SARS-CoV-2 and its emerging variants continue to pose a significant global public health threat. The SARS-CoV-2 main protease (M^pro) is a critical target for the development of antiviral agents that can inhibit viral replication and transcription. In this study, we identified chebulagic acid (CHLA), isolated from Terminalia chebula Retz., as a potent non-peptidomimetic and non-covalent M^pro inhibitor. CHLA exhibited intermolecular interactions and provided significant protection to Vero E6 cells against a range of SARS-CoV-2 variants, including the wild-type, Delta, Omicron BA.1.1, BA.2.3, BA.4, and BA.5, with EC₅₀ values below 2 μmol/L. Moreover, in vivo studies confirmed the antiviral efficacy of CHLA in K18-hACE2 mice. Notably, CHLA bound to a unique groove at the interface between M^pro domains I and II, which was revealed by the high-resolution crystal structure (1.4 Å) of the M^pro-CHLA complex, shrinking the substrate binding pocket of M^pro and inducing M^pro aggregation. CHLA was proposed to act as an allosteric inhibitor. Pharmacokinetic profiling and safety assessments underscore CHLA's potential as a promising broad-spectrum antiviral candidate. These findings report a novel binding site on M^pro and identify antiviral activity of CHLA, providing a robust framework for lead compounds discovery and elucidating the underlying molecular mechanisms of inhibition.

Objective To investigate the killing effect of the recombinant oncolytic influenza virus OvFlu-GM-CSF,constructed using reverse genetics(RG)technology,in combination with chemotherapy drug,gemcitabine(GEM),against pancreatic cancer cells.Methods The recombinant oncolytic virus OvFlu-GM-CSF was successfully rescued using RG technology in our previous study.The virus was then comprehensively characterized through chicken red blood cell hemagglutination assay,transmission electron microscopy,and viral replication assay.CCK-8 assay was utilized to determine the impact of OvFlu-GM-CSF viruses[multiplicities of infection(MOI):0,0.1,1.0,3.0]on the survival rate of pancreatic cancer cell lines(Panc02,PANC-1,SW1990,BxPC-3)and normal pancreatic ductal epithelial cells(HPDE6-C7)after treatment for 24,48 or 72 h.Using a subcutaneous tumor-bearing mouse model of pancreatic cancer,36 female C57BL/6 mice(6 weeks old)were randomly divided into PBS group,recombinant oncolytic virus group,GEM group,and the combined treatment group,with 9 mice in each group.PBS(100 μL/animal)or OvFlu-GM-CSF virus(1× 107PFU/100 μL)was given to the mice of the corresponding groups through intratumoral injection,while GEM(100 mg/kg)was injected intraperitoneally,once per 3 days,for totally 9 times.Changes in tumor volume and survival rate were monitored.Multi-immunofluorescence staining was employed to analyze T cell infiltration and proliferation in the tumor tissues.HE staining was performed to observe the pathological changes in major organs(heart,liver,lungs,kidneys and brain),and the serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured to evaluate the safety of the recombinant oncolytic virus.Results The recombinant oncolytic influenza virus OvFlu-GM-CSF has a hemagglutination titer of 28,typical morphological features of influenza virus,and can selectively replicate within pancreatic cancer cells.At the cellular level,the viruses demonstrated a significant selective cytotoxic effect on Panc02,PANC-1,SW1990,and BxPC-3 cells under the conditions of 48 h post-infection and MOI=3.0,when compared to 48 h post-infection and MOI=0(P＜0.01).The cell survival rate was gradually decreased with the increase in MOI value and the extension of infection time(P＜0.01),but the viruses showed no significant effect on normal pancreatic ductal epithelial cells(HPDE6-C7).In the pancreatic cancer tumor-bearing mouse model,the combined treatment of the viruses+GEM significantly reduced the tumor volume than simple virus treatment and simple GEM treatment(P＜0.01),and enhanced the infiltration of T cells in the tumor tissues.No obvious pathological changes were observed in the above-mentioned major organs.Additionally,there were no significant differences in the serum levels of ALT and AST in the OvFlu-GM-CSF group,GEM group,and OvFlu-GM-CSF+GEM group compared to the PBS group.Conclusion RS technology-constructed recombinant oncolytic influenza virus OvFlu-GM-CSF,when combined with the chemotherapeutic agent GEM,enhances the cytotoxic efficacy against pancreatic cancer cells and effectively activates the host's anti-tumor immune response.

