Liver transcriptome response to hyperthermic stress in three distinct chicken lines.

BACKGROUND: High ambient temperatures cause stress in poultry, especially for broiler lines, which are genetically selected for rapid muscle growth. RNA-seq technology provides powerful insights into environmental response from a highly metabolic tissue, the liver. We investigated the effects of acute (3 h, 35 °C) and chronic (7d of 35 °C for 7 h/d) heat stress on the liver transcriptome of 3-week-old chicks of a heat-susceptible broiler line, a heat-resistant Fayoumi line, and their advanced intercross line (AIL). RESULTS: Transcriptome sequencing of 48 male chickens using Illumina HiSeq 2500 technology yielded an average of 33.9 million, 100 base-pair, single-end reads per sample. There were 8 times more differentially expressed genes (DEGs) (FDR < 0.05) in broilers (n = 627) than Fayoumis (n = 78) when comparing the acute-heat samples to the control (25 °C) samples. Contrasting genetic lines under similar heat treatments, the highest number of DEGs appeared between Fayoumi and broiler lines. Principal component analysis of gene expression and analysis of the number of DEGs suggested that the AIL had a transcriptomic response more similar to the Fayoumi than the broiler line during acute heat stress. The number of DEGs also suggested that acute heat stress had greater impact on the broiler liver transcriptome than chronic heat stress. The angiopoietin-like 4 (ANGPTL4) gene was identified as differentially expressed among all 6 contrasts. Ingenuity Pathway Analysis (IPA) created a novel network that combines the heat shock protein family with immune response genes. CONCLUSIONS: This study extends our understanding of the liver transcriptome response to different heat exposure treatments in distinct genetic chicken lines and provides information necessary for breeding birds to be more resilient to the negative impacts of heat. The data strongly suggest ANGPTL4 as a candidate gene for improvement of heat tolerance in chickens.

Transcriptome Response of Liver and Muscle in Heat-Stressed Laying Hens.

Exposure to high ambient temperature has detrimental effects on poultry welfare and production. Although changes in gene expression due to heat exposure have been well described for broiler chickens, knowledge of the effects of heat on laying hens is still relatively limited. In this study, we profiled the transcriptome for pectoralis major muscle (n = 24) and liver (n = 24), during a 4-week cyclic heating experiment performed on layers in the early phase of egg production. Both heat-control and time-based contrasts were analyzed to determine differentially expressed genes (DEGs). Heat exposure induced different changes in gene expression for the two tissues, and we also observed changes in gene expression over time in the control animals suggesting that metabolic changes occurred during the transition from onset of lay to peak egg production. A total of 73 DEGs in liver were shared between the 3 h heat-control contrast, and the 4-week versus 3 h time contrast in the control group, suggesting a core set of genes that is responsible for maintenance of metabolic homeostasis regardless of the physiologic stressor (heat or commencing egg production). The identified DEGs improve our understanding of the layer's response to stressors and may serve as targets for genetic selection in the future to improve resilience.

Evaluation of thrombolysis using tissue plasminogen activator in lower extremity deep venous thrombosis with concomitant femoral-popliteal venous segment involvement.

OBJECTIVE: Current guidelines recommend thrombolytic therapy for iliofemoral deep venous thrombosis (DVT). Anticoagulation is the standard treatment for femoral-popliteal and tibial-level DVT. The objective of this study was to evaluate the efficacy of catheter-directed thrombolysis (CDT) using tissue plasminogen activator vs standard anticoagulation alone in patients with lower extremity DVT involving the femoral-popliteal segment. METHODS: A retrospective review was performed of patients referred to the vascular surgery service with lower extremity DVT from 2006 to 2015. Patients who had DVT involving the femoral-popliteal segment were identified, including some patients who had concomitant involvement of iliofemoral and tibial veins. Patients with pure iliofemoral and tibial vein DVT were excluded from this analysis. Review of medical records, follow-up ultrasound studies, hypercoagulable panel, and venography were performed. Comparison of outcomes between patients who received thrombolytic therapy using tissue plasminogen activator and patients who received standard anticoagulation alone was performed. The primary outcomes measured were restoration of patency of the femoral-popliteal segment at 3 months, incidence of post-thrombotic syndrome (PTS), and valvular dysfunction. Secondary outcomes were incidence of bleeding, in-hospital mortality, and pulmonary embolism. RESULTS: The study cohort was composed of 191 patients (CDT, n = 89; anticoagulation alone, n = 102) who met inclusion criteria. Most patients with thrombus involving the femoral-popliteal segment also had proximal venous segment involvement, with 93% of the patient cohort having proximal iliofemoral DVT. Patients who did not receive CDT were older (mean age of 64 years vs 51 years; P < .001) and had more associated comorbidities, such as diabetes, immobility, and cancer. A significant number of patients who received CDT had a positive family history for DVT (21.3% vs 8.8%; P = .023), and it was more likely to be their first episode of DVT (73.0% vs 55.9%; P = .016). Patients who received CDT were more likely to have restoration of patency (74.7% vs 11.1%; P < .001) and lower incidence of PTS (21.3% vs 73.4%; P < .001) and valvular dysfunction (23.0% vs 66.7%; P < .001) compared with patients who were treated with anticoagulation alone. Incidence of bleeding was significantly more for patients treated with anticoagulation alone (14.7% vs 5.6%; P = .018) compared with patients who received CDT. On multivariate analysis, age was the predominant risk factor for bleeding. There was no significant difference in mortality and pulmonary embolism. CONCLUSIONS: In patients with acute proximal DVT and concomitant femoral-popliteal venous segment involvement, CDT resulted in superior patency at 3 months and less PTS and valvular reflux. This was achieved without increase in bleeding complications compared with anticoagulation alone. Age was the major factor predictive of bleeding in either group. The results of this study may not be applicable to patients with pure femoral-popliteal venous segment DVT because only 3% of patients had this finding.

