Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture.

Chromosome conformation capture (3C) methods measure the spatial proximity between DNA elements in the cell nucleus. Many methods have been developed to sample 3C material, including the Capture-C family of protocols. Capture-C methods use oligonucleotides to enrich for interactions of interest from sequencing-ready 3C libraries. This approach is modular and has been adapted and optimized to work for sampling of disperse DNA elements (NuTi Capture-C), including from low cell inputs (LI Capture-C), as well as to generate Hi-C like maps for specific regions of interest (Tiled-C) and to interrogate multiway interactions (Tri-C). We present the design, experimental protocol and analysis pipeline for NuTi Capture-C in addition to the variations for generation of LI Capture-C, Tiled-C and Tri-C data. The entire procedure can be performed in 3 weeks and requires standard molecular biology skills and equipment, access to a next-generation sequencing platform, and basic bioinformatic skills. Implemented with other sequencing technologies, these methods can be used to identify regulatory interactions and to compare the structural organization of the genome in different cell types and genetic models.

IL-1-driven stromal-neutrophil interactions define a subset of patients with inflammatory bowel disease that does not respond to therapies.

Current inflammatory bowel disease (IBD) therapies are ineffective in a high proportion of patients. Combining bulk and single-cell transcriptomics, quantitative histopathology and in situ localization across three cohorts of patients with IBD (total n = 376), we identify coexpressed gene modules within the heterogeneous tissular inflammatory response in IBD that map to distinct histopathological and cellular features (pathotypes). One of these pathotypes is defined by high neutrophil infiltration, activation of fibroblasts and vascular remodeling at sites of deep ulceration. Activated fibroblasts in the ulcer bed display neutrophil-chemoattractant properties that are IL-1R, but not TNF, dependent. Pathotype-associated neutrophil and fibroblast signatures are increased in nonresponders to several therapies across four independent cohorts (total n = 343). The identification of distinct, localized, tissular pathotypes will aid precision targeting of current therapeutics and provides a biological rationale for IL-1 signaling blockade in ulcerating disease.

RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course.

Bovine tuberculosis, caused by infection with members of the Mycobacterium tuberculosis complex, particularly Mycobacterium bovis, is a major endemic disease affecting cattle populations worldwide, despite the implementation of stringent surveillance and control programs in many countries. The development of high-throughput functional genomics technologies, including RNA sequencing, has enabled detailed analysis of the host transcriptome to M. bovis infection, particularly at the macrophage and peripheral blood level. In the present study, we have analysed the transcriptome of bovine whole peripheral blood samples collected at -1 week pre-infection and +1, +2, +6, +10, and +12 weeks post-infection time points. Differentially expressed genes were catalogued and evaluated at each post-infection time point relative to the -1 week pre-infection time point and used for the identification of putative candidate host transcriptional biomarkers for M. bovis infection. Differentially expressed gene sets were also used for examination of cellular pathways associated with the host response to M. bovis infection, construction of de novo gene interaction networks enriched for host differentially expressed genes, and time-series analyses to identify functionally important groups of genes displaying similar patterns of expression across the infection time course. A notable outcome of these analyses was identification of a 19-gene transcriptional biosignature of infection consisting of genes increased in expression across the time course from +1 week to +12 weeks post-infection.

TreeSummarizedExperiment: a S4 class for data with hierarchical structure.

Data organized into hierarchical structures (e.g., phylogenies or cell types) arises in several biological fields. It is therefore of interest to have data containers that store the hierarchical structure together with the biological profile data, and provide functions to easily access or manipulate data at different resolutions. Here, we present TreeSummarizedExperiment, a R/S4 class that extends the commonly used SingleCellExperiment class by incorporating tree representations of rows and/or columns (represented by objects of the phylo class). It follows the convention of the SummarizedExperiment class, while providing links between the assays and the nodes of a tree to allow data manipulation at arbitrary levels of the tree. The package is designed to be extensible, allowing new functions on the tree (phylo) to be contributed. As the work is based on the SingleCellExperiment class and the phylo class, both of which are popular classes used in many R packages, it is expected to be able to interact seamlessly with many other tools.

Orchestrating single-cell analysis with Bioconductor.

Recent technological advancements have enabled the profiling of a large number of genome-wide features in individual cells. However, single-cell data present unique challenges that require the development of specialized methods and software infrastructure to successfully derive biological insights. The Bioconductor project has rapidly grown to meet these demands, hosting community-developed open-source software distributed as R packages. Featuring state-of-the-art computational methods, standardized data infrastructure and interactive data visualization tools, we present an overview and online book (https://osca.bioconductor.org) of single-cell methods for prospective users.

