Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine the effects of diet and age on gut microbial composition, ascertain the metagenomic profile of the gut microbes, and relate these to gut barrier function and inflammation in children 7 to 18 years of age. Objective 2: No longer applies. Objective 3: Using samples collected from rural African children aged 1- 2 years at risk for environmental enteropathy (EE), identify a panel of human mRNAs in fecal samples indicative of EE, evaluate this panel as a biomarker for EE in other populations, test whether micronutrient and/or fish oil supplements can reduce EE, explore the microbiome of children with and without EE, and correlate the mRNA panel markers with child growth parameters. Subobjective 3A: Using samples collected from rural African children at risk for EE, identify a panel of human mRNAs in fecal samples indicative of EE, correlate the mRNA panel markers with the L:M test and child growth parameters. Subobjective 3B: Determine whether micronutrient and/or fish oil supplements can reduce EE in rural African children aged 1-3 years. Subobjective 3C: Explore the microbiome of the gut in children with EE, looking for characteristic patterns of bacterial populations, or the presence of novel viruses, which may provide clues regarding the stimuli of the chronic gut inflammation. Approach (from AD-416): Gut microbes account for 90% of the cells and 99% of the genetic material in humans. The gut microbiome (bacteria and their genes) has coevolved with humans to serve a critical symbiotic role in maintaining health yet, the presence of an abnormal microbiome composition may contribute to chronic health disorders like allergic, circulatory, and inflammatory bowel diseases, functional abdominal pain disorders, and obesity. Little is known about the development of the child gut microbiome, the influence of diet, and how variations in the gut microbiome affect health. The long-term goal of this research is to understand the interaction between the gut microbiome and the health of children. The objectives center around gaining a better understanding of the gut microbiome, how it is affected by diet and age, its associations with gut health in the developed and developing worlds, and obesity risk. Through our studies, we anticipate an increased understanding of how diet and age influence gut microbial population composition, and in turn, alter physiological functioning and promote health. Additionally, insight will be gained into potential mechanisms by examining gut microbial genes and potential gene products (microbiome) and exploring relationships with physiologic outcomes (gut barrier function and inflammatory state) that may promote or impair health. This information will lead to improved understanding how foods and their components support health and reduce disease risk and can be used in formulating nutrition policies and government programs. The results will be of benefit to children and adolescents and can be used as a guide for similar investigations in adults. For Objective 1, children ages 7-12 years were recruited. The children kept a 2-week diary in which they recorded when/if they had abdominal pain, when they stooled, and the character of their stool (constipation/ normal/diarrhea). They also kept a 3-day diary of what they ate. During the time they kept these diaries, they collected stool samples and underwent a test to evaluate the integrity of the intestinal lining. The stools were analyzed to measure the types of bacteria present and the level of inflammation in the intestine. The integrity of the intestinal lining was also measured in the siblings and parents of a similar group of children (those with/without abdominal pain). This year we completed analysis of the types of bacteria present in the stools and analysis of the integrity of the intestinal lining. We currently are using very sophisticated statistical analyses to combine all the data (abdominal pain, stooling, food intake, bacteria in the stool, integrity of the intestinal lining, and the level of inflammation in the intestine). By combining these data, we will learn how the diet and the bacteria in the intestine can influence whether a child has abdominal pain, an impaired intestinal lining, and/or inflammation in the intestine. For Objective 3A, stool samples were collected from a group of about 400 children from Malawi aged 2-5 years. These samples were carefully analyzed for a certain class of genetic molecules originating from the human tissue that lines the gastrointestinal tract, rather than the bacteria or food in the gastrointestinal tract. The analyses were done in such a way that any and all genetic material was detected, rather than just a few of these molecules. Eighteen key molecules were identified and specific tests for just these were created. The specific testing was then used to create a test that rates gut health in rural African children. This test can now be applied to other populations or to see if efforts to improve gut health are effective. The information was made public so other researchers can use this test. For Objective 3B, a group of rural African (Malawi) children aged 1-3 years entered into a clinical trial to determine whether giving them additional vitamins and minerals or additional vitamins, minerals and fish oil improved their gut health and linear growth. The daily supplement was added to the standard porridge