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Objective:To establish and validate a fluorescence focus assay (FFA) for rapid titration of Japanese encephalitis virus (JEV) and to evaluate its application in the production of Japanese encephalitis vaccine.Methods:Recombinant JEV non-structural protein 1 (NS1) was expressed in a prokaryotic expression system. After purification, JEV-NS1 was used to immunize rabbits to induce polyclonal antibody. FFA was established with the polyclonal antibody to titer JEV. The accuracy of FFA was validated by comparing with plaque assay, and the specificity, precision, linearity, range and robustness of FFA were also validated. Twenty-eight batches of live-attenuated Japanese encephalitis vaccine were titrated with FFA and plaque assay to analyze the relationship between the two assays.Results:FFA established with polyclonal antibody against JEV-NS1 could be used to titrate JEV, and there was no cross reaction with other viruses (tick-borne encephalitis virus, yellow fever virus, coxsackievirus A2, coxsackievirus A4). Results of the validation tests showed that FFA met the requirement of quality control for live-attenuated Japanese encephalitis vaccine. FFA was more consistency than plaque assay.Conclusions:The established FFA could be used for virus titration in the production of live-attenuated Japanese encephalitis vaccine.
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Objective:To evaluate the efficacy of linked color imaging (LCI) for the colorectal polyp detection, especially detection of adenoma.Methods:A retrospective analysis was conducted on the patients who underwent LCI or white light imaging(WLI) mode of LASEREO colonoscopy from May 2018 to March 2019.The differences of the detection rates in the global polyps, adenomatous polyps, flat polyps, small polyps (≤5 mm) and right-sided polyps under two modes were compared. Color differences between the adenomatous polyps and surrounding mucosa (ΔE) were examined under two modes, based on L *a *b * color space by the Commission Internationale de L′Eclairage in 1976. Results:The global polyp detection rate, especially adenoma detection rate, in LCI group was higher than that in WLI group(45.53% VS 32.83%, P=0.038; 53.65% VS 39.62%, P=0.009). The color difference(ΔE) between adenomatous polyps and surrounding mucosa in LCI group was significantly higher than that in WLI group(27.24±8.67 VS 15.28±6.68, P<0.001). In addition, the detection rates of flat polyps, small polyps and right-sided polyps in LCI group were higher than those in WLI group (61.98% VS 47.17%, P=0.005; 60.94% VS 42.77%, P=0.001; 45.83% VS 32.70%, P=0.012). Conclusion:LCI can effectively improve the colorectal polyp detection rate, especially adenoma detection rate, which is worthy of clinical application.
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Objective@#To investigate the regulation of emodin on microRNA expressions in mouse model with sepsis by GeneChip microRNA array.@*Methods@#Forty two c57 mice were randomly (random number) divided into 3 groups: sham operation group (sham group, n=14),sepsis group(n=14) and emodin group (n=14). The sepsis model of the mouse was subjected to cecal ligation and puncture (CLP). Mice in emodin group received intraperitoneal injection of emodin (40 mg/kg) half an hour before operation and every 12 hours after CLP. All mice were sacrificed 48 h after surgery and part of the ileum were removed for intestine tissue stained with hematoxylin eosin. The levels of tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6) and intestinal fatty acid binding protein (I-FABP) in peripheral blood were measured by enzyme-linked immunosorbent assay (ELISA). Total RNA of ileum tissues was extracted from the sepsis group and emodin group, and then subjected to miRNA microarray. The results of microarray were further verified by quantitative real-time PCR (qRT-PCR). Target genes of differentially expressed miRNAs were predicted and subjected to gene ontology (GO) and KEGG pathway enrichment analysis. Data of multi-groups were analyzed by one way variance (ANOVA) and inter-group comparisons were made by SNK-q tests. Mann-Whitney U test was used when homogeneity of variance were not met. The value of P<0.05 was considered statistically significant.@*Results@#Compared to the sepsis group, the levels of serum TNF-α, IL-6 and I-FABP in the emodin group were decreased significantly. MiRNA microarray showed that 23 miRNAs were differentially expressed in the emodin group as compared to the sepsis group, among which 17 miRNAs were up-regulated and 6 miRNAs were down-regulated. qRT-PCR results were consistent with miRNA array data. Target genes of 10 selected miRNAs were predicted and subjected to bioinformatic analysis. A total of 3 410 target genes were significantly enriched in 2 072 GO biological process terms, 246 GO cellular component items and 277 GO molecular function terms. Moreover, KEGG pathway analysis showed that these differential miRNAs were involved in the regulation of PI3K-Akt signaling pathway, MAPK signaling pathway, and TNF signaling pathway.@*Conclusions@#Emodin can regulate the expression of multiple microRNAs, and play an important role in protecting the intestinal mucosal barrier via a variety of targets and pathways.
