Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that acts as a key player in the HCV replication complex. Understanding the interplay between the viral and cellular components of the HCV replication complex could provide new insight for prevention of the progression of HCV-associated hepatocellular carcinoma (HCC). In this study, the NS5B protein was used as the bait in a pulldown assay to screen for NS5B-interacting proteins that are present in Huh7 hepatoma cell lysates. After mass spectrophotometric analysis, fatty acid synthase (FASN) was found to interact with NS5B. Coimmunoprecipitation and double staining assays further confirmed the direct binding between NS5B and FASN. The domain of NS5B that interacts with FASN was also determined. Moreover, FASN was associated with detergent-resistant lipid rafts and colocalized with NS5B in active HCV replication complexes. In addition, overexpression of FASN enhanced HCV expression in Huh7/Rep-Feo cells, while transfection of FASN small interfering RNA (siRNA) or treatment with FASN-specific inhibitors decreased HCV replication and viral production. Notably, FASN directly increased HCV NS5B RdRp activity in vitro . These results together indicate that FASN interacts with NS5B and modulates HCV replication through a direct increase of NS5B RdRp activity. FASN may thereby serve as a target for the treatment of HCV infection and the prevention of HCV-associated HCC progression.

We assessed the use of high-resolution melting (HRM) analysis for the rapid identification of influenza A virus subtypes and the detection of newly emerging virus variants. The viral matrix gene was amplified by LightCycler real-time reverse transcription-PCR (RT-PCR) in the presence of the LCGreen I fluorescent dye. Upon optimization of the assay conditions, all the major influenza A virus subtypes, including H1N1, H3N2, H5N1, H7N3, and H9N2, were amplifiable by this method and had a PCR product length of 179 bp. Real-time RT-PCR of in vitro-transcribed H3N2 RNA revealed a standard curve for quantification with a linear range (correlation coefficient = 0.9935) across at least 8 log units of RNA concentrations and a detection limit of 10 ³ copies of viral RNA. We performed HRM analysis of the PCR products with the HR-1 instrument and used the melting profiles as molecular fingerprints for virus subtyping. The virus subtypes were identified from the high-resolution derivative plot obtained by heteroduplex formation between the PCR products of the viral isolates tested and those of the reference viral isolates. The melting profiles were consistent with minimal interassay variability. Hence, an HRM database and a working protocol were established for the identification of these five influenza A virus subtypes. When this protocol was used to test 21 clinical influenza A virus isolates, the results were comparable to those obtained by RT-PCR with hemagglutinin-specific primer sets. Sequence variants of the clinical isolates ( n = 4) were also revealed by our HRM analytical scheme. This assay requires no multiplexing or hybridization probes and provides a new approach for influenza A virus subtyping and genetic screening of virus variants in a clinical virology laboratory.

Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptorβ(RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown thatde novoDNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed thatRARBhypermethylation was involved in the areca-associated oral carcinogenesis.

The Chinese herb modified Yi Guan Jian (mYGJ) is an effective regimen that is usually used in outpatients with chronic liver diseases such as fibrosis and cirrhosis. However, the mechanism for the action of mYGJ on liver fibrosis is not yet clear. In this study, we found that mYGJ induced hepatic stellate cells (HSCs) apoptosis concomitant with the downregulation of Bcl‐2 expression and slight elevation of Bax level. Moreover, the reactive oxygen species (ROS) were generated in the early stages of mYGJ‐induced HSCs apoptosis to facilitate calcium and cytochrome c release from the mitochondria to cytosol. Subsequently, caspase 9 and caspase 3 were activated. Furthermore, the activation of ER stress‐associated caspase 12 in HSCs was also evaluated. Together, we report the first evidence‐based study to demonstrate that mYGJ decoction induces HSCs apoptosis through ROS accumulation and the intrinsic apoptosis pathway. These findings provide rationale for further clinical investigation of traditional Chinese medicine recipes against liver fibrosis.