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Objective: The purpose of this study was to determine whether activation of NF-kappaB and expression of COX-2 are associated with neutrophil infiltration in systemic vascular tissue of women with preeclampsia.Study Design: Subcutaneous fat biopsies were obtained at cesarean section or abdominal surgery from preeclamptic women (n = 7), normal pregnant women (n = 6), and normal nonpregnant women (n = 5). Resistance-sized vessels (10 to 200 microm) in subcutaneous fat were evaluated using immunohistochemical staining for: (1) CD66b, a neutrophil antigen, (2) nuclear factor-kappaB (NF-kappaB), a transcription factor for genes of inflammation, and (3) cyclooxygenase-2 (COX-2), an inflammatory gene product.Results: The percentage of vessels which showed staining for CD66b, NF-kappaB, and COX-2 was significantly greater for preeclamptic patients as compared to normal nonpregnant or normal pregnant patients. In preeclamptic patients, vessel staining for NF-kappaB and COX-2 was present in both endothelium and in vascular smooth muscle. Leukocytes in the lumen and adhered to endothelium also stained for NF-kappaB and COX-2. Activation of NF-kappaB and expression of COX-2 were coincident with neutrophil flattening and adherence to endothelium and infiltration into the intimal space.Conclusion: These data demonstrate activation of NF-kappaB and expression of COX-2 in systemic vasculature of women with preeclampsia, and they demonstrate that this vascular inflammation is linked with neutrophil infiltration. Neutrophil release of toxic substances, such as reactive oxygen species, TNFalpha and thromboxane, could be responsible for vasoconstriction and vascular dysfunction. These data clearly place preeclampsia in the category of an inflammatory disease associated with immune dysfunction. [ABSTRACT FROM AUTHOR]

Background: Neuroimaging studies of major depression have typically been conducted using group-level approaches. However, given interindividual differences in brain systems, there is a need for individualized approaches to brain systems mapping and putative links toward diagnosis, symptoms, and behavior., Methods: We used an iterative parcellation approach to map individualized brain systems in 328 participants from a multisite, placebo-controlled clinical trial. We hypothesized that participants with depression would show abnormalities in salience, control, default, and affective systems, which would be associated with higher levels of self-reported anhedonia, anxious arousal, and worse cognitive performance. Within hypothesized brain systems, we compared patch sizes (number of vertices) between depressed and healthy control groups. Within depressed groups, abnormal patches were correlated with hypothesized clinical and behavioral measures., Results: Significant group differences emerged in hypothesized patches of 1) the lateral salience system (parietal operculum; t 326  = -3.11, p = .002) and 2) the control system (left medial posterior prefrontal cortex region; z = -3.63, p < .001), with significantly smaller patches in these regions in participants with depression than in healthy control participants. Results suggest that participants with depression with significantly smaller patch sizes in the lateral salience system and control system regions experience greater anxious arousal and cognitive deficits., Conclusions: The findings imply that neural features mapped at the individual level may relate meaningfully to diagnosis, symptoms, and behavior. There is strong clinical relevance in taking an individualized brain systems approach to mapping neural functional connectivity because these associated region patch sizes may help advance our understanding of neural features linked to psychopathology and foster future patient-specific clinical decision making., (Copyright © 2024 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

A voluntary control programme for Johne's disease, the Irish Johne's Control Programme (IJCP) has been implemented in Ireland since 2017. The objective of this observational study was to assess Irish beef and dairy farmers' Johne's disease knowledge, implemented management practices and IJCP opinions. A questionnaire open to dairy and beef farmers was distributed via social media and email. In total 126 responses were used for this study; these responses came from mostly young farmers (18-25 years old) and represent a small proportion of the total number of dairy and beef farmers in Ireland whose average age is 55.Most respondents claimed to know what Johne's disease was (73%; 92/126) and associated the disease to loss of body condition (68%; 78/114) and diarrhoea (59%; 67/114). Twenty-eight respondents (mostly dairy farmers; 22/28) reported positive cases in their premises. And 38% reported to implement management practices to prevent Johne's disease transmission within or into their herd (i.e. management of milk for calf consumption and isolation of Johne's test-positive or newly purchased stock; 47/124).Eighteen percent (22/125) of respondents were, at the time of questionnaire or previously, members of the IJCP. The main benefits reported by some of the participating farmers were identification of positive cases (29%; 4/14), and management of milk for calf consumption (21%; 3/14). While the main disadvantage was inaccurate testing methods (50%; 10/20). The main reasons reported for the lack of participation in the IJCP were not being aware of the programme (52%; 53/102) and not having a Johne's disease problem on the farm (48%; 49/102).In conclusion, this study suggests that while young farmers are aware of Johne's disease, their participation in the IJCP is limited and could benefit from further promotion. Studies representing the wider farming community in Ireland are warranted to gather non-biased input and contribute to Johne's disease control in Ireland., (© 2023. Veterinary Ireland.)

There are few early biomarkers to identify pregnancies at risk of preeclampsia (PE) and abnormal placental function. In this cross-sectional study, we utilized targeted ultra-performance liquid chromatography-ESI MS/MS and a linear regression model to identify specific bioactive lipids that serve as early predictors of PE. Plasma samples were collected from 57 pregnant women prior to 24-weeks of gestation with outcomes of either PE (n = 26) or uncomplicated term pregnancies (n = 31), and the profiles of eicosanoids and sphingolipids were evaluated. Significant differences were revealed in the eicosanoid, (±)11,12 DHET, as well as multiple classes of sphingolipids; ceramides, ceramide-1-phosphate, sphingomyelin, and monohexosylceramides; all of which were associated with the subsequent development of PE regardless of aspirin therapy. Profiles of these bioactive lipids were found to vary based on self-designated race. Additional analyses demonstrated that PE patients can be stratified based on the lipid profile as to PE with a preterm birth linked to significant differences in the levels of 12-HETE, 15-HETE, and resolvin D1. Furthermore, subjects referred to a high-risk OB/GYN clinic had higher levels of 20-HETE, arachidonic acid, and Resolvin D1 versus subjects recruited from a routine, general OB/GYN clinic. Overall, this study shows that quantitative changes in plasma bioactive lipids detected by ultra-performance liquid chromatography-ESI-MS/MS can serve as an early predictor of PE and stratify pregnant people for PE type and risk., Competing Interests: Conflict of interest All authors of this article declare that they have no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutrophils expressing cyclooxygenase-2 (COX-2) extensively infiltrate maternal blood vessels in preeclampsia, associated with vascular inflammation. Because pregnancy neutrophils also express protease-activated receptor 1 (PAR-1, F2R thrombin receptor), which they do not in non-pregnant subjects, they can be activated by proteases. We tested the hypothesis that aspirin at a dose sufficient to inhibit COX-2 would reduce inflammatory responses in preeclampsia neutrophils. Neutrophils were isolated from normal pregnant and preeclamptic women at approximately 30 weeks' gestation. Normal pregnancy neutrophils were treated with elastase, a protease elevated in preeclampsia, or elastase plus aspirin to inhibit COX-2, or elastase plus pinane thromboxane, a biologically active structural analog of thromboxane and a thromboxane synthase inhibitor. Preeclamptic pregnancy neutrophils were treated with the same doses of aspirin or pinane thromboxane. Confocal microscopy with immunofluorescence staining was used to determine the cellular localization of the p65 subunit of nuclear factor-kappa B (NF-κB) and media concentrations of thromboxane were measured to evaluate the inflammatory response. In untreated neutrophils of normal pregnant women, p65 was localized to the cytosol. Upon stimulation with elastase, p65 translocated from the cytosol to the nucleus coincident with increased thromboxane production. When neutrophils were co-treated with aspirin or pinane thromboxane, elastase was not able to cause nuclear translocation of p65 or increase thromboxane. In untreated neutrophils of preeclamptic women, the p65 subunit was present in the nucleus and thromboxane production was elevated, but when preeclamptic neutrophils were treated with aspirin or pinane thromboxane, p65 was cleared from the nucleus and returned to the cytosol along with decreased thromboxane production. These findings suggest that COX-2 is a downstream mediator of PAR-1 and demonstrate that PAR-1- mediated inflammation can be inhibited by aspirin. Given the extensive and ubiquitous expression of PAR-1 and COX-2 in preeclamptic women, consideration should be given to treating women with preeclampsia using a dose of aspirin sufficient to inhibit COX-2.

