PTBP1 suppresses porcine epidemic diarrhea virus replication via inducing protein degradation and IFN production.

Porcine epidemic diarrhea virus (PEDV) causes severe morbidity and mortality among newborn piglets. It significantly threatens the porcine industry in China and around the globe. To accelerate the developmental pace of drugs or vaccines against PEDV, a deeper understanding of the interaction between viral proteins and host factors is crucial. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), is crucial for controlling RNA metabolism and biological processes. The present work focused on exploring the effect of PTBP1 on PEDV replication. PTBP1 was upregulated during PEDV infection. The PEDV nucleocapsid (N) protein was degraded through the autophagic and proteasomal degradation pathways. Moreover, PTBP1 recruits MARCH8 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) for N protein catalysis and degradation through selective autophagy. Furthermore, PTBP1 induces the host innate antiviral response via upregulating the expression of MyD88, which then regulates TNF receptor-associated factor 3/ TNF receptor-associated factor 6 expression and induces the phosphorylation of TBK1 and IFN regulatory factor 3. These processes activate the type Ⅰ IFN signaling pathway to antagonize PEDV replication. Collectively, this work illustrates a new mechanism related to PTBP1-induced viral restriction, where PTBP1 degrades the viral N protein and induces type Ⅰ IFN production to suppress PEDV replication.

Comprehensive cognition of yak (Bos grunniens) AIFM2 gene and its anti-ferroptosis role in bisphenol A-induced fetal fibroblast model.

Apoptosis-inducing factor mitochondrion-associated 2 (AIFM2) has been identified as a gene with anti-ferroptosis properties. To explore whether AIFM2 exerts anti-ferroptosis role in yaks (Bos grunniens), we cloned yak AIFM2 gene and analyzed its biological characteristics. The coding region of AIFM2 had 1122 bp and encoded 373 amino acids, which was conserved in mammals. Next, RT-qPCR results showed an extensive expression of AIMF2 in yak tissues. Furthermore, we isolated yak skin fibroblasts (YSFs) and established a bisphenol A (BPA)-induced ferroptosis model to further investigate the role of AIFM2. BPA elevated oxidative stress (reactive oxygen species, ROS) and lipid peroxidation (malondialdehyde, MDA and BODIPY), and reduced cell viability and antioxidant capacity (glutathione, GSH), with the severity depending on the dosage. Of note, a supplement of Ferrostatin-1 (Fer), an inhibitor of ferroptosis, restored the previously mentioned indicators. Subsequently, we constructed an AIFM2 overexpression vector and designed AIFM2 specific interfering siRNAs, which were transfected into YSFs. The results showed that overexpressing AIFM2 alleviated ferroptosis, characterizing by significant changes of cell viability, ROS, BODIPY, MDA and GSH. Meanwhile, interfering AIFM2 aggravated ferroptosis, demonstrating the critical anti-ferroptosis role of the yak AIFM2 gene. This study shed light on further exploring the molecular mechanism of AIFM2 in plateau adaptability.

Construction of Lentiviral Vectors Carrying Six Pluripotency Genes in Yak to Obtain Yak iPSC Cells.

Yak is an excellent germplasm resource on the Tibetan Plateau and is able to live in high-altitude areas with hypoxic, cold, and harsh environments. Studies on induced pluripotent stem cells (iPSCs) in large ruminants commonly involve a combination strategy involving six transcription factors, Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28 (OSKMNL). This strategy tends to utilize genes from the same species to optimize pluripotency maintenance. In this study, we cloned the six pluripotency genes (OSKMNL) from yak and constructed a multi-cistronic lentiviral vector carrying these genes. This vector efficiently delivered the genes into yak fibroblasts, aiming to promote the reprogramming process. We verified that the treated cells had several pluripotency characteristics, marking the first successful construction of a lentiviral system carrying yak pluripotency genes. This achievement lays the foundation for subsequent establishment of yak iPSCs and holds significant implications for yak-breed improvement and germplasm-resource conservation.

The Protective Effect of Quercetin against the Cytotoxicity Induced by Fumonisin B1 in Sertoli Cells.

