Dejun Ji

Transcriptome MicroRNA Profiling of Bovine Mammary Glands Infected with Staphylococcus aureus

MicroRNAs are small non-coding RNA molecules that are important regulators of gene expression at the post-transcriptional level. miRNAs impact the processes of cell proliferation, differentiation and apoptosis. Thus, the regulation of miRNA expression profiles associated with mastitis will be conducive for its control. In this study, Staphylococcus aureus (S. aureus) was administered to the mammary gland of Chinese Holstein cows to construct a bacteria-type mastitis model. Total RNA was isolated from bovine mammary gland tissue samples from the S. aureus-induced mastitis group and controls. miRNAs were analyzed using Solexa sequencing and bioinformatics processing for the experimental group and control group. Two miRNA libraries were constructed respectively. A total of 370 known bovine miRNAs and 341 novel mi RNAs were detected for the S. aureus and 358 known bovine miRNAs and 232 novel miRNAs for control groups. A total of 77 miRNAs in the S. aureus group showed significant differences compared to the control group. GO (Gene Ontology) analysis showed these target genes were involved in the regulation of cells, binding, etc., while KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that these genes were enriched in endocytosis, and olfactory transduction pathways involved in cancer. These results provide an experimental basis to reveal the cause and regulatory mechanism of mastitis and also suggest the potential of miRNAs to serve as biomarkers for the diagnosis of mastitis in dairy cows.

Polymorphisms of the IL8 gene correlate with milking traits, SCS and mRNA level in Chinese Holstein

To explore the relation of Interleukin-8 (IL8) gene polymorphism with immunity against mastitis in dairy cow, the polymorphism of IL8 gene was investigated in 610 Chinese Holstein cow from 30 bull families in a dairy farm in Shanghai using polymerase chain reaction–single strand conformation polymorphism (PCR–SSCP) technique, and milk yield, milk fat percentage, milk protein percentage, 305 day corrected milk yield, 305 day milk fat yield, 305 day milk protein yield and somatic cell score (SCS) were measured and analyzed, and the mRNA levels of IL8 genotypes in blood were detected by real-time PCR. The results showed that three genotypes, KK, KA and AA were detected with frequencies of 0.187, 0.451, and 0.362, respectively. The gene frequencies of K and A were 0.412 and 0.588, respectively. The significant association of the IL8 mutations with milk yield, 305 day milk protein yield, 305 day corrected milk yield and 305 day milk fat yield, SCS, and significant association with milk protein percentage were identified, while their association with milk fat percentage were not significant. KK had higher milk yield, 305 day milk protein yield, 305 day corrected milk yield and 305 day milk fat yield than AA or KA, and the least square mean of SCS of KK was significantly lower than that of AA or KA. AA had significant lower milk protein yield than KK or KA. The relative IL8 mRNA level of KK in blood was the highest. These findings demonstrated that IL8 genotype significantly correlated with mastitis resistance and the locus could be a useful genetic marker for mastitis resistance selection and breeding in Chinese Holstein.

Association of SCD1 and DGAT1 SNPs with the intramuscular fat traits in Chinese Simmental cattle and their distribution in eight Chinese cattle breeds.

Intramuscular fat (IMF) is a key parameter for evaluation of nutritional quality of beef, with its endogenous synthesis regulated by stearoyl CoA desaturase (SCD1) and diacylglycerol-acyl transferase 1 (DGAT1) genes in cattle. The object of this research was to evaluate the effect of SCD1 and DGAT1 polymorphisms on IMF trait in beef cattle and to estimate the frequency distribution of SNPs in the two genes in Chinese cattle populations. The SCD1 and DGAT1 polymorphisms were detected by PCR-single strand conformation polymorphism (PCR-SSCP) method in Chinese Simmental cattle and their associations with IMF traits were analyzed using the general linear model (GLM). The frequency distribution of SNPs in SCD1 and DGAT1 genes were detected by PCR-SSCP method and analyzed in seven other cattle populations. The results showed significant associations of SNPs SCD1-878, SCD1-762, and DGAT1 10433 and 10434 with IMF (%) and shearing force values (SFV; kg) in Chinese Simmental cattle. A haplotype combining SCD1-878C, SCD1-762T, and DGAT1 10433 and 10434-GC had the highest IMF, marbling score and shearing force. The polymorphic investigation indicated that the frequency of SCD1-878C or SCD1-762T was significantly higher in Chinese southern cattle (Leiqiong, Yunnan High pump, BMY or Minnan Cattle) than in Chinese northern cattle (Chinese Simmental, Luxi Cattle, Bohai Black or Chinese Holstein), while the frequency of DGAT1 10433 and 10434-GC in Chinese indigenous breed (Leiqiong, Yunnan High pump, BMY, Luxi Cattle, Bohai Black, or Minnan Cattle) was significantly lower than breeds with imported blood (Chinese Simmental or Chinese Holstein). These findings demonstrated that both the SCD1 and DGAT1 SNPs were prospect genetic markers for IMF traits, and the SCD1 SNPs could be used as a genetic marker for southern or northern blood in Chinese cattle.

