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Data Treatment for LC-MS Untargeted Analysis. LC-MS非靶向分析的数据处理。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4334-1_5
Mar Garcia-Aloy, Johannes Rainer, Pietro Franceschi
{"title":"Data Treatment for LC-MS Untargeted Analysis.","authors":"Mar Garcia-Aloy, Johannes Rainer, Pietro Franceschi","doi":"10.1007/978-1-0716-4334-1_5","DOIUrl":"https://doi.org/10.1007/978-1-0716-4334-1_5","url":null,"abstract":"<p><p>Liquid Chromatography-Mass Spectrometry (LC-MS) untargeted experiments require complex bioinformatic strategies to extract information from the experimental data. Here we discuss the \"data preprocessing,\" the set of procedures performed on the raw data to produce a data matrix which will be the starting point for the subsequent statistical analysis. Data preprocessing is a crucial step on the path to knowledge extraction, which should be carefully controlled and optimized in order to maximize the output of any untargeted metabolomics investigation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2891 ","pages":"91-108"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984021","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
Structural Dynamics of SNARE Complex Assembly in the Ribbon Synapses Observed by smFRET. smFRET观察带状突触中SNARE复合物组装的结构动力学。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_13
Alexa L Andrzejewski, Joshua Ferrar, Marla Kratzer-Charron, Mark E Bowen, Ucheor B Choi
{"title":"Structural Dynamics of SNARE Complex Assembly in the Ribbon Synapses Observed by smFRET.","authors":"Alexa L Andrzejewski, Joshua Ferrar, Marla Kratzer-Charron, Mark E Bowen, Ucheor B Choi","doi":"10.1007/978-1-0716-4314-3_13","DOIUrl":"https://doi.org/10.1007/978-1-0716-4314-3_13","url":null,"abstract":"<p><p>Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique for studying the structural dynamics of protein molecules or detecting interactions between protein molecules in real time. Due to the high sensitivity in spatial and temporal resolution, smFRET can decipher sub-populations within heterogeneous native state conformations, which are generally lost in traditional measurements due to ensemble averaging. In addition, the single-molecule reconstitution allows protein molecules to be observed for an extensive period of time and can recapitulate the geometry of the cellular environment to retain biological function. Here we provide a detailed method of using smFRET to monitor the conformational dynamics of syntaxin-3b from the ribbon synapses during assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"185-196"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979104","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
Dissecting SNARE-Mediated Exocytosis in RBL-2H3 Mast Cells. 剖析RBL-2H3肥大细胞中SNARE介导的外吞作用
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_10
Pratikshya Adhikari, Hao Xu
{"title":"Dissecting SNARE-Mediated Exocytosis in RBL-2H3 Mast Cells.","authors":"Pratikshya Adhikari, Hao Xu","doi":"10.1007/978-1-0716-4314-3_10","DOIUrl":"https://doi.org/10.1007/978-1-0716-4314-3_10","url":null,"abstract":"<p><p>SNARE-dependent mast cell (MC) exocytosis causes the release of a wide variety of mediators with important physiological/pathological consequences. Unlike synaptic transmission in the brain, which relies primarily on one set of exocytic SNAREs (i.e., Syntaxin1, SNAP-25, and VAMP2), MCs produce a multitude of exocytic SNAREs that can form a minimum of 8 distinct sets of fusogenic trans-SNARE complexes. Here we describe the genetic approaches we have developed to dissect the specific roles of these SNAREs in RBL-2H3 cells, a widely utilized model for studying MC signaling and exocytosis.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"149-166"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979139","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
Electron Tomography of Organelles and Vesicles in the Investigation of SNARE Function and Localization. 研究 SNARE 功能和定位的细胞器和囊泡电子断层扫描。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_17
Nicholas A Bright, J Paul Luzio
{"title":"Electron Tomography of Organelles and Vesicles in the Investigation of SNARE Function and Localization.","authors":"Nicholas A Bright, J Paul Luzio","doi":"10.1007/978-1-0716-4314-3_17","DOIUrl":"https://doi.org/10.1007/978-1-0716-4314-3_17","url":null,"abstract":"<p><p>Electron tomography can provide additional morphological information not easily obtained by conventional transmission electron microscopy of thin sections. It uses a goniometer stage in the electron microscope to tilt the specimen and collect a series of 2D images from different orientations, which are combined to provide a 3D volume tomogram and a colored reconstruction of the morphological feature(s) of interest. Here we describe the protocols for its use in visualizing changes in organelle morphology after depletion of the SNARE proteins VAMP7 and VAMP8 and to study VAMP7 localization on endolysosomes/lysosomes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"237-247"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979141","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
