Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

Francisella tularensis is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance

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

Background: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93–1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94–1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93–1·05; p=0·79). Interpretation: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

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

Background: In this study, we aimed to evaluate the effects of tocilizumab in adult patients admitted to hospital with COVID-19 with both hypoxia and systemic inflammation. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. Those trial participants with hypoxia (oxygen saturation <92% on air or requiring oxygen therapy) and evidence of systemic inflammation (C-reactive protein ≥75 mg/L) were eligible for random assignment in a 1:1 ratio to usual standard of care alone versus usual standard of care plus tocilizumab at a dose of 400 mg–800 mg (depending on weight) given intravenously. A second dose could be given 12–24 h later if the patient's condition had not improved. The primary outcome was 28-day mortality, assessed in the intention-to-treat population. The trial is registered with ISRCTN (50189673) and ClinicalTrials.gov (NCT04381936). Findings: Between April 23, 2020, and Jan 24, 2021, 4116 adults of 21 550 patients enrolled into the RECOVERY trial were included in the assessment of tocilizumab, including 3385 (82%) patients receiving systemic corticosteroids. Overall, 621 (31%) of the 2022 patients allocated tocilizumab and 729 (35%) of the 2094 patients allocated to usual care died within 28 days (rate ratio 0·85; 95% CI 0·76–0·94; p=0·0028). Consistent results were seen in all prespecified subgroups of patients, including those receiving systemic corticosteroids. Patients allocated to tocilizumab were more likely to be discharged from hospital within 28 days (57% vs 50%; rate ratio 1·22; 1·12–1·33; p<0·0001). Among those not receiving invasive mechanical ventilation at baseline, patients allocated tocilizumab were less likely to reach the composite endpoint of invasive mechanical ventilation or death (35% vs 42%; risk ratio 0·84; 95% CI 0·77–0·92; p<0·0001). Interpretation: In hospitalised COVID-19 patients with hypoxia and systemic inflammation, tocilizumab improved survival and other clinical outcomes. These benefits were seen regardless of the amount of respiratory support and were additional to the benefits of systemic corticosteroids. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

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

ANALYSIS OF ON-DEMAND FORECASTING METHODOLOGY FOR FUTURE CONTRACT ACTIONS AT NAVAL SURFACE WARFARE CENTER, PORT HUENEME DIVISION (NSWC PHD)

For the past decade and continuing today, Naval Surface Warfare Center, Port Hueneme Division (NSWC PHD) has been in a perpetual state of emergency. Inaccurate forecasting for future contract actions contained in our Long Range Acquisition Forecast (LRAF) has left the NSWC PHD acquisition workforce unprepared to face increasing demands. As a result, more pending contract actions are unknown until the need date arrives and an emergency effort must be initiated to complete the action. This emergent trend can be found throughout the Department of the Navy (DON). It is imperative that a more accurate forecasting model be utilized within NSWC PHD to capture the demand signal of the acquisition workforce. This thesis reviewed the policy and procedures of the U.S. federal government including those within the Department of Defense (DOD), DON and Navy Sea Systems Command (NAVSEA), and NSWC PHD to understand the procedure for capturing the demand for future contract actions. This research found that the current methodology in place for the LRAF is heavily dependent on the requirement generator and historical references, which do not cover all contracting data at NSWC PHD. This paper identified a path for the acquisition workforce to incorporate data-driven analytics to its forecasting models to more accurately represent demand for that workforce. This research begins the process of moving toward the data-driven forecasting mentioned and determining the first steps forward.Civilian, Department of the NavyCivilian, Department of the NavyCivilian, Department of the NavyApproved for public release. Distribution is unlimited

Effect of standardized training on the reliability of the Cochrane risk of bias assessment tool: a prospective study.

BACKGROUND The Cochrane risk of bias tool is commonly criticized for having a low reliability. We aimed to investigate whether training of raters, with objective and standardized instructions on how to assess risk of bias, can improve the reliability of the Cochrane risk of bias tool. METHODS In this pilot study, four raters inexperienced in risk of bias assessment were randomly allocated to minimal or intensive standardized training for risk of bias assessment of randomized trials of physical therapy treatments for patients with knee osteoarthritis pain. Two raters were experienced risk of bias assessors who served as reference. The primary outcome of our study was between-group reliability, defined as the agreement of the risk of bias assessments of inexperienced raters with the reference assessments of experienced raters. Consensus-based assessments were used for this purpose. The secondary outcome was within-group reliability, defined as the agreement of assessments within pairs of inexperienced raters. We calculated the chance-corrected weighted Kappa to quantify agreement within and between groups of raters for each of the domains of the risk of bias tool. RESULTS A total of 56 trials were included in our analysis. The Kappa for the agreement of inexperienced raters with reference across items of the risk of bias tool ranged from 0.10 to 0.81 for the minimal training group and from 0.41 to 0.90 for the standardized training group. The Kappa values for the agreement within pairs of inexperienced raters across the items of the risk of bias tool ranged from 0 to 0.38 for the minimal training group and from 0.93 to 1 for the standardized training group. Between-group differences in Kappa for the agreement of inexperienced raters with reference always favored the standardized training group and was most pronounced for incomplete outcome data (difference in Kappa 0.52, p < 0.001) and allocation concealment (difference in Kappa 0.30, p = 0.004). CONCLUSIONS Intensive, standardized training on risk of bias assessment may significantly improve the reliability of the Cochrane risk of bias tool

