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 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 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.

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.

Objective To analyze the dynamic change in placental growth factor(PLGF)level in patients with preeclampsia(PE)and its role in predicting pregnancy outcomes.Methods A retrospective cohort study was conducted on 198 PE patients admitted to Department of Obstetrics of Tianjin Beichen Hospital from January 2021 to January 2024.Among them,151 cases were identified as non-severe PE and 47 cases as severe PE.During the same period,another 100 healthy pregnant women were included in the department and served as the control group.According to the final pregnancy status of the PE patients,they were divided into an adverse outcome group(88 cases)and a good outcome group(110 cases).Locally Weighted Scatterplot Smoothing(LOWESS)was used to analyze the relationship between vascular endothelial function indicators and PLGF level.Logistic regression was employed to identify the influencing factors for adverse pregnancy outcomes.Receiver operating characteristic(ROC)curve was plotted to evaluate the value of PLGF level for predicting adverse pregnancy outcomes in patients with non-severe PE and severe PE.Results The PLGF level was in a peak shape at different gestational weeks,relatively low at 28～29+6 weeks of gestation,gradually increasing with the increment of gestational weeks,reaching a peak at 30～31+6 weeks of gestation,and then gradually decreased(P<0.05).The PLGF level was gradually reduced in the control group,non-severe PE group,and severe PE group in turn(P<0.05).LOWESS analysis showed that the PLGF level exhibited certain nonlinear relationship with soluble fms-like tyrosine kinase receptor 1(sFlt-1),vascular endothelial growth factor(VEGF),nitric oxide(NO),thrombomodulin(TM),thromboxane A2(TXA2),and Von Willebrand factor(VwF).The results of logistic regression analysis indicated that after gradually eliminating collinearity confounding factors and adjusting for each covariate,there was still an independent correlation between the PLGF level at 28～29+6 gestational weeks and pregnancy outcome(OR=0.593,95%CI:0.583～0.778,P<0.001).After converting the PLGF level into a binary variable,there was an independent correlation between high PLGF level and pregnancy outcome(OR=0.773,95%CI:0.671～0.885,P<0.001).Compared with the quintile with the lowest PLGF(Q1),as the PLGF level gradually increased(Q2 to Q5),the correlation effect values were(OR=0.765,95%CI:0.639～0.837,P=0.092),(OR=0.752,95%CI:0.624～0.818,P=0.056),(OR=0.696,95%CI:0.615～0.813,P=0.027),and(OR=0.642,95%CI:0.591～0.733,P<0.001),and the trend test was statistically significant(Ptrend<0.001).ROC curve analysis revealed that the area under the ROC curve(AUC)of serum PLGF level at 28-29+6 gestational weeks in predicting adverse pregnancy outcomes in non-severe PE patients was 0.830(95%CI:0.635～0.917),the optimal critical threshold was 75.47 ng/L,the sensitivity was 88.37%,the specificity was 71.30%,the positive predictive value was 55.07%,and the negative predictive value was 93.90%.Its AUC value of the level for the prediction in patients with severe PE was 0.874(95%CI:0.677～0.923),the optimal critical threshold was 38.53 ng/L,the sensitivity was 88.89%,the specificity was 50.00%,the positive predictive value was 97.56%,and the negative predictive value was 16.67%.Conclusion At 28～29+6 gestational weeks,PLGF has an independent correlation with pregnancy outcomes and shows a good predictive performance for adverse pregnancy outcomes in patients with non-severe PE and severe PE.

BACKGROUND:There is a significant correlation between sperm DNA fragmentation index and fertilization,embryonic development potential,embryo implantation,miscarriage,and offspring safety.However,its clinical reference value is affected by many factors,resulting in extremely limited clinical significance.This study took live birth as the outcome,corrected other confounding factors through propensity score matching,constructed the best clinical cutoff value of sperm DNA fragmentation index and live birth,and conducted internal and external tests on it,which has good predictive value and clinical application efficiency. OBJECTIVE:To investigate the reference threshold and offspring short-term security of in vitro fertilization-embryo transfer sperm DNA fragmentation index based on live birth. METHODS:A total of 1 921 patients who received in vitro fertilization and embryo transfer in Changzhou Maternal and Child Health Area Hospital from May 2019 to May 2021 were selected.On the basis of tendency matching tolerance of 0.02 and propensity score matching of 1:1,540 cases were successfully matched in each live birth group and non-live birth group,and the model group was established.135 patients who received in vitro fertilization and embryo transfer in Nanxishan Hospital of Guangxi Zhuang Autonomous Region were selected as the external validation group.The optimal clinical cutoff value of sperm DNA fragmentation index for live birth was investigated by the receiver operating characteristic curve.The accuracy and clinical application efficacy of the cutoff value were evaluated by restricted cubic spline curve,standard curve,clinical decision curve,clinical impact curve and internal and external validation tests. RESULTS AND CONCLUSION:(1)The DNA fragmentation index of sperm in the non-live birth group was significantly higher than that in the live birth group and had a significant negative correlation with live birth(r=-0.444,P<0.001).(2)Receiver operating characteristic curve results showed that the optimal cut-off value of DNA fragmentation index for live birth was 24.33%;the area under the curve was 0.775(0.746,0.804);the specificity was 72.60%;the sensitivity was 78.90%,and the accuracy was 75.70%.(3)Restricted cubic spline curve fitting the results of Logistic regression showed that when the sperm DNA fragmentation index was greater than 24.57%,the risk of clinical non-live birth increased.(4)The probability of Logistic regression analysis results showed that sperm DNA fragmentation index was a risk factor for live birth[OR(95%CI)=0.916(0.904,0.928),P<0.001],and when sperm DNA fragmentation index was greater than 27.78%,the probability of clinical live birth would be less than 50%.With the increase of sperm DNA fragmentation index by 1 unit,the probability of a live birth fell by 8.4%.(5)Internal and external to the validation of the clinical cutoff value showed that the cutoff point had certain clinical predictive value and accuracy.(6)Clinical decision curve and clinical impact curve results exhibited that the prediction model based on the clinical cut-off value had the maximum clinical net benefit value when the threshold probability was 0.22-0.73,and the ratio of loss to gain within the threshold probability range was always less than 1,which confirmed that the prediction model had good clinical application effectiveness.(7)The results of sperm DNA fragmentation index and offspring short-term security analysis showed that sperm DNA fragmentation index had no significant differences with preterm birth,body weight,deformity and sex.(8)These findings suggest that the optimal clinical cut-off value of sperm DNA fragmentation index for in vitro fertilization-embryo transfer live birth was 24.33%.The established clinical prediction model has good differentiation,accuracy and clinical application effectiveness.Sperm DNA fragmentation index has no significant impact on offspring short-term security,but large samples and long-term follow-up evaluation are still needed.

