2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This CRIS research unit focuses on the nutritional regulation of cell and organ growth, differentiation, and development and is composed of five individual research projects:. 1)intestinal amino acid requirements in neonates;. 2)nutritional regulation of tissue anabolism in neonates; 3)consequences of perinatal undernutrition for satellite cell function and skeletal muscle growth;. 4)nutrient regulation of blood and blood vessel formation; and. 5)nutritional influences on gastrointestinal function in health and disease. This research unit falls within Components 1 (Nutrient Requirements) and 7 (Bioavailability of Nutrients and Food Components) of National Program 107 – Human Nutrition. Additionally, this research adheres to ARS Strategic Plan Goal #4 Improve the Nation's Nutrition and Health, specifically Objective 4.1: Promote Healthier Individual Food Choices and Lifestyles and Prevent Obesity; Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods; and Determine Food Consumption Patterns of Americans. Project 1: Intestinal amino acid requirements in neonates Optimum nutrition from fetal life through to puberty is critical for sustaining the appropriate growth and functional development of the organism. "Optimum" requires that we know how an organism uses specific nutrients at different stages of development. This research project addresses the underlying metabolic and physiological processes that determine the amino acid requirements in infants. Specifically, it examines the metabolic fate and quantitative significance of the amino acids, methionine and cysteine, used by the gastrointestinal tract in neonates fed enterally and parenterally. This is important because the high metabolic activity of the intestine consumes a significant proportion of the dietary amino acid intake and, thereby, can limit their availability for somatic growth. Thus, the metabolic fate and functional requirements of indispensable amino acids within the intestines is critical to understanding the metabolic basis of infant amino acid requirements. The project has four objectives: (1) quantify the intestinal metabolic fate of methionine derived from the diet and systemic circulation in enterally fed neonatal animal models; (2) quantify the intestinal metabolic fate of cysteine derived from the diet and systemic circulation in enterally fed neonatal piglets; (3) quantify the intestinal metabolic fate of parenterally administered methionine and cysteine in TPN-fed neonatal piglets; and (4) establish the cellular localization and metabolic significance of sulfur amino acid (SAA) metabolism in the maintenance of intestinal epithelial cell redox status and cell survival. Project 2: Nutritional regulation of tissue anabolism in neonates Researchers are identifying the mechanism by which specific nutrients regulate the synthesis of tissue proteins and how this changes with development. This research project will examine how protein synthesis in neonates is regulated by branched chain amino acids and the intracellular mechanisms that regulate the response. The studies are quantifying the changes in translation initiation and protein synthesis that occur in vivo in response to variations in circulating levels of branched chain amino acids, other amino acids, glucose, and insulin. How stage of development and feeding status influence these processes are also being examined. This work will provide valuable information to improve strategies for the nutritional management of low birth weight infants. Project 3: Consequences of perinatal undernutrition for satellite cell function and muscle growth Our research will examine how the intrauterine nutritional environment of the fetus and nutrition in early postnatal life influence skeletal muscle development. Specifically, it aims to identify how the development of skeletal muscles is compromised in a rat model when perinatal nutrition is suboptimal. This is important because there are data that indicate that inadequate muscle growth in early life can never be entirely reversed. This may have significant implications for the precocious development of a number of detrimental, chronic conditions in adulthood, such as glucose intolerance, sarcopenia, obesity and osteoporosis. The objectives of the project are: (1) to determine the effects of undernutrition either during fetal development or postnatally on satellite cell replicative capacity; (2) to clarify the roles of insulin-like growth factor-I (IGF-I) and hepatocyte growth factor (HGF) in the nutritional regulation of satellite cell replication; (3) to identify the mechanism that relates ribosomal formation to satellite cell replication; (4) to delineate the significance of glucocorticoids in mediating the effects of intrauterine malnutrition on skeletal muscle growth in the fetus. Achieving these objectives will enable us to develop preventive and therapeutic strategies for those individuals who, because they were inappropriately nourished during the perinatal period, are at greater risk for the development of chronic adult diseases, such as glucose intolerance, sarcopenia, obesity and osteoporosis. These disorders constitute the most common chronic medical problems in our adult population today, and their treatment is a substantial economic burden on society. Project 4: Nutrient regulation of blood and blood vessel formation Most embryos that die prior to birth do so for one of three reasons:. 