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.

Objective To provide a scoping review of studies on the application of intelligent healthcare for continuity management in liver transplant patients,in order to provide a reference for the future development of intelligent liver transplant management.Methods A systematic search of relevant studies was conducted in PubMed,Embase,CINAHL,Web of Science,Cochrane Library,Scopus,CNKI,Wanfang Database,VIP Database,and CBM from the establishment of the databases to 30 September 2024.Following this search,the included literature was screened,summarized,and analyzed.Results A total of 19 papers were included.The intervention forms included application,intelligent medical device,and remote rehabilitation platform.The content elements included transplant health management,rehabilitation program planning,health data monitoring,telemedicine consultation,psychosocial support,and follow-up supervision.The evaluation indicators included physiological indicators,self-management ability,health-related quality of life,transplantation outcomes,patient satisfaction,and feasibility indicators.Conclusion The application of intelligent healthcare in the continuity management of liver transplantation patients is feasible and effective,and it is recommended that healthcare professionals integrate the diverse practice forms of intelligent healthcare,deepen the content elements and refine the evaluation indicator system based on the actual clinical situation,in order to improve the quality of the continuity management of liver transplantation patients.

Aim To investigate the effect of icariin(ICA)on chronic ulcerative colitis(UC)in mice.Methods Male SPF-grade C57BL/6J mice were ran-domly divided into the control group,model group,low-dose ICA group,and high-dose ICA group.Except for the control group,the rest of the mice were established as chronic UC models.Expressions of tight junction proteins,inflammatory factors,fibrosis markers,macro-phage markers,and MAPK signaling pathways in the colonic epithelium of each group were determined by Western blot,qPCR,immunohistochemistry;the degree of intestinal damage and fibrosis was detected by HE and Masson staining;the co-localization of different macrophage markers with related factors was examined by immunofluorescence.Results Treatment of ICA significantly improved the general condition of chronic UC mice,decreased the infiltration of M1-type macro-phages and the secretion of pro-inflammatory factors,inhibited the MAPK signaling pathway,reduced the co-localization expression of M1-type macrophages with the MAPK signaling pathway,and downregulated the infiltration of M2-type macrophages and the secretion of pro-fibrotic factors in the colon.Conclusion ICA mitigates the inflammatory damage and fibrosis of colon in chronic UC mice.

Aim To investigate the effects of breast cancer-related depression model on microglial activa-tion and neuronal function via the NLRP3-COX-2 sig-naling pathway.Methods In the cell experiment,BV2 and HT22 cells were co-cultured to observe the polarization of BV2 cells and the injury of HT22 cells.In the animal experiment,behavioral experiments were used to evaluate the depressive behaviors of mice.HE staining and tumor weight were used to evaluate the changes of axillary tumors.ELISA was used to detect the contents of CA153,CA125,CEA,as well as the changes in the contents of 5-HT,DA and others in the serum and hippocampus.RT-qPCR and immunohisto-chemistry were used to detect the mRNA and protein expression of IL-1 β,IL-18 and other indicators,re-spectively.Immunofluorescence was used to detect CD1 1b and Iba-1.Nissl staining and transmission elec-tron microscopy were used to observe the hippocampal neurons.Results The results of cell experiments showed that compared with the model group,the pro-tein contents and mRNA expression levels of NLRP3,COX-2,and IL-1 β in the NLRP3 knockdown group were all decreased.The results of animal experiments showed that compared with the model group,the be-havioral performance of the experimental group was im-proved,and the mRNA and protein expression levels of IL-1 β,IL-18,and NLRP3 were decreased.Conclu-sion BCRD may mediate the polarization of microglia and the injury of hippocampal neurons through the NL-RP3-COX-2 signaling pathway.

Objective To design an 8-channel gene analyzer to take the place of the widely used gene analyzer with problems in inconvenient consumable replacement and short storage time of electrophoresis polymer.Methods The 8-channel gene analyzer had its mechanical components composed of an automatic sample loading table,a polymer injection module,a high-voltage temperature control module,an optical module and an integrated U box,its electrical control system made up of a host computer(an embedded computer)and three slave computers(a sampling control board,a polymer injection control board and a high-voltage temperature control board).The automatic sample loading table involved in four motors and transmission systems for x,y,z directions and optical alignment,the transmission systems adopted mainly belt drive mode and the optical alignment motor had its threads with an anti-backlash structure;the polymer injuection module was manipulated by the polymer injection control board,and the polymer block was made of highly transparent acrylic material;the high-voltage temperature control module realized the regulation of electrophoresis voltage and the detection of electrophoresis current by the low-ripple precision high-voltage power supply,and controlled the temperature of the heating furnace by the proportional-integral-differential(PID)algorithm;the optical module consisted of an excitation module and a light-receiving module,which had the base of the reflector made of low expansion coefficient alloy material;the integrated U box had the electrophoresis polymer,capillary array,polymer block and anode buffer in a plastic housing;the host computer had the data acquisition software programmed with C# and C++,and the slave computers were controlled by STM32 SCM.Results The 8-channel gene analyzer had no significant differences with the widely used ABI3500 gene analyzer in resolution,precision accuracy and clinical results.Conclusion The 8-channel gene analyzer gains advantages in consumable replacement and storage time of electrophoresis polymer,and can meet the requirements for gene sequencing.[Chinese Medical Equipment Journal,2025,46(2):24-30]

