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.Department of Agriculture, Food and the MarineEuropean Commission - Seventh Framework Programme (FP7)Science Foundation IrelandUniversity College Dubli

Comparative functional genomics and the bovine macrophage response to strains of the Mycobacterium genus

Mycobacterial infections are major causes of morbidity and mortality in cattle and are also potential zoonotic agents with implications for human health. Despite the implementation of comprehensive animal surveillance programs, many mycobacterial diseases have remained recalcitrant to eradication in several industrialized countries. Two major mycobacterial pathogens of cattle are Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis (MAP), the causative agents of bovine tuberculosis (BTB) and Johne's disease (JD), respectively. BTB is a chronic, granulomatous disease of the respiratory tract that is spread via aerosol transmission, while JD is a chronic granulomatous disease of the intestines that is transmitted via the fecal-oral route. Although these diseases exhibit differential tissue tropism and distinct complex etiologies, both M. bovis and MAP infect, reside, and replicate in host macrophages - the key host innate immune cell that encounters mycobacterial pathogens after initial exposure and mediates the subsequent immune response. The persistence of M. bovis and MAP in macrophages relies on a diverse series of immunomodulatory mechanisms, including the inhibition of phagosome maturation and apoptosis, generation of cytokine-induced necrosis enabling dissemination of infection through the host, local pathology, and ultimately shedding of the pathogen. Here, we review the bovine macrophage response to infection with M. bovis and MAP. In particular, we describe how recent advances in functional genomics are shedding light on the host macrophage-pathogen interactions that underlie different mycobacterial diseases. To illustrate this, we present new analyses of previously published bovine macrophage transcriptomics data following in vitro infection with virulent M. bovis, the attenuated vaccine strain M. bovis BCG, and MAP, and discuss our findings with respect to the differing etiologies of BTB and JD

RNA-seq transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis

Bovine tuberculosis, caused by infection with 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 gene expression microarrays and RNA-sequencing (RNA-seq), 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 analyzed the peripheral blood leukocyte (PBL) transcriptome of eight natural M. bovis-infected and eight age- and sex-matched non-infected control Holstein-Friesian animals using RNA-seq. In addition, we compared gene expression profiles generated using RNA-seq with those previously generated using the high-density Affymetrix(®) GeneChip(®) Bovine Genome Array platform from the same PBL-extracted RNA. A total of 3,250 differentially expressed (DE) annotated genes were detected in the M. bovis-infected samples relative to the controls (adjusted P-value ≤0.05), with the number of genes displaying decreased relative expression (1,671) exceeding those with increased relative expression (1,579). Ingenuity(®) Systems Pathway Analysis (IPA) of all DE genes revealed enrichment for genes with immune function. Notably, transcriptional suppression was observed among several of the top-ranking canonical pathways including Leukocyte Extravasation Signaling. Comparative platform analysis demonstrated that RNA-seq detected a larger number of annotated DE genes (3,250) relative to the microarray (1,398), of which 917 genes were common to both technologies and displayed the same direction of expression. Finally, we show that RNA-seq had an increased dynamic range compared to the microarray for estimating differential gene expression

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro

BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. RESULTS: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. CONCLUSIONS: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA

Meta-Analysis of Mitochondrial DNA Reveals Several Population Bottlenecks during Worldwide Migrations of Cattle

Several studies have investigated the differentiation of mitochondrial DNA in Eurasian, African and American cattle as well as archaeological bovine material. A global survey of these studies shows that haplogroup distributions are more stable in time than in space. All major migrations of cattle have shifted the haplogroup distributions considerably with a reduction of the number of haplogroups and/or an expansion of haplotypes that are rare or absent in the ancestral populations. The most extreme case is the almost exclusive colonization of Africa by the T1 haplogroup, which is rare in Southwest Asian cattle. In contrast, ancient samples invariably show continuity with present-day cattle from the same location. These findings indicate strong maternal founder effects followed by limited maternal gene flow when new territories are colonized. However, effects of adaptation to new environments may also play a rol

Development and Characterisation of High Surface Energy Microstructured Sol-gel Coatings for Sensing Applications

