Proteomic profiling identifies novel proteins for genetic risk of severe COVID-19: the Atherosclerosis Risk in Communities Study.

BACKGROUND: Genome-wide association studies have identified six genetic variants associated with severe COVID-19, yet the mechanisms through which they may affect disease remains unclear. We investigated proteomic signatures related to COVID-19 risk variants rs657152 (ABO), rs10735079 (OAS1/OAS2/OAS3), rs2109069 (DPP9), rs74956615 (TYK2), rs2236757 (IFNAR2) and rs11385942 (SLC6A20/LZTFL1/CCR9/FYCO1/CXCR6/XCR1) as well as their corresponding downstream pathways that may promote severe COVID-19 in risk allele carriers and their potential relevancies to other infection outcomes. METHODS: A DNA aptamer-based array measured 4870 plasma proteins among 11 471 participants. Linear regression estimated associations between the COVID-19 risk variants and proteins with correction for multiple comparisons, and canonical pathway analysis was conducted. Cox regression assessed associations between proteins identified in the main analysis and risk of incident hospitalized respiratory infections (2570 events) over a 20.7-year follow-up. RESULTS: The ABO variant rs657152 was associated with 84 proteins in 7241 white participants with 24 replicated in 1671 Black participants. The TYK2 variant rs74956615 was associated with ICAM-1 and -5 in white participants with ICAM-5 replicated in Black participants. Of the 84 proteins identified in the main analysis, seven were significantly associated with incident hospitalized respiratory infections including Ephrin type-A receptor 4 (hazard ratio (HR): 0.87; P = 2.3 × 10-11) and von Willebrand factor type A (HR: 1.17; P = 1.6x10-13). CONCLUSIONS: Novel proteomics signatures and pathways for COVID-19-related risk variants TYK2 and ABO were identified. A subset of these proteins predicted greater risk of incident hospitalized pneumonia and respiratory infections. Further studies to examine these proteins in COVID-19 patients are warranted.

Hearing intervention versus health education control to reduce cognitive decline in older adults with hearing loss in the USA (ACHIEVE): a multicentre, randomised controlled trial.

BACKGROUND: Hearing loss is associated with increased cognitive decline and incident dementia in older adults. We aimed to investigate whether a hearing intervention could reduce cognitive decline in cognitively healthy older adults with hearing loss. METHODS: The ACHIEVE study is a multicentre, parallel-group, unmasked, randomised controlled trial of adults aged 70-84 years with untreated hearing loss and without substantial cognitive impairment that took place at four community study sites across the USA. Participants were recruited from two study populations at each site: (1) older adults participating in a long-standing observational study of cardiovascular health (Atherosclerosis Risk in Communities [ARIC] study), and (2) healthy de novo community volunteers. Participants were randomly assigned (1:1) to a hearing intervention (audiological counselling and provision of hearing aids) or a control intervention of health education (individual sessions with a health educator covering topics on chronic disease prevention) and followed up every 6 months. The primary endpoint was 3-year change in a global cognition standardised factor score from a comprehensive neurocognitive battery. Analysis was by intention to treat. This trial was registered at ClinicalTrials.gov, NCT03243422. FINDINGS: From Nov 9, 2017, to Oct 25, 2019, we screened 3004 participants for eligibility and randomly assigned 977 (32·5%; 238 [24%] from ARIC and 739 [76%] de novo). We randomly assigned 490 (50%) to the hearing intervention and 487 (50%) to the health education control. The cohort had a mean age of 76·8 years (SD 4·0), 523 (54%) were female, 454 (46%) were male, and most were White (n=858 [88%]). Participants from ARIC were older, had more risk factors for cognitive decline, and had lower baseline cognitive scores than those in the de novo cohort. In the primary analysis combining the ARIC and de novo cohorts, 3-year cognitive change (in SD units) was not significantly different between the hearing intervention and health education control groups (-0·200 [95% CI -0·256 to -0·144] in the hearing intervention group and -0·202 [-0·258 to -0·145] in the control group; difference 0·002 [-0·077 to 0·081]; p=0·96). However, a prespecified sensitivity analysis showed a significant difference in the effect of the hearing intervention on 3-year cognitive change between the ARIC and de novo cohorts (pinteraction=0·010). Other prespecified sensitivity analyses that varied analytical parameters used in the total cohort did not change the observed results. No significant adverse events attributed to the study were reported with either the hearing intervention or health education control. INTERPRETATION: The hearing intervention did not reduce 3-year cognitive decline in the primary analysis of the total cohort. However, a prespecified sensitivity analysis showed that the effect differed between the two study populations that comprised the cohort. These findings suggest that a hearing intervention might reduce cognitive change over 3 years in populations of older adults at increased risk for cognitive decline but not in populations at decreased risk for cognitive decline. FUNDING: US National Institutes of Health.

