Microbiome features associated with performance measures in athletic and non-athletic individuals: A case-control study.

The influence of human gut microbiota on health and disease is now commonly appreciated. Therefore, it is not surprising that microbiome research has found interest in the sports community, hoping to improve health and optimize performance. Comparative studies found new species or pathways that were more enriched in elites than sedentary controls. In addition, sport-specific and performance-level-specific microbiome features have been identified. However, the results remain inconclusive and indicate the need for further assessment. In this case-control study, we tested two athletic populations (i.e. strength athletes, endurance athletes) and a non-athletic, but physically active, control group across two acute exercise bouts, separated by a 2-week period, that measured explosive and high intensity fitness level (repeated 30-s all-out Wingate test (WT)) and cardiorespiratory fitness level (Bruce Treadmill Test). While we did not identify any group differences in alpha and beta diversity or significant differential abundance of microbiome components at baseline, one-third of the species identified were unique to each group. Longitudinal sample (pre- and post-exercise) analysis revealed an abundance of Alistipes communis in the strength group during the WT and 88 species with notable between-group differences during the Bruce Test. SparCC recognized Bifidobacterium longum and Bifidobacterium adolescentis, short-chain fatty acid producers with probiotic properties, species strongly associated with VO2max. Ultimately, we identified several taxa with different baseline abundances and longitudinal changes when comparing individuals based on their VO2max, average power, and maximal power parameters. Our results confirmed that the health status of individuals are consistent with assumptions about microbiome health. Furthermore, our findings indicate that microbiome features are associated with better performance previously identified in elite athletes.

Robustness of cancer microbiome signals over a broad range of methodological variation.

In 2020, we identified cancer-specific microbial signals in The Cancer Genome Atlas (TCGA) [1]. Multiple peer-reviewed papers independently verified or extended our findings [2-12]. Given this impact, we carefully considered concerns by Gihawi et al. [13] that batch correction and database contamination with host sequences artificially created the appearance of cancer type-specific microbiomes. (1) We tested batch correction by comparing raw and Voom-SNM-corrected data per-batch, finding predictive equivalence and significantly similar features. We found consistent results with a modern microbiome-specific method (ConQuR [14]), and when restricting to taxa found in an independent, highly-decontaminated cohort. (2) Using Conterminator [15], we found low levels of human contamination in our original databases (~1% of genomes). We demonstrated that the increased detection of human reads in Gihawi et al. [13] was due to using a newer human genome reference. (3) We developed Exhaustive, a method twice as sensitive as Conterminator, to clean RefSeq. We comprehensively host-deplete TCGA with many human (pan)genome references. We repeated all analyses with this and the Gihawi et al. [13] pipeline, and found cancer type-specific microbiomes. These extensive re-analyses and updated methods validate our original conclusion that cancer type-specific microbial signatures exist in TCGA, and show they are robust to methodology.

Alkaline Phosphatase Relieves Colitis in Obese Mice Subjected to Forced Exercise via Its Anti-Inflammatory and Intestinal Microbiota-Shaping Properties.

Intestinal alkaline phosphatase (IAP) is an enzyme that plays a protective role in the gut. This study investigated the effect of IAP treatment on experimental colitis in mice subjected to forced exercise on a high-fat diet. C57BL/6 mice with TNBS colitis were fed a high-fat diet and subjected to forced treadmill exercise with or without IAP treatment. Disease activity, oxidative stress, inflammatory cytokines, and gut microbiota were assessed. Forced exercise exacerbated colitis in obese mice, as evidenced by increased disease activity index (DAI), oxidative stress markers, and proinflammatory adipokines and cytokines. IAP treatment significantly reduced these effects and promoted the expression of barrier proteins in the colonic mucosa. Additionally, IAP treatment altered the gut microbiota composition, favoring beneficial Verrucomicrobiota and reducing pathogenic Clostridia and Odoribacter. IAP treatment ameliorates the worsening effect of forced exercise on murine colitis by attenuating oxidative stress, downregulating proinflammatory biomarkers, and modulating the gut microbiota. IAP warrants further investigation as a potential therapeutic strategy for ulcerative colitis.