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium species, is prevalent in crops and animal feed, posing significant health risks to livestock and humans. FB1 induces oxidative stress in Sertoli cells, destroys testicular structure, and affects spermatogenesis. However, methods to mitigate the reproductive toxicity of FB1 in testes remain unknown. Quercetin, a natural flavonoid antioxidant, may offer protective benefits. This study investigated the protective effects and mechanisms of quercetin against FB1-induced reproductive toxicity in TM4 cells (a Sertoli cell line). The results indicated that 40 µM quercetin improved cell viability, reduced apoptosis, and preserved cell functions. Quercetin also decreased reactive oxygen species (ROS) levels in TM4 cells exposed to FB1, enhanced the expression of antioxidant genes, and improved mitochondrial membrane potential. Compared with FB1 alone, the combination of quercetin and FB1 increased ATP levels, as well as pyruvate and lactic acid, the key glycolysis products. Furthermore, this combination elevated the mRNA and protein expression of glycolysis-related genes, including glucose-6-phosphate isomerase 1 (Gpi1), hexokinase 2 (Hk2), aldolase (Aldoa), pyruvate kinase, muscle (Pkm), lactate dehydrogenase A (Ldha) and phosphofructokinase, liver, B-type (Pfkl). Quercetin also boosted the activity of PKM and LDHA, two crucial glycolytic enzymes. In summary, quercetin mitigates FB1-induced toxicity in TM4 cells by reducing ROS levels and enhancing glycolysis. This study offers new insights into preventing and treating FB1-induced toxic damage to the male reproductive system and highlights the potential application of quercetin.

Nuclear ribonucleoprotein RALY targets virus nucleocapsid protein and induces autophagy to restrict porcine epidemic diarrhea virus replication.

Porcine epidemic diarrhea virus (PEDV) causes diarrhea and dehydration in pigs and leads to great economic losses in the commercial swine industry. However, the underlying molecular mechanisms of host response to viral infection remain unclear. In the present study, we investigated a novel mechanism by which RALY, a member of the heterogeneous nuclear ribonucleoprotein family, significantly promotes the degradation of the PEDV nucleocapsid (N) protein to inhibit viral replication. Furthermore, we identified an interaction between RALY and the E3 ubiquitin ligase MARCH8 (membrane-associated RING-CH 8), as well as the cargo receptor NDP52 (nuclear dot protein 52 kDa), suggesting that RALY could suppress PEDV replication by degrading the viral N protein through a RALY-MARCH8-NDP52-autophagosome pathway. Collectively, these results suggest a preventive role of RALY against PEDV infection via the autophagy pathway and open up the possibility of inducing RALY in vivo as an effective prophylactic and preventive treatment for PEDV infection.

hnRNP K Degrades Viral Nucleocapsid Protein and Induces Type I IFN Production to Inhibit Porcine Epidemic Diarrhea Virus Replication.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteric coronavirus currently spreading in several nations and inflicting substantial financial damages on the swine industry. The currently available coronavirus vaccines do not provide adequate protection against the newly emerging viral strains. It is essential to study the relationship between host antiviral factors and the virus and to investigate the mechanisms underlying host immune response against PEDV infection. This study shows that heterogeneous nuclear ribonucleoprotein K (hnRNP K), the host protein determined by the transcription factor KLF15, inhibits the replication of PEDV by degrading the nucleocapsid (N) protein of PEDV in accordance with selective autophagy. hnRNP K was found to be capable of recruiting the E3 ubiquitin ligase, MARCH8, aiming to ubiquitinate N protein. Then, it was found that the ubiquitinated N protein could be delivered into autolysosomes for degradation by the cargo receptor NDP52, thereby inhibiting PEDV proliferation. Moreover, based on the enhanced MyD88 expression, we found that hnRNP K activated the interferon 1 (IFN-1) signaling pathway. Overall, the data obtained revealed a new mechanism of hnRNP K-mediated virus restriction wherein hnRNP K suppressed PEDV replication by degradation of viral N protein using the autophagic degradation pathway and by induction of IFN-1 production based on upregulation of MyD88 expression. IMPORTANCE The spread of the highly virulent PEDV in many countries is still leading to several epidemic and endemic outbreaks. To elucidate effective antiviral mechanisms, it is important to study the relationship between host antiviral factors and the virus and to investigate the mechanisms underlying host immune response against PEDV infection. In the work, we detected hnRNP K as a new host restriction factor which can hinder PEDV replication through degrading the nucleocapsid protein based on E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. In addition, via the upregulation of MyD88 expression, hnRNP K could also activate the interferon (IFN) signaling pathway. This study describes a previously unknown antiviral function of hnRNP K and offers a new vision toward host antiviral factors that regulate innate immune response as well as a protein degradation pathway against PEDV infection.

