The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1)encoded by Epstein-Barr virus(pshLMP1),and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells,we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis,RT-PCR and western blot.pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method.Furthermore,the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors.According to our research,we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells,and also provides a novel application of RNA interference technology against-EBV.

CD36 promotes tubular ferroptosis by regulating the ubiquitination of FSP1 in acute kidney injury

Reactive oxidative species(ROos)production-driven ferroptosis plays a role in acute kidney injury(Akl).However,its exact molecular mechanism is poorly understood.Scavenger receptor CD36 has important roles in oxidizing lipids,lipid accumulation,metabolic syndrome,and insulin resistance in chronic kidney disease,but its roles remain unexplored in AKl.The present study investigated the role and mechanism of CD36 in regulating proximal tubular cell ferroptosis and AKl.The expression of CD36 was found to be significantly up-regulated in AKI renal tissues and correlated with renal function,which might serve as an independent biomarker for AKl patients.Moreover,in adult mice subjected to AKl,deletion of CD36(CD36-/-)induced tubular cell Ros accumulation,ferroptosis activation,and renal injury.Mechanistically,combining LC-MS/MS,co-IP,and ubiquitination analyses revealed that CD36 could specifically bind to ferroptosis suppressor protein 1(FSP1)and regulate its ubiquitination at sites K16 and K24,leading to FSP1 degradation and progression of ferroptosis in AKl.The present results emphasize a novel mechanism of CD36 in cisplatin-induced AKl.The discovery of the special CD36 roles in promoting ferroptosis and AKI development by regulating the ubiquitination of FSP1 in proximal tubular cells may be potential therapeutic targets for AKl.Moreover,CD36 may play a key role in the progression of AKl.Therefore,targeting CD36 may provide a promising treatment option for AKI.

S-phase arrest after vincristine treatment may promote hepatitis B virus replication

AIM:To observe the effect of vincristine on hepatitis B virus(HBV) replication in vitro and to study its possible mechanisms.METHODS:Vincristine was added to the cultures of two cell lines stably expressing HBV.Then,the levels of hepatitis B surface antigen(HBs Ag),hepatitis B e antigen(HBe Ag),and hepatitis B core antigen(HBc Ag) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay and Western blot.The HBV pregenome RNA(pg RNA) was detected using reverse transcription-PCR and realtime fluorescent quantitative PCR(RT-q PCR),and viral DNA was detected using Southern blot and RT-q PCR.Cell proliferation after drug treatment was detected using the Brd U incorporation test and the trypan blue exclusion assay.Cell cycle and cell apoptosis were examined using flow cytometry and Western blot.RESULTS:Vincristine up-regulated HBV replication directly in vitro in a dose-dependent manner,and 24-h exposure to 0.1 μmol/L vincristine induced more than 4-fold and 3-fold increases in intracellular HBV DNA and the secretion of viral DNA,respectively.The expression of HBV pg RNA,intracellular HBs Ag and HBc Ag,and the secretion of HBe Ag were also increased significantly after drug treatment.Most importantly,vincristine promoted the cell excretion of HBV nucleocapsids instead of HBV Dane particles,and the nucleocapsids are closely related to the HBV pathogenesis.Furthermore,vincristine inhibited the proliferation of cells stably expressing HBV.The higher the concentration of the drug,the more significant the inhibition of the cell proliferation and the stronger the HBV replication ability in cells.Flow cytometry indicated that cell cycle arrest at S-phase was responsible for the cell proliferation inhibition.CONCLUSION:Vincristine has a strong stimulatory effect on HBV replication and induces cell cycle arrest,and cell proliferation inhibition may be conducive to viral replication.

