Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation.

The progression and malignancy of many tumors are associated with increased tissue stiffness. Conversely, the oncogenically transformed cells can be confined in soft stroma. Yet, the underlying mechanisms by which soft matrix confines tumorigenesis and metastasis remain elusive. Here, we show that pancreatic cancer cells are suppressed in the soft extracellular matrix, which is associated with YAP1 degradation through autophagic-lysosomal pathway rather than Hippo signal mediated proteasome pathway. In the soft stroma, PTEN is upregulated and activated, which consequently promotes lysosomal biogenesis, leading to the activation of cysteine-cathepsins for YAP1 degradation. In vitro, purified cathepsin L can directly digest YAP1 under acidic conditions. Lysosomal stress, either caused by chloroquine or overexpression of cystatin A/B, results in YAP1 accumulation and malignant transformation. Likewise, liver fibrosis induced stiffness can promote malignant potential in mice. Clinical data show that down-regulation of lysosomal biogenesis is associated with pancreatic fibrosis and stiffness, YAP1 accumulation, and poor prognosis in PDAC patients. Together, our findings suggest that soft stroma triggers lysosomal flux-mediated YAP1 degradation and induces cancer cell dormancy.

Comparative efficacy and safety of tenofovir amibufenamide vs tenofovir alafenamide in the initial 48-week treatment of high viral load chronic hepatitis B: A single-centre retrospective study.

BACKGROUND/AIM: Tenofovir amibufenamide (TMF) employs innovative ProTide technology and a methylation strategy to enhance the lipid solubility and plasma stability of the amide bond, providing advantages over tenofovir alafenamide (TAF). Despite promising Phase III clinical trial results demonstrating its antiviral efficacy, real-world data on TMF remains scarce. This study evaluates the antiviral efficacy and safety of TMF compared to TAF as the initial treatment in patients with high viral loads of chronic hepatitis B (CHB). METHODS: We retrospectively collected clinical data from March 1 2022 to June 30 2022 for highly viremic CHB patients who received either TMF (n = 58) or TAF (n = 32) as their initial monotherapy at Beijing YouAn Hospital. To understand the efficacy and safety of TMF over 48 weeks, we compared the virological response rates and HBeAg/HBsAg serological clearance rates between TMF and TAF groups. Also, the changes in serum creatinine, eGFR and serum lipid levels were assessed. RESULTS: Baseline median HBV DNA levels were 7.85 (6.89, 8.36) IgIU/ml for TMF and 7.44 (6.89, 8.03) IgIU/ml for TAF. Median ALT levels were 102.0 (56.0, 210.0) U/L for TMF and 195.0 (73.5, 371.0) U/L for TAF, with HBeAg positivity rates of 70.7% and 75.0%, respectively. At 48 weeks, virological response rates (HBV DNA <10 IU/ml) were 43.5% (20/46) for TMF and 42.9% (12/28) for TAF (p = 1.000). ALT normalization rates were 87.9% for TMF and 90.6% for TAF (p = .969), and HBeAg serological clearance rates were 21.1% and 18.2%, respectively (p = 1.000). No patients achieved HBsAg clearance. Compared with the baseline, LDL-C levels increased, while eGFR decreased, with no significant differences in serum creatinine, triglycerides and total cholesterol levels noted at week 48 for both TMF and TAF groups. CONCLUSION: TMF demonstrates comparable antiviral efficacy to TAF when used as initial therapy in highly viremic CHB patients, with similar impacts on renal function and lipid profiles.

Unveiling Ionized Interfacial Water-Induced Localized H* Enrichment for Electrocatalytic Nitrate Reduction.

Electrocatalytic nitrate reduction reaction (NO3RR) is a process that requires the participation of eight electrons and nine protons. The regulation of active hydrogen (H*) supply and a deep understanding of related processes are necessary for improving the ammonia yield rate and Faradaic efficiency (FE). Herein, we synthesized a series of atomically precise copper-halide clusters Cu2X2(BINAP)2 (X=Cl, Br, I), among which the Cu2Cl2(BINAP)2 cluster shows the optimal ammonia FE of 94.0 % and an ammonia yield rate of 373 µmol h-1 cm-2. In situ experiments and theoretical calculations reveal that halogen atoms, especially Cl in Cu2Cl2(BIANP)2, can significantly affect the distance of alkali metal-ionized water on the catalyst surface, which can promote the water dissociation to enhance the localized H* enrichment for the continues hydrogenation of nitrate to ammonia. This work explains the role of H* in the hydrogenation process of NO3RR and the importance of localized H* enrichment strategy for improving the FEs.

