Risk management and empirical study of the doctor-patient relationship: based on 1790 litigation cases of medical damage liability disputes in China.

BACKGROUND: Compensation for medical damage liability disputes (CMDLD) seriously hinders the healthy development of hospitals and undermines the harmony of the doctor-patient relationships (DPR). Risk management in the DPR has become an urgent issue of the day. The study aims to provide a comprehensive description of CMDLD in China and explore its influencing factors, and make corresponding recommendations for the management of risks in the DPR. METHODS: This study extracted data from the China Judgment Online - the official judicial search website with the most comprehensive coverage. Statistical analysis of 1,790 litigation cases of medical damage liability disputes (COMDLD) available from 2015 to 2021. RESULTS: COMDLD generally tended to increase with the year and was unevenly distributed by regions; the compensation rate was 52.46%, the median compensation was 134,900 yuan and the maximum was 2,234,666 yuan; the results of the single factor analysis showed that there were statistically significant differences between the compensation for different years, regions, treatment attributes, and trial procedures (P < 0.05); the correlation analysis showed that types of hospitals were significantly negatively associated with regions (R=-0.082, P < 0.05); trial procedures were significantly negatively correlated with years (R=-0.484, P < 0.001); compensat- ion was significantly positively correlated with years, regions, and treatment attributes (R = 0.098-0.294, P < 0.001) and negatively correlated with trial procedures (R=-0.090, P < 0.01); regression analysis showed that years, treatment attributes, and regions were the main factors affecting the CMDLD (P < 0.05). CONCLUSIONS: Years, regions, treatment attributes, and trial procedures affect the outcome of CMDLD. This paper further puts forward relevant suggestions and countermeasures for the governance of doctor-patient risks based on the empirical results. Including rational allocation of medical resources to narrow the differences between regions; promoting the expansion and sinking of high-quality resources to improve the level of medical services in hospitals at all levels; and developing a third-party negotiation mechanism for medical disputes to reduce the cost of medical litigation.

Molecular Characteristics of JAK2 and Its Effect on the Milk Fat and Casein Synthesis of Ovine Mammary Epithelial Cells.

In addition to its association with milk protein synthesis via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, JAK2 also affects milk fat synthesis. However, to date, there have been no reports on the effect of JAK2 on ovine mammary epithelial cells (OMECs), which directly determine milk yield and milk contents. In this study, the coding sequence (CDS) region of ovine JAK2 was cloned and identified and its tissue expression and localization in ovine mammary glands, as well as its effects on the viability, proliferation, and milk fat and casein levels of OMECs, were also investigated. The CDS region of ovine JAK2, 3399 bp in length, was cloned and its authenticity was validated by analyzing its sequence similarity with JAK2 sequences from other animal species using a phylogenetic tree. JAK2 was found to be expressed in six ovine tissues, with the highest expression being in the mammary gland. Over-expressed JAK2 and three groups of JAK2 interference sequences were successfully transfected into OMECs identified by immunofluorescence staining. When compared with the negative control (NC) group, the viability of OMECs was increased by 90.1% in the pcDNA3.1-JAK2 group. The over-expression of JAK2 also increased the number and ratio of EdU-labeled positive OMECs, as well as the expression levels of three cell proliferation marker genes. These findings show that JAK2 promotes the viability and proliferation of OMECs. Meanwhile, the triglyceride content in the over-expressed JAK2 group was 2.9-fold higher than the controls and the expression levels of four milk fat synthesis marker genes were also increased. These results indicate that JAK2 promotes milk fat synthesis. Over-expressed JAK2 significantly up-regulated the expression levels of casein alpha s2 (CSN1S2), casein beta (CSN2), and casein kappa (CSN3) but down-regulated casein alpha s1 (CSN1S1) expression. In contrast, small interfered JAK2 had the opposite effect to JAK2 over-expression on the viability, proliferation, and milk fat and milk protein synthesis of OMECs. In summary, these results demonstrate that JAK2 promotes the viability, proliferation, and milk fat synthesis of OMECs in addition to regulating casein expression in these cells. This study contributes to a better comprehension of the role of JAK2 in the lactation performance of sheep.

Screening and identification of lncRNAs in preadipocyte differentiation in sheep.

