Development and validation of a lifestyle risk index to screen for metabolic syndrome and its components in two multi-ethnic cohorts.

BACKGROUND: Metabolic syndrome (MetS) is a precursor to cardiovascular diseases and type 2 diabetes. Existing MetS prediction models relied heavily on biochemical measures and those based on non-invasive predictors such as lifestyle behaviours were limited. We aim to (1) develop a weighted lifestyle risk index for MetS and (2) externally validate this index using two Asian-based cohorts in Singapore. METHODS: Using data from the Multi-Ethnic Cohort (MEC) 1 (n = 2873, 41% male), multiple logistic regression was used to identify predictors associated with MetS. A weighted lifestyle risk index was generated using coefficients of the selected predictors in the development cohort (MEC1). Subsequently, the performance of the lifestyle risk index in predicting the occurrence of MetS within 10 years was assessed by discrimination and calibration in an external validation cohort (MEC2) (n = 6070, 43% male). RESULTS: A lifestyle risk index for MetS with nine predictors was developed (age, sex, ethnicity, having a family history of diabetes, BMI, diet, physical activity, smoking status, and screen time). This index demonstrated acceptable discrimination in the development cohort [AUC (95% CI) = 0.74 (0.71, 0.76)] and the validation cohort [AUC (95% CI) = 0.79 (0.77, 0.81)]. CONCLUSION: This lifestyle risk index exhibits potential for risk stratification in population-based screening programmes. Future research could apply a similar methodology to develop disease-specific lifestyle risk indices using nationwide registry-based data.

Risk prediction models for type 2 diabetes using either fasting plasma glucose or HbA1c in Chinese, Malay, and Indians: Results from three multi-ethnic Singapore cohorts.

AIMS: To assess three well-established type 2 diabetes (T2D) risk prediction models based on fasting plasma glucose (FPG) in Chinese, Malays, and Indians, and to develop simplified risk models based on either FPG or HbA1c. METHODS: We used a prospective multiethnic Singapore cohort to evaluate the established models and develop simplified models. 6,217 participants without T2D at baseline were included, with an average follow-up duration of 8.3 years. The simplified risk models were validated in two independent multiethnic Singapore cohorts (N = 12,720). RESULTS: The established risk models had moderate-to-good discrimination (area under the receiver operating characteristic curves, AUCs 0.762 - 0.828) but a lack of fit (P-values < 0.05). Simplified risk models that included fewer predictors (age, BMI, systolic blood pressure, triglycerides, and HbA1c or FPG) showed good discrimination in all cohorts (AUCs ≥ 0.810), and sufficiently captured differences between the ethnic groups. While recalibration improved fit the simplified models in validation cohorts, there remained evidence of miscalibration in Chinese (p ≤ 0.012). CONCLUSIONS: Simplified risk models including HbA1c or FPG had good discrimination in predicting incidence of T2D in three major Asian ethnic groups. Risk functions with HbA1c performed as well as those with FPG.

Molecular modelling and site-directed mutagenesis provide insight into saccharide pyruvylation by the Paenibacillus alvei CsaB enzyme.

Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic ß-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and KM and kcat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue.

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

Replacing dietary carbohydrates and refined grains with different alternatives and risk of cardiovascular diseases in a multi-ethnic Asian population.

BACKGROUND: Cardiovascular diseases (CVDs) are a major cause of deaths worldwide, but prospective data on dietary risk factors for CVD in South and Southeast Asian populations are sparse. OBJECTIVE: We aimed to evaluate the relation between macronutrient and food intakes and incidence of major adverse cardiovascular events (MACEs) in a multi-ethnic Asian population. METHODS: We used data from the Singapore Multi-Ethnic Cohort (n = 12,408), a prospective cohort of ethnic Chinese, Malay, and Indian adults aged 21‒65 y recruited between 2004 and 2010. Dietary intakes were assessed using a validated food-frequency questionnaire, and the incidence of MACEs was ascertained through data linkage with national registries. A Cox proportional hazards model was used to evaluate the associations between dietary intakes and MACE risk. RESULTS: Over a mean (SD) follow-up of 10.1 (2.3) y, 746 incident cases of MACEs were documented. We observed a direct association between carbohydrate intake and MACE risk (highest vs. lowest quartile, HR: 1.35; 95% CI: 1.07, 1.71; P-trend = 0.001). Replacing 5% of energy from carbohydrate with polyunsaturated fat (HR: 0.79; 95% CI: 0.69, 0.91) but not saturated fat (HR: 0.95; 95% CI: 0.84, 1.09) was significantly associated with a lower MACE risk. In terms of food groups, replacing 1 serving/d of refined grains with fruit (HR: 0.93; 95% CI: 0.87, 0.99), vegetables (HR: 0.92; 95% CI: 0.85, 1.00), and dairy (HR: 0.90; 95% CI: 0.82, 0.98) was associated with lower MACE risk. Cholesterol intake was associated with a higher MACE risk in ethnic Indians (highest vs. lowest quartile, HR: 2.40; 95% CI: 1.53, 3.75; P-trend < 0.001) but not in ethnic Malay or Chinese (P-interaction = 0.015). CONCLUSIONS: Moderating carbohydrate intakes by increasing polyunsaturated fat intake and replacing refined grains with fruits, vegetables, and dairy was associated with lower MACE risk in Asian populations.

