首页 > 最新文献

Methods in molecular biology最新文献

英文 中文
Efficient and Sensitive Sample Preparation, Separations, and Data Acquisition for Label-Free Single-Cell Proteomics. 用于无标记单细胞蛋白质组学的高效、灵敏的样品制备、分离和数据采集。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3934-4_7
Thy Truong, Ximena Sanchez-Avila, Kei G I Webber, S Madisyn Johnston, Ryan T Kelly

We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass spectrometry data acquisition. The Tecan Uno Single Cell Dispenser provides rapid cell isolation and nanoliter-volume reagent dispensing within 384-well PCR plates. A newly developed sample processing workflow achieves cell lysis, protein denaturation, and digestion in 1 h with a single reagent dispensing step. Low-flow liquid chromatography coupled with wide-window data-dependent acquisition results in the quantification of nearly 3000 proteins per cell using an Orbitrap Exploris 480 mass spectrometer. This approach greatly broadens accessibility to sensitive single-cell proteome profiling for nonspecialist laboratories.

我们介绍了一种灵敏高效的无标记单细胞蛋白质组学工作流程,该流程横跨样品制备、液相色谱分离和质谱数据采集。Tecan Uno 单细胞分装仪可在 384 孔 PCR 板内快速进行细胞分离和纳升容量试剂分装。新开发的样品处理工作流程只需一个试剂分配步骤,即可在 1 小时内完成细胞裂解、蛋白质变性和消化。低流量液相色谱法与宽窗口数据采集相结合,使用 Orbitrap Exploris 480 质谱仪对每个细胞的近 3000 种蛋白质进行定量。这种方法大大拓宽了非专业实验室进行灵敏单细胞蛋白质组分析的途径。
{"title":"Efficient and Sensitive Sample Preparation, Separations, and Data Acquisition for Label-Free Single-Cell Proteomics.","authors":"Thy Truong, Ximena Sanchez-Avila, Kei G I Webber, S Madisyn Johnston, Ryan T Kelly","doi":"10.1007/978-1-0716-3934-4_7","DOIUrl":"10.1007/978-1-0716-3934-4_7","url":null,"abstract":"<p><p>We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass spectrometry data acquisition. The Tecan Uno Single Cell Dispenser provides rapid cell isolation and nanoliter-volume reagent dispensing within 384-well PCR plates. A newly developed sample processing workflow achieves cell lysis, protein denaturation, and digestion in 1 h with a single reagent dispensing step. Low-flow liquid chromatography coupled with wide-window data-dependent acquisition results in the quantification of nearly 3000 proteins per cell using an Orbitrap Exploris 480 mass spectrometer. This approach greatly broadens accessibility to sensitive single-cell proteome profiling for nonspecialist laboratories.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2817 ","pages":"67-84"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141437070","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
Profiling Mouse Brain Single-Cell-Type Proteomes Via Adeno-Associated Virus-Mediated Proximity Labeling and Mass Spectrometry. 通过腺相关病毒介导的近距离标记和质谱法分析小鼠大脑单细胞型蛋白质组
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3934-4_10
Him K Shrestha, Huan Sun, Ju Wang, Junmin Peng

Single-cell-type proteomics is an emerging field of research that combines cell-type specificity with the comprehensive proteome coverage offered by bulk proteomics. However, the extraction of single-cell-type proteomes remains a challenge, particularly for hard-to-isolate cells like neurons. In this chapter, we present an innovative technique for profiling single-cell-type proteomes using adeno-associated virus (AAV)-mediated proximity labeling (PL) and tandem-mass-tag (TMT) mass spectrometry. This technique eliminates the need for cell isolation and offers a streamlined workflow, including AAV delivery to express TurboID (an engineered biotin ligase) controlled by cell-type-specific promoters, biotinylated protein purification, on-bead digestion, TMT labeling, and liquid chromatography-mass spectrometry (LC-MS). We examined this method by analyzing distinct brain cell types in mice. Initially, recombinant AAVs were used to concurrently express TurboID and mCherry proteins driven by neuron- or astrocyte-specific promoters, which was validated through co-immunostaining with cellular markers. With biotin purification and TMT analysis, we successfully identified around 10,000 unique proteins from a few micrograms of protein samples with high reproducibility. Our statistical analyses revealed that these proteomes encompass cell-type-specific cellular pathways. By utilizing this technique, researchers can explore the proteomic landscape of specific cell types, paving the way for new insights into cellular processes, deciphering disease mechanisms, and identifying therapeutic targets in neuroscience and beyond.

单细胞型蛋白质组学是一个新兴的研究领域,它将细胞型特异性与大体蛋白质组学提供的全面蛋白质组覆盖范围相结合。然而,单细胞型蛋白质组的提取仍然是一项挑战,尤其是对于神经元等难以分离的细胞。在本章中,我们介绍了一种利用腺相关病毒(AAV)介导的接近标记(PL)和串联质量标签(TMT)质谱分析单细胞型蛋白质组的创新技术。这项技术无需进行细胞分离,工作流程简化,包括利用 AAV 表达由细胞特异性启动子控制的 TurboID(一种工程化生物素连接酶)、生物素化蛋白质纯化、珠上消化、TMT 标记和液相色谱-质谱联用(LC-MS)。我们通过分析小鼠不同的脑细胞类型来检验这种方法。首先,使用重组 AAV 在神经元或星形胶质细胞特异性启动子的驱动下同时表达 TurboID 和 mCherry 蛋白,并通过与细胞标记物的联合免疫染色进行验证。通过生物素纯化和TMT分析,我们成功地从几微克的蛋白质样本中鉴定出了约10,000种独特的蛋白质,而且重现性很高。我们的统计分析显示,这些蛋白质组包含细胞类型特异的细胞通路。利用这项技术,研究人员可以探索特定细胞类型的蛋白质组图谱,为深入了解细胞过程、破译疾病机理以及确定神经科学及其他领域的治疗靶点铺平道路。
{"title":"Profiling Mouse Brain Single-Cell-Type Proteomes Via Adeno-Associated Virus-Mediated Proximity Labeling and Mass Spectrometry.","authors":"Him K Shrestha, Huan Sun, Ju Wang, Junmin Peng","doi":"10.1007/978-1-0716-3934-4_10","DOIUrl":"10.1007/978-1-0716-3934-4_10","url":null,"abstract":"<p><p>Single-cell-type proteomics is an emerging field of research that combines cell-type specificity with the comprehensive proteome coverage offered by bulk proteomics. However, the extraction of single-cell-type proteomes remains a challenge, particularly for hard-to-isolate cells like neurons. In this chapter, we present an innovative technique for profiling single-cell-type proteomes using adeno-associated virus (AAV)-mediated proximity labeling (PL) and tandem-mass-tag (TMT) mass spectrometry. This technique eliminates the need for cell isolation and offers a streamlined workflow, including AAV delivery to express TurboID (an engineered biotin ligase) controlled by cell-type-specific promoters, biotinylated protein purification, on-bead digestion, TMT labeling, and liquid chromatography-mass spectrometry (LC-MS). We examined this method by analyzing distinct brain cell types in mice. Initially, recombinant AAVs were used to concurrently express TurboID and mCherry proteins driven by neuron- or astrocyte-specific promoters, which was validated through co-immunostaining with cellular markers. With biotin purification and TMT analysis, we successfully identified around 10,000 unique proteins from a few micrograms of protein samples with high reproducibility. Our statistical analyses revealed that these proteomes encompass cell-type-specific cellular pathways. By utilizing this technique, researchers can explore the proteomic landscape of specific cell types, paving the way for new insights into cellular processes, deciphering disease mechanisms, and identifying therapeutic targets in neuroscience and beyond.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2817 ","pages":"115-132"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141437150","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
Imaging and Analysis of the Dynamics of Filamentous Actin Structures in Live Endothelial Cells. 活内皮细胞丝状肌动蛋白结构动力学的成像和分析。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3429-5_11
Jerome W Breslin, Zeinab Y Motawe

The ability to view and record the movements of subcellular structures is a powerful tool that has accelerated the discovery and understanding of signaling mechanisms that control microvascular functions such as the control of endothelial permeability. Advances in molecular biology over the past few decades have facilitated the generation of fusion proteins in which fluorescent reporters based upon the structure of green fluorescent protein can be linked to proteins found in human endothelial cells, such as VE-cadherin or β-actin. These fusion proteins have been found to incorporate into structures alongside their native protein counterparts, allowing the dynamic visualization of how these subcellular structures are modified when cells are challenged with stimuli such as inflammatory mediators. The result of such studies has been a much more advanced view of the complex mechanisms by which endothelial cells maintain barrier properties than previously obtained by only viewing fixed cells labeled by immunofluorescence. Here, we describe our protocols that we have used to view the dynamics of actin filaments using time-lapse microscopy to record endothelial cells expressing GFP-actin and the analysis tools available to quantify dynamics of subcellular structures.

观察和记录亚细胞结构运动的能力是一种强大的工具,它加速了对控制微血管功能(如内皮通透性的控制)的信号机制的发现和理解。过去几十年来,分子生物学的进步促进了融合蛋白的产生,其中基于绿色荧光蛋白结构的荧光报告子可以与人类内皮细胞中发现的蛋白质连接,如VE钙粘蛋白或β-肌动蛋白。这些融合蛋白已被发现与它们的天然蛋白对应物一起结合到结构中,从而可以动态可视化当细胞受到诸如炎症介质等刺激时这些亚细胞结构是如何被修饰的。这类研究的结果对内皮细胞维持屏障特性的复杂机制有了更深入的了解,而不是以前只观察免疫荧光标记的固定细胞。在这里,我们描述了我们的方案,我们使用延时显微镜来观察肌动蛋白丝的动力学,以记录表达GFP肌动蛋白的内皮细胞,以及可用于量化亚细胞结构动力学的分析工具。
{"title":"Imaging and Analysis of the Dynamics of Filamentous Actin Structures in Live Endothelial Cells.","authors":"Jerome W Breslin, Zeinab Y Motawe","doi":"10.1007/978-1-0716-3429-5_11","DOIUrl":"10.1007/978-1-0716-3429-5_11","url":null,"abstract":"<p><p>The ability to view and record the movements of subcellular structures is a powerful tool that has accelerated the discovery and understanding of signaling mechanisms that control microvascular functions such as the control of endothelial permeability. Advances in molecular biology over the past few decades have facilitated the generation of fusion proteins in which fluorescent reporters based upon the structure of green fluorescent protein can be linked to proteins found in human endothelial cells, such as VE-cadherin or β-actin. These fusion proteins have been found to incorporate into structures alongside their native protein counterparts, allowing the dynamic visualization of how these subcellular structures are modified when cells are challenged with stimuli such as inflammatory mediators. The result of such studies has been a much more advanced view of the complex mechanisms by which endothelial cells maintain barrier properties than previously obtained by only viewing fixed cells labeled by immunofluorescence. Here, we describe our protocols that we have used to view the dynamics of actin filaments using time-lapse microscopy to record endothelial cells expressing GFP-actin and the analysis tools available to quantify dynamics of subcellular structures.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2711 ","pages":"129-146"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11369499/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41132881","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Bioorthogonal Labeling and Click-Chemistry-Based Visualization of the Tannerella forsythia Cell Wall. 基于生物正交标记和点击化学的连翘单宁细胞壁可视化研究。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3491-2_1
Stephen N Hyland, Sreedevi Chinthamani, Sushanta Ratna, Kimberly A Wodzanowski, Liam-Michael D Sandles, Kiyonobu Honma, Catherine Leimkuhler-Grimes, Ashu Sharma

The objective of this chapter is to provide a detailed protocol for the peptidoglycan (cell wall) labeling of the periodontal pathogen Tannerella forsythia and the development of a laboratory-safe Escherichia coli strain utilizing the N-acetylmuramic acid recycling enzymes AmgK, N-acetylmuramate/N-acetylglucosamine kinase, and MurU, N-acetylmuramate alpha-1-phosphate uridylyltransferase, from T. forsythia. The procedure involves bioorthogonal labeling of bacterial cells with an azido-modified analog of the amino sugar, N-acetylmuramic acid, through "click chemistry" with a fluorescent dye. The protocol is suitable for the generation of fluorescently labeled peptidoglycan molecules for applications in the study of bacterial and peptidoglycan trafficking in the host cells and cell wall recycling in complex microbiomes.

