[Objective] To compare next generation sequencing (NGS) library construction technology between probe hybridization capture and amplicon methods, and analyze the influencing factors of HLA genotyping resolution level and its prospects in clinical applications. [Methods] A total of 207 clinical samples with known typing results and samples from the proficiency testing plan were selected. The conformity rate of HLA genotyping results, allele coverage and typing data analysis indicators were confirmed, and the effects of two library construction methods on the level of HLA genotyping discrimination were compared. [Results] The concordance rate of 207 samples with the feedback results of PT or prior well-characterized HLA genotypes was 100%. Among them, 91 samples were captured using hybridization probe capture method. Compared with the original amplicon method, the hybridization probe capture method can distinguish the alleles of DRB1 and DPB1 that cannot be determined in 13 samples. The allelic imbalance of DRB1, DPA1, and DQB1 loci in 6 samples was resolved. Three samples were found to have missed detection of alleles at the DQA1 and DQB1 loci. [Conclusion] The performance indicators of hybridization probe capture and amplicon performance confirmation meet the requirements of clinical detection of HLA genotyping, which provides an experimental method and basis for clinical application.

OBJECTIVES: To investigate the role of ferroptosis in diquat-induced acute kidney injury (AKI) and its molecular mechanisms. METHODS: Transgenic zebrafish models with Tg (Eco.Tshb:EGFP) labeling of the renal tubules and Tg (lyz:dsRed2) labeling of the neutrophils were both divided into control group, gentamicin (positive control) group, diquat poisoning group, ferroptosis inhibitor group. The indicators of kidney injury, inflammatory response, and ferroptosis were examined in the zebrafish, and the changes in expressions of voltage-dependent anion-selective channel protein 1 (VDAC1) and mitochondrial ferritin (FTMT) were detected using Western blotting. RESULTS: AKI induced by diquat exhibited a significant dose-effect relationship, and the severity of injury was proportional to the exposure concentration. Diquat also caused marked oxidative stress and inflammatory responses in the zebrafish models. Rhodamine metabolism assay and HE staining revealed significantly declined glomerular filtration function of the zebrafish as diquat exposure concentration increased. Immunofluorescence staining highlighted significant changes in the expressions of ferroptosis markers GPX4 and FTH1 in zebrafish renal tissues following diquat exposure. In diquat-exposed zebrafish, treatment with ferrostatin-1, a ferroptosis inhibitor, obviously upregulated GPX4 and downregulated FTH1 expressions and improved the metabolic rate of glucan labeled with rhodamine B. Diquat exposure significantly upregulated the expression of VDAC1 and FTMT in zebrafish, and the application of ferrostatin-1 and VBIT-12 (a VDAC1 inhibitor) both caused pronounced downregulation of FTMT expression. CONCLUSIONS Ferroptosis is a critical mechanism underlying diquat-induced AKI, in which VDAC1 and FTMT play important regulatory roles, suggesting their potential as therapeutic target for AKI caused by diquat.
Animals ; Zebrafish ; Ferroptosis/drug effects* ; Acute Kidney Injury/chemically induced* ; Diquat/toxicity* ; Animals, Genetically Modified ; Voltage-Dependent Anion Channel 1/metabolism* ; Ferritins/metabolism* ; Oxidative Stress

Instrument separation is a critical complication during root canal therapy, impacting treatment success and long-term tooth preservation. The etiology of instrument separation is multifactorial, involving the intricate anatomy of the root canal system, instrument-related factors, and instrumentation techniques. Instrument separation can hinder thorough cleaning, shaping, and obturation of the root canal, posing challenges to successful treatment outcomes. Although retrieval of separated instrument is often feasible, it carries risks including perforation, excessive removal of tooth structure and root fractures. Effective management of separated instruments requires a comprehensive understanding of the contributing factors, meticulous preoperative assessment, and precise evaluation of the retrieval difficulty. The application of appropriate retrieval techniques is essential to minimize complications and optimize clinical outcomes. The current manuscript provides a framework for understanding the causes, risk factors, and clinical management principles of instrument separation. By integrating effective strategies, endodontists can enhance decision-making, improve endodontic treatment success and ensure the preservation of natural dentition.
Humans ; Root Canal Therapy/adverse effects* ; Consensus ; Root Canal Preparation/adverse effects*

