[Show abstract][Hide abstract] ABSTRACT: Human tissue plasminogen activator (tPA) belongs to the serine protease family. It converts plasminogen into plasmin and is used clinically to treat thrombosis. Human tPA is composed of 527 amino acids residues and contains 17 disulfide bonds. Escherichia coli has been used only rarely for the efficient production of recombinant tPA. However, the functional expression of full-length tPA that contains multiple disulfide bonds on an industrial scale remains challenging. Here, we describe the soluble expression and characterization of full-length tPA by auto-induction in E. coli.
We achieved optimal levels of gene expression, minimized negative effects related to the production of heterologous proteins, and optimized cytoplasmic yields. Three different E. coli strains, BL21 (DE3), Rosetta, and Origami 2, could express tPA using an auto-induction mechanism. In addition, similar yields of recombinant protein were produced at temperatures of 33, 35, and 37°C. The E. coli strain origami 2 could increase disulfide bond formation in cytoplasmic tPA and produce purified soluble recombinant protein (~0.9 mg/l medium). The full-length tPA was monomeric in solution, and fibrin plate assays confirmed that the recombinant tPA displayed serine protease activity.
This is the first report that describes the heterologous expression of correctly folded active full-length tPA. This could provide valuable information for using prokaryotic auto-induction expression systems to produce tPA at industrial and pharmaceutical levels without in vitro refolding during the production step.
[Show abstract][Hide abstract] ABSTRACT: Human rhinovirus (HRV) is not only responsible for at least one-half of all common colds but is also associated with bronchitis, bronchiolitis, pneumonia, and acute asthma exacerbation. However, the impact of different HRV types and viral load on disease severity has not been thoroughly elucidated.
From January 2012 to September 2014, 1742 nasopharyngeal aspirate specimens from hospitalized children with lower respiratory tract infections (LRTIs) were analyzed by quantitative HRV-specific real-time polymerase chain reaction (PCR).
Among these 1742 children, HRV (407/1742, 23%) was the second most common viral agent following RSV. HRV-A, HRV-B, HRV-C, and HRV untyped were detected in 229 (56%), 27 (7%), 100 (25%), and 51 (13%) specimens, respectively. Except children who experienced wheezing were more common in the HRV-C detection group than in the HRV-A detection group, there were no other significant differences between the two groups, including the percent of children diagnosed with severe diseases. Logistic regression models demonstrated that there was no difference in disease severity among HRV types. In HRV-A detection group, when children was under 2 years of age, the viral load was higher in the severe group than in the non-severe group, but in the HRV-C detection group, there was no difference.
HRV was frequently present in hospitalized children with LRTIs in Chongqing, China. The disease severity for HRV-C and HRV-A was similar. A high load of HRV-A in the lower respiratory tract might be connected with disease severity in children under 2 years of age.
[Show abstract][Hide abstract] ABSTRACT: Viral genotype shift in chronic hepatitis B (CHB) patients during antiviral therapy has been reported, but the underlying mechanism remains elusive.
38 CHB patients treated with ADV for one year were selected for studying genotype shift by both deep sequencing and Sanger sequencing method.
Sanger sequencing method found that 7.9% patients showed mixed genotype before ADV therapy. In contrast, all 38 patients showed mixed genotype before ADV treatment by deep sequencing. 95.5% mixed genotype rate was also obtained from additional 200 treatment-naïve CHB patients. Of the 13 patients with genotype shift, the fraction of the minor genotype in 5 patients (38%) increased gradually during the course of ADV treatment. Furthermore, responses to ADV and HBeAg seroconversion were associated with the high rate of genotype shift, suggesting drug and immune pressure may be key factors to induce genotype shift. Interestingly, patients with genotype C had a significantly higher rate of genotype shift than genotype B. In genotype shift group, ADV treatment induced a marked enhancement of genotype B ratio accompanied by a reduction of genotype C ratio, suggesting genotype C may be more sensitive to ADV than genotype B. Moreover, patients with dominant genotype C may have a better therapeutic effect. Finally, genotype shifts was correlated with clinical improvement in terms of ALT.
Our findings provided a rational explanation for genotype shift among ADV-treated CHB patients. The genotype and genotype shift might be associated with antiviral efficiency.
