Jove-Journal of Visualized Experiments最新文献

Tunneling nanotubes (TNTs) are thin, actin-based intercellular conduits that enable long-range transfer of organelles and signaling cargo. Although widely reported across multiple cell types, their presence in human skin cells has not been well described. This article describes a standardized protocol to detect and characterize TNTs in vitro between human epidermal keratinocytes and dermal fibroblasts. The method involves preparing a co-culture of primary cells, gentle fixation to preserve fragile TNTs, membrane labeling with wheat germ agglutinin, F-actin staining with phalloidin, and systematic z-stack imaging by inverted confocal microscopy to distinguish TNTs suspended above the substratum from adherent filopodia. Optional immunostaining for α-tubulin allows assessment of microtubule incorporation. TNTs are defined by three features: thin, straight protrusions connecting two or more cells, the presence of F-actin, and continuity across cell pairs in serial optical sections. Representative results demonstrate TNTs linking dermal-dermal, epidermal-epidermal, and dermal-epidermal pairs, with variable cytoskeletal composition (F-actin alone or F-actin plus α-tubulin). Critical steps include gentle fixation, use of fresh reagents, and acquisition of sufficient z-planes to avoid misclassification, while common artifacts include TNT breakage and incomplete staining. Together, these optimized steps enable reproducible TNT detection in skin cell systems and offer a methodological basis for future investigation of TNT-mediated communication in skin biology and regeneration.

Congestion in IoT-enabled wireless sensor networks (WSNs) degrades packet delivery, latency, and energy usage, impairing the network, especially under bursty and heterogeneous traffic conditions. This protocol illustrates an intelligent congestion control technique that combines hybrid data aggregation, adaptive scheduling, and a neuro-fuzzy decision engine to efficiently handle network load. The method involves first generating simulation data, creating topologies of different node densities, and setting up traffic patterns using NS-2.35. Packet traces are obtained for each scenario to allow reproducible evaluation. The protocol workflow refers to the combination of two mechanisms: (1) hybrid aggregation, which combines packets in time- and count-based windows while retaining priority labels, and (2) adaptive scheduling, which handles dual priority queues via weighted round robin. A neuro-fuzzy controller always evaluates buffer occupancy, link quality, channel utilization, residual energy, and traffic priority. Taking these inputs, it regulates aggregation depth, queue weights, and transmission decisions by fuzzy inference and neuro-adaptive learning. Performance measurement tasks encompass the calculation of packet delivery ratio, end-to-end latency, throughput, node-level energy consumption, and network lifetime. Statistical analyses are performed across multiple runs to check the reliability of the results. The approach reveals better performance in the simulation compared to the baseline schemes. This protocol offers a reproducible framework for exploring hybrid congestion control methods that enable energy-efficient, scalable, and QoS-aware operation in IoT-enabled WSN environments.

This study aims to investigate the therapeutic potential and mechanisms of electroacupuncture (EA) in alleviating vascular dysfunction caused by high-salt diets, with a focus on gut microbiota modulation. We randomly divided 21 rats into Normal Control, High-Salt Model, and EA Intervention groups (n = 7 per group). The "high-salt blood stasis syndrome" model in Wistar rats was induced by intragastric perfusion of 12% NaCl. Treatments were performed between 17:00 and 19:00 Beijing time: rats were restrained with a soft cloth, followed by 20-min EA stimulation at BL23 and KI3. After the experiment, rat blood, blood vessels, and fecal samples were collected. Vascular effects were evaluated via coagulation function test, hemorheological analysis, and arterial histopathological assessment. 16S rDNA sequencing was used to analyze the composition and abundance changes of gut microbiota. The findings revealed that chronic high-salt intake disrupted blood viscosity, damaged vascular endothelial integrity, and caused gut microbial dysbiosis. Remarkably, EA treatment effectively reversed these pathological changes. Correlation analysis further identified specific gut microbiota (e.g., Lachnospiraceae and Ruminococcaceae) that strongly correlated with altered blood viscosity parameters (P < 0.05). Therefore, the present study concludes that high-salt diet-induced vasculopathy can be reversed by EA treatment, which may be attributed to EA's ability to regulate gut microbiota involved in bile acid metabolism and short-chain fatty acid synthesis.

