Jove-Journal of Visualized Experiments最新文献

{"title":"Optimization of In vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring.","authors":"Tina Vodopivec Seravalli, Janja Skok, Tjaša Marušič, Rok Sekirnik","doi":"10.3791/67503","DOIUrl":"https://doi.org/10.3791/67503","url":null,"abstract":"<p><p>In vitro transcription reaction (IVT) is a complex, multi-component enzymatic synthesis of mRNA from a linear DNA template, catalyzed by an RNA polymerase, e.g. T7. Due to the high cost of IVT reagents,  IVT is a critical step in the mRNA drug substance production process and has been the focus of intense optimization in the field. To decrease the cost of mRNA production, reagents must be utilized optimally. Effective optimization necessitates a comprehensive understanding of the impact of individual reagents on reaction kinetics, i.e., the consumption of nucleoside triphosphates (NTPs) and the production of mRNA. Traditionally, low-throughput, end-point analytical techniques have been used for the analysis of mRNA. Though such methods give valuable information on mRNA content, at-line, near real-time analytics is needed to fully understand IVT reaction. We demonstrate how a liquid chromatography analytical method that separates NTPs, as well as pDNA and mRNA, can be used in near real-time to study IVT reaction kinetics. With the knowledge of the influence of different IVT components on kinetics and yield, rapid chromatographic analysis can be used to convert batch IVT reaction into a fed-batch mode that further increases productivity and reduces the overall cost of the reaction.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144035636","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":"Three-Dimensional Printing Assisted Physician-Modified Fenestrated Endografts for Thoracic Endovascular Aortic Aneurysm Repair in Zone 1.","authors":"Zihe Zhao, Yuexue Han, Chaohui Pan, Jianhang Hu, Reyaguli Keyoumu, Tianxin Li, Yi Jin, Yuliya Panteleeva, Tao Tang, Zhao Liu","doi":"10.3791/67893","DOIUrl":"https://doi.org/10.3791/67893","url":null,"abstract":"<p><p>Aortic arch aneurysm is a life-threatening cardiovascular disorder that requires timely medical intervention. Aneurysms in Zone 1 typically involve multiple branch arteries, making repair challenging. Open surgical repair often results in significant surgical trauma, massive blood loss, and prolonged operative time. With advancements in endovascular technology, fenestrated/branched thoracic endovascular aortic repair (F/B TEVAR) has been employed for aortic arch repair and branch artery reconstruction. Stent grafts for F/B TEVAR require personalized modification and fabrication based on patient anatomy. Physician-modified fenestrated endografts (PMEGs) offer a feasible approach for personalized aortic arch aneurysm repair in Zone 1. However, fabricating PMEGs demands a thorough understanding of anatomy and extensive experience, making it challenging for most surgeons. To simplify this process, three-dimensional printing is used to assist in precise fenestration. PMEGs guided by three-dimensional printing enhance branch artery patency and reduce post-operative endoleaks following F/B TEVAR. Further follow-up is necessary to assess the long-term benefits and efficacy of this technique.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144057825","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":"An Immature Murine Model of Reversible Unilateral Ureteral Obstruction.","authors":"Ping Li, Qi-Tong Guo, Mei-Iing Chen, Chen-Xi Jia, Xing Liu, Tao Lin, Da-Wei He, De-Ying Zhang, Guang-Hui Wei","doi":"10.3791/67238","DOIUrl":"https://doi.org/10.3791/67238","url":null,"abstract":"<p><p>Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143992419","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":"Importance of Jumping Ability in Handball Throwing Speed and Accuracy.","authors":"Jaime Tuquet, Elena Mainer Pardos, Antonio Cartón-Llorente, Alberto Roso-Moliner, Gustavo García-Buendía, Demetrio Lozano","doi":"10.3791/67443","DOIUrl":"https://doi.org/10.3791/67443","url":null,"abstract":"<p><p>Throwing the ball to score a goal in handball is a critical action for success in offensive play. Speed and accuracy are two determining factors for successful throws. While there is substantial research on the importance of these factors, discrepancies exist regarding their relationship. This study aims to analyze the impact of jumping ability on throwing speed and accuracy across different competitive levels in handball. Thirty-five male handball players were recruited from amateur and professional levels. Jumping ability was assessed and throwing speed and accuracy were measured