Methods in molecular biology最新文献_第10页

Analysis of Enzymatic Activity of Matrix Metalloproteinases by Collagen Zymography Utilizing Extracted Collagen from Bovine Muscle Tissue. 利用牛肌肉组织胶原提取，用胶原酶谱法分析基质金属蛋白酶的酶活性。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4478-2_9

Larissa A Koulicoff, Michael D Chao

引用次数: 0

Purification and Ultramicroscopic Observation of the Influenza A Virus Ribonucleoprotein Complex. 甲型流感病毒核糖核蛋白复合物的纯化及超微观察。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4326-6_7

Masahiro Nakano, Takeshi Noda

引用次数: 0

Techniques to Determine Mammalian Sperm Capacitation. 测定哺乳动物精子获能的技术。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4406-5_31

Joan E Rodríguez Gil, Olga Blanco-Prieto

{"title":"Techniques to Determine Mammalian Sperm Capacitation.","authors":"Joan E Rodríguez Gil, Olga Blanco-Prieto","doi":"10.1007/978-1-0716-4406-5_31","DOIUrl":"10.1007/978-1-0716-4406-5_31","url":null,"abstract":"The detection of the achievement of the capacitation status in a sperm sample is a very important asset for optimizing most reproductive techniques centered on semen, from freezing to \"in vitro\" fertilization. However, there is not a single, simple test that can determine the precise capacitation of a sample. This implies that a combined panel of separate tests focused on separate aspects of sperm function must be carried out to obtain a precise knowledge of the functional status of the sample. This work deals with a brief explanation of the most important techniques applied at these moments to determine sperm capacitation, with an emphasis not on the description of each technique, but on the advantages, disadvantages, and main purposes taking into account practical aspects such as the precise target by which a laboratory wants to determine capacitation. In this way, the main aim of this work is to give a practical guide for practitioners of laboratories from separate objectives, from standard semen quality analysis to molecular and/or mechanistic studies of sperm function, for choosing the most adequate tests to determine capacitation basing on the intended precise targets chosen in each case.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2897 ","pages":"463-495"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143811997","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Quality Assurance in Metabolomics and Metabolic Profiling. 代谢组学和代谢谱的质量保证。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4334-1_2

Jennifer A Kirwan, Ulrike Bruning, Jonathan D Mosley

引用次数: 0

Analysis of Transient Receptor Potential Ion Channels in ME/CFS. ME/CFS瞬时受体电位离子通道分析。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4498-0_6

Natalie Eaton-Fitch, Katsuhiko Muraki, Etianne Martini Sasso, Chandi Magawa, Sonya Marshall-Gradisnik

{"title":"Analysis of Transient Receptor Potential Ion Channels in ME/CFS.","authors":"Natalie Eaton-Fitch, Katsuhiko Muraki, Etianne Martini Sasso, Chandi Magawa, Sonya Marshall-Gradisnik","doi":"10.1007/978-1-0716-4498-0_6","DOIUrl":"https://doi.org/10.1007/978-1-0716-4498-0_6","url":null,"abstract":"This chapter provides a comprehensive overview of methodologies currently employed to study ion channels, particularly transient receptor potential melastatin 3 (TRPM3) in the context of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Sample preparation involves the collection of whole blood, separation of peripheral blood mononuclear cells (PBMCs) via density gradient centrifugation, and isolation of natural killer (NK) cells. Protein expression analysis utilizes flow cytometry, liquid chromatography-mass spectrometry (LC-MS), western blotting, and immunofluorescence techniques. Functional analysis focuses on calcium imaging and electrophysiology techniques to investigate ion channel responses to pharmacological stimuli. The authors highlight that some experimental protocols included within this chapter require specialized training and equipment. In order to replicate these protocols extended training is advised, specifically when attempting electrophysiology experimentation. The use of advanced techniques for detailed analysis provides insights into ion channel function and potential implications in the pathomechanism of ME/CFS offering avenues for further research and therapeutic exploration.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2920 ","pages":"83-99"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144078799","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Quantitative Proteomics on Immune Cells of ME/CFS Patients Using SWATH-MS. 应用SWATH-MS对ME/CFS患者免疫细胞进行定量蛋白质组学研究。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4498-0_8

Abhishek Kumar, Katie Peppercorn, Torsten Kleffmann

引用次数: 0

Real-Time Measurement of Mitochondrial Function and Glycolysis in Lymphoblastoid Cell Lines. 淋巴母细胞样细胞系线粒体功能和糖酵解的实时测量。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4498-0_11