Neutrophils, which extensively infiltrate maternal systemic blood vessels in preeclampsia, express protease-activated receptor 1 (PAR-1) but only during pregnancy. Neutrophils are generally considered to be non-specific in their response, but the pregnancy-specific expression of PAR-1 could result in a gene expression profile unique to pregnancy, which could help explain why the maternal inflammatory response in preeclampsia is systemic rather than localized. We sought to determine if gene expression of pregnancy neutrophils would differ if stimulated by a protease versus bacterial lipopolysaccharide (LPS). We isolated neutrophils from normal pregnant women at 30 weeks' gestation and cultured them with elastase or LPS. We used elastase because it is a protease elevated in women with preeclampsia, and it activates pregnancy neutrophils via PAR-1. RNA was isolated from the neutrophils for sequencing of the transcriptomes. We discovered many differences in the gene expression profiles. For example, exposure to elastase resulted in three times more uniquely expressed genes than LPS, and the number of significantly differentially upregulated and downregulated genes was greater for elastase. Analysis of canonical pathways revealed similarities for innate immunity but also differences. LPS treatment enriched more pathways, but elastase activated more genes in each pathway. Elastase treatment enriched the MAPK signaling pathway, whereas LPS did not. This is significant because MAPK is a key mediator of transcriptional responses. These findings indicate that protease stimulation of pregnancy neutrophils results in a different profile than stimulation with LPS, which may help explain why the sterile inflammatory response of preeclampsia is systemic and unique to pregnancy.

Neutrophils extensively infiltrate maternal blood vessels in preeclampsia. This could explain why multiple organs are affected in this enigmatic disorder. Lipid peroxides produced by the placenta are probably the first factors that activate neutrophils as they circulate through the intervillous space, but then a second factor specific to pregnancy comes into play, protease-activated receptor 1. The only time neutrophils express protease-activated receptor 1 is during pregnancy. This means that neutrophils can be activated by a mechanism specific to pregnancy, that is, by proteases. Two proteases that are elevated in preeclampsia and activate protease-activated receptor 1 are matrix metalloproteinase-1 and neutrophil elastase. There is an 8-fold increase in vascular protease-activated receptor 1 expression in women with preeclampsia, and protease-activated receptor 1 is also expressed on the placenta, a pregnancy-specific tissue. The question arises if the pregnancy-specific expression of protease-activated receptor 1 is essential to the pathophysiology of preeclampsia. Protease activation of protease-activated receptor 1 in neutrophils of women with normal pregnancies causes activation of RhoA kinase. RhoA kinase phosphorylates nuclear factor-kappa B causing its translocation from the cytosol into the nucleus, increasing the expression of inflammatory genes. This signaling pathway is blocked by inhibition of either protease-activated receptor 1 or RhoA kinase activity. In contrast, neutrophils obtained from preeclamptic women are already activated, with nuclear factor-kappa B localized in the nucleus. Surprisingly, inhibition of either protease-activated receptor 1 or RhoA kinase results in an efflux of nuclear factor-kappa B from the nucleus back into the cytoplasm. Cyclooxygenase-2 seems to be a downstream mediator between protease-activated receptor 1 and RhoA kinase because aspirin inhibits the nuclear translocation of nuclear factor-kappa B and inhibits neutrophil production of superoxide, thromboxane, and tumor necrosis factor alpha. Currently, low-dose aspirin is the standard of care to prevent preeclampsia in high-risk women. Generally, the actions of low-dose aspirin are attributed to selective inhibition of maternal platelet thromboxane production. However, a recent study showed that beneficial effects extend to the placenta, where aspirin corrected the imbalance of increased thromboxane and reduced prostacyclin and oxidative stress. Selective inhibition of placental thromboxane is possible because thromboxane and prostacyclin are compartmentalized. Thromboxane is produced by trophoblast cells and prostacyclin by endothelial cells, so as aspirin crosses the placenta, its levels decline, sparing prostacyclin. Placental oxidative stress is attenuated because cyclooxygenase-2 inhibition decreases the generation of reactive oxygen species to decrease the formation of isoprostanes. The clinical manifestations of preeclampsia can be explained by protease activation of protease-activated receptor 1 in different tissues. In neutrophils, it can account for their activation and inflammatory response. In vascular tissue, protease-activated receptor 1 activation leads to enhanced vascular reactivity to angiotensin II to cause hypertension. In the placenta, it leads to oxidative stress, increased soluble fms-like tyrosine kinase, and thromboxane production. Activation of protease-activated receptor 1 on endothelial cells causes contraction, leading to edema and proteinuria, and activation on platelets leads to coagulation abnormalities. As proteases that activate protease-activated receptor 1 are elevated in the circulation of women with preeclampsia, consideration should be given to the inhibition of protease-activated receptor 1 as a treatment. Recently, The Food and Drug Administration (FDA) approved a protease-activated receptor 1 inhibitor, creating an opportunity to test whether protease-activated receptor 1 inhibition can prevent and/or treat preeclampsia, but a standard dose of aspirin might be just as effective by blocking its downstream actions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

DNA methylation is an epigenetic mechanism controlling gene expression, and reduced methylation is associated with increased gene expression. We hypothesized that IL-17 cytokines are regulated by DNA methylation, are elevated in the circulation of preeclamptic women, and stimulate vascular neutrophil chemokine expression, which could account for vascular infiltration of neutrophils in preeclampsia. We found significantly reduced DNA methylation of IL17A, IL17E, and IL17F genes in omental arteries of preeclamptic women, significantly reduced methylation of IL2, which regulates IL-17-producing T-lymphocytes, and significantly reduced methylation of genes encoding neutrophil chemokines and TNFα receptors related to lymphocyte function. Maternal plasma levels of IL-17A were significantly elevated in the second trimester of preeclamptic pregnancy as compared to normal pregnancy. To test if methylation regulates IL-17 cytokines, a lymphocyte cell line (Jurkat) was cultured with a hypomethylating agent. Hypomethylation increased expression of IL17E (aka IL25), IL17F, and IL2. IL17A was not expressed by Jurkat cells. To test the potential role of IL-17 cytokines in vascular neutrophil infiltration associated with preeclampsia, human vascular smooth muscle cells were cultured with IL-17 cytokines. IL-17A, but not IL-17E or IL-17F, increased gene expression of neutrophil chemokines (IL-8, CXCL5, and CXCL6) that are increased in vascular smooth muscle of preeclamptic women. The monocyte chemokine, CCL-2, was not increased. TNFα also increased neutrophil chemokines. IL-17 cytokines are regulated by DNA methylation; IL-17A is elevated in preeclampsia and stimulates expression of neutrophil chemokines in vascular smooth muscle. IL-17A could be responsible for vascular infiltration of neutrophils in preeclampsia., (© 2021. Society for Reproductive Investigation.)

Neutrophils are activated and extensively infiltrate blood vessels in preeclamptic women. To identify genes that contribute to neutrophil activation and infiltration, we analyzed the transcriptomes of circulating neutrophils from normal pregnant and preeclamptic women. Neutrophils were collected at 30 weeks' gestation and RNA and DNA were isolated for RNA sequencing and 5-hydroxy-methylcytosine (5-hmC) sequencing as an index of dynamic changes in neutrophil DNA methylation. Women with normal pregnancy who went on to develop mild preeclampsia at term had the most uniquely expressed genes (697) with 325 gene ontology pathways upregulated, many related to neutrophil activation and function. Women with severe preeclampsia who delivered prematurely had few pathways up- or downregulated. Cluster analysis revealed that gene expression in women with severe preeclampsia was an inverse mirror image of gene expression in normal pregnancy, while gene expression in women who developed mild preeclampsia was remarkably different from both. DNA methylation marks, key regulators of gene expression, are removed by the action of ten-eleven translocation (TET) enzymes, which oxidize 5-methylcytosines (5mCs), resulting in locus-specific reversal of DNA methylation. DNA sequencing for 5-hmC revealed no differences among the three groups. Genome-wide DNA methylation revealed extremely low levels in circulating neutrophils suggesting they are de-methylated. Collectively, these data demonstrate that neutrophil gene expression profiles can distinguish different preeclampsia phenotypes, and in the case of mild preeclampsia, alterations in gene expression occur well before clinical symptoms emerge. These findings serve as a foundation for further evaluation of neutrophil transcriptomes as biomarkers of preeclampsia phenotypes. Changes in DNA methylation in circulating neutrophils do not appear to mediate differential patterns of gene expression in either mild or severe preeclampsia.

