Objective　To detect clustered regularly interspaced short palindromic repeats (CRISPR) in Listeria monocytogenes, and analyze the structure and homology of CRISPR loci. Methods Totally 34 strains of Listeria monocytogenes isolated in our laboratory were identified, PCR amplified and sequenced. The repeat sequence structure and spacer sequence homology in CRISPR loci were analyzed by bioinformatics software. Results A total of 7 CRISPR loci were detected in 34 strains. The mutation rate of the first 2 and last 2 bases of the Repeat sequence of CRISPR loci was higher, while the mutation rate of the middle part was lower. Seven CRISPR sites form eight CRISPR structural types, among which the Repeat sequences of CRISPR1 and CRISPR2 are relatively conserved, while the Repeat sequences of CRISPR1 and CRISPR5 can form dumbbell shaped secondary structures. The number of Spacer sequences contained in each CRISPR site ranges from 2 to 15, with an average of 2.43. The 136 Spacer sequences detected were not only homologous to Listeria plasmids and bacteriophages, but also homologous to uncultured virus sequences, staphylococcal bacteriophages, and Listeria innocua. The same CRISPR genotype did not show large-scale clustering, but some strains in the same year were in the same evolutionary cluster with close genetic relationships. Conclusion The CRISPR structure of Listeria monocytogenes in this study exhibits high specificity, and its homology with bacteriophages provides a theoretical basis for the application of bacteriophages in the control and prevention of Listeria monocytogenes.

Objective To develop an active monitoring model for adverse drug events(ADEs)related to antimicrobial use in children based on the China hospital pharmacovigilance system(CHPS).Methods Trigger items for the active monitoring model were initially drafted through a review of relevant literature,adverse reaction databases,and drug label warnings,and subsequently refined using the Delphi method.A retrospective analysis was performed on pediatric inpatients who received antibiotics at Anning First People's Hospital between January 1 and December 31,2024.The detection rate and positive predictive value(PPV)of the active monitoring model were calculated and compared with spontaneous ADE reports from the same period.Risk factors for ADEs were further analyzed using logistic regression.Results 25 trigger items were established for the active monitoring model.Among 1,784 cases,233 ADEs were identified,yielding a detection rate of 13.06%(233/1,784).The spontaneous reporting rate of adverse events during the same period was 1.85%(33/1,784).The difference between the two was statistically significant(P<0.001).There were 727 positive trigger events,with 299 cases of ADE detected,resulting in an overall PPV of 41.13%(299/727).Logistic regression revealed that antibiotic use exceeding 3 days(OR=1.454,95%CI:1.012-2.088)was significantly associated with ADE occurrence.Conclusion Compared with conventional spontaneous reporting,the active monitoring model can significantly improve the detection rate of ADEs of pediatric antimicrobial drugs and achieve active and real-time monitoring of drug adverse events.

