A comparative analysis of the clinical symptoms of benign paroxysmal positional vertigo between older and young and middle-aged patients / 中华内科杂志

Objective: To compare the differences in clinical symptoms and the time required for diagnosis of benign paroxysmal positional vertigo (BPPV) between older patients and young and middle-aged patients in the structured inquiry of dizziness history. Methods: The medical records of 6 807 patients diagnosed with BPPV from the Vertigo Database of Vertigo Clinical Diagnosis, Treatment, and Research Center of Beijing Tiantan Hospital, Capital Medical University, between January 2019 and October 2021 were retrospectively analyzed. The data included basic demographic information, clinical symptoms in a structured medical history questionnaire, and the time interval from the appearance of BPPV symptoms to diagnosis consultation. The patients were divided into the young and middle-aged group (<65 years old) and the older group (≥65 years old). The differences in clinical symptoms and consultation time were compared between these two groups. Categorical variables were represented by numbers (%), and compared using Chi-squared tests or Fisher's exact probability test for analysis; whereas, continuous variables conforming to normal distribution were represented by mean±standard deviation. Both data groups were compared and analyzed by Student's t-test. Results: The mean age of the older group was 65-92 (71±5) years, while the mean age of the middle-aged group was 18-64 (49±12) years. The incidence of vertigo (42.5% vs. 49.1%, χ2=23.69, P<0.001); vertigo triggered by changes in position of the head or body (52.4% vs. 58.7%, χ2=22.31, P<0.001); and autonomic symptoms (10.1% vs. 12.4%, χ2=7.09, P=0.008) were lower, but hearing loss (11.8% vs. 7.8%, χ2=27.36, P<0.001) and sleep disorders (18.5% vs. 15.2%, χ2=11.13, P=0.001) were higher in the older group than in the young and middle-aged group. The time from the appearance of dizziness to diagnosis was commonly longer in the older patient group than the other group (55.0% vs. 38.5%, χ2=55.95, P<0.001). Conclusions: Older patients with BPPV have more atypical symptoms and complex concomitant symptoms than young and middle-aged patients. For older patients with dizziness, positional testing is needed to confirm the possibility of BPPV even if the clinical symptoms are atypical.

Molecular Cloning,Sequence Analysis,and Expression of Pepsinogens C Gene from Adult Alligator Sinensis Stomach / 中国医学科学院学报

Objective To investigate the molecular clone and structural features of pepsinogen C(PGC) gene in the stomach of Alligator sinensis,explore the phylogenetic relationships and tissue distribution,and analyze the variation of PGC expression in the stomachs of adult Alligator sinensis at different life stages. Methods The full-length cDNA of PGC gene of Alligator sinensis was cloned by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends and then sequenced.The physical and chemical parameters and advanced structures of the PGC protein were predicted by bioinformatics methods and tools.The PGC amino acid sequences of the Alligator sinensis and other vertebrates were compared by Clustal X software.The neighbor-joining phylogenetic tree was built by MEGA 6 software.Immunohistochemistry was used to locate PGC in the gastric mucosa of Alligator sinensis.The variation of the PGC mRNA levels in the stomach at different life stages was detected by quantitative real-time polymerase chain reaction.Results Reverse transcription polymerase chain reaction and rapid amplification of cDNA ends revealed a 1568 bp cDNA full-length sequence containing 1167 bp open reading frame,which encoded 388 amino acids.The PGC gene of Alligator sinensis had been deposited in the GenBank Data Libraries under the accession number of KY799383.Bioinformatics analysis predicted that the Alligator sinensis PGC had a theoretical relative molecular mass of 41 998 with a theoretical isoelectric point of 4.16.In addition,the three-dimensional structure of the PGC was constructed by homology modeling to predict its active site with two essential aspartyl residues and six essential cysteine residues involved in forming three disulphide bonds.The neighbor-joining phylogenetic tree of vertebrates from the amino acids sequences of PGC showed all crocodiles were clustered as a group,and the PGC of Alligator sinensis was the closest to Alligator mississippiensis.Alligator sinensis PGC was specifically expressed in the gastric mucosa,and its expressions significantly differed during reproduction and hibernation significantly(P<0.05).Conclusions Alligator sinensis PGC gene is highly conserved in evolution.Its protein is a gastric specific digestive proteinase that belongs to a aspartic proteinase family.