Omega-3 Polyunsaturated Fatty Acids are not associated with Peripheral Artery Disease in a Meta-Analysis from the Multi-Ethnic Study of Atherosclerosis and Atherosclerosis Risk in Communities Study Cohorts.

BACKGROUND: Research suggests omega-3 polyunsaturated fatty acids (PUFAs) exert favorable effects on several biological processes involved in the development and progression of atherosclerotic cardiovascular disease (ASCVD). However, studies examining the relationship between omega-3 PUFAs and peripheral artery disease (PAD) are scarce. OBJECTIVES: We evaluated the associations between omega-3 PUFAs and incident PAD in a meta-analysis of the Multi-Ethnic Study of Atherosclerosis (MESA) and Atherosclerosis Risk in Communities (ARIC) study cohorts. METHODS: Omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were measured at baseline for all MESA (n = 6495) and Minnesota ARIC participants (n = 3612). Incident clinical PAD events (MESA n = 106; ARIC n = 149) identified primarily through ICD discharge codes were assessed through follow-up of each cohort. Associations between omega-3 PUFAs (EPA, DHA, and EPA+DHA) and incident PAD were modeled in MESA and ARIC as quartiles and continuously using Cox proportional hazards regression, respectively. A fixed-effects meta-analysis was conducted to evaluate associations in the 2 cohorts combined. RESULTS: In the fully adjusted model, in 10,107 participants, no significant associations were observed between EPA, DHA, or EPA+DHA, and incident PAD modeled as quartiles or continuously for either MESA or ARIC cohorts separately or in the meta-analysis after a follow-up of approximately 15 y. CONCLUSION: This study is consistent with previous literature indicating that the beneficial effects of omega-3 PUFAs on the markers of ASCVD may not translate to a clinically meaningful decrease in PAD risk.

Proteomics Analysis of Genetic Liability of Abdominal Aortic Aneurysm Identifies Plasma Neogenin and Kit Ligand: The ARIC Study.

BACKGROUND: Genome-wide association studies have reported 23 gene loci related to abdominal aortic aneurysm (AAA)-a potentially lethal condition characterized by a weakened dilated vessel wall. This study aimed to identify proteomic signatures and pathways related to these risk loci to better characterize AAA genetic susceptibility. METHODS: Plasma concentrations of 4870 proteins were determined using a DNA aptamer-based array. Linear regression analysis estimated the associations between the 23 risk alleles and plasma protein levels with adjustments for potential confounders in a race-stratified analysis of 1671 Black and 7241 White participants. Significant proteins were then evaluated for their prediction of clinical AAA (454 AAA events in 11 064 individuals), and those significantly associated with AAA were further interrogated using Mendelian randomization analysis. RESULTS: Risk variants proximal to PSRC1-CELSR2-SORT1, PCIF1-ZNF335-MMP9, RP11-136O12.2/TRIB1, ZNF259/APOA5, IL6R, PCSK9, LPA, and APOE were associated with 118 plasma proteins in Whites and 59 were replicated in Black participants. Novel associations with clinical AAA incidence were observed for kit ligand (HR, 0.59 [95% CI, 0.42-0.82] for top versus first quintiles) and neogenin (HR, 0.64 [95% CI, 0.46-0.88]) over a median 21.2-year follow-up; neogenin was also associated with ultrasound-detected asymptomatic AAA (N=4295; 57 asymptomatic AAA cases). Mendelian randomization inverse variance weighted estimates suggested that AAA risk is promoted by lower levels of kit ligand (OR per SD=0.67; P=1.4×10-5) and neogenin (OR per SD=0.50; P=0.03). CONCLUSIONS: Low levels of neogenin and kit ligand may be novel risk factors for AAA development in potentially causal pathways. These findings provide insights and potential targets to reduce AAA susceptibility.

Antithrombin, Protein C, and Protein S: Genome and Transcriptome-Wide Association Studies Identify 7 Novel Loci Regulating Plasma Levels.

BACKGROUND: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total. METHODS: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. RESULTS: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (GCKR, BAZ1B, and HP-TXNL4B) and 1 with PS levels (ORM1-ORM2). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (FCGRT), 1 with PC (GOLM2), and 1 with PS (MYL7). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17, and HP genes in antithrombin regulation. CONCLUSIONS: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.

Menthol and Other Flavor Chemicals in Cigarettes from Vietnam and the Philippines.