Objective To develop a nomogram model based on the nucleotide-binding oligomer-ization domain-like receptor protein 3(NLRP3)/caspase-1 signaling pathway to predict the value of secondary vascular dementia(VaD)after acute cerebral infarction(ACI)following thrombolytic ther-apy.Methods A total of 289 patients with ACI admitted to Yuncheng Central Hospital Affiliated to Shanxi Medical University from March 2021 to March 2024 were selected as study subjects,and were divided into VaD group(n=60)and non-VaD group(n=229)based on the occurrence of VaD.Rele-vant clinical data were compared between the two groups,and influencing factors for secondary VaD af-ter ACI were analyzed.A nomogram prediction model was constructed and validated.Results The proportions of patients with a history of smoking,hypertension and coronary heart disease,infarction in key areas,responsible large vessel stenosis ≥50％,leukoaraiosis,and thrombolysis benefit were higher in the VaD group than in the non-VaD group(P＜0.05).Logistic regression analysis showed that the degree of responsible vessel stenosis,infarctionin key areas,leukoaraiosis,and the expression of NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),and caspase-1,which are related to the NLRP3/caspase-1 signaling pathway,were independent influencing factors for sec-ondary VaD after ACI(P＜0.05).Receiver operating characteristic(ROC)curve,calibration curve,and decision curve analysis(DCA)indicated that the nomogram model had high discrimina-tion and predictive efficacy,with significant positive net benefit.Conclusion The independent in-fluencing factors for secondary VaD after ACI include the degree of responsible vessel stenosis,in-farction in key areas,leukoaraiosis,and the expression of NLRP3,ASC,and caspase-1 related to the NLRP3/caspase-1 signaling pathway.The nomogram prediction model established based on these indicators has high predictive value and clinical efficacy.

Objective:The predictive model of cardiac arrest in the emergency room was constructed and validated based on Logistic regression.Methods:This study was a retrospective cohort study. Patients admitted to the emergency room of the First Affiliated Hospital of Xinjiang Medical University from January 2020 to July 2021 were included. The general information, vital signs, clinical symptoms, and laboratory examination results of the patients were collected, and the outcome was cardiac arrest within 24 hours. The patients were randomly divided into modeling and validation group at a ratio of 7:3. LASSO regression and multivariable logistic regression were used to select predictive factors and construct a prediction model for cardiac arrest in the emergency room. The value of the prediction model was evaluated using the area under the receiver operator characteristic curve (AUC), calibration curve, and decision curve analysis (DCA).Results:A total of 784 emergency room patients were included in the study, 384 patients occurred cardiac arrest. The 10 variables were ultimately selected to construct a risk prediction model for cardiac arrest: Logit( P)= -4.503+2.159×modified early warning score (MEWS score)+2.095×chest pain+1.670×abdominal pain+ 2.021×hematemesis+2.015×cold extremities+5.521×endotracheal intubation+0.388×venous blood lactate-0.100×albumin+0.768×K ++0.001×D-dimer. The AUC of the model group was 0.984 (95% CI: 0.976-0.993) and that of the validation group was 0.972 (95% CI: 0.951-0.993). This prediction model demonstrates good calibration, discrimination, and clinical applicability. Conclusions:Based on the MEWS score, chest pain, abdominal pain, hematemesis, cold extremities, tracheal intubation, venous blood lactate, albumin, K +, and D-dimer, a predictive model for cardiac arrest in the in-hospital emergency room was constructed to predict the probability of cardiac arrest in emergency room patients and adjust the treatment strategy in time.

