Statistical Inference of In Vivo Properties of Human DNA Methyltransferases from Double-Stranded Methylation Patterns

DNA methyltransferases establish methylation patterns in cells and transmit these patterns over cell generations, thereby influencing each cell's epigenetic states. Three primary DNA methyltransferases have been identified in mammals: DNMT1, DNMT3A and DNMT3B. Extensive in vitro studies have investigated key properties of these enzymes, namely their substrate specificity and processivity. Here we study these properties in vivo, by applying novel statistical analysis methods to double-stranded DNA methylation patterns collected using hairpin-bisulfite PCR. Our analysis fits a novel Hidden Markov Model (HMM) to the observed data, allowing for potential bisulfite conversion errors, and yields statistical estimates of parameters that quantify enzyme processivity and substrate specificity. We apply this model to methylation patterns established in vivo at three loci in humans: two densely methylated inactive X (Xi)-linked loci ( and ), and an autosomal locus (), where methylation densities are tissue-specific but moderate. We find strong evidence for a high level of processivity of DNMT1 at and , with the mean association tract length being a few hundred base pairs. Regardless of tissue types, methylation patterns at are dominated by DNMT1 maintenance events, similar to the two Xi-linked loci, but are insufficiently informative regarding processivity to draw any conclusions about processivity at that locus. At all three loci we find that DNMT1 shows a strong preference for adding methyl groups to hemi-methylated CpG sites over unmethylated sites. The data at all three loci also suggest low (possibly 0) association of the de novo methyltransferases, the DNMT3s, and are consequently uninformative about processivity or preference of these enzymes. We also extend our HMM to reanalyze published data on mouse DNMT1 activities in vitro. The results suggest shorter association tracts (and hence weaker processivity), and much longer non-association tracts than human DNMT1 in vivo

Testing the FMR1 Promoter for Mosaicism in DNA Methylation among CpG Sites, Strands, and Cells in FMR1-Expressing Males with Fragile X Syndrome

Variability among individuals in the severity of fragile X syndrome (FXS) is influenced by epigenetic methylation mosaicism, which may also be common in other complex disorders. The epigenetic signal of dense promoter DNA methylation is usually associated with gene silencing, as was initially reported for FMR1 alleles in individuals with FXS. A paradox arose when significant levels of FMR1 mRNA were reported for some males with FXS who had been reported to have predominately methylated alleles. We have used hairpin-bisufite PCR, validated with molecular batch-stamps and barcodes, to collect and assess double-stranded DNA methylation patterns from these previously studied males. These patterns enable us to distinguish among three possible forms of methylation mosaicism, any one of which could explain FMR1 expression in these males. Our data indicate that cryptic inter-cell mosaicism in DNA methylation can account for the presence of FMR1 mRNA in some individuals with FXS

Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper.

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the "gut sac" method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner

A mobile phone-based care model for outpatient cardiac rehabilitation: the care assessment platform (CAP)

<p>Abstract</p> <p>Background</p> <p>Cardiac rehabilitation programs offer effective means to prevent recurrence of a cardiac event, but poor uptake of current programs have been reported globally. Home based models are considered as a feasible alternative to avoid various barriers related to care centre based programs. This paper sets out the study design for a clinical trial seeking to test the hypothesis that these programs can be better and more efficiently supported with novel Information and Communication Technologies (ICT).</p> <p>Methods/Design</p> <p>We have integrated mobile phones and web services into a comprehensive home- based care model for outpatient cardiac rehabilitation. Mobile phones with a built-in accelerometer sensor are used to measure physical exercise and WellnessDiary software is used to collect information on patients' physiological risk factors and other health information. Video and teleconferencing are used for mentoring sessions aiming at behavioural modifications through goal setting. The mentors use web-portal to facilitate personal goal setting and to assess the progress of each patient in the program. Educational multimedia content are stored or transferred via messaging systems to the patients phone to be viewed on demand. We have designed a randomised controlled trial to compare the health outcomes and cost efficiency of the proposed model with a traditional community based rehabilitation program. The main outcome measure is adherence to physical exercise guidelines.</p> <p>Discussion</p> <p>The study will provide evidence on using mobile phones and web services for mentoring and self management in a home-based care model targeting sustainable behavioural modifications in cardiac rehabilitation patients.</p> <p>Trial registration</p> <p>The trial has been registered in the Australian New Zealand Clinical Trials Registry (ANZCTR) with number ACTRN12609000251224.</p