INTRODUCTION: Tobacco product flavors can increase product appeal, adolescent initiation and experimentation, and difficulty quitting. Flavored tobacco products are not restricted in Vietnam or the Philippines despite the high smoking prevalence among those 15 years of age and older (24% and 23%, respectively). There are no published reports to our knowledge on the levels of flavor chemicals in the cigarettes sold in these two countries. METHODS: Cigarettes were purchased in Vietnam (32 brand variants) and the Philippines (19 brand variants) during 2020. Chemical analyses gave the mg/filter, mg/rod, and mg/stick (= mg/(filter + rod)) values for 180 individual flavor chemicals. Values were calculated for menthol, clove-related compounds, and "other flavor chemicals" (OFCs). RESULTS: Five flavor groupings were found among the brand variants purchased in Vietnam: menthol + OFCs (n = 15), OFCs only (n = 8), nonflavored (n = 7), menthol + OFCs with a clove flavorant (n = 1) and menthol only (n = 1). Three flavor groupings were found among the brand variants purchased in the Philippines: menthol + OFCs (n = 10), nonflavored (n = 5), and menthol only (n = 4). CONCLUSIONS: A range of flavored cigarette products are being offered by tobacco companies in Vietnam and the Philippines, presumably to maximize cigarette sales. Regulation of flavor chemicals should be considered in these two countries. IMPLICATIONS: Article 9 of the WHO Framework Convention on Tobacco Control (FCTC), ratified by both Vietnam and the Philippines, states that "there is no justification for permitting the use of ingredients, such as flavoring agents, which help make tobacco products attractive." Flavors increase product appeal, adolescent initiation and experimentation, and difficulty quitting. These analyses found that cigarettes purchased in Vietnam and the Philippines contained menthol and other flavor chemicals. Tobacco companies are offering multiple flavor chemical profiles and nominally nonflavored versions in these countries; regulation of flavor chemicals should be considered in these two countries.

Anemia, Iron Deficiency, and Cause-Specific Mortality: The Atherosclerosis Risk in Communities (ARIC) Study.

INTRODUCTION: Anemia is a risk factor for all-cause mortality in older adults. Iron deficiency may also be associated with adverse outcomes, independent of its role in causing anemia. This study tested the hypotheses that anemia, and low ferritin among non-anemic participants, were associated with all-cause and cause-specific mortality in a community-based cohort of older adults. METHODS: Fasting blood was obtained from 5,070 ARIC participants (median age: 75) in 2011-2013. Anemia was defined by hemoglobin concentrations <12 g/dL in women and <13 g/dL in men. We classified 4,020 non-anemic participants by quartiles of plasma ferritin, measured by the SomaScan proteomics platform. Cox proportional hazards regression was used. RESULTS: Over a median of 7.5 years, there were 1147 deaths, including 357 due to cardiovascular disease (CVD), 302 to cancer and 132 to respiratory disease. Compared to those with normal hemoglobin, participants with anemia had a higher risk of all-cause mortality [hazard ratio 1.81 (95% CI: 1.60-2.06)], and mortality due to CVD [1.77 (1.41-2.22)], cancer [1.81 (1.41-2.33)], and respiratory disease [1.72 (1.18-2.52)] in demographics-adjusted models. In fully adjusted models, associations with all-cause mortality [1.37 (1.19-1.58)] and cause-specific mortality were attenuated. In non-anemic participants, lower ferritin levels were not associated with all-cause or cause-specific mortality, though associations were observed among participants with lesser evidence of inflammation and for cancer mortality in men only. CONCLUSION: Anemia is an important risk factor in older adults and may contribute to mortality due to CVD, cancer, and respiratory disease. Our results do not provide evidence that iron deficiency, independent of anemia, is a risk factor for mortality in this population.

Hearing loss and cognition: A protocol for ensuring speech understanding before neurocognitive assessment.

INTRODUCTION: Many neurocognitive evaluations involve auditory stimuli, yet there are no standard testing guidelines for individuals with hearing loss. The ensuring speech understanding (ESU) test was developed to confirm speech understanding and determine whether hearing accommodations are necessary for neurocognitive testing. METHODS: Hearing was assessed using audiometry. The probability of ESU test failure by hearing status was estimated in 2679 participants (mean age: 81.4 ± 4.6 years) using multivariate logistic regression. RESULTS: Only 2.2% (N = 58) of participants failed the ESU test. The probability of failure increased with hearing loss severity; similar results were observed for those with and without mild cognitive impairment or dementia. DISCUSSION: The ESU test is appropriate for individuals who have variable degrees of hearing loss and cognitive function. This test can be used prior to neurocognitive testing to help reduce the risk of hearing loss and compromised auditory access to speech stimuli causing poorer performance on neurocognitive evaluation.

