查找使用阿拉丁产品的科研文献

CDC42 supports HBV entry by NTCP translocation to the plasma membrane and macropinocytosis

Shuzhi Cui,Wei Gao,Yuxin Chen,Yi Xu,Zhifang Li,Yu Wei,Yaming Jiu

CDC42 is a member of Rho GTPase family that regulates various biological processes and its activity can be hijacked by invading pathogens. Here, we discovered that the level of active CDC42 in hepatocytes positively correlates with the entry capacity of hepatitis B virus (HBV). Mechanistically, CDC42 activation effectively promotes the transport of the viral receptor sodium taurocholate co-transporting polypeptide (NTCP) to the plasma membrane via Rab11 dependent recycling endosomal pathway. NTCP interacts with Rab11 and activation of CDC42 signaling reinforces the interaction between NTCP and Rab11. We further show that clathrin mediated endocytosis (CME), the known HBV entry pathway, is independent of CDC42 activity. Intriguingly, we reveal that CDC42 dependent macropinocytosis is a route for HBV entry, which is equally essential for viral infection as CME. Together, our findings uncover new mechanisms for HBV entry that involve unrecognized functions of CDC42 and suggest that Rho GTPase signaling might represent a potential target for antiviral therapy.

Desmethoxycurcumin aids IFNα’s anti-HBV activity by antagonising CRYAB reduction and stabilising IFNAR1 protein

Jinlai Wei,Xichuan Deng,Wenying Dai,Lingxin Xie,Guangyuan Zhang,Xinyue Fan,Xinyue Li,Zhixing Jin,Qin Xiao,Tingting Chen

Betaine-assisted recombinase polymerase assay with enhanced specificity

Guang-Cheng Luo,Ting-Ting Yi,Bing Jiang,Xiao-lan Guo,Guo-Yuan Zhang

Recombinase polymerase amplification (RPA) is a widespread isothermal amplification method and regarded as an excellent candidate to replace polymerase chain reaction. However, the specificity of RPA is not always satisfactory when the sample contains amounts of background DNA . Herein, we report a novel RPA method named betaine-assisted RPA (B-RPA) that uses inexpensive betaine to avoid nonspecific amplification effectively. Result show that nonspecific amplification is prone to occur in RPA if the primers have not been rigorously refined, especially in detecting samples with large amounts of background DNA. This problem has been addressed by adding betaine to the RPA reactions. Our data show that the addition of 0.8 M betaine can significantly increase specificity and efficiency simultaneously. This B-RPA method is also used to detect hepatitis B virus DNA in clinical plasma samples, thereby demonstrating the clinical practicability of B-RPA.

PreS/2-21-Guided siRNA Nanoparticles Target to Inhibit Hepatitis B Virus Infection and Replication.

Lixia Gao,Jie Yang,Jutao Feng,Ziying Liu,Ying Dong,Jiangyan Luo,Liangzhentian Yu,Jiamei Wang,Hongying Fan,Weifeng Ma,Tiancai Liu

A viable therapy is needed to overcome the deadlock of the incurable chronic hepatitis B (CHB). The prolonged existence of covalently closed circular DNA (cccDNA) and integrated HBV DNA in the nucleus of hepatocytes is the root cause of CHB. As a result, it is critical to successfully suppress HBV DNA replication and eliminate cccDNA. RNA interference has been proven in recent research to silence the expression of target genes and thereby decrease HBV replication. However, siRNA is susceptible to be degraded by RNA enzymes in vivo , making it difficult to deliver successfully and lacking of tissue targeting. To exploit the advantages of siRNA technology while also overcoming its limitations, we designed a new strategy and prepared biomimetic nanoparticles that were directed by PreS/2-21 peptides and precisely loaded HBV siRNA. Experiments on these nanoparticles in vitro and in vivo revealed that they are tiny, stable, safe and highly targetable, with high inhibitory effects on HBV DNA, pgRNA, cccDNA, HBeAg and HBsAg. PreS/2-21-directed nanoparticles loaded with HBV gene therapy drugs are expected to be promising for the treatment of CHB.