OBJECTIVE: To investigate the clinical and genetic characteristics of a Chinese pedigree affected with Neurodevelopmental disorder with absent language and variable seizures (NEDALVS) due to variant of WASF1 gene, and to review the literature on NEDALVS associated with WASF1 gene variants. METHODS: A 4-year-and-8-month-old boy with NEDALVS diagnosed at Linyi People's Hospital in July 2024 due to "discovering language development delay for more than 2 years" and his family members were selected as the study subjects. Clinical data of the family members were collected. Peripheral venous blood samples were collected from family members. Whole-exome sequencing (WES) was performed, and candidate variants were verified, by Sanger sequencing. Pathogenicity of candidate variant was classified according to the Standards and Guidelines for the Interpretation of Sequence Variants established by the American College of Medical Genetics and Genomics (ACMG). Using the MUpro website, SWISS-MODEL, PyMOL, Clustal X, PolyPhen-2, and Mutation Taster software, bioinformatics analysis of protein three-dimensional structure modeling for gene mutations, cross-species conservation of mutant amino acids, and pathogenicity prediction of mutation sites. Relevant literature was retrieved from databases such as CNKI, Wanfang Data Knowledge Service Platform, and PubMed, and the clinical phenotypes and genotypes of patients with WASF1 gene mutations reported in the literature were summarized and analyzed. This study was approved by the Medical Ethics Committee of Linyi People's Hospital (Ethics No.: YX200303). RESULTS: The proband, a 4-year and 8-month-old male, mainly presented with delayed language and motor development, accompanied by autistic behaviors; the proband's younger brother was 2 years and 7 months old at the time of consultation, mainly presented with delayed language and motor development, accompanied by short stature; the proband's mother mainly presents with limited language expression and poor interpersonal interaction; the proband's maternal grandmother mainly presents with soliloquizing?behavior. The results of WES showed that the proband carried a heterozygous mutation c.214C>T (p.Arg72Cys) in the WASF1 gene, and this site has not been recorded in the database. Sanger sequencing confirmed that the proband's younger brother, mother, and maternal grandmother had harbored the same variant. Based on the guidelines from the ACMG, this variant was rated as likely pathogenic (PM2_Supporting+PP1+PP3+PP4). Through SWISS-MODEL homology modeling and PyMOL structure visualization analysis, it was further confirmed that this variant can lead to a decrease in protein stability. Amino acid sequence conservation analysis of the WASF1 protein using Clustal X software suggested that the c.214C>T (p.Arg72Cys) variant has caused replacement of a highly conserved amino acid. According to the results of PolyPhen-2 and Mutation Taster, the p.Arg72Cys variant was predicted to be a hazardous. By following the retrieval strategy set in this study, a total of 5 research articles regarding to patients with NEDALVS caused by WASF1 gene mutations were retrieved, which involved 15 patients. Combining the proband and their family members discovered in this study, there were a total of 19 NEDALVS patients. The main clinical features included: motor developmental delay (100%, 17/17), language/intellectual developmental delay (100%, 17/17), epilepsy (64.7%, 11/17), autistic behavior (76.5%, 13/17), hypotonia (70.6%, 12/17), abnormal electroencephalogram (64.7%, 11/17), and short stature (17.6%, 3/17). All 19 patients had heterozygous mutations, with 8 mutation sites. Missense mutations were the most common, accounting for 84.2% (16/19). CONCLUSION A pathogenic variant of the WASF1 gene was identified in a pedigree affected with NEDALVS. Discovery of the novel variant has, expanded the mutational spectrum of the WASF1 gene.
Child, Preschool ; Female ; Humans ; Infant ; Male ; China ; Exome Sequencing ; Mutation ; Neurodevelopmental Disorders/genetics* ; Pedigree ; Seizures/genetics* ; East Asian People/genetics*

OBJECTIVE: To investigate the clinical and genetic characteristics of a family with Vissers-Bodmer Syndrome (VIBOS) and to review the relevant literature on VIBOS caused by CNOT1 gene variants. METHODS: A child diagnosed with VIBOS due to "growth retardation for over 6 years" at the Linyi People's Hospital on March 1, 2024 and her family members were selected as the study subjects. Clinical data of the family were collected. Peripheral venous blood samples were collected from the family members. Whole-exome sequencing (WES) was performed on the proband's peripheral blood, and Sanger sequencing was used for verification of the candidate variant in the family. Pathogenicity of the candidate variant was classified according to the "Standards and Guidelines for the Interpretation of Sequence Variants" established by the American College of Medical Genetics and Genomics American College of Medical Genetics (ACMG). Bioinformatics analysis, including pathogenicity prediction using Mutation Taster, three-dimensional protein structure modeling using SWISS-MODEL, and functional impact assessment using PyMOL, was performed. Relevant literature on VIBOS patients due to variants of the CNOT1 gene was retrieved from databases such as CNKI, Wanfang Data, and PubMed. The clinical phenotypes and genotypes of the patients were summarized. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.: YX200303). RESULTS: The proband, a 6-year-and-7-month-old female, presented with short stature, distinctive facial features (esotropia, hypertelorism, prominent nasolabial folds), webbed neck, clinodactyly, and intellectual disability. WES revealed that she has carried a heterozygous c.736delG (p.V246*) variant of the CNOT1 gene, which was unreported previously. The proband's father exhibited borderline intellectual function but no short stature or distinctive facial features. Sanger sequencing confirmed that he has carried the same heterozygous variant. According to the ACMG guidelines, this genetic variant was predicted as "likely pathogenic" (PVS1+PM2_Supporting). The c.736delG (p.V246*) variant was predicted to have a deleterious effect by Mutation Taster. Subsequent homology modeling using SWISS-MODEL, coupled with structural visualization and comparison using PyMOL, confirmed that it may cause premature termination of translation and produce a truncated protein. Literature search has retrieved five articles on VIBOS due to CNOT1 gene variants, which included 45 cases. Together with the proband and her father, the common clinical features among these 47 patients included distinctive facial features (83.0%, 39/47), speech delay (70.2%, 33/47), motor delay (70.2%, 33/47), intellectual disability (59.6%, 28/47), and short stature (48.9%, 23/47). In terms of the types of the variants, missense variants were the most common (47.4%, 18/38), followed by frameshift variants (21.0%, 8/38). The variant sites have mainly located in exons 7, 25, and 31. No significant genotype-phenotype correlation was noted. CONCLUSION The c.736delG (p.V246*) frameshift variant of the CNOT1 gene is likely the genetic etiology of VIBOS in this proband. The clinical manifestations of the proband were more severe than in her fathers, which suggested phenotypic variability associated with this variant. This study has provided new evidence for the understanding of the genetic basis of VIBOS.
Child ; Female ; Humans ; Male ; Exome Sequencing ; Intellectual Disability/genetics* ; Mutation ; Pedigree ; Transcription Factors/genetics* ; East Asian People/genetics*

Objective:To investigate clinical efficacy of narrow-band ultraviolet B (NB-UVB) irradiation around vitiliginous lesions in the treatment of refractory vitiligo.Methods:A total of 126 patients with refractory vitiligo were retrospectively collected from Department of Dermatology, the First Affiliated Hospital of Nanjing Medical University from June 2019 to November 2020. The patients were treated with NB-UVB irradiation around vitiliginous lesions after partial covering (perilesional irradiation group) , or conventional NB-UVB irradiation (conventional irradiation group) , twice a week for 3 consecutive months. After the treatment, the efficacy was evaluated. By using the propensity score method, the lesions in the 2 groups were matched at a ratio of 1∶1. Univariate and multivariate logistic regression analyses and stratified analysis were used to analyze the clinical efficacy of NB-UVB irradiation around vitiliginous lesions in the treatment of refractory vitiligo.Results:Totally, there were 420 skin lesions in the perilesional irradiation group and 257 in the conventional irradiation group, and 190 lesions were enrolled into each group by propensity-score matching. Before and after the matching, the response rates were both significantly higher in the perilesional irradiation group (71.9%, 67.9%, respectively) than in the conventional irradiation group (31.9%, 30.0%, respectively, both P < 0.05) . After the propensity-score matching, both univariate and multivariate logistic regression analyses showed significant differences in the efficacy between the perilesional irradiation group and conventional irradiation group ( OR = 4.9, 95% CI: 3.2, 7.6, P < 0.001; OR = 12.0, 95% CI: 6.5, 22.3, P < 0.001, respectively) . Vitiliginous lesions were classified according to hair types and irradiation methods: before the matching, there were 187 vitiliginous lesions with white hairs treated with the conventional irradiation and 246 treated with the perilesional irradiation, and there were 70 vitiliginous lesions with black hairs treated with the conventional irradiation and 174 treated with the perilesional irradiation; after the matching, 140 vitiliginous lesions with white hairs and 50 with black hairs were enrolled into each radiation group. Stratified analysis showed that the response rates of vitiliginous lesions with white hairs were significantly higher in the perilesional irradiation group (77.6%, 72.8%, respectively) than in the conventional irradiation group before and after the matching (19.3%, 20.7%, respectively, both P < 0.01) ; for the vitiliginous lesions with black hairs, there was no significant difference in the response rate between the 2 groups ( P = 0.908) . Conclusion:The efficacy of NB-UVB irradiation around vitiliginous lesions is superior to the conventional irradiation in the treatment of refractory vitiligo, especially vitiliginous lesions with white hairs.