Studies of preadipocyte differentiation and fat deposition in sheep have mainly focused on functional genes, and with no emphasis placed on the role that long non-coding RNAs (lncRNAs) may have on the activity of those genes. Here, the expression profile of lncRNAs in ovine preadipocyte differentiation was investigated and the differentially expressed lncRNAs were screened on day 0 (D0), day 2(D2) and day 8(D8) of ovine preadipocyte differentiation, with their target genes being predicted. The competing endogenous RNA (ceRNA) regulatory network was constructed by GO and KEGG enrichment analysis for functional annotation, and some differentially expressed lncRNAs were randomly selected to verify the RNA-Seq results by RT-qPCR. In the study, a total of 2517 novel lncRNAs and 3943 known lncRNAs were identified from ovine preadipocytes at the three stages of differentiation, with the highest proportion being intergenic lncRNAs. A total of 3455 lncRNAs were expressed at all three stages of preadipocyte differentiation, while 214, 226 and 228 lncRNAs were uniquely expressed at day 0, day 2 and day 8, respectively. By comparing the expression of the lncRNAs between the three stages of differentiation stages, a total of 405, 272 and 359 differentially expressed lncRNAs were found in D0-vs-D2, D0-vs-D8, and D2-vs-D8, respectively. Functional analysis revealed that the differentially expressed lncRNAs were enriched in signaling pathways related to ovine preadipocyte differentiation, such as mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase protein kinase B (PI3K-Akt) pathway, and the transforming growth factor beta (TGF-ß) pathway. In summary, lncRNAs from preadipocytes at different stages of differentiation in sheep were identified and screened using RNA-Seq technology, and the regulatory mechanisms of lncRNAs in preadipocyte differentiation and lipid deposition were explored. This study provides a theoretical reference for revealing the roles of lncRNAs in ovine preadipocyte differentiation and also offers a theoretical basis for further understanding the regulatory mechanisms of ovine preadipocyte differentiation.

Molecular Genetic Characteristics of the Hoxc13 Gene and Association Analysis of Wool Traits.

Homobox C13 (Hoxc13) is an important transcription factor in hair follicle cycle development, and its deletion had been found in a variety of animals leading to abnormal hair growth and disruption of the hair follicle system. In this study, we used immunofluorescence, immunohistochemistry, real-time fluorescence quantitative PCR (RT-qPCR), and Kompetitive Allele-Specific PCR (KASP) genotyping to investigate molecular genetic characteristics of the Hoxc13 gene in Gansu alpine fine-wool sheep. The results revealed that Hoxc13 was significantly expressed during both the anagen and catagen phases (p < 0.05). It was found to be highly expressed predominantly in the dermal papillae and the inner and outer root sheaths, showing a distinct spatiotemporal expression pattern. Two single nucleotide polymorphisms (SNPs) in the exon 1 of Hoxc13, both the individual locus genotypes and the combined haplotypes were found to be correlated with wool length (p < 0.05). It was determined the mutations led to changes in mRNA expression, in which higher expression of this gene was related with longer wool length. In summary, this unique spatiotemporal expression pattern of the Hoxc13 gene may regulate the wool length of Gansu alpine fine-wool sheep, which can be used as a molecular genetic marker for wool traits and thus improve the breed.

Spatiotemporal Expression Characterization of KRTAP6 Family Genes and Its Effect on Wool Traits.

Keratin-related proteins (KAPs) are structural components of wool fibers and are thought to play a key role in regulating the physical and mechanical properties of fibers. Among all KAP genes (KRTAPs), KRTAP6 gene family (KRTAP6-1, KRTAP6-2, KRTAP6-3, KRTAP6-4, and KRTAP6-5) is a very important member with high polymorphism and notable association with some wool traits. In this study, we used real-time fluorescence quantitative PCR (RT-qPCR) and in situ hybridization to investigate spatiotemporal expression of KRTAP6s. The results revealed that KRTAP6 family genes were significantly expressed during anagen compared to other stages (p < 0.05). And it was found the five genes were expressed predominantly in the dermal papillae, inner and outer root sheaths, and showed a distinct spatiotemporal expression pattern. Also, it was found that KRTAP6-1 and KRTAP6-5 mRNA expression was negatively correlated with wool mean fiber diameter (MFD) and mean staple strength (MSS) (p < 0.05). In summary, the KRTAP6 family genes share a similar spatiotemporal expression pattern. And KRTAP6-1 and KRTAP6-5 may regulate the MFD and MSS of Gansu Alpine fine-wool sheep wool by changing the expression.