A Galactose-Engineered Dual-Responsive Nanocarrier for ASO/CRISPR-Cas9 Delivery to Inhibit HBV Replication

Liuxian Chen,Qin Huang,Yongjie Liu,Ke Chen,Qing Yang,Hua Tang,Dan Wang,Zhenrong Tang

Complete hepatitis B virus (HBV) cure is hindered primarily by the stable persistence of covalently closed circular DNA (cccDNA). Gene editing approaches to eradicate HBV by targeting cccDNA face challenges and limitations due to suboptimal editing efficiency and substantial off-target effects. Herein, a combinatorial therapeutic strategy is developed that integrates CRISPR/Cas9-mediated cccDNA disruption with an antisense oligonucleotide (ASO)-targeted degradation of pregenomic RNA (pgRNA). To overcome delivery challenges, a hepatocyte-targeting nanocarrier (UACPG) is engineered, featuring low immunogenicity, high payload capacity, and dual-stimuli responsiveness. The UACPG platform enabled liver-specific delivery through surface-conjugated targeting ligands, followed by on-demand release of Cas9 ribonucleoprotein complexes and ASO via RNase H-dependent degradation and near-infrared (NIR) light activation. The results demonstrated that UACPG can effectively reduce HBV replication and viral antigen levels, while significantly lowering cccDNA in hydrodynamic HBV-infected mouse models, with no significant off-target effects observed. This nanocarrier achieved the spatiotemporally controlled release of gene-editing systems in vitro and in vivo, significantly inhibiting the replication of HBV, thereby establishing an innovative technological platform for developing curative HBV therapies.

Amplification-free detection of HBV DNA mediated by CRISPR-Cas12a using surface-enhanced Raman spectroscopy

Yuwan Du,Shuaifeng Ji,Qingyang Dong,Jiang Wang,Dianpeng Han,Zhixian Gao

Nucleic acid markers have been widely used in the detection of various virus-related diseases, including hepatitis B virus (HBV), which is spreading worldwide. The trans -activated CRISPR-Cas system has shown excellent sensitivity and specificity in nucleic acid detection. However, nucleic acid testing usually requires amplification of the target nucleic acid for more accurate and specific detection; furthermore, current nucleic acid assays are time-consuming, costly, and are limited by non-specific cross-reactivity. We developed an amplification-free viral DNA biosensor-based diagnostic method that uses a clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas)-based approach with surface enhanced Raman spectroscopy . This method can specifically identify the target site by changing the crRNA sequence. In addition, the incubation period and development of the disease can be determined by quantitative detection of viral DNA. This system could achieve rapid and highly sensitive detection of HBV DNA within 50 min and vast detection range from 0.1 pM to 1 nM. Therefore, a combined CRISPR/Cas12a-SERS-based assay would improve the sensitivity of detection in assays using multiple biomarkers. In conclusion, our CRISPR/Cas12a-based biosensor would enable rapid, simple, and sensitive detection of HBV nucleic acids.

A Highly Sensitive Silicon Nanowire Array Field Effect Transistor Biosensor for Detecting HBV-DNA and AFP

Peng Sun,Mingbin Liu,Yongxin Zhang,Chaoran Liu,Xun Yang

Chronic hepatitis B poses a serious threat to human health and life, and early diagnosis is essential to improving patient cure rates. Hepatitis B virus (HBV) and Alpha-fetoprotein (AFP) are two key biomarkers for diagnosing chronic hepatitis B. In this study, we propose a silicon nanowire array field effect transistor (SiNW-array FET) biosensor that enables highly sensitive, real-time, and low-cost joint detection of both HBV and AFP. The SiNW-array FET is fabricated using traditional micro-nano fabrication techniques such as self-limiting oxidation and anisotropic etching, and its morphology and electrical properties were tested. The results show that the diameters of the fabricated silicon nanowires (SiNWs) are uniform and the SiNW-array FET exhibits a strong output signal and high signal-to-noise ratio. Through specific chemical modification on the surface of SiNWs, the SiNW-array FET is highly sensitive and specific to HBV-DNA fragments and AFP, with ultralow detection limits of 0.1 fM (HBV-DNA) and 0.1 fg/mL (AFP). The detection curve of the SiNW-array FET exhibits good linearity within the HBV-DNA concentration range of 0.1 fM to 100 pM and AFP concentration range of 0.1 fg/mL to 1000 pg/mL. More importantly, the device could also detect HBV-DNA successfully in serum samples, laying a solid foundation for the highly sensitive clinical detection of chronic hepatitis B.

