A RAC-GEF network critical for early intestinal tumourigenesis.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease

Effect of pre-exposure use of hydroxychloroquine on COVID-19 mortality: a population-based cohort study in patients with rheumatoid arthritis or systemic lupus erythematosus using the OpenSAFELY platform.

BACKGROUND: Hydroxychloroquine has been shown to inhibit entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into epithelial cells in vitro, but clinical studies found no evidence of reduced mortality when treating patients with COVID-19. We aimed to evaluate the effectiveness of hydroxychloroquine for prevention of COVID-19 mortality, as opposed to treatment for the disease. METHODS: We did a prespecified observational, population-based cohort study using national primary care data and linked death registrations in the OpenSAFELY platform, which covers approximately 40% of the general population in England, UK. We included all adults aged 18 years and older registered with a general practice for 1 year or more on March 1, 2020. We used Cox regression to estimate the association between ongoing routine hydroxychloroquine use before the COVID-19 outbreak in England (considered as March 1, 2020) compared with non-users of hydroxychloroquine and risk of COVID-19 mortality among people with rheumatoid arthritis or systemic lupus erythematosus. Model adjustment was informed by a directed acyclic graph. FINDINGS: Between Sept 1, 2019, and March 1, 2020, of 194 637 people with rheumatoid arthritis or systemic lupus erythematosus, 30 569 (15·7%) received two or more prescriptions of hydroxychloroquine. Between March 1 and July 13, 2020, there were 547 COVID-19 deaths, 70 among hydroxychloroquine users. Estimated standardised cumulative COVID-19 mortality was 0·23% (95% CI 0·18 to 0·29) among users and 0·22% (0·20 to 0·25) among non-users; an absolute difference of 0·008% (-0·051 to 0·066). After accounting for age, sex, ethnicity, use of other immunosuppressive drugs, and geographical region, no association with COVID-19 mortality was observed (HR 1·03, 95% CI 0·80 to 1·33). We found no evidence of interactions with age or other immunosuppressive drugs. Quantitative bias analyses indicated that our observed associations were robust to missing information for additional biologic treatments for rheumatological disease. We observed similar associations with the negative control outcome of non-COVID-19 mortality. INTERPRETATION: We found no evidence of a difference in COVID-19 mortality among people who received hydroxychloroquine for treatment of rheumatological disease before the COVID-19 outbreak in England. Therefore, completion of randomised trials investigating pre-exposure prophylactic use of hydroxychloroquine for prevention of severe outcomes from COVID-19 are warranted. FUNDING: Medical Research Council

ChIP-chip versus ChIP-seq: Lessons for experimental design and data analysis

<p>Abstract</p> <p>Background</p> <p>Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq) allows genome-wide discovery of protein-DNA interactions such as transcription factor bindings and histone modifications. Previous reports only compared a small number of profiles, and little has been done to compare histone modification profiles generated by the two technologies or to assess the impact of input DNA libraries in ChIP-seq analysis. Here, we performed a systematic analysis of a modENCODE dataset consisting of 31 pairs of ChIP-chip/ChIP-seq profiles of the coactivator CBP, RNA polymerase II (RNA PolII), and six histone modifications across four developmental stages of <it>Drosophila melanogaster</it>.</p> <p>Results</p> <p>Both technologies produce highly reproducible profiles within each platform, ChIP-seq generally produces profiles with a better signal-to-noise ratio, and allows detection of more peaks and narrower peaks. The set of peaks identified by the two technologies can be significantly different, but the extent to which they differ varies depending on the factor and the analysis algorithm. Importantly, we found that there is a significant variation among multiple sequencing profiles of input DNA libraries and that this variation most likely arises from both differences in experimental condition and sequencing depth. We further show that using an inappropriate input DNA profile can impact the average signal profiles around genomic features and peak calling results, highlighting the importance of having high quality input DNA data for normalization in ChIP-seq analysis.</p> <p>Conclusions</p> <p>Our findings highlight the biases present in each of the platforms, show the variability that can arise from both technology and analysis methods, and emphasize the importance of obtaining high quality and deeply sequenced input DNA libraries for ChIP-seq analysis.</p

Anthrax Toxins Inhibit Neutrophil Signaling Pathways in Brain Endothelium and Contribute to the Pathogenesis of Meningitis

