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Background: Maternal nutrition in pregnancy has been linked to offspring health in early and later life, with changes to DNA methylation (DNAm) proposed as a mediating mechanism. Objective: We investigated intervention-associated DNAm changes in children whose mothers participated in 2 randomized controlled trials of micronutrient supplementation before and during pregnancy, as part of the EMPHASIS (Epigenetic Mechanisms linking Preconceptional nutrition and Health Assessed in India and sub-Saharan Africa) study (ISRCTN14266771). Design: We conducted epigenome-wide association studies with blood samples from Indian (n = 698) and Gambian (n = 293) children using the Illumina EPIC array and a targeted study of selected loci not on the array. The Indian micronutrient intervention was food based, whereas the Gambian intervention was a micronutrient tablet. Results: We identified 6 differentially methylated CpGs in Gambians [2.5-5.0% reduction in intervention group, all false discovery rate (FDR)

Size at birth is known to be influenced by various fetal and maternal factors including genetic effects. South Asians have a high burden of low birthweight and cardiometabolic diseases, yet studies of common genetic variations underpinning these phenotypes are lacking. We generated independent, weighted fetal genetic score (fGS) and maternal genetic score (mGS) from 196 birthweight-associated variants identified in Europeans and conducted association analysis with various fetal birth parameters and anthropometric and cardiometabolic traits measured at different follow-up stages (5-6 years’ intervals) from seven Indian and Bangladeshi cohorts of South Asian ancestry. The results from above cohorts were compared with South Asians in UK BioBank and The Exeter Family Study of Childhood Health, a European ancestry cohort. Birthweight increased by 50.7g and 33.6g per standard deviation of fGS (p = 9.1x10-11) and mGS (p = 0.003) respectively in South Asians. A relatively weaker maternal genetic score effect compared to Europeans indicates possible different intrauterine exposures between Europeans and South Asians. Birthweight was strongly associated with body size in both childhood and adolescence (p = 3x10-5 - 1.9x10-51), however, fetal genetic score was associated with body size in childhood only (p < 0.01) and with head circumference, fasting glucose and triglycerides in adults (p < 0.01). The substantially smaller newborn size in South Asians with comparable fetal genetic effect to Europeans on birthweight suggests a significant role of factors related to fetal growth that were not captured by the present genetic scores. These factors may include different environmental exposures, maternal body size, health and nutritional status etc. Persistent influence of genetic loci on size at birth and adult metabolic syndrome in our study supports a common genetic mechanism partly explaining associations between early development and later cardiometabolic health in various populations, despite marked differences in phenotypic and environmental factors in South Asians.

OBJECTIVES: Background: The FUT2 gene is responsible for the secretion of ABO blood type antigens into the body fluids (saliva, mucous, urine, tears, breast milk, sweat, and semen). Those who secrete the antigens into body fluids are call secretors, those who do not are called non-secretors. Hypothesis: GWAS studies have reported FUT gene variants to be associated with circulating vitamin-B12 (Vit-B12) concentrations. Missense mutations in the FUT2 gene result in a non-secretor phenotype. Thus, the secretory status of an individual may affect circulating vitamin-B12 concentrations over and above the genotype. METHODS: Materials and Methods: We included 780 participants (271 children, 282 mothers, and 227 fathers) from Pune Maternal Nutrition Study (PMNS). We measured the secretor status of individuals in saliva by hemagglutination test. A total of eight genetic variants including six SNPs from the FUT2 gene (rs492602, rs681343, rs281377, rs601338, rs1800027, and rs602662) and two SNPs from the FUT6 gene (rs3760776 and rs3760775) from our previous GWAS study were correlated with circulating vitamin-B12 levels. We tested the associations of FUT gene variants with secretor status phenotype and of the secretor phenotype with circulating vit-B12, folate, and ferritin concentrations in addition to H.pylori antibody levels. RESULTS: Results and Discussion: We found 33% of participants were non-secretors compared to 20% reported in Western Caucasian populations. Non-secretors had higher vitamin-B12 concentrations but not of folate and ferritin, vitamin-B12 associations were over and above FUT genotypes. Non-secretors showed a higher response to Vit-B12 supplementation. We found a FUT2 haplotype () to be strongly associated with Vit-B12 concentrations and non-secretor status. Non-secretors had lower H.pylori antibody concentrations. FUT6 genotype and haplotype were associated with Vit-B12 concentrations but not with secretor status and H.pylori antibody levels. CONCLUSIONS: Our data suggest that secretor status may influence Vit-B12 concentrations through susceptibility to H.pylori infection and possibly other gut microbiota. A higher frequency of non-secretors in Indians could offer a selective advantage against Vit-B12 deficiency. FUNDING SOURCES: BBSRC, UK; MRC, UK; WELLCOME TRUST, UK; DBT, India; ICMR India

