Jove-Journal of Visualized Experiments最新文献

{"title":"Vacuum-Assisted Breast Biopsy and Excision for Breast Lesions: A Systematic Review and Meta-analysis of Malignant Upgrade in B3 Lesions.","authors":"Xuejing Wang","doi":"10.3791/74198","DOIUrl":"https://doi.org/10.3791/74198","url":null,"abstract":"<p><p>Vacuum-assisted breast biopsy (VAB/VABB) and vacuum-assisted excision (VAE) serve distinct diagnostic and therapeutic roles, requiring pathway-specific evaluation of their outcomes. This systematic review reconstructed a report-level and outcome-level dataset from 26 publications, of which 18 were reviewed in full text and 8 remained incompletely retrieved. Reports without complete full text were retained for narrative context but excluded from quantitative synthesis. Five full-text studies contributed 98 malignant upgrades among 844 B3 lesions after diagnostic VAB/VABB. The pooled proportion was 11.23% (95% confidence interval [CI], 7.22%-17.04%; 95% prediction interval [PI], 4.56%-25.06%; I² = 60.8%). Three full-text studies of second-line VAE reported study-specific upgrade proportions from 5.1% to 21.4%; these estimates were not pooled because selection pathways and case mix differed substantially. Post-VAE radiologic removal, absence of residual lesion, and recurrence-free outcomes ranged from 86.0% to 99.2% across five full-text cohorts but were not combined because they represented different endpoints and follow-up intervals. Only one fully retrieved study provided reconstructable diagnostic 2 × 2 data, precluding a diagnostic meta-analysis or hierarchical model. Certainty was very low for the principal observational outcomes and low for the single randomized comparison. The evidence supports pathway-specific interpretation and multidisciplinary decision-making, not universal superiority over core needle biopsy or equivalence to surgical excision.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149216957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enrichment of Retinal Endothelial Cells and Müller Glia Enable Comprehensive scRNA-Seq Profiling of the Blood-Retinal Barrier.","authors":"Johannes Robert Fleischer, Melanie Schwämmle, Jan Niklas Neß, Jakob Arnold, Malte Jung, Sagar, Günther Schlunck, Hansjürgen Agostini, Felicitas Bucher","doi":"10.3791/73625","DOIUrl":"https://doi.org/10.3791/73625","url":null,"abstract":"<p><p>Obtaining high-quality single-cell suspensions from neural tissues such as the murine retina remains technically challenging due to cellular fragility, tight intercellular junctions, and the heterogeneous composition of the tissue. These challenges are further compounded when studying rare cell populations, including vascular endothelial cells and Müller glia, which each account for less than 2% and are underrepresented in standard dissociation workflows, often lost during processing. Here, we present an optimized method for isolating murine retinal cells that preserves cellular integrity and enables selective enrichment of sparse populations for downstream single-cell RNA sequencing (scRNA-seq). Our protocol integrates gentle enzymatic dissociation with mechanical trituration to maximize cell viability and minimize transcriptional artifacts. We further incorporate targeted enrichment strategies, using fluorescence-activated cell sorting (FACS), to increase the relative abundance of vascular endothelial and Müller glial cells without compromising overall transcriptomic diversity. Moreover, we tested antibody-based cell hashing to enable sample multiplexing to improve cost efficiency. We demonstrate that this approach yields high-quality single-cell suspensions with improved representation of rare retinal cell types, enabling more robust and reproducible scRNA-seq analyses. This method provides a practical framework for researchers aiming to investigate the vascular and glial cell components of the retina at single-cell resolution and can be adapted to other neural tissues in which rare cell populations are of interest.