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Recent studies have established cell type- specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-kappaB. In each of these studies, inhibitors of NF-kappaB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-kappaB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-kappaB activation and apoptosis in cultured cell lines. To address whether Sindbis virus- induced NF-kappaB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-kappaB activity. Complete suppression of virus-induced NF-kappaB activity neither prevents nor potentiates Sindbis virus-induced apoptosis. In contrast, inhibition of NF-kappaB activity before infection inhibits Sindbis virus-induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-kappaB activity, regulates expression of gene products required for Sindbis virus-induced death. Furthermore, we show that in the same cell line, NF-kappaB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-kappaB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-kappaB activity relative to the death stimulus.

Background: The microbiota-derived short chain fatty acid butyrate has been shown to lower blood pressure (BP) in rodent studies. Nonetheless, the net effect of butyrate on hypertension in humans remains uncovered. In this study, for the first time, we aimed to determine the effect of oral butyrate on BP in patients with hypertension., Methods: We performed a double-blind randomized placebo-controlled trial including 23 patients with hypertension. Antihypertensive medication was discontinued for the duration of the study with a washout period of 4 weeks before starting the intervention. Participants received daily oral capsules containing either sodium butyrate or placebo with an equivalent dosage of sodium chloride for 4 weeks. The primary outcome was daytime 24-hour systolic BP. Differences between groups over time were assessed using linear mixed models (group-by-time interaction)., Results: Study participants (59.0±3.7 years; 56.5% female) had an average baseline office systolic BP of 143.5±14.6 mm Hg and diastolic BP of 93.0±8.3 mm Hg. Daytime 24-hour systolic and diastolic BP significantly increased over the intervention period in the butyrate compared with the placebo group, with an increase of +9.63 (95% CI, 2.02-17.20) mm Hg in daytime 24-hour systolic BP and +5.08 (95% CI, 1.34-8.78) mm Hg in diastolic BP over 4 weeks. Butyrate levels significantly increased in plasma, but not in feces, upon butyrate intake, underscoring its absorption., Conclusions: Four-week treatment with oral butyrate increased daytime systolic and diastolic BP in subjects with hypertension. Our findings implicate that butyrate does not have beneficial effects on human hypertension, which warrants caution in future butyrate intervention studies., Registration: URL: https://onderzoekmetmensen.nl/; Unique identifier: NL8924., Competing Interests: M. Nieuwdorp is on the scientific board at Caelus Pharmaceuticals, the Netherlands. S.L. Hazen reports being named as a coinventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics, being a paid consultant formerly for Procter & Gamble, and currently being with Zehna Therapeutics. He also reports having received research funds from Procter & Gamble, Zehna Therapeutics, and Roche Diagnostics and being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland HeartLab, a wholly owned subsidiary of Quest Diagnostics, Procter & Gamble, and Zehna Therapeutics. The other authors report no conflicts.

Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing β1- versus β2-adrenergic receptors (β1AR and β2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of β2AR, but not β1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on β2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical β2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR., (© 2024. The Author(s).)

Background: Growing evidence indicates that trimethylamine N -oxide, a gut microbial metabolite of dietary choline and carnitine, promotes both cardiovascular disease and chronic kidney disease risk. It remains unclear how circulating concentrations of trimethylamine N -oxide and its related dietary and gut microbe-derived metabolites (choline, betaine, carnitine, γ-butyrobetaine, and crotonobetaine) affect incident heart failure (HF)., Methods: We evaluated 11 768 participants from the Cardiovascular Health Study and the Multi-Ethnic Study of Atherosclerosis with serial measures of metabolites. Cox proportional hazard models were used to examine the associations between metabolites and incident HF, adjusted for cardiovascular disease risk factors., Results: In all, 2102 cases of HF occurred over a median follow-up of 15.9 years. After adjusting for traditional risk factors, higher concentrations of trimethylamine N -oxide (hazard ratio, 1.15 [95% CI, 1.09-1.20]; P <0.001), choline (hazard ratio, 1.44 [95% CI, 1.26-1.64]; P <0.001), and crotonobetaine (hazard ratio, 1.24 [95% CI, 1.16-1.32]; P <0.001) were associated with increased risk for incident HF. After further adjustment for renal function (potential confounder or mediator), these associations did not reach Bonferroni-corrected statistical significance ( P =0.01, 0.049, and 0.006, respectively). Betaine and carnitine were nominally associated with a higher incidence of HF ( P <0.05). In exploratory analyses, results were similar for subtypes of HF based on left ventricular ejection fraction, and associations appeared generally stronger among Black and Hispanic/Latino versus White adults, although there were no interactions for any metabolites with race., Conclusions: In this pooled analysis of 2 well-phenotyped, diverse, community-based cohorts, circulating concentrations of gut microbe-derived metabolites such as trimethylamine N -oxide, choline, and crotonobetaine were independently associated with a higher risk of developing HF., Registration: URL: https://www.clinicaltrials.gov/; Unique identifiers: NCT00005133 and NCT00005487., Competing Interests: Dr Tang is a consultant for Sequana Medical, Cardiol Therapeutics, Genomics plc, Zehna Therapeutics, WhiteSwell, Boston Scientific, CardiaTec Biosciences, Intellia Therapeutics, Bristol Myers Squibb, Alleviant Medical, Alexion Pharmaceuticals, BioCardia, and Salubris Biotherapeutics and has received honorarium from Springer Nature, Belvoir Media Group, and American Board of Internal Medicine. Drs Hazen and Z. Wang report being named as co-inventors on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics. Drs Hazen and Z. Wang report having received royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland Heart Lab, a fully owned subsidiary of Quest Diagnostics, and Procter & Gamble. Dr Hazen is a paid consultant for Zehna Therapeutics and Proctor & Gamble and has received research funds from Zehna Therapeutics, Proctor & Gamble, Pfizer Inc, and Roche Diagnostics. Dr Bäckhed reports receiving research support from Biogaia AB, is founder and shareholder of Implexion Pharma AB and Roxbiosens Inc, and is on the scientific advisory board for Bactolife A/S. Dr Psaty reported serving on the Steering Committee of the Yale Open Data Access Project funded by Johnson & Johnson. Dr Mozaffarian reported receiving personal fees from Acasti Pharma Inc, America’s Test Kitchen, Barilla, Cleveland Clinic Foundation, Danone SA, GOED (Global Organization for EPA and DHA Omega-3s), and Motif FoodWorks; serving on the scientific advisory board for Beren Therapeutics GmbH, Brightseed, Calibrate, DayTwo (ended June 2020), Elysium Health, Filtricine Inc, Foodome, HumanCo, January Inc, Perfect Day Inc, Season, and Tiny Organics; and receiving chapter royalties from UpToDate outside the submitted work. The other authors report no conflicts.