Investigation of Causal Effects of Protein Biomarkers on Cardiovascular Disease in Persons With HIV.

BACKGROUND: There is an incompletely understood increased risk for cardiovascular disease (CVD) among people with HIV (PWH). We investigated if a collection of biomarkers were associated with CVD among PWH. Mendelian randomization (MR) was used to identify potentially causal associations. METHODS: Data from follow-up in 4 large trials among PWH were used to identify 131 incident CVD cases and they were matched to 259 participants without incident CVD (controls). Tests of associations between 460 baseline protein levels and case status were conducted. RESULTS: Univariate analysis found CLEC6A, HGF, IL-6, IL-10RB, and IGFBP7 as being associated with case status and a multivariate model identified 3 of these: CLEC6A (odds ratio [OR] = 1.48, P = .037), HGF (OR = 1.83, P = .012), and IL-6 (OR = 1.45, P = .016). MR methods identified 5 significantly associated proteins: AXL, CHI3L1, GAS6, IL-6RA, and SCGB3A2. CONCLUSIONS: These results implicate inflammatory and fibrotic processes as contributing to CVD. While some of these biomarkers are well established in the general population and in PWH (IL-6 and its receptor), some are novel to PWH (HGF, AXL, and GAS6) and some are novel overall (CLEC6A). Further investigation into the uniqueness of these biomarkers in PWH and the role of these biomarkers as targets among PWH is warranted.

Proteomic analysis of diabetes genetic risk scores identifies complement C2 and neuropilin-2 as predictors of type 2 diabetes: the Atherosclerosis Risk in Communities (ARIC) Study.

AIMS/HYPOTHESIS: Genetic predisposition to type 2 diabetes is well-established, and genetic risk scores (GRS) have been developed that capture heritable liabilities for type 2 diabetes phenotypes. However, the proteins through which these genetic variants influence risk have not been thoroughly investigated. This study aimed to identify proteins and pathways through which type 2 diabetes risk variants may influence pathophysiology. METHODS: Using a proteomics data-driven approach in a discovery sample of 7241 White participants in the Atherosclerosis Risk in Communities Study (ARIC) cohort and a replication sample of 1674 Black ARIC participants, we interrogated plasma levels of 4870 proteins and four GRS of specific type 2 diabetes phenotypes related to beta cell function, insulin resistance, lipodystrophy, BMI/blood lipid abnormalities and a composite score of all variants combined. RESULTS: Twenty-two plasma proteins were identified in White participants after Bonferroni correction. Of the 22 protein-GRS associations that were statistically significant, 10 were replicated in Black participants and all but one were directionally consistent. In a secondary analysis, 18 of the 22 proteins were found to be associated with prevalent type 2 diabetes and ten proteins were associated with incident type 2 diabetes. Two-sample Mendelian randomisation indicated that complement C2 may be causally related to greater type 2 diabetes risk (inverse variance weighted estimate: OR 1.65 per SD; p=7.0 × 10-3), while neuropilin-2 was inversely associated (OR 0.44 per SD; p=8.0 × 10-3). CONCLUSIONS/INTERPRETATION: Identified proteins may represent viable intervention or pharmacological targets to prevent, reverse or slow type 2 diabetes progression, and further research is needed to pursue these targets.

Racial and Ethnic Differences in All-Cause and Cardiovascular Disease Mortality: The MESA Study.

