Objective:To explore the impact of various fixatives on the nuclear-cytoplasmic localization of Yes-associated protein (YAP) in human corneal epithelial cells under hyperosmotic stress condition.Methods:Immortalized human corneal epithelial cells were divided into control group and hypertonic group.After 1 day of normal culture, cells of the hypertonic group were exposed to hyperosmotic medium at 450 mOsM by adding sodium chloride for 1 hour.No special treatment was given to the control group.Both groups of cells were fixed with four different fixatives, including 4% paraformaldehyde (PFA), -20 ℃ precooled absolute ethanol, -20 ℃ precooled methanol-acetone 1∶1 mixture, and Zamboni fixative solution for 20 minutes.Subsequent to fixation, immunofluorescent staining procedures were performed to identify the intracellular localization of YAP in the two groups.Results:After fixation with 4% PFA, human corneal epithelial cells showed normal morphology with YAP mainly in the nucleus in both groups, and there was no significant difference in the mean nuclear YAP fluorescence intensity between the two groups ( t=1.803, P=0.121).After fixation with absolute ethanol, cells showed some degree of shrinkage and deformation, diffuse YAP fluorescence staining with YAP-positive signals mainly localized in the cytoplasm in both groups, and the mean nuclear YAP fluorescence intensity was slightly decreased in the hypertonic group compared with the control group, but the difference was not statistically significant ( t=0.803, P=0.453).After fixation with methanol-acetone 1∶1 mixture, cells were crenulated with YAP mainly in the cytoplasm, and the mean nuclear YAP fluorescence intensity in the hypertonic group was slightly decreased compared with the control group, but the difference was not statistically significant ( t=1.067, P=0.327).After fixation with Zamboni solution, the cell structure was complete and clearly outlined, and the YAP nucleoplasmic translocation phenomenon could be clearly observed in cells in different states.The mean nuclear YAP fluorescence intensity in the hypertonic group was 197.5±34.5, which was significantly higher than 62.2±10.0 in the control group ( t=7.530, P<0.001). Conclusions:In the immunofluorescence staining experiment, the nucleoplasmic localization of YAP in corneal epithelial cells is affected by different fixative treatments.Zamboni fixative is better than 4% PFA, absolute ethanol, and methanol-acetone 1∶1 mixture in observing nuclear translocation of YAP after hypertonic stimulation.

Due to physiological and metabolic characteristics and disease characteristics being different from adults, such as the high incidence of infectious diseases, children are at a high-risk of acute kidney injury (AKI) related to anti-bacterial agents, and there were many challenges in prevention and treatment. The hot issues in the research of anti-bacterial agent-related AKI in children, including the study of nephrotoxicity mechanism of anti-bacterial agents, the exploration of early biomarkers, and the optimization of treatment strategies, are expounded in this article. In the future, it is recommended to strengthen researches on child specificity, deepen basic researches, optimize monitoring plans for pediatric treatment drugs, and establish a pediatric drug monitoring system, so as to provide references for rational clinical drug use of the anti-bacterial agents and further researches on AKI.