Comparison of the rescue efficiency of Sendai virus minigenome mediated by CMV and T7 promoter / 病毒学报

In this study, we constructed the plasmid of Sendai virus (SeV) BB1 strain minigenome with Gaussia luciferase (Gluc) as reporter and compared the rescue efficiency of SeV minigenome mediated by T7 promoter with that by CMV promoter. Firstly, the sequence was designed and synthesized which contained hammerhead ribozyme, sequence composed of the trailer, untranslated region of L gene, untranslated region of N gene, and the leader from SeV, and mutant hepatitis delta virus ribozyme sequence. Then, the synthesized sequence was inserted into pVAX1 containing CMV and T7 promoters and the general vector for SeV minigenome pVAX-miniSeV was obtained. Furthermore, pVAX-miniSeV-Gluc (+) and pVAX-miniSeV-Gluc(-) were obtained by inserting Gluc gene into pVAX-miniSeV. From the supernatant of BHK-21 cell transfected with pVAX-miniSeV-Gluc(+), high level of Gluc expression was detection indicating the normal transcription function of CMV promoter. pVAX-SeV-miniGluc(-) and plasmids expressing N,P and L protein of SeV were co-transfected into BST T7/5 cell which derived from BHK-21 and expressed T7 RNA polymerase stably. And high expression of Gluc was found, which indicated that SeV minigenome was efficiently rescued. However, we failed to repeat the result on BHK-21 cell, implying that T7 promoter and CMV promoter may have different effects on the rescue efficiency of SeV minigenome. Therefore, we further constructed SeV minigenome vectors pT7-miniSeV-Gluc (-) and pCMV-miniSeV-Gluc(-) with single promoter of T7 or CMV. Then, these vectors were transfected into BSR T7/ 5 cells respectively accompanied with the N, P, and L protein expression vectors. The result demonstrated that high expression of Gluc was found in the group of pT7-miniSeV-Gluc(-), which failed in the group of pCMV-miniSeV-Gluc(-). It indicated that T7 promoter significantly increased the rescue efficiency of SeV minigenome. We successfully constructed a SeV minigenome vector with secreted luciferase gene as report er and proved T7 promoter can enhance the rescue efficiency of SeV minigenome, which provides basis for construction of infectious clone containing SeV full-length genome.

Study on the differences of two mouse models of hepatitis B virus infection by transduction with rAAV8-1. 3HBV / 病毒学报

We recently developed a mouse model of hepatitis B virus (HBV) chronic infection by intravenous (i.v.) injection with rAAV8-1. 3HBV to C57BL/6 mice. To define the responses of different mouse strains after injection with rAAV8-1. 3HBV, we intravenously injected rAAV8-1. 3HBV at doses of 4 x10(9) (Viral genome,vg), 4 x 10(10) vg and 4 x 10(11) vg to C57BL/6 and BALB/c mice,respectively, and determined the levels of serum HBV antigen and antibody by ELISA,serum viral DNA by real-time PCR,and HBcAg expression in liver by immunohistochemical staining. For C57BL/6 mouse strain with injection of rAAV8-1. 3HBV at three doses, 100% of the mice carried HBV for more than 8 months. The levels of serum HBsAg and HBeAg, serum viral DNA and HBcAg-positive hepatocytes increased in a rAAV8-1. 3HBV dose-dependent manner. For C57BL/6 mice injected with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg,over 40% of hepatocytes expressed HBcAg and serum viral DNA reached over 10(5) IU/mL. No HBV antibody was detected in sera of C57BL/6 mice. For BALB/c mice with injection of rAAV8-1. 3HBV at three doses, serum HBeAg, serum viral DNA and HBcAg-positive hepatocytes persisted for more than 8 months, but serum HBsAg declined remarkably at 2 weeks after injection. The levels of serum HBeAg and HBcAg-positive hepatocytes in BALB/c mice increased in a rAAV8-1. 3HBV dose-dependent manner. Injection with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg resulted in over 50% of BALB/c mice hepatocytes expressing HBcAg. Serum anti-HBsAg were detected in BALB/c mice with rAAV8-1. 3HBV injection at the dose of 4 x10 (10) vg. In conclusion, both C57BL/6 and BALB/c strains can be developed to chronic HBV infection mouse models by i. v. injection with rAAV8-1. 3HBV at doses of 4 x10(9) - 4 x 10(11) vg and the levels of HBV replication increase in a rAAV8-1. 3HBV dose-dependent manner. In contrast to C57BL/6 strain, the BALB/c mice carry out humoral immunity to HBsAg, but fail to mediate HBV clearance.

