Favorable Antiviral Effect of Metformin on Severe Acute Respiratory Syndrome Coronavirus 2 Viral Load in a Randomized, Placebo-Controlled Clinical Trial of Coronavirus Disease 2019.

BACKGROUND: Metformin has antiviral activity against RNA viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The mechanism appears to be suppression of protein translation via targeting the host mechanistic target of rapamycin pathway. In the COVID-OUT randomized trial for outpatient coronavirus disease 2019 (COVID-19), metformin reduced the odds of hospitalizations/death through 28 days by 58%, of emergency department visits/hospitalizations/death through 14 days by 42%, and of long COVID through 10 months by 42%. METHODS: COVID-OUT was a 2 × 3 randomized, placebo-controlled, double-blind trial that assessed metformin, fluvoxamine, and ivermectin; 999 participants self-collected anterior nasal swabs on day 1 (n = 945), day 5 (n = 871), and day 10 (n = 775). Viral load was quantified using reverse-transcription quantitative polymerase chain reaction. RESULTS: The mean SARS-CoV-2 viral load was reduced 3.6-fold with metformin relative to placebo (-0.56 log10 copies/mL; 95% confidence interval [CI], -1.05 to -.06; P = .027). Those who received metformin were less likely to have a detectable viral load than placebo at day 5 or day 10 (odds ratio [OR], 0.72; 95% CI, .55 to .94). Viral rebound, defined as a higher viral load at day 10 than day 5, was less frequent with metformin (3.28%) than placebo (5.95%; OR, 0.68; 95% CI, .36 to 1.29). The metformin effect was consistent across subgroups and increased over time. Neither ivermectin nor fluvoxamine showed effect over placebo. CONCLUSIONS: In this randomized, placebo-controlled trial of outpatient treatment of SARS-CoV-2, metformin significantly reduced SARS-CoV-2 viral load, which may explain the clinical benefits in this trial. Metformin is pleiotropic with other actions that are relevant to COVID-19 pathophysiology. CLINICAL TRIALS REGISTRATION: NCT04510194.

Multi-omic approach associates blood methylome with bronchodilator drug response in pediatric asthma.

BACKGROUND: Albuterol is the drug most widely used as asthma treatment among African Americans despite having a lower bronchodilator drug response (BDR) than other populations. Although BDR is affected by gene and environmental factors, the influence of DNA methylation is unknown. OBJECTIVE: This study aimed to identify epigenetic markers in whole blood associated with BDR, study their functional consequences by multi-omic integration, and assess their clinical applicability in admixed populations with a high asthma burden. METHODS: We studied 414 children and young adults (8-21 years old) with asthma in a discovery and replication design. We performed an epigenome-wide association study on 221 African Americans and replicated the results on 193 Latinos. Functional consequences were assessed by integrating epigenomics with genomics, transcriptomics, and environmental exposure data. Machine learning was used to develop a panel of epigenetic markers to classify treatment response. RESULTS: We identified 5 differentially methylated regions and 2 CpGs genome-wide significantly associated with BDR in African Americans located in FGL2 (cg08241295, P = 6.8 × 10-9) and DNASE2 (cg15341340, P = 7.8 × 10-8), which were regulated by genetic variation and/or associated with gene expression of nearby genes (false discovery rate < 0.05). The CpG cg15341340 was replicated in Latinos (P = 3.5 × 10-3). Moreover, a panel of 70 CpGs showed good classification for those with response and nonresponse to albuterol therapy in African American and Latino children (area under the receiver operating characteristic curve for training, 0.99; for validation, 0.70-0.71). The DNA methylation model showed similar discrimination as clinical predictors (P > .05). CONCLUSIONS: We report novel associations of epigenetic markers with BDR in pediatric asthma and demonstrate for the first time the applicability of pharmacoepigenetics in precision medicine of respiratory diseases.

Admixture mapping of severe asthma exacerbations in Hispanic/Latino children and youth.

