Identification of a recombinant equine coronavirus in donkey, China.

Equine coronavirus (ECoV) was first identified in the USA and has been previously described in several countries. In order to test the presence of ECoV in China, we collected 51 small intestinal samples from donkey foals with diarrhoea from a donkey farm in Shandong Province, China between August 2020 and April 2021. Two samples tested positive for ECoV and full-length genome sequences were successfully obtained using next-generation sequencing, one of which was further confirmed by Sanger sequencing. The two strains shared 100% sequence identity at the scale of whole genome. Bioinformatics analyses further showed that the two Chinese strains represent a novel genetic variant of ECoV and shared the highest sequence identity of 97.05% with the first identified ECoV strain - NC99. In addition, it may be a recombinant, with the recombination region around the NS2 gene. To our knowledge, this is the first documented report of ECoV in China, highlighting its risk to horse/donkey breeding. In addition, its potential risk to public health also warrants further investigation.

Genomes reveal selective sweeps in kiang and donkey for high-altitude adaptation.

Over the last several hundred years, donkeys have adapted to high-altitude conditions on the Tibetan Plateau. Interestingly, the kiang, a closely related equid species, also inhabits this region. Previous reports have demonstrated the importance of specific genes and adaptive introgression in divergent lineages for adaptation to hypoxic conditions on the Tibetan Plateau. Here, we assessed whether donkeys and kiangs adapted to the Tibetan Plateau via the same or different biological pathways and whether adaptive introgression has occurred. We assembled a de novo genome from a kiang individual and analyzed the genomes of five kiangs and 93 donkeys (including 24 from the Tibetan Plateau). Our analyses suggested the existence of a strong hard selective sweep at the EPAS1 locus in kiangs. In Tibetan donkeys, however, another gene, i.e., EGLN1, was likely involved in their adaptation to high altitude. In addition, admixture analysis found no evidence for interspecific gene flow between kiangs and Tibetan donkeys. Our findings indicate that despite the short evolutionary time scale since the arrival of donkeys on the Tibetan Plateau, as well as the existence of a closely related species already adapted to hypoxia, Tibetan donkeys did not acquire adaptation via admixture but instead evolved adaptations via a different biological pathway.

MicroRNA-137 inhibits autophagy and chemosensitizes pancreatic cancer cells by targeting ATG5.

PURPOSE: Autophagy play an important role in tumor chemotherapy resistance. It has been reported that miR-137 expression was reducedand involved in the regulation of sensitivity of PC cells to chemotherapy. However, little is known about the underlying molecular mechanisms. In this study, we hypothesized that miR-137 might sensitize PC cells to chemotherapy thought regulating cell autophagy. METHODS: Cell survival was determined with MTT assay. Apoptotic cells were assessed with flow cytometric analysis. Fluorescence intensity of GFP-LC3 and RFP-GFP-LC3 were examined with immunofluorescence analysis to determine the autophagy and autophagic flux level. Western blotting assay was used to determine protein expression levels of LC3II/LC3I, P62, FUNDC1 and ATG5. mRNA expression level of miR-137 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Dual-luciferase reporter assay was used to evaluate the directly binding of miR-137 with its targets. Xenograft model was setup to evaluate tumor growth. RESULTS: The results showed that doxorubicin (Dox) induced autophagy but downregulated the expression level of miR-137 in pancreatic cancer (PC) cells. In turn, overexpression of miR-137 enhanced the effect of Dox on decreasing cell survival, inducing cell apoptosis and inhibiting autophagy rather than influencing autophagic flux in PC cells. Further mechanistic study identified that ATG5 was a direct target of miR-137. Moreover, overexpression of ATG5 dramatically reversed the promotion of apoptosis and inhibition of autophagy mediated by higher expression level of miR-137. We also demonstrated that miR-137 sensitized PANC-1 cells to Dox through inhibiting ATG5 and autophagy in vivo. CONCLUSIONS: Our findings demonstrated for the first time that miR-137 was able to promote sensitivity of PC cells to chemotherapy via inhibition of autophagy mediated by ATG5. Therefore, miR-137 may act as a potential therapeutic target for pancreatic cancer.

Ideal cardiovascular health and the subclinical impairments of cardiovascular diseases: a cross-sectional study in central south China.