BACKGROUND: Despite improvements in population health, marked racial and ethnic disparities in longevity and cardiovascular disease (CVD) mortality persist. This study aimed to describe risks for all-cause and CVD mortality by race and ethnicity, before and after accounting for socioeconomic status (SES) and other factors, in the MESA study (Multi-Ethnic Study of Atherosclerosis). METHODS: MESA recruited 6814 US adults, 45 to 84 years of age, free of clinical CVD at baseline, including Black, White, Hispanic, and Chinese individuals (2000-2002). Using Cox proportional hazards modeling with time-updated covariates, we evaluated the association of self-reported race and ethnicity with all-cause and adjudicated CVD mortality, with progressive adjustments for age and sex, SES (neighborhood SES, income, education, and health insurance), lifestyle and psychosocial risk factors, clinical risk factors, and immigration history. RESULTS: During a median of 15.8 years of follow-up, 22.8% of participants (n=1552) died, of which 5.3% (n=364) died of CVD. After adjusting for age and sex, Black participants had a 34% higher mortality hazard (hazard ratio [HR], 1.34 [95% CI, 1.19-1.51]), Chinese participants had a 21% lower mortality hazard (HR, 0.79 [95% CI, 0.66-0.95]), and there was no mortality difference in Hispanic participants (HR, 0.99 [95% CI, 0.86-1.14]) compared with White participants. After adjusting for SES, the mortality HR for Black participants compared with White participants was reduced (HR, 1.16 [95% CI, 1.01-1.34]) but still statistically significant. With adjustment for SES, the mortality hazards for Chinese and Hispanic participants also decreased in comparison with White participants. After further adjustment for additional risk factors and immigration history, Hispanic participants (HR, 0.77 [95% CI, 0.63-0.94]) had a lower mortality risk than White participants, and hazard ratios for Black participants (HR, 1.08 [95% CI, 0.92-1.26]) and Chinese participants (HR, 0.81 [95% CI, 0.60-1.08]) were not significantly different from those of White participants. Similar trends were seen for CVD mortality, although the age- and sex-adjusted HR for CVD mortality for Black participants compared with White participants was greater than all-cause mortality (HR, 1.72 [95% CI, 1.34-2.21] compared with HR, 1.34 [95% CI, 1.19-1.51]). CONCLUSIONS: These results highlight persistent racial and ethnic differences in overall and CVD mortality, largely attributable to social determinants of health, and support the need to identify and act on systemic factors that shape differences in health across racial and ethnic groups.

Eugenol, menthol and other flavour chemicals in kreteks and 'white' cigarettes purchased in Indonesia.

BACKGROUND: Flavoured tobacco products are not restricted in Indonesia, a country with about 68 million adults who smoke. Most use clove-mixed tobacco cigarettes ('kreteks'); non-clove ('white') cigarettes are also available. Although the use of flavour chemicals has been identified by WHO as promoting tobacco use, little has been reported for Indonesia about the levels of flavourants in either kreteks or 'white cigarettes'. METHODS: 22 kretek brand variants and nine 'white' cigarette brand variants were purchased in Indonesia during 2021/2022; one of the kretek packs contained three colour-coded variants, giving a total sample number of 24 for the kreteks. Chemical analyses gave the mg/stick (=mg/(filter+rod)) values for 180 individual flavour chemicals that included eugenol (a clove-flavoured compound), four other clove-related compounds and menthol. RESULTS: Eugenol was present at significant levels in all 24 kreteks (2.8-33.8 mg/stick), but was essentially absent in all of the cigarettes. Menthol was present in 14 of 24 kreteks, with levels ranging from 2.8 to 12.9 mg/stick, and in five of the nine cigarettes, with levels ranging from 3.6 to 10.8 mg/stick. Other flavour chemicals were also found in many of the kretek and cigarette samples. CONCLUSIONS: In this small sample, we found numerous variations of flavoured tobacco products offered by multinational and national companies in Indonesia. Given the body of evidence that flavours make tobacco products more appealing, regulation of clove-related compounds, menthol and other flavour chemicals should be considered in Indonesia.

Multi-ethnic GWAS and fine-mapping of glycaemic traits identify novel loci in the PAGE Study.

AIMS/HYPOTHESIS: Type 2 diabetes is a growing global public health challenge. Investigating quantitative traits, including fasting glucose, fasting insulin and HbA1c, that serve as early markers of type 2 diabetes progression may lead to a deeper understanding of the genetic aetiology of type 2 diabetes development. Previous genome-wide association studies (GWAS) have identified over 500 loci associated with type 2 diabetes, glycaemic traits and insulin-related traits. However, most of these findings were based only on populations of European ancestry. To address this research gap, we examined the genetic basis of fasting glucose, fasting insulin and HbA1c in participants of the diverse Population Architecture using Genomics and Epidemiology (PAGE) Study. METHODS: We conducted a GWAS of fasting glucose (n = 52,267), fasting insulin (n = 48,395) and HbA1c (n = 23,357) in participants without diabetes from the diverse PAGE Study (23% self-reported African American, 46% Hispanic/Latino, 40% European, 4% Asian, 3% Native Hawaiian, 0.8% Native American), performing transethnic and population-specific GWAS meta-analyses, followed by fine-mapping to identify and characterise novel loci and independent secondary signals in known loci. RESULTS: Four novel associations were identified (p < 5 × 10-9), including three loci associated with fasting insulin, and a novel, low-frequency African American-specific locus associated with fasting glucose. Additionally, seven secondary signals were identified, including novel independent secondary signals for fasting glucose at the known GCK locus and for fasting insulin at the known PPP1R3B locus in transethnic meta-analysis. CONCLUSIONS/INTERPRETATION: Our findings provide new insights into the genetic architecture of glycaemic traits and highlight the continued importance of conducting genetic studies in diverse populations. DATA AVAILABILITY: Full summary statistics from each of the population-specific and transethnic results are available at NHGRI-EBI GWAS catalog ( https://www.ebi.ac.uk/gwas/downloads/summary-statistics ).

