The duck gastrointestinal tract (GIT) harbors an abundance of microorganisms that play an important role in duck health and production. Here, we constructed the first relatively comprehensive duck gut microbial gene catalog (24 million genes) and 4437 metagenome-assembled genomes using 375 GIT metagenomic samples from four different duck breeds across five intestinal segments under two distinct rearing conditions. We further characterized the intestinal region-specific microbial taxonomy and their assigned functions, as well as the temporal development and maturation of the duck gut microbiome. Our metagenomic analysis revealed the similarity within the microbiota of the foregut and hindgut compartments, but distinctive taxonomic and functional differences between distinct intestinal segments. In addition, we found a significant shift in the microbiota composition of newly hatched ducks (3 days), followed by increased diversity and enhanced stability across growth stages (14, 42, and 70 days), indicating that the intestinal microbiota develops into a relatively mature and stable community as the host duck matures. Comparing the impact of different rearing conditions (with and without water) on duck cecal microbiota communities and functions, we found that the bacterial capacity for lipopolysaccharide biosynthesis was significantly increased in ducks that had free access to water, leading to the accumulation of pathogenic bacteria and antibiotic-resistance genes. Taken together, our findings expand the understanding of the microbiome signatures linked to intestinal regional, temporal development, and rearing conditions in ducks, which highlight the significant impact of microbiota on poultry health and production.

The life cycle of genome builds spans interlocking pillars of assembly, annotation, and comparative genomics to drive biological insights. While tools exist to address each pillar separately, there is a growing need for tools to integrate different pillars of a genome project holistically. For example, comparative approaches can provide quality control of assembly or annotation; genome assembly, in turn, can help to identify artifacts that may complicate the interpretation of genome comparisons. The JCVI library is a versatile Python-based library that offers a suite of tools that excel across these pillars. Featuring a modular design, the JCVI library provides high-level utilities for tasks such as format parsing, graphics generation, and manipulation of genome assemblies and annotations. Supporting genomics algorithms like MCscan and ALLMAPS are widely employed in building genome releases, producing publication-ready figures for quality assessment and evolutionary inference. Developed and maintained collaboratively, the JCVI library emphasizes quality and reusability.

Rapid and accurate diagnostic tests are fundamental for improving patient outcomes and combating infectious diseases. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas12a-based detection system has emerged as a promising solution for on-site nucleic acid testing. Nonetheless, the effective design of CRISPR RNA (crRNA) for Cas12a-based detection remains challenging and time-consuming. In this study, we propose an enhanced crRNA design system with deep learning for Cas12a-mediated diagnostics, referred to as EasyDesign. This system employs an optimized convolutional neural network (CNN) prediction model, trained on a comprehensive data set comprising 11,496 experimentally validated Cas12a-based detection cases, encompassing a wide spectrum of prevalent pathogens, achieving Spearman's ρ = 0.812. We further assessed the model performance in crRNA design for four pathogens not included in the training data: Monkeypox Virus, Enterovirus 71, Coxsackievirus A16, and Listeria monocytogenes. The results demonstrated superior prediction performance compared to the traditional experiment screening. Furthermore, we have developed an interactive web server (https://crispr.zhejianglab.com/) that integrates EasyDesign with recombinase polymerase amplification (RPA) primer design, enhancing user accessibility. Through this web-based platform, we successfully designed optimal Cas12a crRNAs for six human papillomavirus (HPV) subtypes. Remarkably, all the top five predicted crRNAs for each HPV subtype exhibited robust fluorescent signals in CRISPR assays, thereby suggesting that the platform could effectively facilitate clinical sample testing. In conclusion, EasyDesign offers a rapid and reliable solution for crRNA design in Cas12a-based detection, which could serve as a valuable tool for clinical diagnostics and research applications.