本章的目的是提供一种详细的方案,用于牙周病原体连翘单胞菌的肽聚糖(细胞壁)标记,并利用N-乙酰胞壁酸循环酶AmgK、N-乙酰胞膜酸/N-乙酰葡糖胺激酶和MurU、N-乙酰细胞壁酸α-1-磷酸尿苷基转移酶开发实验室安全的大肠杆菌菌株,来自连翘。该程序包括通过荧光染料的“点击化学”,用叠氮修饰的氨基糖类似物N-乙酰壁酸对细菌细胞进行生物正交标记。该方案适用于产生荧光标记的肽聚糖分子,用于研究宿主细胞中的细菌和肽聚糖运输以及复杂微生物群中的细胞壁循环。
{"title":"Bioorthogonal Labeling and Click-Chemistry-Based Visualization of the Tannerella forsythia Cell Wall.","authors":"Stephen N Hyland, Sreedevi Chinthamani, Sushanta Ratna, Kimberly A Wodzanowski, Liam-Michael D Sandles, Kiyonobu Honma, Catherine Leimkuhler-Grimes, Ashu Sharma","doi":"10.1007/978-1-0716-3491-2_1","DOIUrl":"10.1007/978-1-0716-3491-2_1","url":null,"abstract":"<p><p>The objective of this chapter is to provide a detailed protocol for the peptidoglycan (cell wall) labeling of the periodontal pathogen Tannerella forsythia and the development of a laboratory-safe Escherichia coli strain utilizing the N-acetylmuramic acid recycling enzymes AmgK, N-acetylmuramate/N-acetylglucosamine kinase, and MurU, N-acetylmuramate alpha-1-phosphate uridylyltransferase, from T. forsythia. The procedure involves bioorthogonal labeling of bacterial cells with an azido-modified analog of the amino sugar, N-acetylmuramic acid, through \"click chemistry\" with a fluorescent dye. The protocol is suitable for the generation of fluorescently labeled peptidoglycan molecules for applications in the study of bacterial and peptidoglycan trafficking in the host cells and cell wall recycling in complex microbiomes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2727 ","pages":"1-16"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11513787/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41204777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Organic Supplementation of Vaccinium corymbosum Micropropagation Media. 蔓越橘微繁殖培养基的有机添加剂。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3782-1_11
Marcelina Krupa-Małkiewicz, Ireneusz Ochmian, Monika Figiel-Kroczyńska

The best Vaccinium corymbosum plant growth under in vitro conditions can be achieved by using the right composition and pH of the medium. For the initial phase of in vitro culture, a combination of cytokinins-mostly zeatin-can usually be used. Organic supplementation of the medium enables the use of a replacement for the expensive natural cytokinin used in micropropagation of highbush blueberry. This chapter describes the experiments with silicon Hydroplus™ Actisil (Si), coconut water (CW), and different pH (5.0; 5.5, and 6.0) as a stress factor. The addition of 200 mg dm-3 silicon solution and 15% coconut water strongly stimulated highbush blueberry plant growth in vitro. Moreover, silicon solution benefits the negative effects of higher pH of the medium used for micropropagation of V. corymbosum. Maximum vegetative development of blueberry explants was obtained at pH 5.

在离体培养条件下,使用正确的培养基成分和酸碱度可以使越橘植物生长得最好。在离体培养的初始阶段,通常可以使用细胞分裂素(主要是玉米素)的组合。在培养基中添加有机物可以替代高丛蓝莓微繁殖中使用的昂贵的天然细胞分裂素。本章介绍了使用硅 Hydroplus™ Actisil(Si)、椰子水(CW)和不同 pH 值(5.0、5.5 和 6.0)作为胁迫因子的实验。添加 200 mg dm-3 硅溶液和 15%的椰子水能强烈刺激高丛蓝莓植物的离体生长。此外,硅溶液还能缓解用于蓝莓微繁殖的培养基 pH 值升高所带来的负面影响。蓝莓外植体在 pH 值为 5 时获得最大的无性发育。
{"title":"Organic Supplementation of Vaccinium corymbosum Micropropagation Media.","authors":"Marcelina Krupa-Małkiewicz, Ireneusz Ochmian, Monika Figiel-Kroczyńska","doi":"10.1007/978-1-0716-3782-1_11","DOIUrl":"10.1007/978-1-0716-3782-1_11","url":null,"abstract":"<p><p>The best Vaccinium corymbosum plant growth under in vitro conditions can be achieved by using the right composition and pH of the medium. For the initial phase of in vitro culture, a combination of cytokinins-mostly zeatin-can usually be used. Organic supplementation of the medium enables the use of a replacement for the expensive natural cytokinin used in micropropagation of highbush blueberry. This chapter describes the experiments with silicon Hydroplus™ Actisil (Si), coconut water (CW), and different pH (5.0; 5.5, and 6.0) as a stress factor. The addition of 200 mg dm<sup>-3</sup> silicon solution and 15% coconut water strongly stimulated highbush blueberry plant growth in vitro. Moreover, silicon solution benefits the negative effects of higher pH of the medium used for micropropagation of V. corymbosum. Maximum vegetative development of blueberry explants was obtained at pH 5.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2788 ","pages":"197-207"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140857104","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
Precision Genome Editing with CRISPR-Cas9. 利用 CRISPR-Cas9 进行精准基因组编辑。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3782-1_21
Shahroz Rahman, Abdul Rehman Ikram, Farrukh Azeem, Muhammad Tahir Ul Qamar, Tayyaba Shaheen, Mehboob-Ur-Rahman

The CRISPR/Cas9 system is a revolutionary technology for genome editing that allows for precise and efficient modifications of DNA sequences. The system is composed of two main components, the Cas9 enzyme and a guide RNA (gRNA). The gRNA is designed to specifically target a desired DNA sequence, while the Cas9 enzyme acts as molecular scissors to cut the DNA at that specific location. The cell then repairs the digested DNA, either through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in either indels or precise modifications of DNA sequences with broad implications in biotechnology, agriculture, and medicine. This chapter provides a practical approach for utilizing CRISPR/Cas9 in precise genome editing, including identifying the target gene sequence, designing gRNA and protein (Cas9), and delivering the CRISPR components to target cells.

CRISPR/Cas9 系统是一种革命性的基因组编辑技术,可对 DNA 序列进行精确、高效的修改。该系统由 Cas9 酶和引导 RNA(gRNA)两大部分组成。gRNA 专为所需的 DNA 序列而设计,而 Cas9 酶则像分子剪刀一样在特定位置剪切 DNA。然后,细胞通过非同源末端连接(NHEJ)或同源定向修复(HDR)来修复被消化的 DNA,从而产生嵌合或精确修饰的 DNA 序列,对生物技术、农业和医学产生广泛影响。本章介绍了利用 CRISPR/Cas9 进行精确基因组编辑的实用方法,包括确定目标基因序列、设计 gRNA 和蛋白质 (Cas9),以及将 CRISPR 组件输送到目标细胞。
{"title":"Precision Genome Editing with CRISPR-Cas9.","authors":"Shahroz Rahman, Abdul Rehman Ikram, Farrukh Azeem, Muhammad Tahir Ul Qamar, Tayyaba Shaheen, Mehboob-Ur-Rahman","doi":"10.1007/978-1-0716-3782-1_21","DOIUrl":"10.1007/978-1-0716-3782-1_21","url":null,"abstract":"<p><p>The CRISPR/Cas9 system is a revolutionary technology for genome editing that allows for precise and efficient modifications of DNA sequences. The system is composed of two main components, the Cas9 enzyme and a guide RNA (gRNA). The gRNA is designed to specifically target a desired DNA sequence, while the Cas9 enzyme acts as molecular scissors to cut the DNA at that specific location. The cell then repairs the digested DNA, either through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in either indels or precise modifications of DNA sequences with broad implications in biotechnology, agriculture, and medicine. This chapter provides a practical approach for utilizing CRISPR/Cas9 in precise genome editing, including identifying the target gene sequence, designing gRNA and protein (Cas9), and delivering the CRISPR components to target cells.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2788 ","pages":"355-372"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140870653","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
Multicellular Tumoroids for Investigating Cancer Stem-Like Cells in the Heterogeneous Tumor Microenvironment. 用于研究异质肿瘤微环境中癌症干样细胞的多细胞肿瘤实体
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_8
Kathleen M Burkhard, Geeta Mehta

Cancer stem-like cells (CSC) are a major contributing factor to chemoresistance, tumor recurrence, and poor survival outcomes in patients across cancer types. Signaling from non-tumor cells in the tumor microenvironment (TME) enriches for and supports CSC. This complex cell-cell signaling in the heterogeneous TME presents a challenge for patient survival; however, it also presents an opportunity to develop new targeted therapies that can inhibit survival of CSC. In this chapter, we report a multicellular tumoroid model which can be used to investigate the interactions between cancer cells and non-tumor cells in the TME to better understand the contribution of various cell types to cancer cell phenotypes, as well as the underlying mechanisms involved. The following methods allow for each cell type to be distinguished using FACS and studied individually. Gene expression can be analyzed for cancer cells, as well as the other non-tumor cells using qPCR following sorting. The response to chemotherapeutic agents and expression of stem markers can be determined for cancer cells using flow cytometry, excluding the other cell types to get an accurate view of the cancer cells. Furthermore, the viability of non-tumor cells can be analyzed as well to determine if there are cytotoxic effects of the drugs on non-tumor cells. Thus, the multicellular tumoroid model will reveal the interactions between the CSC and non-tumor cells in the heterogenous TME, resulting in discoveries in the fields of cancer biology, novel targeted therapies, and personalized drug screening for precision medicine.

癌症干样细胞(CSC)是导致各种癌症患者出现化疗耐药性、肿瘤复发和生存率低下的主要因素。肿瘤微环境(TME)中来自非肿瘤细胞的信号富集并支持癌干细胞。异质性肿瘤微环境中这种复杂的细胞-细胞信号传导对患者的生存提出了挑战;然而,它也为开发可抑制 CSC 生存的新型靶向疗法提供了机会。在本章中,我们将报告一种多细胞类肿瘤模型,该模型可用于研究TME中癌细胞与非肿瘤细胞之间的相互作用,从而更好地了解各种细胞类型对癌细胞表型的贡献以及相关的潜在机制。以下方法可使用 FACS 对每种细胞类型进行区分和单独研究。分选后可使用 qPCR 分析癌细胞和其他非肿瘤细胞的基因表达。使用流式细胞术可确定癌细胞对化疗药物的反应和干标志物的表达,同时排除其他细胞类型,以准确了解癌细胞的情况。此外,还可以分析非肿瘤细胞的存活率,以确定药物是否对非肿瘤细胞产生细胞毒性作用。因此,多细胞类肿瘤模型将揭示异质肿瘤组织间质干细胞和非肿瘤细胞之间的相互作用,从而在癌症生物学、新型靶向疗法和用于精准医疗的个性化药物筛选等领域有所发现。
{"title":"Multicellular Tumoroids for Investigating Cancer Stem-Like Cells in the Heterogeneous Tumor Microenvironment.","authors":"Kathleen M Burkhard, Geeta Mehta","doi":"10.1007/978-1-0716-3730-2_8","DOIUrl":"10.1007/978-1-0716-3730-2_8","url":null,"abstract":"<p><p>Cancer stem-like cells (CSC) are a major contributing factor to chemoresistance, tumor recurrence, and poor survival outcomes in patients across cancer types. Signaling from non-tumor cells in the tumor microenvironment (TME) enriches for and supports CSC. This complex cell-cell signaling in the heterogeneous TME presents a challenge for patient survival; however, it also presents an opportunity to develop new targeted therapies that can inhibit survival of CSC. In this chapter, we report a multicellular tumoroid model which can be used to investigate the interactions between cancer cells and non-tumor cells in the TME to better understand the contribution of various cell types to cancer cell phenotypes, as well as the underlying mechanisms involved. The following methods allow for each cell type to be distinguished using FACS and studied individually. Gene expression can be analyzed for cancer cells, as well as the other non-tumor cells using qPCR following sorting. The response to chemotherapeutic agents and expression of stem markers can be determined for cancer cells using flow cytometry, excluding the other cell types to get an accurate view of the cancer cells. Furthermore, the viability of non-tumor cells can be analyzed as well to determine if there are cytotoxic effects of the drugs on non-tumor cells. Thus, the multicellular tumoroid model will reveal the interactions between the CSC and non-tumor cells in the heterogenous TME, resulting in discoveries in the fields of cancer biology, novel targeted therapies, and personalized drug screening for precision medicine.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2777 ","pages":"99-122"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119948","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
Prostate Cancer Organoids for Tumor Modeling and Drug Screening. 用于肿瘤建模和药物筛选的前列腺癌组织块。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_10
Amani Yehya, Fatima Ghamlouche, Sana Hachem, Wassim Abou-Kheir

Prostate cancer (PCa) is the second most common malignancy and the fifth leading cause of cancer death in men worldwide. Despite its prevalence, the highly heterogenic PCa has shown difficulty to establish representative cell lines that reflect the diverse phenotypes and different stages of the disease in vitro and hence hard to model in preclinical research. The patient-derived organoid (PDO) technique has emerged as a groundbreaking three-dimensional (3D) tumor modeling platform in cancer research. This versatile assay relies on the unique ability of cancer stem cells (CSCs) to self-organize and differentiate into organ-like mini structures. The PDO culture system allows for the long-term maintenance of cancer cells derived from patient tumor tissues. Moreover, it recapitulates the parental tumor features and serves as a superior preclinical model for in vitro tumor representation and personalized drug screening. Henceforth, PDOs hold great promise in precision medicine for cancer. Herein, we describe the detailed protocol to establish and propagate organoids derived from isolated cell suspensions of PCa patient tissues or cell lines using the 3D semisolid Matrigel™-based hanging-drop method. In addition, we highlight the relevance of PDOs as a tool for evaluating drug efficacy and predicting tumor response in PCa patients.

前列腺癌(PCa)是全球第二大最常见的恶性肿瘤,也是导致男性癌症死亡的第五大原因。尽管前列腺癌发病率很高,但这种高度异源性的前列腺癌很难在体外建立能反映其不同表型和不同阶段的代表性细胞系,因此很难在临床前研究中建立模型。患者衍生类器官(PDO)技术已成为癌症研究中一种突破性的三维(3D)肿瘤建模平台。这种多功能检测方法依赖于癌症干细胞(CSCs)自我组织和分化成器官样微型结构的独特能力。PDO 培养系统可长期保持来自患者肿瘤组织的癌细胞。此外,它还能再现亲代肿瘤的特征,是体外肿瘤表征和个性化药物筛选的理想临床前模型。因此,PDOs 在癌症精准医疗方面大有可为。在此,我们介绍了使用基于三维半固体 Matrigel™ 的悬滴法建立和繁殖源自 PCa 患者组织或细胞系分离细胞悬液的器官组织的详细方案。此外,我们还强调了 PDOs 作为 PCa 患者药物疗效评估和肿瘤反应预测工具的相关性。
{"title":"Prostate Cancer Organoids for Tumor Modeling and Drug Screening.","authors":"Amani Yehya, Fatima Ghamlouche, Sana Hachem, Wassim Abou-Kheir","doi":"10.1007/978-1-0716-3730-2_10","DOIUrl":"10.1007/978-1-0716-3730-2_10","url":null,"abstract":"<p><p>Prostate cancer (PCa) is the second most common malignancy and the fifth leading cause of cancer death in men worldwide. Despite its prevalence, the highly heterogenic PCa has shown difficulty to establish representative cell lines that reflect the diverse phenotypes and different stages of the disease in vitro and hence hard to model in preclinical research. The patient-derived organoid (PDO) technique has emerged as a groundbreaking three-dimensional (3D) tumor modeling platform in cancer research. This versatile assay relies on the unique ability of cancer stem cells (CSCs) to self-organize and differentiate into organ-like mini structures. The PDO culture system allows for the long-term maintenance of cancer cells derived from patient tumor tissues. Moreover, it recapitulates the parental tumor features and serves as a superior preclinical model for in vitro tumor representation and personalized drug screening. Henceforth, PDOs hold great promise in precision medicine for cancer. Herein, we describe the detailed protocol to establish and propagate organoids derived from isolated cell suspensions of PCa patient tissues or cell lines using the 3D semisolid Matrigel™-based hanging-drop method. In addition, we highlight the relevance of PDOs as a tool for evaluating drug efficacy and predicting tumor response in PCa patients.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2777 ","pages":"135-144"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119949","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
Surface Markers for the Identification of Cancer Stem Cells. 用于识别癌症干细胞的表面标记。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_4
Tasfik Ul Haque Pronoy, Farhadul Islam, Vinod Gopalan, Alfred King-Yin Lam

