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.

Medical academic conferences represent one of the important carriers of scientific and technological exchanges， promoting the progress of medical science and technology and the dissemination of knowledge. However， the surge in the number of conferences has not been matched by a corresponding improvement in academic quality， leading to widespread doubts about their academic nature. The standardization of medical academic conferences urgently requires clarifying the core value standards of high-quality medical academic conferences from the perspective of academic ethics. Based on the perspective of academic ethics and employing Bourdieu’s field theory， this study systematically analyzed the functional positioning and value norms of medical academic conferences. It argued that they were committed to “pursuing truth” in knowledge production at the individual level， focused on “pursuing goodness” in communication and dissemination at the academic community level， and strove for “pursuing innovation” in innovative influence at the social level. Taking a professional annual conference as a typical case， this study further delved into the operational processes and internal mechanisms of academic annual conferences in the medical field. On this basis， optimization recommendations were proposed， including strengthening the principle of academic priority， promoting equal participation mechanisms， fostering innovative development models， and other aspects， with a view to providing a theoretical basis and practical guidance for the high-quality development of medical academic conferences.

Objective To observe the clinical efficacy of Xiaoyu Sanjie Prescription,which is derived from classical prescriptions Chaihu Guizhi Ganjiang Tang and Huoluo Xiaoling Dan,in treating middle-risk lung nodules of phlegm blended with blood stasis type.Methods A total of 104 cases of patients with middle-risk lung nodules of phlegm blended with blood stasis type who met the inclusion criteria were randomly divided into the trial group and the control group,with 52 cases in each group.Eventually,a total of 97 cases completed the trial for epidemic outbreak,of which 48 cases were in the trial group and 49 cases were in the control group.Both groups received health training,and then the control group was only given regular follow-up,while the trial group was treated with Xiaoyu Sanjie Prescription orally.The course of treatment covered three months.The changes in traditional Chinese medicine(TCM)syndrome scores and the area of maximum lung nodule of patients in the two groups before and after treatment were observed.After treatment,overall TCM syndrome efficacy and overall western medicine efficacy as well as their efficacies for various nodules types in the two groups were assessed.Results(1)After treatment,the distribution of the grade of TCM syndrome scores in the trial group was improved significantly when compared with that before treatment(P<0.01),while that in the control group showed no significant improvement(P>0.05),and the intergroup comparison after treatment showed that the improvement in the trial group was significantly superior to that in the control group(P<0.01).(2)The total effective rate of overall TCM syndrome efficacy in the trial group was 81.25%(39/48),and that in the control group was 20.41%(10/49);the intergroup comparison(tested by rank sum test)showed that the overall TCM syndrome efficacy in the trial group was significantly superior to that in the control group(P<0.01).In terms of the efficacy for various nodule types,the trial group had stronger TCM syndrome efficacy for multiple nodules,mixed solid nodules,pure ground glass nodules and solid nodules than the control group,in particular the efficacy for multiple nodules and mixed solid nodules,and the differences were all statistically significant(P<0.05 or P<0.01).(3)After treatment,the area of the maximum lung nodule in the trial group was significantly reduced(P<0.01),whereas there was no significant reduction in the control group compared with that before treatment(P>0.05).Statistically significant difference was shown in the post-treatment area between the two groups and in the pre-and post-treatment difference of the area between the two groups(P<0.05 or P<0.01),which suggested that the trial group's effect on the reduction of maximum lung nodule area was significantly superior to that of the control group.(4)With regard to the efficacy of western medicine,on the whole,the total effective rate of overall western medicine efficacy in the trial group was 45.83%(22/48),while that in the control group was 6.12%(3/49),and the intergroup comparison(tested by rank sum test)showed that the overall western medicine efficacy in the trial group was significantly superior to that in the control group(P<0.01).The western medicine efficacy for multiple nodules in the trial group was superior to that in the control group(P<0.01),while no statistically significant difference was presented in western medicine efficacy for mixed solid nodules,solid nodules,and pure ground glass nodules between the two groups(P>0.05).Conclusion Xiaoyu Sanjie Prescription is effective on relieving the clinical symptoms of patients with middle-risk lung nodules of phlegm blended with blood stasis type,and is effective on stabilizing,reducing or even eliminating some of the lung nodules.The compatibility principle of the formula deserves further discussion.