these children consume and given for 6 months. Children that received the vitamins and minerals with or without fish did improve their gut health, but their linear growth was similar. This information is now used to justify the distribution of vitamins and minerals to rural African children as a means to improve gut health. For Objective 3C, rural African children aged 6-12 months had their stool tested using a novel method developed in a previous objective, for the detection of specific pieces of genetic material originating from the human tissue in the intestinal tract. The intent was to measure their gut health. Gut health is important because it is one of the major determinants of child growth and development. The test gave plausible results, but the specific pieces of genetic material were not optimal to cleanly categorize the gut health in this age group. Accomplishments 01 Impaired integrity of the intestinal lining and inflammation in the intestine are related to diet. Between 10-15% of school age children and adults worldwide suffer from intermittent chronic abdominal pain. This pain is sometimes associated with constipation and/or diarrhea and the economic cost of this condition in the United States (for adults) has been calculated to be $30 billion per year. Scientists in Houston, Texas conducted research that showed that impaired integrity of the intestinal lining and/or inflammation in the intestine are associated with abdominal pain conditions and that carbohydrate ingested is linked to both. This relationship between dietary carbohydrate and impaired integrity of the intestinal lining and inflammation in the intestine is not associated with obesity. Studies by other researchers have shown that, in addition to abdominal pain, impaired integrity of the intestinal lining and/or inflammation in the intestine have been associated with a number of health problems such as liver disease, depression, and pancreatic disease; thus, our results have relevance to other disorders associated with impaired integrity of the intestinal lining. These results add to the understanding of how diet can affect the intestinal lining and contribute to poor health.
Impacts
(N/A)
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine the effects of diet and age on gut microbial composition, ascertain the metagenomic profile of the gut microbes, and relate these to gut barrier function and inflammation in children 7 to 18 years of age. Objective 2: Develop a new de novo assembly method to assemble short sequence reads into long contiguous reads (contigs). Apply this method to assemble gut microbiome sequence reads. Develop a statistical method to cluster contigs and quantify abundance of these clusters. Perform genetic association testing for haplotype-microbiome interactions that affect the risk of childhood obesity. Objective 3: Using samples collected from rural African children aged 1- 2 years at risk for environmental enteropathy (EE), identify a panel of human mRNAs in fecal samples indicative of EE, evaluate this panel as a biomarker for EE in other populations, test whether micronutrient and/or fish oil supplements can reduce EE, explore the microbiome of children with and without EE, and correlate the mRNA panel markers with child growth parameters. Subobjective 3A: Using samples collected from rural African children at risk for EE, identify a panel of human mRNAs in fecal samples indicative of EE, correlate the mRNA panel markers with the L:M test and child growth parameters. Subobjective 3B: Determine whether micronutrient and/or fish oil supplements can reduce EE in rural African children aged 1-3 years. Subobjective 3C: Explore the microbiome of the gut in children with EE, looking for characteristic patterns of bacterial populations, or the presence of novel viruses, which may provide clues regarding the stimuli of the chronic gut inflammation. Approach (from AD-416): Gut microbes account for 90% of the cells and 99% of the genetic material in humans. The gut microbiome (bacteria and their genes) has coevolved with humans to serve a critical symbiotic role in maintaining health yet, the presence of an abnormal microbiome composition may contribute to chronic health disorders like allergic, circulatory, and inflammatory bowel diseases, functional abdominal pain disorders, and obesity. Little is known about the development of the child gut microbiome, the influence of diet, and how variations in the gut microbiome affect health. The long-term goal of this research is to understand the interaction between the gut microbiome and the health of children. The objectives center around gaining a better understanding of the gut microbiome, how it is affected by diet and age, its associations with gut health in the developed and developing worlds, and obesity risk. Through our studies, we anticipate an increased understanding of how diet and age influence gut microbial population composition, and in turn, alter physiological functioning and promote health. Additionally, insight will be gained into potential mechanisms by examining gut microbial genes and potential gene products (microbiome) and exploring relationships with physiologic outcomes (gut barrier function and inflammatory state) that may promote or impair health. This information will lead to improved understanding how foods and their components support health and reduce disease risk and can be used in formulating nutrition