BACKGROUND: In the USA, genetically admixed populations have the highest asthma prevalence and severe asthma exacerbations rates. This could be explained not only by environmental factors but also by genetic variants that exert ethnic-specific effects. However, no admixture mapping has been performed for severe asthma exacerbations. OBJECTIVE: We sought to identify genetic variants associated with severe asthma exacerbations in Hispanic/Latino subgroups by means of admixture mapping analyses and fine mapping, and to assess their transferability to other populations and potential functional roles. METHODS: We performed an admixture mapping in 1124 Puerto Rican and 625 Mexican American children with asthma. Fine-mapping of the significant peaks was performed via allelic testing of common and rare variants. We performed replication across Hispanic/Latino subgroups, and the transferability to non-Hispanic/Latino populations was assessed in 1001 African Americans, 1250 Singaporeans and 941 Europeans with asthma. The effects of the variants on gene expression and DNA methylation from whole blood were also evaluated in participants with asthma and in silico with data obtained through public databases. RESULTS: Genomewide significant associations of Indigenous American ancestry with severe asthma exacerbations were found at 5q32 in Mexican Americans as well as at 13q13-q13.2 and 3p13 in Puerto Ricans. The single nucleotide polymorphism (SNP) rs1144986 (C5orf46) showed consistent effects for severe asthma exacerbations across Hispanic/Latino subgroups, but it was not validated in non-Hispanics/Latinos. This SNP was associated with DPYSL3 DNA methylation and SCGB3A2 gene expression levels. CONCLUSIONS: Admixture mapping study of asthma exacerbations revealed a novel locus that exhibited Hispanic/Latino-specific effects and regulated DPYSL3 and SCGB3A2.

Novel insights into the whole-blood DNA methylome of asthma in ethnically diverse children and youth.

BACKGROUND: The epigenetic mechanisms of asthma remain largely understudied in African Americans and Hispanics/Latinos, two populations disproportionately affected by asthma. We aimed to identify markers, regions and processes with differential patterns of DNA methylation (DNAm) in whole blood by asthma status in ethnically diverse children and youth, and to assess their functional consequences. METHODS: DNAm levels were profiled with the Infinium MethylationEPIC or HumanMethylation450 BeadChip arrays among 1226 African Americans or Hispanics/Latinos and assessed for differential methylation per asthma status at the CpG and region (differentially methylated region (DMR)) level. Novel associations were validated in blood and/or nasal epithelium from ethnically diverse children and youth. The functional and biological implications of the markers identified were investigated by combining epigenomics with transcriptomics from study participants. RESULTS: 128 CpGs and 196 DMRs were differentially methylated after multiple testing corrections, including 92.3% and 92.8% novel associations, respectively. 41 CpGs were replicated in other Hispanics/Latinos, prioritising cg17647904 (NCOR2) and cg16412914 (AXIN1) as asthma DNAm markers. Significant DNAm markers were enriched in previous associations for asthma, fractional exhaled nitric oxide, bacterial infections, immune regulation or eosinophilia. Functional annotation highlighted epigenetically regulated gene networks involved in corticosteroid response, host defence and immune regulation. Several implicated genes are targets for approved or experimental drugs, including TNNC1 and NDUFA12. Many differentially methylated loci previously associated with asthma were validated in our study. CONCLUSIONS: We report novel whole-blood DNAm markers for asthma underlying key processes of the disease pathophysiology and confirm the transferability of previous asthma DNAm associations to ethnically diverse populations.

Dissecting and tuning primer editing by proofreading polymerases.

Proofreading polymerases have 3' to 5' exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards allow next-generation sequencing-based enzymological measurements, reveal the extent of editing, and allow the comparison of different polymerases and cycling conditions. We demonstrate that proofreading polymerases edit PCR primers in a concentration-dependent manner, and we examine whether primer editing exhibits any sequence specificity. In addition, we use these standards to show that primer editing is tunable through the incorporation of phosphorothioate linkages. Finally, we demonstrate the ability of primer editing to robustly rescue the drop-out of taxa with 16S rRNA gene-targeting primer mismatches using mock communities and human skin microbiome samples.

Leukocyte telomere length, cancer incidence and all-cause mortality among Chinese adults: Singapore Chinese Health Study.

Telomeres play a key role in chromosomal maintenance and stability. To date, few studies have investigated the association of leukocyte telomere length with risk of cancer incidence and all-cause mortality in a large prospective cohort, particularly of the Asian population. Relative telomere lengths in genomic DNA from peripheral blood samples were quantified using a validated quantitative real-time PCR among 26 540 middle-aged or older Chinese adults. Hazard ratios (HRs) and 95% confidence intervals (CIs) of cancer and deaths by quintiles of telomere length were calculated using the Cox proportional hazards regression method with adjustment for age, sex and other potential confounders. After baseline blood collection, 4353 persons developed cancer and 7609 died. Participants with the longest decile of telomeres had a 26% (95% CI: 11%-44%) higher risk of total cancer incidence compared to the shortest decile after controlling for age, sex and other potential founders (Ptrend < .0001). In contrast, longer telomeres were associated with lower risk of all-cause mortality (HR = 0.93; 95% CI: 0.84-1.03), noncancer death (HR = 0.81; 95% CI: 0.71-0.92), specifically, death from chronic obstructive pulmonary disease and pneumonia (HR = 0.79, 95% CI: 0.70-0.89) and digestive diseases (HR = 0.60, 95% CI: 0.42-0.88). Our findings demonstrated that longer telomeres are associated with increased risk of cancer development overall and several common cancer types including breast, rectal, prostate, pancreatic cancer and lung adenocarcinoma. Our study also confirmed that longer telomeres are associated with a reduced risk of noncancer related death.

