Jove-Journal of Visualized Experiments最新文献

{"title":"Modeling Alcohol Consumption in Rodents Using Two-Bottle Choice Home Cage Drinking and Microstructural Analysis.","authors":"Shikun Hou, Nathaly M Arce Soto, Elizabeth J Glover","doi":"10.3791/67486","DOIUrl":"10.3791/67486","url":null,"abstract":"<p><p>Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are provided with access to two drinking bottles, one of which contains regular tap water and the other ethanol, with daily intake measured by change in bottle weight over the 24 h period. Consequently, this approach requires no specialized laboratory equipment. While such ease of implementation is likely the greatest contributor to its widespread adoption by preclinical alcohol researchers, the resolution of drinking data acquired using this approach is limited by the number of times the researcher measures bottle weight (e.g., once daily). However, the desire to examine drinking patterns in the context of overall intake, pharmacological interventions, and neuronal manipulations has prompted the development of home cage lick detection systems that can acquire data at the level of individual licks. Although a number of these systems have been developed recently, the open-source system, LIQ HD (Lick Instance Quantifier Home cage Device), has garnered significant attention in the field for its affordability and user friendliness. Although exciting, this system was designed for use in mice. Here, we review appropriate procedures for standard and lickometer-equipped two-bottle choice home cage drinking. We also introduce methods for adapting the LIQ HD system to rats including hardware modifications to accommodate larger cage size and a redesigned 3D printed bottle holder compatible with standard off-the-shelf drinking bottles. Using this approach, researchers can examine daily drinking patterns in addition to levels of intake in many rats in parallel thereby increasing the resolution of acquired data with minimal investment in additional resources. These methods provide researchers with the flexibility to use either standard bottles or a lickometer-equipped apparatus to interrogate the neurobiological mechanisms underlying alcohol drinking depending on their precise experimental needs.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711519","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":"Production and Optimization of LTE, a Leishmania tarentolae Derived Cell-Free Protein Expression System for Recombinant Protein Production.","authors":"Wayne A Johnston, Juan Alfaro Palma, Kirill Alexandrov","doi":"10.3791/65592","DOIUrl":"https://doi.org/10.3791/65592","url":null,"abstract":"<p><p>This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate Leishmania tarentolae, referred to as Leishmania Translational Extract or LTE. Although this organism originally evolved as a parasite of geckos, it can be cultivated easily and inexpensively in flasks or bioreactors. Unlike Leishmania major, it is non-pathogenic to humans and does not require special laboratory precautions. Another advantage of using Leishmania for CFPS is that the addition of a single antisense oligonucleotide to the CFPS, targeting a conserved splice leader sequence on the 5'-end of all protein-coding RNAs, can suppress endogenous protein expression. We provide procedures for cell disruption and lysate processing, which have been simplified and improved compared to previous versions. These procedures start with simple flask cultures. Additionally, we explain how to introduce genetic information using vectors containing species-independent translation initiation sites (SITS) and how to perform straightforward batch optimization and quality control to ensure consistent protein expression quality.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711541","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":"Mitochondrial Respiration Quantification in Yeast Whole Cells.","authors":"Gerardo M Nava, Andres Carrillo-Garmendia, Juan Carlos González-Hernández, Luis Alberto Madrigal-Perez","doi":"10.3791/67186","DOIUrl":"https://doi.org/10.3791/67186","url":null,"abstract":"<p><p>Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to different nutritional and environmental conditions give valuable information to elucidate molecular mechanisms. Oxidative phosphorylation is the primary source of ATP in most cells, and mitochondrial respiration activity is a key component of oxidative phosphorylation, maintaining mitochondrial membrane potential for ATP synthesis. Mitochondrial respiration is often studied in isolated mitochondria that are missing the cellular context. Here, we present a method for quantifying mitochondrial respiration in yeast-intact cells. This method applies to any yeast species, although it has been generally used for Saccharomyces cerevisiae cells. First, the yeast growth in specific conditions is tested. Then, cells are washed and resuspended in deionized water with a 1:1 ratio (w/v). Cells are then placed in an oximeter chamber with constant stirring, and a Clark electrode is used to quantify oxygen consumption. Some molecules are sequentially placed into the chamber and selected according to this effect on the electron transport chain or ATP synthesis. ATPase inhibitor oligomycin is first added to measure respiration coupled to ATP synthesis. Afterward, an uncoupler is used to measure the maximal respiratory capacity. Finally, a mix of electron transport chain inhibitors is added to discard oxygen consumption unrelated to mitochondrial respiration. Data are analyzed using a linear regression to obtain the slope, representing the oxygen consumption rate. The advantage of this method is that it is specific for yeast mitochondrial respiration, maintaining the cellular context. It is essential to highlight that inhibitors used in mitochondrial respiration quantification could vary between yeast species.