Myocyte enhancer factor 2C: an osteoblast transcription factor identified by DMSO enhanced mineralization

Free to read on publisher website Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem-cell mediated therapies for fracture and other orthopaedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of simulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase (ALP) activity and extracellular matrix mineralization. Furthermore, similar DMSO mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1 we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased ALP activity and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. Flow on knockdown of bone specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts

Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines

Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenylamidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationshipsfrom comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site

Internal control genes for quantitative RT-PCR expression analysis in mouse osteoblasts, osteoclasts and macrophages

<p>Abstract</p> <p>Background</p> <p>Real-time quantitative RT-PCR (qPCR) is a powerful technique capable of accurately quantitating mRNA expression levels over a large dynamic range. This makes qPCR the most widely used method for studying quantitative gene expression. An important aspect of qPCR is selecting appropriate controls or normalization factors to account for any differences in starting cDNA quantities between samples during expression studies. Here, we report on the selection of a concise set of housekeeper genes for the accurate normalization of quantitative gene expression data in differentiating osteoblasts, osteoclasts and macrophages. We implemented the use of geNorm, an algorithm that determines the suitability of genes to function as housekeepers by assessing expression stabilities. We evaluated the expression stabilities of 18S, ACTB, B2M, GAPDH, HMBS and HPRT1 genes.</p> <p>Findings</p> <p>Our analyses revealed that 18S and GAPDH were regulated during osteoblast differentiation and are not suitable for use as reference genes. The most stably expressed genes in osteoblasts were ACTB, HMBS and HPRT1 and their geometric average constitutes a suitable normalization factor upon which gene expression data can be normalized. In macrophages, 18S and GAPDH were the most variable genes while HMBS and B2M were the most stably expressed genes. The geometric average of HMBS and B2M expression levels forms a suitable normalization factor to account for potential differences in starting cDNA quantities during gene expression analysis in macrophages. The expression stabilities of the six candidate reference genes in osteoclasts were, on average, more variable than that observed in macrophages but slightly less variable than those seen in osteoblasts. The two most stably expressed genes in osteoclasts were HMBS and B2M and the genes displaying the greatest levels of variability were 18S and GAPDH. Notably, 18S and GAPDH were the two most variably expressed control genes in all three cell types. The geometric average of HMBS, B2M and ACTB creates an appropriate normalization factor for gene expression studies in osteoclasts.</p> <p>Conclusion</p> <p>We have identified concise sets of genes suitable to use as normalization factors for quantitative real-time RT-PCR gene expression studies in osteoblasts, osteoclasts and macrophages.</p

A randomised controlled trial of a digital intervention (Renewed) to support symptom management, wellbeing and quality of life in cancer survivors

Background: Many cancer survivors following primary treatment have prolonged poor quality of life.Aim: To determine the effectiveness of a bespoke digital intervention to support cancer survivors.Design: Pragmatic parallel open randomised trial.Setting: UK general practices.Methods: People having finished primary treatment (&lt;= 10 years previously) for colo-rectal, breast or prostate cancers, with European-Organization-for-Research-and-Treatment-of-Cancer QLQ-C30 score &lt;85, were randomised by online software to: 1) detailed ‘generic’ digital NHS support (‘LiveWell’;n=906), 2) a bespoke complex digital intervention (‘Renewed’;n=903) addressing symptom management, physical activity, diet, weight loss, distress, or 3) ‘Renewed-with-support’ (n=903): ‘Renewed’ with additional brief email and telephone support. Results: Mixed linear regression provided estimates of the differences between each intervention group and generic advice: at 6 months (primary time point: n’s respectively 806;749;705) all groups improved, with no significant between-group differences for EORTC QLQ-C30, but global health improved more in both intervention groups. By 12 months there were: small improvements in EORTC QLQ-C30 for Renewed-with-support (versus generic advice: 1.42, 95% CIs 0.33-2.51); both groups improved global health (12 months: renewed: 3.06, 1.39-4.74; renewed-with-support: 2.78, 1.08-4.48), dyspnoea, constipation, and enablement, and lower NHS costs (generic advice £265: in comparison respectively £141 (153-128) and £77 (90-65) lower); and for Renewed-with-support improvement in several other symptom subscales. No harms were identified.Conclusion: Cancer survivors quality of life improved with detailed generic online support. Robustly developed bespoke digital support provides limited additional short term benefit, but additional longer term improvement in global healthenablement and symptom management, with substantially lower NHS costs.<br/