A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2.

BACKGROUND: The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. RESULTS: Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach. CONCLUSIONS: The tailed amplicon method we describe represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.

Leukocyte telomere length in relation to risk of lung adenocarcinoma incidence: Findings from the Singapore Chinese Health Study.

Telomeres are crucial in the maintenance of chromosome integrity and genomic stability. Critically short telomeres can trigger programed cell death while cells with longer telomeres may have increased likelihood of replicative errors, resulting in genetic mutations and chromosomal alterations, and ultimately promoting oncogenesis. Data on telomere length and lung cancer risk from large prospective cohort studies are spare. Relative telomere length in peripheral blood leukocytes was quantified using a validated monochrome multiplex quantitative polymerase chain reaction (qPCR) method in 26,540 participants of the Singapore Chinese Health Study. After a follow-up of 12 years, 654 participants developed lung cancer including 288 adenocarcinoma, 113 squamous cell carcinoma and 253 other/unknown histological type. The Cox proportional hazard regression was used to estimate hazard ratio (HR) and 95% confidence interval (CI). HR of lung adenocarcinoma for individuals in the highest comparing the lowest 20 percentile of telomere length was 2.84 (95% CI 1.94-4.14, ptrend < 0.0001). This positive association was present in never smokers (ptrend < 0.0001), ever smokers (ptrend = 0.0010), men (ptrend = 0.0003), women (ptrend < 0.0001), and in shorter (ptrend = 0.0002) and longer (ptrend = 0.0001) duration of follow-up. There was no association between telomere length and risk of squamous cell carcinoma or other histological type of lung cancer in all or subgroups of individuals. The agreement of results from this prospective cohort study with those of previous prospective studies and Mendelian randomization studies suggest a possible etiological role of telomere length in the development of lung adenocarcinoma.

Characterizing the genetic basis of transcriptome diversity through RNA-sequencing of 922 individuals.

Understanding the consequences of regulatory variation in the human genome remains a major challenge, with important implications for understanding gene regulation and interpreting the many disease-risk variants that fall outside of protein-coding regions. Here, we provide a direct window into the regulatory consequences of genetic variation by sequencing RNA from 922 genotyped individuals. We present a comprehensive description of the distribution of regulatory variation--by the specific expression phenotypes altered, the properties of affected genes, and the genomic characteristics of regulatory variants. We detect variants influencing expression of over ten thousand genes, and through the enhanced resolution offered by RNA-sequencing, for the first time we identify thousands of variants associated with specific phenotypes including splicing and allelic expression. Evaluating the effects of both long-range intra-chromosomal and trans (cross-chromosomal) regulation, we observe modularity in the regulatory network, with three-dimensional chromosomal configuration playing a particular role in regulatory modules within each chromosome. We also observe a significant depletion of regulatory variants affecting central and critical genes, along with a trend of reduced effect sizes as variant frequency increases, providing evidence that purifying selection and buffering have limited the deleterious impact of regulatory variation on the cell. Further, generalizing beyond observed variants, we have analyzed the genomic properties of variants associated with expression and splicing and developed a Bayesian model to predict regulatory consequences of genetic variants, applicable to the interpretation of individual genomes and disease studies. Together, these results represent a critical step toward characterizing the complete landscape of human regulatory variation.

Genome-wide imputation study identifies novel HLA locus for pulmonary fibrosis and potential role for auto-immunity in fibrotic idiopathic interstitial pneumonia.