Identification of microRNA transcriptome throughout the lifespan of yak (Bos grunniens) corpus luteum.

The corpus luteum (CL) is a temporary organ that plays a critical role for female fertility by maintaining the estrous cycle. MicroRNA (miRNA) is a class of non-coding RNAs involved in various biological processes. However, there exists limited knowledge of the role of miRNA in yak CL. In this study, we used high-throughput sequencing to study the transcriptome dynamics of miRNA in yak early (eCL), middle (mCL) and late-stage CL (lCL). A total of 6,730 miRNAs were identified, including 5,766 known and 964 novels miRNAs. Three miRNAs, including bta-miR-126-3p, bta-miR-143 and bta-miR-148a, exhibited the highest expressions in yak CLs of all the three stages. Most of the miRNAs were 20-24 nt in length and the peak was at 22 nt. Besides, most miRNAs with different lengths displayed significant uracil preference at the 5'-end. Furthermore, 1,067, 280 and 112 differentially expressed (DE) miRNAs were found in eCL vs. mCL, mCL vs. lCL, and eCL vs. lCL, respectively. Most of the DE miRNAs were down-regulated in the eCL vs. mCL and eCL vs. lCL groups, and up-regulated in the mCL vs. lCL group. A total of 18,904 target genes were identified, with 18,843 annotated. Pathway enrichment analysis of the DE miRNAs target genes illustrated that the most enriched cellular process in each group included pathways in cancer, PI3K-Akt pathway, endocytosis, and focal adhesion. A total of 20 putative target genes in 47 DE miRNAs were identified to be closely associated with the formation, function or regression of CL. Three DE miRNAs, including bta-miR-11972, novel-miR-619 and novel-miR-153, were proved to directly bind to the 3'-UTR of their predicated target mRNAs, including CDK4, HSD17B1 and MAP1LC3C, respectively. Both of these DE miRNAs and their target mRNAs exhibited dynamic expression profiles across the lifespan of yak CL. This study presents a general basis for understanding of the regulation of miRNA on yak CL and also provides a novel genetic resource for future analysis of the gene network during the estrous cycle in the yak.

Dynamic transcriptome analysis of Maiwa yak corpus luteum during the estrous cycle.

Maiwa yak is a special breed of animal living on the Qinghai-Tibet Plateau, which has great economic value, but its fertility rate is low. The corpus luteum (CL) is a temporary tissue that plays a crucial role in maintaining the physiological cycle. However, little is known about the transcriptome profile in Maiwa yak CL. In the present study, the transcriptome of Maiwa yak CL at early (EYCL), middle (MYCL) and late-stages (LYCL) was studied employing high-throughput sequencing. A total of 25,922 transcripts were identified, including 22,277 known as well as 3,645 novel ones. Furthermore, 690 and 212 differentially expressed (DE) mRNAs were detected in the EYCL vs. MYCL and MYCL vs. LYCL groups, respectively. KEGG pathway enrichment analysis of DEGs illustrated that the most enriched pathway was PI3K-Akt pathway. Furthermore, twenty-six DEGs were totally found to be associated with different biological processes of CL development. One of these genes, PGRMC1, displayed a dynamical expression trend during the lifespan of yak CL. The knockdown of PGRMC1 in luteinized yak granulosa cells resulted in defective steroidogenesis. In conclusion, this study analyzed the transcriptome profiles in yak CL of different stages, and provided a novel database for analyzing the gene network in yak CL.HIGHLIGHTSThe manuscript analyzed the transcriptome profiles in yak CL during the estrous cycle.Twenty-six DEGs were found to be associated with the development or function of CL.One of the DEGs, PGRMC1, was found to be responsible for steroidogenesis in luteinized yak granulosa cells.

RFRP-3 Influences Apoptosis and Steroidogenesis of Yak Cumulus Cells and Compromises Oocyte Meiotic Maturation and Subsequent Developmental Competence.

