Identification of novel molecular markers of mastitis caused by Staphylococcus aureus using gene expression profiling in two consecutive generations of Chinese Holstein dairy cattle

Background: Mastitis in dairy cows caused by Staphylococcus aureus is a major problem hindering economic growth in dairy farms worldwide. It is difficult to prevent or eliminate due to its asymptomatic nature and long persistence of infection. Although transcriptomic responses of bovine mammary gland cells to pathogens that cause mastitis have been studied, the common responses of peripheral blood leukocytes to S. aureus infection across two consecutive generations of dairy cattle have not been investigated.Methods: In the current study, RNA-Seq was used to profile the transcriptomes of peripheral blood leukocytes sampled from S. aureus-infected mothers and their S. aureus-infected daughters, and also healthy non-infected mothers and their healthy daughters. Differential gene expression was evaluated as follows: 1) S. aureus-infected cows versus healthy non-infected cows(S vs. H, which include all the mothers and daughters), 2) S. aureus-infected mothers versus healthy non-infected mothers(SM vs. HM), and 3) S. aureus-infected daughters versus healthy noninfected daughters(SMD vs. HMD).Results: Analysis of all identified expressed genes in the four groups(SM, SMD, HM, and HMD) showed that EPOR,IL9, IFNL3, CCL26, IL26 were exclusively expressed in both the HM and HMD groups, and that they were significantly(P < 0.05) enriched for the cytokine-cytokine receptor interaction pathway. A total of 17, 13 and 10 differentially expressed genes(DEGs)(FDR Padj. < 0.1 and |FC| > 1.2) were detected in the three comparisons, respectively. DEGs with P < 0.05 and |FC| > 2 were used for functional enrichment analyses. For the S vs. H comparison, DEGs detected included CCL20, IL13 and MMP3, which are associated with the IL-17 signaling pathway. In the SM vs. HM and SMD vs. HMD comparisons, five(BLA-DQB, C1 R, C2, FCGR1 A, and KRT10) and six(BLA-DQB, C3 AR1, CFI, FCAR, FCGR3 A, and LOC10498484) genes, respectively, were involved in the S. aureus infection pathway.Conclusions: Our study provides insights into the transcriptomic responses of bovine peripheral blood leukocytes across two generations of cattle naturally infected with S. aureus. The genes highlighted in this study could serve as expression biomarkers for mastitis and may also contain sequence variation that can be used for genetic improvement of dairy cattle for resilience to mastitis.

Tumor necrosis factor-alpha(TNF-α) in spotted halibut Verasper variegatus at the embryonic and metamorphic stages

As an aquatic fish,the spotted halibut Verasper variegatus is highly susceptible to bacterial and virus infections.Tumor necrosis factor-alpha(TNF-α)as a cytokine could control the inflammatory responses.The functions of TNF-αin many species have been widely studied,particularly in mammals.However,little is known about the TNF-αfunctions in V.variegatus.We first cloned and sequenced the TNF-αgene in V.variegatus(VvTNF-α).The two conserved cysteine residues,transmembrane sequence,Thr-Leu motif,and TNF family signature,as well as the TA-rich motifs of its proteins related to inflammatory responses had high similarity to those of the other teleost and mammalian TNF-α.The phylogenetic analysis showed that VvTNF-αwas consistent with TNF-αgenes of other vertebrates.The VvTNF-αtranscripts were extensively distributed in the peripheral blood leukocytes(PBLs),spleen,and gill,indicating that the VvTNF-αhad a role in immune function.Furthermore,treatment with pathogen-associated molecular patterns(PAMPs)could induce a rapid and significant increase of VvTNF-αin the PBLs,which reveals that VvTNF-αdoes participate in the host immune responses against bacterial and viral pathogens.We found that VvTNF-αhad an interesting expression pattern during metamorphosis,showing that the flatfish TNF-αmay have some novel functions during specific developmental stages.In addition,the 3 D structure prediction of VvTNF-αprovided an indication of how it is likely to interact with other proteins.Therefore,VvTNF-αhas multiple functions,and provides valuable information to explore novel functions of TNF-α.

Novel Functions of Interleukin-1 Beta in Spotted Halibut(Verasper variegatus) at Its Embryonic and Metamorphic Stages

Interleukin-lbeta （IL-1β） is a cytokine involved in the control of inflammatory responses. The functions of IL-1β have been extensively studied in many vertebrates. However, few studies focused on the functions of IL-1β in the spotted halibut （Verasper variegatus）, an endangered aquatic fish that easily suffers from bacterial and viral infections. We first cloned and sequenced the IL-1β gene from V. variegatus （VvIL-1β）. Its conservative interleukin-1 propeptide and interleukin-1/18 domains corresponding regions, characteristics of the IL-1 protein family and TA-rich motifs relating to inflammatory responses were similar to those of the IL-1β genes from other teleost and mammals. The phylogenetic analysis showed that VvIL-1β was homologous to the IL-1β gene of other vertebrates. Treatment with pathogen-associated molecular patterns （PAMPs） induced a significant and rapid up-regulation of VvIL-1β expression in the peripheral blood leukocytes （PBLs）, revealing that VvIL-1β is actively involved in the host ilmnune responses against bacterial and viral pathogens. The expression level of VvIL-1β was relatively high in the PBLs, gill, and intestine, indicating the immunological function of VvIL-1β . VvIL-1β also had interesting expression patterns at specific developmental stages, implying that the IL-1β of flatfishes may have some novel functions during embryonic development and metamorphosis. The 3D structure prediction also provided information about how VvIL-113 interacts with its receptor protein. These results suggested that VvIL-1β may have multiple functions in V. variegatus and provided us valuable information for understanding the novel functions of IL- 1β.

Methylation status of DJ-1 in leukocyte DNA of Parkinson’s disease patients

Background:DJ-1 has been thought as a candidate biomarker for Parkinson’s disease(PD).It was found reduced in PD brains,CSF and saliva,although there were conflicting results.How DJ-1 expression may be regulated is not clear.Recently,blood-based DNA methylation represents a highly promising biomarker for PD by regulating the causative gene expression.Thus,in this study,we try to explore whether blood-based DNA methylation of DJ-1 could be used as a biomarker to differentiate PD patients from normal control(NC),and whether DNA methylation could regulate DJ-1 expression in a SH-SY5Y cell model.Methods:Forty PD patients and 40 NC were recruited in this study.DNA was extracted from peripheral blood leukocytes(PBLs).Methylation status of two CpG islands(CpG1 and CpG2)in promoter region of DJ-1 was explored by bisulfite specific PCR-based sequencing method.Methylation inhibitor 5-Aza-dC was used to treat SH-SY5Y cell line,DJ-1 level was detected in both mRNA and protein level.Results:CpG sites in these two CpG islands(CpG1 and CpG2)of DJ-1 were unmethylated in both PD and NC group.In SH-SY5Y cell model treated by methylation inhibitor,there was no significant change of DJ-1 expression in either mRNA level or protein level.Conclusions:Our results indicated that DNA methylation inhibitor didn’t alter DJ-1 gene expression in SH-SY5Y cell model,and DNA methylation of DJ-1 promoter region in PBLs level might not be an efficient biomarker for PD patients.