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During prolonged wetting of the fleece, proliferation of bacterial flora often dominated by Pseudomonas aeruginosa or Dermatophilus congolensis can induce dermatitis and fleece damage termed fleece rot and dermatophilosis respectively, which predispose sheep to blowfly strike. A large research effort in the 1980s and 1990s on vaccines to control fleece rot and dermatophilosis met with limited success. This review examines theoretical and technological advances in microbial ecology, pathogenesis, immunology, vaccine development and the characterisation of microbial virulence factors that create new opportunities for development of vaccines against these diseases. Genomic technologies have now created new opportunities for examining microbial dynamics and pathogen virulence in dermatitis. An effective vaccine requires the combination of appropriate antigens with an adjuvant that elicits a protective immune response that ideally provides long-lasting protection in the field. A clinical goal informed by epidemiological, economic and animal welfare values is needed as a measure of vaccine efficacy. Due to dependence of fleece rot and dermatophilosis on sporadic wet conditions for their expression, vaccine development would be expedited by in vitro correlates of immune protection. The efficacy of vaccines is influenced by genetic and phenotypic characteristics of the animal. Advances in understanding vaccine responsiveness, immune defence in skin and immune competence in sheep should also inform any renewed efforts to develop new fleece rot and dermatophilosis vaccines. The commercial imperatives for new vaccines are likely to continue to increase as the animal welfare expectations of society intensify and reliance on pharmacotherapeutics decrease due to chemical resistance, market pressures and societal influences. Vaccines should be considered part of an integrated disease control strategy, in combination with genetic selection for general immune competence and resistance to specific diseases, as well as management practices that minimise stress and opportunities for disease transmission. The strategy could help preserve the efficacy of pharmacotherapeutics as tactical interventions to alleviate compromised welfare when adverse environmental conditions lead to a break down in integrated strategic disease control. P. aeruginosa and D. congolensis are formidable pathogens and development of effective vaccines remains a substantial challenge.

Fleece rot develops following prolonged wetting of sheep when bacterial proliferation in wool and on skin induces exudation of serum proteins onto the skin surface and causes damage to wool follicles and fibres. These processes create an attractive environment for blowflies to lay eggs, leading to body strike. Current reliance on insecticides for prevention and treatment of fly strike is being increasingly challenged by development of insecticide resistance. This review examines the large body of past research on the bacterial causes of fleece rot, the genetics of sheep susceptibility and resistance, the characteristics of the resulting immune defence reactions, and attempts to control fleece rot by vaccination. The high dependence on weather conditions for expression of fleece rot hampers studies on the disease. Normal skin and wool are populated by a dynamic microbial community. During adverse environmental conditions, natural resistance to fleece rot associated with physical characteristics of wool and skin can be overwhelmed and a complex mix of bacteria flourishes. Prolonged hydration alone, and in combination with bacterial exoproducts, induces dermatitis and exudation of immunoglobulins and other serum proteins onto the skin surface. Pathogens do not usually penetrate the epidermis. Nonetheless, during prolonged skin hydration, sheep can become sensitised to fleece rot pathogens and produce antibodies. Antibody titres rise late within a typical (3 week) case of fleece rot. High naturally acquired antibody titres may contribute to resistance to fleece rot. In contrast to some evidence for a protective role of antibody, there is little evidence for innate or adaptive cellular immune responses contributing to protection against fleece rot pathogens. Previous attempts to develop vaccines have met with mixed success. Nonetheless, there remain prospects for development of a new vaccine to control fleece rot. Further knowledge on the microbial ecology of normal and wet skin would assist this endeavour and may help identify other control strategies.

Lumpy wool (dermatophilosis) develops following prolonged wetting of sheep when bacterial proliferation in wool and on skin induce an exudative dermatitis, causing a superficial skin lesion and damage to wool follicles and fibres. The incidence of dermatophilosis is strongly dependent on wet and warm weather and, hence, infection is sporadic. While older animals are less at risk than are lambs, it is unclear whether this reflects naturally acquired immune resistance or the maturation of skin and wool fibres. Dermatophilosis directly causes wool production losses and it also is a risk factor for blowfly strike, which has a substantial economic impact and increasing challenges associated with current control procedures. This review assessed research on the bacterial causes of lumpy wool, the characteristics of the resulting immune defence reactions in sheep, current control strategies, and limitations of previous attempts to control lumpy wool by sheep vaccination.

usc There are critical molecular mechanisms that can be activated to induce myocardial repair, and in humans this is most efficient during fetal development. The timing of heart development in relation to birth and the size/electrophysiology of the heart are similar in humans and sheep, providing a model to investigate the repair capacity of the mammalian heart and how this can be applied to adult heart repair. Myocardial infarction was induced by ligation of the left anterior descending coronary artery in fetal (105 days gestation when cardiomyocytes are proliferative) and adolescent sheep (6 mo of age when all cardiomyocytes have switched to an adult phenotype). An ovine gene microarray was used to compare gene expression in sham and infarcted (remote, border and infarct areas) cardiac tissue from fetal and adolescent hearts. The gene response to myocardial infarction was less pronounced in fetal compared with adolescent sheep hearts and there were unique gene responses at each age. There were also region-specific changes in gene expression between each age, in the infarct tissue, tissue bordering the infarct, and tissue remote from the infarction. In total, there were 880 genes that responded to MI uniquely in the adolescent samples compared with 170 genes in the fetal response, as well as 742 overlap genes that showed concordant direction of change responses to infarction at both ages. In response to myocardial infarction, there were specific changes in genes within pathways of mitochondrial oxidation, muscle contraction, and hematopoietic cell lineages, suggesting that the control of energy utilization and immune function are critical for effective heart repair. The more restricted gene response in the fetus may be an important factor in its enhanced capacity for cardiac repair Refereed/Peer-reviewed

Myocardial infarction is a primary contributor towards the global burden of cardiovascular disease. Rather than repairing the existing damage of myocardial infarction, current treatments only address the symptoms of the disease and reducing the risk of a secondary infarction. Cardiac regenerative capacity is dependent on cardiomyocyte proliferation, which concludes soon after birth in humans and precocial species such as sheep. Human fetal cardiac tissue has some ability to repair following tissue damage, whereas a fully matured human heart has minimal capacity for cellular regeneration. This is in contrast to neonatal mice and adult zebrafish hearts, which retain the ability to undergo cardiomyocyte proliferation and can regenerate cardiac tissue after birth. In mice and zebrafish models, microRNAs (miRNAs) have been implicated in the regulation of genes involved in cardiac cell cycle progression and regeneration. However, the significance of miRNA regulation in cardiomyocyte proliferation for humans and other large mammals, where the timing of heart development in relation to birth is similar, remains unclear. miRNAs may be valuable targets for therapies that promote cardiac repair after injury. Therefore, elucidating the role of specific miRNAs in large animals, where heart development closely resembles that of humans, remains vitally important for identifying therapeutic targets that may be translated into clinical practice focused on tissue repair.