RF amide-related peptide 3 (RFRP-3), a mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), is identified to be a novel inhibitory endogenous neurohormonal peptide that regulates mammalian reproduction by binding with specific G protein-coupled receptors (GPRs) in various species. Herein, our objectives were to explore the biological functions of exogenous RFRP-3 on the apoptosis and steroidogenesis of yak cumulus cells (CCs) and the developmental potential of yak oocytes. The spatiotemporal expression pattern and localization of GnIH/RFRP-3 and its receptor GPR147 were determined in follicles and CCs. The effects of RFRP-3 on the proliferation and apoptosis of yak CCs were initially estimated by EdU assay and TUNEL staining. We confirmed that high-dose (10-6 mol/L) RFRP-3 suppressed viability and increased the apoptotic rates, implying that RFRP-3 could repress proliferation and induce apoptosis. Subsequently, the concentrations of E2 and P4 were significantly lower with 10-6 mol/L RFRP-3 treatment than that of the control counterparts, which indicated that the steroidogenesis of CCs was impaired after RFRP-3 treatment. Compared with the control group, 10-6 mol/L RFRP-3 treatment decreased the maturation of yak oocytes efficiently and subsequent developmental potential. We sought to explore the potential mechanism of RFRP-3-induced apoptosis and steroidogenesis, so we observed the levels of apoptotic regulatory factors and hormone synthesis-related factors in yak CCs after RFRP-3 treatment. Our results indicated that RFRP-3 dose-dependently elevated the expression of apoptosis markers (Caspase and Bax), whereas the expression levels of steroidogenesis-related factors (LHR, StAR, 3ß-HSD) were downregulated in a dose-dependent manner. However, all these effects were moderated by cotreatment with inhibitory RF9 of GPR147. These results demonstrated that RFRP-3 adjusted the expression of apoptotic and steroidogenic regulatory factors to induce apoptosis of CCs, probably through binding with its receptor GPR147, as well as compromised oocyte maturation and developmental potential. This research revealed the expression profiles of GnIH/RFRP-3 and GPR147 in yak CCs and supported a conserved inhibitory action on oocyte developmental competence.

ß-Nicotinamide Mononucleotide Alleviates Hydrogen Peroxide-Induced Cell Cycle Arrest and Death in Ovarian Granulosa Cells.

Maintaining normal functions of ovarian granulosa cells (GCs) is essential for oocyte development and maturation. The dysfunction of GCs impairs nutrition supply and estrogen secretion by follicles, thus negatively affecting the breeding capacity of farm animals. Impaired GCs is generally associated with declines in Nicotinamide adenine dinucleotide (NAD+) levels, which triggers un-controlled oxidative stress, and the oxidative stress, thus, attack the subcellular structures and cause cell damage. ß-nicotinamide mononucleotide (NMN), a NAD+ precursor, has demonstrated well-known antioxidant properties in several studies. In this study, using two types of ovarian GCs (mouse GCs (mGCs) and human granulosa cell line (KGN)) as cell models, we aimed to investigate the potential effects of NMN on gene expression patterns and antioxidant capacity of both mGCs and KGN that were exposed to hydrogen peroxide (H2O2). As shown in results of the study, mGCs that were exposed to H2O2 significantly altered the gene expression patterns, with 428 differentially expressed genes (DEGs) when compared with those of the control group. Furthermore, adding NMN to H2O2-cultured mGCs displayed 621 DEGs. The functional enrichment analysis revealed that DEGs were mainly enriched in key pathways like cell cycle, senescence, and cell death. Using RT-qPCR, CCK8, and ß-galactosidase staining, we found that H2O2 exposure on mGCs obviously reduced cell activity/mRNA expressions of antioxidant genes, inhibited cell proliferation, and induced cellular senescence. Notably, NMN supplementation partially prevented these H2O2-induced abnormalities. Moreover, these similar beneficial effects of NMN on antioxidant capacity were confirmed in the KGN cell models that were exposed to H2O2. Taken together, the present results demonstrate that NMN supplementation protects against H2O2-induced impairments in gene expression pattern, cell cycle arrest, and cell death in ovarian GCs through boosting NAD+ levels and provide potential strategies to ameliorate uncontrolled oxidative stress in ovarian GCs.

