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.

Objective:To detect the expression levels of 12 cytokines in the plasma of patients with primary biliary cholangitis (PBC) and explore their association with PBC disease activity and ursodeoxycholic acid (UDCA) therapeutic response.Methods:This study enrolled 127 patients with PBC who visited Peking Union Medical College Hospital between December 2021 and November 2023 (PBC group) and 32 healthy controls who underwent physical examinations during the same period (control group). The expression of 12 cytokines was measured using flow cytometry, and compared between groups. Spearman correlation analysis was performed to assess the relationship between cytokine levels and five laboratory indicators reflecting PBC disease activity [alkaline phosphatase (ALP), glutamyl transpeptidase (GGT), total bilirubin (TBil), aspartate aminotransferase (AST), and total bile acid (TBA)]. Furthermore, the receiver operating characteristic (ROC) curve analysis was performed to evaluate the effectiveness of cytokines in distinguishing between patients who responded to UDCA treatment and those who did not.Results:The plasma interleukin (IL)-8 levels between the PBC group and healthy controls showed no significant differences, and the plasma IFN-γ levels in PBC patients were significantly higher than those in health controls ( P<0.05). Spearman analysis showed that IL-8 was positively correlated with ALP, GGT, TBil, AST, and TBA ( R2=0.348, 0.401, 0.406, 0.495, 0.417; all P<0.01), and negative correlation was observed between IFN-γand ALP, GGT, and TBA levels ( R2=-0.265, -0.253, -0.232; all P<0.05). The plasma IL-8 level in 52 PBC patients who did not respond to UDCA treatment was significantly higher, while the IFN-γ level was significantly lower than those in 56 PBC patients who responded to UDCA treatment (both P<0.05). ROC analysis showed that the area under the curve for distinguishing plasma IL-8 and IFN-γlevels between PBC patients responding to UDCA treatment and not is 0.631 and 0.783, respectively. The sensitivities were 87.5% and 90.5%, and the specificities were 44.2% and 57.9%, respectively. Conclusion:The levels of plasma IL-8 and IFN-γ are correlated with the disease activity of PBC and can be used to reflect the therapeutic responses to UDCA in PBC patients.

Objective:The performance of GeneXpert MTB/RIF (Xpert), DNA isothermal amplification fluorescence assay (DNA isothermal amplification) and RNA simultaneous amplification and testing (SAT-TB) were evaluated by detecting Mycobacterium tuberculosis in the pus samples of superficial tuberculous lymphadenitis and skeletal tuberculosis, two common extrapulmonary tuberculosis. Methods:Cross-sectional study. A total of 242 patients with suspected superficial tuberculous lymphadenitis and skeletal tuberculosis admitted to Shaanxi Provincial Tuberculosis Prevention and Control Hospital were collected from January 2022 to December 2023 and pus samples were taken from the lesions for examination. Among them, 210 patients were laboratory confirmed or clinically diagnosed with tuberculosis, 108 patients with superficial tuberculous lymphadenitis and 102 patients with skeletal tuberculosis. 32 patients without tuberculosis. Mycobacterium Tuberculosis culture, Xpert, DNA isothermal amplification, and SAT-TB detection were performed on the pus samples of all patients. The detection results were statistically analyzed using SPSS26.0 and MedCalc software. The detection effectiveness of several different detection methods for Mycobacterium tuberculosis in pus samples was compared, and P<0.05 was considered statistically significan. Results:The sensitivity of Mycobacterium Tuberculosis culture, Xpert, DNA isothermal amplification and SAT-TB in TB patients were 25.2% (53/210), 88.6% (186/210), 81.9% (172/210) and 61.0% (128/210), respectively. The area under curve (AUC) values of the four detection methods in the diagnosis of tuberculosis were 0.626, 0.943, 0.910 and 0.805, respectively. The AUC value of tuberculosis culture in the diagnosis of tuberculosis was significantly lower than that of the three molecular diagnostic techniques ( P<0.05). The AUC value of DNA isothermal amplification was significantly higher than that of SAT-TB detection method ( P<0.05). The positive tuberculosis culture was used as a reference standard where the sensitivity of Xpert, DNA isothermal amplification and SAT-TB reached 96.2%(51/53), 90.6%(48/53) and 86.8%(46/53), respectively. There was no significant difference in sensitivity among the three molecular diagnostic techniques ( P>0.05). In Xpert MTB positive patients, the positive rate of DNA isothermal amplification was 90.3% (168/186), and there was good consistency between DNA isothermal amplification and Xpert MTB detection results ( Kappa=0.765, P<0.001). Xpert detected 27 cases of rifampicin resistance, the resistance rate was 12.9% (27/210). Conclusion:GeneXpert and DNA isothermal amplification have high sensitivity and specificity in the samples of extrapulmonary tuberculosis pus, and the results of DNA isothermal amplification and GeneXpert MTB/RIF detection are highly consistent. In tuberculosis screening, DNA isothermal amplification fluorescence detection can be used for preliminary screening to reduce the detection cost.