This study investigates the development of high surface energy photoreactive organic inorganic hybrid sol-gel coatings for the microstructuration of high-resolution microfluidic platforms and optofluidic biosensor platforms by standard photolithography processes. To achieve this, the first step of our work consisted of identifying the fundamental physico chemical processes governing the structuration and surface properties of hybrid organic inorganic sol-gel coatings. For this purpose, a reference material based on the combination of an organosilane (3-Methacryloxypropytrimethoxysilane, MAPTMS) and a transition metal (zirconium propoxide, ZPO), was firstly developed and characterised. It was highlighted that chemical, physical and combined physical and chemical processes can be performed to impact the structure, morphology and surface properties of hybrid sol-gel coatings. Therefore, our work progressed towards the investigations of chemical strategies that may impact the general properties of hybrid coatings, with a specific objective on the alteration of their surface properties. For this purpose, 3 strategies have identified including (1) to alter the content of transition metal, (2) to vary the hydrolysis degree and (3) to form core-shell nanoparticle by the surface functionalisation of the reference material during its preparation along with the curing process of the coatings. The materials were characterised employing a set of structural, thermal and surface characterisations techniques namely Contact Angle measurements (CA), DLS, DSC, FTIR, 29Si-NMR. Fundamentally, a triangular relationship between the wettability, the condensation and curing process of the coatings was taking place. More specifically, the wettability was governed by the occurrence of parallel and competitive hydroxylation and condensation processes of the coatings. Having performed the identified chemical strategies, our work has progressed towards the investigations of physical and physico-chemical treatments of the final coatings. Here, the effects of air-plasma, nitrogen-plasma and plasma treatments combined with post-silane ii surface functionalisation were performed and the durability of the treatments investigated. Although hydrophobic recovery was observed for all materials, it was found that air-plasma enabled to achieve the most stable surface properties due to the formation of hydrophilic hydroxyl groups at the surface of the coatings. The next step of the work focussed on the microstructuration fabrication of a microfluidic platform. The photolithography fabrication conditions were established to enable the successful preparation of well-defined microchannels with resolutions ranging from 50 to 500 microns. Having developed our microfluidic platform, our work concentrated on developing strategies to integrate an optical transducer onto the platform to enable the fabrication of an optofluidic device that may be applied as biosensor, thus demonstrating the potential of our technology for biosensing applications. The biosensor design we proposed consisted of integrated optical waveguides onto microfluidics that would also be fabricated employing a photolithography process. The fabrications conditions of the optofluidic platform were established by considering the required optical conditions that enable efficient light propagation in the waveguides, which can be used as an optical excitation to fluorophores located within sensor spots in the microchannels. The successful demonstration of concept of the optofluidic-based biosensor concept was successfully performed by recording optical emissions of biomolecules fluorophores under optical excitations with the optical waveguides integrated on the microfluidic platform. The work reported in this thesis has been multidisciplinary requiring chemistry, physics, biotechnology and engineering competencies which have been synergised for the development of the first “whole hybrid sol-gel optofluidic biosensor platform”. It is also showing the potential of the proposed technology for applications where functional microstructured coatings are required

Cyclosporin A and steroid therapy in sixty-six cadaver kidney recipients

From nine to 18 months ago, 66 patients were given 67 randomly matched cadaveric kidneys with cyclosporin A and steroid therapy. Nine of the recipients were undergoing retransplantation. The over-all kidney survival rate to date has been 77.6 per cent, and 78.8 per cent of the recipients are dialysis-free. The patient mortality in this learning phase was 13.3 per cent. Nephrotoxicity, hepatotoxicity and other side-effects of cyclosporin A could usually be dealt with by dosage adjustments, making feasible the chronic use of this agent. One B-cell immunoblastic sarcoma was encountered which was monoclonal. It was not responsible for death. Another patient had a perforation of the intestine from a lymphoproliferative reaction in which the B cells were polyclonal. After jejunal resection a year ago, there were no further complications. This lesion was not classified as a lymphoma. Both lymphoproliferative lesions were associated with a rise in antibody to viral capsid antigen of Epstein-Barr virus. Results of this study have verified the effectiveness and relative safety of cyclosporin A with steroids for immunosuppression in human recipients of cadaveric kidneys

Interrogation of modern and ancient genomes reveals the complex domestic history of cattle

The analysis of mitochondrial and nuclear DNA sequence polymorphisms from modern cattle populations has had a profound impact on our understanding of the events surrounding the domestication of cattle. From these studies, it has been possible to distinguish between pre- and post-domestic genetic differentiation, supporting previous assertions from archaeological studies and, in some cases, revealing novel aspects of the demographic history of cattle. Analyses of genetic material retrieved from the remains of extinct ancestral wild cattle have also added valuable layers of information pertaining to cattle domestic origins; however, information from these investigations have, in general, been limited to small, variable portions of the mitochondrial genome owing to technical challenges associated with the retrieval and amplification of ancient DNA. In recent years, however, new high-throughput, massively parallel genomics technology platforms, such as single-nucleotide polymorphism (SNP) genotyping arrays and next-generation sequencing (NGS), have provided a new impetus to the studies of genetic variation in extant and ancient cattle. Arrays of SNP have facilitated high-resolution genetic surveys of global cattle populations and detection of ancient and recent genomic selective sweeps. Next-generation sequencing analyses of modern and ancient cattle hold great promise for identifying and cataloging of pre- and post-domestication patterns of genomic variation and correlating this with natural and artificial selection processes