1)failure to implant or establish fetal-maternal connection;. 2)inability to produce blood cells to sustain embryonic life; or. 3)inadequate circulatory system. Our current knowledge of the molecular regulation of these processes and our understanding of how specific nutrients regulate these processes is very limited. Gaining such insights will greatly aid in understanding how maternal diet influences pregnancy outcome, and how it could be altered to prevent embryonic death. In our studies, we will examine the molecular events that lead to the formation of blood vessels and, more specifically, how nutrients such as retinoic acid, regulate this process. Blood vessel formation will be studied in the mouse embryo model in mice that are genetically deficient in retinoic acid; thus, the impact of retinoic acid deficiency on vascular development can be studied directly. Understanding how blood vessels form and how retinoic acid controls this process will provide important insights that can be applied to controlling the aberrant vessel formation that underlies much prevalent pathology including tumorigenesis, diabetes, and arteriosclerosis. This research project will contribute to the national program mission to understand how nutrients regulate cell and organ growth during development by revealing how retinoids regulate the formation of blood and blood vessels in the embryo. The long-term goal of these studies is to understand, on a cellular and molecular level, the events leading to vascular development and hematopoiesis. We further aim to determine how nutrients, such as Vitamin A, contribute to the regulation of these processes. Project 5: Nutritional influences on gastrointestinal function in health and disease Recurrent abdominal pain in children and irritable bowel syndrome (IBS) in adults are debilitating conditions present in a significant number of individuals. These conditions often lead to significant disability (e.g., school absence) with adverse economic impact (expensive medical evaluations, loss of job productivity because of parent time away from work). From 30% to 66% of children with recurrent abdominal pain will go on to develop the irritable bowel syndrome in adulthood. Irritable bowel syndrome is more prevalent than heart disease and hypertension combined, and its economic impact has been calculated to be almost $8 billion per year. Our research will develop nutritional strategies to minimize the occurrence of recurrent abdominal pain in children, with specific emphasis on the effects of probiotics, fiber (Objective. 1)and sugars (Objective 2). The resulting measurements will identify the mechanisms whereby these dietary components impact those gastrointestinal functions that are responsible for the symptoms of recurrent abdominal pain (Objective 3). The development of effective treatments in childhood recurrent abdominal pain is important because it is likely to reduce the chances of developing IBS in adulthood. 2.List by year the currently approved milestones (indicators of research progress) Project 1: Intestinal amino acid requirements in neonates Year 1 (FY2005) Completion of animal infusion studies and preliminary plasma analysis of isotopic tracers and quantify the intestinal metabolic fate of methionine derived from the diet and systemic circulation in enterally fed neonatal piglets. Year 2 (FY2006) Completion of animal infusion studies and preliminary plasma analysis of isotopic tracers described and quantify the intestinal metabolic fate of cysteine derived from the diet and systemic circulation in enterally fed neonatal piglets. Development of cell culture studies to assess the localization and extent of intestinal methionine metabolism. Year 3 (FY2007) Completion of animal infusion studies and preliminary plasma analysis of isotopic tracers described in and quantify the intestinal metabolic fate of parenterally administered methionine and cysteine in parenterally fed neonatal piglets Continued development of cell culture studies to assess the localization and extent of intestinal methionine metabolism. Year 4 (FY2008) Perform cell culture studies and biochemical assessments of epithelial methionine metabolism to establish the cellular localization and metabolic