The Difficult Case of Crystallization and Structure Solution for the ParC55 Breakage-Reunion Domain of Topoisomerase IV from Streptococcus pneumoniae

BACKGROUND: Streptococcus pneumoniae is the major cause of community-acquired pneumonia and is also associated with bronchitis, meningitis, otitis and sinusitis. The emergence and increasing prevalence of resistance to penicillin and other antibiotics has led to interest in other anti-pneumonococcal drugs such as quinolones that target the enzymes DNA gyrase and topoisomerase IV. During crystallization and in the avenues to finding a method to determine phases for the structure of the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae, obstacles were faced at each stage of the process. These problems included: majority of the crystals being twinned, either non-diffracting or exhibiting a high mosaic spread. The crystals, which were grown under conditions that favoured diffraction, were difficult to flash-freeze without loosing diffraction. The initial structure solution by molecular replacement failed and the approach proved to be unviable due to the complexity of the problem. In the end the successful structure solution required an in-depth data analysis and a very detailed molecular replacement search. METHODOLOGY/PRINCIPAL FINDINGS: Crystal anti-twinning agents have been tested and two different methods of flash freezing have been compared. The fragility of the crystals did not allow the usual method of transferring the crystals into the heavy atom solution. Consequently, it was necessary to co-crystallize in the presence of the heavy atom compound. The multiple isomorphous replacement approach was unsuccessful because the 7 cysteine mutants which were engineered could not be successfully derivatized. Ultimately, molecular replacement was used to solve the structure by sorting through a large number of solutions in space group P1 using CNS. CONCLUSIONS/SIGNIFICANCE: The main objective of this paper is to describe the obstacles which were faced and overcome in order to acquire data sets on such difficult crystals and determine phases for successful structure solution

Gender inequalities during COVID-19

This is the final version. Available on open access from SAGE Publications via the DOI in this record The onset of the global COVID-19 pandemic put a halt to progress toward gender equality and, instead, exacerbated existing gender inequalities across domains—from gendered divisions of labour to economic stability. In this paper we document some of the most glaring gender inequalities that have arisen in the COVID-19 pandemic and discuss how social psychological theories and research—including work on gender stereotypes and roles, responses to threat, precarious masculinity, perceptions of risk, and backlash—can help to explain the roots of these inequalities. In doing so, we use a broad definition of gender and consider relevant intersections of identity. Finally, we present three key considerations for research on gender inequalities moving forward. Namely, the need for social psychologists to (a) challenge binary conceptualizations of gender, (b) broaden the focus of research on gender inequalities, and (c) adopt an intersectional lens to address systemic inequalities in the wake of COVID-19.European Commissio

Career Barriers and Motivations for Women and Men Working in Disaster Risk Reduction: A Snapshot in the Asia-Pacific Region

This is the final version. Available on open access from the United Nations Office for Disaster Risk Reduction via the DOI in this recordThis publication explores enablers and barriers to women’s careers and leadership aspirations in disaster risk reduction (DRR), the ways in which workplace outcomes are different for women and men, the understanding of what predicts these differential outcomes and what opportunities there are to redress them. The results of this research suggest the day-to-day experiences of DRR professionals shape their career motivations and ambition, their well-being and their desire to stay within the profession. While there are many commonalities in women and men’s experiences in DRR, there are also key differences. The report identified barriers to women’s career advancement, well-being and longevity in DRR including but not limited to: women’s lower willingness to sacrifice for their careers (likely influenced by lower expectations that these sacrifices will be rewarded), care demands placed on women with children, organisational constraints on women’s workplace authenticity, lower work-life balance and lower quality professional relationships with co-workers and especially senior colleagues. The study also uncovered barriers to men’s well-being in DRR, particularly in regard to their experiences of bullying, burnout and desire to leave the profession. Importantly, the findings also offer insight into what organisations and the DRR profession more broadly can do to improve women and men’s career outcomes in DRR

Preliminary Studies of Magnetic NDE Techniques for Identifying Neutron Embrittlement of Pressure Vessel Steel

In operating nuclear reactors, the steel pressure vessel is exposed to neutron irradiation. The high energy part (\u3e1 MeV) of this irradiation, over a long period, makes the steel brittle and susceptible to rupture

A microbial platform for renewable propane synthesis based on a fermentative butanol pathway

Background Propane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli. Results We report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L). Conclusions This study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde

Experimental characterization of the hypersonic flow around a cuboid

Understanding the hypersonic flow around faceted shapes is important in the context of the fragmentation and demise of satellites undergoing uncontrolled atmospheric entry. To better understand the physics of such flows, as well as the satellite demise process, we perform an experimental study of the Mach 5 flow around a cuboid geometry in the University of Manchester High SuperSonic Tunnel. Heat fluxes are measured using infrared thermography and a 3D inverse heat conduction solution, and flow features are imaged using schlieren photography. Measurements are taken at a range of Reynolds numbers from 40.0×103 to 549×103. The schlieren results suggest the presence of a separation bubble at the windward edge of the cube at high Reynolds numbers. High heat fluxes are observed near corners and edges, which are caused by boundary-layer thinning. Additionally, on the side (off-stagnation) faces of the cube, we observe wedge-shaped regions of high heat flux emanating from the windward corners of the cube. We attribute these to vortical structures being generated by the strong expansion around the cube’s corners. We also observe that the stagnation point of the cube is off-centre of the windward face, which we propose is due to sting flex under aerodynamic loading. Finally, we propose a simple method of calculating the stagnation point heat flux to a cube, as well as relations which can be used to predict hypersonic heat fluxes to cuboid geometries such as satellites during atmospheric re-entry

Molecular Genetics of T Cell Development

T cell development is guided by a complex set of transcription factors that act recursively, in different combinations, at each of the developmental choice points from T-lineage specification to peripheral T cell specialization. This review describes the modes of action of the major T-lineage-defining transcription factors and the signal pathways that activate them during intrathymic differentiation from pluripotent precursors. Roles of Notch and its effector RBPSuh (CSL), GATA-3, E2A/HEB and Id proteins, c-Myb, TCF-1, and members of the Runx, Ets, and Ikaros families are critical. Less known transcription factors that are newly recognized as being required for T cell development at particular checkpoints are also described. The transcriptional regulation of T cell development is contrasted with that of B cell development, in terms of their different degrees of overlap with the stem-cell program and the different roles of key transcription factors in gene regulatory networks leading to lineage commitment

A new estimation of the recent tropospheric molecular hydrogen budget using atmospheric observations and variational inversion

This paper presents an analysis of the recent tropospheric molecular hydrogen (H2) budget with a particular focus on soil uptake and European surface emissions. A variational inversion scheme is combined with observations from the RAMCES and EUROHYDROS atmospheric networks, which include continuous measurements performed between mid-2006 and mid-2009. Net H2 surface flux, then deposition velocity and surface emissions and finally, deposition velocity, biomass burning, anthropogenic and N2 fixation-related emissions were simultaneously inverted in several scenarios. These scenarios have focused on the sensibility of the soil uptake value to different spatio-temporal distributions. The range of variations of these diverse inversion sets generate an estimate of the uncertainty for each term of the H2 budget. The net H2 flux per region (High Northern Hemisphere, Tropics and High Southern Hemisphere) varies between −8 and +8 Tg yr−1. The best inversion in terms of fit to the observations combines updated prior surface emissions and a soil deposition velocity map that is based on bottom-up and top-down estimations. Our estimate of global H2 soil uptake is −59±9 Tg yr−1. Forty per cent of this uptake is located in the High Northern Hemisphere and 55% is located in the Tropics. In terms of surface emissions, seasonality is mainly driven by biomass burning emissions. The inferred European anthropogenic emissions are consistent with independent H2 emissions estimated using a H2/CO mass ratio of 0.034 and CO emissions within the range of their respective uncertainties. Additional constraints, such as isotopic measurements would be needed to infer a more robust partition of H2 sources and sinks

Appetite suppressants and valvular heart disease - a systematic review

Background Although appetite suppressants have been implicated in the development of valvular heart disease, the exact level of risk is still uncertain. Initial studies suggested that as many as 1 in 3 exposed patients were affected, but subsequent research has yielded substantially different figures. Our objective was to systematically assess the risk of valvular heart disease with appetite suppressants. Methods We accepted studies involving obese patients treated with any of the following appetite suppressants: fenfluramine, dexfenfluramine, and phentermine. Three types of studies were reviewed: controlled and uncontrolled observational studies, and randomized controlled trials. Outcomes of interest were echocardiographically detectable aortic regurgitation of mild or greater severity, or mitral regurgitation of moderate or greater severity. Results Of the 1279 patients evaluated in seven uncontrolled cohort studies, 236 (18%) and 60 (5%) were found to have aortic and mitral regurgitation, respectively. Pooled data from six controlled cohort studies yielded, for aortic regurgitation, a relative risk ratio of 2.32 (95% confidence intervals 1.79 to 3.01, p < 0.00001) and an attributable rate of 4.9%, and for mitral regurgitation, a relative risk ratio of 1.55 (95% confidence intervals 1.06 to 2.25, p = 0.02) with an attributable rate of 1.0%. Only one case of valvular heart disease was detected in 57 randomized controlled trials, but this was judged unrelated to drug therapy. Conclusions The risk of valvular heart disease is significantly increased by the appetite suppressants reviewed here. Nevertheless, when considering all the evidence, valvulopathy is much less common than suggested by the initial, less methodologically rigorous studies