OBJECTIVES/GOALS: To determine in vitro mechanisms by which fruits and vegetables (FV) contribute to colon barrier development in Latin American infants. We hypothesize that simulated colonic fermentation of FVs will stimulatein vitro cell barrier function by activating the hypoxia-inducible factor (HIF) pathway in colonocytes. METHODS/STUDY POPULATION: FVs consumed by US-based Latin American infants 6-12 months old (identified from NHANES-What We Eat in America Surveys) will be combined with human breast-milk samples from women self-identified as Hispanic or non-Hispanic, and then subjected to in vitro digestion and anaerobic colonic fermentation using human feces. FV fermenta will be incubated with Caco2 monolayers to measure in vitro cell permeability and protein levels of cellular tight junction, metabolic, and HIF signaling enzymes. To examine their effects in vivo, FVs identified to modulate in vitro barrier function, will be fed (5% freeze dried powder) to wild-type mice and the above parameters will be examined. If in vivo effects are found, intestinal specific HIF knockout mice will be used to examine the role of HIF signaling in mediating these effects. RESULTS/ANTICIPATED RESULTS: We expect that fermenta derived from human milk and FVs will reduce in vitro gut permeability in Caco2 monolayers by increasing gene and protein expression of the HIF signaling complex relative to fermenta of human milk alone. This will be reflected with higher cellular trans-epithelial resistance and greater expression levels of tight junction proteins. We expect FV powder consumption will similarly increase in vivo gut permeability and expression of related genes in mice as compared to mice fed diets without FVs. As we expect an increase in HIF signaling in the colon, we expect that FV powder consumption will not enhance in vivo gut permeability in mice colons with an intestinal specific knockout of HIF. DISCUSSION/SIGNIFICANCE: Data from this study will provide mechanistic evidence to help clinicians promote relevant FVs recommendations for Latin American infants and families. Due to the link between gut permeability and obesity, our next step will be to conduct a dietary intervention in this population.