Phylogenetic Relationships and Status Quo of Colonies for Gayal Based on Analysis of Cytochrome b Gene Partial Sequences

Thirty-three mutations and four different haplotypes were found when cytochrome b(Cytb) gene partial sequences of 12 gayals were analyzed. Together with sequences of Bos indicus, Bos taurus, Bos grunniens and Bos gaurus with Bubalus bubalis as the out group, the partial sequences of Cytb gene of gayals were aligned and base composition and nucleotide variation of Cytb gene were analyzed. The phylogenetic trees were constructed by the NJ method and the MP method respectively, both supporting almost the same topology. Gayal is an independent species of Bos from Bos indicus, Bos taurus and Bos gaurus. The results also indicate that a great proportion of gayal bloodline was invaded by other species, and the protection of gayal is facing a formidable situation.

Heme Oxygenase-2 Suppress TNF-α and IL6 Expression via TLR4/MyD88-Dependent Signaling Pathway in Mouse Cerebral Vascular Endothelial Cells

Heme oxygenase (HO) represents an intrinsic antiinflammatory system based on its ability to inhibit expression of proinflammatory cytokines. The constitutive isoform heme oxygenase-2 (HO-2) has high expression and activity in cerebral microvascular endothelial cells (CMVEC). This study was undertaken to evaluate the role of HO-2 in regulation of TLR4/MyD88-dependent signaling and to study the effect of HO-2 on the expression and secretion of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and Interleukin-6 (IL6) in CMVEC. HO-2 short hairpin RNA (shRNA) and HO-2 overexpression plasmids were used to observe the effect of HO-2 on proinflammatory cytokines in CMVEC in vitro, and the results showed that the messenger RNA (mRNA) and protein levels of TNF-α and IL6 were increased and decreased, respectively, compared with control groups. LPS-stimulated TNF-α and IL6 mRNA and protein were also reduced in CMVEC treated with an inhibitor of TLR4 signaling, CLI-095, or HO-2 overexpression. CLI-095 and HO-2 overexpression both reduced TLR4 expression in CMVEC, and HO-2 shRNA blocked these effects of CLI-095. CLI-095 and HO-2 overexpression potently suppressed TLR4/MyD88-dependent proinflammatory cytokine expression in CMVEC. These results suggest that HO-2 plays an important role in protecting CMVEC against cytokine-mediated inflammation.

Analysis on genetic diversity and isolation mechanism by distance of different ecological type sheep breeds in Mongolia sheep group.

In the present study with Tan sheep, small-tailed Han sheep, Hu sheep, Tong sheep, and Wadi sheep, we detected the distribution of gene frequency of several microsatellite sites in different chromosomes, the result showed that: 1) Hu sheep was in the status of Hardy-Weinberg extreme disequilibrium (P < 0.01), while populations including Tong sheep, small-tailed Han sheep, Tan sheep, and Wadi sheep were in Hardy-Weinberg equilibrium (P > 0.05). 2) Variance analysis of the heterozygosity and polymorphic information content at microsatellite makers showed that there were not significant differences among populations as to heterozygosity and PIC (P > 0.05), as to effective number of alleles there were not significant differences both among Tan sheep, Hu sheep, Tong sheep, and Wadi sheep, and between Wadi sheep and small-tailed sheep (P > 0.05), but between the former three populations and the latter two populations, there were significant differences (0.01<P < 0.05). The variation levels of small-tailed Han sheep was the highest in the five populations based on microsatellite maker data, subsequently followed by Wadi sheep, Tong sheep, Tan sheep, and then Hu sheep. 3) The phylogenetic relationships of the five sheep populations in this study did not meet the mechanism of isolation by distance, and the genetic differentiation relationships among five sheep populations were not closely linearly correlative with their geography distribution. Our findings supported related records in literature:The five populations originated on different time stage from the primogenitor population and communicated genetically with each other thereafter in the process of natural and artificial selection and on different ecological environment.

Migration and accumulation of primordial germ cells of early embryo in quail.