Assaying Paenibacillus alvei CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release.

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a ß-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium's cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s-1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s-1 were revealed. P. alvei CsaB was inactive on synthetic pNP-ß-d-ManNAc and ß-d-ManNAc-ß-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

Diet Quality and Lower Refined Grain Consumption are Associated With Less Weight Gain in a Multi-Ethnic Asian Adult Population.

BACKGROUND: The prevalence of obesity is increasing in many Asian countries. However, longitudinal data on the impacts of dietary factors on weight gain in Asian populations are sparse. OBJECTIVES: We evaluated the relationship between changes in intakes of nutrients, foods, and beverages and diet quality and long-term changes in body weight. METHODS: We used data (n = 3064) from the Singapore Multi-Ethnic Cohort, a prospective cohort including Chinese, Indian, and Malay residents aged 21-65 years. Dietary intakes were assessed using an FFQ, and body weight and waist circumference were measured during health examinations. Diet quality was evaluated using the Dietary Approaches to Stop Hypertension (DASH) and Alternative Healthy Eating Index (AHEI-2010) scores. Data were collected at baseline (2004-2010) and follow-up (2011-2016), with a mean follow-up of 6.0 years. Linear regression was used to assess the associations between dietary changes and weight change, adjusted for socio-demographic and lifestyle variables. RESULTS: Improvements in dietary quality scores (DASH, -0.34 kg per 5 points; AHEI-2010, -0.35 kg per 10 points) and replacement of carbohydrates with protein (-0.44 kg per 5% of energy) were significantly associated with less weight gain. Increased intakes of white rice (+0.25 kg per serving/d), soft drinks (+0.69 kg), red meat (+0.58 kg), and poultry with skin (+0.74 kg) were directly associated with weight gain. The replacement of 1 serving per day of white rice with whole grains (-0.68 kg), vegetables (-0.33 kg), poultry without skin (-0.79 kg), and eggs (-0.87 kg) was associated with less weight gain. Similar associations were observed between changes in dietary factors and changes in waist circumference. CONCLUSIONS: Among Asian adults, increasing dietary quality, reducing soft drink consumption, and replacing white rice with whole grains, vegetables, and selected high-protein foods was associated with less weight gain.

Making novel staple foods the norm: perspectives from adult consumers with and without diabetes.

Novel staple foods are staple foods that are modified with the purpose of improving their nutritional properties. However, consumers' acceptance towards novel staple foods remains to be evaluated, especially in Asian populations where staple foods like white rice are a major source of energy. The objective of this study was to explore consumers' attitudes and perceptions towards novel staple foods in a multi-ethnic Asian population. We conducted 11 focus group discussions, with 37 healthy participants and 22 participants with diabetes recruited through newspaper, email and poster advertisement and in-person recruitment at a clinic. Thematic analysis using the general inductive approach was performed. We found that participants' conceptual understanding of the modification process affected their acceptance towards novel staple foods. Plant-based modifications were considered natural and acceptable while genetic modification and use of chemicals were unnatural and undesirable. Participants expected novel staple foods to be more expensive and less tasty and this was largely due to their perceptions and experiences with healthy eating. Participants with diabetes or family history of diabetes were generally more willing to compromise taste and cost for healthier staple foods, and this appeared to be driven by concerns about diabetes and its related co-morbidities. The appearance of food was an important influence on participants' initial impression of the food, which appeared to be mediated by taste expectations. Participants' trust of novel staple foods was largely influenced by their trust in food industry, governmental authorities and nutrition science that was mediated through pathways of information and food safety.

Attitudes and beliefs regarding food in a multi-ethnic Asian population and their association with socio-demographic variables and healthy eating intentions.

BACKGROUND: Attitudes and beliefs regarding food are known to influence dietary behaviours and may reflect cultural traditions. Therefore, understanding the food culture in the population is important to inform culturally relevant and acceptable public health interventions. OBJECTIVE: (1) To evaluate attitudes towards pleasure and health with regard to eating and its association with healthy eating intentions. (2) To assess traditional beliefs about 'hot-cold balance' and its influence on food choice. METHODS: We conducted a cross-sectional study using an online research panel comprising of 787 male and female adults of Chinese, Malay and Indian ethnicity recruited from population-based cohorts and public housing estates in Singapore. A self-administered questionnaire was used to assess beliefs and attitudes towards food. To evaluate the association of beliefs and attitudes with socio-demographic determinants and potential consequences for healthy eating intentions, multivariable logistic regression was used with adjustment for age, gender and ethnicity. RESULTS: Beliefs about the importance of considering hot-cold balance for food choice were common among all three Asian ethnic groups and most prominent among the Chinese ethnic group (56.3% Malay, 64.5% Indian and 72.4% Chinese). The majority of participants (73.8%) often consider the effects of diet on health, but also value eating pleasure greatly (84.9%). Older age was associated with a shift in attitudes from valuing eating pleasure towards health. Valuing the importance of food for health was associated with a wide range of healthy eating intentions, whereas highly valuing eating pleasure was associated with a lower intention to reduce salt intake. CONCLUSION: In a cosmopolitan Asian population, both eating for health and eating for pleasure were valued highly and traditional beliefs regarding the hot-cold balance were held by the majority.