OBJECTIVES: To investigate the mechanism by which circ_EPHB4 regulates temozolomide (TMZ) sensitivity of glioma cells through the miR-424-5p/Wnt3 signal axis. METHODS: We detected the expression levels of circ_EPHB4, miR-424-5p and Wnt3 mRNA in glioma specimens from 25 patients with primary glioma and 25 patients experiencing relapse following temozolomide-based chemotherapy and in TMZ-sensitive and -resistant glioma A172 and SHG44 cells with circ_EPHB4 knockdown using qRT-PCR, and Wnt3 protein expression level was detected with Western blotting. Cell viability, colony-forming ability, and apoptosis of the cells with circ_EPHB4 knockdown were assessed, and the targeted regulation relationship between circ_EPHB4, miR-424-5p, and Wnt3 was verified by dual luciferase reporter assay and RNA immunoprecipitation (RIP) experiments. The effect of circ_EPHB4 knockdown on tumorigenesis of glioma cells was evaluated in subcutaneous tumor-bearing nude mouse models. RESULTS: The expression of circ_EPHB4 was significantly increased in glioma tissues and cells as compared with normal neural tissues and astrocytes (P=0.014). In TMZ-resistant glioma cells, circ_EPHB4 knockdown resulted in an obvious reduction of IC50 value of TMZ, inhibited cell colony formation, and promoted cell apoptosis, and these effects were reversed by miR-424-5p knockdown. The expressions of miR-424-5p and circ_EPHB4 were negatively correlated in glioma tissues (P=0.011). MiR-424-5p knockdown also attenuated the effect of circ_EPHB4 knockdown on expressions of PCNA, P-gp, MRP1 and bax. CONCLUSIONS Circ_EPHB4 regulates Wnt3 expression through "sponge adsorption" of miR-424-5p, thereby modulating TMZ-resistant glioblastoma cell clonogenesis, apoptosis, and TMZ sensitivity, suggesting the potential of circ_EPHB4 as a therapeutic target for reversing drug resistance of gliomas.
MicroRNAs/genetics* ; Humans ; Temozolomide ; Glioma/genetics* ; Animals ; Mice, Nude ; Cell Line, Tumor ; Wnt3 Protein/metabolism* ; Mice ; Apoptosis ; RNA, Circular ; Drug Resistance, Neoplasm ; Brain Neoplasms/pathology* ; Signal Transduction

OBJECTIVES: To investigate the oncogenic role of circular RNA circ_EPHB4 in glioma and its molecular mechanism. METHODS: Microarray analysis was performed to identify the differentially expressed circRNAs in glioma tissues. The effects of circ_EPHB4 on glioma cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and tumorigenicity in vivo were assessed using scratch wound healing assay, Transwell invasion assay and nude mouse models bearing subcutaneous tumors. RNA immunoprecipitation (RIP), RNA stability assays, and gene overexpression and silencing techniques were employed to validate the synergistic regulatory effect of circ_EPHB4 and the N6-methyladenosine (m⁶A) reader protein YTHDF3 on Wnt3 expression. RESULTS: Circ_EPHB4 was significantly overexpressed by 2.3 folds (|log2FC|=1.2, P<0.01) in glioma tissues compared to the adjacent tissues, and by 2.5 folds in glioma cell line U373 compared to normal cells (P<0.001). Overexpression of circ_EPHB4 significantly enhanced migration and invasion of glioma cells, and promoted the expressions of EMT markers N-cadherin and vimentin. In the tumor-bearing mouse models, the tumor volume in circ_EPHB4 overexpression group was significantly greater than that in the control group, and the lung metastatic foci increased by 4.2 folds. Overexpression of circ_EPHB4 promoted oncogenesis by upregulating Wnt3 expression, while YTHDF3 extended the half-life of Wnt3 mRNA in an m⁶A-dependent manner. Simultaneous knockdown of circ_EPHB4 and YTHDF3 resulted in an obvious reduction of Wnt3 mRNA expression by up to 47% compared to its level following knocking down either circ_EPHB4 or YTHDF3 alone. CONCLUSIONS Circ_EPHB4 and YTHDF3 promote glioma progression by jointly targeting the Wnt3 signaling pathway, which may provide a new therapeutic strategy for gliomas.
Glioma/genetics* ; Humans ; Animals ; Cell Line, Tumor ; RNA-Binding Proteins/genetics* ; RNA, Circular ; Epithelial-Mesenchymal Transition ; Mice, Nude ; Cell Movement ; Wnt3 Protein/genetics* ; Mice ; Disease Progression ; Adenosine/metabolism* ; Brain Neoplasms/metabolism* ; Gene Expression Regulation, Neoplastic

Neutralizing antibodies have been designed to specifically target and bind to the receptor binding domain (RBD) of spike (S) protein to block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus from attaching to angiotensin converting enzyme 2 (ACE2). This study reports a distinctive nanobody, designated as VHH21, that directly catalyzes the S-trimer into an irreversible transition state through postfusion conformational changes. Derived from camels immunized with multiple antigens, a set of nanobodies with high affinity for the S1 protein displays abilities to neutralize pseudovirion infections with a broad resistance to variants of concern of SARS-CoV-2, including SARS-CoV and BatRaTG13. Importantly, a super-resolution screening and analysis platform based on visual fluorescence probes was designed and applied to monitor single proteins and protein subunits. A spontaneously occurring dimeric form of VHH21 was obtained to rapidly destroy the S-trimer. Structural analysis via cryogenic electron microscopy revealed that VHH21 targets specific conserved epitopes on the S protein, distinct from the ACE2 binding site on the RBD, which destabilizes the fusion process. This research highlights the potential of VHH21 as an abzyme-like nanobody (nanoabzyme) possessing broad-spectrum binding capabilities and highly effective anti-viral properties and offers a promising strategy for combating coronavirus outbreaks.
Single-Domain Antibodies/immunology* ; Spike Glycoprotein, Coronavirus/metabolism* ; SARS-CoV-2/immunology* ; Animals ; Humans ; Antibodies, Neutralizing/immunology* ; Camelus ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Angiotensin-Converting Enzyme 2

ObjectiveTo collate and compare the characteristics and differences in the methods for constructing animal models of different subtypes of gastroesophageal reflux disease （GERD） based on literature， providing a reference for researchers in this field regarding animal model construction. MethodsExperimental studies related to GERD including reflux esophagitis （RE）， nonerosive reflux disease （NERD） and Barrett's esophagus （BE） model construction from January 1， 2014 to January 27， 2024， were retrieved from databases such as CNKI， Wanfang， VIP， Web of Science， and Pubmed. Information on animal strains， genders， modeling methods including disease-syndrome combination models， modeling cycles were extracted； for studies with model evaluation， the methods of model evaluation were also extracted； then analyzing all those information. ResultsA total of 182 articles were included. SD rats were most frequently selected when inducing animal models of RE （88/148， 59.46%） and NERD （9/14， 64.29%）. For BE， C57BL/6 mice were most commonly used （11/20， 55.00%）. Male animals （RE： 111/135， 82.22%； NERD： 11/14， 78.57%； BE： 10/12， 83.33%） were the most common gender among the three subtypes. The key to constructing RE animal models lies in structural damage to the esophageal mucosal layer， gastric content reflux， or mixed reflux， among which forestomach ligation + incomplete pylorus ligation （42/158， 26.58%） was the most common modeling method； the key to constructing NERD animal models lies in micro-inflammation of the esophageal mucosa， visceral hypersensitivity， and emotional problems， and intraperitoneal injection of a mixed suspension of ovalbumin and aluminum hydroxide combined with acid perfusion in the lower esophagus （8/14， 57.14%） was the most common modeling method； the key to constructing BE animal models lies in long-term inflammatory stimulation of the esophageal mucosa and bile acid reflux， and constructing interleukin 2-interleukin 1β transgenic mice （7/25， 28.00%） was the most common modeling method. Adverse psychological stress was the most common method for inducing liver depression. ConclusionsThe construction key principles and methodologies for RE， NERD， and BE animal models exhibit significant differences. Researchers should select appropriate models based on subtype characteristics （e.g.， RE focusing on structural damage， NERD emphasizing visceral hypersensitivity）. Current studies show insufficient exploration of traditional Chinese medicine disease-syndrome combination models. Future research needs to optimize syndrome modeling approaches （e.g.， composite etiology simulation） and establish integrated Chinese-Western medicine evaluation systems to better support mechanistic investigations of traditional Chinese medicine.

Objective To explore TCM syndrome distribution law in patients with overlapping non-erosive reflux disease(NERD)and epigastric pain syndrome(EPS)gastrointestinal symptoms.Methods A multi-center,cross-sectional study was conducted to investigate the general information of 800 patients with overlapping NERD and EPS gastrointestinal symptoms in four hospitals,such as gender,age,body mass index(BMI)and four diagnostic information of TCM.Descriptive frequency analysis,factor analysis and clustering analysis were used to summarize the TCM syndrome types and distribution characteristics.Results The average age of 800 patients with overlapping NERD and EPS gastrointestinal symptoms was(44.50±14.43)years old,the average BMI was(23.17±4.80)kg/m2,and the male to female ratio was 3:5.Frequency of 95 TCM symptoms/signs≥20%.18 common factor variables were obtained based on factor analysis,and the cumulative contribution rate was 67.11%.The first three syndrome elements of disease location were liver,stomach and spleen,and the disease nature syndrome elements were qi stagnation,qi deficiency and yin deficiency.Based on the clustering analysis of 18 common factor variables,combined with expert discussion,four main TCM syndrome types were obtained,which were liver-stomach stagnation heat syndrome(213 cases,26.63%),spleen-stomach damp heat syndrome(209 cases,26.13%),spleen-stomach deficiency and cold qi stagnation syndrome(190 cases,23.75%)and qi-phlegm stagnation syndrome(188 cases,23.50%).There was no significant difference in the distribution of TCM syndrome types among patients with different genders,ages and BMI values(P>0.05).Patients with a course of disease≥2 years and those residing long-term north of the Qinling-Huaihe Line showed a significantly higher prevalence of spleen-stomach dampness-heat syndrome(P<0.05).Conclusion The syndrome elements of disease location of overlapping NERD and EPS gastrointestinal symptoms are mainly liver,stomach and spleen.The TCM syndrome types are liver-stomach stagnation heat syndrome,spleen-stomach damp heat syndrome,spleen-stomach deficiency cold qi stagnation syndrome and qi-phlegm stagnation syndrome.The course of disease and the regional differences between north and south may be the influencing factors of the distribution of syndrome types.

Objective To explore TCM syndrome distribution law in patients with overlapping non-erosive reflux disease(NERD)and epigastric pain syndrome(EPS)gastrointestinal symptoms.Methods A multi-center,cross-sectional study was conducted to investigate the general information of 800 patients with overlapping NERD and EPS gastrointestinal symptoms in four hospitals,such as gender,age,body mass index(BMI)and four diagnostic information of TCM.Descriptive frequency analysis,factor analysis and clustering analysis were used to summarize the TCM syndrome types and distribution characteristics.Results The average age of 800 patients with overlapping NERD and EPS gastrointestinal symptoms was(44.50±14.43)years old,the average BMI was(23.17±4.80)kg/m2,and the male to female ratio was 3:5.Frequency of 95 TCM symptoms/signs≥20%.18 common factor variables were obtained based on factor analysis,and the cumulative contribution rate was 67.11%.The first three syndrome elements of disease location were liver,stomach and spleen,and the disease nature syndrome elements were qi stagnation,qi deficiency and yin deficiency.Based on the clustering analysis of 18 common factor variables,combined with expert discussion,four main TCM syndrome types were obtained,which were liver-stomach stagnation heat syndrome(213 cases,26.63%),spleen-stomach damp heat syndrome(209 cases,26.13%),spleen-stomach deficiency and cold qi stagnation syndrome(190 cases,23.75%)and qi-phlegm stagnation syndrome(188 cases,23.50%).There was no significant difference in the distribution of TCM syndrome types among patients with different genders,ages and BMI values(P>0.05).Patients with a course of disease≥2 years and those residing long-term north of the Qinling-Huaihe Line showed a significantly higher prevalence of spleen-stomach dampness-heat syndrome(P<0.05).Conclusion The syndrome elements of disease location of overlapping NERD and EPS gastrointestinal symptoms are mainly liver,stomach and spleen.The TCM syndrome types are liver-stomach stagnation heat syndrome,spleen-stomach damp heat syndrome,spleen-stomach deficiency cold qi stagnation syndrome and qi-phlegm stagnation syndrome.The course of disease and the regional differences between north and south may be the influencing factors of the distribution of syndrome types.

Objective To investigate the molecular mechanism of ALKBH5 reducing sepsis-induced myocardial dysfunction(SIMD).Methods The expression levels of ALKBH5 and TRAF1 in the blood of 50 SIMD patients and 50 healthy individuals were detected using reverse transcription fluorescence quantitative polymerase chain reaction(RT-qPCR),and the correlation between their expression levels was analyzed by person analysis;In vitro experiments,H9C2 myocardial cells were divided into 7 groups according to over expression of TARF1 and knockdown ALKBH5.The molecular mechanism of ALKBH5 targeting TRAF1 to regulate lipopolysaccharide(LPS)induced myocardial cell damage was studied through experiments such as CCK8,ELISA,and Western blot;In the in vivo experiment of rats,LPS induced rats were divided into 6 groups according to over expression of TARF1 and knockdown ALKBH5.Experimental methods such as colorimetry,ELISA,Western blot,HE staining,and immuno-histochemistry were used to study the mechanism of ALKBH5 targeting TRAF1 through NF-κB pathway in reduc-ing myocardial cell damage.Results The expression levels of ALKBH5 and TRAF 1 were downregulated in SIMD,and the Pearson analysis showed a positive correlation between them(P<0.001);In vitro experiments showed that overexpression of TRAF1 promotes cell proliferation,inhibits the expression of inflammatory factors and proteins involved in the NF-κB pathway,and knockdown ALKBH5 obtain the opposite resulst;In vivo experi-ments in rats showed that knockdown ALKBH5 promotes injury in cardiomyocytes,expression of inflammatory factors and NF-κB-related pathway proteins,and nuclear translocation of NF-κB p65 protein,but the overexpression of TRAF 1 yielded the opposite results.Conclusion ALKBH5 increases the stability of TRAF1 by reducing its meth-ylation,thereby inhibiting NF-κB pathway,thereby reducing SIMD.

Small ubiquitin-related modifier(SUMOylation)is a dynamic post-translational modification that maintains cardiac function and can protect against a hypertrophic response to cardiac pressure overload.However,the function of SUMOylation after myocardial infarction(MI)and the molecular details of heart cell responses to SUMO1 deficiency have not been determined.In this study,we demonstrated that SUMO1 protein was inconsistently abundant in different cell types and heart regions after MI.However,SUMO1 knockout significantly exacerbated systolic dysfunction and infarct size after myocardial injury.Single-nucleus RNA sequencing revealed the differential role of SUMO1 in regulating heart cells.Among cardiomyocytes,SUMO1 deletion increased the Nppa+Nppb+Ankrd1+cardiomyocyte subcluster pro-portion after MI.In addition,the conversion of fibroblasts to myofibroblasts subclusters was inhibited in SUMO1 knockout mice.Importantly,SUMO1 loss promoted proliferation of endothelial cell subsets with the ability to reconstitute neovascularization and expressed angiogenesis-related genes.Computational analysis of ligand/receptor interactions suggested putative pathways that mediate cardiomyocytes to endothelial cell communication in the myocardium.Mice preinjected with cardiomyocyte-specific AAV-SUMO1,but not the endothelial cell-specific form,and exhibited ameliorated cardiac remodeling following MI.Collectively,our results identified the role of SUMO1 in cardiomyocytes,fibroblasts,and endothelial cells after Ml.These findings provide new insights into SUMO1 involvement in the patho-genesis of MI and reveal novel therapeutic targets.

Antibodies, Neutralizing ; Antibodies, Viral/isolation & purification* ; B-Lymphocytes/virology* ; COVID-19/virology* ; Humans ; Immunity/genetics* ; RNA, Viral/immunology* ; SARS-CoV-2/pathogenicity* ; Single-Cell Analysis

Objective:To explore the application value of microsurgical tethered cord release in children with tethered cord syndrome under perioperative electro-neurophysiology monitoring.Methods:Ninety-six patients with tethered cord syndrome accepted tethered cord release in our hospital from January 2015 to December 2019 were chosen in our study; perioperative electro-neurophysiology monitoring was performed to evaluate whether there was neurological impairment. The surgical results, neuroelectrophysiological monitoring results, and follow-up results were retrospectively analyzed.Results:In these 96 patients, symptoms disappeared in 45 patients, improved in 34, not improved in 10, worsened in 3, and tethered again in 4 patients, with a total effective rate of 82.6%. No death was noted. Preoperative neuroelectrophysiological monitoring showed abnormal sensory and motor functions of lower limbs in 40 patients, and postoperative monitoring showed abnormal sensory and motor functions of lower limbs in 6 patients. Follow up was performed for an average of 13 months; symptoms improved in 79 patients, stabilized in 10 patients, and aggravated in 7 patients.Conclusion:In children with tethered cord syndrome, tethered cord release should be performed as soon as possible regardless of early neurological injury; perioperative electro-neurophysiology monitoring can protect spinal cord function, prevent nerve function injury, improve the surgical safety and improve the prognosis of these children.