Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

The OSCAR-IB Consensus Criteria for Retinal OCT Quality Assessment

Retinal optical coherence tomography (OCT) is an imaging biomarker for neurodegeneration in multiple sclerosis (MS). In order to become validated as an outcome measure in multicenter studies, reliable quality control (QC) criteria with high inter-rater agreement are required

Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells

The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering β-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear β-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active β-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation.We acknowledge funding from an EPSRC Platform grant which supported McMurray and a Wellcome Trust project grant which supported Wann and McMurray. Wann is now supported on an ARUK project grant. Thompson was funded by a BBSRC PhD studentshi

BRIT1/MCPH1 links chromatin remodelling to DNA damage response

To detect and repair damaged DNA, DNA damage response proteins need to overcome the barrier of condensed chromatin to gain access to DNA lesions1. ATP-dependent chromatin remodeling is one of the fundamental mechanisms used by cells to relax chromatin in DNA repair2–3. However, the mechanism mediating their recruitment to DNA lesions remains largely unknown. BRIT1 (also known as MCPH1) is an early DNA damage response protein that is mutated in human primary microcephaly4–8. We report here a previously unknown function of BRIT1 as a regulator of ATP-dependent chromatin remodeling complex SWI/SNF in DNA repair. Upon DNA damage, BRIT1 increases its interaction with SWI/SNF through the ATM/ATR-dependent phosphorylation on the BAF170 subunit. This increase of binding affinity provides a means by which SWI/SNF can be specifically recruited to and maintained at DNA lesions. Loss of BRIT1 causes impaired chromatin relaxation owing to reduced association of SWI/SNF with chromatin. This explains the decreased recruitment of repair proteins to DNA lesions and reduced efficiency of repair in BRIT1-deficient cells, resulting in impaired survival from DNA damage. Our findings, therefore, identify BRIT1 as a key molecule that links chromatin remodeling with DNA damage response in the control of DNA repair, and its dysfunction contributes to human disease

Safety and immunogenicity of a self-amplifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging trial

Background: Lipid nanoparticle (LNP) encapsulated self-amplifying RNA (saRNA) is a novel technology formulated as a low dose vaccine against COVID-19. Methods: A phase I first-in-human dose-ranging trial of a saRNA COVID-19 vaccine candidate LNP-nCoVsaRNA, was conducted at Imperial Clinical Research Facility, and participating centres in London, UK, between 19th June to 28th October 2020. Participants received two intramuscular (IM) injections of LNP-nCoVsaRNA at six different dose levels, 0.1-10.0μg, given four weeks apart. An open-label dose escalation was followed by a dose evaluation. Solicited adverse events (AEs) were collected for one week from enrolment, with follow-up at regular intervals (1-8 weeks). The binding and neutralisation capacity of anti-SARS-CoV-2 antibody raised in participant sera was measured by means of an anti-Spike (S) IgG ELISA, immunoblot, SARS-CoV-2 pseudoneutralisation and wild type neutralisation assays. (The trial is registered: ISRCTN17072692, EudraCT 2020-001646-20). Findings: 192 healthy individuals with no history or serological evidence of COVID-19, aged 18-45 years were enrolled. The vaccine was well tolerated with no serious adverse events related to vaccination. Seroconversion at week six whether measured by ELISA or immunoblot was related to dose (both p<0.001), ranging from 8% (3/39; 0.1μg) to 61% (14/23; 10.0μg) in ELISA and 46% (18/39; 0.3μg) to 87% (20/23; 5.0μg and 10.0μg) in a post-hoc immunoblot assay. Geometric mean (GM) anti-S IgG concentrations ranged from 74 (95% CI, 45-119) at 0.1μg to 1023 (468-2236) ng/mL at 5.0μg (p<0.001) and was not higher at 10.0μg. Neutralisation of SARS-CoV-2 by participant sera was measurable in 15% (6/39; 0.1μg) to 48% (11/23; 5.0μg) depending on dose level received. Interpretation: Encapsulated saRNA is safe for clinical development, is immunogenic at low dose levels but failed to induce 100% seroconversion. Modifications to optimise humoral responses are required to realise its potential as an effective vaccine against SARS-CoV-2. Funding: This study was co-funded by grants and gifts from the Medical Research Council UKRI (MC_PC_19076), and the National Institute Health Research/Vaccine Task Force, Partners of Citadel and Citadel Securities, Sir Joseph Hotung Charitable Settlement, Jon Moulton Charity Trust, Pierre Andurand, Restore the Earth