Implementation of guidelines to reduce the amount of biodegradable municipal waste (BMW) sent to landfill has created a need in the waste-management industry to investigate possible methods of accelerating biostabilisation of residual BMW. The effect of commercially feasible manipulations (lime and green waste (GW)) on the rate of biostabilisation of the fine (<20 mm) fraction of residual BMW was investigated. The physical and chemical attributes of the composted wastes were measured, and their bacterial communities profiled using traditional culture-based methods. In addition, ammonia-oxidising microbes were monitored during the biostabilisation process using molecular profiling methods. Addition of GW accelerated biostabilisation, reduced conductivity and increased the levels of ammonia-oxidising bacterial (AOB) and archaeal (AOA) genes. The best stability was noted in the dual (Lime + GW) treatment, which was under the limit of 13 mmol O 2 kg DM -1 h -1 recommended by the Irish compost standard. Biostabilised wastes met recommendations for source-segregated compost for pH (6-8) and pathogens ( E. coli and Salmonella ), but not heavy metals, indicating their unsuitability for uses other than landfill cover. Levels of AOA genes (log 3-6 g -1 DM) were higher than AOB (log 1-6 g -1 DM, indicating AOA may contribute more to potential ammonia oxidation in residual BMW composting.

The road to low-dose aspirin therapy for the prevention of preeclampsia began in the 1980s with the discovery that there was increased thromboxane and decreased prostacyclin production in placentas of preeclamptic women. At the time, low-dose aspirin therapy was being used to prevent recurrent myocardial infarction and other thrombotic events based on its ability to selectively inhibit thromboxane synthesis without affecting prostacyclin synthesis. With the discovery that thromboxane was increased in preeclamptic women, it was reasonable to evaluate whether low-dose aspirin would be effective for preeclampsia prevention. The first clinical trials were very promising, but then two large multi-center trials dampened enthusiasm until meta-analysis studies showed aspirin was effective, but with caveats. Low-dose aspirin was most effective when started <16 weeks of gestation and at doses >100 mg/day. It was effective in reducing preterm preeclampsia, but not term preeclampsia, and patient compliance and patient weight were important variables. Despite the effectiveness of low-dose aspirin therapy in correcting the placental imbalance between thromboxane and prostacyclin and reducing oxidative stress, some aspirin-treated women still develop preeclampsia. Alterations in placental sphingolipids and hydroxyeicosatetraenoic acids not affected by aspirin, but with biologic actions that could cause preeclampsia, may explain treatment failures. Consideration should be given to aspirin's effect on neutrophils and pregnancy-specific expression of protease-activated receptor 1, as well as additional mechanisms of action to prevent preeclampsia.

Sexual dimorphism, the phenomenon whereby males and females of the same species are distinctive in some aspect of appearance or size, has previously been documented in cattle for traits such as growth rate and carcass merit using a quantitative genetics approach. No previous study in cattle has attempted to document sexual dimorphism at a genome level; therefore, the objective of the present study was to determine whether genomic regions associated with size and muscularity in cattle exhibited signs of sexual dimorphism. Analyses were undertaken on 10 linear-type traits that describe the muscular and skeletal characteristics of both males and females of five beef cattle breeds: 1,444 Angus (AA), 6,433 Charolais (CH), 1,129 Hereford, 8,745 Limousin (LM), and 1,698 Simmental. Genome-wide association analyses were undertaken using imputed whole-genome sequence data for each sex separately by breed. For each single-nucleotide polymorphism (SNP) that was segregating in both sexes, the difference between the allele substitution effect sizes for each sex, in each breed separately, was calculated. Suggestively (P ≤ 1 × 10-5) sexually dimorphic SNPs that were segregating in both males and females were detected for all traits in all breeds, although the location of these SNPs differed by both trait and breed. Significantly (P ≤ 1 × 10-8) dimorphic SNPs were detected in just three traits in the AA, seven traits in the CH, and three traits in the LM. The vast majority of all segregating autosomal SNPs (86% in AA to 94% in LM) had the same minor allele in both males and females. Differences (P ≤ 0.05) in allele frequencies between the sexes were observed for between 36% (LM) and 66% (AA) of the total autosomal SNPs that were segregating in both sexes. Dimorphic SNPs were located within a number of genes related to muscularity and/or size including the NAB1, COL5A2, and IWS1 genes on BTA2 that are located close to, and thought to be co-inherited with, the MSTN gene. Overall, sexual dimorphism exists in cattle at the genome level, but it is not consistent by either trait or breed., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

While breeding indexes exist globally to identify candidate parents of the next generation, fewer tools exist that provide guidance on the expected monetary value of young animals. The objective of the present study was therefore to develop the framework for a cattle decision-support tool which incorporates both the genetic and non-genetic information of an animal and, in doing so, better predict the potential market value of an animal, whatever the age. Two novel monetary indexes were constructed and their predictive ability of carcass value was compared to that of the Irish national Terminal breeding index, typical of other terminal indexes used globally. A constructed Harvest index was composed of three carcass-related traits [i.e., 1) carcass weight, 2) carcass conformation and 3) carcass fat, each weighted by their respective economic value] and aimed at purchasers of animals close to harvest; the second index, termed the Calf index, also included docility and feed intake (weighted by their respective economic value), thus targeting purchasers of younger calves for growing (and eventually harvesting). Genetic and non-genetic fixed and random effect model solutions from the Irish national genetic evaluations underpinned all indexes. The two novel indexes were formulated using three alternative estimates of an animal's total merit for comparative purposes: 1) an index based solely on the animal's breed solutions, 2) an index which also included within-breed animal differences, and 3) an index which, as well as considering additive and non-additive genetic effects, also included non-genetic effects (referred to as production values [PVs]). As more information (i.e., within breed effects and subsequently non-genetic effects) was included in the total merit estimate, the correlations strengthened between the two proposed indexes and the animal's calculated carcass market value; the correlation coefficients almost doubled in strength when total merit was based on PV-based estimates as compared to the breed solutions alone. Including phenotypic live-weight data, collected during the animal's life, strengthened the predictive ability of the indexes further. Based on the results presented, the proposed indexes may fill the void in decision support when purchasing or selling cattle. In addition, given the dynamic nature of indexes, they have the potential to be updated in real-time as information becomes available., (Copyright © 2020. Published by Elsevier B.V.)

Low-dose aspirin, which selectively inhibits thromboxane synthesis, is now standard of care for the prevention of preeclampsia in at risk women, but some women still develop preeclampsia despite an aspirin regimen. To explore the "aspirin failures," we undertook a comprehensive evaluation of placental lipids to determine if abnormalities in non-aspirin sensitive lipids might help explain why some women on low-dose aspirin develop preeclampsia. We studied placentas from women with normal pregnancies and women with preeclampsia. Placental villous explants were cultured and media analyzed by mass spectrometry for aspirin-sensitive and non-aspirin-sensitive lipids. In women who developed severe preeclampsia and delivered preterm, there were significant elevations in non-aspirin-sensitive lipids with biologic actions that could cause preeclampsia. There were significant increases in 15- and 20-hydroxyeicosatetraenoic acids and sphingolipids: D-e-C 18:0 ceramide, D-e-C 18:0 sphingomyelin, D-e-sphingosine-1-phosphate, and D-e-sphinganine-1-phosphate. With regard to lipids sensitive to aspirin, there was no difference in placental production of thromboxane or prostacyclin, but prostaglandins were lower. There was no difference for isoprostanes, but surprisingly, anti-inflammatory omega 3 and 6 PUFAs were increased. In total, 10 of 30 eicosanoids and 5 of 42 sphingolipids were abnormal in women with severe early onset preeclampsia. Lipid changes in women with mild preeclampsia who delivered at term were of lesser magnitude with few significant differences. The placenta produces many aspirin-sensitive and non-aspirin-sensitive lipids. Abnormalities in eicosanoids and sphingolipids not sensitive to aspirin might explain why some aspirin-treated women develop preeclampsia.

