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.

The STAR tool was used to evaluate and analyze the science, transparency, and applicability of Chinese pathology guidelines and consensus published in medical journals in 2022. There were a total of 18 pathology guidelines and consensuses published in 2022, including 1 guideline and 17 consensuses. The results showed that the guideline score was 21.83 points, lower than the overall guideline average (43.4 points). Consensus ratings scored an average of 27.87 points, on par with the overall consensus level (28.3 points). Areas that scored above the overall level were "conflict of interest" and "working groups", while areas that scored below the overall level were "proposals", "funding", "evidence", "consensus approaches" and "accessibility". To sum up, the formulation of pathology guidelines and consensuses in 2022 is not standardized, and the evidence retrieval process, evidence evaluation methods and grading criteria for recommendations on clinical issues are not provided in the formulation process; the process and method for reaching consensus are not provided, the plan is lacking, and registration is not carried out. It is therefore suggested that guidelines/consensus makers in the field of pathology should attach importance to evidence-based medical evidence, strictly follow guideline formulation methods and processes, further improve the scientific, applicable and transparent guidelines/consensuses in the field, and better provide support for clinicians and patients.

Objective To explore the action mechanism of tauroursodeoxycholic acid(TUDCA)promoting intracellular clearance of Burkholderia pseudomallei(B.pseudomallei)in RAW264.7 macrophages.Methods After TUDCA of different concentrations were used to treat RAW264.7 cells pre-infected with B.pseudomallei for 8 h or not,flow cytometry was applied to detect the apoptosis of the infected and control cells.In addition,another endoplasmic reticulum stress(ERS)inhibitor 4-PBA was used to detect the apoptosis and proliferation of host cells after B.pseudomallei infection with Annexin-V/PI double staining and MTT cell proliferation assay.Furthermore,after transfected with CHOP siRNA,Western blotting and flow cytometry were employed to detect the effect of TUDCA on the expression levels of Caspase-3 and Caspase-12 and the changes in apoptotic rate after B.pseudomallei infection,respectively.Finally,the effect of TUDCA on intracellular multiplication of infected RAW264.7 cells were observed to estimate the CFU value in the presence and absence of CHOP siRNA.Results Under different concentrations of TUDCA,100 or 200 μmol/L TUDCA significantly reduced B.pseudomallei-induced apoptosis in RAW264.7 cells(P＜0.05).Meanwhile,both TUDCA and 4-PBA treatment could decrease the apoptosis induced by B.pseudomallei infection by ERS(P＜0.05).Further,the expression levels of Caspase-3 and Caspase-12 were obviously increased after B.pseudomallei infection compared with uninfected groups,but their expression levels in the siCHOP group was significantly lower than that in the siC group.Besides,flow cytometry also showed that TUDCA could reduce apoptosis induced by B.pseudomallei infection(P＜0.05),but no significant effect of TUDCA on apoptosis was observed under CHOP knockdown.Finally,intracellular CFU assay indicated that TUDCA treatment promoted the host cell clearance of B.pseudomallei(P＜0.05),but no such effect was observed in siCHOP group.Conclusion In B.pseudomallei infected RAW264.7 cells,TUDCA promotes the intracellular clearance of the bacteria by inhibiting ERS-induced apoptosis.

Objective:To investigate the efficacy and safety of ixazomib combined with thalidomide and dexamethasone in the treatment of multiple myeloma(MM).Methods:The clinical data of 60 MM patients admitted to our center from January 2019 to June 2022 were analyzed retrospectively,including 43 newly diagnosed patients and 17 patients with recurrence and progression.All patients were treated with ixazomib combined with thalidomide and dexamethasone,and completed 2 to 7 treatment cycles.Results:The overall response rate(ORR)of all patients was 98.3％.Among them,53 patients completed 4 treatment cycles,and the ORR was 86.8％.Seventeen patients completed the whole treatment cycle,with curative effect reaching 88.2％achieving very good partial response and above,and 52.9％achieving complete response and above.Albumin and β2-microglobulin of all patients had been improved rapidly after treatment.The deadline was August 31,2022.The median follow-up time was 14(3-24)months,and overall survival(OS)rate was 86.67％.The OS rate of patients with recurrence and progression was significantly lower than that of newly diagnosed patients(P＜0.05).The most common adverse reaction of hematology was lymphopenia(53.3％),followed by anemia(33.3％).The most common non-hematological adverse reaction was fatigue(68.33％),followed by peripheral neuropathy(31.67％).Conclusion:Ixazomib combined with thalidomide and dexamethasone is effective in the treatment of MM,with good short-term efficacy,survival and safety.However,its long-term efficacy needs further observation.