Publisher Correction: Orchestrating single-cell analysis with Bioconductor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The bovine alveolar macrophage DNA methylome is resilient to infection with Mycobacterium bovis.

DNA methylation is pivotal in orchestrating gene expression patterns in various mammalian biological processes. Perturbation of the bovine alveolar macrophage (bAM) transcriptome, due to Mycobacterium bovis (M. bovis) infection, has been well documented; however, the impact of this intracellular pathogen on the bAM epigenome has not been determined. Here, whole genome bisulfite sequencing (WGBS) was used to assess the effect of M. bovis infection on the bAM DNA methylome. The methylomes of bAM infected with M. bovis were compared to those of non-infected bAM 24 hours post-infection (hpi). No differences in DNA methylation (CpG or non-CpG) were observed. Analysis of DNA methylation at proximal promoter regions uncovered >250 genes harbouring intermediately methylated (IM) promoters (average methylation of 33-66%). Gene ontology analysis, focusing on genes with low, intermediate or highly methylated promoters, revealed that genes with IM promoters were enriched for immune-related GO categories; this enrichment was not observed for genes in the high or low methylation groups. Targeted analysis of genes in the IM category confirmed the WGBS observation. This study is the first in cattle examining genome-wide DNA methylation at single nucleotide resolution in an important bovine cellular host-pathogen interaction model, providing evidence for IM promoter methylation in bAM.

The Short Chain Fatty Acid Butyrate Imprints an Antimicrobial Program in Macrophages.

Host microbial cross-talk is essential to maintain intestinal homeostasis. However, maladaptation of this response through microbial dysbiosis or defective host defense toward invasive intestinal bacteria can result in chronic inflammation. We have shown that macrophages differentiated in the presence of the bacterial metabolite butyrate display enhanced antimicrobial activity. Butyrate-induced antimicrobial activity was associated with a shift in macrophage metabolism, a reduction in mTOR kinase activity, increased LC3-associated host defense and anti-microbial peptide production in the absence of an increased inflammatory cytokine response. Butyrate drove this monocyte to macrophage differentiation program through histone deacetylase 3 (HDAC3) inhibition. Administration of butyrate induced antimicrobial activity in intestinal macrophages in vivo and increased resistance to enteropathogens. Our data suggest that (1) increased intestinal butyrate might represent a strategy to bolster host defense without tissue damaging inflammation and (2) that pharmacological HDAC3 inhibition might drive selective macrophage functions toward antimicrobial host defense.

Invasive Salmonella exploits divergent immune evasion strategies in infected and bystander dendritic cell subsets.

Non-typhoidal Salmonella (NTS) are highly prevalent food-borne pathogens. Recently, a highly invasive, multi-drug resistant S. Typhimurium, ST313, emerged as a major cause of bacteraemia in children and immunosuppressed adults, however the pathogenic mechanisms remain unclear. Here, we utilize invasive and non-invasive Salmonella strains combined with single-cell RNA-sequencing to study the transcriptome of individual infected and bystander monocyte-derived dendritic cells (MoDCs) implicated in disseminating invasive ST313. Compared with non-invasive Salmonella, ST313 directs a highly heterogeneous innate immune response. Bystander MoDCs exhibit a hyper-activated profile potentially diverting adaptive immunity away from infected cells. MoDCs harbouring invasive Salmonella display higher expression of IL10 and MARCH1 concomitant with lower expression of CD83 to evade adaptive immune detection. Finally, we demonstrate how these mechanisms conjointly restrain MoDC-mediated activation of Salmonella-specific CD4+ T cell clones. Here, we show how invasive ST313 exploits discrete evasion strategies within infected and bystander MoDCs to mediate its dissemination in vivo.

RNA Sequencing (RNA-Seq) Reveals Extremely Low Levels of Reticulocyte-Derived Globin Gene Transcripts in Peripheral Blood From Horses (Equus caballus) and Cattle (Bos taurus).

