Estrogen Suppresses Cytokines Release in cc4821 Neisseria meningitidis Infection via TLR4 and ERß-p38-MAPK Pathway.

Estrogen has long been known to possess immune-modulatory effects in diseases, and multiple pathological conditions show great sex disparities. However, the impact of estrogen in Neisseria meningitidis infection has not been determined. The present study aimed to investigate the role of estrogen in N. meningitidis infection and the molecular mechanism. We selected 35 N. meningitidis isolates representing different clonal complexes (cc), serogroups, and isolation sources to infect the HBMEC cell line. Results showed that the expression of estrogen receptor (ER) ß in N. meningitidis-infected cells was downregulated compared with that in normal cells. The expression of ERß induced by invasive isolates was lower than that in carriers. Serogroup C isolates induced the lowest expression of ERß compared with serogroup A and B isolates. We used four cc4821 N. meningitidis isolates to infect two kinds of host cells (human brain microvascular endothelial cells and meningeal epithelial cells). The results showed that 17 ß-estradiol (E2) could inhibit the release of inflammatory factors interleukin (IL)-6, IL-8, and tumor necrosis factor-α after N. meningitidis infection via TLR4. E2 could inhibit the activation of the p38-MAPK signal pathway induced by N. meningitidis infection through binding to ERß, and significantly inhibit the release of inflammatory factors in N. meningitidis-infected host cells. This study demonstrated that estrogen plays a protective role in N. meningitidis infection. ERß is potentially associated with the release of inflammatory cytokines in N. meningitidis infection, which sheds light on a possible therapeutic strategy for the treatment of invasive diseases caused by N. meningitidis.

Metagenomic deep sequencing obtains taxonomic and functional profiles of Haemaphysalis longicornis that vary in response to different developmental stages and sexes.

Ticks can transmit numerous pathogens and harbor diverse microbial communities. Considerable progress has been made in the characterization of the bacterial profiles of ticks, whereas other members of tick microbiota (such as fungi and viruses) and the functional characteristics of ticks warrant further exploration. To investigate the taxonomic and functional profiles and explore potential pathogens they were carrying, samples of different developmental stages and of both sexes of Haemaphysalis longicornis were collected in the present study and the metagenomic deep sequencing method was applied. Metagenomic deep sequencing results revealed that bacteria were predominant, followed by fungi, viruses, archaea and metazoans. Proteobacteria was the dominant phylum in the microbiota of H. longicornis. The abundance of microbial species varied significantly among groups, the bacteria of nymphs and female adults demonstrated unique characteristics, and the microbial community of males overlapped with those of nymphs and females. Functional annotation results demonstrated that the metagenomic sequences of the three groups were classified under metabolism, genetic information processing, environmental information processing and cellular processes. Differences in functional characteristics were observed in both the pathways composition and abundance of carbohydrate-active enzymes. Furthermore, whole metagenome sequencing helped to elucidate the diversity of pathogens carried by H. longicornis, which may facilitate further research attempting to prevent and control tick-borne diseases.

Targeting ADAR1 suppresses progression and peritoneal metastasis of gastric cancer through Wnt / ß-catenin pathway.

Objective: Peritoneal metastasis frequently occurs in advanced gastric cancer, which is typically not eligible for radical surgery. Here, this study observed the function and regulatory mechanism of ADAR1 in peritoneal metastasis of gastric cancer. Methods: ADAR1, CALR and ß-catenin proteins were detected in normal mucosa, primary gastric cancer, metastatic lymph node and metastatic omentum tissues by immunohistochemistry, western blot, and immunofluorescence. After silencing ADAR1 by siADAR1, the effect and mechanism of ADAR1 on gastric cancer metastasis were observed in nude mouse models of gastric cancer with peritoneal metastasis as well as HGC-27 and AGS gastric cancer cells. Result: Our results showed that ADAR1 was significantly up-regulated in gastric cancer, metastatic lymph node and metastatic omentum tissues. Its up-regulation was significantly correlated to lymph node metastasis and peritoneal metastasis. Silencing ADAR1 significantly reduced the volume of peritoneal metastatic tumors and weakened oncogene CALR expression, Wnt / ß-catenin pathway and epithelial-mesenchymal transition (EMT) process in vivo. Furthermore, ADAR1 knockdown distinctly suppressed cell viability, colony formation and migration of HGC-27 and AGS cells and ameliorated the effects of Wnt pathway activator on tumor progression. The similar findings were investigated when treated with ADAR1 inhibitor 8-Azaadenosine. Conclusion: Collectively, this study identified a novel oncogenic function of ADAR1 in peritoneal metastasis of gastric cancer via Wnt / ß-catenin pathway. Hence, ADAR1 could be a novel marker and therapeutic target against gastric cancer metastasis.

Microbiota assessment across different developmental stages of Dermacentor silvarum (Acari: Ixodidae) revealed stage-specific signatures.