Anthrax meningitis is the main neurological complication of systemic infection with Bacillus anthracis approaching 100% mortality. The presence of bacilli in brain autopsies indicates that vegetative bacteria are able to breach the blood-brain barrier (BBB). The BBB represents not only a physical barrier but has been shown to play an active role in initiating a specific innate immune response that recruits neutrophils to the site of infection. Currently, the basic pathogenic mechanisms by which B. anthracis penetrates the BBB and causes anthrax meningitis are poorly understood.Using an in vitro BBB model, we show for the first time that B. anthracis efficiently invades human brain microvascular endothelial cells (hBMEC), the single cell layer that comprises the BBB. Furthermore, transcriptional profiling of hBMEC during infection with B. anthracis revealed downregulation of 270 (87%) genes, specifically key neutrophil chemoattractants IL-8, CXCL1 (Gro alpha) and CXCL2 (Gro beta), thereby strongly contrasting hBMEC responses observed with other meningeal pathogens. Further studies using specific anthrax toxin-mutants, quantitative RT-PCR, ELISA and in vivo assays indicated that anthrax toxins actively suppress chemokine production and neutrophil recruitment during infection, allowing unrestricted proliferation and dissemination of the bacteria. Finally, mice challenged with B. anthracis Sterne, but not the toxin-deficient strain, developed meningitis.These results suggest a significant role for anthrax toxins in thwarting the BBB innate defense response promoting penetration of bacteria into the central nervous system. Furthermore, establishment of a mouse model for anthrax meningitis will aid in our understanding of disease pathogenesis and development of more effective treatment strategies

Genetic variation in a member of the laminin gene family affects variation in body composition in Drosophila and humans

<p>Abstract</p> <p>Background</p> <p>The objective of the present study was to map candidate loci influencing naturally occurring variation in triacylglycerol (TAG) storage using quantitative complementation procedures in <it>Drosophila melanogaster</it>. Based on our results from <it>Drosophila</it>, we performed a human population-based association study to investigate the effect of natural variation in <it>LAMA5 </it>gene on body composition in humans.</p> <p>Results</p> <p>We identified four candidate genes that contributed to differences in TAG storage between two strains of <it>D. melanogaster</it>, including <it>Laminin A </it>(<it>LanA</it>), which is a member of the α subfamily of laminin chains. We confirmed the effects of this gene using a viable <it>LanA </it>mutant and showed that female flies homozygous for the mutation had significantly lower TAG storage, body weight, and total protein content than control flies. <it>Drosophila LanA </it>is closely related to human <it>LAMA5 </it>gene, which maps to the well-replicated obesity-linkage region on chromosome 20q13.2-q13.3. We tested for association between three common single nucleotide polymorphisms (SNPs) in the human <it>LAMA5 </it>gene and variation in body composition and lipid profile traits in a cohort of unrelated women of European American (EA) and African American (AA) descent. In both ethnic groups, we found that SNP rs659822 was associated with weight (EA: <it>P </it>= 0.008; AA: <it>P </it>= 0.05) and lean mass (EA: <it>P= </it>0.003; AA: <it>P </it>= 0.03). We also found this SNP to be associated with height (<it>P </it>= 0.01), total fat mass (<it>P </it>= 0.01), and HDL-cholesterol (<it>P </it>= 0.003) but only in EA women. Finally, significant associations of SNP rs944895 with serum TAG levels (<it>P </it>= 0.02) and HDL-cholesterol (<it>P </it>= 0.03) were observed in AA women.</p> <p>Conclusion</p> <p>Our results suggest an evolutionarily conserved role of a member of the laminin gene family in contributing to variation in weight and body composition.</p

Comparative Transcriptional Profiling of Bacillus cereus Sensu Lato Strains during Growth in CO2-Bicarbonate and Aerobic Atmospheres