Background In food animal agriculture, there is a need to identify the mechanisms that can improve the efficiency of muscle growth and protein accretion. Callipyge sheep provide excellent machinery since the up-regulation of DLK1 and RTL1 results in extreme postnatal muscle hypertrophy in distinct muscles. The aim of this study is to distinguish the genes that directly respond to DLK1 and RTL1 signaling from the genes that change as the result of muscle specific effects. Results The quantitative PCR results indicated that DLK1 expression was significantly increased in hypertrophied muscles but not in non-hypertrophied muscles. However, RTL1 was up-regulated in both hypertrophied and non-hypertrophied muscles. Five genes, including PARK7, DNTTIP1, SLC22A3, METTL21E and PDE4D, were consistently co-expressed with DLK1, and therefore were possible transcriptional target genes responding to DLK1 signaling. Treatment of myoblast and myotubes with DLK1 protein induced an average of 1.6-fold and 1.4-fold increase in Dnttip1 and Pde4d expression respectively. Myh4 expression was significantly elevated in DLK1-treated myotubes, whereas the expression of Mettl21e was significantly increased in the DLK1-treated myoblasts but reduced in DLK1-treated myotubes. DLK1 treatment had no impact on Park7 expression. In addition, Park7 and Dnttip1 increased Myh4 and decreased Myh7 promoter activity, resemble to the effects of Dlk1. In contrast, expression of Mettl21e increased Myh7 and decreased Myh4 luciferase activity. Conclusion The study provided additional supports that RTL1 alone was insufficient to induce muscle hypertrophy and concluded that DLK1 was likely the primary effector of the hypertrophy phenotype. The results also suggested that DNTTIP1 and PDE4D were secondary effector genes responding to DLK1 signaling resulting in muscle fiber switch and muscular hypertrophy in callipyge lamb. Electronic supplementary material The online version of this article (10.1186/s12864-018-4682-1) contains supplementary material, which is available to authorized users.

Aim: Characterizing the response to myocardial infarction (MI) in the regenerative sheep fetus heart compared to the post-natal non-regenerative adolescent heart may reveal key morphological and molecular differences that equate to the response to MI in humans. We hypothesized that the immediate response to injury in (a) infarct compared with sham, and (b) infarct, border, and remote tissue, in the fetal sheep heart would be fundamentally different to the adolescent, allowing for repair after damage Methods: We used a sheep model of MI induced by ligating the left anterior descending coronary artery. Surgery was performed on fetuses (105 days) and adolescent sheep (6 months). Sheep were randomly separated into MI (n = 5) or Sham (n = 5) surgery groups at both ages. We used magnetic resonance imaging (MRI), histological/immunohistochemical staining, and qRT-PCR to assess the morphological and molecular differences between the different age groups in response to infarction Results: Magnetic resonance imaging showed no difference in fetuses for key functional parameters; however there was a significant decrease in left ventricular ejection fraction and cardiac output in the adolescent sheep heart at 3 days post-infarction. There was no significant difference in functional parameters between MRI sessions at Day 0 and Day 3 after surgery. Expression of genes involved in glucose transport and fatty acid metabolism, inflammatory cytokines as well as growth factors and cell cycle regulators remained largely unchanged in the infarcted compared to sham ventricular tissue in the fetus, but were significantly dysregulated in the adolescent sheep. Different cardiac tissue region-specific gene expression profiles were observed between the fetal and adolescent sheep Conclusion: Fetuses demonstrated a resistance to cardiac damage not observed in the adolescent animals. The manipulation of specific gene expression profiles to a fetal-like state may provide a therapeutic strategy to treat patients following an infarction Refereed/Peer-reviewed

G-quadruplexes (G4) are highly stable tetra-stranded DNA secondary structures known to mediate gene regulation and to trigger genomic instability events during replication. G4 structural stability can be affected by DNA methylation and oxidation modifications; thus nutrients such as folate that have the ability to alter these processes could potentially modify the genomic occurrence of G4 elements. Hela cells were cultured in a range of folate concentrations or in the presence or absence of 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor. G4 structures were then quantified by immunofluorescence using an automated quantitative imaging system. G4 frequency in Hela cells and nuclei area mean were increased in 20nM folate medium compared with 2000nM folate, as well as in the presence of 5-aza-2'-deoxycytidine when compared to cells non-exposed to 5-aza-2'-deoxycytidine. These changes were exacerbated when pyridostatin, a G4 stabilising ligand, was added to the culture medium. G4 intensity in Hela cells cultured in deficient folate condition with pyridostatin was highly correlated with DNA damage as measured by γH2AX immunofluorescence (r = 0.71). This study showed for the first time that cellular G4 balance is modifiable by low folate concentrations and that these changes may occur as a consequence of DNA hypomethylation. Although the exact mechanism by which these changes occur is unclear, these findings establish the possibility that nutrients could be utilised as a tool for sustaining genome integrity by modifying G4 frequency at a cellular level.

Domestication fundamentally reshaped animal morphology, physiology and behaviour, offering the opportunity to investigate the molecular processes driving evolutionary change. Here we assess sheep domestication and artificial selection by comparing genome sequence from 43 modern breeds (Ovis aries) and their Asian mouflon ancestor (O. orientalis) to identify selection sweeps. Next, we provide a comparative functional annotation of the sheep genome, validated using experimental ChIP-Seq of sheep tissue. Using these annotations, we evaluate the impact of selection and domestication on regulatory sequences and find that sweeps are significantly enriched for protein coding genes, proximal regulatory elements of genes and genome features associated with active transcription. Finally, we find individual sites displaying strong allele frequency divergence are enriched for the same regulatory features. Our data demonstrate that remodelling of gene expression is likely to have been one of the evolutionary forces that drove phenotypic diversification of this common livestock species., M.N.-S. and Q.N. are funded by the CSIRO Science Excellence Research Office. M.P.-E. was funded by a CSIRO McMaster Visiting Fellowship. Mouflon genome sequences were generated by the NextGen Consortium, as part of the European Union’s Seventh Framework Programme (FP7/2010-2014 grant 244356).

