Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Death

The death of islet β cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank hyperglycemia, especially in type 1 diabetes. The development of sensitive and reliable biomarkers of β cell death may allow for early therapeutic intervention to prevent or delay the development of diabetes. Recently, several groups including our own have reported that cell-free, differentially methylated DNA encoding preproinsulin (INS) in the circulation is correlated to β cell death in pre-type 1 diabetes and new-onset type 1 diabetes. Here, we present a step-by-step protocol using digital PCR for the measurement of cell-free INS DNA that is differentially methylated at cytosine at position -69 bp (relative to the transcriptional start site). We demonstrate that the assay can distinguish between methylated and unmethylated cytosine at position -69 bp, is linear across several orders of magnitude, provides absolute quantitation of DNA copy numbers, and can be applied to samples of human serum from individuals with new-onset type 1 diabetes and disease-free controls. The protocol described here can be adapted to any DNA species for which detection of differentially methylated cytosines is desired, whether from circulation or from isolated cells and tissues, and can provide absolute quantitation of DNA fragments

Dynamic equilibrium sets atomic content of galaxies across cosmic time

We analyze 88 independent high-resolution cosmological zoom-in simulations of disk galaxies in the NIHAO simulations suite to explore the connection between the atomic gas fraction and angular momentum of baryons throughout cosmic time. The study is motivated by the analytic model of \citet{obreschkow16}, which predicts a relation between the atomic gas fraction $f_{\rm atm}$ and the global atomic stability parameter $q \equiv j\sigma / (GM)$, where $M$ and $j$ are the mass and specific angular momentum of the galaxy (stars+cold gas) and $\sigma$ is the velocity dispersion of the atomic gas. We show that the simulated galaxies follow this relation from their formation ($z\simeq4$) to present within $\sim 0.5$ dex. To explain this behavior, we explore the evolution of the local Toomre stability and find that $90\%$--$100\%$ of the atomic gas in all simulated galaxies is stable at any time. In other words, throughout the entire epoch of peak star formation until today, the timescale for accretion is longer than the timescale to reach equilibrium, thus resulting in a quasi-static equilibrium of atomic gas at any time. Hence, the evolution of $f_{\rm atm}$ depends on the complex hierarchical growth history primarily via the evolution of $q$. An exception are galaxies subject to strong environmental effects.Comment: 12 pages, 7 figures; accepted to Ap

Effects of gut chemistry in marine bivalves on the assimilation of metals from ingested sediment particles

Bioavailability and uptake of trace metals by benthic animals are often assumed to be limited by authigenic sulfide minerals because of their low metal solubilities and reactivities under sedimentary conditions. However, digestive processes and gut conditions such as Eh, pH, and enzyme or surfactant activity, can affect the release of ingested metals in the gut and control uptake. In a series of laboratory experiments with the deposit-feeding clam, Macoma balthica and the suspension-feeding mussel, Mytilus edulis, we assessed assimilation efficiencies (AE) of radioisotopes of Ag, Cd and Co associated with acid-volatile sulfide (AVS), iron oxide (re-oxidized AVS), and reduced and oxidized natural sediment. To evaluate controls on AE, we measured the gut passage time (GPT) of ingested particles, gut Eh, pH, and extraction of Ag, Cd, and Co from particles into gut juice. In general, the overall trends of AEs and metal extraction were Co \u3e Cd ≥ Ag. AEs, metal extraction, and GPTs were higher in M. balthica than in M. edulis in most cases. M. balthica tended overall to take up metals more readily from oxidized than reduced natural sediment, whereas M. edulis did the opposite for Co and Cd. AEs of metals in reoxidized AVS (Fe-oxides) were generally similar to oxic sediment (Ag being the exception for M. edulis). In M. balthica, there was no significant difference in AEs from AVS and Fe-oxide particles for Cd (14 -20%) or Co (27-35%), but AEs for Ag from AVS particles were greater in large clams (28%) than small clams (15%). There were generally poor correlations between AEs of metals and metal release in gut juice. Low pH and moderate reducing conditions facilitated dissolution of AVS- and iron oxide-bound metal in the guts of both animals. The GPTs (64 h) for Co associated with AVS particles in M. edulis were an order of magnitude greater than for Ag and Cd, or for Co associated with other particle types. Overall, no single mechanism appears to control metal AE in marine bivalves and in vitro studies of metal dissolution in gut juice do not completely mimic the complex digestive processes operating in vivo, and thus cannot fully explain metal assimilation in these animals