Recombinant adeno-associated virus type 8 mediated dual-luciferase gene expression in mouse / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc).</p><p><b>METHODS</b>rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro. BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im). Then Glue activities in blood were measured,the whole-body images for Flue activities were performed and Flue activities of tissue lysate were also detected.</p><p><b>RESULTS</b>rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells. The Glue was mainly detected in the culture media while the Flue was mainly detected within cells. The blood Glue activities of mice received rAAV8-Gluc/Fluc by iv or im peaked at 10-20 d post injection and persisted for at least 120d. The blood Gluc activities increased at the rAAV8-Gluc/Fluc dose-dependent manner. For mice received rAAV8 by iv, Fluc mainly expressed in liver and minor Fluc expression was also found in cardiac muscle and skeletal muscle. For mice received rAAV8-Gluc/Fluc by im, Fluc mainly expressed in local skeletal muscle and secondly in liver.</p><p><b>CONCLUSION</b>rAAV8-Gluc/Fluc has been prepared successfully and its mediating transgene expression in mice has been investigated. This research will facilitate preclinical studies for rAAV8-mediated gene therapy.</p>

Effects of Lin28a and Lin28b on let-7 family activity / 病毒学报

In this report, we study the effects of over-expression of Lin28a and Lin28b on let-7 family activity in HeLaS3. Firstly, we constructed pAAV2neo-Lin28a and pAAV2neo-Lin28b to express Lin28a and Lin28b, respectively. Then, pAAV2neo-Lin28a and pAAV2neo-Lin28b were transfected into HeLaS3, selected with G418 and obtained cell lines, HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b, to express Lin28a and Lin28b stably. Thereafter, we constructed eight plasmid vectors for detection of let-7 family activity based on pAAV2neo-Gluc-(Fluc). These vectors were further packaged into recombinant adeno-associated viral vectors (rAAV) which were used as sensors, nominated as Asensors, to detect inhibition activity of miRNA at post-transcriptional level. Subsequently, with HeLaS3 as a control, we assayed expression levels of Lin28a and Lin28b by Western blot, detected expression levels of let-7 family by QRT-PCR, and tested let-7 family activity by Asensors in HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b. Results demonstrated that both HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b could express Lin28a and Lin28b effectively. Compared with HeLaS3, the expression level of let-7 family except let-7e declined in HeLaS3/pAAV2neo-Lin28a. But declining extent among members of let-7 family was different. The let-7 family activity also decreased while the decreasing extent varied among members. Furthermore, the activity level was not consistent with its expression level for the same member in let-7 family. Compared with HeLaS3, both expression level and activity level of let-7 family in HeLaS3/ pAAV2neo-Lin28b were decreased. However, the decreasing extent of let-7 family expression changes was larger than that of HeLaS3/pAAV2neo-Lin28a while the decreasing extent of activity changes was similar. In this study, we established a method to detect and compare post-transcriptional inhibition level mediated by miRNA complementary targets. We firstly clarified the effect of Lin28a and Lin28b on let-7 family activity profile and found that this effect was not the same as that at expression level of let-7 family, suggesting that it was more comprehensive to understand miRNA regulation roles to detect both miRNA expression and activity. This paves a way for further research on mechanism of regulation of let-7 family.

