Molecular evolution of bovine Toll-like receptor 2 suggests substitutions of functional relevance

<p>Abstract</p> <p>Background</p> <p>There is accumulating evidence that polymorphism in Toll-like receptor (<it>TLR) </it>genes might be associated with disease resistance or susceptibility traits in livestock. Polymorphic sites affecting TLR function should exhibit signatures of positive selection, identified as a high ratio of non-synonymous to synonymous nucleotide substitutions (ω). Phylogeny based models of codon substitution based on estimates of ω for each amino acid position can therefore offer a valuable tool to predict sites of functional relevance. We have used this approach to identify such polymorphic sites within the bovine <it>TLR2 </it>genes from ten <it>Bos indicus </it>and <it>Bos taurus </it>cattle breeds. By analysing <it>TLR2 </it>gene phylogeny in a set of mammalian species and a subset of ruminant species we have estimated the selective pressure on individual sites and domains and identified polymorphisms at sites of putative functional importance.</p> <p>Results</p> <p>The ω were highest in the mammalian TLR2 domains thought to be responsible for ligand binding and lowest in regions responsible for heterodimerisation with other TLR-related molecules. Several positively-selected sites were detected in or around ligand-binding domains. However a comparison of the ruminant subset of <it>TLR2 </it>sequences with the whole mammalian set of sequences revealed that there has been less selective pressure among ruminants than in mammals as a whole. This suggests that there have been functional changes during ruminant evolution. Twenty newly-discovered non-synonymous polymorphic sites were identified in cattle. Three of them were localised at positions shaped by positive selection in the ruminant dataset (Leu227Phe, His305Pro, His326Gln) and in domains involved in the recognition of ligands. His326Gln is of particular interest as it consists of an exchange of differentially-charged amino acids at a position which has previously been shown to be crucial for ligand binding in human TLR2.</p> <p>Conclusion</p> <p>Within bovine TLR2, polymorphisms at amino acid positions 227, 305 and 326 map to functionally important sites of TLR2 and should be considered as candidate SNPs for immune related traits in cattle. A final proof of their functional relevance requires further studies to determine their functional effect on the immune response after stimulation with relevant ligands and/or their association with immune related traits in animals.</p

Genetic selection for fast growth generates bone architecture characterised by enhanced periosteal expansion and limited consolidation of the cortices but a diminution in the early responses to mechanical loading.

International audienceBone strength is, in part, dependent on a mechanical input that regulates the (re)modelling of skeletal elements to an appropriate size and architecture to resist fracture during habitual use. The rate of longitudinal bone growth in juveniles can also affect fracture incidence in adulthood, suggesting an influence of growth rate on later bone quality. We have compared the effects of fast and slow growth on bone strength and architecture in the tibiotarsi of embryonic and juvenile birds. The loading-related biochemical responses (intracellular G6PD activity and NO release) to mechanical load were also determined. Further, we have analysed the proliferation and differentiation characteristics of primary tibiotarsal osteoblasts from fast and slow-growing strains. We found that bones from chicks with divergent growth rates display equal resistance to applied loads, but weight-correction revealed that the bones from juvenile fast growth birds are weaker, with reduced stiffness and lower resistance to fracture. Primary osteoblasts from slow-growing juvenile birds proliferated more rapidly and had lower alkaline phosphatase activity. Bones from fast-growing embryonic chicks display rapid radial expansion and incomplete osteonal infilling but, importantly, lack mechanical responsiveness. These findings are further evidence that the ability to respond to mechanical inputs is crucial to adapt skeletal architecture to generate a functionally appropriate bone structure and that fast embryonic and juvenile growth rates may predispose bone to particular architectures with increased fragility in the adult. (C) 2009 Elsevier Inc. All rights reserved

Characterization of the fine specificity of bovine CD8 T-cell responses to defined antigens from the protozoan parasite Theileria parva

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes

CKCS CEL data

The dataset is microrarray data collected from canine RNA samples. The experimental design of this project is to perform comparative analysis (unpaired one-way ANOVA) between normal canine mitral valves, late stage Myxomatous mitral valves from a mixed breed population and late stage Myxomatous mitral valves from Cavalier King Charles Spaniels (CKCS). At the time of deposit, the data creators are working on a draft manuscript with which this dataset is associated, whose draft title is "Comparative transcriptomic profiling of myxomatous mitral valve disease in the cavalier King Charles spaniel".Markby, Greg. (2020). CKCS CEL data, [dataset]. University of Edinburgh. College of Medicine & Veterinary Medicine. The Roslin institute. https://doi.org/10.7488/ds/2754

Landcover Classification of Luambe National Park, Zambia

Luambe National Park (LNP) is a small, remote and relatively undeveloped national park in the Luangwa Valley, eastern Zambia. Baseline ecological data have been lacking and few publications relating to the ecology of the national park and surrounding game management area (GMA) exist. The aim of this work was to produce an accurate landcover classification that could be used as a baseline dataset for monitoring ecological health in the park. Fuzzy set theory was used to classify remotely sensed Landsat 7 ETM+ imagery with a spatial resolution of 30m. A ground survey to collect training and test data was conducted in August and September 2005. The most recent L1G Landsat dataset was obtained from the Global Land Cover Facility maintained by the University of Maryland (acquisition date 04/10/2001, path 170, row 069, cloud cover 0%). Bands one, two, three, four, five and seven were used for the classification. Erdas Imagine 8.4 (Leica Geosystems AG, Atlanta, USA) was used to perform a supervised classification using the maximum likelihood classifier with activation of the fuzzy classification function. The eight-layered output dataset was then processed to a single-layer hard classification using the fuzzy convolution facility. An error matrix was produced and producer’s and user’s accuracies calculated for each class. Nine landcover classes were identified and the overall accuracy of the classification was 71.2% (95% CI: 65.3-76.7%). The overall kappa statistic was 0.67 and the estimator of kappa (KS) for stratified random sampling was 0.74. This dataset contains the landcover classification for the national park area only. The dataset for the national park and surrounding game management areas can be found using the identifier http://hdl.handle.net/10672/60

