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ABSTRACT: BACKGROUND: Shigella is a major pathogen responsible for bacillary dysentery, a severe form of shigellosis. Severity of the disease depends on the virulence of the infecting strain. Shigella pathogenicity is a multi-gene phenomenon, involving the participation of genes on an unstable large virulence plasmid and chromosomal pathogenicity islands. RESULTS: A multiplex PCR (mPCR) assay was developed to detect S. flexneri 2a from rural regions of Zhengding (Hebei Province, China). We isolated and tested 86 strains using our mPCR assay, which targeted the ipaH, ial and set1B genes. A clinical strain of S. flexneri 2a 51 (SF51) containing ipaH and ial, but lacking set1B was found. The virulence of this strain was found to be markedly decreased. Further testing showed that the SF51 strain lacked pic. To investigate the role of pic in S. flexneri 2a infections, a pic knockout mutant (SF301-[increment]pic) and two complementation strains, SF301-[increment]pic/pPic and SF51/pPic, were created. Differences in virulence for SF51, SF301-[increment]pic, SF301-[increment]pic/pPic, SF51/pPic and S. flexneri 2a 301 (SF301) were compared. Compared with SF301, both SF51 and SF301-[increment]pic exhibited lower levels of Hela cell invasion and resulted in reduced keratoconjunctivitis, with low levels of tissue damage seen in murine eye sections. The virulence of SF301-[increment]pic and SF51 was partially recovered in vitro and in vivo through the addition of a complementary pic gene. CONCLUSIONS: The pic gene appears to be involved in an increase in pathogenicity of S. flexneri 2a. This gene assists with bacterial invasion into host cells and alters inflammatory reactions.
BMC Microbiology 02/2013; 13(1):31. · 3.04 Impact Factor
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Fangyou Yu,
Qunhua Ying,
Chun Chen,
Tingjian Li,
Baixing Ding,
Ying Liu,
Yuanyuan Lu,
Zhiqiang Qin,
Chris Parsons,
Cassandra Salgado, Di Qu,
Jingye Pan,
Liangxing Wang
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ABSTRACT: Outbreaks caused by Klebsiella pneumoniae producing carbapenemases and other β-lactamases have been reported. Four neonates admitted to a neonatal intensive care unit (NICU) in a Chinese hospital developed respiratory infection while receiving intensive care. In all four cases, multidrug-resistant K. pneumoniae was isolated from multiple respiratory specimens, leading to additional characterization of these organisms and investigation of the local environment in the NICU. Multiple β-lactamase genes, including bla(TEM-1), bla(IMP-4), bla(DHA-1) and bla(CTX-M-14), as well as the quinolone resistance gene qnrB4, were harboured by transferable plasmids from all four clinical isolates. Furthermore, PFGE confirmed that three of the four clinical isolates from the patients and three K. pneumoniae isolates collected from the hands of health-care workers and an incubator in the NICU belonged to the same PFGE cluster, indicating that an outbreak due to multidrug-resistant K. pneumoniae carrying bla(IMP-4) and bla(DHA-1) occurred in this NICU. As far as is known, this is the first report of the co-existence of bla(IMP-4) and bla(DHA-1) in the same K. pneumoniae isolate. These data suggest that additional precautions are needed to prevent outbreaks of infection caused by multidrug-resistant K. pneumoniae resulting from environmental exposure in NICUs.
Journal of Medical Microbiology 03/2012; 61(Pt 7):984-9. · 2.50 Impact Factor
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ABSTRACT: A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.
Virology 03/2012; 427(2):98-106. · 3.35 Impact Factor
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Ren-zheng Huang,
Li-kang Zheng,
Hua-yong Liu,
Bin Pan,
Jian Hu,
Tao Zhu,
Wei Wang,
Dan-bin Jiang,
Yang Wu,
You-cong Wu,
Shi-qing Han, Di Qu
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ABSTRACT: To evaluate the efficacies of six derivatives of Compound 2, a novel YycG histidine kinase inhibitor with the thiazolidione core structure in the treatment of medical device-related biofilm infections.
