Construction of Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast Cells

The FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18- and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans which might play an important role on lipometabolism. To reveal the function of fat1 gene in bovine fetal fibroblast cells and gain the best cell nuclear donor for transgenic bovines, the codon of fat1 sequence was optimized based on the codon usage frequency preference of bovine muscle protein, and directionally cloned into the eukaryotic expression vector pEF-GFP. After identifying by restrictive enzyme digests with AatII/XbaI and sequencing, the fusion plasmid pEF-GFP-fat1 was identified successfully. The pEF-GFP-fat1 vector was transfected into bovine fetal fibroblast cells mediated by Lipofectamine2000TM. The positive bovine fetal fibroblast cells were selected by G418 and detected by RT-PCR. The results showed that a 1,234 bp transcription was amplified by reverse transcription PCR and the positive transgenic fat1 cell line was successfully established. Then the expression level of fat1 gene in positive cells was detected using quantitative PCR, and the catalysis efficiency was detected by gas chromatography. The results demonstrated that the catalysis efficiency of fat1 was significantly high, which can improve the total PUFAs rich in EPA, DHA and DPA. Construction and expression of pEF-GFP-fat1 vector should be helpful for further understanding the mechanism of regulation of fat1 in vitro. It could also be the first step in the production of fat1 transgenic cattle

Analysis of DNA Methylation in Various Swine Tissues

DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

High-throughput hemodialysis on the clinical efficacy and micro-inflammatory state, calcium and phosphorus metabolism, heart and kidney function in patients with end-stage renal disease

To compare the clinical efficacy, heart and kidney function, calcium and phosphorus metabolism, serological indicators, and the effects of micro-inflammatory status after two hemodialysis treatments to evaluate the best treatment for patients with end-stage renal disease. According to the criteria for inclusion and exclusion, collected in the Department of Nephrology, third People's Hospital of Gansu Province, patients were selected and received treatment between July 2019 and July 2021. A total of 60 cases were randomized. The urea nitrogen (BUN), blood creatinine (Scr), calcium and phosphorus metabolism levels, inflammation-related factors, and serum-related indicators of the two groups of patients before and after the treatment were detected for half a year. The effective rate (83.33%) of the observation group was higher than that of the control group (66.67%). After treatment, the iPTH, β2-MG, Hcy of the observation group were lower than those of the control group, and ALB was higher than that of the control group. The inflammation-related index observation group was significantly lower than the control However, the difference in Scr and BNU index levels between the two groups of patients after treatment was not so obvious that they could not be evaluated. In terms of all indicators and parameters, high-flux hemodialysis can better treat ESRD and improve the heart and kidney function of patients

Differential Expression of , , and Genes in Various Adipose Tissues and Muscle from Yanbian Yellow Cattle and Yan Yellow Cattle

The objective of this study was to investigate the correlation between cattle breeds and deposit of adipose tissues in different positions and the gene expressions of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), and Acyl-CoA dehydrogenase (ACADM), which are associated with lipid metabolism and are valuable for understanding the physiology in fat depot and meat quality. Yanbian yellow cattle and Yan yellow cattle reared under the same conditions display different fat proportions in the carcass. To understand this difference, the expression of PPARγ, FASN, and ACADM in different adipose tissues and longissimus dorsi muscle (LD) in these two breeds were analyzed using the Real-time quantitative polymerase chain reaction method (qRT-PCR). The result showed that PPARγ gene expression was significantly higher in adipose tissue than in LD in both breeds. PPARγ expression was also higher in abdominal fat, in perirenal fat than in the subcutaneous fat (p<0.05) in Yanbian yellow cattle, and was significantly higher in subcutaneous fat in Yan yellow cattle than that in Yanbian yellow cattle. On the other hand, FASN mRNA expression levels in subcutaneous fat and abdominal fat in Yan yellow cattle were significantly higher than that in Yanbian yellow cattle. Interestingly, ACADM gene shows greater fold changes in LD than in adipose tissues in Yan yellow cattle. Furthermore, the expressions of these three genes in lung, colon, kidney, liver and heart of Yanbian yellow cattle and Yan yellow cattle were also investigated. The results showed that the highest expression levels of PPARγ and FASN genes were detected in the lung in both breeds. The expression of ACADM gene in kidney and liver were higher than that in other organs in Yanbian yellow cattle, the comparison was not statistically significant in Yan yellow cattle