Deletion of IV a2 gene from adenoviral genome by lambda-Red recombinase system and packaging of the recombinant adenovirus / 病毒学报

This investigation is to delete the most of the coding sequence (1104 bp) of the IV a2 gene in an adenovirus genome by a lambda-Red recombinase system-mediated PCR-targeting approach and rescue a recombinant adenovirus with IV a2 deletion. First, the template pAK of PCR targeting, containing kanamycin cassette, was constructed. Then, a linear fragment for PCR targeting, which had 39 bp homologous arms at both of its terminus, was amplified by PCR from the pAK. The pFG140 and the linear fragment were electroporated into E. coli BW25113/pIJ790 sequentially and the recombinant pFG140-deltaIV a2 (1104) was established by homologous recombination between the linear fragment and the pFG140 with aid of X-Red recombinase. The precise deletion of 1 104 bp fragment from IV a2 was confirmed by restriction endonucleases digestion and DNA sequencing. ORF of IV a2 was amplified by PCR from pFG140 and then cloned into the pAAV2neo vector. The recombinant adenovirus Ad5delta IV a2 (1104) was rescued by co-transfection of pFG140-deltaIV a2 (1104) and pAAV2neo-IV a2 into HEK293 cells. It was shown by Western Blot that IV a2 could not be detected in the Ad5deltaIV a2 (1104)- infected HEK293 cells. This study established a PCR-targeting strategy for manipulating adenovirus genome directly by a lambda-Red recombinase system, and a recombinant adenovirus with IV a2 deletion was obtained.

Establishment of hepatitis B virus (HBV) chronic infection mouse model by in vivo transduction with a recombinant adeno-associated virus 8 carrying 1. 3 copies of HBV genome (rAAN8-1. 3HBV) / 病毒学报

In this report, we developed a HBV infection model in C57BL/6 mouse line by in vivo injection of a recombinant adeno-associated virus 8 vector carrying 1. 3 copies of HBV genome (ayw subtype) (rAAV8-1. 3HBV). We firstly prepared and purified the rAAV8-1. 3HBV and then injected it into three C57BL/6 mice with the dose of 2 x 10e11vg, respectively. HBsAg and HBeAg were assayed in sera collected at different time points post injection. Ten weeks post injection, the three mice were sacrificed and blood and liver tissue were taken for assay. Copies of HBV DNA were detected by real time PCR and the way of HBV DNA replication was identified by PCR. Subsequently, detection of HBV antigen by immunohistochemistry and pathology analysis of liver tissue of mice were performed. The results suggested that expression of HBsAg and HBeAg lasted for at least 10 weeks in mice sera. Among mice injected with rAAV8-1. 3HBV, HBsAg levels were showed an 'increasing-decreasing-increasing' pattern (the lowest level at the 4th week post injection), while HBeAg levels were kept high and relatively stable. HBV DNA copies were 4.2 x 10(3), 3.6 x 10(3), 2.5 x 10(3) copies/mL in sera and 8.0 x 10(6), 5.7 x 10(6), 2.6 x 10(6) copies/g in hepatic tissues of three mice, respectively. We found that the linear 1. 3HBV DNA in the rAAV8-1. 3HBV could self form into circular HBV genome and replicate in livers of HBV transfected mice. HBsAg and HBcAg were both positive in liver tissue of mice injected with rAAV8-1. 3HBV and no obvious pathological characters were found in liver of mice injected with rAAV8-1. 3HBV. In conclusion, we successfully developed a HBV chronic infection model in C57BL/6 mouse line by in vivo transduction with the recombinant virus rAAV8-1. 3HBV, in which HBV genes could be continuously expressed and replicated over 10 weeks, and paved a way for further characterization of the human chronic hepatitis B virus infection and evaluation of vaccine and anti-HBV agents.