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149216996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment of Rat Endometriosis Model by Autologous Endometrial Transplantation and Proof-of-Concept Pharmacological Validation with Elagolix.","authors":"Yanan Wang, Yanan Li, Yawen Li, Yunlong Hou, Tianyu Kang, Kaixin Li, Xue Wang, Xueying Chen, Mengnan Li","doi":"10.3791/74321","DOIUrl":"https://doi.org/10.3791/74321","url":null,"abstract":"<p><p>Endometriosis (EM) is a common chronic gynecological disorder in women of reproductive age, yet its pathogenesis remains unclear, and current therapeutic options are limited by significant side effects. Endometriosis itself increases the risk of premature ovarian failure. Long-term lack of standardized treatment may accelerate the decline of ovarian function and prematurely trigger the aging process of the reproductive system. Reliable animal models are therefore essential for investigating disease mechanisms and evaluating novel treatments. This study presents a detailed protocol for establishing an endometriosis model in Sprague‑Dawley (SD) rats via autologous endometrial transplantation, and evaluates the therapeutic efficacy of the FDA‑approved gonadotropin‑releasing hormone (GnRH) antagonist Elagolix. The method involved excising uterine tissue, isolating endometrial fragments, and suturing them onto the peritoneal wall. At 21 days post‑surgery, ectopic lesions with typical cystic morphology had successfully formed in 18 out of 20 rats (90% success rate). Following model validation, Elagolix administration (100 mg/kg daily, oral gavage for 4 weeks) significantly reduced final lesion volume compared with the model group (inhibition rate: 86.7%), decreased serum levels of follicle‑stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2), and suppressed peritoneal cytokine levels, including tumor necrosis factor‑α (TNF‑α), vascular endothelial growth factor (VEGF), and interleukin‑6 (IL‑6). Histopathological examination revealed glandular atrophy, reduced epithelial proliferation, and diminished inflammatory infiltration in Elagolix‑treated lesions. The model demonstrated several advantages, including operational simplicity, high stability, and avoidance of immune rejection. The pharmacological response to GnRH antagonism in this model demonstrated biological activity consistent with the known mechanism of Elagolix, although pain-related and behavioral outcomes were not evaluated. Collectively, this model provides a standardized and reliable platform for mechanistic studies and preclinical drug efficacy assessment in endometriosis.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149217063","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"p53 Immunophenotype and Clinical Features Associated with Endometrial Polyp Recurrence in Women with Metabolic Syndrome: A Retrospective Cohort Study.","authors":"Shasha Liu, Li Cai, Lili Geng, Lanting Pang, Hua Xu, Na Chen, Xiaodan Li, Haiying Li","doi":"10.3791/73553","DOIUrl":"https://doi.org/10.3791/73553","url":null,"abstract":"<p><p>Endometrial polyp recurrence after hysteroscopic resection remains clinically relevant in women with metabolic syndrome. In this retrospective cohort study, we evaluated the associations of a prespecified study-defined diffuse-strong p53 immunohistochemical phenotype, Ki-67 expression, and selected clinical characteristics with 24-month recurrence and explored the incremental discrimination provided by the IHC markers. The analytic cohort included 180 women with metabolic syndrome (MetS) who underwent hysteroscopic resection of histologically confirmed endometrial polyps (EPs) and had ascertainable recurrence status through 24 months. Associations with recurrence were assessed using a prespecified multivariable logistic model and a supportive multivariable Cox model, while exploratory prediction performance was evaluated using repeated stratified cross-validation. During 24 months of follow-up, 58 of 180 women (32.2%) had documented recurrence. Diffuse-strong p53 phenotype (adjusted odds ratio (aOR) 2.74, 95% CI 1.32-5.68), multiple polyps (aOR 2.74, 95% CI 1.25-6.02), AUB (aOR 2.29, 95% CI 1.12-4.65), and endometrial thickness (aOR 1.25 per mm, 95% CI 1.05-1.48) were independently associated with recurrence. Ki-67 ≥ 10% showed a directionally positive but statistically uncertain adjusted association (aOR 1.85, 95% CI 0.88-3.90). Supportive Cox analysis showed associations of diffuse-strong p53 phenotype, multiple polyps, AUB, and endometrial thickness with time to recurrence, whereas the estimate for Ki-67 remained statistically uncertain. The integrated model showed modestly higher repeated cross-validated discrimination than the clinical-only model (AUC 0.734 vs. 0.679; ΔAUC 0.056, 95% CI 0.006-0.105). Diffuse-strong p53 phenotype and selected clinical characteristics were associated with recurrence, whereas the contribution of Ki-67 remained exploratory; the modest incremental discrimination of the integrated model requires external validation before clinical application.