The minimal inhibitory concentration (MIC) of the derivatives was determined using the macrodilution broth method, and the minimal bactericidal concentration (MBC) was obtained via sub-culturing 100 μL from each negative tube from the MIC assay onto drug-free Mueller-Hinton agar plates. Biofilm-killing effect for immature (6 h-old) biofilms was examined using a semiquantitative plate assay, and the effect on mature (24 h-old) biofilms was observed under a confocal laser scanning microscope (CLSM).
The derivatives potently suppressed the growth of Staphylococcus epidermidis. The MIC values of the derivatives H2-10, H2-12, H2-20, H2-29, H2-27, and H2-28 on S epidermidis ATCC 35984 were 24.3, 6.5, 6.2, 3.3, 3.1, and 1.5 μg/mL, respectively. The MBC values of these derivatives were 48.6, 52.2, 12.4, 52.6, 12.4, and 6.2 μg/mL, respectively. The derivatives killed all bacteria in immature (6 h-old) biofilms and eliminated the biofilm proliferation. The derivatives also displayed strong bactericidal activities toward cells in mature (24 h-old) biofilms, whereas they showed low cytotoxicity and hemolytic activity toward Vero cells and human erythrocytes.
The bactericidal and biofilm-killing activities of the new anti-YycG compounds were significantly better than the parent Compound 2.
Acta Pharmacologica Sinica 03/2012; 33(3):418-25. · 1.95 Impact Factor
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Yang Wu,
Jiaxue Wang,
Tao Xu,
Jingran Liu,
Wenqi Yu,
Qiang Lou,
Tao Zhu,
Nianan He,
Haijing Ben,
Jian Hu,
Friedrich Götz, Di Qu
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ABSTRACT: Due to its ability to form biofilms on medical devices, Staphylococcus epidermidis has emerged as a major pathogen of nosocomial infections. In this study, we investigated the role of the two-component signal transduction system ArlRS in regulating S. epidermidis biofilm formation. An ArlRS-deficient mutant, WW06, was constructed using S. epidermidis strain 1457 as a parental strain. Although the growth curve of WW06 was similar to that of SE1457, the mutant strain was unable to form biofilms in vitro. In a rabbit subcutaneous infection model, sterile disks made of polymeric materials were implanted subcutaneously followed with inoculation of WW06 or SE1457. The viable bacteria cells of WW06 recovered from biofilms on the embedded disks were much lower than that of SE1457. Complementation of arlRS genes expression from plasmid in WW06 restored biofilm-forming phenotype both in vivo and in vitro. WW06 maintained the ability to undergo initial attachment. Transcription levels of several genes involved in biofilm formation, including icaADBC, sigB, and sarA, were decreased in WW06, compared to SE1457; and icaR expression was increased in WW06, detected by real-time reverse-transcription PCR. The biofilm-forming phenotype was restored by overexpressing icaADBC in WW06 but not by overexpressing sigB, indicating that ArlRS regulates biofilm formation through the regulation of icaADBC. Gel shift assay showed that ArlR can bind to the promoter region of the ica operon. In conclusion, ArlRS regulates S. epidermidis biofilm formation in an ica-dependent manner, distinct from its role in S. aureus.