PLoS ONE 06/2015; 10(6):e0131337. DOI:10.1371/journal.pone.0131337 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To describe the Hepatitis B e antigen(HBeAg) seroconversion related mutation profiles of the basal core promoter(BCP)/precore regions in e antigen seroconverted child patients, a cohort of 245 child patients with CHB and a control patients group of 92 adult patients with CHB were recruited. The mutation frequencies of six nucleotides or nucleotide combinations including nucleotide (nt)1896, nt1762/1764, nt1752, nt1846, nt1899 and nt1753 showed significant differences between HBeAg positive and HBeAg-negative child patients groups. The frequencies of these HBeAg seroconversion-related mutations were significantly lower in HBeAg-negative children with CHB than in HBeAg-negative adults with CHB, especially for the mutation G1896A (41.1% vs 91.7%, P<0.001), and the average number of BCP/precore region mutations in samples from HBeAg-negative child patients was also obviously lower than in HBeAg-negative adult patients(3.62±3.03 vs 4.89 ±2.09, P<0.001), suggesting less impact of mutations in the BCP/precore region on HBeAg seroconversion in child patients than adult patients.
PLoS ONE 03/2015; 10(3):e0120733. DOI:10.1371/journal.pone.0120733 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in basal core promoter (BCP) and precore regions of hepatitis B virus (HBV) are associated with course and treatment outcomes of chronic HBV infection. While BCP and precore mutation analysis have been carried out in adult patients between different genotypes, this analysis has rarely been performed for chronically infected children.
The aim of this study was to assess the mutation profiles of BCP and precore regions in different HBV genotypes in chronically infected children.
A cohort of 245 children and 92 adults with chronic HBV infection was included in this study. BCP and precore regions were analyzed by PCR amplification and sequenced.
Ten nucleotide positions, including nt1679, nt1721, nt1753, nt1757, nt1758, nt1762, nt1764, nt1775, nt1856 and nt1858 in BCP/precore regions of HBV genome, showed obviously higher frequencies of mutation in genotype C subjects than in genotype B subjects among children, while there were only three positions, including nt1679, nt1758 and nt1775 showing higher mutation frequencies in genotype C subjects than in genotype B subjects in adults. Several combined mutations were obviously highly distributed in children with chronic HBV genotype C infection, such as G1721A/A1775G/T1858C triple mutation; a novel combined mutation type, exclusively detected in children with chronic HBV genotype C infection. In addition, G1721A/A1775G/T1858C combined mutation was associated with higher viral load and lower age distribution.
The mutation ratio difference between genotypes B and C in children was higher than that of adults and several combined mutations were exclusively detected in children with chronic HBV genotype C infection associated with higher viral load.
[Show abstract][Hide abstract] ABSTRACT: Studies on molecular mechanisms of the persist infection of hepatitis B virus have been hampered by a lack of a robust animal model. We successfully established a simple, versatile, and reproducible HBV persist infection model in vitro and in vivo with the circularized HBV DNA. The cells and mice were transfected or injected with circularized HBV DNA and pAAV/HBV1.2, respectively. At the indicated time, the cells, supernatants, serum samples, and liver tissues were collected for virological and serological detection. Both in vitro and in vivo, the circularized HBV DNA and pAAV/HBV1.2 could replicate and transcribe efficiently, but the infection effect of the former was superior to the latter (p < 0.05). The injection of circularized HBV genome DNA into the mice robustly supported HBV infection and approximately 80% of HBV infected mice established persistent infection for at least 10 weeks. This study demonstrated that the infection efficiency and replication ability of the circularized structure of HBV DNA overmatched that of the expression plasmid containing the linear structure of HBV DNA in vitro and in vivo. Meanwhile, this research results could provide useful tools and methodology for further study of pathogenic mechanisms and potential antiviral treatments of human chronic HBV infection in vitro and in vivo.
