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Objective@#To investigate the pyroptosis induced by different enteroviruses in human neuroblastoma cells SH-SY5Y and the differences among them.@*Methods@#SH-SY5Y cells were infected with nine strains of enterovirus respectively, including enterovirus A71 (EV-A71), Coxsackievirus A (CA), Coxsackievirus B (CB), Echovirus (Echo). The cellular morphology of infected and control groups were observed and activity of Caspase-1 of infected and control groups were detected by flow cytometry at 48 h post infection.@*Results@#The activity of Caspase-1 induced by EV-A71 was higher than control (P<0.001), and the activity of Caspase-1 induced by EV-A71 isolated from severe case was significantly higher than that induced by EV-A71 isolated from common case (P<0.001). The activity of Caspase-1 induced by CA was at a lower level. The activity of Caspase-1 induced by CB and Echo were both significantly higher than that induced by EV-A71 and control (P<0.001). Cytopathic effects (CPE) were found to be related with the activity of Caspase-1.@*Conclusions@#EV-A71, CB and Echo all could induce pyroptosis mediated by Caspase-1 in SH-SY5Y cells, but the ability of CA to induce pyroptosis was at a lower level, which may provide evidences for further study on mechanism of neuropathy caused by enterovirus.
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Objective@#To analyze the molecular epidemiology, genetic variations and evolution of enterovirus 71 (EV71) strains isolated in Jiangsu Province from 2009 to 2018.@*Methods@#Statistical methods were used to analyze the data about epidemiological characteristics and results of pathogen detection in cases with EV71 infection in Jiangsu Province from 2009 to 2018. The complete VP1 sequences of 80 EV71 strains were amplified and sequenced for analysis of diversity and phylogenesis.@*Results@#A total of 41 858 enterovirus-positive hand, foot and mouth disease cases were reported in Jiangsu Province from 2009 to 2018. EV71 was the predominant pathogen, accounting for 36.52%, and responsible for most of the severe cases. However, the percentage of EV71 among all pathogens gradually decreased over time. EV71 infection reached the peak in April to June and mainly occurred in children aged six months to five years old with higher incidence in males than in females. In terms of regional distribution, EV71 infections were characterized by area clustering in Jiangsu Province, mainly detected in Nanjing, Suzhou, Wuxi and Lianyungang. The 80 EV71 isolates belonged to C4a genotype. Nucleotide differences between them and three vaccine strains (H07, FY23 and FY7VP5)were 0.6%-5.5%, 0.8%-5.7% and 1.9%-6.9% and amino acid difference were 0-1.4%, 0.3%-2.0% and 0.3%-2.0%, respectively. Amino acid mutations in the epitopes of the 80 EV71 strains did not marked by years or regions.@*Conclusions@#EV71 strains showed obvious epidemiological characteristics in time, population and regional distribution in Jiangsu Province from 2009 to 2018.All of the 80 EV71 isolates belonged to C4a subgenotype. The nucleotide sequences between them and the vaccine strains varied greatly, but the homology of amino acids was relatively high, indicating the existence of some synonymous mutations and no risk of antigenic drift. This study would provide reference for EV71 vaccination in Jiangsu Province.
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Objective To analyze the molecular epidemiology, genetic variations and evolution of enterovirus 71 (EV71) strains isolated in Jiangsu Province from 2009 to 2018. Methods Statistical meth-ods were used to analyze the data about epidemiological characteristics and results of pathogen detection in cases with EV71 infection in Jiangsu Province from 2009 to 2018. The complete VP1 sequences of 80 EV71 strains were amplified and sequenced for analysis of diversity and phylogenesis. Results A total of 41858 enterovirus-positive hand, foot and mouth disease cases were reported in Jiangsu Province from 2009 to 2018. EV71 was the predominant pathogen, accounting for 36. 52％, and responsible for most of the severe cases. However, the percentage of EV71 among all pathogens gradually decreased over time. EV71 infection reached the peak in April to June and mainly occurred in children aged six months to five years old with higher incidence in males than in females. In terms of regional distribution, EV71 infections were character-ized by area clustering in Jiangsu Province, mainly detected in Nanjing, Suzhou, Wuxi and Lianyungang. The 80 EV71 isolates belonged to C4a genotype. Nucleotide differences between them and three vaccine strains (H07,FY23 and FY7VP5) were 0. 6％-5. 5％, 0. 8％-5. 7％ and 1. 9％-6. 9％ and amino acid difference were 0-1. 4％, 0. 3％-2. 0％ and 0. 3％-2. 0％, respectively. Amino acid mutations in the epitopes of the 80 EV71 strains did not marked by years or regions. Conclusions EV71 strains showed ob-vious epidemiological characteristics in time, population and regional distribution in Jiangsu Province from 2009 to 2018. All of the 80 EV71 isolates belonged to C4a subgenotype. The nucleotide sequences between them and the vaccine strains varied greatly, but the homology of amino acids was relatively high, indicating the existence of some synonymous mutations and no risk of antigenic drift. This study would provide reference for EV71 vaccination in Jiangsu Province.