Comprehensive Functional Annotation of Metagenomes and Microbial Genomes Using a Deep Learning-Based Method.

Comprehensive protein function annotation is essential for understanding microbiome-related disease mechanisms in the host organisms. However, a large portion of human gut microbial proteins lack functional annotation. Here, we have developed a new metagenome analysis workflow integrating de novo genome reconstruction, taxonomic profiling, and deep learning-based functional annotations from DeepFRI. This is the first approach to apply deep learning-based functional annotations in metagenomics. We validate DeepFRI functional annotations by comparing them to orthology-based annotations from eggNOG on a set of 1,070 infant metagenomes from the DIABIMMUNE cohort. Using this workflow, we generated a sequence catalogue of 1.9 million nonredundant microbial genes. The functional annotations revealed 70% concordance between Gene Ontology annotations predicted by DeepFRI and eggNOG. DeepFRI improved the annotation coverage, with 99% of the gene catalogue obtaining Gene Ontology molecular function annotations, although they are less specific than those from eggNOG. Additionally, we constructed pangenomes in a reference-free manner using high-quality metagenome-assembled genomes (MAGs) and analyzed the associated annotations. eggNOG annotated more genes on well-studied organisms, such as Escherichia coli, while DeepFRI was less sensitive to taxa. Further, we show that DeepFRI provides additional annotations in comparison to the previous DIABIMMUNE studies. This workflow will contribute to novel understanding of the functional signature of the human gut microbiome in health and disease as well as guiding future metagenomics studies. IMPORTANCE The past decade has seen advancement in high-throughput sequencing technologies resulting in rapid accumulation of genomic data from microbial communities. While this growth in sequence data and gene discovery is impressive, the majority of microbial gene functions remain uncharacterized. The coverage of functional information coming from either experimental sources or inferences is low. To solve these challenges, we have developed a new workflow to computationally assemble microbial genomes and annotate the genes using a deep learning-based model DeepFRI. This improved microbial gene annotation coverage to 1.9 million metagenome-assembled genes, representing 99% of the assembled genes, which is a significant improvement compared to 12% Gene Ontology term annotation coverage by commonly used orthology-based approaches. Importantly, the workflow supports pangenome reconstruction in a reference-free manner, allowing us to analyze the functional potential of individual bacterial species. We therefore propose this alternative approach combining deep-learning functional predictions with the commonly used orthology-based annotations as one that could help us uncover novel functions observed in metagenomic microbiome studies.

Sequence-structure-function relationships in the microbial protein universe.

For the past half-century, structural biologists relied on the notion that similar protein sequences give rise to similar structures and functions. While this assumption has driven research to explore certain parts of the protein universe, it disregards spaces that don't rely on this assumption. Here we explore areas of the protein universe where similar protein functions can be achieved by different sequences and different structures. We predict ~200,000 structures for diverse protein sequences from 1,003 representative genomes across the microbial tree of life and annotate them functionally on a per-residue basis. Structure prediction is accomplished using the World Community Grid, a large-scale citizen science initiative. The resulting database of structural models is complementary to the AlphaFold database, with regards to domains of life as well as sequence diversity and sequence length. We identify 148 novel folds and describe examples where we map specific functions to structural motifs. We also show that the structural space is continuous and largely saturated, highlighting the need for a shift in focus across all branches of biology, from obtaining structures to putting them into context and from sequence-based to sequence-structure-function based meta-omics analyses.

Homology Modeling of the G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are therapeutically important family of membrane proteins. Despite growing number of experimental structures available for GPCRs, homology modeling remains a relevant method for studying these receptors and for discovering new ligands for them. Here we describe the state-of-the-art methods for modeling GPCRs, starting from template selection, through fine-tuning sequence alignment to model refinement.

Deep embeddings to comprehend and visualize microbiome protein space.

