ObjectiveTo evaluate the detection specificity for clinical samples and the detection capability for standard substances of five commercially available multiplex polymerase chain reaction (PCR) nucleic acid detection kits (hereinafter referred to as the kits) for respiratory pathogens, and to provide a reference for selecting appropriate detection kits for multi-pathogen nucleic acid testing of respiratory infections. MethodsA total of 60 respiratory pathogen-positive clinical samples with known redults were selected and tested using the five kits (labeled as A, B, C, D, and E). The detection rates and Kappa coefficients were calculated to evaluate the consistency between the results from these kits and those from single-pathogen PCR kits. According to the limit of detection (LOD) provided by the kits, standard substances of respiratory pathogens (including 12 types such as influenza virus, Mycoplasma pneumoniae, and Bordetella pertussis) were diluted to four concentrations (250, 500, 1 000, and 2 000 copies·mL⁻¹). All five kits were used for detection to evaluate their respective detection capabilities. ResultsCompared with the results from single-pathogen PCR kits, the five tested kits demonstrated good consistency (all Kappa >0.80). Among them, Kit A had the highest detection rate (100.00%), followed by Kits C and E (98.33%), and then Kits B and D (95.00%). All five kits showed a relatively low false negative rate (FNR) for samples with a cycle threshold (Ct) value ≤35 (≤2.38%). However, for samples with Ct values>35, the FNR increased accordingly(average FNR=6.67%, P=0.029). Kit C exhibited the highest detection sensitivity for the tested standard substances (average LOD: 458.33 copies·mL⁻¹), followed by Kit D, then Kits A/E, and finally Kit B. ConclusionThe five multiplex PCR kits showed good consistency with single-pathogen detection results, but each had its own performance emphasis. Kit A, with the highest detection rate and high throughput, is suitable for targeted viral screening. Kit B, covering the broadest pathogen spectrum (including fungi/bacteria), is suitable for comprehensive respiratory pathogen screening. Kits C, D and E, are applicable for rapid detection. It is important to note that the detection efficacy of all kits decreases for low viral load samples with Ct values >35. In practical application, selection should be based on specific screening objectives, throughput requirements, and sample types.

Chimeric antigen receptor T-cell (CAR-T) therapy, a revolutionary technology in tumor immunotherapy, has achieved breakthrough progress in the treatment of hematological malignancies. To date, multiple CAR-T products have been approved for clinical use worldwide, but progress in their application to solid tumor treatment has been slow mainly due to the highly complex biological characteristics of solid tumors. In recent years, the continuous identification of novel targets in solid tumors, continuous innovations in genetic engineering technologies, growing maturity of combination therapeutic strategies, and rapid advancement in artificial intelligence have expanded the potential applications of CAR-T therapy in solid tumor treatment, highlighting its broad translational prospects. This article systematically reviews recent clinical progress in CAR-T therapy for solid tumors, comprehensively analyzes core challenges and corresponding countermeasures, and outlines future directions.

Objective The study aims to investigate the impact of microRNA-874-3p(miR-874-3p)regulation of plakophilin 3(PKP3)on the malignant biological behavior of lung adenocarcinoma cells and its underlying mechanism.Methods Immunohistochemistry and immunocytochemistry were used to detect the expression of PKP3 in lung adenocarcinoma tissue microarray and lung adenocarcinoma cell line A549 cells respectively,and the relationship between PKP3 and clinicopathological features of lung adenocarcinoma patients was analyzed.Select lung adenocarcinoma cell line A549,the experiment was divided into A549 cell group(blank control group),miR-NC group(transfected with miR-NC)and miR-mimics group(transfected with miR-874-3p mimics),sh-NC group(control group transfected with PKP3 silencing plasmid),sh-PKP3 group(transfected with PKP3 silencing plasmid),miR+pcDNA-PKP3 group(transfected with miR-874-3P mimics+pcDNA-PKP3,rescue group)and miR+pcDNA-NC group(transfected with miR-874-3p mimics+pcDNA-NC).The proliferation,invasion,migration and apoptosis of cells in each group were detected.ENCORI database was used to predict the upstream gene of PKP3,and dual luciferase assay was used to detect the targeting relationship between miR-874-3p and PKP3.MAPK/mTOR pathway-related proteins were detected by Western blotting.Results The expression of PKP3 in lung adenocarcinoma tissue was significantly higher than that in adjacent tissues.The high expression of PKP3 was related to clinical stage,tumor size,and lymph node metastasis(P＜0.05).Compared with the human normal lung epithelial cells(BEAS-2B),the expression of PKP3 in A549 cells increased significantly,and the expression of miR-874-3p decreased(P＜0.05).Overexpression of miR-874-3p decreased the PKP3 expression level(P＜0.05).Compared with the control group,both overexpression of miR-874-3p and silenced PKP3 inhibited the cloning and invasion ability of A549 cells,caused cell cycle arrest,and decreased the expression levels of cyclin dependent kinase 4(CDK4),cyclin D1,cyclin E1 proteins in A549 cells(P＜0.05).The expressions of Bax protein and Caspase-3 protein were up-regulated(P＜0.05),and apoptosis increased.Overexpression of PKP3 could reverse the biological behavior of overexpression of miR-874-3p.Overexpression of miR-874-3p and silencing of PKP3 significantly decreased the expressions of P38 MAPK and mTOR phosphorylated proteins.Conclusion MiR-874-3p can negatively regulate PKP3 expression and inhibit the malignant biological behavior of A549 cells through MAPK/mTOR pathway.