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OBJECTIVE: To compare the maternal and infant outcomes of pregnant women infected with human immunodeficiency virus(HIV)treated with different regimens of highly active antiretroviral therapy(HAART).METHODS: For pregnant women infected with the human immunodeficiency virus(HIV)who received antiviral therapy and delivered in the Eighth Peple's Hospital of Guangzhu between May 2015 and June 2018,they will be grouped according to different treatment options. The pregnant women's body weight,CD4+T lymphocytes,white blood cells,hemoglobin,serum albumin,neonatal body weight and adverse pregnancy outcomes were compared and analyzed.RESULTS:(1)There was no significantly statistical difference between the two groups of pregnant women in terms of body weight,white blood cells,hemoglobin or serum albumin(P>0.05).(2)The changes of CD4+T lymphocytes in the two groups of pregnant women before and after treatment were statistically different(P0.05).(4)There was no significantly statistical difference in the incidence of premature birth,premature rupture of fetal membrane,low birth weight,low amniotic fluid,fetal malformation or neonatal asphyxia between the two groups(P>0.05).Until December 2018,there were no positive reports of HIVRNA and HIV antibody detection in two groups of infants.CONCLUSION: The two HAART schemes have no significant difference in the influence on nutritional status,immune status or maternal and infant outcomes of HIV-infected pregnant women,and they are both effective and feasible,and vertical transmission of HIV from mother to child can be blocked.
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We developed a method for detecting encephalitis and meningitis virus by using multiplex PCR combined with invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles.Primers were designed based on the conservative regions of encephalitis and meningitis virus (Eastern equine encephalitis virus,EEEV;Western equine encephalomyelitis virus,WEEV;West Nile virus,WNV;Nipah virus,NiPA;Japanese encephalitis virus,JEV).Multiplex PCR system,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were established to detect different encephalitis and meningitis virus in one reaction.Tick-borne encephalitis virus (TBEV),St Louis encephalitis virus (StLEV),Chikungunya virus (CHIKV) and Dengue virus(DV) were used to test its specificity.Quantitative RNA transcribed in vitro and PCR fragments were used to assess its sensitivity.Clinical specimens collected from JEV patients were detected by this method.A method for detecting encephalitis and meningitis virus by using multiplex PCR,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were successfully established.This method can detect targeted pathogens specifically,and it has no cross reaction with TBEV,StLEV,CHIKV and DV.The detecting limitation for different targets was 103 copies/μL.Clinical samples were positive for JEV nucleic acids for above assay.The method presented here has characteristic of high specificity,sensitivity and throughput.The results can be observed by visual inspection.This method has broad application prospects in pathogen detection.
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Objective To evaluate the practicability of using CRISPR/Cas9 genome editing tech-nology for inhibition of hepatitis B virus ( HBV) replication. Methods Two sgRNA targeting sites were de-signed for the S region of HBV genome. The CRISPR/Cas9 expression plasmids specific for HBV were con-structed and then transfected into a cell line expressing HBV genome(HepG2-N10). The cytotoxicity of cells transfected with different expression plasmids were detected by MTT assay. The levels of hepatitis B surface antigen ( HBsAg ) were determined by using chemiluminescent immunoassay ( CLIA ) . The expression of HBV at mRNA level was analyzed by quantitative real-time PCR ( qRT-PCR) . The qPCR was performed for the detection of extracellular and intracellular HBV DNA. The next-generation sequencing ( NGS) Illumina MiSeq Platform was used to analyze HBV genome editing. Results No significant cytotoxic effects were de-tected in HepG2-N10 cells transfected with different expression plasmids. Compared with the cells carrying pCas-Guide-GFP-Scramble, the levels of HBsAg in the supernatants of transfected cell culture harboring pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were decreased by 24. 2% (P0. 05), respectively. The levels of HBsAg in cells transfected with pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were respectively decreased by 16. 4% (P>0. 05) and 32. 1% (P>0. 05) as compared with that of pCas-Guide-GFP-Scramble transfected group. The expression of HBV at mRNA level was inhibited as indica-ted by the results of qRT-PCR. Moreover, the levels of extracellular HBV DNA were respectively suppressed by 23% (P>0. 05) and 35% (P0. 05) and 18% (P>0. 05). Different types of insertion/deletion mutation were de-tected in HBV genome by high-throughput sequencing. Conclusion HBV-specific CRISPR/Cas9 system could inhibit the expression of HBV gene and the replication of virus. Therefore, the CRISPR/Cas9 genome editing technology might be used as a potential tool for the treatment of persistent HBV infection.
