Systemic neutrophils are triggered by respiratory Bacillus Calmette- Guérin and mediate pulmonary mycobacterial clearance in synergy with the triggering receptor expressed on myeloid cells 1.

Tuberculosis remains a threat to public health. The only approved vaccine, Bacillus Calmette-Guérin (BCG), is administered intradermally and provides limited protection, and its effect on innate immunity via the respiratory route has not been fully elucidated. A mouse model with genetically depleted TREM1 and seven-color flow cytometry staining were used to characterize the comprehensive immune response induced by respiratory BCG, through evaluating organ bacterial loads, lung histopathology, and lung immunohistochemistry. During respiratory BCG infection, the murine lungs displayed effective bacterial clearance. Notably, marked differences in neutrophils were observed between thymus and bone marrow cells, characterized by a significant increase in the expression of the triggering receptor expressed on myeloid cells 1 (TREM1). Subsequently, upon depletion of TREM1, a reduction in pulmonary neutrophils was observed, which further exacerbated bacterial loads and resulted in worsened pathology following respiratory BCG infection. In summary, up-regulated expression of TREM1 in rapidly increasing circulating neutrophil by pulmonary BCG is required for an efficient host response to BCG infection, and suggests the important role of TREM1 in neutrophil-related pulmonary bacteria clearance and pathology.

Aristolochia mimics stink bugs to repel vertebrate herbivores via TRPA1 activation.

Mimicry is the phenomenon in which one species (the mimic) closely resembles another (the model), enhancing its own fitness by deceiving a third party into interacting with it as if it were the model. In plants, mimicry is used primarily to gain fitness by withholding rewards from mutualists or deterring herbivores cost-effectively. While extensive work has been documented on putative defence mimicry, limited investigation has been conducted in the field of chemical mimicry. In this study, we used field experiments, chemical analyses, behavioural assays, and electrophysiology, to test the hypothesis that the birthwort Aristolochia delavayi employs chemical mimicry by releasing leaf scent that closely resembles stink bug defensive compounds and repels vertebrate herbivores. We show that A. delavayi leaf scent is chemically and functionally similar to the generalized defensive volatiles of stink bugs and that the scent effectively deters vertebrate herbivores, likely through the activation of TRPA1 channels via (E)-2-alkenal compounds. This study provides an unequivocal example of chemical mimicry in plants, revealing intricate dynamics between plants and vertebrate herbivores. Our study underscores the potency of chemical volatiles in countering vertebrate herbivory, urging further research to uncover their potentially underestimated importance.

Bioinformatics analysis of differentially expressed proteins in alcoholic fatty liver disease treated with recombinant human cytoglobin.

Cytoglobin (Cygb) is a globin molecule that is ubiquitously expressed in all tissues and has a protective role under oxidative stress. It has also been demonstrated to be effective in the treatment of alcoholic fatty liver disease (AFLD). In order to study the molecular mechanisms underlying its beneficial effects for the treatment of alcoholic liver, two­dimensional electrophoresis and mass spectrometric analysis were performed on serum and liver tissues from an in vivo rat model of AFLD. A total of 26 differentially expressed proteins were identified in the serum and 20 differentially expressed proteins were identified in liver specimens. Using online bioinformatics tools, it was indicated that these differentially expressed proteins were primarily associated with pathways including binding and uptake of ligands by scavenger receptors, response to corticosteroid, plasma lipoprotein remodeling, regulation of complement cascade, hydrogen peroxide catabolic process, as well as response to nutrient and monosaccharide. The present results suggested that recombinant human Cygb exerts its role in the treatment of AFLD primarily through affecting nutrient metabolism, monocarboxylic acid biosynthesis, regulation of glutathione expression, plasma lipoprotein remodeling and removal of metabolic waste from the blood.

Impact on growth, oxidative stress, and apoptosis-related gene transcription of zebrafish after exposure to low concentration of arsenite.

Low concentrations of arsenic (As) contamination in aquatic environment is a worldwide issue, which is of great concern. To evaluate the impact of low concentrations of As on zebrafish, we measured the growth, antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), oxidative damage (malondialdehyde, MDA) and apoptosis-related genes (nrf2, p53 and c-jun) of adult zebrafish after exposing to different AsIII concentrations (0, 10, 50, 100 or 150 µg L-1) for 28 d. Results indicated that exposure to low AsIII concentrations decreased the zebrafish weight by 14%, increased the activities of SOD and CAT by 23-41% and 31-59%, decreased the contents of MDA by 29-54%, and modulated transcription of apoptosis related genes. Our study showed that chronic exposure to AsIII concentrations <150 µg L-1 generated oxidative stress and damage on zebrafish, and altered apoptosis-related genes in zebrafish.

The influence of ESR1 rs9340799 and ESR2 rs1256049 polymorphisms on prostate cancer risk.

Estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) may play a role in the development of prostate cancer. Many studies focused on ESR1 rs9340799 and ESR2 rs1256049 polymorphisms to explore associations with prostate cancer risk. These studies showed inconsistent and conflicting results. The aim of this meta-analysis was to investigate the pooled association of ESR1 rs9340799 and ESR2 rs1256049 polymorphisms with prostate cancer risk. A systematic literature search was conducted to identify related studies (up to February 2014) in several online databases including PubMed, Google Scholar, CNKI and Wanfang online libraries. A total of 16 eligible articles were enrolled in this updated meta-analysis. The result suggested that ESR1 rs9340799 polymorphism was significantly associated with prostate cancer in overall populations (GG+GA vs. AA: P = 0.002; G vs. A: P = 0.004), Caucasians (GG+GA vs. AA: P = 0.008; G vs. A: P = 0.016) and Africans (GG+GA vs. AA: P = 0.005; G vs. A: P = 0.006), but not in Asians (GG+GA vs. AA: P = 0.462; G vs. A: P = 0.665). The result also showed that there was a significant association between ESR2 rs1256049 polymorphism and prostate cancer in Caucasians (AA+AG vs. GG: P = 0.016; A vs. G: P = 0.005), but no association in overall populations (AA+AG vs. GG: P = 0.826; A vs. G: P = 0.478), Asians (AA+AG vs. GG: P = 0.177; A vs. G: P = 0.703) and Africans (AA+AG vs. GG: P = 0.847; A vs. G: P = 0.707). The cumulative meta-analysis and sensitivity analysis showed the results were robust. In conclusion, this meta-analysis indicated that ESR1 rs9340799 polymorphism was associated with prostate cancer risk in overall populations, Caucasians and Africans, while ESR2 rs1256049 polymorphism was associated with prostate cancer risk in Caucasians. However, the biological mechanisms need to be further investigated.

Production of mouse monoclonal antibodies against Helicobacter pylori Catalase and mapping the antigenic epitope by phage display library.

The Catalase of Helicobacter pylori (H. pylori) helps bacteria to protect themselves from oxygen toxicity and damage and have been identified an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against Catalase and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, MAbs were produced by the hybridoma technique using recombinant Catalase--GST as the immunogen and were immunoscreened against phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 34 phage clones were randomly selected and their specificity to mAb was verified by sandwich and competitive inhibition ELISA. Fifteen phage clones were sequenced and their amino acids were deduced. One mimotope (SVSLPYANLATH) showed good match with Catalase protein at 394-405aa and the serum of mice induced by the phage clone clearly recognized Catalase protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Catalase would provide an alternative approach for the development of a vaccine for H. pylori.

[A novel system for producing lentiviral vectors].

The aim was to develop a cell culture system capable of producing high titer lentiviral vector stocks with recombinant vaccinia viruses as helpers. BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL and the envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamine2000. After 4 days incubation, the culture supernatant of lentiviral vectors was collected and judged by RT-PCR and the Western blot, the results showed that lentiviral vectors were found in the culture supernatant; approximately (11.71 +/- 0.80) x 10(11) copies of lentiviral vector RNA were present per mL of cell culture supernatant, as detected by Real-time PCR; the vector stocks with titers was up to (1.3 +/- 0.18) x 10(8) tu/mL, as detected by flow cytometry , which is one order of magnitude higher than the output of classical manufacture system. These results suggest that the new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established. It provides the basis for the future development of industrial application.

Production of mouse monoclonal antibodies against Helicobacter pylori Lpp20 and mapping the antigenic epitope by phage display library.

Lpp20, an outer membrane protein of Helicobacter pylori (H. pylori), has been identified as an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against it and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, the Lpp20 gene was obtained from H. pylori genomic DNA by PCR (GenBank accession no. DQ106902), cloned into pGEX-4T-1 vector and expressed in Escherichia coli (E. coli) as a recombinant fusion protein with glutathione-S-transferase (GST), which was purified by GST-affinity chromatography. mAbs were produced by the hybridoma technique using Lpp20-GST as the immunogen. Using mAb as the target molecule and immunoscreening phage-displayed random dodecapeptide library (Ph.D.-12), the positive phage clones were sequenced and analyzed. Phage clones were chosen to immunize mice to evaluate the potential of phagotopes as effective vaccines. One mimotope (SWPLYSDASGLG) showed a good match with the Lpp20 proteins at 114-117aa (DASG) and the serum of mice induced by the phage clone clearly recognized Lpp20 protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Lpp20 providing an alternative approach for the diagnosis and development of a vaccine for H. pylori.

[Therapeutic effect of Ginkgo biloba leaf extract on hypercholestrolemia in children with nephrotic syndrome].

OBJECTIVE: To explore the therapeutic effects of the extract of Ginkgo biloba leaf on hypercholestrolemia in children with primary nephritic syndrome (NS). METHODS: Thirty-five children with NS were randomized into 2 groups for treatment with prednisone plus Ginkgo biloba leaf extract (18 cases) or with prednisone plus dipyridamole (17 cases) for 8 weeks. After completion of the treatments, the therapeutic effects were evaluated and the changes in the blood biochemical markers assayed. RESULTS: The 8-week treatment with the extract significantly ameliorated the clinical symptoms and blood biochemistry as compared with prednisone plus dipyridamole group (P<0.01). The levels of urinic protein and blood lipid in Ginkgo leaf group were significantly lower than those in prednisome plus dipyridamole group (P<0.05). CONCLUSION: The extract from Ginkgo biloba leaf can lower blood lipid levels and urinic protein in children with NS and improve their clinical syptoms and the renal function, therefore has much clinical value as an adjuvant treatment of steroid therapy in such children.

[Cloning, expression and identification of catalase of Helicobacter pylori].

AIM: To construct the recombinant plasmid containing catalase (KatA) of Helicobacter pylori (Hp), analyze its nucleic acid sequence, express it in E. coli and study its antigenicity. METHODS: KatA fragments were amplified from Hp chromosomal DNA by PCR. Its T-A was cloned, sequenced and compared with other HP strains on the GenBank. Then the gene cloned into pGEX-4T-1 fusion expression vector was expressed in E. coli and purified by GST-affinity chromatography. The purified product was used to identify 29 stains of mouse anti Hp monoclonal antibodies and analyze antigenicity with serum of Hp-infected patients by Western blot. RESULTS: KatA fragments were composed of 1,515 bp (GenBank No. DQ333889) and the nucleotide homology with other Hp strains on the GenBank was 96%-97%. 85 kDa of the recombinant KatA-pGEX-4T-1 was expressed in E. coli. 4 of 29 anti-Hp mouse monoclonal antibodies were against KatA. Western blot analysis proved that KatA was specifically recognized in the serum of Hp-infected patients. CONCLUSION: The recombinant KatA has original antigenicity. It is of great value to clinical sero-diagnosis and vaccine study of Hp.

[Preparation and identification of monoclonal antibodies against Helicobacter pylori].

OBJECTIVE: To prepare and identify monoclonal antibodies (mAbs) against Helicobacter pylori (Hp). METHODS: BALB/c mice were immunized with the supernatant and precipitation of cultured Hp after ultrasonication and mAbs were obtained by means of hybridoma technique. The resultant mAbs was evaluated for subtype, titer, affinity, and further identified with Lpp20, HspA, urease A, CagA, urease B, and catalase prepared by recombinant expression. RESULTS: Totally 34 hybridoma cell lines were established which secreted specific mAbs, including 31 against the supernatant and 3 against the precipitation of Hp, and the prepared mAbs showed specific reaction against Lpp20 (3 strains), HspA (2 strains), urease A (4 strains), CagA (1 strain), urease B (5 strains), and catalase (2 strains) antigens, respectively. The mAbs was all identified as immunoglobulin G1 (IgG1) and theirs titer in the culture supernatant and ascites was 1:16 to 1:32 and 1:32000 to 1:64000 respectively with affinity constants (K(aff)) ranging from 1 x 10(-10) to 5.2 x 10(-12) mol/L. CONCLUSION: The mAbs specially against Hp have been obtained, which may facilitate further study of detection and vaccine development of Hp.

[Preparation of a monoclonal antibodies against Plasmodium falciparum glutamate dehydrogenase and establishment of colloidal gold-immunochromatographic assay].

OBJECTIVE: To prepare a monoclonal antibodies (mAbs) against glutamate dehydrogenase (GDH) of Plasmodium falciparum (FCC1/HN strain) and establish colloidal gold-immunochromatographic assay (GICA) for diagnosis of Plasmodium falciparum malaria. METHODS: Recombinant GDH was used to immunize Balb/C mice and the mAbs against GDH were prepared using hybridoma technique followed by identification of IgG isotype and its affinity. Protein-G affinity chromatography was employed to purify the antibodies, which were labeled with colloidal gold for establishment of GICA for Plasmodium falciparum detection. RESULTS: Six mAbs were obtained and identified as IgG1(kappa) of IgG isotypes with affinity constants (Kaff) ranging from 1 x 10(-8) to 2.8 x 10(-10). GICA had a sensitivity of 86.66%; and specificity of 96.43%; for Plasmodium falciparum detection compared with routine microscopic examination. CONCLUSION: The established GICA is rapid and accurate for Plasmodium falciparum detection with such potential utility as for instant diagnosis of Plasmodium falciparum malaria.

A vaccinia replication system for producing recombinant hepatitis C virus.

AIM: To develop a cell culture system capable of producing high titer hepatitis C virus (HCV) stocks with recombinant vaccinia viruses as helpers. METHODS: Two plasmids were used for the generation of recombinant HCV: one containing the full-length HCV cDNA cloned between T7 promoter and T7 terminator of pOCUS-T7 vector, and the other containing the HCV polyprotein open reading frame (ORF) directly linked to a vaccinia late promoter in PSC59. These two plasmids were co-transfected into BHK21 cells, which were then infected with vTF7-3 recombinant vaccinia helper viruses. RESULTS: After 5 d of incubation, approximately 3.6X10(7) copies of HCV RNA were present per milliliter of cell culture supernatant, as detected by fluorescence quantitative RT-PCR (FQ-PCR). The yield of recombinant HCV using this cell system increased 100- to 1 000- fold compared to in vitro- transcribed HCV genomic RNA or selective subgenomic HCV RNA molecule method. CONCLUSION: This cell culture system is capable of producing high titer recombinant HCV.

[Soluble expression of Plasmodium falciparum glutamate dehydrogenase in Escherichia coli, and its purification and identification].

OBJECTIVE: To make soluble expression of Plasmodium falciparum (FCC1/HN) glutamate dehydrogenase (GDH) in Escherichia coli, purification and immunocompetence identification of the recombinant non-fusion GDH. METHODS: The GDH gene was cloned into prokaryotic expression vector pET23 (a) to form recombinant expression vector pET23 (a)/GDH. pET23(a)/GDH was transformed into E. coli BL21 (DE3). Induced by IPTG (isopropyl-beta D-thiogalactoside), GDH was highly expressed in the supernatant after sonication. The soluble recombinant GDH was purified by Source-Q and Source-S chromatography. Enzyme-linked immunosorbent assay and Western blotting were carried out to identify the immunocompetence of the purified product. RESULTS: SDS-PAGE analysis showed that the soluble GDH protein accounted for approximately 15% of the total bacterial protein. By two-step ion-exchange chromatography, the purity of GDH reached more than 90% and the GDH possessed high antigenicity. CONCLUSION: The soluble expression of GDH results in an integral three-dimensional structure epitope with high biological activity.

[Establishment and identification of recombinant NS-1 cell strain that stably expresses chimeric HBc containing HBV multiepitope short peptides].

OBJECTIVE: To establish recombinant NS-1 cell strain that is capable of stable expression of chimeric HBc particle containing HBV multi epitope short peptides. METHODS: The recombinant plasmid, pHBc-Mep, was transfected into NS-1 cells via Lipofectamine, and the recombinant cell strain was screened with G418 and subclone screening. The expression products of the cells were examined by RT-PCR, ELISA, indirect immunofluorescence assay (IFA) and Western blotting. RESULTS: The results of RT-PCR, ELISA, IFA and Western blotting demonstrated that the recombinant protein HBc-Mep was expressed in the screened cells after continuous cloning for 3 times, but not in cells transfected with pcDNA3.1 or nontransfected cells. CONCLUSION: The recombinant cell strain stably expressing chimeric HBc particle containing multi epitope short peptides of HBV, designated as NS/HBc-Mep, has been established successfully.

[Cloning and expression of hepatitis C core protein gene].

OBJECTIVE: To express hepatitis C virus (HCV) core protein gene fragment in E. coli. METHODS: A fragment of HCV core gene sequence 357 bp in length was amplified by PCR, digested with EcoRI+Hind III and inserted to the plasmid vector pET-32a to construct recombinant HCVc/pET-32a plasmid, which was transformed into E.coli BL-21 and induced by IPTG for its expression. The expressed proteins obtained were identified by SDS-PAGE and Western blotting. RESULTS: The core sequence of HCV was amplified and after IPTG induction, a fusion protein of 32,000 was resulted exhibiting specific reaction with HCV-positive serum and high antigenicity. CONCLUSION: It is possible to efficiently express HCV core protein in E.coli.

[Cloning of hepatitis C virus open reading frame sequence and non structural protein expressions of the virus].

OBJECTIVE: To induce the expressions of the constituent proteins of hepatitis C virus (HCV) using a vaccinia virus expression system. METHODS: The open reading frame (ORF) sequence encoding HCV large polyprotein precursor was cloned into a vaccinia virus promoter to construct the recombinant plasmid pVHCV, which was subsequently used, along with a control plasmid (pSC59) in a parallel experiment, to transfect BHK21 cells via Lipofectamine 2000 reagent, followed by infection of the cells with vTF7-3 vaccinia viruses. After a 48-h culture, the expressions of HCV nonstructural proteins NS3 and NS5a in the cells were detected using Western blotting and immunofluorescence assay. RESULTS: Western blotting analysis presented the nonstructural proteins NS3 and NS5a detected in pVHCV-transfected BKH21 cells as two bands with molecular weights of 70,000 and 56,000, respectively. In the immunofluorescence assay, intense granular fluorescence signal was detected in the cytoplasm of pVHCV-transfected cells, irrespective of the use of either NS3- or NS5a- specific mouse monoclonal antibody. CONCLUSION: HCV nonstructural proteins NS3 and NS5a can be expressed in BKH21 cells.

[Preparation and identification of phage-displayed ICAM-1-mimic peptide].

AIM: To obtain bioactive ICAM-1 mimetic peptide. METHODS: Phages displaying P1 and P2 were prepared by phage amplication and PEG precipitation. The binding between phage-displayed peptides and anti-ICAM-1 mAb 15.2 was evaluated by sandwich ELISA and competitive ELISA. Bioactivities of P1 and P2 was detected by immunohistochemical staining. RESULTS: Phage-displayed peptides P1 and P2 could specifically bind to mAb 15.2, and the binding could be competitively inhibited by ICAM-1. Immunohistochemical staining showed that P1 and P2 could mimic the binding of ICAM-1 to its receptor LFA-1. CONCLUSION: Phage-displayed peptides P1 and P2 are bioactive just as native ICAM-1.

[Application of phage antibody library technology: problems and their solutions].

In spite of the wide application of phage antibody library technology in antibody engineering, problems are often present especially in the key steps, for instance, library construction, screening and expression of the antibody. The authors conducted an analysis of these problems and thereby proposes their solutions.