International Journal of Molecular Sciences 03/2015; 16(3):5141-5160. DOI:10.3390/ijms16035141 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dysregulation of miRNA is widely involved in human cancers, including hepatocellular carcinoma (HCC). Array data for miRNAs indicated that miR-331-3p might be one of the disorderly expressed miRNAs in HCC cell lines, but the function of miR-331-3p in HCC remains unclear. In this study, quantitative real time polymerase chain reaction (qRT-PCR) results indicated that miR-331-3p was up-regulated in HepG2.2.15 cells, Ad-HBV-HepG2 cells and pCH9/3091transfected SMMC7721 cells compared with their control group, respectively. miRNA target prediction software was used, and VHL was found to be one of the target genes of miR-331-3p. qRT-PCR and western blot analysis indicated VHL expression was decreased when miR-331-3p was over-expressed and increased when miR-331-3p was inhibited in SMMC7721 cells. The luciferase reporter activity was inhibited in SMMC7721 cells when co-transfected with miR-331-3p expression vector and VHL 3'-UTR wild type vector and increased in HepG2.2.15 transfected with miR-331-3p inhibitor compared to its control group respectively. When co-transfected with miR-331-3p expression vector and VHL 3'-UTR mutated type vector in SMMC7721 cells the luciferase reporter activity was recovered. All of these results show that HBV up-regulated miR-331-3p expression in HCC cell lines and miR-331-3p could inhibit VHL expression by directly targeting its 3'-UTR. This provided useful information in exploring the mechanism of HCC induced by HBV infection.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are a new class of well-conserved small noncoding RNAs that mediate posttranscriptional gene regulation. Hepatitis B virus (HBV) causes various liver diseases, including chronic hepatitis, liver cirrhosis and hepatocellular cancer. Recent data have indicated HBV alters miRNAs expression patterns, but the underlying mechanisms have not been fully established so far. Here, we provide a hypothesis that HBV alters the expressions of miRNAs by playing a role in the microRNA production process. In this study, we demonstrate that HBV downregulates miRNAs processor DGCR8 mRNA and protein expression in stable and transient HBV-expressing cells. HBV downregulates DGCR8 expression by inhibiting its promoter activity, and HBs and HBx may be involved in this process. Ectopic expression and knockdown of YY1 revealed that YY1 suppresses the activity of the DGCR8 promoter, while YY1 expression is significantly upregulated by HBV. In conclusion, our data show that HBV proteins repress DGCR8 promoter activity by upregulating the expression of transcription factor YY1. This provides a new insight into the mechanism of HBV-induced miRNA dysregulation.
Archives of Virology 11/2014; 160(3). DOI:10.1007/s00705-014-2286-x · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and children under five years of age. The novel genotype ON1 has a 72-nucleotide duplication, which is the largest duplicated genome portion of RSV. Whether the ON1 genotype will follow the pattern of the BA genotype, which has a 60-nucleotide duplication, and become the predominant RSV-A strain is a global concern. To obtain information regarding the prevalence of the ON1 genotype in Chongqing in Southwestern China, we examined the circulation pattern of RSV-A identified over four consecutive years (June 2009 to August 2013). In this study, 312 (12%) RSV-A strains were isolated from 2601 nasopharyngeal aspirates, and partial G gene was sequenced successfully in 250 isolates. Of the sequenced Chongqing RSV-A isolates, 237 (94.8%) strains were the NA1 genotype, 4 (1.6%) strains were the NA3 genotype, 4 (1.6%) strains were the NA4 genotype, 1 (0.4%) strain was the GA1 genotype, and 4 (1.6%) strains were identified as the ON1 genotype. Analysis of the distribution, phylogeny, and evolution of the ON1 strains that were collected globally until December 2013 revealed that the ON1 genotype has rapidly disseminated across the world under positive selection pressures. Future studies will determine whether this new genotype will continue to spread and become the dominant strain of RSV-A worldwide. These findings may contribute to the understanding of RSV evolution and to the potential development of a vaccine against RSV.
[Show abstract][Hide abstract] ABSTRACT: Background
Human bocavirus is a newly discovered parvovirus. Multiple studies have confirmed the presence of human bocavirus1 (HBoV1) in respiratory tract samples of children. The viral load, presentation of single detection and its role as a causative agent of severe respiratory tract infections have not been thoroughly elucidated.
We investigated the presence of HBoV1 by quantitative polymerase chain reaction (PCR) of nasopharyngeal aspirate specimens from 1229 children hospitalized for respiratory tract infections. The samples were analyzed for 15 respiratory viruses by PCR and 7 respiratory viruses by viral culture.
At least one virus was detected in 652 (53.1%) of 1229 children, and two or more viruses were detected in 266 (21.6%) children. HBoV1 was detected in 127 children (10.3%), in which 66/127 (52%) of the cases were the only HBoV1 virus detected. Seasonal variation was observed with a high HBoV1 infection rate in summer. A cutoff value of 107 copies/mL was used to distinguish high and low HBoV1 viral loads in the nasopharyngeal aspirates. High viral loads of HBoV1 were noted predominantly in the absence of other viral agents (28/39, 71.8%) whereas there was primarily co-detection in cases of low HBoV1 viral loads (50/88, 56.8%). There were no differences in the clinical symptoms and severity between HBoV1 single detection and co-detection. In cases of HBoV1 single detection, the high viral load group was more prevalent among children with dyspnea and wheezing than was the low viral load group (42.9% vs. 23.7%, P = 0.036; 60.7% vs. 31.6%, P = 0.018). In clinical severity, a significant difference was recorded (25.0% vs. 5.3%, P = 0.003) between high viral load and low viral load groups. Of the HBoV1 positive patients associated with severe respiratory tract infections, 10/18 (55.6%) patients belonged to the HBoV1 high viral load group, and 7/10 (70%) patients had cases of HBoV1 single detection.
HBoV1 at a high viral load is not frequently found in co-detection with other respiratory viruses, and a single detection with a high viral load could be an etiological agent of severe respiratory tract infections.
[Show abstract][Hide abstract] ABSTRACT: A simple technique for the identification of common genotypes of the hepatitis B virus (HBV) remains to be identified. The present study was conducted to establish such a methodology. Four plasmids of genotypes A‑D and 123 clinical serum specimens of HBV‑infected patients were genotyped. HBV genotypes would be detected successfully when the HBV genotype reached a viral load of 1 x 103 copies/ml or the BC genotype mixed samples reached a 5% level. The lower limit of detection of HBV DNA in serum specimens was determined to be 2.14x102 IU/ml. The assay sensitivity and specificity were 100% and the consistency was demonstrated to reach as high as 90.24 and 100% compared with that of the DNA sequencing and cloning. The frequencies of the genotypes B, C, BC, BD and BCD were found to be 65.0, 23.6, 7.3, 3.3 and 0.8%, respectively. The accuracy of detection of the mixed infections was also higher using the rapid and simple SNaPshot method compared with that achieved with the DNA sequencing methods. The results of the present study indicated that the SNaPshot technique accurately distinguishes the HBV genotypes A‑D and is able to be readily applied as a monitoring tool in HBV prognosis and treatment.
Molecular Medicine Reports 07/2014; 10(3). DOI:10.3892/mmr.2014.2372 · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RAB GTPase 5A (RAB5A), a member of the Rab subfamily of small GTPases, acts as an oncogene and has been associated with various key cellular functions, including cell growth, differentiation, apoptosis and angiogenesis. Recently, it has been reported that the Rab5a gene is involved in the progression of cancer. Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers, and it is usually associated with persistent hepatitis B virus (HBV) infections. Emerging evidence suggests that HBV alters microRNA (miRNA) expression profiles, but the mechanisms underlying this process have not yet been fully elucidated. Here, we examine how HBV affects the production of miR-101-1, which has been shown to be downregulated in HCC. We found that HBV could repress miR-101-3p by inhibiting its promoter activity. Downregulation of miR-101-3p promoted cancer cell growth and migration, and a specific miR-101-3p inhibitor was able to enhance proliferation and migration. Moreover, we identified Rab5a was one of the target genes of miR-101-3p in HBV-related HCC. Forced expression of miR-101-3p in liver cell lines resulted in a marked reduction of the expression of Rab5a at both the mRNA and protein level by directly targeting the 3'untranslated region of Rab5a. Overexpression of Rab5a resulted in a reversal of the suppression of proliferation and migration of SMMC-7721 cells mediated by miR-101-3p. Taken together, our data show that HBV can downregulate miR-101-3p expression by inhibiting its promoter activity and that downregulation of miR-101-3p promotes HCC cell proliferation and migration by targeting Rab5a. This provides new insights into the mechanisms of HBV-related HCC pathogenesis.
