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.

BACKGROUND:Degenerative scoliosis is defined as a condition that occurs in adulthood with a coronal cobb angle of the spine>10° accompanied by sagittal deformity and rotational subluxation,which often produces symptoms of spinal cord and nerve compression,such as lumbar pain,lower limb pain,numbness,weakness,and neurogenic claudication.The finite element method is a mechanical analysis technique for computer modelling,which can be used for spinal mechanics research by building digital models that can realistically restore the human spine model and design modifications. OBJECTIVE:To review the application of finite element method in the etiology and treatment of degenerative scoliosis. METHODS:The literature databases CNKI,PubMed,and Web of Science were searched for articles on the application of finite element method in degenerative scoliosis published before October 2023.Search terms were"finite element analysis,biomechanics,stress analysis,degenerative scoliosis,adult spinal deformity"in Chinese and English.Fifty-four papers were finally included. RESULTS AND CONCLUSION:(1)The biomechanical findings from the degenerative scoliosis model constructed using the finite element method were identical to those from the in vivo experimental studies,which proves that the finite element method has a high practical value in degenerative scoliosis.(2)The study of the etiology and treatment of degenerative scoliosis by the finite element method is conducive to the prevention of the occurrence of the scoliosis,slowing down the progress of the scoliosis,the development of a more appropriate treatment plan,the reduction of complications,and the promotion of the patients'surgical operation.(3)The finite element method has gradually evolved from a single bony structure to the inclusion of soft tissues such as muscle ligaments,and the small sample content is increasingly unable to meet the research needs.(4)The finite element method has much room for exploration in degenerative scoliosis.

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 understand the prevalence of elevated blood pressure and its association with dietary patterns in children and adolescents in China, providing evidence for developing dietary intervention of hypertension in children and adolescents. Methods: Data were derived from the China Children s Nutrition and Health System Survey and Application Project(2019-2021). A stratified cluster random sampling method was used to include 7 933 participants from 28 survey sites in seven major regions of Northeast, North, Northwest, East, Central, South and Southwest China. Multivariate Logistic regression models were used to analyze associations between demographic characteristics, nutritional status and elevated blood pressure. Exploratory factor analysis identified dietary patterns, which were divided into three quartile groups (T3, T2, T1) based on factor scores (compliance for dietary pattern) from high to low, and multivariate Logistic regression model assessed the correlation between elevated blood pressure and dietary patterns. Results: The prevalence of elevated blood pressure was 15.4% among Chinese children aged 7-17 years. Significant differences were observed across nutritional status (reference: underweight; normal weight: OR =1.57; overweight: OR = 2.61 ; obesity: OR =3.85), urban/rural residence (reference: rural; urban: OR =0.86), and paternal education (reference: junior high school and below; bachelor degree or above: OR =0.68) ( P <0.05). The detection rates of high blood pressure in T3 group children and adolescents with four dietary patterns (staple food, animal based food, snacks, vegetables and fruits) were 15.7%, 14.6%, 16.8%, and 15.8%, respectively. After adjusting for residence, paternal education, and nutritional status, the "snack dietary pattern" (mainly candy, sugar sweetened beverages, and processed snacks) showed positive associations with elevated blood pressure in T2 ( OR =1.21) and T3 ( OR =1.19) tertiles ( P <0.05). Conclusions The snack dietary pattern is a related factor for elevated blood pressure in children and adolescents. Restricting unhealthy snack intake may promote cardiovascular health.