Integrated host and viral transcriptome analyses reveal pathology and inflammatory response mechanisms to ALV-J injection in SPF chickens.

Avian leukosis virus (ALV) is detrimental to poultry health and causes substantial economic losses from mortality and decreased performance. Because tumorigenesis is a complex mechanism, the regulatory architecture of the immune system is likely to include the added dimensions of modulation by miRNAs and long-noncoding RNA (lncRNA). To characterize the response to ALV challenge, we developed a novel methodology that combines four datasets: mRNA expression and the associated regulatory factors of miRNA and lncRNA, and ALV gene expression. Specific Pathogen-Free (SPF) layer chickens were infected with ALV-J or maintained as non-injected controls. Spleen samples were collected at 40 days post injection (dpi), and sequenced. There were 864 genes, 7 miRNAs and 17 lncRNAs differentially expressed between infected and non-infected birds. The combined analysis of the 4 RNA expression datasets revealed that ALV infection is detected by pattern-recognition receptors (TLR9 and TLR3) leading to a type-I IFN mediated innate immune response that is modulated by IRF7 and IRF1. Co-expression network analysis of mRNA with miRNA, lncRNA and virus genes identified key elements within the complex networks utilized during ALV response. The integration of information from the host transcriptomic, epigenetic and virus response also has the potential to provide deeper insights into other host-pathogen interactions.

Large chromosome deletions, duplications, and gene conversion events accumulate with age in normal human colon crypts.

Little is known about the types and numbers of mutations that may accumulate in normal human cells with age. Such information would require obtaining enough DNA from a single cell to accurately carry out reliable analysis despite extensive amplification; and complete genomic coverage under these circumstances is difficult. We have compared colon crypts, which are putatively clonal and contain ~2000 cells each, to determine how much somatic genetic variation occurs in vivo (without ex vivo cell culturing). Using high-density SNP microarrays, we find that chromosome deletions, duplications, and gene conversions were significantly more frequent in colons from the older individuals. These changes affected lengths ranging from 73 kb to 46 Mb. Although detection requires progeny of a single mutant stem cell to reach niche dominance over neighboring stem cells, none of the deletions appear likely to confer a selective advantage. Mutations can become fixed randomly during stem cell evolution through neutral drift in normal human crypts. The fact that chromosomal changes are detected in individual crypts with increasing age suggests that either such changes accumulate with age or single stem cell dominance increases with age, and the former is more likely. This progressive genome-wide divergence of human somatic cells with age has implications for aging and disease in multicellular organisms.

Heterogeneity and randomness of DNA methylation patterns in human embryonic stem cells.

DNA methylation has been proposed to be important in many biological processes and is the subject of intense study. Traditional bisulfite genomic sequencing allows detailed high-resolution methylation pattern analysis of each molecule with haplotype information across a few hundred bases at each locus, but lacks the capacity to gather voluminous data. Although recent technological developments are aimed at assessing DNA methylation patterns in a high-throughput manner across the genome, the haplotype information cannot be accurately assembled when the sequencing reads are short or when each hybridization target only includes one or two cytosine-phosphate-guanine (CpG) sites. Whether a distinct and nonrandom DNA methylation pattern is present at a given locus is difficult to discern without the haplotype information, and the DNA methylation patterns are much less apparent because the data are often obtained only as methylation frequencies at each CpG site with some of these methods. It would facilitate the interpretation of data obtained from high-throughput bisulfite sequencing if the loci with nonrandom DNA methylation patterns could be distinguished from those that are randomly methylated. In this study, we carried out traditional genomic bisulfite sequencing using the normal diploid human embryonic stem (hES) cell lines, and utilized Hamming distance analysis to evaluate the existence of a distinct and nonrandom DNA methylation pattern at each locus studied. Our findings suggest that Hamming distance is a simple, quick, and useful tool to identify loci with nonrandom DNA methylation patterns and may be utilized to discern links between biological changes and DNA methylation patterns in the high-throughput bisulfite sequencing data sets.