We tested a novel hypothesis that elevated levels of proteases in the maternal circulation of preeclamptic women activate neutrophils due to their pregnancy-specific expression of protease-activated receptor 1 (PAR-1). Plasma was collected longitudinally from normal pregnant and preeclamptic women and analyzed for MMP-1 and neutrophil elastase. Neutrophils were isolated for culture and confocal microscopy. Omental fat was collected for immunohistochemistry. Circulating proteases were significantly elevated in preeclampsia. Confocal microscopy revealed that tet methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, and p65 subunit of NF-κB were strongly localized to the nucleus of untreated neutrophils of preeclamptic women, but in untreated neutrophils of normal pregnant women they were restricted to the cytosol. Treatment of normal pregnancy neutrophils with proteases activated PAR-1, leading to activation of RhoA kinase (ROCK), which triggered translocation of TET2 and p65 from the cytosol into the nucleus, mimicking the nuclear localization in neutrophils of preeclamptic women. IL-8, an NF-κB-regulated gene, increased in association with TET2 and p65 nuclear localization. Co-treatment with inhibitors of PAR-1 or ROCK prevented nuclear translocation and IL-8 did not increase. Treatment of preeclamptic pregnancy neutrophils with inhibitors emptied the nucleus of TET2 and p65, mimicking the cytosolic localization of normal pregnancy neutrophils. Expression of PAR-1 and TET2 were markedly increased in omental fat vessels and neutrophils of preeclamptic women. We conclude that elevated levels of circulating proteases in preeclamptic women activate neutrophils due to their pregnancy-specific expression of PAR-1 and speculate that TET2 DNA de-methylation plays a role in the inflammatory response.

Meticulous culling decisions, coupled with careful breeding decisions, are fundamental to shifting a population distribution in the favorable direction and improving profit per cow. Nevertheless, there is a paucity of easy-to-use dynamic tools to aid in culling decisions in beef cattle. The motivation for the present study was to develop a monetary-based culling tool, here referred to as the Beef Female's Profit Potential (BFPP), to identify females for culling. The BFPP reflects the expected lifetime profitability of an individual female in a herd for the expected remainder of her lifetime; this profit included that of the beef female herself as well as her progeny. The BFPP index framework was composed of 4 subindexes reflecting the value of an animal: (1) as a nulliparae (this was voided if the cow had already calved), (2) for the remainder of her current parity, (3) summed across each of her expected remaining parities, and (4) when she is retained within the herd and not voluntarily culled. Each subindex was comprised of different components reflecting both genetic and non-genetic effects associated with each female. Transition matrices predicting the expected longevity of each female and their expected month of calving were also utilized in calculating the expected remaining lifetime profitability of each female. The BFPP index was validated on 21,102 beef cows as well as their harvested progeny from 875 herds by stratifying the cows, within herd, into 4 strata based on their BFPP. The mean of the within-herd correlation between the BFPP and the Irish national replacement (i.e., breeding) index was, on average, 0.45 indicating the shortcomings of the breeding index as a culling tool. Cows within the top BFPP stratum had a genetic expectation of accruing almost an additional €36 profit per calving, relative to cows within the worst stratum; when validated on the cow's own calving interval and survival performance as well as their progeny's carcass performance, the actual phenotypic value was estimated to be an additional €32 profit per calving. A proportion of this additional profit was due to the harvested progeny of the high BFPP cows having, on average, heavier, more conformed carcasses with less fat cover relative to their poor BFPP contemporaries. This BFPP framework is a useful and easy-to-use tool to aid in producer decision making on the choice of females to voluntarily cull but also on which replacement heifers to graduate into the mature herd., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sneathia amnii is a poorly characterized emerging pathogen that has been implicated in amnionitis and urethritis. We found that S. amnii damages fetal membranes, and we identified and purified a cytotoxic exotoxin that lyses human red blood cells and damages cells from fetal membranes. The gene appears to be cotranscribed with a second gene that encodes a protein with identity to two-partner system transporters, suggesting that it is the "A," or secreted component of a type Vb system. The toxin is 1,881 amino acids with a molecular weight of approximately 200 kDa. It binds to red blood cell membranes and forms pores with a diameter of 2.0 to 3.0 nm, resulting in osmolysis. Because it appears to be the "A" or passenger component of a two-partner system, we propose to name this novel cytotoxin/hemolysin CptA for cytopathogenic toxin component A. IMPORTANCE Sneathia amnii is a very poorly characterized emerging pathogen that can affect pregnancy outcome and cause urethritis and other infections. To date, nothing is known about its virulence factors or pathogenesis. We have identified and isolated a cytotoxin, named CptA for cytopathogenic toxin, component A, that is produced by S. amnii CptA is capable of permeabilizing chorionic trophoblasts and lysing human red blood cells and, thus, may play a role in virulence. Except for small domains conserved among two-partner secretion system passenger proteins, the cytotoxin exhibits little amino acid sequence homology to known toxins. In this study, we demonstrate the pore-forming activity of this novel toxin., (Copyright © 2020 American Society for Microbiology.)

Linear type traits describing the skeletal characteristics of an animal are moderately to strongly genetically correlated with a range of other performance traits in cattle including feed intake, reproduction traits and carcass merit; thus, type traits could also provide useful insights into the morphological differences among animals underpinning phenotypic differences in these complex traits. The objective of the present study was to identify genomic regions associated with five subjectively scored skeletal linear traits, to determine if these associated regions are common in multiple beef and dairy breeds, and also to determine if these regions overlap with those proposed elsewhere to be associated with correlated performance traits. Analyses were carried out using linear mixed models on imputed whole genome sequence data separately in 1,444 Angus, 1,129 Hereford, 6,433 Charolais, 8,745 Limousin, 1,698 Simmental, and 4,494 Holstein-Friesian cattle, all scored for the linear type traits. There was, on average, 18 months difference in age at assessment of the beef versus the dairy animals. While the majority of the identified quantitative trait loci (QTL), and thus genes, were both trait-specific and breed-specific, a large-effect pleiotropic QTL on BTA6 containing the NCAPG and LCORL genes was associated with all skeletal traits in the Limousin population and with wither height in the Angus. Other than that, little overlap existed in detected QTLs for the skeletal type traits in the other breeds. Only two QTLs overlapped the beef and dairy breeds; both QTLs were located on BTA5 and were associated with height in both the Angus and the Holstein-Friesian, despite the difference in age at assessment. Several detected QTLs in the present study overlapped with QTLs documented elsewhere that are associated with carcass traits, feed intake, and calving difficulty. While most breeding programs select for the macro-traits like carcass weight, carcass conformation, and feed intake, the higher degree of granularity with selection on the individual linear type traits in a multi-trait index underpinning the macro-level goal traits, presents an opportunity to help resolve genetic antagonisms among morphological traits in the pursuit of the animal with optimum performance metrics., (Copyright © 2020 Doyle, Berry, Veerkamp, Carthy, Walsh, Evans and Purfield.)

Background: Linear type traits, which reflect the muscular characteristics of an animal, could provide insight into how, in some cases, morphologically very different animals can yield the same carcass weight. Such variability may contribute to differences in the overall value of the carcass since primal cuts vary greatly in price; such variability may also hinder successful genome-based association studies. Therefore, the objective of our study was to identify genomic regions that are associated with five muscularity linear type traits and to determine if these significant regions are common across five different breeds. Analyses were carried out using linear mixed models on imputed whole-genome sequence data in each of the five breeds, separately. Then, the results of the within-breed analyses were used to conduct an across-breed meta-analysis per trait., Results: We identified many quantitative trait loci (QTL) that are located across the whole genome and associated with each trait in each breed. The only commonality among the breeds and traits was a large-effect pleiotropic QTL on BTA2 that contained the MSTN gene, which was associated with all traits in the Charolais and Limousin breeds. Other plausible candidate genes were identified for muscularity traits including PDE1A, PPP1R1C and multiple collagen and HOXD genes. In addition, associated (gene ontology) GO terms and KEGG pathways tended to differ between breeds and between traits especially in the numerically smaller populations of Angus, Hereford, and Simmental breeds. Most of the SNPs that were associated with any of the traits were intergenic or intronic SNPs located within regulatory regions of the genome., Conclusions: The commonality between the Charolais and Limousin breeds indicates that the genetic architecture of the muscularity traits may be similar in these breeds due to their similar origins. Conversely, there were vast differences in the QTL associated with muscularity in Angus, Hereford, and Simmental. Knowledge of these differences in genetic architecture between breeds is useful to develop accurate genomic prediction equations that can operate effectively across breeds. Overall, the associated QTL differed according to trait, which suggests that breeding for a morphologically different (e.g. longer and wider versus shorter and smaller) more efficient animal may become possible in the future.