policies and government programs. The results will be of benefit to children and adolescents and can be used as a guide for similar investigations in adults. For Objective 1, fecal bacterial samples have been sequenced, gut barrier function analyses have been carried out, as have the fecal measures of gut neuroimmune inflammation. A preliminary evaluation of the data suggests that children who complain of abdominal pain with eating have increased gut inflammation compared with children who do not have meal related pain. An additional finding is that the level of neuroimmune activation differs between boys and girls; a finding not described previously. A manuscript with these findings was submitted for consideration for publication. Similarly, gut barrier function is impaired in children who have diet related pain but a preliminary evaluation of the data does not show a correlation with the degree of gut neuroimmune activation. We currently are in the process of analyzing the dietary records so we can examine the role of diet composition in these findings. Once the fecal bacterial samples have been analyzed, we will integrate the data into the results from the dietary records, gut barrier and neuroimmune measures. For Objective 2 we slightly modified the existing sub-objective of assembling microbiome to developing new statistical methods to quantify microbiome diversity. We applied the method to analyze existing microbiome dataset in Human Microbiome Project (HMP), and our method can recapitulate relationship reported by HMP, but our method does not rely on mapping reads to reference genomes. Instead we used matrices to represent nucleotides and turn each reads to a number. The past year was dedicated to the study of optimal representation and whether such an optimal representation exists. This project works very well and evolves to become a thesis project of a PhD student in my lab. For Objective 3A the analyses method was fully developed and validated, this work has been published in a peer-reviewed journal, so that other scientists can reliably use it. The process of selecting a smaller panel of RNA probes to measure gut health and predict linear growth is complete and also published. This enabled us to categorize future gut health and growth correctly 80-85% of the time. This entire objective is complete. We have moved on to a similar exercise for infants living in rural Africa. For Objective 3B, the clinical trial has been completed, and it showed that in vulnerable populations of young children in rural Africa, dietary supplementation with multivitamins and fish oil did not reduce gut inflammation. Laboratory assessment of samples is complete and mathematical models informing of us as to the key determinants of child growth are still being developed. For Objective 3C, we continue our analysis of 147 stools from 49 children collected at three different time points over a 6-month period. These children had dual sugar absorption test at each time point to determine there Environmental Enteric Dysfunction (EED) status. As seen in other studies, the bacteriophage communities in these are dominated by families Microviridae and Caudovirales. For the majority of the samples, Caudovirales is the most abundant bacteriophage family. As Caudovirales is the dominant family, our subsequent analyses focused within this family. We first examined the effect of age on Caudovirales alpha diversity (Shannon) and richness. Caudovirales diversity and richness increased as children aged. Next we explored if EED status effects Caudovirales richness and/or diversity while also taking into account age. Interestingly, Caudovirales Shannon diversity decreased with age in children with EED, whereas in children without EED or with moderate EED Caudovirales diversity increased. We are now examining this finding at lower taxonomic levels and looking for relationships with the bacterial communities in these samples. Accomplishments 01 New test for children's gut health. Rural African children often are stunted because of gut inflammation. Children's Nutrition Research Center researchers based in rural Malawi, Africa worked closely with 300 young children to compare their gut health measured by two different tests. Traditionally, gut health is measured by a sugar absorption test and this test was compared to a panel of small molecules that indicate cellular function in the gut. The comparison found that either test can be used to identify poor gut health. With this test we can more easily identify children who need interventions to improve their gut health in a less expensive way. 02 Immune system activation may identify children who may respond to dietary treatment for abdominal pain. Activation of the immune system and gut nerves account for pain related to certain meals. Up to 15% of children worldwide experience chronic abdominal pain and in a large percent of these cases the pain is related to diet. Researchers at the Children's Nutrition Research Center in Houston, Texas have discovered that in many of these children there is activation of the immune system and nerves within the gut that appear to be responsible for the pain. Using tests to detect this activation, we can begin to identify children for whom specific changes in diet will relieve their pain. These findings are important as it provides a nutritional change that may relieve chronic abdominal pain in this young population worldwide.
