Triptolide Induces Growth Inhibition and Apoptosis of Human Laryngocarcinoma Cells by Enhancing p53 Activities and Suppressing E6-Mediated p53 Degradation

Triptolide, an active compound extracted from Chinese herb Leigongteng (Tripterygium wilfordii Hook F.), shows a broad-spectrum of anticancer activity through its cytotoxicity. However, the efficacy of triptolide on laryngocarcinoma rarely been evaluated, and the mechanism by which triptolide-induced cellular apoptosis is still not well understood. In this study, we found that triptolide significantly inhibited the laryngocarcinoma HEp-2 cells proliferation, migration and survivability. Triptolide induces HEp-2 cell cycle arrest at the G1 phase and apoptosis through intrinsic and extrinsic pathways since both caspase-8 and -9 are activated. Moreover, triptolide enhances p53 expression by increasing its stability via down-regulation of E6 and E6AP. Increased p53 transactivates down-stream target genes to initiate apoptosis. In addition, we found that short time treatment with triptolide induced DNA damage, which was consistent with the increase in p53. Furthermore, the cytotoxicity of triptolide is decreased by p53 knockdown or use of caspases inhibitor. In conclusion, our results demonstrated that triptolide inhibits cell proliferation and induces apoptosis in laryngocarcinoma cells by enhancing p53 expression and activating p53 functions through induction of DNA damage and suppression of E6 mediated p53 degradation. These studies indicate that triptolide is a potential anti-laryngocarcinoma drug.     

Comparison of Endoscopic Ultrasonography and Multislice Spiral Computed Tomography for the Preoperative Staging of Gastric Cancer - Results of a Single Institution Study of 610 Chinese Patients

Background

This study compared the performance of endoscopic ultrasonography (EUS) and multislice spiral computed tomography (MSCT) in the preoperative staging of gastric cancer.

Methodology/Principal Findings

A total of 610 patients participated in this study, all of whom had undergone surgical resection, had confirmed gastric cancer and were evaluated with EUS and MSCT. Tumor staging was evaluated using the Tumor-Node-Metastasis (TNM) staging and Japanese classification. The results from the imaging modalities were compared with the postoperative histopathological outcomes. The overall accuracies of EUS and MSCT for the T staging category were 76.7% and 78.2% (P=0.537), respectively. Stratified analysis revealed that the accuracy of EUS for T1 and T2 staging was significantly higher than that of MSCT (P<0.001 for both) and that the accuracy of MSCT in T3 and T4 staging was significantly higher than that of EUS (P<0.001 and 0.037, respectively). The overall accuracy of MSCT was 67.2% when using the 13th edition Japanese classification, and this percentage was significantly higher than the accuracy of EUS (49.3%) and MSCT (44.6%) when using the 6th edition UICC classification (P<0.001 for both values).

Conclusions/Significance

Our results demonstrated that the overall accuracies of EUS and MSCT for preoperative staging were not significantly different. We suggest that a combination of EUS and MSCT is required for preoperative evaluation of TNM staging.     

Molecular Epidemiologic Characterization of a Clustering HCV Infection Caused by Inappropriate Medical Care in Heyuan City of Guangdong,China

Background

From November 2011 to January 2012, a number of clustering cases of HCV infection were reported in Zijin County, Heyuan City, Guangdong, China. Most patients in the clustering cases suspected that they could be infected due to inappropriate medical care in the clinic located at the Xiangshui road. However, the molecular epidemiology of the clustering cases remains unknown.

Methodology

The residents, living at Xiangshui Road, with HCV antibody positive reported from 2011 and 2012 were recruited. A survey of the HCV infected individuals from the clustering cases was conducted. Each participant underwent a questionnaire defining demographic characteristics and health care history. HCV serological test and viral load test were performed to confirm the infection status. Molecular phylogenetic analysis and Bayesian coalescence analysis were conducted to further confirm the HCV subtype distribution and to reconstruct the associated demographic history and time-scaled phylogeny among the clustering cases.