Objective To target at the NKG2A-HLA-E inhibitory axis,a pluripotent stem cell(iPSC)-derived geneti-cally engineered natural killer cells(NK cells)with NKG2A knockout(NKG2A KO-iNK)were prepared and then their tumor-killing efficacy was evaluated in vitro.Methods NKG2A was knocked out in iPSCs using gene-editing technology.These cells were then differentiated into NKG2A KO-iNK cells.Surface markers at each differentiation stage were analyzed by flow cytometry.Western blot confirmed NKG2A knockout,and flow cytometry assessed expres-sion of activating receptors(NKG2D)and natural cytotoxicity receptors(NKp30,NKp44,NKp46)in NKG2A KO-iNK cells.Cytotoxic activity against tumor cell lines with varying human leukocyte antigen E(HLA-E)expression level was evaluated via lactate dehydrogenase(LDH)release assay.Results Co-transfection of iPSCs with Cas9 pro-tein and three small-guide RNAs(sgRNAs)targeting at exons 1 and 2 of the KLRC1 gene(encoding NKG2A)suc-cessfully generated monoclonal NKG2A-knockout iPSCs(NKG2A KO-iPSCs)with a single T-base insertion in exon 1.During iPSC differentiation into NK cells,CD34 expression reached 30％-50％at the embryoid body(EB)stage(day 8),while CD56 and CD 16 expression exceeded 80％by day 28.Western blot confirmed complete NKG2A knockout in NKG2A KO-iNK cells.Flow cytometry revealed comparable expression level of activating receptor NKG2D and cytotox-icity receptors(NKp30,NKp44,NKp46)between NKG2A KO-iNK and wild-type iNK(WT-iNK)cells.The LDH assay results indicated that the cytotoxic activity of NKG2A KO-iNK cells against the HLA-E highly-expressed B-cell precursor leukemia cell line Nalm6 cells was significantly higher than that of WT-iNK cells,while there was no signif-icant difference between them and human myeloma cell line H929 cells with low HLA-E expression and human hepa-tocellular carcinoma cell line HepG2 cells with almost no HLA-E expression.Interferon-γ(IFN-γ)pretreatment up regulated HLA-E expression in Nalm6 cells,further amplifying NKG2A KO-iNK-mediated cytotoxicity.Conclusions By disrupting the NKG2A-HLA-E inhibitory axis,NKG2A KO-iNK cells exhibit markedly enhanced in vitro cytotoxic-ity against HLA-E-high tumor cells.This result highlights their potential function as a novel adoptive cell therapy strategy for cancers reliant on HLA-E-mediated immune evasion.

Objective To investigate the molecular mechanism of the induction of gastric cancer cell line AGS death by p97 inhibitor eeyarestatin Ⅰ(Eer Ⅰ).Methods AGS cells were treated with Eer Ⅰ.Then liquid chro-matography-mass spectrometry was used to perform proteome analysis for screening differentially expressed proteins and to find underlying signaling pathways.At the same time,the proteins of related pathway were investi-gated by protein immunoblotting.Cell proliferation was detected using the CCK-8 test kit;Cell apoptosis was detec-ted using TUNEL staining test kit;Liperfluo probe was used to detect ferroptosis-related lipid peroxides.Results In Eer Ⅰ treatment group,there were significant changes in proteins(fold change＞1.5 and P＜0.05),in which 125 proteins were increased and 132 proteins were decreased.The enrichment analysis of these DEPs showed that Eer Ⅰ might significantly affect cell apoptosis and ferroptosis-related signaling pathways.Furthermore,Eer Ⅰcould increase genomic DNA fragmentation related to cell apoptosis,increase of lipid peroxides in the ferroptosis pathway,causes changes in cell death related proteins,and inhibit the proliferation of gastric cancer cells.Conclusions p97 inhibitor Eer Ⅰ can induce cell apoptosis and ferroptosis in AGS cells,thereby inhibiting tumor cell proliferation.

Objective To explore the correlation between remnant cholesterol level and CI-AKI in elderly patients with ACS and concomitant T2DM following PCI.Methods A retrospective case-control study was conducted on 759 elderly T2DM patients with ACS undergoing PCI in the Affi-liated Hospital of Xuzhou Medical University between January 2020 and December 2023.The pa-tients were randomly allocated into a training group(n=608)and a validation group(n=151)in a ratio of 8∶2.Based on CI-AKI diagnostic criteria,the training group was further stratified into a CI-AKI subgroup(n=95)and a non-CI-AKI subgroup(n=513).Multivariate logistic regression analysis was used to identify the independent risk factors for CI-AKI.Results The remnant cho-lesterol level was significantly higher in the CI-AKI subgroup than the non-CI-AKI subgroup[0.78(0.51,1.07)mmol/L vs 0.57(0.40,0.81)mmol/L,P＜0.01].Red blood cell count,remnant cholesterol,age,uric acid,log-transformed systemic immune-inflammatory(SII)index,and cysta-tin C were identified as independent predictors for CI-AKI in T2DM patients with ACS after PCI(P＜0.01).ROC curve analysis demonstrated that the model combining remnant cholesterol with other independent predictors exhibited superior discriminative performance in both the training group(AUC=0.818,95％CI:0.773-0.863)and validation group(AUC=0.787,95％CI:0.686-0.889)when compared to the models excluding remnant cholesterol(P＜0.01).Conclusion Rem-nant cholesterol is an independent predictor of CI-AKI in elderly patients with ACS and T2DM af-ter PCI.A risk prediction model based on remnant cholesterol and other independent risk factors demonstrates enhanced predictive performance for postoperative CI-AKI in the patients.