significance of sulfur amino acid (SAA) metabolism in the maintenance of intestinal epithelial cell redox status and cell survival. Year 5 (FY2009) Complete cell culture studies and biochemical assessments of epithelial methionine metabolism to establish the cellular localization and metabolic significance of sulfur amino acid (SAA) metabolism in the maintenance of intestinal epithelial cell redox status and cell survival. Project 2: Nutritional regulation of tissue anabolism in neonates Year 1 (FY2005) Determine the effect of leucine on protein synthesis and translation initiation factors activation. Year 2 (FY2006) Determine the effect of other amino acids on protein synthesis and translation initiation; determine the effect of signaling inhibitors on glucose-stimulated protein synthesis; determine the effect of development on abundance and feeding induced activation and protein-protein interaction of PTEN, PDK1, PKB, TSC1-2, Rheb, and mTOR. Year 3 (FY2007) Determine the effect of glucose on the intracellular signaling pathways leading to protein synthesis and determine the effect of development on abundance and feeding induced activation and protein-protein interaction of PTEN, PDK1, PKB, TSC1-2, Rheb, and mTOR. Year 4 (FY2008) Identify the effect of development on nutrient and insulin signaling proteins that regulate protein synthesis and determine rapamycin's effect on protein synthesis and translation initiation. Year 5 (FY2009) Determine the role of insulin and amino acids in the developmental change in the feeding-induced activation of insulin and nutrient signaling proteins leading to protein synthesis. Project 3: Consequences of perinatal undernutrition for satellite cell function and muscle growth Year 1 (FY2005) Establish technique for isolating satellite cells from muscles, and culture procedures to measure in vitro proliferation rates. Compare the consequences of intrauterine versus postnatal undernutrition on the growth in skeletal muscle mass, satellite cell number, and replicative capacity upon nutritional rehabilitation. Year 2 (FY2006) Establish technique for isolating satellite cells from muscles, and culture procedures to measure in vitro proliferation rates. Compare the consequences of intrauterine versus postnatal undernutrition on the growth in skeletal muscle mass, satellite cell number, and replicative capacity upon nutritional rehabilitation. Year 3 (FY 2007) Complete muscle measurements. Assess plasma and muscle IGF-I, HGF, and myostatin levels, as well as the abundance and degree of activation of teh respective muscle receptors in response to the nutritional treatments. Develop, produce, and test in vitro myogenic cDNA expression plasmids for HGF and IGF-I. Verify efficacy of in vivo gene transfer and expression of peptides from HGF and IGF expression plasmids. Compare growth of skeletal muscle mass in pups subjected to postnatal malnutrition followed by post-weaning nutritional rehabilitation with or without augmented expression of local IGF-I and/or HGF induced by in vivo gene transfer. Year 4 (FY 2008) Complete analytical measurements. Evaluate data. Perform animal component of protocol as previously described for Objective 1, but include two additional treatments to distinguish the roles of glucocorticoids and protein deficiency on the impairment of intrauterine muscle growth. Year 5 (FY 2009) Complete analysis and assessment of skeletal muscle mass from fetus exposed to variable levels of glucocorticoids. Evaluate data from Objective 4. Project 4: Nutrient regulation of blood and blood vessel formation Year 1 (FY2005) Measure levels of cellular apoptosis and proliferation in Raldh2-/- and WT embryos and yolk sacs. Assess the level of visceral endoderm differentiation and measure its expression of soluble effectors in WT and Raldh2-/- yolk sac tissues. Year 2 (FY2006) Determine the extent to which yolk sac progenitors from Raldh2-/- mutants and WT embryos form hematopoietic colonies in vitro. Measure the levels of differentiated blood cells formed in Raldh2-/- and WT embryos/yolk sacs in vivo and in colony forming assays in vitro. Year 3 (FY2007) Complete gene and protein expression analyses. Attempt to rescue the vascular defects in Raldh2-/- mutants with soluble effectors down-regulated in response to retinoic acid deficiency. Measure the restoration of morphological defects, cellular proliferation and gene expression in rescued mutants. Year 4 (FY2008) Determine the hierarchy of signaling molecules involved in endodermal induction of vascular development in the mesoderm. Measure levels of transcription factors and signaling molecules, which induce or are associated with, embryonic hematopoiesis in Raldh2-/- and WT embryos. Determine whether retinoid-regulated factors induce hematopoietic cell fates in WT mesodermal progenitors that we have recently isolated and