BACKGROUND: Ideal cardiovascular health (CVH) is related to a low cardiovascular disease risk profile. This study aimed to investigate CVH metrics with both the biomarkers and markers of subclinical impairments of cardiovascular diseases (CVDs) in subjects from central south China. METHODS: The ideal CVH score (non-smoking status; ideal body mass index; regular physical activity; healthy diet; and an optimal serum cholesterol, blood pressure, and glucose profile; 1 point for each; total score: 0-7) was collected from 3009 participants without a history of CVDs. Subclinical biomarkers were assessed using C-reactive protein, homocysteine, and microalbuminuria. The presence of subclinical disease markers was defined as having at least one of the following: increased carotid intima-media thickness, carotid plaque, left ventricular hypertrophy, left ventricular systolic dysfunction, or a reduced ankle-brachial index. The association of biomarkers and markers with the CVH score was evaluated using multivariate logistic regression and linear regression analyses. RESULTS: Only 0.2% of the study participants met all 7 ideal CVH metrics (CVH score = 7). Compared to the female participants, the male participants had poorer CVH profiles and a higher incidence of subclinical lesions (P < 0.05). In the fully adjusted models, per 1-unit increase in the CVH score was inversely associated with the biomarker levels (ß = -0.092 - -0.224, P < 0.05 for all) and the odds of the presence of markers (odds ratio, 0.808; 95% confidence interval, 0.755-0.865). Similar relationships were observed in the gender subgroups and were stronger in the females. CONCLUSION: A clear inverse association was observed between the biomarkers or markers of subclinical impairments and the CVH score in a central south Chinese population, implying the importance of ideal CVH for the primordial prevention of CVDs.

Correction: Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis.

[This corrects the article DOI: 10.1371/journal.pone.0159719.].

Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis.

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine-cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5' splicing and alternative 3' splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis.

[Functional haplotypes of INCENP affect promoter activity and bovine semen quality].

To explore the association between single nucleotide polymorphisms (SNPs) in the promoter region of the inner centromere protein (INCENP) gene and bovine semen quality, the haplotypes in 250 Chinese Holstein bulls were detected using PCR-RFLP method in this study. Two SNPs (g.-556 G>T, rs 136823901 and g.-692 C>T, rs 211010999) and three haplotypes (CG, TT, TG) were identified in the promoter region of INCENP. The genotype frequency and allele frequency of these two SNPs as well as the correlation between different SNP haplotype combinations and bovine semen quality were then analyzed. Our results showed that fresh sperm motility of the GT genotype was significantly higher than that of the GG genotype (P<0.05) at the SNP site g.-556 G>T, while fresh and frozen-thawed sperm motilities of the haplotype combinations H1H1(CCGG), H1H3(CTGT), H2H3(TTGT) and H3H3(TTTT) were significantly higher than that of H1H2 (P<0.05). To further study the possible mechanisms by which g.-556 G>T and g.-692 C>T affect semen quality, three haplotype plasmids were respectively transfected into MLTC-1 cells. The TG haplotype demonstrated the highest luciferase activity, suggesting that g.-556 G>T and g.-692 C>T are functional mutations which could regulate INCENP gene expression by affecting promoter activity and thus affect semen quality.

Association between reversal of multidrug resistance by methyl jasmonate and P-glycoprotein ATPase activity in hepatocellular carcinoma.

OBJECTIVE: To study the effects of methyl jasmonate on multidrug resistance in a mouse model of hepatocellular carcinoma. METHODS: Multidrug resistant H22 (H22/FAP) hepatocellular carcinoma cells were produced in vitro by continuous exposure to increasing doses of doxorubicin, cisplatin and 5-fluorouracil (FAP regimen). Cell toxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay. Survival time was calculated for BALB/c mice that received intraperitoneal injections of H22/FAP cells followed by treatment with methyl jasmonate or verapamil in combination with FAP for 7 days. Adenosine triphosphate (ATP) hydrolysis was used to measure the activity of permeability-glycoprotein (P-gp) ATPase activity in plasma membranes. RESULTS: The MTT assay showed that methyl jasmonate significantly enhanced the cytotoxicity of the FAP regimen in multidrug resistant H22/FAP cells. Methyl jasmonate (10 mg/kg and 5 mg/kg) combined with FAP significantly increased survival time in BALB/c mice by 44.25% and 48.01%, respectively, compared with FAP. Methyl jasmonate increased P-gp ATPase activity. CONCLUSION: The combined use of methyl jasmonate and the FAP regimen might be a novel strategy for overcoming the multidrug resistance often observed in hepatocellular carcinoma.

[Delorme procedure for full-thickness rectal prolapse: a report of 25 cases].

