2013 Annual Report
1a.Objectives (from AD-416):
Obj. 1: No longer applies. Obj. 2: Investigate the pathways and nutritional modulation of methyl group production in under- and normal weight pregnant women. Sub-Obj 2A. Determine whole body protein kinetics, methionine kinetics and transmethylation, an index of methyl production and utilization, serine and glycine fluxes, indices of production rates of methyl group precursors, and conversion of serine to glycine, and glycine to CO2, indices of methyl group supply from these precursors to the transmethylation pathway, in under- and normal-weight pregnant women. Sub-Obj 2B. Determine the effect of dietary supplementation with sulfur amino acid-rich whey protein vs. legume/cereal protein on methionine production and transmethylation rate and on serine and glycine fluxes in underweight pregnant women. Sub-Obj 2C. Determine methionine kinetics and transmethylation rates during the first trimester in groups of underweight pregnant women with either normal or low plasma vitamin B12 concentration, after dietary supplementation with Vitamin B12. Sub-Obj 2D. Determine methionine kinetics and transmethylation rates in underweight pregnant women with either normal or low plasma folate concentration after dietary supplementation with folate.Obj. 3: Investigate differences in bowel flora, antioxidant capacity, and mitochondrial integrity between severely malnourished and well-nourished children. Sub-Obj 3A. Measure the populations of bacterial divisions and species in bowel flora populations in children as well as bowel flora diversity with edematous as well as non-edematous SCU and in well-nourished children. Sub-Obj 3B. Measure antioxidant capacity and mitochondrial integrity, as well as characterize the immune system in children with edematous vs. non-edematous SCU. Obj. 4: Initiate a pilot study of genetic susceptibility to ESCM. Obj. 5: Conduct exploratory analyses of the relationship between risk of ESCM and individual genetic variation. Obj. 6: Evaluate population-specific genetic variation. Obj. 7: Characterize the developmental profile of the GI microbiome and transcriptome in healthy, term infants. Obj. 8: Compare the effect of breast versus bottle-feeding on the development of GI microbiome and lactose digestion/absorption. Obj. 9: Profile changes in the GI microbiome in response to the introduction of weaning foods such as dietary starch in the form of cereal. Obj. 10: detect gene-gene/environment interactions.
1b.Approach (from AD-416):
Whole body protein kinetics, methionine production and transmethylation, serine and glycine fluxes, and conversion of serine to glycine and glycine to carbon dioxide will be measured in groups of Indian women with low (=18.5) and normal (>18.5 = 25) BMI between 10 and 12 weeks of pregnancy and again at 26-28 weeks. These measurements plus maternal gestational weight gain, neonate gestational age, birth weight, length, and head circumference will be repeated in groups with BMIs =18.5 after dietary supplement with more energy and protein and in those women with low blood vitamin B12 and folate, after 16 weeks of supplementation with vitamin B12 and folate. Additional studies will evaluate 6- to 24-month twins who are at high risk for malnutrition. Stool samples will be collected in a disposable diaper for multiplex pyrosequencing of bacterial 16S rRNA genes present in gut microbial communities and pyrosequencing of total community DNA (the gut microbiome). A second study will be performed in 50 severely undernourished, 6- to 12-month-old children who are receiving therapeutic food to promote rapid catch-up growth. Antioxidant capacity will be assessed by whole blood glutathione, erythrocyte superoxide dismutase, erythrocyte glutathione peroxidase, and serum oxidized proteins. Mitochondrial integrity will be assessed by lactate and the copy numbers of mitochondrial DNA/RNA in peripheral monocytes, measured by real time duplex nucleic acid sequence-based amplification. To assess how immune response varies with nutritional state, a panel of 27 cytokines will be assessed. Collect data on genetic susceptibility to ESCM and other phenotypes that result from malnutrition using cutting-edge genomics tools and methods in human genetics. Cutting edge technology will be utilized to address this significant global health/nutritional concern. Utilize models to identify genotype associations.
Studies to achieve subobjective 2A, have been completed. We also expanded subobjective 2C to 20 subjects per group and included a third group of 20 subjects that will not be supplemented with either milk or vitamin B12. Currently 52 subjects out of 60 have been studied.
With regard to Objective 3B, to measure antioxidant capacity and mitochondrial integrity in children with marasmus and kwashiorkor with and without HIV infection, we have begun to analyze all specimens and research samples. There has been a significant change in the field of science/medicine that fundamentally reduces the importance of this research. Now most cases of HIV infection in infants in Malawi are prevented using maternal antiretroviral therapy. Thus there are no longer the abundance of children with HIV and no availability of children to be studied as a comparison group.