Genome sequences for hundreds of mammalian species are available, but an understanding of their genomic regulatory regions, which control gene expression, is only beginning. A comprehensive prediction of potential active regulatory regions is necessary to functionally study the roles of the majority of genomic variants in evolution, domestication, and animal production. We developed a computational method to predict regulatory DNA sequences (promoters, enhancers and transcription factor binding sites) in production animals (cows and pigs) and extended its broad applicability to other mammals. The method utilizes human regulatory features identified from thousands of tissues, cell lines, and experimental assays to find homologous regions that are conserved in sequences and genome organization and are enriched for regulatory elements in the genome sequences of other mammalian species. Importantly, we developed a filtering strategy, including a machine learning classification method, to utilize a very small number of species-specific experimental datasets available to select for the likely active regulatory regions. The method finds the optimal combination of sensitivity and accuracy to unbiasedly predict regulatory regions in mammalian species. Furthermore, we demonstrated the utility of the predicted regulatory datasets in cattle for prioritizing variants associated with multiple production and climate change adaptation traits, and identifying potential genome editing targets.

Heritable trait variation within a population of organisms is largely governed by DNA variations that impact gene transcription and protein function. Identifying genetic variants that affect complex functional traits is a primary aim of population genetics studies, especially in the context of human disease and agricultural production traits. The identification of alleles directly altering mRNA expression and thereby biological function is challenging due to difficulty in isolating direct effects of cis-acting genetic variations from indirect trans-acting genetic effects. Allele specific gene expression or allelic imbalance in gene expression (AI) occurring at heterozygous loci provides an opportunity to identify genes directly impacted by cis-acting genetic variants as indirect trans-acting effects equally impact the expression of both alleles. However, the identification of genes showing AI in the context of the expression of all genes remains a challenge due to a variety of technical and statistical issues. The current study focuses on the discovery of genes showing AI using single nucleotide polymorphisms as allelic reporters. By developing a computational and statistical process that addressed multiple analytical challenges, we ranked 5,809 genes for evidence of AI using RNA-Seq data derived from brown adipose tissue samples from a cohort of late gestation fetal lambs and then identified a conservative subgroup of 1,293 genes. Thus, AI was extensive, representing approximately 25% of the tested genes. Genes associated with AI were enriched for multiple Gene Ontology (GO) terms relating to lipid metabolism, mitochondrial function and the extracellular matrix. These functions suggest that cis-acting genetic variations causing AI in the population are preferentially impacting genes involved in energy homeostasis and tissue remodelling. These functions may contribute to production traits likely to be under genetic selection in the population. Refereed/Peer-reviewed

Molecular mechanisms that are associated with age-related denervation and loss of skeletal muscle mass and function (sarcopenia) are described for female C57Bl/6J mice aged 3, 15, 24, 27 and 29 months (m). Changes in mRNAs and proteins associated with myofibre denervation and protein metabolism in ageing muscles are reported, across the transition from healthy adult myofibres to sarcopenia that occurs between 15 and 24 m. This onset of sarcopenia at 24 m, corresponded with increased expression of genes associated with neuromuscular junction denervation including Chnrg, Chrnd, Ncam1, Runx1, Gadd45a and Myog. Sarcopenia in quadriceps muscles also coincided with increased protein levels for Igf1 receptor, Akt and ribosomal protein S6 (Rps6) with increased phosphorylation of Rps6 (Ser235/236) and elevated Murf1 mRNA and protein, but not Fbxo32: many of these changes are also linked to denervation. Global transcription profiling via microarray analysis confirmed these functional themes and highlighted additional themes that may be a consequence of pathology associated with sarcopenia, including changes in fatty acid metabolism, extracellular matrix structure and protein catabolism. Ageing was also associated with increased global gene expression variance, consistent with decreased control of gene regulation.

Sarcopenia is associated with adverse health outcomes. This study investigated whether skeletal muscle gene expression was associated with lean mass and grip strength in community-dwelling older men. Utilising a cross-sectional study design, lean muscle mass and grip strength were measured in 88 men aged 68-76 years. Expression profiles of 44 genes implicated in the cellular regulation of skeletal muscle were determined. Serum was analysed for circulating cytokines TNF (tumour necrosis factor), IL-6 (interleukin 6, IFNG (interferon gamma), IL1R1 (interleukin-1 receptor-1). Relationships between skeletal muscle gene expression, circulating cytokines, lean mass and grip strength were examined. Participant groups with higher and lower values of lean muscle mass (n = 18) and strength (n = 20) were used in the analysis of gene expression fold change. Expression of VDR (vitamin D receptor) [fold change (FC) 0.52, standard error for fold change (SE) ± 0.08, p = 0.01] and IFNG mRNA (FC 0.31; SE ± 0.19, p = 0.01) were lower in those with higher lean mass. Expression of IL-6 (FC 0.43; SE ± 0.13, p = 0.02), TNF (FC 0.52; SE ± 0.10, p = 0.02), IL1R1 (FC 0.63; SE ± 0.09, p = 0.04) and MSTN (myostatin) (FC 0.64; SE ± 0.11, p = 0.04) were lower in those with higher grip strength. No other significant changes were observed. Significant negative correlations between serum IL-6 (R = -0.29, p = 0.005), TNF (R = -0.24, p = 0.017) and grip strength were demonstrated. This novel skeletal muscle gene expression study carried out within a well-characterized epidemiological birth cohort has demonstrated that lower expression of VDR and IFNG is associated with higher lean mass, and lower expression of IL-6, TNF, IL1R1 and myostatin is associated with higher grip strength. These findings are consistent with a role of proinflammatory factors in mediating lower muscle strength in community-dwelling older men.

The callipyge phenotype in sheep involves substantial postnatal muscle hypertrophy and other changes to carcass composition. A single nucleotide polymorphism in the DLK1-DIO3 imprinted gene cluster alters gene expression of the paternal allele-specific protein-coding genes and several maternal allele-specific long noncoding RNA and microRNA when the mutation is inherited in cis. The inheritance pattern of the callipyge phenotype is polar overdominant because muscle hypertrophy only occurs in heterozygous animals that inherit a normal maternal allele and the callipyge SNP on the paternal allele (+/C). We examined the changes of gene expression of four major transcripts from the DLK1-DIO3 cluster and four myosin isoforms during the development of muscle hypertrophy in the semimembranosus as well as in the supraspinatus that does not undergo hypertrophy. The homozygous (C/C) animals had an intermediate gene expression pattern for the paternal allele-specific genes and two myosin isoforms, indicating a biological activity that was insufficient to change muscle mass. Transcriptome analysis was conducted by RNA sequencing in the four callipyge genotypes. The data show that homozygous animals (C/C) have lower levels of gene expression at many loci relative to the other three genotypes. A number of the downregulated genes are putative targets of the maternal allele-specific microRNA with gene ontology, indicating regulatory and cell signaling functions. These results suggest that the trans-effect of the maternal noncoding RNA and associated miRNA is to stabilize the expression of a number of regulatory genes at a functional, but low level to make the myofibers of homozygous (C/C) lambs less responsive to hypertrophic stimuli of the paternal allele-specific genes.