Impacts
(N/A)
Publications
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine the effects of diet and age on gut microbial composition, ascertain the metagenomic profile of the gut microbes, and relate these to gut barrier function and inflammation in children 7 to 18 years of age. Objective 2: Develop a new de novo assembly method to assemble short sequence reads into long contiguous reads (contigs). Apply this method to assemble gut microbiome sequence reads. Develop a statistical method to cluster contigs and quantify abundance of these clusters. Perform genetic association testing for haplotype-microbiome interactions that affect the risk of childhood obesity. Objective 3: Using samples collected from rural African children aged 1- 2 years at risk for environmental enteropathy (EE), identify a panel of human mRNAs in fecal samples indicative of EE, evaluate this panel as a biomarker for EE in other populations, test whether micronutrient and/or fish oil supplements can reduce EE, explore the microbiome of children with and without EE, and correlate the mRNA panel markers with child growth parameters. Subobjective 3A: Using samples collected from rural African children at risk for EE, identify a panel of human mRNAs in fecal samples indicative of EE, correlate the mRNA panel markers with the L:M test and child growth parameters. Subobjective 3B: Determine whether micronutrient and/or fish oil supplements can reduce EE in rural African children aged 1-3 years. Subobjective 3C: Explore the microbiome of the gut in children with EE, looking for characteristic patterns of bacterial populations, or the presence of novel viruses, which may provide clues regarding the stimuli of the chronic gut inflammation. Approach (from AD-416): Gut microbes account for 90% of the cells and 99% of the genetic material in humans. The gut microbiome (bacteria and their genes) has coevolved with humans to serve a critical symbiotic role in maintaining health yet, the presence of an abnormal microbiome composition may contribute to chronic health disorders like allergic, circulatory, and inflammatory bowel diseases, functional abdominal pain disorders, and obesity. Little is known about the development of the child gut microbiome, the influence of diet, and how variations in the gut microbiome affect health. The long-term goal of this research is to understand the interaction between the gut microbiome and the health of children. The objectives center around gaining a better understanding of the gut microbiome, how it is affected by diet and age, its associations with gut health in the developed and developing worlds, and obesity risk. Through our studies, we anticipate an increased understanding of how diet and age influence gut microbial population composition, and in turn, alter physiological functioning and promote health. Additionally, insight will be gained into potential mechanisms by examining gut microbial genes and potential gene products (microbiome) and exploring relationships with physiologic outcomes (gut barrier function and inflammatory state) that may promote or impair health. This information will lead to improved understanding how foods and their components support health and reduce disease risk and can be used in formulating nutrition policies and government programs. The results will be of benefit to children and adolescents and can be used as a guide for similar investigations in adults. For Objective 1, we finished recruiting children for our study. We recruited a total of 509 children between 7 to 18 years of age. We used a dietary questionnaire to obtain information from the children as to what they were eating at the time they were studied because diet can affect the types of bacteria present in the gut. We have collected stool samples to analyze for the types of bacteria present in the guts of the children. We also collected samples of urine that will allow us to measure how well the gut barrier functions to keep out potentially harmful substances from the body. Our next step is to determine the types of bacteria present. We also are developing new techniques to help us measure gut barrier function more accurately in the future. In Objective 2, we developed a novel method to quantify short sequencing reads without the need of reference genomes. This is desirable in that in metagenomics reference genomes are either incomplete or unreliable. We have a software package that takes reads (or contigs) as input and output genetic distance between two samples. The inferred pairwise distance can recapitulate the sample relatedness using data obtained from the human microbiome project (HMP). Moreover, the initial analysis was based on reference genomes and half of the sequence reads were discarded. We applied our method on the discarded half of the reads and, quite interestingly, we recovered