Objective:This study aims to explore the synergistic effects of DIP and other medical insurance supply-side policies.Method:City A that has piloted DIP reform was set as the treatment group,and City B without reform was set as the control group.A total of 1 120 public medical institution samples from 2019 to 2022 were collected.The total medical expenses during hospitalization and some structural expenses were analyzed using DID method.Result:DIP had a significant inhibitory effect on the medical expenses,and the expenses of checkups and examinations during hospitalization in city A,but had no impact on the drug and the material expenses during hospitalization.Conclusion:DIP played a significant cost control role and effectively controlled the total medical expenses during hospitalization.The synergistic effects of price adjustment of medical services policy and national centralized drug/material procurement policy on cost control were insufficient.DIP synergized with other supply-side policies to promote rational medical cost structure.It is suggested that medical insurance departments should focus on the synergistic effects of medical insurance supply-side policies to jointly improve the efficiency of medical insurance fund utilization.

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

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

Animals ; SARS-CoV-2 ; Antibodies, Neutralizing ; Macaca mulatta ; COVID-19/prevention & control* ; Antibodies, Viral ; Vaccines

ObjectiveTo investigate the effect of porcine large intestine-processed Dahuang （Radix et Rhizoma Rhei） on defecation in constipation model mice and the possible mechanism. MethodsFifty Kunming mice were randomized to blank group （n=10） and model group （n=40）. Loperamide suspension at the dose of 8 mg/（kg·d） was given by gavage for four consecutive days to establish a model of constipation. The 24 successfully modeled mice were randomly divided into model group， processed Dahuang group， lactulose group， raw Dahuang group， with six mice in each group. Moreover， six randomly selected mice were chosen as control group. Since the fifth day， 8 mg/（kg·d） of loperamide suspension by gavage was given to the model group， processed Dahuang group， raw Dahuang group， and lactulose group； two hours later， the processed and raw Dahuang groups were administered with 0.6 g/（kg·d） of processed and raw Dahuang suspension， respectively， while the lactulose group was given 0.6 g/（kg·d） of latulose suspension， and the blank group and the model group were given 0.2 ml/10 g of distilled water by gavage， all for four days. The general condition， body weight after the last gavage， number of fecal particles within six hours， fecal wet weight， fecal water content ratio， intestinal propulsion rate and colonic histology changes by HE staining of each group were detected. ResultsThe body weight of the mice in the raw Dahuang group was significantly lighter than that in the other groups （P＜0.05 or P＜0.01）. The number of fecal particles， fecal wet weight and intestinal propulsion rate of mice significantly decreased in the model group than in the blank group （P＜0.05 or P＜0.01）. Compared to those in the model group， the number of fecal particles and fecal wet weight in the processed Dahuang group， lactulose group and raw Dahuang group significantly increased， and the fecal water content ratio in the raw Dahuang group increased as well （P＜0.05 or P＜0.01）. Compared to those in the processed Dahuang group， the number of fecal particles and fecal wet weight in the raw Dahuang group decreased， while the fecal water content ratio increased （P＜0.05 or P＜0.01）， and the fecal water content ratio in the lactulose group increased significantly （P＜0.05）. The intestinal propulsion rate in the processed Dahuang group was higher than that in the model group， lactulose group and raw Dahuang group （P＜0.05 or P＜0.01）. Histopathological analysis showed that the colonic crypts and goblet cells in the blank group were normal and clear， and the colonic muscular layer was thicker. The colonic crypts of the mice in the model group were damaged， with reduced goblet cells to varying degrees and changed colonic muscularis. In the lactulose group and raw Dahuang group， part of the crypts were broken， and the goblet cells were damaged to varying degrees， while in the processed Dahuang group， still the colonic tissue structure of the mice was relatively clear， and the colonic crypts and goblet cells were relatively normal， with thickened muscular layer of the colon. ConclusionPorcine large intestine-processed Dahuang could improve defecation in constipation model mice， and reduce the drastic purgation function of raw Dahuang， for which the mechanism may be related to the protection of colon histopathological damage.

Objective To study the effect of mitochondrial reactive oxygen species(ROS)on male fertility and its application value in assisted reproduction so as to provide a new method for fertility assessment and etiological treatment of male infertility.Methods The relationship between sperm ROS and male infertility,sperm nuclear DNA integrity and sperm membrane function was analyzed.Then the relationships of the ROS of sperms for fertiliza-tion with fertilization rate,cleavage rate,embryo rate and pregnancy outcome were analyzed.Results(1)The percentage of the high ROS in sperm membrane of the male infertility group was significantly higher than that of the normal fertility group(P<0.01),but there was no statistical difference in the percentage of the high ROS in the sperm mitochondria between the two groups.(2)The normal rate of sperm membrane function in the normal group was significantly higher than that in the abnormal group(P<0.01),but there was no significant difference in the sperm DNA fragmentation index(DFI)between the groups.(3)There was significant difference in the fertilization rate between the IVF control group and IVF observation group,but no significant difference was seen in the cleavage rate,excellent embryo rate and clinical pregnancy rate between the groups.Conclusion Sperm ROS is related to male fertility to some extent.Abnormal ROS may lead to impaired sperm membrane function and affect sperm fertilization ability,thus affecting male fertility.The detection of ROS in sperm can be used as a new method to evaluate male fertility and provide a basis for diagnosis or treatment of male infertility.For patients with ART or infertility,the appropriate time to conceive can be selected according to the sperm ROS level.