Relationship of depression, disability, and family caregiver attitudes to the quality of life of Kuwaiti persons with multiple sclerosis: a controlled study

<p>Abstract</p> <p>Background</p> <p>Assessment of subjective quality of life (QOL) of persons with multiple sclerosis (MS) could facilitate the detection of psychosocial aspects of disease that may otherwise go unrecognized. The objectives of the study were to (i) compare the QOL ratings of relapsing remitting (RRMS) and progressive (PMS) types of MS with those of a general population group and the impression of their family caregivers; and (ii) assess the association of demographic, clinical, treatment, depression, and caregiver variables with patients' QOL.</p> <p>Methods</p> <p>Consecutive clinic attendees at the national neurology hospital were assessed with the 26 -item WHOQOL Instrument, Beck's Depression Inventory and Expanded Disability Scale. Caregivers rated their impression of patients' QOL and attitudes to patients' illness.</p> <p>Results</p> <p>The 170 patients (60 m, 109 f) consisted of 145(85.3%) with RRMS and 25 with PMS, aged 32.4(SD 8.8), age at onset 27.1(7.7), EDSS score 2.9 (1.8), and 76% were employed. The patients were predominantly dissatisfied with their life circumstances. The RRMS group had higher QOL domain scores (P < 0.001), and lower depression(P > 0.05) and disability (P < 0.0001) scores than the PMS group. Patients had significantly lower QOL scores than the control group (P < 0.001). Caregiver impression was significantly correlated with patients' ratings. Depression was the commonest significant covariate of QOL domains. When we controlled for depression and disability scores, differences between the two MS groups became significant for only one (out of 6) QOL domains. Patients who were younger, better educated, employed, felt less sick and with lesser side effects, had higher QOL. The predictors of patients' overall QOL were disability score, caregiver impression of patients' QOL, and caregiver fear of having MS.</p> <p>Conclusion</p> <p>Our data indicate that MS patients in stable condition and with social support can hope to have better QOL, if clinicians pay attention to depression, disability, the impact of side effects of treatment and family caregiver anxieties about the illness. The findings call for a regular program of psychosocial intervention in the clinical setting, to address these issues and provide caregiver education and supports, in order to enhance the quality of care.</p

In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position–, cell type–, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs

The effect of an electronic cognitive aid on the management of ST-elevation myocardial infarction during caesarean section: a prospective randomised simulation study

Background Cognitive aids have come to be viewed as promising tools in the management of perioperative critical events. The majority of published simulation studies have focussed on perioperative crises that are characterised by time pressure, rare occurrence, or complex management steps (e.g., cardiac arrest emergencies, management of the difficult airway). At present, there is limited information on the usefulness of cognitive aids in critical situations with moderate time pressure and complexity. Intraoperative myocardial infarction may be an emergency to which these limitations apply. Methods Anaesthetic teams were allocated to control (no cognitive aid; n = 10) or intervention (cognitive aid provided; n = 10) groups. The primary aim of this study was to compare cognitive aid versus memory for intraoperative ST-elevation myocardial infarction (STEMI) management in a simulation of caesarean delivery under spinal anaesthesia. We identified nine evidence-based metrics of essential care from current guidelines and subdivided them into mandatory (high level of evidence; no interference with surgery) and optional (lower class of recommendation; possible impact on surgery) tasks. Six clinically relevant tasks were added by consensus. Implementation of these steps was measured by scoring task items in a binary fashion (yes/no). The interval between the diagnosis of STEMI and the first contact with the cardiac catheterisation lab was measured. To determine whether or not the cognitive aid had prompted an action, participants from the cognitive aid group were interviewed during debriefing on every single treatment step. At the end of the simulation, session participants were asked to complete a survey. Results The presence of the cognitive aid did not shorten the time interval until the cardiac catheterisation lab was contacted. The availability of the cognitive aid improved task performance in the tasks identified from the guidelines (93% vs. 69%; p < 0.001) as well as overall task performance (87.5% vs. 59%; p < 0.001). The observed difference in performance can be attributed to the use of the cognitive aid, as performance from memory alone would have been comparable across both groups. Trainees appeared to derive greater benefit from the cognitive aid than did consultants and nurses. Conclusions The management of intraoperative ST-elevation myocardial infarction can be improved if teams use a cognitive aid. Trainees appeared to derive greater benefit from the cognitive aid than did consultants and nurses