Despite intensive preventive cardiovascular disease (CVD) efforts, substantial residual CVD risk remains even for individuals receiving all guideline-recommended interventions. Niacin is an essential micronutrient fortified in food staples, but its role in CVD is not well understood. In this study, untargeted metabolomics analysis of fasting plasma from stable cardiac patients in a prospective discovery cohort (n = 1,162 total, n = 422 females) suggested that niacin metabolism was associated with incident major adverse cardiovascular events (MACE). Serum levels of the terminal metabolites of excess niacin, N1-methyl-2-pyridone-5-carboxamide (2PY) and N1-methyl-4-pyridone-3-carboxamide (4PY), were associated with increased 3-year MACE risk in two validation cohorts (US n = 2,331 total, n = 774 females; European n = 832 total, n = 249 females) (adjusted hazard ratio (HR) (95% confidence interval) for 2PY: 1.64 (1.10-2.42) and 2.02 (1.29-3.18), respectively; for 4PY: 1.89 (1.26-2.84) and 1.99 (1.26-3.14), respectively). Phenome-wide association analysis of the genetic variant rs10496731, which was significantly associated with both 2PY and 4PY levels, revealed an association of this variant with levels of soluble vascular adhesion molecule 1 (sVCAM-1). Further meta-analysis confirmed association of rs10496731 with sVCAM-1 (n = 106,000 total, n = 53,075 females, P = 3.6 × 10 -18 ). Moreover, sVCAM-1 levels were significantly correlated with both 2PY and 4PY in a validation cohort (n = 974 total, n = 333 females) (2PY: rho = 0.13, P = 7.7 × 10 -5 ; 4PY: rho = 0.18, P = 1.1 × 10 -8 ). Lastly, treatment with physiological levels of 4PY, but not its structural isomer 2PY, induced expression of VCAM-1 and leukocyte adherence to vascular endothelium in mice. Collectively, these results indicate that the terminal breakdown products of excess niacin, 2PY and 4PY, are both associated with residual CVD risk. They also suggest an inflammation-dependent mechanism underlying the clinical association between 4PY and MACE., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Importance: The key atherosclerotic TMAO originates from the initial gut microbial conversion of L -carnitine and other dietary compounds into TMA. Developing therapeutic strategies to block gut microbial TMA production needs a detailed understanding of the different production mechanisms and their relative contributions. Recently, we identified a two-step anaerobic pathway for TMA production from L -carnitine through initial conversion by some microbes into the intermediate γBB which is then metabolized by other microbes into TMA. Investigational studies of this pathway, however, are limited by the lack of single microbes harboring the whole pathway. Here, we engineered E. fergusonii strain to harbor the whole two-step pathway and optimized the expression through cloning a specific chaperone from the original host. Inoculating germ-free mice with this recombinant E. fergusonii is enough to raise serum TMAO to pathophysiological levels upon L-carnitine feeding. This engineered microbe will facilitate future studies investigating the contribution of this pathway to cardiovascular disease., Competing Interests: Drs. Hazen and Wang report being named as co-inventors on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics. Drs. Hazen and Wang report having received royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland Heart Lab, a fully owned subsidiary of Quest Diagnostics, and Procter & Gamble. Dr. Hazen is a paid consultant for Procter & Gamble, and Zehna Therapeutics, and has received research funds from Procter & Gamble, Pfizer Inc., Roche Diagnostics and Zehna Therapeutics. Jennifer A. Buffa reports having received royalty payments from Procter & Gamble. The other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Aims: Little is known about associations of trimethylamine N-oxide (TMAO), a novel gut microbiota-generated metabolite of dietary phosphatidylcholine and carnitine, and its changes over time with all-cause and cause-specific mortality in the general population or in different race/ethnicity groups. The study aimed to investigate associations of serially measured plasma TMAO levels and changes in TMAO over time with all-cause and cause-specific mortality in a multi-ethnic community-based cohort., Methods and Results: The study included 6,785 adults from the Multi-Ethnic Study of Atherosclerosis. TMAO was measured at baseline and year 5 using mass spectrometry. Primary outcomes were adjudicated all-cause mortality and cardiovascular disease (CVD) mortality. Secondary outcomes were deaths due to kidney failure, cancer, or dementia obtained from death certificates. Cox proportional hazards models with time-varying TMAO and covariates assessed the associations with adjustment for sociodemographics, lifestyles, diet, metabolic factors, and comorbidities. During a median follow-up of 16.9 years, 1704 participants died and 411 from CVD. Higher TMAO levels associated with higher risk of all-cause mortality [hazard ratio (HR): 1.12, 95% confidence interval (CI): 1.08-1.17], CVD mortality (HR: 1.09, 95% CI: 1.00-1.09), and death due to kidney failure (HR: 1.44, 95% CI: 1.25-1.66) per inter-quintile range, but not deaths due to cancer or dementia. Annualized changes in TMAO levels associated with higher risk of all-cause mortality (HR: 1.10, 95% CI: 1.05-1.14) and death due to kidney failure (HR: 1.54, 95% CI: 1.26-1.89) but not other deaths., Conclusion: Plasma TMAO levels were positively associated with mortality, especially deaths due to cardiovascular and renal disease, in a multi-ethnic US cohort., Competing Interests: Conflict of interest Z.W. and S.L.H. report being named as co-inventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics. S.L.H. also reports being a paid consultant for Zehna Therapeutics. S.L.H. reports having received research funds from Procter & Gamble, Zehna Therapeutics and Roche Diagnostics. Z.W. and S.L.H. report being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland Heart Lab, and Procter & Gamble, and S.L.H. from Zehna Therapeutics. B.M.P. serves on the Steering Committee of the Yale Open Data Access Project funded by Johnson & Johnson. D. Mozaffarian reports research funding from the National Institutes of Health, the Gates Foundation, The Rockefeller Foundation, Vail Innovative Global Research, and Kaiser Permanente; personal fees from Acasti Pharma and Barilla; scientific advisory board, Beren Therapeutics, Brightseed, Calibrate, Elysium Health, Filtricine, HumanCo, Instacart, January Inc, Perfect Day, Tiny Organics, and (ended) Discern Dx, Day Two, and Season Health; stock ownership in Calibrate and HumanCo; and chapter royalties from UpToDate. The other authors report no relationships with industry., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recent studies show gut microbiota-dependent metabolism of dietary phenylalanine into phenylacetic acid (PAA) is critical in phenylacetylglutamine (PAGln) production, a metabolite linked to atherosclerotic cardiovascular disease (ASCVD). Accordingly, microbial enzymes involved in this transformation are of interest. Using genetic manipulation in selected microbes and monocolonization experiments in gnotobiotic mice, we identify two distinct gut microbial pathways for PAA formation; one is catalyzed by phenylpyruvate:ferredoxin oxidoreductase (PPFOR) and the other by phenylpyruvate decarboxylase (PPDC). PPFOR and PPDC play key roles in gut bacterial PAA production via oxidative and non-oxidative phenylpyruvate decarboxylation, respectively. Metagenomic analyses revealed a significantly higher abundance of both pathways in gut microbiomes of ASCVD patients compared with controls. The present studies show a role for these two divergent microbial catalytic strategies in the meta-organismal production of PAGln. Given the numerous links between PAGln and ASCVD, these findings will assist future efforts to therapeutically target PAGln formation in vivo., Competing Interests: Declaration of interests S.L.H., Z.W., and J.T.A. report being named as co-inventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and/or therapeutics and being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland HeartLab, a wholly owned subsidiary of Quest Diagnostics, P&G, and Zehna Therapeutics. S.L.H. and J.T.A. report being paid consultants for Zehna Therapeutics. S.L.H. also reports having received research funds from P&G, Roche Diagnostics, and Zehna Therapeutics. The other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The gut microbiota-dependent metabolite phenylacetylgutamine (PAGln) is both associated with atherothrombotic heart disease in humans, and mechanistically linked to cardiovascular disease pathogenesis in animal models via modulation of adrenergic receptor signaling., Methods: Here we examined both clinical and mechanistic relationships between PAGln and heart failure (HF). First, we examined associations among plasma levels of PAGln and HF, left ventricular ejection fraction, and N-terminal pro-B-type natriuretic peptide in 2 independent clinical cohorts of subjects undergoing coronary angiography in tertiary referral centers (an initial discovery US Cohort, n=3256; and a validation European Cohort, n=829). Then, the impact of PAGln on cardiovascular phenotypes relevant to HF in cultured cardiomyoblasts, and in vivo were also examined., Results: Circulating PAGln levels were dose-dependently associated with HF presence and indices of severity (reduced ventricular ejection fraction, elevated N-terminal pro-B-type natriuretic peptide) independent of traditional risk factors and renal function in both cohorts. Beyond these clinical associations, mechanistic studies showed both PAGln and its murine counterpart, phenylacetylglycine, directly fostered HF-relevant phenotypes, including decreased cardiomyocyte sarcomere contraction, and B-type natriuretic peptide gene expression in both cultured cardiomyoblasts and murine atrial tissue., Conclusions: The present study reveals the gut microbial metabolite PAGln is clinically and mechanistically linked to HF presence and severity. Modulating the gut microbiome, in general, and PAGln production, in particular, may represent a potential therapeutic target for modulating HF., Registration: URL: https://clinicaltrials.gov/; Unique identifier: NCT00590200 and URL: https://drks.de/drks&#95;web/; Unique identifier: DRKS00020915.

Background: Effects of animal source foods (ASF) on atherosclerotic cardiovascular disease (ASCVD) and underlying mechanisms remain controversial. We investigated prospective associations of different ASF with incident ASCVD and potential mediation by gut microbiota-generated trimethylamine N-oxide, its L-carnitine-derived intermediates γ-butyrobetaine and crotonobetaine, and traditional ASCVD risk pathways., Methods: Among 3931 participants from a community-based US cohort aged 65+ years, ASF intakes and trimethylamine N-oxide-related metabolites were measured serially over time. Incident ASCVD (myocardial infarction, fatal coronary heart disease, stroke, other atherosclerotic death) was adjudicated over 12.5 years median follow-up. Cox proportional hazards models with time-varying exposures and covariates examined ASF-ASCVD associations; and additive hazard models, mediation proportions by different risk pathways., Results: After multivariable-adjustment, higher intakes of unprocessed red meat, total meat, and total ASF associated with higher ASCVD risk, with hazard ratios (95% CI) per interquintile range of 1.15 (1.01-1.30), 1.22 (1.07-1.39), and 1.18 (1.03-1.34), respectively. Trimethylamine N-oxide-related metabolites together significantly mediated these associations, with mediation proportions (95% CI) of 10.6% (1.0-114.5), 7.8% (1.0-32.7), and 9.2% (2.2-44.5), respectively. Processed meat intake associated with a nonsignificant trend toward higher ASCVD (1.11 [0.98-1.25]); intakes of fish, poultry, and eggs were not significantly associated. Among other risk pathways, blood glucose, insulin, and C-reactive protein, but not blood pressure or blood cholesterol, each significantly mediated the total meat-ASCVD association., Conclusions: In this large, community-based cohort, higher meat intake associated with incident ASCVD, partly mediated by microbiota-derived metabolites of L-carnitine, abundant in red meat. These novel findings support biochemical links between dietary meat, gut microbiome pathways, and ASCVD.