Objective To establish models for real-time dynamic monitoring of intracellular cytoplasmic adenosine triphosphate(ATP)and mitochondrial ATP levels in cells in order to study the changes in metabolic processes.Methods The lentiviral plasmids of the cytoplasmic chemogenetic green fluorescent protein(GFP)ATP probe(Chemo-G)and those of the mitochondrial-localized chemogenetic GFP ATP probe(mito-Chemo-G)were constructed before being transfected into HEK293T together with the helper plasmids,respectively.Chemo-G and mito-Chemo-G lentiviruses were obtained.HeLa cells were infected with the lentivirus.Puromycin resistance selection and flow cytometry cell sorting were employed to identify and isolate the infected HeLa cells.The growth and GFP expressions of HeLa cells were observed.A live-cell imaging system was used for continuous imaging of the cells,with stimuli added at specific time points to alter intracellular ATP levels to observe changes in the fluorescence intensity of the ATP probe.Results Lentiviral plasmids containing Chemo-G and mito-Chemo-G sequences were constructed.Two cell lines which could stably express Chemo-G and mito-Chemo-G were established that exhibited strong growth and accurate intracellular fluorescence localization.Live-cell imaging revealed that after the addition of 2-deoxy-D-glucose(2-DG)into HeLa-Chemo-G,the fluorescence resonance energy transfer(FRET)/GFP ratio showed a decrease that was partially reversed by the addition of glucose.The FRET/GFP ratio increased after histamine stimulation,but rapidly decreased after the addition of oligomycin.Conclusion The Chemo-G and mito-Chemo-G lentiviral vectors and stably transfected cell lines HeLa-Chemo-G and HeLa-mito-Chemo-G are constructed,which provides reliable experimental models for studying cellular metabolism and changes in intracellular ATP levels.

Objective To explore the application value of combining the ultrasound breast imaging reporting and data system(BI-RADS)classification with serum fibroblast growth factor receptor 1(FGFR1)and growth differentiation factor 3(GDF3)in the differential diagnosis of benign and malignant breast masses.Methods A total of 159 patients with breast masses were selected and divided into the benign mass group(n=83)and the malignant mass group(n=76)based on postoperative pathological diagnosis.All patients underwent ultrasound examination,and enzyme-linked immunosorbent assay(ELISA)was applied to detect serum levels of FGFR1 and GDF3.Receiver operating characteristic(ROC)curve was applied to analyze the diagnostic value of ultrasound BI-RADS classification and serum FGFR1 and GDF3 levels for benign and malignant breast masses.Kappa test was applied to analyze the consistency between various diagnostic methods and pathological diagnosis.Results The serum levels of FGFR1 and GDF3,the proportions of irregular morphology,unclear boundaries,spiculation,microcalcifications,blood flow grade Ⅱ-Ⅲ and posterior echo attenuation,RI and PI were higher in the malignant tumor group than those in the benign tumor group(P＜0.05).The area under the curve(AUC)of FGFR1,GDF3 and ultrasound BI-RADS classification in the differential diagnosis of benign and malignant breast masses separately and in combination was 0.802(95%CI:0.732-0.871),0.817(95%CI:0.751-0.884),0.848(95%CI:0.784-0.912)and 0.956(95%CI:0.918-0.993),respectively.The combined diagnosis was more effective than that of the individual diagnosis of each indicator.The consistency between the individual and combined diagnosis of benign and malignant breast masses and pathological diagnosis showed that the Kappa values were 0.517,0.514,0.688 and 0.912,respectively,with the highest consistency observed in the combined diagnosis(P＜0.05).Conclusion Ultrasound BI-RADS classification combined with serum FGFR1 and GDF3 has high application value in the differential diagnosis of benign and malignant breast masses.