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PURPOSE: Squamous cell carcinoma (SCC) of the tongue has a relatively high incidence of all oral cancers. Some studies have reported a relationship between intraoral dental prosthesis and SCC of the tongue; however, this relationship remains controversial. The purpose of this study was to investigate the relationship between SCC of the tongue and the positional aspects of dental prosthesis using a retrospective analysis. MATERIALS AND METHODS: A total of 439 patients with SCC of the tongue were diagnosed and treated in the Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital. Patients were treated over a 12.5-year period ranging from January 1, 2001 to June 30, 2013. Statistical analysis was performed to examine potential differences between the groups. RESULTS: The number of patients with a crown and/or a bridge (134, 63.5%) was significantly different than the number of patients without a prosthesis (77, 36.5%). Even after accounting for different types of prostheses such as crowns, bridges, and dentures, no significant differences were observed between the position of the prosthesis and the location of the SCC of the tongue, with significance defined as a P-value less than .05 by the Pearson-Chi square test. CONCLUSION: Patients with crowns and/or bridges exhibited more frequent SCC of the tongue compared with patients without these prosthesis. These data support the hypothesis that mechanical trauma and galvanic phenomena play a role in the etiology of SCC of the tongue.
Subject(s)
Humans , Carcinoma, Squamous Cell , Crowns , Dental Prosthesis , Dentures , Incidence , Mouth Neoplasms , Prostheses and Implants , Retrospective Studies , Seoul , Surgery, Oral , TongueABSTRACT
PURPOSE: Squamous cell carcinoma (SCC) of the tongue has a relatively high incidence of all oral cancers. Some studies have reported a relationship between intraoral dental prosthesis and SCC of the tongue; however, this relationship remains controversial. The purpose of this study was to investigate the relationship between SCC of the tongue and the positional aspects of dental prosthesis using a retrospective analysis. MATERIALS AND METHODS: A total of 439 patients with SCC of the tongue were diagnosed and treated in the Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital. Patients were treated over a 12.5-year period ranging from January 1, 2001 to June 30, 2013. Statistical analysis was performed to examine potential differences between the groups. RESULTS: The number of patients with a crown and/or a bridge (134, 63.5%) was significantly different than the number of patients without a prosthesis (77, 36.5%). Even after accounting for different types of prostheses such as crowns, bridges, and dentures, no significant differences were observed between the position of the prosthesis and the location of the SCC of the tongue, with significance defined as a P-value less than .05 by the Pearson-Chi square test. CONCLUSION: Patients with crowns and/or bridges exhibited more frequent SCC of the tongue compared with patients without these prosthesis. These data support the hypothesis that mechanical trauma and galvanic phenomena play a role in the etiology of SCC of the tongue.
Subject(s)
Humans , Carcinoma, Squamous Cell , Crowns , Dental Prosthesis , Dentures , Incidence , Mouth Neoplasms , Prostheses and Implants , Retrospective Studies , Seoul , Surgery, Oral , TongueABSTRACT
Objective To generate a comD gene knock-out mutant of Streptococcus pneumoniae,and determine the correlation of comD gene and the bacterial resistance against β-lactam antibiotics and understand the effect of closantel down-regulating comD, comE and comC mRNA levels. Methods A suicide plasmid pEVP3comD was constructed for comD gene knock-out and a comD gene knock-out mutant (comD-)was generated through homologous recombination and insertion inactivation. PCR and immunofluorescence method were used to identify the comD- mutant and real-time fluorescence quantitative PCR was applied to detect the changes of comD, comE and comC mRNA levels before and after closantel treatment in comD-mutant and wild-type strain. Double agar dilution method was performed to determine the sensitivity of comD- mutant and wild-type strain to penicillin G and cefotaxime. Results The comD gene in genome DNA of the generated comD- mutant was inactivated by sequencing and immunofluorescence detection. 50 μ mol/L or 100 μmol/L closantel had a function to down-regulate the comD, comE and comC mRNA levels ( P ＜ 0. 05) whereas 25 μmol/L closantel did not. Both the MIC values of penicillin G and cefotaxime inhibiting comD- mutant were 32 μg/ml which was significantly higher than that of wild-type strain (0.06 μg/ml and 1 μg/ml). Conclusion In this study a comD gene knock-out mutant of S. pneumoniae was successfully generated. There is a close correlation between comD gene and β-lactam antibiotics resistance of S. pneumoniae. Closantel has a function to inhibit the competence formation of S. pneumoniae through down-regulating the transcription levels of comD, comE and comC genes.