Laser generated electron transport experiment in a novel wire nail target

The transport of high intensity (2x1020 W/cm2) laser generated relativistic electrons with a solid target has been studied in a novel geometry. The targets were 20 um diameter solid copper wires, coated with ~ 2um of titanium, with an 80 um diameter hemispherical termination. They were illuminated by an ~500fs, ~200J pulse of 1.053um laser light focused to a ~ 20um diameter spot centered on the flat face of the hemisphere. K-alpha fluorescence from the Cu and Ti regions was imaged together with extreme ultraviolet (X-UV) emission at 68 and 256eV. Results showed a quasi exponential decline in K-alpha emission along the wire over a distance of a few hundred microns from the laser focus, consistent with bulk Ohmic inhibition of the relativistic electron transport. Weaker Ka and X-UV emission on a longer scale length showed limb brightening suggesting a transition to enhanced transport at the surface of the wire

Utilization and Harmonization of Adult Accelerometry Data: Review and Expert Consensus.

PURPOSE: This study aimed to describe the scope of accelerometry data collected internationally in adults and to obtain a consensus from measurement experts regarding the optimal strategies to harmonize international accelerometry data. METHODS: In March 2014, a comprehensive review was undertaken to identify studies that collected accelerometry data in adults (sample size, n ≥ 400). In addition, 20 physical activity experts were invited to participate in a two-phase Delphi process to obtain consensus on the following: unique research opportunities available with such data, additional data required to address these opportunities, strategies for enabling comparisons between studies/countries, requirements for implementing/progressing such strategies, and value of a global repository of accelerometry data. RESULTS: The review identified accelerometry data from more than 275,000 adults from 76 studies across 36 countries. Consensus was achieved after two rounds of the Delphi process; 18 experts participated in one or both rounds. The key opportunities highlighted were the ability for cross-country/cross-population comparisons and the analytic options available with the larger heterogeneity and greater statistical power. Basic sociodemographic and anthropometric data were considered a prerequisite for this. Disclosure of monitor specifications and protocols for data collection and processing were deemed essential to enable comparison and data harmonization. There was strong consensus that standardization of data collection, processing, and analytical procedures was needed. To implement these strategies, communication and consensus among researchers, development of an online infrastructure, and methodological comparison work were required. There was consensus that a global accelerometry data repository would be beneficial and worthwhile. CONCLUSIONS: This foundational resource can lead to implementation of key priority areas and identification of future directions in physical activity epidemiology, population monitoring, and burden of disease estimates.This work, and authors involved in this work were supported by the UK Medical Research Council (grants MC_UU_12015/3 and MRC Centenary Award to KWi, SB); the British Heart Foundation (grant FS/12/58/29709 to KWi); the Australian Heart Foundation (grant PH 12B 7054 to GNH); the Australian National Health and Medical Research Council (Fellowship to NO; Program grant to NO; NHMRC Centre for Research Excellence Grant in the Translational Science of Sedentary Behaviour APP1041056 to GNH, NO, DD); an Australian Postgraduate Award (to SS); The Coca-Cola Company, Body Media, U.S. National Institutes of Health, and Technogym (to SB); MRC, Chartered Society of Physiotherapy, EPSRC, Greater Manchester Academic Health Science Network (to MG); Australian Research Council (Future Fellowship: FT100100918 to DD).This is the final published version. It first appeared at http://dx.doi.org/10.1249/MSS.000000000000066

xclim: xarray-based climate data analytics

xclim is a Python library that enables computation of climate indicators over large, hetero- geneous data sets. It is built using xarray objects and operations, can seamlessly benefit from the parallelization handling provided by dask, and relies on community conventions for data formatting and metadata attributes. xclim is meant as a tool to facilitate both climate science research and the delivery of operational climate services and products. In addition to climate indicator calculations, xclim also includes utilities for bias correction and statistical adjustment, ensemble analytics, model diagnostics, data quality assurance, and metadata standards compliance

Energy Injection for Fast Ignition

In the fast ignition concept, assembled fuel is ignited through a separate high intensity laser pulse. Fast Ignition targets facilitate this ignition using a reentrant cone. It provides clear access through the overlaying coronal plasma, and controls the laser plasma interaction to optimize hot-electron production and transport into the compressed plasma. Recent results suggest that the cone does not play any role in guiding light or electrons to its tip, and coupling to electrons can be reduced by a small amount of preplasma. This puts stringent requirements on the ignition laser focusing, pointing, and prepulse