PLoS ONE 01/2012; 7(7):e40041. · 4.09 Impact Factor
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Fangyou Yu,
Chun Chen,
Qiang Chen,
Xiaojun Yu,
Baixing Ding,
Lehe Yang,
Qiaoqiao Li,
Zhiqiang Qin,
Chris Parsons,
Xueqing Zhang,
Liming Zhang, Di Qu,
Liangxing Wang,
Jingye Pan
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ABSTRACT: Human illnesses caused by Salmonella species often require antibiotic treatment, and antibiotic resistance for Salmonella species is conferred through multiple mechanisms. Salmonella enterica Thompson, a pathogen commonly infecting poultry, causes human infection following food-borne transmission, and mechanisms of antibiotic resistance for this pathogen have not been well characterized. We isolated Salmonella enterica Thompson (Salmonella enterica Thompson NC24) from the stool of a 3-year-old patient with diarrhea in Nanchang, China. The isolate demonstrated resistance to cephalosporins and fluoroquinolones, including ceftazidime, cefotaxime, cefoxitin, ciprofloxacin and levofloxacin with mean inhibitory concentrations of 128, 16, 128, 8 and 8 µg/mL, respectively. Plasmids containing blaTEM-1b and blaDHA-1 genes encoding AmpC β-lactamases were identified and readily transferable to E. coli. Mutations in quinolone resistance-determining regions were also identified for gyrA (DNA gyrase) and parC (topoisomerase IV). To our knowledge, this is the first report identifying transmissible plasmid-mediated AmpC β-lactamases for Salmonella enterica Thompson, and these findings have significant implications for the development of antibiotic resistance for this pathogen within the food supply, transmission of resistance to other enteric pathogens, and the treatment of human infections caused by this organism.
Journal of Medical Microbiology 11/2011; 61(Pt 3):460-2. · 2.50 Impact Factor
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ABSTRACT: Human enterovirus 71 (EV71) is the primary pathogen of hand, foot, and mouth disease (HFMD). EV71 infection may lead to neurologic damage, with higher incidence of fatality compared with other HFMD pathogens. An effective drug or vaccine against EV71 infection is currently unavailable. It is desirable to determine the pathogen of HFMD accurately and quickly for early treatment. In the current study, reverse-transcription and loop-mediated isothermal amplification (RT-LAMP) technology were developed to detect EV71. The efficacy of detecting EV71 was compared with regular nested reverse-transcription polymerase chain reaction (RT-PCR). After detecting 108 clinical specimens, results showed that RT-LAMP can specifically detect EV71, but not Coxsackie virus A16, and exhibited a specificity of 100% and a sensitivity of 97.1%, which was higher than regular RT-PCR. The findings indicate that RT-LAMP is a practical method for EV71 diagnostic applications, particularly in small county institutes of medical service. The detection ability of RT-LAMP was significantly affected by cryopreservation as the clinical specimens were repeatedly subject to freezing and thawing treatments.
Diagnostic microbiology and infectious disease 09/2011; 71(3):244-51. · 2.45 Impact Factor
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ABSTRACT: Treating biofilm infections on implanted medical devices is formidable, even with extensive antibiotic therapy. The aim of this study was to investigate whether ultrasound (US)-targeted microbubble (MB) destruction (UTMD) could enhance vancomycin activity against Staphylococcus epidermidis RP62A biofilms. Twelve-hour biofilms were treated with vancomycin combined with UTMD. The vancomycin and MB (SonoVue) were used at concentrations of 100 μg/ml and 30% (vol/vol), respectively, in studies in vitro. After US exposure (0.08 MHz, 1.0 W/cm(2), 50% duty cycle, and 10-min duration), the biofilms were cultured at 37 °C for another 12 h. The results showed that many micropores were found in biofilms treated with vancomycin combined with UTMD. Biofilm densities (A(570) values) and the viable counts of S. epidermidis recovered from the biofilm were significantly decreased compared with those of any other groups. Furthermore, the highest percentage of dead cells was found, using confocal laser scanning microscopy, in the biofilm treated with vancomycin combined with UTMD. The viable counts of bacteria in biofilms in an in vivo rabbit model also confirmed the enhanced effect of vancomycin combined with UTMD. UTMD may have great potential for improving antibiotic activity against biofilm infections.
Antimicrobial Agents and Chemotherapy 08/2011; 55(11):5331-7. · 4.84 Impact Factor
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Qiang Lou,
Tao Zhu,
Jian Hu,
Haijing Ben,
Jinsong Yang,
Fangyou Yu,
Jingran Liu,
Yang Wu,
Adrien Fischer,
Patrice Francois,
Jacques Schrenzel, Di Qu
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ABSTRACT: Staphylococcus epidermidis (SE) has emerged as one of the most important causes of nosocomial infections. The SaeRS two-component signal transduction system (TCS) influences virulence and biofilm formation in Staphylococcus aureus. The deletion of saeR in S. epidermidis results in impaired anaerobic growth and decreased nitrate utilization. However, the regulatory function of SaeRS on biofilm formation and autolysis in S. epidermidis remains unclear.