Comparison of aggregate and individual participant data approaches to meta-analysis of randomised trials : An observational study

BACKGROUND: It remains unclear when standard systematic reviews and meta-analyses that rely on published aggregate data (AD) can provide robust clinical conclusions. We aimed to compare the results from a large cohort of systematic reviews and meta-analyses based on individual participant data (IPD) with meta-analyses of published AD, to establish when the latter are most likely to be reliable and when the IPD approach might be required. METHODS AND FINDINGS: We used 18 cancer systematic reviews that included IPD meta-analyses: all of those completed and published by the Meta-analysis Group of the MRC Clinical Trials Unit from 1991 to 2010. We extracted or estimated hazard ratios (HRs) and standard errors (SEs) for survival from trial reports and compared these with IPD equivalents at both the trial and meta-analysis level. We also extracted or estimated the number of events. We used paired t tests to assess whether HRs and SEs from published AD differed on average from those from IPD. We assessed agreement, and whether this was associated with trial or meta-analysis characteristics, using the approach of Bland and Altman. The 18 systematic reviews comprised 238 unique trials or trial comparisons, including 37,082 participants. A HR and SE could be generated for 127 trials, representing 53% of the trials and approximately 79% of eligible participants. On average, trial HRs derived from published AD were slightly more in favour of the research interventions than those from IPD (HRAD to HRIPD ratio = 0.95, p = 0.007), but the limits of agreement show that for individual trials, the HRs could deviate substantially. These limits narrowed with an increasing number of participants (p < 0.001) or a greater number (p < 0.001) or proportion (p < 0.001) of events in the AD. On average, meta-analysis HRs from published AD slightly tended to favour the research interventions whether based on fixed-effect (HRAD to HRIPD ratio = 0.97, p = 0.088) or random-effects (HRAD to HRIPD ratio = 0.96, p = 0.044) models, but the limits of agreement show that for individual meta-analyses, agreement was much more variable. These limits tended to narrow with an increasing number (p = 0.077) or proportion of events (p = 0.11) in the AD. However, even when the information size of the AD was large, individual meta-analysis HRs could still differ from their IPD equivalents by a relative 10% in favour of the research intervention to 5% in favour of control. We utilised the results to construct a decision tree for assessing whether an AD meta-analysis includes sufficient information, and when estimates of effects are most likely to be reliable. A lack of power at the meta-analysis level may have prevented us identifying additional factors associated with the reliability of AD meta-analyses, and we cannot be sure that our results are generalisable to all outcomes and effect measures. CONCLUSIONS: In this study we found that HRs from published AD were most likely to agree with those from IPD when the information size was large. Based on these findings, we provide guidance for determining systematically when standard AD meta-analysis will likely generate robust clinical conclusions, and when the IPD approach will add considerable value

An empirical investigation of intuitions about uptake

Since Austin’s introduction of the locutionary-illocutionary-perlocutionary distinction, it has been a matter of debate within speech act theory whether illocutionary acts like promising, warning, refusing and telling require audience ‘uptake’ in order to be performed. Philosophers on different sides of this debate have tried to support their positions by appealing to hypothetical scenarios, designed to elicit intuitive judgements about the role of uptake. However, philosophers’ intuitions appeared to remain deadlocked, while laypeople’s intuitions have not yet been probed. To begin rectifying that, we ran two experiments probing lay intuitions about the implications of uptake failure. Overall, we found that participants’ responses were skewed towards agreement that speech acts were performed, despite the lack of uptake. There were, however, significant differences across the four different speech act types we investigated (with the highest levels of agreement found for refusing, followed by warning, then telling, and finally promising). We also obtained evidence of complex effects relating to the (high or low) stakes involved in the scenarios. While this study only represents an initial exploration of intuitions about uptake, our results form a basis for further research into their nature and significance, across a range of speech acts, scenarios, and experimental designs

Small molecule inhibitors of Late SV40 Factor (LSF) abrogate hepatocellular carcinoma (HCC): evaluation using an endogenous HCC model

