Division of Nutrition

Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis.

We have identified a novel gene expressed in murine embryonic stem (ES) cells and in a restricted, tissue-specific pattern during mouse development. The gene is also expressed in blood vessels; hence, we have named it asrij (Sanskrit; asRˆij = blood). The gene encodes a novel conserved, predicted transmembrane protein of 247 amino acids, which is localized to lysosomes and endosomes. During ES cell-derived blood vessel formation in vitro, Asrij expression precedes and partially overlaps with the vascular markers Flk-1 and PECAM. During development, Asrij is expressed predominantly in mouse embryonic blood vessels. The asrij transcript is alternatively spliced, and its expression is regulated in a tissue-specific manner. An asrij splice variant that is enriched in the adult mouse brain encodes a protein of 196 amino acids. Asrij can serve as an early stem cell marker that is down-regulated in nonvascular tissues. Our data indicate that Asrij belongs to a novel class of conserved proteins with a complex developmental profile and suggests multiple functions for the gene. Development Dynamics 227:578–586, 2003. © 2003 Wiley-Liss, Inc.

Background: Low-quality dietary protein intake and vitamin B-12 deficiency could interact to decrease methionine transmethylation and remethylation rates during pregnancy and may affect epigenetic modifications of the fetal genome. Objective: The objective of this randomized, partially open-labeled intervention trial was to examine the effect of supplemental high-quality protein and vitamin B-12 on third-trimester methionine kinetics in pregnant Indian women with a low vitamin B-12 status. Methods: Pregnant women with low serum vitamin B-12 concentrations (<200 pmol/L) were randomly assigned to 1 of 3 groups: the first group received balanced protein-energy supplementation of 500 mL milk/d plus a 10-mg vitamin B-12 tablet/d (M+B-12 group; n = 30), the second group received milk (500 mL/d) plus a placebo tablet (M+P group; n = 30), and the third group received a placebo tablet alone (P group; n = 33). Third-trimester fasting plasma amino acid kinetics were measured by infusing 1-13 C,methyl-2 H 3-methionine, ring-2 H 5-phenylalanine, ring-2 H 4-tyrosine,1-13 C-glycine, and 2,3,3-2 H 3 , 15 N-serine in a subset of participants. Placental mRNA expression of genes involved in methionine pathways, placental long interspersed nuclear elements 1 (LINE-1) methylation, and promoter methylation levels of vascular endothelial growth factor (VEGF) were analyzed.

The importance of the vasculature in tumor progression and metastasis is widely accepted. Studies on the regula-tion of blood vessel formation have potential implications on devising pro-and anti-angiogenic therapies for com-bating ischemic diseases or cancer, respectively. ...

Background: Low-quality dietary protein intake and vitamin B-12 deficiency could interact to decrease methionine transmethylation and remethylation rates during pregnancy and may affect epigenetic modifications of the fetal genome.Objective: The objective of this randomized, partially open-labeled intervention trial was to examine the effect of supplemental high-quality protein and vitamin B-12 on third-trimester methionine kinetics in pregnant Indian women with a low vitamin B-12 status.Methods: Pregnant women with low serum vitamin B-12 concentrations (&lt;200 pmol/L) were randomly assigned to 1 of 3 groups: the first group received balanced protein-energy supplementation of 500 mL milk/d plus a 10-μg vitamin B-12 tablet/d (M+B-12 group; n = 30), the second group received milk (500 mL/d) plus a placebo tablet (M+P group; n = 30), and the third group received a placebo tablet alone (P group; n = 33). Third-trimester fasting plasma amino acid kinetics were measured by infusing 1-(13...

The pathophysiology of diabetic nephropathy (DN) in type 2 diabetes (T2D) patients is minimally understood. We compared untargeted high-resolution accurate mass (HRAM) orbitrap-based plasma metabolomic profiles of 31 T2D-DN (with estimated glomerular filtration rate ≤80 mL/min/1.73 m2), 29 T2D and 30 normal glucose tolerance (NGT) Indian men. Of the 939 plasma metabolites that were differentially abundant amongst the NGT, T2D and T2D-DN (ANOVA, False Discovery Rate – FDR adjusted p-value 

Venous plasma metabolomics is a potent and highly sensitive tool for identifying and measuring metabolites of interest in human health and disease. Accurate and reproducible insights from such metabolomic studies require extreme care in removing preanalytical confounders; one of these is the duration of tourniquet application when drawing the venous blood sample. Using an untargeted plasma metabolomics approach, we evaluated the effect of varying durations of tourniquet application on the variability in plasma metabolite concentrations in five healthy female subjects. Tourniquet application introduced appreciable variation in the metabolite abundances: 73% of the identified metabolites had higher temporal variation compared to interindividual variation [Intra-Class Correlation (ICC) &gt; 0.50]. As such, we recommend tourniquet application for minimal duration and to wait for 5 min with the needle in situ after removing the tourniquet, to reduce hemostasis-induced variability and fal...

Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis. Conflict of interest: The authors have declared that no conflict of interest exists.

Antivascular agents have become a standard of treatment for many malignancies. However, most of them target the VEGF pathway and lead to refractoriness. To improve the diversity of options for antivascular therapy, we applied a high-throughput screen for small molecules targeting cell adhesion. We then assayed the resulting antiadhesion hits in a transgenic zebrafish line with endothelial expression of EGFP ( Tg(fli1:EGFP) y1 ) to identify nontoxic molecules with antivascular activity selective to neovasculature. This screen identified dehydro-α-lapachone (DAL), a natural plant product. We found that DAL inhibits vessel regeneration, interferes with vessel anastomosis, and limits plexus formation in zebrafish. Furthermore, DAL induces vascular pruning and growth delay in orthotopic mammary tumors in mice. We show that DAL targets cell adhesion by promoting ubiquitination of the Rho-GTPase Rac1, which is frequently up-regulated in many different cancers.

Objectives Leptin (LEP) is a vital placental hormone that is known to affect different aspects of placental function and fetal development. The present study aimed to determine the association of placental LEP transcript abundance with maternal, placental, and newborn parameters. Subjects/methods In this retrospective case-control study, placental samples (n = 105) were collected from small (SGA) and appropriate (AGA) for gestational age full-term singleton pregnancies (n = 44 SGA and n = 61 AGA). Placental transcript abundance of LEP was assessed by real-time quantitative PCR after normalization to a reference gene panel. LEP methylation was measured using a quantitative MethyLight assay in a subset of samples (n = 54). Results Placental LEP transcript abundance was negatively and significantly associated with placental weight (β = −3.883, P = 0.015). This association continued to be significant in the SGA group (β = −10.332, P = 0.001), both in female (β = −15.423, P = 0.021) and male births (β = −10.029, P = 0.007). LEP transcript abundance was not associated with LEP methylation levels (Spearman's ρ = 0.148, P = 0.287). Conclusion We conclude that placental upregulation of LEP is an integral and fetal sex-independent component of placental growth restriction, which can be potentially targeted through maternal dietary modifications to improve fetoplacental growth.

Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain reaction (qRT-PCR) based quantitative gene expression assays. Selection of stably expressed reference genes is therefore crucial for ensuring reproducibility of such assays. However, there is a complete dearth of data on stability of commonly used reference genes in Peripheral Blood Mononuclear Cells (PBMCs) from Type 2 diabetes mellitus (T2DM) patients. We have evaluated the gene expression stability of 4 widely used reference genes (Beta-actin, ACTB; Peptidylprolyl Isomerase B, PPIB; Tyrosine 3 Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, YWHAZ; and Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH); in PBMCs from 39 T2DM patients and 47 normoglycemic (NGT) subjects. ACTB and YWHAZ were found to be the most stable genes in PBMCs from T2DM patients and therefore, can be recommended as suitable reference genes...

Aims: Placental physiology and morphology is critically regulated by DNA methylation. As such, placental global DNA methylation and transcript abundance of placental DNA methyltransferases (DNMT1 and DNMT3A) may relate to placental and fetal growth in human pregnancies. We aimed to test correlations of human fetoplacental parameters and birth weight with the placental expression of DNA methyltransferases (DNMT1 and DNMT3A) and placental global methylation. Subjects and methods: Placentae (n ¼ 109) were collected from small-(SGA) and appropriate-(AGA) for gestational age full-term singleton pregnancies (n ¼ 56 SGA and 53 AGA). Placentae and neonates were weighed at birth. Realtime quantitative PCR was performed to assess placental transcript abundance of DNMT1, DNMT3A and DNTMT3B normalized to a panel of reference genes. LINE-1 methylation was measured using a quantitative MethyLight assay in a subset of samples (n ¼ 68). Associations of placental transcript abundances of DNMT1, DNMT3A and DNMT3B and of LINE-1 methylation levels with maternal, placental and neonatal parameters were tested. Results: Placental DNMT1 transcript abundance associated positively with placental weight (b ¼ 10.21, P ¼ 0.013). This association was specific to the AGA births (b ¼ 12.77, P ¼ 0.022) and was absent in the SGA births. Association of DNMT1 expression with placental weight and birth weight within the AGA births was specific to the female gender (Birth weight: b ¼ 83.61, P ¼ 0.043; Placental weight: b ¼ 23.92, P ¼ 0.025). Placental DNMT1 transcript levels were not different according to SGA status or gender. Placental DNMT3A transcript levels and LINE-1 methylation levels did not show any associations with maternal, placental and neonatal parameters. Conclusions: Placental DNMT1 expression was found to be associated positively with placental weight and birth weight, specifically in the female AGA births. Thus, we hypothesize that placental DNMT1 participates in fetoplacental growth in a fetal gender-specific manner.