Objective:To analyze functional changes in macrophages in mouse models of vulvovaginal candidiasis (VVC) by modulating indoleamine 2,3-dioxygenase 1 (IDO1) .Methods:Forty specific-pathogen-free female ICR mice were randomly divided into 4 groups using a complete randomization method: a blank group, a VVC model group, a VVC model + 1-methyltryptophan (1-MT) group (referred to as the 1-MT group), a VVC model + interferon-γ (IFN-γ) group (referred to as the IFN-γ group), with 10 mice in each group. Except for the blank group, all the mice were injected subcutaneously with estradiol benzoate oil solution in the abdomen every other day for 6 days prior to modeling to induce pseudoestrus; after successful induction of pseudoestrus, the mice were inoculated vaginally with Candida albicans suspensions at a concentration of 2 × 10 9 CFU/ml once a day for 5 days to establish VVC mouse models, and subcutaneous injections of estradiol benzoate oil solution were continued simultaneously to maintain the pseudoestrus state; 1 day before inoculation with fungal suspensions, mice in the 1-MT group and IFN-γ group were pretreated with 1-MT and IFN-γ respectively, followed by once-daily same intervention for 6 consecutive days; mice in the blank group and VVC model group were intraperitoneally injected with physiological saline solution once a day for 6 consecutive days. On the 5th day of modeling, vaginal conditions in mice were observed and vaginal symptoms were scored; the vaginal lavage fluid was collected for smear microscopy and fungal colony counting; then, the mice were sacrificed, the vaginal tissues were collected and subjected to hematoxylin-eosin (HE) staining and periodic acid-Schiff (PAS) staining, and the expression of IDO1 and the macrophage surface marker F4/80 was determined in the vaginal tissues by an immunofluorescence method; real-time fluorescence-based quantitative PCR (qPCR) was performed to determine mRNA expression levels of IDO1, inducible nitric oxide synthase (iNOS), and arginase 1 (Arg-1) in the vaginal tissues, and Western blot analysis to determine the IDO1 protein expression in the vaginal tissues. One-way analysis of variance was used to analyze the differences in indices among groups, and Tukey test was used for multiple comparisons. Results:Smear microscopic examination of the vaginal lavage fluid on the 5th day of modeling showed elongated hyphae with a few spores in the VVC model group, a large number of elongated hyphae aggregating in clusters with surrounding spores in the 1-MT group, and a few thin and short hyphae with a large number of spores in the IFN-γ group. Compared with the VVC model group (360.0 ± 15.9), the fungal colony counts in the vaginal lavage fluid significantly increased in the 1-MT group (523.7 ± 67.7, P = 0.002), but significantly decreased in the IFN-γ group (258.3 ± 27.57, P = 0.026). HE staining and PAS staining showed small abscess formation in the epidermis and predominant infiltration of neutrophils throughout the epidermal and dermal layers with a large number of spores and a few hyphae in the VVC model group; thickened epidermis and diffuse inflammatory infiltration predominated by neutrophils in the dermis were seen in the 1-MT group, with a large number of hyphae and spores aggregating into clusters, which were predominated by hyphae; in the IFN-γ group, spores aggregated in the epidermis without obvious hyphae, and a small amount of inflammatory cells predominated by neutrophils infiltrated the dermis. Immunofluorescence assay revealed that the relative fluorescence intensities of IDO1 and F4/80 were highest in the IFN-γ group and the 1-MT group, respectively. Western blot analysis revealed that the IDO1 protein expression in the VVC model group was significantly higher than that in the blank group ( P < 0.001) and the 1-MT group ( P < 0.05), but significantly lower than that in the IFN-γ group ( P < 0.05). qPCR showed that iNOS mRNA expression significantly increased in the VVC model group compared with the blank group ( P < 0.01), and increased in the IFN-γ group compared with the blank group, VVC model group and 1-MT group (all P < 0.001); Arg-1 mRNA expression significantly increased in the VVC model group compared with the blank group ( P < 0.001) and IFN-γ group ( P < 0.01), and increased in the 1-MT group compared with the blank group, VVC model group, and IFN-γ group (all P < 0.001) . Conclusion:In the VVC mouse models, upregulation of IDO1 may cause macrophage polarization toward the M1 phenotype, and inhibition of IDO1 may cause increased macrophage recruitment and exacerbate the inflammatory response.

Objective:This study aims to evaluate the performance of digital PCR (dPCR) detecting multiple and single copies genes of the Epstein-Barr virus (EBV) for nucleic acid quantification and explore their applicability in clinical settings.Methods:Compared the sensitivity, specificity, precision, lower limit of detection (LoD), and linearity for multicopy BamHI-W dPCR and single-copy EBNA1 dPCR systems. Linear regression analysis using the least squares method was employed to evaluate the linearity. Additionally, we analyzed plasma samples from 182 patients with suspected EBV-related diseases between January and July 2022 at the Southern Medical University Southern Hospital, using both dPCR and quantitative PCR (qPCR) for EBV DNA quantification. Linear regression analysis using the least squares method was conducted to assess their quantitative correlation.Results:The dPCR systems for both multicopy and single-copy genes showed excellent linearity ( R 2 values of 0.992 and 0.997, respectively, both P<0.001). The LoD were 188 IU/ml for BamHI-W gene and 358 IU/ml for EBNA1 gene dPCR systems. The logarithmic coefficient of variation ( CV) values for high-concentration samples (1 000 000 IU/ml) were 0.34% and 0.21% for the BamHI-W gene and EBNA1 gene dPCR assays, respectively, while for low-concentration samples (5 000 IU/ml) were 0.98% and 0.64%, respectively. In the detection of seven common clinical infectious pathogens and EBV positive samples, only EBV-positive samples yielded positive signals in the dPCR detection system, with no cross-reaction with other pathogens. In 182 samples, the positive detection rates were 47.80% (87/182) for BamHI-W gene and 35.16% (64/182) for EBNA1 gene dPCR, compared to 43.41% (79/182) for qPCR. Linear correlation analysis with qPCR showed R2 values of 0.837 for BamHI-W gene and 0.763 for EBNA1 gene dPCR (both P<0.001). The BamHI-W gene copy number ranged from 3 to 18 copies per clinical sample, with patient-specific variations. There was a high consistency in viral load trends between the multicopy BamHI-W gene and single-copy EBNA1 gene dPCR systems within individual patients. Conclusions:The dPCR methods detecting EBV multiple and single copies genes showed high sensitivity, specificity, precision, and quantitative accuracy, suitable for clinical sample analysis. The multicopy BamHI-W gene dPCR method notably enhances detection sensitivity and can be used as a supplement to current EBV DNA load detection methods, especially in low-concentration samples. For within-patient EBV DNA monitoring, the multicopy gene method proves more effective, while inter-patient comparisons might necessitate single-copy gene methods or normalize them using the same standard.