Understanding the function of microbial proteins is essential to reveal the clinical potential of the microbiome. The application of high-throughput sequencing technologies allows for fast and increasingly cheaper acquisition of data from microbial communities. However, many of the inferred protein sequences are novel and not catalogued, hence the possibility of predicting their function through conventional homology-based approaches is limited, which indicates the need for further research on alignment-free methods. Here, we leverage a deep-learning-based representation of proteins to assess its utility in alignment-free analysis of microbial proteins. We trained a language model on the Unified Human Gastrointestinal Protein catalogue and validated the resulting protein representation on the bacterial part of the SwissProt database. Finally, we present a use case on proteins involved in SCFA metabolism. Results indicate that the deep learning model manages to accurately represent features related to protein structure and function, allowing for alignment-free protein analyses. Technologies that contextualize metagenomic data are a promising direction to deeply understand the microbiome.

Treatment With Multi-Species Probiotics Changes the Functions, Not the Composition of Gut Microbiota in Postmenopausal Women With Obesity: A Randomized, Double-Blind, Placebo-Controlled Study.

Probiotics are known to regulate host metabolism. In randomized controlled trial we aimed to assess whether interventions with probiotic containing following strains: Bifidobacterium bifidum W23, Bifidobacterium lactis W51, Bifidobacterium lactis W52, Lactobacillus acidophilus W37, Levilactobacillus brevis W63, Lacticaseibacillus casei W56, Ligilactobacillus salivarius W24, Lactococcus lactis W19, and Lactococcus lactis W58 affect gut microbiota to promote metabolic effects. By 16S rRNA sequencing we analyzed the fecal microbiota of 56 obese, postmenopausal women randomized into three groups: (1) probiotic dose 2.5 × 109 CFU/day (n = 18), (2) 1 × 1010 CFU/day (n = 18), or (3) placebo (n = 20). In the set of linear mixed-effects models, the interaction between pre- or post-treatment bacterial abundance and time on cardiometabolic parameters was significantly (FDR-adjusted) modified by type of intervention (26 and 19 three-way interactions for the pre-treatment and post-treatment abundance, respectively), indicating the modification of the bio-physiological role of microbiota by probiotics. For example, the unfavorable effects of Erysipelotrichi, Erysipelotrichales, and Erysipelotrichaceae on BMI might be reversed, but the beneficial effect of Betaproteobacteria on BMI was diminished by probiotic treatment. Proinflammatory effect of Bacteroidaceae was alleviated by probiotic administration. However, probiotics did not affect the microbiota composition, and none of the baseline microbiota-related features could predict therapeutic response as defined by cluster analysis. Conclusions: Probiotic intervention alters the influence of microbiota on biochemical, physiological and immunological parameters, but it does not affect diversity and taxonomic composition. Baseline microbiota is not a predictor of therapeutic response to a multispecies probiotic. Further multi-omic and mechanistic studies performed on the bigger cohort of patients are needed to elucidate the cardiometabolic effect of investigated probiotics in postmenopausal obesity.

Individuals with substance use disorders have a distinct oral microbiome pattern.

BACKGROUND: Substance use disorder emerges from a complex interaction between genetic predisposition, life experiences, exposure, and subsequent adaptation of biological systems to the repeated use of drugs. Recently, investigators have proposed that the human microbiota may play a role in brain health and disease. In particular, the human oral microbiome is a distinct and diverse ecological niche with its composition influenced by external factors such as lifestyle, diet, and oral hygiene. This investigation examined whether individuals with substance use disorder (SU) show a different oral microbiome pattern and whether this pattern is sufficient to delineate the SU group from healthy comparison (HC) subjects. METHODS: Participants were a sub-sample (N â€‹= â€‹177) of the Tulsa 1000 (T-1000) project. We analyzed 123 SU and 54 HC subjects using 16S rRNA marker gene sequencing to characterize the oral microbiome. RESULTS: The groups differed significantly based on the UniFrac distance, a phylogenetic-based measure of beta diversity, but did not differ in alpha diversity. Using a machine learning approach, microbiome features combined with socio-demographic variables successfully categorized group membership with 87%-92% accuracy, even after controlling for external factors such as smoking or alcohol consumption. SU individuals with relatively lower diversity also reported higher levels of negative reinforcement experiences associated with their primary substance of abuse. CONCLUSIONS: Oral microbiome features are useful to sufficiently differentiate SU from HC subjects. There is some evidence that subjects whose drug use is driven by negative reinforcement show an impoverished oral microbiome. Taken together, the oral microbiome may help to understand the dysfunctional biological processes that promote substance use or may be pragmatically useful as a risk or severity biological marker.