the pairwise sample relatedness using the discarded reads, although the results are not identical to those inferred from the undiscarded half reads. We also applied our method to a RNA sequencing datasets, and results showed that our method can reliably quantify transcriptome differences between samples. With regard to Objective 3 much was accomplished this fiscal year. For Objective 3A the analyses method was fully developed and validated, this work has been published in a peer- reviewed journal, so that other scientists can reliably use it. The process of selecting a smaller panel of RNA probes to measure gut health and predict linear growth is complete and also published. This enabled us to categorize future gut health and growth correctly 80-85% of the time. For Objective 3B, the clinical trial has been completed, and it showed that in vulnerable populations of young children in rural Africa, dietary supplementation with multivitamins and fish oil did not reduce gut inflammation. Laboratory assessment of samples is almost complete and mathematical models informing of us as to the key determinants of child growth are being developed. For Objective 3C, in a stool sample from a child with poor gut health, we discovered a new virus which we named Statovirus C1 (Stool associated Tombus-like viruses). We have now published a manuscript detailing this new viral taxon. Additionally, we have now expanded our analysis to stools from 53 children collected at three different time points over a 6-month period. We have identified the presence of many DNA and RNA eukaryotic viruses as well as bacteriophage in these samples. Analysis is still underway, but we have found that children with poor gut health have statistically significant (p=0.0009) more Anellovirus reads in their stool than children with good gut health. Anelloviruses are commonly found in human samples and they currently have no known disease association. However, Anellovirus load has previously been associated with host immune status. Accomplishments 01 Dietary fiber reduces belly pain risk in children. Belly pain is a common complaint in children and up to 20% of school age children have pain frequently. Researchers in Houston, Texas completed a study in children (7-18 years of age) that showed that adding fiber to the diet can reduce the number of episodes of belly pain. By providing extra fiber to their diet, the children had less belly pain and the fiber did not disrupt the types of bacteria found in the intestine. These findings are important and provide potential solutions to relieving pain through ones diet. 02 Bacteria in the gut influences stooling pattern. Constipation is a common problem in children. Methane is produced by bacteria in the gut and excreted in breath. Researchers in Houston, Texas measured the amount of methane gas excreted in the breath of children ages 7-17 years. They discovered that how fast food moved along the gastrointestinal tract was related to the amount of methane produced. These results suggest that the types of bacteria in the gut can affect the risk of constipation. 03 Altered stooling habits are related to the development of long-term belly pain. Belly pain is a common complaint in children and up to 20% of school age children have pain frequently. Researchers in Houston, Texas discovered that children whose belly pain was related to their stooling habits were more likely to have long-term pain than those children who had belly pain that did not relate to their stooling habit. Bacteria in the intestine can affect stooling habit, suggesting that in some children, belly pain likely is related to the type of bacteria present in the intestine. 04 Stooling pattern of normal children defined. There is little information about the normal stooling pattern of healthy children. Researchers in Houston, Texas examined a large group of healthy children and described for the first time how often they stooled and whether they had constipation or diarrhea. By defining normal in healthy children, future studies will be able to examine how diet, bacteria, and other factors influence stooling pattern and gastrointestinal health. 05 A novel method to quantify sequencing data. For many applications of next generation sequencing, challenges remain due to unavailable or low quality reference genomes which often cause half of the samples that cannot be mapped. Researchers in Houston, Texas developed a novel method to quantify sequencing data without the need of reference genomes by reconstructing phylogenetic relationship between different bacterial species. Our method recapitulates genetic diversities in Human Microbiome Project which collects hundreds of metagenomics samples from various sites. Our analysis revealed the contribution of unmapped reads in metagenomics studies and will aid researchers for future studies.
