Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation.

G protein-coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here, we describe the design of a generalizable and cell lysate-based method that leverages the interaction between an agonist-activated GPCR and a conformation-specific binder to reconstitute split nanoluciferase (NanoLuc) in vitro. This tool, In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), has broad applications. We have demonstrated IGNiTR's use with three Gs-coupled GPCRs, two Gi-coupled GPCRs and three classes of conformation-specific binders: nanobodies, miniG proteins, and G protein peptidomimetics. As an in vitro method, IGNiTR enables the use of synthetic G protein peptidomimetics and provides easily scalable and portable reagents for characterizing GPCRs and ligands. We tested three diverse applications of IGNiTR: (1) proof-of-concept GPCR ligand screening using dopamine receptor D1 IGNiTR; (2) detection of opioids for point-of-care testing; and (3) characterizing GPCR functionality during Nanodisc-based reconstitution processes. Due to IGNiTR's unique advantages and the convenience of its cell lysate-based format, this tool will find extensive applications in GPCR ligand detection, screening, and GPCR characterization.

The Relationship Between Trimethylamine-N-Oxide and Prevalent Cardiovascular Disease in a Multiethnic Population Living in Canada.

BACKGROUND: Microflora-dependent trimethylamine-N-oxide (TMAO) formation, which results from intake of choline and L-carnitine-rich food, shows promise as a predictor of cardiovascular disease (CVD) risk, but these associations have not been examined in ethnically diverse populations. In a multiethnic population-based study of adults in Canada, we assessed the stability of TMAO and L-carnitine in stored serum samples and their association with intimal medial thickness, prevalent risk factors, and clinical events. METHODS: In a randomly sampled cross-sectional study of 1286 Canadians, fasting serum samples were collected and stored. In 292 consecutive individuals (99 CVD cases and 193 unmatched control subjects), L-carnitine and TMAO concentrations were assessed using validated analytical approaches. RESULTS: The mean (± SD) TMAO level was 1.998 ± 3.13 µM and L-carnitine was 42.29 ± 11.35 µM. The relative levels of the samples did not appreciably change after 3 freeze-thaw cycles (coefficient of variation, 5.6% and 4.7%, respectively). No significant association between L-carnitine levels and prevalent CVD was found, with adjustment for covariates (odds ratio, 1.57; 95% confidence interval, 0.58-4.26; P trend = 0.65), for highest vs lowest quintile group. TMAO levels showed a significant, graded association with prevalent CVD (odds ratio, 3.17; 95% confidence interval, 1.05-9.51; P trend = 0.02). After further adjustment for diabetes status, meat, fish, and cholesterol intake, the association remained significant. No significant association between carotid intimal medial thickness and L-carnitine (P = 0.64) or TMAO (P = 0.18) was found. CONCLUSIONS: Serum TMAO and L-carnitine analysis on stored samples is reliable. Our findings support an association between TMAO with prevalent CVD in a multiethnic population. This finding requires replication in larger studies in which dietary intake and stored serum samples exist.

Contribution of common non-synonymous variants in PCSK1 to body mass index variation and risk of obesity: a systematic review and meta-analysis with evidence from up to 331 175 individuals.

Polymorphisms rs6232 and rs6234/rs6235 in PCSK1 have been associated with extreme obesity [e.g. body mass index (BMI) ≥ 40 kg/m(2)], but their contribution to common obesity (BMI ≥ 30 kg/m(2)) and BMI variation in a multi-ethnic context is unclear. To fill this gap, we collected phenotypic and genetic data in up to 331 175 individuals from diverse ethnic groups. This process involved a systematic review of the literature in PubMed, Web of Science, Embase and the NIH GWAS catalog complemented by data extraction from pre-existing GWAS or custom-arrays in consortia and single studies. We employed recently developed global meta-analytic random-effects methods to calculate summary odds ratios (OR) and 95% confidence intervals (CIs) or beta estimates and standard errors (SE) for the obesity status and BMI analyses, respectively. Significant associations were found with binary obesity status for rs6232 (OR = 1.15, 95% CI 1.06-1.24, P = 6.08 × 10(-6)) and rs6234/rs6235 (OR = 1.07, 95% CI 1.04-1.10, P = 3.00 × 10(-7)). Similarly, significant associations were found with continuous BMI for rs6232 (ß = 0.03, 95% CI 0.00-0.07; P = 0.047) and rs6234/rs6235 (ß = 0.02, 95% CI 0.00-0.03; P = 5.57 × 10(-4)). Ethnicity, age and study ascertainment significantly modulated the association of PCSK1 polymorphisms with obesity. In summary, we demonstrate evidence that common gene variation in PCSK1 contributes to BMI variation and susceptibility to common obesity in the largest known meta-analysis published to date in genetic epidemiology.

