Investigating the basis of substrate recognition in the pC221 relaxosome

The nicking of the origin of transfer (oriT) is an essential initial step in the conjugative mobilization of plasmid DNA. In the case of staphylococcal plasmid pC221, nicking by the plasmid-specific MobA relaxase is facilitated by the DNA-binding accessory protein MobC; however, the role of MobC in this process is currently unknown. In this study, the site of MobC binding was determined by DNase I footprinting. MobC interacts with oriT DNA at two directly repeated 9 bp sequences, mcb1 and mcb2, upstream of the oriT nic site, and additionally at a third, degenerate repeat within the mobC gene, mcb3. The binding activity of the conserved sequences was confirmed indirectly by competitive electrophoretic mobility shift assays and directly by Surface Plasmon Resonance studies. Mutation at mcb2 abolished detectable nicking activity, suggesting that binding of this site by MobC is a prerequisite for nicking by MobA. Sequential site-directed mutagenesis of each binding site in pC221 has demonstrated that all three are required for mobilization. The MobA relaxase, while unable to bind to oriT DNA alone, was found to associate with a MobC–oriT complex and alter the MobC binding profile in a region between mcb2 and the nic site. Mutagenesis of oriT in this region defines a 7 bp sequence, sra, which was essential for nicking by MobA. Exchange of four divergent bases between the sra of pC221 and the related plasmid pC223 was sufficient to swap their substrate identity in a MobA-specific nicking assay. Based on these observations we propose a model of layered specificity in the assembly of pC221-family relaxosomes, whereby a common MobC:mcb complex presents the oriT substrate, which is then nicked only by the cognate MobA

Strategies and challenges associated with recruiting retirement village communities and residents into a group exercise intervention

Background: Randomized controlled trials (RCTs) provide the highest level of scientific evidence, but successful participant recruitment is critical to ensure the external and internal validity of results. This study describes the strategies associated with recruiting older adults at increased falls risk residing in retirement villages into an 18-month cluster RCT designed to evaluate the effects of a dual-task exercise program on falls and physical and cognitive function. Methods: Recruitment of adults aged ≥65 at increased falls risk residing within retirement villages (size 60–350 residents) was initially designed to occur over 12 months using two distinct cohorts (C). Recruitment occurred via a three-stage approach that included liaising with: 1) village operators, 2) independent village managers, and 3) residents. To recruit residents, a variety of different approaches were used, including distribution of information pack, on-site presentations, free muscle and functional testing, and posters displayed in common areas. Results: Due to challenges with recruitment, three cohorts were established between February 2014 and April 2015 (14 months). Sixty retirement villages were initially invited, of which 32 declined or did not respond, leaving 28 villages that expressed interest. A total of 3947 individual letters of invitation were subsequently distributed to residents of these villages, from which 517 (13.1%) expressions of interest (EOI) were received. Across three cohorts with different recruitment strategies adopted there were only modest differences in the number of EOI received (10.5 to 15.3%), which suggests that no particular recruitment approach was most effective. Following the initial screening of these residents, 398 (77.0%) participants were deemed eligible to participate, but a final sample of 300 (58.0% of the 517 EOI) consented and was randomized; 7.6% of the 3947 residents invited. Principal reasons for not participating, despite being eligible, were poor health, lack of time and no GP approval. Conclusion: This study highlights that there are significant challenges associated with recruiting sufficient numbers of older adults from independent living retirement villages into an exercise intervention designed to improve health and well-being. Trial registration: Australian New Zealand Clinical Trials Registry: ACTRN12613001 161718. Date registered 23rd October 2013

Very Early Optical Afterglows of Gamma-Ray Bursts: Evidence for Relative Paucity of Detection

Very early observations with the Swift satellite of gamma-ray burst (GRB) afterglows reveal that the optical component is not detected in a large number of cases. This is in contrast to the bright optical flashes previously discovered in some GRBs (e.g. GRB 990123 and GRB 021211). Comparisons of the X-ray afterglow flux to the optical afterglow flux and prompt gamma-ray fluence is used to quantify the seemingly deficient optical, and in some cases X-ray, light at these early epochs. This comparison reveals that some of these bursts appear to have higher than normal gamma-ray efficiencies. We discuss possible mechanisms and their feasibility for explaining the apparent lack of early optical emission. The mechanisms considered include: foreground extinction, circumburst absorption, Ly-alpha blanketing and absorption due to high redshift, low density environments, rapid temporal decay, and intrinsic weakness of the reverse shock. Of these, foreground extinction, circumburst absorption, and high redshift provide the best explanations for most of the non-detections in our sample. There is tentative evidence of suppression of the strong reverse shock emission. This could be because of a Poynting-flux-dominated flow or a pure non-relativistic hydrodynamical reverse shock.Comment: 22 pages, 5 figures. Accepted for publication in Ap

Effectiveness of dual-task functional power training for preventing falls in older people: Study protocol for a cluster randomised controlled trial