Structure-based protein function prediction using graph convolutional networks.

The rapid increase in the number of proteins in sequence databases and the diversity of their functions challenge computational approaches for automated function prediction. Here, we introduce DeepFRI, a Graph Convolutional Network for predicting protein functions by leveraging sequence features extracted from a protein language model and protein structures. It outperforms current leading methods and sequence-based Convolutional Neural Networks and scales to the size of current sequence repositories. Augmenting the training set of experimental structures with homology models allows us to significantly expand the number of predictable functions. DeepFRI has significant de-noising capability, with only a minor drop in performance when experimental structures are replaced by protein models. Class activation mapping allows function predictions at an unprecedented resolution, allowing site-specific annotations at the residue-level in an automated manner. We show the utility and high performance of our method by annotating structures from the PDB and SWISS-MODEL, making several new confident function predictions. DeepFRI is available as a webserver at https://beta.deepfri.flatironinstitute.org/ .

Gut microbiome in serious mental illnesses: A systematic review and critical evaluation.

Schizophrenia and bipolar disorder (BD) are associated with debilitating psychiatric and cognitive dysfunction, worse health outcomes, and shorter life expectancies. The pathophysiological understanding of and therapeutic resources for these neuropsychiatric disorders are still limited. Humans harbor over 1000 unique bacterial species in our gut, which have been linked to both physical and mental/cognitive health. The gut microbiome is a novel and promising avenue to understand the attributes of psychiatric diseases and, potentially, to modify them. Building upon our previous work, this systematic review evaluates the most recent evidence of the gut microbiome in clinical populations with serious mental illness (SMI). Sixteen articles that met our selection criteria were reviewed, including cross-sectional cohort studies and longitudinal treatment trials. All studies reported alterations in the gut microbiome of patients with SMI compared to non-psychiatric comparison subjects (NCs), and beta-diversity was consistently reported to be different between schizophrenia and NCs. Ruminococcaceae and Faecalibacterium were relatively decreased in BD, and abundance of Ruminococcaceae was reported across several investigations of SMI to be associated with better clinical characteristics. Lactic acid bacteria were relatively more abundant in SMI and associated with worse clinical outcomes. There was very limited evidence for the efficacy of probiotic or prebiotic interventions in SMI. As microbiome research in psychiatry is still nascent, the extant literature has several limitations. We critically evaluate the current data, including experimental approaches. There is a need for more unified methodological standards in order to arrive at robust biological understanding of microbial contributions to SMI.

Gut microbiome in Schizophrenia: Altered functional pathways related to immune modulation and atherosclerotic risk.