Objective To explore the factors affecting the prognosis of patients with symptomatic severe intracranial artery stenosis after interventional therapy,and to construct a nomogram prediction model accordingly.Methods The clinical data of 121 patients with symptomatic severe intracranial artery stenosis,who received interventional treatment at the First People's Hospital of Zhengzhou from June 1,2021 to October 31,2024,were retrospectively analyzed.The general data,characteristics of vascular lesions,treatment-related factors and prognosis of patients were collected.According to the modified Rankin scale(mRS)score,the patients were divided into good prognosis group(mRS score ≤2 points)and poor prognosis group(mRS score＞2 points).The clinical data were compared between the two groups.Multivariate logistic regression analysis was used to identify and determine independent factors affecting patient's outcomes,to construct a nomogram prediction model and to validate this model.Results Among the 121 patients,31(25.61％)had poor prognosis and 90(74.38％)had good prognosis.The postoperative 3-month mRS score was lower than preoperative mRS score value(P＞0.05).There were significant differences in hypertension history,stenosis site,stenosis degree,collateral circulation state,interventional therapeutic mode,cholesterol level,platelet count,lesion length and preoperative NIHSS score(P＜0.05)between the poor prognosis group and the good prognosis group.Multivariate analysis showed that hypertension history,stenosis degree,collateral circulation status,cholesterol level,platelet count,lesion's length and preoperative NIHSS score were the independent influencing factors for the prognosis of patients with symptomatic severe intracranial artery stenosis.The predicted AUC of the nomogram model was 0.931(95％ CI=0.873-0.989),and the calibration curve showed that the predicted value was in good agreement with the actual value.Conclusion Hypertension history,stenosis degree,collateral circulation status,cholesterol level,platelet count,lesion length and preoperative NIHSS score are the important influencing factors for the prognosis of patients with symptomatic severe intracranial artery stenosis.The nomogram prediction model constructed in this study shows a high accuracy in predicting the prognosis of patients,and it can provide important reference for clinical decision-making.

Objective To explore the effect of Sangxing Zhike Formula in rats with cough vari-ant asthma(CVA)and its possible mechanism based on the cyclic adenosine monophosphate(cAMP)/cystic fibrosis transmembrane conductance regulator(CFTR)pathway.Methods SD rats were randomly divided into control group,model group,dexamethasone group(0.5 mg/kg)and low-,medium-,high-dose Sangxing Zhike Formula groups(9.6,19.2 and 38.4 g/kg)using a ran-dom number table method,with 9 rats in each group.Except for the control group,CVA rat models were established in the other groups.Rats in each group were administered the drug by gavage once a day for 14 consecutive days.The general conditions of rats in each group were observed.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)in rat serum.Hematoxylin-eosin(HE)staining and periodic acid-Schiff(PAS)staining were used to observe the pathological changes in rat lung and bronchial tissues,and the acid-base balance of airway surface liquid(ASL)was measured.Western blot and real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)were used to detect the expression levels of pro-tein kinase A(PKA),CFTR protein and their mRNA in lung tissues of rats.Results Compared with the control group,rats in the model group showed listlessness,dull fur,slow weight gain,a significantly expanded area of alveolar septal consolidation,and a large number of inflammatory cell infiltrations around the bronchi.Compared with the model group,rats in each intervention group had better general conditions and reduced inflammatory infiltrations in lung tissues and bronchial lu-mens.Compared with the control group,the serum levels of IL-1 β and IL-18 in the model group were increased,and the pH values of ASL in the model group,low-dose Sangxing Zhike Formula group,medium-dose Sangxing Zhike Formula group,and high-dose Sangxing Zhike Formula group were all decreased,with statistically significant differences(P＜0.05).Compared with the model group,the serum levels of IL-1β and IL-18 in each intervention group were decreased,and the pH values of ASL in the dexamethasone group,medium-dose Sangxing Zhike Formula group,and high-dose Sangxing Zhike Formula group were increased,with statistically significant differences(P＜0.05).Compared with the control group,the expressions of PKA protein and PKA mRNA in the model group,low-dose Sangxing Zhike Formula group,and medium-dose Sangxing Zhike Formula group were all decreased,and the expressions of CFTR protein and CFTR mRNA in the model group and each intervention group were all decreased,with statistically significant differences(P＜0.05).Compared with the model group,the expressions of PKA protein and PKA mRNA in the dexametha-sone group and high-dose Sangxing Zhike Formula group were increased,and the expressions of CFTR protein and CFTR mRNA in the high-dose Sangxing Zhike Formula group were increased,with statistically significant differences(P＜0.05).Conclusion Sangxing Zhike Formula can im-prove the general conditions of CVA rats,regulate the acid-base balance of ASL,reduce airway in-flammatory cell infiltration andairway remodeling,and decrease the levels of inflammatory factors IL-1β and IL-18.Its mechanism may be related to the cAMP/CFTR pathway.