Principal Findings

The molecular phylogenetic analysis revealed that only two HCV subtypes, 2a and 6a, were found among the clustering cases. There was no close HCV subtype evolutionary relation was observed among patients from the same family. The 6a cluster showed higher viral loads than the 2a cluster. In addition, the Bayesian skyline plot analysis showed that both the HCV 2a and 6a subtype infections among the Heyuan cases experienced an “expansion-diminishment-expansion” featured dissemination. The 2a clustering infection occurred in 2004, and the 6a clustering cases originated in 2006.

Conclusions

The molecular epidemiological characters imply that the inappropriate medical practices were possibly associated with the clustering HCV cases in Heyuan City during 2011, 2012. Latent HCV subtypes 2a and 6a infection may cause the prevalence and become a new public health issue in Guangdong, China.     

Identifying Neural Patterns of Functional Dyspepsia Using Multivariate Pattern Analysis: A Resting-State fMRI Study

Background

Previous imaging studies on functional dyspepsia (FD) have focused on abnormal brain functions during special tasks, while few studies concentrated on the resting-state abnormalities of FD patients, which might be potentially valuable to provide us with direct information about the neural basis of FD. The main purpose of the current study was thereby to characterize the distinct patterns of resting-state function between FD patients and healthy controls (HCs).

Methodology/Principal Findings

Thirty FD patients and thirty HCs were enrolled and experienced 5-mintue resting-state scanning. Based on the support vector machine (SVM), we applied multivariate pattern analysis (MVPA) to investigate the differences of resting-state function mapped by regional homogeneity (ReHo). A classifier was designed by using the principal component analysis and the linear SVM. Permutation test was then employed to identify the significant contribution to the final discrimination. The results displayed that the mean classifier accuracy was 86.67%, and highly discriminative brain regions mainly included the prefrontal cortex (PFC), orbitofrontal cortex (OFC), supplementary motor area (SMA), temporal pole (TP), insula, anterior/middle cingulate cortex (ACC/MCC), thalamus, hippocampus (HIPP)/parahippocamus (ParaHIPP) and cerebellum. Correlation analysis revealed significant correlations between ReHo values in certain regions of interest (ROI) and the FD symptom severity and/or duration, including the positive correlations between the dmPFC, pACC and the symptom severity; whereas, the positive correlations between the MCC, OFC, insula, TP and FD duration.

Conclusions

These findings indicated that significantly distinct patterns existed between FD patients and HCs during the resting-state, which could expand our understanding of the neural basis of FD. Meanwhile, our results possibly showed potential feasibility of functional magnetic resonance imaging diagnostic assay for FD.     

Genetic Variation in Interleukin 28B and Response to Antiviral Therapy in Patients with Dual Chronic Infection with Hepatitis B and C Viruses

Concurrent infection with hepatitis C virus (HCV) and hepatitis B virus (HBV) was not uncommon in China. To date, information on predictors of response to treatment of dually-infected HCV/HBV is limited. The aim of this study was to evaluated whether determination of the interleukin 28B (IL-28B) polymorphism statuses sufficient to predict treatment response of interferon (IFN)-based therapy in patients chronically infected with both hepatitis B and C viruses. We investigated the role of IL28B variations (rs8099917 and rs12979860) in response to IFN-based treatment and evaluated its association with the risk of the null virological response (NVR) in HCV /HBV dually-infected patients. We found that the overall distributions of the genotypes among the sustained virological response (SVR), NVR groups were significantly different (P<0.001): patients with the rs8099917 TG genotype had an increased risk of NVR (odds ratio [OR] =2.37 95% confidence interval [CI] =1.16–4.83, P =0.017), and those with the GG genotype had a further increased risk of NVR (OR=4.23, 95% CI =1.17-15.3, P=0.027). The rs12979860 allele was also highly associated with treatment failure (CT/TT vs. CC; OR =2.04, 95%CI =1.05-3.97, P =0.037). Moreover, we found that IL28B rs8099917 G variants (TG+GG) interact with HCV genotype 1(G1) to result in higher risk of NVR (P=0.009), and that they are also associated with HBV DNA reactivation (TG+GG vs. TT, P=0.005). Furthermore, multivariate regression analysis show that the rs8099917 G allele was the most important factor significantly associated with a NVR in HCV G1 patients. This study suggest that IL28B genotyping may be a valid pretreatment predictor of which patients are likely to respond to treatment in this group of difficult-to-treat HCV/HBV dually-infected patients.     

Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas,and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

Hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) has a poor prognosis with high in-hospital mortality. Hepatic and circulating inflammatory cytokines, such as fibrinogen like protein 2 (fgl2), FasL/Fas, and TNFα/TNFR1, play a significant role in the pathophysiology of ACLF. This study aimed to investigate the therapeutic effect of recombinant adenoviral vectors carrying constructed DNA code for non-native microRNA (miRNA) targeting mouse fgl2 (mfgl2) or both mFas and mTNFR1 on murine hepatitis virus (MHV)-3-induced fulminant hepatitis in BALB/cJ mice. Artificial miRNA eukaryotic expression plasmids against mfgl2, mFas, and mTNFR1 were constructed, and their inhibitory effects on the target genes were confirmed in vitro. pcDNA6.2-mFas-mTNFR1- miRNA，which expresses miRNA against both mFas and mTNFR1 simultaneously，was constructed. To construct a miRNA adenovirus expression vector against mfgl2, pcDNA6.2-mfgl2-miRNA was cloned using Gateway technology. Ad-mFas-mTNFR1- miRNA was also constructed by the same procedure. Adenovirus vectors were delivered by tail-vein injection into MHV-3-infected BALB/cJ mice to evaluate the therapeutic effect. 8 of 18 (44.4%) mice recovered from fulminant viral hepatitis in the combined interference group treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA. But only 4 of 18 (22.2%) mice receiving Ad-mfgl2-miRNA and 3 of 18 (16.7%) mice receiving Ad-mFas-mTNFR1- miRNA survived. These adenovirus vectors significantly ameliorated inflammatory infiltration, fibrin deposition, hepatocyte necrosis and apoptosis, and prolonged survival time. Our data illustrated that combined interference using adenovirus-mediated artificial miRNAs targeting mfgl2, mFas, and mTNFR1 might have significant therapeutic potential for the treatment of fulminant hepatitis.     

The External Validity of Randomized Controlled Trials of Hypertension within China: from the Perspective of Sample Representation

Objective

To explore external validity of randomized controlled trials (RCTs) of hypertension within China from the view of sample representation.

Methods

Comprehensive literature searches were performed in Medline, Embase, Cochrane Central Register of Controlled Trials (CCTR) et al and advanced search strategies were used to locate hypertension RCTs as well as observational studies conducted in China during 1996 to 2009 synchronously. The risk of bias in RCTs and observational studies was assessed by two modified scales respectively, and then both types of studies with 3 or more grading scores were included for the purpose of evaluating of external validity. Following that the study characteristics relative to sample representation were extracted from RCTs and observational studies synchronously, and the later were taken as external references for validating sample representation of RCTs.

Results

226 hypertension RCTs and 21 observational studies were included for final analysis. Comparing samples with observational studies, the mean age of samples within RCTs was 54.46 years, significantly lower than that of observational studies (66.35 years) (P=0.002). The average disease course in patients of RCTs was 3.89 years and grade III hypertensive patients accounted for 17%; both were lower than that of the observational studies (12.96 years, P<0.001; 34%, P=0.026 respectively). In addition, the proportions of patients with complications due to heart failure, stroke, diabetes, or coronary heart disease in RCTs were 8%, 5%, 12% and 11% correspondingly, all of which were significantly less than that of observational studies (11%, 18%, 17% and 29%).

Conclusion

Sample characteristics within hypertension RCTs were significantly different from those in observational studies. The samples in most RCTs were under-represented. It’s feasible to take samples of observational studies as a mirror of the actual composition of hypertension patients in the real world, if the reporting of observational studies is abundant and available.     

Overexpression of an Acidic Endo-β-1,3-1,4-glucanase in Transgenic Maize Seed for Direct Utilization in Animal Feed

Background

Incorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed.

Methodology/Principal Findings

Here we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0–8.0, which is in agreement with the normal environment of digestive tract.

Conclusion/Significance

Our study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.     

Production of a Dominant-Negative Fragment Due to G3BP1 Cleavage Contributes to the Disruption of Mitochondria-Associated Protective Stress Granules during CVB3 Infection

Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the Picornaviridae family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3C^pro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.