Epigenetic Age and the Risk of Incident Atrial Fibrillation.

BACKGROUND: The most prominent risk factor for atrial fibrillation (AF) is chronological age; however, underlying mechanisms are unexplained. Algorithms using epigenetic modifications to the human genome effectively predict chronological age. Chronological and epigenetic predicted ages may diverge in a phenomenon referred to as epigenetic age acceleration (EAA), which may reflect accelerated biological aging. We sought to evaluate for associations between epigenetic age measures and incident AF. METHODS: Measures for 4 epigenetic clocks (Horvath, Hannum, DNA methylation [DNAm] PhenoAge, and DNAm GrimAge) and an epigenetic predictor of PAI-1 (plasminogen activator inhibitor-1) levels (ie, DNAm PAI-1) were determined for study participants from 3 population-based cohort studies. Cox models evaluated for associations with incident AF and results were combined via random-effects meta-analyses. Two-sample summary-level Mendelian randomization analyses evaluated for associations between genetic instruments of the EAA measures and AF. RESULTS: Among 5600 participants (mean age, 65.5 years; female, 60.1%; Black, 50.7%), there were 905 incident AF cases during a mean follow-up of 12.9 years. Unadjusted analyses revealed all 4 epigenetic clocks and the DNAm PAI-1 predictor were associated with statistically significant higher hazards of incident AF, though the magnitudes of their point estimates were smaller relative to the associations observed for chronological age. The pooled EAA estimates for each epigenetic measure, with the exception of Horvath EAA, were associated with incident AF in models adjusted for chronological age, race, sex, and smoking variables. After multivariable adjustment for additional known AF risk factors that could also potentially function as mediators, pooled EAA measures for 2 clocks remained statistically significant. Five-year increases in EAA measures for DNAm GrimAge and DNAm PhenoAge were associated with 19% (adjusted hazard ratio [HR], 1.19 [95% CI, 1.09-1.31]; P<0.01) and 15% (adjusted HR, 1.15 [95% CI, 1.05-1.25]; P<0.01) higher hazards of incident AF, respectively. Mendelian randomization analyses for the 5 EAA measures did not reveal statistically significant associations with AF. CONCLUSIONS: Our study identified adjusted associations between EAA measures and incident AF, suggesting that biological aging plays an important role independent of chronological age, though a potential underlying causal relationship remains unclear. These aging processes may be modifiable and not constrained by the immutable factor of time.

Diabetes Duration and Subclinical Myocardial Injury: The Atherosclerosis Risk in Communities Study (ARIC).

BACKGROUND: Diabetes exerts adverse effects on the heart, and a longer diabetes duration is associated with greater heart failure risk. We studied diabetes duration and subclinical myocardial injury, as reflected by high-sensitivity cardiac troponin (hs-cTnT). METHODS: We analyzed 9052 participants without heart failure or coronary heart disease (mean age 63 years, 58% female, 21% Black, 15% with diabetes) at The Atherosclerosis Risk in Communities Study (ARIC) Visit 4 (1996 to 1998). Diabetes duration was calculated based on diabetes status at Visits 1 (1987 to 1989) through 4, or using self-reported age of diabetes diagnosis prior to Visit 1. We used multinomial logistic regression to determine the association of diabetes duration with increased (≥14 ng/L) or detectable (≥6 ng/L) Visit 4 hs-cTnT, relative to undetectable hs-cTnT, adjusted for demographics and cardiovascular risk factors. RESULTS: The prevalence of increased Visit 4 hs-cTnT was higher in persons with longer diabetes duration, from 12% for those with diabetes 0 to <5 years up to 31% among those with diabetes for ≥15 years (P for trend <0.0001). New onset diabetes at Visit 4 was associated with 1.92× higher relative risk (95% CI, 1.27-2.91) of increased hs-cTnT than no diabetes. Longer diabetes duration was associated with greater myocardial injury, with duration ≥15 years associated with 9.29× higher risk (95% CI, 5.65-15.29) for increased hs-cTnT and 2.07× (95% CI, 1.24-3.16) for detectable hs-cTnT, compared to no diabetes. CONCLUSIONS: Longer diabetes duration is strongly associated with subclinical myocardial injury. Interventional studies are needed to assess whether the prevention and delay of diabetes onset can mitigate early myocardial damage.

Disposable Puff Bar Electronic Cigarettes: Chemical Composition and Toxicity of E-liquids and a Synthetic Coolant.

