ABSTRACT OF SUPPLEMENTAL RESEARCH This proposal is submitted in response to PA-20-227 (Administrative Supplements for Research on Dietary Supplements). The Specific Aims of parent award R21 AI144521 are (1) to design, fabricate and test micro- engineered ingestible capsules for sampling the gastro-intestinal tract; (2) to test the capsules in pigs. The parent project seeks to demonstrate the feasibility of non-invasively sampling the gastro-intestinal tract in a realistic animal model and to validate the ingestible capsule technology. Non-invasive sampling of the gastro-intestinal tract will advance the study of the intestinal microbiome and its beneficial functions. We are seeking supplemental funding to leverage research conducted under the parent award to study the effect of dietary supplements on the bacterial microbiota populating the gastro-intestinal tract. Although the intestinal microbiota's response to various perturbations, like diet, antibiotics or inflammatory conditions has been studied, the bulk of this research has been conducted in humans and rodents. For obvious reasons, most of these studies rely on the analysis of fecal DNA and fecal metabolites, which do not closely mirror changes in more proximal sections of the GI tract, like the jejunum, ileum and ascending colon. Supplemental funding would allow us to: 1) increase the number of pigs to study the effect of 3 commonly used prebiotic supplements (inulin, pectin, cellulose) on the microbiota populating different organs of the GI tract, 2) expand the scope of the project beyond the bacterial microbiota to the intestinal epithelium and its transcriptional response to diet. To non-invasively investigate the response of the intestinal epithelium to changes in diet, we are proposing to use RNA-Seq to analyze the "exfoliome", i.e., the transcriptome of exfoliated epithelial cells excreted in feces. To assess the quality of the exfoliome, we will compare exfoliome RNA-Seq data with RNA- Seq data obtained from epithelial cells collected directly from different GI organs after the animal has been sacrificed. The analysis of the pig intestinal exfoliome seeks to evaluate whether changes in enterocyte gene expression are detectable non-invasively by sequencing fecal cDNA. Although supplementary experiments expand the scope of the parent award to include dietary intervention, this research is not dependent on the optimization of the ingestible capsule. Pigs used for testing the capsules will be leveraged to investigate the following Supplemental Specific Aims: 1) to assess the impact of inulin, pectin and cellulose on the bacterial microbiota sampled in vivo using an ingestible capsule and collected post-mortem from different GI organs; 2) to apply RNA-Seq to evaluate the feasibility of characterizing the transcriptome of exfoliated enterocytes from pigs fed control and test diet. Thus, supplemental experiments will use a well-defined dietary intervention to expand the scope of the parent award in two directions; 1) to assess the effect of diet on commensal bacterial which are not present in fecal DNA; 2) to test the feasibility of non- invasively characterizing the enterocyte transcriptome and its response to dietary perturbation.

We are proposing supplemental experiments to leverage ongoing research aiming to develop a non- invasive method to sample the gastro-intestinal tract of a live animal. Supplemental funding will allow us to evaluate how the bacterial populations in the intestine respond to a change in diet and test a novel method for its ability to detect the response of intestinal cells to such changes.