Maternal and newborn health profile in a first nations community in Canada.

OBJECTIVES: We sought to characterize maternal health profiles and birth outcomes among First Nations people living in Southern Ontario. METHODS: We performed a retrospective chart review of all 453 women from the Six Nations Reserve, Ontario, who were pregnant between 2005 and 2010. Maternal health behaviours, past medical history, physical measurements, birth outcomes, and newborn characteristics were abstracted. Key maternal and newborn characteristics were compared with those of a cohort of non-First Nations women recruited from nearby Hamilton, Ontario. RESULTS: The average age of women in the study cohort was 25.1 ± 6.2 (mean ± SD) years, and 75.8% were multiparous. The mean pre-pregnancy BMI was 28.3 ± 6.6 kg/m(2), and the average weight gain in pregnancy was 14.9 ± 8.3 kg. Mean weight gain during pregnancy was inversely associated with pre-pregnancy BMI, and 57.1% of women gained more than the recommended weight. The prevalence of type 2 diabetes or gestational diabetes was 4.7%, hypertension was present before or during pregnancy in 5.6%, and 35% used tobacco during pregnancy. The mean gestational age at delivery was 39.5 ± 1.7 weeks and the mean crude birth weight was 3619 ± 557 g. The main determinants of newborn weight included sex of the newborn, pre-pregnancy BMI, and weight gain during pregnancy. Compared with a contemporary cohort of 622 non-First Nations mothers and newborns, First Nations mothers were, on average, younger (25.1 vs. 32.1 years; P < 0.001), had a higher mean pre-pregnancy BMI (28.3 vs. 26.8 kg/m(2); P < 0.001), and were more likely to use tobacco during pregnancy (35.0% vs. 14.4%; P < 0.001). First Nations newborns had significantly higher mean birth weight (+176 grams) and length (+2.3 cm) than non-First Nations newborns. CONCLUSION: First Nations mothers from the Six Nations Reserve tended to have a high pre-pregnancy BMI, tended to gain more than the recommended weight during pregnancy, and commonly used tobacco during pregnancy. Programs to prevent overweight/obesity and excess weight gain during pregnancy and to minimize smoking are required among women of child-bearing age in this community.

Objectifs : Nous avons cherché à caractériser les profils de santé maternelle et les issues de l'accouchement chez les peuples des Premières Nations vivant dans le sud de l'Ontario. Méthodes : Nous avons mené une analyse rétrospective des dossiers des 453 femmes de la Six Nations Reserve, en Ontario, qui étaient enceintes entre 2005 et 2010. Les comportements de santé maternelle, les antécédents médicaux, les mesures physiques, les issues de l'accouchement et les caractéristiques néonatales ont fait l'objet d'un résumé. Les caractéristiques maternelles et néonatales clés ont été comparées à celles d'une cohorte de femmes n'étant pas issues des Premières Nations qui ont été recrutées tout près, à Hamilton, en Ontario. Résultats : L'âge moyen des femmes de la cohorte d'étude était de 25,1 ± 6,2 ans (moyenne ± σ) et 75,8 % d'entre elles étaient multipares. L'IMC prégrossesse moyen était 28,3 ± 6,6 kg/m2 et le gain pondéral moyen pendant la grossesse était de 14,9 ± 8,3 kg. Le gain pondéral moyen pendant la grossesse était inversement proportionnel à l'IMC prégrossesse et 57,1 % des femmes ont dépassé le gain pondéral recommandé. La prévalence du diabète de type 2 ou du diabète gestationnel était de 4,7 %, une hypertension était présente avant ou pendant la grossesse chez 5,6 % des participantes et 35 % d'entre elles ont consommé du tabac pendant la grossesse. L'âge gestationnel moyen au moment de l'accouchement était de 39,5 ± 1,7 semaines et le poids de naissance brut moyen était de 3 619 ± 557 g. Parmi les principaux déterminants du poids néonatal, on trouvait le sexe du nouveau-né, l'IMC prégrossesse et le gain pondéral pendant la grossesse. Par comparaison avec une cohorte contemporaine de 622 mères et nouveau-nés n'étant pas issus des Premières Nations, les mères issues de ces dernières étaient, en moyenne, plus jeunes (25,1 vs 32,1 ans; P < 0,001), présentaient un IMC prégrossesse moyen plus élevé (28,3 vs 26,8 kg/m2; P < 0,001) et étaient plus susceptibles de consommer du tabac pendant la grossesse (35,0 % vs 14,4 %; P < 0,001). Les nouveau-nés issus des Premières Nations présentaient une longueur (+2,3 cm) et un poids de naissance (+176 grammes) moyens considérablement plus élevés, par comparaison avec les nouveau-nés n'étant pas issus des Premières Nations. Conclusion : Les mères issues des Premières Nations de la Six Nations Reserve ont eu tendance à présenter un IMC prégrossesse élevé et à dépasser le gain pondéral recommandé pendant la grossesse, en plus de consommer couramment du tabac pendant cette période. Des programmes qui visent la prévention de la surcharge pondérale / de l'obésité et du gain pondéral excédentaire pendant la grossesse, et qui visent à minimiser le tabagisme s'avèrent requis pour les femmes en âge de procréer de cette communauté.