Construction of a dual-luciferase co-expression vector and its characteristics in vitro and in vivo / 病毒学报

A novel dual luciferase expression vector was designed and its expression characteristics were studied in vitro and in vivo. Firstly, the Gluc and Fluc genes were connected via the TaV 2A sequence by overlaping PCR, and inserted into the expression vector pAAV2neoCAG, obtaining the recombinant plasmid pAAV2neoCAG-Gluc-2A-Fluc. Then pAAV2neoCAG-Gluc-2A-Fluc was transfected into BHK21 cells and the activity of Gluc and Fluc in the supernatant and cell lysates were assayed respectively. Results showed that both Gluc and Fluc were expressed successfully. The Gluc was mainly detected in the culture media while the Fluc was mostly within cells. The activity of Gluc in the supernatant increased gradually with time while the Fluc activity in cells almost kept stable. To investigate the expression of pAAV2neoCAG-Gluc-2A-Fluc in vivo, the plasmid was hydrodynamically injected into BALB/c mice through tail vein. The Gluc activity was assayed in a small volume of blood taken by tail vein at different time points. Results showed that Gluc was expressed stably at least 7 days. Live bioluminescence imaging technology was used to compare the expression characteristics of Gluc and Fluc. Whole body imaging was seen when coelenterazine, a specific substrate for Gluc, was injected, and the imaging signals decreased rapidly within 10 minutes. Liver imaging was showed when Flue specific substrate named D-Luciferin was injected, and the imaging remained stable at least for half an hour. The dual luciferase expression vector pAAV2neoCAG-Gluc-2A-Fluc combines the advantages of the secreted report gene Gluc and the non-secreted report gene Fluc, and will provide a new tool for cell labeling and tracing.

Cloning and expression of the six coding genes of sendai virus BB1 strain / 病毒学报

Six genes for nucleoprotein, phosphoprotein, matrix protein, hemagglutinin neuramindase protein, fusion protein and large protein were obtained by reverse transcription and PCR methods based on our previous work of sequencing full length genome of sendai virus BB1 strain (DQ219803 in GenBank). Sequencing showed the six genes were completely identical to that we reported. In order to supply the function necessary for rescuing and packaging of sendai virus vector in trans, the N, P, M, F, HN and L genes were separately cloned into an adenoviral shuttle expression vector pDC316 resulting in six recombinant adenoviral plasimds. Six replicating defective recombinant adenoviruses Ad5-N, Ad5-P, Ad5-M, Ad5-F, Ad5-HN and Ad5-L were obtained by separately cotransfection of pDC316 carrying N, P, M, F, HN and L genes with the adenoviral genomic plasmid pBHGloxdeltaE1, 3Cre into HEK293cells. Restrictive enzymatic results indicated that the six recombinant plasmids were correctly constructed. PCR results showed the recombinant adenoviruses contained the respective SeV genes . Western blotting as well as immunofluorescence assay indicated the expression of the corresponding proteins of sendai virus. These work laid the basis for the construction of the full length genome plasmid of sendai virus BB1 strain and the setup of SeV virus vector system based on SeV BB1 strain.

Two HCV vaccines based on adeno-associated virus vectors elicited durable and effective immune responses in mice / 病毒学报

Recombinant adeno-associated viruses (rAAV) vectors have been shown to mediate long-term transgene expression in mice and nonhuman primates. We have adapted viral vector system based on two rAAV vectors, namely rAAV1 and rAAV2. We have generated rAAV vectors expressing the envelope glycoprotein (E1 and E2) derived from Chinese HCV patient (genotype 1b) and used these to immunize BALB/c mice. We detected the total antibody titer by IFA and neutralizing antibody (nAb) using in vitro HCV neutralizing assays based on HCV pseudotyped particles. Furthermore, IFN-gamma ELISpot assay was used to assess the T cellular response against HCV at 12 weeks after rAAV1-E1E2 immunization. We also analyzed HCV envelope glycoprotein expression in muscle of rAAV1-E1E2 immunized mice. Our data showed: (i) rAAV1 directed long-term expression of HCV genes in mice; (ii) immunized intramuscularly with a single dose of rAAV elicited durable and effective immune responses in mice; and (iii) Moreover, rAAV1-E1E2 induced higher total antibody and nAb titers than rAAV2-E1E2 did. These data suggest that rAAV1 vectors could stimulate robust, durable, and effective immune responses against HCV.