Cancer stem cells have genetic and functional characteristics which can turn them resistant to standard cancer therapeutic targets. Identification of these cells is challenging and is done mainly by detecting the expression of antigens specific to stem cells. Currently, there is a significant number of surface markers available which can detect cancer stem cells by directly targeting the specific antigens present in cells. These markers possess differential expression patterns and sub-localizations in cancer stem cells compared to nonneoplastic and somatic cells. In addition to these biomarkers, multiple analytical methods and techniques, including functional assays, cell sorting, filtration approaches, and xenotransplantation methods, are used to identify cancer stem cells. This chapter will overview the functional significance of cancer stem cells, their biological correlations, specific markers, and detection methods.

癌症干细胞具有遗传和功能特征,可使其对标准癌症治疗目标产生抗药性。这些细胞的识别具有挑战性,主要通过检测干细胞特异性抗原的表达来实现。目前,有大量表面标记物可通过直接靶向细胞中存在的特定抗原来检测癌症干细胞。与非肿瘤细胞和体细胞相比,这些标记物在癌症干细胞中具有不同的表达模式和亚定位。除这些生物标记物外,还有多种分析方法和技术,包括功能测试、细胞分拣、过滤法和异种移植法,用于识别癌症干细胞。本章将概述癌症干细胞的功能意义、生物学相关性、特异性标志物和检测方法。
{"title":"Surface Markers for the Identification of Cancer Stem Cells.","authors":"Tasfik Ul Haque Pronoy, Farhadul Islam, Vinod Gopalan, Alfred King-Yin Lam","doi":"10.1007/978-1-0716-3730-2_4","DOIUrl":"10.1007/978-1-0716-3730-2_4","url":null,"abstract":"<p><p>Cancer stem cells have genetic and functional characteristics which can turn them resistant to standard cancer therapeutic targets. Identification of these cells is challenging and is done mainly by detecting the expression of antigens specific to stem cells. Currently, there is a significant number of surface markers available which can detect cancer stem cells by directly targeting the specific antigens present in cells. These markers possess differential expression patterns and sub-localizations in cancer stem cells compared to nonneoplastic and somatic cells. In addition to these biomarkers, multiple analytical methods and techniques, including functional assays, cell sorting, filtration approaches, and xenotransplantation methods, are used to identify cancer stem cells. This chapter will overview the functional significance of cancer stem cells, their biological correlations, specific markers, and detection methods.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2777 ","pages":"51-69"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119950","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
Conformational Heterogeneity of β-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy. 利用正交自旋标签和脉冲 ESR 光谱原位观测 β 管膜蛋白的构象异质性
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3734-0_15
Sophie Ketter, Aathira Gopinath, Benesh Joseph

Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of β-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd3+-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34 GHz), the data analysis, and interpretation.

革兰氏阴性细菌的外膜蛋白(OMPs)参与了细胞的许多基本功能。它们紧紧包裹在不对称脂质双分子层的外膜中。电子自旋共振(ESR)光谱技术与位点定向自旋标记(SDSL)相结合,可直接观察这些蛋白质在其原生环境中的结构和构象动态。在这里,我们描述了一种利用硝基氧化物、三芳甲基(triarylmethyl)和基于 Gd3+ 的自旋标签对离体外膜和完整大肠杆菌中的β管膜蛋白进行位点定向自旋标记的方案。此外,还介绍了随后的连续波(CW)和正交脉冲电子-电子双共振(PELDOR)测量,以及 Q 波段(34 GHz)的实验设置、数据分析和解释。
{"title":"Conformational Heterogeneity of β-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy.","authors":"Sophie Ketter, Aathira Gopinath, Benesh Joseph","doi":"10.1007/978-1-0716-3734-0_15","DOIUrl":"10.1007/978-1-0716-3734-0_15","url":null,"abstract":"<p><p>Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of β-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd<sup>3+</sup>-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34 GHz), the data analysis, and interpretation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2778 ","pages":"237-257"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119955","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
期刊
Methods in molecular biology
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1