Aims: Gut microbiota and their generated metabolites impact the host vascular phenotype. The metaorganismal metabolite trimethylamine N-oxide (TMAO) is both associated with adverse clinical thromboembolic events, and enhances platelet responsiveness in subjects. The impact of TMAO on vascular Tissue Factor (TF) in vivo is unknown. Here, we explore whether TMAO-enhanced thrombosis potential extends beyond TMAO effects on platelets, and is linked to TF. We also further explore the links between gut microbiota and vascular endothelial TF expression in vivo., Methods and Results: In initial exploratory clinical studies, we observed that among sequential stable subjects (n = 2989) on anti-platelet therapy undergoing elective diagnostic cardiovascular evaluation at a single-site referral centre, TMAO levels were associated with an increased incident (3 years) risk for major adverse cardiovascular events (MACE) (myocardial infarction, stroke, or death) [4th quartile (Q4) vs. Q1 adjusted hazard ratio (HR) 95% confidence interval (95% CI), 1.73 (1.25-2.38)]. Similar results were observed within subjects on aspirin mono-therapy during follow-up [adjusted HR (95% CI) 1.75 (1.25-2.44), n = 2793]. Leveraging access to a second higher risk cohort with previously reported TMAO data and monitoring of anti-platelet medication use, we also observed a strong association between TMAO and incident (1 year) MACE risk in the multi-site Swiss Acute Coronary Syndromes Cohort, focusing on the subset (n = 1469) on chronic dual anti-platelet therapy during follow-up [adjusted HR (95% CI) 1.70 (1.08-2.69)]. These collective clinical data suggest that the thrombosis-associated effects of TMAO may be mediated by cells/factors that are not inhibited by anti-platelet therapy. To test this, we first observed in human microvascular endothelial cells that TMAO dose-dependently induced expression of TF and vascular cell adhesion molecule (VCAM)1. In mouse studies, we observed that TMAO-enhanced aortic TF and VCAM1 mRNA and protein expression, which upon immunolocalization studies, was shown to co-localize with vascular endothelial cells. Finally, in arterial injury mouse models, TMAO-dependent enhancement of in vivo TF expression and thrombogenicity were abrogated by either a TF-inhibitory antibody or a mechanism-based microbial choline TMA-lyase inhibitor (fluoromethylcholine)., Conclusion: Endothelial TF contributes to TMAO-related arterial thrombosis potential, and can be specifically blocked by targeted non-lethal inhibition of gut microbial choline TMA-lyase., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Importance: Little is known about the association of trimethylamine N-oxide (TMAO), a novel plasma metabolite derived from L-carnitine and phosphatidylcholine, and related metabolites (ie, choline, betaine, carnitine, and butyrobetaine) with risk of death among older adults in the general population., Objective: To investigate the associations of serial measures of plasma TMAO and related metabolites with risk of total and cause-specific death (ie, deaths from cardiovascular diseases [CVDs] and non-CVDs) among older adults in the US., Design, Setting, and Participants: This prospective cohort study involved 5333 participants from the Cardiovascular Health Study-a community-based longitudinal cohort of adults aged 65 years or older-who were followed up from June 1, 1989, to December 31, 2015. Participants were from 4 communities in the US (Forsyth County, North Carolina; Sacramento County, California; Washington County, Maryland; and Allegheny County, Pennsylvania). Data were analyzed from March 17 to June 23, 2021., Exposures: Plasma TMAO, choline, betaine, carnitine, and butyrobetaine levels were measured using stored samples from baseline (June 1, 1989, to May 31, 1990, or November 1, 1992, to June 31, 1993) and follow-up examination (June 1, 1996, to May 31, 1997). Measurements were performed through stable-isotope dilution liquid chromatography with tandem mass spectrometry using high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry., Main Outcomes and Measures: Deaths (total and cause specific) were adjudicated by a centralized Cardiovascular Health Study events committee based on information from medical records, laboratory and diagnostic reports, death certificates, and/or interviews with next of kin. The associations of each metabolite with mortality were assessed using Cox proportional hazards regression models., Results: Among 5333 participants in the analytic sample, the mean (SD) age was 73 (6) years; 2149 participants (40.3%) were male, 3184 (59.7%) were female, 848 (15.9%) were African American, 4450 (83.4%) were White, and 35 (0.01%) were of other races (12 were American Indian or Alaska Native, 4 were Asian or Pacific Islander, and 19 were of other races or ethnicities). During a median follow-up of 13.2 years (range, 0-26.9 years), 4791 deaths occurred. After adjustment for potential confounders, the hazard ratios for death from any cause (ie, total mortality) comparing extreme quintiles (fifth vs first) of plasma concentrations were 1.30 (95% CI, 1.17-1.44) for TMAO, 1.19 (95% CI, 1.08-1.32) for choline, 1.26 (95% CI, 1.15-1.40) for carnitine, and 1.26 (95% CI, 1.13-1.40) for butyrobetaine. Plasma betaine was not associated with risk of death. The extent of risk estimates was similar for CVD and non-CVD mortality., Conclusions and Relevance: In this cohort study, plasma concentrations of TMAO and related metabolites were positively associated with risk of death. These findings suggest that circulating TMAO is an important novel risk factor associated with death among older adults.

Purpose: L-Carnitine is abundant in animal source foods, particularly red meat, and circulating L-carnitine may be related to the incidence of coronary heart disease (CHD). We investigated whether long-term changes in plasma L-carnitine over 10 years were associated with the CHD incidence and also examined joint associations of carnitine-rich red meat consumption and L-carnitine changes on the subsequent risk of CHD., Methods: This prospective nested case-control study included 772 healthy women at baseline (386 incident CHD cases and 386 healthy controls). Plasma L-carnitine levels were measured both at the first (1989-90) and second blood collections (2000-02). Incident cases of CHD were prospectively followed from the date of the second blood collection through 2016., Results: Overall, a greater increase in L-carnitine from the first to the second time point was related to a higher risk of CHD, regardless of the initial L-carnitine levels (relative risk: 1.36 (95% CI 0.999, 1.84) per 1-SD increase). The 10-year changes in L-carnitine were positively associated with red meat consumption over time, and women with higher red meat intake (≥ 36 g/day) and with greater increases in L-carnitine had a 1.86 (95% CI 1.13, 3.09) times increased risk of CHD, as compared to those with lower red meat intake and lesser increases in L-carnitine., Conclusion: Long-term increases in L-carnitine levels were associated with the subsequent incidence of CHD, especially among women with higher intake of red meat. Our results suggest the importance of atherogenic L-carnitine changes and dietary intakes over time in the prevention of CHD., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO - ) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN - ), yielding CNO - and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on "lipid-poor" apoA-I forms via the nonenzymatic CNO - pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma., Competing Interests: Conflict of interest S. L. H. reports being named as coinventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics, being a paid consultant for Procter & Gamble and Zehna Therapeutics, having received research funds from Procter & Gamble, Zehna Therapeutics, and Roche Diagnostics, and being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland HeartLab, a wholly owned subsidiary of Quest Diagnostics, Procter & Gamble, and Zehna Therapeutics. All the other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The heightened cardiovascular disease (CVD) risk observed among omnivores is thought to be linked, in part, to gut microbiota-dependent generation of trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the interrelationship of γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. In the present study, we show that plasma γBB levels in individuals from a clinical cohort (n = 2,918) are strongly associated with incident CVD event risks. Culture of human faecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine → γBB → TMA transformation, elevate TMAO levels and enhance thrombosis potential in recipients after arterial injury. RNA-sequencing analyses of E. timonensis identified a six-gene cluster, herein named the γBB utilization (gbu) gene cluster, which is upregulated in response to γBB. Combinatorial cloning and functional studies identified four genes (gbuA, gbuB, gbuC and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when coexpressed in Escherichia coli. Finally, reanalysis of samples (n = 113) from a clinical, randomized diet, intervention study showed that the abundance of faecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine → γBB → TMA → TMAO transformation in hosts and contributes to CVD risk., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

