This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.

A lack of relevant disease models for Campylobacter jejuni has long been an obstacle to research into this common enteric pathogen. Here we used an infant rabbit to study C. jejuni infection, which enables us to define several previously unknown but key features of the organism. C. jejuni is capable of systemic invasion in the rabbit, and developed a diarrhea symptom that mimicked that observed in many human campylobacteriosis. The large intestine was the most consistently colonized site and produced intestinal inflammation, where specific cytokines were induced. Genes preferentially expressed during C. jejuni infection were screened, and acs, cj1385, cj0259 seem to be responsible for C. jejuni invasion. Our results demonstrates that the infant rabbit can be used as an alternative experimental model for the study of diarrheagenic Campylobacter species and will be useful in exploring the pathogenesis of other related pathogens.

Campylobacter jejuni is a prevalent foodborne pathogen worldwide. Human infection by C. jejuni primarily arises from contaminated poultry meats. Genes expressed in vivo may play an important role in the pathogenicity of C. jejuni. We applied an immunoscreening method, in vivo-induced antigen technology (IVIAT), to identify in vivo-induced genes during human infection by C. jejuni. An inducible expression library of genomic proteins was constructed from sequenced C. jejuni NCTC 11168 and was then screened using adsorbed, pooled human sera obtained from clinical patients. We successfully identified 24 unique genes expressed in vivo. These genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, and other processes. We selected six genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results showed that the selected six genes were significantly upregulated in vivo but not in vitro. In short, these identified in vivo-induced genes may contribute to human infection of C. jejuni, some of which may be meaningful vaccine candidate antigens or diagnosis serologic markers for campylobacteriosis. IVIAT may present a significant and efficient method for understanding the pathogenicity mechanism of Campylobacter and for finding targets for its prevention and control.

Background: In polycystic ovary syndrome (PCOS), ovarian physiology is tightly linked to the metabolic disturbances observed in this disease. Vitamin D (VD) plays an important role in the regulation of ovulatory dysfunction and can influence genes involved in steroidogenesis in granulosa cells (GCs). However, its role in the proliferation and apoptosis of ovarian GCs is unclear. The present study aimed to investigate the role of in the hormone synthesis, proliferation, and apoptosis of ovarian GCs. Methods:The abnormal expression of miRNAs in ovarian tissues of VD-deficient mice was analyzed using transcriptome sequencing. The direct target of miR-196b-5p was predict and confirmed by bioinformatics analysis and the dual-luciferase reporter assay. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect the levels of miR-196b-5p, cell proliferation was detected via the CCK8 assay, and cell apoptosis and reactive oxygen species (ROS) were measured via flow cytometry. The levels of radixin (RDX), leucine rich repeat containing 17 (LRRC17), aromatase (CYP19A1), and glucose transporter 4 (GLUT4) were detected by performing RT-qPCR or western blot.Results: We found that miR-196b-5p was significantly downregulated among the 672 miRNAs that were differentially expressed (DE) in VD-deficient mice. In addition, the results demonstrated that downregulated expression of miR-196b-5p significantly increased the level of RDX and LRRC17, and reduced expression of miR-196b-5p significantly promoted ovarian GC apoptosis and inhibited cell proliferation. Downregulated expression of miR-196b-5p promoted cellular ROS production and inhibited sex hormone production and glucose uptake. Transfection with miR-196b-5p mimics significantly increased the expression of CYP19A1 and GLUT4 and decreased the RDX and LRRC17 levels in ovarian GCs. Conclusions:This study shows that miR-196b-5p can regulate the oxidative stress (OS), glucose uptake, and steroid production pathway of GCs, thus promoting follicular development and maturation. This is a step towards a feasible treatment for PCOS.

Vitamin D (VD) is not only associated with bone growth and development, but is also closely associated with numerous other pathological conditions. The present study aimed to investigate the effect of microRNA (miRNA/miR)-378d on ovarian granulosa cells by regulating the round spermatid basic protein 1 (Rsbn1) in the absence of VD. The abnormal expression of miRNAs in ovarian tissues of the VD-deficient mouse was analyzed using transcriptome sequencing. miR-378d, glucose transporter 4 (Glut4) and aromatase (Cyp19a) expression levels were examined via reverse transcription-quantitative (RT-q)PCR and western blotting. The expression levels of Rsbn1, Glut4 and Cyp19a were detected in transfected mouse ovarian granulosa cells. The targeting regulation between miR-378d and Rsbn1 was verified using double reporter gene assay and functional rescue experiments. Among the 672 miRNAs that were differentially expressed, cluster analysis revealed that 17 were significantly upregulated and 16 were significantly downregulated. Moreover, miR-378d showed significant upregulation, which was further verified via RT-qPCR. It was identified that the protein expression level of Rsbn1 was significantly downregulated. Furthermore, Glut4 mRNA expression was significantly decreased in the mimic group but markedly increased in the inhibitor group. By contrast, the mRNA expression levels of Rsbn1 and Cyp19a did not demonstrate any significant difference. The western blotting results indicated that the protein expression levels of Rsbn1 and Glut4 were decreased and increased, respectively, while Cyp19a did not show any significant change. In addition, the double reporter gene experiments confirmed that Rsbn1 was the target gene of miR-378d. Collectively, the present results demonstrated that miR-378d was abnormally overexpressed in the ovarian tissues of the VD-deficient mice, and that miR-378d could inhibit Glut4 production by targeting Rsbn1, which may lead to insulin resistance.