Impacts
(N/A)
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine the effects of diet and age on gut microbial composition, ascertain the metagenomic profile of the gut microbes, and relate these to gut barrier function and inflammation in children 7 to 18 years of age. Objective 2: Develop a new de novo assembly method to assemble short sequence reads into long contiguous reads (contigs). Apply this method to assemble gut microbiome sequence reads. Develop a statistical method to cluster contigs and quantify abundance of these clusters. Perform genetic association testing for haplotype-microbiome interactions that affect the risk of childhood obesity. Objective 3: Using samples collected from rural African children aged 1- 2 years at risk for environmental enteropathy (EE), identify a panel of human mRNAs in fecal samples indicative of EE, evaluate this panel as a biomarker for EE in other populations, test whether micronutrient and/or fish oil supplements can reduce EE, explore the microbiome of children with and without EE, and correlate the mRNA panel markers with child growth parameters. Subobjective 3A: Using samples collected from rural African children at risk for EE, identify a panel of human mRNAs in fecal samples indicative of EE, correlate the mRNA panel markers with the L:M test and child growth parameters. Subobjective 3B: Determine whether micronutrient and/or fish oil supplements can reduce EE in rural African children aged 1-3 years. Subobjective 3C: Explore the microbiome of the gut in children with EE, looking for characteristic patterns of bacterial populations, or the presence of novel viruses, which may provide clues regarding the stimuli of the chronic gut inflammation. Approach (from AD-416): Gut microbes account for 90% of the cells and 99% of the genetic material in humans. The gut microbiome (bacteria and their genes) has coevolved with humans to serve a critical symbiotic role in maintaining health yet, the presence of an abnormal microbiome composition may contribute to chronic health disorders like allergic, circulatory, and inflammatory bowel diseases, functional abdominal pain disorders, and obesity. Little is known about the development of the child gut microbiome, the influence of diet, and how variations in the gut microbiome affect health. The long-term goal of this research is to understand the interaction between the gut microbiome and the health of children. The objectives center around gaining a better understanding of the gut microbiome, how it is affected by diet and age, its associations with gut health in the developed and developing worlds, and obesity risk. Through our studies, we anticipate an increased understanding of how diet and age influence gut microbial population composition, and in turn, alter physiological functioning and promote health. Additionally, insight will be gained into potential mechanisms by examining gut microbial genes and potential gene products (microbiome) and exploring relationships with physiologic outcomes (gut barrier function and inflammatory state) that may promote or impair health. This information will lead to improved understanding how foods and their components support health and reduce disease risk and can be used in formulating nutrition policies and government programs. The results will be of benefit to children and adolescents and can be used as a guide for similar investigations in adults. For Objective 1, we continue to recruit a large number of children 7 to 18 years of age so that we may have a robust size data set for our study. We developed a dietary questionnaire to obtain the child�s dietary history and have described the use of the questionnaire in a publication. In a subset of children (n=33), we examined the impact of additional dietary fiber on the composition of the gut microbiome using whole genome shotgun sequencing which will allow us to infer potential alterations in metabolic pathways engendered by changes in the gut microbiome. In terms of microbial population composition, there were trends toward an increased relative abundance of bacteria belonging to the phylum Bacteroidetes and a decreased relative abundance of Firmicutes when fiber supplementation was given. Similar trends were observed at the class level with potential enrichment in bacteria belonging to the class Bacteroidia and the Placebo group in bacteria belonging to the class Clostridia. These data have been published. We are delaying the main analysis of the samples until we have finished recruiting subjects so that we may sequence all samples simultaneously and eliminate the risk of run to run variation. The studies of barrier function are proceeding in a timely fashion. For Objective 2, we continue to develop algorithms and computer programs to assemble short reads into long contigs. Currently our program can reliably assemble contigs of 2000-3000 base pairs from paired-end reads with each read 100 base pairs and insert lengths of 300-500 base pairs. Jointly