Objective To establish a sandwich enzyme-linked immunosorbent assay (ELISA) method for the quantitative detection of Chromogranin A (CHGA) in saliva, and to explore its preliminary application in the testing of saliva samples. Methods Recombinant human CHGA protein was used to immunize BALB/c mice, and monoclonal antibodies (mAbs) were prepared and screened using conventional hybridoma technology. A double-antibody sandwich ELISA detection method was constructed, and the matrix effect of saliva samples was optimized. This method was then applied to detect the concentration of CHGA in the saliva of stressed individuals. Results Twenty-one stable hybridoma cell lines secreting high affinity anti-human CHGA antibodies were obtained. A pair of detection antibodies with the best effect was selected, and the optimal coating concentration was determined to be 10 μg/mL, with the optimal dilution of detection antibodies being 1:32 000. The accuracy and reproducibility of this method were verified, with both intra-batch and inter-batch variation coefficients less than 15×, and the recovery rate between 80× and 120×. The matrix effect was further optimized to make it suitable for saliva sample detection. Saliva samples from individuals in different stress states were collected, and the CHGA levels were detected using the method established in this study, indicating its potential to reflect the intensity of stress. Conclusion A reliable saliva CHGA ELISA detection method has been successfully established, and its potential as a biomarker in stress-related research has been preliminarily explored.
Saliva/metabolism* ; Enzyme-Linked Immunosorbent Assay/methods* ; Humans ; Animals ; Mice, Inbred BALB C ; Mice ; Chromogranin A/immunology* ; Antibodies, Monoclonal/immunology* ; Female ; Male ; Reproducibility of Results ; Adult

The regulatory processes in developmental biology research are significantly influenced by long non-coding RNAs (lncRNAs). However, the dynamics of lncRNA expression during human tooth development remain poorly understood. In this research, we examined the lncRNAs present in the dental epithelium (DE) and dental mesenchyme (DM) at the late bud, cap, and early bell stages of human fetal tooth development through bulk RNA sequencing. Developmental regulators co-expressed with neighboring lncRNAs were significantly enriched in odontogenesis. Specific lncRNAs expressed in the DE and DM, such as PANCR, MIR205HG, DLX6-AS1, and DNM3OS, were identified through a combination of bulk RNA sequencing and single-cell analysis. Further subcluster analysis revealed lncRNAs specifically expressed in important regions of the tooth germ, such as the inner enamel epithelium and coronal dental papilla (CDP). Functionally, we demonstrated that CDP-specific DLX6-AS1 enhanced odontoblastic differentiation in human tooth germ mesenchymal cells and dental pulp stem cells. These findings suggest that lncRNAs could serve as valuable cell markers for tooth development and potential therapeutic targets for tooth regeneration.
Humans ; RNA, Long Noncoding/metabolism* ; Odontogenesis/genetics* ; Tooth Germ/embryology* ; Cell Differentiation ; Gene Expression Regulation, Developmental ; Mesoderm/metabolism* ; Tooth/embryology* ; Gene Expression Profiling ; Sequence Analysis, RNA ; Dental Pulp/cytology*

Identification of disease-specific cell subtypes (DSCSs) has profound implications for understanding disease mechanisms, preoperative diagnosis, and precision therapy. However, achieving unified annotation of DSCSs in heterogeneous single-cell datasets remains a challenge. In this study, we developed the gPRINT algorithm (generalized approach for cell subtype identification with single cell's voicePRINT). Inspired by the principles of speech recognition in noisy environments, gPRINT transforms gene position and gene expression information into voiceprints based on ordered and clustered gene expression phenomena, obtaining unique "gene print" patterns for each cell. Then, we integrated neural networks to mitigate the impact of background noise on cell identity label mapping. We demonstrated the reproducibility of gPRINT across different donors, single-cell sequencing platforms, and disease subtypes, and its utility for automatic cell subtype annotation across datasets. Moreover, gPRINT achieved higher annotation accuracy of 98.37% when externally validated based on the same tissue, surpassing other algorithms. Furthermore, this approach has been applied to fibrosis-associated diseases in multiple tissues throughout the body, as well as to the annotation of fibroblast subtypes in a single tissue, tendon, where fibrosis is prevalent. We successfully achieved automatic prediction of tendinopathy-specific cell subtypes, key targets, and related drugs. In summary, gPRINT provides an automated and unified approach for identifying DSCSs across datasets, facilitating the elucidation of specific cell subtypes under different disease states and providing a powerful tool for exploring therapeutic targets in diseases.
Humans ; Algorithms ; Single-Cell Analysis ; Databases, Genetic ; Molecular Sequence Annotation