The saeRS genes of SE1457 were deleted by homologous recombination. The saeRS deletion mutant, SE1457ΔsaeRS, exhibited increased biofilm formation that was disturbed more severely (a 4-fold reduction) by DNase I treatment compared to SE1457 and the complementation strain SE1457saec. Compared to SE1457 and SE1457saec, SE1457ΔsaeRS showed increased Triton X-100-induced autolysis (approximately 3-fold) and decreased cell viability in planktonic/biofilm states; further, SE1457ΔsaeRS also released more extracellular DNA (eDNA) in the biofilms. Correlated with the increased autolysis phenotype, the transcription of autolysis-related genes, such as atlE and aae, was increased in SE1457ΔsaeRS. Whereas the expression of accumulation-associated protein was up-regulated by 1.8-fold in 1457ΔsaeRS, the expression of an N-acetylglucosaminyl transferase enzyme (encoded by icaA) critical for polysaccharide intercellular adhesin (PIA) synthesis was not affected by the deletion of saeRS.
Deletion of saeRS in S. epidermidis resulted in an increase in biofilm-forming ability, which was associated with increased eDNA release and up-regulated Aap expression. The increased eDNA release from SE1457ΔsaeRS was associated with increased bacterial autolysis and decreased bacterial cell viability in the planktonic/biofilm states.
BMC Microbiology 06/2011; 11:146. · 3.04 Impact Factor
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ABSTRACT: Mouse hepatitis virus (MHV) produces a series of subgenomic RNAs for viral protein expression. As a prototype coronavirus, MHV has been explored extensively and is often used to express foreign proteins. Previously, a 13-residue deletion in the MHV spike (S) protein endodomain was found to reduce syncytium formation dramatically while inhibiting virus replication slightly. In this study, the effects of the S mutation on MHV infectivity and foreign protein expression were further examined in rat or mouse L2, NIH/3T3 and Neuro-2a cells. The replacement of the MHV 2a/haemagglutinin-esterase gene with a membrane-anchored protein hook (HK) and replacement of gene 4 with EGFP did not change the adaptability and cytopathology of recombinant viruses in these cells. However, the cytopathic effect of the recombinants with the partial S deletion was reduced significantly in these cells. The replication and foreign protein expression of the S-mutated recombinants were found to be more efficient in L2 cells than in Neuro-2a and NIH/3T3 cells. Meanwhile, the distribution patterns of HK and EGFP expressed by the recombinant viruses were similar to those in cells transfected with a eukaryotic expression vector. These results suggest that the partial deletion in the S endodomain may increase the usefulness of MHV as a viral vector by attenuation and maintaining foreign protein expression.
Journal of virological methods 03/2011; 172(1-2):46-53. · 2.13 Impact Factor
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ABSTRACT: Rational designed novel thiazolidiones were synthesized and evaluated for antibiofilm activity. The active derivatives were not only potent inhibitors of Staphylococcus epidermidis biofilm growth but also efficient antibacterial agents. 3f showed 4-fold higher activity (6.25 μM) in the biofilms dispersal assay and significantly higher antibacterial activity (MIC 3.125 μM) in comparison to the 3-(5-((6- (ethoxycarbonyl)-5-(benzo[1,3]dioxol-5-yl)-3-oxo-7-phenyl- thiazolo[3,2-a]pyrimidin-2(5H)-ylidene)methyl)furan-2-yl)benzoic acid (1).