The preparation of monoclonal antibodies against AAV2 and study on their characterizations / 病毒学报

7 strains of stable cell lines secreting monoclonal antibodies against AAV2 capsids were obtained by immunizing BALB/C mice with highly purified recombinant adeno-associated virus. Among them, the monoclonal antibodies B10 and G4 had neutralizing activity, and their subtypes were IgG1 and IgG2a, respectively. The binding characterizations of the two neutralizing antibodies were studied. Both B10 and G4 showed serotype specific binding activities to rAAV2 virus particles other than AAV1, AAV5, and AAV8, and the binding could not be blocked by heparin. After incubating with the two antibodies separately, rAAV2 viruses could still bind to sensitive cell line BHK-21, suggesting that the binding sites of the two antibodies to rAAV2 located at different positions on viral particle surface from the primary receptor binding sites of AAV2. Western blotting assay showed that B10 could bind to VP1, VP2 and VP3 of rAAV2. However, G4 bound none of them. The results suggested that B10 recognized a linear epitope of AAV2 capsid, whereas G4 probably recognized a conformational epitope on the surface of AAV2 virus particle. The two antibodies with different characteristics provided valuable tools for AAV2 virus particles detection and infection processes.

Study on the association of cytochrome P450 polymorphisms and the risk of esophageal cancer: a meta-analysis / 中华流行病学杂志

Objective To examine the association between CYP1A1 polymorphisms (MspI and Ile/Val) and esophageal cancer (EC) by systematically reviewing the risk of the original studies. Methods Data from 16 papers (8 for MspI, 14 for Ile/Val) regarding case-control studies on the association of cytochrome P450 polymorphisms and risk of esophageal cancer was analyzed by dominant model (variant genotype vs. wild-type genotype) through meta-analysis. Stratified analysis was carried out according to the pathological types. Results In systematical analysis, CYP1A1 MspI variant genotype (TC+CC) had no association with EC risk (OR=1.17,95%CI: 0.82-1.66). Similar results were observed in esophageal squamous-cell carcinoma(ESCC) (OR=1.17,95%CI: 0.82-1.69) and esophageal adenocarcinoma (EAC) (OR=1.39,95% CI: 0.67-2.09). Individuals with the CYP1A1 Ile/Val variant genotype (Ile/Val + Val/Val) had an increased risk for EC, when comparing with wild type (Iie/Iie ), with an OR of 1.39 (95 %CI: 1.07-1.80). CYP1A1 Ile/Val variant genotype could increase the risk of ESCC (OR=1.43,95%CI:1.07-1.91) but no significant association was found with EAC (OR=1.20,95%CI:0.62-2.30). Conclusion CYP1A1 gene polymorphism Ile/Val might have played a role in the development of ESCC but CYP1A1 MspI polymorphism might not be associated with the susceptibility of EC.

Comparison of TTR and CMV promoters in vivo and in vitro via a secreted luciferase reporter system / 病毒学报

GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.

DNA repair gene xeroderma pigmentosum group D 751 polymorphism and the risk on esophageal cancer:a Meta-analysis / 中华流行病学杂志

Objective To explore the association between XPD codon 751 polymorphism and esophageal cancer (EC) by systematically reviewing the risk of the original studies. Methods A comprehensive search was conducted to identify all case-control studies of XPD codon 751 polymorphism and EC risk. Meta-analysis was applied with Rev Man 4.2 software for calculation of pooled OR value (with 95%C1)of EC, esophageal squamous cell carcinoma (ESCC)and esophageal adenocarcinoma (EAC). Results Of the 12 case-control studies selected for this Meta-analysis, a total of 2558 EC cases and 5122 controls were included. Compared with the wild-type homozygote Lys/Lys, the pooled Odds Ratios (with 95% CI) of Lys/Gln, Gin/Gin, (Lys/Gln + Gln/Gln) genotypes of XPD codon 751 polymorphism for EC risk were 1.19(1.05, 1.34), 1.22(0.86, 1.74), 1.20(1.01,1.42), respectively. In a stratified analysis, a total of 1417 ESCC cases and 2312 controls were included, and individuals carrying Lys/Gln genotype or (Lys/Gln+Gln/Gln) had 1.22-fold or 1.24-fold excess risks for ESCC compared with those carrying Lys/Lys genotype. A total of 935 EAC cases and 2604 controls were included, and none of the genotype of XPD codon 751 genetic polymorphism was found to be related to EAC. Conclusion Both heterogyzote Lys/Gln and (Lys/Gln + Gln/Gln) for XPD codon 751 genetic polymorphism were associated with an increased risk of developing esophageal cancer. Furthermore, heterogyzote Lys/Gln and (Lys/Gln + Gln/Gln) for XPD codon 751 genetic polymorphism might have increased the risk of ESCC, but have no association with EAC.