Ovine KRTAP36-2: A New Keratin-Associated Protein Gene Related to Variation in Wool Yield.

Keratin-associated proteins (KAPs) are structural components of wool fibres. High-glycine/tyrosine (HGT)-KAPs are a subset of the KAP family, and their abundance in fibres varies. In this study, we report the discovery of an ovine HGT-KAP gene to which we assigned the name KRTAP36-2. Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analyses revealed four variants of this gene in a screening population of 170 sheep from a variety of breeds. The DNA sequencing of the variants revealed four single-nucleotide polymorphisms (SNPs) and a dinucleotide deletion. Three of these SNPs were in the coding region, and one of these was non-synonymous and potentially led to the amino acid substitution p.Cys27Gly near the middle of the protein. The remaining SNP was located near the putative TATA box, and the di-nucleotide deletion was near the putative transcription initiation site. The effect of this variation in KRTAP36-2 was investigated in 274 Southdown × Merino lambs that were the progeny of five sires. Variation was only found to be associated with wool yield, that is, the proportion of the greasy fleece that remained as clean fleece upon scouring (expressed as a percentage). This may have some value in increasing wool production.

Functional differentiation of the ovine preadipocytes -insights from gene expression profiling.

The preadipocytes differentiation is a vital process of lipogenesis; exploring the molecular mechanisms of lipogenesis contributes to improve the meat quality and final commercial income. Lipogenesis has been widely reported in other livestock, but little is known about the gene expression profiles at different stages during preadipocytes differentiation in sheep. In this study, ovine preadipocytes were cultured in vitro and then induced to begin differentiation. Then, the gene expression profiles of preadipocytes collected on day 0 (D0), day 2 (D2), and day 8 (D8) of differentiation were analyzed by RNA-seq technology. According to the findings, 2254 differentially expressed genes (DEGs) were found in D2 vs D0; 1817 DEGs and 1902 DEGs were found in D8 vs D0 and D8 vs D2, respectively. The DEGs were found to be enriched in several biological processes, including focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, steroid biosynthesis, and MAPK signaling pathway, according to Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The regulatory network of the DEGs related to ovine preadipocytes differentiation was systematically constructed, which showed that hub genes might modulate ovine preadipocytes differentiation. In summary, preadipocyte differentiation is regulated by several key genes, including ACACB, CXCL6, SREBF1, INSIG1, APOE, GJA1, CDH11, SYNE1, PCSK1, S100A4, FN1, PLIN2, CXCL6, FN1, PTX3, and FABP3. This study provides a deeper knowledge of the roles of genes in sheep lipogenesis by revealing global gene expression profiles during preadipocyte differentiation.

Identification and Characterization of Circular RNAs (circRNAs) Using RNA-Seq in Two Breeds of Cashmere Goats.

Circular RNA (circRNA) is a type of non-coding RNA generated from back-splicing the reactions of linear RNA. It plays an important role in various cellular and biological processes. However, there are few studies about the regulatory effect of circRNAs on cashmere fiber traits in cashmere goats. In this study, the expression profiles of circRNAs in skin tissue were compared between Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats, with a significant difference in cashmere fiber yield, cashmere fiber diameter, and cashmere fiber color, using RNA-seq. A total of 11,613 circRNAs were expressed in the caprine skin tissue, and their type, chromosomal distribution, and length distribution were characterized. A total of 115 up-regulated circRNAs and 146 down-regulated circRNAs in LC goats were screened compared to ZB goats. The authenticity of 10 differentially expressed circRNAs was validated by detecting their expression levels and the head-to-tail splice junction using RT-PCR and DNA sequencing, respectively. The parent genes of differentially expressed circRNA were mainly enriched in some Gene Ontology (GO) terms and pathways related to cashmere fiber traits, such as the canonical Wnt signaling pathway, which is involved in the regulation of cell promotion, stem cell proliferation, Wnt signaling pathway regulation, epithelial morphogenesis, MAPK signaling pathway, and cell adhesion molecules pathway. Eight differentially expressed circRNAs were further selected to construct a circRNA-miRNA network, and some miRNAs that were previously reported as related to fiber traits were found in the network. This study provides a deep understanding of the roles of circRNAs in the regulation of cashmere fiber traits in cashmere goats and the involvement of differential splicing in phenotypic expression according to breed and region.