L -alpha glycerylphosphorylcholine (GPC), a nutritional supplement, has been demonstrated to improve neurological function. However, a new study suggests that GPC supplementation increases incident stroke risk thus its potential adverse effects warrant further investigation. Here we show that GPC promotes atherosclerosis in hyperlipidemic Apoe -/- mice. GPC can be metabolized to trimethylamine N-oxide, a pro-atherogenic agent, suggesting a potential molecular mechanism underlying the observed atherosclerosis progression. GPC supplementation shifted the gut microbial community structure, characterized by increased abundance of Parabacteroides , Ruminococcus , and Bacteroides and decreased abundance of Akkermansia , Lactobacillus , and Roseburia , as determined by 16S rRNA gene sequencing. These data are consistent with a reduction in fecal and cecal short chain fatty acids in GPC-fed mice. Additionally, we found that GPC supplementation led to an increased relative abundance of choline trimethylamine lyase ( cutC )-encoding bacteria via qPCR. Interrogation of host inflammatory signaling showed that GPC supplementation increased expression of the proinflammatory effectors CXCL13 and TIMP-1 and activated NF-κB and MAPK signaling pathways in human coronary artery endothelial cells. Finally, targeted and untargeted metabolomic analysis of murine plasma revealed additional metabolites associated with GPC supplementation and atherosclerosis. In summary, our results show GPC promotes atherosclerosis through multiple mechanisms and that caution should be applied when using GPC as a nutritional supplement.

Background/objectives: Alterations in gut microbiota have been linked to obesity and impaired lipid metabolism. Lipoproteins are heterogeneous, and lipoprotein subspecies containing apolipoprotein C-III (apoCIII) have adverse associations with obesity and related cardiometabolic abnormalities. We investigated associations of weight-loss diet-induced decreases in atherogenic gut-microbial metabolites, trimethylamine N-oxide (TMAO) and L-carnitine, with improvements in atherogenic lipoproteins containing apoCIII among patients with obesity., Subjects/methods: This study included overweight and obese adults who participated in a 2-year weight-loss dietary intervention, the POUNDS Lost trial. Blood levels of TMAO and L-carnitine were measured at baseline and 6 months after the intervention; 6-month changes in the metabolites were calculated. We evaluated 2-year changes in lipid profiles (n = 395) and cholesterol [Chol] in lipoprotein (very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)) subfractions defined by the presence or absence of apoCIII (n = 277)., Results: The initial (6-month) decrease in L-carnitine was significantly associated with long-term (2-year) reductions in non-HDL-Chol and LDL-Chol (p < 0.05). Also, the decrease in L-carnitine was significantly related to decreases in Chol in LDL with apoCIII (p = 0.034) and Chol in [LDL + VLDL] with apoCIII (p = 0.018). We found significant interactions between dietary fat and TMAO on changes in LDL-Chol (P interaction  = 0.013) and Chol in [LDL + VLDL] with apoCIII (P interaction  = 0.0048); a greater increase in TMAO was related to lesser improvements in the lipoprotein outcomes if participants consumed a high-fat compared to a low-fat diet., Conclusions: Changes in TMAO and L-carnitine induced by weight-loss diets were associated with long-term improvements in atherogenic lipoproteins containing apoCIII, implicating that these metabolic changes might be predictive of an individual's response to the dietary treatment to modify the unfavorable lipid profiles in obese patients. Dietary fat intake might modify associations of TMAO changes with long-term improvements of atherogenic cholesterol metabolism in overweight and obese adults. CLINICALTRIALS., Gov Identifier: NCT00072995., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background Trimethylamine N-oxide (TMAO) is a gut microbiota-dependent metabolite of dietary choline, L-carnitine, and phosphatidylcholine-rich foods. On the basis of experimental studies and patients with prevalent disease, elevated plasma TMAO may increase risk of atherosclerotic cardiovascular disease (ASCVD). TMAO is also renally cleared and may interact with and causally contribute to renal dysfunction. Yet, how serial TMAO levels relate to incident and recurrent ASCVD in community-based populations and the potential mediating or modifying role of renal function are not established. Methods and Results We investigated associations of serial measures of plasma TMAO, assessed at baseline and 7 years, with incident and recurrent ASCVD in a community-based cohort of 4131 (incident) and 1449 (recurrent) older US adults. TMAO was measured using stable isotope dilution liquid chromatography-tandem mass spectrometry (laboratory coefficient of variation, <6%). Incident ASCVD (myocardial infarction, fatal coronary heart disease, stroke, sudden cardiac death, or other atherosclerotic death) was centrally adjudicated using medical records. Risk was assessed by multivariable Cox proportional hazards regression, including time-varying demographics, lifestyle factors, medical history, laboratory measures, and dietary habits. Potential mediating effects and interaction by estimated glomerular filtration rate (eGFR) were assessed. During prospective follow-up, 1766 incident and 897 recurrent ASCVD events occurred. After multivariable adjustment, higher levels of TMAO were associated with a higher risk of incident ASCVD, with extreme quintile hazard ratio (HR) compared with the lowest quintile=1.21 (95% CI, 1.02-1.42; P -trend=0.029). This relationship appeared mediated or confounded by eGFR (eGFR-adjusted HR, 1.07; 95% CI, 0.90-1.27), as well as modified by eGFR ( P -interaction <0.001). High levels of TMAO were associated with higher incidence of ASCVD in the presence of impaired renal function (eGFR <60 mL/min per 1.73 m 2 : HR, 1.56 [95% CI, 1.13-2.14]; P -trend=0.007), but not normal or mildly reduced renal function (eGFR ≥60 mL/min per 1.73 m 2 : HR, 1.03 [95% CI, 0.85-1.25]; P -trend=0.668). Among individuals with prior ASCVD, TMAO associated with higher risk of recurrent ASCVD (HR, 1.25 [95% CI, 1.01-1.56]; P -trend=0.009), without significant modification by eGFR. Conclusions In this large community-based cohort of older US adults, serial measures of TMAO were associated with higher risk of incident ASCVD, with apparent modification by presence of impaired renal function and with higher risk of recurrent ASCVD.

Clinical studies have demonstrated associations between circulating levels of the gut-microbiota-derived metabolite trimethylamine-N-oxide (TMAO) and stroke incident risk. However, a causal role of gut microbes in stroke has not yet been demonstrated. Herein we show that gut microbes, through dietary choline and TMAO generation, directly impact cerebral infarct size and adverse outcomes following stroke. Fecal microbial transplantation from low- versus high-TMAO-producing human subjects into germ-free mice shows that both TMAO generation and stroke severity are transmissible traits. Furthermore, employing multiple murine stroke models and transplantation of defined microbial communities with genetically engineered human commensals into germ-free mice, we demonstrate that the microbial cutC gene (an enzymatic source of choline-to-TMA transformation) is sufficient to transmit TMA/TMAO production, heighten cerebral infarct size, and lead to functional impairment. We thus reveal that gut microbiota in general, specifically the metaorganismal TMAO pathway, directly contributes to stroke severity., Competing Interests: Declaration of interests S.L.H. reports being named as co-inventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics, being a paid consultant for Procter & Gamble, having received research funds from Procter & Gamble and Roche Diagnostics, and being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland HeartLab, a subsidiary of Quest Diagnostics, and Procter & Gamble. The other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Recent studies have shown low high-density lipoprotein cholesterol (HDL-C) and dysregulated lipid metabolism in chronic thromboembolic pulmonary hypertension (CTEPH). Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-C and mediates most of its functions. We hypothesize that ApoA-1 and its oxidative state might be more sensitive biomarkers in CTEPH. Plasma levels of HDL-C, ApoA-I, paraoxonase-1 enzyme activity (PON1), and the oxidized dysfunctional ApoA-I (oxTrp72-ApoA-I) were measured in patients with CTEPH and compared to those in healthy controls. Association with markers of disease severity in CTEPH was assessed. We included a total of 61 patients with CTEPH (age: 61.2 ± 15 years; male 52.5%) and 28 control subjects (age: 60.1 ± 8 years; male 59.3%). When adjusting for age, sex, body mass index, and statin use, ApoA-I was lower in CTEPH compared to controls (CTEPH:125.2 ± 27 mg/dl; control:158.3 ± 29.4 mg/dl; p  < 0.001), but HDL-C levels were not statistically different. There were no significant differences in PON and oxTrp72-ApoA-I/ApoA-I ratio. In exploratory analyses, ApoA-I was associated with mean right atrial pressure (r s  = -0.32, p  = 0.013) and N-terminal pro B-type natriuretic peptide (r s  = -0.31, p  = 0.038). There were no significant associations between HDL-C, PON1, or oxTrp72-ApoA-I/ApoA-I ratio and markers of disease severity. We conclude that ApoA-I is a more sensitive biomarker than HDL-C in CTEPH, and may be associated with right heart dysfunction., (© The Author(s) 2021.)