Distinctive Pattern of Metal Deposition in Neurologic Wilson Disease: Insights From 7T Susceptibility-Weighted Imaging.

BACKGROUND AND OBJECTIVES: Noninvasive and accurate biomarkers of neurologic Wilson disease (NWD), a rare inherited disorder, could reduce diagnostic error or delay. Excessive subcortical metal deposition seen on susceptibility imaging has suggested a characteristic pattern in NWD. With submillimeter spatial resolution and increased contrast, 7T susceptibility-weighted imaging (SWI) may enable better visualization of metal deposition in NWD. In this study, we sought to identify a distinctive metal deposition pattern in NWD using 7T SWI and investigate its diagnostic value and underlying pathophysiologic mechanism. METHODS: Patients with WD, healthy participants with monoallelic ATP7B variant(s) on a single chromosome, and health controls (HCs) were recruited. NWD and non-NWD (nNWD) were defined according to the presence or absence of neurologic symptoms during investigation. Patients with other diseases with comparable clinical or imaging manifestations, including early-onset Parkinson disease (EOPD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and neurodegeneration with brain iron accumulation (NBIA), were additionally recruited and assessed for exploratory comparative analysis. All participants underwent 7T T1, T2, and high-resolution SWI scanning. Quantitative susceptibility mapping and principal component analysis were performed to illustrate metal distribution. RESULTS: We identified a linear signal intensity change consisting of a hyperintense strip at the lateral border of the globus pallidus in patients with NWD. We termed this feature "hyperintense globus pallidus rim sign." This feature was detected in 38 of 41 patients with NWD and was negative in all 31 nNWD patients, 15 patients with EOPD, 30 patients with MSA, 15 patients with PSP, and 12 patients with NBIA; 22 monoallelic ATP7B variant carriers; and 41 HC. Its sensitivity to differentiate between NWD and HC was 92.7%, and specificity was 100%. Severity of the hyperintense globus pallidus rim sign measured by a semiquantitative scale was positively correlated with neurologic severity (ρ = 0.682, 95% CI 0.467-0.821, p < 0.001). Patients with NWD showed increased susceptibility in the lenticular nucleus with high regional weights in the lateral globus pallidus and medial putamen. DISCUSSION: The hyperintense globus pallidus rim sign showed high sensitivity and excellent specificity for diagnosis and differential diagnosis of NWD. It is related to a special metal deposition pattern in the lenticular nucleus in NWD and can be considered as a novel neuroimaging biomarker of NWD. CLASSIFICATION OF EVIDENCE: The study provides Class II evidence that the hyperintense globus pallidus rim sign on 7T SWI MRI can accurately diagnose neurologic WD.

Stepwise structural evolution toward robust carboranealkynyl-protected copper nanocluster catalysts for nitrate electroreduction.

Atomically precise metal nanoclusters (NCs) are emerging as idealized model catalysts for imprecise metal nanoparticles to unveil their structure-activity relationship. However, the directional synthesis of robust metal NCs with accessible catalytic active sites remains a great challenge. In this work, we achieved bulky carboranealkynyl-protected copper NCs, the monomer Cu13·3PF6 and nido-carboranealkynyl bridged dimer Cu26·4PF6, with fair stability as well as accessible open metal sites step by step through external ligand shell modification and metal-core evolution. Both Cu13·3PF6 and Cu26·4PF6 demonstrate remarkable catalytic activity and selectivity in electrocatalytic nitrate (NO3-) reduction to NH3 reaction, with the dimer Cu26·4PF6 displaying superior performance. The mechanism of this catalytic reaction was elucidated through theoretical computations in conjunction with in situ FTIR spectra. This study not only provides strategies for accessing desired copper NC catalysts but also establishes a platform to uncover the structure-activity relationship of copper NCs.

Electrocatalytic Nitrate Reduction on Metallic CoNi-Terminated Catalyst with Industrial-Level Current Density in Neutral Medium.

Green ammonia synthesis through electrocatalytic nitrate reduction reaction (eNO3RR) can serve as an effective alternative to the traditional energy-intensive Haber-Bosch process. However, achieving high Faradaic efficiency (FE) at industrially relevant current density in neutral medium poses significant challenges in eNO3RR. Herein, with the guidance of theoretical calculation, a metallic CoNi-terminated catalyst is successfully designed and constructed on copper foam, which achieves an ammonia FE of up to 100% under industrial-level current density and very low overpotential (-0.15 V versus reversible hydrogen electrode) in a neutral medium. Multiple characterization results have confirmed that the maintained metal atom-terminated surface through interaction with copper atoms plays a crucial role in reducing overpotential and achieving high current density. By constructing a homemade gas stripping and absorption device, the complete conversion process for high-purity ammonium nitrate products is demonstrated, displaying the potential for practical application. This work suggests a sustainable and promising process toward directly converting nitrate-containing pollutant solutions into practical nitrogen fertilizers.

Peg-IFNα combined with hepatitis B vaccination contributes to HBsAg seroconversion and improved immune function.

PURPOSE: The purpose of this study was to investigate immunological variations between a group that received the hepatitis B vaccine and a non-vaccine group. We focused on a cohort that achieved HBsAg seroclearance after Peg-IFNα treatment of CHB. METHODS: We enrolled twenty-eight individuals who achieved HBsAg seroclearance after Peg-IFNα treatment. They were divided into two groups: a vaccine group (n = 14) and a non-vaccine group (n = 14). We assessed lymphocyte subpopulations, B cell- and T cell-surface costimulatory/inhibitory factors, cytokines and immunoglobulin levels were detected at different time points to explore immune-function differences between both groups. RESULTS: The seroconversion rate in the vaccine group at 24 weeks post-vaccination was 100%, which was significantly higher (p = 0.006) than that of the non-vaccine group (50%). Additionally, more individuals in the vaccine group exhibited anti-HBs levels exceeding 100 IUs/L and 300 IUs/L compared to the non-vaccine group (p < 0.05). The vaccine group demonstrated significantly increase total B cells and class-switched B cells at 24 weeks and plasma cells, CD80+B cells, Tfh cells, and ICOS+Tfh cell at 12 weeks, compared with baseline levels (p < 0.05). Conversely, Bregs (CD24+CD27+ and CD24+CD38high) decreased significantly at 24 weeks (p < 0.05). None of the above changes were statistically significance in the non-vaccine group (p > 0.05). Total IgG increased significantly in the vaccine group, and IL-2, IL-5, and IL-6 concentrations increased significantly at week 24 (p < 0.05). Differences in various types of cytokines and immunoglobulins in the plasma of the non-vaccine group were not significant (p > 0.05). Anti-HBs titers positively correlated with Th1/Th2 cells at 24 weeks (r = 0.448 and 0.458, respectively, p = 0.022 and 0.019, respectively), and negatively with CD24+CD38highBreg cells (r = -0.402, p = 0.042). CONCLUSIONS: After achieving HBsAg seroclearance through Peg-IFNα treatment for CHB, administering the hepatitis B vaccine significantly increased anti-HBs-seroconversion rates and antibody levels. We also observed significant immunological differences between the vaccine and non-vaccine groups. Specifically, the vaccine group exhibited significant increases in B cells, plasma cells, and Tfh cells, while Breg levels was significantly lower. These immunological changes are likely conducive to the production of anti-HBs antibodies. However, in the non-vaccine group, the observed changes were not significantlly significant.

Tandem Nitrate-to-Ammonia Conversion on Atomically Precise Silver Nanocluster/MXene Electrocatalyst.

Electrocatalytic reduction of nitrate (NO3 RR) to synthesize ammonia (NH3 ) provides a competitive manner for carbon neutrality and decentralized NH3 synthesis. Atomically precise nanoclusters, as an advantageous platform for investigating the NO3 RR mechanisms and actual active sites, remain largely underexplored due to the poor stability. Herein, we report a (NH4 )9 [Ag9 (mba)9 ] nanoclusters (Ag9 NCs) loaded on Ti3 C2 MXene (Ag9 /MXene) for highly efficient NO3 RR performance towards ambient NH3 synthesis with improved stability in neutral medium. The composite structure of MXene and Ag9 NCs enables a tandem catalysis process for nitrate reduction, significantly increasing the selectivity and FE of NH3 . Besides, compared with individual Ag9 NCs, Ag9 /MXene has better stability with the current density performed no decay after 108 hours of reaction. This work provides a strategy for improving the catalytic activity and stability of atomically precise metal NCs, expanding the mechanism research and application of metal NCs.