Decidual macrophages secrete proteases that activate protease-activated receptor 1 (PAR-1). We hypothesized that activation of the inflammatory response by bacteria is amplified by proteases, initiating labor. In addition, we hypothesized that commensal bacteria trigger an inflammatory response by activating NF-κB and TET methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, via a protease amplified PAR-1, RhoA kinase (ROCK) pathway. To evaluate these hypotheses, we compared responses of mononuclear cells with Lactobacillus crispatus, prevalent in the vaginal microbiome of women of European ancestry, with L. iners and Fusobacterium nucleatum, which are more prevalent in vaginal samples collected from African-American women. Decidual tissue was collected at term not-in-labor (TNL), term labor (TL), spontaneous preterm labor (sPTL), and infected preterm labor (iPTL) and immunostained for PAR-1, TET2, and CD14. Mononuclear cells and THP-1 macrophage cells were treated with bacteria and elastase, a known activator of PAR-1. The inflammatory response was monitored by confocal microscopy of TET2 and the p65 subunit of NF-κB, as well as IL-8 production. Decidual staining for PAR-1, TET2, and CD14 increased TNL < TL < sPTL < iPTL. All treatments stimulated translocation of TET2 and p65 from the cytosol to the nucleus and increased IL-8, but L. iners and F. nucleatum caused more robust responses than L. crispatus. Inhibition of PAR-1 or ROCK prevented TET2 and p65 nuclear translocalization and increases in IL-8. Our findings demonstrate that proteases amplify the inflammatory response to commensal bacteria. The more robust response to bacteria prevalent in African-American women may contribute to racial disparities in preterm birth.

Sustainable dairy cow performance relies on coevolution in the development of breeding and management strategies. Tailoring breeding programs to herd performance metrics facilitates improved responses to breeding decisions. Although herd-level raw metrics on performance are useful, implicitly included within such statistics is the mean herd genetic merit. The objective of the present study was to quantify the expected response from selection decisions on additive and nonadditive merit by herd performance metrics independent of herd mean genetic merit. Performance traits considered in the present study were age at first calving, milk yield, calving to first service, number of services, calving interval, and survival. Herd-level best linear unbiased estimates (BLUE) for each performance trait were available on a maximum of 1,059 herds, stratified as best, average, and worst for each performance trait separately. The analyses performed included (1) the estimation of (co)variance for each trait in the 3 BLUE environments and (2) the regression of cow-level phenotypic performance on either the respective estimated breeding value (EBV) or the heterosis coefficient of the cow. A fundamental assumption of genetic evaluations is that 1 unit change in EBV equates to a 1 unit change in the respective phenotype; results from the present study, however, suggest that the realization of the change in phenotypic performance is largely dependent on the herd BLUE for that trait. Herds achieving more yield, on average, than expected from their mean genetic merit, had a 20% greater response to changes in EBV as well as 43% greater genetic standard deviation relative to herds within the worst BLUE for milk yield. Conversely, phenotypic performance in fertility traits (with the exception of calving to first service) tended to have a greater response to selection as well as a greater additive genetic standard deviation within the respective worst herd BLUE environments; this is suggested to be due to animals performing under more challenging environments leading to larger achievable gains. The attempts to exploit nonadditive genetic effects such as heterosis are often the basis of promoting cross-breeding, yet the results from the present study suggest that improvements in phenotypic performance is largely dependent on the environment. The largest gains due to heterotic effects tended to be within the most stressful (i.e., worst) BLUE environment for all traits, thus suggesting the heterosis effects can be beneficial in mitigating against poorer environments., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Deregulated immune response to a dysbiotic resident microflora within the oral cavity leads to chronic periodontal disease, local tissue destruction, and various systemic complications. To preserve tissue homeostasis, inflammatory signaling pathways involved in the progression of periodontitis must be tightly regulated. A20 (TNFAIP3), a ubiquitin-editing enzyme, has emerged as one of the key regulators of inflammation. Yet, the function of A20 in the oral mucosa and the biological pathways in which A20 mitigates periodontal inflammation remain elusive. Using a combination of in vivo and ex vivo disease models, we report in this study that A20 regulates inflammatory responses to a keystone oral bacterium, Porphyromonas gingivalis , and restrains periodontal inflammation through its effect on NF-κB signaling and cytokine production. Depletion of A20 using gene editing in human macrophage-like cells (THP-1) significantly increased cytokine secretion, whereas A20 overexpression using lentivirus infection dampened the cytokine production following bacterial challenge through modulating NF-κB activity. Similar to human cells, bone marrow-derived macrophages from A20-deficient mice infected with P. gingivalis displayed increased NF-κB activity and cytokine production compared with the cells isolated from A20-competent mice. Subsequent experiments using a murine ligature-induced periodontitis model showed that even a partial loss of A20 promotes an increased inflammatory phenotype and more severe bone loss, further verifying the critical function of A20 in the oral mucosa. Collectively, to our knowledge, these findings reveal the first systematic evidence of a physiological role for A20 in the maintenance of oral tissue homeostasis as a negative regulator of inflammation., (Copyright © 2019 by The American Association of Immunologists, Inc.)

A dispersive liquid-liquid microextraction (DLLME) method, combined with HPLC-UV detection, was developed for the extraction and preconcentration of δ-tocopherol from bovine milk. This method was used to study the effect of supplementing cow feed with the seaweed Ascophyllum nodosum on vitamin content in milk. The optimal experimental conditions were determined: 200 μL of chloroform (extraction solvent), 1.0 mL of ethanol (dispersive solvent), 5 mL of water (aqueous phase). Under these optimal conditions the DLLME method provided linearity in the range 0.01 μg/mL to 8 μg/mL with R 2 values of 0.998. Limit of detection (LOD) was 0.01 μg/mL, while the enrichment factor was 89. Cow feed that was supplemented with Ascophyllum nodosum was shown to increase δ-tocopherol levels from 3.82 μg/mL to 5.96 μg/mL., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Genetic evaluations decompose an observed phenotype into its genetic and nongenetic components; the former are termed BLUP with the solutions for the systematic environmental effects in the statistical model termed best linear unbiased estimates (BLUE). Geneticists predominantly focus on the BLUP and rarely consider the BLUE. The objective of this study, however, was to define and quantify the association between 8 herd-level characteristics and BLUE for 6 traits in dairy herds, namely (1) age at first calving, (2) calving to first service interval (CFS), (3) number of services, (4) calving interval (CIV), (5) survival, and (6) milk yield. Phenotypic data along with the fixed and random effects solutions were generated from the Irish national multi-breed dairy cow fertility genetic evaluations on 3,445,557 cows; BLUE for individual contemporary groups were collapsed into mean herd-year estimates. Data from 5,707 spring-calving herds between the years 2007 and 2016 inclusive were retained; association analyses were undertaken using linear mixed multiple regression models. Pearson coefficient correlations were used to quantify the relationships among individual trait herd-year BLUE, and transition matrices were used to understand the dynamics of mean herd BLUE estimates over years. Based on the mean annual trends in raw, BLUP, and BLUE, it was estimated that BLUE were associated with at least two-thirds of the improvement in CIV and milk production over the past 10 yr. Milk recording herds calved heifers for the first time on average 15 d younger, had an almost 2 d longer CFS but 2.3 d shorter CIV than non-milk-recording herds. Larger herd sizes were associated with worse BLUE for both CFS and CIV. Expanding herds and herds that had the highest proportion of cows born on the farm itself, on average, calved heifers younger and had shorter CIV. By separating the raw performance of a selection of herds into their respective BLUE and BLUP, it was possible to identify herds with inferior management practices that were being compensated by superior genetics; similarly, herds were identified with superior BLUE, but because of their inferior genetic merit, were not reaching their full potential. This suggests that BLUE could have a pivotal role in a tailored decision support tool that would enable producers to focus on the most limiting factor hindering them from achieving their maximum performance., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Matrix metalloproteinase-1 (MMP-1) and neutrophil elastase are proteolytic enzymes involved in tissue remodeling, but a role for them as uterotonic agents has not been considered. However, both these proteases activate protease-activated receptor 1 (PAR-1) that mediates thrombin-induced contractions. Matrix metalloproteinase-1 and elastase are products of neutrophils that infiltrate intrauterine tissues at the time of labor, so we tested the hypothesis that these proteases might be novel uterotonic agents acting via PAR-1. Decidual tissue was collected from fetal membranes of term not-in-labor (TNL), term labor (TL), and preterm labor (PTL) women and analyzed for gene and protein expression of MMP-1 and neutrophil elastase. Contractile effects of MMP-1 and elastase were tested with uterine strips of day 19 and 20 gestation rats. Expression of MMP-1 and neutrophil elastase was increased in TL and PTL as compared to TNL. Expression of both the pro- and active enzymes of MMP-1 increased progressively from TNL to TL to PTL. Tumor necrosis factor-α, a neutrophil product, increased MMP-1 in decidual and myometrial cells. Both MMP-1 and elastase stimulated strong contractions of myometrial strips, which were prevented by inhibition of PAR-1 and inhibition of inositol trisphosphate receptor or calcium channel blockade. Indomethacin did not prevent protease-induced contractions. These data suggest that MMP-1 and neutrophil elastase may be important but heretofore unrecognized players in stimulating uterine contractions at the time of labor, and they may explain why indomethacin delays, but does not prevent, PTL because indomethacin inhibits the prostaglandin component but not the protease component of labor.