Background: Falls are a major public health concern with at least one third of people aged 65 years and over falling at least once per year, and half of these will fall repeatedly, which can lead to injury, pain, loss of function and independence, reduced quality of life and even death. Although the causes of falls are varied and complex, the age-related loss in muscle power has emerged as a useful predictor of disability and falls in older people. In this population, the requirements to produce explosive and rapid movements often occurs whilst simultaneously performing other attention-demanding cognitive or motor tasks, such as walking while talking or carrying an object. The primary aim of this study is to determine whether dual-task functional power training (DT-FPT) can reduce the rate of falls in community-dwelling older people. Methods/Design: The study design is an 18-month cluster randomised controlled trial in which 280 adults aged =65 years residing in retirement villages, who are at increased risk of falling, will be randomly allocated to: 1) an exercise programme involving DT-FPT, or 2) a usual care control group. The intervention is divided into 3 distinct phases: 6 months of supervised DT-FPT, a 6-month 'step down' maintenance programme, and a 6-month follow-up. The primary outcome will be the number of falls after 6, 12 and 18 months. Secondary outcomes will include: lower extremity muscle power and strength, grip strength, functional assessments of gait, reaction time and dynamic balance under single- and dual-task conditions, activities of daily living, quality of life, cognitive function and falls-related self-efficacy. We will also evaluate the cost-effectiveness of the programme for preventing falls. Discussion: The study offers a novel approach that may guide the development and implementation of future community-based falls prevention programmes that specifically focus on optimising muscle power and dual-task performance to reduce falls risk under 'real life' conditions in older adults. In addition, the 'step down' programme will provide new information about the efficacy of a less intensive maintenance programme for reducing the risk of falls over an extended period. Trial registration: Australian New Zealand Clinical Trials Registry: ACTRN12613001161718. Date registered 23 October 2013

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro

The isolation of plasmid-protein relaxation complexes from bacteria is indicative of the plasmid nicking-closing equilibrium in vivo that serves to ready the plasmids for conjugal transfer. In pC221 and pC223, the components required for in vivo site- and strand-specific nicking at oriT are MobC and MobA. In order to investigate the minimal requirements for nicking in the absence of host-encoded factors, the reactions were reconstituted in vitro. Purified MobA and MobC, in the presence of Mg(2+) or Mn(2+), were found to nick at oriT with a concomitant phosphorylation-resistant modification at the 5′ end of nic. The position of nic is consistent with that determined in vivo. MobA, MobC, and Mg(2+) or Mn(2+) therefore represent the minimal requirements for nicking activity. Cross-complementation analyses showed that the MobC proteins possess binding specificity for oriT DNA of either plasmid and are able to complement each other in the nicking reaction. Conversely, nicking by the MobA proteins is plasmid specific. This suggests the MobA proteins may encode the nicking specificity determinant

Effectiveness of dual-task functional power training for preventing falls in older people: study protocol for a cluster randomised controlled trial

Background Falls are a major public health concern with at least one third of people aged 65 years and over falling at least once per year, and half of these will fall repeatedly, which can lead to injury, pain, loss of function and independence, reduced quality of life and even death. Although the causes of falls are varied and complex, the age-related loss in muscle power has emerged as a useful predictor of disability and falls in older people. In this population, the requirements to produce explosive and rapid movements often occurs whilst simultaneously performing other attention-demanding cognitive or motor tasks, such as walking while talking or carrying an object. The primary aim of this study is to determine whether dual-task functional power training (DT-FPT) can reduce the rate of falls in community-dwelling older people. Methods/Design The study design is an 18-month cluster randomised controlled trial in which 280 adults aged ≥65 years residing in retirement villages, who are at increased risk of falling, will be randomly allocated to: 1) an exercise programme involving DT-FPT, or 2) a usual care control group. The intervention is divided into 3 distinct phases: 6 months of supervised DT-FPT, a 6-month \u27step down\u27 maintenance programme, and a 6-month follow-up. The primary outcome will be the number of falls after 6, 12 and 18 months. Secondary outcomes will include: lower extremity muscle power and strength, grip strength, functional assessments of gait, reaction time and dynamic balance under single- and dual-task conditions, activities of daily living, quality of life, cognitive function and falls-related self-efficacy. We will also evaluate the cost-effectiveness of the programme for preventing falls. Discussion The study offers a novel approach that may guide the development and implementation of future community-based falls prevention programmes that specifically focus on optimising muscle power and dual-task performance to reduce falls risk under \u27real life\u27 conditions in older adults. In addition, the \u27step down\u27 programme will provide new information about the efficacy of a less intensive maintenance programme for reducing the risk of falls over an extended period. Trial registration Australian New Zealand Clinical Trials Registry: ACTRN12613001161718 webcite. Date registered 23 October 2013

Very early optical afterglows of gamma-ray bursts: evidence for relative paucity of detection,”

ABSTRACT Very early observations with the Swift satellite of -ray burst (GRB) afterglows reveal that the optical component is not detected in a large number of cases. This is in contrast to the bright optical flashes previously discovered in some GRBs (e.g., GRB 990123 and GRB 021211). Comparisons of the X-ray afterglow flux to the optical afterglow flux and prompt -ray fluence is used to quantify the seemingly deficient optical, and in some cases X-ray, light at these early epochs. This comparison reveals that some of these bursts appear to have higher than normal -ray efficiencies. We discuss possible mechanisms and their feasibility for explaining the apparent lack of early optical emission. The mechanisms considered include, foreground extinction, circumburst absorption, Ly blanketing and absorption due to high-redshift, low-density environments, rapid temporal decay, and intrinsic weakness of the reverse shock. Of these, foreground extinction, circumburst absorption, and high redshift provide the best explanations for most of the nondetections in our sample. There is tentative evidence of suppression of the strong reverse shock emission. This could be because of a Poynting flux-dominated flow or a pure nonrelativistic hydrodynamic reverse shock