Summary The ruminant developmental transition from late foetus to lamb is associated with marked changes in skeletal muscle structure and function that reflect programming for new physiological demands following birth. To determine whether epigenetic changes are involved in this transition, we investigated the genomic architecture of the chromatin modification, histone 3 lysine 27 trimethylation (H3K27me3), which typically regulates early life developmental processes; however, its role in later life processes is unclear. Chromatin immunoprecipitation coupled with next‐generation sequencing was used to map H3K27me3 nucleosomes in ovine longissimus lumborum skeletal muscle at 100 days of gestation and 12 weeks post‐partum. In both states, H3K27me3 modification was associated with genes, transcription start sites and CpG islands and with transcriptional silencing. The H3K27me3 peaks consisted of two major categories, promoter specific and regional, with the latter the dominant feature. Genes encoding homeobox transcription factors regulating early life development and genes involved in neural functions, particularly gated ion channels, were strongly modified by H3K27me3. Gene promoters differentially modified by H3K27me3 in the foetus and lamb were enriched for gated ion channels, which may reflect changes in neuromuscular function. However, most modified genes showed no changes, indicating that H3K27me3 does not have a large role in late muscle maturation. Notably, promyogenic transcription factors were strongly modified with H3K27me3 but showed no differences between the late gestation foetus and lamb, likely reflecting their lack of involvement in the myofibre fusion process occurring in this transition. H3K27me3 is a major architectural feature of the epigenetic landscape of ruminant skeletal muscle, and it comments on gene transcription and gene function in the context of late skeletal muscle development.

Background Body weight (BW) is an important trait for meat production in sheep. Although over the past few years, numerous quantitative trait loci (QTL) have been detected for production traits in cattle, few QTL studies have been reported for sheep, with even fewer on meat production traits. Our objective was to perform a genome-wide association study (GWAS) with the medium-density Illumina Ovine SNP50 BeadChip to identify genomic regions and corresponding haplotypes associated with BW in Australian Merino sheep. Methods A total of 1781 Australian Merino sheep were genotyped using the medium-density Illumina Ovine SNP50 BeadChip. Among the 53 862 single nucleotide polymorphisms (SNPs) on this array, 48 640 were used to perform a GWAS using a linear mixed model approach. Genotypes were phased with hsphase; to estimate SNP haplotype effects, linkage disequilibrium blocks were identified in the detected QTL region. Results Thirty-nine SNPs were associated with BW at a Bonferroni-corrected genome-wide significance threshold of 1 %. One region on sheep (Ovis aries) chromosome 6 (OAR6) between 36.15 and 38.56 Mb, included 13 significant SNPs that were associated with BW; the most significant SNP was OAR6_41936490.1 (P = 2.37 × 10−16) at 37.69 Mb with an allele substitution effect of 2.12 kg, which corresponds to 0.248 phenotypic standard deviations for BW. The region that surrounds this association signal on OAR6 contains three genes: leucine aminopeptidase 3 (LAP3), which is involved in the processing of the oxytocin precursor; NCAPG non-SMC condensin I complex, subunit G (NCAPG), which is associated with foetal growth and carcass size in cattle; and ligand dependent nuclear receptor corepressor-like (LCORL), which is associated with height in humans and cattle. Conclusions The GWAS analysis detected 39 SNPs associated with BW in sheep and a major QTL region was identified on OAR6. In several other mammalian species, regions that are syntenic with this region have been found to be associated with body size traits, which may reflect that the underlying biological mechanisms share a common ancestry. These findings should facilitate the discovery of causative variants for BW and contribute to marker-assisted selection. Electronic supplementary material The online version of this article (doi:10.1186/s12711-015-0142-4) contains supplementary material, which is available to authorized users.

Rtl1 and Dlk1 are two genes in an imprinted gene cluster on mouse chromosome 12. Rtl1 is a paternally expressed gene encoding a retrotransposon-like protein, but as yet, there is no evidence that a protein is produced by this gene although a transcript is produced. Dlk1 is also paternally expressed and it encodes a plasma membrane-bound protein known to act as a negative regulator of adipogenesis. Genes at other imprinted loci often show coordinate expression and may interact at a functional level. We tested the hypothesis that Dlk1 and Rtl1 are functionally related. Rtl1 and Dlk1 mRNA expression patterns show similar time courses of down-regulation during induced adipogenesis of 3T3L1 cells. Despite the coordinate down-regulation of Dlk1 and Rtl1 mRNA levels, Rtl1 over-expression did not affect the time course of adipogenesis or Dlk1 expression. It is concluded that these two genes are neither directly nor indirectly functionally related.

Pharmacokinetics of leptin in mammals has received limited attention and only one study has examined more than two time points and this was in ob/ob mice. This study is the first to observe the distribution of leptin over a time course in female mice. A physiologic dose (12 ng) of radiolabelled leptin was injected in adult female mice via the lateral tail vein and tissues were dissected out and measured for radioactivity over a time course up to two hours. Major targets for administered leptin included the liver, kidneys, gastrointestinal tract and the skin while the lungs had high concentrations of administered leptin per gram of tissue. Leptin was also found to enter the lumen of the digestive tract intact from the plasma. Very little of the dose (