OBJECTIVE: To evaluate the use of Delorme procedure for full-thickness rectal prolapse. METHODS: A series of 25 patients with full-thickness rectal prolapse were treated by Delorme procedure in four institutions between March 2005 and June 2010. The clinicopathological data were analyzed retrospectively. RESULTS: There were 9 males and 16 females. The mean age was 52(46-72) years old. All the procedures were successfully performed. There were no perioperative deaths. The mean operative time was 65(45-150) min. The intraoperative bleeding was 58(20-200) ml. The mean length of hospital stay was 8.5(5-14) days. Anastomosis dehiscence occurred in 1 patient at post-operative day 7 who was managed under anesthesia. Minor complications occurred in 8(32%) patients, including urinary retention(n=3), intractable pain(n=1), and bowel obstruction(n=4). The follow up time ranged from 2 to 6 years with a median of 3.5 years. Prolapse recurrence was observed in 1(4%) patient during the follow up. The remission rates of fecal incontinence, constipation, bleeding were 37.5%(6/16), 45.5%(5/11), and 15.4%(2/11), respectively. The Wexner incontinence score significantly decreased (median, 5.0 vs. 9.0, P<0.01). The resting pressure and maximum squeeze pressure increased significantly after surgery, while the initial volume and maximal tolerance volume decreased significantly(All P<0.01). CONCLUSIONS: Delorme procedure is safe and easy to perform. The anorectal function is improved after surgery. Therefore it should be considered the procedure of choice for rectal prolapsed.

[Polymorphism of POU1F1 gene and PRL gene and their combined effects on milk performance traits in Chinese Holstein cattle].

Three novel SNPs were found by DNA sequencing, PCR-RFLP and CRS-PCR methods were used for genotyping in 979 Chinese Holstein cattle. One SNP, G1178C, was identified in exon 2 of POU1F1 gene. Two novel SNPs, A906G and A1134G, were identified in 5'-flanking regulatory region (5'-UTR) of PRL gene. The association between polymorphisms of the two genes and milk performance traits were analyzed with PROC GLM of SAS. The results showed that GC genotype at 1178 locus of POU1F1 gene was advantageous for milk yield, milk protein yield, and milk fat yield. AG genotype at 906 locus was advantageous for milk yield. There was no significant difference between 1134 locus and milk performance traits of 5'-UTR of PRL gene. Analysis of genotype combination effect on milk production traits showed that the effect of combined genotype was not simple sum of single genotypes and the effects of gene pyramiding seemed to be more important in molecular breeding.

Two novel SNPs in HSF1 gene are associated with thermal tolerance traits in Chinese Holstein cattle.

Heat-shock transcription factors (HSFs) play an important role in regulating heat stress response. The activation of heat-shock protein (HSP) genes is mediated by HSFs, which bind to promoters of HSP genes. In this research, two novel single nucleotide polymorphisms, T909C and G4693T, and their association with thermal tolerance were investigated in 951 Chinese Holstein cattle. Linkage disequilibrium and haplotype construction were analyzed using SHEsis software. Four haplotypes were constructed, and nine haplotype combinations were found. Potassium content in erythrocytes (PCE), decreased rate of milk production (R), rectal temperature (RT), and heat-tolerance coefficient (HTC) were selected for the thermotolerance index. Association analysis showed that thermal tolerance in Chinese Holstein cattle was significantly affected by T909C and G4693T. The PCE of cows with CC or TC genotype was lower than that of TT at the 909 position (p < 0.05). Cows with TT genotype had lower PCE (p < 0.01) and higher HTC (p < 0.05) at the 4693 position. Cows with H2H4 haplotype combination had lower PCE (p < 0.01), R (p < 0.05) and RT (p < 0.05) and higher HTC (p < 0.05) than those with H1H3 haplotype combination. Bioinformatic analysis predicted that the 4693 position was located in the microRNA-binding (bta-miR-484) region. Quantitative reverse transcription-polymerase chain reaction demonstrated that 4693-T mutation caused the disruption of microRNA target binding, resulting in the relief of the transcriptional repression, which, in turn, resulted in increased expression. Thus, the HSF1 gene is useful in dairy cattle thermal tolerant breeding.

[ShRNA against NSP4 gene inhibits the proliferation of bovine rotavirus in vitro].

