n-3 Polyunsaturated fatty acids (n-3 PUFA) are important for human health. Alternative resources of n-3 PUAFs created by transgenic domestic animals would be an economic approach. In this study, we generated a mfat-1 transgenic cattle expressed a Caenorhabditis elegans gene, mfat-1, encoding an n-3 fatty acid desaturase. Fatty acids analysis of tissue and milk showed that all of the examined n-3 PUAFs were greatly increased and simultaneously the n-6 PUAFs decreased in the transgenic cow. A significantly reduction of n-6/n-3 ratios (P<0.05) in both tissue and milk were observed.

Defensins as one of the most abundant classes of antimicrobial peptides are an essential part of the innate immunity that has evolved in most living organisms from lower organisms to humans. To identify specific defensins as interesting antifungal leads, in this study, we constructed a more rigorous benchmark dataset and the iDPF-PseRAAAC server was developed to predict the defensin family and subfamily. Using reduced dipeptide compositions were used, the overall accuracy of proposed method increased to 95.10% for the defensin family, and 98.39% for the vertebrate subfamily, which is higher than the accuracy from other methods. The jackknife test shows that more than 4% improvement was obtained comparing with the previous method. A free online server was further established for the convenience of most experimental scientists at http://wlxy.imu.edu.cn/college/biostation/fuwu/iDPF-PseRAAAC/index.asp. A friendly guide is provided to describe how to use the web server. We anticipate that iDPF-PseRAAAC may become a useful high-throughput tool for both basic research and drug design.

The success of cloned animal “Dolly Sheep” demonstrated the somatic cell nuclear transfer (SCNT) technique holds huge potentials for mammalian asexual reproduction. However, the extremely poor development of SCNT embryos indicates their molecular mechanism remain largely unexplored. Deciphering the spatiotemporal patterns of gene expression in SCNT embryos is a crucial step toward understanding the mechanisms associated with nuclear reprogramming. In this study, a valuable transcriptome recourse of SCNT embryos was firstly established, which derived from different inter-/intra donor cells. The gene co-expression analysis identified 26 cell-specific modules, and a series of regulatory pathways related to reprogramming barriers were further enriched. Compared to the intra-SCNT embryos, the inter-SCNT embryos underwent only complete partially reprogramming. As master genome trigger genes, the transcripts related to TFIID subunit, RNA polymerase and mediators were incomplete activated in inter-SCNT embryos. The inter-SCNT embryos only wasted the stored maternal mRNA of master regulators, but failed to activate their self-sustained pathway of RNA polymerases. The KDM family of epigenetic regulator also seriously delayed in inter-SCNT embryo reprogramming process. Our study provided new insight into understanding of the mechanisms of nuclear reprogramming.

Cloned animals generated by somatic cell nuclear transfer (SCNT) have been reported for many years; however, SCNT is extremely inefficient, and zygotic genome activation (ZGA) is required for SCNT‐mediated somatic cell reprogramming. To identify candidate factors that facilitate ZGA in SCNT‐mediated reprogramming, we performed siRNA‐repressor and mRNA‐inducer screenings, which reveal Dux, Dppa2, and Dppa4 as key factors enhancing ZGA in SCNT. We show that direct injection of ZGA inducers has no significant effect on SCNT blastocyst formation; however, following the establishment of an inducible Dux transgenic mouse model, we demonstrate that transient overexpression of Dux not only improves SCNT efficiency but also increases that of chemically induced pluripotent stem cell reprogramming. Moreover, transcriptome profiling reveals that Dux‐treated SCNT embryos are similar to fertilized embryos. Furthermore, transient overexpression of Dux combined with inactivation of DNA methyltransferases (Dnmts) further promotes the full embryonic development of SCNT‐derived animals. These findings enhance our understanding of ZGA‐regulator function in somatic reprogramming.

Myostatin (MSTN) is mostly expressed in skeletal muscle and plays crucial roles in the negative regulation of muscle mass development. The methylation and demethylation of myogenesis-specific genes are major regulatory factors in muscle satellite cell differentiation. The present study was designed to investigate the mechanism of myogenic differentiation regulated by MSTN mutation (MT) and the methylation/demethylation state of downstream genes. The results showed that, in the MSTN-/+ satellite cells, a higher myotube fusion index and a larger myotube length were observed compared to the wild type controls; the genes associated with myogenesis were all up-regulated compared to the WT controls. The methylation of the promoters and gene bodies of PAX3, PAX7, MyoD, and MyoG were all down-regulated, while the expression of the key demethylase TET1 was significantly promoted. ChIP-qPCR was used to demonstrate that the SMAD2/SMAD3 complex combined with the promoter of TET1 to inhibit the activity of TET1 promoter, indicating that MSTN may regulate TET1 via SMAD2/SMAD3. The overexpression of TET1 in wild type cells promoted myogenic differentiation, increased the myotube index, and reduced the methylation of the associated genes. On the contrary, the knockdown of TET1 in the MSTN mutant cells resulted in the opposite phenomena as in the overexpressed cells. In conclusion, the myostatin mutant showed an increased transcriptional activity of TET1, inducing higher levels of demethylation and improving the transcriptional activity levels of myogenic differentiation-associated genes. The binding of SMAD2/SMAD3 directly to the TET1 promoter region indicated that the MSTN mutant demethylated the myogenesis-specific genes by up-regulating TET1, which is directly controlled by SMAD2/SMAD3.