OBJECTIVE To establish the fingerprint of Bushen ningshen ointment， determine the contents of its major constituents， and investigate its in vitro antioxidant activity. METHODS High performance liquid chromatography （HPLC） fingerprints of 10 batches of Bushen ningshen ointment were established. Similarity evaluation and identification of common peaks were subsequently performed. The contents of 10 components such as salidroside were determined using the same HPLC method. Using the scavenging rates against 2，2′-azino bis（3-ethylbenzothiazoline-6-sulfonic acid） （ABTS） and 1，1-diphenyl-2- picrylhydrazyl （DPPH） radicals， as well as ferric ion reducing antioxidant power （FRAP） as indicators， the anti-oxidant activity of the ointment was evaluated； grey relational analysis and partial least squares regression were conducted using SIMCA 14.1 software to establish the spectrum-effect relationship. RESULTS The fingerprint chromatogram of 10 batches of Bushen ningshen ointment contained 24 common peaks， with similarity values all exceeding 0.96. Eleven peaks were identified as adenosine （peak 1）， salidroside （peak 4）， morroniside （peak 6）， catechin （peak 7）， paeoniflorin （peak 10）， spinosin （peak 11）， ferulic acid （peak 12）， isoquercitrin （peak 13）， E-mail：wli1743@163.com verbascoside （peak 14）， paeonol （peak 23）， and emodin （peak 24）. Content determination results showed that the average contents of salidroside， morroniside， catechin， paeoniflorin， spinosin， ferulic acid， isoquercitrin， verbascoside， paeonol， and emodin were 0.725， 1.962， 0.214， 3.395， 0.124， 0.107， 0.286， 0.019， 0.034 and 0.067 mg/g， respectively. The antioxidant potency composite index （APC） for the 10 batches ranged from 85.08% to 96.35%. Spectrum-effect relationship analysis indicated that all 24 common peaks were positively correlated with the antioxidant capacity. Seventeen peaks had variable importance in projection values ＞1， specitically peaks 2， 5， 6， 7， 9， 10， 13- 21， 23， and 24. CONCLUSIONS This study successfully established the HPLC fingerprint and content determination method for Bushen ningshen ointment. The compounds represented by the 17 common peaks such as morroniside may be the active components contributing to its antioxidant effects.

BACKGROUND: Epoxyeicosatrienoic acids (EETs), which are metabolites of arachidonic acid catalyzed by cytochrome P450 epoxygenase, are degraded into inactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). Many studies have revealed that sEH gene deletion exerts protective effects against diabetes. Vascular calcification is a common complication of diabetes, but the potential effects of sEH on diabetic vascular calcification are still unknown. METHODS: The level of aortic calcification in wild-type and Ephx2-/- C57BL/6 diabetic mice induced with streptozotocin was evaluated by measuring the aortic calcium content through alizarin red staining, immunohistochemistry staining, and immunofluorescence staining. Mouse vascular smooth muscle cell lines (MOVAS cells) treated with β-glycerol phosphate (0.01 mol/L) plus advanced glycation end products (50 mg/L) were used to investigate the effects of sEH inhibitors or sEH knockdown and EETs on the calcification of vascular smooth muscle cells, which was detected by Western blotting, alizarin red staining, and Von Kossa staining. RESULTS: sEH gene deletion significantly inhibited diabetic vascular calcification by increasing levels of EETs in the aortas of mice. EETs (especially 11,12-EET and 14,15-EET) efficiently prevented the osteogenic transdifferentiation of MOVAS cells by decreasing nidogen-2 (NID2) expression. Interestingly, suppressing sEH activity by small interfering ribonucleic acid or specific inhibitors did not block osteogenic transdifferentiation of MOVAS cells induced by β-glycerol phosphate and advanced glycation end products. NID2 overexpression significantly abolished the inhibitory effect of sEH gene deletion on diabetic vascular calcification. Moreover, NID2 overexpression mediated by adeno-associated virus 9 vectors markedly increased insulin-like growth factor 2 (IGF2) and phospho-ERK1/2 expression in MOVAS cells. Overall, sEH gene knockout inhibited diabetic vascular calcification by decreasing aortic NID2 expression and, then, inactivating the downstream IGF2-ERK1/2 signaling pathway. CONCLUSIONS sEH gene deletion markedly inhibited diabetic vascular calcification through repressed osteogenic transdifferentiation of vascular smooth muscle cells mediated by increased aortic EET levels, which was associated with decreased NID2 expression and inactivation of the downstream IGF2-ERK1/2 signaling pathway.
Animals ; Mice ; Vascular Calcification/metabolism* ; Mice, Inbred C57BL ; Epoxide Hydrolases/metabolism* ; Diabetes Mellitus, Experimental/genetics* ; Male ; Gene Deletion ; MAP Kinase Signaling System/genetics* ; Cell Line ; Immunohistochemistry ; Muscle, Smooth, Vascular/metabolism* ; Signal Transduction/genetics* ; Mice, Knockout