Multifaceted Influence of Histone Deacetylases on DNA Damage Repair:Implications for Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most commonly diagnosed cancers and a leading cause of cancerrelated mortality worldwide,but its pathogenesis remains largely unknown.Nevertheless,genomic instability has been recognized as one of the facilitating characteristics of cancer hallmarks that expedites the acquisition of genetic diversity.Genomic instability is associated with a greater tendency to accumulate DNA damage and tumor-specific DNA repair defects,which gives rise to gene mutations and chromosomal damage and causes oncogenic transformation and tumor progression.Histone deacetylases(HDACs)have been shown to impair a variety of cellular processes of genome stability,including the regulation of DNA damage and repair,reactive oxygen species generation and elimination,and progression to mitosis.In this review,we provide an overview of the role of HDAC in the different aspects of DNA repair and genome instability in HCC as well as the current progress on the development of HDAC-specific inhibitors as new cancer therapies.

Durability of Hepatitis B surface antigen seroclearance in patients experienced nucleoside analogs or interferon monotherapy: A real-world data from Electronic Health Record

Little is known about the difference in durability of HBsAg seroclearance induced by nucleoside analogs (NAs) or by interferon (IFN). A real-world, retrospective cohort study was conducted. Patients were assigned into two groups: NAs monotherapy-induced HBsAg seroclearance subjects and IFN monotherapy induced-HBsAg seroclearance subjects. A total of 198 subjects, comprised by 168 NAs monotherapy-induced and 30 IFN monotherapy-induced, who achieved HBsAg seroclearance were included in this study. The one-year probabilities of confirmed HBsAg seroclearance were significantly different in patients with NAs monotherapy and IFN monotherapy (0.960 (with 95% CI 0.922–0.999) vs. 0.691 (with 95% CI 0.523–0.913), log-rank-P = 4.04e-4). 73.3% (11 of 15) HBsAg recurrence occurred within one year after HBsAg seroclearance. The one-year probabilities of confirmed HBsAg seroclearance were higher in IFN monotherapy patients with anti-HBs than in IFN monotherapy patients without anti-HBs (0.839 (with 95% CI 0.657–1.000) vs. 0.489 (with 95% CI 0.251–0.953), log-rank test, P = 0.024). Our study thus provided novel insights into the durability of HBsAg seroclearance induced by NAs or IFN monotherapy. In particular, the HBsAg seroreversion rate was relatively high in IFN monotherapy subjects. The presence of anti-HBs was significantly correlated with a longer durability of functional cure induced by IFN treatment. And one-year follow-up in HBsAg seroclearance achieved individuals is proper for averting HBsAg seroreversion and other liver disease.

The molecular pathogenic role of inflammatory stress in dysregulation of lipid homeostasis and hepatic steatosis

Non-alcoholic Fatty Liver Disease(NAFLD)is becoming the leading cause of chronic liver injury in developed countries and China.Chronic systemic inflammation plays a decisive role and is fundamental in the progression of NAFLD from simple steatosis(SS)toward higher risk nonalcoholic steatohepatitis(NASH)states.However,the exact mechanisms by which inflammation leading to NASH are incompletely understood.In this review,we focus the role of the cross talk between inflammation and lipid homeostasis on the progression of NAFLD.

Efficacy of rapid antigen self-testing for SARS-CoV-2 screening:Real-world evidence from a prospective cohort study

Antigen rapid diagnostic tests(Ag-RDTs)have been considered and implemented as an important diagnostic and screening tool to identify SARS-CoV-2 infections in community settings.1 Ag-RDTs are less sensitive,particularly in asymptomatic populations,compared with laboratorybased viral nucleic acid amplification tests(NAATs)such as reverse transcription polymerase chain reaction(RT-PCR).2 However,taking into account the facts that Ag-RDTs are effective for identifying most contagious individuals,they are faster and less expensive than RT-PCR,as well as that RT-PCR could produce positive results for weeks to months after the infection,2 WHO recommends Ag-RDTs be offered as COVID-19 self-testing for screening purposes in addition to professionally administered testing services regardless of the community transmission level.