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149217190","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Machine Learning and Post-Hoc Feature Attribution for Predicting Cardiac MRI Motion Artifacts: A Retrospective Cohort Study.","authors":"Ya Li, Qiuyi Cai, Lin Li, Jianlin Li, Dun Pan, Jian Liu","doi":"10.3791/72876","DOIUrl":"https://doi.org/10.3791/72876","url":null,"abstract":"<p><p>Anxiety-induced motion artifacts frequently compromise image quality during non-sedated cardiac magnetic resonance imaging (CMR), leading to rescans and increased healthcare burdens. This retrospective cohort study hypothesized that combining multidimensional psychological and physiological indicators could accurately predict artifact occurrence. Baseline data from 514 adult patients, encompassing 15 clinical features, were systematically analyzed. Four machine learning algorithms were trained and evaluated using the area under the receiver operating characteristic curve (AUC). The eXtreme Gradient Boosting model demonstrated the highest discriminative ability (AUC = 0.875). To provide clinical transparency, SHapley Additive exPlanations (SHAP) analysis was applied, revealing that state anxiety (evaluated via the State-Trait Anxiety Inventory State subscale, STAI-State), maximum breath-hold duration, and heart rate variability were primarily associated with artifact risk. The novelty of this approach lies in uncovering non-linear threshold effects-such as a sharp risk escalation when STAI-State scores exceeded 45-and complex interactions between physical capacity and claustrophobia. In conclusion, this interpretable machine learning framework offers a robust tool for patient-specific risk stratification, suggesting that preemptive and targeted nursing interventions could be optimized based on individualized algorithmic predictions.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149217224","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spatial Neighborhood Construction and NICHES Connectomics for Differential Ligand-Receptor Analysis in Fibrotic Human Lung.","authors":"Diego Perez Aracena, Nuoya Wang, Micha Sam Brickman Raredon","doi":"10.3791/70816","DOIUrl":"https://doi.org/10.3791/70816","url":null,"abstract":"<p><p>Spatial transcriptomics enables measurement of gene expression within intact tissue architecture, yet most analytical workflows emphasize cell-type classification over cellular interaction. Workflows that do infer intercellular signaling typically aggregate inferred interactions across clusters, regions, or spots, and fix neighborhood definitions, ligand-receptor (LR) databases, and scoring functions that users cannot easily inspect or change. The unmet need is therefore an analysis workflow that preserves signaling inference at the resolution of individual cell-cell pairs, constrains candidate interactions using measured physical distance, and exposes every modeling choice as a user-set parameter. Here, we describe a computational protocol that meets these requirements by inferring LR signaling at cell-cell pair resolution, grounded in physical tissue space, from imaging-based spatial transcriptomics data. The workflow defines spatial neighborhoods and constructs a directed connectomics graph in which edges represent the set of inferred LR interactions between cells. Using the NICHES (Niche Interactions and Communication Heterogeneity in Extracellular Signaling) package, three complementary representations are derived: a Cell-to-Cell (C2C) edge-level connectome, a Cell-to-Neighborhood (C2N) outgoing signaling summary, and a Neighborhood-to-Cell (N2C) incoming signaling summary. A parallel Neighborhood Composition assay characterizes the local tissue architecture of the spatial neighborhoods. The framework is modular and tunable: users may adjust neighborhood definitions, select alternative LR databases, and modify scoring and aggregation methods. These node- and edge-aligned data layers support visualization, clustering, and differential analysis of spatially organized signaling patterns. Applied to fibrotic lung tissue, the workflow segregates architecturally defined neighborhoods and identifies differential LR mechanisms that distinguish fibrotic regions from surrounding tissue. This protocol provides a reproducible framework for studying spatially organized intercellular communication across tissues and disease contexts.