RNA-Seq Reveals the Roles of Long Non-Coding RNAs (lncRNAs) in Cashmere Fiber Production Performance of Cashmere Goats in China.

Long non-coding RNAs (lncRNAs) are a kind of non-coding RNA being >200 nucleotides in length, and they are found to participate in hair follicle growth and development and wool fiber traits regulation. However, there are limited studies reporting the role of lncRNAs in cashmere fiber production in cashmere goats. In this study, Liaoning cashmere (LC) goats (n = 6) and Ziwuling black (ZB) goats (n = 6) with remarkable divergences in cashmere yield, cashmere fiber diameter, and cashmere color were selected for the construction of expression profiles of lncRNAs in skin tissue using RNA sequencing (RNA-seq). According to our previous report about the expression profiles of mRNAs originated from the same skin tissue as those used in the study, the cis and trans target genes of differentially expressed lncRNAs between the two caprine breeds were screened, resulting in a lncRNA-mRNA network. A total of 129 lncRNAs were differentially expressed in caprine skin tissue samples between LC goats and ZB goats. The presence of 2 cis target genes and 48 trans target genes for the differentially expressed lncRNAs resulted in 2 lncRNA-cis target gene pairs and 93 lncRNA-trans target gene pairs. The target genes concentrated on signaling pathways that were related to fiber follicle development, cashmere fiber diameter, and cashmere fiber color, including PPAR signaling pathway, metabolic pathways, fatty acid metabolism, fatty acid biosynthesis, tyrosine metabolism, and melanogenesis. A lncRNA-mRNA network revealed 22 lncRNA-trans target gene pairs for seven differentially expressed lncRNAs selected, of which 13 trans target genes contributed to regulation of cashmere fiber diameter, while nine trans target genes were responsible for cashmere fiber color. This study brings a clear explanation about the influences of lncRNAs over cashmere fiber traits in cashmere goats.

The Identification and Characteristics of miRNAs Related to Cashmere Fiber Traits in Skin Tissue of Cashmere Goats.

microRNAs (miRNAs) are involved in the regulation of biological phenomena by down-regulating the expression of mRNAs. In this study, Liaoning cashmere (LC) goats (n = 6) and Ziwuling black (ZB) goats (n = 6) with different cashmere fiber production performances were selected. We supposed that miRNAs are responsible for the cashmere fiber trait differences. To test the hypothesis, the expression profiles of miRNAs from the skin tissue of the two caprine breeds were compared using small RNA sequencing (RNA-seq). A total of 1293 miRNAs were expressed in the caprine skin samples, including 399 known caprine miRNAs, 691 known species-conserved miRNAs, and 203 novel miRNAs. Compared with ZB goats, 112 up-regulated miRNAs, and 32 down-regulated miRNAs were found in LC goats. The target genes of the differentially expressed miRNAs were remarkably concentrated on some terms and pathways associated with cashmere fiber performance, including binding, cell, cellular protein modification process, and Wnt, Notch, and MAPK signaling pathways. The miRNA-mRNA interaction network found that 14 miRNAs selected may contribute to cashmere fiber traits regulation by targeting functional genes associated with hair follicle activities. The results have reinforced others leading to a solid foundation for further investigation of the influences of individual miRNAs on cashmere fiber traits in cashmere goats.

Integrated transcriptome analysis reveals roles of long non-coding RNAs (lncRNAs) in caprine skeletal muscle mass and meat quality.