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711516","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":"Metabolomic Analysis of Barley by Gas Chromatography/Mass Spectrometry.","authors":"Peter J Wieber, Gabrielle Feber, Patrick J Hrdlicka, Zachary Kayler","doi":"10.3791/67175","DOIUrl":"https://doi.org/10.3791/67175","url":null,"abstract":"<p><p>Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe and quantify the impacts of drought on grain and other agricultural products. The identified metabolites may allow for the identification of the biochemical response that allows the plant to tolerate stressful environments. The methodology presented herein allowed for the total metabolomic analysis of barley flour using gas chromatography/mass spectrometry (GC/MS). Barley flour metabolite extracts were fractionated into four fractions based on polarity. To allow for analysis by GC/MS, metabolites were derivatized to increase volatility and metabolite separation: fatty acids esters were derivatized into fatty acid methyl esters; sugars were oximated into their straight chain form; and metabolites with hydroxyl groups were converted to their corresponding silyl ethers. The derivatized samples were injected into the GC/MS and the generated mass spectra were used for metabolite identification by comparing the generated spectra to the National Institute of Standards and Technology (NIST) Tandem Mass Spectra library. The method described here can also be used to examine the total metabolome for other plants, furthering our understanding of the biochemical responses of stressed plants.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711457","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":"Fentanyl Analog Screening using LC-TIMS-TOF MS/MS.","authors":"Andrew R Forero, Lilian Valadares Tose, Matthew Willetts, Melvin A Park, Elisa N Shoff, Francisco Fernandez-Lima","doi":"10.3791/65562","DOIUrl":"10.3791/65562","url":null,"abstract":"<p><p>The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its analogs are the major contributors to fatal and nonfatal overdoses in the United States. Most recent cases of fentanyl-related overdose are linked to illicitly manufactured fentanyl and its associated extreme potency. In the present work, we describe a high-throughput analytical protocol for the screening of fentanyl analogs. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and tandem mass spectrometry allow for the separation and assignment of hundreds of fentanyl analogs from a single sample in a single scan. The described approach takes advantage of the recent development of data-dependent acquisition and data-independent acquisition using parallel accumulation in the mobility trap followed by sequential fragmentation using collision-induced dissociation. The fentanyl analogs are confidently assigned based on their retention time, mobility, and MS fragmentation pattern.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711368","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":"Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics.","authors":"Darian T Carroll, Sarah R Lindsley, Melissa Kirigiti, Alex Buko, Angela Jones, Paul Kievit, Maureen Gannon","doi":"10.3791/66849","DOIUrl":"10.3791/66849","url":null,"abstract":"<p><p>One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, such as combined single-cell RNA sequencing and metabolomics. The method in the current study utilizes flash-frozen pancreatic islets from fetal non-human primates collected over a span of two years for input into single-nucleus RNA sequencing and ATAC sequencing assays. The cytosolic fraction generated during nuclear extraction was retained for downstream capillary electrophoresis-mass spectrometry and subsequent metabolite quantification. This method allows for bulk analysis of metabolites that contribute to the changing transcriptomic and epigenomic landscapes within experimental conditions. It is applicable to many tissue types and maximizes the amount of information that can be extracted from samples that are not readily available. As the contribution of metabolism to the establishment of cellular identity via epigenetic modifications becomes more appreciated, techniques that allow for identifying the contribution of metabolites in specific cell types are timely and necessary.