Tracheobronchial adenoid cystic carcinoma （TACC） is a low-grade malignant tumor originating from the airway mucosa. Despite its slow progression，it is characterized by high invasiveness，frequent recurrence，and a strong tendency for metastasis. Preclinical studies have shown that vascular-targeted therapy holds significant potential. However，an effective systemic treatment for TACC has not been established yet. This study explored TACC from the perspective of "Feiji" in traditional Chinese medicine （TCM） as the starting point. It deeply investigated the mechanisms of abnormal collaterals and tumor vascular recruitment and further elaborated on the theoretical connection between abnormal collaterals and tumor vascular recruitment. Firstly，collateral hyperactivity led to disordered and erratic pulmonary collaterals. Their abnormal structures were similar to the disorderly and tortuous nature of tumor （pseudo）angiogenesis. This resulted in imbalances in the functions of circulation，perfusion，and reverse injection of the pulmonary collaterals，and then led to unrestrained collateral dysfunction and the accumulation of pathogenic factors. Secondly，the remodeling of the extracellular matrix （ECM） and epithelial-mesenchymal transition （EMT） in TACC were critical processes in vascular co-option （VCO），representing the micro-level manifestation of the displacement of nutrient and defense. During this process，ECM remodeling made TACC cells more likely to hijack normal blood vessels，creating a complex vascular microenvironment conducive to tumor growth. In terms of treatment，this study proposed a TCM strategy of "regulating collaterals to expel pathogenic factors and nourishing collaterals to strengthen the healthy Qi"，and listed potential TCM. These were intended to regulate the Qi and blood in the collaterals，repair the functions of abnormal collaterals，and intervene in the vascular recruitment process of TACC. Future research should focus on improving the TCM clinical syndrome characteristics of TACC. Through modern molecular biology techniques，it is necessary to deeply analyze the micro-level pattern of vascular recruitment in TACC. This would enrich the understanding of the profound connection between abnormal collaterals and tumor vascular recruitment，providing empirical evidence for TCM-targeted therapies for vascular recruitment in TACC.

Background: s/Aims: Non-invasive models stratifying clinically significant portal hypertension (CSPH) are limited. Herein, we developed a new non-invasive model for predicting CSPH in patients with compensated cirrhosis and investigated whether carvedilol can prevent hepatic decompensation in patients with high-risk CSPH stratified using the new model. Methods: Non-invasive risk factors of CSPH were identified via systematic review and meta-analysis of studies involving patients with hepatic venous pressure gradient (HVPG). A new non-invasive model was validated for various performance aspects in three cohorts, i.e., a multicenter HVPG cohort, a follow-up cohort, and a carvediloltreating cohort. Results: In the meta-analysis with six studies (n=819), liver stiffness measurement and platelet count were identified as independent risk factors for CSPH and were used to develop the new “CSPH risk” model. In the HVPG cohort (n=151), the new model accurately predicted CSPH with cutoff values of 0 and –0.68 for ruling in and out CSPH, respectively. In the follow-up cohort (n=1,102), the cumulative incidences of decompensation events significantly differed using the cutoff values of <–0.68 (low-risk), –0.68 to 0 (medium-risk), and >0 (high-risk). In the carvediloltreated cohort, patients with high-risk CSPH treated with carvedilol (n=81) had lower rates of decompensation events than non-selective beta-blockers untreated patients with high-risk CSPH (n=613 before propensity score matching [PSM], n=162 after PSM). Conclusions Treatment with carvedilol significantly reduces the risk of hepatic decompensation in patients with high-risk CSPH stratified by the new model.

Lumbosacral radiculopathy refers to the pain syndrome caused by inflammation or mechanical compression of the lumbar nerve root, mainly manifested as low back pain, and radiating to the lower limbs in cutaneous mode, which can be accompanied by numbness, paresthesia, tingling, muscle weakness and loss of specific reflexes and other symptoms, which not only bring physical pain and life inconvenience to the patients, but also bring huge economic burden to the social medical care. Selective nerve root block(SNRB), as a safe, effective, low-cost, precise and minimally invasive clinical technique, can accurately intervene in specific nerve roots and quickly relieve pain symptoms by reducing inflammation and improving the surrounding environment of nerves. However, there are still many challenges and controversies in practice, such as precise targeting requirements, drug selection, potential risks and complications, and differences in efficacy among different patient populations. The purpose of this review is to systematically review and analyze the existing research results on SNRB, so as to provide useful reference and guidance for the further development of this field.

Accurate timing of myelination is crucial for the proper functioning of the central nervous system. Here, we identified a de novo heterozygous mutation in TMEM63A (c.1894G>A; p. Ala632Thr) in a 7-year-old boy exhibiting hypomyelination. A Ca²⁺ influx assay suggested that this is a loss-of-function mutation. To explore how TMEM63A deficiency causes hypomyelination, we generated Tmem63a knockout mice. Genetic deletion of TMEM63A resulted in hypomyelination at postnatal day 14 (P14) arising from impaired differentiation of oligodendrocyte precursor cells (OPCs). Notably, the myelin dysplasia was transient, returning to normal levels by P28. Primary cultures of Tmem63a^-/- OPCs presented delayed differentiation. Lentivirus-based expression of TMEM63A but not TMEM63A_A632T rescued the differentiation of Tmem63a^-/- OPCs in vitro and myelination in Tmem63a^-/- mice. These data thus support the conclusion that the mutation in TMEM63A is the pathogenesis of the hypomyelination in the patient. Our study further demonstrated that TMEM63A-mediated Ca²⁺ influx plays critical roles in the early development of myelin and oligodendrocyte differentiation.
Animals ; Cell Differentiation/physiology* ; Oligodendroglia/metabolism* ; Mice, Knockout ; Mice ; Male ; Myelin Sheath/metabolism* ; Humans ; Child ; Cells, Cultured ; Oligodendrocyte Precursor Cells/metabolism*