Journal Of Visualized Experiments[JOURNAL]
Sun
200 papers
An Explainable Privacy Preserving Multimodal Ensemble Framework For Skin Lesion Classification.
Koul A, Singh NP
J Vis Exp · 2026 Jun · PMID 42371851 · Publisher ↗
Among dermatological diseases, skin cancer is among the most life-threatening. Early and accurate diagnosis is important for improving a patient's prognosis. Nevertheless, traditional AI-based diagnostic methods face sev...
Among dermatological diseases, skin cancer is among the most life-threatening. Early and accurate diagnosis is important for improving a patient's prognosis. Nevertheless, traditional AI-based diagnostic methods face several challenges, including privacy concerns, limited interpretability, and a severe class imbalance in multi-class skin lesion datasets. To overcome these challenges, the proposed paper proposes a privacy-aware, explainable multimodal skin lesion classification model that combines complex deep learning models and an ensemble modeling approach with explainable artificial intelligence methods. Experimental evaluation is conducted using publicly available HAM10000 benchmark data on multi-class skin lesion classification that can be accessed by means of Kaggle Hub, distributed over seven clinically significant lesion classes (akiec, bcc, bkl, df, mel, nv, vasc). To balance the data, a class-balancing technique is used to boost the minority classes. The EfficientNet B4, DenseNet201, and MobileNetv2 are used to extract deep feature representations, afterward combined with salient clinical metadata to create a robust multimodal feature space. These multimodal features are used to train XGBoost, LightGBM, Deep Neural Classifier (DNC) that resulted classification accuracies of 92%, 90% with 94% respectively. A stacked ensemble strategy is applied to combine the outputs of XGBoost, LightGBM, and Deep Neural Classifier (DNC), which leads to an improvement in accuracy of 96%. Model interpretability techniques provide feature-level explanations that increase transparency. The experimental findings proved the practicality of the suggested framework in terms of efficiency with clinically relevant real-life classification of skin lesions.
De Novo vs. Recurrent Hepatitis B After Liver Transplantation: Clinical Profiles And Functional Cure.
Hou W, Chen X
J Vis Exp · 2026 Jun · PMID 42371850 · Publisher ↗
This study compares the clinical characteristics and functional cure rates of de novo (DNH) versus recurrent hepatitis B virus (HBV) infections following liver transplantation. Data from 150 adult patients diagnosed with...
This study compares the clinical characteristics and functional cure rates of de novo (DNH) versus recurrent hepatitis B virus (HBV) infections following liver transplantation. Data from 150 adult patients diagnosed with post-transplant HBV between 2010 and 2024 were analyzed, explicitly excluding individuals receiving anti-HBc-positive donor grafts. Participants were stratified by pre-transplant serology into DNH (n = 36) and recurrent HBV (n = 114) cohorts. Clinically, the DNH group experienced more severe acute hepatic injury upon infection, evidenced by elevated median peak bilirubin levels and a remarkably higher incidence of hepatitis B e antigen (HBeAg) positivity at initial diagnosis (86.1% vs. 9.6%, p < 0.001). Despite this severe acute presentation, DNH patients exhibited robust hepatic recovery following antiviral intervention, achieving alanine aminotransferase (ALT) normalization rates comparable to the recurrent group (50.0% vs. 48.2%, p = 1.000). Functional cure, explicitly defined as sustained hepatitis B surface antigen (HBsAg) loss, was achieved in 20.67% (31/150) of the total cohort. Time-to-event Kaplan-Meier survival analysis demonstrated that the recurrent cohort achieved functional cure significantly earlier and at a higher cumulative probability (Log-rank p = 0.037). Crucially, multivariate Cox proportional hazards regression established that combination therapy with hepatitis B immunoglobulin (HBIG) served as a robust independent predictor of definitive functional cure (Adjusted Hazard Ratio = 4.21, p = 0.002), successfully overcoming initial baseline disparities. In conclusion, while DNH manifests as a severe acute infection due to pre-transplant immune naivety, recurrent HBV is associated with a more favorable trajectory toward functional cure. These findings support the implementation of personalized antiviral management, establishing that integrating nucleos(t)ide analogs with low-dose HBIG significantly optimizes definitive serological outcomes.
Fructus xanthii Extract Alleviates Osteoarthritis by Preserving Cartilage Integrity.
Dou X, Li X, Wang H … +9 more , Li M, Zhang W, Xu H, Zhang Y, Zhang E, Ma H, Zhao Y, Zou L, Yin L
J Vis Exp · 2026 Jun · PMID 42371840 · Publisher ↗
Osteoarthritis (OA) is a progressive degenerative joint disorder for which current pharmacological and surgical interventions mainly relieve symptoms rather than reverse the underlying pathological changes, highlighting...
