Deep mutational scanning of hepatitis B virus reveals a mechanism for cis-preferential reverse transcription.

Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two overlapping reading frames, and during evolution, multiple selective pressures can act on shared nucleotides. This study combines an RNA-based HBV cell culture system with deep mutational scanning (DMS) to uncouple cis- and trans-acting sequence requirements in the HBV genome. The results support a leaky ribosome scanning model for polymerase translation, provide a fitness map of the HBV polymerase at single-nucleotide resolution, and identify conserved prolines adjacent to the HBV polymerase termination codon that stall ribosomes. Further experiments indicated that stalled ribosomes tether the nascent polymerase to its template RNA, ensuring cis-preferential RNA packaging and reverse transcription of the HBV genome.

Inclisiran: a new generation of lipid-lowering siRNA therapeutic.

Atherosclerotic heart disease (AHD) is a major cause of morbidity and mortality worldwide. Lowering low-density lipoprotein cholesterol (LDL-C) levels is a key strategy to prevent and treat AHD. Inclisiran is a novel siRNA drug that targets proprotein convertase subtilisin/kexin type 9 (PCSK9) gene expression and reduces LDL-C levels with only two or three injections per year. This review summarizes the mechanism, efficacy, safety, and applications of Inclisiran in various populations and settings, based on recent literature. It also compares Inclisiran with other lipid-lowering drugs, especially other PCSK9 inhibitors. We conclude that Inclisiran is a promising lipid-lowering agent that can provide convenience and effectiveness for patients with high cardiovascular risk. However, some challenges and limitations remain for Inclisiran, such as its long-term safety and efficacy, its cost-effectiveness and accessibility, and its interactions and synergies with other drugs. These issues need further investigation and evaluation in future studies.

An RNA-based system to study hepatitis B virus replication and evaluate antivirals.

Hepatitis B virus (HBV) chronically infects an estimated 300 million people, and standard treatments are rarely curative. Infection increases the risk of liver cirrhosis and hepatocellular carcinoma, and consequently, nearly 1 million people die each year from chronic hepatitis B. Tools and approaches that bring insights into HBV biology and facilitate the discovery and evaluation of antiviral drugs are in demand. Here, we describe a method to initiate the replication of HBV, a DNA virus, using synthetic RNA. This approach eliminates contaminating background signals from input virus or plasmid DNA that plagues existing systems and can be used to study multiple stages of HBV replication. We further demonstrate that this method can be uniquely applied to identify sequence variants that confer resistance to antiviral drugs.

[Relationships between stoichiometric characteristics of soil enzymatic activities and environmental factors in parallel valleys of western Yunnan, China.] / æ»‡è¥¿å¹¶æµæ²³è°·åŒºåœŸå£¤é ¶æ´»æ€§åŒ–å­¦è®¡é‡å­¦ç‰¹å¾ä¸ŽçŽ¯å¢ƒå› å­çš„å ³ç³».

The valleyes of Hengduan Mountains contain the landscapes with high heterogeneity as well as high diversity of climate and vegetation types. To explore the soil cycling of four elements (C, N, P, S) across the parallel valleys of Nujiang River, Lancang River, Jinsha River and Yuanjiang River in western Yunnan, we collected top soils (0-10 cm) in forests, grasslands, and croplands. The activities of soil enzymes, including ß-glucosidase (BG), ß-N-acetylglucosaminidase (NAG), acid phosphatase (AP), and sulfatase (SU), which drive the soil C, N, P and S cycling, were determined. We analyzed the relationships of soil enzymatic activities and their stoichiometric characteristics with environmental factors. The activities of both AP and NAG had significant difference among different basins and different land types. The activities of AP, BG, NAG and SU were significantly positively related with each other. From southeast to northwest, the activities of BG, NAG, and SU increased with the altitude. Across all basins, the ecoenzymatic ratios of soils always ranked as AP:SU > BG:SU > NAG:SU > BG:NAG > BG:AP > NAG:AP. Compared with forest and grassland soil, cropland soils in each watershed had a higher BG:NAG and a lower NAG:AP (except Yuanjiang River basin). Moreover, AP:SU, BG:SU and NAG:SU of cropland soils were lower than those of forest and grassland in Yuanjiang River basin. However, they were higher than forest and lower than grassland in both Lancang River basin and Jinsha River basin. Soil enzyme activities and enzymatic stoichiometry were affected by physicochemical properties of soil, climate, and location, with the most contribution from soil physicochemical properties. Agricultural land use significantly affected the stoichiometry of C:N:P acquiring enzymes in soils by reducing the activity of N-degrading enzymes relative, resulting in the increases of BG:NAG and the decreases of NAG:AP. Agricultural activities had limited effects on other enzymatic stoichiometries.

RICTOR/mTORC2 affects tumorigenesis and therapeutic efficacy of mTOR inhibitors in esophageal squamous cell carcinoma.

Dysregulation of mTORC1/mTORC2 pathway is observed in many cancers and mTORC1 inhibitors have been used clinically in many tumor types; however, the mechanism of mTORC2 in tumorigenesis is still obscure. Here, we mainly explored the potential role of mTORC2 in esophageal squamous cell carcinoma (ESCC) and its effects on the sensitivity of cells to mTOR inhibitors. We demonstrated that RICTOR, the key factor of mTORC2, and p-AKT (Ser473) were excessively activated in ESCC and their overexpression is related to lymph node metastasis and the tumor-node-metastasis (TNM) phase of ESCC patients. Furthermore, we found that mTORC1/ mTORC2 inhibitor PP242 exhibited more efficacious anti-proliferative effect on ESCC cells than mTORC1 inhibitor RAD001 due to RAD001-triggered feedback activation of AKT signal. Another, we demonstrated that down-regulating expression of RICTOR in ECa109 and EC9706 cells inhibited proliferation and migration as well as induced cell cycle arrest and apoptosis. Noteworthy, knocking-down stably RICTOR significantly suppresses RAD001-induced feedback activation of AKT/PRAS40 signaling, and enhances inhibition efficacy of PP242 on the phosphorylation of AKT and PRAS40, thus potentiates the antitumor effect of RAD001 and PP242 both in vitro and in vivo. Our findings highlight that selective targeting mTORC2 could be a promising therapeutic strategy for future treatment of ESCC.

Uncovering the biosynthetic potential of rare metagenomic DNA using co-occurrence network analysis of targeted sequences.

Sequencing of DNA extracted from environmental samples can provide key insights into the biosynthetic potential of uncultured bacteria. However, the high complexity of soil metagenomes, which can contain thousands of bacterial species per gram of soil, imposes significant challenges to explore secondary metabolites potentially produced by rare members of the soil microbiome. Here, we develop a targeted sequencing workflow termed CONKAT-seq (co-occurrence network analysis of targeted sequences) that detects physically clustered biosynthetic domains, a hallmark of bacterial secondary metabolism. Following targeted amplification of conserved biosynthetic domains in a highly partitioned metagenomic library, CONKAT-seq evaluates amplicon co-occurrence patterns across library subpools to identify chromosomally clustered domains. We show that a single soil sample can contain more than a thousand uncharacterized biosynthetic gene clusters, most of which originate from low frequency genomes which are practically inaccessible through untargeted sequencing. CONKAT-seq allows scalable exploration of largely untapped biosynthetic diversity across multiple soils, and can guide the discovery of novel secondary metabolites from rare members of the soil microbiome.