Causal relationship between adiponectin and metabolic traits: a Mendelian randomization study in a multiethnic population.

BACKGROUND: Adiponectin, a secretagogue exclusively produced by adipocytes, has been associated with metabolic features, but its role in the development of the metabolic syndrome remains unclear. OBJECTIVES: We investigated the association between serum adiponectin level and metabolic traits, using both observational and genetic epidemiologic approaches in a multiethnic population assembled in Canada. METHODS: Clinical data and serum adiponectin level were collected in 1,157 participants of the SHARE/SHARE-AP studies. Participants were genotyped for the functional rs266729 and rs1260326 SNPs in ADIPOQ and GCKR genes. RESULTS: Adiponectin level was positively associated with HDL cholesterol and negatively associated with body mass index, waist-to-hip ratio, triglycerides, fasting glucose, fasting insulin, systolic and diastolic pressure (all P<0.002). The rs266729 minor G allele was associated with lower adiponectin and higher HOMA-IR (P = 0.004 and 0.003, respectively). The association between rs266729 SNP and HOMA-IR was no longer significant after adjustment for adiponectin concentration (P = 0.10). The rs266729 SNP was associated with HOMA-IR to an extent that exceeded its effect on adiponectin level (0.15 SD 95% C.I. [0.06, 0.24], P<0.001). There was no significant interaction between rs266729 SNP and ethnicity on adiponectin or HOMA-IR. In contrast, the SNP rs1260326 in GCKR was associated with HOMA-IR (P<0.001), but not with adiponectin level (P = 0.67). CONCLUSION: The association of the functional promoter polymorphism rs266729 with lower serum adiponectin and increased insulin resistance in diverse ethnic groups may suggest a causal relationship between adiponectin level and insulin resistance.

Aboriginal birth cohort (ABC): a prospective cohort study of early life determinants of adiposity and associated risk factors among Aboriginal people in Canada.

BACKGROUND: Aboriginal people living in Canada have a high prevalence of obesity, type 2 diabetes, and cardiovascular disease (CVD). To better understand the pre and postnatal influences on the development of adiposity and related cardio-metabolic factors in adult Aboriginal people, we will recruit and follow prospectively Aboriginal pregnant mothers and their children - the Aboriginal Birth Cohort (ABC) study. METHODS/DESIGN: We aim to recruit 300 Aboriginal pregnant mothers and their newborns from the Six Nations Reserve, and follow them prospectively to age 3 years. Key details of environment and health including maternal nutrition, glucose tolerance, physical activity, and weight gain will be collected. At birth, cord blood and placenta samples will be collected, as well as newborn anthropometric measurements. Mothers and offspring will be followed annually with serial measurements of diet and physical activity, growth trajectory, and adiposity. DISCUSSION: There is an urgent need to understand maternal and child factors that underlie the early development of adiposity and type 2 diabetes in Aboriginal people. The information generated from this cohort will assist the Six Nations community in developing interventions to prevent early adiposity in Aboriginal children.

Computationally estimated apolipoproteins B and A1 in predicting cardiovascular risk.