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711538","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":"A Rapid, Simple Workflow for Quantification of External Adult Drosophila Structures.","authors":"Laura Martins, Ramy Wong, Fredrick J Larabee, Bree K Grillo-Hill","doi":"10.3791/67485","DOIUrl":"https://doi.org/10.3791/67485","url":null,"abstract":"<p><p>The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It is a simple structure of repeating unit eyes, termed ommatidia, that is used to characterize genetic interactions and gene functions. Mutations that affect eye architecture can be easily detected and analyzed; hence, this system is frequently used in under-resourced institutions. Further phenotypic analysis often includes a Scanning Electron Microscope (SEM) to generate high-magnification images suitable for quantitative analysis. However, SEMs are expensive and require costly reagents; sample preparation spans days; and, often, they need full-time staff for sample preparation and instrument maintenance. This limits their utility at under-resourced institutions or during budgetary austerity. In entomology, the use of high-resolution digital imaging technology is a common practice for the identification and characterization of species. This paper describes a method that combines strategies and allows for high-resolution digital imaging of adult Drosophila structures and quantitative analysis using the open software ImageJ. The workflow is a rapid and student-friendly alternative that remedies the limitations of underfunded and under-resourced research facilities with a cost-effective and rapid approach to quantitative phenotypic analysis.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711422","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":"Microfluidics in Assessing Platelet Function.","authors":"Emily Mihalko, Abiha Abdullah, Lara Hoteit, David Akinbode, Susan Shea, Matthew D Neal","doi":"10.3791/67214","DOIUrl":"https://doi.org/10.3791/67214","url":null,"abstract":"<p><p>Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711459","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":"Spinal Hernia Repair and Cauda Equina Repositioning after Lumbar Decompression under Three-Dimensional Microscopy: A Case Report and Literature Review.","authors":"Zhenhao Zhang, YiBo Dong, YiZhou Xie, HanXiang Yang, Xiaohong Fan, Yang Yu","doi":"10.3791/67045","DOIUrl":"10.3791/67045","url":null,"abstract":"<p><p>Cauda equina herniation (CEH) is a relatively rare and severe perioperative complication that may occur after lumbar spine surgery. Here, we present a case report of a 36-year-old female patient who experienced CEH after an endoscopic L5-S1 laminectomy and discectomy. The patient presented with right L5-S1 radiculopathy that correlated with findings in medical imaging and physical examination. Subsequently, she underwent endoscopic L5-S1 laminotomy and discectomy. A day after the operation, the patient developed urine leakage, hematochezia, aggravated constipation, and found no relief from pain in the right lower limb. MRI revealed cerebrospinal fluid leakage at the surgical site. After consultation with the urology and anorectal department, the patient was fitted with a urinary catheter, prescribed hemorrhoid medication, and underwent anal sphincter training as recommended by the doctor. After 1 week of treatment, the patient's urinary function returned to normal, but constipation persisted while the pain in the right lower limb eased. After 5 months, the patient was hospitalized due to radiating pain in both lower limbs and constipation. An MRI revealed herniation of the L5/S1 nerve root sac. Subsequently, L5/S1 total laminectomy decompression and dural sac repair were performed under a three-dimensional (3D) microscopy. Postoperatively, lower limb pain and constipation were alleviated. CEH following spinal endoscopy, though rare, demands significant clinical attention. The successful outcome in this case illustrates the value of surgical revision under 3D microscopic guidance, offering a viable strategy for patients presenting with this complication.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711569","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":"Local Hyperthermia for Warts Treatment.","authors":"Yun Pan, Xiao-Li Chen, Na Tan, Ai-Jun Chen","doi":"10.3791/67384","DOIUrl":"https://doi.org/10.3791/67384","url":null,"abstract":"<p><p>Warts, benign epidermal proliferations, are a direct result of human papillomavirus (HPV) infection, specifically targeting the keratinocytes within the stratum corneum of the skin. The development of warts is the most common clinical manifestation of HPV, with plantar warts, condylomata acuminata, and common warts being the most frequently observed types. These growths can be unsightly and sometimes painful, affecting the quality of life for those afflicted. Although various treatments are available, ranging from topical medications to surgical procedures, the quest for a treatment that is both safe and effective while minimizing invasiveness continues. This is particularly crucial for populations with heightened risk factors, such as immuno-compromised individuals. In the clinical need for minimally invasive treatments, local hyperthermia has emerged as a promising therapeutic strategy for wart management. As demonstrated in various studies, local hyperthermia is effective as a standalone treatment, offering a valuable alternative for patients seeking less intrusive therapeutic options.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711451","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}