The increased prevalence of obesity and related comorbidities is a major public health problem. While genetic factors undoubtedly play a role in determining individual susceptibility to weight gain and obesity, the identified genetic variants only explain part of the variation. This has led to growing interest in understanding the potential role of epigenetics as a mediator of gene-environment interactions underlying the development of obesity and its associated comorbidities. Initial evidence in support of a role of epigenetics in obesity and type 2 diabetes mellitus (T2DM) was mainly provided by animal studies, which reported epigenetic changes in key metabolically important tissues following high-fat feeding and epigenetic differences between lean and obese animals and by human studies which showed epigenetic changes in obesity and T2DM candidate genes in obese/diabetic individuals. More recently, advances in epigenetic methodologies and the reduced cost of epigenome-wide association studies (EWAS) have led to a rapid expansion of studies in human populations. These studies have also reported epigenetic differences between obese/T2DM adults and healthy controls and epigenetic changes in association with nutritional, weight loss, and exercise interventions. There is also increasing evidence from both human and animal studies that the relationship between perinatal nutritional exposures and later risk of obesity and T2DM may be mediated by epigenetic changes in the offspring. The aim of this review is to summarize the most recent developments in this rapidly moving field, with a particular focus on human EWAS and studies investigating the impact of nutritional and lifestyle factors (both pre- and postnatal) on the epigenome and their relationship to metabolic health outcomes. The difficulties in distinguishing consequence from causality in these studies and the critical role of animal models for testing causal relationships and providing insight into underlying mechanisms are also addressed. In summary, the area of epigenetics and metabolic health has seen rapid developments in a short space of time. While the outcomes to date are promising, studies are ongoing, and the next decade promises to be a time of productive research into the complex interactions between the genome, epigenome, and environment as they relate to metabolic disease. Refereed/Peer-reviewed

G-quadruplexes (G4) are highly stable tetra-stranded secondary DNA structures known to mediate gene regulation. These structures are resolved by DNA helicases and are believed to be a causal factor in the phenotype of premature ageing disorders following mutations in DNA helicase genes. The relevance of G4 structures in ageing may be further implicated by their dynamic relationship with DNA modification mechanisms. When DNA methylation and oxidation occur at the vicinity of G4 elements, they can affect the stability of G4 structures which may in turn mediate gene expression resulting in deleterious effects on genome integrity. Therefore, the influence of nutritional deficiencies or excess on oxidation and methylation mechanisms may be contributing factors affecting the stability of G4 structures and their balance in the human genome. We propose that dietary nutrients such as folate and antioxidants may play a beneficial role in reducing G4-induced DNA damage through changes in G4 structure stability. The current knowledge advocates the importance of resolving G4 structures by DNA helicases for sustained genome integrity, and the existence of stability changes in G4 structures when associated with DNA methylation and oxidation modifications.

Immunological control of cutaneous myiasis of sheep caused by Lucilia cuprina larvae has been an elusive goal. Antibody to antigens derived from the peritrophic membrane can stunt or kill larvae in a dose dependent fashion. Thus efficacy of vaccines employing these antigens may be limited by the amount of antibody in skin available for ingestion by larvae. The potential for elevating antibody concentrations in skin by intradermal immunisation with the recombinant peritrophic membrane antigens peritrophin-44, peritrophin-48 and peritrophin-95 was therefore examined. Using within-animal comparisons, specific antibody was significantly higher in skin transudates from locally immunised sites than from adjacent adjuvant control sites. It was concluded that cutaneous immunisation may assist immunological control of blowfly larvae.

Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8+ T cell epitopes by CD11c+ dendritic cells in draining lymph nodes and early priming of antigen-specific CD8+ T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8+ T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection., Author Summary Maintenance proteins of viruses establishing latent infections regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The Epstein-Barr virus maintenance protein, EBNA1, has recently been shown to contain unusual G-quadruplex structures within its repeat mRNA that reduces its translational efficiency. In this study we assess how modification of the EBNA1 mRNA repeat sequence to destabilize the native G-quadruplex structures and thereby increase translation, impacts on the activation of EBNA1-specific T cells in vivo. Mice primed with viral vectors encoding a more efficiently translated EBNA1 mRNA revealed increased trafficking of EBNA1-specific T cells, an enhanced functional profile and increased expression of transcription factors providing evidence for a potential link between mRNA translational efficiency and antigen presentation in vivo and the resultant impact on the functional programming of effector T cells. These findings suggest a novel approach to therapeutic development through the use of antisense strategies or small molecules targeting EBNA1 mRNA structure.

The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. Using muscle fibre typing immunohistochemistry, we confirmed muscle specific effects and demonstrated that affected muscles had greater prevalence and hypertrophy of type 2X fast twitch glycolytic fibres and decreased representation of types 1, 2C, 2A and/or 2AX fibres. To investigate potential systemic effects of the mutation, proton NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. Microarray analysis of the perirenal adipose tissue depot did not reveal a transcriptional effect of the mutation in this tissue. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness.

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) includes a unique glycine-alanine repeat domain that inhibits the endogenous presentation of cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by blocking proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated diseases. Here, we show that cotranslational ubiquitination combined with N-end rule targeting enhances the intracellular degradation of EBNA1, thus resulting in a dramatic reduction in the half-life of the antigen. Using DNA expression vectors encoding different forms of ubiquitinated EBNA1 for in vivo studies revealed that this rapid degradation, remarkably, leads to induction of a very strong CTL response to an EBNA1-specific CTL epitope. Furthermore, this targeting also restored the endogenous processing of HLA class I-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific CTLs. These observations provide, for the first time, evidence that the glycine-alanine repeat-mediated proteasomal block on EBNA1 can be reversed by specifically targeting this antigen for rapid degradation resulting in enhanced CD8+ T cell-mediated recognition in vitro and in vivo.

The peritrophic matrix lines the midgut of most insects and has important roles in digestion, protection of the midgut from mechanical damage and invasion by micro-organisms. Although a few intrinsic peritrophic matrix proteins have been characterised, no direct homologues of any of these proteins have been found in other insect species, even closely related species, suggesting that the peritrophic matrix proteins show considerable sequence divergence. We now report the identification of the cDNA and genomic DNA sequences of a Chrysomya bezziana homologue of the Lucilia cuprina intrinsic peritrophic matrix protein, peritrophin-48. The gene for C. bezziana peritrophin-48 spans 1315 bp and consists of three exons (65, 560 and 690 bp, respectively) separated by introns of 566 and 72 bp. The transcriptional start site, identified by a consensus of cDNA clones and primer extension analysis, is probably located 58 bp upstream from the start codon. However, there may be multiple start sites for transcription. Two potential TATA boxes and a consensus arthropod transcription initiator are located within 134 bp of sequence upstream of the putative transcriptional start site suggesting that this region contains the gene promoter. Immuno-fluorescence localization demonstrated that C. bezziana peritrophin-48 was localised to the larval peritrophic matrix. Protein fold recognition analysis indicated structural similarities between peritrophin-48 and wheatgerm lectin. As wheatgerm lectin binds chitin, this result suggested that C. bezziana peritrophin-48 may also bind chitin, a constituent of the peritrophic matrix. Chitin binding studies with a recombinant peritrophin-48 protein confirmed that it binds chitin. A Drosophila melanogaster homologue of peritrophin-48 encoded in an EST and a genomic sequence was also identified. The pairwise percentage identities of the deduced amino acid sequences for the peritrophin-48 homologues from the three higher Dipteran species were relatively low, ranging between 32 and 42%. Despite this sequence variability, the predicted structure of these proteins, dictated by five domains, each containing a characteristic distribution of six cysteines, was strictly conserved. It is concluded that considerable sequence variation can be tolerated in this protein because of the constraints imposed on the structure of the protein by an extensive disulphide bonded framework.