Pulsed-electromagnetic-field (PEMF) treatment was found to enhance cellular differentiation of the mouse preosteoblast, MC3T3-E1, to a more osteoblastic phenotype. Differentiation genes such as Alp, BSPI, cFos, Ibsp, Osteocalcin, Pthr1 and Runx2 showed increased expression in response to PEMF stimulation. Detailed molecular mechanisms linking PEMF to the activation of these genes are limited. Two adenosine receptors known to be modulated in response to PEMF, Adora2A and Adora3, were functionally impaired by CRISPR-Cas9-mediated gene disruption, and the consequences of which were studied in the context of PEMF-mediated osteoblastic differentiation. Disruption of Adora2A resulted in a delay of Alp mRNA expression, but not alkaline phosphatase protein expression, which was similar to that found in wild type cells. However, Adora3 disruption resulted in significantly reduced responses at both the alkaline phosphatase mRNA and protein levels throughout the PEMF stimulation period. Defects observed in response to PEMF were mirrored using a chemically defined growth and differentiation-inducing media (DM). Moreover, in cells with Adora2A disruption, gene expression profiles showed a blunted response in cFos and Pthr1 to PEMF treatment; whereas cells with Adora3 disruption had mostly blunted responses in AlpI, BSPI, Ibsp, Osteocalcin and Sp7 gene activation. To demonstrate specificity for Adora3 function, the Adora3 open reading frame was inserted into the ROSA26 locus in Adora3 disrupted cells culminating in rescued PEMF responsiveness and thereby eliminating the possibility of off-target effects. These results lead us to propose that there are complementary and parallel positive roles for adenosine receptor A2A and A3 in PEMF-mediated osteoblast differentiation., Competing Interests: The authors have read the journal’s policy and have the following competing interests: NZ is an employee of and own stock in Orthofix Medical Inc. and EIW and JTR are consultants and own stock in Orthofix Medical Inc. (https://www.orthofix.com/). There are no patents, products in development associated with this research to declare. The pulsed electromagnetic signal (PhysioStim®, Orthofix Medical Inc., Lewisville TX) used in this study is FDA-approved for the clinical treatment of long bone non-unions. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background/aim: Chemoresistance is a major consequence of multicycle chemotherapy and can be attributed to constitutive activation of pro-survival signaling pathways. Nitric oxide is a ubiquitous signaling molecule which has been shown to inhibit several pathways involved with survival signaling in cancer cells. We have previously demonstrated the anti-tumor activity of a nitric oxide-donor, nitrosylcobalamin (NO-Cbl), mediated by increased expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. We also demonstrated that a functional Apo2L/TRAIL receptor is necessary for the induction of cell death by NO-Cbl and the Apo2L/TRAIL death receptor DR4 (TRAIL R1) is S-nitrosylated. The aim of the study was to examine the effects of nitric oxide (NO) on nuclear factor kappa B (NF-κB) and determine whether nitric oxide could sensitize drug-resistant melanomas to Apo2L/TRAIL via inhibition of NF-κB or inhibitor kappa B kinase (IKK)., Materials and Methods: Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by the TUNEL assay. The activation status of NF-κB was established by assaying luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity., Results: NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL, but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα., Conclusion: NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of nitric oxide to inhibit NF-κB and potentiate the effects of chemotherapeutic agents, such as Apo2L/TRAIL, represents a promising anti-cancer combination based on recent clinical investigations of anti-TRAIL antibodies for cancer treatment strategies., (Copyright © 2020 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Previously, we reported apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), has potent anti-melanoma activity. We used DNA microarray and bioinformatics to interrogate gene expression profiles of tumors from apoA-I expressing (A-I Tg +/- ) versus apoA-I-null (A-I KO) animals to gain insights into mechanisms of apoA-I tumor protection. Differential expression analyses of 11 distinct tumors per group with > 1.2-fold cut-off and a false discovery rate adjusted p < 0.05, identified 176 significant transcripts (71 upregulated and 105 downregulated in A-I Tg +/- versus A-I KO group). Bioinformatic analyses identified the mevalonate and de novo serine/glycine synthesis pathways as potential targets for apoA-I anti-tumor activity. Relative to A-I KO, day 7 B16F10L melanoma tumor homografts from A-I Tg +/- exhibited reduced expression of mevalonate-5-pyrophosphate decarboxylase ( Mvd ), a key enzyme targeted in cancer therapy, along with a number of key genes in the sterol synthesis arm of the mevalonate pathway. Phosphoglycerate dehydrogenase ( Phgdh ), the first enzyme branching off glycolysis into the de novo serine synthesis pathway, was the most repressed transcript in tumors from A-I Tg +/- . We validated our mouse tumor studies by comparing the significant transcripts with adverse tumor markers previously identified in human melanoma and found 45% concordance. Our findings suggest apoA-I targets the mevalonate and serine synthesis pathways in melanoma cells in vivo , thus providing anti-tumor metabolic effects by inhibiting the flux of biomolecular building blocks for macromolecule synthesis that drive rapid tumor growth., Competing Interests: CONFLICTS OF INTEREST Dr. Hazen reports being named as co-inventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics. Dr. Hazen also reports being a paid consultant for P&G, having received research funds from P&G and Roche Diagnostics, and being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland HeartLab and P&G. All remaining authors declare that they have no conflicts of interest with the contents of this article.

Objective: Gut microbial metabolism of dietary choline, a nutrient abundant in a Western diet, produces trimethylamine (TMA) and the atherothrombosis- and fibrosis-promoting metabolite TMA-N-oxide (TMAO). Recent clinical and animal studies reveal that elevated TMAO levels are associated with heightened risks for both cardiovascular disease and incident chronic kidney disease development. Despite this, studies focusing on therapeutically targeting gut microbiota-dependent TMAO production and its impact on preserving renal function are limited. Approach and Results: Herein we examined the impact of pharmacological inhibition of choline diet-induced gut microbiota-dependent production of TMA, and consequently TMAO, on renal tubulointerstitial fibrosis and functional impairment in a model of chronic kidney disease. Initial studies with a gut microbial choline TMA-lyase mechanism-based inhibitor, iodomethylcholine, confirmed both marked suppression of TMA generation, and consequently TMAO levels, and selective targeting of the gut microbial compartment (ie, both accumulation of the drug in intestinal microbes and limited systemic exposure in the host). Dietary supplementation of either choline or TMAO significantly augmented multiple indices of renal functional impairment and fibrosis associated with chronic subcutaneous infusion of isoproterenol. However, the presence of the gut microbiota-targeting inhibitor iodomethylcholine blocked choline diet-induced elevation in TMAO, and both significantly improved decline in renal function, and significantly attenuated multiple indices of tubulointerstitial fibrosis. Iodomethylcholine treatment also reversed many choline diet-induced changes in cecal microbial community composition associated with TMAO and renal functional impairment., Conclusions: Selective targeting of gut microbiota-dependent TMAO generation may prevent adverse renal structural and functional alterations in subjects at risk for chronic kidney disease.

Apolipoprotein A-I (apoA-I) is the major protein constituent of high-density lipoprotein (HDL) and a target of myeloperoxidase-dependent oxidation in the artery wall. In atherosclerotic lesions, apoA-I exhibits marked oxidative modifications at multiple sites, including Trp 72 Site-specific mutagenesis studies have suggested, but have not conclusively shown, that oxidative modification of Trp 72 of apoA-I impairs many atheroprotective properties of this lipoprotein. Herein, we used genetic code expansion technology with an engineered Saccharomyces cerevisiae tryptophanyl tRNA-synthetase (Trp-RS):suppressor tRNA pair to insert the noncanonical amino acid 5-hydroxytryptophan (5-OHTrp) at position 72 in recombinant human apoA-I and confirmed site-specific incorporation utilizing MS. In functional characterization studies, 5-OHTrp 72 apoA-I (compared with WT apoA-I) exhibited reduced ABC subfamily A member 1 (ABCA1)-dependent cholesterol acceptor activity in vitro (41.73 ± 6.57% inhibition; p < 0.01). Additionally, 5-OHTrp 72 apoA-I displayed increased activation and stabilization of paraoxonase 1 (PON1) activity (μmol/min/mg) when compared with WT apoA-I and comparable PON1 activation/stabilization compared with reconstituted HDL (WT apoA-I, 1.92 ± 0.04; 5-OHTrp 72 apoA-I, 2.35 ± 0.0; and HDL, 2.33 ± 0.1; p < 0.001, p < 0.001, and p < 0.001, respectively). Following injection into apoA-I-deficient mice, 5-OHTrp 72 apoA-I reached plasma levels comparable with those of native apoA-I yet exhibited significantly reduced (48%; p < 0.01) lipidation and evidence of HDL biogenesis. Collectively, these findings unequivocally reveal that site-specific oxidative modification of apoA-I via 5-OHTrp at Trp 72 impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both in vitro and in vivo .

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and β2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors., Competing Interests: Declaration of Interests S.L.H. reports being named as co-inventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics, being a paid consultant for P&G, having received research funds from P&G and Roche Diagnostics, and being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland HeartLab, Quest Diagnostics, and P&G. The other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: A gut-microbial metabolite, trimethylamine N-oxide (TMAO), has been associated with coronary atherosclerotic burden. No previous prospective study has addressed associations of long-term changes in TMAO with coronary heart disease (CHD) incidence., Objectives: The purpose of this study was to investigate whether 10-year changes in plasma TMAO levels were significantly associated with CHD incidence., Methods: This prospective nested case-control study included 760 healthy women at baseline. Plasma TMAO levels were measured both at the first (1989 to 1990) and the second (2000 to 2002) blood collections; 10-year changes (Δ) in TMAO were calculated. Incident cases of CHD (n = 380) were identified after the second blood collection through 2016 and were matched to controls (n = 380)., Results: Regardless of the initial TMAO levels, 10-year increases in TMAO from the first to second blood collection were significantly associated with an increased risk of CHD (relative risk [RR] in the top tertile: 1.58 [95% confidence interval (CI): 1.05 to 2.38]; RR per 1-SD increment: 1.33 [95% CI: 1.06 to 1.67]). Participants with elevated TMAO levels (the top tertile) at both time points showed the highest RR of 1.79 (95% CI: 1.08 to 2.96) for CHD as compared with those with consistently low TMAO levels. Further, we found that the ΔTMAO-CHD relationship was strengthened by unhealthy dietary patterns (assessed by the Alternate Healthy Eating Index) and was attenuated by healthy dietary patterns (p interaction = 0.008)., Conclusions: Long-term increases in TMAO were associated with higher CHD risk, and repeated assessment of TMAO over 10 years improved the identification of people with a higher risk of CHD. Diet may modify the associations of ΔTMAO with CHD risk., (Copyright © 2020 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Post-translational modifications (PTMs) of protein tyrosine (Tyr) residues can serve as a molecular fingerprint of exposure to distinct oxidative pathways and are observed in abnormally high abundance in the majority of human inflammatory pathologies. Reactive oxidants generated during inflammation include hypohalous acids and nitric oxide-derived oxidants, which oxidatively modify protein Tyr residues via halogenation and nitration, respectively, forming 3-chloroTyr, 3-bromoTyr, and 3-nitroTyr. Traditional methods for generating oxidized or halogenated proteins involve nonspecific chemical reactions that result in complex protein mixtures, making it difficult to ascribe observed functional changes to a site-specific PTM or to generate antibodies sensitive to site-specific oxidative PTMs. To overcome these challenges, we generated a system to efficiently and site-specifically incorporate chloroTyr, bromoTyr, and iodoTyr, and to a lesser extent nitroTyr, into proteins in both bacterial and eukaryotic expression systems, relying on a novel amber stop codon-suppressing mutant synthetase (haloTyrRS)/tRNA pair derived from the Methanosarcina barkeri pyrrolysine synthetase system. We used this system to study the effects of oxidation on HDL-associated protein paraoxonase 1 (PON1), an enzyme with important antiatherosclerosis and antioxidant functions. PON1 forms a ternary complex with HDL and myeloperoxidase (MPO) in vivo . MPO oxidizes PON1 at tyrosine 71 (Tyr71), resulting in a loss of PON1 enzymatic function, but the extent to which chlorination or nitration of Tyr71 contributes to this loss of activity is unclear. To better understand this biological process and to demonstrate the utility of our GCE system, we generated PON1 site-specifically modified at Tyr71 with chloroTyr and nitroTyr in Escherichia coli and mammalian cells. We demonstrate that either chlorination or nitration of Tyr71 significantly reduces PON1 enzymatic activity. This tool for site-specific incorporation of halotyrosine will be critical to understanding how exposure of proteins to hypohalous acids at sites of inflammation alters protein function and cellular physiology. In addition, it will serve as a powerful tool for generating antibodies that can recognize site-specific oxidative PTMs.

Lipoproteins are supramolecular assemblies of proteins and lipids with dynamic characteristics critically linked to their biological functions as plasma lipid transporters and lipid exchangers. Among them, spherical high-density lipoproteins are the most abundant forms of high-density lipoprotein (HDL) in human plasma, active participants in reverse cholesterol transport, and associated with reduced development of atherosclerosis. Here, we employed elastic incoherent neutron scattering (EINS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine the average particle dynamics and protein backbone local mobility of physiologically competent discoidal and spherical HDL particles reconstituted with human apolipoprotein A-I (apoA-I). Our EINS measurements indicated that discoidal HDL was more dynamic than spherical HDL at ambient temperatures, in agreement with their lipid-protein composition. Combining small-angle neutron scattering (SANS) with contrast variation and MS cross-linking, we showed earlier that the most likely organization of the three apolipoprotein A-I (apoA-I) chains in spherical HDL is a combination of a hairpin monomer and a helical antiparallel dimer. Here, we corroborated those findings with kinetic studies, employing hydrogen-deuterium exchange mass spectrometry (HDX-MS). Many overlapping apoA-I digested peptides exhibited bimodal HDX kinetics behavior, suggesting that apoA-I regions with the same amino acid composition located on different apoA-I chains had different conformations and/or interaction environments., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Objective: Type 2 diabetes is related to obesity and altered bone health, and both are affected by gut microbiota. We examined associations of weight loss diet-induced changes in a gut microbiota-related metabolite trimethylamine N-oxide (TMAO), and its precursors (choline and l-carnitine), with changes in bone mineral density (BMD) considering diabetes-related factors., Research Design and Methods: In the 2-year Preventing Overweight Using Novel Dietary Strategies trial (POUNDS Lost), 264 overweight and obese participants with measurement of BMD by DXA scan were included in the present analysis. The participants were randomly assigned to one of four diets varying in macronutrient intake. Association analysis was performed in pooled participants and different diet groups. Changes in blood levels of TMAO, choline, and l-carnitine from baseline to 6 months after the dietary intervention were calculated., Results: We found that a greater reduction in plasma levels of TMAO from baseline to 6 months was associated with a greater loss in whole-body BMD at 6 months and 2 years ( P = 0.03 and P = 0.02). The greater reduction in TMAO was also associated with a greater loss in spine BMD ( P = 0.005) at 2 years, independent of body weight changes. The associations were not modified by baseline diabetes status and glycemic levels. Changes in l-carnitine, a precursor of TMAO, showed interactions with dietary fat intake in regard to changes of spine BMD and hip BMD at 6 months (all P < 0.05). Participants with the smallest decrease in l-carnitine showed less bone loss in the low-fat diet group than the high-fat diet group ( P spine = 0.03 and P hip = 0.02)., Conclusions: TMAO might protect against BMD reduction during weight loss, independent of diet interventions varying in macronutrient content and baseline diabetes risk factors. Dietary fat may modify the relation between change in plasma l-carnitine level and changes in BMD. Our findings highlight the importance of investigating the relation between TMAO and bone health in patients with diabetes., (© 2019 by the American Diabetes Association.)

