In-fusion cloning technology,as a revolutionary and efficient molecular biology tool,has been applied in multiple research fields such as basic biology,biotechnology,and biomedicine.In this article,we introduce a teaching reform project suitable for undergraduate students in the course of"Molecular Bi-ology Laboratory",which utilizes in-Fusion cloning technology to construct a prokaryotic expression vector for alkaline phosphatase mutant genes.Through specific teaching cases,we systematically explored the design and implementation of experimental projects,and focused on analyzing the key and difficult points of the teaching content.Our teaching practice has found that the implementation of this educational re-form project has achieved very good results in enhancing students' core biological literacy,bioinformatics skills,research thinking,and innovation abilities.At the same time,the application of this technology can significantly improve the quality of experimental teaching,providing new ideas and practical refer-ences for promoting the reform and innovation of National First-Class Courses.

Aim To explore the role and mechanism of epimedokoreanin B(EKB)in HepaRG cell pyroptosis through endoplasmic reticulum stress and NLRP1-me-diated pyroptosis pathway.Methods The effect of EKB on the viability of HepaRG cells at different con-centrations was determined by MTT assay,and the cell growth status was recorded by Incucyte.Four groups of HepaRG cells were set up.The control group was cul-tured with complete medium for 24 h;the drug admin-istration group was cultured with three concentration gradients of 6.25,12.5 and 25 μmol·L-1 of EKB for 24 h.Western blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins and pyroptosis-related proteins in the cells of each group.Results HepaRG cells showed cytotoxicity at a concentration of 6.25 μmol·L-1 for 24 h,and the half maximal inhibitory concentration(IC50)was 12.41 μmol·L-1.Incucyte recordings of the cell growth status showed that the cells in the control group were in good growth status,and the vesicular pyropto-sis cells appeared in the different concentrations of EKB and the cells swelled and ruptured after 24 h.Western blot showed that the protein expression levels of endoplasmic reticulum stress-related proteins pERK,eIF-2α,ATF-4,GRP78,and CHOP significantly in-creased in HepaRG cells at 25 μmol·L-1 of EKB compared with the control group.The proteins of the classical pathway of cellular pyroptosis mediated by NLRP1,caspase-1,cleaved caspase-1,GSDMD,GS-DMD-N significantly increased in HepaRG cells.Con-clusion EKB administration induces HepaRG cell py-roptosis,and EKB activates HepaRG cells to undergo endoplasmic reticulum stress and activates the NLRP1/caspase-1/GSDMD-mediated pyroptosis pathway.

In-fusion cloning technology,as a revolutionary and efficient molecular biology tool,has been applied in multiple research fields such as basic biology,biotechnology,and biomedicine.In this article,we introduce a teaching reform project suitable for undergraduate students in the course of"Molecular Bi-ology Laboratory",which utilizes in-Fusion cloning technology to construct a prokaryotic expression vector for alkaline phosphatase mutant genes.Through specific teaching cases,we systematically explored the design and implementation of experimental projects,and focused on analyzing the key and difficult points of the teaching content.Our teaching practice has found that the implementation of this educational re-form project has achieved very good results in enhancing students' core biological literacy,bioinformatics skills,research thinking,and innovation abilities.At the same time,the application of this technology can significantly improve the quality of experimental teaching,providing new ideas and practical refer-ences for promoting the reform and innovation of National First-Class Courses.

Aim To explore the role and mechanism of epimedokoreanin B(EKB)in HepaRG cell pyroptosis through endoplasmic reticulum stress and NLRP1-me-diated pyroptosis pathway.Methods The effect of EKB on the viability of HepaRG cells at different con-centrations was determined by MTT assay,and the cell growth status was recorded by Incucyte.Four groups of HepaRG cells were set up.The control group was cul-tured with complete medium for 24 h;the drug admin-istration group was cultured with three concentration gradients of 6.25,12.5 and 25 μmol·L-1 of EKB for 24 h.Western blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins and pyroptosis-related proteins in the cells of each group.Results HepaRG cells showed cytotoxicity at a concentration of 6.25 μmol·L-1 for 24 h,and the half maximal inhibitory concentration(IC50)was 12.41 μmol·L-1.Incucyte recordings of the cell growth status showed that the cells in the control group were in good growth status,and the vesicular pyropto-sis cells appeared in the different concentrations of EKB and the cells swelled and ruptured after 24 h.Western blot showed that the protein expression levels of endoplasmic reticulum stress-related proteins pERK,eIF-2α,ATF-4,GRP78,and CHOP significantly in-creased in HepaRG cells at 25 μmol·L-1 of EKB compared with the control group.The proteins of the classical pathway of cellular pyroptosis mediated by NLRP1,caspase-1,cleaved caspase-1,GSDMD,GS-DMD-N significantly increased in HepaRG cells.Con-clusion EKB administration induces HepaRG cell py-roptosis,and EKB activates HepaRG cells to undergo endoplasmic reticulum stress and activates the NLRP1/caspase-1/GSDMD-mediated pyroptosis pathway.

Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype–phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.

It is of great significance to construct an information platform for performance appraisal of tertiary public hospitals to realize real-time monitoring and intervention of appraisal indicators, which is conducive to the transmission of responsibility for index optimization to departments and medical groups, and to promote the fine management of hospitals and promote high-quality development.This paper introduced the practice and effect of building a performance appraisal information platform for tertiary public hospitals in four aspects: accurate data filling, timely dynamic monitoring, visual display and safety management since 2022. At the same time, suggestions were put forward for platform optimization from four aspects: data quality control, co-construction and sharing, promotion and application, and system integration, in order to provide reference for other hospitals.

AIM To study the effects of Dianxianqing Granules on Tau protein in a mouse model of Alzheimer's disease (AD).METHODS The mice expressing P301S mutant Tau variant were randomly divided into the model group,the MCC950 group (NLRP3 inhibitor,10 mg/kg),the Dianxianqing Granules group (12.48 g/kg),the MCC950+Dianxianqing Granules group,in contrast to the C57BL/6 mice of the control group.After 5 months of administration,the mice had their learning and memory ability tested by Y maze test and Morris water maze test;their cerebral morphological changes observed by HE staining;their cerebral expressions of Caspase-1 and GSDMD proteins detected by immunohistochemical method;their expression of cerebral Tau protein detected by immunofluorescence;and their cerebral expressions of Tau,p-Tau (ser202),p-Tau (thr205),NLRP3,Caspase-1,IL-1β and IL-18 detected by Western blot.RESULTS Compared with the control group,the model group displayed decreased rate of spontaneous alternate reaction and times of crossing platform (P<0.05,P<0.01);abnormal hippocampal morphology,decreased number of neurons,increased cerebral positive expressions of Caspase-1 and GSDMD (P<0.05);deposition of a large number of brown granules in cytoplasm,and increased protein expressions of Tau,p-Tau (ser202),p-Tau (thr205),NLRP3,Caspase-1,IL-1βand IL-18 in the hippocampus and the cortex (P<0.05,P<0.01).Compared with the model group,the group intervened with Dianxianqing Granules demonstrated both increased rate of spontaneous alternate reaction and times of crossing platform (P<0.05);complete and normal morphology of the brain,a diversity of fine neurons,reduced cerebral positive expressions of Caspase-1 and GSDMD (P<0.05);and decreased protein expressions of Tau,p-Tau (ser202),p-Tau (thr205),NLRP3,Caspase-1,IL-1β and IL-18 in the hippocampus and the cortex (P<0.05,P<0.01).CONCLUSION Dianxianqing Granules may inhibit Tau protein expression in the mouse model of AD via NLRP3/Caspase-1 pathway.

Objective To investigate the significance of plasma levels of insulin-like growth factor(IGF)-1 and insulin-like growth factor binding protein(IGFBP)-3 in predicting acute respiratory distress syndrome(ARDS)and prognosis in critical patients.Methods A totally of 131 critical patients in intensive care unit(ICU)of the First Affiliated Hospital of Wenzhou Medical University were reviewed.Plasma concentrations of IGF-1,IGFBP-3,blood biochemistry,procalcitonin(PCT),lactic acid(LAC)and blood albumin were measured in enrolled patients.The 60-day fatality of enrolled patients was calculated.The differences between ARDS group and control group,as well as 60-day dead group and survival group were compared.Results Plasma IGF-1 and IGFBP-3 in ARDS group were significantly lower than those in control group,while plasma PCT was higher than that in control group.Plasma levels of IGF-1 and IGFBP-3 in dead group were significantly lower than those in survival group.Multivariate Logistic regression analysis and receiver operating characteristic curve results showed that IGF-1 area under curve(AUC)was 0.770,sequential organ failure assessment(SOFA)(AUC=0.692)and PCT(AUC=0.710)were independent risk factors for ARDS in critical patients.IGF-1(AUC=0.807),IGFBP-3(AUC=0.759)and SOFA score(AUC=0.859)were independent risk factors for death in critical patients.Conclusion The plasma levels of IGF-1 and IGFBP-3 in critica patients are significantly decreased,which may be an important factor for ARDS risk and fatality in critical patients.