For Objectives 4-6, we completed a pilot study of genome-wide DNA methylation contrasting children with kwashiorkor and marasmus. The pilot study data showed a dramatic difference in methylation at thousands of loci. Children with kwashiorkor showed deficient DNA methylation, suggesting a global impact of their severe malnutrition. The methylation abnormalities appear to preferentially affect genes with roles in energy metabolism, but many other genes are also affected. The methylation abnormality was corrected in study subjects where the DNA was obtained >10 years after survival from the malnutrition events. These results provide the justification for a larger, more definitive analysis of epigenetic correlates of childhood malnutrition. We will pursue an Integrative Genomic Analysis – a highly novel translational study design – that will allow us to identify the loci that may "drive" the body's response to severe malnutrition. We have recently published a critical paper demonstrating proof of principle of the Integrative Genomics study design. In this paper we use data from clinical trials of seasonal influenza vaccine to show how investigators can use combined genetic and transcriptomics data to analyze a complex physiological response to a medical intervention. We also developed a novel statistical framework for the data analysis, causality analysis, and power modeling. This approach will be used in our planned future studies of severe childhood malnutrition.
For objectives 7-9, we published a manuscript describing the types of gut bacteria found in healthy children 7-12 years of age. Studies by other researchers have suggested that after 2 to 3 years of age, there is little change in the types of gut bacteria. However, our results cast doubt on these earlier findings that were observed in smaller studies. We have published our findings and currently are analyzing additional results (from 40 children) to understand better, how and why the gut bacteria continue to change from infancy into adulthood. We have been unable to carry out such studies in infants (objective.
7)due to a lack of research funds whereas we do have funds to study children. For objective 8, we prepared a manuscript that describes a questionnaire that we developed to have children and their families report the foods they eat (dietary records), how much they eat, and whether any of the foods cause stomach and/or intestinal disturbances. The information that we obtain from these records is currently being compared to the types of gut bacteria in children so we can understand better the relationship between diet, the types of gut bacteria present, and possible indigestion problems.
For objective 9, we have completed collecting samples from 50 children who were studied before and after they took a fiber supplement. We are awaiting final analysis of their samples to measure how fiber in the diet affects not only the types of gut bacteria but also the bacterial genes and what products these genes are responsible for making. For objective 10, our lab is growing with additional personnel who are working on a novel method to assemble genomes from short sequencing reads to large contigs (contiguous read sequences). We are also working on the Bayesian graphical model and are assisting with bioinformatics on collaborative projects with other CNRC researchers. We had a book chapter published recently regarding whole-genome multi-snp analysis. Additionally, a manuscript is under review and two others are in final preparation.
Does gut bacteria change with age? The trillions of bacteria in our gut are critically important in helping us digest food, make important nutrients that are vital to our health, and help our immune systems develop and work properly. Scientists at the Children's Nutrition Research Center in Houston, Texas, learned that, in contrast to what was previously thought, the types of bacteria in our gut continue to evolve throughout childhood. These findings will help researchers better understand the factors that control digestion and utilization of dietary nutrients. Additionally scientists will better understand the role of gut bacteria in the changes in the immune system that occur with age.
Gut bacteria are related to symptoms after eating. Evidence suggests that diet is an important factor in how a person feels after eating, but many unanswered questions remain. Scientists at the Children's Nutrition Research Center in Houston, Texas, found that the types of bacteria in our gut may be related to symptoms of indigestion such as belly pain after eating certain foods. By evaluating the diet of children at different ages as well as their gut bacteria, we will begin to understand more fully the way diet and the gut bacteria are interrelated across different child ages and their impact on indigestion. That will help us to design healthier diets that also are less likely to cause stomach upset.
Increasing dietary fiber changes the population of bacteria in the gut. Individuals are encouraged to increase fiber in our diet because studies suggest that it benefits our health. Studies 50 years ago suggested that the gut bacteria help us digest the fiber we eat. However, by using cutting edge molecular techniques, we can understand how dietary fiber and bacteria interact in ways never before possible. Scientists at the Children's Nutrition Research Center in Houston, Texas, learned that eating fiber causes dramatic shifts in the types of gut bacteria. Using other advanced techniques, we are currently studying how this change in gut bacteria population affects our health. Findings from this work will help researchers develop correlations of specific gut bacteria genes may have on our health.
Role of bacteria in severe malnutrition. Scientists are puzzled as to why some children fed the same diet develop severe malnutrition, while others do not. Researchers at the Children's Nutrition Research Center in Houston, Texas, looked at bacteria from twins where one of the twins developed severe malnutrition while the other remained well nourished were identified by deep sequencing of stool specimens. Very different bacterial populations were seen between the twins, and a distinct pattern of bacteria was seen in stool from children with severe malnutrition. This insight of different bacterial populations despite similiar diets may lead to new treatments for malnutrition that not only provide missing nutrients, but change bacterial populations in the gut.