characterized. Year 5 (FY2009) Complete data collection, analyses, and manuscript preparation for all objectives. Project 5: Nutritional influences on gastrointestinal function in health and disease Year 1 (2005) This is a randomized double-blind protocol that entails the recruitment of 180 human subjects (45 subjects in 2005) with the necessary characteristics to qualify for the study. The efficacy of treatment for 4 weeks with a probiotic, fiber, or a placebo on symptoms of recurrent abdominal pain will be assessed (Objective 1). Tests will be performed that will measure GI transit time, carbohydrate malabsorption, GI integrity, and GI inflammation. Subjects who do not respond positively to treatments in Objective 1 will be tested in Objective 2 in which the effect of a sugar elimination diet on symptoms will be assessed. Year 2 (2006) This is a randomized double-blind protocol that entails the recruitment of 180 human subjects (45 subjects in 2006) with the necessary characteristics to qualify for the study. The efficacy of treatment for 4 weeks with a probiotic, fiber, or a placebo on symptoms of recurrent abdominal pain will be assessed (Objective 1). Tests will be performed that will measure GI transit time, carbohydrate malabsorption, GI integrity, and GI inflammation. Subjects who do not respond positively to treatments in Objective 1 will be tested in Objective 2 in which the effect of a sugar elimination diet on symptoms will be assessed. Year 3 (2007) This is a randomized double-blind protocol that entails the recruitment of 180 human subjects (45 subjects in 2007) with the necessary characteristics to qualify for the study. The efficacy of treatment for 4 weeks with a probiotic, fiber, or a placebo on symptoms of recurrent abdominal pain will be assessed (Objective 1). Tests will be performed that will measure GI transit time, carbohydrate malabsorption, GI integrity, and GI inflammation. Subjects who do not respond positively to treatments in Objective 1 will be tested in Objective 2 in which the effect of a sugar elimination diet on symptoms will be assessed. Year 4 (2008) This is a randomized double-blind protocol that entails the recruitment of 180 human subjects (45 subjects in 2008) with the necessary characteristics to qualify for the study. The efficacy of treatment for 4 weeks with a probiotic, fiber, or a placebo on symptoms of recurrent abdominal pain will be assessed (Objective 1). Tests will be performed that will measure GI transit time, carbohydrate malabsorption, GI integrity, and GI inflammation. The subjects' pain episodes and stooling patterns will also be tracked. Subjects who do not respond positively to treatments in Objective 1 will be tested in Objective 2 in which the effect of a sugar elimination diet on symptoms will be assessed. Year 5 (FY2009) Recruitment of a total of 180 human subjects with the necessary characteristics to qualify for the study will be complete. The effects of the various treatments on the perceived indices of pain will be related to the corollary measures of gastrointestinal function, to determine the mechanism responsible for the observed responses (Objective 3). The results will be disseminated through publications and presentation to healthcare workers who deal with this population of children. 4a.List the single most significant research accomplishment during FY 2006. Project 1: Intestinal amino acid requirements in neonates Importance of Dietary Methionine Metabolism in the Gut Researchers at the Children's Nutrition Research Center at Houston, TX, completed a test of how key dietary sulfur amino acids (methionine, homocysteine, and cysteine) are used by the infant gut. Through the process of transmethylation, both homocysteine and cysteine are made from methionine; cysteine is an essential ingredient used to combat cellular stress; however, the overproduction of homocysteine has been linked to heart attack, stroke, and cell stress in infants and adults. CNRC researchers used infant animals as a model of the human infant to trace the use of methionine in the gut and the whole body of infant subjects using methionine stable isotopes. Researchers discovered that 20% of the dietary methionine is used by the gut for normal growth and function; more importantly, researchers showed for the first time that the gut is an important organ that converts methionine to both homocysteine and cysteine. In addition, our results showed that the gut is capable of converting homocysteine back to methionine by a process known as remethylation, and this process is important since remethylation is a critical process to minimize overproduction of harmful homocysteine in the body. The findings have important implications: enhancing strategies to improve the use of methionine ingested by infants; understanding that the gut may play a key role in the overproduction