Advanced LC-IMS-MS Protocol for Holistic Metabolite Analysis in Wine and Grape Samples. 用于葡萄酒和葡萄样品中整体代谢物分析的先进 LC-IMS-MS 程序。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4334-1_13
Vania Sáez, Sara Ferrero-Del-Teso, Fulvio Mattivi, Urska Vrhovsek, Panagiotis Arapitsas
{"title":"Advanced LC-IMS-MS Protocol for Holistic Metabolite Analysis in Wine and Grape Samples.","authors":"Vania Sáez, Sara Ferrero-Del-Teso, Fulvio Mattivi, Urska Vrhovsek, Panagiotis Arapitsas","doi":"10.1007/978-1-0716-4334-1_13","DOIUrl":"https://doi.org/10.1007/978-1-0716-4334-1_13","url":null,"abstract":"<p><p>The final aim of metabolomics is the comprehensive and holistic study of the metabolome in biological samples. Therefore, the use of instruments that enable the analysis of metabolites belonging to various chemical classes in a wide range of concentrations is essential, without compromising on robustness, resolution, sensitivity, specificity, and metabolite annotation. These characteristics are crucial for the analysis of very complex samples, such as wine, whose metabolome is the result of the sum of metabolites derived from grapes, yeast(s), bacteria(s), and chemical or physical modification during winemaking. In recent years, a big advantage, in this direction, was the hardware developments on hyphenated instruments that enable the integration of liquid chromatography (LC), ion mobility spectrometry (IMS), and mass spectrometry (MS). This chapter describes an LC-IMS-MS protocol for the analysis of wine and grape samples as well as the use of IMS data in metabolite annotation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2891 ","pages":"239-256"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984011","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
Magnetic Resonance Imaging Techniques for Investigating the MCT8-Deficient Brain in Murine Disease Models. 研究小鼠疾病模型中 MCT8 缺失脑部的磁共振成像技术
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4252-8_12
Marina Guillén-Yunta, Ana Guadaño-Ferraz, Víctor Valcárcel-Hernández
{"title":"Magnetic Resonance Imaging Techniques for Investigating the MCT8-Deficient Brain in Murine Disease Models.","authors":"Marina Guillén-Yunta, Ana Guadaño-Ferraz, Víctor Valcárcel-Hernández","doi":"10.1007/978-1-0716-4252-8_12","DOIUrl":"10.1007/978-1-0716-4252-8_12","url":null,"abstract":"<p><p>Magnetic resonance imaging (MRI) techniques have emerged as powerful tools for unraveling the pathophysiology of rare diseases, mainly due to their pivotal role in early diagnosis, disease characterization, and treatment monitoring in a non-invasive manner. In this chapter, we will review two essential MRI tools used for studying and evaluating the pathophysiology of Allan-Herndon-Dudley Syndrome or MCT8 deficiency, a rare disease caused by inactivating mutations in the SLC16A2 gene, encoding for the thyroid hormone-specific transmembrane transporter MCT8. These two MRI techniques are time-of-flight magnetic resonance angiography (TOF-MRA) and diffusion tensor imaging (DTI).</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2876 ","pages":"175-186"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142695706","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
Patterning Functional Proteins in Ultrabithorax-Based Materials. 功能蛋白在超胸腔基材料中的应用。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4322-8_17
Britt Faulk, Amanda Jons, Brandon Look Fong, Maximillian Lara, Andrew R Irion, Sarah E Bondos
{"title":"Patterning Functional Proteins in Ultrabithorax-Based Materials.","authors":"Britt Faulk, Amanda Jons, Brandon Look Fong, Maximillian Lara, Andrew R Irion, Sarah E Bondos","doi":"10.1007/978-1-0716-4322-8_17","DOIUrl":"https://doi.org/10.1007/978-1-0716-4322-8_17","url":null,"abstract":"<p><p>The ability to add bioactivities, such as cell signaling or ligand recognition, to biomaterials has generated the potential to include multiple bioactivities into a single material. In some cases, it is desirable to localize these activities to different areas of the biomaterial, creating functional patterns. While photolithography and 3D printing have been effective techniques for patterning functions in many materials, patterning remains a challenge in materials composed of protein, in part due to how these materials are artificially assembled. Protein fibers are often produced from protein films that co-acervate at the air-water interface. This chapter describes methods to leverage this coacervation process to pattern materials, using the Drosophila melanogaster Hox protein Ultrabithorax (Ubx) as a model self-assembling protein. Through gene fusion, Ubx and a functional protein are produced as a single polypeptide, capable of both forming materials and performing the activity of interest. This functionality is retained in the final materials. In this chapter, we describe how to use multiple Ubx fusion proteins to not only imbue the final materials with multiple functions, but also to create macroscale patterns of the appended proteins in fibrous protein-based materials. These patterned materials include striped fibers, bifunctional-faced fibers, gradient fibers, and core-shell fibers.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2889 ","pages":"245-256"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142914976","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