using a high-performance sports radar. Significant differences (p < 0.05) were found between amateur and professional groups in countermovement jump (CMJ) and throwing speed jump. The comparative analysis revealed significant differences in throwing speed and in correct and incorrect throws, with large to very large effect sizes. Pearson's r correlation indicated significant correlations between throwing speed jump and CMJ, countermovement jump left (CMJL), and countermovement jump right (CMJR), and between throwing speed deficit and CMJL in the total sample. The results show that professional handball players exhibit higher throwing accuracy and speed compared to amateurs. Throwing speed and accuracy are positively correlated with performance in male handball players.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144065030","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":"Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes.","authors":"Nicholas Kusi-Appauh, Stefan H Mueller, Stephen F Ralph, Olga Yurieva, Michael E O'Donnell, Jacob S Lewis, Lisanne M Spenkelink","doi":"10.3791/68080","DOIUrl":"https://doi.org/10.3791/68080","url":null,"abstract":"<p><p>The ability of proteins involved in eukaryotic DNA replication to overcome obstacles - such as protein and DNA 'roadblocks' - is critical for ensuring faithful genome duplication. G-quadruplexes are higher-order nucleic acid structures that form in guanine-rich regions of DNA and have been shown to act as obstacles, interfering with genomic maintenance pathways. This study introduces a real-time, fluorescence microscopy-based method to observe DNA polymerase interactions with G-quadruplex structures. Short, primed DNA oligonucleotides containing a G-quadruplex were immobilized on functionalized glass coverslips within a microfluidic flow cell. Fluorescently labeled DNA polymerases were introduced, allowing their behavior and stoichiometry to be monitored over time. This approach enabled the observation of polymerase behavior as it was stalled by a G-quadruplex. Specifically, using fluorescently labeled yeast polymerase δ, it was found that upon encountering a G-quadruplex, the polymerase undergoes a continuous cycle of binding and unbinding. This single-molecule assay can be adapted to study interactions between various DNA-maintenance proteins and obstacles on the DNA substrate.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143992423","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":"Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion.","authors":"Elizabeth Ortiz, Kyaw Hsu Thway, Gabriela Ortiz-Soto, Paulina Yao, Jonathan A Kelber","doi":"10.3791/67902","DOIUrl":"https://doi.org/10.3791/67902","url":null,"abstract":"<p><p>Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144042403","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":"Eye Tracking During A Complex Aviation Task For Insights Into Information Processing.","authors":"Naila Ayala, Suzanne Kearns, Elizabeth Irving, Shi Cao, Ewa Niechwiej-Szwedo","doi":"10.3791/66886","DOIUrl":"https://doi.org/10.3791/66886","url":null,"abstract":"<p><p>Eye tracking has been used extensively as a proxy to gain insight into the cognitive, perceptual, and sensorimotor processes that underlie skill performance. Previous work has shown that traditional and advanced gaze metrics reliably demonstrate robust differences in pilot expertise, cognitive load, fatigue, and even situation awareness (SA). This study describes the methodology for using a wearable eye tracker and gaze mapping algorithm that captures naturalistic head and eye movements (i.e., gaze) in a high-fidelity flight motionless simulator. The method outlined in this paper describes the area of interest (AOI)-based gaze analyses, which provides more context related to where participants are looking, and dwell time duration, which indicates how efficiently they are processing the fixated information. The protocol illustrates the utility of a wearable eye tracker and computer vision algorithm to assess changes in gaze behavior in response to an unexpected in-flight emergency. Representative results demonstrated that gaze was significantly impacted when the emergency event was introduced. Specifically, attention allocation, gaze dispersion, and gaze sequence complexity significantly decreased and became highly concentrated on looking outside the front window and at the airspeed gauge during the emergency scenario (all p values < 0.05). The utility and limitations of employing a wearable eye tracker in a high-fidelity motionless flight simulation environment to understand the spatiotemporal characteristics of gaze behavior and its relation to information processing in the aviation domain are discussed.