We recently reported genetic variants associated with birth weight and their effect on future cardiometabolic risk in Europeans. Despite a higher burden of low birth weight and cardiometabolic disorders, such studies are lacking in South Asians. We generated fetal and maternal genetic scores (fGS and mGS) from 196 birth weight-associated variants identified in Europeans and conducted association analysis with various birth measures and serially measured anthropometric and cardiometabolic traits from seven Indian and Bangladeshi cohorts. Although fGS and mGS were comparable to Europeans, birth weight was substantially smaller suggesting strong environmental constraints on fetal growth in South Asians. Birth weight increased by 50.7g and 33.6g per standard deviation fGS (P=9.1×10−11) and mGS (P=0.003) in South Asians. The fGS was further associated with childhood body size and head circumference, fasting glucose, and triglycerides in adults (P

We recently reported genetic variants associated with birth weight and their effect on future cardiometabolic risk in Europeans. Despite a higher burden of low birth weight and cardiometabolic disorders, such studies are lacking in South Asians. We generated fetal and maternal genetic scores (fGS and mGS) from 196 birth weight-associated variants identified in Europeans and conducted association analysis with various birth measures and serially measured anthropometric and cardiometabolic traits from seven Indian and Bangladeshi cohorts. Although fGS and mGS were comparable to Europeans, birth weight was substantially smaller suggesting strong environmental constraints on fetal growth in South Asians. Birth weight increased by 50.7g and 33.6g per standard deviation fGS (P=9.1x10-11) and mGS (P=0.003) in South Asians. The fGS was further associated with childhood body size and head circumference, fasting glucose, and triglycerides in adults (P

The Fourier-transform infrared (FT-IR) spectroscopic approach has been employed to understand the recombinant human G-CSF (rhG-CSF) protein accumulation, secondary structure, and thermal stability in Escherichia coli grown under a temperature shift strategy (37 and 28°C) in various media formulations. The choline + sodium pyruvate (37°C) and sodium pyruvate (28°C) formulations have shown the highest inclusion body (IB) accumulation of 0.41 and 0.46 mg/mL, respectively. Furthermore, insights on the structure of the rhG-CSF within IBs and intact cells have been investigated through secondary structure analysis. Thermal stability experiments were also carried out to explain the pattern of the second derivative structure of rhG-CSF. The studies showed that choline + sodium pyruvate formulation has preserved the protein secondary structure even at 82°C. Overall, the FT-IR spectroscopic technique can also be adopted to accelerate the characterization of other recombinant therapeutic proteins of E. coli origin.

Recombinant proteins are frequently hampered by aggregation during the refolding and purification process. A simple and rapid method for in vitro refolding and purification of recombinant human parathyroid hormone (rhPTH 1–34) expressed in Escherichia coli with protein folding size exclusion chromatography (PF-SEC) was developed in the present work. Discrete effects of potential solution additives such as urea, polypolyethylene glycol, proline, and maltose on the refolding with simultaneous purification of rhPTH were investigated. The results of individual additives indicated that both maltose and proline had remarkable influences on the efficiency of refolding with a recovery yield of 65 and 66 % respectively. Further, the synergistic effect of these additives on refolding was also explored. These results demonstrate that the additive combinations are more effective for inhibiting protein aggregation during purification of rhPTH in terms of recovery yield, purity, and specific activity. The maltose and proline combination system achieved the highest renatured rhPTH having a recovery yield of 78 %, a purity of ≥99 %, and a specific activity of 3.31 × 103 cAMP pM/cell respectively, when compared to the classical dilution method yield (41 %) and purity (97 %). In addition, the role of maltose and proline in a combined system on protein aggregation and refolding has been explained. The molecular docking (in silico) scores of maltose (−10.91) and proline (−9.0) support the in vitro results.

This work, for the first time reports the optimization of rhG-CSF in Escherichia coli using novel media supplements. The rhG-CSF production was optimized using Plackett–Burman (P–B) and Central composite design (CCD) operated in an integrated process. Three novel media supplements (sodium pyruvate, methylglucoside and choline) along with two conventional essential elements (sodium citrate and magnesium sulphate) were screened with a P–B design. An integrated application of CCD represented a statistically significant response (R2 = 98.56 % and P < 0.0001) for the factors under the study, that suggested a quadratic model for the prediction of rhG-CSF. The CCD results reveal the highest rhG-CSF production of 31.88 µg/mL with optimized components comprising 1.75 g/L of sodium pyruvate, 1.5 g/L of methylglucoside, 2.215 g/L of sodium citrate, 1.7 g/L of choline and 0.036 g/L of magnesium sulphate.

Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0 M) and arginine (0.6 M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6 × 108 IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF.