Identification of long non-protein coding RNAs in chicken skeletal muscle using next generation sequencing

AbstractVertebrate genomes encode thousands of non-coding RNAs including short non-coding RNAs (such as microRNAs) and long non-coding RNAs (lncRNAs). Chicken (Gallus gallus) is an important model organism for developmental biology, and the recently assembled genome sequences for chicken will facilitate the understanding of the functional roles of non-coding RNA genes during development. The present study concerns the first systematic identification of lncRNAs using RNA-Seq to sample the transcriptome during chicken muscle development. A computational approach was used to identify 281 new intergenic lncRNAs in the chicken genome. Novel lncRNAs in general are less conserved than protein-coding genes and slightly more conserved than random non-coding sequences. The present study has provided an initial chicken lncRNA catalog and greatly increased the number of chicken ncRNAs in the non-protein coding RNA database. Furthermore, the computational pipeline presented in the current work will be useful for characterizing lncRNAs obtained from deep sequencing data

The cardiomyocyte disrupts pyrimidine biosynthesis in non-myocytes to regulate heart repair

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair

Systematic identification and characterization of chicken (Gallus gallus) ncRNAs

Recent studies have demonstrated that non-coding RNAs (ncRNAs) play important roles during development and evolution. Chicken, the first genome-sequenced non-mammalian amniote, possesses unique features for developmental and evolutionary studies. However, apart from microRNAs, information on chicken ncRNAs has mainly been obtained from computational predictions without experimental validation. In the present study, we performed a systematic identification of intermediate size ncRNAs (50–500 nt) by ncRNA library construction and identified 125 chicken ncRNAs. Importantly, through the bioinformatics and expression analysis, we found the chicken ncRNAs has several novel features: (i) comparative genomic analysis against 18 sequenced vertebrate genomes revealed that the majority of the newly identified ncRNA candidates is not conserved and most are potentially bird/chicken specific, suggesting that ncRNAs play roles in lineage/species specification during evolution. (ii) The expression pattern analysis of intronic snoRNAs and their host genes suggested the coordinated expression between snoRNAs and their host genes. (iii) Several spatio-temporal specific expression patterns suggest involvement of ncRNAs in tissue development. Together, these findings provide new clues for future functional study of ncRNAs during development and evolution

A systematic analysis of the skeletal muscle miRNA transcriptome of chicken varieties with divergent skeletal muscle growth identifies novel miRNAs and differentially expressed miRNAs

Abstract Background Functional studies have demonstrated that microRNAs (miRNAs or miRs) play critical roles in a wide spectrum of biological processes including development and disease pathogenesis. To investigate the functional roles that miRNAs play during chicken skeletal muscle development, the miRNA transcriptomes of skeletal muscles from broiler and layer chickens were profiled using Solexa deep sequencing. Results Some miRNAs have multiple isoforms and several miRNAs* are present at higher levels than their corresponding miRNAs. Thirty three novel and 189 known chicken miRNAs were identified using computational approaches. Subsequent miRNA transcriptome comparisons and real-time PCR validation experiments revealed 17 miRNAs that were differentially expressed between broilers and layers, and a number of targets of these miRNAs have been implicated in myogenesis regulation. Using integrative miRNA target-prediction and network-analysis approaches an interaction network of differentially expressed and muscle-related miRNAs and their putative targets was constructed, and miRNAs that could contribute to the divergent muscle growth of broiler and layer chickens by targeting the ACVR2B gene were identified, which can causes dramatic increases in muscle mass. Conclusions The present study provides the first transcriptome profiling-based evaluation of miRNA function during skeletal muscle development in chicken. Systematic predictions aided the identification of potential miRNAs and their targets, which could contribute to divergent muscle growth in broiler and layer chickens. Furthermore, these predictions generated information that can be utilized in further research investigating the involvement of interaction networks, containing miRNAs and their targets, in the regulation of muscle development.</p

Cardiac fibroblast proliferation rates and collagen expression mature early and are unaltered with advancing age.