Objective:To investigate the safety and clinical efficacy of laparoscopy dominated approaches to two different local resections for duodenal stromal tumors.Methods:From May 2015 to May 2021 25 duodenal stromal tumors cases were allocated to wedged resection group (8 cases) and segmental resection (17cases).Results:Compared with the segmental resection group, the operative time in the wedge resection group was significantly shorter [(202±43) min vs. (299±128) min, t=-2.814, P=0.010]. The intraoperative blood loss was 20 (10-50) ml in the wedge resection group and 30 (15-100) ml in the segmental resection group ( t=-1.128, P>0.05). Patients in the wedge resection group had a significantly shorter postoperative hospital stay, 7(9-11) days vs. 14 (10-28) days, t=-2.66, P=0.008. There was no difference in the incidence of postoperative complications and gastric emptying disorders between the two groups ( P>0.05). Conclusion:In spite of laparoscopic,robotic or open approaches, wedge resection and segmental resection based on anatomic location for duodenal stromal tumors are both safe and satisfactory.

Objective To evaluate the effect of the treatment with low-concentration hydrogen peroxide (H2O2) on the adhesive function of and autophagy in human melanocytes,and to screen long noncoding RNAs (lncRNAs) related to autophagy.Methods Melanocytes were isolated from foreskins of healthy males after circumcision,and subjected to cultivation.Melanocytes at exponential growth phase were divided into 3 groups:control group receiving no treatment,H2O2 group treated with 400 μ mol/L H2O2,and H2O2 + NAC group pretreated with 4 mmol/L NAC for 2 hours followed by the treatment with 400 μmol/L H2O2.After 5-day treatment,immunofluorescence study was performed to determine the expression of Ecadherin,microtubule-associated protein 1 light chain 3 (LC3)and p62,and Western blot analysis to determine the expression of autophagy-related protein LC3 and p62.Cell structures and autophagosomes were observed by transmission electron microscopy,and autophagy-related lncRNAs were screened using gene chip technology.Statistical analysis was done with Graphpad Prism 6 software using one-way analysis of variance for comparison among groups,and Tukey's test for multiple comparisons.Results Under the confocal microscopy,the H2O2 group showed significantly decreased fluorescence intensity of E-cadherin and LC3 in the melanocytes and decreased number of autophagosomes in melanocytes,but significantly increased fluorescence intensity of p62 compared with the control group and H2O2 + NAC group.Western blot analysis showed that the LC3-Ⅱ/LC3-Ⅰ ratio in the melanocytes was significantly lower in the H2O2 group (0.604 ± 0.012) than in the control group (1.200 ± 0.081,q =7.718,P ＜ 0.01) and H2O2 + NAC group (1.017 ± 0.062,q =5.076,P ＜ 0.05),while the p62/β-actin ratio in the melanocytes was significantly higher in the H2O2 group (0.881 ± 0.079) than in the control group (0.456 ± 0.121,q =4.847,P ＜ 0.05) and H2O2 + NAC group (0.492 ± 0.049,q =4.439,P ＜ 0.05).There were no significant differences in the LC3-Ⅱ/LC3-Ⅰ ratio or p62/β-actin ratio between the H2O2 + NAC group and control group (P ＞ 0.05).Gene chip technology showed that 18 autophagy-related lncRNAs were associated with premature senescence of melanocytes and differentially expressed in the H2O2 group compared with the control group,and the autophagy-related lncRNA NONHSAT190308.1 (＞ 10-fold increase) was screened out.Conclusion Lowconcentration H2O2 can decrease the expression of E-cadherin and the level of autophagy in melanocytes,and can up-regulate the expression of autophagy-associated lncRNA NONHSAT190308.1.