ELUCIDATION OF MECHANISMS GENERATING 5-HYDROXYMETHYLCYTOSINE (5hmC) IN MAMMALIAN MITOCHONDRIA

DNA methylation plays a pivotal role in governing cellular processes including genomic imprinting, gene expression, and development. Recently, the Tet family of methylcytosine dioxygenases(Tet1, Tet2 and Tet3) was found to catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate in the pathway of DNA demethylation. Tet enzymes catalyze this hydroxylation in a 2-oxoglutarate and Fe2+ dependent manner. We have recently reported significant levels of 5mC and 5hmC modification in immunoprecipitates of mammalian mitochondrial DNA(mtDNA). We provide the first evidence that a DNA Methyltransferase-1 isoform (mtDNMT1) translocates to the mitochondria using an N-terminal mitochondrial targeting sequence. mtDNMT1 expression is upregulated by NRF1 and PGC1α, master regulators of mitochondrial biogenesis and function, as well as by loss of p53. Altered mtDNMT1 expression asymmetrically affects mtDNA transcription. We are now pursuing the role of Tet proteins in generating 5hmC in mtDNA. Using an in vitro enzyme assay, we have successfully detected Tet activity in crude and percoll purified mitochondrial fractions of HCT116 cells. Mitoprot analysis on Tet family predicts that Tet1 may be translocated to the mitochondria. Immunoblot analysis indicates that a band of expected size(235kDa) is present on immunoblots of mitochondrial fraction from mouse embryonic stem cells with an antibody directed against Tet1. This band, however, is not protected from trypsin treatment of mitochondria indicating that Tet1 may not be transported to the mitochondrial matrix. The putative Tet1 mitochondrial targeting sequence (MTS) fails to carry heterologous protein to the mitochondria. Knock out of Tet1 in mouse ES cells also does not alter 5hmC signal in hydroxyMeDIP assay. We now seek to determine if Tet2/Tet3 may be involved in 5hmC generation. In the nucleus, 5hmC serves as an intermediate in the process of DNA demethylation through the combined action of cytidine deaminases and the base excision repair pathway. We plan to investigate if 5hmC holds the same functional significance in the mitochondria as it does in the nucleus. Our overall goal is to understand epigenetic regulation of normal mitochondrial function and changes that occur in diseases involving mitochondrial dysfunction such as ischemic heart disease, neurodegenerative diseases like Parkinsons disease, and cancer

A Comparison of Homogenization vs. Enzymatic Lysis for Microbiome Profiling in Clinical Endoscopic Biopsy Tissue Samples

Identification of the human microbiome has proven to be of utmost importance with the emerging role of bacteria in various physiological and pathological processes. High throughput sequencing strategies have evolved to assess the composition of the microbiome. To identify possible bias that may exist in the processing of tissue for whole genome sequencing (WGS), it is important to evaluate the extraction method on the overall microbial content and composition. Here we compare two different methods of extraction, homogenization vs. enzymatic lysis, on gastric, esophageal and colorectal biopsies and survey the microbial content and composition using WGS and quantitative PCR (qPCR). We examined total bacterial content using universal 16S rDNA qPCR as well as the abundance of three phyla (Actinobacter, Firmicutes, Bacteroidetes) and one genus (Fusobacterium). We found minimal differences between the two extraction methods in the overall community structure. Furthermore, based on our qPCR analysis, neither method demonstrated preferential extraction of any particular clade of bacteria, nor significantly altered the detection of Gram-positive or Gram-negative organisms. However, although the overall microbial composition remained very similar and the most prevalent bacteria could be detected effectively using either method, the precise community structure and microbial abundances between the two methods were different, primarily due to variations in detection of low abundance genus. We also demonstrate that the homogenization extraction method provides higher microbial DNA content and higher read counts from human tissue biopsy samples of the gastrointestinal tract

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Effect of probiotic mouthrinse on dental plaque accumulation: A randomized controlled trial

Introduction: Dental caries and inflammatory periodontal diseases result from the accumulation of many different bacteria that form dental plaque, a naturally acquired bacterial biofilm that develops on the teeth. Periodontal diseases are one of the most prevalent oral diseases affecting more than 50% of Indian community. Materials and Method: A two months randomised controlled trial was conducted among ninety school children aged between 13 and 15 years, from a hostel located in Davangere city. The 90 study subjects who fulfilled inclusion and exclusion criteria were randomly divided into 3 groups namely Placebo, Chlorhexidine and Probiotic groups. Plaque scores were recorded at baseline (0 day), on 15 th day (after 14 days of intervention) and 3 weeks (after discontinuation of intervention). Statistical analysis was done using one way ANOVA and paired ′t′ test and P value less than 0.05 was considered statistically significant. Results: There was no statistically significant difference between groups at baseline. On 15 th day and after 3 weeks, plaque scores were significantly higher in placebo group compared to probiotic group. On 15 th day and after 3 weeks, plaque scores were higher in chlorhexidine group compared to probiotic group but difference was not statistically significant. Conclusion: Probiotic mouth rinse was more effective for inhibition of dental plaque accumulation after 14 days of intervention and also after 3 weeks of discontinuation of intervention