Metabolic diversity in commensal protists regulates intestinal immunity and trans-kingdom competition.

The microbiota influences intestinal health and physiology, yet the contributions of commensal protists to the gut environment have been largely overlooked. Here, we discover human- and rodent-associated parabasalid protists, revealing substantial diversity and prevalence in nonindustrialized human populations. Genomic and metabolomic analyses of murine parabasalids from the genus Tritrichomonas revealed species-level differences in excretion of the metabolite succinate, which results in distinct small intestinal immune responses. Metabolic differences between Tritrichomonas species also determine their ecological niche within the microbiota. By manipulating dietary fibers and developing in vitro protist culture, we show that different Tritrichomonas species prefer dietary polysaccharides or mucus glycans. These polysaccharide preferences drive trans-kingdom competition with specific commensal bacteria, which affects intestinal immunity in a diet-dependent manner. Our findings reveal unappreciated diversity in commensal parabasalids, elucidate differences in commensal protist metabolism, and suggest how dietary interventions could regulate their impact on gut health.

Ultra-deep sequencing of Hadza hunter-gatherers recovers vanishing gut microbes.

The gut microbiome modulates immune and metabolic health. Human microbiome data are biased toward industrialized populations, limiting our understanding of non-industrialized microbiomes. Here, we performed ultra-deep metagenomic sequencing on 351 fecal samples from the Hadza hunter-gatherers of Tanzania and comparative populations in Nepal and California. We recovered 91,662 genomes of bacteria, archaea, bacteriophages, and eukaryotes, 44% of which are absent from existing unified datasets. We identified 124 gut-resident species vanishing in industrialized populations and highlighted distinct aspects of the Hadza gut microbiome related to in situ replication rates, signatures of selection, and strain sharing. Industrialized gut microbes were found to be enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. This unparalleled view of the Hadza gut microbiome provides a valuable resource, expands our understanding of microbes capable of colonizing the human gut, and clarifies the extensive perturbation induced by the industrialized lifestyle.

A microbiome-targeting fibre-enriched nutritional formula is well tolerated and improves quality of life and haemoglobin A1c in type 2 diabetes: A double-blind, randomized, placebo-controlled trial.

AIMS: To investigate a prebiotic fibre-enriched nutritional formula on health-related quality of life and metabolic control in type 2 diabetes. MATERIALS AND METHODS: This was a 12-week, double-blind, placebo-controlled study with an unblinded dietary advice only comparator arm. Participants were randomized 2:1:1 to a prebiotic fibre-enriched nutritional formula (Active), a placebo fibre-absent nutritional formula (Placebo), or non-blinded dietary advice alone (Diet). Primary endpoint was change in core Type 2 Diabetes Distress Assessment System (cT2-DDAS) at week 12. Glycated haemoglobin (HbA1c) change was a key secondary endpoint. RESULTS: In total, 192 participants were randomized. Mean age was 54.3 years, HbA1c 7.8%, and body mass index 35.9 kg/m2 . At week 12, cT2-DDAS reduced significantly in Active versus Placebo (-0.4, p = .03), and HbA1c was reduced significantly in Active vs Placebo (-0.64%, p = .01). Gut microbiome sequencing revealed that the relative abundance of two species of butyrate-producing bacteria (Roseburia faecis and Anaerostipes hadrus) increased significantly in Active vs. Placebo. CONCLUSIONS: A microbiome-targeting nutritional formula significantly improved cT2-DDAS and HbA1c, suggesting the potential for prebiotic fibre as a complement to lifestyle and/or pharmaceutical interventions for managing type 2 diabetes.

Butyrate Differentiates Permissiveness to Clostridioides difficile Infection and Influences Growth of Diverse C. difficile Isolates.

