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Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore, the objective was to study the effects of SCFAs on biomarkers of ISC activity, differentiation, barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET), propionate (PROP), or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments, but increased with ACET. As a result of BUT and PROP treatments, there was an increase in differentiation markers for enterocyte, Paneth, goblet, and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3β, Reg3γ, and Defb1 were stimulated by BUT and PROP, but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures, although tight junctions were influenced by BUT and PROP, but not ACET in monolayer format. Furthermore, BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall, individual SCFAs are potent stimulators of cellular gene expression, however, PROP and especially BUT show great efficacy for driving cell differentiation and gene expression.

Background Interactions between diet, stress and the gut microbiome are of interest as a means to modulate health and performance. Here, in vitro fermentation was used to explore the effects of a sudden change in diet, 21 days sole sustenance on the Meal, Ready-to-Eat (MRE) U.S. military combat ration, on inter-species competition and functional potential of the human gut microbiota. Human fecal samples collected before and after MRE intervention or consuming a habitual diet (HAB) were introduced to nutrient-rich media supplemented with starch for in vitro fermentation under ascending colon conditions. 16S rRNA amplicon and Whole-metagenome sequencing (WMS) were used to measure community composition and functional potential. Specific statistical analyses were implemented to detect changes in relative abundance from taxa, genes and pathways. Results Differential changes in relative abundance of 11 taxa, Dorea, Lachnospira , Bacteroides fragilis , Akkermansia muciniphila , Bifidobacterium adolescentis , Betaproteobacteria , Enterobacteriaceae , Bacteroides egerthii , Ruminococcus bromii , Prevotella , and Slackia, and nine Carbohydrate-Active Enzymes, specifically GH13_14, over the 24 h fermentation were observed as a function of the diet intervention and correlated to specific taxa of interest. Conclusions These findings suggest that consuming MRE for 21 days acutely effects changes in gut microbiota structure in response to carbohydrate but may induce alterations in metabolic capacity. Additionally, these findings demonstrate the potential of starch as a candidate supplemental strategy to functionally modulate specific gut commensals during stress-induced states.

Previous studies on capsaicin, the bioactive compound in chili peppers, have shown that it may have a beneficial effect in vivo when part of a regular diet. These positive health benefits, including an anti-inflammatory potential and protective effects against obesity, are often attributed to the gut microbial community response to capsaicin. However, there is no consensus on the mechanism behind the protective effect of capsaicin. In this study, we used an in vitro model of the human gut microbiota to determine how regular consumption of capsaicin impacts the gut microbiota. Using a combination of NextGen sequencing and metabolomics, we found that regular capsaicin treatment changed the structure of the gut microbial community by increasing diversity and certain SCFA abundances, particularly butanoic acid. Through this study, we determined that the addition of capsaicin to the in vitro cultures of the human gut microbiome resulted in increased diversity of the microbial community and an increase in butanoic acid. These changes may be responsible for the health benefits associated with CAP consumption.

The consumption of probiotics is widely encouraged due to reports of their positive effects on human health. In particular, Lacticaseibacillus rhamnosus strain GG (LGG) is an approved probiotic that has been reported to improve health outcomes, especially for gastrointestinal disorders. However, how LGG cooperates with the gut microbiome has not been fully explored. To understand the interaction between LGG and its ability to survive and grow within the gut microbiome, this study introduced LGG into established microbial communities using an in vitro model of the colon. LGG was inoculated into the simulated ascending colon and its persistence in, and transit through the subsequent transverse and descending colon regions was monitored over two weeks. The impact of LGG on the existing bacterial communities was investigated using 16S rRNA sequencing and short-chain fatty acid analysis. LGG was able to engraft and proliferate in the ascending region for at least 10 days but was diminished in the transverse and descending colon regions with little effect on short-chain fatty acid abundance. These data suggest that the health benefits of the probiotic LGG rely on its ability to transiently engraft and modulate the host microbial community.

Spider silks are protein-based \"biopolymer\" filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to \"biomimic\" the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