Objective:To examine the clinical outcomes of patients undergoing total thoracoscopic aortic-mitral double-valve replacement.Methods:This is a retrospective case series study. The clinical data of 50 patients who underwent double-valve replacement under a total thoracoscopic two-port approach from November 2021 to August 2022 in the Department of Cardiovascular Surgery, Fujian Medical University Union Hospital were retrospectively analyzed. There were 32 males and 18 females, with an age of (55.3±8.8) years (range: 21 to 62 years). Among them, 36 cases had rheumatic heart disease and 14 cases had infective endocarditis. The 3 rd intercostal space between the right anterior axillary line and the midclavicular line was selected as the main operating hole, the total thoracoscopic double-valve replacement were successfully carried out. Baseline data, intraoperative information, surgical outcomes, and postoperative complications were collected for all patients. Results:The cardiopulmonary bypass time was (168.2±30.9) minutes (range: 125 to 187 minutes), the aortic cross-clamping time was (118.8±16.5) minutes (range: 96 to 147 minutes). Five patients received bioprosthetic valves, and 45 received mechanical prosthetic valves. Postoperative mechanical ventilation lasted (9.6±3.4) hours (range: 5.1 to 14.2 hours), the ICU stay was (24.8±7.3) hours (range: 16.3 to 30.1 hours), and the postoperative hospital stay was (6.5±1.2) days (range: 5.0 to 8.0 days). Four patients received red blood cell transfusions of (2.7±0.9) units (range: 2 to 4 units), and the postoperative chest drainage volume was (222.1±56.3) ml (range: 175 to 289 ml). No deaths occurred intraoperatively or in the early postoperative period. One patient required reoperation due to bleeding in the aortic incision. Three patients had mild to moderate paravalvular leakage around the prosthetic aortic valve, with no cases of third-degree atrioventricular block or conversions to median sternotomy.Conclusions:The early outcomes of total thoracoscopic double valve replacement surgery are satisfactory, demonstrating safety and efficacy. This surgical approach expands the scope of total thoracoscopic cardiac surgery, which warrants further investigation and research.

OBJECTIVE To set up normal ranges for indexes in embryo-fetal development toxicity studies in Sprague-Dawley(SD)rats and to establish a background database to provide reference for the embryo-fetal development toxicity evaluation of drugs.METHODS The data on embryonic develop-ment and fetal growth from embryo-fetal development toxicity studies(11 items)conducted by our center between 2013 and 2022 was statistically analyzed,involving 205 pregnant rats and 3037 fetuses in total,with the mean and standard deviation,coefficient of variation and 95%confidence interval calculated.The indexes included body mass,body mass gain and food consumption during pregnancy,pregnancy outcomes(pregnancy rate,average corpora lutea,average Implant sites,average live conceptuses,live conceptuse rate,resorption rate and dead conceptuse rate),fetal growth and development(fetal mass,placental mass and sex ratio),appearance abnormality rate,visceral abnormality rate,and skeletal abnormality rate.RESULTS The mass of pregnant rats trended up during gestation,with significant increases in the late period.Food consumption increased along with gestation.Caesarean section was conducted on gestation day 20,and the pregnancy rate was 93.2%.The average corpora lutea,Implant sites and live conceptuses were 18.0±3.2,15.9±2.8 and 14.8±3.0,respectively.The live conceptuse rate was 93.4%while the total dead embryo rate was 6.6%.The average mass of fetuses and placenta were respectively 3.6±0.3 and(0.6±0.3)g,and the fetal sex ratio(male/female)was 0.94.The incidence of fetal appearance abnormalities was about 0.2%,and that of soft tissue abnormalities was approximately 0.8%.The rate of skeletal abnormalities was about 1.2%,with higher incidence of non-ossification and incomplete ossification mostly identified on sternum and hyoid bone.The numbers of ossifications of metacarpal bones,metatarsal bones and sacrococcygeal vertebrae were 7.0±0.7,8.0±0.1 and 7.4±0.5,respectively.The rate of ossification of sternumⅠtoⅣwas higher,with an average of about 98.6%-99.9%.The ossification rates of sternum Ⅴ and Ⅵ were(68.0±28.4)%and(82.8±23.9)%.CONCLUSION The background database of indexes in the embryo-fetal development toxicity study on SD rats is established for our GLP laboratory,which provides reference for reproductive toxicity studies.