The larvae of the fly Lucilia cuprina cause a cutaneous myiasis in mammalian hosts, particularly sheep. The glycoprotein, peritrophin-95, isolated from Lucilia cuprina larval peritrophic matrix, is a candidate vaccine antigen. This protein induced an immune response in vaccinated sheep that inhibited larval growth. Recombinant forms of peritrophin-95 were produced in bacteria and baculovirus-infected insect cells. The bacterial protein was not glycosylated and incorrectly folded whereas the insect cell-expressed protein was glycosylated and probably correctly folded. Sheep immunised with purified native peritrophin-95 generated strong larval growth inhibitory activity in their sera, whereas sheep immunised with either recombinant form of peritrophin-95 generated only relatively weak inhibitory activity. Ingested ovine antibodies to native peritrophin-95 mediated the anti-larval growth activity and this was independent of the presence of ovine complement. The activity was associated with IgG(1) and IgG(2) but not IgM. There were strong antibody responses to both the correctly folded native peritrophin-95 polypeptide and the oligosaccharides present on this glycoprotein. Immuno-affinity isolation of antibody to the peritrophin-95 polypeptide and antibody to peritrophin-95 oligosaccharides demonstrated that the larval growth inhibitory activity resided with both antibodies. Lectin blots and ELISA data showed substantial differences between the oligosaccharides attached to native peritrophin-95 and insect cell-expressed recombinant peritrophin-95. It was concluded that the oligosaccharides attached to native peritrophin-95 and its unique polypeptide structure are essential for the induction of larval growth inhibitory activity in the sera of sheep vaccinated with this antigen.

The peritrophic matrix is a prominent feature of the digestive tract of most insects, but its function, formation, and even its composition remain contentious. This matrix is a molecular sieve whose toughness and elasticity are generated by glycoproteins, proteoglycans, and chitin fibrils. We now describe a small, highly conserved protein, peritrophin-15, which is an abundant component of the larval peritrophic matrices of the Old World screwworm fly, Chrysomya bezziana, and sheep blowfly, Lucilia cuprina. Their deduced amino acid sequences code for a 8-kDa secreted protein characterized by a highly conserved and novel register of six cysteines. Two Drosophila homologues have also been identified from unannotated genomic sequences. Recombinant peritrophin-15 binds strongly and specifically to chitin; however, the stoichiometry of binding is low (1:10,000 N-acetyl glucosamine). We propose that peritrophin-15 caps the ends of the chitin polymer. Immunogold studies localized peritrophin-15 to the peritrophic matrix and specific vesicles in cells of the cardia, the small organ of the foregut responsible for peritrophic matrix synthesis. The vesicular contents are disgorged at the base of microvilli underlying the newly formed peritrophic matrix. This is the first time that the process of synthesis and integration of a peritrophic matrix protein into the nascent peritrophic matrix has been observed.

The gut of most insects is lined with a peritrophic matrix that facilitates the digestive process and protects insects from invasion by micro-organisms and parasites. It is widely accepted that the matrix is composed of chitin, proteins and proteoglycans. Here we critically re-examine the chitin content of the typical type 2 peritrophic matrix from the larvae of the fly Lucilia cuprina using a range of techniques. Many of the histochemical and biochemical techniques indicate the presence of chitin, although they are often adversely influenced by the presence of highly glycosylated proteins, a principal component of the matrix. The alkali-stable fraction, which is used as an indicator of the maximum chitin content in a biological sample, is only 7.2% of the weight of the matrix. Larvae fed on the potent chitin synthase inhibitor polyoxin D or the chitin-binding agent Calcofluor White, showed strong concentration-dependent inhibition of larval weight and survival but no discernible effects on the matrix structure. A bacterial endochitinase fed to larvae had no effect on larval growth and no observable effect in vitro on the structure of isolated peritrophic matrix. RT-PCR did not detect a chitin synthase mRNA in cardia, the tissue from which PM originates. It is concluded that chitin is a minor structural component of the type 2 peritrophic matrix of this insect.

In mature male sheep, the level of nutrition acutely influences the secretion of reproductive hormones. The mechanism involved is not fully understood but findings in humans and laboratory rodents would suggest a major role for leptin that is secreted from adipose tissue and then travels via the circulation to the central nervous system. Before we can begin to test this hypothesis, we need to be able to measure leptin concentrations in blood plasma and cerebrospinal fluid. We have therefore developed a radioimmunoassay using antibodies raised against biologically active recombinant bovine-ovine leptin. Using this assay, we found that plasma concentrations of leptin were highly correlated to back-fat thickness and to the ratio of back-fat thickness to liveweight, in female and castrated male sheep. Plasma concentrations of leptin were higher in female sheep than in castrated or intact male sheep. Serial samples (every 5 min) suggested that the secretion of leptin in male sheep is episodic but it does not appear to show clear pulsatility, increases post-prandially, or a diurnal rhythm. Leptin concentrations in both plasma and cerebrospinal fluid increased within 5 days in male sheep fed a diet with a high content of energy and protein that also stimulates the secretion of LH pulses. These data suggest that in sheep, as in other species, leptin production is correlated with the mass of adipose tissue and that the hormone passes from the circulation to the cerebrospinal fluid and then to hypothalamic sites. There, it may affect appetite and perhaps GnRH secretion. The role of leptin in the link between nutrition and reproduction needs further investigation.

The peritrophic matrix (or peritrophic membrane) lines the gut of most insects at one or more stages of the life cycle. It has important roles in the facilitation of the digestive processes in the gut and the protection of the insect from invasion by microorganisms and parasites. The traditional view of the peritrophic matrix as a relatively insert sieve, composed largely of proteins and glycosaminoglycans embedded in a chitinous matrix, is under revision as more is learned about the molecular characteristics of the peritrophic matrix proteins. This review summarizes emerging knowledge of the main protein constituents of the peritrophic matrix. The availability of the first sequences of integral peritrophic matrix proteins has coincided with the explosion of information in sequence databases. It is therefore possible to examine common structural themes in this family of proteins as well as in proteins of unknown location and function from a variety of other insects, nematodes and viruses. The review concludes with speculation about the biological functions of the proteins in this matrix.

