is the focus of the current study. Herein, 67 dietary phytochemicals were screened using a green fluorescent protein (GFP)-microtubule-associated protein light chain 3 (LC3)-red fluorescent protein (RFP)-LC3ΔG probe for the quantitative assessment of autophagic degradation. Among them, isorhamnetin, chrysoeriol, 2,2′,4′-trihydroxychalcone, and zerumbone enhanced the autophagy flux in HeLa cells. Meanwhile, analysis of the structure–activity relationships indicated that the 3′-methoxy-4′-hydroxy group on the B-ring in the flavone skeleton and an ortho-phenolic group on the chalcone B-ring were crucial for phytochemicals activities. These active compounds were also effective in colon carcinoma Caco-2 cells, and some of them increased the expression of p62 protein, a typical substrate of autophagic proteolysis, indicating that phytochemicals impact p62 levels in autophagy-dependent and/or -independent manners. In addition, these compounds were characterized by distinct modes of action. While isorhamnetin and chrysoeriol enhanced autophagy in an mTOR signaling-dependent manner, the actions of 2,2′,4′-trihydroxychalcone and zerumbone were independent of mTOR signaling. Hence, these dietary phytochemicals may prove effective as potential preventive or therapeutic strategies for lifestyle-related diseases

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Immortalization of Fetal Bovine Colon Epithelial Cells by Expression of Human Cyclin D1, Mutant Cyclin Dependent Kinase 4, and Telomerase Reverse Transcriptase: An In Vitro Model for Bacterial Infection.

Cattle are the economically important animals in human society. They are essential for the production of livestock products such as milk and meats. The production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. To prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. In this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin D1, mutant cyclin dependent kinase 4 (CDK4), and human telomerase reverse transcriptase (TERT). The established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. Furthermore, we exposed the established cell line to two strains of Salmonella enterica and EHEC. Interestingly, S. Typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than S. Enteritidis. Quantitative RT-PCR revealed that gene expression of Toll-like receptor 1 (TLR1), TLR 2 and TLR 3, whereas TLR 4, 5 and 6 were not detectable in established cells. Our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection

Fluorescence microscopic images of cells subjected to the adhesion and invasion assay.

<p><i>S</i>. Typhimurium (a-d: low-power fields and e-h; high-power fields), <i>S</i>. Enteritidis (i-l) after infection, and non-infected control (m-p)) in BFCE-K4DT cells (passage number = 15). Total bacteria were stained green (a, e, i, and m; black arrows), adherent bacteria were stained red (b, f, j, n; white arrows), nuclei of cells were stained blue, and merge images of extracellular bacteria (stars) and intracellular bacteria (black arrows) appear yellow and green respectively (d, h, l, p). Each staining was carried for 3 times and representative pictures were shown here.</p

Results of the immunoblotting analysis and TRAP assay.

<p>(A) Detection of CDK4 and cyclin D1 by immunoblotting. Lane 1, BFCE primary cells; Lanes 2 and 3, BFCE-K4DT cells. (B) Detection of telomerase activity by TRAP assay. Lane 1, 1 kb ladder marker; lane 2, CHAPS buffer as a negative control; lane 3, 293T cells as a positive control; lane 4, BFCE primary cells; lane 5 and 6, BFCE-K4DT cells. The 6-bp ladders were detectable in lanes of positive control and BFCE-K4DT cells. Each experiment was performed in duplicates after cloning (primary cells: 2 times passage, K4DT cells: 15 times passage) and representative data were shown here.</p

Toll-like receptors (TLRs) gene expression in established BFCE-K4DT cells.

<p>+: positive,</p><p>-: negative</p><p>Toll-like receptors (TLRs) gene expression in established BFCE-K4DT cells.</p

The efficiency of virus infection, cell morphology, and population doubling of the established BFCE-K4DT cells.

<p>(A) Green fluorescence was detectable in primary cells transfected with CSII-CMV-EGFP vector (upper panels), while cells transfected with CSII-CMV-TERT vector were not fluoresce (lower panels). Scale bars: 50 μm. (B) The results of the population doubling assay in BFCE primary cells (squares) and BFCE-K4DT cells (diamonds) were plotted. Experiments were carried out in triplicate, and the averages and standard deviations (SD) were calculated. (C) The morphologies of the established cell line after cloning (after 15 times passage). BFCE-K4DT cells show an epithelial-like rounded shape in low-density culture (upper panels) and sheet-like morphology in high-density culture (lower panels). Scale bars: 100 μm.</p

Fluorescent immunohistochemical staining for detection of epithelial markers.

<p>BFCE-K4DT cells (passage number = 15) and BFCE-Primary cells (passage number = 2) were positive for E-cadherin and Cytokeratin-8, whereas BFF NCC cells (passage number was unknown) were negative for both markers. Scale bars: 50 μm. Each staining was carried for 3 times and representative pictures were shown here.</p

Karyotype analysis of established BFCE-K4DT cells.

<p>All mitotic chromosome spreads (50 of 50 examined) from BFCE-K4DT cells showed a 2n = 60XY pattern. This analysis was performed by using cloned cells after 15 times passage.</p