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: and Purpose Limited evidence exists on the effectiveness of endovascular treatment (EVT) for acute posterior circulation tandem lesion (PCTL). This study aimed to explore the role of extracranial vertebral artery (VA) stenting in patients with PCTL stroke undergoing EVT. Methods: Individual patient data were pooled from the BASILAR (EVT for Acute Basilar Artery Occlusion Study) and PERSIST (Posterior Circulation Ischemic Stroke) registries. Patients with PCTLs who underwent EVT were included in the present cohort and divided into the stenting and nonstenting groups based on the placement of extracranial VA stents. The primary efficacy outcome was the modified Rankin Scale (mRS) scores at 90 days and 1 year. Safety outcomes included 24-hour symptomatic intracranial hemorrhage (sICH) and all-cause mortality at 90 days and 1 year post-surgery. Results: A combined dataset of 1,320 patients with posterior circulation artery occlusion, including 263 (19.9%) with tandem lesions, of whom 217 (median age, 65 years; 82.9% male) met the inclusion criteria for the analysis. The stenting group had 84 (38.7%) patients, while the non-stenting group had 133 (61.3%). After adjustment for the potential confounders, extracranial VA stenting was associated with favorable shifts in mRS scores at both 90 days (adjusted common odds ratio [OR], 2.30; 95% confidence interval [CI], 1.23–4.28; P<0.01) and 1 year (adjusted OR [aOR], 2.04; 95% CI [1.05–3.97]; P=0.04), along with lower rate of mortality at both 90 days (aOR, 0.45; 95% CI [0.21–0.93]; P=0.01) and 1 year (aOR, 0.36; 95% CI [0.16–0.79]; P=0.01), with no significant difference in sICH incidence (aOR, 0.35; 95% CI [0.06–1.98]; P=0.24). Conclusion Extracranial VA stenting during EVT may improve functional outcomes and reduce mortality in patients with PCTL strokes.

Objective To verify the applicability of a pig STR multiplex amplification system for detecting processed foods containing pork and their digestive samples,and to evaluate its potential in food safety and forensic biological evidence analysis.Methods DNA profiles were obtained using the pig STR amplification system from food samples with different levels of processing(raw pork,boiled pork,fried pork,and sausage)and from digestive samples(rat gastric contents).The influence of processing methods on DNA integrity was assessed.The uniformity of large-scale processed ham products,the consistency of DNA profiles from different parts of the same sample,and the DNA degradation patterns after rat digestion were examined.Results STR profiling of pig DNA was successful in all tested samples.Short fragments showed high amplification stability,while long fragment signals weakened with increasing processing complexity.In processed ham products,DNA profiles were consistent across all sampled parts,with fragment drift within±0.5 bp.Analysis of rat gastric contents showed slight DNA degradation within 2 hours;after 3 hours,long fragment signals weakened,and after 4 hours,some loci signals were lost.Conclusion The pig STR multiplex amplification system exhibits excellent performance in detecting processed pork products and their digestive samples.It can meet the requirements of food traceability and forensic biological evidence analysis for processed pork,providing new insights for the advancement of forensic testing techniques in this field.

