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.

To verify whether chaperones can promote the soluble expression of PlpE in Escherichia coli and whether the expressed protein is active,prokaryotic expression and Western blot detection were performed.The results showed that:The PlpE prokaryotic expression vector pET-32a(+)-plpE was expressed as inclusion body,and the expression form was not changed by changing the concentration of inducer,induction time and temperature.The companion proteins pG-KJE8,pGro7,pKJE7 and pG-Tf2 were co-expressed with pET-32a(+)-plpE in Eschierichia coli expres-sion system,respectively.When the final concentration of IPTG of 0.5 mmol/L,L-arabinose of 0.5 g/L or tetracycline of 5.0 μg/L were added as inducers and induced at 37 ℃ for 8 h,the results showed that the molecular companion pGro7 could change the expression of rp-PlpE from inclu-sion body to soluble expression.pG-KJE8,pKJE7 and pG-Tf2 had no effect on the expression of rp-PlpE.The soluble rp-PlpE can react specifically with the positive serum of Mannheimia haemolyti-ca.Therefore,the study showed that the co-expression of the chaperone protein pGro7 can make the rp-PlpE protein express in a soluble form,and the purified protein exhibits reactogenicity.These findings lay the foundation for the establishment of a subunit vaccine and serological diagno-sis methods for Mannheimia haemolytica.

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 explore the relationship between overactive bladder(OAB)and neuropsychological characteristics in patients with cerebral small-vessel disease(CSVD).Methods A total of 208 patients with CSVD admitted to the First Affiliated Hospital of Shihezi University from December 2023 to November 2024 were selected as research subjects.They were divided into OAB group(44 cases)and non-OAB group(164 cases)according to overactive bladder symptom score.The general information and imaging data of two groups of patients were compared.The cognitive function,anxiety and depression of patients were evaluated by using Montreal cognitive assessment(MoCA),digit span test(DST),auditory verbal learning test(AVLT),verbal fluency test(VFT),clock drawing test(CDT),Hamilton anxiety scale(HAMA)and Hamilton depression scale(HAMD).Logistic regression analysis was used to analyze the influencing factors of OAB in patients with CSVD.Pearson correlation analysis was used to analyze the correlation between imaging indicators and anxiety symptoms of patients in OAB group.Results The HAMA and HAMD scores of patients in OAB group were higher than those in non-OAB group(P＜0.05),but there were no statistically significant differences in MoCA,DST,AVLT,VFT,and CDT scores between two groups(P＞0.05).There was a statistically significant difference in the incidence of anxiety among patients with different degrees of OAB(P＜0.05).Multivariate Logistic regression analysis after adjusting for confounding factors showed that anxiety was an independent influencing factor for OAB in patients with CSVD(P＜0.05).The periventricular white matter hyperintensity of patients in OAB group was negatively correlated with the HAMA score(P＜0.05),while basal ganglia lacunar infarction,centrum semiovale lacunar infarction,basal ganglia enlarged perivascular space and centrum semiovale enlarged perivascular space were all positively correlated with the HAMA score(P＜0.05).Conclusion The anxiety and depression symptoms of patients with CSVD accompanied by OAB were more severe than those of non-OAB patients.Anxiety may be an independent influencing factor for the occurrence of OAB in patients with CSVD.

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 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 In this paper,the ethnic patent medicines(EPMs)in the Chinese Pharmacopeia,National Basic Medical Insurance,Employment Injury Insurance and Maternity Insurance Medicine List,and National Essential Medicine List(re-ferred to as"one pharmacopeia and two lists")were counted and analyzed.Methods The EPMs in"one pharmacopeia and two lists"were summarized,and the information of manufacturers,medicinal materials used in EPMs,and their current quality standards were collated,and their indicative function,adverse reactions,contraindications,and precautions were analyzed.The modern research level of the above EPMs was analyzed by searching relevant literature on CNKI and PubMed.Results A total of 142 EPMs in"one pharmacopeia and two lists"were included,and 68 of the EPMs had exclusive manufacturers and 98.75％of the medicinal materials had provincial-level or higher quality standards.There were issues such as the expression of indicative function in the instruction being mixed between medical terms,91.55％ of the EPMs lacking sufficient information on adverse re-actions,contraindications,and precautions,and modern basic research was weak.Conclusions In the development of EPMs,it is important to improve information on adverse reactions,contraindications,and precautions in the instructions.In expressing indic-ative function,it is necessary to avoid mixing medical terms,which is more conducive to the clinical promotion of EPMs.

