AIM: To develop a novel gene-delivery therapeutic based on CRISPR/Cas9 genome editing technology capable of specifically targeting and knocking out the VEGFA gene, thereby achieving sustained suppression of VEGFA expression in retinal pigment epithelial(RPE)cells and providing a new strategy for gene therapy in retinal neovascular diseases.METHODS:Single guide RNAs targeting the human VEGFA gene for knockout were designed, and corresponding recombinant plasmids were constructed. A novel polymer(PTEE)was used to encapsulate the plasmids to prepare a PTEE-loaded anti-VEGFA plasmid(PLAP)gene delivery system. PTEE materials at concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 μg/μL were co-incubated with ARPE-19 cells, and the biocompatibility of PTEE was evaluated using the cell counting kit-8(CCK-8)assay. Recombinant plasmids expressing green fluorescent protein(GFP)were constructed. Lipofectamine 3000 and jetOPTIMUS®DNA transfection reagents were used as control groups, and PTEE nanomaterials were used as the experimental group to encapsulate the plasmids. When the cell confluence reached 80%, the formulations were transfected into ARPE-19 and 293T cells. GFP expression was observed under light microscopy, and the transfection efficiencies of each group were compared. ARPE-19 cells were induced under hypoxia, and PLAP was transfected into the cells. The expression level of VEGFA was detected by enzyme-linked immunosorbent assay(ELISA)to evaluate the efficacy of this novel gene delivery system.RESULTS: After co-incubation of ARPE-19 cells with different concentrations of PTEE for 24 h and 48 h, no significant effect on cell viability was observed in any group. The transfection efficiency of PLAP in ARPE-19 cells was higher than that in the Lipo3000 and jetOPTIMUS groups, with statistically significant differences(P<0.01). Hypoxia for 6 h significantly induced the upregulation of VEGFA mRNA expression in ARPE-19 cells, and under hypoxic conditions, the PTEE group exhibited a significant inhibitory effect on VEGFA expression(P<0.01).CONCLUSION:PLAP exhibits favorable biocompatibility and prominent VEGFA inhibitory effects in vitro, making it a potential candidate drug for gene therapy of retinal neovascular diseases.

Tissue-resident memory T cells(TRM)are a key immune memory cell type responsible for the recurrence of many skin diseases.TRM cells remain in the skin tissue following infection or injury and maintain immune memory by forming a memory reservoir.When the same or similar external stimuli are encountered again,TRM cells can be rapidly activated,releasing inflammatory cytokines and cytotoxic substances,leading to local tissue damage and/or disease recurrence.The formation and function of TRM cells are regulated by various factors,such as T cell receptor signals,cytokines,co-stimulatory molecules,etc.Furthermore,TRM cells may exhibit different phenotypes and functions in different types of skin diseases.Therefore,understanding the characteristics and regulatory mechanisms of TRM cells is of great significance for studying the mechanisms of skin disease recurrence as well as for developing therapeutic strategies targeting TRM cells.

Based on the cost accounting series system issued by relevant national departments,it proposes the methods and steps for cost accounting of medical service projects and DRG disease groups.Using mathematical derivation,it proves the linkage relationship among medical service item,DRG disease group costs and charging prices under the income distribution coefficient method,and provides examples to verify.The purpose is to clarify the data logic under this method,summarize the data rules of using the accounting method to carry out cost accounting,provide inspiration,suggestions,and specific implementation paths reference for hospitals and governments,propose optimization and improvement suggestions for the shortcomings of this method,further improve and promote the accounting method,provide data references for pricing and DRG payment reform of medical service projects,and accelerate the process of widely applying cost accounting results.

In terms of the complexity of the"left"and"right"concepts,these directional terms not only indicate spatial orientation but also embody hierarchical order,official titles,systemic imbalances,and temporal sequences.This study explains the medical theory of"liver is born on the left and lung is hidden on the right,"and reveals its implications for ascending and descending of qi movement pathways,sequential patterns,non-equilibrium states,and the centrality of the middle,thus linking the connotation of this theory and its application.In view of this,the theory of"liver is born on the left and lung is hidden on the right"can be used in the clinical diagnosis and treatment of qi-blood-body fluid and emotional diseases,and guides disease prevention and treatment,as well as enhances the subjective initiative of health maintenance and longevity.This study not only deepens the modern interpretation of the classical theory but also provides novel perspectives for its application in disease prevention and treatment.