RNA-seq has emerged as an important technology for measuring gene expression in peripheral blood samples collected from humans and other vertebrate species. In particular, transcriptomics analyses of whole blood can be used to study immunobiology and develop novel biomarkers of infectious disease. However, an obstacle to these methods in many mammalian species is the presence of reticulocyte-derived globin mRNAs in large quantities, which can complicate RNA-seq library sequencing and impede detection of other mRNA transcripts. A range of supplementary procedures for targeted depletion of globin transcripts have, therefore, been developed to alleviate this problem. Here, we use comparative analyses of RNA-seq data sets generated from human, porcine, equine, and bovine peripheral blood to systematically assess the impact of globin mRNA on routine transcriptome profiling of whole blood in cattle and horses. The results of these analyses demonstrate that total RNA isolated from equine and bovine peripheral blood contains very low levels of globin mRNA transcripts, thereby negating the need for globin depletion and greatly simplifying blood-based transcriptomic studies in these two domestic species.

iSEE: Interactive SummarizedExperiment Explorer.

Data exploration is critical to the comprehension of large biological data sets generated by high-throughput assays such as sequencing. However, most existing tools for interactive visualisation are limited to specific assays or analyses. Here, we present the iSEE (Interactive SummarizedExperiment Explorer) software package, which provides a general visual interface for exploring data in a SummarizedExperiment object. iSEE is directly compatible with many existing R/Bioconductor packages for analysing high-throughput biological data, and provides useful features such as simultaneous examination of (meta)data and analysis results, dynamic linking between plots and code tracking for reproducibility. We demonstrate the utility and flexibility of iSEE by applying it to explore a range of real transcriptomics and proteomics data sets.

Comparative 'omics analyses differentiate Mycobacterium tuberculosis and Mycobacterium bovis and reveal distinct macrophage responses to infection with the human and bovine tubercle bacilli.

Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis in a range of mammals, including humans. A key feature of MTBC pathogens is their high degree of genetic identity yet distinct host tropism. Notably, while Mycobacterium bovis is highly virulent and pathogenic for cattle, the human pathogen M. tuberculosis is attenuated in cattle. Previous research also suggests that host preference amongst MTBC members has a basis in host innate immune responses. To explore MTBC host tropism, we present in-depth profiling of the MTBC reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv at both the global transcriptional and the translational level via RNA-sequencing and SWATH MS. Furthermore, a bovine alveolar macrophage infection time course model was used to investigate the shared and divergent host transcriptomic response to infection with M. tuberculosis H37Rv or M. bovis AF2122/97. Significant differential expression of virulence-associated pathways between the two bacilli was revealed, including the ESX-1 secretion system. A divergent transcriptional response was observed between M. tuberculosis H37Rv and M. bovis AF2122/97 infection of bovine alveolar macrophages, in particular cytosolic DNA-sensing pathways at 48 h post-infection, and highlights a distinct engagement of M. bovis with the bovine innate immune system. The work presented here therefore provides a basis for the identification of host innate immune mechanisms subverted by virulent host-adapted mycobacteria to promote their survival during the early stages of infection.

Experimental infection of cattle with Mycobacterium tuberculosis isolates shows the attenuation of the human tubercle bacillus for cattle.

The Mycobacterium tuberculosis complex (MTBC) is the collective term given to the group of bacteria that cause tuberculosis (TB) in mammals. It has been reported that M. tuberculosis H37Rv, a standard reference MTBC strain, is attenuated in cattle compared to Mycobacterium bovis. However, as M. tuberculosis H37Rv was isolated in the early 1930s, and genetic variants are known to exist, we sought to revisit this question of attenuation of M. tuberculosis for cattle by performing a bovine experimental infection with a recent M. tuberculosis isolate. Here we report infection of cattle using M. bovis AF2122/97, M. tuberculosis H37Rv, and M. tuberculosis BTB1558, the latter isolated in 2008 during a TB surveillance project in Ethiopian cattle. We show that both M. tuberculosis strains caused reduced gross pathology and histopathology in cattle compared to M. bovis. Using M. tuberculosis H37Rv and M. bovis AF2122/97 as the extremes in terms of infection outcome, we used RNA-Seq analysis to explore differences in the peripheral response to infection as a route to identify biomarkers of progressive disease in contrast to a more quiescent, latent infection. Our work shows the attenuation of M. tuberculosis strains for cattle, and emphasizes the potential of the bovine model as a 'One Health' approach to inform human TB biomarker development and post-exposure vaccine development.

Plasma Metabolomics Implicates Modified Transfer RNAs and Altered Bioenergetics in the Outcomes of Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a heterogeneous disorder with high mortality. METHODS: We conducted a comprehensive study of plasma metabolites using ultraperformance liquid chromatography mass spectrometry to identify patients at high risk of early death, to identify patients who respond well to treatment, and to provide novel molecular insights into disease pathogenesis. RESULTS: Fifty-three circulating metabolites distinguished well-phenotyped patients with idiopathic or heritable PAH (n=365) from healthy control subjects (n=121) after correction for multiple testing (P<7.3e-5) and confounding factors, including drug therapy, and renal and hepatic impairment. A subset of 20 of 53 metabolites also discriminated patients with PAH from disease control subjects (symptomatic patients without pulmonary hypertension, n=139). Sixty-two metabolites were prognostic in PAH, with 36 of 62 independent of established prognostic markers. Increased levels of tRNA-specific modified nucleosides (N2,N2-dimethylguanosine, N1-methylinosine), tricarboxylic acid cycle intermediates (malate, fumarate), glutamate, fatty acid acylcarnitines, tryptophan, and polyamine metabolites and decreased levels of steroids, sphingomyelins, and phosphatidylcholines distinguished patients from control subjects. The largest differences correlated with increased risk of death, and correction of several metabolites over time was associated with a better outcome. Patients who responded to calcium channel blocker therapy had metabolic profiles similar to those of healthy control subjects. CONCLUSIONS: Metabolic profiles in PAH are strongly related to survival and should be considered part of the deep phenotypic characterization of this disease. Our results support the investigation of targeted therapeutic strategies that seek to address the alterations in translational regulation and energy metabolism that characterize these patients.

GOexpress: an R/Bioconductor package for the identification and visualisation of robust gene ontology signatures through supervised learning of gene expression data.

BACKGROUND: Identification of gene expression profiles that differentiate experimental groups is critical for discovery and analysis of key molecular pathways and also for selection of robust diagnostic or prognostic biomarkers. While integration of differential expression statistics has been used to refine gene set enrichment analyses, such approaches are typically limited to single gene lists resulting from simple two-group comparisons or time-series analyses. In contrast, functional class scoring and machine learning approaches provide powerful alternative methods to leverage molecular measurements for pathway analyses, and to compare continuous and multi-level categorical factors. RESULTS: We introduce GOexpress, a software package for scoring and summarising the capacity of gene ontology features to simultaneously classify samples from multiple experimental groups. GOexpress integrates normalised gene expression data (e.g., from microarray and RNA-seq experiments) and phenotypic information of individual samples with gene ontology annotations to derive a ranking of genes and gene ontology terms using a supervised learning approach. The default random forest algorithm allows interactions between all experimental factors, and competitive scoring of expressed genes to evaluate their relative importance in classifying predefined groups of samples. CONCLUSIONS: GOexpress enables rapid identification and visualisation of ontology-related gene panels that robustly classify groups of samples and supports both categorical (e.g., infection status, treatment) and continuous (e.g., time-series, drug concentrations) experimental factors. The use of standard Bioconductor extension packages and publicly available gene ontology annotations facilitates straightforward integration of GOexpress within existing computational biology pipelines.

Integrated computational prediction and experimental validation identifies promiscuous T cell epitopes in the proteome of Mycobacterium bovis.

The discovery of novel antigens is an essential requirement in devising new diagnostics or vaccines for use in control programmes against human tuberculosis (TB) and bovine tuberculosis (bTB). Identification of potential epitopes recognised by CD4+ T cells requires prediction of peptide binding to MHC class-II, an obligatory prerequisite for T cell recognition. To comprehensively prioritise potential MHC-II-binding epitopes from Mycobacterium bovis, the agent of bTB and zoonotic TB in humans, we integrated three binding prediction methods with the M. bovisproteome using a subset of human HLA alleles to approximate the binding of epitope-containing peptides to the bovine MHC class II molecule BoLA-DRB3. Two parallel strategies were then applied to filter the resulting set of binders: identification of the top-scoring binders or clusters of binders. Our approach was tested experimentally by assessing the capacity of predicted promiscuous peptides to drive interferon-γ secretion from T cells of M. bovis infected cattle. Thus, 376 20-mer peptides, were synthesised (270 predicted epitopes, 94 random peptides with low predictive scores and 12 positive controls of known epitopes). The results of this validation demonstrated significant enrichment (>24 %) of promiscuously recognised peptides predicted in our selection strategies, compared with randomly selected peptides with low prediction scores. Our strategy offers a general approach to the identification of promiscuous epitopes tailored to target populations where there is limited knowledge of MHC allelic diversity.

High-throughput transcriptomics reveals common and strain-specific responses of human macrophages to infection with Mycobacterium abscessus Smooth and Rough variants.

BACKGROUND: Mycobacterium abscessus (MAB) is an emerging pathogen causing pulmonary infections in those with inflammatory lung disorders, such as Cystic Fibrosis (CF), and is associated with the highest fatality rate among rapidly growing mycobacteria (RGM). Phenotypically, MAB manifests as either a Smooth (MAB-S) or a Rough (MAB-R) morphotype, which differ in their levels of cell wall glycopeptidolipids (GPLs) and in their pathogenicity in vivo. As one of the primary immune cells encountered by MAB, we sought to examine the early transcriptional events within macrophages, following infection with both MAB-S or MAB-R. RESULTS: We sampled the transcriptomes (mRNA and miRNA) of THP-1-derived macrophages infected with both MAB-R and MAB-S at 1, 4 and 24 h post-infection (hpi) using RNA-seq. A core set of 606 genes showed consistent expression profiles in response to both morphotypes, corresponding to the early transcriptional response to MAB. The core response is type I Interferon (IFN)-driven, involving the NF-κB and MAPK signaling pathways with concomitant pro-inflammatory cytokine production, and network analysis identified STAT1, EGR1, and SRC as key hub and bottleneck genes. MAB-S elicited a more robust transcriptional response at both the mRNA and miRNA levels, which was reflected in higher cytokine levels in culture supernatants. The transcriptional profiles of macrophages infected with both morphotypes were highly correlated, however, and a direct comparison identified few genes to distinguish them. Most of the induced miRNAs have previously been associated with mycobacterial infection and overall miRNA expression patterns were similarly highly correlated between the morphotypes. CONCLUSIONS: The report here details the first whole transcriptome analysis of the early macrophage response to MAB infection. The overall picture at the early stages of macrophage infection is similar to that of other mycobacteria, reflected in a core type I IFN and pro-inflammatory cytokine response. Large-scale transcriptional differences in the host response to the different MAB morphotypes are not evident in the early stages of infection, however the subset of genes with distinct expression profiles suggest potentially interesting differences in internal trafficking of MAB within macrophages.

Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle.

BACKGROUND: Domestication of the now-extinct wild aurochs, Bos primigenius, gave rise to the two major domestic extant cattle taxa, B. taurus and B. indicus. While previous genetic studies have shed some light on the evolutionary relationships between European aurochs and modern cattle, important questions remain unanswered, including the phylogenetic status of aurochs, whether gene flow from aurochs into early domestic populations occurred, and which genomic regions were subject to selection processes during and after domestication. Here, we address these questions using whole-genome sequencing data generated from an approximately 6,750-year-old British aurochs bone and genome sequence data from 81 additional cattle plus genome-wide single nucleotide polymorphism data from a diverse panel of 1,225 modern animals. RESULTS: Phylogenomic analyses place the aurochs as a distinct outgroup to the domestic B. taurus lineage, supporting the predominant Near Eastern origin of European cattle. Conversely, traditional British and Irish breeds share more genetic variants with this aurochs specimen than other European populations, supporting localized gene flow from aurochs into the ancestors of modern British and Irish cattle, perhaps through purposeful restocking by early herders in Britain. Finally, the functions of genes showing evidence for positive selection in B. taurus are enriched for neurobiology, growth, metabolism and immunobiology, suggesting that these biological processes have been important in the domestication of cattle. CONCLUSIONS: This work provides important new information regarding the origins and functional evolution of modern cattle, revealing that the interface between early European domestic populations and wild aurochs was significantly more complex than previously thought.

RNA sequencing provides exquisite insight into the manipulation of the alveolar macrophage by tubercle bacilli.

Mycobacterium bovis, the agent of bovine tuberculosis, causes an estimated $3 billion annual losses to global agriculture due, in part, to the limitations of current diagnostics. Development of next-generation diagnostics requires a greater understanding of the interaction between the pathogen and the bovine host. Therefore, to explore the early response of the alveolar macrophage to infection, we report the first application of RNA-sequencing to define, in exquisite detail, the transcriptomes of M. bovis-infected and non-infected alveolar macrophages from ten calves at 2, 6, 24 and 48 hours post-infection. Differentially expressed sense genes were detected at these time points that revealed enrichment of innate immune signalling functions, and transcriptional suppression of host defence mechanisms (e.g., lysosome maturation). We also detected differentially expressed natural antisense transcripts, which may play a role in subverting innate immune mechanisms following infection. Furthermore, we report differential expression of novel bovine genes, some of which have immune-related functions based on orthology with human proteins. This is the first in-depth transcriptomics investigation of the alveolar macrophage response to the early stages of M. bovis infection and reveals complex patterns of gene expression and regulation that underlie the immunomodulatory mechanisms used by M. bovis to evade host defence mechanisms.