Whole-exome mutational landscape of metastasis in patient-derived hepatocellular carcinoma cells

In order to explore the genomic basis for liver cancer metastasis,whole-exome sequencing(WES)was performed on patient-derived hepatocellular carcinoma(HCC)cell lines with differential metastatic potentials and analyzed their clonal evolution relationships.An evolutionary tree based on genomic single nucleotide polymorphism(SNP)was constructed in MegaX software.The WES data showed that the average percentage of heterogeneous mutations in each HCC cell lines was 16.55%(range,15.38%e18.17%).C:G>T:A and T:A>C:G somatic transitions were the two most frequent substitutions.In these metastatic HCC cell lines,non-silent gene mutations were found in 21.88%of known driver genes and 10 classical signaling pathways.The protein interaction network was constructed by STRING,and hub genes were found in the shared trunk mutation genes and the heterogeneous branch mutations respectively.In cBioPortal database,some of the selected hub genes were found to be associated with poor overall survival(OS)of HCC patients.Among the mutated HCC driver genes,a novel KEAP1 mutation with a homozygous frameshift truncation at the c-terminal Nrf2 binding region was detected and verified in MHCC97-H and HCC97LM3 cells.In conclusion,WES data demonstrate that HCC cell lines from tumor biopsy specimens of the same patient have obtained different metastatic potentials through repeated selection in rodents in vivo,and they do indeed have a genetic relationship at the genomic level.

Protective Effect of Neonatal Hepatitis B Vaccine Against HBV Breakthrough Infection in Children with Leukemia:A Real-world Study

Background and Aims:Hepatitis B vaccine is the most effective preventive measure against hepatitis B virus(HBV)infection.However,the risk of HBV breakthrough infection in fully immunized children(neonatal hepatitis B immunization)who receive immunosuppressive therapy and transfusion of blood components is not well characterized.In this real-world study,we aimed to investigate the immune protection conferred by neonatal hepatitis B vaccine in children with acute lymphoblastic leukemia(ALL)who were treated with immunosuppressive therapy and blood component transfusions.Methods:Children with ALL who had received all three doses of neonatal hepatitis B vaccine were included in this study.HBV seromarkers were detected before and after the initiation of immunosuppressive therapy.Results:A total of 1,011 children with ALL who were fully vaccinated against hepatitis B in infancy before the initiation of immunosuppressive therapy were eligible for inclusion.HBV infection was detected in four of 410 children(0.98%)with an HBsAg test after the initiation of immunosuppressive therapy.The median interval from treatment initiation was 19 months.Conclusions:Three doses of neonatal hepatitis B vaccine conferred adequate protection.In endemic regions,there is a low risk of HBV breakthrough infection in fully immunized children with immunosuppressive therapy.

CK1α upregulates the IFNAR1 expression to prompt the anti-HBV effect of type Ⅰ IFN in hepatoma carcinoma cells

Casein kinase 1α(CK1α) mediates the phosphorylation and degradation of interferon-α/β receptor 1(IFNAR1) in response to viral infection. However, how CK1α regulates hepatitis B virus(HBV) replication and the anti-HBV effects of IFN-α are less reported. Here we show that CK1α can interact with IFNAR1 in hepatoma carcinoma cells and increased the abundance of IFNAR1 by reducing the ubiquitination levels in the presence of HBV.Furthermore, CK1α promotes the IFN-α triggered JAK-STAT signaling pathway and consequently enhances the antiviral effects of IFN-α against HBV replication. Our results collectively provide evidence that CK1α positively regulates the anti-HBV activity of IFN-α in hepatoma carcinoma cells, which would be a promising therapeutic target to improve the effectiveness of IFN-α therapy to cure CHB.

Complementary Presence of HBV Humoral and T-cell Response Provides Protective Immunity after Neonatal Immunization

Background and Aims:Hepatitis B vaccination is the most cost effective way to prevent hepatitis B virus(HBV)infection.Hepatitis B vaccine(HepB)efficacy is usually assessed by an-ti-hepatitis B surface antigen(HBsAg)level,but there are few reports of humoral and cellular immune responses to HepB in children after neonatal vaccination.Methods:A group of 100 children with a history of primary hepatitis B immunization were included in this study to evaluate the efficacy of HepB.Blood samples were obtained from 80 children before,and 41 children after,a single HepB booster dose.Children with low anti-HBsAg(HBs)titers of<100 mIU/mL received a booster dose after giving their informed consent.Anti-HBsAg,T-cell re-sponse and percentage of B-cell subsets were assayed before and after the booster.Results:Of the 80 children,81.36%had positive T cell and anti-HBsAg responses at baseline.After the booster dose,the anti-HBsAg titer(p<0.0001),positive HBsAg-specific T-cell response(p=0.0036),and spot-form-ing cells(p=0.0003)increased significantly.Compared with pre-existing anti-HBsAg titer<10 mIU/mL,the anti-HBsAg(p=0.0005)and HBsAg-specific T-cell responses(p<0.0001)increased significantly in preexisting anti-HBsAg titer between 10 and 100 mIU/mL group.Change of the HBV-specific hu-moral response was the reverse of the T-cell response with age.Peripheral blood lymphocytes,B cells,and subset fre-quency decreased.Conclusions:HBV immunization protec-tion persisted at least 13 years after primary immunization because of the complementary presence of HBV-specific hu-moral antibodies and a T-cell immune response.One dose of a HepB booster induced protective anti-HBsAg and promoted an HBsAg-specific T-cell response.In HBV endemic regions,a HepB booster is recommended to children without anti-HBsAg because of effectiveness in HBV prevention.

Opinion on the Policy of Lifting Restrictions to Entry Under the Circumstance of the COVID-19 Pandemic

One of the serious consequences of the coronavirus disease 2019(COVID-19)pandemic is the extreme restriction on international contacts.Communication between some countries has almost been frozen for a period of time,which is a manifestation of“social distancing”,a strategy for epidemic prevention and control,among countries.In the context of economic globalization,“social distancing”has caused serious adverse effects,including poor communication in society,the economy and personnel,reduced efficiency of resource allocation,and increased costs.Therefore,people should take necessary measures to restore international contact and eliminate these adverse effects as soon as possible.

Detection of HBV DNA integration in plasma cell-free DNA of different HBV diseases utilizing DNA capture strategy

The landscape of hepatitis B virus(HBV)integration in the plasma cell-free DNA(cfDNA)of HBV-infected patients with different stages of liver diseases[chronic hepatitis B(CHB),liver cirrhosis(LC),and hepatocellular carcinoma(HCC)]remains unclear.In this study,we developed an improved strategy for detecting HBV DNA integration in plasma cfDNA,based on DNA probe capture and next-generation sequencing.Using this optimized strategy,we successfully detected HBV integration events in chimeric artificial DNA samples and HBV-infected HepG2-NTCP cells at day one post infection,with high sensitivity and accuracy.The characteristics of HBV integration events in the HBV-infected HepG2-NTCP cells and plasma cfDNA from HBV-infected individuals(CHB,LC,and HCC)were further investigated.A total of 112 and 333 integration breakpoints were detected in the HepG2-NTCP cells and 22 out of 25(88%)clinical HBV-infected samples,respectively.In vivo analysis showed that the normalized number of support unique sequences(nnsus)in HCC was significantly higher than in CHB or LC patients(P values<0.05).All integration breakpoints are randomly distributed on human chromosomes and are enriched in the HBV genome around nt 1800.The majority of integration breakpoints(61.86%)are located in the gene-coding region.Both non-homologous end-joining(NHEJ)and microhomology-mediated end-joining(MMEJ)interactions occurred during HBV integration across the three different stages of liver diseases.Our study provides evidence that HBV DNA integration can be detected in the plasma cfDNA of HBV-infected patients,including those with CHB,LC,or HCC,using this optimized strategy.

Classifying hepatitis B therapies with insights from covalently closed circular DNA dynamics

The achievement of a functional cure for chronic hepatitis B(CHB)remains limited to a minority of patients treated with currently approved drugs.The primary objective in developing new anti-HBV drugs is to enhance the functional cure rates for CHB.A critical prerequisite for the functional cure of CHB is a substantial reduction,or even eradication of covalently closed circular DNA(cccDNA).Within this context,the changes in cccDNA levels during treatment become as a pivotal concern.We have previously analyzed the factors influencing cccDNA dynamics and introduced a preliminary classification of hepatitis B treatment strategies based on these dynamics.In this review,we employ a systems thinking perspective to elucidate the fundamental aspects of the HBV replication cycle and to rationalize the classification of treatment strategies according to their impact on the dynamic equilibrium of cccDNA.Building upon this foundation,we categorize current anti-HBV strategies into two distinct groups and advocate for their combined use to significantly reduce cccDNA levels within a well-defined timeframe.

Role of TMS5： staphylococcal multidrug-efflux protein QacA

Background QacA, a main exporter mediating the multidrug-resistance of Staphylococcus aureus to a vanety of antiseptics and disinfectants, possesses a topology of 14 a-helical transmembrane segments （TMS）. Our study aimed to determine the importance and topology of amino acid residues in and flanking the cytoplasmic end of TMS5. Methods Site-directed mutagenesis was used to mutate 5 residues, including L146, A147, V148, W149 and S150, into cysteine. A minimum inhibitory concentration （MIC） and transport assay with or without N-ethylmaleimide （NEM） were performed to analyse the function of these mutants. Results All of the mutants showed comparable protein expression levels. MIC analysis suggested that mutant W149C showed low resistance levels to the drugs, but the mutations at L146, A147, V148, and S150C had little or no effect on the resistance level. And the results of the fluorimetdc transport assay were in agreement with those of MIC analysis, that is to say, W149C did not allow transport to the substrates to be tested, while the other mutants retained significant transport ability. The reaction of the different mutant proteins with Fluorescein-NEM revealed that the mutant L146C was highly reactive with NEM; the W149C and S150C mutants were moderately reactive; A147C was barely reactive and V148C showed no reactivity. Conclusions The study identified that residues W149 and S150 situated at the interface of the aqueous： lipid junction as functionally important residues, probably involved in the substrate binding and translocation of QacA.

A potent neutralizing antibody provides protection against SARS-CoV-2 Omicron and Delta variants via nasal delivery

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is still rapidly spreading worldwide.Many drugs and vaccines have been approved for clinical use show efficacy in the treatment and prevention of SARS-CoV-2 infections.However,the emergence of SARS-CoV-2 variants of concern(VOCs),such as Delta(B.1.617.2)and the recently emerged Omicron(B.1.1.529),has seriously challenged the application of current therapeutics.Therefore,there is still a pressing need for identification of new broad-spectrum antivirals.Here,we further characterized a human antibody(58G6),which we previously isolated from a patient,with a broadly authentic virus-neutralizing activity that inhibits the Delta and Omicron variants with half-maximal inhibitory concentrations(ICso)of 1.69 ng/ml and 54.31 ng/ml,respectively.58G6 shows prophylactic and therapeutic effcacy in hamsters challenged with the Delta and Omicron variants through nasal delivery.Notably,a very low dosage(2 mg/kg daily)of 58G6 efficiently prevented Omicron variant replication in the lungs.These advantages may overcome the efficacy limitation of currently approved neutralizing antibodies that can be administered only by intravenous injection.In general,58G6 is a promising prophylactic and therapeutic candidate against current circulating VOCs and even future emerging mutants.To the best of our knowledge,58G6 is one of the most potent neutralizing antibodies against Omicron,with a broader spectrum than those approved for clinical use.58G6 could be developed as a nebulized therapy,which would be more cost effective and user friendly and enhance the clinical outcome comparedto thatobtainedwithdirect nasaldelivery.

A novel phenotypic assay of hepatitis B virus polymerase with extensive site-specific mutagenesis

Dear Editor,A common reason for drug failure during long-term treatment of chronic hepatitis B with nucleot(s)ide analogues(NUCs)is the emergence of drug resistance(Das et al.,2001).Most primary NUCs-resistant mutations identified in clinical samples have been limited to a minority of amino acids(usually less