Whole first-instar Lucilia cuprina larvae were homogenised and sequentially extracted with a series of buffers of progressively more severe solubilising power. The final extract, using a buffer containing 6 M-urea, was fractionated by preparative isoelectric focussing. At each step in this process, protein fractions were tested in sheep vaccination trials for their ability to induce immune responses affecting the growth of L. cuprina larvae which fed on the sera from vaccinated sheep. One isoelectric focussing fraction (pH 5.9-6.7) containing a number of larval proteins induced an immune response which inhibited the growth of larvae by a mean of 84 +/- 7% in an in vitro feeding bioassay. The recovery of larvae after feeding on sera from sheep vaccinated with this fraction was significantly reduced by 35 +/- 13%. This antilarval effect was shown to be mediated by ingested ovine antibodies. Immunofluorescence and immunogold localisations showed that the immune response was directed at proteins from the larval peritrophic membrane, larval cuticle and, to lesser extent, basement membranes and microvilli of digestive epithelial cells. Electron microscopic examination of larvae feeding on sera from sheep vaccinated with this fraction showed that the normally semi-permeable peritrophic membrane was blocked on the luminal side by an electron-lucent layer of undefined composition. It is postulated that this layer prevents nutrients from moving from the gut to the underlying digestive epithelial cells, thereby starving the larvae. The sera from sheep vaccinated with another isoelectric focussing fraction (pH 3.4-5.5) reduced the mean larval weight by 56 +/- 13% without significant effects on larval survival.

The gut of most insects is lined with a semi-permeable peritrophic membrane (or peritrophic matrix) composed of chitin, proteoglycans and proteins. Despite the probable importance of the peritrophic membrane in facilitating the digestive process and protecting insects from invasion by micro-organisms and parasites, there has been little characterization of the specific components and their interactions within this acellular structure. Here we report the characterization of an integral peritrophic membrane glycoprotein, peritrophin-48, from the larvae of the fly Lucilia cuprina, a primary agent of cutaneous myiasis in sheep. Peritrophin-48 was purified from peritrophic membrane obtained by larval culture and its location within the peritrophic membrane determined by immuno-fluorescence and immuno-gold localizations. The cDNA coding for peritrophin-48 was cloned and sequenced. The deduced amino acid sequence codes for a protein of 375 amino acids containing an amino-terminal signal sequence followed by five similar, but non-identical domains, each approximately 65–70 amino acids in length and characterised by a specific register of six cysteines. The deduced amino acid sequence shows significant similarity to two other peritrophic membrane proteins, peritrophin-95 and peritrophin-44, from the same species. A reverse transcriptase-PCR approach indicated that there are several highly related peritrophin-48 genes expressed in each individual. Reverse transcriptase-PCR also demonstrated the expression of peritrophin-48 in all three larval instars and adults but not pupae or eggs. Peritrophin-48 was expressed only by the cardia and by the larval midgut. A simple structural model of a basic unit of a type 2 peritrophic membrane is presented.

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsia (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.

The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

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Background We have recently described a method for the construction of an informative gene expression correlation landscape for a single tissue, longissimus muscle (LM) of cattle, using a small number (less than a hundred) of diverse samples. Does this approach facilitate interspecies comparison of networks? Findings Using gene expression datasets from LM samples from a single postnatal time point for high and low muscling sheep, and from a developmental time course (prenatal to postnatal) for normal sheep and sheep exhibiting the Callipyge muscling phenotype gene expression correlations were calculated across subsets of the data comparable to the bovine analysis. An “Always Correlated” gene expression landscape was constructed by integrating the correlations from the subsets of data and was compared to the equivalent landscape for bovine LM muscle. Whilst at the high level apparently equivalent modules were identified in the two species, at the detailed level overlap between genes in the equivalent modules was limited and generally not significant. Indeed, only 395 genes and 18 edges were in common between the two landscapes. Conclusions Since it is unlikely that the equivalent muscles of two closely related species are as different as this analysis suggests, within tissue gene expression correlations appear to be very sensitive to the samples chosen for their construction, compounded by the different platforms used. Thus users need to be very cautious in interpretation of the differences. In future experiments, attention will be required to ensure equivalent experimental designs and use cross-species gene expression platform to enable the identification of true differences between different species.

Selective breeding programs aiming to increase the productivity and profitability of the sheep meat industry use elite, progeny tested sires. The broad genetic traits of primary interest in the progeny of these sires include skeletal muscle yield, fat content, eating quality and reproductive efficiency. Natural mutations in sheep that enhance muscling have been identified, while a number of genome scans have identified and confirmed quantitative trait loci for skeletal muscle traits. The detailed phenotypic characteristics of sheep carrying these mutations or quantitative trait loci affecting skeletal muscle show a number of common biological themes, particularly changes in developmental growth trajectories, alterations of whole animal morphology and a shift towards fast twitch glycolytic fibres. The genetic, developmental and biochemical mechanisms underpinning the actions of some of these genetic variants are described. This review critically assesses this research area, identifies gaps in knowledge and highlights mechanistic linkages between genetic polymorphisms and skeletal muscle phenotypic changes. This knowledge may aid the discovery of new causal genetic variants and in some cases lead to the development of biochemical and immunological strategies aimed at enhancing skeletal muscle.

Background In livestock populations the genetic contribution to muscling is intensively monitored in the progeny of industry sires and used as a tool in selective breeding programs. The genes and pathways conferring this genetic merit are largely undefined. Genetic variation within a population has potential, amongst other mechanisms, to alter gene expression via cis- or trans-acting mechanisms in a manner that impacts the functional activities of specific pathways that contribute to muscling traits. By integrating sire-based genetic merit information for a muscling trait with progeny-based gene expression data we directly tested the hypothesis that there is genetic structure in the gene expression program in ovine skeletal muscle. Results The genetic performance of six sires for a well defined muscling trait, longissimus lumborum muscle depth, was measured using extensive progeny testing and expressed as an Estimated Breeding Value by comparison with contemporary sires. Microarray gene expression data were obtained for longissimus lumborum samples taken from forty progeny of the six sires (4-8 progeny/sire). Initial unsupervised hierarchical clustering analysis revealed strong genetic architecture to the gene expression data, which also discriminated the sire-based Estimated Breeding Value for the trait. An integrated systems biology approach was then used to identify the major functional pathways contributing to the genetics of enhanced muscling by using both Estimated Breeding Value weighted gene co-expression network analysis and a differential gene co-expression network analysis. The modules of genes revealed by these analyses were enriched for a number of functional terms summarised as muscle sarcomere organisation and development, protein catabolism (proteosome), RNA processing, mitochondrial function and transcriptional regulation. Conclusions This study has revealed strong genetic structure in the gene expression program within ovine longissimus lumborum muscle. The balance between muscle protein synthesis, at the levels of both transcription and translation control, and protein catabolism mediated by regulated proteolysis is likely to be the primary determinant of the genetic merit for the muscling trait in this sheep population. There is also evidence that high genetic merit for muscling is associated with a fibre type shift toward fast glycolytic fibres. This study provides insight into mechanisms, presumably subject to strong artificial selection, that underpin enhanced muscling in sheep populations.