In terms of the complexity of the"left"and"right"concepts,these directional terms not only indicate spatial orientation but also embody hierarchical order,official titles,systemic imbalances,and temporal sequences.This study explains the medical theory of"liver is born on the left and lung is hidden on the right,"and reveals its implications for ascending and descending of qi movement pathways,sequential patterns,non-equilibrium states,and the centrality of the middle,thus linking the connotation of this theory and its application.In view of this,the theory of"liver is born on the left and lung is hidden on the right"can be used in the clinical diagnosis and treatment of qi-blood-body fluid and emotional diseases,and guides disease prevention and treatment,as well as enhances the subjective initiative of health maintenance and longevity.This study not only deepens the modern interpretation of the classical theory but also provides novel perspectives for its application in disease prevention and treatment.

Objective To examine the phenotypic characteristics of Ecto-5'-nucleotidase(NtSe/CD73)gene knockout mice in restenosis of blood vessels after vein transplantation so as to identify potential targets for early diagnosis and drug treatment of vascular restenosis after clinical coronary artery bypass surgery.Methods CD73 gene knockout mice aged 8-10 weeks were used as the experimental group,and the littermate wild-type mice were used as the control group.Using the inferior vena cava of mice as a donor,we transplanted to the right carotid artery of allogeneic mice with a trocar method to establish a mouse inferior vena cava carotid artery vascular transplantation model.At the 4th week post-model establishment,we systematically evaluated the patency of the transplanted blood vessels,the formation of the vascular intima,the proliferation of the media,the morphology of the elastin layer,and the expression of inflammatory factors.The vascular smooth muscle cells(VSMCs)from the inferior vena cava of mice were isolated in vitro,and the migration and proliferation were assessed with CD73 inhibitor adenosine 5'-(alpha,beta-methylene)diphosphate(APCP).Results In CD73 knockout mice,neointima formation was impaired,the elastic fiber layer was disrupted and lost,and medial smooth muscle cells proliferated more actively.These changes ultimately led to decreased vascular wall elasticity and increased blood flow resistance.The immunohistochemical staining results suggest that in CD73 gene knockout mice the transplanted vein tissue showed extensive infiltration of Mac-2 positive macrophages,and the expression of cytokines interleukin-1 β(IL-1 β),IL-6,and transforming growth factor β(TGF-β)was significantly increased.CD73 deficiency exacerbated inflammatory responses and vascular remodeling in venous tissues.Scratch wound healing and cell proliferation assays revealed that CD73 inhibition promoted VSMCs proliferation,yet concurrently impaired their migratory capacity.Conclusion Knockout of the CD73 gene exacerbates vascular remodeling and inflammatory response in vein grafts,offering crucial insights for the precise diagnosis and targeted treatment of patients with CD73 gene defects undergoing coronary artery bypass surgery in clinical practice.

Objective To identify and cluster non-O1/O139 Vibrio cholerae(NOVC)using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS),and evaluate the feasibility of MALDI-TOF MS as a method for the identification and clustering of NOVC.Methods The NOVC was identified by the MALDI-TOF MS equipped with V5 database,V12 database,highly pathogenic bacteria database,and V5 database combined with self-built spectrum projection and analyzed by the principal com-ponent analysis(PCA)and main spectrum projection(MSP)clustering.Results The NOVC was incorrectly identified as Vibrio al-bensis by the MALDI-TOF MS equipped with V5 database or V12 database,while the MALDI-TOF MS equipped with highly pathogenic bacteria database or V5 database combined with self-built spectrum projection could correctly identify NOVC.The PCA clustering of MALDI-TOF MS could distinguish NOVC from other bacterial strains and refine the differentiation of NOVC species to show the dis-tance relationship between NOVC species.Some spectrum projections of NOVC were extremely similar to the reference strains used to establish the database,and MSP clustering could not distinguish the differences between NOVC species.Conclusion The identifica-tion ability of MALDI-TOF MS for NOVC is limited by its database.The MALDI-TOF MS equipped with highly pathogenic bacteria da-tabase or V5 database combined with the self-built spectrum projection can accurately identify NOVC.The PCA clustering of MALDI-TOF MS has certain reference significance for the intra-and inter-species identification and homology analysis of NOVC.

To enhance hospital operational management capability,the sample hospital initiated a financial talent development program aligned with current development needs and policy directions.This program established a talent development framework encompassing hierarchical training,employment mechanisms,and digital empowerment,forming a three-tier financial talent echelon comprising senior,intermediate,and junior professionals.It effectively facilitated the transformation of financial functions toward"strategic support,business service,risk prevention and control,and value creation".It analyzes the hospital's practical experience in financial talent development,aiming to offer insights for financial workforce development in public hospitals.These efforts synergize with the modernization of governance systems and capabilities in health economics,driving high-quality development in public healthcare institutions.

