The Escherichia coli RnlA–RnlB toxin–antitoxin complex: production, characterization and crystallization

The Escherichia coli rnlAB operon encodes a toxin–antitoxin module that is involved in protection against infection by bacteriophage T4. The full-length RnlA–RnlB toxin–antitoxin complex as well as the toxin RnlA were purified to homogeneity and crystallized. When the affinity tag is placed on RnlA, RnlB is largely lost during purification and the resulting crystals exclusively comprise RnlA. A homogeneous preparation of RnlA–RnlB containing stoichiometric amounts of both proteins could only be obtained using a His tag placed C-terminal to RnlB. Native mass spectrometry and SAXS indicate a 1:1 stoichiometry for this RnlA–RnlB complex. Crystals of the RnlA–RnlB complex belonged to space group C2, with unit-cell parameters a = 243.32, b = 133.58, c = 55.64 Å, β = 95.11°, and diffracted to 2.6 Å resolution. The presence of both proteins in the crystals was confirmed and the asymmetric unit is likely to contain a heterotetramer with RnlA2:RnlB2 stoichiometry

Real-time dynamics of the formation of hydrated electrons upon irradiation of water clusters with extreme ultraviolet light

Free electrons in a polar liquid can form a bound state via interaction with the molecular environment. This so-called hydrated electron state in water is of fundamental importance e.g.~in cellular biology or radiation chemistry. Hydrated electrons are highly reactive radicals that can either directly interact with DNA or enzymes, or form highly excited hydrogen (H∗) after being captured by protons. Here, we investigate the formation of the hydrated electron in real-time employing XUV femtosecond pulses from a free electron laser, in this way observing the initial steps of the hydration process. Using time-resolved photoelectron spectroscopy we find formation timescales in the low picosecond range and resolve the prominent dynamics of forming excited hydrogen states

The Debrisoft ® monofilament debridement pad for use in acute or chronic wounds: A NICE medical technology guidance

As part of its Medical Technology Evaluation Programme, the National Institute for Health and Care Excellence (NICE) invited a manufacturer to provide clinical and economic evidence for the evaluation of the Debrisoft ® monofilament debridement pad for use in acute or chronic wounds. The University of Birmingham and Brunel University, acting as a consortium, was commissioned to act as an External Assessment Centre (EAC) for NICE, independently appraising the submission. This article is an overview of the original evidence submitted, the EAC’s findings and the final NICE guidance issued. The sponsor submitted a simple cost analysis to estimate the costs of using Debrisoft® to debride wounds compared with saline and gauze, hydrogel and larvae. Separate analyses were conducted for applications in home and applications in a clinic setting. The analysis took an UK National Health Service (NHS) perspective. It incorporated the costs of the technologies and supplementary technologies (such as dressings) and the costs of their application by a district nurse. The sponsor concluded that Debrisoft® was cost saving relative to the comparators. The EAC made amendments to the sponsor analysis to correct for errors and to reflect alternative assumptions. Debrisoft® remained cost saving in most analyses and savings ranged from £77 to £222 per patient compared with hydrogel, from £97 to £347 compared with saline and gauze, and from £180 to £484 compared with larvae depending on the assumptions included in the analysis and whether debridement took place in a home or clinic setting. All analyses were severely limited by the available data on effectiveness, in particular a lack of comparative studies and that the effectiveness data for the comparators came from studies reporting different clinical endpoints compared with Debrisoft®. The Medical Technologies Advisory Committee made a positive recommendation for adoption of Debrisoft® and this has been published as a NICE medical technology guidance (MTG17).The Birmingham and Brunel Consortium is funded by NICE to act as an External Assessment Centre for the Medical Technologies Evaluation Programme

Estimands and their estimators for clinical trials Impacted by the COVID-19 pandemic: a report from the NISS Ingram Olkin Forum Series on unplanned clinical trial disruptions