Analysis of the tandem-repeat polymorphisms in DC-SIGNR alleles among drug users population with or without HIV/HCV infection / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the distribution of DC-SIGN/DC-SIGNR alleles among drug user (DUs) populations with or without HIV/HCV infection in Shenzhen, and to evaluate the role of these alleles in the construction of genetic resistance to HIV or HCV and screen out the anti-HIV/HCV gene in Shenzhen.</p><p><b>METHODS</b>All 500 DU blood samples were collected from Shenzhen Detoxification Center, including 313 from injected drug users (IDUs). All samples were screened for HIV and HCV antibody by means of ELISA. The genomic DNA were extracted and amplified by PCR. The neck domain repeat regions of DC-SIGN/DC-SIGNR were sequenced directly from the PCR products to confirm the amplification for some samples and all positive PCR products were analyzed by agarose gel electrophoresis.</p><p><b>RESULTS</b>Of 500 samples, 97 were found HIV positive, all of which were IDUs and HCV positive. The total positive rate of HCV among all HIV negative DU was 57.57% (232/403), and it was 63.89% (138/216) among IDUs; in comparing to the 50.26% (94/187) of DUs with other manners there showed significant difference (chi(2) = 7.61, P = 0.0058). Among HIV + DUs, there was a higher proportion of patient with the DC-SIGNR 5/6 and 5/8 (Fisher's exact, P = 0.043 and P = 0.034) with statistical significance; there was no statistically significant difference between HCV + and HCV-DUs and no significant difference between IDUs and other DUs for the DC-SIGNR polymorphism.</p><p><b>CONCLUSION</b>The results might indicate that DC-SIGN/DC-SIGNR polymorphism might not influence the susceptibility to HCV. Genotype 5/6 might probably have a relation with HIV infection, but still need further investigation for the low frequency.</p>

Development of infectious pseudo-particle harboring three subtypes hepatitis C virus glycoproteins and their application in neutralization assays / 病毒学报

In this study, three expression vectors encoding unmodified glycoproteins E1 and E2 from H77 (1a), Hebei (1b) and JFH1 (2a) strains were constructed to form pVRC-H77-E1E2, pVRC-HeBei-E1E2 and pVRC-JFH1-E1E2 expressing constructs. The protein expression was confirmed by immunofluorescene assay(IFA) and Western blot. The Lentiviral vector has the ability to package the cellular membrane into pseudo-particles. The plasmid expressing HCV E1-E2 glycoproteins in native form was co-transfected into 293FT cells with a lentiviral packaging plasmid (pHR'CMV delta R8.2)and a self-inactivated (SIN) transfer plasmid (pCS-CG) containing a reporter EGFP gene to produce infectious HCV pseudo-particles(pp). Flow cytometry assays showed that the HCVpp could infect Huh7 and Huh7-CD81, and the infectivity in Huh7-CD81 was about 2-3 times higher than that in Huh7 cells. Meanwhile, HCVpp could neither infect non-liver cells, for example, the 293 cells, nor HepG2 cell . Titration of HCVpp by p24 ELISA assay or infection assay showed that this HCVpp may contain 5-25 ng/mL p24 or 10(4)-10(5) TU (transducing unit)/ ml. An in vitro HCV neutralizing assays based on HCVpp (1a, 1b, 2a) were then established using AP33, a monoclone antibody with cross-neutralizing ability to different HCV strains. The neutralizing ability of the antibodies from HCV infected patients was further studied with this HCVpp system. In summary, three kinds of HCVpp (1a, 1b, 2a subtype) were successfully developed; In vitro HCV neutralizing assays based on HCVpp and SIN lentiviral system were established. This system paves a way for characterization of early steps of HCV infection (host tropisms, receptor binding, membrane fusion, et al. ) or screening anti-HCV drugs (such as inhibitor to virus entry). This system can be further applied to assess the human immune responses in HCV patients or evaluate HCV vaccine candidates.