BACKGROUND: Fibrotic idiopathic interstitial pneumonias (fIIP) are a group of fatal lung diseases with largely unknown etiology and without definitive treatment other than lung transplant to prolong life. There is strong evidence for the importance of both rare and common genetic risk alleles in familial and sporadic disease. We have previously used genome-wide single nucleotide polymorphism data to identify 10 risk loci for fIIP. Here we extend that work to imputed genome-wide genotypes and conduct new RNA sequencing studies of lung tissue to identify and characterize new fIIP risk loci. RESULTS: We performed genome-wide genotype imputation association analyses in 1616 non-Hispanic white (NHW) cases and 4683 NHW controls followed by validation and replication (878 cases, 2017 controls) genotyping and targeted gene expression in lung tissue. Following meta-analysis of the discovery and replication populations, we identified a novel fIIP locus in the HLA region of chromosome 6 (rs7887 P meta = 3.7 × 10(-09)). Imputation of classic HLA alleles identified two in high linkage disequilibrium that are associated with fIIP (DRB1*15:01 P = 1.3 × 10(-7) and DQB1*06:02 P = 6.1 × 10(-8)). Targeted RNA-sequencing of the HLA locus identified 21 genes differentially expressed between fibrotic and control lung tissue (Q < 0.001), many of which are involved in immune and inflammatory response regulation. In addition, the putative risk alleles, DRB1*15:01 and DQB1*06:02, are associated with expression of the DQB1 gene among fIIP cases (Q < 1 × 10(-16)). CONCLUSIONS: We have identified a genome-wide significant association between the HLA region and fIIP. Two HLA alleles are associated with fIIP and affect expression of HLA genes in lung tissue, indicating that the potential genetic risk due to HLA alleles may involve gene regulation in addition to altered protein structure. These studies reveal the importance of the HLA region for risk of fIIP and a basis for the potential etiologic role of auto-immunity in fIIP.

Next generation sequencing in endocrine practice.

With the completion of the Human Genome Project and advances in genomic sequencing technologies, the use of clinical molecular diagnostics has grown tremendously over the last decade. Next-generation sequencing (NGS) has overcome many of the practical roadblocks that had slowed the adoption of molecular testing for routine clinical diagnosis. In endocrinology, targeted NGS now complements biochemical testing and imaging studies. The goal of this review is to provide clinicians with a guide to the application of NGS to genetic testing for endocrine conditions, by compiling a list of established gene mutations detectable by NGS, and highlighting key phenotypic features of these disorders. As we outline in this review, the clinical utility of NGS-based molecular testing for endocrine disorders is very high. Identifying an exact genetic etiology improves understanding of the disease, provides clear explanation to families about the cause, and guides decisions about screening, prevention and/or treatment. To illustrate this approach, a case of hypophosphatasia with a pathogenic mutation in the ALPL gene detected by NGS is presented.

5-HTP inhibits eosinophilia via intracellular endothelial 5-HTRs; SNPs in 5-HTRs associate with asthmatic lung function.

Background: Previous research showed that 5-hydroxytryptophan (5HTP), a metabolic precursor of serotonin, reduces allergic lung inflammation by inhibiting eosinophil migration across endothelial monolayers. Objective: It is unknown if serotonin receptors are involved in mediating this 5HTP function or if serotonin receptor (HTR) single nucleotide polymorphisms (SNPs) associate with lung function in humans. Methods: Serotonin receptor subtypes were assessed by qPCR, western blot, confocal microscopy, pharmacological inhibitors and siRNA knockdown. HTR SNPs were assessed in two cohorts. Results: Pharmacological inhibition or siRNA knockdown of the serotonin receptors HTR1A or HTR1B in endothelial cells abrogated the inhibitory effects of 5HTP on eosinophil transendothelial migration. In contrast, eosinophil transendothelial migration was not inhibited by siRNA knockdown of HTR1A or HTR1B in eosinophils. Surprisingly, these HTRs were intracellular in endothelial cells and an extracellular supplementation with serotonin did not inhibit eosinophil transendothelial migration. This is consistent with the inability of serotonin to cross membranes, the lack of selective serotonin reuptake receptors on endothelial cells, and the studies showing minimal impact of selective serotonin reuptake inhibitors on asthma. To extend our HTR studies to humans with asthma, we examined the CHIRAH and GALA cohorts for HTR SNPs that affect HTR function or are associated with behavior disorders. A polygenic index of SNPs in HTRs was associated with lower lung function in asthmatics. Conclusions: Serotonin receptors mediate 5HTP inhibition of transendothelial migration and HTR SNPs associate with lower lung function. These results may serve to aid in design of novel interventions for allergic inflammation.

Association between celiac disease and iron deficiency in Caucasians, but not non-Caucasians.