Based on the NSP4 sequence of bovine rotavirus (BRV), the shRNA was designed and synthesized, and a shRNA recombinant lenti-virus vector RNAi-H1-89 was constructed. The recombinant RNAi-H1-89 Lenti-virus was packaged by transfecting the 293T cell with the recombinant vector RNAi-H1-89 and two helper plasmids using lipofectamine, and then used to infect MA104 cells. The MA104 cells were further infected with BRV strain G6 24h post-infection, with the LacZ shRNA recombinant lenti-virus as control. Thirty-six hours later, the CPE of the infected cells was observed under microscope, shRNA of NSP4 gene inhibited CPE in MA104 cell; the shRNA against NSP4 gene also inhibited NSP4 gene expression by RT-PCR, The virus titer in the cell culture supernatant was significant lower compared with the control group. The above results showed that RNAi-H1-89 against NSP4 gene could specifically silence NSP4 gene expression, and inhibit the proliferation of BRV.

[Genetic polymorphism in 5'-flanking region of the lactoferrin gene and its associations with mastitis in Chinese Holstein cows].

Bovine lactoferrin (LF) is a multifunctional glycoprotein found in milk, which acts mainly as a defense factor in the mammary gland. Polymorphism has been found in the bovine LF gene. However, there is no report on genetic polymorphism of LF gene and its associations with mastitis in dairy cattle. In this study, the promoter fragment of LF gene containing -926(G/A), -915(T/G), -478(/G), and +72(T/C) mutations were genotyped by the PCR-RFLP and CRS-PCR method. Two hundred and sixty-eight Chinese Holstein cows were screened. Least square linear model (LSM) analysis was applied to evaluate the associations of LF gene with somatic cell score (SCS). The results indicated that the SCS was significantly affected by -478(/G) and +72(T/C), but not by the other two loci (P >0.05). The SCS of cow with genotype AB in +72(T/C) position was significantly lower than that of genotype AA (P<0.01) or AB (P<0.05). In position -478(/G), the cow with genotype CC showed significantly lower SCS in contrast to cow with genotype CD and DD (P < 0.01). In conclusion, genotype AB in position +72(T/C) and genotype CC in position -478(/G) of LF gene were advantageous genotype, which can be used as candidate markers for mastitis resistance selection in dairy cattle.

[Genetic polymorphism of HSP70-1 gene and its correlation with resistance to mastitis in Chinese Holstein].

The polymorphisms of HSP70-1 gene in 253 Chinese Holstein dairy cows were studied, and the association between the polymorphisms and somatic cell score (SCS) were analyzed. PCR-SSCP, PCR-RFLP and DNA sequencing were used to investigate mutations in the coding region of HSP70-1 gene. The G-->A-->C mutation at 1 623 bp and G-->A mutation at 2 409 bp were found and both of them were silence mutations that caused no alteration in amino acid sequence. Chi-square test showed both loci were'nt at Hardy-Weinberg disequilibrium in Chinese Holstein. In the meanwhile, the association of 2 409 locus and SCS was not significant. However, the polymorphism at 1623 locus affected SCS significantly (P<0.05). The SCS of genotype CC was significantly lower than that of genotype AG and GG (P<0.05), so genotype CC was mastitis resistant. These results suggest that genotype CC of HSP70-1 gene may be used as a molecular and genetic marker to improve the phenotype of anti-mastitis in Chinese Holstein dairy cows.

[Genetic polymorphism of Nramp1 gene and correlation with mastitis in Holstein cattle].

In this research, PCR-SSCP technique was used to analyze the polymorphisms of the exon 11 of Nramp1 gene in Chinese Holstein cattle (n=344), and correlation between polymorphisms of Nramp1 with somatic cell score (SCS) and milk production traits was analyzed. The results show that three genotypes namely AA, BB, and AB were detected. Allele A was predominant and the frequencies of alleles A and B were estimated to be 0.767 and 0.233, respectively. Chi-square test indicated that the polymorphic locus in Chinese Holstein fitted Hardy-Weinberg equilibrium (P>0.05). Sequencing analysis showed two polymorphic sites at positions 200 bp (C/G) and 254 bp (T/G), which resulted in amino acid alteration Ala356Pro and Leu374Met. The least squares means of SCS in Holstein cattle was lower for genotype AA than that for genotypes AB and BB (P<0.05). The least squares means of milk yield of genotype AA and AB were higher than that for genotype BB (P<0.05, P<0.01, respectively). Genotype AA was beneficial to mastitis resistance. This suggested that Nramp1 may be a candidate gene responsible for mastitis in Holstein cattle.

[Genetic polymorphism of k-casein gene exon 4 and its cor-relation with milk production traits in Chinese Holsteincows.].

