Exercise-Conditioned Plasma and DNMT3B Concentration in PBMCs

DNA methylation is modifiable by acute and chronic exercise. DNA methyltransferases (DNMT) catalyze this process; however, there is a lack of literature concerning the specific mechanisms by which exercise‐induced modifications occur. Interleukin 6 (IL‐6) stimulation of various cell lines has been shown to augment DNMT expression and nuclear translocation, which suggests a possible pathway by which exercise is able to elicit changes in epigenetic enzymes. The present study sought to elucidate the response of thede novomethyltransferases DNMT3A and DNMT3B to circulatory factors found in plasma isolated from whole blood before and after 120‐min of treadmill running at an intensity of 60% of individual velocity at(v) interspersed with 30‐sec sprints at 90% of vevery 10‐min. Peripheral blood mononuclear cells (PBMCs) isolated from a resting participant were incubated with plasma isolated from exercising participants (n=10) or recombinant IL‐6 (rIL‐6), followed by nuclear protein extraction and quantification of DNMT3A and DNMT3B concentrations. Nuclear concentrations of DNMT3B significantly decreased following the experimental protocol (P=0.03), with no change observed in DNMT3A (P=0.514).Various concentrations of rIL‐6 caused an elevation in both DNMT3A and DNMT3B nuclear concentration compared with the blank control. The conflicting results between exercising and rIL‐6 conditions suggests that IL‐6 does regulate DNMT nuclear transport, however, other plasma mediators may also exert significant influence on the nuclear concentrations of these enzymes

Minimal muscle damage after a marathon and no influence of beetroot juice on inflammation and recovery

This study examined whether beetroot juice (BTJ) would attenuate inflammation and muscle damage following a marathon. Using a double blind, independent group’s design, 34 runners (~16 previous marathons completed) consumed either BTJ or an isocaloric placebo (PLA) for 3 days following a marathon. Maximal isometric voluntary contractions (MIVC), countermovement jumps (CMJ), muscle soreness, serum cytokines, leucocytosis, creatine kinase (CK), high sensitivity C-reactive protein (hs-CRP) and aspartate aminotransferase (AST) were measured pre, post, and on the 2 days after the marathon. CMJ and MIVC were reduced after the marathon (P0.05). Muscle soreness was increased in the day after the marathon (BTJ; 45±48 vs. PLA; 46±39 mm) and had returned to baseline by day 2, irrespective of supplementation (P=0.694). Cytokines (Interleukin-6; IL-6, interleukin-8, tumour necrosis factor-α) were increased immediately post-marathon but apart from IL-6 had returned to baseline values by day 1 post. No interaction effects were evident for IL-6 (P=0.213). Leucocytes increased 1.7 fold after the race and remained elevated 2 days post, irrespective of supplement (P<0.0001). CK peaked at 1 day post marathon (BTJ: 965±967 & PLA: 1141±979 IU·L-1) and like AST and hs-CRP, was still elevated 2 days after the marathon (P<0.05); however, no group differences were present for these variables. Beetroot juice did not attenuate inflammation or reduce muscle damage following a marathon, possibly because most of these indices were not markedly different from baseline values in the days after the marathon

Potential Associations between Severity of Infection and the Presence of Virulence-Associated Genes in Clinical Strains of Staphylococcus aureus

