Jove-Journal of Visualized Experiments最新文献

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, the durable response remains limited. Recent studies have shown that the epigenetic landscape of preinfusion CART cell products can influence response to therapy, and gene editing has been proposed as a solution. However, more work needs to be done to determine the optimal gene editing strategy. Genome-wide CRISPR screens have become popular tools to both investigate mechanisms of resistance and optimize gene editing strategies. Yet their application to primary cells presents many challenges. Here we describe a method to complete a genome-wide CRISPR knockout screen in CART cells from healthy donors. As a proof-of-concept model, we designed this method to investigate the development of exhaustion in CART cells targeting the CD19 antigen. However, we believe that this approach can be used to address a variety of mechanisms of resistance to therapy in different CAR constructs and tumor models.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. Traditionally, research in this area has relied on animal experiments, but there's a growing demand for alternative methods that adhere to the 3R principles-replace, reduce, and refine. Porcine organoids have emerged as a promising tool, offering a more accurate in vitro replication of the in vivo conditions than traditional cell models. One major challenge with intestinal organoids is their inward-facing apical surface and outward-facing basolateral surface. This limitation can be overcome by creating two-dimensional (2D) organoid layers on transwell inserts (from here on referred to as insert(s)), providing access to both surfaces. In this study, we successfully developed two-dimensional cultures of porcine jejunum and colon organoids. The cultivation process involves two key phases: First, the formation of a cellular monolayer, followed by the differentiation of the cells using tailored media. Cellular growth is tracked by measuring transepithelial electrical resistance, which stabilizes by day 8 for colon organoids and day 16 for jejunum organoids. After a 2-day differentiation phase, the epithelium is ready for analysis. To quantify and track active electrogenic transport processes, such as chloride secretion, we employ the Ussing chamber technique. This method allows for real-time measurement and detailed characterization of epithelial transport processes. This innovative in vitro model, combined with established techniques like the Ussing chamber, provides a robust platform for physiologically characterizing the porcine GIT within the 3R framework. It also opens opportunities for investigating pathophysiological mechanisms and developing potential therapeutic strategies.

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella focus on prevalence rather than quantification, which limits their utility in assessing contamination levels and risks. This study introduces a novel most probable number (MPN) assay designed to quantify Salmonella in ready-to-cook poultry products, such as chicken cordon bleu. The method involves washing the poultry sample, concentrating the rinse through centrifugation, and serially diluting it in a 48-well block. The MPN assay is integrated with the loop-mediated isothermal amplification (LAMP) method to provide a sensitive, accurate, and rapid quantification of Salmonella contamination within the same timeframe as existing Food Safety and Inspection Service (FSIS) protocols. Results show a strong linear correlation between the MPN-LAMP measurements and theoretical inoculation levels (R² = 0.933). However, variability at lower concentrations highlights challenges in accurately detecting Salmonella at these levels, with the practical lower limit of detection estimated at approximately 300 CFU/g. Potential refinements to improve the protocol's applicability include increasing the quantity sampled to further improve the limit of detection, optimizing enrichment media formulations, and expanding molecular detection to target multiple Salmonella serovars. Overall, this study presents a practical tool for the food industry, enabling reliable quantification of Salmonella contamination in poultry products, contributing to improved food safety and public health.

Laparoscopic cholecystectomy (LC) is the gold-standard treatment for cholelithiasis and cholecystitis. In difficult cases with severe inflammation and adhesions, the risk of bile duct injury (BDI) is significantly higher. Precise identification of anatomical biliary structures is essential to prevent such injuries. Conventional intraoperative visualization techniques (IVT) have limited clinical application due to their complexity, increased trauma, and high error rates. Near-infrared fluorescence (NIRF) imaging, utilizing indocyanine green (ICG) as a fluorescent dye, has emerged as an innovative IVT technique. It is increasingly recognized as a feasible, safe, and effective approach for LC. However, the efficacy of NIRF in difficult LC procedures remains unclear, and the optimal timing and dosage of ICG administration are yet to be established. This article outlines the main steps for performing fluorescence-guided difficult LC in a patient with acute gangrenous cholecystitis and evaluates the imaging effects of NIRF in various scenarios. The patient was positioned supine, with four trocars placed. Upon switching to fluorescence mode, the fluorescently labeled bile ducts were readily identified. Following fluorescence guidance, Calot's triangle was carefully dissected. The cystic duct (CD) and cystic artery (CA) were individually identified and clipped before the gallbladder was extracted. Finally, the surgical field was inspected in fluorescence mode to detect bile leakage. With satisfactory ICG imaging and a smooth procedure, the patient's postoperative recovery was uneventful. NIRF is a safe and effective technology that shows great promise for future clinical applications.