Biophysical mechanisms of single-cell interactions with microtopographical cues

Biophysical cues encoded in the extracellular matrix (ECM) are increasingly being explored to control cell behavior in tissue engineering applications. Recently, we showed that cell adhesion to microtopographical structures (“micropegs”) can suppress proliferation in a manner that may be blunted by inhibiting cellular contractility, suggesting that this effect is related to altered cell-scaffold mechanotransduction. We now directly investigate this possibility at the microscale through a combination of live-cell imaging, single-cell mechanics methods, and analysis of gene expression. Using time-lapse imaging, we show that when cells break adhesive contacts with micropegs, they form F-actin-filled tethers that extend and then rupture at a maximum, critical length that is greater than trailing-edge tethers observed on topographically flat substrates. This critical tether length depends on myosin activation, with inhibition of Rho-associated kinase abolishing topography-dependent differences in tether length. Using cellular de-adhesion and atomic force microscopy indentation measurements, we show that the micropegs enhance cell-scaffold adhesive interactions without changing whole-cell elasticity. Moreover, micropeg adhesion increases expression of specific mechanotransductive genes, including RhoA GTPase and myosin heavy chain II, and, in myoblasts, the functional marker connexin 43. Together, our data support a model in which microtopographical cues alter the local mechanical microenvironment of cells by modulating adhesion and adhesion-dependent mechanotransductive signaling

Neck circumference is associated with adipose tissue content in thigh skeletal muscle in overweight and obese premenopausal women

Neck circumference (NC) has been proposed as a simple and practical tool, independently associated with cardiometabolic risk factors. However, the association of NC with inter-muscular adipose tissue (IMAT) is still to be determined. We aimed to examine the association of NC with thigh IMAT, and visceral adipose tissue (VAT) measured with computed tomography (CT) in overweight/obese women. 142 premenopausal overweight and obese Caucasian women participated in this crosssectional study. NC was measured with an inextensible metallic tape above the thyroid cartilage according to International Society for Advancement of Kinanthropometry protocol. Thigh IMAT and VAT volumes were measured with a single cross-sectional CT. Regarding the covariates, fat mass (FM) was assessed with dual-energy x-ray absorptiometry and physical activity was objectively measured with accelerometry. NC was positively associated with thigh IMAT and VAT volumes (standardized β coefcient: β=0.45, P-value= ≤0.001, β=0.60, P=≤0.001; respectively), which persisted after adjusting for age, height, overall FM or moderate-to-vigorous physical activity. Our fndings show that NC is associated with thigh IMAT volume in overweight and obese premenopausal Caucasian women, regardless of the amount of lower-body fatness. These results suggest underscoring the relevance of NC as a marker of adipose tissue content in thigh skeletal muscle.Portuguese Foundation for Science and Technology Sapiens 358007/99Oeiras City CouncilBecel PortugalRoche Pharmaceuticals PortugalCompal PortugalUniversity of Granada Plan Propio de Investigacion 2016 -Excellence actions: Unit of Excellence on Exercise and Health (UCEES)Junta de AndaluciaEuropean Union (EU) SOMM17/6107/UGRFundacion Carolina C.201657496

Xenobiotic metabolizing enzyme gene polymorphisms predict response to lung volume reduction surgery

<p>Abstract</p> <p>Background</p> <p>In the National Emphysema Treatment Trial (NETT), marked variability in response to lung volume reduction surgery (LVRS) was observed. We sought to identify genetic differences which may explain some of this variability.</p> <p>Methods</p> <p>In 203 subjects from the NETT Genetics Ancillary Study, four outcome measures were used to define response to LVRS at six months: modified BODE index, post-bronchodilator FEV₁, maximum work achieved on a cardiopulmonary exercise test, and University of California, San Diego shortness of breath questionnaire. Sixty-four single nucleotide polymorphisms (SNPs) were genotyped in five genes previously shown to be associated with chronic obstructive pulmonary disease susceptibility, exercise capacity, or emphysema distribution.</p> <p>Results</p> <p>A SNP upstream from glutathione S-transferase pi (<it>GSTP1</it>; p = 0.003) and a coding SNP in microsomal epoxide hydrolase (<it>EPHX1</it>; p = 0.02) were each associated with change in BODE score. These effects appeared to be strongest in patients in the non-upper lobe predominant, low exercise subgroup. A promoter SNP in <it>EPHX1 </it>was associated with change in BODE score (p = 0.008), with the strongest effects in patients with upper lobe predominant emphysema and low exercise capacity. One additional SNP in <it>GSTP1 </it>and three additional SNPs in <it>EPHX1 </it>were associated (p < 0.05) with additional LVRS outcomes. None of these SNP effects were seen in 166 patients randomized to medical therapy.</p> <p>Conclusion</p> <p>Genetic variants in <it>GSTP1 </it>and <it>EPHX1</it>, two genes encoding xenobiotic metabolizing enzymes, were predictive of response to LVRS. These polymorphisms may identify patients most likely to benefit from LVRS.</p