Objective To provide a scoping review of studies on the application of intelligent healthcare for continuity management in liver transplant patients,in order to provide a reference for the future development of intelligent liver transplant management.Methods A systematic search of relevant studies was conducted in PubMed,Embase,CINAHL,Web of Science,Cochrane Library,Scopus,CNKI,Wanfang Database,VIP Database,and CBM from the establishment of the databases to 30 September 2024.Following this search,the included literature was screened,summarized,and analyzed.Results A total of 19 papers were included.The intervention forms included application,intelligent medical device,and remote rehabilitation platform.The content elements included transplant health management,rehabilitation program planning,health data monitoring,telemedicine consultation,psychosocial support,and follow-up supervision.The evaluation indicators included physiological indicators,self-management ability,health-related quality of life,transplantation outcomes,patient satisfaction,and feasibility indicators.Conclusion The application of intelligent healthcare in the continuity management of liver transplantation patients is feasible and effective,and it is recommended that healthcare professionals integrate the diverse practice forms of intelligent healthcare,deepen the content elements and refine the evaluation indicator system based on the actual clinical situation,in order to improve the quality of the continuity management of liver transplantation patients.

Objective To compare the value of three-dimensional tomography ultrasound imaging(3D-TUI)and MRI for diagnosng early cesarean scar pregnancy(CSP).Methods Seventy-six patients who had gestational week not more than 12 weeks and with clinically suspected CSP were retrospectively enrolled.According to surgical and postoperative pathological findings,the patients were categorized into CSP group(n=65)and non-CSP group(n=11).The diagnostic performance of 3D-TUI,MRI and the combination of these two for CSP,also the depiction of CSP characteristics of 3D-TUI and MRI were compared.Results The sensitivity,specificity,accuracy,positive predictive value and negative predictive value of combination of 3D-TUI and MRI for diagnosing CSP was 96.92％,90.91％,96.05％,98.44％and 83.33％,respectively,with the accuracy higher than that of 3D-TUI alone(P＜0.05).No significant difference of diagnostic efficacy was found between 3D-TUI and MRI(all P＞0.05).3D-TUI showed a higher accuracy rate than MRI for displaying yolk sac,embryo,villi invasion into local scars and implantation of villi into muscle layer(all P＜0.05),whereas MRI was more accurate for diagnosing intrauterine bleeding(P＜0.05).Conclusion 3D-TUI could be used as the preferred initial imaging examination for early CSP.Combining with MRI could further improve its diagnostic accuracy.

This study evaluated the potential of OPC-167832 as a new method for the treatment of Mycobacterium fortuitum infec-tion.Drug sensitivity tests were conducted with the broth microdilution method to determine the minimum inhibitory concentration(MIC)of OPC-167832 against standard strains of M.fortuitum and 44 clinical isolates of M.fortuitum.A DprE1 overexpression strain was constructed,and the effect in the MIC of OPC-167832 against M.fortuitum were explored.Intracellular germicidal tests and checkerboard tests were conducted to verify the ability of OPC-167832 to kill intracellular M.fortuitum,and its interaction with five drugs:amikacin,clarithromycin,imipenem,moxifloxacin,and clofazimine.The MIC50 and MIC90 against 44 clinical isolates of M.fortuitum were 0.031 25 μg/mL and 0.062 5μg/mL,respectively.The epidemiological cut-off value(ECOFF)was 0.062 5 μg/mL.Overexpression of DprE1 led to resistance to OPC-167832 in M.fortuitum.After 24 hours of incubation,the intracellular bacterial in-hibition rate of OPC-167832 at a 1 μg/mL concentration was 81.37%,exceeding the 74.05%inhibition rate of amikacin at a 1 μg/mL concentration.OPC-167832 showed strong inhibitory activity against M.fortuitum in vitro and in macrophages,and might provide a promising treatment for M.fortuitum infection.