AIM To study the effects of Dianxianqing Granules on Tau protein in a mouse model of Alzheimer's disease (AD).METHODS The mice expressing P301S mutant Tau variant were randomly divided into the model group,the MCC950 group (NLRP3 inhibitor,10 mg/kg),the Dianxianqing Granules group (12.48 g/kg),the MCC950+Dianxianqing Granules group,in contrast to the C57BL/6 mice of the control group.After 5 months of administration,the mice had their learning and memory ability tested by Y maze test and Morris water maze test;their cerebral morphological changes observed by HE staining;their cerebral expressions of Caspase-1 and GSDMD proteins detected by immunohistochemical method;their expression of cerebral Tau protein detected by immunofluorescence;and their cerebral expressions of Tau,p-Tau (ser202),p-Tau (thr205),NLRP3,Caspase-1,IL-1β and IL-18 detected by Western blot.RESULTS Compared with the control group,the model group displayed decreased rate of spontaneous alternate reaction and times of crossing platform (P<0.05,P<0.01);abnormal hippocampal morphology,decreased number of neurons,increased cerebral positive expressions of Caspase-1 and GSDMD (P<0.05);deposition of a large number of brown granules in cytoplasm,and increased protein expressions of Tau,p-Tau (ser202),p-Tau (thr205),NLRP3,Caspase-1,IL-1βand IL-18 in the hippocampus and the cortex (P<0.05,P<0.01).Compared with the model group,the group intervened with Dianxianqing Granules demonstrated both increased rate of spontaneous alternate reaction and times of crossing platform (P<0.05);complete and normal morphology of the brain,a diversity of fine neurons,reduced cerebral positive expressions of Caspase-1 and GSDMD (P<0.05);and decreased protein expressions of Tau,p-Tau (ser202),p-Tau (thr205),NLRP3,Caspase-1,IL-1β and IL-18 in the hippocampus and the cortex (P<0.05,P<0.01).CONCLUSION Dianxianqing Granules may inhibit Tau protein expression in the mouse model of AD via NLRP3/Caspase-1 pathway.

Aim To explore the effect of γ-ray on the mRNA,protein expression levels and metabolic activity level of the key drug metabolic enzyme CYP3A1 in rat liver. Methods Wistar rats were randomly divided into control group, 24 h post-radiation group and 72 h post-radiation group. The experimental group was exposed to total body irradiation of single 6 Gy γ-ray. Blood was collected from the orbital venous plexus for blood routine examination and biochemical analysis 24 h and 72 h after irradiation, and liver tissue was prepared for quantifying expression of CYP3A1 mRNA and liver-specific microRNA (miR-122-5p) through RT-PCR. The expression level of CYP3A1 protein was analyzed by Western blot, and the metabolic activity level of CYP3A1 detected by the specific substrate midazolam combined with LC-MS method. Results Com¬pared with the control group, the weights of the rats in the radiation group significantly decreased, and the number of white blood cells was markedly reduced. Simultaneously, the activities of alanine aminotrans-ferase and alkaline phosphatase continuously descended, as well as the levels of total bilirubin and bile acid significantly increased, which indicated that the liver may be damaged after radiation. The relative expression of CYP3A1 mRNA continued to increase significantly 24 h and 72 h after irradiation. CYP3A1 protein expression and metabolic activity levels showed an obvious increasing trend 24 h after irradiation, and rose significantly 72 h after irradiation compared with the control group. At the same time, the expression of miR-122-5p in liver of rats in the 24 h and 72 h post-radiation group continued to decrease rapidly compared with the control group. Conclusions γ-ray radiation may arouse damage effect on liver, which leads to the continuous up-regulation of the mRNA and protein expression levels of the capital metabolic enzyme CYP3A1 in liver tissue, as well as the elevation of the metabolic activity level. The regulatory mechanism might be related to miR-122-5p.