Objective:To explore the impact of various fixatives on the nuclear-cytoplasmic localization of Yes-associated protein (YAP) in human corneal epithelial cells under hyperosmotic stress condition.Methods:Immortalized human corneal epithelial cells were divided into control group and hypertonic group.After 1 day of normal culture, cells of the hypertonic group were exposed to hyperosmotic medium at 450 mOsM by adding sodium chloride for 1 hour.No special treatment was given to the control group.Both groups of cells were fixed with four different fixatives, including 4% paraformaldehyde (PFA), -20 ℃ precooled absolute ethanol, -20 ℃ precooled methanol-acetone 1∶1 mixture, and Zamboni fixative solution for 20 minutes.Subsequent to fixation, immunofluorescent staining procedures were performed to identify the intracellular localization of YAP in the two groups.Results:After fixation with 4% PFA, human corneal epithelial cells showed normal morphology with YAP mainly in the nucleus in both groups, and there was no significant difference in the mean nuclear YAP fluorescence intensity between the two groups ( t=1.803, P=0.121).After fixation with absolute ethanol, cells showed some degree of shrinkage and deformation, diffuse YAP fluorescence staining with YAP-positive signals mainly localized in the cytoplasm in both groups, and the mean nuclear YAP fluorescence intensity was slightly decreased in the hypertonic group compared with the control group, but the difference was not statistically significant ( t=0.803, P=0.453).After fixation with methanol-acetone 1∶1 mixture, cells were crenulated with YAP mainly in the cytoplasm, and the mean nuclear YAP fluorescence intensity in the hypertonic group was slightly decreased compared with the control group, but the difference was not statistically significant ( t=1.067, P=0.327).After fixation with Zamboni solution, the cell structure was complete and clearly outlined, and the YAP nucleoplasmic translocation phenomenon could be clearly observed in cells in different states.The mean nuclear YAP fluorescence intensity in the hypertonic group was 197.5±34.5, which was significantly higher than 62.2±10.0 in the control group ( t=7.530, P<0.001). Conclusions:In the immunofluorescence staining experiment, the nucleoplasmic localization of YAP in corneal epithelial cells is affected by different fixative treatments.Zamboni fixative is better than 4% PFA, absolute ethanol, and methanol-acetone 1∶1 mixture in observing nuclear translocation of YAP after hypertonic stimulation.

Nocturnal enuresis (NE) is a common pediatric disease with unclear pathogenesis.The prevailing hypothesis is that NE is caused by a discrepancy between nocturnal urine output and bladder capacity and sleep-wake disturbances.Monosymptomatic nocturnal enuresis (MNE) with nocturnal polyuria is a common subtype of NE, which can be effectively treated by Desmopressin.However, it is possible that some children may demonstrate an inability to respond favorably to this form of therapy and become resistant to it, which significantly impacts their physical and mental health and overall quality of life.In this article, recent advances in the pathogenesis and treatment of Desmopressin-resistant MNE with nocturnal polyuria in children were reviewed to provide a reference for its treatment.

Due to physiological and metabolic characteristics and disease characteristics being different from adults, such as the high incidence of infectious diseases, children are at a high-risk of acute kidney injury (AKI) related to anti-bacterial agents, and there were many challenges in prevention and treatment. The hot issues in the research of anti-bacterial agent-related AKI in children, including the study of nephrotoxicity mechanism of anti-bacterial agents, the exploration of early biomarkers, and the optimization of treatment strategies, are expounded in this article. In the future, it is recommended to strengthen researches on child specificity, deepen basic researches, optimize monitoring plans for pediatric treatment drugs, and establish a pediatric drug monitoring system, so as to provide references for rational clinical drug use of the anti-bacterial agents and further researches on AKI.