of homocysteine in the blood. These results may give scientists new clues into how overproduction of homocysteine in the gut may contribute to major gut diseases, especially inflammatory bowel disease and cancer. [NP107 Components 1 (Nutrient Requirements)] 4b.List other significant research accomplishment(s), if any. Project 1: Intestinal Amino Acid Requirements in Neonates Understanding Body Composition in Newborns Fed Intravenously Researchers at the Children's Nutrition Research Center at Houston, TX, completed a study designed to examine the impact of chronic intravenous nutrition on body composition and metabolism using newborn animal models. A large percentage of premature infants are fed intravenously during the first month of life, yet there is little information as to how such nutrition affects their body composition and nutrient metabolism. The study measured growth of body constituents using dual energy X-ray absorption (DEXA) measurements and glucose metabolism by intravenous glucose tolerance test in newborn animal models fed exclusivly intravenously for 2 weeks after birth. The results showed that newborns exclusivly fed intravenously deposited less lean tissue and developed fatty liver disease hepatic steatosis and insulin resistance compared to those fed by the enteral method. These novel findings may have important implications for the long-term development of obesity or type 2 diabetes in premature infants. [NP107-Component 1 (Nutrient Requirements)] Project 2: Nutritional regulation of tissue anabolism in neonates Effect of Amino Acids on Protein Synthesis and Translation Initiation Factor Activation Additional knowledge is needed to improve strategies for the nutritional management of low birth weight infants. Researchers at the Children's Nutrition Research Center, Houston, TX, have demonstrated that the branched chain amino acid, leucine, stimulates protein synthesis by increasing translation initiation factor activation. Our studies concluded that this effect is specific for leucine and its alpha-keto acid and that the other branched chain amino acids have no effect. Additionally, the response to leucine is not sustained because the circulating concentrations of other amino acids fall as they are used for protein synthesis. The stimulation of protein synthesis by leucine can be maintained, however, when circulating amino acids levels are maintained by infusion. Such accomplishments provides valuable information to improve strategies for the nutritional management of low birth weight infants. [NP107-Component 7 (Bioavailability of Nutrients and Food Components)] Developmental Regulation of the Activation of Signaling Components Leading to Translation Initiation A more thorough understanding of the regulatory processes of skeletal muscle growth in newborns is needed. Research conducted at the Children's Nutrition Research Center has demonstrated that many of the nutrient- and insulin-signaling components that are involved in the regulation of protein synthesis in skeletal muscle are developmentally regulated. Researchers have indicated that in skeletal muscle the activation of some of the negative regulators of protein synthesis is low, and the activation of some of the positive regulators is high in young pigs, consistent with the elevated rates of protein synthesis in neonatal muscle. The developmental changes in the abundance and activation of these signaling components likely contribute to the high rate of protein synthesis and more rapid gain in skeletal muscle mass in neonates. [NP107-Component 7 (Bioavailability of Nutrients and Food Components)] Project 3: Consequences of perinatal undernutrition for satellite cell function and muscle growth Impaired Muscle Growth following Undernutrition in the Mouse Much of the basic research on the perinatal origins of adult diseases has been performed in rats, yet if mice could be used, a greater variety of genetic methods and molecular reagents would be available. However, no data is available to establish if skeletal muscle growth in the mouse is impacted in the same way as it is in the rat. Thus, Children's Nutrition Research Center researchers designed diets and performed in mice an experiment similar to that described for the rat. Researchers have established that when mouse dams are fed low-protein diets (8% protein) during pregnancy and lactation, postnatal growth of skeletal muscle in the offspring is severely compromised. Nutritional rehabilitation from 3 weeks of age does not restore muscle mass. These results demonstrate that the effects of perinatal undernutrition on skeletal muscle growth in the mouse are similar to other species that have been studied, thus our researchers may perform the proposed studies, modified as necessary, in mice. [NP107-Component 1 (Nutrient Requirements)] Satellite Cell Numbers are Reduced