OBJECTIVE To observe the effect of acupuncture combined with a modified Zhengan Xifeng Decoction on the early clinical symptoms and oxidative stress status of patients with hypertensive intracerebral hemorrhage(HICH)of liver-kidney yin defi-ciency syndrome.METHODS A total of 60 patients with hypertensive intracerebral hemorrhage(liver-kidney yin deficiency syn-drome)who successfully received 24-hour acute-phase treatment at the Department of Neurosurgery,Acupuncture and Rehabilitation Department,and wards of Kunshan Hospital Affiliated to Nanjing University of Chinese Medicine(Kunshan Traditional Chinese Medi-cine Hospital)from July 2021 to January 2023 were recruited.A randomization table generated by SPSS 22.0 software was used to di-vide the patients into a control group and an observation group,with 30 patients in each group.The control group received symptomatic treatment,conventional rehabilitation training,and modified Zhengan Xifeng Decoction,while the observation group received additional acupuncture treatment on the basis of the control group.The treatment course was 1 month for both groups.Clinical effective rate,TCM syndrome scores,motor and neurological function scores[Activity of Daily Living(ADL)scale,National Institutes of Health Stroke Scale(NIHSS),Modified Ashworth Scale(MAS),Simplified Fugl-Meyer Assessment(FMA)scale],improvement in hematoma lesions,Fraction anisotropy(FA)ratio(rFA),and serum levels of antioxidant stress response factors Kelch-like ECH-associated protein 1(Keap1)and nuclear factor-E2-related factor 2(Nrf2)were observed before and after treatment in both groups.RESULTS After treat-ment,the clinical effective rate in the observation group was significantly higher than that in the control group(P<0.05);the TCM syn-drome scores in both groups decreased significantly(P<0.05),with the observation group showing better results than the control group(P<0.05);the ADL and FMA scores in both groups reduced significantly(P<0.05,P<0.01),while the NIHSS and Ashworth Scale scores increased significantly(P<0.05,P<0.01),with the observation group showing better improvement than the control group(P<0.05);serum Keap1 levels decreased significantly in both groups(P<0.01),while Nrf2 levels enhanced significantly(P<0.05,P<0.01),with the observation group showing better improvement than the control group(P<0.05);imaging studies showed that after treat-ment,the residual hematoma volume decreased significantly in both groups(P<0.05,P<0.01),and the rFA value increased signifi-cantly(P<0.05,P<0.01),with the observation group showing better results than the control group(P<0.01).CONCLUSION On the basis of standard treatment for the acute phase,acupuncture combined with Zhengan Xifeng Decoction can significantly improve the early clinical symptoms of HICH patients with liver-kidney yin deficiency,reduce oxidative stress levels,and promote the recovery of nerve and motor functions.

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.

In order to understand the infection status of Escherichia coli(E.coli)in different popu-lations of chickens,E.coli was isolated and identified from 1-day-old chickens,dead embryos,clini-cal dead chickens from 3 hatcheries and 9 broiler farms in Yantai area,and 1 950 samples from e-liminated breeder chickens;the homology of the isolated strains was analyzed by pulsed field gel e-lectrophoresis;virulence genes and serotypes were identified by PCR;pathogenicity was determined by challenge chicken embryos.Drug resistance of the strain was determined by drug sensitivity test.Results showed that a total of 116 strains of E.coli were isolated,of which 97 strains were successfully classified into 66 band types,15 virulence genes and 5 serotype were detected by PCR,and 56,32 and 28 strains were divided into high,medium and low pathogenic strains by chicken embryo challenge test.Results indicated that E.coli from clinically dead chickens and eliminated breeder chickens had higher pathogenicity and drug resistance.This study provides a reference ba-sis for the prevention and control of colibacillosis in chicken farms.

Objective:To analyze the clinical characteristics of pulmonary mucormycosis (PM) in children with leukemia, and explore its diagnosis and treatment strategies and prognosis.Methods:In this case-series study, the clinical data of 19 children who were diagnosed with leukemia complicated by PM at the First Affiliated Hospital of Zhengzhou University from January 2020 to June 2024 were retrospectively analyzed.Their gender, age, type of leukemia, clinical manifestations, chest CT findings, bronchoscopy results, etiology, treatment regimens, and prognosis were summarized.The patients were divided into a survival group and a death group.The clinical data were compared between the 2 groups.Categorical variables were compared using the Fisher′s exact test, and continuous variables were compared using the Wilcoxon rank-sum test.Results:A total of 19 children aged 2 to 17 years with leukemia complicated by PM were involved, including 9 boys and 10 girls, with most cases (15 cases) occurring in the autumn and winter.The median time from the first leukemia chemotherapy to infection with mucor was 1 month.Clinical manifestations mainly included fever (100%), cough (94.7%), hemoptysis (42.1%), and pneumothorax (21.1%).Chest CT findings primarily showed extensive lung consolidation (84.2%), pleural effusion (84.2%), pulmonary nodules (78.9%), halo signs (73.7%), cavitation (36.8%), crescent signs (26.3%), and reverse halo signs (21.1%).Fourteen children underwent bronchoscopy, which primarily revealed tracheal obstruction, pale or congested mucosa, mucosal edema, mucosal necrosis, bronchial stenosis, and bronchial cavitary fistulas.Mucor was detected using various methods including metagenomic next-generation sequencing (mNGS), cultures, and lung tissue microscopy.Patients were mainly treated with intravenous administration of Amphotericin B in different formulations combined with oral Posaconazole.Three patients also underwent surgical resection of the affected lung lobe in addition to medical treatment.After treatment, 14 patients had a good prognosis, while 5 patients died.The causes of death were massive hemoptysis in 2 cases, severe respiratory failure in 1 case, and treatment withdrawn due to critical condition in 2 cases.There were no statistically significant differences in age, gender, type of leukemia, whether to undergo bronchoscopy and surgery, time from the first chemotherapy to onset of infection, presence of comorbid infection, chest CT characteristics, and time to start treatment between survival and death groups(all P>0.05). Conclusions:There is no specificity in clinical manifestations of leukemia complicated by PM, which, however, still exhibits some characteristics.mNGS plays a crucial role in early diagnosis.Intravenous administration of Amphotericin B combined with oral Posaconazole and surgical intervention are an effective treatment regimen.