N6‐methyladenosine (m6A) methylation is the most common and abundant modification on mammalian messenger RNA (mRNA) and regulates the pluripotency of embryonic stem cells (ESCs). Research has shown that melatonin plays a fundamental role in DNA and histone modifications. However, the effect of melatonin on RNA modification is unknown. Here, for the first time, we investigated the effect of melatonin on m6A modifications in long‐term‐cultured ESCs. Pluripotency studies indicated that 10 μmol/L melatonin sufficiently maintained ESCs with stemness features over 45 passages (more than 90 days). Notably, treatment of ESCs with melatonin led to a significant decrease in the nuclear presence of m6A methyltransferase complex and decreased global m6A modification. Depletion of melatonin receptor 1 (MT1) by CRISPR/Cas9 significantly reduced the effects of melatonin on ESC pluripotency and m6A modification. Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) revealed that melatonin promotes stabilization of core pluripotency factors, such as Nanog, Sox2, Klf4, and c‐Myc, by preventing m6A‐dependent mRNA decay. Using cell signaling pathway profiling systems, melatonin was shown to regulate m6A modification predominantly through the MT1‐JAK2/STAT3‐Zfp217 signal axis. This study reveals a new dimension regarding melatonin regulation of gene expression at the RNA level.

During the process of embryonic development in mammals, epigenetic modifications must be erased and reconstructed. In particular, the trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcriptional repression and contributes to the maintenance of the pluripotent embryos. In this study, we determined that the global levels of the H3K27me3 marker were elevated in MII oocyte chromatin and decrease to minimal levels at the 8-cell and morula stages. When the blastocyst hatched, H3K27me3 was re-established in the inner cell mass. We also determined that H3K27me3-specific demethylases, UTX and JMJD3, were observed at high transcript and protein levels in mouse preimplantation embryos. In the activated oocytes, when the H3K27me3 disappeared at the 8-cell stage, the UTX (but not JMJD3) protein levels were undetectable. Using RNA interference, we suppressed UTX and JMJD3 gene expression in the embryos and determined that the functions of UTX and JMJD3 were complementary. When JMJD3 levels were decreased by RNA interference, the embryo development rate and quality were improved, but the knockdown of UTX produced the opposite results. Understanding the epigenetic mechanisms controlling preimplantation development is critical to comprehending the basis of embryonic development and to devise methods and approaches to treat infertility.

Exosomal micro (mi)RNAs have been suggested to have important roles in abdominal obesity, and to be associated with metabolic alterations via posttranscriptional regulation of target genes. However, exosomal miRNA profiles in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) have rarely been investigated. In the present study, microarray data were obtained from the Gene Expression Omnibus database with the following accession numbers: GSE68885 (exosomal miRNAs in SAT obtained from seven patients with obesity and five lean patients), GSE50574 (exosomal miRNAs in VAT obtained from seven patients with obesity and five lean patients) and GSE29718 [mRNAs in SAT (obtained from seven patients with obesity and eight lean patients) and VAT (obtained from three patients with obesity and two lean patients)]. Differentially expressed (DE)-miRNAs and differentially expressed genes (DEGs) were identified using the Linear Models for Microarray Data method, and mRNA targets of DE-miRNAs were predicted using the miRWalk2.0 database. Potential functions of DE-miRNA target genes were determined using the Database for Annotation, Visualization and Integrated Discovery. As a result, 10 exosomal DE-miRNAs were identified in SAT between patients with obesity and lean patients, while 58 DE-miRNAs were identified in VAT between patients with obesity and lean patients. miRNA (miR)-4517 was revealed to be a downregulated exosomal miRNA between SAT and VAT, while the other DE-miRNAs were SAT-(e.g. hsa-miR-3156-5p and hsa-miR-4460) or VAT-(e.g. hsa-miR-582-5p, hsa-miR-566 and miR-548) specific. Following overlapping with the target genes of DE-miRNAs, only one DEG [cluster of differentiation 86 (CD86)] was identified in SAT samples, whereas 25 DEGs (e.g. fibroblast growth factor 2 (FGF2), FOS like 2, AP-1 transcription factor subunit (FOSL2); and adenosine monophosphate deaminase 3 (AMPD3)] were identified in VAT samples. CD86 was revealed to be regulated by hsa-miR-3156-5p; whereas FGF2, FOSL2 and AMPD3 were revealed to be regulated by hsa-miR-582-5p, hsa-miR-566 and miR-548, respectively. Functional enrichment analysis demonstrated that these target genes may be associated with inflammation. In conclusion, exosomal miRNAs may represent underlying therapeutic targets for the treatment of abdominal obesity and metabolic disorders via regulation of inflammatory genes.