Linear type traits describing the skeletal, muscular, and functional characteristics of an animal are routinely scored on live animals in both the dairy and beef cattle industries. Previous studies have demonstrated that genetic parameters for certain performance traits may differ between breeds; no study, however, has attempted to determine if differences exist in genetic parameters of linear type traits among breeds or sexes. Therefore, the objective of the present study was to determine if genetic covariance components for linear type traits differed among five contrasting cattle breeds, and to also investigate if these components differed by sex. A total of 18 linear type traits scored on 3,356 Angus (AA), 31,049 Charolais (CH), 3,004 Hereford (HE), 35,159 Limousin (LM), and 8,632 Simmental (SI) were used in the analysis. Data were analyzed using animal linear mixed models which included the fixed effects of sex of the animal (except in the investigation into the presence of sexual dimorphism), age at scoring, parity of the dam, and contemporary group of herd-date of scoring. Differences (P < 0.05) in heritability estimates, between at least two breeds, existed for 13 out of 18 linear type traits. Differences (P < 0.05) also existed between the pairwise within-breed genetic correlations among the linear type traits. Overall, the linear type traits in the continental breeds (i.e., CH, LM, SI) tended to have similar heritability estimates to each other as well as similar genetic correlations among the same pairwise traits, as did the traits in the British breeds (i.e., AA, HE). The correlation between a linear function of breeding values computed conditional on covariance parameters estimated from the CH breed with a linear function of breeding values computed conditional on covariance parameters estimated from the other breeds was estimated. Replacing the genetic covariance components estimated in the CH breed with those of the LM had least effect but the impact was considerable when the genetic covariance components of the AA were used. Genetic correlations between the same linear type traits in the two sexes were all close to unity (≥0.90) suggesting little advantage in considering these as separate traits for males and females. Results for the present study indicate the potential increase in accuracy of estimated breeding value prediction from considering, at least, the British breed traits separate to continental breed traits.

The cause of spontaneous preterm labor (sPTL) is not known, but it could be due to epigenetic alterations that increase the sensitivity of decidual tissue to inflammatory stimuli. We collected decidual tissue from women at term not in labor (TNL), women at term in labor (TL), and women with sPTL. Illumina Infinium HumanMethylation450 BeadChip analysis revealed significantly reduced DNA methylation for TLR-2 and TLR-9 in sPTL as compared to TL. Immunohistochemical staining documented significantly increased expression of TLR-2 and TLR-9 in decidual tissue of women with sPTL as compared to TL or TNL. TLR expression was not present in decidual cells, but localized to tissue leukocytes as revealed by staining for CD14, a macrophage antigen, and neutrophil elastase. Microarray analysis of inflammatory genes to assess innate immune response demonstrated marked increases in expression of inflammatory cytokines and chemokines in women with TL as compared to TNL. However, when sPTL was compared to TL, there was a further increase in inflammatory cytokines, and a remarkable increase in neutrophil chemokines. These results suggest that epigenetic mechanisms could play a role in increasing leukocyte infiltration, and increasing the sensitivity of decidual tissue to inflammatory stimuli that could precipitate labor prematurely., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Trisomy 21 (T21) is the most common chromosome abnormality in humans and is associated with a spectrum of phenotypes, including cognitive impairment, congenital heart defects and immune system defects. In addition, T21 is also associated with abnormalities of fetal membranes including chorioamniotic separation, delayed fusion of the chorioamniotic membranes, defects in syncytiotrophoblast formation, as well as amniocyte senescence. There is evidence indicating miRNAs encoded by sequences on chromosome 21 (Chr-21) are involved in several of the cognitive and neurological phenotypes of T21, but the role of Chr-21 derived miRNAs in fetal membrane abnormalities associated with T21 has not been investigated. In the current study, we determined the expression patterns of three miRNAs derived from a cluster on Chr-21 - hsa-miR-99a, hsa-miR-125b and hsa-let-7c in chorioamniotic membranes obtained from term pregnancies with spontaneous rupture (n = 20). Tissue and location specific expression patterns within the chorioamniotic membranes were identified. The rupture zone in the choriodecidua had distinct expression patterns compared to other fetal membrane locations. Despite the increased gene dosage associated with T21, the expression of all three miRNAs was reduced in cultured T21 amniocytes as compared to cultured euploid amniocytes. In silico analysis of experimentally validated targets of the three miRNAs suggest these Chr-21 derived miRNAs play a potential role in fetal membrane rupture and the fetal membrane defects associated with T21., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Matrix metalloproteinase 1 (MMP-1) is an activator of protease-activated receptor 1 (PAR-1), which is known to mediate the release of endothelin 1 (ET-1) in endothelial cells and activate the RhoA kinase (ROCK) pathway. Recently, we reported increased serum and vascular MMP-1 in women with preeclampsia and hypothesized that the action of MMP-1 on PAR-1 might have vasoconstrictive effects. Resistance-sized omental arteries obtained from normal pregnant women were mounted on a myograph system and perfused with MMP-1 in a dose range of 0.025 to 25 ng/mL or with angiotensin II (Ang II) in a dose range of 0.001 to 10 µmol/L in the presence of intraluminal MMP-1 (2.5 ng/mL) perfusion. Angiotensin II dose response was also performed with omental arteries from women with preeclampsia. Matrix metalloproteinase 1 caused dose-dependent vasoconstriction in endothelium-intact, but not in endothelium-denuded, vessels from normal pregnant women, which was blocked by inhibitors of PAR-1 and ET-1 type A receptor blocker. Intraluminal perfusion with a constant amount of MMP-1 enhanced vessel reactivity to Ang II, which was blocked by inhibitors of PAR-1, ROCK, and ET-1. Enhanced vascular reactivity to Ang II was observed in endothelium-intact, but not in endothelium-denuded, arteries of women with preeclampsia. Inhibitors of PAR-1, ROCK, and ET-1 blocked enhanced vascular reactivity to Ang II in endothelium-intact preeclamptic arteries. These data demonstrate that MMP-1 has potent vasoconstrictor effects and the ability to enhance vascular reactivity to vasoconstrictor hormones, which are mediated by an endothelial PAR-1, ROCK, and ET-1 pathway. Increased circulating levels of MMP-1 and its increased expression in systemic vessels of women with preeclampsia may contribute to the development of maternal hypertension., (© The Author(s) 2015.)