Dermacentor silvarum is an important vector of tick-borne pathogens. The microbiota of ticks has been recognized to influence their development, fitness, and reproduction as well as the acquisition, establishment and transmission of pathogens. Eggs, larvae, nymphs and adults (females and males) of D. silvarum were used in this study to evaluate microbial community and diversity across different developmental stages. The results demonstrated that some developmental stages host different bacterial species, confirming that each stage of development could have a specific associated microbiota. Proteobacteria was the dominant phylum in most stages, while Actinobacteria was the most abundant in nymphs. The abundance of Brevibacterium significantly increased from the egg stage to the following stages, and there was a sharp increase in the abundance of this genus among nymphs. Eggs showed the highest microbial richness and diversity, and the microbial community of eggs exhibited high similarity to that of females. Moreover, the high abundance of Coxiella in eggs and females further indicated that members of this genus are vertically transmitted symbionts. Nymphs showed a microbial composition distinct from those of the other stages, and the microbial community associated with males possessed higher species richness and greater bacterial diversity than that of females. Further investigations are needed to determine the functions of the microbiota and provide information enabling a better understanding of the diversity of the microbial community at different life stages.

Characterization of microbiota diversity of field-collected Haemaphysalis longicornis (Acari: Ixodidae) with regard to sex and blood meals.

Haemaphysalis longicornis is a prominent tick species in China, and the major vector of an emerging tick-borne disease: severe fever with thrombocytopenia syndrome (SFTS). Microbiome diversity of ticks is influenced by several factors. In this study, we investigated microbiome diversity in field-collected female and male H. longicornis ticks and compared the microbial composition of fed and unfed ticks and of those feeding on different hosts using barcode sequencing of V3-V4 region of 16S RNA gene. Regardless of sex, host, and feeding status; the highest abundance among all samples was found for the genus Coxiella. The relative numbers of Coxiella sequences decreased with the length of the blood feeding, whereas the numbers of Staphylococcus and Corynebacterium increased gradually. The dominance of Coxiella across all samples indicates that it is an obligate symbiont of H. longicornis. Overall, higher microbiome richness was detected in male ticks than in female ticks. Fed ticks showed a more diverse microbe composition than unfed ticks, and ticks fed on goats exhibited the highest diversity. These findings of this study can serve as a basis for future studies of microbiota biology and interactions between the microbes and pathogens of H. longicornis.

Aloe Polysaccharides Inhibit Influenza A Virus Infection-A Promising Natural Anti-flu Drug.

Influenza A virus causes periodic outbreaks and seriously threatens human health. The drug-resistant mutants have shown an epidemic trend because of the abuse of chemical drugs. Aloe polysaccharides (APS) extracted from Aloe vera leaves have evident effects on the therapy of virus infection. However, the activity of APS in anti-influenza virus has yet to be investigated. Here, we refined polysaccharides from A. vera leaf. In vitro test revealed that APS could inhibit the replication of a H1N1 subtype influenza virus, and the most obvious inhibitory effect was observed in the viral adsorption period. Transmission electron microscopy indicated that APS directly interacted with influenza virus particles. Experiments on PR8 (H1N1) virus infection in mice demonstrated that APS considerably ameliorated the clinical symptoms and the lung damage of the infected mice, and significantly reduced the virus loads and mortality. Our findings provided a theoretical basis for the development of novel natural anti-influenza agents.

Anti-proliferation effects of interferon-gamma on gastric cancer cells.

IFN-γ plays an indirect anti-cancer role through the immune system but may have direct negative effects on cancer cells. It regulates the viability of gastric cancer cells, so we examined whether it affects their proliferation and how that might be brought about. We exposed AGS, HGC-27 and GES-1 gastric cancer cell lines to IFN-γ and found significantly reduced colony formation ability. Flow cytometry revealed no effect of IFN-γ on apoptosis of cell lines and no effect on cell aging as assessed by ß-gal staining. Microarray assay revealed that IFN-γ changed the mRNA expression of genes related to the cell cycle and cell proliferation and migration, as well as chemokines and chemokine receptors, and immunity-related genes. Finally, flow cytometry revealed that IFN-γ arrested the cells in the G1/S phase. IFN-γ may slow proliferation of some gastric cancer cells by affecting the cell cycle to play a negative role in the development of gastric cancer.

Evaluation of fetal skeletal malformations in deoxynivalenol-treated mice using microarray analysis.