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<p><b>OBJECTIVE</b>To construct prokaryotic expression systems of TCS genes comD/comE/comC of Streptococcus pneumoniae, and to determine the correlation of ComD and ComC with the drug resistance.</p><p><b>METHODS</b>The entire comD, comE and comC genes were amplified by PCR and their prokaryotic expression systems were established by routine genetic engineering technique. SDS-PAGE and Bio-Rad Agarose Image Analyzor was applied to measure the outputs of target recombinant proteins rComD, rComE and rComC. Rabbits were immunized with these recombinant proteins to prepare antisera. The resistance of S.pneumoniae strains to penicillin and cefotaxime was examined after ComD and ComC were blocked by antisera.</p><p><b>RESULT</b>Compared with the reported sequences, similarities of nucleotide and amino acid sequences of the cloned comD, comE and comC genes were 98.4% approximately 99.3% and 99.1% approximately 100%, respectively. The constructed engineering bacteria E.coli BL21DE3(pET42a-comD), E.coli BL21DE3(pET42a-comE) and E.coli BL21DE3(pET42a-comC) were able to efficiently express the target recombinant proteins and the outputs of rComD, rComE and rComC were 28%, 25% and 35% of the total bacterial proteins, respectively. The double immunodiffusion titers of rabbit antisera against rComD, rComE or rComC were 1:4, 1:4 and 1:8, respectively. After the ComD and/or ComC were blocked by the antisera, the cefotaxime-sensitive S. pneumoniae strains became to resistant to antibiotics but there were no changes for cefotaxime-resistant strains and resistance to penicillin for all tested strains.</p><p><b>CONCLUSION</b>The prokaryotic expression systems of S.pneumoniae comD/come/comC genes have been successfully constructed, and the study also indicates that both the ComD and ComC are involved in the drug resistance of S. pneumoniae to cefotaxime.</p>
Subject(s)
Animals , Rabbits , Bacterial Proteins , Genetics , Cefotaxime , Pharmacology , Escherichia coli , Genetics , Metabolism , Recombinant Proteins , Genetics , Recombination, Genetic , Signal Transduction , Streptococcus pneumoniae , Genetics , beta-Lactam Resistance , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To construct prokaryotic expression systems of Streptococcus pneumoniae ciaH and ciaR genes,and to determine their correlation with drug resistance.</p><p><b>METHODS</b>The total length of ciaH and ciaR genes was amplified by PCR and their prokaryotic expression systems were established by using routine genetic engineering technique. SDS-PAGE was applied to measure the outputs of target recombinant proteins rCiaH and rCiaR. Rabbits antisera and IgGs against rCiaH and rCiaR were prepared. The resistance to penicillin and cefotaxime of S.pneumoniae strains was examined after CiaH and CiaR were extracellularly and intracellularly blocked by the IgGs.</p><p><b>RESULT</b>The homogeneity of nucleotide and amino acid sequences of the cloned ciaH and ciaR genes with the reported sequences was 99.9-100% and 100%, respectively. The recombinant bacteria E.coli BL21DE3pET42a-ciaH and E.coli BL21DE3pET42a-ciaR were able to express the target recombinant proteins rCiaH and rCiaR with efficiency. The outputs of rCiaH and rCiaR were 33% and 45% of the total bacterial proteins, respectively. The double immunodiffusion titers of rCiaH antiserum,rCiaR antiserum,rCiaH-IgG and rCiaR-IgG were 1:4,1:4,1:1 and 1:1, respectively. After CiaH was extracellularly or intracellularly blocked by CiaH-IgG, and CiaR was intracellularly blocked by CiaR-IgG, the penicillin-sensitive or cefotaxime-sensitive strains developed resistance to the two antibiotics; but the blocks did not change that of penicillin-resisting or cefotaxime-resisting strains.</p><p><b>CONCLUSION</b>The prokaryotic expression systems of S. pneumoniae ciaH/ciaR genes have been successfully constructed in this study. Both the CiaH and CiaR may be involved in penicillin and cefotaxime resistance of the bacterium.</p>