[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: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.

Objective:To evaluate the value of left echography (LVO) in the diagnosis of false ventricular aneurysm complicated with mural thrombus.Methods:The clinical data of 10 patients with suspected pseudoventricular aneurysm examined by thoracic echocardiography (TTE) in Wuhan Asian Heart Hospital from January 2018 to March 2024 were retrospectively analyzed. All patients underwent LVO examination to further diagnose pseudoventricular tumor and whether it was complicated with mural thrombosis. Computed tomography angiography (CTA) or cardiac magnetic resonance (CMR) examination was used as the gold standard to analyze the diagnostic value of LVO in the diagnosis of pseudoventricular tumor.Results:Among the 10 suspected pseudoventricular tumors examined by TTE, LVO detected 6 cases of left ventricular pseudoaneurysm and 1 case of right ventricular pseudoaneurysm; CTA confirmed that 6 cases of left ventricular pseudoaneurysm detected by LVO were correctly diagnosed, 1 case of right ventricular pseudoaneurysm was misdiagnosed, CMR diagnosed right ventricular diverticula, LVO diagnosis accuracy was 6/7, and 4 cases of thrombi were detected. The detection rate was 4/4. The maximum transverse diameter of the tumor body of the communicating mouth/false ventricular aneurysm was 0.46±0.04. 1 patient underwent coronary artery bypass grafting and resection of false ventricular aneurysm. 1 patient underwent coronary artery interventional stent surgery; 4 routine conservative drug treatment, follow-up observation; One case of right ventricular diverticulum did not require special treatment.Conclusions:LVO contrast agent can clearly show the tumor body and location, measure the tumor entrance and size, and show mural thrombus. It is the first choice for the identification of false ventricular tumor. The diverticula was similar to the image of false ventricular aneurysm, and the sensitivity and specificity of right ventricular wall motion were higher in CMR than in LVO.

Acute myocarditis(AM)may rapidly progress to fulminant myocarditis(FM),but lacks special clinical presentation.Multimodal echocardiography combined conventional transthoracic echocardiography,two-dimensional and three-dimensional speckle tracking imaging,myocardial contrast echocardiography and so on is helpful to detecting AM in early stage and assessing the severity,being of great value for clinical decision-making and prognostic evaluation.The research progresses of multimodal echocardiography in AM were reviewed in this article.

To summarize the nursing care of a patient with acute myocardial infarction after double knee joint replacement due to alkaptonuria.The main nursing points were as follows:strict condition monitoring,being alert to the occurrence of cardiac complications;strengthening thrombus and bleeding management to prevent related complications;early progressive rehabilitation exercise to promote functional rehabilitation of the affected limb;implementing analgesic management to improve pain symptoms;carrying out nutrition assessment and personalized nutrition support;paying attention to humanistic care,targeted psychological nursing.After careful treatment and nursing care,the patient was discharged from the hospital.After 3 months of telephone follow-up,the patient recovered well.