BACKGROUND & AIMS: Celiac disease is an increasingly recognized disorder in Caucasian populations of European origin. Little is known about its prevalence in non-Caucasians. Although it is thought to be a cause of iron-deficiency anemia, little is known about the extent to which celiac disease contributes to iron deficiency in Caucasians, and especially non-Caucasians. We analyzed samples collected from participants in the Hemochromatosis and Iron Overload Screening study to identify individuals with iron deficiency and to assess the frequency of celiac disease. METHODS: We analyzed serum samples from white men (≥25 y) and women (≥50 y) who participated in the Hemochromatosis and Iron Overload Screening study; cases were defined as individuals with iron deficiency (serum ferritin level, ≤12 µg/L) and controls were those without (serum ferritin level, >100 µg/L in men and >50 µg/L in women). All samples also were analyzed for human recombinant tissue transglutaminase immunoglobulin A; positive results were confirmed by an assay for endomysial antibodies. Patients with positive results from both celiac disease tests were presumed to have untreated celiac disease, and those with a positive result from only 1 test were excluded from analysis. We analyzed HLA genotypes and frequencies of celiac disease between Caucasians and non-Caucasians with iron deficiency. RESULTS: Celiac disease occurred in 14 of 567 cases (2.5%) and in only 1 of 1136 controls (0.1%; Fisher exact test, P = 1.92 × 10(-6)). Celiac disease was more common in Caucasian cases (14 of 363; 4%) than non-Caucasian cases (0 of 204; P = .003). Only 1 Caucasian control and no non-Caucasian controls had celiac disease. The odds of celiac disease in individuals with iron deficiency was 28-fold (95% confidence interval, 3.7-212.8) that of controls; 13 of 14 cases with celiac disease carried the DQ2.5 variant of the HLA genotype. CONCLUSIONS: Celiac disease is associated with iron deficiency in Caucasians. Celiac disease is rare among non-Caucasians-even among individuals with features of celiac disease, such as iron deficiency. Celiac disease also is rare among individuals without iron deficiency. Men and postmenopausal women with iron deficiency should be tested for celiac disease.

Genetic and environmental predictors of serum 25-hydroxyvitamin D concentrations among middle-aged and elderly Chinese in Singapore.

Vitamin D is known for maintaining Ca homeostasis and bone structure, and may also decrease susceptibility to chronic and infectious diseases. However, data on vitamin D status and its predictors among Southeast Asian populations are limited. We evaluated the distribution and determinants (genetic and environmental) of serum 25-hydroxyvitamin D (25(OH)D) concentrations among 504 middle-aged and elderly participants (aged 45-74 years) in the Singapore Chinese Health Study. Data on dietary and other lifestyle factors were collected by trained interviewers. Serum 25(OH)D concentrations and genetic polymorphisms in vitamin D metabolism pathway enzymes (cytochrome P450 (CYP) 2R1, 3A4, 27B1, 24A1; vitamin D binding protein (also known as group-specific component, GC); and vitamin D receptor) were measured using stored biospecimens. Mean 25(OH)D concentration was 68·8 nmol/l. Serum 25(OH)D concentrations were positively associated with dietary vitamin D intake, and inversely associated with hours spent sitting at work. BMI was not associated with 25(OH)D concentrations. CYP2R1 rs10741657, rs12794714, rs1993116; CYP3A4 rs2242480; and GC rs4588, rs7041, rs16847015, rs2298849 were statistically significantly associated with 25(OH)D concentrations. Individuals with the Gc2-2 haplotype (rs4588AA/rs7041TT) had statistically significantly lower 25(OH)D concentrations compared to all other Gc haplotypes (P-trend < 0·001). The majority of participants (86 %) had 25(OH)D concentrations ≥ 50 nmol/l, which is consistent with the 2011 Institute of Medicine (US) recommendation for bone health, and 32 % had concentrations of ≥ 75 nmol/l that are thought to be required for broader health effects. Dietary vitamin D intake, hours spent indoors at work and genetic variation in CYP2R1, CYP3A4 and GC are significant predictors of 25(OH)D concentrations among Singapore Chinese.

Mycobacterium tuberculosis Requires the Outer Membrane Lipid Phthiocerol Dimycocerosate for Starvation-Induced Antibiotic Tolerance.