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus playing a critical role in regulating the methane cycle. The wetlands in South Texas, which are subject to frequent weather events, fluctuating salinity levels, and anthropogenic activities due to climate change, influence methane cycling. Despite the ecological importance of these processes, methane cycling in South Texas coastal wetlands remains insufficiently explored. To address this gap, we developed and optimized a method for detecting genes related to methanogens and methanotrophs, including mcrA as a biomarker for methanogens and pmoA1, pmoA2, and mmoX as biomarkers for methanotrophs. Additionally, this study aimed to visualize the spatial and temporal distribution patterns of methanogen and methanotroph abundance utilizing the geographic information system (GIS) software ArcGIS Pro. The integration of these molecular techniques with advanced geospatial visualization provided critical insights into the spatial and temporal distribution of methanogen and methanotroph communities across South Texas wetlands. Thus, the methodology established in this study offers a robust framework for mapping microbial dynamics in wetlands, enhancing our understanding of methane cycling under varying environmental conditions, and supporting broader ecological and environmental change studies.

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research due to their role in regulating intercellular communication, potentially serving as valuable biomarkers for cardiovascular disease. However, the EV proteome and its potential as a biomarker in cardiovascular diagnostics remain poorly understood. This protocol presents a standardized method for the isolation and quantification of plasma-derived EV and the analysis of their protein cargo using plasma samples from patients presenting to the Chest Pain Unit of a large university hospital. Following routine phlebotomy, EV are isolated from plasma by differential ultracentrifugation. The enrichment of specific EV marker proteins in EV isolates is visualized by immunoblotting, and average size distribution and plasma EV concentrations are quantified by nanoparticle tracking analysis. Finally, ultra-performance liquid chromatography-tandem mass spectrometry is employed for label-free analysis of the EV proteome. This protocol thus provides a comprehensive approach to study and use plasma-derived EV as potential carriers of critical biological information as well as to explore their potential as novel biomarkers.

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both naïve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

Radical endoscopic thyroidectomy (ET) offers superior cosmetic outcomes and enhanced visibility of the surgical field compared to open surgery. However, the thyroid's unique physiological functions and intricate surrounding anatomy may result in various surgical complications. Mixed reality (MR), a real-time holographic visualization technology, enables the creation of highly realistic 3D models in the real world and facilitates multiple human-computer interactions. MR can be utilized for both preoperative evaluation and intraoperative navigation. First, semi-automatic 3D reconstruction of the neck from enhanced computed tomography images is performed using 3Dslicer. Next, the 3D model is imported into Unity3D to create a virtual hologram that can be displayed on an MR helmet-mounted display (HMD). During surgery, surgeons can wear the MR HMD to locate lesions and surrounding anatomy through the virtual hologram. In this study, patients requiring radical ET were randomly assigned to either the experimental group or the control group. Surgeons performed MR-assisted radical ET in the experimental group. A comparative analysis of surgical outcomes and the results of scales was conducted. This study successfully developed the neck 3D model and the virtual hologram. According to the NASA Task Load Index Scale, the experimental group exhibited significantly higher scores in 'Own Performance' and lower scores in 'Effort' compared to the control group (p = 0.002). Additionally, on the Likert Subjective Evaluation Scale, the mean scores for all questions exceeded 3. Although the incidence of surgical complications was lower in the experimental group than in the control group, the differences in surgical outcomes were not statistically significant.MR is beneficial for enhancing performance and alleviating the burden of surgeons during the perioperative period. Furthermore, MR has demonstrated the potential to enhance the safety of ET. Therefore, it is essential to further investigate the surgical applications of MR.