Jove-Journal of Visualized Experiments最新文献

{"title":"Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut.","authors":"Pascal Benz, Masina Plenge, Simon Wagner, Gemma Mazzuoli-Weber","doi":"10.3791/67666","DOIUrl":"https://doi.org/10.3791/67666","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442427","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":"Modified Most Probable Number Assay to Quantify Salmonella in Raw and Ready-to-Cook Chicken Products.","authors":"Yiping He, Joseph Capobianco, Gretchen Dykes, Cheryl M Armstrong, Chin-Yi Chen, Katrina Counihan, Joe Lee, Sue Reed, Shannon Tilman","doi":"10.3791/67910","DOIUrl":"https://doi.org/10.3791/67910","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442446","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":"The Role of Indocyanine Green Fluorescence in Complex Laparoscopic Cholecystectomy Navigation.","authors":"Luyao Zhang, Xialei Liu, Baojia Zou, Jian Li, Chaonong Cai, Peiping Li","doi":"10.3791/67562","DOIUrl":"https://doi.org/10.3791/67562","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442454","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":"Visualizing Methane-Cycling Microbial Dynamics in Coastal Wetlands.","authors":"Shahida Ferdousee, Mohammad Sadman Alam, Myung Hwangbo, Jongsun Kim","doi":"10.3791/67715","DOIUrl":"https://doi.org/10.3791/67715","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442471","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":"Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery.","authors":"Gabriel Jakob Trauner, Dominika Bernath-Nagy, Melek Sükran Kalinyaprak, Sabine Merker, Marcin Luzarowski, Florian Leuschner, Norbert Frey, Evangelos Giannitsis, Jona Benjamin Krohn","doi":"10.3791/67083","DOIUrl":"https://doi.org/10.3791/67083","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442239","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":"Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung.","authors":"Daisy A Hoagland, Ruth A Franklin","doi":"10.3791/67648","DOIUrl":"10.3791/67648","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442233","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 Reality Assisted Radical Endoscopic Thyroidectomy.","authors":"Peng Zhang, Yifan Ke, Jiezhong Wu, Jia Qi, Wenchao Li, Weiling Huang, Peng Sun, Long Zou, Yuqi Jiang, Kunpeng Hu","doi":"10.3791/67522","DOIUrl":"https://doi.org/10.3791/67522","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442391","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":"Quantitative PCR-based Assay to Measure Sonic Hedgehog Signaling in Cellular Model of Ciliogenesis.","authors":"Priyadarshini Halder, Shubhra Majumder","doi":"10.3791/67407","DOIUrl":"https://doi.org/10.3791/67407","url":null,"abstract":"<p><p>Ciliopathy refers to a collection of multisystemic and polygenic human diseases and disorders that arise due to aberration of structure or function of motile or non-motile (primary) cilia that are microtubule-based cell protrusions. Primary cilia (PC) are present in most human epithelial and endothelial tissues and serve as a hub for physiological signal transduction, including Sonic Hedgehog (SHH) signaling. Various situations demand examining the function of PC, which may be achieved by measuring the canonical SHH signaling pathway that is almost exclusively mediated by PC. Here, a quantitative PCR (qPCR) based technique is developed to measure the transcriptional level of SHH pathway genes in quiescent hTERT-RPE1 (or RPE1) cells that mostly contain PC. Quiescence in RPE1 cells is achieved by serum starvation for 48 h, while activation of the SHH pathway is promoted by a specific agonist. This cell culture-based assay is easy to follow and sensitive. Successful demonstration of this assay in ciliated RPE1 cells indicates its immense potential as an in vitro assay to examine the proper function of PC upon genetic or epigenetic alteration of one or multiple genes, which may be associated with various ciliopathies.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442450","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":"Evaluating Salidroside as a Therapeutic Agent for Vascular Calcification Using Network Pharmacology and Experimental Rat Models.","authors":"Han-Ying Xu, Xiao-Lei Tang, Peng-Fei Li, Dong-Mei Zhang, Guang Ta, Jing Lu, Jian Wang","doi":"10.3791/67728","DOIUrl":"https://doi.org/10.3791/67728","url":null,"abstract":"<p><p>Vascular calcification (VC) is a critical pathological condition associated with significant morbidity and mortality. This study employs a hybrid approach of network pharmacology and molecular biology to delineate the therapeutic mechanisms of salidroside (SAL), an active compound from Rhodiola crenulata, against VC. Through database mining and network analysis, 388 SAL targets intersecting with 2871 VC-associated targets were identified, resulting in 208 common targets. A protein-protein interaction (PPI) network constructed via the String database and topological analysis in Cytoscape 3.9.1 pinpointed 10 key targets, including IL6, TNF, TP53, IL1B, HIF1A, CASP3, and STAT3, among others. The identified genes were concentrated in the lipid and atherosclerosis pathways, indicating that the improvement of VC by SAL may occur through the regulation of abnormal expression of lipid and inflammatory factors. It was also found that SAL inhibits the abnormal expression of inflammatory factors, thereby activating the JAK2/STAT3 pathway to intervene in the progression of VC. The JAK2/STAT3 pathway is a key molecular mechanism by which SAL prevents further deterioration of VC. Functional enrichment analyses revealed the involvement of these targets in inflammatory responses and lipid metabolism, pivotal pathways in VC. In vivo studies in rats demonstrated SAL's efficacy in mitigating dyslipidemia and vascular inflammation, with improved serum lipid profiles and reduced vascular calcium deposition. The mechanistic exploration, grounded in Western blot analysis, demonstrated salidroside's ability to regulate the JAK2/STAT3 signaling pathway, highlighting its potential as a modulator in this critical molecular mechanism and offering a potential therapeutic target for VC. The strength of this research lies in its methodological rigor, integrating computational predictions with in vivo validations. This comprehensive approach establishes a robust framework for exploring the therapeutic mechanisms of natural compounds in combating VC.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442587","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":"Translating Extracellular Electron Transfer Activities with Organic Electrochemical Transistors.","authors":"Yang Gao, Yuchen Zhou, Xudong Ji, Austin J Graham, Christopher M Dundas, Ismar E Miniel Mahfoud, Bailey M Tibbett, Benjamin Tan, Gina Partipilo, Ananth Dodabalapur, Jonathan Rivnay, Benjamin K Keitz","doi":"10.3791/67928","DOIUrl":"https://doi.org/10.3791/67928","url":null,"abstract":"<p><p>Extracellular electron transfer (EET) is a process through which certain microorganisms can transfer electrons across their cell membranes to external electron acceptors, linking cellular metabolism to their environment. While Geobacter and Shewanella have been the primary models for EET research, emerging studies reveal that EET-active species are also associated with fermentation and the human gut microbiome. Leveraging the ability of EET to bridge biological and electronic systems, we present a protocol for using organic electrochemical transistors (OECTs) to translate microbial EET activity into easily detectable electrical signals. This system enables the use of cellular responses to external stimuli for biosensing and biocomputing applications. Specifically, we demonstrated the de-doping of the p-type poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT: PSS) channel in the OECT is driven by cellular EET from Shewanella oneidensis. By transcriptionally controlling EET flux by genetic circuits, we establish the biosensing capability of this hybrid OECT system to detect chemical stimuli, such as inducer molecules. Furthermore, we introduce plasmid-based Boolean logic gates within the cells, allowing them to process environmental signals and drive current changes in the OECTs, further demonstrating the biocomputing potential of these devices. This method provides a novel interface between biological systems and electronics, enabling future high-throughput screening, biosensing, and biocomputing applications.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442424","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}