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143992421","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":"5/6 Nephrectomy Using Sharp Bipolectomy Via Midline Laparotomy in Rats.","authors":"Alexander Studier-Fischer, Berkin Özdemir, Caelan Max Haney, Luisa Egen, Maurice Stephan Michel, Niklas Westhoff, Markus Mieth, Zoltan Czigany, Gabriel Alexander Salg, Henrik Nienhüser, Franck Billmann, Karl-Friedrich Kowalewski","doi":"10.3791/67322","DOIUrl":"https://doi.org/10.3791/67322","url":null,"abstract":"<p><p>Chronic kidney disease (CKD) affects over 10% of the global population, amounting to more than 800 million individuals worldwide. The advances in the treatment of CKD have had a significant impact on patient outcomes. While in the past, CKD was often considered a death sentence, with many patients succumbing to the complications of end-stage renal disease, it is now increasingly being managed as a chronic condition with the availability of dialysis and kidney transplantation, as well as new pharmaceutical developments such as SGLT2 inhibitors or nonsteroidal mineralocorticoid receptor antagonists. Yet, there continues to be a growing demand for further exploration into the pathophysiological processes and potential therapeutic interventions. Reliable biological models play a crucial role in facilitating this research. Given the multifaceted nature of renal disease, which encompasses not only cell biology but also vascular microanatomy and endocrine signaling, an appropriate model must possess a level of biological complexity that only an animal model can offer, rendering rodents an obvious choice. This manuscript, therefore, provides an intricate, systematic protocol for surgically reducing renal parenchyma through midline laparotomy and combined total and partial nephrectomy in rats for survival as well as non-survival applications. It emphasizes the critical role of precise surgical techniques in ensuring consistent and reliable outcomes. Prime examples of potential applications for this model include biomolecular and pharmaceutical studies as well as the development of innovative intraoperative imaging modalities, such as hyperspectral imaging, to objectively visualize and differentiate renal malperfusion.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144041142","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":"Stochastic Semi-active Control Method of Structure Based on Magnetorheological Dampers Considering Time Delay.","authors":"Zhenkai Zhang, Zhiwei Bao, Wenyong Ma, Peifang Sun","doi":"10.3791/68259","DOIUrl":"https://doi.org/10.3791/68259","url":null,"abstract":"<p><p>The use of Magnetorheological (MR) dampers in semi-active control systems faces a key challenge: time delay caused by feedback processes, which reduces the reliability of civil engineering structures under stochastic excitations. This paper proposes a Stochastic Optimal Semi-active Control method with time delay compensation (SOSC-PSO), leveraging the Physical Stochastic Optimal control theory (PSO) to address this issue and maintain structural reliability. The proposed method derives the semi-active control force as a function of both current and previous states, compensating for time delays in the control process. To optimize control effectiveness, key parameters are tuned based on a reliability criterion for the system. Validation analyses on single-degree-of-freedom and multi-degree-of-freedom structures under stochastic seismic excitations show that time delays significantly impair the performance of MR dampers. However, the SOSC-PSO method with time delay compensation significantly improves control effectiveness, and with optimized parameters, it enhances the reliability of the structural control system beyond methods without parameter optimization.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144021401","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":"In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase.","authors":"Isalira Peroba Ramos, Bruna Barreira, João Gabriel Regis Sobral, Rita de Cássia Dos Passos Ferraz da Silva, Hyago da Silva Medeiros Elidio, Isabele Barbieri, Bárbara Souza Neil Magalhães, Wanderley de Souza, Cristina Henriques","doi":"10.3791/67050","DOIUrl":"https://doi.org/10.3791/67050","url":null,"abstract":"<p><p>American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease. By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR. After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR. Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143993711","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}