Long non-coding RNAs (lncRNAs) play important roles in the growth and development of skeletal muscle. However, there is limited information on goats. In this study, expression profiles of lncRNAs in Longissimus dorsi muscle from Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats with divergent meat yield and meat quality were compared using RNA-sequencing. Based on our previous microRNA (miRNA) and mRNA profiles obtained from the same tissues, the target genes and binding miRNAs of differentially expressed lncRNAs were obtained. Subsequently, lncRNA-mRNA interaction networks and a ceRNA network of lncRNA-miRNA-mRNA were constructed. A total of 136 differentially expressed lncRNAs were identified between the two breeds. Fifteen cis target genes and 143 trans target genes were found for differentially expressed lncRNAs, and they were enriched in muscle contraction, muscle system process, muscle cell differentiation, and p53 signaling pathway. A total of 69 lncRNA-trans target gene pairs were constructed, with close relationship with muscle development, intramuscular fat deposition, and meat tenderness. A total of 16 lncRNA-miRNA-mRNA ceRNA pairs were identified, of which some reportedly associated with skeletal muscle development and fat deposition were found. The study will provide an improved understanding of the roles of lncRNAs in caprine meat yield and meat quality.

LncRNA and Protein Expression Profiles Reveal Heart Adaptation to High-Altitude Hypoxia in Tibetan Sheep.

The Tibetan sheep has an intricate mechanism of adaptation to low oxygen levels, which is influenced by both genetic and environmental factors. The heart plays a crucial role in the adaptation of Tibetan sheep to hypoxia. In the present study, we utilized transcriptomic and proteomic technologies to comprehensively analyze and identify the long non-coding RNAs (lncRNAs), genes, proteins, pathways, and gene ontology (GO) terms associated with hypoxic adaptation in Tibetan sheep at three different altitudes (2500 m, 3500 m, and 4500 m). By integrating the differentially expressed (DE) lncRNA target genes, differentially expressed proteins (DEPs), and differentially expressed genes (DEGs), we were able to identify and characterize the mechanisms underlying hypoxic adaptation in Tibetan sheep. Through this integration, we identified 41 shared genes/proteins, and functional enrichment analyses revealed their close association with lipid metabolism, glycolysis/gluconeogenesis, and angiogenesis. Additionally, significant enrichment was observed in important pathways such as the PPAR signaling pathway, glycolysis/gluconeogenesis, the oxoacid metabolic process, and angiogenesis. Furthermore, the co-expression network of lncRNAs and mRNAs demonstrated that lncRNAs (MSTRG.4748.1, ENSOART00020025894, and ENSOART00020036371) may play a pivotal role in the adaptation of Tibetan sheep to the hypoxic conditions of the plateau. In conclusion, this study expands the existing database of lncRNAs and proteins in Tibetan sheep, and these findings may serve as a reference for the prevention of altitude sickness in humans.

MicroRNA-200c Affects Milk Fat Synthesis by Targeting PANK3 in Ovine Mammary Epithelial Cells.

Milk fat is the foremost nutrient of milk and a vital indicator in evaluating milk quality. Accumulating evidence suggests that microRNAs (miRNAs) are involved in the synthesis of milk fat. The miR-200c is closely related to lipid metabolism, but little is known about its effect on the synthesis of milk fat in MECs of ewes. Herein, the effect of miR-200c on the proliferation of ovine mammary epithelial cells (MECs) and its target relationship with a predicted target gene were investigated. The regulatory effects of miR-200c on the expression of the target genes and the content of triglycerides in ovine MECs were further analyzed. The results revealed that the expression level of miR-200c was differentially expressed in both eight tissues selected during lactation and in mammary gland tissues at different physiological periods. Overexpression of miR-200c inhibited the viability and proliferation of ovine MECs, while inhibition of miR-200c increased cell viability and promoted the proliferation of ovine MECs. Target gene prediction results indicated that miR-200c would bind the 3'UTR region of pantothenate kinase 3 (PANK3). Overexpression of miR-200c reduced the luciferase activity of PANK3, while inhibition of miR-200c increased its luciferase activity. These findings illustrated that miR-200c could directly interact with the target site of the PANK3. It was further found that overexpression of miR-200c reduced the expression levels of PANK3 and, thus, accelerated the synthesis of triglycerides. In contrary, the inhibitor of miR-200c promoted the expression of PANK3 that, thus, inhibited the synthesis of triglycerides in ovine MECs. Together, these findings revealed that miR-200c promotes the triglycerides synthesis in ovine MECs via increasing the lipid synthesis related genes expression by targeting PANK3.

MicroRNA-381 Regulates Proliferation and Differentiation of Caprine Skeletal Muscle Satellite Cells by Targeting PTEN and JAG2.