European journal of medicinal chemistry 03/2011; 46(3):819-24. · 3.27 Impact Factor
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ABSTRACT: PhoQ/PhoP is an important two-component system that regulates Shigella virulence. We explored whether the PhoQ/PhoP system is a promising target for new antibiotics against S. flexneri infection. By using a high-throughput screen and enzymatic activity coupled assay, four compounds were found as potential PhoQ inhibitors. These compounds not only inhibited the activity of SF-PhoQc autophosphorylation but also displayed high binding affinities to the SF-PhoQc protein in the Surface Plasmon Resonance response. A S. flexneri cell invasion assay showed that three of these potential PhoQ inhibitors inhibit the invasion of HeLa cells by S. flexneri 9380. In a Mouse Sereny test, mice inoculated with S. flexneri 9380 pre-treated with the potential PhoQ inhibitors 1, 2, 3 or 4 displayed no inflammation, whereas mice inoculated with S. flexneri 9380 alone displayed severe keratoconjunctival inflammation. All four potential PhoQ inhibitors showed no significant cytotoxicity or hemolytic activity. These data suggest that the four potential PhoQ inhibitors inhibited the virulence of S. flexneri and that PhoQ/PhoP is a promising target for the development of drugs against S. flexneri infection.
PLoS ONE 01/2011; 6(8):e23100. · 4.09 Impact Factor
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Jian Hu,
Tao Xu,
Tao Zhu,
Qiang Lou,
Xueqin Wang,
Yang Wu,
Renzheng Huang,
Jingran Liu,
Huayong Liu,
Fangyou Yu,
Baixing Ding,
Yalin Huang,
Wenyan Tong, Di Qu
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ABSTRACT: Because there is no effective antibiotic to eradicate Staphylococcus epidermidis biofilm infections that lead to the failure of medical device implantations, the development of anti-biofilm vaccines is necessary. Biofilm formation by S. epidermidis requires accumulation-associated protein (Aap) that contains sequence repeats known as G5 domains, which are responsible for the Zn(2+)-dependent dimerization of Aap to mediate intercellular adhesion. Antibodies against Aap have been reported to inhibit biofilm accumulation. In the present study, three monoclonal antibodies (MAbs) against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5) were generated. MAb(18B6) inhibited biofilm formation by S. epidermidis RP62A to 60% of the maximum, while MAb(25C11) and MAb(20B9) enhanced biofilm accumulation. All three MAbs aggregated the planktonic bacteria to form visible cell clusters. Epitope mapping revealed that the epitope of MAb(18B6), which recognizes an identical area within AapBrpt constructs from S. epidermidis RP62A, was not shared by MAb(25C11) and MAb(20B9). Furthermore, all three MAbs were found to affect both Aap expression and extracellular polymeric substance (EPS, including extracellular DNA and PIA) biosynthesis in S. epidermidis and enhance the cell accumulation. These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections.
PLoS ONE 01/2011; 6(6):e20918. · 4.09 Impact Factor
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ABSTRACT: Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown.
In the present study, we demonstrated that lytSR knock-out in S. epidermidis did not alter susceptibility to Triton X-100 induced autolysis. Quantitative murein hydrolase assay indicated that disruption of lytSR in S. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between S. epidermidis strain 1457 and its lytSR mutant. Compared to the wild-type counterpart, 1457ΔlytSR produced slightly more biofilm, with significantly decreased dead cells inside. Microarray analysis showed that lytSR mutation affected the transcription of 164 genes (123 genes were upregulated and 41 genes were downregulated). Specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated. Impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457ΔlytSR, which is consistent with the microarray data.
The preliminary results suggest that in S. epidermidis LytSR two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. Based on the microarray data, it appears that lytSR inactivation induces a stringent response. In addition, LytSR may indirectly enhance biofilm formation by altering the metabolic status of the bacteria.