Aim To investigate the regulatory mecha-nisms of donepezil on the expression and enzymatic ac-tivity of cytochrome P450 3A4(CYP3A4),elucidate the synergistic impact of hypoxia on CYP3A4 function,and reveal its potential association with drug-induced cardiotoxicity,particularly QT interval prolongation.Methods Western blot,co-immunoprecipitation,and gene knockdown techniques were employed to evaluate the effects of donepezil and hypoxia on CYP3A4 pro-tein expression.CYP3A4 enzymatic activity was as-sessed using an in vitro incubation system with rat liver microsomes combined with high-performance liquid chromatography(HPLC),and the half-maximal inhib-itory concentration(IC50)was determined.Results Donepezil(10 μmol·L-1)and hypoxia reduced CYP3A4 protein expression to 31.75％and 45.90％of the control levels,respectively.Both interventions activated the gp78-mediated ubiquitin-proteasome path-way,significantly increasing CYP3A4 ubiquitination levels by 2.1-fold compared to the control group,thereby promoting proteasomal degradation.Donepezil inhibited CYP3A4 enzyme activity with an IC50 of 83.4μmol·L-1,and hypoxia synergistically enhanced this inhibitory effect,reducing the IC50 to 20.79 μmol·L-1.Conclusion Donepezil downregulates CYP3A4 function through dual mechanisms involving ubiquitin-mediated proteasomal degradation and direct enzymatic inhibition.Hypoxia potentiates this effect,leading to impaired metabolism of CYP3A4 substrate drugs,ele-vated plasma drug concentrations(1.6-2.3-fold in-crease compared to normal metabolic conditions),and an increased risk of QT interval prolongation and other forms of cardiotoxicity.

Objective:To investigate the mechanism by which microRNA-183 (miR-183) regulates the progression of diabetic nephropathy (DN) through targeting phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and modulating the AKT signaling pathway, and to identify potential therapeutic targets for DN.Methods:(1) Bioinformatic analysis of miRNA expression: MiRNA expression datasets from diabetic nephropathy (DN) and control samples were obtained from the Gene Expression Omnibus database. Differential expression analysis was performed, and differentially expressed miRNAs (DEmiRNAs) were identified using thresholds of an absolute log 2 (fold changes) >1 and an adjusted P-value<0.05. The results were visualized in a volcano plot and a heatmap. (2) Animal model establishment and in vivo interventional studies: A DN rat model was induced by administration of a high-fat/high-sucrose diet combined with an intraperitoneal injection of streptozotocin. Rats were randomly assigned into four groups ( n=10 per group) using a random number table: control group, DN model group, miR-183 inhibitor negative control (NC) group, and miR-183 inhibitor group. The latter two groups received tail vein injections of the miR-183 inhibitor NC or the miR-183 inhibitor, respectively, for eight consecutive weeks. Parameters including fasting blood glucose, 24-hour urinary protein excretion, urinary albumin excretion rate (UAER), serum creatinine, and blood urea nitrogen (BUN) were measured. Renal histopathological changes were assessed by HE and PAS staining. Furthermore, the expression of candidate miRNAs from patient data was validated, and the mechanism of action of miR-183 was investigated using quantitative real-time PCR and Western blotting. (3) In vitro mechanistic investigations in cultured podocytes: Mouse podocyte clone-5 (MPC5) cells were cultured in vitro and subjected to the following conditions: normal glucose (5.3 mmol/L glucose), high glucose (30 mmol/L glucose), and osmotic control (5.3 mmol/L glucose+19.5 mmol/L mannitol). Cells in the logarithmic growth phase were transfected with the miR-183 inhibitor (100 nmol/L), miR-183 mimic (50 nmol/L), or their corresponding negative controls. A dual-luciferase reporter assay was conducted to validate the binding interaction between miR-183 and the 3'-untranslated region (3'UTR) of PTEN. The effects of miR-183 on the AKT signaling pathway, apoptosis-related proteins, and cell viability were evaluated by quantitative real-time PCR, Western blotting, and the cell counting kit-8 assay, respectively. Results:MiR-183 expression was markedly upregulated in renal tissues from DN patients and DN model rats (both P<0.05). Inhibition of miR-183 significantly reduced renal miR-183 levels by 90.2% ( P<0.01), decreased fasting blood glucose by 65.3% ( P<0.01), and improved renal function parameters, including reductions in urinary protein (40.3%), blood urea nitrogen (32.1%), urinary albumin excretion rate (22.5%), and serum creatinine (40.2%) (all P<0.01). Histological analyses showed attenuation of glomerular lesions and glycogen accumulation. Bioinformatic prediction and experimental validation identified PTEN as a direct target of miR-183, confirmed by dual-luciferase assays. In vitro, miR-183 inhibition increased PTEN expression, reduced AKT phosphorylation, promoted podocyte proliferation, and suppressed apoptosis (upregulation of Bcl-2 and downregulation of cleaved-caspase-3). These effects were abolished upon PTEN knockdown. Conclusions:miR-183 aggravates DN by targeting PTEN and activating the AKT signaling pathway. Inhibition of miR-183 improves renal function and reduces podocyte apoptosis, suggesting miR-183 as a potential therapeutic target for DN.