Deoxynivalenol (DON [vomitoxin]), one of trichothecene mycotoxins produced by the fungus Fusarium, is commonly detected in cereal foods across the world. DON induces diverse toxic effects in humans and animals, including emesis and diarrhea, anorexia, and immunotoxicity, and impaired maternal reproduction and fetal development. Recently, the teratogenic potential of DON has been shown and has received much attention. DON can cause various skeletal deformities in fetuses, but the underlying mechanisms have not yet been fully examined. In this study, fetal skeletal malformations in DON-treated maternal mice were thoroughly investigated using microarray assay. The results showed that DON administration caused various skeletal defects in fetuses, including misaligned or fused sternebrae and vertebrae, divided or fused ribs and polydactyly, hemivertebrae, short toes, and tail anomalies. Microarray analysis showed that 282 genes, including 148 downregulated and 134 upregulated genes, were abnormally expressed in fetal vertebral bones after maternal DON exposure. These identified genes can be classified into several categories: skeletal development, carcinogenesis, nervous disorders, sperm development and embryogenesis, and inflammation. Of these, 6 genes, mostly related to bone development, were intentionally selected for further validation using real-time reverse transcription-Polymerase Chain Reaction (RT-PCR). It was confirmed that the mRNA expression of 4 genes, i.e., fibrillin-1, Col9A2, 3'-phosphoadenosine 5'-phosphosulfate synthase 2, and Pax1, was upregulated significantly by DON administration, whereas that of 2 other genes, Runx2 and parathyroid hormone-like hormone, was downregulated significantly. Taken together, the results of our study suggest that altered expression of these 6 genes plays a critical role in fetal skeletal deformities induced by DON and thus they are worthy of further investigation.

[Construction of a recombinant eukaryotic expression vector containing HSV 2gD and its immune responses].

AIM: To construct and express pcDNA3.1-IL-18-HSV P6 recombinant DNA vaccine, and to observe the immune responses of pcDNA3.1-IL-18-HSV P6 genetic vaccine. METHODS: Genes of P6 and IL-18 were cloned into the eukaryotic expression vector pcDNA3.1(-) respectively, and confirmed by enzyme digestion and sequence analysis, the recombinant plasmid pcDNA3.1-IL-18-HSVP6 was transformed into CHO cells. The expressed protein was characterized by indirect immunofluorescence and Western blotting. The recombinant plasmid pcDNA3.1-IL-18-HSVP6 was used to inoculate BALB/c mice by intramuscular injection for three times, once a week. One week after the last vaccination, the levels of specific IgG antibody, IFN-γ and IL-18 were detected by ELISA; One month after the last vaccination, spleen cells of vaccinated mice were separated and proliferation of spleen lymphocytes were detected by MTT assay. RESULTS: Recombinant pcDNA3.1-IL-18-HSV P6 plasmid was confirmed by enzyme digestion and DNA sequencing. After inoculated by pcDNA3.1-IL-18-HSV P6 vaccine, the mice could produce higher level of splenocytes proliferation and secrected higher level of IFN-γ, IL-18 and specific antibody. CONCLUSION: DNA vaccine of pcDNA3.1-IL-18-HSV P6 has been successfully constructed, and it can effectively induce humoral and cellular immune responses, which provided a basis for constructing new type of DNA vaccine.

The variable codons of H5N1 avian influenza A virus haemagglutinin genes.

We investigated the selection pressures on the haemagglutinin genes of H5N1 avian influenza viruses using fixed effects likelihood models. We found evidence of positive selection in the sequences from isolates from 1997 to 2007, except viruses from 2000. The haemagglutinin sequences of viruses from southeast Asia, Hong Kong and mainland China were the most polymorphic and had similar nonsynonymous profiles. Some sites were positively selected in viruses from most regions and a few of these sites displayed different amino acid patterns. Selection appeared to produce different outcomes in viruses from Europe, Africa and Russia and from different host types. One position was found to be positively selected for human isolates only. Although the functions of some positively selected positions are unknown, our analysis provided evidence of different temporal, spatial and host adaptations for H5N1 avian influenza viruses.

Detection of Escherichia coli O157 using equal-length double-stranded fluorescence probe in a real-time polymerase chain reaction assay.

BACKGROUND: Enterohemorrhagic Escherichia coli (E. coli) O157 is a dangerous pathogen, which causes bloody diarrhea and severe hemolytic uremic syndrome (HUS). Although several assay systems based on real-time polymerase chain reaction (PCR) have been integrated to detect this pathogen, most of them are not specific. We report a real-time quantitative PCR method targeting rfbE, a gene specifically expressed in E. coli O157. This method can therefore be used to diagnose enterohemorrhagic Escherichia coli (E. coli) O157. METHODS: A nucleic acid based diagnostic assay system, combining equal-length double-stranded fluorescence probe technique and real-time PCR, was developed to detect E. coli O157. This assay system take advantage of the highly conserved rfbE O-antigen synthesis gene, and a pair of fluorescence-quenching probes complementary to rfbE gene were used in a real-time PCR to quantify the presence of the pathogen. RESULTS: The specificity of the diagnostic method was assessed by comparing test results on 14 different related pathogens including common E. coli, enteroinvasive Escherichia coli (EIEC), Salmonella, Shigella and E. coli O157. The detection limit of the method was determined using 10-fold serial dilutions of an E. coli O157 standard sample, and as few as 1.49 x 10(3) CFU/ml could be detected. All E. coli with serotype O157, which expresses rfbE gene, were positive in this assay, while all other species without rfbE gene expression were negative. CONCLUSIONS: By combining equal-length double-stranded fluorescence probe technique and real-time PCR, we have developed a simple, rapid, specific and sensitive method to detect E. coli O157.