from Undernutrition in the Mouse Satellite cell replication is necessary for muscle growth, and a more thorough understanding of its process is needed. A practical approach to quantify satellite cells and myonuclei in the intact muscle is critical in any effort to determine the effects of perinatal malnutrition on muscle growth. This was not readily available for satellite cells. Researchers at the Children's Nutrition Research Center have systematically evaluated a number of satellite-cell specific markers by immunofluorescent staining of mouse muscle frozen sections and visualization by confocal microscopy. Researchers have optimized a technique that uses an antibody to the satellite-specific marker in conjunction with markers for the basal lamina (laminin) and nuclei (DAPI) to visualize and quantify the various nuclear populations in muscle. Data indicate that at weaning, the number of satellite cells is reduced in pups suckled on malnourished dams, and that the response to realimentation is not sufficient to promote catch-up growth. [NP107-Component 1 (Nutrient Requirements)] Project 4: Nutrient regulation of blood and blood vessel formation Retinoic Acid is Key for Proper Embryonic Development Researchers at the Children's Nutrition Research Center, Houston, TX, discovered that during embryonic development, retinoic acid is needed for the specialization of endothelial cells into cells within the endothelium that have the capacity to generate all blood cell lineages, or hemogenic endothelium. Researchers discovered that retinoic acid plays a critical role in the specification of endothelial cells. These research findings further scientist's understanding of the role of specific nutrients in embryonic development and lend insight into when during development it will be critically important to monitor and regulate the availability of retinoic acid. CNRC researchers made this discovery by studying the developing mouse embryo, and will continue studies in this model to further our knowledge of vascular development and hematopoiesis. [NP107-Component 1 (Nutrient Requirements)] 4c.List significant activities that support special target populations. None. 5.Describe the major accomplishments to date and their predicted or actual impact. This research unit falls within Components 1 (Nutrient Requirements) and 7 (Bioavailability of Nutrients and Food Components) of National Program 107 – Human Nutrition. Additionally, this research adheres to ARS Strategic Plan Goal #4 Improve the Nation's Nutrition and Health, specifically Objective 4.1: Promote Healthier Individual Food Choices and Lifestyles and Prevent Obesity; Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods; and Determine Food Consumption Patterns of Americans. Project 1: Intestinal amino acid requirements in neonates Researchers have completed a study designed to quantify the extent of dietary methionine metabolism by the gut using an animal model as a model for the human infant. Scientists traced the metabolism in the gut and whole body using highly sensitive 13C- and 2H-labeled methionine isotopes as a marker and discovered that 20% of the dietary methionine is metabolized or used by the gut for normal growth and function. More importantly, researchers showed for the first time that the gut is an important organ that converts methionine to homocysteine and cysteine. These findings have implications for the determining the metabolic availability of dietary methionine in infant formulas. The consumers of the scientific information from the lab's scientific publications and presentations include pediatricians, neonatologists, gastroenterologists, pediatric gastroenterologists, dieticians, and academic nutritionist with an interest in dietary nutrient requirements and methionine metabolism. The findings address the need to understand the biological basis for dietary amino acid requirements in infants. Dietary Recommended Intakes for amino acids in infants are based on factorial estimates and represent at best indirect measures of nutrient requirements. Project 2: Nutritional regulation of tissue anabolism in neonates CNRC researchers have demonstrated that the increase in the branched chain amino acid, leucine, that occurs after a meal acts as a nutrient signal to rapidly stimulate protein synthesis in muscle of young animals. A sustained response to leucine requires that other amino acids be available as substrates for protein synthesis. This work will provide important new information on the potential for using leucine supplementation to optimize the nutritional management of low birth weight infants. Project 3: Consequences of perinatal undernutrition for satellite cell function and muscle growth Researchers have demonstrated that mouse responds to perinatal undernutrition in much the same way as other species that have been studied. This is an important observation because it means that the vast array of genetic tools and molecular reagents available can be applied to address the biological question relating to the long-term effects of perinatal nutrition. Project 4: Nutrient regulation of blood and blood vessel formation Scientists at the Children's Nutrition Research Center in Houston, TX, have gained knowledge of the molecular role(s) of specific nutrients during blood and blood vessel development that will provide insights into the impact of maternal diet on the developing embryo. We are also in the process of discovering novel, retinoic acid-regulated genes that modulate blood and vascular development, and will determine whether these genes will be useful for the treatment of vascular and hematopoietic defects in adults. We anticipate pregnant women and physicians who care for them will benefit from this work. Other scientists will also gain a better understanding of the molecular regulation of blood and blood vessel development, and the impact of specific nutrients on these processes. Project 5: Nutritional influences on gastrointestinal function in health and disease Our ongoing preliminary work has demonstrated that children with recurrent abdominal pain have evidence of gastrointestinal dysfunction as well as disturbances in autonomic nervous system functioning. As these data are analyzed further we will be able to design potential interventions that will improve the health of these children. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Project 2: Nutritional regulation of tissue anabolism in neonates The scientific results of this project are being disseminated to other scientists in publications and in abstracts / presentations at scientific meetings. The information from the work is published as it becomes available for consideration of implementation for the years of this project. Practical results, applied to humans and animals, will be forthcoming as they are indicated by the research results. The long delay results from the extensive testing for safety and efficiency necessary before acceptance of technologies applied to humans and animals. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Project 1: Intestinal amino acid requirements in neonates DG Burrin Invited presentation entitled “Application of Piglet Models to Explore the Pathobiology and Treatment of Pediatrics GI Disease," Bristol-Meyers Squibb, Mead Johnson Nutritionals, Evansville, IN, August 2005. DG Burrin Invited presentation entitled “Nutritional Significance of Gut Sulfur Amino Acid Metabolism,” Department of Biochemistry, Memorial University, St. Johns, Newfoundland. February 2006. DG Burrin Invited presentation entitled “Nutritional Significance of Gut Methionine Metabolism,” Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada. March 2006. DG Burrin Invited presentation entitled “Nutritional Support of the Neonatal Infant,” Department of Human Nutrition, Royal Veterinary and Agricultural University, Copenhagen, Denmark. May 2006. DG Burrin Invited presentation entitled “Metabolic Fate of Amino Acids in the Gut,” Department of Cell and Organism Biology, Lund, Sweden. June 2006. Project 2: Nutritional regulation of tissue anabolism in neonates Suryawan A and Davis TA. Activation of signaling components leading to translation initiation in skeletal muscle of neonatal pigs is regulated by feeding and development. NIH sponsored Nutrient Sensing, Insulin Signaling, and Hamartoma Syndromes Workshop, Bethesda, MD, 2006. Review Publications Bohnsack, B.L., Lai, L., Dolle, P., Hirschi, K.K. 2004. Signaling heirarchy downstream of retinoic acid that independently regulates vascular remodeling and endothelial cell proliferation. Genes and Development. 18(11):1345-1358. Huss, W.J., Lai, L., Barrios, R.J., Hirschi, K.K., Greenberg, N.M. 2004. Retinoic acid slows progression and promotes apoptosis of spontaneous prostate cancer. The Prostate. 61(2):142-152. Teitelbaum, D.H., Tracy, T.F., Aouthmany, M.M., Llanos, A., Brown, M.B., Yu, S., Brown, M.R., Shulman, R.J., Hirschl, R.B., Derusso, P.A., Cox, J., Dahlgren, J., Groner, J.I., Strouse, P.J. 2005. Use of cholecystokinin-octapeptide for the prevention of parenteral nutrition-associated cholestasis. Pediatrics. 115(5):1332-1340. Stoll, B., Price, P.T., Reeds, P.J., Chang, X., Henry, J.F., Van Goudoever, J.B., Holst, J.J., Burrin, D.G. 2006. Feeding an elemental diet vs a milk-based formula does not decrease intestinal mucosal growth in infant pigs. Journal of Parenteral and Enteral Nutrition. 30:32-39. Lai, L., Bohnsack, B.L., Niederreither, K., Hirschi, K.K. 2003. Retinoic acid regulates endothelial cell proliferation during vasculogenesis. Development. 