The COVID-19 pandemic continues to affect the conduct of clinical trials globally. Complications may arise from pandemic-related operational challenges such as site closures, travel limitations and interruptions to the supply chain for the investigational product, or from health-related challenges such as COVID-19 infections. Some of these complications lead to unforeseen intercurrent events in the sense that they affect either the interpretation or the existence of the measurements associated with the clinical question of interest. In this article, we demonstrate how the ICH E9(R1) Addendum on estimands and sensitivity analyses provides a rigorous basis to discuss potential pandemic-related trial disruptions and to embed these disruptions in the context of study objectives and design elements. We introduce several hypothetical estimand strategies and review various causal inference and missing data methods, as well as a statistical method that combines unbiased and possibly biased estimators for estimation. To illustrate, we describe the features of a stylized trial, and how it may have been impacted by the pandemic. This stylized trial will then be re-visited by discussing the changes to the estimand and the estimator to account for pandemic disruptions. Finally, we outline considerations for designing future trials in the context of unforeseen disruptions

Recommendations for the use of next-generation sequencing (NGS) for patients with metastatic cancers: a report from the ESMO Precision Medicine Working Group

Next-generation sequencing (NGS) allows sequencing of a high number of nucleotides in a short time frame at an affordable cost. While this technology has been widely implemented, there are no recommendations from scientific societies about its use in oncology practice. The European Society for Medical Oncology (ESMO) is proposing three levels of recommendations for the use of NGS. Based on the current evidence, ESMO recommends routine use of NGS on tumour samples in advanced non-squamous non-small-cell lung cancer (NSCLC), prostate cancers, ovarian cancers and cholangiocarcinoma. In these tumours, large multigene panels could be used if they add acceptable extra cost compared with small panels. In colon cancers, NGS could be an alternative to PCR. In addition, based on the KN158 trial and considering that patients with endometrial and small-cell lung cancers should have broad access to anti-programmed cell death 1 (anti-PD1) antibodies, it is recommended to test tumour mutational burden (TMB) in cervical cancers, well- and moderately-differentiated neuroendocrine tumours, salivary cancers, thyroid cancers and vulvar cancers, as TMB-high predicted response to pembrolizumab in these cancers. Outside the indications of multigene panels, and considering that the use of large panels of genes could lead to few clinically meaningful responders, ESMO acknowledges that a patient and a doctor could decide together to order a large panel of genes, pending no extra cost for the public health care system and if the patient is informed about the low likelihood of benefit. ESMO recommends that the use of off-label drugs matched to genomics is done only if an access programme and a procedure of decision has been developed at the national or regional level. Finally, ESMO recommends that clinical research centres develop multigene sequencing as a tool to screen patients eligible for clinical trials and to accelerate drug development, and prospectively capture the data that could further inform how to optimise the use of this technology

The influence of feature selection methods on accuracy, stability and interpretability of molecular signatures

Motivation: Biomarker discovery from high-dimensional data is a crucial problem with enormous applications in biology and medicine. It is also extremely challenging from a statistical viewpoint, but surprisingly few studies have investigated the relative strengths and weaknesses of the plethora of existing feature selection methods. Methods: We compare 32 feature selection methods on 4 public gene expression datasets for breast cancer prognosis, in terms of predictive performance, stability and functional interpretability of the signatures they produce. Results: We observe that the feature selection method has a significant influence on the accuracy, stability and interpretability of signatures. Simple filter methods generally outperform more complex embedded or wrapper methods, and ensemble feature selection has generally no positive effect. Overall a simple Student's t-test seems to provide the best results. Availability: Code and data are publicly available at http://cbio.ensmp.fr/~ahaury/

The Effect of Provider Density on Lung Cancer Survival Among Blacks and Whites in the United States

IntroductionLung cancer mortality rates may vary with access to specialty providers and local resources. We sought to examine the effect of access to care, using density of lung cancer care providers, on lung cancer mortality among blacks and whites in the United States.MethodsWe examined U.S. county-level data for age-adjusted lung cancer mortality rates from 2003 to 2007. Our primary independent variable was per capita number of thoracic oncologic providers, adjusting for county-level smoking rates, socioeconomic status, and other geographic factors. Data were obtained from 2009 Area Resource File, National Center for Health Statistics, and the County Health Rankings Project.ResultsProviders of lung cancer care were unevenly distributed among the U.S. counties. For example, 41.4% of the U.S. population reside in counties with less than four thoracic surgeons per 100,000 people, 23.4% in counties with 4 to 15 surgeons per 100,000 people, and 35.3% in counties with more than 15 surgeons per 100,000 people. Geographically, 4.3% of whites compared with 11.2% of blacks lived in high lung cancer mortality zones. Lung cancer mortality did not vary by density of thoracic surgeons or oncology services; however, higher primary care provider density was associated with lung cancer mortality reduction of 4.1 per 100,000 for whites.ConclusionVariation in provider density for thoracic oncology in the United States was not associated with a difference in lung cancer mortality. Lower mortality associated with higher primary care provider density suggests that equitable access to primary care may lead to reduced cancer disparities