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Objective To investigate the regulation of emodin on microRNA expressions in mouse model with sepsis by GeneChip microRNA array.Methods Forty two c57 mice were randomly (random number) divided into 3 groups:sham operation group (sham group,n=14),sepsis group(n=14) and emodin group (n=14).The sepsis model of the mouse was subjected to cecal ligation and puncture (CLP).Mice in emodin group received intraperitoneal injection of emodin (40 mg/kg) half an hour before operation and every 12 hours after CLP.All mice were sacrificed 48 h after surgery and part of the ileum were removed for intestine tissue stained with hematoxylin eosin.The levels of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6) and intestinal fatty acid binding protein (I-FABP) in peripheral blood were measured by enzyme-linked immunosorbent assay (ELISA).Total RNA of ileum tissues was extracted from the sepsis group and emodin group,and thensubjected to miRNA microarray.The results of microarray were further verified by quantitative real-time PCR (qRT-PCR).Target genes of differentially expressed miRNAs were predicted and subjected to gene ontology (GO) and KEGG pathway enrichment analysis.Data of multi-groups were analyzed by one way variance (ANOVA) and inter-group comparisons were made by SNK-q tests.Mann-Whitney U test was used when homogeneity of variance were not met.The value of P＜0.05 was considered statistically significant.Results Compared to the sepsis group,the levels of serum TNF-α,IL-6 and I-FABP in the emodin group were decreased significantly.MiRNA microarray showed that 23 miRNAs were differentially expressed in the emodin group as compared to the sepsis group,among which 17 miRNAs were up-regulated and 6 miRNAs were down-regulated.qRT-PCR results were consistent with miRNA array data.Target genes of 10 selected miRNAs were predicted and subjected to bioinformatic analysis.A total of 3 410 target genes were significantly enriched in 2 072 GO biological process terms,246 GO cellular component items and 277 GO molecular function terms.Moreover,KEGG pathway analysis showed that these differential miRNAs were involved in the regulation of PI3K-Akt signaling pathway,MAPK signaling pathway,and TNF signaling pathway.Conclusions Emodin can regulate the expression of multiple microRNAs,and play an important role in protecting the intestinal mucosal barrier via a variety of targets and pathways.
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Objective To provide the diagnostic proof for a suspected Chinese family with BS,and NOD2 gene mutation types and clinical features were analyzed in this study.Methods Nine members (4 males and 5 females) of this family were enrolled.To clarify the genotype,the whole exome sequencing by next-generation sequencing from the proband and his parents was performed,and all members were subjected to Sanger sequencing.For the newly discovered NOD2 missense mutation,its pathological predictions were conducted online by adopting polyphen software.Clinical data of affected cases diagnosed by NOD2 analysis were collected to analyze illustrate the clinical features.Results (1)The proband of the family was a 5-year-old Chinese Han boy,who had the clinical triad of dermatitis,polyarthritis and uveitis.The body temperature and C-reactive protein (CRP) was normal.Besides the proband,2 members were diagnosed as BS by means of NOD2 analysis.The coexistence of 2 missense mutations was detected.One novel mutation was c.1981G ＞ C,p.A661 P,and another previously reported one was c.2006A ＞ G,p.H669A.(2) Mutations identified in the male proband were inherited from his father.Tracing the other pedigree members,it was disclosed that his grandmother had the heterozygous dual NOD2 mutations.The proband displayed a phenotype featuring the symptom triad of granulomatous dermatitis,symmetric arthritis,and recurrent uveitis,with normal temperature and CRP level.Conclusions The coexistence of A661P and H669A mutations in NOD2 caused BS in a Chinese pedigree,which derived from the proband's grandmother.This is the first report of A661P mutation in NOD2 in a Chinese pedigree of this disease.The proband has multi hydatoncus surrounding multi-joints,but no persistent fever and no elevated CRP,which may help to differentiate BS from other inherited autoinllammatory diseases in clinical settings.