Unexpected Replication Boost by Simeprevir for Simeprevir-Resistant Variants in Genotype 1a Hepatitis C Virus.

Simeprevir is a novel NS3/4A protease inhibitor (PI) of hepatitis C virus (HCV). The baseline polymorphism NS3-Q80K is frequently observed in genotype (GT) 1a HCV and often associated with treatment failure in simeprevir-containing regimens. We aimed to elucidate mechanisms of treatment failure due to NS3-Q80K. We included a Q80R mutation in our study and generated a series of Huh-7.5 cell lines, each of which harbored either wild-type GT 1a strain H77S.3 or the Q80K or Q80R variant. The cells were cultured with increasing concentrations of simeprevir, and NS3 domain sequences were determined. The mutations identified by sequence analyses were subsequently introduced into H77S.3. The sensitivity of each mutant to the NS3/4A PIs simeprevir, asunaprevir, grazoprevir, and paritaprevir was analyzed. We introduced the mutations into GT 1b strain N.2 and compared the sensitivity to simeprevir with that of GT 1a strain H77S.3. While simeprevir treatment selected mutations at residue D168, such as D168A/V in the wild-type virus, an additional mutation at residue R155, R155K, was selected in Q80K/R variants at simeprevir concentrations of <2.5 µM. Sensitivity analyses showed that simeprevir concentrations of <1 µM significantly boosted the replication of Q80K/R R155K variants. Interestingly, this boost was not observed with the other NS3/4A PIs or in Q80R R155Q/G/T/W variants or GT 1b isolates. The boosted replication of the Q80K+R155K variant by simeprevir could be related to treatment failure in simeprevir-containing antiviral treatments in GT 1a HCV-infected patients with the NS3-Q80K polymorphism. This result provides new insight into how resistance-associated variants can cause treatment failure.

Two new furanone derivatives from the endophytic fungus Byssochlamys sp. and their cytotoxic activities.

Two new furanone derivatives, byssochlanones A-B (1-2) were purified from the endophytic fungus Byssochlamys sp. isolated from the wetland plant, Phragmites australis. Their structures were elucidated on the basis of extensive spectroscopic analyses. Compounds 1-2 represented typical furanone analogues which are not common in natural products. The absolute configuration of compounds 1-2 were identified through quantum-chemical electronic circular dichroism (ECD) calculation compared with their experimental CD. In addition, compounds 1-2 were tested for their cytotoxic activities against HCT-8 and Hela cancer cell lines, and compound 2 showed moderate activity against HCT-8 cells with IC50 value of 21.3 µM.

Probing subwavelength in-plane anisotropy with antenna-assisted infrared nano-spectroscopy.

Infrared nano-spectroscopy based on scattering-type scanning near-field optical microscopy (s-SNOM) is commonly employed to probe the vibrational fingerprints of materials at the nanometer length scale. However, due to the elongated and axisymmetric tip shank, s-SNOM is less sensitive to the in-plane sample anisotropy in general. In this article, we report an easy-to-implement method to probe the in-plane dielectric responses of materials with the assistance of a metallic disk micro-antenna. As a proof-of-concept demonstration, we investigate here the in-plane phonon responses of two prototypical samples, i.e. in (100) sapphire and x-cut lithium niobate (LiNbO3). In particular, the sapphire in-plane vibrations between 350 cm-1 to 800 cm-1 that correspond to LO phonon modes along the crystal b- and c-axis are determined with a spatial resolution of < λ/10, without needing any fitting parameters. In LiNbO3, we identify the in-plane orientation of its optical axis via the phonon modes, demonstrating that our method can be applied without prior knowledge of the crystal orientation. Our method can be elegantly adapted to retrieve the in-plane anisotropic response of a broad range of materials, i.e. subwavelength microcrystals, van-der-Waals materials, or topological insulators.

LsGRP1, a class II glycine-rich protein of Lilium, confers plant resistance via mediating innate immune activation and inducing fungal programmed cell death.