A disrupted "dysbiotic" gut microbiome engenders susceptibility to the diarrheal pathogen Clostridioides difficile by impacting the metabolic milieu of the gut. Diet, in particular the microbiota-accessible carbohydrates (MACs) found in dietary fiber, is one of the most powerful ways to affect the composition and metabolic output of the gut microbiome. As such, diet is a powerful tool for understanding the biology of C. difficile and for developing alternative approaches for coping with this pathogen. One prominent class of metabolites produced by the gut microbiome is short-chain fatty acids (SCFAs), the major metabolic end products of MAC metabolism. SCFAs are known to decrease the fitness of C. difficile in vitro, and high intestinal SCFA concentrations are associated with reduced fitness of C. difficile in animal models of C. difficile infection (CDI). Here, we use controlled dietary conditions (8 diets that differ only by MAC composition) to show that C. difficile fitness is most consistently impacted by butyrate, rather than the other two prominent SCFAs (acetate and propionate), during murine model CDI. We similarly show that butyrate concentrations are lower in fecal samples from humans with CDI than in those from healthy controls. Finally, we demonstrate that butyrate impacts growth in diverse C. difficile isolates. These findings provide a foundation for future work which will dissect how butyrate directly impacts C. difficile fitness and will lead to the development of diverse approaches distinct from antibiotics or fecal transplant, such as dietary interventions, for mitigating CDI in at-risk human populations. IMPORTANCE Clostridioides difficile is a leading cause of infectious diarrhea in humans, and it imposes a tremendous burden on the health care system. Current treatments for C. difficile infection (CDI) include antibiotics and fecal microbiota transplant, which contribute to recurrent CDIs and face major regulatory hurdles, respectively. Therefore, there is an ongoing need to develop new ways to cope with CDI. Notably, a disrupted "dysbiotic" gut microbiota is the primary risk factor for CDI, but we incompletely understand how a healthy microbiota resists CDI. Here, we show that a specific molecule produced by the gut microbiota, butyrate, is negatively associated with C. difficile burdens in humans and in a mouse model of CDI and that butyrate impedes the growth of diverse C. difficile strains in pure culture. These findings help to build a foundation for designing alternative, possibly diet-based, strategies for mitigating CDI in humans.

Ultra-deep Sequencing of Hadza Hunter-Gatherers Recovers Vanishing Gut Microbes.

The gut microbiome is a key modulator of immune and metabolic health. Human microbiome data is biased towards industrialized populations, providing limited understanding of the distinct and diverse non-industrialized microbiomes. Here, we performed ultra-deep metagenomic sequencing and strain cultivation on 351 fecal samples from the Hadza, hunter-gatherers in Tanzania, and comparative populations in Nepal and California. We recover 94,971 total genomes of bacteria, archaea, bacteriophages, and eukaryotes, 43% of which are absent from existing unified datasets. Analysis of in situ growth rates, genetic pN/pS signatures, high-resolution strain tracking, and 124 gut-resident species vanishing in industrialized populations reveals differentiating dynamics of the Hadza gut microbiome. Industrialized gut microbes are enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. This unparalleled view of the Hadza gut microbiome provides a valuable resource that expands our understanding of microbes capable of colonizing the human gut and clarifies the extensive perturbation brought on by the industrialized lifestyle.

Assessing the effects of alternative plant-based meats v. animal meats on biomarkers of inflammation: a secondary analysis of the SWAP-MEAT randomized crossover trial.

Alternative plant-based meats have grown in popularity with consumers recently and researchers are examining the potential health effects, or risks, from consuming these products. Because there have been no studies to date that have specifically assessed the health effects of plant-based meats on biomarkers of inflammation, the purpose of this work was to conduct a secondary analysis of the Study With Appetizing Plantfood - Meat Eating Alternatives Trial (SWAP-MEAT). SWAP-MEAT was a randomised crossover trial that involved generally healthy adults eating 2 or more servings of plant-based meats per day for 8 weeks (i.e. Plant phase) followed by 2 or more servings of animal meats per day for 8 weeks (i.e. Animal phase). Results of linear mixed-effects models indicated only 4 out of 92 biomarkers reached statistical significance. The results were contrary to our hypothesis, since we expected relative improvements in biomarkers of inflammation from the plant-based meats.