assembling multiple species is feasible. The program scales linearly with the number of reads and is not sensitive to the number of species. But assembled contigs may contain reads from different species. This is a challenge that appears to be difficult to overcome when assembling short reads. With regard to Objective 3 much was accomplished this fiscal year. For objective 3A the analyses method was fully developed and validated, this work has been published in a peer-reviewed journal, so that other scientists can reliably use it. The process of selecting a smaller panel of RNA probes to measure gut health and predict liner growth is underway. For objective 3B, the clinical trial has been completed, and it showed that in a vulnerable population of young children in rural Africa, dietary supplementation with multivitamins and fish oil did not reduce gut inflammation. Laboratory assessment of samples is ongoing. For objective 3C analysis is still ongoing, but in the stools from the children at a single time point, the genus enterovirus (member of the picornavirus family) was the most prevalent and was found more frequently in the environmental enteropathy (EE) samples. We additionally, discovered a new virus tentatively named Statovirus C (Stool associated Tombus-like virus) in one of samples from a child with environmental enteric dysfunction (EED). Interestingly, we have now detected viruses related to this in stools from macaques, mice, and cattle. A manuscript describing this new viral family is in preparation. Accomplishments 01 Clinical trial to reduce gut inflammation. Rural African children often are stunted because of gut inflammation. Children's Nutrition Research Center researchers based in rural Malawi, Africa worked closely with 250 young children to see that they received either a daily combination of fish oil and vitamins or a placebo, a harmless substance with no active ingredients. The children had their gut health measured with a urine test while receiving the supplement and found that the fish oil and multivitamins did not improve gut health in this population. Having conducted this work using the most powerful clinical assessment tool, the randomized controlled clinical trial, the results constitute powerful evidence suggesting these simple dietary interventions are not enough to reduce gut inflammation in this population. 02 Identification of viruses in stool in children with gut inflammation. Researchers are interested in determining if children with gut inflammation have differences in the viruses in their gut compared to children that do not have inflammation. To do this, Children's Nutrition Research Center researchers based in Malawi, Africa worked to determine what viruses are present by a novel genetic sequencing method and the use of our custom software to interpret data. We have examined stool samples from children with gut inflammation and children without. We have detected numerous viruses in these samples including new viruses that have never before been known to exist. 03 Food intolerances in healthy children. A lack of knowledge exists regarding why otherwise healthy children have food intolerances. Using our food questionnaire, researchers at the Children's Nutrition Research Center in Houston, Texas investigated whether healthy children perceive that they have intolerances to certain foods. We found that 62. 5% of children 7-18 years of age identify, most commonly, with two foods to which they have an adverse reaction which led 41% of them to avoid certain foods. Moving forward, we plan to investigate whether the types of gut bacteria play a role in causing these intolerances given the importance of gut bacteria in helping to digest and process certain foods. 04 Changes in the types of gut bacteria with age. A lack of knowledge exists regarding if there are changes in the types of gut bacteria with age and what relationship they may have to human disease. In a preliminary study, researchers at the Children's Nutrition Research Center in Houston, Texas surveyed the types of gut bacteria present in children 7-12 years of age and compared the types of bacteria present to those found in adults based upon data from the human (adult) microbiome project. Previous studies have suggested that by 3-4 years of age the gut bacteria in children are similar to those in adults however, investigating a larger number of children with more precise techniques, we found that the gut population of children in this age group is still undergoing development. Very importantly, when we examined the genetic potential of the gut bacteria, we found that children's gut bacterial communities were enriched in functions which may support ongoing development, while adult communities were enriched in functions associated with inflammation, obesity, and increased risk of adiposity. These observations suggest an opportunity to seek ways to alter the development of the gut bacterial population to help prevent adult onset diseases.