In this research, we have identified primordial germ cells (PGCs) in quail embryo using Quail Hemangioblastic Lineage (QH1) monoclonal antibody analysis. Quail PGCs originated from the opaca of unincubated blastodisc, and then transferred to the pellucida and the germinal crescent. At 27 hours post-incubation, a few PGCs first appeared in blood vessels of the pellucida, where many PGCs accumulated at 36 hours post-incubation. The PGCs scattered or clustered from head to omphalo mesenteric and mainly settled down in the mesenchymal blood vessels of head at 45 hours post-incubation. The size of PGCs population increased significantly (P<0.05) from stage XII (12.8±4.82 µm) to primitive streak stage (106.7±8.74 µm) and from Head process stage (95.8±19.74 µm) to tenth somite stage (199.4±19.97 µm). It is concluded that the PGCs scattered in the head area before migration to the germinal crescent and distributed randomly in both gland. The number of PGCs varied at different stages with two peaks, primitive streak stage (18 hours post-incubation) and tenth somite (36 hours post-incubation).

The analysis of genetic diversity and differentiation of six Chinese cattle populations using microsatellite markers.

A total of 321 individuals from six cattle populations of four species in a bovine subfamily in China were studied using 12 pairs of microsatellite markers. The genetic diversities within and between populations were calculated. The phylogenetic trees were constructed by (delta mu)(2) and DA distances, and the divergence times between populations were estimated by (delta mu)(2). Altogether, 144 microsatellite alleles were detected including 24 private alleles and nine shared alleles. Chinese Holstein had the largest number of private alleles (10), whereas, Bohai black and Buffalo had the smallest number of private alleles (2). Chinese Holstein showed the highest genetic variability. Its observed number of alleles (Na), mean effective number of alleles (MNA), and mean heterozygosity (He) were 7.7500, 4.9722, and 0.7719, respectively, whereas, the Buffalo and Yak showed low genetic variability. In the phylogenetic trees, Luxi and Holstein grouped first, followed by Bohai and Minnan. Yak branched next and buffalo emerged as the most divergent population from other cattle populations. Luxi and Bohai were estimated to have diverged 0.039-0.105 million years ago (MYA), however, buffalo and Holstein diverged 0.501-1.337 MYA. The divergence time of Yak versus Minnan, Holstein and buffalo was 0.136-0.363, 0.273-0.729, and 0.326-0.600 MYA, respectively.

The immunosuppression mechanism of hypodermin A on complement

Hypodermin A (HA), a serine protease secreted by first-instar larvae of Hypoderma lineatum (Diptera: Oestridae) is associated with inflammatory and the specific immune responses in cattle hosts. In the present study, the cDNA sequence of HA was synthesized, and found to have fifteen amino acids which differed from the sequence available in GenBank. We then examined the association between recombinant HA and guinea-pig complement component 3 (C3) through a co-immunoprecipitation assay. Cos7 cells stably expressing HA were generated, and were found to be more resistant to lysis by guinea-pig C3 than the controls. HA was also able to degrade the C6 and C5b-9 of guinea-pig C3. The presumed DNA binding site of HA with guinea-pig C3 was detected by an electrophoretic mobility shift assay (EMSA). In contrast, after stable transfection, mHA was unable to reduce the amount of C3 or to inhibit its cytotoxicity, while HA could degrade guinea-pig C3 and inhibit the complement pathway. The findings suggest that recombinant HA could serve as an immunosuppressive agent against organ rejection after xenotransplantation.

Behavior differentiation between wild Japanese quail, domestic quail, and their first filial generation

The number of wild quail has dramatically reduced in China and reached a state of endangerment with the deterioration of the environment in recent years. In this study, we examined the ecological behaviors of quails in the cage to determine the differentiation level between wild Japanese quail and domestic quail, to detect the relationship between quail behavior and evolutionary differentiation and to analyze the possibility of restoring effective size of wild population. With the on-the-spot observations and measurements, the behaviors of 3 categories of quail, namely wild Japanese quail from the Weishan Lake area in China, domestic quail, and their first filial generation (F(1)) were studied. Domestic quail differed from wild Japanese quail in morphological pattern and ecological behaviors, including some indexes of figure type and egg, vocalization, aggression and fighting, and mating, but wild Japanese quail and domestic quail could succeed in mating and reproducing fertile hybrid offspring. There were significant differences between domestic quail and wild Japanese quail in reproductive traits, involved mating times, fertility rate, hatching rate, and hatching rate of fertilized eggs (P < 0.05). The first filial generation presented significant difference from the wild Japanese quail in vocalization, aggression and fighting, mating, hatching rate, hatching rate of fertilized eggs, and some egg indexes (P < 0.05) and significantly differ from the domestic quail in vocalization, hatching rate, and hatching rate of fertilized eggs (P < 0.05). Evolutionary differentiation between wild quail and domestic quail was still at a relatively low level because no reproductive isolation existed. The advantages of the F(1) hybrids in reproductive capacity, fertilization, and hatching recommend that releasing hybrids instead of domestic quails to the wild would be a more effective way to restore the effective size of wild quail population if necessary.