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 investigate the clinical significance of CD105 and erythropoietin-producing hepatocellular receptor A2 (EPHA2) expressions in breast invasive ductal carcinoma and the effects of the combination of diabetes mellitus on CD105 and EPHA2 expressions.Methods:A retrospective case series study was conducted. A total of 74 patients with breast invasive ductal carcinoma in Affiliated Hospital of Jiangsu University from June 2019 to June 2024 were selected, and paraffin specimens from the patients after surgery were collected. Immunohistochemistry SP method was used to detect the expressions of CD105 and EPHA2 proteins in specimens. CD105 expression was expressed as the number of CD105 labeled microvessels, and EPHA2 expression was expressed as the proportion of EPHA2 positive expression area. The correlation of CD105 and EPHA2 expressions with the clinicopathological characteristics and diabetes mellitus of patients, as well as the relationship between the expressions of CD105 and EPHA2 were analyzed. Cox proportional hazards model was used to make univariate and multivariate analysis of the factors influencing overall survival of patients.Results:All 74 patients were female. The median age was 60 years old, 44 patients (59.46%) had tumor grade ≥ grade 3, 36 patients (48.65%) had tumor diameter ≥ 2 cm, 28 patients (37.84%) had lymph node metastasis, 30 patients (40.54%) had nerve vessel invasion, and 32 patients (43.24%) had diabetes mellitus. There were statistically significant differences in the proportion of patients with different age, tumor diameter, TNM stage, lymph node metastasis or not and nerve vessel invasion or not between diabetes mellitus group and non-diabetes mellitus group (all P < 0.05). The number of CD105 marking the microvessel was (32±9) and (24±8), respectively in diabetes mellitus group and non-diabetes mellitus group, and the difference was statistically significant ( t = 3.63, P < 0.010); the positive expression area proportion of EPHA2 was (19±5)% and (15±4)%, respectively, and the difference was statistically significant ( t = 3.85, P < 0.010). The expression of CD105 was related to the duration of diabetes mellitus, tumor diameter, TNM stage and lymph node metastasis or not (all P < 0.05), and the expression of EPHA2 was related to tumor diameter and TNM stage (all P < 0.05). Pearson correlation analysis showed a positive correlation between CD105 and EPHA2 expression ( r = 0.75, P < 0.001). The differences in overall survival of patients with or without diabetes mellitus and patients with different CD105 and EPHA2 expressions were statistically significant (all P < 0.05). Multivariate analysis showed that CD105 expression ( HR = 1.10, 95% CI: 1.04-1.16, P = 0.001) and EPHA2 expression ( HR = 1.35, 95% CI: 1.10-1.66, P = 0.005) were independent factors influencing the overall survival of patients with breast invasive ductal carcinoma. Conclusions:The expressions of CD105 and EPHA2 are independent prognostic factors in patients with breast invasive ductal carcinoma, and diabetes mellitus can promote the expressions of CD105 and EPHA2, which may increase the risk of poor prognosis.