Objective:To assess the effectiveness of Buzzy? device in alleviating pain and anxiety in children during puncture-related operation,and to provide more comprehensive intervention option for puncture-related management of children.Methods:Randomized controlled trials(RCTs)about the application of Buzzy? device in puncture-related pain and anxiety of children were searched from China National Knowledge Infrastructure(CNKI),Wanfang,VIP,PubMed,Embase and Web of Science(WOS)databases.The retrieval time was from the establishment of the database to July 2023.In the interventions measures,the control groups used different methods that included standard care group,ShotBlocker device group and distraction card group.The obtained 528 relevant literatures were implemented literature selection and data extraction by two independent reviewers according to the established inclusion and exclusion criteria.RevMan5.4 and Stata17.0 software were adopted to conduct Meta-analysis for the pain and anxiety of main outcome indicators,and the success rate of the first puncture,parents'satisfaction and adverse events of secondary outcome indicators in the literatures.Results:A total of 20 eligible studies that could meet the conditions were included,involving 2 876 children,including 1 110 children in the Buzzy? device group and 1 766 children in the control group.The Buzzy? device group was better than the standard care group and the distraction card group in alleviating puncture-related operation pain in children,and the differences of that among them were statistical significance(Z=7.18,2.13,P<0.05),and was better than the standard care group in anxiety,and the difference was statistical significance(Z=5.00,P<0.05).The Buzzy? device group and the distraction card group were better than standard care group and Shotblocker device group in satisfaction,and the difference was significant(x2=63.236,P<0.05).The interventions in the Buzzy? device group and other groups did not have effect on the success rate of the first puncture and incidence of adverse event.Conclusion:The Buzzy? device can alleviate pain and anxiety of children during puncture operation,and improve children's satisfaction for puncture operation.It does not affect the success rate of puncture,and there is not adverse events in its operation.

Objective:To explore the diagnostic value of the macrophage activation syndrome/systemic juvenile idiopathic arthritis（MS）score in macrophage activation syndrome（MAS）associated with systemic juvenile idiopathic arthritis（sJIA），and to provide a reference for clinical work.Methods:This study was a retrospective case-control analysis，conducted on the patients initially diagnosed as sJIA-associated with MAS and admitted into the Department of Rheumatology and Immunology of Children's Hospital Affiliated to Xi 'an Jiaotong University from July 1st，2016 to June 30th，2023. All of the patients met the diagnostic criteria for patients with MAS associated with sJIA according to the 2016 European Alliance of Associations for Rheumatology（EULAR）/American College of Rheumatology（ACR）/Pediatric Rheumatology International Trials Organization（PRINTO）standards. The basic information at baseline，clinical manifestations，and auxiliary examination results were collected. The MS score was applied to re-evaluate the children diagnosed as sJIA-associated with MAS. When the MS score ≥-2.1，the possibility of sJIA with MAS was high. Thirty cases of sJIA without MAS were randomly selected as the control group.Results:There were 28 cases in the MAS group，including 13 males（46.43%）and 15 females（53.57%），with an average age of（7.51±4.01）years. Compared with the control group，the MAS group were significantly more likely to have high fever（ χ2=8.539， P=0.003），hepatomegaly（ χ2=11.621， P<0.001），splenomegaly（ χ2=11.710， P<0.001）and neurological involvement（ χ2=27.619， P<0.001），with the differences being statistically significant. Meanwhile，there were statistically significant differences between the two groups in terms of white blood cell count（ Z=-4.001， P<0.001），neutrophil count（ Z=-3.659， P<0.001），platelet count（ Z=-4.687， P<0.001），albumin level（ Z=-4.018， P<0.001），alanine aminotransferase（ Z=-3.846， P<0.001），aspartate aminotransferase（ Z=-5.932， P<0.001），lactate dehydrogenase（ Z=-6.150， P<0.001），triglycerides（ Z=-5.874， P<0.001），fibrinogen（ Z=-5.808， P<0.001），ferritin（ Z=-5.280， P<0.001），erythrocyte sedimentation rate（ Z=-3.971， P<0.001），ferritin/erythrocyte sedimentation rate（ Z=-5.433， P<0.001），reduction of two-line cells in blood（ χ2=11.408， P<0.001）and the presence of hemophagocytosis in bone marrow smears（ χ2=28.260， P<0.001）. Moreover，there was a statistically significant difference in MS scores between the two groups（ Z=-6.148， P<0.001），with higher MS scores in the MAS group. Nevertheless，this study showed the median MS scores of both groups ≥-2.1. Conclusion:The MS score was significant to a certain degree as reference for the diagnosis of MAS，and this study showed that the MS score in the MAS group was significantly higher than the control group. However，the median MS scores in both groups were no less than -2.1. This might be related to the influence of factors during the assessment，which made it necessary to optimize the cutoff values of the MS score. Therefore，prospective studies should be carried out on the role of MS score in early identification of MAS.