Nocturnal enuresis (NE) is a common pediatric disease with unclear pathogenesis.The prevailing hypothesis is that NE is caused by a discrepancy between nocturnal urine output and bladder capacity and sleep-wake disturbances.Monosymptomatic nocturnal enuresis (MNE) with nocturnal polyuria is a common subtype of NE, which can be effectively treated by Desmopressin.However, it is possible that some children may demonstrate an inability to respond favorably to this form of therapy and become resistant to it, which significantly impacts their physical and mental health and overall quality of life.In this article, recent advances in the pathogenesis and treatment of Desmopressin-resistant MNE with nocturnal polyuria in children were reviewed to provide a reference for its treatment.

Objective:To assess the efficiency of modified enrichment method for cell-free fetal DNA (cffDNA) through purified superparamagnetic beads during non-invasive prenatal testing (NIPT).Methods:A total of 26 252 pregnant women undergoing NIPT at the Maternal and Child Health Care Hospital of Haidian District from December 2017 to September 2022 were recruited and randomly assigned into the conventional group ( n = 10 573) and the modified enrichment group ( n = 15 679), who were then subjected to the screening and enrichment of the cffDNA using a conventional and modified technique, respectively. High-risk pregnant women detected by NIPT were subjected to invasive prenatal diagnosis. All women were followed up for their pregnancy outcomes, and the detection efficacy of the two methods was compared in terms of fragment size, concentration of cffDNA, duplicate detection rate, and indices of clinical laboratory tests. Results:The fragment size of the main peak of the cell-free DNA library of the modified enrichment group was significantly lower than that of the conventional group [267 (264, 269) bp vs. 294 (292, 296) bp, P<0.01], while the concentration of cffDNA was significantly higher [21.86% (17.61%, 26.36%) vs. 9.08% (6.87%, 11.87%), P<0.01]. In addition, the duplicate detection rate (0.740% vs. 2.02%, χ2=83.90, P<0.01) and detection failure rate (0.006% vs. 0.057%, P<0.05) in the modified enrichment group were significantly lower than those of the conventional group. The combined positive predictive value (PPV) in both high-risk (64.3% vs. 76.1%) and low-risk (35.3% vs. 45.5%) pregnant women from the modified enrichment group was slightly lower than those from the conventional group, though no significant difference was detected. There was one false negative case for trisomy 21 among the high-risk pregnant women from the conventional group, and no false negative case was found in the modified enrichment group. Conclusion:The modified technique to screen and enrich the cffDNA has significantly enhanced the relative concentration of cffDNA and reduced the failure and duplication detection rate of NIPT, which has significantly reduced the incidence of false negative cases due to the low concentration of cffDNA, and greatly increased the overall detection efficacy of NIPT.

Objective:To investigate the current status of literature reading report and clinical research ability among professional postgraduate students in pediatrics, and to provide a reference for cultivating high-quality pediatric talents.Methods:A total of 91 professional postgraduate students majoring in pediatrics were selected as research subjects from five affiliated hospitals of Zhengzhou University, and according to whether they participated in literature reading report, they were divided into participating group with 38 students and non-participating group with 53 students. The method of questionnaire combined with interview was used to investigate the implementation of literature reading report, the willingness to participate in literature reading report, and the current status of clinical research ability. SPSS 25.0 was used to perform the chi-square test and the Wilcoxon rank-sum test.Results:In the participating group, 60.53% (23/38) of the students thought that there was a significant improvement in clinical research ability after literature reading report, and in the non-participating group, 94.12% (48/51) of the students wanted to carry out literature reading report. Compared with the non-participating group, the participating group had significantly higher publication rate, project participation rate, academic exchange rate, and literature reading quantity ( P<0.05) and significantly better abilities of literature search and reading, professional English, and PPT presentation ( P<0.05), while there were no significant differences between the two groups in the scores of statistical analysis ability, project design ability, clinical thinking ability, and evidence-based medicine ability ( P>0.05). Conclusions:Literature reading report can promote the improvement in clinical research ability among professional postgraduate students in pediatrics, and it should be carried out regularly for a long time with adherence to the principle of clinical orientation and emphasis on the reading of methodology and statistics in literature.