The host–microbe interaction is critical for intestinal homeostasis. By‐products from microbial metabolism of unabsorbed dietary components have been studied increasingly as potential contributors to health and disease. In vitro fermentation systems provide a way to simulate microbial activity and by‐product production of the colon using human fecal samples. Objectives of the study were to determine how clarified supernatants from two different fermentation conditions affect markers of cell proliferation, differentiation, barrier function, and immune function in a human‐induced pluripotent (iPSC) colon organoid model. SCFA and BCFA's of the supernatants were analyzed and were similar to known in vivo concentrations. Molecular results showed 25% of the clarified supernatant from batch fermentation led to a more physiological intestinal phenotype including increased markers of differentiation, including alkaline phosphatase, chromogranin A, SCFA transport monocarboxylate transporter‐1, (6.2‐fold, 2.1‐fold, and 1.8‐fold, respectively; p < 0.05). Mucin production (mucin‐2, mucin‐4) was increased in cells treated with 25% supernatant, as observed by confocal microscopy. In addition, increased tight junction expression (claudin‐3) was noted by immunofluorescence in 25% supernatant‐ treated cells. A dose–response increase in barrier function was observed over the 72‐h time course, with a twofold increase in transepithelial electrical resistance (TER) in the 25% group compared to the control group (p < 0.05). To further investigate host effects, clarified supernatants from a continuous multistage fermentation representing the ascending (AC), transverse (TC), and descending (DC) colonic domains were utilized and some regional differences were observed including increased markers of inflammation (IL‐1β, 6.15 pg/ml; IL‐6, 27.58 pg/ml; TNFα, 4.49 pg/ml; p < 0.05) in DC‐treated samples only. Overall, clarified supernatants represent a valuable model to examine effects of microbial by‐products on host intestinal development and function and future efforts will be designed to further understand microbial communities and metabolites, along with additional host response measures.

In the present research, we investigated changes in the gut metabolome that occurred in response to the administration of the Laticaseibacillus rhamnosus strain GG (LGG). The probiotics were added to the ascending colon region of mature microbial communities established in a human intestinal microbial ecosystem simulator. Shotgun metagenomic sequencing and metabolome analysis suggested that the changes in microbial community composition corresponded with changes to metabolic output, and we can infer linkages between some metabolites and microorganisms. The in vitro method permits a spatially-resolved view of metabolic transformations under human physiological conditions. By this method, we found that tryptophan and tyrosine were mainly produced in the ascending colon region, while their derivatives were detected in the transverse and descending regions, revealing sequential amino acid metabolic pathways along with the colonic tract. The addition of LGG appeared to promote the production of indole propionic acid, which is positively associated with human health. Furthermore, the microbial community responsible for the production of indole propionic acid may be broader than is currently known.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among DoD organizations and to facilitate resource, material and information sharing among consortium members, which includes collaborators in academia and industry. The 2023 annual symposium was a hybrid meeting held in Washington DC on 26–27 September 2023 concurrent with the virtual attendance, with oral and poster presentations and discussions centered on microbiome-related topics within five broad thematic areas: 1) Environmental Microbiome Characterization; 2) Microbiome Analysis; 3) Human Microbiome Characterization; 4) Microbiome Engineering; and 5) In Vitro and In Vivo Microbiome Models. Collectively, the symposium provided an update on the scope of current DoD and DoD-affiliated microbiome research efforts and fostered collaborative opportunities. This report summarizes the presentations and outcomes of the 7th annual TSMC symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among DoD organizations and to facilitate resource, material and information sharing amongst consortium members, which includes collaborators in academia and industry. The 6th Annual TSMC Symposium was a hybrid meeting held in Fairlee, Vermont on 27–28 September 2022 with presentations and discussions centered on microbiome-related topics within seven broad thematic areas: (1) Human Microbiomes: Stress Response; (2) Microbiome Analysis & Surveillance; (3) Human Microbiomes Enablers & Engineering; (4) Human Microbiomes: Countermeasures; (5) Human Microbiomes Discovery - Earth & Space; (6) Environmental Micro & Myco-biome; and (7) Environmental Microbiome Analysis & Engineering. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the activities and outcomes from the 6th annual TSMC symposium.

Polyphenols and fermentable fibers have shown favorable effects on gut microbiota composition and metabolic function. However, few studies have investigated whether combining multiple fermentable fibers or polyphenols may have additive beneficial effects on gut microbial states. Here, an in vitro fermentation model, seeded with human stool combined from 30 healthy volunteers, was supplemented with blends of polyphenols (PP), dietary fibers (FB), or their combination (PPFB) to determine influence on gut bacteria growth dynamics and select metabolite changes. PP and FB blends independently led to significant increases in the absolute abundance of select beneficial taxa, namely Ruminococcus bromii, Bifidobacterium spp., Lactobacillus spp., and Dorea spp. Total short-chain fatty acid concentrations, relative to non-supplemented control (F), increased significantly with PPFB and FB supplementation but not PP. Indole and ammonia concentrations decreased with FB and PPFB supplementation but not PP alone while increased antioxidant capacity was only evident with both PP and PPFB supplementation. These findings demonstrated that, while the independent blends displayed selective positive impacts on gut states, the combination of both blends provided an additive effect. The work outlines the potential of mixed substrate blends to elicit a broader positive influence on gut microbial composition and function to build resiliency toward dysbiosis.