Robust variation in infant gut microbiome assembly across a spectrum of lifestyles.

Infant microbiome assembly has been intensely studied in infants from industrialized nations, but little is known about this process in nonindustrialized populations. We deeply sequenced infant stool samples from the Hadza hunter-gatherers of Tanzania and analyzed them in a global meta-analysis. Infant microbiomes develop along lifestyle-associated trajectories, with more than 20% of genomes detected in the Hadza infant gut representing novel species. Industrialized infants-even those who are breastfed-have microbiomes characterized by a paucity of Bifidobacterium infantis and gene cassettes involved in human milk utilization. Strains within lifestyle-associated taxonomic groups are shared between mother-infant dyads, consistent with early life inheritance of lifestyle-shaped microbiomes. The population-specific differences in infant microbiome composition and function underscore the importance of studying microbiomes from people outside of wealthy, industrialized nations.

The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans.

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

A randomized crossover trial on the effect of plant-based compared with animal-based meat on trimethylamine-N-oxide and cardiovascular disease risk factors in generally healthy adults: Study With Appetizing Plantfood-Meat Eating Alternative Trial (SWAP-MEAT).

BACKGROUND: Despite the rising popularity of plant-based alternative meats, there is limited evidence of the health effects of these products. OBJECTIVES: We aimed to compare the effect of consuming plant-based alternative meat (Plant) as opposed to animal meat (Animal) on health factors. The primary outcome was fasting serum trimethylamine-N-oxide (TMAO). Secondary outcomes included fasting insulin-like growth factor 1, lipids, glucose, insulin, blood pressure, and weight. METHODS: SWAP-MEAT (The Study With Appetizing Plantfood-Meat Eating Alternatives Trial) was a single-site, randomized crossover trial with no washout period. Participants received Plant and Animal products, dietary counseling, lab assessments, microbiome assessments (16S), and anthropometric measurements. Participants were instructed to consume ≥2 servings/d of Plant compared with Animal for 8 wk each, while keeping all other foods and beverages as similar as possible between the 2 phases. RESULTS: The 36 participants who provided complete data for both crossover phases included 67% women, were 69% Caucasian, had a mean ± SD age 50 ± 14 y, and BMI 28 ± 5 kg/m2. Mean ± SD servings per day were not different by intervention sequence: 2.5 ± 0.6 compared with 2.6 ± 0.7 for Plant and Animal, respectively (P = 0.76). Mean ± SEM TMAO concentrations were significantly lower overall for Plant (2.7 ± 0.3) than for Animal (4.7 ± 0.9) (P = 0.012), but a significant order effect was observed (P = 0.023). TMAO concentrations were significantly lower for Plant among the n = 18 who received Plant second (2.9 ± 0.4 compared with 6.4 ± 1.5, Plant compared with Animal, P = 0.007), but not for the n = 18 who received Plant first (2.5 ± 0.4 compared with 3.0 ± 0.6, Plant compared with Animal, P = 0.23). Exploratory analyses of the microbiome failed to reveal possible responder compared with nonresponder factors. Mean ± SEM LDL-cholesterol concentrations (109.9 ± 4.5 compared with 120.7 ± 4.5 mg/dL, P = 0.002) and weight (78.7 ± 3.0 compared with 79.6 ± 3.0 kg, P < 0.001) were lower during the Plant phase. CONCLUSIONS: Among generally healthy adults, contrasting Plant with Animal intake, while keeping all other dietary components similar, the Plant products improved several cardiovascular disease risk factors, including TMAO; there were no adverse effects on risk factors from the Plant products.This trial was registered at clinicaltrials.gov as NCT03718988.

Mapping the genomic landscape of CRISPR-Cas9 cleavage.

RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.

The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits.