Methods in molecular biology最新文献_第7页

Subcellular Localization of Geminivirus Proteins by Laser Scanning Confocal Microscopy. 用激光扫描共聚焦显微镜研究双病毒蛋白的亚细胞定位。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4454-6_18

Christiane Eliza Motta Duarte, João Paulo Batista Machado, Bianca Gouveia-Mageste, Fredy Davi Albuquerque Silva, Elizabeth Pacheco Batista Fontes

{"title":"Subcellular Localization of Geminivirus Proteins by Laser Scanning Confocal Microscopy.","authors":"Christiane Eliza Motta Duarte, João Paulo Batista Machado, Bianca Gouveia-Mageste, Fredy Davi Albuquerque Silva, Elizabeth Pacheco Batista Fontes","doi":"10.1007/978-1-0716-4454-6_18","DOIUrl":"10.1007/978-1-0716-4454-6_18","url":null,"abstract":"In eukaryotic cells, the subcellular localization of proteins is inherently linked to their function. Since viruses rely on the host cellular machinery to complete their life cycle, viral proteins are expected to employ the host transport machinery to reach various compartments. Several factors, including the multifunctional nature of viral proteins, the stage of virus infection, and interactions with both viral and host proteins, influence the final destination of viral proteins. For instance, NSP (nuclear shuttle protein) from bipartite begomoviruses and CP (coat protein) from monopartite begomoviruses typically exhibit nuclear localization, yet their subcellular distribution can vary depending on coexpression partners and stage of infection. Virtually all viral proteins display dynamic subcellular distribution patterns that change under their specific functions at different stages of the virus life cycle. Thus, identifying the subcellular distribution of viral proteins is essential for comprehending their multiple roles during infection. This chapter outlines a protocol for efficiently determining the subcellular localization of viral proteins during infection or when expressed with protein partners. The protocol essentially consists of three steps: (i) cloning the viral protein and protein partners fused to fluorescent tags, (ii) transiently expressing the tagged proteins in N. benthamiana leaves, and (iii) determining the subcellular localization of the tagged proteins using confocal microscopy.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2912 ","pages":"205-226"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143597373","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

Assessment of Bacterial Contamination in Semen. 精液中细菌污染的评估

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4406-5_39

Ferran Garriga, Sergi Bonet, Marc Yeste

{"title":"Assessment of Bacterial Contamination in Semen.","authors":"Ferran Garriga, Sergi Bonet, Marc Yeste","doi":"10.1007/978-1-0716-4406-5_39","DOIUrl":"10.1007/978-1-0716-4406-5_39","url":null,"abstract":"Semen contamination is one of the main issues of concern in livestock industry. While the presence of some bacteria in semen is considered as normal, high bacterial loads detrimentally affect sperm quality and fertilizing ability, thus having a negative repercussion on the efficiency of artificial insemination. For this reason, the present chapter focuses on the methods used for the assessment of semen contamination in farm animals. Routinely, bacterial culture is performed to assess the overall degree of contamination in sperm samples; in some cases, however, the identification of bacterial species is needed to determine the optimal antibiotic composition of semen preservation media and address whether those bacteria have developed antibiotic resistance. In addition, other techniques, such as scanning electron microscopy, can be used to evaluate sperm-bacteria interaction. This chapter also discusses the strategies to prevent bacteria contamination in semen. As the ejaculate is not a sterile fluid, media for preservation usually include, as aforementioned, antibiotics to avoid bacterial growth. Because of the mounting restriction in the usage of antibiotics, which is due to the increase of bacterial resistance, other approaches to prevent bacterial contamination of semen include preservation at low temperatures (5 °C) and the inclusion of alternative molecules such as antimicrobial peptides.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2897 ","pages":"591-600"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143811695","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

Live-Cell Single-Molecule Imaging of Influenza A Virus-Receptor Interaction. 甲型流感病毒与受体相互作用的活细胞单分子成像。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4326-6_4

Lukas Broich, Yang Fu, Christian Sieben

引用次数: 0

Bimolecular Fluorescence Complementation (BiFC) Technique for Exocytic Proteins in Murine Hippocampal Neurons. 小鼠海马神经元胞外蛋白的双分子荧光互补技术。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_20

Gözdem Karapinar Kapucu, Thorsten Trimbuch, Christian Rosenmund, Marion Weber-Boyvat

引用次数: 0

High-Resolution Imaging of Intracellular Trafficking of B Cell Receptor Using Specific Hybridization Internalization Probe (SHIP). 利用特异性杂交内化探针（SHIP）对B细胞受体细胞内运输进行高分辨率成像。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4442-3_6

Sara Hernández-Pérez, Pieta K Mattila

{"title":"High-Resolution Imaging of Intracellular Trafficking of B Cell Receptor Using Specific Hybridization Internalization Probe (SHIP).","authors":"Sara Hernández-Pérez, Pieta K Mattila","doi":"10.1007/978-1-0716-4442-3_6","DOIUrl":"10.1007/978-1-0716-4442-3_6","url":null,"abstract":"Recent advancements in microscopy have greatly expanded our understanding of intracellular traffic. Yet, due to the inherent characteristics of B cells, such as their small size and high receptor density on the plasma membrane, visualization of internalized cargo or receptors remains challenging. This challenge is particularly pronounced in the case of the B cell receptor (BCR), where accurate detection of internalized, antigen-bound BCR molecules can be strongly hindered by the signal from the plasma membrane-bound pool of the same molecules.To tackle this issue, we adapted the Specific Hybridization Internalization Probe (SHIP) assay, initially designed for flow cytometry studies, for the study of BCR internalization using microscopy. This assay utilizes a single-stranded DNA (ssDNA) fluorescence internalization probe (FIP) paired with a complementary ssDNA quenching probe that \"turns off\" the signal from the (extracellular) surface-bound BCRs, greatly facilitating the unambiguous identification of internalized (intracellular) receptors. Moreover, the assay is versatile and adaptable to a range of imaging modalities, including live-cell imaging and super-resolution microscopy. SHIP proves to be a valuable tool in the study of intracellular processes, offering enhanced imaging precision for the detection of internalized BCRs.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2909 ","pages":"73-82"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542645","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

Use of Human Acute Myeloid Leukemia Cells to Study Graft-Versus-Leukemia Immunity in Xenogeneic Mouse Models of Graft-Versus-Host Disease. 利用人急性髓系白血病细胞研究移植物抗宿主病小鼠异种模型的移植物抗白血病免疫

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4430-0_17

Charline Faville, Bianca E Silva, Frédéric Baron, Grégory Ehx

{"title":"Use of Human Acute Myeloid Leukemia Cells to Study Graft-Versus-Leukemia Immunity in Xenogeneic Mouse Models of Graft-Versus-Host Disease.","authors":"Charline Faville, Bianca E Silva, Frédéric Baron, Grégory Ehx","doi":"10.1007/978-1-0716-4430-0_17","DOIUrl":"10.1007/978-1-0716-4430-0_17","url":null,"abstract":"Allogeneic hematopoietic cell transplantation (allo-HCT) is the main therapeutic approach for patients with high-risk acute myeloid leukemia (AML), but the rate of relapse remains high and is associated with poor outcomes. Discovering new approaches to maximize the graft-versus-leukemia (GVL) effects while mitigating graft-versus-host disease (GVHD) should therefore be pursued. Because of the difficulties in modeling AML in mice, patient-derived xenotransplantations (PDXs) in immunodeficient NOD-scid-IL2rgnull (NSG) mice are preferred to study the GVL effects. In PDX, AML is typically induced through the intravenous injection of cell lines or leukemic blasts obtained from patients. GVHD and GVL effects are induced by (co)-injecting human T cells or peripheral blood mononuclear cells (PBMCs). While this approach enables the induction of systemic leukemia, notably developing in the spleen and bone marrow of the animals, it can also be associated with difficulties in monitoring the disease, notably by flow cytometry. This can be circumvented by using luciferase-expressing AML cells or transplanting the leukemic cells in Matrigel to generate solid tumors that are easier to monitor. Here, we provide detailed instructions on how to prepare human PBMCs and leukemic cells, transplant them, and monitor the disease in NSG mice.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2907 ","pages":"359-375"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657771","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

Utilization of Clinical Data and Evaluation of Biomarkers in the Investigation of Graft-Versus-Host Disease Outcomes. 移植物抗宿主病预后研究中临床数据的利用和生物标志物的评估。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4430-0_3

Serhat Çelik, Leylagül Kaynar

引用次数: 0

Agroinoculation of Plants with Geminiviruses. 双病毒植物的农接种。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4454-6_5

Anelise F Orílio

引用次数: 0

Untargeted Metabolic Phenotyping by LC-MS. LC-MS的非靶向代谢表型分析。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4334-1_6

Ian D Wilson, Elizabeth Want

引用次数: 0

FRET-Based Sensor for Measuring Adenine Nucleotide Binding to AMPK. 基于fret的测量腺苷核苷酸与AMPK结合的传感器。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4284-9_2

Roland Abi Nahed, Martin Pelosse, Francesco Aulicino, Florine Cottaz, Imre Berger, Uwe Schlattner

引用次数: 0