Tolerance of Mycobacterium tuberculosis to antibiotics contributes to the long duration of tuberculosis (TB) treatment and the emergence of drug-resistant strains. M. tuberculosis drug tolerance is induced by nutrient restriction, but the genetic determinants that promote antibiotic tolerance triggered by nutrient limitation have not been comprehensively identified. Here, we show that M. tuberculosis requires production of the outer membrane lipid phthiocerol dimycocerosate (PDIM) to tolerate antibiotics under nutrient-limited conditions. We developed an arrayed transposon (Tn) mutant library in M. tuberculosis Erdman and used orthogonal pooling and transposon sequencing (Tn-seq) to map the locations of individual mutants in the library. We screened a subset of the library (~1,000 mutants) by Tn-seq and identified 32 and 102 Tn mutants with altered tolerance to antibiotics under stationary-phase and phosphate-starved conditions, respectively. Two mutants recovered from the arrayed library, ppgK::Tn and clpS::Tn, showed increased susceptibility to two different drug combinations under both nutrient-limited conditions, but their phenotypes were not complemented by the Tn-disrupted gene. Whole-genome sequencing revealed single nucleotide polymorphisms in both the ppgK::Tn and clpS::Tn mutants that prevented PDIM production. Complementation of the clpS::Tn ppsD Q291* mutant with ppsD restored PDIM production and antibiotic tolerance, demonstrating that loss of PDIM sensitized M. tuberculosis to antibiotics. Our data suggest that drugs targeting production of PDIM, a critical M. tuberculosis virulence determinant, have the potential to enhance the efficacy of existing antibiotics, thereby shortening TB treatment and limiting development of drug resistance. IMPORTANCE Mycobacterium tuberculosis causes 10 million cases of active TB disease and over 1 million deaths worldwide each year. TB treatment is complex, requiring at least 6 months of therapy with a combination of antibiotics. One factor that contributes to the length of TB treatment is M. tuberculosis phenotypic antibiotic tolerance, which allows the bacteria to survive prolonged drug exposure even in the absence of genetic mutations causing drug resistance. Here, we report a genetic screen to identify M. tuberculosis genes that promote drug tolerance during nutrient starvation. Our study revealed the outer membrane lipid phthiocerol dimycocerosate (PDIM) as a key determinant of M. tuberculosis antibiotic tolerance triggered by nutrient starvation. Our study implicates PDIM synthesis as a potential target for development of new TB drugs that would sensitize M. tuberculosis to existing antibiotics to shorten TB treatment.

Epigenomic response to albuterol treatment in asthma-relevant airway epithelial cells.

BACKGROUND: Albuterol is the first-line asthma medication used in diverse populations. Although DNA methylation (DNAm) is an epigenetic mechanism involved in asthma and bronchodilator drug response (BDR), no study has assessed whether albuterol could induce changes in the airway epithelial methylome. We aimed to characterize albuterol-induced DNAm changes in airway epithelial cells, and assess potential functional consequences and the influence of genetic variation and asthma-related clinical variables. RESULTS: We followed a discovery and validation study design to characterize albuterol-induced DNAm changes in paired airway epithelial cultures stimulated in vitro with albuterol. In the discovery phase, an epigenome-wide association study using paired nasal epithelial cultures from Puerto Rican children (n = 97) identified 22 CpGs genome-wide associated with repeated-use albuterol treatment (p < 9 × 10-8). Albuterol predominantly induced a hypomethylation effect on CpGs captured by the EPIC array across the genome (probability of hypomethylation: 76%, p value = 3.3 × 10-5). DNAm changes on the CpGs cg23032799 (CREB3L1), cg00483640 (MYLK4-LINC01600), and cg05673431 (KSR1) were validated in nasal epithelia from 10 independent donors (false discovery rate [FDR] < 0.05). The effect on the CpG cg23032799 (CREB3L1) was cross-tissue validated in bronchial epithelial cells at nominal level (p = 0.030). DNAm changes in these three CpGs were shown to be influenced by three independent genetic variants (FDR < 0.05). In silico analyses showed these polymorphisms regulated gene expression of nearby genes in lungs and/or fibroblasts including KSR1 and LINC01600 (6.30 × 10-14 ≤ p ≤ 6.60 × 10-5). Additionally, hypomethylation at the CpGs cg10290200 (FLNC) and cg05673431 (KSR1) was associated with increased gene expression of the genes where they are located (FDR < 0.05). Furthermore, while the epigenetic effect of albuterol was independent of the asthma status, severity, and use of medication, BDR was nominally associated with the effect on the CpG cg23032799 (CREB3L1) (p = 0.004). Gene-set enrichment analyses revealed that epigenomic modifications of albuterol could participate in asthma-relevant processes (e.g., IL-2, TNF-α, and NF-κB signaling pathways). Finally, nine differentially methylated regions were associated with albuterol treatment, including CREB3L1, MYLK4, and KSR1 (adjusted p value < 0.05). CONCLUSIONS: This study revealed evidence of epigenetic modifications induced by albuterol in the mucociliary airway epithelium. The epigenomic response induced by albuterol might have potential clinical implications by affecting biological pathways relevant to asthma.