The popularity of disposable fourth-generation electronic cigarettes (ECs) among young adults and adolescents has been increasing since the ban on flavored cartridge EC products such as JUUL. Although the constituents and toxicity of some cartridge-based fourth-generation ECs, such as JUUL, have been studied, limited data exist for other disposable ECs such as Puff. The purpose of this study was to determine flavor chemicals, synthetic coolants, and nicotine concentrations in 16 disposable Puff devices, evaluate the cytotoxicity of the different flavors from the Puff brand using in vitro assays, and investigate the health risks of synthetic coolants in EC products. Gas chromatography/mass spectrometry was used to identify and quantify chemicals in Puff EC fluids. One hundred and twenty-six flavor chemicals were identified in Puff fluids, and 16 were >1 mg/mL. WS-23 (2-isopropyl-N,2,3-trimethylbutyramide) was present in all products, and concentrations ranged from 0.8 to 45.1 mg/mL. WS-3 (N-ethyl-p-menthane-3-carboxamide) concentrations ranged from 1.5 to 16.4 mg/mL in 6/16 products. Nicotine concentrations ranged from 40.6 to 52.4 (average 44.8 mg/mL). All unvaped fluids were cytotoxic at dilutions between 0.1 and 10% in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. The cytotoxicity of Puff fluids was highly correlated with total chemical concentrations, nicotine, WS-23, both synthetic coolants, and synthetic coolants plus ethyl maltol. Lower concentrations of WS-23 than those in the fluids adversely affected cell growth and morphology. Concentrations of synthetic coolants exceeded levels used in consumer products. The margin of exposure data showed that WS-3 and WS-23 concentrations were high enough in Puff products to present a health hazard. Our study demonstrates that disposable Puff ECs have high levels of cytotoxic chemicals. The data support the regulation of flavor chemicals and synthetic coolants in ECs to limit potentially harmful health effects.

Association of Differential Leukocyte Count With Incident Abdominal Aortic Aneurysm Over 22.5 Years: The ARIC Study.

OBJECTIVE: Leukocytes contribute to the development of abdominal aortic aneurysm (AAA). We evaluated whether associations of differential leukocyte counts with AAA persist after accounting for traditional risk factors of AAA. APPROACH AND RESULTS: Among 11 217 adults from the Atherosclerosis Risk in Communities Study, we evaluated associations of differential leukocyte counts at baseline (1987­1989) with incident AAAs over a median follow-up of 22.5 years, using Cox proportional hazards regression. Each differential leukocyte count was categorized into 5 groups­below normal, tertiles within the normal range, and above normal, with the first tertile serving as the referent. We identified 377 incident AAAs through 2011, using hospital discharge diagnoses, linked Medicare records, or death certificates. At baseline, higher neutrophil, monocyte, and eosinophil counts were associated with higher risk of AAA, independent of smoking, other differential leukocyte counts, and other traditional risk factors. The association with incident AAA was the strongest for above normal neutrophil count, with an adjusted hazard ratio (95% CI) of 2.17 (1.29­3.64). Below normal neutrophil, lymphocyte, eosinophil and basophil counts were associated with higher risk of AAA with adjusted hazard ratio (95% CI) between 1.86 (1.04­3.35) and 1.62 (1.10­2.39). CONCLUSIONS: Higher neutrophil, monocyte, and eosinophil counts in midlife are associated with higher risk of AAA, even after accounting for traditional risk factors such as smoking, obesity, and atherosclerosis. This suggests the need to identify nontraditional risk factors and treatment strategies to mitigate the residual risk of AAA conferred by midlife inflammation. Whether immunosuppression is associated with higher risk of AAA needs further investigation.

Gaseous air pollutants and DNA methylation in a methylome-wide association study of an ethnically and environmentally diverse population of U.S. adults.