K-casein gene was regarded as a candidate gene for milk production traits of cows. In this study, a 779 bp fragment of k-casein gene of Chinese Holstein was amplified by polymerase chain reaction (PCR), the polymorphisms of three loci of k-casein gene were detected by PCR-RFLP with restriction endonuclease Taq, Hind, Pst. After sequencing, T/C single nucleotide polymorphism (SNP) was identified at nucleotide 10 891C/A SNP was identified at nucleotide 10 927 and G/A SNP was identified at nucleotide 10 988 in exon4 of k-casein gene. Both alleles (A and B) of three loci were found in the population that showed low polymorphism. The gene frequencies of A and B were 86.03% and 13.97%, respectively. The genotype frequencies of AA, AB, and BB were 73.71%, 24.63%, and 1.66%, respectively. Statistical results of c2 test indicated that three polymorphism sites in the population fitted with Hardy-Weinberg equilibrium (P > 0.05). Meanwhile, the effect of polymorphism of k-casein gene on milk production traits was analyzed. The results indicated that in the three loci, the different genotype of k-casein gene had no significant influence on milk yield and milk protein percent (P > 0.05). The cows with genotypes BB and AB showed higher milk fat percent than those with genotype AA ( P < 0.05 ) ; with genotype AB showed higher fat protein ratio than those with genotype AA ( P < 0.05 ). The polymorphism of the three loci in the experimental population is closely linked. The conclusion is that k-casein B allele can be used as the molecular genetic markers of modifying milk fat percent in Chinese Holstein cows.

[The development and application of method for detecting bovine complex vertebral malformation].

Complex vertebral malformation (CVM), a lethal autosomal recessive inherited defect in Holstein calves, was newly reported worldwide. The molecular cause of CVM was a substitution of guanine by thymine (G-->T) in a solute carrier family 35 member 3 gene (SLC35A3), encoding UDP-N-acetylglucosamine transporter. It was characterized by stillborn, abortion, and premature birth. The objective of this study was to study the actual carrier frequency of the CVM mutation in a population of Chinese Holstein (=Chinese Black-and-White) normal cattle. The normal 436 Holstein cows and 93 Holstein bulls were genotyping by using the Created Restriction Site PCR (CRS-PCR) and Allele-specific PCR (AS-PCR) methods. There were two bulls and one cow in three observed CVM-carriers. In the Holstein dairy cattle and Holstein bull population, the percentages of CVM carriers were estimated as 0.60% and 2.20% respectively. This study provided a more reliable and useful method for extensive screening of CVM and also offers a theoretical basis for molecular diagnosis in Holstein calves.

[Expression, purification of recombinant Luxi yellow cattle IFN-alpha fusion protein and its antiviral activities].

Interferon a gene was cloned from genomic DNA of Chinese Luxi yellow cattle by PCR, and the PCR product was inserted into vector pET32a( + ) to make a recombinant plasmid pET32a( + )/BoIFN-alpha. The expression of BoIFN-alpha in Escherichia coli was induced by addition of IPTG. Sequence analysis showed that the Chinese Luxi yellow cattle IFN-alpha gene is composed of 498 nucleotides, encoding a mature polypeptide of 166 amino acids. Compared with other BoIFN-alpha subtypes, it shares the highest identity of 97.6% to the C-subtype. SDS-PAGE results showed that recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 40 kD and the recombinant proteins accounted for 26.7% of the whole proteins.The expressed product was purified by affinity chromatography with immobilized nickel chelating NTA (Ni-NTA) and its antiviral activities were tested on MDBK/VSV cell system. Its antiviral activities were 5 x 10(5) u/mg on MDBK/VSV cell system. The results showed that the expression plasmid was successfully constructed and BoIFN-alpha C2 protein was expressed in Escherichia coli. Moreover the purification had good effects on antiviral activities.

Behavior of new complexes of tetrakis(4-methoxylphenyl)porphyrin with heavy rare earth elements in reversed-phase high performance liquid chromatography.

An HPLC method has been developed for the separation of new complexes of tetrakis(4-methoxylphenyl)porphyrin (TMOPP) with four heavy rare earth elements (RE = Y, Er, Tm, and Yb). The function of amine and acid in the mobile phase has been investigated and a reasonable explanation is presented. Successful separation of the RE-TMOPP-Cl complexes is accomplished in 10 min with a mobile phase consisting of methanol-water-acetic acid-triethanolamine. The detection limits (S/N= 3) for the four complexes are 0.01 microg/mL. This method is rapid, sensitive, and simple.