Hundreds of microbiota gene expressions are significantly different between healthy and diseased humans. The “bottleneck” preventing a mechanistic dissection of how they affect host biology/disease is that many genes are encoded by nonmodel gut commensals and not genetically manipulatable. Approaches to efficiently identify their gene transfer methodologies and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. This paper will introduce a step-by-step protocol to identify gene transfer conditions and build the gene manipulation tools for nonmodel gut microbes, focusing on Gram-negative Bacteroidia and Gram-positive Clostridia organisms. This protocol enables us to identify gene transfer methods and develop gene manipulation tools without prior knowledge of their genome sequences, by targeting bacterial 16s ribosomal RNAs or expanding their compatible replication origins combined with clustered regularly interspaced short palindromic repeats machinery. Such an efficient and generalizable approach will facilitate functional studies that causally connect gut microbiota genes to host diseases.

Emerging evidence has demonstrated the profound impact of the gut microbiome on cardiovascular diseases through the production of diverse metabolites. Using an animal model of myocardial ischemia–reperfusion (I/R) injury, we found that the prophylactic administration of a well-known probiotic, Bifidobacterium infantis (B. infantis), exhibited cardioprotective effects in terms of preserving cardiac contractile function and preventing adverse cardiac remodeling following I/R and that these cardioprotective effects were recapitulated by its metabolite inosine. Transcriptomic analysis further revealed that inosine mitigated I/R-induced cardiac inflammation and cell death. Mechanistic investigations elucidated that inosine suppressed the production of pro-inflammatory cytokines and reduced the numbers of dendritic cells and natural killer cells, achieved through the activation of the adenosine A2A receptor (A2AR) that when inhibited abrogated the cardioprotective effects of inosine. Additionally, in vitro studies using C2C12 myoblasts revealed that inosine attenuated cell death by serving as an alternative carbon source for adenosine triphosphate (ATP) generation through the purine salvage pathway when subjected to oxygen-glucose deprivation/reoxygenation that simulated myocardial I/R injury. Likewise, inosine reversed the I/R-induced decrease in ATP levels in mouse hearts. Taken together, our findings indicate that B. infantis or its metabolite inosine exerts cardioprotective effects against I/R by suppressing cardiac inflammation and attenuating cardiac cell death, suggesting prophylactic therapeutic options for acute ischemic cardiac injury.

Functional cure for chronic hepatitis B (CHB) remains challenging due to the lack of direct intervention methods for hepatic inflammation. Multi-omics research offers a promising approach to understand hepatic inflammation mechanisms in CHB. A Bayesian linear model linked gene expression with clinical parameters, and population-specific expression analysis (PSEA) refined bulk gene expression into specific cell types across different clinical phases. These models were integrated into our analysis of key factors like inflammatory cells, immune activation, T cell exhaustion, chemokines, receptors, and interferon-stimulated genes (ISGs). Validation through multi-immune staining in liver specimens from CHB patients bolstered our findings. In CHB patients, increased gene expression related to immune cell activation and migration was noted. Marker genes of macrophages, T cells, immune-negative regulators, chemokines, and ISGs showed a positive correlation with serum alanine aminotransferase (ALT) levels but not hepatitis B virus DNA levels. The PSEA model confirmed T cells as the source of exhausted regulators, while macrophages primarily contributed to chemokine expression. Upregulated ISGs (ISG20, IFI16, TAP2, GBP1, PSMB9) in the hepatitis phase were associated with T cell and macrophage infiltration and positively correlated with ALT levels. Conversely, another set of ISGs (IFI44, ISG15, IFI44L, IFI6, MX1) mainly expressed by hepatocytes and B cells showed no correlation with ALT levels. Our study presents a multi-omics analysis integrating bulk transcriptomic, single-cell sequencing data, and clinical data from CHB patients to decipher the cause of intrahepatic inflammation in CHB. The findings confirm that macrophages secrete chemokines like CCL20, recruiting exhausted T cells into liver tissue; concurrently, hepatocyte innate immunity is suppressed, hindering the antiviral effects of ISGs.