Electrochemiluminescence DNA Biosensor for HBV Based on Coralloid Poly(Aniline-Luminol)-MWCNTs and Catalysis of Ferrocene

Ruirui Dong,Yanhui Zhang,Jiali Huang,Marhaba Habibul,Guixin Li

A novel electrochemiluminescence biosensor was presented for the assay of hepatitis B virus (HBV) DNA. Coralloid poly(aniline-luminol)-MWCNTs was prepared on an electrode surface, which was followed by the attachment of capture DNA via amide bond formation. The HBV-DNA and ferrocene labelled DNA hybridized with capture DNA to form a sandwich structure on electrode surface. Ferrocene can effectively increase the signal output of the ECL sensor due to the catalysis of ferrocene on electrochemiluminescence of luminol. The biosensor rendered a linear dependence over the range from 1.0×10 −16 to 1.0×10 −7 M with a low detection limit of 2.4×10 −17 M.

Betaine-assisted recombinase polymerase assay for rapid hepatitis B virus detection

Ting-ting Yi,Han-yun Zhang,Hua Liang,Guo-zhong Gong,Yan Cai

Hepatitis B virus (HBV) is a worldwide epidemic pathogen that causes hepatitis B. On-site screening the HBV infection is of critical importance for preventing and diagnosing HBV infection. In this paper, a simple, visual, and rapid method for on-site detection of HBV-DNA has been developed. This method is based on betaine-assisted recombinase polymerase assay and followed with naked-eye detection via lateral flow assay (BRPA-LF). Result show that nonspecific amplification is prone to occur in recombinase polymerase amplification (RPA) if the assay was performed with serum sample without purification. This problem has been addressed by adding 0.8 M of betaine to the RPA reactions. It was demonstrated that BRPA-LF can detect 1,000 copies of HBV-DNA in 50 μL mixture, and achieved 90% sensitivity and 100% specificity for serum sample detection. These results demonstrated that BRPA-LF can resist serum interference and has great potential for on-site screening of HBV infection.

TRIM28 functions as SUMO ligase to SUMOylate TRAF6 and regulate NF-κB activation in HBV-replicating cells

Yang Yanfang,Wang Tao,Fu Yuyin,Li Xukui,Yu Fuxun

Background Hepatitis B virus (HBV) is a pathogen that poses a serious threat to human health. The interaction between HBV and host has made great progress in recent years. SUMOylation is involved in virus-related cancer progression, but there are fewer studies on the mechanism of SUMOylation on HBV replication and antiviral defense. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a critical adaptor of the NF-κB pathways. Here, we focus on the roles of TRIM28 in regulating TRAF6 SUMOylation in HBV-replicating cells. Methods The SUMO1-modified TRAF6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated TRAF6 was detected by western blot using an anti-TRAF6 antibody. The interaction between TRAF6 and TRIM28 was identified by immunoprecipitation and LC–MS/MS. The modification sites of TRAF6 SUMOylation were identified by amino acid site mutation. Expression and localization of TRAF6 and TRIM28 were assessed by immunohistochemistry and immunofluorescence. The hydrodynamic injection HBV mouse model was used to determine the function of TRIM28-mediated TRAF6 SUMOylation in vivo. Results The results show that the levels of SUMO1-modified TRAF6 are elevated in HBV-replicating cells. Lys453 is a major SUMO1 modification site of TRAF6. There is an antagonistic interaction between SUMOylation and ubiquitination of TRAF6 protein. The SUMO ligase TRIM28 is responsible for catalyzing TRAF6 SUMOylation. Compared to the wild-type TRAF6, its SUMO site mutant TRAF6 K453R promotes NF-κB activation. Moreover, TRIM28 overexpression attenuates TRAF6-mediated NF-κB activation, thereby inhibiting HBV replication in vivo. Conclusions Our findings demonstrate that SUMO ligase TRIM28 affects the ability of TRAF6 on NF-κB activation, nucleocytoplasmic shuttling and HBV replication-related indicators. Our data reveal that TRIM28-mediated SUMOylation of TRAF6 is a novel mechanism to regulate the inflammatory response, which may pave the way for new strategies to control anti-HBV.