Objective:To examine the clinical outcomes of patients undergoing total thoracoscopic aortic-mitral double-valve replacement.Methods:This is a retrospective case series study. The clinical data of 50 patients who underwent double-valve replacement under a total thoracoscopic two-port approach from November 2021 to August 2022 in the Department of Cardiovascular Surgery, Fujian Medical University Union Hospital were retrospectively analyzed. There were 32 males and 18 females, with an age of (55.3±8.8) years (range: 21 to 62 years). Among them, 36 cases had rheumatic heart disease and 14 cases had infective endocarditis. The 3 rd intercostal space between the right anterior axillary line and the midclavicular line was selected as the main operating hole, the total thoracoscopic double-valve replacement were successfully carried out. Baseline data, intraoperative information, surgical outcomes, and postoperative complications were collected for all patients. Results:The cardiopulmonary bypass time was (168.2±30.9) minutes (range: 125 to 187 minutes), the aortic cross-clamping time was (118.8±16.5) minutes (range: 96 to 147 minutes). Five patients received bioprosthetic valves, and 45 received mechanical prosthetic valves. Postoperative mechanical ventilation lasted (9.6±3.4) hours (range: 5.1 to 14.2 hours), the ICU stay was (24.8±7.3) hours (range: 16.3 to 30.1 hours), and the postoperative hospital stay was (6.5±1.2) days (range: 5.0 to 8.0 days). Four patients received red blood cell transfusions of (2.7±0.9) units (range: 2 to 4 units), and the postoperative chest drainage volume was (222.1±56.3) ml (range: 175 to 289 ml). No deaths occurred intraoperatively or in the early postoperative period. One patient required reoperation due to bleeding in the aortic incision. Three patients had mild to moderate paravalvular leakage around the prosthetic aortic valve, with no cases of third-degree atrioventricular block or conversions to median sternotomy.Conclusions:The early outcomes of total thoracoscopic double valve replacement surgery are satisfactory, demonstrating safety and efficacy. This surgical approach expands the scope of total thoracoscopic cardiac surgery, which warrants further investigation and research.

The adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the canonical type IV exporter fold. Herein, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, or ATP analogue (AMP-PNP) at resolutions ranging from 2.8 to 3.5 Å. The structure of IrtAB bound ATP-Mg2+ shows a "head-to-tail" dimer of nucleotide-binding domains (NBDs), a closed amphipathic cavity within the transmembrane domains (TMDs), and a metal ion liganded to three histidine residues of IrtA in the cavity. Cryo-electron microscopy (Cryo-EM) structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB. Moreover, the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transport cycle. This study provides a structural basis to explain the ATP-driven conformational changes that occur in IrtAB.
Siderophores/metabolism* ; Iron/metabolism* ; Mycobacterium tuberculosis/metabolism* ; Cryoelectron Microscopy ; Adenosine Triphosphate/metabolism* ; ATP-Binding Cassette Transporters

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M^pro) and papain like protease (PL^pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851M^pro inhibitors and 205 PL^pro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M^pro and PL^pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M^pro inhibitors and over 20% of PL^pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M^pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
Humans ; Antiviral Agents/chemistry* ; COVID-19 ; COVID-19 Drug Treatment ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Protease Inhibitors/chemistry* ; SARS-CoV-2/enzymology* ; Viral Nonstructural Proteins

Heme/metabolism* ; Cryoelectron Microscopy ; Membrane Transport Proteins ; Escherichia coli Proteins/metabolism*

Antiviral Agents/pharmacology* ; COVID-19 ; Coronavirus 3C Proteases ; Humans ; Lactams ; Leucine ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Nitriles ; Proline ; Protease Inhibitors/pharmacology* ; SARS-CoV-2

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M
Antiviral Agents/therapeutic use* ; Binding Sites ; COVID-19/virology* ; Coronavirus Papain-Like Proteases/metabolism* ; Crystallography, X-Ray ; Drug Evaluation, Preclinical ; Drug Repositioning ; High-Throughput Screening Assays/methods* ; Humans ; Imidazoles/therapeutic use* ; Inhibitory Concentration 50 ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Naphthoquinones/therapeutic use* ; Protease Inhibitors/therapeutic use* ; Protein Structure, Tertiary ; Recombinant Proteins/isolation & purification* ; SARS-CoV-2/isolation & purification*