Bacillus species are spore-forming bacteria that are ubiquitous in the environment and display a range of virulent and avirulent phenotypes. This range is particularly evident in the Bacillus cereus sensu lato group; where closely related strains cause anthrax, food-borne illnesses, and pneumonia, but can also be non-pathogenic. Although much of this phenotypic range can be attributed to the presence or absence of a few key virulence factors, there are other virulence-associated loci that are conserved throughout the B. cereus group, and we hypothesized that these genes may be regulated differently in pathogenic and non-pathogenic strains.Here we report transcriptional profiles of three closely related but phenotypically unique members of the Bacillus cereus group--a pneumonia-causing B. cereus strain (G9241), an attenuated strain of B. anthracis (Sterne 34F(2)), and an avirulent B. cereus strain (10987)--during exponential growth in two distinct atmospheric environments: 14% CO(2)/bicarbonate and ambient air. We show that the disease-causing Bacillus strains undergo more distinctive transcriptional changes between the two environments, and that the expression of plasmid-encoded virulence genes was increased exclusively in the CO(2) environment. We observed a core of conserved metabolic genes that were differentially expressed in all three strains in both conditions. Additionally, the expression profiles of putative virulence genes in G9241 suggest that this strain, unlike Bacillus anthracis, may regulate gene expression with both PlcR and AtxA transcriptional regulators, each acting in a different environment.We have shown that homologous and even identical genes within the genomes of three closely related members of the B. cereus sensu lato group are in some instances regulated very differently, and that these differences can have important implications for virulence. This study provides insights into the evolution of the B. cereus group, and highlights the importance of looking beyond differences in gene content in comparative genomics studies

Inhaled corticosteroid use and risk COVID-19 related death among 966,461 patients with COPD or asthma: an OpenSAFELY analysis

AbstractBackgroundEarly descriptions of the coronavirus outbreak showed a lower prevalence of asthma and COPD than was expected for people diagnosed with COVID-19, leading to speculation that inhaled corticosteroids (ICS) may protect against infection with SARS-CoV-2, and development of serious sequelae. We evaluated the association between ICS and COVID-19 related death using linked electronic health records in the UK.MethodsWe conducted cohort studies on two groups of people (COPD and asthma) using the OpenSAFELY platform to analyse data from primary care practices linked to national death registrations. People receiving an ICS were compared to those receiving alternative respiratory medications. Our primary outcome was COVID-19 related death.FindingsWe identified 148,588 people with COPD and 817,973 people with asthma receiving relevant respiratory medications in the four months prior to 01 March 2020. People with COPD receiving ICS were at a greater risk of COVID-19 related death compared to those receiving a long-acting beta agonist (LABA) and a long-acting muscarinic antagonist (LAMA) (adjusted HR = 1.38, 95% CI = 1.08 – 1.75). People with asthma receiving high dose ICS were at an increased risk of death compared to those receiving a short-acting beta agonist (SABA) only (adjusted HR = 1.52, 95%CI = 1.08 – 2.14); the adjusted HR for those receiving low-medium dose ICS was 1.10 (95% CI = 0.82 – 1.49). Quantitative bias analyses indicated that an unmeasured confounder of only moderate strength of association with exposure and outcome could explain the observed associations in both populations.InterpretationThese results do not support a major role of ICS in protecting against COVID-19 related deaths. Observed increased risks of COVID-19 related death among people with COPD and asthma receiving ICS can be plausibly explained by unmeasured confounding due to disease severity.FundingThis work was supported by the Medical Research Council MR/V015737/1.</jats:sec

Use of non-steroidal anti-inflammatory drugs and risk of death from COVID-19: an OpenSAFELY cohort analysis based on two cohorts.

OBJECTIVES: To assess the association between routinely prescribed non-steroidal anti-inflammatory drugs (NSAIDs) and deaths from COVID-19 using OpenSAFELY, a secure analytical platform. METHODS: We conducted two cohort studies from 1 March to 14 June 2020. Working on behalf of National Health Service England, we used routine clinical data in England linked to death data. In study 1, we identified people with an NSAID prescription in the last 3 years from the general population. In study 2, we identified people with rheumatoid arthritis/osteoarthritis. We defined exposure as current NSAID prescription within the 4 months before 1 March 2020. We used Cox regression to estimate HRs for COVID-19 related death in people currently prescribed NSAIDs, compared with those not currently prescribed NSAIDs, accounting for age, sex, comorbidities, other medications and geographical region. RESULTS: In study 1, we included 536 423 current NSAID users and 1 927 284 non-users in the general population. We observed no evidence of difference in risk of COVID-19 related death associated with current use (HR 0.96, 95% CI 0.80 to 1.14) in the multivariable-adjusted model. In study 2, we included 1 708 781 people with rheumatoid arthritis/osteoarthritis, of whom 175 495 (10%) were current NSAID users. In the multivariable-adjusted model, we observed a lower risk of COVID-19 related death (HR 0.78, 95% CI 0.64 to 0.94) associated with current use of NSAID versus non-use. CONCLUSIONS: We found no evidence of a harmful effect of routinely prescribed NSAIDs on COVID-19 related deaths. Risks of COVID-19 do not need to influence decisions about the routine therapeutic use of NSAIDs