We have identified a novel gene expressed in murine embryonic stem (ES) cells and in a restricted, tissue-specific pattern during mouse development. The gene is also expressed in blood vessels; hence, we have named it asrij (Sanskrit; asRˆij ‫؍‬ blood). The gene encodes a novel conserved, predicted transmembrane protein of 247 amino acids, which is localized to lysosomes and endosomes. During ES cell-derived blood vessel formation in vitro, Asrij expression precedes and partially overlaps with the vascular markers Flk-1 and PECAM. During development, Asrij is expressed predominantly in mouse embryonic blood vessels. The asrij transcript is alternatively spliced, and its expression is regulated in a tissue-specific manner. An asrij splice variant that is enriched in the adult mouse brain encodes a protein of 196 amino acids. Asrij can serve as an early stem cell marker that is down-regulated in nonvascular tissues. Our data indicate that Asrij belongs to a novel class of conserved proteins with a complex developmental profile and suggests multiple functions for the gene.

The discovery of antibiotics and other antimicrobial agents in the 1930s is arguably the most significant therapeutic advance in medical history. Penicillin and the sulfa drugs touched off the search for and discovery of countless derivative compounds and several new antibiotic classes. However, the pace of discovery has slowed down, and there is growing appreciation that much of the low-lying fruit accessible to traditional methods of antimicrobial discovery has been harvested. Combating emerging drug-resistant strains of infectious agents may require the adoption of fresh approaches to drug target validation, small-molecule discovery and safety assessment. The recent development of several infectious disease models in zebrafish raises the possibility of a new paradigm in antimicrobial discovery.

Background: A striking number of low birth weight (LBW) Indian babies are born annually. Previous studies have confirmed the positive association between milk intake and birth weight. However, the relations between protein and vitamin B 12 from milk and birth weight have not been systematically explored. Aims: We examined the relations between birth weight and maternal intake of milk, protein from milk and vitamin B 12 from milk. Methods: This prospective, observational cohort study was conducted in an urban South Indian hospital. The dietary intakes of milk and milk products were assessed using validated food frequency questionnaire and at delivery birth outcomes were measured. The relations between milk products, milk protein, and vitamin B 12 from milk with birth weight and gestational weight gain were assessed in 2036 births with first trimester dietary and delivery data. Results: Median consumption of milk products in the first trimester was 310 g$day À1 and average birth weight was 2876 g. Birth weight was positively associated with intake of milk products and of % protein from milk products (%milk protein) in the first trimester [b ¼ 86.8, 95% confidence interval (CI): 29.1, 144.6; b ¼ 63.1, 95% CI: 10.8, 115.5; P < 0.001 for both]. Intake of milk products and of %milk protein in the third trimester was positively associated with gestational weight gain (GWG) between the second and third trimester (One-way ANOVA, P < 0.001 and ¼ 0.001, respectively). Neither birth weight nor GWG were associated with %vitamin B 12 from milk products. Conclusions: These findings indicate that intake of milk products in the first trimester and especially, protein from milk products is positively associated with birth weight in this South Asian Indian population.

Background: Maternal macronutrient intake is likely to play a pivotal role in fetoplacental growth. Male fetuses grow faster and their growth is more responsive to maternal size. Objective: We assessed the role of fetal sex in modifying the effect of maternal macronutrient intake on the risk of small-for-gestationalage (SGA) birth. Design: This was a prospective, observational cohort study of 2035 births from an urban South Asian Indian population. Maternal intakes of total energy and macronutrients were recorded by validated food-frequency questionnaires. The interaction of trimester 1 macronutrient intake with fetal sex was tested on the outcome of SGA births. Results: The prevalence of SGA was 28%. Trimester 1 macronutrient composition was high in carbohydrate and low in fat (means ± SDs-carbohydrate: 64.6% ± 5.1%; protein: 11.5% ± 1.1%; and fat: 23.9% ± 4.4% of energy). Higher carbohydrate and lower fat consumption were each associated with an increased risk of SGA [adjusted OR (AOR) per 5% of energy (95% CI): carbohydrate: 1.15 (1.01, 1.32); fat: 0.83 (0.71, 0.97)] specifically among male births (males: n = 1047; females: n = 988). Dietary intake of >70% of energy from carbohydrate was also associated with increased risk (AOR: 1.67; 95% CI: 1.00, 2.78), whereas >25% of energy from fat intake was associated with decreased risk (AOR: 0.61; 95% CI: 0.41, 0.90) of SGA in male births. Conclusions: Higher carbohydrate and lower fat intakes early in pregnancy were associated with increased risk of male SGA births. Therefore, we speculate that fetal sex acts as a modifier of the role of maternal periconceptional nutrition in optimal fetoplacental growth.