In our previous study, microRNA (miR)-381 was found to be the most down-regulated miRNA in skeletal muscle of Liaoning cashmere goats with higher skeletal muscle mass, but the molecular mechanism involved remains unclear. In this study, primary caprine skeletal muscle satellite cells (SMSCs) were isolated and identified. We investigated the effect of miR-381 on the viability, proliferation and differentiation of caprine SMSCs, and the target relationships of miR-381 with jagged canonical Notch ligand 2 (JAG2) and phosphatase and tensin homolog (PTEN). Cells isolated were positive for SMSC-specific marker protein Pax7. This suggests that purified SMSCs were obtained. The expression level of miR-381 achieved a peak value on day 4 after SMSC differentiation, and miR-381 also significantly increased the expression levels of myogenic differentiation marker genes: myosin heavy chain (MyHC), myogenin (MyoG) and myocyte enhancer factor 2C (MEF2C) in differentiated SMSCs, the area of MyHC-positive myotubes and the myogenic index. These findings suggest that miR-381 promoted myogenic differentiation of caprine SMSCs. The CCK8 assay and EDU staining analysis showed that miR-381 mimic both inhibited the viability of SMSCs and decreased the percentage of EDU-labeled positive SMSCs. In contrast, miR-381 inhibitor had the opposite effect with miR-381 mimic. A dual luciferase reporter assay verified that miR-381 can target JAG2 and PTEN by binding to the 3'-untranslated regions (3'-UTR) of the genes. The transfection of miR-381 mimic into caprine SMSCs resulted in decreases in expression levels of JAG2 and PTEN, while miR-381 inhibitor increased the two target genes in expression. This is the first study to reveal the biological mechanisms by which miR-381 regulates caprine SMSC activities.

Characteristics and Expression of circ_003628 and Its Promoted Effect on Proliferation and Differentiation of Skeletal Muscle Satellite Cells in Goats.

In our previous a study, circ_003628 was one of the most highly expressed circular RNAs (circRNAs) in the Longissimus dorsi muscle of goats found by RNA-seq, suggesting that the circRNA may be important for caprine muscle growth and development. However, there have been no reports describing the molecular mechanisms by which circ_003628 regulates the activities of goat skeletal muscle satellite cells (SMSCs). In this study, reverse transcriptase-PCR (RT-PCR) and DNA sequencing were used to validate the authenticity of circ_003628, and its characteristics, expression profile and effect on goat SMSCs were also studied using real-time quantitative-PCR (RT-qPCR), EdU, CCK-8 and immunofluorescence assays. Circ_003628 is partially originated from 13 exons, 12 introns and 3'-untranslated regions (UTR) of caprine Myosin Heavy Chain 1 (MYH1), and 25 exons and 5' UTR of Myosin Heavy Chain 4 (MYH4), as well as intergenic sequences between the two genes. A total of 77.07% of circ_003628 were located in the nuclei of goat SMSCs, while 22.93% were expressed in the cytoplasm. The circRNAs were only expressed in triceps brachii, quadriceps femoris and longissimus dorsi muscle tissues in nine caprine tissues investigated, with the highest expression level in longissimus dorsi muscle. The expression level of circ_003628 gradually increased during differentiation periods of goat SMSCs and reached the maximum on day 6 after differentiation. The small interfering RNA of circ_003628 (named si-circ_003628) inhibited the viability and proliferation of goat SMSCs, and also decreased the expression of four cell proliferation marker genes: paired box 7 (Pax7), cyclin-dependent kinase 2 (CDK2), CDK4 and CyclinD1 in goat SMSCs. Transfection of si-circ_003628 significantly decreased the area of MyHC-labeled myotubes of goat SMSCs, as well as the expression levels of three differentiation marker genes: myosin heavy chain (MyHC), myogenin (MyoG), and myocyte enhancer factor 2C (MEF2C). These results suggest that circ_003628 promotes the viability, proliferation, and differentiation of goat SMSCs, and they also provide an improved understanding of the roles of circ_003628 in skeletal muscle growth and development in goats.

Transcriptional analysis of microRNAs related to unsaturated fatty acid synthesis by interfering bovine adipocyte ACSL1 gene.