BMC Microbiology 11/2010; 10:287. · 3.04 Impact Factor
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Penghui Lin,
Tiancen Hu,
Jian Hu,
Wenqi Yu,
Cong Han,
Jian Zhang,
Guangrong Qin,
Kunqian Yu,
Friedrich Götz,
Xu Shen,
Hualiang Jiang, Di Qu
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ABSTRACT: The emergence of multi-drug-resistant strains of Staphylococcus epidermidis emphasizes the need to develop new antibiotics. The unique and essential role of the peptide deformylase (PDF) in catalysing the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria makes it an attractive antibacterial drug target. In the present study, both deformylase homologues from S. epidermidis (SePDF-1 and SePDF-2) were cloned and expressed, and their enzymic activities were characterized. Co(2+)-substituted SePDF-1 exhibited much higher enzymic activity (k(cat)/K(m) 6.3 × 10(4) M(-1) s(-1)) than those of Ni(2+)- and Zn(2+)-substituted SePDF-1, and SePDF-1 showed much weaker binding ability towards Ni(2+) than towards Co(2+) and Zn(2+), which is different from PDF in Staphylococcus aureus (SaPDF), although they share 80 % amino-acid sequence identity. The determined crystal structure of SePDF-1 was similar to that of (SaPDF), except for differences in the metal-binding sites. The other deformylase homologue, SePDF-2, was shown to have no peptide deformylase activity; the function of SePDF-2 needs to be further investigated.
Microbiology 10/2010; 156(Pt 10):3194-202. · 3.06 Impact Factor
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ABSTRACT: A series of novel 2-arylimino-3-aryl-thiazolidine-4-ones was designed, synthesized and tested for in vitro antibiofilm activity against Staphylococcus epidermidis. Among them tested, some compounds with carboxylic acid groups showed good antibiofilm activity. The antibiofilm concentration of 1x was 6.25 microM. The structure-activity relationships revealed that incorporation of 2-phenylfuran moiety could greatly enhance antibiofilm activity of thiazolidine-4-one.
Bioorganic & medicinal chemistry letters 03/2010; 20(8):2461-4. · 2.65 Impact Factor
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ABSTRACT: To investigate the immunomodulatory effects of andrographolide on both innate and adaptive immune responses.
Andrographolide (10 microg/mL in vitro or 1 mg/kg in vivo) was used to modulate LPS-induced classical activated (M1) or IL-4-induced alternative activated (M2) macrophages in vitro and humor immune response to HBsAg in vivo. Cytokine gene expression profile (M1 vs M2) was measured by real-time PCR, IL-12/IL-10 level was detected by ELISA, and surface antigen expression was evaluated by flow cytometry, whereas phosphorylation level of ERK 1/2 and AKT was determined by Western blot. The level of anti-HBs antibodies in HBsAg immunized mice was detected by ELISA, and the number of HBsAg specific IL-4-producing splenocyte was enumerated by ELISPOT.
Andrographolide treatment in vitro attenuated either LPS or IL-4 induced macrophage activation, inhibited both M1 and M2 cytokines expression and decreased IL-12/IL-10 ratio (the ratio of M1/M2 polarization). Andrographolide down-regulated the expression of mannose receptor (CD206) in IL-4 induced macrophages and major histocompability complex/costimulatory molecules (MHC I, CD40, CD80, CD86) in LPS-induced macrophages. Correspondingly, anti-HBs antibody production and the number of IL-4-producing splenocytes were reduced by in vivo administration of andrographolide. Reduced phosphorylation levels of ERK1/2 and AKT were observed in macrophages treated with andrographolide.
Andrographolide can modulate the innate and adaptive immune responses by regulating macrophage phenotypic polarization and Ag-specific antibody production. MAPK and PI3K signaling pathways may participate in the mechanisms of andrographolide regulating macrophage activation and polarization.
Acta Pharmacologica Sinica 02/2010; 31(2):191-201. · 1.95 Impact Factor
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ABSTRACT: Aim: To investigate the immunomodulatory effects of andrographolide on both innate and adaptive immune responses.
Acta Pharmacologica Sinica 01/2010; 31(2):191-201. · 1.95 Impact Factor
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Yanfeng Zhao,
Haijing Ben,
Su Qu,
Xinwen Zhou,
Liang Yan,
Bin Xu,
Shuangcheng Zhou,
Qiang Lou,
Rong Ye,
Tianlun Zhou,
Pengyuan Yang, Di Qu
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ABSTRACT: Hepatitis B virus (HBV) is a major cause of liver infection in human. Because of the lack of an appropriate cell culture system for supporting HBV infection efficiently, the cellular and molecular mechanisms of hepadnavirus infection remain incompletely understood. Duck heptatitis B virus (DHBV) can naturally infect primary duck hepatocytes (PDHs) that provide valuable model systems for studying hepadnavirus infection in vitro. In this report, we explored global changes in cellular protein expression in DHBV infected PDHs by two-dimension gel electrophoresis (2-DE) combined with MALDI-TOF/TOF tandem mass spectrometry (MS/MS).