BACKGROUND: The clinical spectrum of Staphylococcus aureus infection ranges from asymptomatic nasal carriage to osteomyelitis, infective endocarditis (IE) and death. In this study, we evaluate potential association between the presence of specific genes in a collection of prospectively characterized S. aureus clinical isolates and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: Two hundred thirty-nine S. aureus isolates (121 methicillin-resistant S. aureus [MRSA] and 118 methicillin-susceptible S. aureus [MSSA]) were screened by array comparative genomic hybridization (aCGH) to identify genes implicated in complicated infections. After adjustment for multiple tests, 226 genes were significantly associated with severity of infection. Of these 226 genes, 185 were not in the SCCmec element. Within the 185 non-SCCmec genes, 171 were less common and 14 more common in the complicated infection group. Among the 41 genes in the SCCmec element, 37 were more common and 4 were less common in the complicated group. A total of 51 of the 2014 sequences evaluated, 14 non-SCCmec and 37 SCCmec, were identified as genes of interest. CONCLUSIONS/SIGNIFICANCE: Of the 171 genes less common in complicated infections, 152 are of unknown function and may contribute to attenuation of virulence. The 14 non-SCCmec genes more common in complicated infections include bacteriophage-encoded genes such as regulatory factors and autolysins with potential roles in tissue adhesion or biofilm formation

Novel human genetic variants associated with extrapulmonary tuberculosis: a pilot genome wide association study

<p>Abstract</p> <p>Background</p> <p>Approximately 5-10% of persons infected with <it>M. tuberculosis </it>develop tuberculosis, but the factors associated with disease progression are incompletely understood. Both linkage and association studies have identified human genetic variants associated with susceptibility to pulmonary tuberculosis, but few genetic studies have evaluated extrapulmonary disease. Because extrapulmonary and pulmonary tuberculosis likely have different underlying pathophysiology, identification of genetic mutations associated with extrapulmonary disease is important.</p> <p>Findings</p> <p>We performed a pilot genome-wide association study among 24 persons with previous extrapulmonary tuberculosis and well-characterized immune defects; 24 pulmonary tuberculosis patients and 57 patients with <it>M. tuberculosis </it>infection served as controls. The Affymetrix GeneChip Human Mapping Xba Array was used for genotyping; after careful quality control, genotypes at 44,175 single nucleotide polymorphisms (SNPs) were available for analysis. Eigenstrat quantified population stratification within our sample; logistic regression, using results of the Eigenstrat analysis as a covariate, identified significant associations between groups. Permutation testing controlled the family-wise error rate for each comparison between groups. Four SNPs were significantly associated with extrapulmonary tuberculosis compared to controls with <it>M. tuberculosis </it>infection; one (rs4893980) in the gene PDE11A, one (rs10488286) in KCND2, and one (rs2026414) in PCDH15; one was in chromosome 7 but not associated with a known gene. Two additional variants were significantly associated with extrapulmonary tuberculosis compared with pulmonary tuberculosis; one (rs340708) in the gene FAM135B and one in chromosome 13 but not associated with a known gene. The function of all four genes affects cell signaling and activity, including in the brain.</p> <p>Conclusions</p> <p>In this pilot study, we identified 6 novel variants not previously known to be associated with extrapulmonary tuberculosis, including two SNPs more common in persons with extrapulmonary than pulmonary tuberculosis. This provides some support for the hypothesis that the pathogenesis and genetic predisposition to extrapulmonary tuberculosis differs from pulmonary tuberculosis. Further study of these novel SNPs, and more well-powered genome-wide studies of extrapulmonary tuberculosis, is warranted.</p

Effect of 6 months of hybrid closed-loop insulin delivery in adults with type 1 diabetes: a randomised controlled trial protocol