Objective To propose a novel method for verifying the isocenter in radiotherapy based on markers and conduct a preliminary test. Methods A feasibility experiment was conducted on wooden box phantom for radiotherapy resetting. Fifteen groups of displacement data were randomly generated,corresponding to the position deviations of the isocenter in the radiotherapy plan relative to the original isocenter. According to each set of displacement data,with the aid of movable lasers and a CT scanning couch,CT scanning was performed with two sets of markers (3 per set) affixed to the phantom which were corresponding to the original and treatment isocenters,respectively. In the Eclipse treatment planning system,the coordinate data of the original and treatment isocenters were manually verified,and the difference of coordinate data was calculated to obtain the actual displacement value. The treatment isocenter position was finally confirmed by comparing with the actual displacement. In addition,the study attempts to use threshold segmentation algorithm to automatically detect metal markers and obtain coordinate values of the original isocenter on patient-positioned CT images. In the wooden box experiment,the absolute value of the difference between the actual displacement value and the planned displacement value (?d) was used to represent the position accuracy of treatment isocenter,and the deviation value obtained with threshold segmentation algorithm for isocenter detection was ?s. Results The ?d in the X (left-right),Y (superior-inferior) and Z (anterior-superior) directions was (0.83±0.37) mm,0 (0,0.5) mm and (0.45±0.29) mm,respectively. The ?s in the X,Y and Z directions was (0.46±0.22) mm,0 (0,0.5) mm and (0.33±0.29) mm,respectively. The mean values of ?s in 3 directions were all lower than 2 mm,within the range of permissible clinical positioning error. Conclusion The method of automatically verifying treatment isocenter position based on markers is feasible,and the study provides a useful reference for radiotherapy resetting using CT simulator.

Objective To assess the long-term prognosis of nonparoxysmal atrial fibrillation(NPAF)patients after radiofrequency catheter ablation(RFCA)and identify influencing factors.Methods A prospective,randomized controlled trial was conducted from October 2021 to June 2023 at our center.A total of 183 consecutive NPAF patients undergoing RFCA were randomly assigned to the ARNI group(n=91)or control group(n=92).Data collected included baseline characteristics,procedure-related parameters,low voltage areas(LVAs),left atrial mean pressure(LMP),and clinical outcomes at 12 months.The primary endpoint included freedom from atrial fibril-lation recurrence at 12 months post-ablation and secondary endpoints comprised complication rates,mean arterial pressure(MAP),left atrial diameter(LAD),and cardiac function.Results The two groups presented no significant differences in baseline characteristics(P>0.05).The ARNI group demonstrateda significantly higher rate of freedom from AF recurrence compared to the control group at 12 months(79.12%vs.66.30%,HR=0.56,P=0.039).More-over,the ARNI group showed significantly smaller LAD[(40.17±4.72)mm vs.(42.29±6.08)mm,P=0.013)and a significantly lower N-terminal proB-type natriuretic peptide(NT-proBNP)level[98.00(45.00,456.00)vs.128.50(73.50,586.50),P=0.039].Both groups showed no significant differences in complication rates or MAP post-ablation(P>0.05).Cox regression analysis identified ARNI as an independent predictor for maintaining freedom from AF recurrence at 12 months post-ablation(HR=0.524,P=0.038).Subgroup analyses showed that high LMP patients had a lower rate of freedom from AF recurrence than those with normal pressures(51.61%vs.83.47%,P<0.001),and demonstrated significantly improved ARNIoutcomes(64.52%vs.38.71%,P=0.042).Conclusion ARNI improves long-term outcomes for NPAF patients following RFCA,particularly in those with highLMP.

Type 2 diabetes mellitus(T2DM)is a chronic metabolic disorder caused by a combination of genetic and environmental factors.The epigenetic transcriptional modifications after RNA transcription can affect gene expression and glucose homeostasis.N6-methyladenine(m6A),as an RNA methylation modification,plays an important role in the pathogenesis of T2DM.This article reviews the research progress of m6A methylation in T2DM.