BACKGROUND:Slow bone repair and poor bone formation quality are still problems during masquelet technique in the treatment of large segment bone defects.H-type vessels can induce osteogenesis,enhance the local angiogenesis and osteogenesis coupling,and promote bone repair.However,there are few reports on the role of H-type blood vessels in the bone induced membrane.OBJECTIVE:To construct a large segment bone defect model of SD rat tibia,observe the expression characteristics of H-type blood vessels in the bone induced membrane,then to identify the expression peak point of H-type blood vessels in the bone induced membrane and determine the optimal period of bone grafting.METHODS:Sixty SD rats were randomly divided into a control group(n=30)and a model group(n=30)by random number table method.The two groups were further divided into three subgroups at 4,6,and 8 weeks after bone cement implantation,with 10 rats in each group.A 4 mm bone defect model of the right tibia was constructed in both the control and the model groups.Polymethyl methacrylate bone cement was implanted in the model group to induce bone biomembrane formation,while bone cement was not implanted in the control group.At 4,6,and 8 weeks after bone cement implantation,6 rats were randomly selected at each time point.The bone induction membrane tissue was cut from the model group,and the non-bone soft tissue of the corresponding part was cut from the control group.The dynamic expressions of H-type blood vessels in the bone induced membrane were identified by immunofluorescence.The morphological changes of the bone induced membrane were observed by hematoxylin-eosin staining.The formation of blood vessels in the bone induced membrane was observed by angiography.The expression levels of osteoblast-specific transcription factor in the bone induced membrane were detected by immunohistochemistry.Four rats remained at each time point.In the model group,the bone induced membrane was cut open and the bone cement was removed and autologous coccyx was implanted.In the control group,autologous coccyx was implanted in the bone defect area.Micro-CT evaluation of the tibial defect was performed 8 weeks after bone grafting.RESULTS AND CONCLUSION:(1)Immunofluorescence staining showed that the expression of H-type vessels in the model group was most obvious 6 weeks after bone cement implantation,and the expression of H-type vessels in the model group at each time point after bone cement implantation was higher than that in the control group(P＜0.05).(2)Hematoxylin-eosin staining and angiography showed that the number and volume of new blood vessels at each time point after bone cement implantation in the model group were greater than those in the control group(P＜0.05).The order of the number and volume of new blood vessels in the model group was:8 weeks after bone cement implantation＞6 weeks after bone cement implantation＞4 weeks after bone cement implantation.(3)Immunohistochemical staining showed that the positive expression of osteoblast-specific transcription factors at each time point after bone cement implantation in the model group was higher than that in the control group(P＜0.05),and the positive expression of osteoblast-specific transcription factors in the model group was most obvious 6 weeks after bone cement implantation.(4)Micro-CT detection showed that the bone repair effect of the three subgroups in the model group was significantly better than that of the corresponding subgroups in the control group,and the bone repair effect of the subgroup in the model group 6 weeks after bone cement implantation was better than that of the subgroups 4 and 8 weeks after bone cement implantation.The results indicate that H-type blood vessels are dynamically expressed in the bone induced membrane and reached a peak 6 weeks after bone cement implantation.Good bone repair effects can be obtained by the bone induced membrane bone grafting 6 weeks after bone cement implantation.

Objective To investigate the predictive value of pulmonary artery(Pa)to aortic(Ao)diameter ratio(Pa/Ao)for long-term major adverse cardiovascular events(MACEs)in patients with acute decompensated heart failure(ADHF).Methods ADHF patients hospitalized in the Department of Cardiology of Nanjing Drum Tower Hospital from January 2018 to January 2023 were consecutively enrolled.The data of gender,age,past medical history,laboratory examination,echocardiography,chest CT and medication were collected.The diameters of Pa and Ao were measured at the bifurcation of main pulmonary artery on chest CT,and Pa/Ao was calculated.The Kaplan-Meier method was used for survival analysis,and the Log-rank test was used to compare the survival rate between the two groups.Cox proportional hazards regression model was used to analyze the association between Pa/Ao and MACEs,and subgroup analysis was performed according to different age,sex,BMI,B-type natriuretic peptide level,and left ventricular ejection fraction.Results A total of 600 ADHF patients were enrolled,with an average age of 69.6 years and 347(57.8%)males.During a median follow-up of 306(127,624)days,327(54.5%)patients experienced MACEs.The ADHF patients were divided into Pa/Ao<0.93 group and Pa/Ao≥0.93 group according to the analysis of maximum selection rank statistics.Kaplan-Meier curve showed that the incidence of MACEs in Pa/Ao≥0.93 group was significantly higher than that in Pa/Ao<0.93 group(PLog-rank<0.001).Multivariate Cox regression analysis showed that Pa/Ao was an independent predictor of MACEs in ADHF patients(HR=11.62,95%CI:4.91～27.50,P<0.001).Subgroup analysis showed that Pa/Ao had predictive value for different ADHF populations(all P<0.05).Conclusion Elevated Pa/Ao is a predictor of long-term MACEs in ADHF patients.

