Disease-Relevant Single Cell Photonic Signatures Identify S100ß Stem Cells and their Myogenic Progeny in Vascular Lesions.

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100ß+ mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- ß1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100ß+ vSCs and identified the presence of S100ß+vSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.

A dual targeted ß-defensin and exome sequencing approach to identify, validate and functionally characterise genes associated with bull fertility.

Bovine fertility remains a critical issue underpinning the sustainability of the agricultural sector. Phenotypic records collected on >7,000 bulls used in artificial insemination (AI) were used to identify 160 reliable and divergently fertile bulls for a dual strategy of targeted sequencing (TS) of fertility-related ß-defensin genes and whole exome sequencing (WES). A haplotype spanning multiple ß-defensin genes and containing 94 SNPs was significantly associated with fertility and functional analysis confirmed that sperm from bulls possessing the haplotype showed significantly enhanced binding to oviductal epithelium. WES of all exons in the genome in 24 bulls of high and low fertility identified 484 additional SNPs significantly associated with fertility. After validation, the most significantly associated SNP was located in the FOXJ3 gene, a transcription factor which regulates sperm function in mice. This study represents the first comprehensive characterisation of genetic variation in bovine ß-defensin genes and functional analysis supports a role for ß-defensins in regulating bull sperm function. This first application of WES in AI bulls with divergent fertility phenotypes has identified a novel role for the transcription factor FOXJ3 in the regulation of bull fertility. Validated genetic variants associated with bull fertility could prove useful for improving reproductive outcomes in cattle.

Capturing goats: documenting two hundred years of mitochondrial DNA diversity among goat populations from Britain and Ireland.

The domestic goat (Capra hircus) plays a key role in global agriculture, being especially prized in regions of marginal pasture. However, the advent of industrialized breeding has seen a dramatic reduction in genetic diversity within commercial populations, while high extinction rates among feral herds have further depleted the reservoir of genetic variation available. Here, we present the first survey of whole mitochondrial genomic variation among the modern and historical goat populations of Britain and Ireland using a combination of mtDNA enrichment and high throughput sequencing. Fifteen historical taxidermy samples, representing the indigenous 'Old Goat' populations of the islands, were sequenced alongside five modern Irish dairy goats and four feral samples from endangered populations in western Ireland. Phylogenetic and network analyses of European mitochondrial variation revealed distinct groupings dominated by historical British and Irish samples, which demonstrate a degree of maternal genetic structure between the goats of insular and continental Europe. Several Irish modern feral samples also fall within these clusters, suggesting continuity between these dwindling populations and the ancestral 'Old Goats' of Ireland and Britain.

Sperm-Coating Beta-Defensin 126 Is a Dissociation-Resistant Dimer Produced by Epididymal Epithelium in the Bovine Reproductive Tract.

Beta-defensins are innate immune molecules, often described as antimicrobial peptides because of their bactericidal activity and are now known to have diverse additional functions, including cell signaling, chemoattraction, immunoregulation, and reproduction. In humans and primates, beta-defensin 126 has been shown to regulate the ability of sperm to swim through cervical mucus and to protect sperm from attack by the female immune system during transit toward the oviduct. Bovine beta-defensin 126 (BBD126) is the ortholog of human defensin 126, and computational analysis here revealed significant conservation between BBD126 and other mammalian orthologs at the N-terminus, although extensive sequence differences were detected at the C-terminus, implying possible species-specific roles for this beta-defensin in reproduction. We had previously demonstrated preferential expression of this and related beta-defensin genes in the bovine male reproductive tract, but no studies of bovine beta-defensin proteins have been performed to date. Here, we analyzed BBD126 protein using a monoclonal antibody (a-BBD126) generated against a 14 amino acid peptide sequence from the secreted fragment of BBD126. The specificity of a-BBD126 was validated by testing against the native form of the peptide recovered from bovine caudal epididymal fluid and recombinant BBD126 generated using a prokaryotic expression system. Western blot analysis of the native and recombinant forms showed that BBD126 exists as a dimer that was highly resistant to standard methods of dissociation. Immunohistochemical staining using a-BBD126 demonstrated BBD126 protein expression by epithelial cells of the caudal epididymis and vas deferens from both mature and immature bulls. BBD126 could also be seen (by confocal microscopy) to coat caudal sperm, with staining concentrated on the tail of the sperm cells. This study is the first to demonstrate beta-defensin 126 protein expression in the bovine reproductive tract and on bull sperm. Its dissociation-resistant dimeric structure is likely to have important functional implications for the role of BBD126 in bovine reproduction.