Objective:To establish a linkage prediction model for human leukocyte antigen (HLA) DPA1-DPB1 and validate it by using clinical data and follow-up data from unrelated allogeneic hematopoietic stem cell transplantation donors and recipients, and to explore the clinical application value of the prediction model in transplantation prognosis.Methods:This is a retrospective study. Leveraging the artificial neural network algorithm of NetMHCⅡpan and the DPA1-DPB1 haplotype linkage database of the Chinese population established in our previous research, and incorporating the amino acid FASTA data of DPA1-DPB1 of all known sequences newly published by the Latest International Immunogenetics/Human Leukocyte Antigens, 47 DPA1-DPB1 linkage models were established. Employing next-generation sequencing technology based on the hybridization capture library construction method, HLA genotyping tests for HLA-A, -B, -C, DRB1, DQB1, DQA1, DRB3/4/5, DPB1, and DPA1 (9 loci) were performed on 250 donor-recipients pairs who underwent unrelated-donor hematopoietic stem cell transplantation in the Department of Hematology of the First Affiliated Hospital of Soochow University between January 2016 and September 2021. HLA typing data and clinical information of transplant donors and recipients were retrospectively analyzed to assess and predict the impact of permissive and non-permissive linkage mismatches of DPA1-DPB1 on transplantation prognosis. The Kaplan-Meier method with the log-rank test was applied to compare the survival curves of overall survival (OS) rates between different groups. Additionally, a competing risks model was utilized to compare the cumulative incidence of grade Ⅱ-Ⅳ acute graft-versus-host disease and non-relapse mortality (NRM) across groups. The area under the receiver operating characteristic curve was employed to compare the predictive performance of the established prediction model with that of the T-cell epitope (TCE) model.Results:According to the different hydrophilic and hydrophobic properties of amino acids, the DPA1-DPB1 linkage model is categorized into types Ⅰ-Ⅳ: type I consists of 6 hydrophobic types at P1-P8 plus hydrophilic type at P9; type Ⅱ includes 17 hydrophobic types; type Ⅲ comprises 9 amphiphilic types; and type Ⅳ consists of 15 hydrophilic types. According to the prediction model, DPA1-matched and DPB1-mismatched donor-recipient cases were classed into P1-matched or P1-mismatched groups. Compared with fully matched DPA1 and DPB1 cases, P1-mismatched patients had a 2-year OS rate of 75% (12/16) versus 96.2%(25/26) (χ2=4.13, P=0.04), and a NRM rate of 4/16 versus 0 (χ2=7.05, P<0.01). However, there was no statistically significant difference in the 2-year OS and NRM rates compared to DPA1 and DPB1 cases ( P>0.05). The prediction model established in this study demonstrated a larger area under the receiver operating characteristic curve for predicting the 2-year OS rate compared with the DPB1 TCE model ( Z=0.71, P=0.48). In donor-recipient cases where both DPA1 and DPB1 were mismatched, the 2-year OS rates decreased and the NRM increased in both P1-matched and P1-mismatched cases compared with fully matched DPA1 and DPB1. Moreover, P1-mismatched patients had a worse prognosis compared to P1-matched patients. Conclusion:The DPA1-DPB1 linkage prediction model established based on high-throughput next-generation sequencing technology can be used to predict the impact of HLA-DP mismatches on OS and NRM in transplantation, and the prediction performance is superior to the TCE model.