Higher TP53BP2 expression is associated with HBsAg loss in peginterferon-α-treated patients with chronic hepatitis B.

BACKGROUND & AIMS: HBsAg loss is only observed in a small proportion of patients with chronic hepatitis B (CHB) who undergo interferon treatment. Investigating the host factors crucial for functional cure of CHB can aid in identifying individuals who would benefit from peginterferon-α (Peg-IFNα) therapy. METHODS: We conducted a genome-wide association study (GWAS) by enrolling 48 patients with CHB who achieved HBsAg loss after Peg-IFNα treatment and 47 patients who didn't. In the validation stage, we included 224 patients, of whom 90 had achieved HBsAg loss, to validate the identified significant single nucleotide polymorphisms. To verify the functional involvement of the candidate genes identified, we performed a series of in vitro and in vivo experiments. RESULTS: GWAS results indicated a significant association between the rs7519753 C allele and serum HBsAg loss in patients with CHB after Peg-IFNα treatment (p = 4.85 × 10-8, odds ratio = 14.47). This association was also observed in two independent validation cohorts. Expression quantitative trait locus analysis revealed higher hepatic TP53BP2 expression in individuals carrying the rs7519753 C allele (p = 2.90 × 10-6). RNA-sequencing of liver biopsies from patients with CHB after Peg-IFNα treatment revealed that hepatic TP53BP2 levels were significantly higher in the HBsAg loss group compared to the HBsAg persistence group (p = 0.035). In vitro and in vivo experiments demonstrated that loss of TP53BP2 decreased interferon-stimulated gene levels and the anti-HBV effect of IFN-α. Mechanistically, TP53BP2 was found to downregulate SOCS2, thereby facilitating JAK/STAT signaling. CONCLUSION: The rs7519753 C allele is associated with elevated hepatic TP53BP2 expression and an increased probability of serum HBsAg loss post-Peg-IFNα treatment in patients with CHB. TP53BP2 enhances the response of the hepatocyte to IFN-α by suppressing SOCS2 expression. IMPACT AND IMPLICATIONS: Chronic hepatitis B (CHB) remains a global public health issue. Although current antiviral therapies are more effective in halting disease progression, only a few patients achieve functional cure for hepatitis B with HBsAg loss, highlighting the urgent need for a cure for CHB. This study revealed that the rs7519753 C allele, which is associated with high expression of hepatic TP53BP2, significantly increases the likelihood of serum HBsAg loss in patients with CHB undergoing Peg-IFNα treatment. This finding not only provides a promising predictor for HBsAg loss but identifies a potential therapeutic target for Peg-IFNα treatment. We believe our results are of great interest to a wide range of stakeholders based on their potential clinical implications.

CRISPR/Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection.

BACKGROUND/PURPOSE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner. METHODS: K. pneumoniae (KP)-, K. pneumoniae carbapenemase (KPC)- and New Delhi metallo-ß-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation. RESULTS: Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/µL with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 101 copies/µL with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1 CFU/cm2 KP could be detected. CONCLUSION: We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.

The potential serum sphingolipid biomarkers for distinguishing Wilson disease.

BACKGROUND: The diagnosis of Wilson's disease (WD) remains a challenging endeavor in clinical practice. Serum sphingolipids play a significant role in the development of liver disease. In this study, we examined the serum sphingolipid profile in patients with WD and explored the potential diagnostic utility of serum sphingolipid metabolites. These metabolites may aid in distinguishing WD patients from healthy controls and identifying those with a risk of cirrhosis. METHODS: This study consecutively enrolled 26 WD patients and 88 healthy controls. We utilized high-performance liquid chromatography-tandem mass spectrometry to analyze a panel of 88 serum sphingolipid metabolites. The data were analyzed by multivariate statistical methods. RESULTS: Among the 88 sphingolipids metabolites analyzed, 17 sphingolipids were observed significant differences between WD and HC groups (all P < 0.05). Notably, five sphingolipids, namely S1P (d18:1), Cer (d18:2/21:0), SM41:2, sph(d18:1), and Cer (d18:2/22:0), each with an AUC exceeding 0.9, emerged as potential biomarkers for WD. Additionally, in the comparison between WD patients with and without cirrhosis, 24 sphingolipid metabolites exhibited significant differences (all P < 0.05). We identified Cer(d18:1/20:0), Cer(d18:2/22:0), Cer(d18:2/24:0), Cer(d18:2/20:0), and Cer(d18:2/18:0), each with an AUC exceeding 0.9, as potential serological markers for WD patients with cirrhosis. CONCLUSION: For enhanced clinical applicability, we propose considering Cer (d18:2/22:0) as a predictive marker applicable to both WD patients and their susceptibility to cirrhosis. This particular ceramide has exhibited strong diagnostic and predictive performance. These findings have the potential to facilitate non-invasive WD diagnosis.

Clinical study of hepatitis B vaccine in achieving hepatitis B surface antibody seroconversion in patients with functional cure.

Hepatitis B Surface Antigen (HBsAg) seroclearance is the highest treatment goal recommended by the current guidelines for hepatitis B. Levels of antibodies to HBsAg (anti-HBs) are strongly associated with HBsAg recurrence, but hepatitis B vaccination may increase the anti-HBs seroconversion rate and reduce recurrence. We conducted a retrospective clinical study to ascertain the effect of this vaccination on the seroconversion rate and levels of protective anti-HBs after HBsAg. In this retrospective study, we distributed a questionnaire through an online survey platform to collect information related to hepatitis B vaccination in patients with functional cure of hepatitis B with Interferon-α (IFNα) therapy. We enrolled 320 patients who achieved functional cure from IFNα therapy. Of these, 219 patients had received hepatitis B vaccination according to their personal preference and drug accessibility after HBsAg seroclearance, whereas the remaining 101 patients did not receive hepatitis B vaccination. The anti-HBs seroconversion rate of 78.1% in the vaccinated group was significantly greater than that in the unvaccinated group (41.6%) (p < 0.001). Stratified comparisons with anti-HBs of ≥ 100 IU/L and ≥ 300 IU/L showed that both proportions in the vaccinated group were greater than those in the unvaccinated group (71.2% vs. 32.7% and 56.2% vs. 17.8%, respectively, all p-values < 0.001). Logistic regression analysis showed that the odds ratio of vaccination was 4.427, which was the strongest influencing factor for anti-HBs, reaching 100 IU/L or higher. Hepatitis B vaccination in patients after HBsAg seroclearance not only increased the anti-HBs seroconversion rate but also significantly increased antibody levels, with good safety, indicating the clinical value of vaccine therapy for patients with functional cure.

CRISPR/Cas13a-Assisted accurate and portable hepatitis D virus RNA detection.

BACKGROUND & AIMS: Hepatitis delta virus (HDV) infection accelerates the progression of chronic hepatitis B virus (HBV) infection, posing a large economic and health burden to patients. At present, there remains a lack of accurate and portable detection methods for HDV RNA. Here, we aim to establish a convenient, rapid, highly sensitive and specific method to detect HDV RNA using CRISPR-Cas13a technology. METHODS: We established fluorescence (F) and lateral flow strip (L) assays based on CRISPR-Cas13a combined with RT-PCR and RT-RAA for HDV RNA detection, respectively. we conducted a cohort study of 144 patients with HDV-IgG positive to evaluate the CRISPR-Cas13a diagnostic performance for identifying HDV in clinical samples, compared to RT-qPCR and RT-ddPCR. RESULTS: For synthetic HDV RNA plasmids, the sensitivity of RT-PCR-CRISPR-based fluorescence assays was 1 copy/µL, higher than that of RT-qPCR (10 copies/µL) and RT-ddPCR (10 copies/µL); for HDV RNA-positive samples, the sensitivity of RT-RAA-CRISPR-based fluorescence and lateral flow strip assays was 10 copies/µL, as low as that of RT-qPCR and RT-ddPCR, and the assay took only approximately 85 min. Additionally, the positivity rates of anti-HDV IgG-positive samples detected by the RT-qPCR, RT-ddPCR, RT-PCR-CRISPR fluorescence and RT-RAA-CRISPR lateral flow strip methods were 66.7% (96/144), 76.4% (110/144), 81.9% (118/144), and 72.2% (104/144), respectively. CONCLUSIONS: We developed a highly sensitive and specific, as well as a portable and easy CRISPR-based assay for the detection of HDV RNA, which could be a prospective measure for monitoring the development of HDV infection and evaluating the therapeutic effect.

Performance of Hepatitis Delta Virus (HDV) RNA Testing for the Diagnosis of Active HDV Infection: Systematic Review and Meta-analysis.

Background and Aims: Hepatitis delta virus (HDV) is a defective virus and causes severe liver disease. Several HDV RNA assays have been developed, however the diagnostic efficacy remains unclear.This systematic review and meta-analysis aims to evaluate the diagnostic accuracy of HDV RNA assays to aid in the diagnosis of active hepatitis D. Methods: The PubMed, Embase, and Cochrane Library databases were systematically searched from the beginning to June 31, 2022. Information on the characteristics of the literature and data on sensitivity, specificity, and area under curve (AUC) of the receiver operating characteristic (ROC) were extracted. Stata 14.0 was used for meta-analysis of the combined sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio. Results: A total of 10 studies were included in the meta-analysis. The summary sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio of HDV RNA assays for HDV diagnosis were 0.92 (95% CI: 0.87-0.95), 0.90 (95% CI: 0.86-0.93), 7.74 (95% CI: 5.31-11.29), 0.10 (95% CI: 0.06-0.18) and 99.90 (95% CI: 47.08-211.99), respectively. The AUC of the pooled ROC curve was 0.95 (95% CI: 0.92-0.96). Conclusions: The results show that HDV RNA assays had high diagnostic performance. However, that is limited by the number and quality of studies. Standard protocols for the development of assays by manufacturers and larger studies on the use of the assays are needed.

ATP7B R778L mutant hepatocytes resist copper toxicity by activating autophagy and inhibiting necroptosis.

Wilson's disease (WD) is an inherited disease characterized by copper metabolism disorder caused by mutations in the adenosine triphosphatase copper transporting ß gene (ATP7B). Currently, WD cell and animal model targeting the most common R778L mutation in Asia is lacking. In addition, the mechanisms by which hepatocytes resist copper toxicity remain to be further elucidated. In this study, we aimed to construct a novel WD cell model with R778L mutation and dissected the molecular basics of copper resistance. A novel HepG2 cell line stably expressing the ATP7B R778L gene (R778L cell) was constructed. The expression of necroptosis- and autophagy-related molecules was detected by PCR and Western blot (WB) in wild-type (WT) HepG2 and R778L cells with or without CuSO4 treatment. In addition, we detected and compared the levels of autophagy and necroptosis in CuSO4-treated R778L cells with the activation and inhibition of autophagy. Moreover, the mRNA and protein levels of autophagy and necroptosis signaling molecules were compared in R778L cells with the overexpression and knockdown of Unc-51 Like Autophagy Activating Kinase 1 (ULK1) and Autophagy Related 16 Like 1 (ATG16L1). We successfully constructed an R778L mutation HepG2 cell line. CuSO4 triggered the enhanced expression of autophagy and necroptosis signaling molecules in WT HepG2 cells and R778L cells. Remarkably, higher levels of autophagy and necroptosis were observed in R778L cells compared with those in WT cells. Autophagy activation led to weakened necroptosis mediated by RIPK3 and MLKL, conversely, autophagy inhibition brought about enhanced necroptosis. At the molecular level, ULK1- and ATG16L1 overexpression resulted in reduced necroptosis levels and vice versa. ULK1- and ATG16L1-mediated autophagy activation protects hepatocytes against RIPK3- and MLKL-mediated necroptosis in our new WD cell model treated with CuSO4. Targeted therapy by autophagy activation or necroptosis inhibition may be a novel and effective strategy to treat WD.

IP10 and Anti-HBc can Predict Virological Relapse and HBsAg Loss in Chronic Hepatitis B Patients after Nucleos(t)ide Analog Discontinuation.

INTRODUCTION: To assess predictive ability of serum interferon-inducible protein 10 (IP10) and hepatitis B core antibody (anti-HBc) levels for virological relapse (VR) and hepatitis B surface antigen (HBsAg) loss after nucleos(t)ide analog (NA) discontinuation. METHODS: In this multicenter prospective study, overall 139 patients were followed up for 24 months after NA discontinuation. RESULTS: End of treatment (EOT) IP10 and anti-HBc were 29.2 (5.1-66.4) pg/mL and 193.6 (136.9-221.4) IU/mL. EOT IP10 and anti-HBc were independent predictors for VR and HBsAg loss in Cox regression analysis. Cumulative rates of VR in patients with EOT IP10 > 26.99 pg/mL was 31.9% (vs. 70.1%, hazard ratio [HR] 2.998, p < 0.001). Cumulative incidences of VR in patients with EOT anti-HBc ≤141.35 IU/mL was 49.1% (vs. 60.6%, HR 2.99, p < 0.001). Cumulative probabilities of VR was 16.7% in patients with EOT IP10 > 26.99 pg/mL plus anti-HBc ≤141.35 IU/mL (vs. 73.6%, HR 6.464, p < 0.001). Cumulative probabilities of HBsAg loss in patients with EOT IP10 > 93.5 pg/mL was 46.2% (vs. 4.7%, HR 10.94, p < 0.001). Cumulative probabilities of HBsAg loss in patients with EOT anti-HBc ≤78.42 IU/mL were 47.1% (vs. 5%, HR 12.27, p < 0.001). Patients with EOT IP10 > 93.5 pg/mL plus anti-HBc ≤78.42 IU/mL had the highest 24-month cumulative HBsAg loss rate (53.8% vs. 4%, HR 16.83, p < 0.001). CONCLUSION: High EOT IP10 and low EOT anti-HBc levels were related to both lower risk of VR and higher probability of HBsAg loss.

Lower end of treatment HBsAg and HBcrAg were associated with HBsAg loss after nucleos(t)ide analog cessation.

BACKGROUND: Since hepatitis B surface antigen (HBsAg) loss is rarely achieved with nucleos(t)ide analog (NA) treatment, most patients require life-long NA treatment. Previous studies have shown that some patients remain virologically responsive even after NA cessation. However, there is still controversy surrounding whether NA discontinuation increases the HBsAg loss rate. Therefore, this study aimed to assess the cumulative rate of HBsAg loss and identify the predictors of HBsAg loss after NA discontinuation. METHODS: This multicenter prospective study included HBV e antigen (HBeAg)-positive patients without cirrhosis from 12 hospitals in China who met the inclusion criteria. The enrolled patients stopped NA and were followed up with clinical and laboratory assessments every 3 months for 24 months after NA cessation or until clinical relapse (CR) occurred. RESULTS: Overall, 158 patients were classified into two groups. Group A included patients with HBsAg positivity at NA cessation (n = 139), and Group B included patients with HBsAg negativity at NA cessation (n = 19). In Group A, the 12-month and 24-month cumulative rates of HBsAg loss were4.3%and 9.4%, respectively. End of treatment (EOT) HBsAg (hazard ratio (HR) = 0.152, P < 0.001) and EOT hepatitis B core-related antigen (HBcrAg) (HR = 0.257, P = 0.001) were associated with HBsAg loss. The areas under the receiver operating characteristic curves for EOT HBsAg and HBcrAg levels were 0.952 (P < 0.001) and 0.765 (P < 0.001), respectively. Patients with EOT HBsAg ≤ 135 IU/mL (59.2% vs. 1.3%, P < 0.001) or HBcrAg ≤ 3.6 logU/mL (17% vs. 5.4%, P = 0.027) had a higher 24-month cumulative HBsAg loss rate. In Group B, none of the patients experienced virological relapse after NA cessation. Only 1 (5.3%) patient had HBsAg reversion. CONCLUSIONS: EOT HBsAg ≤ 135 IU/mL or HBcrAg ≤ 3.6 logU/mL can be used to identify patients with a higher likelihood of HBsAg loss after NA cessation. Patients with HBsAg negativity after NA cessation have favorable clinical outcomes, and HBsAg loss was durable in most cases.

Analysis of clinical characteristics of thyroid disorders in patients with chronic hepatitis B treated with pegylated-interferon alpha.

BACKGROUND: Thyroid disorders (TD) is a common complication of pegylated-interferon alpha (Peg-IFNα) therapy. Few studies have investigated the relationship between TD and the efficacy of interferon therapy for chronic hepatitis B (CHB). Therefore, we analyzed the clinical characteristics of TD in patients with CHB treated with Peg-IFNα, and evaluated the correlation between TD and Peg-IFNα treatment efficacy. METHODS: In this retrospective study, the clinical data of 146 patients with CHB receiving Peg-IFNα therapy were collected and analyzed. RESULTS: During the course of Peg-IFNα therapy, positive conversion of thyroid autoantibodies and TD occurred in 7.3% (85/1158) and 8.8% (105/1187) patients, respectively, and was diagnosed more often in women. The most common thyroid disorder was hyperthyroidism (53.3%), followed by subclinical hypothyroidism (34.3%). We found that thyroid function returned to normal in 78.7% of patients with CHB, and thyroid antibody levels returned to the negative range in approximately 50% of patients after interferon treatment cessation. Only 25% of patients with clinical TD required treatment. Compared with patients with hypothyroidism/subclinical hypothyroidism, patients with hyperthyroidism/subclinical hyperthyroidism showed greater reduction and seroclearance of hepatitis B surface antigen (HBsAg) levels. CONCLUSIONS: TD are not an absolute contraindication for interferon therapy; however, patients should be monitored closely during interferon therapy. In pursuit of functional cure, a balance between efficacy and safety must be achieved.

Serum HBsAg and HBcrAg is associated with inflammation in HBeAg-positive chronic hepatitis B patients.

Backgrounds & aims: Liver inflammation is the main risk factor for developing liver fibrosis, cirrhosis, and even hepatocellular carcinoma in chronic hepatitis B (CHB) patients. To replace biopsy, additional non-invasive biomarkers to diagnose and grade liver necroinflammation are urgently required in clinical practice. Method: Ninety-four CHB patients, including 74 HBeAg-positive and 20 HBeAg-negative patients, were enrolled and started entecavir or adefovir therapy. Serum HBV RNA, HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), ALT and AST levels, as well as intrahepatic HBV DNA and cccDNA were measured at baseline and during treatment. Liver inflammation was assessed at baseline and month 60 by liver biopsy. Inflammation regression was defined as a ≥1-grade decrease according to the Scheuer scoring system. Results: In HBeAg-positive CHB patients, at baseline, serum HBsAg and HBcrAg levels negatively correlated with inflammation grade, while ALT and AST levels positively correlated with inflammation grade. AST plus HBsAg exhibited excellent diagnostic ability for significant inflammation with an AUROC of 0.896. After 60 months of antiviral treatment, almost all the patients' liver inflammation ameliorated to G1, and no patients had inflammation progression. Conclusion: Besides ALT and AST, serum HBsAg and HBcrAg correlated with inflammation grade in HBeAg-positive CHB patients before NAs treatment. Moreover, the combination of HBsAg and AST exhibited excellent diagnostic ability for significant inflammation.

CRISPR/Cas13a-assisted rapid and portable HBV DNA detection for low-level viremia patients.

BACKGROUND & AIMS: The WHO declared to eliminate hepatitis B virus (HBV) by 2030. However, an increasing number of patients are presenting with low-level viremia (LLV) with the widespread use of antiviral medications. The diagnostic efficiency and coverage area of HBV infection are low. Hence, this study intended to drive the HBV infection detection to effectively adaptable for any small to medium-sized laboratory or field survey. METHODS: We established, optimized, and evaluated a colloidal gold test strip for detection of HBV DNA based on CRISPR/Cas13a combined with recombinase-aided amplification (RAA) technology. Furthermore, 180 HBV-infected patients (including patients with different viral loads, LLV patients and dynamic plasma samples of patients on antiviral therapy) were enrolled for clinical validation. RESULTS: The strip detection of HBV DNA was established based on RAA-CRISPR-Cas13a technology with a sensitivity of 101 copies/µL and a specificity of 100%. HBV DNA gradient concentration plasmids and clinical samples were effectively identified by this approach. The positive coincidence rate for LLV patients was 87%, while the negative coincidence rate was 100%. The positive coincidence rate reached 100% in LLV patients (viral loading >100 IU/mL). The sensitivity, specificity, positive predictive agreement (PPA) and negative predictive agreement (NPA) values of dynamic plasma detection in patients on antiviral therapy were 100%, 92.15%, 93.75%, and 100%, respectively. CONCLUSIONS: We develop rapid and portable RAA-CRISPR/Cas13a-based strip of HBV DNA detection for LLV patients. This study provides a visual and faster alternative to current PCR-based diagnosis for HBV infection.