Single-Round Infectious Particle Antiviral Screening Assays for the Japanese Encephalitis Virus.

Japanese Encephalitis virus (JEV) is a mosquito-borne flavivirus with a positive-sense single-stranded RNA genome that contains a big open reading frame (ORF) flanked by 5'- and 3'- untranslated regions (UTRs). Nearly 30,000 JE cases with 10,000 deaths are still annually reported in East Asia. Although the JEV genotype III vaccine has been licensed, it elicits a lower protection against other genotypes. Moreover, no effective treatment for a JE case is developed. This study constructed a pBR322-based and cytomegaloviruses (CMV) promoter-driven JEV replicon for the production of JEV single-round infectious particles (SRIPs) in a packaging cell line expressing viral structural proteins. Genetic instability of JEV genome cDNA in the pBR322 plasmid was associated with the prokaryotic promoter at 5' end of the JEV genome that triggers the expression of the structural proteins in E. coli. JEV structural proteins were toxic E. coli, thus the encoding region for structural proteins was replaced by a reporter gene (enhanced green fluorescent protein, EGFP) that was in-frame fused with the first eight amino acids of the C protein at N-terminus and the foot-and-mouth disease virus (FMDV) 2A peptide at C-terminus in a pBR322-based JEV-EGFP replicon. JEV-EGFP SRIPs generated from JEV-EGFP replicon-transfected packaging cells displayed the infectivity with cytopathic effect induction, self-replication of viral genomes, and the expression of EGFP and viral proteins. Moreover, the combination of JEV-EGFP SRIP plus flow cytometry was used to determine the half maximal inhibitory concentration (IC50) values of antiviral agents according to fluorescent intensity and positivity of SRIP-infected packaging cells post treatment. MJ-47, a quinazolinone derivative, significantly inhibited JEV-induced cytopathic effect, reducing the replication and expression of JEV-EGFP replicon in vitro. The IC50 value of 6.28 µM for MJ-47 against JEV was determined by the assay of JEV-EGFP SRIP infection in packaging cells plus flow cytometry that was more sensitive, effective, and efficient compared to the traditional plaque assay. Therefore, the system of JEV-EGFP SRIPs plus flow cytometry was a rapid and reliable platform for screening antiviral agents and evaluating antiviral potency.

Antiviral Potential of a Novel Compound CW-33 against Enterovirus A71 via Inhibition of Viral 2A Protease.

Enterovirus A71 (EV-A71) in the Picornaviridae family causes hand-foot-and-mouth disease, aseptic meningitis, severe central nervous system disease, even death. EV-A71 2A protease cleaves Type I interferon (IFN)-α/ß receptor 1 (IFNAR1) to block IFN-induced Jak/STAT signaling. This study investigated anti-EV-A7l activity and synergistic mechanism(s) of a novel furoquinoline alkaloid compound CW-33 alone and in combination with IFN-ß Anti-EV-A71 activities of CW-33 alone and in combination with IFN-ß were evaluated by inhibitory assays of virus-induced apoptosis, plaque formation, and virus yield. CW-33 showed antiviral activities with an IC50 of near 200 µM in EV-A71 plaque reduction and virus yield inhibition assays. While, anti-EV-A71 activities of CW-33 combined with 100 U/mL IFN-ß exhibited a synergistic potency with an IC50 of approximate 1 µM in plaque reduction and virus yield inhibition assays. Molecular docking revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory effect of CW33 on in vitro enzymatic activity of recombinant 2A protease IC50 = 53.1 µM). Western blotting demonstrated CW-33 specifically inhibiting 2A protease-mediated cleavage of IFNAR1. CW-33 also recovered Type I IFN-induced Tyk2 and STAT1 phosphorylation as well as 2\',5\'-OAS upregulation in EV-A71 infected cells. The results demonstrated CW-33 inhibiting viral 2A protease activity to reduce Type I IFN antagonism of EV-A71. Therefore, CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative approaches to treat EV-A71 infection.

S100A8 as potential salivary biomarker of oral squamous cell carcinoma using nanoLC-MS/MS.

BACKGROUND: Oral squamous cell carcinoma (OSCC) shows low 5-year survival; early treatment greatly reduces mortality and morbidity. Saliva is a non-invasive sample, with good potential to discover biomarkers for early detection. METHODS: NanoLC-MS/MS served to analyze saliva proteome from control subjects (n=35) and OSCC patients T1 (n=29), T2 (n=36), T3 (n=14) and T4 (n=21) stages. Identified biomarkers were verified by Western blot and ELISA assays. RESULTS: NanoLC-MS/MS analysis of salivary proteins between 10 and 15kDa identified S100A8, hemoglobin delta and gamma-G globin in T3 and T4 stage OSCC as well as S100A7 in T1 and T2 stage OSCC. Western blot and ELISA indicated positive correlation between salivary S100A8 increment and tumor size stage. High level of S100A8 appeared in 3.4, 13.9, 92.9, and 100% of saliva OSCC patients with T1, T2, T3, and T4 stages, respectively. Significant increase of salivary S100A7 was observed in 20.7% and 11.1% of those with T1 and T2, respectively. AUROC curve indicated high sensitivity, specificity and accuracy of S100A8-based ELISA as a detector. CONCLUSIONS: NanoLC-MS/MS, Western blot and ELISA manifested salivary S100A8 as a specific and sensitive marker for detection of OSCC patients. Salivary S100A8 protein could be applicable in developing OSCC diagnostics.

Proteomic identification of salivary transferrin as a biomarker for early detection of oral cancer.

Oral cancer has a low five-year survival rate. Early detection of oral cancer could reduce the mortality and morbidity associated with this disease. Saliva, which can be sampled non-invasively and is less complex than blood, is a good potential source of oral cancer biomarkers. Proteomic analysis of saliva from oral cancer patients and control subjects was performed to identify salivary biomarkers of early stage oral cancer in humans. The protein profile of pooled salivary samples from patients with oral squamous cell carcinoma (OSCC) or OSCC-free control subjects was analyzed using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. Potential biomarkers were verified by Western blotting and ELISA assays. Transferrin levels were elevated in the saliva of OSCC patients as determined using 2DE followed by MALDI-TOF MS and confirmed by MALDI-TOF/TOF MS, Western blotting and ELISA. The increase in salivary transferrin levels in OSCC patients strongly correlated with the size and stage of the tumor. The area under the receiver-operating characteristics curves showed that salivary transferrin-based ELISA was highly specific, sensitive and accurate for the early detection of oral cancer. We have identified salivary transferrin as a biomarker for the detection of early stage oral cancer. This finding provides a promising basis for the development of a non-invasive diagnostic test for early stage oral cancer.

[Clinical experience of orthodontic treatment on 36 cases with congenital lower incisor missing].

OBJECTIVE: To explore the treatment plan in patients with congenital lower incisor missing. METHODS: Thirty-six patients with congenital lower incisor missing were included in the investigation. The missing location, missing quantity, malocclusion type and treatment method were analyzed. Bolton index of two groups were analyzed. The first group was congenital missing one lower incisor who treated by extracting two upper first premolars and one lower first premolar. The second group was congenital missing two lower incisors who treated by exacting two upper first premolars. RESULTS: The therapeutic effect of 36 cases were better. Overall ratio in two groups were 91.70% +/- 1.85% and 92.83% +/- 2.74%, anterior ratio were 81.69% +/- 2.12% and 85.46% +/- 2.39%, anterior tooth-size discrepancy were (2.16 +/- 0.64) mm and (4.27 +/- 1.14) mm. There were no significance difference on overall ratio in two groups (P > 0.05). There were significance difference in anterior ratio and anterior tooth-size discrepancy (P < 0.01). CONCLUSION: For patients with congenital missing one lower incisor who need extraction in upper and lower jaws, it is less affection on Bolton index that extracting one premolar at non-missing side than extracting one center incisor in mandible.

[Clinic study of root resorption caused by edgewise appliance combined with flat bite plate treatment ].

OBJECTIVE: To study the root resorption caused by edgewise appliance combined with flat bite plate treatment. METHODS: 65 patients with edgewise technique and flat bite plate treatment were chosen. All patients were taken panoramic radiograph before flat bite plate treatment, immediately after flat bite plate treatment and 6 months after flat bite plate treatment. Root resorption grade of mandibular permanent incisors were analyzed. RESULTS: There were statistical difference in the root resorption grade of three groups (P < 0.05), but there was no statistical difference between male and female (P > 0.05). CONCLUSION: Flat bite plate can cause root resorption of mandibular permanent incisors, but the root resorption is slight.