Objective This study aims to achieve high-yield functional expression of the human voltage-gated potassium channel K_V3.1 using an Escherichia coli cell-free protein synthesis system, thereby providing a novel synthetic approach for drug screening, structural analysis and functional characterization of K_V3.1. Methods K_V3.1 was expressed in an Escherichia coli cell-free protein synthesis system for 10 hours in the presence of peptide surfactant A₆K. The secondary structure of K_V3.1 was analyzed by circular dichroism spectroscopy. The potassium channel activity of the recombinant protein liposome K_V3.1-A₆K was investigated using fluorescent dyes Oxonol VI as indicators, which are capable of reflecting alterations in membrane potential. Results Soluble K_V3.1 protein was successfully synthesized, achieving a purified yield of up to 1.2 mg/mL via an Escherichia coli cell-free protein synthesis system. Circular dichroism spectroscopy revealed that K_V3.1 exhibited characteristic α-helical secondary structures. Membrane potential fluorescence assays demonstrated that the K_V3.1-A₆K proteoliposomes, which were reconstructed with surfactant peptide A₆K, exhibited remarkable potassium ion permeability. Conclusion This study successfully achieved high-yield expression of human K_V3.1 with activity using an Escherichia coli-based cell-free protein synthesis system. This innovative method not only significantly enhances the expression yield of K_V3.1, but also maintains its functional activity, thereby establishing a novel and efficient synthetic platform for drug screening and advancing our understanding of structure-function relationships in K_V3.1 research.
Humans ; Escherichia coli/metabolism* ; Shaw Potassium Channels/biosynthesis* ; Cell-Free System ; Circular Dichroism ; Protein Biosynthesis ; Recombinant Proteins/metabolism* ; Membrane Potentials ; Shab Potassium Channels

Approximately 60% of colorectal cancer (CRC) patients exhibit TP53 mutations, which are strongly associated with tumor progression, chemotherapy resistance, and an unfavorable prognosis. However, targeting p53 has historically been challenging, and currently, there are no approved p53-based therapeutics for clinical use worldwide. In this study, we discovered that ubiquitin carboxyl terminal hydrolase L3 (UCHL3) plays a crucial role in high-level glycolysis, enhanced stem-like properties, and 5-fluorouracil (5-FU) chemoresistance in TP53-mutant CRC by exerting its deubiquitinating enzyme activity to stabilize α-enolase (ENO1) protein. Notably, we identified a newly Food and Drug Administration (FDA)-approved drug, pacritinib, that potently suppresses UCHL3 expression by blocking the janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway in TP53-mutant CRC. Furthermore, Pacritinib was demonstrated to effectively inhibit glycolysis and improve the sensitivity to 5-FU chemotherapy in TP53-mutant CRC. Our findings suggest that targeting the JAK2-STAT3-UCHL3-ENO1 axis is a promising strategy to suppress glycolysis and enhance the efficacy of 5-FU chemotherapy in TP53-mutant CRC. Pacritinib shows potential for clinical application in the treatment of TP53-mutant CRC.

Early correction of childhood malocclusion is timely managing morphological, structural, and functional abnormalities at different dentomaxillofacial developmental stages. The selection of appropriate imaging examination and comprehensive radiological diagnosis and analysis play an important role in early correction of childhood malocclusion. This expert consensus is a collaborative effort by multidisciplinary experts in dentistry across the nation based on the current clinical evidence, aiming to provide general guidance on appropriate imaging examination selection, comprehensive and accurate imaging assessment for early orthodontic treatment patients.
Humans ; Malocclusion/diagnostic imaging* ; Child ; Consensus

Objective:To explore the imaging characterization of mandibular third molar (M3) and mandibular second molar (M2) and their associated areas using cone beam CT (CBCT).Methods:A total of 226 images of patients with concomitant mandibular second and third molars in the Department of Oral and Maxillofacial Medical Imaging of Nanjing Stomatology Hospital were selected to retrospectively analyze their clinical information and imaging manifestations from January 2020 to January 2024, and their clinical information and imaging performances were analyzed. Clinical information included patient′s age, gender, and chief complaint; dental tissues (crowns, roots) and periodontal tissues (periodontium, alveolar bone) of the mandibular second molar were evaluated based on their CBCT, and the position (high, medium, low), type of obstruction, dental tissues, periodontal tissues and bone margins of the alveolar bone between them, extent (concave, oblique, flat) and depth of bone defects, and periodontal membrane imaging changes of the mandibular third molars etc.Results:There were 104 cases of mandibular M2 and M3 as the main complaints and 122 cases of non-main complaints in this study. According to the imaging aspects, the mandibular M2 and M3 associated regions with abnormal CBCT imaging manifestations were mainly categorized into six sagittal representation phenotypes (types Ⅰ-Ⅵ); among them, middle-aged and elderly patients≥45 years of age more often showed abnormal imaging phenotypes (75%), and the peripheral bone defects were more severe. The correlation between the type and height of the mandibular M3 obstruction (high:medium:low=42∶110∶74) and the imaging typology of the mandibular M2 and M3 association regions was statistically significant ( P<0.001). The bone defects between mandibular M2 and M3 of the young patients≤25 years of age were predominantly of the oblique type of resorption (41/69 cases), and the least of the flat type (1/69 cases), whereas flat type could reached 27.8% in the middle-aged and elderly patients≥45 years of age, and the circumferential angle of the peripheral bone defects in the mandibular M2 and M3 regions showed strong correlation with age ( P<0.001), typology ( P<0.001), and the height of the obstruction ( P=0.004). The correlation between defect depth and obstruction height for the same fractal type was statistically significant in the age groups≤25 years and 26-44 years ( P≤25 years=0.0385, P26~44 years=0.032). There was a significant correlation between mandibular M3 with different types of obstruction, especially proximal-medial (34/43 cases) and horizontal obstruction (8/43 cases), and neighboring M2 suffering from caries ( P=0.004). The correlation between obstruction height (median 46.3%, low 50.0%) and M2 extra-root resorption (80 cases) was statistically significant ( P<0.001). Conclusions:In this study, we explored the imaging characteristics of the mandibular M2 and M3 association region using CBCT, and established a six-type sagittal classification system for the M2 and M3 association area, providing a reproducible framework for the systematic assessment of the overall status of this region. Further analysis revealed that the incidence of radiographic abnormalities in the bone tissue of this area significantly increased with age. Concurrently, the study confirmed that the impaction status of mandibular M3 is associated with an elevated risk of dental and periodontal diseases affecting M2. These specific findings provide important insights into the pathological relationships within the mandibular M2\M3 region and support clinical decision-making.

Objective:To explore the imaging characterization of mandibular third molar (M3) and mandibular second molar (M2) and their associated areas using cone beam CT (CBCT).Methods:A total of 226 images of patients with concomitant mandibular second and third molars in the Department of Oral and Maxillofacial Medical Imaging of Nanjing Stomatology Hospital were selected to retrospectively analyze their clinical information and imaging manifestations from January 2020 to January 2024, and their clinical information and imaging performances were analyzed. Clinical information included patient′s age, gender, and chief complaint; dental tissues (crowns, roots) and periodontal tissues (periodontium, alveolar bone) of the mandibular second molar were evaluated based on their CBCT, and the position (high, medium, low), type of obstruction, dental tissues, periodontal tissues and bone margins of the alveolar bone between them, extent (concave, oblique, flat) and depth of bone defects, and periodontal membrane imaging changes of the mandibular third molars etc.Results:There were 104 cases of mandibular M2 and M3 as the main complaints and 122 cases of non-main complaints in this study. According to the imaging aspects, the mandibular M2 and M3 associated regions with abnormal CBCT imaging manifestations were mainly categorized into six sagittal representation phenotypes (types Ⅰ-Ⅵ); among them, middle-aged and elderly patients≥45 years of age more often showed abnormal imaging phenotypes (75%), and the peripheral bone defects were more severe. The correlation between the type and height of the mandibular M3 obstruction (high:medium:low=42∶110∶74) and the imaging typology of the mandibular M2 and M3 association regions was statistically significant ( P<0.001). The bone defects between mandibular M2 and M3 of the young patients≤25 years of age were predominantly of the oblique type of resorption (41/69 cases), and the least of the flat type (1/69 cases), whereas flat type could reached 27.8% in the middle-aged and elderly patients≥45 years of age, and the circumferential angle of the peripheral bone defects in the mandibular M2 and M3 regions showed strong correlation with age ( P<0.001), typology ( P<0.001), and the height of the obstruction ( P=0.004). The correlation between defect depth and obstruction height for the same fractal type was statistically significant in the age groups≤25 years and 26-44 years ( P≤25 years=0.0385, P26~44 years=0.032). There was a significant correlation between mandibular M3 with different types of obstruction, especially proximal-medial (34/43 cases) and horizontal obstruction (8/43 cases), and neighboring M2 suffering from caries ( P=0.004). The correlation between obstruction height (median 46.3%, low 50.0%) and M2 extra-root resorption (80 cases) was statistically significant ( P<0.001). Conclusions:In this study, we explored the imaging characteristics of the mandibular M2 and M3 association region using CBCT, and established a six-type sagittal classification system for the M2 and M3 association area, providing a reproducible framework for the systematic assessment of the overall status of this region. Further analysis revealed that the incidence of radiographic abnormalities in the bone tissue of this area significantly increased with age. Concurrently, the study confirmed that the impaction status of mandibular M3 is associated with an elevated risk of dental and periodontal diseases affecting M2. These specific findings provide important insights into the pathological relationships within the mandibular M2\M3 region and support clinical decision-making.

Objective To investigate the longitudinal association between alcohol abstinence and accelerated biological aging among middle-aged and older adults and to explore the potential effect modifiers influencing the association.Methods Utilizing the clinico-biochemical and anthropometric data from the baseline and first repeat survey of the UK Biobank(UKB),we employed the Klemera and Doubal method(KDM)to construct the biological age(BA)and calculate BA acceleration.Change analysis based on multivariate linear regression models was employed to explore the association between changes in alcohol abstinence and changes in BA acceleration.Age,sex,smoking status,tea and coffee consumption,and body mass index were considered as the stratification factors for conducting stratified analysis.Results A total of 5 412 participants were included.Short-term alcohol abstinence(β=1.00,95%confidence interval[CI]:0.15-1.86)was found to accelerate biological aging when compared to consistent never drinking,while long-term abstinence(β=-0.20,95%CI:-1.12-0.71)did not result in a significant acceleration of biological aging.Body mass index may be a potential effect modifier.Conclusion Short-term alcohol abstinence was associated with accelerated biological aging,but the effect gradually diminishes over extended periods of abstinence.

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer death worldwide. It is urgent to develop new drugs to improve the prognosis of ESCC patients. Here, we found benzydamine, a locally acting non-steroidal anti-inflammatory drug, had potent cytotoxic effect on ESCC cells. Benzydamine could suppress ESCC proliferation in vivo and in vitro. In terms of mechanism, CDK2 was identified as a target of benzydamine by molecular docking, pull-down assay and in vitro kinase assay. Specifically, benzydamine inhibited the growth of ESCC cells by inhibiting CDK2 activity and affecting downstream phosphorylation of MCM2, c-Myc and Rb, resulting in cell cycle arrest. Our study illustrates that benzydamine inhibits the growth of ESCC cells by downregulating the CDK2 pathway.
Humans ; Benzydamine ; Esophageal Neoplasms/drug therapy* ; Esophageal Squamous Cell Carcinoma/drug therapy* ; Molecular Docking Simulation ; Phosphorylation ; Cell Proliferation ; Cell Line, Tumor ; Apoptosis ; Cyclin-Dependent Kinase 2

Deficiency of natural killer (NK) cells shows a significant impact on tumor progression and failure of immunotherapy. It is highly desirable to boost NK cell immunity by upregulating active receptors and relieving the immunosuppressive tumor microenvironment. Unfortunately, mobilization of NK cells is hampered by poor accumulation and short retention of drugs in tumors, thus declining antitumor efficiency. Herein, we develop an acid-switchable nanoparticle with self-adaptive aggregation property for co-delivering galunisertib and interleukin 15 (IL-15). The nanoparticles induce morphology switch by a decomposition-metal coordination cascade reaction, which provides a new methodology to trigger aggregation. It shows self-adaptive size-enlargement upon acidity, thus improving drug retention in tumor to over 120 h. The diameter of agglomerates is increased and drug release is effectively promoted following reduced pH values. The nanoparticles activate both NK cell and CD8⁺ T cell immunity in vivo. It significantly suppresses CT26 tumor in immune-deficient BALB/c mice, and the efficiency is further improved in immunocompetent mice, indicating that the nanoparticles can not only boost innate NK cell immunity but also adaptive T cell immunity. The approach reported here provides an innovative strategy to improve drug retention in tumors, which will enhance cancer immunotherapy by boosting NK cells.

BACKGROUND: Metastasis is the main cause of tumor-associated death and mainly responsible for treatment failure of breast cancer. Autophagy accelerates tumor metastasis. In our work, we aimed to investigate the possibility of microRNAs (miRNAs) which participate in the regulation of autophagy to inhibit tumor metastasis. METHODS: MiRNA array and comprehensive analysis were performed to identify miRNAs which participated in the regulation of autophagy to inhibit tumor metastasis. The expression levels of miR-3653 in breast cancer tissues and cells were detected by quantitative real-time polymerase chain reaction. In vivo and in vitro assays were conducted to determine the function of miR-3653. The target genes of miR-3653 were detected by a dual luciferase reporter activity assay and Western blot. The relationship between miR-3653 and epithelial-mesenchymal transition (EMT) was assessed by Western blot. Student's t -test was used to analyze the difference between any two groups, and the difference among multiple groups was analyzed with one-way analysis of variance and a Bonferroni post hoc test. RESULTS: miR-3653 was downregulated in breast cancer cells with high metastatic ability, and high expression of miR-3653 blocked autophagic flux in breast cancer cells. Clinically, low expression of miR-3653 in breast cancer tissues (0.054 ± 0.013 vs . 0.131 ± 0.028, t  = 2.475, P  = 0.014) was positively correlated with lymph node metastasis (0.015 ± 0.004 vs . 0.078 ± 0.020, t  = 2.319, P  = 0.023) and poor prognosis ( P  < 0.001). miR-3653 ameliorated the malignant phenotypes of breast cancer cells, including proliferation, migration (MDA-MB-231: 0.353 ± 0.013 vs . 1.000 ± 0.038, t  = 16.290, P  < 0.001; MDA-MB-468: 0.200 ± 0.014 vs . 1.000 ± 0.043, t  = 17.530, P  < 0.001), invasion (MDA-MB-231: 0.723 ± 0.056 vs . 1.000 ± 0.035, t  = 4.223, P  = 0.013; MDA-MB-468: 0.222 ± 0.016 vs . 1.000 ± 0.019, t  = 31.050, P  < 0.001), and colony formation (MDA-MB-231: 0.472 ± 0.022 vs . 1.000 ± 0.022, t  = 16.620, P  < 0.001; MDA-MB-468: 0.650 ± 0.040 vs . 1.000 ± 0.098, t  = 3.297, P  = 0.030). The autophagy-associated genes autophagy-related gene 12 ( ATG12 ) and activating molecule in beclin 1-regulated autophagy protein 1 ( AMBRA1 ) are target genes of miR-3653. Further studies showed that miR-3653 inhibited EMT by targeting ATG12 and AMBRA1 . CONCLUSIONS Our findings suggested that miR-3653 inhibits the autophagy process by targeting ATG12 and AMBRA1 , thereby inhibiting EMT, and provided a new idea and target for the metastasis of breast cancer.
Cell Line, Tumor ; Epithelial-Mesenchymal Transition/genetics* ; MicroRNAs/metabolism* ; Autophagy/genetics* ; Genes, Regulator ; Gene Expression Regulation, Neoplastic/genetics* ; Cell Proliferation/genetics* ; Cell Movement/genetics* ; Neoplasms/genetics*

Nucleic acid drugs are highly applicable for cancer immunotherapy with promising therapeutic effects, while targeting delivery of these drugs to disease lesions remains challenging. Cationic polymeric nanoparticles have paved the way for efficient delivery of nucleic acid drugs, and achieved stimuli-responsive disassembly in tumor microenvironment (TME). However, TME is highly heterogeneous between individuals, and most nanocarriers lack active-control over the release of loaded nucleic acid drugs, which will definitely reduce the therapeutic efficacy. Herein, we have developed a light-controllable charge-reversal nanoparticle (LCCN) with controlled release of polyinosinic-polycytidylic acid [Poly(I:C)] to treat triple negative breast cancer (TNBC) by enhanced photodynamic immunotherapy. The nanoparticles keep suitably positive charge for stable loading of Poly(I:C), while rapidly reverse to negative charge after near-infrared light irradiation to release Poly(I:C). LCCN-Poly(I:C) nanoparticles trigger effective phototoxicity and immunogenic cell death on 4T1 tumor cells, elevate antitumor immune responses and inhibit the growth of primary and abscopal 4T1 tumors in mice. The approach provides a promising strategy for controlled release of various nucleic acid-based immune modulators, which may enhance the efficacy of photodynamic immunotherapy against TNBC.