Long-chain fatty acyl-CoA synthase 1 (ACSL1) plays a vital role in the synthesis and metabolism of fatty acids. The proportion of highly unsaturated fatty acids in beef not only affects the flavor and improves the meat's nutritional value. In this study, si-ACSL1 and NC-ACSL1 were transfected in bovine preadipocytes, respectively, collected cells were isolated on the fourth day of induction, and then RNA-Seq technology was used to screen miRNAs related to unsaturated fatty acid synthesis. A total of 1,075 miRNAs were characterized as differentially expressed miRNAs (DE-miRNAs), of which the expressions of 16 miRNAs were upregulated, and that of 12 were downregulated. Gene ontology analysis indicated that the target genes of DE-miRNAs were mainly involved in biological regulation and metabolic processes. Additionally, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis identified that the target genes of DE-miRNAs were mainly enriched in metabolic pathways, fatty acid metabolism, PI3K-Akt signaling pathway, glycerophospholipid metabolism, fatty acid elongation, and glucagon signaling pathway. Combined with the previous mRNA sequencing results, several key miRNA-mRNA targeting relationship pairs, i.e., novel-m0035-5p-ACSL1, novel-m0035-5p-ELOVL4, miR-9-X-ACSL1, bta-miR-677-ACSL1, miR-129-X-ELOVL4, and bta-miR-485-FADS2 were screened via the miRNA-mRNA interaction network. Thus, the results of this study provide a theoretical basis for further research on miRNA regulation of unsaturated fatty acid synthesis in bovine adipocytes.

Expression and Variations in EPAS1 Associated with Oxygen Metabolism in Sheep.

Endothelial PAS domain protein 1 gene (EPAS1) is a member of the HIF gene family. This gene encodes a transcription factor subunit that is involved in the induction of oxygen-regulated genes. Several studies have demonstrated that a mutation in EPAS1 could affect oxygen sensing, polycythemia, and hemoglobin level. However, whether EPAS1 mutation affects sheep oxygen metabolism is still unknown. Therefore, we explored the relationship between the variation of EPAS1 and oxygen metabolism in sheep. In this study, variations in ovine EPAS1 exon 15 were investigated in 332 Tibetan sheep and 339 Hu sheep by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. In addition, we studied the effect of these variations on blood gas in 176 Tibetan sheep and 231 Hu sheep. Finally, the mRNA expression of EPAS1 in six tissues of Hu sheep and Tibetan sheep living at different altitudes (2500 m, 3500 m, and 4500 m) was analyzed by real-time quantitative PCR (RT-qPCR). Four alleles (A, B, C, and D) were detected, and their distributions highly differed between Tibetan sheep and Hu sheep. In Tibetan sheep, B was the dominant allele, and C and D alleles were rare, whereas all four alleles were common in Hu sheep. Six single nucleotide polymorphisms (SNPs) were identified between the four alleles and one of them was non-synonymous (p.F606L). While studying the blood gas levels in Tibetan sheep and Hu sheep, one variant region was found to be associated with an elevated pO2 and sO2, which suggested that variations in EPAS1 are associated with oxygen metabolism in sheep. RT-qPCR results showed that EPAS1 was expressed in the six tissues of Hu sheep and Tibetan sheep at different altitudes. In addition, the expression of EPAS1 in four tissues (heart, liver, spleen, and longissimus dorsi muscle) of Hu sheep was lower than that in Tibetan sheep from three different altitudes, and the expression of EPAS1 was positively correlated with the altitude. These results indicate that the variations and expression of EPAS1 is closely related to oxygen metabolism.

Sequence Variation in Caprine KRTAP6-2 Affects Cashmere Fiber Diameter.