Osteoarthritis (OA) is a progressive degenerative joint disorder for which current pharmacological and surgical interventions mainly relieve symptoms rather than reverse the underlying pathological changes, highlighting the urgent need for novel therapeutic agents. This protocol aimed to investigate the potential chondroprotective effects of Fructus Xanthii extract against OA. First, non-targeted metabolomics profiling was conducted using ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) to characterize the chemical constituents of the extract. An integrated strategy combining network pharmacology and transcriptomic mining was then applied to identify bioactive components and putative targets of the extract, followed by cross-analysis with OA-related dysregulated genes to obtain core target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to annotate the main biological processes and signaling pathways modulated by the extract. For in vivo validation, zebrafish cartilage injury models and mouse OA models were used to evaluate the chondroprotective effects of the extract. RNA sequencing (RNA-seq) was further adopted to analyze transcriptomic alterations in chondrocytes after treatment, focusing on OA-associated pathways and gene expression profiles; enzyme-linked immunosorbent assay (ELISA) was subsequently performed to verify the expression levels of key proteins/receptors screened by RNA-seq, so as to further confirm the regulatory effects of Fructus Xanthii extract on core OA targets. This integrated multi-omics and experimental protocol provides a systematic approach to explore the potential of Fructus Xanthii extract as a promising botanical candidate for OA intervention.
Combined Vacuum-Assisted Excision and Thermal Ablation for Symptomatic Benign Thyroid Nodules: A Pilot Study.
Guo R, Liu Y, Zhao J … +1 more , Qian L
J Vis Exp · 2026 Jun · PMID 42371838 · Publisher ↗
Thyroid nodules are a common clinical condition, while thermal ablation (TA) has emerged as a minimally invasive alternative to surgery. Single-session ablation often fails to achieve complete symptomatic resolution in l...
Thyroid nodules are a common clinical condition, while thermal ablation (TA) has emerged as a minimally invasive alternative to surgery. Single-session ablation often fails to achieve complete symptomatic resolution in large, hypervascular nodules. Post-ablation carbonization further limits volumetric resorption, necessitating optimized therapeutic strategies. This pilot study aimed to evaluate the feasibility, safety, and preliminary efficacy of combined vacuum-assisted excision and thermal ablation (VAE-TA) for symptomatic benign thyroid nodules refractory to initial ablation. From December 2023 to January 2025, five patients underwent VAE-TA at a single center. Inclusion criteria included persistent symptoms ≥12 months post-initial ablation and confirmed benignity via fine-needle aspiration. The procedure involved ultrasound-guided vacuum-assisted excision of post-ablation fibrotic tissue followed by microwave ablation. Primary outcomes included feasibility and safety, while secondary outcomes focused on volume reduction rate (VRR) and symptom improvement. Patients were followed up at 1 and 6 months. These preliminary findings suggest that VAE-TA may be a feasible, minimally invasive salvage approach for selected symptomatic benign thyroid nodules with incomplete response to initial ablation, although larger, controlled studies with longer follow-up are required.
Cell-Free In Vitro mRNA Translation In Drosophila Lysates.
Baranda-Ávila N, Valdes-Barreto M, Reza P … +2 more , Vélez DE, Hernández G
J Vis Exp · 2026 Jun · PMID 42371835 · Publisher ↗
Cell-free mRNA in vitro translation has played a crucial role in the understanding of the protein synthesis process during gene expression across eukaryotes. The development of lysates from different systems for in vitro...
Cell-free mRNA in vitro translation has played a crucial role in the understanding of the protein synthesis process during gene expression across eukaryotes. The development of lysates from different systems for in vitro translation has been instrumental in studying the roles of most components of the translation machinery and in dissecting many steps of the protein synthesis process. Different aspects of translation and translational control have been studied using Drosophila melanogaster lysates. This paper provides detailed protocols for preparing synthetic mRNA templates, growing large-scale living fly cultures, preparing translation-competent lysates from Drosophila embryos, and performing cell-free in vitro translation reactions. This protocol is suitable for any species of the Drosophila genus-melanogaster, virilis, pseudoscura, grimshawi, hydei, etc. We further compare this protocol to other translation systems and discuss limitations, critical steps, and troubleshooting. Representative outcomes are included to help assess protocol performance and reproducibility. Finally, we discuss potential applications in biotechnology and research.
Effect of Inhaled Corticosteroids on Clinical Efficacy and Blood Glucose In COPD: A Retrospective Cohort Study.