To compare gene expression among bovine tissues, large bovine RNA-seq datasets were used, comprising 280 samples from 10 different bovine tissues (uterine endometrium, granulosa cells, theca cells, cervix, embryos, leucocytes, liver, hypothalamus, pituitary, muscle) and generating 260 Gbases of data. Twin approaches were used: an information-theoretic analysis of the existing annotated transcriptome to identify the most tissue-specific genes and a de-novo transcriptome annotation to evaluate general features of the transcription landscape. Expression was detected for 97% of the Ensembl transcriptome with at least one read in one sample and between 28% and 66% at a level of 10 tags per million (TPM) or greater in individual tissues. Over 95% of genes exhibited some level of tissue-specific gene expression. This was mostly due to different levels of expression in different tissues rather than exclusive expression in a single tissue. Less than 1% of annotated genes exhibited a highly restricted tissue-specific expression profile and approximately 2% exhibited classic housekeeping profiles. In conclusion, it is the combined effects of the variable expression of large numbers of genes (73%-93% of the genome) and the specific expression of a small number of genes (<1% of the transcriptome) that contribute to determining the outcome of the function of individual tissues.

Obesity is a risk factor for preeclampsia, but the reason for this risk is unknown. Neutrophils infiltrate into systemic blood vessels of both obese and preeclamptic women. Neutrophils are a major source of myeloperoxidase (MPO), which is associated with hypertension. We tested the hypothesis that systemic vasculature of both obese and preeclamptic women will have a significant presence of MPO as a result of neutrophil infiltration. We found that immunohistochemical staining of MPO was significantly greater in subcutaneous fat blood vessels of obese women than overweight women, which was significantly greater than normal weight women. Expression of MPO was significantly greater in maternal blood vessels of preeclamptic women than normal pregnant or normal nonpregnant women. In general, when vessels of overweight or normal pregnant women were stained it was primarily for leukocytes in the lumen and not infiltrated into the vessel. In contrast, in obese and preeclamptic women staining was present for leukocytes in the lumen, flattened, and adhered to the endothelium and infiltrated into the vessel wall. There was also extensive diffuse staining for MPO in vessels of obese and preeclamptic women. In conclusion, both obese and preeclamptic women have increased presence of MPO in systemic vasculature as a result of neutrophil infiltration. We speculate that obese women may be at risk of preeclampsia because their vasculature is already prone to hypertension., (© The Author(s) 2014.)

The objective was to characterise the hormonal composition of follicular fluid from mares with distinct anovulatory-cystic follicles. Follicular fluid was aspirated from six mares that presented with cystic follicles and from pre-ovulatory follicles of five normal mares (controls). Differences in progesterone, oestradiol, testosterone, IGF-I and IGF binding were analysed using Fisher's exact test. There were greater (P < 0.03) follicular fluid oestradiol concentrations in normal follicles and the testosterone concentration of the cystic fluid was greater (P < 0.05) than that of the normal fluid. There also was a greater (P < 0.03) percentage of IGF-I binding and lower (P < 0.02) IGF-I concentrations in the fluid collected from the cystic structures compared with the fluid from normal follicles. Despite the limited number of animals, the fact that fluid aspirated from cystic follicles had higher testosterone and lower oestradiol concentrations could be of diagnostic value when a practitioner wants to distinguish between a cystic and non-cystic persistent follicle. The research reported here also indicates a likely role for the IGF system in the pathogenesis of the development and maintenance of anovulatory follicular structures in mare ovaries., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Follicular fluid (FF), an important microenvironment for the development of oocytes, contains many proteins that are glycosylated with N-linked glycans. This study aimed i) to present an initial analysis of the N-linked glycan profile of bovine FF using hydrophilic interaction liquid chromatography, anion exchange chromatography, high performance liquid chromatography (HPLC)-based separations and subsequent liquid chromatography-mass spectrometry/mass spectrometry analysis; ii) to determine differences in the N-glycan profile between FF from dominant and subordinate follicles from dairy heifers and lactating dairy cows and iii) to identify alterations in the N-glycan profile of FF during preovulatory follicle development using newly selected, differentiated (preovulatory) and luteinised dominant follicles from dairy heifers and lactating cows. We found that the majority of glycans on bovine FF are based on biantennary hypersialylated structures, where the glycans are sialylated on both the galactose and N-acetylglucosamine terminal sugars. A comparison of FF N-glycans from cows and heifers indicated higher levels of nonsialylated glycans with a lower proportion of sialylated glycans in cows than in heifers. Overall, as the follicle develops from Selection, Differentiation and Luteinisation in both cows and heifers, there is an overall decrease in sialylated structures on FF N-glycans., (© 2014 Society for Reproduction and Fertility.)

Previous studies have documented that ovarian antral follicle count (AFC) is positively correlated with number of healthy follicles and oocytes in ovaries (ovarian reserve), as well as ovarian function and fertility in cattle. However, environmental factors (e.g., nutrition, steroids) during pregnancy in cattle and sheep can reduce AFC in offspring. The role that genetic and environmental factors play in influencing the variability in AFC and, correspondingly, the size of the ovarian reserve, ovarian function, and fertility, are, however, poorly understood. The present study tests the hypothesis that variability in AFC in offspring is influenced not only by genetic merit but also by the dam age and lactation status (lactating cows vs. nonlactating heifers) and milk production during pregnancy. Antral follicle count was assessed by ultrasonography in 445 Irish Holstein-Friesian dairy cows and 522 US Holstein-Friesian dairy heifers. Heritability estimates for AFC (± standard error) were 0.31 ± 0.14 and 0.25 ± 0.13 in dairy cows and heifers, respectively. Association analysis between both genotypic sire data and phenotypic dam data with AFC in their daughters was performed using regression and generalized linear models. Antral follicle count was negatively associated with genetic merit for milk fat concentration. Also, AFC was greater in offspring of dams that were lactating (n=255) compared with nonlactating dams (n=89) during pregnancy and was positively associated with dam milk fat concentration and milk fat-to-protein ratio. In conclusion, AFC in dairy cattle is a moderately heritable genetic trait affected by age or lactation status and milk quality but not by level of dam's milk production during pregnancy., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Severe prenatal undernutrition is usually associated with low birth weights in offspring and disorders including hypertension, obesity, and diabetes. Whether alterations in maternal nutrition insufficient to impair birth weight or prenatal growth impact the cardiovascular, stress, or metabolic systems is unknown. In addition, little is known about the effects of maternal dietary restriction on development of the reproductive system in mammals. Here, we use the bovine model, which has a gestational length and birth rate similar to humans, to show that offspring from nutritionally restricted dams (during the first trimester) were born with identical birth weights and had similar postnatal growth rates (to 95 wk of age), puberty, glucose metabolism, and responses to stress compared to offspring from control mothers. However, an increase in maternal testosterone concentrations was detected during dietary restriction, and these dams had offspring with a diminished ovarian reserve (as assessed by a reduction in antral follicle count, reduced concentrations of anti-Müllerian hormone, and increased follicle-stimulating hormone concentrations), enlarged aorta, and increased arterial blood pressure compared with controls. Our study links transient maternal undernutrition and enhanced maternal androgen production with a diminished ovarian reserve as well as potential suboptimal fertility, enlarged aortic trunk size, and enhanced blood pressure independent of alterations in birth weight, postnatal growth, or stress response and glucose tolerance. The implications are that relatively mild transient reductions in maternal nutrition during the first trimester of pregnancy (even those that do not affect gross development) should be avoided to ensure healthy development of reproductive and cardiovascular systems in offspring.

Activation of the death receptor Fas has been reported to produce a two-phase intracellular Ca(2+) release response in coronary arterial myocytes (CAMs), which consists of local Ca(2+) bursts via lysosomal transient potential receptor-mucolipin 1 (TRP-ML1) channels and consequent Ca(2+) release from the sarcoplasmic reticulum (SR). The present study was designed to explore the molecular mechanism by which lysosomal Ca(2+) bursts are coupled with SR Ca(2+) release in mouse CAMs and to determine the functional relevance of this lysosome-associated two-phase Ca(2+) release to apoptosis, a common action of Fas activation with Fas ligand (FasL). By confocal microscopy, we found that transfection of CAMs with TRP-ML1 small interfering (si)RNA substantially inhibited FasL (10 ng/ml)-induced lysosome Ca(2+) bursts and consequent SR Ca(2+) release. In contrast, transfection of CAMs with plasmids containing a full-length TRP-ML1 gene enhanced FasL-induced two-phase Ca(2+) release. We further demonstrated that FasL significantly increased the colocalization of the lysosomal marker Lamp1 with ryanodine receptor 3 and enhanced a dynamic trafficking of lysosomes to the SR. When CAMs were treated with TRP-ML1 siRNA, FasL-induced interactions between the lysosomes and SR were substantially blocked. Functionally, FasL-induced apoptosis and activation of calpain and calcineurin, the Ca(2+) sensitive proteins that mediate apoptosis, were significantly attenuated by silencing TRP-ML1 gene but enhanced by overexpression of TRP-ML1 gene. These results suggest that TRP-ML1 channel-mediated lysosomal Ca(2+) bursts upon FasL stimulation promote lysosome trafficking and interactions with the SR, leading to apoptosis of CAMs via a Ca(2+)-dependent mechanism.