BACKGROUND:The highest incidence of spinal fracture is in the thoracolumbar segment,and its symptoms are back pain,posterior convexity deformity,activity limitation,or with spinal cord nerve injury causing lower limb pain,numbness,and even paraplegia and other complications.The finite element method is a digital computer modeling technique,which can simulate the physical model and carry out force analysis realistically.OBJECTIVE:To review the application of finite element method in thoracolumbar spine fractures.METHODS:We searched the Chinese and English literature databases PubMed,Web of Science,and CNKI for relevant literature on the application of the finite element analysis method in spinal thoracolumbar fracture published before March 2024.The search terms in Chinese and English were:finite element analysis methods,biomechanical phenomena,stress analysis,thoracolumbar fractures,spinal fractures.Finally,55 papers were included.RESULTS AND CONCLUSION:(1)The exploration of thoracolumbar fractures caused by different etiologies(osteoporotic,traumatic,and pathological)through the finite element method is conducive to a deeper understanding of the biomechanics of various types of thoracolumbar fractures,and to improve the individualized and fine-tuned treatment of thoracolumbar fractures.(2)The finite element analysis of a single sample or a small number of samples has the chance,and a larger number of samples are required for the future finite element analysis to reduce the chance caused by the sample.(3)The rigid structure of bones alone cannot meet the biomechanical working conditions of the integrity of the physical object,and future finite element models need to incorporate all the structures of the physical object(e.g.,soft tissues,such as muscles and ligaments)as far as possible.(4)The finite element method has been used in more studies on osteoporotic and traumatic thoracolumbar spine fractures,which will need to be more in-depth in the future,and less in the field of pathologic thoracolumbar fractures,which has a wider scope for future research.

Objective:To explore the potential role of lipoprotein-associated phospholipase A2 (Lp-PLA2) and components of metabolic syndrome (MS) in the early progression of carotid arteriosclerosis.Methods:The study was a cross-sectional study. Urban participants undergoing routine health check-ups were enrolled from all 11 prefecture-level cities in Zhejiang Province between January and December 2022. General clinical information was obtained through interviews, and data on MS was collected from the clinical health examinations. Serum Lp-PLA? levels were measured in all participants. All participants were divided into 3 groups according to the results of the carotid ultrasound: the normal group, the intima thickening group with carotid intima thickening change and the plaque group. Multivariable logistic regression models were used to evaluate the associations of Lp-PLA2, MS, and the components of MS with early carotid atherosclerosis. Receiver operating characteristic (ROC) curve analyses were performed to assess the diagnostic performance of combining Lp-PLA2 with MS and the cumulative number of MS components for early carotid atherosclerosis.Results:A total of 4 009 urban adults undergoing routine health check-ups were enrolled (mean age (48.9±8.46) years, 2 665(66.5 %)male). Of these, 1 398 were in the normal group, 1 650 in the intima thickening group, and 961 in the plaque group. Multivariable logistic regression demonstrated that Lp-PLA2 was independently associated with early carotid atherosclerosis ( OR=1.34, 95% CI: 1.11-1.63, P=0.003). Lp-PLA2 also showed independent positive associations with both carotid intima thickening and carotid plaque formation, with the latter being more pronounced (both P<0.05). MS was independently and positively associated with early carotid atherosclerosis ( OR=1.48, 95 % CI: 1.20-1.84, P<0.001), as well as with intima thickening and carotid plaque formation, with the association being stronger for the latter (both P<0.05). Furthermore, the strength of the association increased progressively with the number of MS components ( P<0.001), especially for carotid plaques formation (both P<0.001). Multivariable logistic regression revealed that, compared with individuals without MS and low Lp-PLA2 levels, the risk of early carotid atherosclerosis was increased in those with high Lp-PLA2 alone, MS alone, or both conditions concurrently, with the highest risk observed when both were present (all P<0.05). ROC analyses demonstrated that the combination of elevated Lp-PLA2 with 3, 4, or 5 MS components yielded good diagnostic performance for early carotid atherosclerosis ( AUC=0.869, 0.888, and 0.889, respectively), intima thickening ( AUC=0.844, 0.860, and 0.845, respectively), and carotid plaque formation ( AUC=0.899, 0.924, and 0.968, respectively) in urban health-screening participants. Conclusions:Lp-PLA2, MS, and the number of MS components were independently and positively associated with early carotid atherosclerosis in urban health chek-up populations. The combination of MS components and Lp-PLA2 provided favorable diagnostic performance for the detection of early carotid atherosclerosis.