Evidence that Listeria innocua modulates its membrane's stored curvature elastic stress, but not fluidity, through the cell cycle.

This paper reports that the abundances of endogenous cardiolipin and phosphatidylethanolamine halve during elongation of the Gram-positive bacterium Listeria innocua. The lyotropic phase behaviour of model lipid systems that describe these modulations in lipid composition indicate that the average stored curvature elastic stress of the membrane is reduced on elongation of the cell, while the fluidity appears to be maintained. These findings suggest that phospholipid metabolism is linked to the cell cycle and that changes in membrane composition can facilitate passage to the succeding stage of the cell cycle. This therefore suggests a means by which bacteria can manage the physical properties of their membranes through the cell cycle

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria

<p>Abstract</p> <p>Background</p> <p>The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria.</p> <p>Results</p> <p>Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning.</p> <p>Conclusion</p> <p>The residual <it>att </it>sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.</p

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

State-of-the-art detection of Mycobacterium tuberculosis in blood during tuberculosis infection using phage technology

Introduction/Abstract: Tuberculosis (TB), an aerosol-transmitted infection caused by Mycobacterium tuberculosis (Mtb), remains the commonest cause of death globally, from an infectious bacterial disease. Nine years on from the launch of the World Health Organization (WHO)’s END-TB strategy, disease incidence rates are stubbornly unchanged [1]. While this represents, in part, a reversal of improving trends caused by the COVID-19 pandemic, it also reflects the fragility and inadequacy of healthcare systems to sustain TB control [2]. Although multifactorial, a key reason for this is the ineffectiveness of existing clinical tools to meet the two key objectives of the END-TB strategy—(i) early diagnosis and treatment of TB disease (to limit onward transmission); and (ii) disease prevention through screening for asymptomatic TB infection (TBI). Meeting both objectives will rely on the development of new biomarkers with high accuracy, but the global nature of the TB problem also requires that new tests are rapid, low cost and can be measured in patients by sampling from universally accessible sites. In this review, we will present the accumulating evidence for circulating Mtb in both TB disease and asymptomatic TBI and discuss the potential utility of novel bacteriophage-based technology for blood-based detection of Mtb

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-2

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p> ●; no sites) were grown to mid-log phase in Tris Minimal Succinate medium with 5 μg mlCm and 0.5% xylose. Bacteria were washed and resuspended in an equal volume of medium containing 5 μg mlCm and 1% glucose. Aliquots were placed in a 96 well microtitre plate and incubated at 37°C in a Tecan Genios Pro. Panel A: fluorescence (solid symbols, (Relative Fluorescence Units; RFU) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3004 (□, ■; 4 sites), pSB3002 (□, ▲; 2 sites) and pSB3000 (○, ●; no sites). Panel B: luminescence (solid symbols, Relative Light Units; RLU) and absorbance (open symbols) were measured at 10 minute intervals. Plasmids used were pSB3014 (□, ■; 4 sites), pSB3012 (□, ▲; 2 sites) and pSB3010 (○, ●; no sites). Data is presented as % maximal signal to allow direct comparison of repression kinetics despite the fact that light levels from each construct were different

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-0

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p>; 4 sites) were grown in LB medium containing 0.5% (w/v) xylose at 37°C. Growth (OD 600 nm; open symbols) and luminescence (Relative Light Units; RLU closed symbols) were monitored over time. Reporter gene data are presented as RLU/ODto account for increasing cell number during the experiment

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-3

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p>operon under the control of the Ppromoter (see Table 2). Cells were grown in MWB at 37°C and luminescence (closed symbols, Relative Light Units; RLU)) and growth measurements (open symbols) were taken at intervals. Reporter gene data are presented as RLU/ODto account for increasing cell number during the experiment

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-1

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p> ●; no sites) were grown to mid-log phase in Tris Minimal Succinate medium with 5 μg mlCm and then diluted 1/20 into fresh medium supplemented with 0.5% xylose. Triplicate samples were placed in a 96 well microtitre plate and incubated at 37°C in a Tecan Genios Pro. Panel A: fluorescence (solid symbols, Relative Fluorescence Units; RFU) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3004 (□, ■; 4 sites), pSB3002 (□, ▲; 2 sites) and pSB3000 (○, ●; no sites). Panel B. luminescence (solid symbols, Relative Light Units; RLU)) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3014 (□, ■; 4 sites), pSB3012 (△, ▲; 2 sites) and pSB3010 (○, ●; no sites). Data is presented as % maximal signal to allow direct comparison of expression kinetics despite the fact that light levels from each construct were different