OBJECTIVE: Apolipoproteins B (apoB) and A1 (apoA1) may be better markers of atherosclerosis than serum lipids. We used computational methods to estimate apoB and apoA1 from serum total cholesterol, HDL-cholesterol and triglycerides and tested their clinical value in comparison to measured apoB and apoA1 values. METHODS: ApoB and apoA1 were measured with standard methods and estimated based on neural network regression models in 2166 young adult with data on carotid artery intima-media thickness (cIMT). RESULTS: Correlations between estimated and measured apoB and apoA1 were r = 0.98 and r = 0.95, respectively. ApoB/apoA1-ratio (both measured and estimated) associated with cIMT in multivariable models, and predicted cIMT at all levels of LDL-cholesterol concentration. Strong correlations between the estimated apolipoproteins and those measured from fasting samples were replicated in over 15,000 Caucasian subjects (r = 0.93-0.96 for apoB and r = 0.91-0.92 for apoA1). Correlations with cIMT were replicated in over 2000 individuals. Estimated apoB/apoA1-ratio calculated from non-fasting lipids in over 20,000 individuals in the INTERHEART study was better than any of the cholesterol measures for estimation of the myocardial risk. CONCLUSIONS: Serum cholesterol, HDL-cholesterol and triglycerides can be used to compute clinically useful estimates of apoB and apoA1. Using this methodology, estimates of apolipoproteins could be routinely added to laboratory reports to complement lipoprotein lipids in risk assessment.

Contextual determinants of health behaviours in an aboriginal community in Canada: pilot project.

BACKGROUND: Rapid change in food intake, physical activity, and tobacco use in recent decades have contributed to the soaring rates of obesity, type 2 diabetes and cardiovascular disease (CVD) in Aboriginal populations living in Canada. The nature and influence of contextual factors on Aboriginal health behaviours are not well characterized. METHODS: To describe the contextual determinants of health behaviours associated with cardiovascular risk factors on the Six Nations reserve, including the built environment, access and affordability of healthy foods, and the use of tobacco.In this cross-sectional study, 63 adults from the Six Nations Reserve completed the modified Neighbourhood Environment Walkability Scale (NEWS), questionnaire assessing food access and availability, tobacco pricing and availability, and the Environmental Profile of Community Health (EPOCH) tool. RESULTS: The structured environment of Six Nations Reserve scored low for walkability, street connectivity, aesthetics, safety, and access to walking and cycling facilities. All participants purchased groceries off-reserve, although fresh fruits and vegetables were reported to be available and affordable both on and off-reserve. On average $151/week is spent on groceries per family. Ninety percent of individuals report tobacco use is a problem in the community. Tobacco is easily accessible for children and youth, and only three percent of community members would accept increased tobacco taxation as a strategy to reduce tobacco access. CONCLUSIONS: The built environment, access and affordability of healthy food and tobacco on the Six Nations Reserve are not perceived favourably. Modification of these contextual factors described here may reduce adverse health behaviours in the community.

BRCA2 variants and cardiovascular disease in a multi-ethnic study.

BACKGROUND: Germline mutations of BRCA1/2 are associated with hereditary breast and ovarian cancer. Recent data suggests excess mortality in mutation carriers beyond that conferred by neoplasia, and recent in vivo and in vitro studies suggest a modulatory role for BRCA proteins in endothelial and cardiomyocyte function. We therefore tested the association of BRCA2 variants with clinical cardiovascular disease (CVD). METHODS: Using data from 1,170 individuals included in two multi-ethnic population-based studies (SHARE and SHARE-AP), the association between BRCA2 variants and CVD was evaluated. 15 SNPs in BRCA2 with minor allele frequencies (MAF) > 0.01 had been previously genotyped using the cardiovascular gene-centric 50 k SNP array. 115 individuals (9.8%) reported a CVD event, defined as myocardial infarction (MI), angina, silent MI, stroke, and angioplasty or coronary artery bypass surgery. Analyses were adjusted for age and sex. The SNPs rs11571836 and rs1799943 were subsequently genotyped using the MassARRAY platform in 1,045 cases of incident MI and 1,135 controls from the South Asian subset of an international case-control study of acute MI (INTERHEART), and rs11571836 was imputed in 4,686 cases and 4500 controls from the Pakistan Risk of Myocardial Infarction Study (PROMIS). RESULTS: Two BRCA2 SNPs, rs11571836 and rs1799943, both located in untranslated regions, were associated with lower risk of CVD (OR 0.47 p = 0.01 and OR 0.56 p = 0.03 respectively) in the SHARE studies. Analysis by specific ethnicities demonstrated an association with CVD for both SNPs in Aboriginal People, and for rs11571836 only in South Asians. No association was observed in the European and Chinese subgroups. A non-significant trend towards an association between rs11571836 and lower risk of MI was observed in South Asians from INTERHEART [OR = 0.87 (95% CI: 0.75-1.01) p = 0.068], but was not evident in PROMIS [OR = 0.96 (95% CI: 0.90-1.03) p = 0.230]. Meta-analysis of both case-control studies resulted in a combined OR of 0.94 (95% CI: 0.89-1.004, p = 0.06). CONCLUSIONS: Although there was an association between two SNPs in BRCA2 and CVD in a multi-ethnic population, these results were not replicated in two South Asian case-control studies of incident MI. Future studies exploring the association between BRCA variants and cardiovascular disorders are needed to clarify the role, if any, for BRCA variants in CVD pathogenesis.