This study presents a reproducible porcine cardiac arrest model utilizing an implantable cardioverter-defibrillator (ICD) for reliable induction and cardioversion of ventricular fibrillation (VF), as well as intracardiac ECG monitoring during resuscitation. Large animal models are vital for translational research in cardiac arrest; however, conventional VF induction and cardioversion techniques -- primarily using external electrodes and defibrillation -- are often hampered by motion artifacts, inconsistent conversion rates, and high animal resource use. This protocol describes repeated cycles of VF induction and cardioversion within the same animal, providing precise control while minimizing physiological stress and animal numbers, in accordance with the 3R principles (Replacement, Reduction, and Refinement). The ICD-based method allows for accurate rhythm monitoring by intracardiac ECG throughout resuscitation, with improved artifact resistance compared to surface ECG. Eleven pigs underwent repeated VF induction and cardioversion, with high success rates and recovery of spontaneous circulation. The model enables standardized and reliable data acquisition for cardiac arrest studies, ensuring experimental consistency and facilitating reproducible investigation of pathophysiology and resuscitation strategies while minimizing animal use.

This study introduces a Hybrid Quantum K-Means Clustering Algorithm with automatic cluster detection for classifying cancerous and non-cancerous gene expression data. The method employs Quantum Multi-Feature Mapping for state encoding, Swap Test-based quantum distance estimation, and Quantum Gradient-Based Optimization to dynamically identify the optimal number of clusters by minimizing intra-cluster variance. Initial centroids are selected through a probability-proportional distance strategy, improving stability and accuracy. Applied to breast cancer datasets, the approach surpasses the existing quantum K-Means algorithm, achieving a Silhouette Score of 0.641 (compared to 0.601), a Calinski-Harabasz Index of 766.57 (compared to 617.65), and a Davies-Bouldin Index of 0.659 (compared to 0.704). These results indicate superior cluster compactness and separation. Although the proposed algorithm exhibits slightly higher time complexity O (N×Kmax×Mobs) due to iterative optimization, it significantly outperforms predefined-K quantum K-Means in clustering accuracy, error reduction, and practical feasibility. Its efficiency in handling high-dimensional data and resilience to quantum noise highlights its potential for real-world bioinformatics applications, particularly in cancer classification using gene expression profiles.

Elastin-like polypeptides (ELP) are engineered biopolymers built from repetitive pentapeptide sequences that mimic motifs found in mammalian tropoelastin. Their unique characteristics make them ideal candidates for a wide array of biomedical applications, ranging from drug and gene delivery to tissue engineering and targeted molecular imaging. Conventional purification approaches from Escherichia coli (E. coli) expression can be ineffective for ELP due to the formation of inclusion bodies. Other methods, such as inverse transition cycling (ITC), utilize the lower critical solution temperature (LCST) properties of ELP to separate it from contaminants such as lipopolysaccharides (LPS), but typically require multiple heating and cooling steps that are time-consuming and can result in low recoveries depending on the sequence, concentration, and molecular weight of the ELP construct. To tackle these challenges, we have developed an organic solvent-based extraction-precipitation workflow that exploits the intrinsic hydrophobicity of ELP to enable rapid, robust, and broadly applicable purification directly from E. coli cell pellets. This method uses polar organic solvents to aid in cell disruption and selectively solubilize ELP in a single step. A subsequent precipitation step effectively removes residual organic solvents, low-molecular-weight impurities, and endotoxins, yielding highly pure ELP with LPS levels below 1 EU/mL in under 3 h. Atomic force microscopy data suggest that ELP-fusion proteins purified in this manner can self-assemble into reverse micelle-like structures that retain fusion protein function. This rapid purification method offers researchers a straightforward and potentially scalable way to purify ELP, creating new possibilities for using ELP and their fusion proteins as flexible building blocks for material and biomedical applications.