Identification of miRNAs associated with the follicular-luteal transition in the ruminant ovary

Sequence and tissue distribution of ovine miRNALittle is known about the involvement of microRNAs (miRNAs) in the follicular-luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles (diameter, 4.0-5.5 mm) pre-ovulatory follicles (6.0-7.0 mm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs which expression decreased in association with the follicular-luteal transition and eight miRNAs which expression increased during this transition. Expression profiles were confirmed by Northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identifies a subset of miRNAs which are potentially important regulators of the follicular-luteal transition.Clinton, Michael. (2013). Identification of miRNAs associated with the follicular-luteal transition in the ruminant ovary, 2010-2012 [Dataset]. University of Edinburgh. Roslin Institute. http://dx.doi.org/10.7488/ds/148

Comparative profiling of murine CLN1 Thalamus and Cortex at early and late disease timepoints

CLN1 disease or Infantile neuronal ceroid lipofuscinosis is a fatal inherited neurodegenerative lysosomal storage disease of early childhood with no currently available therapy. CLN1 disease is caused by mutations in the CLN1 gene, which encodes the enzyme Palmitoyl Protein Thioesterase-1 (PPT-1). We recently found significant spinal pathology in Ppt1-deficient (Ppt1-/-) mice and human CLN1 disease, that contributes to clinical outcome, and precedes the onset of similar changes in the brain of these mice. To further understand the underlying mechanisms that cause such regional vulnerability, we used quantitative proteomic profiling to analyze the cortex and spinal cord tissue from wildtype (WT) and Ppt1-/- mice at early (3 months) and late (7 month) timepoints respectively. Tandem Mass Tagged (TMT) proteomic analysis revealed a significant early inflammatory response as well as changes in mitochondrial function, cell-signalling pathways and developmental processes in the spinal cords of Ppt1-/- mice at 3 months, as compared to their wildtype counterparts. Taken together, these early and progressive pathological and related proteomic changes in this largely unexplored region of the CNS may help explain the limited success of previous brain-directed therapies for CLN1 disease. More importantly, these data fundamentally change our understanding of the progressive, site-specific disease pathogenesis in CLN1 disease, and highlight the importance of early inflammation. This will greatly impact our approach to the timing and targeting of future therapeutic trials for the neuronal ceroid lipofuscinoses (NCLs). The proteomic data generated in the process of this research has been uploaded for general access here.Wishart, Thomas. (2020). Comparative profiling of murine CLN1 Thalamus and Cortex at early and late disease timepoints, 2018-2019 [dataset]. University of Edinburgh (UK) and Washington University (St Louis, Missouri)

Construction of OOEP_IVT, OOEP_CAG, OOEP_VASA and ROSA26_gRNAs plasmids

These pdf files outline the construction of the four CRISPR/Cas9 gene drive DNA plasmid constructs developed during Gus McFarlane's PhD (2016 to 2020). These files provide important supplementary information to his PhD thesis, which is titled "CRISPR-based gene drives for pest control".McFarlane, Gus. (2019). Construction of OOEP_IVT, OOEP_CAG, OOEP_VASA and ROSA26_gRNAs plasmids, [image]. University of Edinburgh. Roslin Institute. https://doi.org/10.7488/ds/2744

M cells and the FAE

Transcriptomic data base on gene expression by mouse M cells Identification of Novel Genes Selectively Expressed in the Follicle-Associated Epithelium from the Meta-Analysis of Transcriptomics Data from Multiple Mouse Cell and Tissue PopulationsThe follicle-associated epithelium (FAE) overlying the Peyer’s patches and the microfold (M) cells within it are important sites of antigen transcytosis across the intestinal epithelium. We obtained a large number of gene expression data files from a range of different primary mouse cells and cell lines and subjected these data to network-based cluster analysis using Biolayout Express3D. Using this meta-analysis approach we identified a transcriptional signature that distinguished the FAE from a large collection of mouse cells and tissues. A co-expressed cluster of 21 FAE-specific genes was identified, and analysis of the transcription factor binding site motifs in their promoter regions indicated that these genes shared an underlying transcriptional programme. This cluster contained known FAE- (Anxa10, Ccl20, Psg18 and Ubd) and M-cell- (Gp2) specific genes, suggesting that the others were novel FAE-specific genes. Some of these novel candidate genes were expressed highly by the FAE and M cells (Calcb, Ces3b, Clca2 and Gjb2), and others only by the FAE (Ascl2, Cftr, Fgf15, Gpr133, Kcna1, Kcnj15, Mycl1, Pgap1 and Rps6kl). We also identified a subset of novel FAE-related genes that were induced in the intestinal epithelium after receptor activator of NF-κB ligand (RANKL)-stimulation. These included Mfge8 which was specific to FAE enterocytes. This study provides new insight into the FAE transcriptome. Further characterisation of the candidate genes identified here will aid the identification of novel regulators of cell function in the FAE