Epigenetic mechanisms may underlie air pollution-health outcome associations. We estimated gaseous air pollutant-DNA methylation (DNAm) associations using twelve subpopulations within Women's Health Initiative (WHI) and Atherosclerosis Risk in Communities (ARIC) cohorts (n = 8397; mean age 61.3 years; 83% female; 46% African-American, 46% European-American, 8% Hispanic/Latino). We used geocoded participant address-specific mean ambient carbon monoxide (CO), nitrogen oxides (NO2; NOx), ozone (O3), and sulfur dioxide (SO2) concentrations estimated over the 2-, 7-, 28-, and 365-day periods before collection of blood samples used to generate Illumina 450 k array leukocyte DNAm measurements. We estimated methylome-wide, subpopulation- and race/ethnicity-stratified pollutant-DNAm associations in multi-level, linear mixed-effects models adjusted for sociodemographic, behavioral, meteorological, and technical covariates. We combined stratum-specific estimates in inverse variance-weighted meta-analyses and characterized significant associations (false discovery rate; FDR<0.05) at Cytosine-phosphate-Guanine (CpG) sites without among-strata heterogeneity (PCochran's Q > 0.05). We attempted replication in the Cooperative Health Research in Region of Augsburg (KORA) study and Normative Aging Study (NAS). We observed a -0.3 (95% CI: -0.4, -0.2) unit decrease in percent DNAm per interquartile range (IQR, 7.3 ppb) increase in 28-day mean NO2 concentration at cg01885635 (chromosome 3; regulatory region 290 bp upstream from ZNF621; FDR = 0.03). At intragenic sites cg21849932 (chromosome 20; LIME1; intron 3) and cg05353869 (chromosome 11; KLHL35; exon 2), we observed a -0.3 (95% CI: -0.4, -0.2) unit decrease (FDR = 0.04) and a 1.2 (95% CI: 0.7, 1.7) unit increase (FDR = 0.04), respectively, in percent DNAm per IQR (17.6 ppb) increase in 7-day mean ozone concentration. Results were not fully replicated in KORA and NAS. We identified three CpG sites potentially susceptible to gaseous air pollution-induced DNAm changes near genes relevant for cardiovascular and lung disease. Further harmonized investigations with a range of gaseous pollutants and averaging durations are needed to determine the effect of gaseous air pollutants on DNA methylation and ultimately gene expression.

The prospective association between periodontal disease and brain imaging outcomes: The Atherosclerosis Risk in Communities study.

AIM: We investigate if periodontal disease is prospectively associated with cerebrovascular and neurodegenerative markers of dementia and Alzheimer's pathology. MATERIALS AND METHODS: N = 1306 participants (Visit 5 mean age = 76.5 [standard deviation = 5.4] years) in the Atherosclerosis Risk in Communities study with completed dental exams at Visit 4 underwent brain magnetic resonance imaging scans at Visit 5 while N = 248 underwent positron emission tomography scans. Participants were classified as edentulous or, among the dentate, by the modified Periodontal Profile Class. Brain volumes were regressed on periodontal status in linear regressions. Cerebrovascular measures and ß-amyloid positivity were regressed on periodontal status in logistic regressions. RESULTS: Periodontal disease was not associated with brain volumes, microhaemorrhages, or elevated ß-amyloid. Compared with periodontally healthy individuals, odds ratios [95% confidence interval] for all-type infarcts were 0.37 [0.20, 0.65] for severe tooth loss and 0.56 [0.31, 0.99] for edentulous participants. CONCLUSIONS: Within the limitations of this study, periodontal disease was not associated with altered brain volumes, microhaemorrhages, or ß-amyloid positivity. Tooth loss was associated with lower odds of cerebral infarcts.

Flavour chemicals, synthetic coolants and pulegone in popular mint-flavoured and menthol-flavoured e-cigarettes.

BACKGROUND: The Food and Drug Administration (FDA) has recently banned flavours from pod-style electronic cigarettes (e-cigarettes), except for menthol and tobacco. JUUL customers have quickly discovered that flavoured disposable e-cigarettes from other manufacturers, such as Puff, are readily available. Our goal was to compare flavour chemicals, synthetic coolants and pulegone in mint-flavoured/menthol-flavoured e-cigarettes from JUUL and Puff, evaluate the cytotoxicity of the coolants and perform a cancer risk assessment for pulegone, which is present in both JUUL pods and disposable Puff products. METHODS: Identification and quantification of chemicals were performed using gas chromatography/mass spectrometry. Cytotoxicity of the coolants was evaluated with BEAS-2B cells using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cancer risk of pulegone was calculated using the margin of exposure (MOE). RESULTS: Menthol was the dominant flavour chemical (>1 mg/mL) in all products from both manufacturers. Minor flavour chemicals (<1 mg/mL) differed in the JUUL and Puff fluids and may produce flavour accents. The concentrations of WS-3 and WS-23 were higher in Puff than in JUUL. WS-23 was cytotoxic in the MTT assay at concentrations 90 times lower than concentrations in Puff fluids. The risk of cancer (MOE<10 000) was greater for mint than for menthol products and greater for Puff than for JUUL. CONCLUSIONS: Switching from flavoured JUUL to Puff e-cigarettes may expose users to increased harm due to the higher levels of WS-23 and pulegone in Puff products. Cancer risk may be reduced in e-cigarettes by using pure menthol rather than mint oils to produce minty-flavoured e-cigarette products.