Objective Zinc finger protein 335 (Zfp335) plays a crucial role in the early development of thymic T cells and the differentiation of peripheral T cell subpopulations. The objective of this study is to investigate the role and underlying mechanisms of Zfp335 in the regulation of regulatory T cell (Treg) within tumor immunity. Methods The Zfp335 gene was specifically knocked out in Treg using tamoxifen (Zfp335^fl/fl FOXP3^creERT2), and the MC38 tumor model was established. On the 7th day after tumor inoculation, tumor size was observed and measured. Tumor size was monitored and recorded daily starting from day 7 post-inoculation. On day 12, tumors were harvested, and the proportions of CD4⁺ T cells, CD8⁺ T cells, and Treg were analyzed by flow cytometry. Additionally, the mitochondrial function of effector regulatory T cell (eTreg) was assessed. Results From day 10 post-tumor inoculation, tumor volume in the Zfp335^CKO group was significantly reduced compared to that of the wild-type (WT) group. Furthermore, the infiltration of CD4⁺ and CD8⁺ T cells, along with their respective effector cells, was significantly higher in the Zfp335^CKO group than in the WT group. The proportions of CD4⁺ and CD8⁺ T cells producing interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were also significantly increased in the Zfp335^CKO group compared to that of the WT group. In addition, the percentage of CD8⁺ T cells secreting granzyme B (GzmB) was significantly higher in the Zfp335^CKO group than that in the WT group. In contrast, the proportion of Treg and inducible T cell co-stimulator (ICOS)⁺ Treg in the Zfp335^CKO group was significantly lower than that in the WT group. Finally, the expression level of Mitotracker Deep Red in eTreg from the Zfp335^CKO group was significantly reduced compared to that in the WT group. Conclusion During tumorigenesis, the specific deletion of Zfp335 impairs Treg activation, which is related to decreased mitochondrial function in eTreg. In Zfp335^CKO mice. Tumors exhibit increased infiltration of effector T cells, accompanied by elevated levels of cytotoxic cytokines, ultimately enhancing resistance to tumor progression.
Animals ; T-Lymphocytes, Regulatory/metabolism* ; Mice ; CD8-Positive T-Lymphocytes/immunology* ; Neoplasms/genetics* ; Cell Line, Tumor ; Mice, Inbred C57BL ; Mice, Knockout ; DNA-Binding Proteins/genetics* ; Female

Objective:To investigate the relationship between sarcopenic obesity(SO), defined by various obesity indicators, and mortality risk in older adults based on a prospective cohort from multiple nursing homes.Methods:Sarcopenia was diagnosed according to the 2019 consensus of the Asian Working Group for Sarcopenia.Obesity was defined using six different indicators: waist circumference(WC), waist-hip ratio(WHR), waist-height ratio(WHTR), body mass index(BMI), visceral fat area(VFA), and percentage of body fat(PBF).A prospective cohort of adults aged 60 and above was established across 15 nursing homes in Zigong City, with annual follow-ups on survival status conducted over two consecutive years.Cox proportional hazards regression models were employed to analyze the association between SO, defined by different obesity indicators, and mortality risk, calculating hazard ratios( HR)and their 95% confidence intervals( CI). Results:A total of 695 older adults were included in the study, of whom 67.1% were male.During the 2-year follow-up period, 88 participants died.The prevalence of SO as defined by WC, WHR, WHTR, BMI, VFA, PBF was found to be 17.6%, 30.1%, 48.9%, 3.0%, 28.3%, and 58.7%, respectively.When SO was defined using WC, WHR, WHTR, BMI, and VFA, there was no statistically significant difference in mortality rates between the SO and non-SO groups.However, when defined by PBF, the SO group exhibited a significantly higher mortality rate compared to the non-SO group(16.9% vs.6.6%, P<0.01).Cox proportional hazards regression analysis revealed that, compared to the non-SO group, the SO group defined by PBF had a significantly increased mortality risk( HR=2.81, 95% CI: 1.67-4.73, P<0.001).After adjusting for potential confounding factors, the mortality risk for the SO group remained significantly higher than that of the non-SO group( HR=1.97, 95% CI: 1.14-3.38, P=0.015). Conclusions:The prevalence of SO varies significantly across different obesity indicators.SO defined by PBF is significantly associated with mortality risk in nursing home residents.This study provides new evidence for further optimizing the diagnostic criteria for SO in this population.