Micro-engineered capsules for spatial sampling of microbiome in vivo

实施研究摘要 本提案是根据PA-20-227（膳食营养研究管理补充材料）提交的。 补充）。母合同R21 AI 144521的具体目标是：（1）设计、制造和测试微 用于胃肠道取样的工程化可摄取胶囊;（2）在猪中测试胶囊。 母项目旨在证明非侵入性胃肠道采样的可行性， 一个真实的动物模型，并验证可摄入胶囊技术。非侵入性采样 胃肠道将推进肠道微生物组及其有益功能的研究。我们 寻求补充资金，以利用在母基金下进行的研究，研究 膳食补充剂对胃肠道细菌微生物群的影响。虽然肠道 微生物群对各种扰动的反应，如饮食，抗生素或炎症条件， 研究，这项研究的大部分已经在人类和啮齿动物中进行。由于显而易见的原因，大多数 这些研究依赖于对粪便DNA和粪便代谢物的分析，这并不能密切反映出 胃肠道的更近端部分，如空肠、回肠和升结肠。补充资金 将使我们：1）增加猪的数量，以研究3种常用益生元的效果 补充剂（菊粉，果胶，纤维素）对胃肠道不同器官的微生物群的影响，2）扩大 该项目的范围超出了细菌微生物群到肠上皮及其转录 对饮食的反应。为了非侵入性地研究肠上皮对饮食变化的反应，我们 提出使用RNA-Seq来分析“脱落组”，即，脱落上皮细胞的转录组 经粪便排泄。为了评估脱落组的质量，我们将比较脱落组RNA-Seq数据与RNA-测序数据。 在动物已经被消化后，从直接从不同GI器官收集的上皮细胞获得的Seq数据。 牺牲了对猪肠道脱落层的分析旨在评估肠上皮细胞基因的变化是否 表达可通过对粪便cDNA进行测序而非侵入性地检测。虽然补充实验 扩大父母奖励的范围，包括饮食干预，这项研究不依赖于 可摄取胶囊的优化。将利用用于检测胶囊的猪来研究 以下补充具体目的：1）评估菊粉、果胶和纤维素对植物生长的影响， 使用可摄入胶囊在体内取样的细菌微生物群， 2）应用RNA-Seq评估表征转录组的可行性 来自饲喂对照和试验饮食的猪的脱落肠细胞的切片。因此，补充实验将使用 明确的饮食干预，以扩大父母奖的范围在两个方向; 1）评估 饮食对粪便DNA中不存在的肠道细菌的影响; 2）测试非 侵袭性地表征肠上皮细胞转录组及其对饮食扰动的反应。

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.