Cardiac fibrosis is a pathophysiologic hallmark of the aging heart, but little is known about how fibroblast proliferation and transcriptional programs change throughout the life span of the organism. Using EdU pulse labeling, we demonstrated that more than 50% of cardiac fibroblasts were actively proliferating in the first day of postnatal life. However, by 4 weeks, only 10% of cardiac fibroblasts were proliferating. By early adulthood, the fraction of proliferating cardiac fibroblasts further decreased to approximately 2%, where it remained throughout the rest of the organism's life. We observed that maximal changes in cardiac fibroblast transcriptional programs and, in particular, collagen and ECM gene expression both in the heart and cardiac fibroblast were maximal in the newly born and juvenile animal and decreased with organismal aging. Examination of DNA methylation changes both in the heart and in cardiac fibroblasts did not demonstrate significant changes in differentially methylated regions between young and old mice. Our observations demonstrate that cardiac fibroblasts attain a stable proliferation rate and transcriptional program early in the life span of the organism and suggest that phenotypic changes in the aging heart are not directly attributable to changes in proliferation rate or altered collagen expression in cardiac fibroblasts

Cardiac fibroblast proliferation rates and collagen expression mature early and are unaltered with advancing age

Cardiac fibrosis is a pathophysiologic hallmark of the aging heart, but little is known about how fibroblast proliferation and transcriptional programs change throughout the life span of the organism. Using EdU pulse labeling, we demonstrated that more than 50% of cardiac fibroblasts were actively proliferating in the first day of postnatal life. However, by 4 weeks, only 10% of cardiac fibroblasts were proliferating. By early adulthood, the fraction of proliferating cardiac fibroblasts further decreased to approximately 2%, where it remained throughout the rest of the organism’s life. We observed that maximal changes in cardiac fibroblast transcriptional programs and, in particular, collagen and ECM gene expression both in the heart and cardiac fibroblast were maximal in the newly born and juvenile animal and decreased with organismal aging. Examination of DNA methylation changes both in the heart and in cardiac fibroblasts did not demonstrate significant changes in differentially methylated regions between young and old mice. Our observations demonstrate that cardiac fibroblasts attain a stable proliferation rate and transcriptional program early in the life span of the organism and suggest that phenotypic changes in the aging heart are not directly attributable to changes in proliferation rate or altered collagen expression in cardiac fibroblasts

Rapid Biodegradation of the Organophosphorus Insecticide Chlorpyrifos by Cupriavidus nantongensis X1T

Chlorpyrifos was one of the most widely used organophosphorus insecticides and the neurotoxicity and genotoxicity of chlorpyrifos to mammals, aquatic organisms and other non-target organisms have caused much public concern. Cupriavidus nantongensis X1T, a type of strain of the genus Cupriavidus, is capable of efficiently degrading 200 mg/L of chlorpyrifos within 48 h. This is ~100 fold faster than Enterobacter B-14, a well-studied chlorpyrifos-degrading bacterial strain. Strain X1T can tolerate high concentrations (500 mg/L) of chlorpyrifos over a wide range of temperatures (30&ndash;42 &deg;C) and pH values (5&ndash;9). RT-qPCR analysis showed that the organophosphorus hydrolase (OpdB) in strain X1T was an inducible enzyme, and the crude enzyme isolated in vitro could still maintain 75% degradation activity. Strain X1T can simultaneously degrade chlorpyrifos and its main hydrolysate 3,5,6-trichloro-2-pyridinol. TCP could be further metabolized through stepwise oxidative dechlorination and further opening of the benzene ring to be completely degraded by the tricarboxylic acid cycle. The results provide a potential means for the remediation of chlorpyrifos- contaminated soil and water

Quantitative Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry Method for Comparison of Prochloraz Residue on Garlic Sprouts after Soaking and Spraying Treatment

Prochloraz is a fungicide that is widely used on vegetables to maintain freshness during storage. To ensure that prochloraz is used in a safe way that reduces the levels of residue on the product, we evaluated two treatment methods (soaking and spraying) that are commonly used for garlic sprouts. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for prochloraz residue on garlic sprouts. The linear range of the method was 5&ndash;500 &mu;g/kg and the correlation coefficient was 0.9983. The average recovery range was 88&ndash;94%, and the relative standard deviation range was 2.6&ndash;9.7%. Garlic sprout samples that had been soaked in or sprayed with prochloraz were collected from cold storage facilities in Laixi and Pingdu, China. For the soaked samples, the ranges for the levels of prochloraz residue on the whole garlic sprouts and stems (edible portion) were 15.76&ndash;25.14 mg/kg and 0.58&ndash;1.62 mg/kg, respectively. For the sprayed samples, the ranges for the levels of prochloraz residue on the whole garlic sprouts and stems were 1.85&ndash;7.89 mg/kg and 0.01&ndash;1.29 mg/kg, respectively. The results of this study provide a scientific basis for rationalizing the use of prochloraz and improving the safety of edible garlic sprouts