The CD4(+) T cell methylome contributes to a distinct CD4(+) T cell transcriptional signature in Mycobacterium bovis-infected cattle.

We hypothesised that epigenetic regulation of CD4(+) T lymphocytes contributes to a shift toward a dysfunctional T cell phenotype which may impact on their ability to clear mycobacterial infection. Combined RNA-seq transcriptomic profiling and Reduced Representation Bisulfite Sequencing identified 193 significantly differentially expressed genes and 760 differentially methylated regions (DMRs), between CD4(+) T cells from M. bovis infected and healthy cattle. 196 DMRs were located within 10 kb of annotated genes, including GATA3 and RORC, both of which encode transcription factors that promote TH2 and TH17 T helper cell subsets respectively. Gene-specific DNA methylation and gene expression levels for the TNFRSF4 and Interferon-γ genes were significantly negatively correlated suggesting a regulatory relationship. Pathway analysis of DMRs identified enrichment of genes involved in the anti-proliferative TGF-ß signaling pathway and TGFB1 expression was significantly increased in peripheral blood leukocytes from TB-infected cattle. This first analysis of the bovine CD4(+) T cell methylome suggests that DNA methylation directly contributes to a distinct gene expression signature in CD4(+) T cells from cattle infected with M. bovis. Specific methylation changes proximal to key inflammatory gene loci may be critical to the emergence of a non-protective CD4(+) T cell response during mycobacterial infection in cattle.

A genome-wide association study for somatic cell score using the Illumina high-density bovine beadchip identifies several novel QTL potentially related to mastitis susceptibility.

Mastitis is an inflammation-driven disease of the bovine mammary gland that occurs in response to physical damage or infection and is one of the most costly production-related diseases in the dairy industry worldwide. We performed a genome-wide association study (GWAS) to identify genetic loci associated with somatic cell score (SCS), an indicator trait of mammary gland inflammation. A total of 702 Holstein-Friesian bulls were genotyped for 777,962 single nucleotide polymorphisms (SNPs) and associated with SCS phenotypes. The SCS phenotypes were expressed as daughter yield deviations (DYD) based on a large number of progeny performance records. A total of 138 SNPs on 15 different chromosomes reached genome-wide significance (corrected p-value ≤ 0.05) for association with SCS (after correction for multiple testing). We defined 28 distinct QTL regions and a number of candidate genes located in these QTL regions were identified. The most significant association (p-value = 1.70 × 10(-7)) was observed on chromosome 6. This QTL had no known genes annotated within it, however, the Ensembl Genome Browser predicted the presence of a small non-coding RNA (a Y RNA gene) in this genomic region. This Y RNA gene was 99% identical to human RNY4. Y RNAs are a rare type of non-coding RNA that were originally discovered due to their association with the autoimmune disease, systemic lupus erythematosus. Examining small-RNA sequencing (RNAseq) data being generated by us in multiple different mastitis-pathogen challenged cell-types has revealed that this Y RNA is expressed (but not differentially expressed) in these cells. Other QTL regions identified in this study also encoded strong candidate genes for mastitis susceptibility. A QTL region on chromosome 13, for example, was found to contain a cluster of ß-defensin genes, a gene family with known roles in innate immunity. Due to the increased SNP density, this study also refined the boundaries for several known QTL for SCS and mastitis.

Genome-wide associations for milk production and somatic cell score in Holstein-Friesian cattle in Ireland.