OBJECTIVE: Recombination events are common and serve as the primary driving force of diverse human adenovirus (HAdV), particularly in children with acute respiratory tract infections (ARIs). Therefore, continual monitoring of these events is essential for effective viral surveillance and control. METHODS: Respiratory specimens were collected from children with ARIs between January 2022 and December 2023. The penton base, hexon, and fiber genes were amplified from HAdV-positive specimens and sequenced to determine the virus type. In cases with inconsistent typing results, genes were cloned into the pGEM-T vector to detect recombination events. Metagenomic next-generation sequencing (mNGS) was performed to characterize the recombinant HAdV genomes. RESULTS: Among 6,771 specimens, 277 (4.09%, 277/6,771) were positvie for HAdV, of which 157 (56.68%, 157/277) were successfully typed, with HAdV-B3 being the dominant type (91.08%, 143/157), and 14 (5.05%, 14/277) exhibited inconsistent typing results, six of which belonged to species B. The penton base genes of these six specimens were classified as HAdV-B7, whereas their hexon and fiber genes were classified as HAdV-B3, resulting in a recombinant genotype designated P7H3F3, which closely resembled HAdV-B114. Additionally, a partial gene encoding L1 52/55 kD was identified, which originated from HAdV-B16. CONCLUSION A novel recombinant, P7H3F3, was identified, containing sequences derived from HAdV-B3 and HAdV-B7, which is similar to HAdV-B114, along with additional sequences from HAdV-B16.
Humans ; Adenoviruses, Human/isolation & purification* ; Respiratory Tract Infections/epidemiology* ; Child, Preschool ; Child ; Recombination, Genetic ; Male ; Beijing/epidemiology* ; Infant ; Female ; Phylogeny ; Adenovirus Infections, Human/epidemiology* ; Acute Disease ; Genome, Viral

Objective:To explore the epidemiological characteristics, clinical manifestations, laboratory findings, and imaging features of children with HRV-associated pneumonia, and to analyze the clinical features and risk factors associated with severe HRV pneumonia, providing references for clinical management.Methods:A single-center, retrospective, observational study was conducted, including 1 001 cases of HRV-positive children with pneumonia admitted to the Respiratory Department of the Affiliated Children′s Hospital of Capital Institute of Pediatrics from January 2019 to December 2023. Among them, 584 cases (58.3%) were male and 417 cases (41.7%) were female, with an age range of 0.1 to 14.9 years, a median age of 3.42 years, and a mean age of (3.92±2.75) years. According to clinical guidelines, the cases were divided into a mild pneumonia group (855 cases, 510 males, 345 females) and a severe pneumonia group (146 cases, 73 males, 73 females). Basic information, clinical, laboratory, and imaging data were collected from the electronic medical record system. Comparisons between different age groups, diagnoses, and pneumonia severity groups were performed using the χ2 test. Multivariate logistic regression analysis was used to identify risk factors for the severity of HRV pneumonia. Results:Among the 1 001 cases of HRV-associated bronchopneumonia, 146 cases (14.6%) were severe pneumonia. The age of severe HRV pneumonia patients was significantly higher than that of the mild pneumonia group (5.2 years vs. 3.7 years, t=-6.050, P<0.01). Severe HRV pneumonia had a higher incidence in autumn and winter (60.9%). Severe HRV pneumonia was associated with higher levels of lactate dehydrogenase (LDH), C-reactive protein (CRP), neutrophils, and creatinine (correlation coefficients 0.198, 0.334, 0.104, 0.142, P<0.01), and lower levels of albumin (correlation coefficient 0.308, P<0.01). Multivariate logistic regression analysis showed that co-infection with Streptococcus pneumoniae or Mycoplasma was an independent risk factor for severe HRV pneumonia [ OR=1.611, 95% confidence interval ( CI):1.066-2.435, P<0.05; OR=3.355, 95% CI:2.062-5.458, P<0.01]. Conclusion:The infection rate of HRV is higher in preschool and school-age children. Severe HRV pneumonia is associated with increased levels of LDH, CRP, neutrophils, and creatinine, as well as decreased levels of albumin. Co-infection with Streptococcus pneumoniae or Mycoplasma may be an independent risk factor for severe HRV pneumonia. High-risk children require enhanced monitoring and early intervention to improve prognosis.