Gut microbiota has been implicated as a modifier of childhood growth. Here, 16S rRNA sequencing-based fecal microbiota profiles of 18-24 month old Indian children were evaluated (n = 41), in relation to their anthropometric parameters, intestinal permeability, body composition and total energy expenditure. Pathway analyses were conducted to assess microbial functions related to stunting, underweight and wasting. The fecal microbiota was enriched in Prevotella 9, Bifidobacterium and Escherichia-Shigella. Weight, weight-forage Z-scores (WAZ) and weight-for-length Z-scores (WLZ), along with age, acted as covariates of microbiota variation specifically in boys (n = 23). Bifidobacterium longum subsp longum abundance was positively associated with WAZ while Bifidobacterium bifidum and Bifidobacterium breve abundances were negatively associated with age. The lipopolysaccharide biosynthesis pathway was upregulated in stunted (n = 16) and wasted (n = 8) children. Findings from this study indicate that child sex may be a critical modifier of the role of gut microbiota on childhood growth.

Venous plasma metabolomics is a potent and highly sensitive tool for identifying and measuring metabolites of interest in human health and disease. Accurate and reproducible insights from such metabolomic studies require extreme care in removing preanalytical confounders; one of these is the duration of tourniquet application when drawing the venous blood sample. Using an untargeted plasma metabolomics approach, we evaluated the effect of varying durations of tourniquet application on the variability in plasma metabolite concentrations in five healthy female subjects. Tourniquet application introduced appreciable variation in the metabolite abundances: 73% of the identified metabolites had higher temporal variation compared to interindividual variation [Intra-Class Correlation (ICC) > 0.50]. As such, we recommend tourniquet application for minimal duration and to wait for 5 min with the needle in situ after removing the tourniquet, to reduce hemostasis-induced variability and false flags in interpretation. Metabolomics, or the identification and measurement of metabolites in biological samples, has become an invaluable means for generating new knowledge in terms of markers for prognosis and diagnosis of human pathophysiology as well as of new leads in understanding causal pathways 1-4. Of the various possible sources of samples from human subjects for metabolomic assessments, components of blood (plasma and serum), remain the most relevant and therefore, most studied type of sample 5. However, unlike metabolomics performed with samples generated from in vitro or animal model experiments that are inherently less heterogenous, using plasma or serum metabolomics as a tool in human translational research is subject to the inherent interindividual variability between subjects due to genetic and environmental factors and their interactions. An additional source of variation is pre-analytic factors. Controlling for such variability introducing pre-analytic factors would be key to improving reproducibility of high impact human metabolomic findings such as the use of sarcosine as a biomarker for progression of prostate cancer 6,7. Here, fasting status, type of collection tube, presence of haemolysis, time taken and temperature of sample during processing, temperature and duration of storage of processed samples, and freeze-thaw cycles have been assessed 8-12. Unknown and unaccounted for variation arising due to pre-analytic factors could lead to overestimation of interindividual variation or even intraindividual variation, if the samples have been collected at multiple time points in a longitudinal fashion. However, Agueusop et al. 13 reported remarkable stability of the human serum metabolome over a 4 weeks' time period, when the samples were collected on 3 different days under stringently controlled conditions. An additional, unstudied and potentially significant variation might arise from local tissue hemostasis during venous blood collection due to variable tourniquet application times and associated haemostasis. We could not identify any study that has investigated this effect on metabolomic readouts in the plasma. Therefore, we explored the effect of different durations of tourniquet-induced local hemostasis on fasted venous plasma metabolomic profiles of South Asian Indian women, for a deeper understanding of tourniquet-time related precautions required for reproducible untargeted human metabolomic studies. Our hypothesis was that the number of metabolites differing in abundance from the control, unrestricted blood flow condition would increase in step with the increased time of tourniquet-induced local hemostasis.