Defence-related LsGRP1 is a leaf-specific plant class II glycine-rich protein (GRP) involved in salicylic acid-induced systemic resistance against grey mould caused by necrotrophic Botrytis elliptica in lily (Lilium) cultivar Stargazer. The C-terminal region of LsGRP1 (LsGRP1C ) can inhibit fungal growth in vitro via a mechanism of inducing fungal apoptosis programmed cell death (PCD). In this study, the role of LsGRP1 in induced defence mechanism was investigated using LsGRP1-silenced Stargazer lily and LsGRP1-transgenic Arabidopsis thaliana. LsGRP1 silencing in lily was found to slightly inhibit plant growth and greatly increase the susceptibility to B. elliptica by suppressing callose deposition and early reactive oxygen species (ROS) accumulation. In contrast, LsGRP1-transgenic Arabidopsis showed higher resistance to Botrytis cinerea and also to Pseudomonas syringae pv. tomato DC3000 as compared to the wild type, accompanied with the enhancement of callose deposition and ROS accumulation. Additionally, LsGRP1 silencing increased plant cell death caused by B. elliptica secretion and reduced pathogen-associated molecular pattern (PAMP)-triggered defence activation in Stargazer lily. Consistently, LsGRP1 expression boosted PAMP-triggered defence responses and effector recognition-induced hypersensitive response in Arabidopsis. Moreover, fungal apoptosis PCD triggered by LsGRP1 in an LsGRP1C -dependent manner was demonstrated by leaf infiltration with LsGRP1C -containing recombinant proteins in Stargazer lily. Based on these results, we presume that LsGRP1 plays roles in plant defence via functioning as a pathogen-inducible switch for plant innate immune activation and acting as a fungal apoptosis PCD inducer to combat pathogen attack.

TRAIL inhibits HBV replication and expression by down-regulating liver-enriched transcription factors.

BACKGROUND AND STUDY AIMS: To investigate the role of low-concentration TRAIL on HBV replication and expression. MATERIAL AND METHODS: MTT assay was performed to determine the minimum concentrations of TRAIL protein in HepG2 cell apoptosis. HepG2 cells were transfected by HBV replication plasmid pHBV4.1. After the treatment with low concentration of TRAIL, the culture supernatant was collected to detect HBsAg and HBeAg by ELISA. Proteins were extracted from the resulted cells, followed by total RNA and HBV DNA intermediate replication. Southern Blot and Northern Blot were carried out to detect HBV RNA and HBV DNA replication intermediates, respectively. RT-PCR and Western Blot were carried out to detect gene and protein expressions for HNF4α, PPARα, and RXRα, respectively. RESULTS: 50 ng/ml of TRAIL protein led to significant decline on the secretions of HBsAg and HBeAg. Expression levels of HBV RNA and HBV DNA replication intermediates were significantly decreased too. In addition, gene and protein expressions of HNF4α, PPARα and RXRα also dropped, especially for PPARα whose expressions significantly decreased. CONCLUSION: TRAIL could inhibit HBV replication and expression by downregulating the expressions of liver-enriched transcription factors HNF4α, PPARα, and RXRα.

Direct Synthesis of Large-Scale Multilayer TaSe2 on SiO2/Si Using Ion Beam Technology.

The multilayer 1T-TaSe2 is successfully synthesized by annealing a Se-implanted Ta thin film on the SiO2/Si substrate. Material analyses confirm the 1T (octahedral) structure and the quasi-2D nature of the prepared TaSe2. Temperature-dependent resistivity reveals that the multilayer 1T-TaSe2 obtained by our method undergoes a commensurate charge-density wave (CCDW) transition at around 500 K. This synthesis process has been applied to synthesize MoSe2 and HfSe2 and expanded for synthesis of one more transition-metal dichalcogenide (TMD) material. In addition, the main issue of the process, that is, the excess metal capping on the TMD layers, is solved by the reduction of thickness of the as-deposited metal thin film in this work.

Establishment and Characterization of a New Cell Line Permissive for Hepatitis C Virus Infection.

