Mice fatal pneumonia model induced by less-virulent Streptococcus pneumoniae via intratracheal aerosolization.

Aim: Animal models of fatal pneumonia caused by Streptococcus pneumoniae (Spn) have not been reliably generated using many strains of less virulent serotypes. Materials & methods: Pulmonary infection of a less virulent Spn serotype1 strain in the immunocompetent mice was established via the intratracheal aerosolization (ITA) route. The survival, local and systemic bacterial spread, pathological changes and inflammatory responses of this model were compared with those of mice challenged via the intratracheal instillation, intranasal instillation and intraperitoneal injection routes. Results: ITA and intratracheal instillation both induced fatal pneumonia; however, ITA resulted in better lung bacterial deposition and distribution, pathological homogeneity and delivery efficiency. Conclusion: ITA is an optimal route for developing animal models of severe pulmonary infections.

What is this article about? Streptococcus pneumoniae (Spn), a type of bacteria, can cause serious illness and death in otherwise healthy people. One way that we study pneumonia is using animals. However, pneumonia in animals infected with Spn in the laboratory does not mimic that in humans very well. To study this illness, we need a new way to set up a proper animal model.What were the results? This study set up a method called intratracheal aerosolization (ITA). In ITA, bacteria can form small droplets called aerosols and reach the deepest parts of a mouse's lung. ITA can cause deadly illness in mice infected with Spn, even if the mice are healthy.What do the results of the study mean? The ITA method could be a useful tool to set up animal models of serious pneumonia with less virulent bacteria.

Aedes albopictus salivary adenosine deaminase is an immunomodulatory factor facilitating dengue virus replication.

The Asian tiger mosquito, Aedes albopictus, is an important vector for the transmission of arboviruses such as dengue virus (DENV). Adenosine deaminase (ADA) is a well-characterized metabolic enzyme involved in facilitating blood feeding and (or) arbovirus transmission in some hematophagous insect species. We previously reported the immunologic function of ADA by investigating its effect on mast cell activation and the interaction with mast cell tryptase and chymase. The 2-D gel electrophoresis and mass spectrometry analysis in the current study revealed that ADA is present and upregulated following mosquito blood feeding, as confirmed by qRT-PCR and western blot. In addition, the recombinant ADA efficiently converted adenosine to inosine. Challenging the Raw264.7 and THP-1 cells with recombinant ADA resulted in the upregulation of IL-1ß, IL-6, TNF-α, CCL2, IFN-ß, and ISG15. The current study further identified recombinant ADA as a positive regulator in NF-κB signaling targeting TAK1. It was also found that recombinant Ae. albopictus ADA facilitates the replication of DENV-2. Compared with cells infected by DENV-2 alone, the co-incubation of recombinant ADA with DENV-2 substantially increased IL-1ß, IL-6, TNF-α, and CCL2 gene transcripts in Raw264.7 and THP-1 cells. However, the expression of IFN-ß and ISG15 were markedly downregulated in Raw264.7 cells but upregulated in THP-1 cells. These findings suggest that the immunomodulatory protein, Ae. albopictus ADA is involved in mosquito blood feeding and may modulate DENV transmission via macrophage or monocyte-driven immune response.

Genomics evolution of Jingmen viruses associated with ticks and vertebrates.

Jingmen virus (JMV) associated with ticks and vertebrates have been found to be related to human disease. We obtained the genome of a Jingmen tick virus (JMTV) strain from Rhipicephalus microplus in Guizhou province and compared the genomes of seven JMV species associated with ticks and vertebrates to understand the evolutionary relationships. The topology of the phylogenetic tree of segment 1 and segment 3 is similar, and segment 2 and segment 4 formed two different topologies, with the main differences being between Alongshan virus (ALSV), Takachi virus, Yanggou tick virus and Pteropus lylei jingmen virus (PLJV), and the possibility of genetic reassortment among these viruses. Moreover, we detected recombination within JMTV and between PLJV and ALSV. The genetic reassortment and recombination that occurs during cross-species transmission of these JMV associated with ticks and vertebrates not only complicates their evolutionary relationships, but also raises the risk of these viruses to humans.

Aedes albopictus salivary proteins adenosine deaminase and 34k2 interact with human mast cell specific proteases tryptase and chymase.

When mosquitoes probe to feed blood, they inoculate a mixture of salivary molecules into vertebrate hosts' skin causing acute inflammatory reactions where mast cell-derived mediators are involved. Mosquito saliva contains many proteins with largely unknown biological functions. Here, two Aedes albopictus salivary proteins - adenosine deaminase (alADA) and al34k2 - were investigated for their immunological impact on mast cells and two mast cell-specific proteases, the tryptase and the chymase. Mouse bone marrow-derived mast cells were challenged with increased concentrations of recombinant alADA or al34k2 for 1, 3, and 6 h, and to measure mast cell activation, the activity levels of ß-hexosaminidase and tryptase and secretion of IL-6 were evaluated. In addition, a direct interaction between alADA or al34k2 with tryptase or chymase was investigated. Results show that bone marrow-derived mast cells challenged with 10 µg/ml of alADA secreted significant levels of ß-hexosaminidase, tryptase, and IL-6. Furthermore, both al34k2 and alADA are cut by human tryptase and chymase. Interestingly, al34k2 dose-dependently enhance enzymatic activity of both tryptase and chymase. In contrast, while alADA enhances the enzymatic activity of tryptase, chymase activity was inhibited. Our finding suggests that alADA and al34k2 via interaction with mast cell-specific proteases tryptase and chymase modulate mast cell-driven immune response in the local skin microenvironment. alADA- and al34k2-mediated modulation of tryptase and chymase may also recruit more inflammatory cells and induce vascular leakage, which may contribute to the inflammatory responses at the mosquito bite site.

Roles of genetic and microenvironmental factors in cancer epithelial-to-mesenchymal transition and therapeutic implication.

Epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells lose their cell-cell contacts resulting in the formation of mesenchymal cells with migratory properties. Increasing evidence indicate EMT plays a key role in the invasion, metastasis and therapeutic resistance of cancer and maintenance of the phenotype of cancer stem cells (CSCs), which makes the prognosis of patients worse. The progression of cancer from epithelial tissue towards a malignant phenotype is driven by multiple factors that remodel the tissue architecture. This review summarizes and analyzes current studies of genetic and microenvironmental factors in inducing and maintaining cancer EMT and therapeutic implications. This will enable a better understanding of the contribution of EMT-associated factors to cancer progression and highlights that genetic factors and tumor microenvironment responsible for EMT could be used as attractive targets for therapeutic intervention.

[Identification and Expression of the yellow Gene Family in Aedes aegypti].

Objective: To identify the yellow family genes in Aedes aegypti and analyze the gene structure, phylogenetic evolution and their expression at various developmental stages and in different tissues. Methods: The yellow gene family was identified in Ae. aegypti by blasting the Ae. aegypti genome database with the amino acid sequence of the MRJP domain of Dm-yellow gene of Drosophila melanogaster（GenBank No. AAF45497）. The physico-chemical property and domains were analyzed with the on-line ExPaSy software. The signal peptide was predicted using SignalP4.1 software. Sequence alignment and the phylogenetic tree were made through combined use of DNAstar, MEGA6.0 and GeneDoc. Total RNA was extracted from Ae. aegypti, cDNA was generated, and expression of the yellow family genes at various developmental stages （egg, first to fourth instar, pupa, non-blood-fed female and male mosquitoes） and in different tissues （salivary gland, midgut, fat body, and ovary） was quantified using qRT-PCR. Results: Twelve yellow genes were identified from Ae. aegypti genome: Aa-yellow, Aa-yellow-b, Aa-yellow-c, Aa-yellow-d, Aa-yellow-e, Aa-yellow-f2, Aa-yellow-fb, Aa-yellow-fc, Aa-yellow-g, Aa-yellow-g2, Aa-yellow-h, and Aa-yellow-x. Bioinformatics demonstrated that all covered the MRJP domain and a signal peptide sequence. Sequence alignment revealed low （15%-49%） homology among the proteins, but high homology（60%） in the conserved domain. According to the phylogenetic tree analysis, the encoded 12 YELLOW proteins were classified into 5 subfamilies, and 11 had orthologues in D. melanogaster. qRT-PCR revealed high expression of Aa-yellow-d （0.018 9） and Aa-yellow-x （0.023 5） in male Ae. aegypti （P<0.01 or P<0.05）; high expression of Aa-yellow-fc （0.024 8, 0.034 9） in female Ae. aegypti and in the salivary gland （P<0.01）; high expression of Aa-yellow-f2 （0.093 4） in the second instar stage （P<0.01）; high expression of Aa-yellow （0.562 1）, Aa-yellow-e （0.004 4）, and Aa-yellow-fb （0.008 4） in the third instar stage （P<0.05）; and high expression of Aa-yellow （0.569 4）, Aa-yellow-e （0.027 0）, Aa-yellow-f2 （0.006 5）, Aa-yellow-fb （0.001 0）, Aa-yellow-h （0.084 8） and Aa-yellow-x （0.015 1） in the ovary. Genes other than Aa-yellow-c （0.004 0） and Aa-yellow-x （0.007 4） were hardly expressed in the midgut. Conclusion: The 12 yellow genes identified in the Ae. aegypti genome have low homology, and are differentially expressed at different developmental stages and in tissues.

Antivirulence properties of an antifreeze protein.

As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.

Naturally mutated envelope protein domain I of Chinese B dengue virus attenuated human dendritic cell maturation.

Dengue virus (DENV) can infect human dendritic cells (DCs), and cause a spectrum of clinical symptoms. Envelope protein of DENV contains three distinct domains, including domain I (DI), domain II (DII) and domain III (DIII), and plays important roles in receptor binding and induction of protective antibodies. Previously, a new DENV-2 type virus (named B strain) with eight gene mutations in DI of the envelope protein was isolated from a dengue hemorrhagic fever patient. BALB/c mice infected with DENV B strain showed more prolonged viremia than mice infected with the New Guinea C (NGC) strain. However, the mechanism of prolonged viremia was not determined. In this study, DI proteins derived from B and NGC strains of DENV were expressed in Rosetta (DE3) host bacteria and purified by affinity chromatography after refolding. A flow cytometry-based binding assay and confocal microscopy indicated that both proteins could bind to human DCs induced from peripheral blood mononuclear cells (PBMCs), but DI of the B strain had a lower affinity than DI of the NGC strain, and viable B virus also show less binding efficiency with DCs. In addition, DI of the NGC strain, but not the B strain, induced IL-12 secretion and phenotypic maturation of DCs, such as up-regulated expression of CD80, CD83, CD86 and HLA-DR. NGC strain could induce more virus specific IgM/IgG. These results suggest that the naturally mutated envelope protein DI of the Chinese B strain of DENV cannot induce DC maturation as high efficiency as that of NGC strain, which may be the partial reason that DENV B strain escapes immune recognition and induce prolonged viremia. The mutated B strain envelope protein is not a good candidate for subunit vaccine target.

[Recombinant plasmid constructed and cytotoxicity studied for outer membrane protein LipL32 gene of Leptospira strain 017].

OBJECTIVE: To construct the recombinant plasmid containing the outer membrane protein LipL32 gene of Leptospira strain 017 and to study on the cytotoxicity of the expression protein. METHODS: By the polymerase chain reaction (PCR), the LipL32 gene was amplified from Leptospira strain 017 genome and cloned into pET32a(+) with enzyme digestion, then used to transform E. coli JM109. After induced with IPTG, the target protein was expressed and used to immunize New zealand white rabbit. Western Blotting identified the immunogenicity of the expressed protein. Then the purified and renatured protein was acted on ECV304 cell so as to get its cytotoxicity detected by examining the LDH and NO (nitrogen monoxide) release from cell. RESULTS: The full length of the LipL32 gene about 816 bp was obtained by PCR. The recombinant plasmid was identified by enzyme digestion, PCR and DNA sequencing. After induced with IPTG, the expressed protein existed mainly in the form of inclusion bodies about 52 x 10(3) (relative molecular mass) which was consistent with the expected size of the fused protein. After rabbit immunity, the titre of the produced multiclonal antibody reached 1 : 32 000 measured by ELISA. Western Blotting analysis found a positive band specifically in the target protein position. The release of the LDH and NO of the ECV304 cell treated with LipL32 had significant increase compared with the control group. CONCLUSION: The recombinant plasmid containing LipL32 gene is successfully constructed and can express the target protein in E. coli JM109. The expressed target protein has cytotoxicity.

[RelE toxin protein of Mycobacterium tuberculosis induces growth inhibition of lung cancer A-549 cell].

OBJECTIVE: To investigate whether the RelE toxin protein of mycobacterium tuberculosis has a growth inhibition effect on lung cancer A-549 cell. METHODS: The complete open-reading frame sequences of RelE, RelB and RelBE genes were amplified by PCR with using M. tuberculosis H37Rv genomic DNA as the template. The RelE, RelB and RelBE genes were subcloned into PcDNA3. 1 (+). After being verified with restriction endonuclease digestion and DNA sequence determination, the recombinant vectors were applied to transfect lung cancer A-549 cells by liposome transfection method. By determining the growth curve and protein level of living cells, MTT cell proliferation assay, the apoptosis of cells with HE staining and the apoptosis rate of transient transfection cells detected by Flow Microfluorimetry, it was observed and analyzed whether the RelE toxin protein of mycobacterium tuberculosis had a growth inhibition and apoptosis effects on lung cancer A-549 cell. RESULTS: The cell proliferation rate of lung cancer A-549 cells effected by the RelE toxin protein of mycobacterium tuberculosis was lower than that of the other groups, and this group cells with RelE protein effect showed more sensitive to nutrition starving. HE staining revealed that this group cells which included the transient transfection with RelE expression plasmid appeared to have more apoptosis cells and higher apoptotic rate than other groups did (P < 0.05). CONCLUSION: The RelE toxin protein of mycobacterium tuberculosis has growth inhibition and apoptotic effect on lung cancer A-549 cell.

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

AIM: To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. METHODS: The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. RESULTS: The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. CONCLUSION: The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

[Protein-protein interaction between hypothetical protein Rv1246c and Rv1247c of Mycobacterium tuberculosis].

OBJECTIVE: To investigate the protein-protein interaction between hypothetical protein Rv1246c and Rv1247c of Mycobacterium tuberculosis. METHODS: By PCR technique, the complete open-reading frame sequences of Rv1246c and Rv1247c gene were amplified from the M. tuberculosis H37Rv genomic DNA as template. The PCR-amplified cDNAs of Rv1247c and Rv1246c gene were subcloned into pGBKT7 and pGADT7-Rec vector respectively for constructing recombinant plasmids pGBKT7-Rv1247c and pGADT7-Rv1246c. After verified by restriction endonuclease digestion and DNA sequence determination, the recombinant vectors were used to transform the yeast cell AH109 by lithium acetate method. RESULTS: The yeast cells co-transformed with pGBKT7-Rv1247c and pGADT7-Rv1246c grew on SD/-Ade/-His/-Leu/-Trp plates, and the beta-galactosidase activity assays showed the positive signal. CONCLUSION: The hypothetical protein Rv1246c and Rv1247c could interact with each other in yeast cells.

[Effect of transfected Rv0901 gene on the activity of mice macrophages].

OBJECTIVE: To test the effect of Rv0901 gene of Mycobacterium tuberculosis on the activity of mice macrophages. METHODS: Peritoneal macrophages of mice were isolated and transfected with pcDNA3. 1 or pcDNA3. 1-Rv0901 plasmid DNA, along with the GFP DNA. The effectiveness of the transfection was detected by RT-PCR. The expression of the GFP and the apoptosis ratio of the macrophages were detected by FCM 72 hours after the transfection. The level of nitric oxide and IFN-gamma in cultural supernatant were also measured 72 hours after transfection. RESULTS: All of the macrophages being transfected had the expression of GFP. After being transfected with pcDNA3. 1-Rv0901, the 528 bp gene of Rv0901 was amplified by RT-PCR. The macrophages transfected with pcDNA3. 1-Rv0901 had higher apoptosis ratio [(56.4 +/- 2.0)% vs (19.9 +/- 1.5)%] and released more nitric oxide [(40.4 +/- 3.0) micromol/L vs (27.5 +/- 3.2) micromol/L] and IFN-gamma [(2.11 +/- 0.031) ng/mL vs (0.62 +/- 0.025) ng/mL] in the cultural supernatants than those transfected with pcDNA3. 1 (P < 0.05). CONCLUSION: Transient transfection of pcDNA3. 1-Rv0901 increases the apoptosis ratio of the macrophages, which could increase the release of nitric oxide and IFN-gamma.

[Molecular cloning and expression of hypothetical proteins Rv1494 and Rv1495 of M.tuberculosis H37Rv strain].

OBJECTIVE: The mechanism by which M.tuberculosis persists and survives in host macrophage is not fully understood, however, the M. tuberculosis chromosome-encoded TA loci perform functions possibly of signaling to these processes. To explore the biological functions of M. tuberculosis chromosome-encoded TA loci, the Rv1494 and Rv1495 genes of M.tuberculosis H37Rv strain were cloned and expressed. METHODS: The hypothetical proteins Rv1494 and Rv1495 were bioinformatically analyzed by means of Bioedit software, Dnaman software and Pfam database. The complete open-reading frame sequences of Rv1494 and Rv1495 genes were amplified by PCR using M.tuberculosis H37Rv genomic DNA as the template, and the PCR products were cloned into prokaryotic expression vector pET32a(+), respectively. After induction of expressions in E.coli host strain BL21 (DE3), the recombinant proteins were purified and detected by Western blotting. RESULTS: According to bioinformatic analysis, the hypothetical proteins of Rv1494 and Rv1495 genes shared some homologies with mazEF family, one of E. coli chromosomal TA loci (homology at 26% and 29.5%). Sequence analysis showed that the inserted target genes and its reading frames were completely correct. The recombinant plasmids were induced with IPTG to effectively express the fusion proteins with relative molecular mass coincident with prediction. The specific positive signals were identified from the immunoblots. CONCLUSION: For the first time, the Rv1494 and Rv1495 genes of M.tuberculosis H37Rv strain were cloned and its prokaryotic expression vectors were constructed successfully in this experiment, which may facilitate further functional study of this mazEF-like gene pair.