Archives of Virology 05/2014; 159(9). DOI:10.1007/s00705-014-2084-5 · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic hepatitis B virus (HBV) infection is a worldwide problem and HBV reactivation following anticancer chemotherapy has become an emerging clinical challenge. However, the mechanisms of HBV reactivation following chemotherapy remain unclear. Epirubicin is an anthracycline drug used in chemotherapy to treat numerous types of malignancy, including breast cancer, acute leukemia, malignant lymphoma, lung cancer, ovarian cancer and stomach cancer. Epirubicin acts by intercalating DNA strands and inhibiting DNA and RNA synthesis. In this study, it was demonstrated that epirubicin directly upregulated the levels of in vitro HBV replication in a concentration‑dependent manner. Exposure to epirubicin for 24 h induced >11- and 6-fold increases in the levels of intracellular and secreted HBV DNA, respectively. In concordance with the elevated levels of HBV DNA, the expression levels of HBV pregenomic RNA, intracellular HBV surface and HBV core antigens, and secreted HBV e antigen were significantly increased by treatment with 0.5 µM epirubicin. Notably, epirubicin promoted cellular excretion of HBV nucleocapsids, which are closely associated with the pathological effects of HBV, including acute liver failure. In conclusion, epirubicin exhibited a direct stimulatory effect on HBV replication and this may be a novel mechanism of HBV reactivation following cytotoxic anticancer chemotherapy.
Molecular Medicine Reports 04/2014; 9(4):1345-50. DOI:10.3892/mmr.2014.1973 · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNA-101(miR-101) has been shown to be down-regulated in hepatocellular carcinoma (HCC). The hepatitis B virus (HBV) is a major risk factor in the development and progression of HCC. However, the correlation between HBV and miR-101 has not yet been fully elucidated. In this study, we reported that HBV could repress miR-101-3p by inhibiting its promoter activity and identified the potential effects of miR-101-3p on some important biological properties of HCC cells by targeting Rap1b. Dual-luciferase reporter assays showed that HBV down-regulated miR-101-3p by inhibiting its promoter activity. Down-regulation of miR-101-3p promoted cell proliferation, migration, and reduced apoptosis, and resulted in up-regulation of Rap1b, while overexpression of miR-101-3p inhibited these processes. Moreover, overexpression of Rap1b was able to reverse the suppressed cell proliferation and migration mediated by miR-101-3p. Our data showed that HBV down-regulated miR-101-3p expression by inhibiting its promoter activity, which resulted in up-regulation of Rap1b, and down-regulation of miR-101-3p or up-regulation of Rap1b promoted proliferation and migration of HCC cells. This provides a new understanding of the mechanism of HBV-related HCC pathogenesis and the potential application of miR-101-3p in cancer therapy.
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidence showed that microRNAs are involved in development and progression of multiple tumors. Recent studies have found that miR-181a were dysregulated in several types of cancers, however, the function of miR-181a in hepatocellular carcinoma (HCC) remains unclear. In this study we assessed the potential association between miR-181a, HBV and HCC.
The expression of miR-181a in HBV-expressing cells was determined by using qRT-PCR. Dual-Luciferase reporter Assay, qRT-PCR and western blot were performed to investigate the target genes of miR-181a. The effects of miR-181a on HCC proliferation were analyzed by MTS and colony formation assay. Tumor growth assay was used to analyze the effect of miR-181a on tumor formation.
HBV up-regulated miR-181a expression by enhancing its promoter activity. Overexpression of miR-181a in hepatoma cells promoted cell growth in vitro and tumor formation in vivo. Conversely, inhibition of miR-181a suppressed the proliferation of HBV-expressing cells. Mechanism investigation revealed that miR-181a inhibited the expression of transcription factor E2F5 by specifically targeting its mRNA 3[prime]UTR. Moreover, E2F5 inhibition induced cell growth and rescued the suppressive effect of miR-181a inhibitor on the proliferation of SMMC-7721 cells. Interestingly, we also discovered that HBV could down-regulate E2F5 expression.
Those results strongly suggested that HBV down-regulated E2F5 expression, in part, by up-regulating the expression of miR-181a. Up-regulation of miR-181a by HBV in hepatoma cells may contribute to the progression of HCC possibly by targeting E2F5, suggesting miR-181a plays important role in HCC development.
BMC Cancer 02/2014; 14(1):97. DOI:10.1186/1471-2407-14-97 · 3.36 Impact Factor