The effects of hepadnavirus infection on hepatocytes were investigated in DHBV infected PDHs by the 2-DE analysis. Proteomic profile of PDHs infected with DHBV were analyzed at 24, 72 and 120 h post-infection by comparing with uninfected PDHs, and 75 differentially expressed protein spots were revealed by 2-DE analysis. Among the selected protein spots, 51 spots were identified corresponding to 42 proteins by MS/MS analysis; most of them were matched to orthologous proteins of Gallus gallus, Anas platyrhynchos or other avian species, including alpha-enolase, lamin A, aconitase 2, cofilin-2 and annexin A2, etc. The down-regulated expression of beta-actin and annexin A2 was confirmed by Western blot analysis, and potential roles of some differentially expressed proteins in the virus-infected cells have been discussed.
Differentially expressed proteins of DHBV infected PDHs revealed by 2-DE, are involved in carbohydrate metabolism, amino acid metabolism, stress responses and cytoskeleton processes etc, providing the insight to understanding of interactions between hepadnavirus and hepatocytes and molecular mechanisms of hepadnavirus pathogenesis.
Proteome Science 01/2010; 8:28. · 2.33 Impact Factor
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Fang-you Yu,
Dan Yao,
Jing-ye Pan,
Chong Chen,
Zhi-qiang Qin,
Chris Parsons,
Le-he Yang,
Qiao-qiao Li,
Xue-qing Zhang, Di Qu,
Liang-xing Wang
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ABSTRACT: Recently, production of 16S rRNA methylases by Gram-negative bacilli has emerged as a novel mechanism for high-level resistance to aminoglycosides by these organisms in a variety of geographic locations. Therefore, the spread of high-level aminoglycoside resistance determinants has become a great concern.
Between January 2006 and July 2008, 680 distinct Escherichia coli clinical isolates were collected from a teaching hospital in Wenzhou, China. PCR and DNA sequencing were used to identify 16S rRNA methylase and extended-spectrum beta-lactamase (ESBL) genes, including armA and rmtB, and in situ hybridization was performed to determine the location of 16S rRNA methylase genes. Conjugation experiments were subsequently performed to determine whether aminoglycoside resistance was transferable from the E. coli isolates via 16S rRNA methylase-bearing plasmids. Homology of the isolates harboring 16S rRNA methylase genes was determined using pulse-field gel electrophoresis (PFGE).
Among the 680 E. coli isolates, 357 (52.5%), 346 (50.9%) and 44 (6.5%) isolates were resistant to gentamicin, tobramycin and amikacin, respectively. Thirty-seven of 44 amikacin-resistant isolates harbored 16S rRNA methylase genes, with 36 of 37 harboring the rmtB gene and only one harboring armA. The positive rates of 16S rRNA methylase genes among all isolates and amikacin-resistant isolates were 5.4% (37/680) and 84.1% (37/44), respectively. Thirty-one isolates harboring 16S rRNA methylase genes also produced ESBLs. In addition, high-level aminoglycoside resistance could be transferred by conjugation from four rmtB-positive donors. The plasmids of incompatibility groups IncF, IncK and IncN were detected in 34, 3 and 3 isolates, respectively. Upstream regions of the armA gene contained ISCR1 and tnpU, the latter a putative transposase gene,. Another putative transposase gene, tnpD, was located within a region downstream of armA. Moreover, a transposon, Tn3, was located upstream of the rmtB. Nineteen clonal patterns were obtained by PFGE, with type H representing the prevailing pattern.
A high prevalence of plasmid-mediated rmtB gene was found among clinical E. coli isolates from a Chinese teaching hospital. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene.
BMC Infectious Diseases 01/2010; 10:184. · 3.12 Impact Factor