Mucins are large glycoproteins that have been identified as the main component of various biological gels and lubricants. Their compositions contribute to the viscoelastic properties of mucus secretions. Gel-forming mucins have now been identified in a variety of organisms from marine molluscs to humans and are thought to cover and protect epithelial cells from attachment and entry of pathogens. We employed a new approach using a combination of tryptic digestion and the fractionation by hydrophobic interaction chromatography to enable the isolation and purification of mucins from the Blue Blubber jellyfish, Catostylus mosaicus . The purified proteins were stained with Alcian blue indicating extensive glycosylation. Amino acid composition analysis of the purified protein found that it was enriched for Ala, Glu, Thr, Pro and Val residues (together constituting ~93 mole % of the protein), which is typical for mucins. Consistent with this, monosaccharide composition analysis revealed extensive O-linked oligosaccharides with N-acetylgalactosamine (GalNAc), galactose (Gal) and N-acetylglucosamine (GlcNAc) as major monosaccharide constituents. The purified C. mosaicus mucins inhibited the attachment of an ocular Pseudomonas aeruginosa (PA) isolate (Paer6294) to human corneal epithelial (HCE) cells grown in vitro .

A glycoprotein (BM86) from the gut cells of the cattle tick Boophilus microplus, when used to vaccinate cattle, has been shown to protect cattle from tick infestation. A recombinant BM86 protein is the principal component of a novel tick vaccine currently under development. The nature of the anchorage of BM86 to tick gut epithelial cells has been investigated using BM86 from B. microplus and recombinant BM86 proteins expressed in insect cells using the baculovirus expression system. BM86 from B. microplus and a full length recombinant BM86 are shown to be anchored to the extracellular surface of tick gut epithelial cells and baculovirus-infected insect cells, respectively by a glycosyl-phosphatidyl inositol membrane anchor. A recombinant BM86 truncated by the removal of a hydrophobic region coding for thirty amino acids at the carboxy-terminal end was secreted from baculovirus-infected Sf9 cells. This secreted form of recombinant BM86 showed strong protective activity against ticks in cattle vaccinated with this protein.

Background The developmental transition between the late fetus and a newborn animal is associated with profound changes in skeletal muscle function as it adapts to the new physiological demands of locomotion and postural support against gravity. The mechanisms underpinning this adaption process are unclear but are likely to be initiated by changes in hormone levels. We tested the hypothesis that this developmental transition is associated with large coordinated changes in the transcription of skeletal muscle genes. Results Using an ovine model, transcriptional profiling was performed on Longissimus dorsi skeletal muscle taken at three fetal developmental time points (80, 100 and 120 d of fetal development) and two postnatal time points, one approximately 3 days postpartum and a second at 3 months of age. The developmental time course was dominated by large changes in expression of 2,471 genes during the interval between late fetal development (120 d fetal development) and 1-3 days postpartum. Analysis of the functions of genes that were uniquely up-regulated in this interval showed strong enrichment for oxidative metabolism and the tricarboxylic acid cycle indicating enhanced mitochondrial activity. Histological examination of tissues from these developmental time points directly confirmed a marked increase in mitochondrial activity between the late fetal and early postnatal samples. The promoters of genes that were up-regulated during this fetal to neonatal transition were enriched for estrogen receptor 1 and estrogen related receptor alpha cis-regulatory motifs. The genes down-regulated during this interval highlighted de-emphasis of an array of functions including Wnt signaling, cell adhesion and differentiation. There were also changes in gene expression prior to this late fetal - postnatal transition and between the two postnatal time points. The former genes were enriched for functions involving the extracellular matrix and immune response while the latter principally involved functions associated with transcriptional regulation of metabolic processes. Conclusions It is concluded that during late skeletal muscle development there are substantial and coordinated changes in the transcription of a large number of genes many of which are probably triggered by increased estrogen levels. These changes probably underpin the adaption of muscle to new physiological demands in the postnatal environment.

peer-reviewed Members of the Ty3-Gypsy retrotransposon family are rare in mammalian genomes despite their abundance in invertebrates and some vertebrates. These elements contain a gag-pol-like structure characteristic of retroviruses but have lost their ability to retrotranspose into the mammalian genome and are thought to be inactive relics of ancient retrotransposition events. One of these retrotransposon-like elements, PEG11 (also called RTL1) is located at the distal end of ovine chromosome 18 within an imprinted gene cluster that is highly conserved in placental mammals. The region contains several conserved imprinted genes including BEGAIN, DLK1, DAT, GTL2 (MEG3), PEG11 (RTL1), PEG11as, MEG8, MIRG and DIO3. An intergenic point mutation between DLK1 and GTL2 causes muscle hypertrophy in callipyge sheep and is associated with large changes in expression of the genes linked in cis between DLK1 and MEG8. It has been suggested that over-expression of DLK1 is the effector of the callipyge phenotype; however, PEG11 gene expression is also strongly correlated with the emergence of the muscling phenotype as a function of genotype, muscle type and developmental stage. To date, there has been no direct evidence that PEG11 encodes a protein, especially as its anti-sense transcript (PEG11as) contains six miRNA that cause cleavage of the PEG11 transcript. Using immunological and mass spectrometry approaches we have directly identified the full-length PEG11 protein from postnatal nuclear preparations of callipyge skeletal muscle and conclude that its over-expression may be involved in inducing muscle hypertrophy. The developmental expression pattern of the PEG11 gene is consistent with the callipyge mutation causing recapitulation of the normal fetal-like gene expression program during postnatal development. Analysis of the PEG11 sequence indicates strong conservation of the regions encoding the antisense microRNA and in at least two cases these correspond with structural or functional domains of the protein suggesting co-evolution of the sense and antisense genes.