Impacts
(N/A)
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine the effects of diet and age on gut microbial composition, ascertain the metagenomic profile of the gut microbes, and relate these to gut barrier function and inflammation in children 7 to 18 years of age. Objective 2: Develop a new de novo assembly method to assemble short sequence reads into long contiguous reads (contigs). Apply this method to assemble gut microbiome sequence reads. Develop a statistical method to cluster contigs and quantify abundance of these clusters. Perform genetic association testing for haplotype-microbiome interactions that affect the risk of childhood obesity. Objective 3: Using samples collected from rural African children aged 1- 2 years at risk for environmental enteropathy (EE), identify a panel of human mRNAs in fecal samples indicative of EE, evaluate this panel as a biomarker for EE in other populations, test whether micronutrient and/or fish oil supplements can reduce EE, explore the microbiome of children with and without EE, and correlate the mRNA panel markers with child growth parameters. Subobjective 3A: Using samples collected from rural African children at risk for EE, identify a panel of human mRNAs in fecal samples indicative of EE, correlate the mRNA panel markers with the L:M test and child growth parameters. Subobjective 3B: Determine whether micronutrient and/or fish oil supplements can reduce EE in rural African children aged 1-3 years. Subobjective 3C: Explore the microbiome of the gut in children with EE, looking for characteristic patterns of bacterial populations, or the presence of novel viruses, which may provide clues regarding the stimuli of the chronic gut inflammation. Approach (from AD-416): Gut microbes account for 90% of the cells and 99% of the genetic material in humans. The gut microbiome (bacteria and their genes) has coevolved with humans to serve a critical symbiotic role in maintaining health yet, the presence of an abnormal microbiome composition may contribute to chronic health disorders like allergic, circulatory, and inflammatory bowel diseases, functional abdominal pain disorders, and obesity. Little is known about the development of the child gut microbiome, the influence of diet, and how variations in the gut microbiome affect health. The long-term goal of this research is to understand the interaction between the gut microbiome and the health of children. The objectives center around gaining a better understanding of the gut microbiome, how it is affected by diet and age, its associations with gut health in the developed and developing worlds, and obesity risk. Through our studies, we anticipate an increased understanding of how diet and age influence gut microbial population composition, and in turn, alter physiological functioning and promote health. Additionally, insight will be gained into potential mechanisms by examining gut microbial genes and potential gene products (microbiome) and exploring relationships with physiologic outcomes (gut barrier function and inflammatory state) that may promote or impair health. This information will lead to improved understanding how foods and their components support health and reduce disease risk and can be used in formulating nutrition policies and government programs. The results will be of benefit to children and adolescents and can be used as a guide for similar investigations in adults. For Objective 1 our goal is to determine the effects of diet and age on gut microbial composition and thus far we have currently enrolled 63 healthy children. Stools have been collected for analysis of gut bacteria while the subjects maintained a record of what they had consumed in their diet during the time they collected a stool sample. This will allow us to determine any relationships between the makeup of the diet and its effect on the types of bacteria present in the intestines. We will be able to determine if the intestines contain bacteria that are considered healthy or unhealthy. The children also have undergone testing to evaluate the ability of their intestines to block potentially noxious materials (test of gut barrier function). For example, whether the gut barrier remains healthy and can block out toxic products produced by some bacteria or whether the barrier is not healthy and is �leaky� to noxious materials. We also have tested the assays we will use to measure the level of inflammation in their intestines. When the gut barrier is weak (leaky) the noxious materials like bacterial products can produce inflammation in the intestine which can lead to inflammation in other sites of the body. This is one of the processes thought to be involved in the liver disease associated with obesity. We will continue to enroll additional children and have them undergo the same testing procedures. In Objective 2 we aim to develop a new de novo assembly method to assemble short sequence reads into long contiguous reads (contigs). This will reduce bias in variant calling caused by repetitive regions in genomes. In the proposed application that concerns gut microbiome, the samples contain many species whose genomes are similar. The classical read-mapping approach is difficult to call variants reliably. Our approach on the other hand, aims to assemble many reads together before mapping them onto the reference genomes. This process increases mapping accuracy and hence the quality of variants call. We currently have an initial version of software developed, and we expect to have a beta- version release in 2016. This new software can produce sensible results on simulated data; after de novo assembly, contigs can be mapped onto reference genomes more evenly and more completely, compared to direct mapping reads. The subsequent variant call based on de novo assembled contigs is much more accurate. We recently acquired a high capacity computer that will facilitate further development of this innovative software. For Objective 3, excellent progress has been made, the clinical trial was initiated and the participation of the children complete. Enrolled were 250 children who received either a daily multiple micronutrient + deworming treatment + a 10 day course of high dose zinc or an appropriate placebo. The children tolerated the intervention well, no complications were noted. We are currently analyzing the laboratory samples for intestinal permeability and stools for microbiota. Growth data have not been compared as the blinding code has not been broken yet. As far as identification of a biomarker of gut health and viruses associated with poor gut health, the sample analyses are in progress and no major obstacles have been encountered at this time. Fifty-four genes that are over expressed in environmental enteropathy have been identified and being tested as potential fecal biomarkers. Any animal research conducted have received IACUC approval. Any human research studies conducted have received IRB approval.
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