Hepatitis C virus (HCV) cell culture systems have facilitated the development of efficient direct-acting antivirals against HCV. Huh-7.5, a subline of the human hepatoma cell line Huh-7, has been used widely to amplify HCV because HCV can efficiently replicate in these cells due to a defect in innate antiviral signalling. Recently, we established a novel cell line, KH, derived from human hepatocellular carcinoma, which showed atypical uptake of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) in a Gd-EOB-DTPA-enhanced magnetic resonance imaging study. KH cells expressed hepatocyte markers including microRNA-122 (miR-122) at a lower level than Huh-7.5 cells. We demonstrated that KH cells could support the entire life cycle of HCV; however, HCV replicated at a lower rate in KH cells compared to Huh-7.5 cells, and virus particles produced from KH cells seemed to have some disadvantages in viral assembly compared with those produced from Huh-7.5 cells. KH cells had more robust interferon-stimulated gene expression and induction upon HCV RNA transfection, interferon-α2b addition, and HCV infection than Huh-7.5 cells. Interestingly, both miR-122 supplementation and IRF3 knockout in KH cells boosted HCV replication to a similar level as in Huh-7.5 cells, suggesting that intact innate antiviral signalling and lower miR-122 expression limit HCV replication in KH cells. KH cells will enable a deeper understanding of the role of the innate immune response in persistent HCV infection.

Efficient Suppression of Hepatitis C Virus Replication by Combination Treatment with miR-122 Antagonism and Direct-acting Antivirals in Cell Culture Systems.

Direct-acting antivirals (DAAs) against Hepatitis C virus (HCV) show effective antiviral activity with few side effects. However, the selection of DAA-resistance mutants is a growing problem that needs to be resolved. In contrast, miR-122 antagonism shows extensive antiviral effects among all HCV genotypes and a high barrier to drug resistance. In the present study, we evaluated three DAAs (simeprevir, daclatasvir, and sofosbuvir) in combination with anti-miR-122 treatment against HCV genotype 1a in cell cultures. We found that combination treatments with anti-miR-122 and a DAA had additive or synergistic antiviral effects. The EC50 values of simeprevir in simeprevir-resistant mutants were significantly decreased by combining simeprevir with anti-miR-122. A similar reduction in EC50 in daclatasvir-resistant mutants was achieved by combining daclatasvir with anti-miR-122. Combination treatment in HCV-replicating cells with DAA and anti-miR-122 sharply reduced HCV RNA amounts. Conversely, DAA single treatment with simeprevir or daclatasvir reduced HCV RNA levels initially, but the levels later rebounded. DAA-resistant mutants were less frequently observed in combination treatments than in DAA single treatments. In summary, the addition of miR-122 antagonism to DAA single treatments had additive or synergistic antiviral effects and helped to efficiently suppress HCV replication and the emergence of DAA-resistant mutants.

The acyclic retinoid Peretinoin inhibits hepatitis C virus replication and infectious virus release in vitro.

Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.

Expression of TRAIL in liver tissue from patients with different outcomes of HBV infection.

OBJECTIVES: Hepatitis B virus (HBV) infection triggers the production of TRAIL, suggesting that TRAIL may play a role in liver injury after HBV infection. However, it remains unclear whether TRAIL expression in liver tissue correlates with the extent of liver injury caused by HBV infection. The aim of this article was to investigate the correlation of TRAIL expression and disease severity. METHODS: Liver biopsy specimens were collected from 71 patients with different outcomes of HBV infection, including 25 cases of chronic hepatitis B (CHB), 18 cases of severe hepatitis B (SHB), and 28 cases of liver cirrhosis (LC). Besides, specimens from 33 healthy individuals without detectable liver diseases were used as negative control (NC). The expression of TRAIL was measured by immunohistochemistry. RESULTS: Expression of TRAIL in the HBV-infected patients was higher than that in the NC (P<0.001). Among the patients, TRAIL expression in the ones with CHB was significantly higher than that in NC (P<0.001). However, there was no statistically significant difference between patients with SHB and NC or between the ones with LC and NC (P=0.067 and P=0.178, respectively). Moreover, TRAIL expression in patients with CHB was higher than that in patients with SHB or LC (P<0.001 for both), whereas no statistically significant difference was observed between patients with SHB and the ones with LC (P=0.511). CONCLUSION: TRAIL is involved in the inflammatory and immunoregulatory response after HBV infection. However, there was no significant correlation between expression of TRAIL and the extent of liver injury.