Keratin-associated proteins (KAPs) are a structural component of cashmere fibers and in part determine fiber attributes. The gene encoding the high-glycine/tyrosine KAP6-2 (called KRTAP6-2) has been described in sheep, but it has not been identified goats. In this study, a 252-bp open reading frame with similarity to ovine KRTAP6-2 was found on goat chromosome 1, with its upstream and downstream flanking sequences are closely related with ovine KRTAP6-2 but are clearly distinct from other ovine KRTAP6-n sequences. Polymerase chain reaction amplification followed by single strand conformation polymorphism analysis of this region revealed five distinct banding patterns representing five different sequences (A to E) in 230 Longdong cashmere goats. Eleven diallelic single nucleotide polymorphisms (SNPs), a three-nucleotide sequence variation, and a 12-bp insertion/deletion were found among these five sequences, with most SNPs being either outside the coding region or synonymous. The presence of variant D was found to be associated with decreased mean fiber diameter (MFD; present: 13.26 ± 0.07 µm; absent: 13.55 ± 0.04 µm; p < 0.001), suggesting that variation in KRTAP6-2 may affect fiber diameter and have value as a molecular marker for improving the cashmere fiber diameter trait.

Physiology and Proteomic Basis of Lung Adaptation to High-Altitude Hypoxia in Tibetan Sheep.

The Tibetan sheep is an indigenous animal of the Tibetan plateau, and after a long period of adaptation have adapted to high-altitude hypoxia. Many physiological changes occur in Tibetan sheep as they adapt to high-altitude hypoxia, especially in the lungs. To reveal the physiological changes and their molecular mechanisms in the lungs of Tibetan sheep during adaptation to high altitudes, we selected Tibetan sheep from three altitudes (2500 m, 3500 m, and 4500 m) and measured blood-gas indicators, observed lung structures, and compared lung proteome changes. The results showed that the Tibetan sheep increased their O2-carrying capacity by increasing the hemoglobin (Hb) concentration and Hematocrit (Hct) at an altitude of 3500 m. While at altitude of 4500 m, Tibetan sheep decreased their Hb concentration and Hct to avoid pulmonary hypertension and increased the efficiency of air-blood exchange and O2 transfer by increasing the surface area of gas exchange and half-saturation oxygen partial pressure. Besides these, some important proteins and pathways related to gas transport, oxidative stress, and angiogenesis identified by proteome sequencing further support these physiology findings, including HBB, PRDX2, GPX1, GSTA1, COL14A1, and LTBP4, etc. In conclusion, the lungs of Tibetan sheep are adapted to different altitudes by different strategies; these findings are valuable for understanding the basis of hypoxic adaptation in Tibetan sheep.

MicroRNA-199a-3p regulates proliferation and milk fat synthesis of ovine mammary epithelial cells by targeting VLDLR.

In our previous study, microRNA (miR)-199a-3p was found to be the most upregulated miRNA in mammary gland tissue during the non-lactation period compared with the peak-lactation period. However, there have been no reports describing the function of miR-199a-3p in ovine mammary epithelial cells (OMECs) and the biological mechanisms by which the miRNA affects cell proliferation and milk fat synthesis in sheep. In this study, the effect of miR-199a-3p on viability, proliferation, and milk fat synthesis of OMECs was investigated, and the target relationship of the miRNA with very low-density lipoprotein receptor (VLDLR) was also verified. Transfection with a miR-199a-3p mimic increased the viability of OMECs and the number of Edu-labeled positive OMECs. In contrast, a miR-199-3p inhibitor had the opposite effect with the miR-199a-3p mimic. The expression levels of three marker genes were also regulated by both the miR-199a-3p mimic and miR-199-3p inhibitor in OMECs. Together, these results suggest that miR-199a-3p promotes the viability and proliferation of OMECs. A dual luciferase assay confirmed that miR-199a-3p can target VLDLR by binding to the 3'-untranslated regions (3'UTR) of the gene. Further studies found a negative correlation in the expression of miR-199a-3p with VLDLR. The miR-199a-3p mimic decreased the content of triglycerides, as well as the expression levels of six milk fat synthesis marker genes in OMECs, namely, lipoprotein lipase gene (LPL), acetyl-CoA carboxylase alpha gene (ACACA), fatty acid binding protein 3 gene (FABP3), CD36, stearoyl-CoA desaturase gene (SCD), and fatty acid synthase gene (FASN). The inhibition of miR-199a-3p increased the level of triglycerides and the expression of LPL, ACACA, FABP3, SCD, and FASN in OMECs. These findings suggest that miR-199a-3p inhibited milk fat synthesis of OMECs. This is the first study to reveal the molecular mechanisms by which miR-199a-3p regulates the proliferation and milk fat synthesis of OMECs in sheep.