Objective:To determine the prevalence of self-reported food allergies among children in the grasslands of North China and to analyze its associated risk factors.Methods:In this study, a cross-sectional epidemiological survey method was used to select children under 14 years old by multi-stage, stratified and random cluster sampling in the grassland ecological area of Zhangbei County, Hebei Province, China from May to July 2018. Face-to-face questionnaires were administered to gather food allergy-related information from the participants. Multivariate logistic regression analysis was used to analyze the risk factors associated with self-reported food allergy.Results:A total of 2 086 children completed the survey. The prevalence of self-reported food allergies was 22.0%(459/2 086). The prevalence of multiple food allergies (≥3 types) was 3.1%(64/2 086) versus 16.3% (341/2 086) for a single food allergy among all children. Mango allergy (6.1%, 127/2 086) was the most common, followed by peach allergy (4.1%, 85/2 086). Children who reported food allergies had a significantly higher prevalence of all 4 atopic disorders (eczema, asthma, allergic rhinitis, and allergic conjunctivitis than those without food allergies(35.73% vs. 20.65%, 5.88% vs. 2.77%, 17.86% vs. 7.38%, 16.78% vs. 10.45%, χ2 =44.663 1, 10.434 3, 45.038 3, 13.728 4, all P<0.001).Significantly associated risk factors of food allergy were found to be pollen allergy ( OR: 2.29; 95% CI: 1.80-2.92) and drug allergy ( OR: 1.53; 95% CI: 1.12-2.09). Conclusions:The prevalence of self-reported food allergies among children in the Zhangbei County area of the North China Grassland was relatively high. Pollen allergy and drug allergy are major risk factors.

OBJECTIVE To study the effects of Ro 64-6198,a selective nociceptin/orphanin FQ receptor(NOPR)agonist,on heroin self-administration and drug-seeking behavior in rats.METHODS Rats were trained to self-administer heroin intravenously at a dose of 0.05 mg·kg-1 under a fixed ratio 1(FR1)reinforcement schedule.Heroin motivation was assessed using a progressive ratio(PR)schedule.Firstly,a stable heroin self-administered rat model was established before the effects of Ro 64-6198 on heroin rewarding under the FR1 schedule were observed.After three days of self-administration recovery training,the effects of Ro 64-6198 on heroin reward motivation were observed under the PR3-4 schedule.Following extinction,the reinstatement of heroin seeking induced by either conditioned cues or heroin priming was evaluated in rats withdrawn from self-administration.The expressions of cAMP response element-binding protein(CREB)and brain-derived neurotrophic factor(BDNF)in the ventral tegmental area(VTA)were analyzed using Western blotting,while the expression of the NOPR in neurons in the VTA was examined through immunofluorescence staining.RESULTS Pretreatment with 3 mg·kg-1 Ro 64-6198 significantly reduced active responses and heroin infusions during FR1 testing,as well as decreased breakpoints,indicating reduced motivation under the PR schedule.At a dose of 1 mg·kg-1,Ro 64-6198 markedly attenuated the reinstatement of heroin-seeking behavior induced by conditioned cues or heroin priming.Furthermore,the administration of SB-612111,an NOPR antagonist,blocked the inhibitory effects of Ro 64-6198 on cue-induced heroin-seeking,although SB-612111 alone had no effect on heroin-seeking behavior.Ro 64-6198 treatment also suppressed the reduction of both phos-phorylated CREB(p-CREB)and BDNF levels in the VTA and the decreased expression of NOPR and p-CREB in dopaminergic neurons of the VTA.CONCLUSION These results demonstrate that Ro 64-6198 can mitigate heroin-seeking behavior through NOPR activation and CREB/BDNF pathway in the VTA.This study is expected to offer evidence for its potential as a clinical treatment for heroin addiction and relapse.