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149216924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Clinical Application of Three-dimensional Laparoscopic Artery-first Pancreaticoduodenectomy in Treating Pancreatic Head Malignant Tumors.","authors":"Zhongnan Chen, Yan Zhang","doi":"10.3791/74248","DOIUrl":"https://doi.org/10.3791/74248","url":null,"abstract":"<p><p>Pancreaticoduodenectomy is a technically demanding procedure associated with substantial surgical risk. Conventional laparoscopic approaches have several limitations, including restricted visualization, which can make exposure of the superior mesenteric artery and identification of vessels and lymph nodes challenging. In patients with vascular adhesion or tumor invasion, surgical dissection is particularly difficult and may increase the risk of intraoperative vascular injury and hemorrhage. In addition, poor delineation of anatomical planes may result in incomplete tumor dissection, inadequate lymphadenectomy, and difficult mobilization of the pancreatic head and duodenum, potentially compromising surgical safety and oncological outcomes. To optimize the surgical workflow, this protocol describes a three-dimensional laparoscopic artery-first pancreaticoduodenectomy with the infracolic artery-first approach as the principal technical modification. By combining early arterial exposure and clear delineation of anatomical planes with the stereoscopic visualization of three-dimensional laparoscopy, this technique facilitates identification of vessel-tissue relationships, vascular control, lymphadenectomy, tumor dissection, and tissue mobilization. These features may help reduce inadvertent vascular injury and intraoperative blood loss while enabling complete tumor resection. The protocol provides a standardized surgical approach, particularly applicable to pancreatic head lesions adjacent to or involving major vessels, and is intended to improve the safety and reproducibility of laparoscopic pancreaticoduodenectomy.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149216894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanical Debridement of Dental Implant Surfaces: Residual Dye Coverage and Qualitative Surface Morphology in a Phantom-Head In Vitro Model.","authors":"Buse Bayraktar Ayakta, Kemal Ustun","doi":"10.3791/73564","DOIUrl":"https://doi.org/10.3791/73564","url":null,"abstract":"<p><p>Mechanical debridement of implant surfaces is limited by implant geometry and defect-related access restrictions. This study evaluated post-treatment residual dye coverage after three mechanical debridement protocols in a standardized phantom-head model, testing the null hypothesis that coverage would not differ among protocols within each defect configuration. Sixty titanium implants were coated with permanent dye and subjected to erythritol air-flowing, sodium bicarbonate air-polishing, or nickel-titanium (Ni-Ti) brush instrumentation in simulated Class 1A and Class 1B peri-implant defects (n = 10 per group). Residual dye coverage was quantified from three photographic projections per surface using ImageJ and expressed as the percentage of evaluable projected implant area covered by residual dye, with the implant as the experimental unit. Pre-treatment coverage was standardized procedurally but not measured quantitatively; outcomes therefore represent post-treatment residual coverage rather than contamination removed. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were used for descriptive surface characterization. Residual dye remained on all implants. Within Class 1A defects, implant-level coverage differed among protocols (p < 0.001), erythritol showing greater coverage than sodium bicarbonate (adjusted p < 0.001) and Ni-Ti (adjusted p = 0.014). Coverage also differed within Class 1B defects (p < 0.001), erythritol again exceeding sodium bicarbonate (adjusted p = 0.018) and Ni-Ti (adjusted p < 0.001). Sodium bicarbonate and Ni-Ti did not differ in either configuration (adjusted p = 0.143 and 0.383). On the distal surface of Class 1B defects, Ni-Ti showed lower coverage than both (adjusted p = 0.003 and 0.005). Surface-site distribution also differed within each protocol in Class 1B defects (p ≤ 0.006). Exploratory SEM-EDS observations of individual specimens showed differences in surface appearance. Within the limitations of this in vitro model, residual dye coverage differed by protocol, and no protocol resulted in a dye-free surface.