Objective To establish atopic dermatitis(AD)-like models in BALB/c mice using three chemical inducers,calcipotriol(MC903),2,4-dinitrochlorobenzene(DNCB),and oxazolone(OXA),and to explore the occurrence of ferroptosis in the different models.Methods Healthy 7-week-old female BALB/c mice were divided randomly into eight groups(n=8 mice per group)based on the induction site(ear/dorsal skin)and inducer:ear/dorsal control groups,MC903 ear/dorsal model groups,DNCB ear/dorsal model groups,and OXA ear/dorsal model groups.Models were established by topical application of the respective agents at specified concentrations.Mice in the MC903 ear/dorsal groups underwent continuous induction for 14 d.Mice in the DNCB and OXA ear/dorsal groups were sensitized for 3 consecutive days,4 days after the sensitization was completedand then challenged 12 times on day 8 and every other day for up to day 30.Skin lesions were observed and skin thickness was measured.Plasma levels of reactive oxygen species(ROS),interleukin(IL)-4,interferon(IFN)-γ,and malondialdehyde(MDA)were detected,the skin was examined by histopathological staining and ultrastructural observation,and expression levels of ferroptosis-related proteins(glutathione peroxidase 4(GPX4),ferritin heavy chain 1(FTH1),long-chain-fatty-acid-CoA ligase 4(ACSL4),transferrin receptor 1(TfR1))were detected by Western Blot.Results Compared with each control mice,all model mice exhibited obvious redness,swelling,scratching,desquamation,and rough thickening of the skin,and skin thickness was significantly increased(P＜0.01).ROS,IFN-γ,IL-4,and MDA levels were elevated to varying extents(P＜0.05)and histopathological features,including epidermal hyperplasia,keratinocyte degeneration,dermal vascular congestion,and immune cell infiltration,were detected in model mice.Transmission electron microscopy also revealed mitochondrial membrane rupture,increased density,and cristae reduction.Expression levels of ferroptosis markers were dysregulated,including significantly decreased GPX4/FTH1(P＜0.05)and increased ACSL4/TfR1 expression(P＜0.05).Conclusions All three chemicals successfully induced AD-like phenotypes in BALB/c mice through site-specific applications.Ferroptosis is involved in the pathological process of AD,but heterogeneity exists among inducers and modeling sites.

Objective:To investigate effect of galangin(Gal)on immune function in nephrotic syndrome(NS)rats by regula-ting stromal cell-derived factor-1(SDF-1)/CXC chemokine receptor 4(CXCR4)signaling pathway.Methods:A NS rat model was established by injecting doxorubicin into tail vein.After successful modeling,rats were randomly grouped into Model group,Gal low,medium and high doses groups,activator group.Twelve rats were randomly selected as control group,and each group was given corre-sponding drug by gavage and intraperitoneal injection,once a day for 6 consecutive weeks.Biochemical indicators such as 24-hour urine protein,urea nitrogen(BUN),creatinine(Scr)and total cholesterol(TC)were detected in rats;HE staining was applied to observe pathological damage in rat kidney tissue;spleen and thymus were taken and weighed,and organ index was calculated;flow cytometry was applied to detect levels of CD3+T,CD4+T,CD8+T,Th1 and Th2 cells in rat peripheral blood;ELISA was applied to detect IL-10,IL-2,TNF-α,IgG and IgM in serum;qRT-PCR and Western blot were applied to detect mRNA and protein expressions of SDF-1 and CXCR4.Results:Compared with control group,24-hour urine protein,BUN,Scr,TC,triglyceride(TG)contents,CD8+T,Th1,Th1/Th2,IL-2,TNF-α,SDF-1,CXCR4 mRNA and protein expressions of rats in Model group were obviously in-creased,albumin(ALB)content,spleen index,thymus index,CD3+T,CD4+T,Th2 cell proportions,CD4+T/CD8+T,IL-10,IgG,and IgM levels were obviously reduced(P<0.05);compared with Model group,24-hour urine protein,BUN,Scr,TC,TG contents,CD8+T,Th1,Th1/Th2,IL-2,TNF-α,SDF-1,CXCR4 mRNA and protein expressions of rats in Gal low,medium and high doses groups were gradually decreased,ALB content,spleen index,thymus index,CD3+T,CD4+T,Th2 cell proportions,CD4+T/CD8+T,IL-10,IgG and IgM levels were gradually increased(P<0.05);compared with high-dose Gal group,changes in above indicators in activator group of rats were obviously reversed(P<0.05).Conclusion:Gal may enhance T lymphocyte immune function by inhibiting SDF-1/CXCR4 pathway,thereby alleviating renal injury in NS rats.