Emerging evidence has linked the gut microbiome changes to schizophrenia. However, there has been limited research into the functional pathways by which the gut microbiota contributes to the phenotype of persons with chronic schizophrenia. We characterized the composition and functional potential of the gut microbiota in 48 individuals with chronic schizophrenia and 48 matched (sequencing plate, age, sex, BMI, and antibiotic use) non-psychiatric comparison subjects (NCs) using 16S rRNA sequencing. Patients with schizophrenia demonstrated significant beta-diversity differences in microbial composition and predicted genetic functional potential compared to NCs. Alpha-diversity of taxa and functional pathways were not different between groups. Random forests analyses revealed that the microbiome predicts differentiation of patients with schizophrenia from NCs using taxa (75% accuracy) and functional profiles (67% accuracy for KEGG orthologs, 70% for MetaCyc pathways). We utilized a new compositionally-aware method incorporating reference frames to identify differentially abundant microbes and pathways, which revealed that Lachnospiraceae is associated with schizophrenia. Functional pathways related to trimethylamine-N-oxide reductase and Kdo2-lipid A biosynthesis were altered in schizophrenia. These metabolic pathways were associated with inflammatory cytokines and risk for coronary heart disease in schizophrenia. Findings suggest potential mechanisms by which the microbiota may impact the pathophysiology of the disease through modulation of functional pathways related to immune signaling/response and lipid and glucose regulation to be further investigated in future studies.

QIIME 2 Enables Comprehensive End-to-End Analysis of Diverse Microbiome Data and Comparative Studies with Publicly Available Data.

QIIME 2 is a completely re-engineered microbiome bioinformatics platform based on the popular QIIME platform, which it has replaced. QIIME 2 facilitates comprehensive and fully reproducible microbiome data science, improving accessibility to diverse users by adding multiple user interfaces. QIIME 2 can be combined with Qiita, an open-source web-based platform, to re-use available data for meta-analysis. The following basic protocol describes how to install QIIME 2 on a single computer and analyze microbiome sequence data, from processing of raw DNA sequence reads through generating publishable interactive figures. These interactive figures allow readers of a study to interact with data with the same ease as its authors, advancing microbiome science transparency and reproducibility. We also show how plug-ins developed by the community to add analysis capabilities can be installed and used with QIIME 2, enhancing various aspects of microbiome analyses-e.g., improving taxonomic classification accuracy. Finally, we illustrate how users can perform meta-analyses combining different datasets using readily available public data through Qiita. In this tutorial, we analyze a subset of the Early Childhood Antibiotics and the Microbiome (ECAM) study, which tracked the microbiome composition and development of 43 infants in the United States from birth to 2 years of age, identifying microbiome associations with antibiotic exposure, delivery mode, and diet. For more information about QIIME 2, see https://qiime2.org. To troubleshoot or ask questions about QIIME 2 and microbiome analysis, join the active community at https://forum.qiime2.org. © 2020 The Authors. Basic Protocol: Using QIIME 2 with microbiome data Support Protocol: Further microbiome analyses.

Microbiome analyses of blood and tissues suggest cancer diagnostic approach.

Systematic characterization of the cancer microbiome provides the opportunity to develop techniques that exploit non-human, microorganism-derived molecules in the diagnosis of a major human disease. Following recent demonstrations that some types of cancer show substantial microbial contributions1-10, we re-examined whole-genome and whole-transcriptome sequencing studies in The Cancer Genome Atlas11 (TCGA) of 33 types of cancer from treatment-naive patients (a total of 18,116 samples) for microbial reads, and found unique microbial signatures in tissue and blood within and between most major types of cancer. These TCGA blood signatures remained predictive when applied to patients with stage Ia-IIc cancer and cancers lacking any genomic alterations currently measured on two commercial-grade cell-free tumour DNA platforms, despite the use of very stringent decontamination analyses that discarded up to 92.3% of total sequence data. In addition, we could discriminate among samples from healthy, cancer-free individuals (n = 69) and those from patients with multiple types of cancer (prostate, lung, and melanoma; 100 samples in total) solely using plasma-derived, cell-free microbial nucleic acids. This potential microbiome-based oncology diagnostic tool warrants further exploration.

IL-4Rα Blockade by Dupilumab Decreases Staphylococcus aureus Colonization and Increases Microbial Diversity in Atopic Dermatitis.