BACKGROUND: Contemporary dairy breeding goals have broadened to include, along with milk production traits, a number of non-production-related traits in an effort to improve the overall functionality of the dairy cow. Increased indirect selection for resistance to mastitis, one of the most important production-related diseases in the dairy sector, via selection for reduced somatic cell count has been part of these broadened goals. A number of genome-wide association studies have identified genetic variants associated with milk production traits and mastitis resistance, however the majority of these studies have been based on animals which were predominantly kept in confinement and fed a concentrate-based diet (i.e. high-input production systems). This genome-wide association study aims to detect associations using genotypic and phenotypic data from Irish Holstein-Friesian cattle fed predominantly grazed grass in a pasture-based production system (low-input). RESULTS: Significant associations were detected for milk yield, fat yield, protein yield, fat percentage, protein percentage and somatic cell score using separate single-locus, frequentist and multi-locus, Bayesian approaches. These associations were detected using two separate populations of Holstein-Friesian sires and cows. In total, 1,529 and 37 associations were detected in the sires using a single SNP regression and a Bayesian method, respectively. There were 103 associations in common between the sires and cows across all the traits. As well as detecting associations within known QTL regions, a number of novel associations were detected; the most notable of these was a region of chromosome 13 associated with milk yield in the population of Holstein-Friesian sires. CONCLUSIONS: A total of 276 of novel SNPs were detected in the sires using a single SNP regression approach. Although obvious candidate genes may not be initially forthcoming, this study provides a preliminary framework upon which to identify the causal mechanisms underlying the various milk production traits and somatic cell score. Consequently this will deepen our understanding of how these traits are expressed.

A genome wide association scan of bovine tuberculosis susceptibility in Holstein-Friesian dairy cattle.

BACKGROUND: Bovine tuberculosis is a significant veterinary and financial problem in many parts of the world. Although many factors influence infection and progression of the disease, there is a host genetic component and dissection of this may enlighten on the wider biology of host response to tuberculosis. However, a binary phenotype of presence/absence of infection presents a noisy signal for genomewide association study. METHODOLOGY/PRINCIPAL FINDINGS: We calculated a composite phenotype of genetic merit for TB susceptibility based on disease incidence in daughters of elite sires used for artificial insemination in the Irish dairy herd. This robust measure was compared with 44,426 SNP genotypes in the most informative 307 subjects in a genome wide association analysis. Three SNPs in a 65 kb genomic region on BTA 22 were associated (i.e. p<10(-5), peaking at position 59588069, p = 4.02×10(-6)) with tuberculosis susceptibility. CONCLUSIONS/SIGNIFICANCE: A genomic region on BTA 22 was suggestively associated with tuberculosis susceptibility; it contains the taurine transporter gene SLC6A6, or TauT, which is known to function in the immune system but has not previously been investigated for its role in tuberculosis infection.

A complete mitochondrial genome sequence from a mesolithic wild aurochs (Bos primigenius).

BACKGROUND: The derivation of domestic cattle from the extinct wild aurochs (Bos primigenius) has been well-documented by archaeological and genetic studies. Genetic studies point towards the Neolithic Near East as the centre of origin for Bos taurus, with some lines of evidence suggesting possible, albeit rare, genetic contributions from locally domesticated wild aurochsen across Eurasia. Inferences from these investigations have been based largely on the analysis of partial mitochondrial DNA sequences generated from modern animals, with limited sequence data from ancient aurochsen samples. Recent developments in DNA sequencing technologies, however, are affording new opportunities for the examination of genetic material retrieved from extinct species, providing new insight into their evolutionary history. Here we present DNA sequence analysis of the first complete mitochondrial genome (16,338 base pairs) from an archaeologically-verified and exceptionally-well preserved aurochs bone sample. METHODOLOGY: DNA extracts were generated from an aurochs humerus bone sample recovered from a cave site located in Derbyshire, England and radiocarbon-dated to 6,738+/-68 calibrated years before present. These extracts were prepared for both Sanger and next generation DNA sequencing technologies (Illumina Genome Analyzer). In total, 289.9 megabases (22.48%) of the post-filtered DNA sequences generated using the Illumina Genome Analyzer from this sample mapped with confidence to the bovine genome. A consensus B. primigenius mitochondrial genome sequence was constructed and was analysed alongside all available complete bovine mitochondrial genome sequences. CONCLUSIONS: For all nucleotide positions where both Sanger and Illumina Genome Analyzer sequencing methods gave high-confidence calls, no discrepancies were observed. Sequence analysis reveals evidence of heteroplasmy in this sample and places this mitochondrial genome sequence securely within a previously identified aurochsen haplogroup (haplogroup P), thus providing novel insights into pre-domestic patterns of variation. The high proportion of authentic, endogenous aurochs DNA preserved in this sample bodes well for future efforts to determine the complete genome sequence of a wild ancestor of domestic cattle.