Isolation and characterization of H2-producing strains Enterobacter sp. and Clostridium sp / 生物工程学报

Two hydrogen-producing bacterial strains were newly isolated and identified as Enterobacter sp. Z-16 and Clostridium sp. C-32 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen production of strain Z-16 were achieved as: initial pH7.0, temperature 35 degrees C , sucrose as the favorite substrate. In comparison, The optimum condition for hydrogen production of strain C-32 were obtained as: initial pH8.0, temperature 35 degrees C , maltose as the favorite substrate . Under batch fermentative hydrogen production conditions, the maximal hydrogen conversion rate for strain Z-16 and strain C-32 were 2.68 mol H2/mol sucrose and 2.71mol H2/mol maltose, respectively. Using glucose as substrate, the hydrogen conversion rate of strain Z-16 and strain C-32 were 2.35 and 2.48 mol H2/mol glucose, respectively. This research suggest a good application potential of strain Z-16 and C-32 in the future biological hydrogen production.

The in vivo and in vitro effects on neovascularization of VEGF gene transfer with a new pseudotyped rAAV1 vector / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effects of vascular endothelial growth factor (VEGF) gene transfer with a new pseudotyped recombinant adeno-associated virus (rAAV) vector with AAV serotype 1 capsid protein (rAAV1) vector on neovascularization.</p><p><b>METHODS</b>PBS, rAAV1-GFP and rAAV1-VEGF vectors were added in C2C12 derived myotubes [10(5) vg (vector genomes) per cell]. Transfer efficiency was determined by fluorescent microscope and VEGF protein concentration in the culture media measured by ELISA. Ten days following ischemia in a hindlimb ischemic mouse model, PBS, 3 x 10(11)vg rAAV1-LacZ vectors and rAAV1-VEGF165 vectors were injected in ischemic thigh muscles. VEGF protein at ischemic thigh muscle was measured by ELISA at 1 month after vector infection. Capillaries and arterioles were observed by immunohistochemical analysis at 6 weeks after vector infection.</p><p><b>RESULTS</b>GFP expression was found in 60% - 80% myotubes at 120 hours after rAAV1-GFP infection. VEGF protein peaked at the 3rd day post rAAV1-VEGF infection with an average concentration of (567.7 +/- 16.8) pg/ml. Transfer efficacy in ischemic thigh muscle was 100% one month post rAAV1-LacZ infection. The average concentration of VEGF protein in ischemic skeletal muscles is (205.4 +/- 36.1) pg/mg total protein in rAAV1-VEGF165 treated mice. Extensive angiogenesis [(147.0 +/- 13.3)/mm(2)] and arteriogenesis [(17.0 +/- 1.2)/mm(2)] were observed in ischemic skeletal muscles at 6 weeks post rAAV1-VEGF165 injection.</p><p><b>CONCLUSION</b>Gene transfer with the new pseudotyped rAAV1-VEGF165 vector might be an effective therapeutic approach for ischemic cardiovascular diseases.</p>

Expression of hCTLA4-Ig mediated by adeno-associated virus in porcine islets and their biological activity / 中南大学学报(医学版)

OBJECTIVE@#To evaluate the expression of hCTLA4-Ig and their biological function in newborn porcine islets (NPIs) transfected with AAV-hCTLA4-Ig.@*METHODS@#Cultured NPIs were transfected with AAV-hCTLA4-Ig. The expression of CTLA4-Ig in these NPIs was assayed by RT-PCR and immunocytochemistry. The levels of IL-2, IFN-gamma, and TNF-alpha in the culture medium were assayed by ELISA after these cells the co-cultured with human. The response of glucose-stimulated insulin secretion was observed in the transgene group and the control group.@*RESULTS@#The expressions of CTLA4-Ig mRNA and protein were detected in the transgene group. The levels of cytokines were obviously lower in the transgene group than those in the control group (P0.05).@*CONCLUSION@#AAV mediated hCTLA4-Ig expression in NPIs could inhibit T lymphocyte to produce cytokines, while the endocrine functions of the NPIs were not significantly affected.