Dupilumab is a fully human antibody to interleukin-4 receptor α that improves the signs and symptoms of moderate to severe atopic dermatitis (AD). To determine the effects of dupilumab on Staphylococcus aureus colonization and microbial diversity on the skin, bacterial DNA was analyzed from swabs collected from lesional and nonlesional skin in a double-blind, placebo-controlled study of 54 patients with moderate to severe AD randomized (1:1) and treated with either dupilumab (200 mg weekly) or placebo for 16 weeks. Microbial diversity and relative abundance of Staphylococcus were assessed by DNA sequencing of 16S ribosomal RNA, and absolute S. aureus abundance was measured by quantitative PCR. Before treatment, lesional skin had lower microbial diversity and higher overall abundance of S. aureus than nonlesional skin. During dupilumab treatment, microbial diversity increased and the abundance of S. aureus decreased. Pronounced changes were seen in nonlesional and lesional skin. Decreased S. aureus abundance during dupilumab treatment correlated with clinical improvement of AD and biomarkers of type 2 immunity. We conclude that clinical improvement of AD that is mediated by interleukin-4 receptor α inhibition and the subsequent suppression of type 2 inflammation is correlated with increased microbial diversity and reduced abundance of S. aureus.

Phylogenomics of 10,575 genomes reveals evolutionary proximity between domains Bacteria and Archaea.

Rapid growth of genome data provides opportunities for updating microbial evolutionary relationships, but this is challenged by the discordant evolution of individual genes. Here we build a reference phylogeny of 10,575 evenly-sampled bacterial and archaeal genomes, based on a comprehensive set of 381 markers, using multiple strategies. Our trees indicate remarkably closer evolutionary proximity between Archaea and Bacteria than previous estimates that were limited to fewer "core" genes, such as the ribosomal proteins. The robustness of the results was tested with respect to several variables, including taxon and site sampling, amino acid substitution heterogeneity and saturation, non-vertical evolution, and the impact of exclusion of candidate phyla radiation (CPR) taxa. Our results provide an updated view of domain-level relationships.

The Microbiome and Its Potential for Pharmacology.

The human microbiota (the microscopic organisms that inhabit us) and microbiome (their genes) hold considerable potential for improving pharmacological practice. Recent advances in multi-"omics" techniques have dramatically improved our understanding of the constituents of the microbiome and their functions. The implications of this research for human health, including microbiome links to obesity, drug metabolism, neurological diseases, cancer, and many other health conditions, have sparked considerable interest in exploiting the microbiome for targeted therapeutics. Links between microbial pathways and disease states further highlight a rich potential for companion diagnostics and precision medicine approaches. For example, the success of fecal microbiota transplantation to treat Clostridium difficile infection has already started to redefine standard of care with a microbiome-directed therapy. In this review we briefly discuss the nature of human microbial ecosystems and with pathologies and biological processes linked to the microbiome. We then review emerging computational metagenomic, metabolomic, and wet lab techniques researchers are using today to learn about the roles host-microbial interactions have with respect to pharmacological purposes and vice versa. Finally, we describe how drugs affect the microbiome, how the microbiome can impact drug response in different people, and the potential of the microbiome itself as a source of new therapeutics.

The impact of skin care products on skin chemistry and microbiome dynamics.

BACKGROUND: Use of skin personal care products on a regular basis is nearly ubiquitous, but their effects on molecular and microbial diversity of the skin are unknown. We evaluated the impact of four beauty products (a facial lotion, a moisturizer, a foot powder, and a deodorant) on 11 volunteers over 9 weeks. RESULTS: Mass spectrometry and 16S rRNA inventories of the skin revealed decreases in chemical as well as in bacterial and archaeal diversity on halting deodorant use. Specific compounds from beauty products used before the study remain detectable with half-lives of 0.5-1.9 weeks. The deodorant and foot powder increased molecular, bacterial, and archaeal diversity, while arm and face lotions had little effect on bacterial and archaeal but increased chemical diversity. Personal care product effects last for weeks and produce highly individualized responses, including alterations in steroid and pheromone levels and in bacterial and archaeal ecosystem structure and dynamics. CONCLUSIONS: These findings may lead to next-generation precision beauty products and therapies for skin disorders.