Profiling of open chromatin in developing pig (Sus scrofa) muscle to identify regulatory regions.

There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programs. To address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We used the "Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq)" to identify putative regulatory regions in flash-frozen semitendinosus muscle from 24 male piglets. We collected samples from the smallest-, average-, and largest-sized male piglets from each litter through five developmental time points. Of the 4661 ATAC-Seq peaks identified that represent regions of open chromatin, >50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with downregulation of genes involved in muscle development that were present in small-sized fetal piglets but absent in large-sized fetal piglets at day 90 of gestation. The dataset that we have generated provides a resource for studies of genome regulation in pigs and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programs.

Differentiation Potential of Mesenchymal Stem/Stromal Cells Is Altered by Intrauterine Growth Restriction.

Consistency in clinical outcomes is key to the success of therapeutic Mesenchymal Stem/Stromal cells (MSCs) in regenerative medicine. MSCs are used to treat both humans and companion animals (horses, dogs, and cats). The properties of MSC preparations can vary significantly with factors including tissue of origin, donor age or health status. We studied the effects of developmental programming associated with intrauterine growth restriction (IUGR) on MSC properties, particularly related to multipotency. IUGR results from inadequate uterine capacity and placental insufficiency of multifactorial origin. Both companion animals (horses, dogs, cats) and livestock (pigs, sheep, cattle) can be affected by IUGR resulting in decreased body size and other associated changes that can include, alterations in musculoskeletal development and composition, and increased adiposity. Therefore, we hypothesized that this dysregulation occurs at the level of MSCs, with the cells from IUGR animals being more prone to differentiate into adipocytes and less to other lineages such as chondrocytes and osteocytes compared to those obtained from normal animals. IUGR has consequences on health and performance in adult life and in the case of farm animals, on meat quality. In humans, IUGR is linked to increased risk of metabolic (type 2 diabetes) and other diseases (cardiovascular), later in life. Here, we studied porcine MSCs where IUGR occurs spontaneously, and shows features that recapitulate human IUGR. We compared the properties of adipose-derived MSCs from IUGR (IUGR-MSCs) and Normal (Normal-MSCs) new-born pig littermates. Both MSC types grew clonally and expressed typical MSC markers (CD105, CD90, CD44) at similar levels. Importantly, tri-lineage differentiation capacity was significantly altered by IUGR. IUGR-MSCs had higher adipogenic capacity than Normal-MSCs as evidenced by higher adipocyte content and expression of the adipogenic transcripts, PPARγ and FABP4 (P < 0.05). A similar trend was observed for fibrogenesis, where, upon differentiation, IUGR-MSCs expressed significantly higher levels of COL1A1 (P < 0.03) than Normal-MSCs. In contrast, chondrogenic and osteogenic potential were decreased in IUGR-MSCs as shown by a smaller chondrocyte pellet and osteocyte staining, and lower expression of SOX9 (P < 0.05) and RUNX2 (P < 0.02), respectively. In conclusion, the regenerative potential of MSCs appears to be determined prenatally in IUGR and this should be taken into account when selecting cell donors in regenerative therapy programmes both in humans and companion animals.

Farmer and Veterinary Practices and Opinions Related to Fertility Testing and Pregnancy Diagnosis of UK Dairy Cows.

Dairy cow farming plays an important role in the UK and worldwide economies. Significant challenges are currently being faced regarding sustainability of the dairy industry. Dairy cow subfertility remains an important issue limiting herd productivity, resulting in annual losses of hundreds of millions of pounds in the UK alone. To address this, accurate monitoring of reproductive status and early detection of fertility issues in individual cows is essential. The aim of this study was to gather farmer and veterinarian opinions on current practices and perceived gaps related to diagnosis of fertility issues and pregnancy testing in UK dairy farms. Using online questionnaires, data were collected and analyzed from a total of 40 farmers and 59 veterinarians. The results showed that non-seen bulling checks and ultrasound were the most frequent tools to detect fertility issues, and that most farmers tested post-calving, and often again before or during mating. Most farmers believed that current tests did not meet their expectations, with half of those being willing to pay more than they were currently paying for fertility testing. In regard to pregnancy testing, ultrasound was most commonly used, at 30-50 days post-insemination either in individual or groups of cows. Again, most farmers believed that current tests did not meet their expectations, and a majority would consider paying a higher cost for a test that was better than those currently available. In addition, a majority of farmers would consider using a test that could detect pregnancy within 2 weeks post-insemination, if such test existed, because they believed it would help improve their herds' reproductive performance. Overall, the opinions of farmers and veterinarians indicate that there is significant scope for improving dairy herd fertility monitoring practices in the UK, through development of improved assays that can diagnose pregnancy and infertility earlier, are less disruptive to farm operations and are more cost effective than available tools. They also provide useful information to guide the future development and implementation of better diagnostics for improving reproductive performance of dairy cattle.