Outpatient treatment of COVID-19 and incidence of post-COVID-19 condition over 10 months (COVID-OUT): a multicentre, randomised, quadruple-blind, parallel-group, phase 3 trial.

BACKGROUND: Post-COVID-19 condition (also known as long COVID) is an emerging chronic illness potentially affecting millions of people. We aimed to evaluate whether outpatient COVID-19 treatment with metformin, ivermectin, or fluvoxamine soon after SARS-CoV-2 infection could reduce the risk of long COVID. METHODS: We conducted a decentralised, randomised, quadruple-blind, parallel-group, phase 3 trial (COVID-OUT) at six sites in the USA. We included adults aged 30-85 years with overweight or obesity who had COVID-19 symptoms for fewer than 7 days and a documented SARS-CoV-2 positive PCR or antigen test within 3 days before enrolment. Participants were randomly assigned via 2 × 3 parallel factorial randomisation (1:1:1:1:1:1) to receive metformin plus ivermectin, metformin plus fluvoxamine, metformin plus placebo, ivermectin plus placebo, fluvoxamine plus placebo, or placebo plus placebo. Participants, investigators, care providers, and outcomes assessors were masked to study group assignment. The primary outcome was severe COVID-19 by day 14, and those data have been published previously. Because the trial was delivered remotely nationwide, the a priori primary sample was a modified intention-to-treat sample, meaning that participants who did not receive any dose of study treatment were excluded. Long COVID diagnosis by a medical provider was a prespecified, long-term secondary outcome. This trial is complete and is registered with ClinicalTrials.gov, NCT04510194. FINDINGS: Between Dec 30, 2020, and Jan 28, 2022, 6602 people were assessed for eligibility and 1431 were enrolled and randomly assigned. Of 1323 participants who received a dose of study treatment and were included in the modified intention-to-treat population, 1126 consented for long-term follow-up and completed at least one survey after the assessment for long COVID at day 180 (564 received metformin and 562 received matched placebo; a subset of participants in the metformin vs placebo trial were also randomly assigned to receive ivermectin or fluvoxamine). 1074 (95%) of 1126 participants completed at least 9 months of follow-up. 632 (56·1%) of 1126 participants were female and 494 (43·9%) were male; 44 (7·0%) of 632 women were pregnant. The median age was 45 years (IQR 37-54) and median BMI was 29·8 kg/m2 (IQR 27·0-34·2). Overall, 93 (8·3%) of 1126 participants reported receipt of a long COVID diagnosis by day 300. The cumulative incidence of long COVID by day 300 was 6·3% (95% CI 4·2-8·2) in participants who received metformin and 10·4% (7·8-12·9) in those who received identical metformin placebo (hazard ratio [HR] 0·59, 95% CI 0·39-0·89; p=0·012). The metformin beneficial effect was consistent across prespecified subgroups. When metformin was started within 3 days of symptom onset, the HR was 0·37 (95% CI 0·15-0·95). There was no effect on cumulative incidence of long COVID with ivermectin (HR 0·99, 95% CI 0·59-1·64) or fluvoxamine (1·36, 0·78-2·34) compared with placebo. INTERPRETATION: Outpatient treatment with metformin reduced long COVID incidence by about 41%, with an absolute reduction of 4·1%, compared with placebo. Metformin has clinical benefits when used as outpatient treatment for COVID-19 and is globally available, low-cost, and safe. FUNDING: Parsemus Foundation; Rainwater Charitable Foundation; Fast Grants; UnitedHealth Group Foundation; National Institute of Diabetes, Digestive and Kidney Diseases; National Institutes of Health; and National Center for Advancing Translational Sciences.

Metformin reduces SARS-CoV-2 in a Phase 3 Randomized Placebo Controlled Clinical Trial.