Dnmt2/Trdmt1 as mediator of RNA polymerase II transcriptional activity in cardiac growth

Dnmt2/Trdmt1 is a methyltransferase, which has been shown to methylate tRNAs. Deficient mutants were reported to exhibit various, seemingly unrelated, defects in development and RNA-mediated epigenetic heredity. Here we report a role in a distinct developmental regulation effected by a noncoding RNA. We show that Dnmt2-deficiency in mice results in cardiac hypertrophy. Echocardiographic measurements revealed that cardiac function is preserved notwithstanding the increased dimensions of the organ due to cardiomyocyte enlargement. Mechanistically, activation of the P-TEFb complex, a critical step for cardiac growth, results from increased dissociation of the negatively regulating Rn7sk non-coding RNA component in Dnmt2-deficient cells. Our data suggest that Dnmt2 plays an unexpected role for regulation of cardiac growth by modulating activity of the P-TEFb complex. © 2016 Ghanbarian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

EPAC-Lung:Pooled analysis of circulating tumor cells in advanced non-small cell lung cancer

Background: We assessed the clinical validity of circulating tumor cell (CTC) quantification for prognostication of patients with advanced non-small cell lung cancer (NSCLC) by undertaking a European pooled analysis of individual patient data. This is the largest study of its kind and the first to examine between-centre heterogeneity of CTC identification in NSCLC.Methods: Nine European NSCLC CTC centers were asked to provide reported/unreported anonymised data for patients with advanced NSCLC who participated in CellSearch CTC studies from January 2003 - March 2017. We used Cox regression models, stratified by centre, to establish the association between CTC count and survival. We assessed the added value of CTCs to prognostic clinico-pathological models using likelihood ratio (LR) statistics and c-indices.Results: Seven out of nine eligible centers provided data for 550 eligible patients, including 209 patients whose prognostic information was previously unpublished. CTC counts of ≥ 2 and ≥5 per 7·5 mL were associated with reduced progression-free survival (≥2 CTCs: HR 1.72, p < 0·001; ≥5 CTCs: HR 2.21, p < 0·001) and overall survival (≥2 CTCs: HR 2·18, p < 0·001; ≥5 CTCs: HR 2·75, p < 0·001), respectively. Survival prediction was significantly improved by addition of baseline CTC count to LR clinico-pathological models (log-transformed CTCs p < 0·0001; ≥2 CTCs p < 0·0001; ≥5 CTCs p < 0·0001), while more moderate improvements were observed with the use of c-index models. There was minor evidence of between-center heterogeneity in the effect on PFS, but not OS.No difference in CTC profile was observed between key NSCLC molecular subsets such as EGFR, ALK, and KRAS.Conclusions: These data confirm CTCs as an independent prognostic indicator of progression-free survival and overall survival in advanced NSCLC. CTC count improves prognostication when added to full clinico-pathological predictive models. ≥2 CTCs is an appropriate cutoff to move towards establishing clinical utility

100 Hz ROCS microscopy correlated with fluorescence reveals cellular dynamics on different spatiotemporal scales

Fluorescence techniques dominate the field of live-cell microscopy, but bleaching and motion blur from too long integration times limit dynamic investigations of small objects. High contrast, label-free life-cell imaging of thousands of acquisitions at 160 nm resolution and 100 Hz is possible by Rotating Coherent Scattering (ROCS) microscopy, where intensity speckle patterns from all azimuthal illumination directions are added up within 10 ms. In combination with fluorescence, we demonstrate the performance of improved Total Internal Reflection (TIR)-ROCS with variable illumination including timescale decomposition and activity mapping at five different examples: millisecond reorganization of macrophage actin cortex structures, fast degranulation and pore opening in mast cells, nanotube dynamics between cardiomyocytes and fibroblasts, thermal noise driven binding behavior of virus-sized particles at cells, and, bacterial lectin dynamics at the cortex of lung cells. Using analysis methods we present here, we decipher how motion blur hides cellular structures and how slow structure motions cover decisive fast motions