130(26):6465-6474. Cottrell, J.J., Stoll, B., Buddington, R.K., Stephens, J.E., Cui, L., Chang, X., Burrin, D.G. 2005. Glucagon-like peptide-2 protects against TPN-induced intestinal hexose malabsorption in enterally re-fed piglets. American Journal of Physiology - Gastrointestinal and Liver Physiology. 53:G293-G300. Stephens, J., Stoll, B., Cottrell, J., Chang, X., Helmrath, M., Burrin, D.G. 2006. Glucagon-like peptide-2 acutely increases proximal small intestinal blood flow in TPN-fed neonatal piglets. American Journal of Physiology - Regulatory Integrative & Comparative Physiology. 59:R283-R289. Hornberger, T.A., Stuppard, R., Conley, K.E., Fedele, M.J., Fiorotto, M.L., Chin, E.R., Esser, K.A. 2004. Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism. Biochemical Journal. 380(Pt 3):795-804. Harvey, R.B., Andrews, K., Droleskey, R.E., Kansagra, K.V., Stoll, B., Burrin, D.G., Sheffield, C.L., Anderson, R.C., Nisbet, D.J. 2006. Qualitative and quantitative comparison of gut bacterial colonization in enterally and parenterally fed neonatal pigs. Current Issues in Intestinal Microbiology. 7:61-64. Bohnsack, B.L., Lai, L., Hirschi, K.K. 2005. Signaling hierarchy that regulates endothelial cell proliferation and vascular remodeling during vasculogenesis [abstract]. Journal of Federation of American Societies for Experimental Biology Journal. 19(4):A234. Spencer, A.U., Yu, S., Tracy, T.F., Aouthamany, M.M., Llanos, A., Brown, M.B., Brown, M., Shulman, R.J., Hirschl, R.B., Derusso, P.A., Cox, J., Dahlgren, J., Strouse, P.J., Groner, J.I., Teitelbaum, D.H. 2005. Parenteral nutrition-associated cholestasis in neonates: multivariate analysis of the potential protective effect of taurine. Journal of Parenteral and Enteral Nutrition. 29(5):337-344. Frank, J.W., Escobar, J., Nguyen, H.V., Jobgen, S.C., Davis, T.A., Wu, G. 2006. Oral N-carbamylglutamate (NCG) supplementation increases growth rate in sow-reared piglets [abstract]. The Federation of American Societies for Experimental Biology Conference: Advancing the Biomedical Frontier, April 1-5, 2006, San Francisco, California. 20(4):Part I, p. A425. Orellana, R.A., Escobar, J., Jeyapalan, A.S., Nguyen, H.V., Suryawan, A., Frank, J., Davis, T.A. 2006. Amino acids augment muscle protein synthesis in neonatal pigs during endotoxemia by modulating translation initiation [abstract]. The Federation of American Societies for Experimental Biology Conference: Advancing the Biomedical Frontier, April 1-5, 2006, San Francisco, California. 20(4):Part I, p. A9. Oliver, W.T., Rosenberger, J., Lopez, R., Fiorotto, M.L. 2006. The local IGF-I enhances the sensitivity of muscle protein synthesis to insulin [abstract]. The 88th Annual Meeting of The Endocrine Society, June 24-27, 2006, Boston, Massachusetts. Abstract #OR40-6, p. 134. Suryawan, A., Escobar, J., Frank, J.W., Nguyen, H.V., Liu, C.W., Davis, T.A. 2006. Developmental regulation of the activation of signaling components leading to translation initiation in skeletal muscle of neonatal pigs [abstract]. The Federation of American Societies for Experimental Biology Conference: Advancing the Biomedical Frontier, April 1-5, 2006, San Francisco, California. 20(4):Part I, p. A425. Oliver, W.T., Rosenberger, J., Lopez, R., Fiorotto, M.L. 2006. Local IGF-I enhances the sensitivity of muscle protein synthesis to insulin [abstract]. The Federation of American Societies for Experimental Biology Conference: Advancing the Biomedical Frontier, April 1-5, 2006, San Francisco, California. 20(5):Part II, p. A821. Oliver, W.T., Lopez, R., Yorks, K.A., Fiorotto, M.L. 2006. Regulation of ERK expression by muscle IGF-I "in vivo" determines the capacity for myonuclear proliferation [abstract]. The Federation of American Societies for Experimental Biology Conference: Advancing the Biomedical Frontier, April 1-5, 2006, San Francisco, California. 20(5):Part II, p. A1046. Jeyapalan, A.S. Orellana, R.A., Suryawan, A., Nguyen, H.V., Escobar, J., Frank, J., Davis, T.A. 2006. Stimulation of muscle protein synthesis by glucose in neonates is AMP kinase independent [abstract]. The Federation of American Societies for Experimental Biology Conference: Advancing the Biomedical Frontier, April 1-5, 2006, San Francisco, California. 20(5):Part II, p. A1046. Majka, S.M., Jackson, K.A., Kienstra, K.A., Majesky, M.W., Goodell, M.A., Hirschi, K.K. 2003. Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration. The Journal of Clinical Investigation. 111(1):71-79. Hirschi, K.K., Burt, J.M., Hirschi, K.D., Dai, C. 2003. Gap junction communication mediates transforming growth factor-beta activation and endothelial-induced mural cell differentiation. Circulation Research. 93(5):429-437. Abramowitz, J., Dai, C., Hirschi, K., Dmitrieva, R.I., Doris, P.A., Liu, L., Allen, J.C. 2003. Ouabain- and marinobufagenin-induced proliferation of human umbilical vein smooth muscle cells and a rat vascular smooth muscle cell line, A7r5. Circulation. 108(24):3048-3053. 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