Comparative genome-wide methylation analysis of longissimus dorsi muscles between Japanese black (Wagyu) and Chinese Red Steppes cattle.

DNA methylation is an important epigenetic mechanism involved in expression of genes in many biological processes including muscle growth and development. Its effects on economically important traits are evinced from reported significant differences in meat quality traits between Japanese black (Wagyu) and Chinese Red Steppes cattle, thus presenting a unique model for analyzing the effects of DNA methylation on these traits. In the present study, we performed whole genome DNA methylation analysis in the two breeds by whole genome bisulfite sequencing (WGBS). Overall, 23150 differentially methylated regions (DMRs) were identified which were located in 8596 genes enriched in 9922 GO terms, of which 1046 GO terms were significantly enriched (p<0.05) including lipid translocation (GO: 0034204) and lipid transport (GO: 0015914). KEGG analysis showed that the DMR related genes were distributed among 276 pathways. Correlation analysis found that 331 DMRs were negatively correlated with the expression levels of differentially expressed genes (DEGs) with 21 DMRs located in promoter regions. Our results identified novel candidate DMRs and DEGs correlated with meat quality traits, which will be valuable for future genomic and epigenomic studies of muscle development and for marker assisted selection of meat quality traits

Agouti Signaling Protein and Its Receptors as Potential Molecular Markers for Intramuscular and Body Fat Deposition in Cattle

Transcriptome analyses of bovine muscle tissue differing in intramuscular fat (IMF) content identified agouti signaling protein (ASIP) as a promising candidate gene for fat deposition. The protein is secreted from adipocytes and may serve as a signaling molecule in cross-talk between adipocytes and muscle fibers or other cells. Known receptors for ASIP are the melanocortin receptors (e.g., MC4R) and attractin (ATRN). The present study was conducted to determine relationships between the expression of ASIP and its receptors in different bovine tissues with fat deposition. Adipose tissues, liver, and longissimus muscle tissue were collected from 246 F2-generation bulls (Charolais × Holstein cross) and gene expression was measured with RT-qPCR. During analysis of subcutaneous fat (SCF) of all bulls, 17 animals were identified with a transposon-derived transcript (Exon2C) inserted in the ASIP gene and dramatically increased ASIP mRNA levels. Significant correlations between normalized mRNA values of SCF and phenotypic traits related to fat deposition were found in bulls without Exon2C. Three retrospectively assigned groups [Exon2C, n = 17; high carcass fat (HCF), n = 20; low carcass fat (LCF), n = 20] were further analyzed to verify expression differences and elucidate molecular reasons. Expression of ASIP could be detected in isolated muscle fibers and adipocytes of Exon2C bulls in contrast to HCF and LCF bulls, indicating ectopic ASIP expression if the transposon is present. Among adipose tissues, highest ASIP mRNA levels were measured in SCF with significantly higher values in HCF compared to LCF bulls (1.6-fold, P &lt; 0.05). However, the protein abundance was below the detection limit in all bulls. Potential ASIP receptors were detected in most investigated tissues. The expression of MC4R was higher and of ATRN was lower in several tissues of LCF compared to HCF bulls, whereas MC1R was not differentially expressed. Bulls of the Exon2C group had lower ATRN mRNA values than HCF and LCF bulls in perirenal fat (PF), but higher (P &lt; 0.05) values in muscle. Receptors were also expressed in tissues where ASIP mRNA was not detected. Consequently, those tissues could be targets for ASIP if it circulates