Dysbiosis of the gut microbiota has been implicated in hypertension, and drug–host–microbiome interactions have drawn considerable attention. However, the influence of angiotensin receptor blocker (ARB)-shaped gut microbiota on the host is not fully understood. In this work, we assessed the alterations of blood pressure (BP), vasculatures, and intestines following ARB-modified gut microbiome treatment and evaluated the changes in the intestinal transcriptome and serum metabolome in hypertensive rats. Hypertensive patients with well-controlled BP under ARB therapy were recruited as human donors, spontaneously hypertensive rats (SHRs) receiving normal saline or valsartan were considered animal donors, and SHRs were regarded as recipients. Histological and immunofluorescence staining was used to assess the aorta and small intestine, and 16S rRNA amplicon sequencing was performed to examine gut bacteria. Transcriptome and metabonomic analyses were conducted to determine the intestinal transcriptome and serum metabolome, respectively. Notably, ARB-modified fecal microbiota transplantation (FMT), results in marked decreases in systolic BP levels, collagen deposition and reactive oxygen species accumulation in the vasculature, and alleviated intestinal structure impairments in SHRs. These changes were linked with the reconstruction of the gut microbiota in SHR recipients post-FMT, especially with a decreased abundance of Lactobacillus, Aggregatibacter, and Desulfovibrio. Moreover, ARB-treated microbes contributed to increased intestinal Ciart, Per1, Per2, Per3, and Cipc gene levels and decreased Nfil3 and Arntl expression were detected in response to ARB-treated microbes. More importantly, circulating metabolites were dramatically reduced in ARB-FMT rats, including 6beta-Hydroxytestosterone and Thromboxane B2. In conclusion, ARB-modified gut microbiota exerts protective roles in vascular remodeling and injury, metabolic abnormality and intestinal dysfunctions, suggesting a pivotal role in mitigating hypertension and providing insights into the cross-talk between antihypertensive medicines and the gut microbiome.

Over the years, microbiome research has achieved tremendous advancements driven by culture-independent meta-omics approaches. Despite extensive research, our understanding of the functional roles and causal effects of the microbiome on phenotypes remains limited. In this study, we focused on the rumen metaproteome, combining it with metatranscriptome and metabolome data to accurately identify the active functional distributions of rumen microorganisms and specific functional groups that influence feed efficiency. By integrating host genetics data, we established the potentially causal relationships between microbes-proteins/metabolites-phenotype, and identified specific patterns in which functional groups of rumen microorganisms influence host feed efficiency. We found a causal link between Selenomonas bovis and rumen carbohydrate metabolism, potentially mediated by bacterial chemotaxis and a two-component regulatory system, impacting feed utilization efficiency of dairy cows. Our study on the nutrient utilization functional groups in the rumen of high-feed-efficiency dairy cows, along with the identification of key microbiota functional proteins and their potentially causal relationships, will help move from correlation to causation in rumen microbiome research. This will ultimately enable precise regulation of the rumen microbiota for optimized ruminant production.

A comprehensive immune landscape for Brucella infection is crucial for developing new treatments for brucellosis. Here, we utilized single-cell RNA sequencing (scRNA-seq) of 290,369 cells from 35 individuals, including 29 brucellosis patients from acute (n = 10), sub-acute (n = 9), and chronic (n = 10) phases as well as six healthy donors. Enzyme-linked immunosorbent assays were applied for validation within this cohort. Brucella infection caused a significant change in the composition of peripheral immune cells and inflammation was a key feature of brucellosis. Acute patients are characterized by potential cytokine storms resulting from systemic upregulation of S100A8/A9, primarily due to classical monocytes. Cytokine storm may be mediated by activating S100A8/A9-TLR4-MyD88 signaling pathway. Moreover, monocytic myeloid-derived suppressor cells were the probable contributors to immune paralysis in acute patients. Chronic patients are characterized by a dysregulated Th1 response, marked by reduced expression of IFN-γ and Th1 signatures as well as a high exhausted state. Additionally, Brucella infection can suppress apoptosis in myeloid cells (e.g., mDCs, classical monocytes), inhibit antigen presentation in professional antigen-presenting cells (APCs; e.g., mDC) and nonprofessional APCs (e.g., monocytes), and induce exhaustion in CD8⁺ T/NK cells, potentially resulting in the establishment of chronic infection. Overall, our study systemically deciphered the coordinated immune responses of Brucella at different phases of the infection, which facilitated a full understanding of the immunopathogenesis of brucellosis and may aid the development of new effective therapeutic strategies, especially for those with chronic infection.