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149217208","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mixed Pneumonia with Pulmonary Embolism and Multiple Pathogens Detected in Bronchoalveolar Lavage Fluid: A Case Report.","authors":"Xia Chen","doi":"10.3791/72065","DOIUrl":"https://doi.org/10.3791/72065","url":null,"abstract":"<p><p>Severe community-acquired pneumonia may involve mixed pathogens and concurrent vascular complications. A 72-year-old man presented with fever, nonproductive cough, vomiting, hypoxemia, markedly elevated inflammatory markers, and an elevated D-dimer concentration. Chest computed tomography showed left lower-lobe consolidation and pleural effusion. Computed tomography pulmonary angiography, obtained because of hypoxemia and suspected pulmonary embolism, showed a filling defect in a right upper-lobe pulmonary artery. The patient received antimicrobial therapy prior to bronchoalveolar lavage (BAL). Bronchoscopy was performed in the bronchopulmonary segment corresponding to the radiographic lesion, and approximately 10 mL of bronchoalveolar lavage fluid (BALF) was recovered aseptically for microbiological and cytological testing. An aliquot of at least 3 mL was allocated to a multiplex pathogen-targeted sequencing assay, which detected Chlamydia psittaci, influenza A H3N2, Streptococcus pneumoniae, and additional conditionally pathogenic or commensal organisms. The three principal detections were interpreted in conjunction with the clinical presentation, imaging, inflammatory markers, BALF cytology, conventional testing, and treatment response. The patient improved during antimicrobial and anticoagulant treatment and was discharged on December 3, 2025. At the documented outpatient follow-up on December 17, rivaroxaban was continued for approximately one further month. This case highlights the adjunctive value and interpretive limitations of targeted sequencing in a treated patient. The serial imaging and invasive sampling reflected case-specific decisions and should not be interpreted as a routine protocol for pneumonia.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149217244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"piRNA-Annotated hsa_piR_016984 Regulates Extracellular Matrix Homeostasis in Human Scleral Fibroblasts by Modulating TGF-β1 mRNA Stability.","authors":"Binbin Dai, Ledan Wang, Weiwei Li, Jingjing Ma, Junhong Jiang, Qinzhu Huang","doi":"10.3791/72780","DOIUrl":"https://doi.org/10.3791/72780","url":null,"abstract":"<p><p>Scleral extracellular matrix (ECM) remodeling is a pathological feature of axial myopia. The relationship between small-RNA-associated regulation and cellular responses to mechanical loading remains incompletely understood. This study examines hsa_piR_016984-associated regulation in human scleral fibroblasts under a defined cyclic-stretch regimen. Text mining and bioinformatics identified myopia-associated hub genes. Fibroblasts underwent cyclic stretch (10% elongation, 1 Hz, 48 h) in vitro. Gene expression was evaluated by quantitative polymerase chain reaction (qPCR), and matrix-associated staining was assessed using toluidine blue. Sequencing dataset analysis and aqueous humor qPCR screened differentially expressed piRNA-annotated small RNAs. RNA pull-down, dual-luciferase reporter assays, and transcript time-course measurements examined their relationship with target messenger RNAs (mRNAs). Eight hub genes, including transforming growth factor beta 1 (TGF-β1) and various matrix metalloproteinases (MMPs), were identified via bioinformatic mining. In vitro, mechanical stretch reduced toluidine blue staining intensity, alongside increased MMP expression and decreased TGF-β1 expression, accompanied by a more pronounced decline in TGF-β1 qPCR signals relative to 0 h after actinomycin D addition. The piRNA-annotated small RNA hsa_piR_016984 was significantly downregulated in myopic aqueous humor. RNA pull-down showed TGFB1 mRNA co-enrichment with biotin-labeled hsa_piR_016984 relative to the negative-control probe. Separately, hsa_piR_016984 manipulation was associated with changes in TGFB1 qPCR signals over time and attenuation of the stretch-induced reduction in matrix staining intensity. Co-enrichment does not itself demonstrate direct RNA-RNA base pairing. Exogenous TGF-β1 increased hsa_piR_016984 expression, whereas BI-4659 decreased its expression under the tested in vitro conditions. These changes do not establish direct SMAD-mediated transcriptional regulation or a positive-feedback loop. Conclusions: The findings describe hsa_piR_016984-associated changes in TGF-β1 transcript signals and matrix staining in cultured human scleral fibroblasts under the tested conditions. They do not establish intrinsic mechanosensitivity or effects on myopia progression in vivo. The descriptive time courses, including post-washout measurements, do not establish mRNA half-life differences.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 236","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149217316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}