Tina Katsaros, Daniel Missailidis, Sarah J Annesley

{"title":"Real-Time Measurement of Mitochondrial Function and Glycolysis in Lymphoblastoid Cell Lines.","authors":"Tina Katsaros, Daniel Missailidis, Sarah J Annesley","doi":"10.1007/978-1-0716-4498-0_11","DOIUrl":"https://doi.org/10.1007/978-1-0716-4498-0_11","url":null,"abstract":"Cells require energy in the form of ATP to function. The two main ways in which cells generate energy in mammalian cells is through glycolysis and oxidative phosphorylation (OXPHOS). Glycolysis takes place in the cytosol and involves the breakdown of glucose molecules, generating ATP and pyruvate, while OXPHOS takes place in the mitochondria and is responsible for producing the majority of ATP for the cell. A dysregulation of these cellular processes has been reported in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). In order to understand the mechanisms of the disease, it is imperative to understand how the bioenergetic pathways are altered in ME/CFS. Here we describe a method for measuring mitochondrial function and glycolytic function using the Agilent Seahorse Extracellular Flux Analyzer. We have optimized these assays for use in actively proliferating lymphoblastoid cell lines that are generated from blood cells. This assay measures oxygen consumption rate and extracellular acidification rates providing an overview of mitochondrial function and efficiency and glycolytic rate and capacity, respectively. These assays are performed on live, intact cells, and enable us to view different components and measurements of energy metabolism through the injection of different compounds that stimulate or inhibit various sections of these pathways. The below method details an optimized glycolysis and mitochondrial assay for 96-well plates with modifications noted for use in 24-well plates.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2920 ","pages":"173-202"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144078945","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Plasmid DNA Cleavage Assay with Eukaryotic Topoisomerase II. 真核生物拓扑异构酶ⅱ质粒DNA切割试验。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4550-5_8

Allison J Thomas, Brooke D Latham, Addison K O'Brian, Mattalyn R Hardin, Joseph Deweese

引用次数: 0

Concepts and Methods for Predicting Viral Evolution. 预测病毒进化的概念和方法。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4326-6_14

Matthijs Meijers, Denis Ruchnewitz, Jan Eberhardt, Malancha Karmakar, Marta Łuksza, Michael Lässig

{"title":"Concepts and Methods for Predicting Viral Evolution.","authors":"Matthijs Meijers, Denis Ruchnewitz, Jan Eberhardt, Malancha Karmakar, Marta Łuksza, Michael Lässig","doi":"10.1007/978-1-0716-4326-6_14","DOIUrl":"10.1007/978-1-0716-4326-6_14","url":null,"abstract":"The seasonal human influenza virus undergoes rapid evolution, leading to significant changes in circulating viral strains from year to year. These changes are typically driven by adaptive mutations, particularly in the antigenic epitopes, the regions of the viral surface protein hemagglutinin targeted by human antibodies. Here, we describe a consistent set of methods for data-driven predictive analysis of viral evolution. Our pipeline integrates four types of data: (1) sequence data of viral isolates collected on a worldwide scale, (2) epidemiological data on incidences, (3) antigenic characterization of circulating viruses, and (4) intrinsic viral phenotypes. From the combined analysis of these data, we obtain estimates of relative fitness for circulating strains and predictions of clade frequencies for periods of up to 1 year. Furthermore, we obtain comparative estimates of protection against future viral populations for candidate vaccine strains, providing a basis for pre-emptive vaccine strain selection. Continuously updated predictions obtained from the prediction pipeline for influenza and SARS-CoV-2 are available at https://previr.app .","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2890 ","pages":"253-290"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143073650","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

GMP Compliant Production of Therapeutic Components of Autologous Adipose Tissue. 符合GMP要求的自体脂肪组织治疗成分的生产。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4510-9_24

Mustafa Uguten, Joeri van Boxtel, Hieronymus P Stevens, Martin C Harmsen, Joris A van Dongen

{"title":"GMP Compliant Production of Therapeutic Components of Autologous Adipose Tissue.","authors":"Mustafa Uguten, Joeri van Boxtel, Hieronymus P Stevens, Martin C Harmsen, Joris A van Dongen","doi":"10.1007/978-1-0716-4510-9_24","DOIUrl":"https://doi.org/10.1007/978-1-0716-4510-9_24","url":null,"abstract":"Adipose tissue is a popular source of tissue for cellular therapy in the field of regenerative medicine. The regenerative potential is often ascribed to the presence of stromal vascular fraction (SVF) containing extracellular matrix and multipotent stromal cells secreting a plethora of growth factors to create a regenerative environment. SVF can be isolated by means of enzymatic or mechanical isolation procedures and expanded in culture or directly used intraoperatively. Depending on the clinical use of SVF, specific regulatory requirements are demanded and might classify SVF as an advanced therapy medicinal product (ATMP). As an ATMP, SVF must be manufactured, processed, and controlled according to good manufacturing practice (GMP) guidelines to ensure safety and quality. Subsequently, the GMP standards require extensive validation, process control, and characterization of SVF. Here we report a GMP-compliant production of clinical grade tissue (tSVF) by means of fractionation of adipose tissue (FAT) procedure. Previous validation studies demonstrated tSVF to be safe and feasible for clinical use intraoperatively according to GMP standards with the appropriate release criteria. The presented procedures can be used as a template for the development of an investigational medicinal product dossier to be enclosed in future clinical trials (Fig. 1).","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2922 ","pages":"307-323"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143972048","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0