Transcriptomic study of yak mammary gland tissue during lactation.

Yak milk, a high-quality milk, is one of the best raw materials for dairy products and economically important to pastoral herdsmen. To make a further understanding of the molecular differences in mammary tissues of the yaks with different milk production during lactation, in this study, we took the use of RNA-seq to perform high-throughput sequencing and analysis of the mammary gland transcriptomes of both high-yielding yak and low-yielding yaks during lactation. By the comparison and analysis of the transcriptome data for the mammary gland tissue of high-yielding yak and low-yield yak, 144 differential genes were screened out, of which 49 were upregulated and 95 were downregulated. Further functional analysis indicated that these differential genes involved in multiple classes based on Gene Ontology (GO) and multiple Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The GO analysis showed that the functions of the differential genes are closely related to the carbohydrate metabolism and other biological processes. KEGG pathway analysis revealed that these genes are mostly enriched in the pathway of antigen processing and presentation, phagosome pathway and type I diabetes pathway and enriched followed by extracellular matrix receptor interaction pathway. Moreover, several other pathways related to amino acid metabolism also showed significant enrichment. Here, the mammary gland transcriptomes of high-yielding yak and low-yielding yaks during lactation have for the first time been compared, and the related differential genes have been screened out and analyzed. Our study paves a way for the further elucidation of the basic molecular mechanism of yak mammary gland tissue, and at the same time provides new ideas for improving the milk production of yaks.

Oocyte-Specific Knockout of Histone Lysine Demethylase KDM2a Compromises Fertility by Blocking the Development of Follicles and Oocytes.

The methylation status of histones plays a crucial role in many cellular processes, including follicular and oocyte development. Lysine-specific demethylase 2a (KDM2a) has been reported to be closely associated with gametogenesis and reproductive performance, but the specific function and regulatory mechanism have been poorly characterized in vivo. We found KDM2a to be highly expressed in growing follicles and oocytes of mice in this study. To elucidate the physiological role of Kdm2a, the zona pellucida 3-Cre (Zp3-Cre)/LoxP system was used to generate an oocyte Kdm2a conditional knockout (Zp3-Cre; Kdm2aflox/flox, termed Kdm2a cKO) model. Our results showed that the number of pups was reduced by approximately 50% in adult Kdm2a cKO female mice mating with wildtype males than that of the control (Kdm2aflox/flox) group. To analyze the potential causes, the ovaries of Kdm2a cKO mice were subjected to histological examination, and results indicated an obvious difference in follicular development between Kdm2a cKO and control female mice and partial arrest at the primary antral follicle stage. The GVBD and matured rates of oocytes were also compromised after conditional knockout Kdm2a, and the morphological abnormal oocytes increased. Furthermore, the level of 17ß-estradiol of Kdm2a cKO mice was only 60% of that in the counterparts, and hormone sensitivity decreased as the total number of ovulated and matured oocytes decreased after superovulation. After deletion of Kdm2a, the patterns of H3K36me2/3 in GVBD-stage oocytes were remarkedly changed. Transcriptome sequencing showed that the mRNA expression profiles in Kdm2a cKO oocytes were significantly different, and numerous differentially expressed genes were involved in pathways regulating follicular and oocyte development. Taken together, these results indicated that the oocyte-specific knockout Kdm2a gene led to female subfertility, suggesting the crucial role of Kdm2a in epigenetic modification and follicular and oocyte development.

Transcriptome profile and unique genetic evolution of positively selected genes in yak lungs.

The yak (Bos grunniens), which is a unique bovine breed that is distributed mainly in the Qinghai-Tibetan Plateau, is considered a good model for studying plateau adaptability in mammals. The lungs are important functional organs that enable animals to adapt to their external environment. However, the genetic mechanism underlying the adaptability of yak lungs to harsh plateau environments remains unknown. To explore the unique evolutionary process and genetic mechanism of yak adaptation to plateau environments, we performed transcriptome sequencing of yak and cattle (Bos taurus) lungs using RNA-Seq technology and a subsequent comparison analysis to identify the positively selected genes in the yak. After deep sequencing, a normal transcriptome profile of yak lung that containing a total of 16,815 expressed genes was obtained, and the characteristics of yak lungs transcriptome was described by functional analysis. Furthermore, Ka/Ks comparison statistics result showed that 39 strong positively selected genes are identified from yak lungs. Further GO and KEGG analysis was conducted for the functional annotation of these genes. The results of this study provide valuable data for further explorations of the unique evolutionary process of high-altitude hypoxia adaptation in yaks in the Tibetan Plateau and the genetic mechanism at the molecular level.

Analysis of gene expression profiles reveals the regulatory network of cold-inducible RNA-binding protein mediating the growth of BHK-21 cells.

Cold-inducible RNA-binding protein (Cirp), the first cold-shock protein identified in mammals, is a sensor protein whose expression increases in response to stress. Recent reports have shown that Cirp is involved in cell proliferation, development, circadian modulation under physiological conditions, and tumor formation and progression. However, the molecular mechanisms underlying the activities of Cirp in the mammalian kidney cells remain unclear. In this study, we constructed BHK-21cells overexpressing Cirp (Cirp + BHK-21) knockdown BHK-21 cells (Cirp - BHK-21) to investigate the function of Cirp in cell proliferation. We analyzed the gene expression of Cirp - BHK-21 cells using genome-wide expression microarrays to explore the molecular mechanism of Cirp action. We found that (1) Cirp overexpression significantly enhanced cell proliferation, whereas Cirp knockdown dramatically reduced cell proliferation, suggesting that Cirp is a positive regulator of BHK-21 cell proliferation. (2) Differentially expressed genes in Cirp - BHK-21 and control cells were shown to be involved in many biological processes. (3) Pathway analysis showed that five enriched pathways, namely, Focal adhesion, Mapk, Wnt, Apoptosis, and Cancer-related signaling pathways, were identified as central pathway networks regulated by Cirp. These results can provide new insights into the molecular mechanisms of Cirp function.

New serotypes of porcine teschovirus identified in Shanghai, China.

Teschoviruses are widely endemic and commonly found in pig fecal samples. In this study, we collected fecal specimens from various pig herds and genotyped them based on the VP1 gene. Of 322 samples, 276 were positive, giving a PTV infectivity rate of 85.7 %. PTV4 was the most common serotype found in Shanghai, followed by PTV8 and PTV10. Interestingly, Some Shanghai strains belonging to a new PTV serotype were also isolated. In phylogenetic analysis, PTV SH8 did not correspond to any known serotype. PTV4 and PTV6 showed similar levels of sequence identity to PTV SH8. These data suggest that PTV SH8 is a new serotype, distinct from the new serotype PTV wild boar/WB2C-TV/2011/HUN, which clusters with PTV SH2, SH10, and SH25.

Yak response to high-altitude hypoxic stress by altering mRNA expression and DNA methylation of hypoxia-inducible factors.

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, which play crucial roles in tumor angiogenesis and mammalian development, and regulate the transcription of genes involved in oxygen homeostasis in response to hypoxia. However, information on HIF-1α and HIF-2α in yak (Bos grunniens) is scarce. The complete coding region of yak HIF-2α was cloned, its mRNA expression in several tissues were determined, and the expression levels were compared with those of closely related low-altitude cattle (Bos taurus), and the methylation status of promoter regions were analyzed to better understand the roles of HIF-1α and HIF-2α in domesticated yak. The yak HIF-2α cDNA was cloned and sequenced in the present work reveals the evolutionary conservation through multiple sequence alignment, although 15 bases changed, resulting in 8 amino acid substitutions in the translated proteins in cattle. The tissue-specific expression results showed that HIF-1α is ubiquitously expressed, whereas HIF-2α expression is limited to endothelial tissues (kidney, heart, lung, spleen, and liver) and blood in yak. Both HIF-1α and HIF-2α expressions were higher in yak tissues than in cattle. The HIF-1α expression level is much higher in yak than cattle in these organs, except for the lung (P < 0.05), but the HIF-2α gene is significantly different in the heart, spleen, and kidney (P < 0.05). Furthermore, the methylation levels in the 5' flanking regulatory regions of HIF-1α and HIF-2α in yak kidney were significantly decreased than cattle counterparts (P < 0.05). Identifying these genes and the comparison of different expressions facilitates the understanding of the biological high-altitude hypoxic stress response mechanism and may assist current medical research to understand hypoxia-related diseases.

