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Abstract Mastitis or other infectious diseases have been related to reduced fertility in cattle. Inflammatory cytokines such as tumor necrosis factor α (TNFα) are released in response to infection and may have negative effects on embryo development. In the current study the effect of exposure to TNFα on the development of in vitro fertilized bovine embryos was examined. Indomethacin, a prostaglandin synthesis inhibitor, was used to determine if blockade of prostaglandin synthesis would alter the effects of TNFα. Ovaries were obtained from a local abattoir and immature COC were isolated from 2-10 mm follicles, in vitro matured and fertilized. After fertilization, groups of presumptive zygotes were randomly placed into either control development medium, medium containing 25 ng/mL TNFα or medium containing 25 ng/mL TNFα plus 1 μg/mL indomethacin. The proportion of blastocysts formed was assessed at day 7 of culture. Fewer embryos exposed to TNFα alone reached the blastocyst stage (17.5 ± 2.4%, P < 0.01) compared with controls (30.5 ± 2.4%) or embryos developed in TNFα plus indomethacin (25.8 ± 2.8%). There was no difference between control embryos and embryos developed in TNFα plus indomethacin. These results indicate that TNFα is inhibitory to the in vitro development of bovine embryos and that this inhibition may be mediated by prostaglandins because it can be blocked by indomethacin.

Genetic and epigenetic variations in regulatory enhancer elements increase susceptibility to a range of pathologies. Despite recent advances, linking enhancer elements to target genes and predicting transcriptional outcomes of enhancer dysfunction remain significant challenges. Using 3D chromatin conformation assays, we generated an extensive enhancer interaction dataset for the human pancreas, encompassing more than 20 donors and five major cell types, including both exocrine and endocrine compartments. We employed a network approach to parse chromatin interactions into enhancer-promoter tree models, facilitating a quantitative, genome-wide analysis of enhancer connectivity. With these tree models, we developed a machine learning algorithm to estimate the impact of enhancer perturbations on cell type-specific gene expression in the human pancreas. Orthogonal to our computational approach, we perturbed enhancer function in primary human pancreas cells using CRISPR interference and quantified the effects at the single-cell level through RNA FISH coupled with high-throughput imaging. Our enhancer tree models enabled the annotation of common germline risk variants associated with pancreas diseases, linking them to putative target genes in specific cell types. For pancreatic ductal adenocarcinoma, we found a stronger enrichment of disease susceptibility variants within acinar cell regulatory elements, despite ductal cells historically being assumed as the primary cell-of-origin. Our integrative approach-combining cell type-specific enhancer-promoter interaction mapping, computational models, and single-cell enhancer perturbation assays-produced a robust resource for studying the genetic basis of pancreas disorders.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or 'hubness', positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages, the trophectoderm (TE), the epiblast (EPI) and the primitive endoderm (PrE). Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements via which transcriptional regulators enact these fates remain understudied. To address this gap, we have characterized, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observed extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although there are distinct groups of genes that are irresponsive to topological changes. In each lineage, a high degree of connectivity or "hubness" positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages, compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a novel predictive model for transcriptional regulation (3D-HiChAT), which outperformed models that use only 1D promoter or proximal variables in predicting levels and cell-type specificity of gene expression. Using 3D-HiChAT, we performed genome-wide in silico perturbations to nominate candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments we validated several novel enhancers that control expression of one or more genes in their respective lineages. Our study comprehensively identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to understand lineage-specific transcriptional behaviors., Competing Interests: Conflict of interest statement The authors declare that the above study was conducted in the absence of any commercial, financial, or personal relationships that could have appeared to influence the work reported in this article. All authors have approved the submitted version.

The use of the internal mammary artery (IMA) in coronary artery bypass surgery has increased substantially over the past 20 years, being at present the conduit of choice for most patients. Complications associated with its use occur occasionally and include life-threatening postoperative ischemia or the revascularized myocardium. We reviewed the records of 1,971 consecutive patients who underwent coronary artery bypass grafting over a 5-year period. All operations included an IMA graft to the left anterior descending coronary artery. Twenty-eight of these patients (1.4%) underwent additional placement of a vein graft on the same region as a salvage maneuver for suspected hypoperfusion as a result of IMA failure. All 28 patients showed life-threatening hemodynamic compromise. Twenty-two of the 28 patients (79%) survived. This was the result of immediate surgical correction, which reversed their hemodynamic instability. IMA hypoperfusion was found more frequently in reoperations and in women and diabetic patients. This syndrome is the result of an imbalance between IMA flow and myocardial demand, causing sudden and unexpected myocardial failure. Its detection and expeditious treatment can successfully modify a serious and potentially lethal clinical situation.

Objective: To compare pulmonary function and peripheral organ blood flow in septic pigs receiving high-volume fluid resuscitation or standard-volume fluid resuscitation with similar goals in oxygen delivery., Design: A prospective study comparing 2 groups of septic pigs., Setting: A university animal research laboratory., Subjects: Eleven septic pigs., Interventions: Basal oxygen delivery was increased from 450 to 550 mL/min to at least 600 mL/min by the sixth hour and maintained for 24 hours. From a baseline pulmonary artery occlusion pressure (PAOP) measurement of approximately 6 mm Hg, the high-volume group (n = 5) was treated until a PAOP measurement of 12 mm Hg was reached and the standard-volume group (n = 6) was treated until a PAOP measurement of 8 mm Hg was reached. Blood transfusions and inotropic agents were added as necessary to reach the oxygen delivery goal., Results: The high-volume group had a significantly greater positive fluid balance, greater weight gain, and a higher PAOP but similar intrapulmonary shunt and extravascular lung water as compared with the standard-volume group., Conclusion: Resuscitation with large volumes of fluid in early sepsis with a physiological goal of a higher PAOP to augment oxygen delivery did not cause increased pulmonary edema and oxygenation deficit compared with maintenance of lower cardiac filling pressures.