INTRODUCTION: Manual determination of insulin dosing largely fails to optimise glucose control in type 1 diabetes. Automated insulin delivery via closed-loop systems has improved glucose control in short-term studies. The objective of the present study is to determine the effectiveness of 6 months\u27 closed-loop compared with manually determined insulin dosing on time-in-target glucose range in adults with type 1 diabetes. METHODS AND ANALYSIS: This open-label, seven-centre, randomised controlled parallel group clinical trial will compare home-based hybrid closed-loop versus standard diabetes therapy in Australia. Adults aged &ge;25 years with type 1 diabetes using intensive insulin therapy (via multiple daily injections or insulin pump, total enrolment target n=120) will undertake a run-in period including diabetes and carbohydrate-counting education, clinical optimisation and baseline data collection. Participants will then be randomised 1:1 either to 26 weeks of MiniMed 670G hybrid closed-loop system therapy (Medtronic, Northridge, CA, USA) or continuation of their current diabetes therapy. The hybrid closed-loop system delivers insulin automatically to address basal requirements and correct to target glucose level, while bolus doses for meals require user initiation and carbohydrate estimation. Analysis will be intention to treat, with the primary outcome time in continuous glucose monitoring (CGM) target range (3.9-10.0&thinsp;mmol/L) during the final 3 weeks of intervention. Secondary outcomes include: other CGM parameters, HbA1c, severe hypoglycaemia, psychosocial well-being, sleep, cognition, electrocardiography, costs, quality of life, biomarkers of vascular health and hybrid closed-loop system performance. Semistructured interviews will assess the expectations and experiences of a subgroup of hybrid closed-loop users. ETHICS AND DISSEMINATION: The study has Human Research Ethics Committee approval. The study will be conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. Results will be disseminated at scientific conferences and via peer-reviewed publications

An investigation into exercise-induced modifications to DNA methylation-regulatory enzymes in human peripheral blood mononuclear cells

DNA methylation, an epigenetic modification which can regulate gene transcription independently from alterations to the nucleotide sequence, can be manipulated by lifestyle factors such as diet and exercise, hypothetically reversing aberrant DNA methylation associated with disease pathogenesis. The underlying mechanisms by which these changes occur are currently poorly characterised, however, in vitro data suggest that inflammatory mediators are involved. Furthermore, regular exercise appears to reduce inactivity-associated systemic inflammation, possibly by alterations to the methylome, thereby suggesting a cyclic relationship between exercise, inflammation, and epigenetic modification. The aims of this research programme, therefore, were to: characterise the acute changes that occur to the de novo DNA methyltransferases following exercise in peripheral blood mononuclear cells (PBMCs), and the role of exercise-induced systemic inflammation in this process; investigate how these changes then translate into functional modifications to the methylome; and to determine whether a training programme utilising sedentary individuals manipulates DNA methylation of genes involved in chronic systemic inflammation associated with physical inactivity. Pilot investigations corroborated previous in vitro data that recombinant IL-6 is able to regulate nuclear concentrations of DNMT3A and DNMT3B in PBMCs. In order to isolate the influence of circulating proteins independently from genetic polymorphisms that may influence susceptibility to epigenetic change, cells were stimulated with exercise-conditioned plasma following intense endurance exercise which elicited significant alterations in nuclear concentrations of DNMT3A and DNMT3B. Eccentric exercise, which is typically not associated with elevations in circulating cytokines, did not cause any significant changes in nuclear or cytoplasmic DNMT concentration, or global DNA methylation; this supports the hypothesis that transient systemic elevations in inflammatory cytokines are important regulators of epigenetic modifications associated with exercise. Lack of transcriptional changes in DNMT3A following both exercise training and an acute maximal bout suggests that, in line with in vitro data, that the observed elevations in nuclear DNMT concentration are largely due to cellular relocalisation and not gene expression of this enzyme. It remains to be elucidated whether the training regime, and the subsequent response to an acute maximal bout, is able to elicit differential methylation of IL6, NFκB2, and ASC, however, in vitro stimulation of PBMCs with the cytokines IL-6 and IL-1β did cause significant changes to IL6 promoter methylation, further supporting the role of these proteins in epigenetic regulation. The data presented in this thesis support the postulation that exercise-induced changes to DNA methylation in PBMCs likely occur due to systemic elevations of inflammatory proteins, in particular IL-6, which causes manipulation of de novo DNMT nuclear concentrations due to cellular translocation of the enzymes themselves. While it was not possible to determine whether exercise directly modified gene-specific methylation, in vitro experiments suggest that inflammatory cytokines are able to regulate IL6 promoter methylation in human PBMCs