Radiation-induced skin injury is a common and debilitating complication in cancer therapy, often resulting in delayed wound healing and increased patient discomfort. There are a few therapeutic drugs available for prevention or treatment. Traditional Chinese Medicine, specifically Acorus calamus L., has shown potential in treating various skin disorders, but its efficacy in radiation-induced skin injury remains underexplored. This study used a Sprague-Dawley rat model exposed to 45 Gy radiation to induce skin injury. Rats were treated with 10%, 20%, and 40% Acorus calamus L. extract for 45 days. Wound healing, inflammation, apoptosis, and angiogenesis were assessed using wound healing rates, histopathological analysis, cytokine measurements, TUNEL staining, and immunohistochemistry. Treatment with Acorus calamus L. accelerated wound healing, with the medium-dose group showing the highest healing rate (88.97% at 45 days). Histopathological analysis revealed reduced inflammation, improved collagen organization, and new blood vessel formation. Serum levels of inflammatory cytokines (IL-1β, IL-6, TNF-α) were significantly reduced, and apoptosis was decreased, with modulation of key apoptotic proteins (P53, Bax, Bcl-2). VEGF and bFGF expression were upregulated, promoting angiogenesis and tissue repair. Acorus calamus L. enhances the healing of radiation-induced skin injuries by reducing inflammation, inhibiting apoptosis, and promoting angiogenesis. These findings suggest its potential as a therapeutic agent for managing radiation-induced skin damage, providing a promising alternative for managing radiation-induced skin injuries in clinical oncology. Further studies are needed to clarify its molecular mechanisms.

Thyrotoxicosis is an endocrine disorder characterized by excess thyroid hormones, yet its etiologic links to systemic aging biology remain incompletely defined. Telomere length (TL) reflects cellular senescence and genome stability and has been implicated in multiple complex diseases. We conducted a two-sample Mendelian randomization (MR) study to evaluate the causal effect of genetically predicted TL on the risk of thyrotoxicosis. Genetic instruments for TL were derived from a large genome-wide association study (GWAS) of European ancestry (n > 470,000). Thyrotoxicosis summary statistics were obtained from the latest FinnGen release (≈4,000 cases and >210,000 controls). Primary inverse-variance-weighted analyses indicated that longer genetically proxied TL is associated with a lower risk of thyrotoxicosis, and the direction and magnitude of the effect were consistent across complementary estimators (MR-Egger, weighted median/maximum likelihood, MR-PRESSO, and MR-RAPS). Sensitivity analyses showed no evidence of directional pleiotropy, and Cochran's Q was used to assess heterogeneity. A Steiger directionality test supported the causal flow from TL to thyrotoxicosis. To our knowledge, this work is among the first MR analyses to assess the causal relationship between overall thyrotoxicosis risk and TL using contemporary GWAS resources, extending prior evidence focused on hyperthyroidism-related phenotypes. These findings suggest that cellular aging processes indexed by TL may contribute to thyrotoxicosis susceptibility and motivate future longitudinal and mechanistic studies on telomere biology in thyroid dysfunction.

Labor pain remains a significant challenge during childbirth. While epidural analgesia is highly effective, it is associated with limitations such as breakthrough pain, motor blockade, and epidural-related maternal fever. Auricular acupressure, a non-pharmacological complementary therapy derived from Traditional Chinese Medicine, may modulate pain perception through gate control theory and biochemical mechanisms. This randomized controlled trial assesses whether combining auricular acupressure with epidural analgesia provides superior pain control compared to epidural analgesia alone. Fifty nulliparous women at term requesting epidural analgesia were randomized to receive either true auricular acupressure at specific points (Shenmen, Subcortex, Genitalia, Sympathetic) or sham non-acupoints stimulation. Pain scores were assessed using a Visual Analog Scale at baseline (cervical dilation 2-3 cm), 30 min post-epidural, and at full cervical dilation (10 cm). Secondary outcomes included local anesthetic consumption, incidence of breakthrough pain, and epidural-related maternal fever, Apgar scores, and maternal satisfaction. The protocol emphasizes accurate acupoint localization, blinding procedures, and standardized outcome assessment to ensure reliable results.

Laparoscopic right hemicolectomy (LRH) has been proven to be an effective and safe surgical approach. However, due to the numerous vessels involved in the right colon and the variability of individual anatomy, LRH still faces many challenges. In the early stages, two surgical approaches were reported in the literature: the lateral-to-medial and medial-to-lateral approaches. The lateral-to-medial approach is typically used in open surgery. Currently, with the development of technology, multiple surgical approaches for LRH are available. This paper reports on a caudal approach method, the caudal-to-cranial approach, combined with complete mesocolic excision (CME) and D3 lymph node dissection. The described procedural sequence-prioritizing the establishment of anatomical planes before vascular dissection-may facilitate precise vascular management by first defining the surgical landscape. This approach appears to enhance procedural safety and could potentially reduce operative difficulty by improving anatomical orientation. Our initial experience suggests that this method supports a thorough lymph node dissection, demonstrating its feasibility for performing CME and D3 lymph node dissection.