Farmer and Veterinary Practices and Opinions Related to the Diagnosis of Mastitis and Metabolic Disease in UK Dairy Cows.

Production diseases are highly prevalent in modern dairy herds, resulting in lost productivity and reduced animal welfare. Two important production diseases are mastitis and metabolic disorders. The availability of robust diagnostic tools that can detect animals at early stages of disease is crucial to prevent the high costs associated with lost productivity and the treatment of clinically and/or chronically diseased animals. Despite a variety of diagnostic methods being available to farmers and veterinarians, the incidence of these diseases in UK dairy herds has not changed over the last decade, underscoring the need for improved approaches for early disease detection. To this end, we administered a questionnaire to farmers and veterinarians to understand current diagnostic practices in the UK dairy cow sector, and to gather opinions on the suitability of currently available diagnostic tests in order to identify specific areas where improvement in diagnostic technologies and/or practices are needed. Data from a total of 34 farmers and 42 veterinarians were analyzed. Results indicated that most farmers surveyed used a combination of methods to diagnose mastitis and metabolic disorders, the most popular of which were visual inspection and milk recording somatic cell count data for mastitis, and body condition score and milk ketone testing for metabolic disorders. These preferences were not always in line with veterinarian recommendations of different diagnostic tools. Moreover, veterinarians indicated they were not satisfied with currently available diagnostic tools or how these were implemented by farmers. Both farmers and veterinarians recognized there was substantial room for improvement of current diagnostic tools, particularly in regard to the need to detect disease early. A majority of respondents preferred new diagnostic tests to be suitable for use with milk rather than blood or urine samples, and to yield results within 24 h. Finally, both groups surveyed identified economic cost as the most important barrier for the future uptake of new diagnostic technologies. The information obtained should guide the future development of diagnostic approaches that meet both the expectations of farmers and veterinarians, and help bring about a reduction in the incidence of production diseases in UK dairy herds.

Proinflammatory cytokine induction of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in human adipocytes is mediated by MEK, C/EBPß, and NF-κB/RelA.

CONTEXT: Levels of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which regenerates active glucocorticoids, are selectively elevated in adipose tissue in human obesity and metabolic syndrome, both conditions associated with chronic low-grade inflammation. 11ß-HSD1 expression is induced by proinflammatory cytokines in a variety of cell types, including in human adipocytes differentiated in vitro. OBJECTIVE: Our objective was to determine the mechanisms by which proinflammatory cytokines induce 11ß-HSD1 in human adipocytes. RESULTS: The proinflammatory cytokines IL-1α (10 ng/mL) and TNFα (20 ng/mL) increased 11ß-HSD1 mRNA levels in human primary adipocyte fractions and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes (P<.001). Inhibition of the MAPK/ERK kinase (MEK) attenuated CCAAT/enhancer binding protein (C/EBP) ß phosphorylation at Thr235 and IL-1α/TNFα induction of 11ß-HSD1 (P≤.007). The small interfering RNA-mediated knockdown of C/EBPß and nuclear factor (NF)-κB/RelA or inhibition of NF-κB/RelA also attenuated cytokine induction of 11ß-HSD1 (P≤.001). Moreover, induction of 11ß-HSD1 by IL-1α in SGBS cells was associated with nuclear localization of C/EBPß and NF-κB/RelA. Chromatin immunoprecipitation experiments showed C/EBPß and NF-κB/RelA located to the 11ß-HSD1 promoter in human adipose tissue. Treatment of adipocyte fractions or SGBS adipocytes with metformin or acetylsalicylic acid, which target C/EBPß and NF-κB/RelA signaling, attenuated the IL-1α induction of 11ß-HSD1 (P≤.002). CONCLUSIONS: Increased proinflammatory signaling in inflamed adipose tissue may mediate elevated 11ß-HSD1 expression at this site via MEK, C/EBPß, and NF-κB/RelA. These molecules/signaling pathways are, therefore, potential targets for drugs, including metformin and acetylsalicylic acid, to prevent/decreased up-regulation of 11ß-HSD1 in human obese/metabolic syndrome adipose tissue.