Qiu J, Wu H, Zhu X
J Vis Exp · 2026 Jun · PMID 42371832 · Publisher ↗
Inhaled corticosteroids (ICS) are commonly used in chronic obstructive pulmonary disease (COPD), but their potential association with hyperglycemia remains unclear. Therefore, evaluating the glycemic safety of ICS in non...
Inhaled corticosteroids (ICS) are commonly used in chronic obstructive pulmonary disease (COPD), but their potential association with hyperglycemia remains unclear. Therefore, evaluating the glycemic safety of ICS in non‑diabetic COPD patients has direct clinical importance for preventing potential metabolic complications during long‑term management. This retrospective cohort study aimed to evaluate whether adding ICS to long‑acting β₂‑agonist (LABA) therapy affects clinical efficacy and glycemic parameters in non‑diabetic COPD patients. A total of 150 hospitalized COPD patients (January 2022-December 2024) were included. Based on their documented treatment regimens, patients were divided into a control group (formoterol monotherapy, n = 75) and an observation group (formoterol/budesonide combination, n = 75). Primary outcomes were FEV₁, FEV₁/FVC, fasting plasma glucose (FPG), and HbA1c. Secondary outcomes included CAT score, liver/kidney function tests, arterial blood gases, bone mineral density (BMD), and adverse reactions. Baseline characteristics were balanced between groups (all P > 0.05). At 12 months, the observation group showed greater improvements in FEV₁ and FEV₁/FVC (both P < 0.01) and a larger reduction in CAT score (P < 0.001) compared with controls. No significant differences were observed in FPG (P = 0.837), HbA1c (P = 0.134), liver/kidney parameters, arterial blood gases, BMD, or adverse event incidence (P = 0.754) between the two groups. In this retrospective cohort of non‑diabetic COPD patients, adding ICS to LABA therapy improved lung function and symptoms without statistically significant effects on blood glucose or other safety parameters. These findings suggest that ICS/LABA combination may be a safe and effective option in carefully selected patients, but further prospective studies are needed to confirm the absence of long‑term glycemic risk.
Comparative Effects of Two Periarticular Analgesic Cocktails on Pain Reduction and Inflammatory Response After Unicompartmental Knee Arthroplasty.
Gu Z, Zhang Z
J Vis Exp · 2026 Jun · PMID 42371821 · Publisher ↗
Unicompartmental knee arthroplasty (UKA) serves as the primary surgical treatment for isolated unicompartmental knee osteoarthritis. Clinical data of 108 patients who underwent UKA at Yueyang People's Hospital from Janua...
Unicompartmental knee arthroplasty (UKA) serves as the primary surgical treatment for isolated unicompartmental knee osteoarthritis. Clinical data of 108 patients who underwent UKA at Yueyang People's Hospital from January 2020 to December 2024 were retrospectively screened. After strict case exclusion, 102 patients were categorized into Group A (ropivacaine combined with morphine and compound betamethasone, n = 52) and Group B (ropivacaine combined with ketorolac tromethamine and epinephrine, n = 50). Propensity score matching (PSM) was subsequently performed to balance baseline characteristics, resulting in 46 patients per group after matching. Primary outcomes consisted of resting and active visual analog scale (VAS) scores, 48 h postoperative opioid consumption, and inflammatory biomarkers, including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), white blood cell (WBC), and neutrophil (NEUT) levels. Secondary outcomes covered knee range of motion (ROM), time to initial postoperative ambulation, Knee Injury and Osteoarthritis Outcome Score (KOOS), Barthel Index (BI), and postoperative adverse events. Baseline parameters were well balanced between the two groups following PSM (all P > 0.05). At 48 h postoperatively, Group B presented significantly decreased resting and active VAS scores, as well as lower 48 h opioid consumption (all P < 0.001). Milder elevations of CRP, ESR, WBC, and NEUT were observed in Group B (all P < 0.001). Additionally, Group B demonstrated superior knee functional recovery reflected by better ROM, higher KOOS and BI scores, and shorter time to first ambulation (all P < 0.001). No significant intergroup difference was detected in the incidence of adverse events (P = 0.503). In this single-center retrospective cohort, the ropivacaine-ketorolac-epinephrine cocktail demonstrates superior analgesic and anti-inflammatory properties with a noninferior safety profile. Limited by the single-center, retrospective design, this regimen is preliminarily considered a promising analgesic alternative for UKA patients, and further large-sample, multicenter, prospective studies are required for validation.
Standardized Magnifying Endoscopy with Narrow-Band Imaging-Guided Targeted Biopsy Workflow For Upper Gastrointestinal Precancerous Lesions.