Hydroxychloroquine for prevention of COVID-19 mortality: a population-based cohort study

AbstractBackgroundHydroxychloroquine has been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro, but early clinical studies found no benefit treating patients with coronavirus disease 2019 (COVID-19). We set out to evaluate the effectiveness of hydroxychloroquine for prevention, as opposed to treatment, of COVID-19 mortality.MethodsWe pre-specified and conducted an observational, population-based cohort study using national primary care data and linked death registrations in the OpenSAFELY platform, representing 40% of the general population in England. We used Cox regression to estimate the association between ongoing routine hydroxychloroquine use prior to the COVID-19 outbreak in England and risk of COVID-19 mortality among people with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Model adjustment was informed by a directed acyclic graph.ResultsOf 194,637 patients with RA or SLE, 30,569 (15.7%) received ≥ 2 prescriptions of hydroxychloroquine in the six months prior to 1 March 2020. Between 1 March 2020 and 13 July 2020, there were 547 COVID-19 deaths, 70 among hydroxychloroquine users. Estimated standardised cumulative COVID-19 mortality was 0.23% (95% CI 0.18–0.29) among users and 0.22% (95% CI 0.20–0.25) among non-users; an absolute difference of 0.008% (95% CI –0.051-0.066). After accounting for age, sex, ethnicity, use of other immunuosuppressives, and geographic region, no association with COVID-19 mortality was observed (HR 1.03, 95% CI 0.80–1.33). We found no evidence of interactions with age or other immunosuppressives. Quantitative bias analyses indicated observed associations were robust to missing information regarding additional biologic treatments for rheumatological disease. We observed similar associations with the negative control outcome of non-COVID-19 mortality.ConclusionWe found no evidence of a difference in COVID-19 mortality among patients who received hydroxychloroquine for treatment of rheumatological disease prior to the COVID-19 outbreak in England.Research in contextEvidence before this studyPublished trials and observational studies to date have shown no evidence of benefit of hydroxychloroquine as a treatment for hospitalised patients who already have COVID-19. A separate question remains: whether routine ongoing use of hydroxychloroquine in people without COVID-19 protects against new infections or severe outcomes. We searched MEDLINE/PubMed for pharmacoepidemiological studies evaluating hydroxychloroquine for prevention of severe COVID-19 outcomes. The keywords “hydroxychloroquine AND (COVID OR coronavirus OR SARS-CoV-2) AND (prophyl* OR prevent*) AND (rate OR hazard OR odds OR risk)” were used and results were filtered to articles from the last year with abstracts available. 109 papers were identified for screening; none investigated pre-exposure prophylactic use of hydroxychloroquine for prevention of severe COVID-19 outcomes. Clinical trials of prophylactic use of hydroxychloroquine are ongoing; however, the largest trial does not expect to meet recruitment targets due to “…unjustified extrapolation and exaggerated safety concerns together with intense politicisation and negative publicity.” In the absence of reported clinical trials, evidence can be generated from real-world data to support the need for randomised clinical trials.Added value of this studyIn this cohort study representing 40% of the population of England, we investigated whether routine use of hydroxychloroquine prior to the COVID-19 outbreak prevented COVID-19 mortality. Using robust pharmacoepidemiological methods, we found no evidence to support a substantial benefit of hydroxychloroquine in preventing COVID-19 mortality. At the same time, we have shown no significant harm, and this generates the equipoise to justify continuing randomised trials. We have demonstrated in this study that it is feasible to address specific hypotheses about medicines in a rapid and transparent manner to inform interim clinical decision making and support the need for large-scale, randomised trial data.Implications of all the available evidenceThis is the first study to investigate the ongoing routine use of hydroxychloroquine and risk of COVID-19 mortality in a general population. While we found no evidence of any protective benefit, due to the observational nature of the study, residual confounding remains a possibility. Completion of trials for prevention of severe outcomes is warranted, but prior to the completion of these, we found no evidence to support the use of hydroxychloroquine for prevention of COVID-19 mortality.</jats:sec