Objective To discuss the correlation between free prostate specific antigen(fPSA),serum ferritin(SF),blood uric acid(SUA)levels and Gleason grading in patients with prostate cancer(PCa).Methods The clinical data of 61 patients with prostate biopsy treated in 411 Hospital of Shanghai University from January to December of 2023 were retrospectively analyzed.According to the results of puncture,the patients were divided into benign prostatic hyperpla-sia(BPH)group(31 cases)and PCa group(30 cases).The levels of fPSA,SF and SUA in patients and Gleason grade in biopsy cases were analyzed.The correlation between fPSA,SF and SUA levels and Gleason grade was analyzed by the method of Spearman.And the diagnostic efficacy of fPSA,SF and SUA levels on PCa was analyzed by receiver operating characteristic curve(ROC).Results The levels of fPSA,SF and SUA in PCa group were significantly higher than those in BPH group(P＜0.05).There were statistically significant differences in levels of fPSA SF and SUA in PCA patients with different Gleason grades(P＜0.05).With the specificity reaching 96.08％and sensitivity reaching 94.35％,the ar-ea under the curve(AUC)of the combined fPSA,SF and SUA levels in the diagnosis of PCa was 0.982,which was higher than that of the single fPSA,SF and SUA levels(P＜0.05).SF and SUA levels in PCa patients were positively correlated with Gleason grade(P＜0.05),while fPSA levels were not correlated with Gleason grade(P＞0.05).Conclusion The levels of SF and SUA in PCa patients are positively correlated with Gleason grade,which can be used as an important index to predict Gleason grade in PCa patients.

To construct a recombinant fowl adenovirus 4(FAdV-4)expressing the Cap protein of goose astrovirus genotype 2(GoAstV-2),the expression cassette of Cap gene was inserted into the natural 1 966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cm-FAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line(LMH)to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rF Ad V4-Cap/GoAstV-2.After 15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GoAstV-2 infected LMH cells as template.LMH cells were in-fected with 15th passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15th passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant vi-rus rF AdV4-Cap/Go Ast V-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-in-fection of FAdV-4 and GoAstV-2 in goose.

OBJECTIVE To evaluate the value of matrix-assisted laser desorption ionization time-of-flight-mass spectrometry(MALDI-TOF MS)in direct identification of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae positive for blood culture.METHODS The blood culture bottles that were positive for E.coli or K.pneumoniae were collected from the patients with bloodstream infection who were treated in Zhongshan Hospi-tal,Fudan University from Jul.2021 to Jun.2023.The isolates were collected by using a gel-contact clotting tube,then the tested strains were respectively mixed with 4 μg/ml of imipenem,4 μg/ml of meropenem and 2 μg/ml of ertapenem for coculture and were incubated at 35 ℃ for 4 and 5 hours,finally,the strains were i-dentified by using the mass spectrum.The minimum inhibitory concentrations(MICs)of the three types of drugs were tested by microbroth dilution method and were set as the golden standards for the test.RESULTS Totally 31 strains of E.coli and 28 strains of K.pneumoniae that were positive in blood culture bottles were collected,both of the effective rates of controlled growth of the E.coli strains were 100.00％after the incuba-tion for 4 and 5 hours,and the effective rates of controlled growth of the K.pneumoniae strains were 9 6.43％and 100.00％after the incubation for 4 and hours,respectively.The sensitivity,specificity,positive predictive value and negative predictive value of the K.pneumoniae strains against the three types of drugs were 100.00％after the incubation for 5 hours.The specificity and positive predictive value of the E.coli strains against imipenem,meropenem and ertapenem were 100.00％after the incubation for 5 hours;the sensitivities were 73.58％,78.93％and 78.93％,respectively;the negative predictive values were 70.60％,75.00％and 75.00％,respectively.CONCLUSIONS MALDI-TOF MS is rapid and accurate for direct identification of the carbapenem-resistant E.coli and K.pneumoniae strains positive in blood culture bottles,and the accuracy reaches at 100.00％for the test of drug-resistant K.pneumoniae strains after the incubation for 5 hours.The method may provide evidence for clinical treatment of bloodstream infections induced by carbapenem-resistant Enterobacteriaceae.