Genetic variation in hyaluronan metabolism loci is associated with plasma plasminogen activator inhibitor-1 concentration.

Elevated plasma plasminogen activator inhibitor-1 (PAI-1) concentration is associated with cardiovascular disease risk. PAI-1 is the primary inhibitor of fibrinolysis within both the circulation and the arterial wall, playing roles in both atherosclerosis and thrombosis. To define the heritable component, subjects within the population-based SHARE (Study of Health Assessment and Risk in Ethnic groups) and SHARE-AP (Study of Health Assessment and Risk Evaluation in Aboriginal Peoples) studies, composed of Canadians of South Asian (n = 298), Chinese (n = 284), European (n = 227), and Aboriginal (n = 284) descent, were genotyped using the gene-centric Illumina HumanCVD BeadChip. After imputation, more than 150,000 single nucleotide polymorphisms (SNPs) in more than 2000 loci were tested for association with plasma PAI-1 concentration. Marginal association was observed with the PAI-1 locus itself (SERPINE1; P < .05). However, 5 loci (HABP2, HSPA1A, HYAL1, MBTPS1, TARP) were associated with PAI-1 concentration at a P < 1 × 10(-5) threshold. The protein products of 2 of these loci, hyaluronan binding protein 2 (HABP2) and hyaluronoglucosaminidase 1 (HYAL1), play key roles in hyaluronan metabolism, providing genetic evidence to link these pathways.

Ethnic variation in adiponectin and leptin levels and their association with adiposity and insulin resistance.

OBJECTIVE: To investigate ethnic differences in adiponectin and leptin concentration and to determine whether these adipokines and a high-glycemic index diet account for ethnic variation in insulin resistance. RESEARCH DESIGN AND METHODS: In 1,176 South Asian, Chinese, Aboriginal, and European Canadians, fasting blood samples were drawn, and clinical history and dietary habits including glycemic index/glycemic load were recorded using standardized questionnaires. Insulin resistance was defined using homeostasis model assessment-insulin resistance (HOMA-IR). RESULTS: Adiponectin concentrations were significantly higher in Europeans (adjusted mean 12.94 [95% CI 2.27-13.64]) and Aboriginal people (11.87 [11.19-12.59]) than in South Asians (9.35 [8.82-9.92]) and Chinese (8.52 [8.03-9.03]) (overall P < 0.001). Serum leptin was significantly higher in South Asians (11.82 [10.72-13.04]) and Aboriginal people (11.13 [10.13-12.23]) than in Europeans (9.21 [8.38-10.12]) and Chinese (8.25 [7.48-9.10]). BMI and waist circumference were inversely associated with adiponectin in every group except the South Asians (P < 0.001 for interaction). Adiponectin was inversely and leptin was positively associated with HOMA-IR (P < 0.001). The increase in HOMA-IR for each given decrease in adiponectin was larger among South Asians (P = 0.01) and Aboriginal people (P < 0.001) than among Europeans. A high glycemic index was associated with a larger decrease in adiponectin among South Asians (P = 0.03) and Aboriginal people (P < 0.001) and a larger increase in HOMA-IR among South Asians (P < 0.05) relative to that in other groups. CONCLUSIONS: South Asians have the least favorable adipokine profile and, like the Aboriginal people, display a greater increase in insulin resistance with decreasing levels of adiponectin. Differences in adipokines and responses to glycemic foods parallel the ethnic differences in insulin resistance.