We analyzed 27,578 CpG sites that map to 14,495 genes in omental arteries of normal pregnant and preeclamptic women for DNA methylation status using the Illumina platform. We found 1685 genes with a significant difference in DNA methylation at a false discovery rate of <10% with many inflammatory genes having reduced methylation. Unsupervised hierarchical clustering revealed natural clustering by diagnosis and methylation status. Of the genes with significant methylation differences, 236 were significant at a false discovery rate of <5%. When data were analyzed more stringently to a false discovery rate of <5% and difference in methylation of >0.10, 65 genes were identified, all of which showed reduced methylation in preeclampsia. When these genes were mapped to gene ontology for molecular functions and biological processes, 75 molecular functions and 149 biological processes were overrepresented in the preeclamptic vessels. These included smooth muscle contraction, thrombosis, inflammation, redox homeostasis, sugar metabolism, and amino acid metabolism. We speculate that reduced methylation may contribute to the pathogenesis of preeclampsia and that alterations in DNA methylation resulting from preeclampsia may increase maternal risk of cardiovascular disease later in life.

Lactation in dairy cattle is associated with a multitude of endocrine, metabolic and immunological changes that not only influence animal health, but also affect fertility, and in particular ovarian function. We have previously generated a global transcriptomic profile of bovine follicular tissue using RNA sequencing. This study aimed to: identify key immune-related transcriptional changes that occur during follicle differentiation and luteinisation using ingenuity pathway analysis (IPA); and determine if a compromised model of stress (i.e. lactation) influences the temporal expression of these genes. Ovarian follicular tissue from Holstein-Friesian non-lactating heifers (n=17) and lactating cows (n=16) was compared at three stages of preovulatory follicle development: (A) the newly selected dominant follicle in the luteal phase (Selection); (B) the follicular phase before the LH surge (Differentiation), and (C) the preovulatory phase after the LH surge (Luteinisation). IPA revealed an over-representation of immune-related pathways in theca compared with granulosa cells during differentiation; these were related to leucocyte extravasation and chemotaxis. Conversely, luteinisation was characterised by over-representation of immune-related pathways in granulosa compared with theca cells; these were related to inflammation and innate immune response. Notably, comparison of follicles from heifers and lactating cows revealed a large number of differentially expressed genes associated with immune cell subpopulations and chemotaxis. In conclusion, identification of immune-related canonical pathways during follicle development supports the hypothesis that ovulation is an inflammatory event. This process is influenced by the physiological status of lactation and likely contributes to compromised peri-ovulatory follicle function by impairing the inflammatory process of ovulation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Maternal vascular dysfunction is a hallmark of preeclampsia. A recently described vascular phenotype of preeclampsia involves increased expression of matrix metalloproteinase-1 (MMP-1) in endothelial cells, vascular smooth muscle, and infiltrating neutrophils. In contrast, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) and collagen type Iα 1 is either reduced or not changed in the vessels, suggesting an imbalance in vessel collagen degradation and synthesis in preeclampsia. In the present study, we explored the possible contribution of DNA methylation to the altered expression of genes involved in collagen metabolism. We assayed the differences in DNA methylation in omental arteries from normal pregnant and preeclamptic women, and determined whether reduced DNA methylation increases the expression of MMP-1 in cultured vascular smooth muscle cells and a neutrophil-like cell line, HL-60. Several MMP genes, including MMP1 and MMP8, were significantly less methylated in preeclamptic omental arteries, whereas TIMP and COL genes either were significantly more methylated or had no significant change in their DNA methylation status compared with normal pregnancy. Experimentally induced DNA hypomethylation increased MMP-1 expression in cultured vascular smooth muscle cells and MMP-1 cells. Our findings suggest that epigenetic regulation contributes to the imbalance in genes involved in collagen metabolism in blood vessels of preeclamptic women., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: Recently, it has been proposed that supplementation with l-Arginine reduces the incidence of preeclampsia in high risk women, but the molecular mechanisms involved in the protective effect need to be determined. In addition, a critical role of l-Arginine in endothelial cell survival during oxidative stress, and the participation of neutrophils in the induction of oxidative stress during preeclampsia have been suggested., Objectives: To address if supplementation with l-arginine provides antioxidant defense in human vascular endothelial cells., Methods: Human vascular endothelial cells (HUVECs) were isolated from umbilical cord veins obtained from healthy women underwent cesarean sections at term, with no evidence of hypertension disorders through the pregnancy. HUVECs were cultured in EndoGro media with LS supplement kit and 1% antibiotic with (n=10) or without 200uM l-Arginine (n=10). Confluent HUVECs were stimulated with neutrophils activated with 50umol/L arachidonic acid (1:16 ratio of neutrophil/cells). After incubation, cells were rinsed in PBS and harvested for RNA and protein extraction. Reverse transcription was performed using the RT(2) First Strand kit, and expression gene profiling was generated using the RT(2) Profiler PCR Array Human Oxidative Stress and Antioxidant Defense that includes the expression profile of 84 genes related to the oxidative pathway. Expression results were analyzed with the RT(2) Profiler PCR Array Data Analysis Template v3.0 and two different lists of fold change in gene expression were generated: (1) HUVEC+neutrophils vs HUVEC+l-Arginine + neutrophils and (2) HUVEC vs HUVEC+neutrophils. Validation of the expression assays was performed using western blots or ELISAS for proteins expressed by selected genes., Results: Fold up- or down gene regulation are shown in Table 1. Forty six genes involved in oxidative stress defense were significantly up-regulated in HUVECs supplemented with l-arginine when were exposed to neutrophils. Interestingly, almost the same genes were down-regulated in non-supplemented HUVECs after neutrophil exposure., Conclusion: Supplementation with l-Arginine upregulates the expression of genes related to antioxidant defense in primary cultures of endothelial cells. This finding provides a novel insight about the molecular mechanisms involved in the protective role of l-Arginine during preeclampsia., (Copyright © 2012. Published by Elsevier B.V.)

Dietary restriction in growing cattle and severe negative energy balance in lactating cows have been associated with altered gonadotropin secretion, reduced follicle diameter, reduced circulating oestradiol concentrations and anovulation. Therefore, we hypothesised that acute dietary restriction would influence the fate and function of the dominant follicle by altering the expression for genes regulating gonadotrophin and IGF response in ovarian follicles. Newly selected dominant follicles were collected 7-8 days after prostaglandin F(2α) (PGF) administration from heifers (n=25) that were individually fed a diet supplying 1.2 maintenance (M; control, n=8) or 0.4 M (restricted, n=17) for a total duration of 18-19 days. Heifers within 0.4 M were ovulatory (n=11) or anovulatory (n=6) depending on whether the dominant follicle present at PGF ovulated or became atretic following luteolysis. Control animals were all ovulatory. Acute dietary restriction decreased IGF-I (P<0.001) and insulin (P<0.05) in circulation; oestradiol (P<0.01) and IGF-I (P<0.01) in follicular fluid; and mRNA for FSHR (P<0.01) in granulosa cells but increased mRNA for IGFBP2 (P<0.05) in theca cells of the newly selected dominant follicle. However, this only led to anovulation when dietary restriction also decreased mRNA for CYP19A1 (P<0.05), IGF2 (P<0.01) and IGF1R (P<0.05) in granulosa cells and LHCGR (P<0.05) in theca cells of follicles collected from heifers fed 0.4 M. These results suggest that the catabolic environment induced by dietary restriction may ultimately cause anovulation by reducing oestradiol synthesis, FSH-responsiveness and IGF signaling in granulosa, and LH-responsiveness in theca cells of dominant follicles., (Copyright © 2012. Published by Elsevier B.V.)