Pro-inflammatory cytokine induction of 11ß-hydroxysteroid dehydrogenase type 1 in A549 cells requires phosphorylation of C/EBPß at Thr235.

11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts inert glucocorticoids into active forms, thereby increasing intracellular glucocorticoid levels, important to restrain acute inflammation. 11ß-HSD1 is induced by pro-inflammatory cytokines in a variety of cells. Here, we show 11ß-HSD1 expression in human A549 epithelial cells is increased by pro-inflammatory cytokines (IL-1α/TNFα) via the P2 promoter of the HSD11B1 gene. Inhibition of p38 MAPK attenuated the pro-inflammatory cytokine induction of mRNA encoding 11ß-HSD1 as well as that encoding C/EBPß. IL-1α/TNFα-induced phosphorylation of C/EBPß at Thr235 was also attenuated by p38 MAPK inhibition suggesting involvement of a p38 MAPK-C/EBPß pathway. siRNA-mediated knock-down of C/EBPß and NF-κB/RelA implicated both transcription factors in the IL-1α/TNFα induction of HSD11B1 mRNA. Transient transfections of HSD11B1 promoter-reporter constructs identified the proximal region of the P2 promoter of HSD11B1 as essential for this induction. IL-1α increased binding of C/EBPß to the HSD11B1 P2 promoter, but this was not observed for NF-κB/RelA, suggesting indirect regulation by NF-κB/RelA. Ectopic expression of mutant chicken C/EBPß constructs unable to undergo phosphorylation at the threonine equivalent to Thr235 attenuated the IL-1α-induction of HSD11B1, whereas mimicking constitutive phosphorylation of Thr235 (by mutation to aspartate) increased basal expression of HSD11B1 mRNA without affecting IL-1α-induced levels. These data clearly demonstrate a role for both C/EBPß and NF-κB/RelA in the pro-inflammatory cytokine induction of HSD11B1 in human epithelial cells and show that p38 MAPK-induced phosphorylation of C/EBPß at Thr235 is critical in this.

Phospholipase Cß3 mediates LH-induced granulosa cell differentiation.

Previous studies showed that under certain conditions LH can stimulate not only adenylate cyclase (AC) but also phospholipase Cß (PLCß) signaling in target cells; however, the physiological involvement of PLCß in LH-induced ovarian follicular cell differentiation has not been determined. To address this, ex vivo expression analyses and specific PLCß targeting were performed in primary bovine granulosa cells. Expression analyses in cells from small (2.0-5.9 mm), medium (6.0-9.9 mm), and ovulatory-size (10.0-13.9 mm) follicles revealed an increase in mRNA and protein levels of heterotrimeric G protein subunits-αs, -αq, -α11, and -αi2 in ovulatory-size follicles, simultaneous with a substantial increase in LH receptor expression. Among the four known PLCß isoforms, PLCß3 (PLCB3) was specifically up-regulated in cells from ovulatory-size follicles, in association with a predominantly cytoplasmic location of PLCB3 in these cells and a significant inositol phosphate response to LH stimulation. Furthermore, RNA interference-mediated PLCB3 down-regulation reduced the ability of LH to induce hallmark differentiation responses of granulosa cells, namely transcriptional up-regulation of prostaglandin-endoperoxide synthase 2 and down-regulation of both aromatase expression and estradiol production. Responses to the AC agonist, forskolin, however, were not affected. In addition, PLCB3 down-regulation did not alter cAMP responses to LH in granulosa cells, ruling out a primary involvement of AC in mediating the effects of PLCB3. In summary, we provide evidence of a physiological involvement of PLCß signaling in ovulatory-size follicles and specifically identify PLCB3 as a mediator of LH-induced differentiation responses of granulosa cells.