Sodium butyrate improves the cloned yak embryo viability and corrects gene expression patterns.

Interspecies somatic cell nuclear transfer (iSCNT), a powerful tool in basic scientific research, has been used widely to increase and preserve the population of endangered species. Yak (Bos grunniens) is one of these species. Development to term of interspecies cloned yak embryos has not been achieved, possibly due to abnormal epigenetic reprogramming. Previous studies have demonstrated that treatment of intraspecies cloned embryos with (NaBu) significantly improves nuclear-cytoplasmic reprogramming and viability in vitro. Therefore, in this study, we evaluated the effect of optimal NaBu concentration and exposure time on preimplantation development of yak iSCNT embryos and on the expression patterns of developmentally important genes. The results showed that 8-cell rate, blastocyst formation rate and total cell number increased significantly compared with their untreated counterparts when yak iSCNT embryos were treated with 5 nM NaBu for 12 h after activation, but that the 2-cell stage embryo rate was not significantly different. The treatment of NaBu also increased significantly the expression levels of Oct-4 and decreased the expression levels of HDAC-2, Dnmt-1 and IGF-1; the expression patterns of these genes were more similar to that of their bovine-yak in vitro fertilization (BY-IVF) counterparts. The results described above indicated that NaBu treatment improved developmental competence in vitro and 'corrected' the gene expression patterns of yak iSCNT embryos.

Effect of DNA Extraction Methods on the Apparent Structure of Yak Rumen Microbial Communities as Revealed by 16S rDNA Sequencing.

To more efficiently identify the microbial community of the yak rumen, the standardization of DNA extraction is key to ensure fidelity while studying environmental microbial communities. In this study, we systematically compared the efficiency of several extraction methods based on DNA yield, purity, and 16S rDNA sequencing to determine the optimal DNA extraction methods whose DNA products reflect complete bacterial communities. The results indicate that method 6 (hexadecyltrimethylammomium bromide-lysozyme-physical lysis by bead beating) is recommended for the DNA isolation of the rumen microbial community due to its high yield, operational taxonomic unit, bacterial diversity, and excellent cell-breaking capability. The results also indicate that the bead-beating step is necessary to effectively break down the cell walls of all of the microbes, especially Gram-positive bacteria. Another aim of this study was to preliminarily analyze the bacterial community via 16S rDNA sequencing. The microbial community spanned approximately 21 phyla, 35 classes, 75 families, and 112 genera. A comparative analysis showed some variations in the microbial community between yaks and cattle that may be attributed to diet and environmental differences. Interestingly, numerous uncultured or unclassified bacteria were found in yak rumen, suggesting that further research is required to determine the specific functional and ecological roles of these bacteria in yak rumen. In summary, the investigation of the optimal DNA extraction methods and the preliminary evaluation of the bacterial community composition of yak rumen support further identification of the specificity of the rumen microbial community in yak and the discovery of distinct gene resources.

Molecular Characterization and Expression Analysis of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Protein-1 Genes in Qinghai-Tibet Plateau Bos grunniens and Lowland Bos taurus.

Insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-1 (IGFBP-1) play a pivotal role in regulating cellular hypoxic response. In this study, we cloned and characterized the genes encoding IGF-1 and IGFBP-1 to improve the current knowledge on their roles in highland Bos grunniens (Yak). We also compared their expression levels in the liver and kidney tissues between yaks and lowland cattle. We obtained full-length 465 bp IGF-1 and 792 bp IGFBP-1, encoding 154 amino acids (AA) IGF-1, and 263 AA IGFBP-1 protein, respectively using reverse transcriptase-polyerase chain reaction (RT-PCR) technology. Analysis of their corresponding amino acid sequences showed a high identity between B. grunniens and lowland mammals. Moreover, the two genes were proved to be widely distributed in the examined tissues through expression pattern analysis. Real-time PCR results revealed that IGF-1 expression was higher in the liver and kidney tissues in B. grunniens than in Bos taurus (p<0.05). The IGFBP-1 gene was expressed at a higher level in the liver (p<0.05) of B. taurus than B. grunniens, but it has a similar expression level in the kidneys of the two species. These results indicated that upregulated IGF-1 and downregulated IGFBP-1 are associated with hypoxia adaptive response in B. grunniens.