Hepatocellular carcinoma (HCC) is a lethal malignancy with high mortality and poor prognosis. Oncogenic transcription factor Late SV40 Factor (LSF) plays an important role in promoting HCC. A small molecule inhibitor of LSF, Factor Quinolinone Inhibitor 1 (FQI1), significantly inhibited human HCC xenografts in nude mice without harming normal cells. Here we evaluated the efficacy of FQI1 and another inhibitor, FQI2, in inhibiting endogenous hepatocarcinogenesis. HCC was induced in a transgenic mouse with hepatocyte-specific overexpression of c-myc (Alb/c-myc) by injecting N-nitrosodiethylamine (DEN) followed by FQI1 or FQI2 treatment after tumor development. LSF inhibitors markedly decreased tumor burden in Alb/c-myc mice with a corresponding decrease in proliferation and angiogenesis. Interestingly, in vitro treatment of human HCC cells with LSF inhibitors resulted in mitotic arrest with an accompanying increase in CyclinB1. Inhibition of CyclinB1 induction by Cycloheximide or CDK1 activity by Roscovitine significantly prevented FQI-induced mitotic arrest. A significant induction of apoptosis was also observed upon treatment with FQI. These effects of LSF inhibition, mitotic arrest and induction of apoptosis by FQI1s provide multiple avenues by which these inhibitors eliminate HCC cells. LSF inhibitors might be highly potent and effective therapeutics for HCC either alone or in combination with currently existing therapies.The present study was supported in part by grants from The James S. McDonnell Foundation, National Cancer Institute Grant R01 CA138540-01A1 (DS), National Institutes of Health Grant R01 CA134721 (PBF), the Samuel Waxman Cancer Research Foundation (SWCRF) (DS and PBF), National Institutes of Health Grants R01 GM078240 and P50 GM67041 (SES), the Johnson and Johnson Clinical Innovation Award (UH), and the Boston University Ignition Award (UH). JLSW was supported by Alnylam Pharmaceuticals, Inc. DS is the Harrison Endowed Scholar in Cancer Research and Blick scholar. PBF holds the Thelma Newmeyer Corman Chair in Cancer Research. The authors acknowledge Dr. Lauren E. Brown (Dept. Chemistry, Boston University) for the synthesis of FQI1 and FQI2, and Lucy Flynn (Dept. Biology, Boston University) for initially identifying G2/M effects caused by FQI1. (James S. McDonnell Foundation; R01 CA138540-01A1 - National Cancer Institute; R01 CA134721 - National Institutes of Health; R01 GM078240 - National Institutes of Health; P50 GM67041 - National Institutes of Health; Samuel Waxman Cancer Research Foundation (SWCRF); Johnson and Johnson Clinical Innovation Award; Boston University Ignition Award; Alnylam Pharmaceuticals, Inc.)Published versio

Addressing multiple modifiable risks through structured community-based Learning Clubs to improve maternal and infant health and infant development in rural Vietnam: protocol for a parallel group cluster randomised controlled trial

Introduction: Optimal early childhood development is an international priority. Risks during pregnancy and early childhood have lasting effects because growth is rapid. We will test whether a complex intervention addressing multiple modifiable risks: maternal nutrition, mental health, parenting capabilities, infant health and development and gender-based violence, is effective in reducing deficient cognitive development among children aged two in rural Vietnam. Methods and analysis: The Learning Clubs intervention is a structured programme combining perinatal stage-specific information, learning activities and social support. It comprises 20 modules, in 19 accessible, facilitated groups for women at a community centre and one home visit. Evidence-informed content is from interventions to address each risk tested in randomised controlled trials in other resource-constrained settings. Content has been translated and culturally adapted for Vietnam and acceptability and feasibility established in pilot testing. We will conduct a two-arm parallel-group cluster-randomised controlled trial, with the commune as clustering unit. An independent statistician will select 84/112 communes in Ha Nam Province and randomly assign 42 to the control arm providing usual care and 42 to the intervention arm. In total, 1008 pregnant women (12 per commune) from 84 clusters are needed to detect a difference in the primary outcome (Bayley Scales of Infant and Toddler Development Cognitive Score \u3c1 SD below standardised norm for 2 years of age) of 15% in the control and 8% in the intervention arms, with 80% power, significance 0.05 and intracluster correlation coefficient 0.03. Ethics and dissemination: Monash University Human Research Ethics Committee (Certificate Number 20160683), Melbourne, Victoria, Australia and the Institutional Review Board of the Hanoi School of Public Health (Certificate Number 017-377IDD- YTCC), Hanoi, Vietnam have approved the trial. Results will be disseminated through a comprehensive multistranded dissemination strategy including peer-reviewed publications, national and international conference presentations, seminars and technical and lay language reports