Current antiviral treatment options for SARS-CoV-2 infections are not available globally, cannot be used with many medications, and are limited to virus-specific targets.1-3 Biophysical modeling of SARS-CoV-2 replication predicted that protein translation is an especially attractive target for antiviral therapy.4 Literature review identified metformin, widely known as a treatment for diabetes, as a potential suppressor of protein translation via targeting of the host mTor pathway.5 In vitro, metformin has antiviral activity against RNA viruses including SARS-CoV-2.6,7 In the COVID-OUT phase 3, randomized, placebo-controlled trial of outpatient treatment of COVID-19, metformin had a 42% reduction in ER visits/hospitalizations/death through 14 days; a 58% reduction in hospitalizations/death through 28 days, and a 42% reduction in Long COVID through 10 months.8,9 Here we show viral load analysis of specimens collected in the COVID-OUT trial that the mean SARS-CoV-2 viral load was reduced 3.6-fold with metformin relative to placebo (-0.56 log10 copies/mL; 95%CI, -1.05 to -0.06, p=0.027) while there was no virologic effect for ivermectin or fluvoxamine vs placebo. The metformin effect was consistent across subgroups and with emerging data.10,11 Our results demonstrate, consistent with model predictions, that a safe, widely available,12 well-tolerated, and inexpensive oral medication, metformin, can be repurposed to significantly reduce SARS-CoV-2 viral load.

Comparative analysis of genome-wide DNA methylation identifies patterns that associate with conserved transcriptional programs in osteosarcoma.

Osteosarcoma is an aggressive tumor of the bone that primarily affects young adults and adolescents. Osteosarcoma is characterized by genomic chaos and heterogeneity. While inactivation of tumor protein p53 (TP53) is nearly universal other high frequency mutations or structural variations have not been identified. Despite this genomic heterogeneity, key conserved transcriptional programs associated with survival have been identified across human, canine and induced murine osteosarcoma. The epigenomic landscape, including DNA methylation, plays a key role in establishing transcriptional programs in all cell types. The role of epigenetic dysregulation has been studied in a variety of cancers but has yet to be explored at scale in osteosarcoma. Here we examined genome-wide DNA methylation patterns in 24 human and 44 canine osteosarcoma samples identifying groups of highly correlated DNA methylation marks in human and canine osteosarcoma samples. We also link specific DNA methylation patterns to key transcriptional programs in both human and canine osteosarcoma. Building on previous work, we built a DNA methylation-based measure for the presence and abundance of various immune cell types in osteosarcoma. Finally, we determined that the underlying state of the tumor, and not changes in cell composition, were the main driver of differences in DNA methylation across the human and canine samples. SIGNIFICANCE: Genome wide comparison of DNA methylation patterns in osteosarcoma across two species lays the ground work for the exploration of DNA methylation programs that help establish conserved transcriptional programs in the context of varied mutational landscapes.

Epigenome-wide association study of lung function in Latino children and youth with asthma.

INTRODUCTION: DNA methylation studies have associated methylation levels at different CpG sites or genomic regions with lung function. Moreover, genetic ancestry has been associated with lung function in Latinos. However, no epigenome-wide association study (EWAS) of lung function has been performed in this population. Here, we aimed to identify DNA methylation patterns associated with lung function in pediatric asthma among Latinos. RESULTS: We conducted an EWAS in whole blood from 250 Puerto Rican and 148 Mexican American children and young adults with asthma. A total of five CpGs exceeded the genome-wide significance threshold of p = 1.17 × 10-7 in the combined analyses from Puerto Ricans and Mexican Americans: cg06035600 (MAP3K6, p = 6.13 × 10-8) showed significant association with pre-bronchodilator Tiffeneau-Pinelli index, the probes cg00914963 (TBC1D16, p = 1.04 × 10-7), cg16405908 (MRGPRE, p = 2.05 × 10-8), and cg07428101 (MUC2, p = 5.02 × 10-9) were associated with post-bronchodilator forced vital capacity (FVC), and cg20515679 (KCNJ6) with post-bronchodilator Tiffeneau-Pinelli index (p = 1.13 × 10-8). However, these markers did not show significant associations in publicly available data from Europeans (p > 0.05). A methylation quantitative trait loci analysis revealed that methylation levels at these CpG sites were regulated by genetic variation in Latinos and the Biobank-based Integrative Omics Studies (BIOS) consortium. Additionally, two differentially methylated regions in REXOC and AURKC were associated with pre-bronchodilator Tiffeneau-Pinelli index (adjusted p < 0.05) in Puerto Ricans and Mexican Americans. Moreover, we replicated some of the previous differentially methylated signals associated with lung function in non-Latino populations. CONCLUSIONS: We replicated previous associations of epigenetic markers with lung function in whole blood and identified novel population-specific associations shared among Latino subgroups.