Effect of standardized training on the reliability of the Cochrane risk of bias assessment tool: a study protocol

BACKGROUND The Cochrane risk of bias (RoB) tool has been widely embraced by the systematic review community, but several studies have reported that its reliability is low. We aim to investigate whether training of raters, including objective and standardized instructions on how to assess risk of bias, can improve the reliability of this tool. We describe the methods that will be used in this investigation and present an intensive standardized training package for risk of bias assessment that could be used by contributors to the Cochrane Collaboration and other reviewers. METHODS/DESIGN This is a pilot study. We will first perform a systematic literature review to identify randomized clinical trials (RCTs) that will be used for risk of bias assessment. Using the identified RCTs, we will then do a randomized experiment, where raters will be allocated to two different training schemes: minimal training and intensive standardized training. We will calculate the chance-corrected weighted Kappa with 95% confidence intervals to quantify within- and between-group Kappa agreement for each of the domains of the risk of bias tool. To calculate between-group Kappa agreement, we will use risk of bias assessments from pairs of raters after resolution of disagreements. Between-group Kappa agreement will quantify the agreement between the risk of bias assessment of raters in the training groups and the risk of bias assessment of experienced raters. To compare agreement of raters under different training conditions, we will calculate differences between Kappa values with 95% confidence intervals. DISCUSSION This study will investigate whether the reliability of the risk of bias tool can be improved by training raters using standardized instructions for risk of bias assessment. One group of inexperienced raters will receive intensive training on risk of bias assessment and the other will receive minimal training. By including a control group with minimal training, we will attempt to mimic what many review authors commonly have to do, that is-conduct risk of bias assessment in RCTs without much formal training or standardized instructions. If our results indicate that an intense standardized training does improve the reliability of the RoB tool, our study is likely to help improve the quality of risk of bias assessments, which is a central component of evidence synthesis

Effect of standardized training on the reliability of the Cochrane risk of bias assessment tool: a study protocol

Abstract Background The Cochrane risk of bias (RoB) tool has been widely embraced by the systematic review community, but several studies have reported that its reliability is low. We aim to investigate whether training of raters, including objective and standardized instructions on how to assess risk of bias, can improve the reliability of this tool. We describe the methods that will be used in this investigation and present an intensive standardized training package for risk of bias assessment that could be used by contributors to the Cochrane Collaboration and other reviewers. Methods/Design This is a pilot study. We will first perform a systematic literature review to identify randomized clinical trials (RCTs) that will be used for risk of bias assessment. Using the identified RCTs, we will then do a randomized experiment, where raters will be allocated to two different training schemes: minimal training and intensive standardized training. We will calculate the chance-corrected weighted Kappa with 95% confidence intervals to quantify within- and between-group Kappa agreement for each of the domains of the risk of bias tool. To calculate between-group Kappa agreement, we will use risk of bias assessments from pairs of raters after resolution of disagreements. Between-group Kappa agreement will quantify the agreement between the risk of bias assessment of raters in the training groups and the risk of bias assessment of experienced raters. To compare agreement of raters under different training conditions, we will calculate differences between Kappa values with 95% confidence intervals. Discussion This study will investigate whether the reliability of the risk of bias tool can be improved by training raters using standardized instructions for risk of bias assessment. One group of inexperienced raters will receive intensive training on risk of bias assessment and the other will receive minimal training. By including a control group with minimal training, we will attempt to mimic what many review authors commonly have to do, that is—conduct risk of bias assessment in RCTs without much formal training or standardized instructions. If our results indicate that an intense standardized training does improve the reliability of the RoB tool, our study is likely to help improve the quality of risk of bias assessments, which is a central component of evidence synthesis

The Fitzroy contaminants project : a study of the nutrient and fine-sediment dynamics of the Fitzroy Estuary and Keppel Bay

The Fitzroy catchment is the largest Queensland catchment discharging to the Great Barrier Reef (GBR) lagoon. Sediments and nutrients together with anthropogenic pollutants originating upstream in the catchment are discharged from the Fitzroy River via the Fitzroy Estuary (FE) and ultimately into Keppel Bay (KB). The estuary and the bay act as natural chemical reactors where the materials delivered undergo chemical and physical transformations before some are deposited and stored in the growing deltaic and beach areas, with the remainder transported eastward to the southern zone of the GBR lagoon. The Reef Water Quality Protection Plan is a recent State–Commonwealth initiative which aims to improve land-use management practices within many Queensland catchments with the intent of reducing the loads of sediments, nutrients and other potentially deleterious substances entering the GBR lagoon. The background knowledge to make realistic predictions of the response of the FE and KB ecosystems to changed sediment and nutrient deliveries did not exist when this project started. The Agricultural Contaminants Project (Project AC) was developed to address these knowledge gaps and to produce a predictive framework to aid managers in the evaluation of various load reduction strategies