Women’s experiences of disrespect and abuse in maternity care facilities in Benue State, Nigeria

Background Disrespect and abuse (D&A) of women in health facilities continues to be a prevailing public health issue in many countries. Studies have reported significantly high prevalence of D&A among women during pregnancy and childbirth in Nigeria, but little is known about women’s perceptions and experiences of D&A during maternity care in the country. The aim of this study was to explore: 1) how women perceived their experiences of D&A during pregnancy, childbirth, and in the postnatal period in Benue State, Nigeria; and 2) how women viewed the impact of D&A on the future use of health facilities for maternity care. Method Five focus group discussions with a sample of 32 women were conducted as part of a qualitative phenomenological study. All the women received maternity care in health facilities in Benue State, Nigeria and had experienced at least one incident of disrespect and abuse. Audio-recorded discussions were transcribed and analysed using a six-stage thematic analysis using NVivo11. Results The participants perceived incidents such as being shouted at and the use of abusive language as a common practice. Women described these incidents as devaluing and dehumanising to their sense of dignity. Some women perceived that professionals did not intend to cause harm by such behaviours. Emerged themes included: (1) ‘normative’ practice; (2) dehumanisation of women; (3) 'no harm intended' and (4) intentions about the use of maternity services in future. The women highlighted the importance of accessing health facilities for safe childbirth and expressed that the experiences of D&A may not impact their intended use of health facilities. However, the accounts reflected their perceptions about the inherent lack of choice and an underlying sense of helplessness. Conclusion Incidents of D&A that were perceived as commonplace carry substantial implications for the provision of respectful maternity care in Nigeria and other similar settings. As a country with one of the highest rates of maternal deaths, the findings point to the need for policy and practice to address the issue urgently through implementing preventive measures, including empowering women to reinforce their right to be treated with dignity and respect, and sensitising health care professionals

The association of depression and all-cause and cause-specific mortality: an umbrella review of systematic reviews and meta-analyses

Background: Depression is a prevalent and disabling mental disorder that frequently co-occurs with a wide range of chronic conditions. Evidence has suggested that depression could be associated with excess all-cause mortality across different settings and populations, although the causality of these associations remains unclear. Methods: We conducted an umbrella review of systematic reviews and meta-analyses of observational studies. PubMed, PsycINFO, and Embase electronic databases were searched through January 20, 2018. Systematic reviews and meta-analyses that investigated associations of depression and all-cause and cause-specific mortality were selected for the review. The evidence was graded as convincing, highly suggestive, suggestive, or weak based on quantitative criteria that included an assessment of heterogeneity, 95% prediction intervals, small-study effects, and excess significance bias. Results: A total of 26 references providing 2 systematic reviews and data for 17 meta-analytic estimates met inclusion criteria (19 of them on all-cause mortality); data from 246 unique studies (N = 3,825,380) were synthesized. All 17 associations had P < 0.05 per random effects summary effects, but none of them met criteria for convincing evidence. Associations of depression and all-cause mortality in patients after acute myocardial infarction, in individuals with heart failure, in cancer patients as well as in samples from mixed settings met criteria for highly suggestive evidence. However, none of the associations remained supported by highly suggestive evidence in sensitivity analyses that considered studies employing structured diagnostic interviews. In addition, associations of depression and all-cause mortality in cancer and post-acute myocardial infarction samples were supported only by suggestive evidence when studies that tried to adjust for potential confounders were considered. Conclusions: Even though associations between depression and mortality have nominally significant results in all assessed settings and populations, the evidence becomes weaker when focusing on studies that used structured interviews and those that tried to adjust for potential confounders. A causal effect of depression on all-cause and cause-specific mortality remains unproven, and thus interventions targeting depression are not expected to result in lower mortality rates at least based on current evidence from observational studies