Ling Z, Jin C, Chu C
J Vis Exp · 2026 Jun · PMID 42371820 · Publisher ↗
Although early detection of upper gastrointestinal precancerous lesions is vital, subtle mucosal variations often limit conventional endoscopic screening. To address this limitation, we present a standardized targeted bi...
Although early detection of upper gastrointestinal precancerous lesions is vital, subtle mucosal variations often limit conventional endoscopic screening. To address this limitation, we present a standardized targeted biopsy protocol using magnifying endoscopy with narrow-band imaging (ME-NBI). This systematic workflow integrates key microstructural and microvascular criteria-specifically demarcation lines, irregular microsurfaces, atypical microvasculature, and marked glandular distortion-to identify high-risk focal areas for mucosal sampling. In a prospective clinical evaluation involving 161 patients, this standardized ME-NBI workflow was compared with a conventional white-light endoscopic assessment. The application of this protocol significantly enhanced the first-pass positive rate, overall biopsy positivity, and the diagnostic yield of high-risk lesions per biopsy sample, while simultaneously reducing the total number of biopsy samples required per patient. Furthermore, the standardized workflow demonstrated high diagnostic performance, achieving an area under the curve (AUC) of 0.928, and yielded strong interobserver agreement. By establishing objective criteria for target selection, this reproducible workflow reduces operator subjectivity, supports early neoplasia detection, and provides a practical framework for clinical implementation.
Anthrone Colorimetric Method for Quantifying Glycogen in Rat Liver and Skeletal Muscle.
Zhu M, Chen W, Li Y … +2 more , Liu Z, Liu Y
J Vis Exp · 2026 Jun · PMID 42371819 · Publisher ↗
Glycogen is a major intracellular glucose reserve in liver and skeletal muscle and plays a fundamental role in carbohydrate metabolism. Hepatic glycogen is essential for preserving systemic glycemic homeostasis, whereas...
Glycogen is a major intracellular glucose reserve in liver and skeletal muscle and plays a fundamental role in carbohydrate metabolism. Hepatic glycogen is essential for preserving systemic glycemic homeostasis, whereas skeletal muscle glycogen supports physical activity and exercise performance. Accurate quantification of tissue glycogen is therefore critical for studies in which glycogen metabolism is dynamically regulated or pathologically altered. Here, we describe a reliable and sensitive anthrone colorimetric method for quantifying glycogen content in rat liver and skeletal muscle. The protocol consists of sequential steps, including alkaline digestion, deproteinization, glycogen precipitation, alcohol washing, and color development using anthrone reagent. The applicability of this method is demonstrated in rats subjected to three physiologically relevant conditions: ad libitum feeding, overnight fasting, and short-term refeeding. These experimental paradigms capture key metabolic states associated with glycogen storage, depletion, and resynthesis. Orthogonal western blot analysis of key enzymes, glycogen synthase and glycogen phosphorylase, provides mechanistic support to the observed metabolic changes. The applicability of this method is further demonstrated in animals treated with epinephrine to model acute stress-induced glycogen breakdown. Collectively, this method provides a wide dynamic range and sufficient sensitivity to quantify tissue glycogen across markedly different physiological states. It offers a reliable tool for investigating glycogen metabolism in vivo and can also be applied to in vitro cell culture models.
Orthodontic Miniscrew Placement in Interradicular Regions using a Chairside Digital CAD-CAM Surgical Guide.
Zhang X, Wang Z, Fan S … +4 more , Liu K, Zhang Z, Wang Y, Wang X
J Vis Exp · 2026 Jun · PMID 42371818 · Publisher ↗
This study aims to introduce a novel chairside digital technique for creating surgical guides to assist in the placement of orthodontic miniscrews. In this clinical validation study, 12 orthodontic miniscrews were placed...
This study aims to introduce a novel chairside digital technique for creating surgical guides to assist in the placement of orthodontic miniscrews. In this clinical validation study, 12 orthodontic miniscrews were placed in six patients using a chairside economical restoration of esthetic ceramics (CEREC) system combined with cone-beam computed tomography (CBCT). A three-dimensional image was generated by integrating CBCT scans with digital dental models acquired via the chairside digital system. The optimal insertion angle and position for orthodontic miniscrews were planned using this image. A surgical guide was then designed in the computer-aided design (CAD) software and manufactured using the computer-aided manufacturing (CAM) system. Preoperative and postoperative CBCT scans obtained from patients were superimposed to compare the angular deviation, entry-point deviation, and endpoint deviation between the planned and actual miniscrew positions. The mean entry-point and endpoint deviations were 2.4 ± 0.7 mm (range: 1.2-3.4 mm) and 2.4 ± 0.6 mm (range: 1.3-3.4 mm), respectively. The mean angular deviation was 3.8° ± 2.3° (range: 0.4°-7.2°). No miniscrew failures were observed during the 6-month follow-up period. This study successfully developed a chairside CAD/CAM-based miniscrew surgical guide method, and its accuracy and stability were preliminarily validated in patients.
Modeling Legal Uncertainty in AI-Generated Content Ownership: A Bayesian Network Approach to Intellectual Property Rights Allocation.
Li Y
J Vis Exp · 2026 Jun · PMID 42371815 · Publisher ↗
The rapid adoption of generative artificial intelligence has exposed limitations in traditional intellectual property frameworks based on human authorship, creating uncertainty in ownership attribution. This study propos...
The rapid adoption of generative artificial intelligence has exposed limitations in traditional intellectual property frameworks based on human authorship, creating uncertainty in ownership attribution. This study proposes a Bayesian Network-based workflow for analyzing ownership of AI-generated content under legal and technical uncertainty. The workflow integrates dataset curation, variable annotation, probabilistic dependency modeling, inference generation, cross-validation, and sensitivity analysis to evaluate relationships among factors, including human contribution, the legality of training data, contractual context, and AI autonomy. The framework was developed using annotated cases derived from a curated AI litigation dataset, publicly available court opinion databases, and AI copyright case trackers. A sensitivity analysis was conducted to examine the influence of legal and technical variables on ownership attribution across different scenarios. The proposed framework supports probabilistic and interpretable reasoning for AI-related intellectual property disputes and provides a structured decision-support approach for legal professionals, policymakers, and AI developers. Results demonstrated stable posterior probability estimates across folds, with ownership prediction consistency exceeding 80% across datasets. The framework enables probabilistic, scenario-based reasoning and provides an interpretable decision-support tool, improving transparency and consistency in resolving ownership disputes for legal professionals, policymakers, and AI developers.
Mechanisms of Qu's Formula 3 for Improving Endometrial Receptivity in PCOS: A Combination of Network Pharmacology and Molecular Dynamics Simulation.
Li Y, Hu W, Liu C … +5 more , Li X, Dai M, Wang F, Zhou J, Qu F
J Vis Exp · 2026 Jun · PMID 42371814 · Publisher ↗
Impaired endometrial receptivity is a major cause of infertility in polycystic ovary syndrome (PCOS). Qu's Formula 3 (QUF3) is a Chinese herbal medicine formula clinically used to improve endometrial receptivity in patie...
Impaired endometrial receptivity is a major cause of infertility in polycystic ovary syndrome (PCOS). Qu's Formula 3 (QUF3) is a Chinese herbal medicine formula clinically used to improve endometrial receptivity in patients with PCOS. This study employed network pharmacology, molecular docking, and molecular dynamics (MD) simulations to investigate the underlying mechanisms. Active constituents of QUF3 were identified using the TCMSP database, and potential targets related to endometrial receptivity and PCOS were retrieved from DrugBank and other resources. A compound-target interaction network and a protein-protein interaction (PPI) network were constructed via Cytoscape to identify key targets. Core targets were subjected to GO and KEGG enrichment analyses. Molecular docking, MD simulations, principal component analysis (PCA), free energy landscape (FEL), and dynamic cross-correlation matrix (DCCM) were used to evaluate binding interactions. From 91 active ingredients and 294 potential drug targets, 60 disease-related targets were identified. Luteolin and sesamin were among the key pharmacodynamic components. Ten core targets were identified: AKT1, EGFR, TNF, TP53, IL6, BCL2, ESR1, IL1B, STAT3, and MMP9. KEGG enrichment revealed 132 signaling pathways, and GO analysis identified 678 entries. MD simulations indicated that the binding between the top five active constituents and their respective targets was stable. PCA, FEL, and DCCM further demonstrated high thermodynamic stability and structural rigidity of these complexes. In conclusion, QUF3 improves endometrial receptivity in PCOS through multicomponent, multitarget, and multipathway interactions. This study provides a theoretical basis, from a computational simulation perspective, for the development of targeted Chinese herbal medicine therapies for PCOS-related infertility, and may have positive implications for improving pregnancy outcomes in women with PCOS in the future.
Single-Cell Mass Spectrometry Imaging Using Desorption Electrospray Ionization Coupled To Orbitrap Mass Spectrometers.
Colwell N, Chen D, Bhusal D … +2 more , Peng Z, Yang Z
J Vis Exp · 2026 Jun · PMID 42371812 · Publisher ↗
Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) enables ambient, matrix-free molecular imaging with minimal sample preparation. This protocol describes a workflow for single-cell MSI of OVCAR-8 ce...
Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) enables ambient, matrix-free molecular imaging with minimal sample preparation. This protocol describes a workflow for single-cell MSI of OVCAR-8 cells using DESI coupled to Orbitrap mass spectrometers. The system integrates a DESI sprayer, a modified mass spectrometer interface, a motorized XYZ stage, and an optical breadboard for high-spatial-resolution imaging. Cells are cultured on gridded glass coverslips, washed with ammonium formate solution to reduce salt interference, dried, and analyzed under ambient conditions. Solvent is delivered continuously using a nano-liquid chromatography system, and nebulizing nitrogen gas is regulated to maintain spray stability. The protocol also describes raster imaging, pixel control, and optical-to-mass spectrometry image registration for single-cell localization. High mass resolution and mass accuracy provided by Orbitrap detection enable differentiation of closely related molecular species and support single-cell metabolomic analysis. This protocol provides an adaptable strategy for implementing high-spatial-resolution single-cell MSI using multiple Orbitrap mass spectrometer platforms.
Protection of Islet Beta Cells from Iron Overload-Induced Injury by Long Non-Coding RNA Maternally Expressed Gene 3.
Liang H, Luo J
J Vis Exp · 2026 Jun · PMID 42371811 · Publisher ↗
Patients with beta-thalassemia major (β-TM) commonly present with abnormal glucose metabolism, and factors such as iron overload, chronic anaemia, hormonal imbalances, liver dysfunction and inflammation-associated insuli...
Patients with beta-thalassemia major (β-TM) commonly present with abnormal glucose metabolism, and factors such as iron overload, chronic anaemia, hormonal imbalances, liver dysfunction and inflammation-associated insulin resistance are considered important contributors to pancreatic islet dysfunction. Long non-coding ribonucleic acids (lncRNAs) are increasingly recognised for their regulatory roles in cell apoptosis and glucose homeostasis. This study explores the function of the lncRNA, maternally expressed gene 3 (MEG3), in iron-induced islet β-cell injury. An iron-overload mouse model was induced via intraperitoneal injection of iron dextran. Iron deposition and tissue damage in the pancreas were evaluated using Prussian blue and hematoxylin and eosin staining, and MEG3 expression was quantified via reverse transcription polymerase chain reaction (RT-PCR). In vitro, MIN6 cells were transfected with MEG3 small interfering RNA to examine apoptosis via flow cytometry, and NF-kappa (ĸ)B signaling pathway activity was assessed by Western blotting. Histological staining confirmed significant iron deposition and structural damage in the pancreatic tissue of iron-overloaded mice. The RT-PCR analysis revealed a marked reduction of MEG3 expression in the pancreas (p<0.05). In vitro, MEG3 knockdown significantly increased apoptosis of MIN6 cells compared with controls. Western blot analysis showed an elevated level of NF-ĸB, indicating activation of the NF-ĸB signaling pathway in MEG3-silenced cells. These findings suggest that MEG3 downregulation may enhance islet β-cell apoptosis via NF-ĸB signaling under iron overload conditions. The MEG3 gene may play a protective role against iron-induced islet β-cell apoptosis, potentially involving modulation of the NF-ĸB pathway. This mechanism may contribute to islet dysfunction in patients with β-TM with iron overload.
Standardized Immunofluorescence Imaging of Tunneling Nanotubes and Mitochondrial Transfer Between Astrocytes and Neurons.
Du X, Deng Y
J Vis Exp · 2026 Jun · PMID 42371809 · Publisher ↗
Mitochondria are essential organelles that regulate energy production, cellular signaling, and metabolic homeostasis in neural cells. Tunneling nanotubes (TNTs) are thin membranous structures that mediate long-distance i...
Mitochondria are essential organelles that regulate energy production, cellular signaling, and metabolic homeostasis in neural cells. Tunneling nanotubes (TNTs) are thin membranous structures that mediate long-distance intercellular communication and facilitate the transfer of cellular components, including mitochondria, between connected cells. Reliable visualization of TNTs and mitochondrial transfer requires careful sample handling because these structures are highly fragile and sensitive to fixation, washing, and imaging conditions. This protocol describes standardized procedures for the fixation, staining, and confocal imaging of TNTs in astrocytes and astrocyte-neuron coculture systems. The workflow includes membrane and cytoskeletal staining for TNT visualization, mitochondrial labeling for tracking mitochondrial localization, and immunofluorescence staining for Miro1 colocalization analysis. Critical steps for preserving TNT morphology, including gentle washing and light-protected handling, are emphasized throughout the procedure. The protocol also outlines imaging approaches for the characterization of TNTs and mitochondria in fixed-cell preparations. These methods provide a reproducible experimental framework for studying TNT formation and mitochondrial transfer between neural cells in vitro.
Screening the Mechanism of Shikonin Against Renal Cell Carcinoma via Network Pharmacology, Molecular Docking, and Cellular Experimental Verification.
Gong WW, Yang Y, Wang SM … +5 more , Zha X, Ruan JH, Zhang L, Wan CL, Zhu ZS
J Vis Exp · 2026 Jun · PMID 42371800 · Publisher ↗
Renal cell carcinoma (RCC) is one of the most common tumors in the urinary system and has the highest mortality rate. Previous investigations have demonstrated that shikonin can treat renal cell carcinoma, but the mechan...
Renal cell carcinoma (RCC) is one of the most common tumors in the urinary system and has the highest mortality rate. Previous investigations have demonstrated that shikonin can treat renal cell carcinoma, but the mechanism remains unclear. Therefore, our study aimed to elucidate the mechanism of shikonin in the treatment of renal cell carcinoma using network pharmacology, molecular docking, and in vitro functional assays, including cell proliferation, migration, and apoptosis, with western blot (WB) validation. Shikonin targets were screened using PharmMapper, SwissTargetPrediction, and other databases, and identified RCC-related targets from Online Mendelian Inheritance in Man (OMIM), GeneCards, and other databases; the potential therapeutic targets were obtained by intersection analysis. A protein-protein interaction (PPI) network was constructed, and Cytoscape was used to screen core targets, while molecular docking was applied to analyze the binding affinity between shikonin and key targets. A total of 374 shikonin targets and 1,087 RCC-related targets were collected, and 98 overlapping target genes were identified. Six core targets (SRC, PIK3CA, PIK3CB, PIK3CD, PTPN11, and PIK3R1) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that shikonin exerted anti-RCC effects mainly by regulating protein kinase activity, HIF-1, and IL-17 signaling pathways. Molecular docking confirmed that shikonin had a strong binding affinity with these core targets. In vitro studies using human RCC Caki‑1 and 786‑O cells demonstrated that shikonin significantly inhibited cell proliferation and migration in a dose‑dependent manner, promoted cell apoptosis, and upregulated caspase‑3 and caspase‑8 activities. Western blot experiments further verified that shikonin modulated the expression of core target proteins and suppressed the HIF-1 signaling pathway. This study systematically elucidates the pharmacological mechanism of shikonin against renal cell carcinoma, providing a theoretical basis for the development and application of shikonin as a novel anti-RCC agent.
Motivation, Pain, and Social Support in Athletic Injury Rehabilitation: A Systematic Review and Narrative Synthesis.
Wang Y
J Vis Exp · 2026 Jun · PMID 42371799 · Publisher ↗
The rehabilitation process from athletic injury to peak performance is influenced by complex psychosocial factors. This systematic review synthesizes evidence regarding the effects of training motivation, pain, and socia...
The rehabilitation process from athletic injury to peak performance is influenced by complex psychosocial factors. This systematic review synthesizes evidence regarding the effects of training motivation, pain, and social support on rehabilitation outcomes. The review was conducted and reported in accordance with the PRISMA 2020 guidelines. A comprehensive search of PubMed, PsycINFO, SPORTDiscus, and Web of Science was performed. Following duplicate removal, two independent reviewers screened titles, abstracts, and full-text articles according to predefined eligibility criteria. Fifteen studies met the inclusion criteria and were included in a narrative synthesis. The findings suggest that motivation, pain, and social support are dynamically interconnected. Social support from family members, clinicians, and particularly peers may serve as a foundational resource that promotes autonomous motivation. In turn, this motivated engagement appears to facilitate adaptive pain coping. Based on the interpretive synthesis of the included studies, athletes who learn to distinguish between "good" pain (indicative of therapeutic progress) and "bad" pain (signaling potential harm) may experience a more favorable rehabilitation trajectory. Available evidence further suggests that such a trajectory is associated with sustained performance outcomes. Conversely, inadequate support may undermine motivation and promote fear-avoidance responses to pain, potentially contributing to poor rehabilitation outcomes or premature career termination in some cases. The therapeutic alliance and peer support emerged as critical mechanisms through which social support may exert its enabling effects. Interventions should therefore adopt a holistic approach that simultaneously strengthens support networks, nurtures autonomous motivation, and promotes adaptive pain appraisal. However, given the interpretive nature of this synthesis and the limitations of the available evidence, these conclusions should be interpreted cautiously. This review proposes an integrated conceptual model illustrating the potential reciprocal interplay among motivation, pain, and social support throughout the rehabilitation process.
