Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: a technical assessment

peer-reviewedRecent advances made in “omics” technologies are contributing to a revolution in livestock selection and breeding practices. Epigenetic mechanisms, including DNA methylation are important determinants for the control of gene expression in mammals. DNA methylation research will help our understanding of how environmental factors contribute to phenotypic variation of complex production and health traits. High-throughput sequencing is a vital tool for the comprehensive analysis of DNA methylation, and bisulfite-based strategies coupled with DNA sequencing allows for quantitative, site-specific methylation analysis at the genome level or genome wide. Reduced representation bisulfite sequencing (RRBS) and more recently whole genome bisulfite sequencing (WGBS) have proven to be effective techniques for studying DNA methylation in both humans and mice. Here we report the development of RRBS and WGBS for use in sheep, the first application of this technology in livestock species. Important technical issues associated with these methodologies including fragment size selection and sequence depth are examined and discussed.AgResearch AR&C grant for funding and Teagasc for providing a short-term overseas training awar

Comparison of N. Atlantic heat storage estimates during the Argo period (1999–2010)

Ocean heat storage is an essential component of the climate system and there is considerable interest in its accurate evaluation. There are a number of heat storage products produced by many different groups. These products are derived from Argo as well as other platforms, for example XBT and CTD, in the last decade. Here we compare two heat storage estimates for the North Atlantic 0–2000 m from 10° to 70° N. One derived solely from Argo data whilst the other is derived from Argo and other platforms. It is found that there is a positive trend in heat storage over the period 1999–2010. This trend is influenced by a strong air–sea interaction event in 2009–2010, and this reduces the upward trend 1999–2008 identified previously. Both data sets are consistent with each other for the layer 0–1000 m on a timescale of beyond 1 yr. There are significant differences at sub-annual time scales and in the layer 1000–2000 m

DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming

<p>Abstract</p> <p>Background</p> <p>Cloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI).</p> <p>Results</p> <p>Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1) showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns.</p> <p>Conclusion</p> <p>These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a good predictor of whether the foetus would develop to term or not.</p

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

peer-reviewedWe 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-β 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

Análise de viabilidade e dimensionamento de um estacionamento solar fotovoltaico para o campus da UFSC em Joinville

TCC (graduação) - Universidade Federal de Santa Catarina. Campus Joinville. Engenharia de Infraestrutura.A migração total de fontes não renováveis para fontes limpas e renováveis ainda não é uma realidade, entretanto com a evolução das tecnologias a energia elétrica proveniente de placas solares ganha cada vez mais espaço no cenário mundial. No âmbito nacional, onde temos condições climáticas privilegiadas para explorar este tipo de energia, ainda temos um baixo aproveitamento na área. Contudo, devido a tendência de crescimentodo setor nos próximos anos algumas empresas já estão percebendo as vantagens para utilização dessa fonte energética e investindo na tecnologia fotovoltaica. A fim de criar uma alternativa diferente, o trabalho propõe o projeto de um estacionamento solar fotovoltaico para o Centro Tecnológico de Joinville (CTJ). O sistema possui uma potência de 380,16kW e tem o retorno do investimento em 65 meses.The total migration of non renewable sources to clean and renewable sources is not a reality yet, in the meantime with the evolution of tecnologies and electric power from solar panels are gaining more and more space in the world scenario. In the national scope, where we have privileged climate conditions to explore this type of energy, we still have a low use in this area. However, due to the growing trend of the sector in the next few years some companies are already realizing the benefits of the usage of this energy resource and investing in photovoltaic energy.In order to create a different alternative, the monograph proposes the project of a solar photovoltaic parking lot for the Technological Center of Joinville (CTJ). The system has a power of 380.16kW and has the return on investment in 65 months

On which timescales do gas transfer velocities control North Atlantic CO2 flux variability?

This is the final version of the article. Available from AGU via the DOI in this record.The North Atlantic is an important basin for the global ocean's uptake of anthropogenic and natural carbon dioxide (CO 2 ), but the mechanisms controlling this carbon flux are not fully understood. The air-sea flux of CO 2 , F, is the product of a gas transfer velocity, k, the air-sea CO 2 concentration gradient, ΔpCO 2 , and the temperature- and salinity-dependent solubility coefficient, α. k is difficult to constrain, representing the dominant uncertainty in F on short (instantaneous to interannual) timescales. Previous work shows that in the North Atlantic, ΔpCO 2 and k both contribute significantly to interannual F variability but that k is unimportant for multidecadal variability. On some timescale between interannual and multidecadal, gas transfer velocity variability and its associated uncertainty become negligible. Here we quantify this critical timescale for the first time. Using an ocean model, we determine the importance of k, ΔpCO 2 , and α on a range of timescales. On interannual and shorter timescales, both ΔpCO 2 and k are important controls on F. In contrast, pentadal to multidecadal North Atlantic flux variability is driven almost entirely by ΔpCO 2 ; k contributes less than 25%. Finally, we explore how accurately one can estimate North Atlantic F without a knowledge of nonseasonal k variability, finding it possible for interannual and longer timescales. These findings suggest that continued efforts to better constrain gas transfer velocities are necessary to quantify interannual variability in the North Atlantic carbon sink. However, uncertainty in k variability is unlikely to limit the accuracy of estimates of longer-term flux variability.This work was supported the RAGNARoCC NERC directed research program (NE/K002546/1, NE/K00249X/1, and NE/K002473/1)

Field measurements of cable self-burial in a sandy marine environment

The world's shallow continental shelves are currently experiencing a rapid pace of development from the growth of offshore renewable energy. The emplacement of infrastructure on the seabed can change the morphology of the bed, the nature of the flow above it and transport of sediment and so complicate the assessment of seabed stability for planning and designing offshore renewable infrastructure. To ascertain how much of an impact these natural processes have on cable stability, we present the first field observations made directly over a section of subsea cable, from two deployments in the Eastern Irish Sea at a location of current and planned offshore windfarms. Profiles of flow, turbulence and suspended sediment concentration were measured over a section of typical high voltage electricity cable. Upon deployment our observations show that sediment was deposited around the cable and self-burial occurred. The rate of deposition varied between surveys dependent on forcing and local bed conditions. Turbulence generated from the cable itself reduced as the embedment depth increased, but the relationship between bed shear stress and suspended sediment concentration was not consistent between surveys. We discuss several processes potentially responsible for the prevalence of deposition around the cable, and the difference in seabed mobility between the surveys

Primary Transgenic Bovine Cells and Their Rejuvenated Cloned Equivalents Show Transgene-Specific Epigenetic Differences

Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine β-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences