PLANET: A Multi-objective Graph Neural Network Model for Protein-Ligand Binding Affinity Prediction.

Predicting protein-ligand binding affinity is a central issue in drug design. Various deep learning models have been published in recent years, where many of them rely on 3D protein-ligand complex structures as input and tend to focus on the single task of reproducing binding affinity. In this study, we have developed a graph neural network model called PLANET (Protein-Ligand Affinity prediction NETwork). This model takes the graph-represented 3D structure of the binding pocket on the target protein and the 2D chemical structure of the ligand molecule as input. It was trained through a multi-objective process with three related tasks, including deriving the protein-ligand binding affinity, protein-ligand contact map, and ligand distance matrix. Besides the protein-ligand complexes with known binding affinity data retrieved from the PDBbind database, a large number of non-binder decoys were also added to the training data for deriving the final model of PLANET. When tested on the CASF-2016 benchmark, PLANET exhibited a scoring power comparable to the best result yielded by other deep learning models as well as a reasonable ranking power and docking power. In virtual screening trials conducted on the DUD-E benchmark, PLANET's performance was notably better than several deep learning and machine learning models. As on the LIT-PCBA benchmark, PLANET achieved comparable accuracy as the conventional docking program Glide, but it only spent less than 1% of Glide's computation time to finish the same job because PLANET did not need exhaustive conformational sampling. Considering the decent accuracy and efficiency of PLANET in binding affinity prediction, it may become a useful tool for conducting large-scale virtual screening.

Analysis of the Binding Sites on BAX and the Mechanism of BAX Activators through Extensive Molecular Dynamics Simulations.

The BAX protein is a pro-apoptotic member of the Bcl-2 family, which triggers apoptosis by causing permeabilization of the mitochondrial outer membrane. However, the activation mechanism of BAX is far from being understood. Although a few small-molecule BAX activators have been reported in the literature, their crystal structures in complex with BAX have not been resolved. So far, their binding modes were modeled at most by simple molecular docking efforts. Lack of an in-depth understanding of the activation mechanism of BAX hinders the development of more effective BAX activators. In this work, we employed cosolvent molecular dynamics simulation to detect the potential binding sites on the surface of BAX and performed a long-time molecular dynamics simulation (50 µs in total) to derive the possible binding modes of three BAX activators (i.e., BAM7, BTC-8, and BTSA1) reported in the literature. Our results indicate that the trigger, S184, and vMIA sites are the three major binding sites on the full-length BAX structure. Moreover, the canonical hydrophobic groove is clearly detected on the α9-truncated BAX structure, which is consistent with the outcomes of relevant experimental studies. Interestingly, it is observed that solvent probes bind to the trigger bottom pocket more stably than the PPI trigger site. Each activator was subjected to unbiased molecular dynamics simulations started at the three major binding sites in five parallel jobs. Our MD results indicate that all three activators tend to stay at the trigger site with favorable MM-GB/SA binding energies. BAM7 and BTSA1 can enter the trigger bottom pocket and thereby enhance the movement of the α1-α2 loop, which may be a key factor at the early stage of BAX activation. Our molecular modeling results may provide useful guidance for designing smart biological experiments to further explore BAX activation and directing structure-based efforts toward discovering more effective BAX activators.

Nutrition and metabolism status alteration in advanced hepatocellular carcinoma patients treated with anti-PD-1 immunotherapy.

PURPOSE: The aim of this study was to evaluate the nutrition and metabolism status alteration during immunotherapy in advanced hepatocellular carcinoma (HCC) patients. METHODS: Patients with advanced HCC who participated in the clinical trials of single-agent anti-PD-1 immunotherapy or sorafenib were retrospectively included. We analyzed self-comparison of the nutritional and metabolic indices of patients in the anti-PD-1 and sorafenib treatment group. We conducted mutual-comparison of the mentioned indices between the disease progression group and disease control group among anti-PD-1 treatment patients. We further analyzed those indices with statistical differences by partial correlation and survival analysis. RESULTS: Both self-comparison before and after treatment in the anti-PD-1 group and mutual-comparison of disease progression and the control group showed significant differences in multiple indices, but we did not observe significant differences in the sorafenib group. Strikingly, albumin (ALB)/prognostic nutritional index (PNI, calculated by serum albumin and lymphocyte count) decreased distinctly in the immunotherapy disease progression group patients. However, changes in ALB/PNI were not significant in disease progression patients from the sorafenib group or in the disease control patients with immunotherapy. Partial correlation analysis suggested that ALB and PNI were positively correlated with the efficacy of immunotherapy. Furthermore, survival analysis showed that the median progression-free survival and median overall survival of patients in the ALB/PNI decreased group were significantly shorter than those of patients from the ALB/PNI increased group. CONCLUSION: Anti-PD-1 immunotherapy might alter the nutritional and metabolic status in advanced HCC patients. We also should pay attention to the nutritional and metabolic status of patients when drug resistance is detected.

Magnesium isoglycyrrhizinate ameliorates concanavalin A-induced liver injury via the p38 and JNK MAPK pathway.

CONTEXT: Acute liver failure is a serious disease caused by a variety of factors, and immunological injury is an important pathological process. Comprehensive liver treatment efficacy is poor, and the mortality rate is high. Magnesium isoglycyrrhizinate (MgIG) is a new glycyrrhizin drug extracted from the traditional Chinese medicine licorice. The mechanism by which MgIG regulates ConcanavalinA (ConA)-induced immunological liver injury in mice is not completely clear. MATERIALS AND METHODS: Immunological liver injury was induced in mice by ConA injection, and the inflammatory macrophages model was induced by lipopolysaccharide (LPS). MgIG was administered 30 min prior to ConA and LPS treatment. The mice in the different groups were sacrificed 12 h after treatment, and macrophages were measured at 30 min, 1 h, and 2 h after induction. Macrophages, liver, and blood samples were then collected for analysis. RESULTS: After drug administration, the MgIG group showed a marked decrease in serum transaminase levels, reduced apoptosis and hepatic inflammatory responses compared to the ConA group. Furthermore, there was a significant reduction in inflammatory cytokine levels in the serum and liver tissue. In vitro, the expression of inflammatory cytokines was distinctly reduced after MgIG administration. In addition, MgIG pretreatment reduced the expression of inflammatory cytokines and regulated the phosphorylation of p38 and JNK proteins in the MAPK pathway. CONCLUSION: These findings demonstrated that MgIG protects against ConA-induced immunological liver injury by markedly alleviating liver inflammation, and this provides guidance for the clinical amelioration of liver inflammation induced by immunological factors.

Activation of EGFR-KLF4 positive feedback loop results in acquired resistance to sorafenib in hepatocellular carcinoma.

Sorafenib is the standard first-line systemic chemotherapeutic drugs for advanced hepatocellular carcinoma (HCC), but acquired resistance to sorafenib is frequently observed in clinical practice. In this study, we first produced three sorafenib resistance (SR) HCC cell lines by using two human HCC cell lines (Hep3B and Huh7) and a human primary HCC cell line. We identified that epidermal growth factor receptor (EGFR) and Kruppel-like factor 4 (KLF4) are dramatically increased in the three SR HCC cell lines. Either inhibition of tyrosine kinase activity of EGFR with Erlotinib/Icotinib or inhibition of KLF4 expression with short hairpin RNA recovered the response of three SR HCC cell lines to sorafenib, suggesting the critical roles of EGFR tyrosine kinase and KLF4 on inducing SR. Luciferase activity and chromatin immunoprecipitation assays further determined that KLF4 promoted EGFR expression through inducing its transcription by directly binding to its promoter. EGFR, conversely, could also promote KLF4 expression through inducing its transcription by binding to its promoter in a tyrosine kinase-dependent manner, suggesting that a positive feedback loop formed by EGFR and KLF4 further amplifies their effects on inducing SR. Up to now, our findings that KLF4 induces the development of SR and it cooperates with EGFR to form a positive feedback loop to amplify their SR-inducing abilities have rarely been reported. Our findings bear possible implications for the improvement of the efficacy of sorafenib in HCC.

A Selectivity Study of FFAR4/FFAR1 Agonists by Molecular Modeling.

FFAR4 has been considered as a potential target for metabolic diseases, including diabetes. Some compounds with biphenyl scaffold, represented by compound SR13 reported by our group, showed significant FFAR4 selectivity. However, the molecular basis for their selectivity has not been definitely disclosed. This study provided insights into the protein-ligand interactions between agonists and FFAR4/FFAR1 by molecular modeling. The important residues identified were consistent with those found in experimental studies. Moreover, the results proposed that the selectivity of SR13 between FFAR4 and FFAR1 depended on whether it can enter the ligand-binding site through the entrance region by adopting its preferential conformation. The big difference between the preferential conformation of SR13 and the narrow entrance region determined its poor agonist activity against FFAR1. These findings will facilitate the further development of selective FFAR4 agonists.

The different expression of caspase-1 in HBV-related liver disease and acts as a biomarker for acute-on-chronic liver failure.

BACKGROUND: Caspase-1 is an evolutionarily conserved enzyme that proteolytically cleaves the precursors of the inflammatory cytokines interleukin 1ß and interleukin 18. However, the role of caspase-1 in determining the severity of acute-on-chronic liver failure (ACLF) has yet to be elucidated. We evaluated the expression levels of caspase-1 in HBV-related liver disease and assessed its utility as a biomarker predicting the severity of ACLF. METHODS: The gene, protein and activity levels of caspase-1 were measured in the liver and/or serum of subjects with HBV-related disease. We also analysed the correlation between the expression levels of caspase-1 and liver injury of ACLF. RESULTS: Compared with the values observed in normal subjects, the relative caspase-1 mRNA and protein levels in livers were decreased in patients with CHB, LC, and HCC but increased in those with ACLF; moreover, ACLF patients had the lowest serum level and hepatic activity of caspase-1 among the five groups. The serum caspase-1 levels in ACLF patients showed a negative correlation with total serum bilirubin and a positive correlation with serum total protein and albumin. Importantly, the serum caspase-1 levels in the surviving group with ACLF were higher than those in the non-surviving group and showed different dynamic trends. Analyses of the area under the receiver operating characteristic curve indicated that caspase-1 (AUC = 0.84, AUC of MELD score = 0.72) may be a useful marker for independently predicting ACLF. CONCLUSION: Caspase-1 is a potential non-invasive biomarker of disease progression and prognosis in ACLF.

Peroxisome proliferator-activated receptor alpha mediates C/EBP homologous protein to protect mice from acute liver failure.

OBJECTIVE: Peroxisome proliferator-activated receptor α (PPARα) activation has been reported to ameliorate liver injury in cases of acute liver failure (ALF). However, its intrinsic protective molecular mechanisms remain largely undetermined. C/EBP homologous protein (CHOP) is an important mediator of lipopolysaccharide (LPS)-induced inflammation. The aim of the present study was to test the hypothesis that PPARα activation alleviates liver inflammation to protect mice from acute liver failure (ALF) mediated by CHOP. METHODS: In a murine model induced by D-galactosamine (D-GalN, 700 mg/kg) and LPS (10 µg/kg), Wy-14643 (6 mg/kg) was administered to activate PPARα. The mice of different groups were killed 6 h after D-GalN/LPS injection, and the liver and blood were collected for analysis. To find out whether PPARα activation protects the liver from injury due to inflammation by regulating CHOP, we used expression plasmid to increase CHOP expression and demonstrated how PPARα mediated CHOP to regulate inflammation in vivo and in vitro. RESULTS: The expression of PPARα was downregulated and the expression of CHOP was upregulated with the development of D-GalN/LPS-induced liver injury. The protective molecular mechanisms of PPARα activation were dependent on the expression of CHOP. Indeed, (1) PPARα activation decreased the expression of CHOP; on the other hand, PPARα knockdown increased the expression of CHOP in vivo; (2) the depressed liver inflammation by PPARα activation was due to the downregulation of CHOP expression, because overexpression of CHOP by transfect plasmid reversed liver protection and increased liver inflammation again; (3) in vitro, PPARα inhibition by siRNA treatment increased the expression of proinflammatory cytokines, and CHOP siRNA co-transfection reversed the expression of proinflammatory cytokines. CONCLUSIONS: Here, we demonstrated that PPARα activation contributes to liver protection and decreases liver inflammation in ALF, particularly through regulating CHOP. Our findings may provide a rationale for targeting PPARα as a potential therapeutic strategy to ameliorate ALF.

[Application of enhanced recovery program in laparoscopic distal pancreatectomy].

Objective: To evaluate the feasibility and safety of applying enhanced recovery after surgery (ERAS) protocol in patients undergoing laparoscopic distal pancreatectomy. Methods: Data of 36 patients undergoing laparoscopic distal pancreatectomy from May 2016 to May 2017 in the First Affiliated Hospital, Zhejiang University School of Medicine were reviewed. The patients were divided into ERAS group (n=12) and control group (n=24). The patients in ERAS group received a series of enhanced recovery procedures, including multimodal analgesia, early off-bed activity and early oral food-taking, etc. Operation time, intraoperative blood loss, time to first flatus, postoperative complications, and length of postoperative hospital stay were evaluated. Results: There were no statistically significant differences in operation time and intraoperative blood loss between ERAS group and control group (all P>0.05). The time to first flatus and length of postoperative hospital stay were significantly shortened in ERAS group (all P<0.05). The ERAS group had lower incidence of postoperative complications (41.7% vs. 66.7%), and the complications in ERAS group tended to be milder, but the differences failed to show statistical significance (all P>0.05). Conclusion: The ERAS protocol for laparoscopic distal pancreatectomy can significantly promote gastrointestinal function recovery and shorten postoperative hospital stay, and may reduce the incidence of postoperative complications.

[Endoplasmic reticulum stress collaborates with lipopolysaccharide to promote the inflammatory response in macrophages].

OBJECTIVE: To investigate the protective mechanism of endoplasmic reticulum stress (ERS) inhibition against inflammation-induced acute liver injury using a mouse model. METHODS: Marrow-derived stem cells were isolated from mouse femur and used to derive macrophages for analysis in experimental inflammation conditions, established by exposure to LPS and consequent activation of TLR4. Tunicamycin, an ERS chemical inducer, was applied to interfere the inflammation model condition.Affect on the inflammation-related factor MAPK was detected by western blot, and affects on gene expression of inflammatory factors were measured by real-time quantitative PCR. Affect on TNFa was also measured by ELISA. RESULTS: Expression of TNFa, IL-6 and IL-1b was induced upon exposure to LPS, with the peak levels being reached at 4 hours of exposure (TNFa, 0.82+/-0.24; IL-1 b, 2.20+/-0.69; IL-6, 0.330+/-0.150). Tunicamycin significantly enhanced the LPS-induced up-regulation of TNFa, IL-6 and IL-1b expression (TNFa, 1.44+/-0.38, t=2.8, P<0.05; IL-1b, 16.063.40, t =7.93, P<0.05; IL-6, 31.1610.60, t=5.08, P<0.05). The tuniamycin treatment also enhanced the LPS-induced up-regulation of the protein expression of phospo-p38, phospho-JNK and phoshpo-ERK. CONCLUSION: ERS collaborates with LPS to promote the TLR4-mediated inflammatory response of macrophages, and this collaboration may be a pathogenic mechanism underlying progressive development of acute liver injury.

Oxidative stress promotes D-GalN/LPS-induced acute hepatotoxicity by increasing glycogen synthase kinase 3ß activity.

OBJECTIVE: Our previous studies have demonstrated that glycogen synthase kinase 3ß (GSK3ß) activity is increased in the progression of acute liver failure (ALF), which aggravates liver injury, while its regulatory mechanism remains elusive. This study is designated to address whether oxidative stress activates GSK3ß to promote ALF. METHODS: In a murine model induced by D-galactosamine (D-GalN) (700 mg/kg) and LPS (10 µg/kg), N-acetylcysteine (300 mg/kg) or SB216763 (25 mg/kg) was used to inhibit oxidative stress or GSK3ß activity, respectively. Serum alanine aminotransferase and aspartate aminotransferase levels were assessed. The parameters of oxidative stress were evaluated in liver tissue. Whether GSK3ß inhibition protects hepatocytes from oxidative stress-induced cell apoptosis was investigated in vitro. Moreover, the activity of GSK3ß was measured in the liver of chronic hepatitis B (CHB) patients and ALF patients. RESULTS: In vivo, N-acetylcysteine ameliorated the D-GalN/LPS-induced hepatotoxicity and reduced GSK3ß activity; GSK3ß inhibition increased hepatic superoxide dismutase activity and the glutathione content, decreased malondialdehyde production in the liver tissues; while GSK3ß inhibition suppressed the JNK activation in the liver and decreased cytochrome c release from mitochondria. In vitro, GSK3ß inhibition lessened hepatocytes apoptosis induced by H2O2 or Antimycin A, as demonstrated by decreased LDH activity, and reduced cleavage of caspase-3 expression. Furthermore, GSK3ß activity in the CHB patients was increased in the phase of ALF. CONCLUSIONS: Results indicate that GSK3ß activation contributes to liver injury by participating in oxidative stress response in ALF and is, therefore, a potential therapeutic target for ALF.

[Role of endoplasmic reticulum stress in D-GalN/LPS-induced acute liver failure].

OBJECTIVE: To study the role of endoplasmic reticulum stress (ERS) in acute liver failure (ALF) using a mouse model of D-Galactosamine/lipopolysaccharide (D-GalN/LPS)-induced ALF. METHODS: The ALF model was established by administering intraperitoneal (i.p.) injections of D-Ga1N (700 mg/kg) and LPS (10 mug/kg) to six C57BL/6 mice. Three of the modeled mice were also administered 4-phenylbutyrate (4-PBA; 100 mg/kg i.p.) at 6 hours before the onset of ALF and served as the intervention group. Non-modeled mice served as controls. All mice were analyzed by western blotting and qRT-PCR to determine the expression levels of ERS-related proteins in liver tissue. Liver function was assessed by measuring levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. Extent of injury to the liver tissue was assessed by hematoxylin-eosin staining and histological analysis. qRT-PCR was also used to detect differences in expression of inflammation-related genes, and western blotting was also used to detect differences in expression of the apoptosis related protein Caspase-3.The extent of apoptosis in liver tissue was assessed by TUNEL assay. RESULTS: The ERS markers GRP78 and GRP94 showed increased expression at both the gene and protein levels which followed progression of ALF. The ERS effector proteins XBP-1, ATF-6 and IRE 1 a involved in the unfolded protein response were activated in the early stages of ALF, and the ERS-induced apoptosis regulators Caspase-12 and CHOP were activated in the late stage of ALF. Inhibition of ERS by 4-PBA intervention protected against injury to liver tissue and function, as evidenced by significantly lower levels of serum ALT and AST and a remarkably decreased extent of histological alterations. Furthermore, the inhibition of ERS suppressed expression of the proinflammatory cytokines TNFa, IL-6 and IL-1 ß, and reduced the extent of hepatocyte apoptosis. CONCLUSION: ERS is activated in the mouse model of D-GalN/LPS-induced ALF. Inhibition of ERS may be protective against liver injury and the mechanism of action may involve reductions in inflammatory and apoptotic factors and/or signaling. Therefore, inhibiting ERS may represent a novel therapeutic approach for treating ALF.

Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism.

Background: Acute liver injury (ALI), which is a type of inflammation-mediated hepatocellular injury, is a clinical syndrome that results from hepatocellular apoptosis and hemorrhagic necrosis. Apoptosis stimulating protein of p53-2 (ASPP2) is a proapoptotic member of the p53 binding protein family. However, the role of ASPP2 in the pathogenesis of ALI and its regulatory mechanisms remain unclear. Methods: The expression of ASPP2 were compared between liver biopsies derived from patients with CHB, patients with ALI, and normal controls. Acute liver injury was modelled in mice by administration of D-GalN/LPS. Liver injury was demonstrated by serum transaminases and histological assessment of liver sections. ASPP2-knockdown mice (ASPP2+/-) were used to determine its role in acute liver injury. Mouse bone marrow macrophages (BMMs) were isolated from wildtype and ASPP2+/- mice and stimulated with LPS, and the supernatant was collected to incubate with the primary hepatocytes. Quantitative real-time PCR and western blot were used to analyze the expression level of target. Results: The expression of ASPP2 was significantly upregulated in the liver tissue of ALI patients and acute liver injury mice. ASPP2+/- mice significantly relieved liver injury through reducing liver inflammation and decreasing hepatocyte apoptosis. Moreover, the conditioned medium (CM) of ASPP2+/- bone marrow-derived macrophages (BMMs) protected hepatocytes against apoptosis. Mechanistically, we revealed that ASPP2 deficiency in BMMs specifically upregulated IL-6 through autophagy activation, which decreased the level of TNF-α to reduce hepatocytes apoptosis. Furthermore, up-regulation of ASPP2 sensitizes hepatocytes to TNF-α-induced apoptosis. Conclusion: Our novel findings show the critical role of ASPP2 in inflammatory immunoregulatory mechanism of ALI and provide a rationale to target ASPP2 as a refined therapeutic strategy to ameliorate acute liver injury.

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.

Does financial globalization promote renewable energy investment? Empirical insights from China.

The increasing integration of financial markets worldwide has brought about significant changes in the investment landscape for renewable energy. However, the connection between financial globalization and renewable energy investment has gotten relatively little consideration. As a result, the analysis's main goal is to determine the asymmetric nexus between financial globalization and renewable energy investment in China, covering the period from 1995 to 2021. The influence of financial globalization on investments in renewable energy has been calculated using the linear and non-linear ARDL frameworks. Both methods analyze the short-run and long-run relationships between financial globalization and renewable energy investment. The linear model highlights the favorable influence of financial globalization on renewable energy investment in the short and long run. On the other side, the non-linear model implies that a rise in financial globalization increases investment in renewable energy in the short and long run, and the fall in financial globalization cause the renewable energy investment to fall only in the long run. In addition, national income help promote renewable energy investment in both the short and long run in linear and non-linear models. Therefore, encouraging international cooperation to develop renewable energy projects through public-private partnerships can increase investment flows and provide greater access to financing.

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.

Supramolecular self-assembled AIE molecules are used in the search for target proteins in norcantharidin.

Norcantharidin (NCTD), a demethylated derivative of cantharidin, is an anticancer active component in traditional Chinese medicine. At present, the main methods for finding its target proteins are pharmacological methods and biophysical screening, which cannot achieve the purpose of efficient and accurate screening. Here we established a new analytical method for specific fishing and assisted imaging for norcantharidin target proteins. For the AIE supramolecule probe, the benzophenone azide (BPA) fluorescent nanoparticles with strong AIE properties were encapsulated in biocompatible DSPE-PEG that covalently coupled with NCTD (named BPA@NCTD NPs). The target proteins of NCTD can be captured by BPA@NCTD NPs, and then be detected to investigate the potential signaling pathways. The screened differential proteins were analysed through the protein and signaling pathway database, and multiple signaling pathways were obtained and verified. The mechanism of norcantharidin in inhibiting the migration and invasion of A549 cells through the P53 signaling pathway was confirmed by Western blot experiments. Our research showed that AIE supramolecule probe BPA@NCTD NPs has the dual functions of specific screening of A549 cells target proteins and biological imaging, which not only offers a good anti-fluorescence quenching ability for the dynamic imaging process of NCTD, but also provides a novel and efficient specific method for efficient analysis of target proteins and signal pathways.

Discovery of Small-Molecule Autophagy Inhibitors by Disrupting the Protein-Protein Interactions Involving Autophagy-Related 5.

One possible strategy for modulating autophagy is to disrupt the critical protein-protein interactions (PPIs) formed during this process. Our attention is on the autophagy-related 12 (ATG12)-autophagy-related 5 (ATG5)-autophagy-related 16-like 1 (ATG16L1) heterotrimer complex, which is responsible for ATG8 translocation from ATG3 to phosphatidylethanolamine. In this work, we discovered a compound with an (E)-3-(2-furanylmethylene)-2-pyrrolidinone core moiety (T1742) that blocked the ATG5-ATG16L1 and ATG5-TECAIR interactions in the in vitro binding assay (IC50 = 1-2 µM) and also exhibited autophagy inhibition in cellular assays. The possible binding mode of T1742 to ATG5 was predicted through molecular modeling, and a batch of derivatives sharing essentially the same core moiety were synthesized and tested. The outcomes of the in vitro binding assay and the flow cytometry assay of those newly synthesized compounds were generally consistent. This work has validated our central hypothesis that small-molecule inhibitors of the PPIs involving ATG5 can tune down autophagy effectively, and their pharmaceutical potential may be further explored.

Diagnostic Efficacy of Serological Antibody Detection Tests for Hepatitis Delta Virus: A Systematic Review and Meta-Analysis.

Background and Aims Coinfection of hepatitis delta virus (HDV) with hepatitis B virus (HBV) causes the most severe form of viral hepatitis, and the global prevalence of HDV infection is underestimated. Although serological testing of anti-HDV antibodies is widely used in the diagnosis of HDV, its diagnostic efficacy remains unclear. This study aimed to evaluate the diagnostic efficacy of HDV serological tests, the results of which may assist in the diagnosis of HDV. Methods Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines were followed. The PubMed, Web of Science and Cochrane Library databases were searched from the beginning to 31 May 2023. Study quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. STATA SE was used for the meta-analysis of the sensitivity, specificity, positive likelihood ratio and negative likelihood ratio. Results Among a total of 1376 initially identified studies, only 12 articles met the final inclusion criteria. The pooled sensitivity and specificity were 1.00 (95% CI: 0.00-1.00) and 0.71 (95% CI: 0.50-0.78) for HDV total antibodies, 0.96 (95% CI: 0.83-0.99) and 0.98 (95% CI: 0.82-1.00) for anti-HDV IgM and 0.95 (95% CI: 0.86-0.98) and 0.96 (95% CI: 0.67-1.00) for anti-HDV IgG. The pooled sensitivity and specificity for HDV serological tests were 0.99 (95% CI: 0.96-1.00) and 0.90 (95% CI: 0.79-0.96). Conclusions This meta-analysis suggests that serological tests have high diagnostic performance in detecting antibodies against HDV, especially in HDV IgM and IgG. However, this conclusion is based on studies of a limited number and quality, and the development of new diagnostic tools with higher precision and reliability is still necessary.

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.