Fluorescence Anisotropy for Monitoring cis- and trans-Membrane Interactions of Synaptotagmin-1. 荧光各向异性监测Synaptotagmin-1的顺膜和跨膜相互作用。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_12
Yongsoo Park
{"title":"Fluorescence Anisotropy for Monitoring cis- and trans-Membrane Interactions of Synaptotagmin-1.","authors":"Yongsoo Park","doi":"10.1007/978-1-0716-4314-3_12","DOIUrl":"https://doi.org/10.1007/978-1-0716-4314-3_12","url":null,"abstract":"<p><p>Vesicle fusion induces neurotransmitter release, orchestrated by synaptotagmin-1 (Syt-1) as a Ca<sup>2+</sup> sensor. However, the precise molecular mechanisms of Syt-1 remain controversial, with various and competing models proposed based on different ionic strengths. Syt-1, residing on the vesicle membrane alongside anionic phospholipids such as phosphatidylserine (PS), undergoes Ca<sup>2+</sup>-induced binding to its own vesicle membrane, known as the cis-interaction, which prevents the trans-interaction of Syt-1 with the plasma membrane. Fluorescence anisotropy offers a methodological advantage for studying protein-membrane interactions. This protocol outlines a method utilizing fluorescence anisotropy to monitor the cis- and trans-membrane interactions of Syt-1, employing both purified native vesicles and plasma membrane-mimicking liposomes (PM-liposomes).</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"175-182"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979144","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
Modular Golden Gate Assembly of Linear DNA Templates for Cell-Free Prototyping. 用于无细胞原型设计的线性 DNA 模板模块化金门组装。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4220-7_11
François-Xavier Lehr, Aukse Gaizauskaite, Katarzyna Elżbieta Lipińska, Sara Gilles, Arpita Sahoo, René Inckemann, Henrike Niederholtmeyer
{"title":"Modular Golden Gate Assembly of Linear DNA Templates for Cell-Free Prototyping.","authors":"François-Xavier Lehr, Aukse Gaizauskaite, Katarzyna Elżbieta Lipińska, Sara Gilles, Arpita Sahoo, René Inckemann, Henrike Niederholtmeyer","doi":"10.1007/978-1-0716-4220-7_11","DOIUrl":"10.1007/978-1-0716-4220-7_11","url":null,"abstract":"<p><p>Cell-free transcription and translation (TXTL) systems have emerged as a powerful tool for testing genetic regulatory elements and circuits. Cell-free prototyping can dramatically accelerate the design-build-test-learn cycle of new functions in synthetic biology, in particular when quick-to-assemble linear DNA templates are used. Here, we describe a Golden-Gate-assisted, cloning-free workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcription units from basic genetic parts of a modular cloning toolbox. Functional DNA templates composed of multiple parts such as promoter, ribosomal binding site (RBS), coding sequence, and terminator are produced in vitro in a one-pot Golden Gate assembly reaction followed by polymerase chain reaction (PCR) amplification. We demonstrate assembly, cell-free testing of promoter and RBS combinations, as well as characterization of a repressor-promoter pair. By eliminating time-consuming transformation and cloning steps in cells and by taking advantage of modular cloning toolboxes, our cell-free prototyping workflow can produce data for large numbers of new assembled constructs within a single day.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2850 ","pages":"197-217"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142372294","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
Studying the Role of HOX Genes in Thrombocyte Development. HOX基因在血栓细胞发育中的作用研究。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4322-8_8
Jabila Mary, Pudur Jagadeeswaran
{"title":"Studying the Role of HOX Genes in Thrombocyte Development.","authors":"Jabila Mary, Pudur Jagadeeswaran","doi":"10.1007/978-1-0716-4322-8_8","DOIUrl":"https://doi.org/10.1007/978-1-0716-4322-8_8","url":null,"abstract":"<p><p>In our laboratory, we study thrombopoiesis and hemostasis using zebrafish as a model organism to unravel the mechanisms of differentiation and development of thrombocytes. We have shown in our earlier work that thrombocytes are functional equivalents of platelets and have transcriptional machinery similar to megakaryocytes. We recently found evidence that hox genes play a role in their development. We used piggyback gene knockdown and thrombocyte quantification assays to understand the influence of these ancient developmental regulators on thrombopoiesis. In this chapter, we describe methods used to discover these hox genes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2889 ","pages":"107-119"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142915038","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
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