Increased ribosomal biogenesis occurs during tissue hypertrophy, but whether ribosomal biogenesis is impaired during atrophy is not known. We show that hyperammonemia, which occurs in diverse chronic disorders, impairs protein synthesis as a result of decreased ribosomal content and translational capacity. Transcriptome analyses, real-time PCR, and immunoblotting showed consistent reductions in the expression of the large and small ribosomal protein subunits (RPL and RPS, respectively) in hyperammonemic murine skeletal myotubes, HEK cells, and skeletal muscle from hyperammonemic rats and human cirrhotics. Decreased ribosomal content was accompanied by decreased expression of cMYC, a positive regulator of ribosomal biogenesis, as well as reduced expression and activity of β-catenin, a transcriptional activator of cMYC. However, unlike the canonical regulation of β-catenin via glycogen synthase kinase 3β (GSK3β)-dependent degradation, GSK3β expression and phosphorylation were unaltered during hyperammonemia, and depletion of GSK3β did not prevent ammonia-induced degradation of β-catenin. Overexpression of GSK3β-resistant variants, genetic depletion of IκB kinase β (IKKβ) (activated during hyperammonemia), protein interactions, and in vitro kinase assays showed that IKKβ phosphorylated β-catenin directly. Overexpressing β-catenin restored hyperammonemia-induced perturbations in signaling responses that regulate ribosomal biogenesis. Our data show that decreased protein synthesis during hyperammonemia is mediated via a novel GSK3β-independent, IKKβ-dependent impairment of the β-catenin-cMYC axis., (Copyright © 2019 American Society for Microbiology.)

Objective: Alterations in gut microbiota have been linked to host insulin resistance, diabetes and impaired amino acid metabolism. We investigated whether changes in gut microbiota-dependent metabolite of trimethylamine N-oxide (TMAO) and its nutrient precursors (choline and L-carnitine) were associated with improvements in glucose metabolism and diabetes-related amino acids in a weight-loss diet intervention., Design: We included 504 overweight and obese adults who were randomly assigned to one of four energy-reduced diets varying in macronutrient intake. The 6-month changes (Δ) in TMAO, choline and L-carnitine levels after the intervention were calculated., Results: Greater decreases in choline and L-carnitine were significantly (p<0.05) associated with greater improvements in fasting insulin concentrations and homeostasis model assessment of insulin resistance (HOMA-IR) at 6 months. The reduction of choline was significantly related to 2-year improvements in glucose and insulin resistance. We found significant linkages between dietary fat intake and ΔTMAO for changes in fasting glucose, insulin and HOMA-IR (p interaction <0.05); a greater increase in TMAO was related to lesser improvements in the outcomes among participants who consumed a high-fat diet. In addition, ΔL-carnitine and Δcholine were significantly related to changes in amino acids (including branched-chain and aromatic amino acids). Interestingly, the associations of ΔTMAO, Δcholine and ΔL-carnitine with diabetes-related traits were independent of the changes in amino acids., Conclusion: Our findings underscore the importance of changes in TMAO, choline and L-carnitine in improving insulin sensitivity during a weight-loss intervention for obese patients. Dietary fat intake may modify the associations of TMAO with insulin sensitivity and glucose metabolism., Trial Registration Number: NCT00072995., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: l-Carnitine, an abundant nutrient in red meat, accelerates atherosclerosis in mice via gut microbiota-dependent formation of trimethylamine (TMA) and trimethylamine N-oxide (TMAO) via a multistep pathway involving an atherogenic intermediate, γ-butyrobetaine (γBB). The contribution of γBB in gut microbiota-dependent l-carnitine metabolism in humans is unknown., Methods: Omnivores and vegans/vegetarians ingested deuterium-labeled l-carnitine (d3-l-carnitine) or γBB (d9-γBB), and both plasma metabolites and fecal polymicrobial transformations were examined at baseline, following oral antibiotics, or following chronic (≥2 months) l-carnitine supplementation. Human fecal commensals capable of performing each step of the l-carnitine→γBB→TMA transformation were identified., Results: Studies with oral d3-l-carnitine or d9-γBB before versus after antibiotic exposure revealed gut microbiota contribution to the initial 2 steps in a metaorganismal l-carnitine→γBB→TMA→TMAO pathway in subjects. Moreover, a striking increase in d3-TMAO generation was observed in omnivores over vegans/vegetarians (>20-fold; P = 0.001) following oral d3-l-carnitine ingestion, whereas fasting endogenous plasma l-carnitine and γBB levels were similar in vegans/vegetarians (n = 32) versus omnivores (n = 40). Fecal metabolic transformation studies, and oral isotope tracer studies before versus after chronic l-carnitine supplementation, revealed that omnivores and vegans/vegetarians alike rapidly converted carnitine to γBB, whereas the second gut microbial transformation, γBB→TMA, was diet inducible (l-carnitine, omnivorous). Extensive anaerobic subculturing of human feces identified no single commensal capable of l-carnitine→TMA transformation, multiple community members that converted l-carnitine to γBB, and only 1 Clostridiales bacterium, Emergencia timonensis, that converted γBB to TMA. In coculture, E. timonensis promoted the complete l-carnitine→TMA transformation., Conclusion: In humans, dietary l-carnitine is converted into the atherosclerosis- and thrombosis-promoting metabolite TMAO via 2 sequential gut microbiota-dependent transformations: (a) initial rapid generation of the atherogenic intermediate γBB, followed by (b) transformation into TMA via low-abundance microbiota in omnivores, and to a markedly lower extent, in vegans/vegetarians. Gut microbiota γBB→TMA/TMAO transformation is induced by omnivorous dietary patterns and chronic l-carnitine exposure., Trial Registration: ClinicalTrials.gov NCT01731236., Funding: NIH and Office of Dietary Supplements grants HL103866, HL126827, and DK106000, and the Leducq Foundation.

Rationale: Gut microbes influence cardiovascular disease and thrombosis risks through the production of trimethylamine N-oxide (TMAO). Microbiota-dependent generation of trimethylamine (TMA)-the precursor to TMAO-is rate limiting in the metaorganismal TMAO pathway in most humans and is catalyzed by several distinct microbial choline TMA-lyases, including the proteins encoded by the cutC/D (choline utilization C/D) genes in multiple human commensals., Objective: Direct demonstration that the gut microbial cutC gene is sufficient to transmit enhanced platelet reactivity and thrombosis potential in a host via TMA/TMAO generation has not yet been reported., Methods and Results: Herein, we use gnotobiotic mice and a series of microbial colonization studies to show that microbial cutC-dependent TMA/TMAO production is sufficient to transmit heightened platelet reactivity and thrombosis potential in a host. Specifically, we examine in vivo thrombosis potential employing germ-free mice colonized with either high TMA-producing stable human fecal polymcrobial communities or a defined CutC-deficient background microbial community coupled with a CutC-expressing human commensal±genetic disruption of its cutC gene (ie, Clostridium sporogenes Δ cutC)., Conclusions: Collectively, these studies point to the microbial choline TMA-lyase pathway as a rational molecular target for the treatment of atherothrombotic heart disease.

Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite that enhances both platelet responsiveness and in vivo thrombosis potential in animal models, and TMAO plasma levels predict incident atherothrombotic event risks in human clinical studies. TMAO is formed by gut microbe-dependent metabolism of trimethylamine (TMA) moiety-containing nutrients, which are abundant in a Western diet. Here, using a mechanism-based inhibitor approach targeting a major microbial TMA-generating enzyme pair, CutC and CutD (CutC/D), we developed inhibitors that are potent, time-dependent, and irreversible and that do not affect commensal viability. In animal models, a single oral dose of a CutC/D inhibitor significantly reduced plasma TMAO levels for up to 3 d and rescued diet-induced enhanced platelet responsiveness and thrombus formation, without observable toxicity or increased bleeding risk. The inhibitor selectively accumulated within intestinal microbes to millimolar levels, a concentration over 1-million-fold higher than needed for a therapeutic effect. These studies reveal that mechanism-based inhibition of gut microbial TMA and TMAO production reduces thrombosis potential, a critical adverse complication in heart disease. They also offer a generalizable approach for the selective nonlethal targeting of gut microbial enzymes linked to host disease limiting systemic exposure of the inhibitor in the host.

Essentials Microbe-dependent production of trimethylamine N-oxide (TMAO) contributes to thrombosis risk. The impact of host flavin monooxygenase 3 (FMO3) modulation on platelet function is unknown. Genetic manipulation of FMO3 in mice alters systemic TMAO levels and thrombosis potential. Genetic manipulation of FMO3 is associated with alteration of gut microbial community structure., Summary: Background Gut microbes play a critical role in the production of trimethylamine N-oxide (TMAO), an atherogenic metabolite that impacts platelet responsiveness and thrombosis potential. Involving both microbe and host enzymatic machinery, TMAO generation utilizes a metaorganismal pathway, beginning with ingestion of trimethylamine (TMA)-containing dietary nutrients such as choline, phosphatidylcholine and carnitine, which are abundant in a Western diet. Gut microbial TMA lyases use these nutrients as substrates to produce TMA, which upon delivery to the liver via the portal circulation, is converted into TMAO by host hepatic flavin monooxygenases (FMOs). Gut microbial production of TMA is rate limiting in the metaorganismal TMAO pathway because hepatic FMO activity is typically in excess. Objectives FMO3 is the major FMO responsible for host generation of TMAO; however, a role for FMO3 in altering platelet responsiveness and thrombosis potential in vivo has not yet been explored. Methods The impact of FMO3 suppression (antisense oligonucleotide-targeting) and overexpression (as transgene) on plasma TMAO levels, platelet responsiveness and thrombosis potential was examined using a murine FeCl 3 -induced carotid artery injury model. Cecal microbial composition was examined using 16S analyses. Results Modulation of FMO3 directly impacts systemic TMAO levels, platelet responsiveness and rate of thrombus formation in vivo. Microbial composition analyses reveal taxa whose proportions are associated with both plasma TMAO levels and in vivo thrombosis potential. Conclusions The present studies demonstrate that host hepatic FMO3, the terminal step in the metaorganismal TMAO pathway, participates in diet-dependent and gut microbiota-dependent changes in both platelet responsiveness and thrombosis potential in vivo., (© 2018 International Society on Thrombosis and Haemostasis.)

Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH- S -transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Using an untargeted metabolomics approach in initial (N = 99 subjects) and replication cohorts (N = 1,162), we discovered and structurally identified a plasma metabolite associated with cardiovascular disease (CVD) risks, N6,N6,N6-trimethyl-L-lysine (trimethyllysine, TML). Stable-isotope-dilution tandem mass spectrometry analyses of an independent validation cohort (N = 2,140) confirmed TML levels are independently associated with incident (3-year) major adverse cardiovascular event risks (hazards ratio [HR], 2.4; 95% CI, 1.7-3.4) and incident (5-year) mortality risk (HR, 2.9; 95% CI, 2.0-4.2). Genome-wide association studies identified several suggestive loci for TML levels, but none reached genome-wide significance; and d9(trimethyl)-TML isotope tracer studies confirmed TML can serve as a nutrient precursor for gut microbiota-dependent generation of trimethylamine (TMA) and the atherogenic metabolite trimethylamine N-oxide (TMAO). Although TML was shown to be abundant in both plant- and animal-derived foods, mouse and human fecal cultures (omnivores and vegans) showed slow conversion of TML to TMA. Furthermore, unlike chronic dietary choline, TML supplementation in mice failed to elevate plasma TMAO or heighten thrombosis potential in vivo. Thus, TML is identified as a strong predictor of incident CVD risks in subjects and to serve as a dietary precursor for gut microbiota-dependent generation of TMAO; however, TML does not appear to be a major microbial source for TMAO generation in vivo.

Increased circulating levels of apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), by genetic manipulation or infusion, protects against melanoma growth and metastasis. Herein, we explored potential roles in melanoma tumorigenesis for host scavenger receptor class B, type 1 (SR-B1), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), all mediators of apoA-I and HDL sterol and lipid transport function. In a syngeneic murine melanoma tumor model, B16F10, mice with global deletion of SR-B1 expression exhibited increased plasma HDL cholesterol (HDLc) levels and decreased tumor volume, indicating host SR-B1 does not directly contribute to HDL-associated anti-tumor activity. In mice with myeloid-specific loss of ABCA1 ( Abca1 -M/-M ; A1 -M/-M ), tumor growth was inhibited by ∼4.8-fold relative to wild type (WT) animals. Abcg1 -M/-M (G1 -M/-M ) animals were also protected by 2.5-fold relative to WT, with no further inhibition of tumor growth in Abca1 / Abcg1 myeloid-specific double knockout animals (DKO). Analyses of tumor-infiltrating immune cells revealed a correlation between tumor protection and decreased presence of the immune suppressive myeloid-derived suppressor cell (MDSC) subsets, Ly-6G + Ly-6C Lo and Ly-6G neg Ly-6C Hi cells. The growth of the syngeneic MB49 murine bladder cancer cells was also inhibited in A1 -M/-M mice. Collectively, our studies provide further evidence for an immune modulatory role for cholesterol homeostasis pathways in cancer., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2-AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or base-catalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate protein-bound 2-AAA from an alternative potential lysine oxidative product, lysine nitrile (LysCN). Using a validated protease-based digestion method coupled with stable isotope dilution LC/MS/MS, we now report protein bound 2-AAA and LysCN are both formed by hypochlorous acid (HOCl) and the MPO/H 2 O 2 /Cl - system of leukocytes. At low molar ratio of oxidant to target protein N ε -lysine moiety, 2-AAA is formed via an initial N ε -monochloramine intermediate, which ultimately produces the more stable 2-AAA end-product via sequential generation of transient imine and semialdehyde intermediates. At higher oxidant to target protein N ε -lysine amine ratios, protein-bound LysCN is formed via initial generation of a lysine N ε -dichloramine intermediate. In studies employing MPO knockout mice and an acute inflammation model, we show that both free and protein-bound 2-AAA, and in lower yield, protein-bound LysCN, are formed by MPO in vivo during inflammation. Finally, both 2-AAA and to lesser extent LysCN are shown to be enriched in human aortic atherosclerotic plaque, a tissue known to harbor multiple MPO-catalyzed protein oxidation products. Collectively, these results show that MPO-mediated oxidation of protein lysyl residues serves as a mechanism for producing 2-AAA and LysCN in vivo. These studies further support involvement of MPO-catalyzed oxidative processes in both the development of atherosclerosis and diabetes risk., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The biochemical mechanisms through which eosinophils contribute to asthma pathogenesis are unclear. Here we show eosinophil peroxidase (EPO), an abundant granule protein released by activated eosinophils, contributes to characteristic asthma-related phenotypes through oxidative posttranslational modification (PTM) of proteins in asthmatic airways through a process called carbamylation. Using a combination of studies we now show EPO uses plasma levels of the pseudohalide thiocyanate (SCN - ) as substrate to catalyze protein carbamylation, as monitored by PTM of protein lysine residues into N ϵ -carbamyllysine (homocitrulline), and contributes to the pathophysiological sequelae of eosinophil activation. Studies using EPO-deficient mice confirm EPO serves as a major enzymatic source for protein carbamylation during eosinophilic inflammatory models, including aeroallergen challenge. Clinical studies similarly revealed significant enrichment in carbamylation of airway proteins recovered from atopic asthmatics versus healthy controls in response to segmental allergen challenge. Protein-bound homocitrulline is shown to be co-localized with EPO within human asthmatic airways. Moreover, pathophysiologically relevant levels of carbamylated protein either incubated with cultured human airway epithelial cells in vitro, or provided as an aerosolized exposure in non-sensitized mice, induced multiple asthma-associated phenotypes including induction of mucin, Th2 cytokines, IFNγ, TGFβ, and epithelial cell apoptosis. Studies with scavenger receptor-A1 null mice reveal reduced IL-13 generation following exposure to aerosolized carbamylated protein, but no changes in other asthma-related phenotypes. In summary, EPO-mediated protein carbamylation is promoted during allergen-induced asthma exacerbation, and can both modulate immune responses and trigger a cascade of many of the inflammatory signals present in asthma., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Apolipoprotein A1 (apoA1) is the major protein component of high-density lipoprotein (HDL) and has well documented anti-inflammatory properties. To better understand the cellular and molecular basis of the anti-inflammatory actions of apoA1, we explored the effect of acute human apoA1 exposure on the migratory capacity of monocyte-derived cells in vitro and in vivo. Acute (20-60 min) apoA1 treatment induced a substantial (50-90%) reduction in macrophage chemotaxis to a range of chemoattractants. This acute treatment was anti-inflammatory in vivo as shown by pre-treatment of monocytes prior to adoptive transfer into an on-going murine peritonitis model. We find that apoA1 rapidly disrupts membrane lipid rafts, and as a consequence, dampens the PI3K/Akt signalling pathway that coordinates reorganization of the actin cytoskeleton and cell migration. Our data strengthen the evidence base for therapeutic apoA1 infusions in situations where reduced monocyte recruitment to sites of inflammation could have beneficial outcomes., Competing Interests: The authors declare that no competing interests exist.

Recent evidence has shown a complex relationship between the gut microbiota, dietary nutrients, and cardiovascular disease (CVD). Trimethylamine-N-oxide (TMAO) production, initiated by the microbiota, has been associated with CVD events. We sought to test if this association exists in HIV-infected persons. After adjusting for aspirin use and CVD risk factors, HIV-infected men were more likely to have coronary stenosis in the second and third TMAO quartiles compared with the first quartile, but did not differ significantly in the fourth quartile. We found an inverted U-shaped association between TMAO levels and the presence of coronary artery stenosis among HIV-infected men.

Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk., (Copyright © 2016 Elsevier Inc. All rights reserved.)