Family Function and Kinesiophobia in Patients after Hip Replacement Surgery: A Cross-Sectional Study on the Mediating Role of Illness Perception.
Tang M, Pan J, Feng J … +3 more , Liu Y, Jiang M, Yang X
J Vis Exp · 2026 Jun · PMID 42371797 · Publisher ↗
This study investigated the association between family function and kinesiophobia in patients after hip replacement surgery and examined whether illness perception mediated this relationship. A total of 200 postoperative...
This study investigated the association between family function and kinesiophobia in patients after hip replacement surgery and examined whether illness perception mediated this relationship. A total of 200 postoperative patients were included. On postoperative day 3, family function, illness perception, and kinesiophobia were assessed using the Family APGAR Index, Brief Illness Perception Questionnaire (BIPQ), and Tampa Scale of Kinesiophobia (TSK-17), respectively. Descriptive, univariate, and correlation analyses were conducted using SPSS software. Mediation analysis was performed using Model 4 of Hayes' PROCESS 3.5 macro. Among the 200 participants, 59% experienced kinesiophobia. Family function was negatively correlated with kinesiophobia, whereas illness perception was positively correlated with kinesiophobia (r = -0.678 and 0.690, respectively; p < 0.001). Higher levels of family support were associated with lower levels of movement-related fear. Illness perception partially mediated the relationship between family function and kinesiophobia (β = -0.208, p < 0.05). The prevalence of postoperative kinesiophobia among patients after hip replacement surgery was high. The findings suggest that better family functioning may be associated with lower kinesiophobia, mediated by more positive illness perceptions. These results indicate that family support and cognitive appraisal should be considered when developing postoperative rehabilitation interventions.
Case Report of Gouty Arthritis Complicated by Septic Arthritis.
Wei X, Yuan J, Zhang Y … +4 more , Wang Z, Hua J, He B, Pei H
J Vis Exp · 2026 Jun · PMID 42340302 · Publisher ↗
This corrects the article 10.3791/69787.
This corrects the article 10.3791/69787.
Pioglitazone in Skin Fibrosis: Mechanistic Rationale and Therapeutic Potential of Pioglitazone for Scarring Dermatoses.
Steevens N, Keller K, Broughton L … +3 more , Stegura C, Beck TC, Valdebran MA
J Vis Exp · 2026 Jun · PMID 42330136 · Publisher ↗
Cutaneous fibrosis - encompassing keloids, hypertrophic scars, localized scleroderma (morphea), and scarring alopecias - remains fundamentally undertreated, with conventional interventions frequently yielding incomplete...
Cutaneous fibrosis - encompassing keloids, hypertrophic scars, localized scleroderma (morphea), and scarring alopecias - remains fundamentally undertreated, with conventional interventions frequently yielding incomplete therapeutic responses and high rates of recurrence. Pioglitazone, a Food and Drug Administration (FDA)-approved peroxisome proliferator-activated receptor γ (PPAR-γ) agonist traditionally utilized for glycemic control in type 2 diabetes mellitus, represents a highly compelling candidate for dermatologic repurposing. Beyond its canonical metabolic functions, robust target engagement of PPAR-γ by pioglitazone suppresses NF-κB-driven cytokine cascades, directly inhibits NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome assembly, promotes macrophage polarization toward tissue-reparative phenotypes, and disrupts profibrotic TGF-β/SMAD signaling architectures. Consequently, the drug exerts a dual mechanism of action, simultaneously attenuating chronic inflammation while inhibiting pathological fibrogenesis. This review expands upon prior analyses by integrating recently published clinical and scientific data, specifically highlighting new translational milestones, including human registry data in progeroid syndromes and randomized controlled trials in cicatricial alopecia. A distinct focus is placed on advancements in localized delivery strategies, such as nanostructured lipid carriers and niosomes, that offer the potential to overcome pioglitazone's inherent physicochemical constraints and thereby maximize dermal target engagement while avoiding systemic toxicities like heart failure and bladder cancer. Furthermore, this analysis synthesizes recent molecular insights connecting fibrosis and inflammation via target engagement biomarkers (e.g., FABP4, CD36) and proposes actionable, biomarker-driven trial designs, including adaptive basket trials, to bridge current gaps in dermatology-specific translation. Demonstrating localized anti-fibrotic and anti-inflammatory efficacy with minimal systemic risk could establish pioglitazone as a mechanism-based, disease-modifying therapy for a broad spectrum of fibrotic and scarring dermatologic disorders.
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