Dual-Shell Chitosan/Ferritin Nanocages Enhance the Stability, Intestinal Transport, and Antiviral Activity of EGCG against HBV

Yixin Dong,Xizhi Yin,Xiangru Shan,Yiqiang Hu,Chuanlong Zhu

Epigallocatechin gallate (EGCG) has shown antiviral potential against the hepatitis B virus (HBV) by restoring lysosomal acidification; however, its application is limited by poor stability and low intestinal permeability. Here, a dual-shell chitosan/ferritin nanosystem (CHE) was developed to enhance the EGCG delivery and functionality. CHE achieved an encapsulation efficiency of 13.61% with a uniform nanoscale size distribution. Compared with free EGCG and single-shell HE, CHE improved storage stability and delayed EGCG release under simulated gastrointestinal conditions. In a Caco-2/HepG2.2.15 or HepAD38 coculture model, CHE enhanced transepithelial transport and intracellular delivery while maintaining epithelial barrier integrity. Mechanistically, CHE promoted lysosomal acidification, increased cathepsin B maturation, and restored autophagic flux, accompanied by reductions in HBV DNA, pgRNA, HBsAg, and HBeAg levels. These findings demonstrate that a dual-shell nanocage integrates stability enhancement with functional intracellular modulation, providing a promising strategy to improve the antiviral efficacy of polyphenols.

Evans Blue Inhibits HBV Replication Through a Dual Antiviral Mechanism by Targeting Virus Binding and Capsid Assembly

Xiao Yu,Liu Chunlan,Tang Wei,Zhang Haiwei,Chen Xulin

Chronic hepatitis B (CHB) is a global health problem caused by human hepatitis B virus (HBV). Current treatment with interferons and nucleos(t)ide analogs (NAs) can cause population tolerance and drug resistance. Therefore, new antiviral drugs, especially those targeting host factors, are urgently needed. Here, we identified Evans blue as a new HBV inhibitor by screening an FDA drug library using Huh7DhNTCP cells and confirmed the antiviral activity in primary human hepatocytes and human sodium taurocholate cotransporting polypeptide (hNTCP)-transfected porcine primary hepatocytes. Our efficacy study showed that Evans blue has an IC50 of 2 μM against HBV infection in Huh7DhNTCP cells, and no apparent toxicity at up to 1000 μM. The IC50 of Evans blue against HBV in primary human hepatocytes was approximately 5 μM. Mechanism studies revealed that Evans blue has a dual anti-HBV effect. It inhibits both the binding of viral preS1 to host cells through the host factor NTCP and the virus capsid assembly by targeting the host factor BK channel. The KD of the direct interaction between Evans blue and NTCP is 8.82E-8 M. Evans blue can suppress capsid assembly at micromolar concentrations by reducing the cytosolic calcium ion concentration. Since the antiviral effects on HBV binding and assembly are both achieved through targeting host factors, Evans blue inhibits the infection of nucleos(t)ide analog drug-resistant HBV strains in Huh7DhNTCP cells. Taken together, our results suggest that Evans blue may be a promising anti-HBV drug candidate in the classes of both entry and assembly inhibitors.