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Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System. 使用 SpyCatcher-SpyTag 系统对外膜蛋白质进行荧光标记。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3734-0_4
Rachael Duodu, Dirk Linke, Jack C Leo

The SpyCatcher-SpyTag system has become a popular and versatile tool for protein ligation. It is based on a small globular protein (SpyCatcher) that binds to a 13-residue peptide (SpyTag), which subsequently leads to the formation of a covalent isopeptide bond. Thus, the reaction is essentially irreversible. Here, we describe how the SpyCatcher-SpyTag system can be used to label surface-exposed bacterial outer membrane proteins, e.g., for topology mapping or fluorescent time-course experiments. We cover using fluorescence measurements and microscopy to measure labeling efficiency using SpyCatcher fused with superfolder GFP in this chapter.

SpyCatcher-SpyTag 系统已成为蛋白质连接方面一种流行的多功能工具。该系统基于一种小球状蛋白质(SpyCatcher)与 13 个残基的多肽(SpyTag)结合,随后形成共价异肽键。因此,该反应基本上是不可逆的。在此,我们将介绍如何利用 SpyCatcher-SpyTag 系统标记表面暴露的细菌外膜蛋白,例如用于拓扑图绘制或荧光时间历程实验。在本章中,我们将介绍如何利用荧光测量和显微镜来测量 SpyCatcher 与超夹层 GFP 融合后的标记效率。
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引用次数: 0
Monitoring the Interaction of the Peptidoglycan with the Bacterial β-Barrel Assembly Machinery. 监测肽聚糖与细菌 β 管组装机制的相互作用
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3734-0_11
Federico Corona, Waldemar Vollmer

Gram-negative bacteria coordinate the biosynthesis of their different cell envelope components. Growth of the outer membrane (OM) requires the essential β-barrel assembly machine (BAM), which inserts OM proteins (OMPs) into the OM. The underlying peptidoglycan (PG) sacculus grows by the insertion of nascent glycan chains. We have previously identified interactions between BAM and PG in E. coli and showed that these interactions coordinate OM biogenesis with PG growth. BAM responds to the maturation state of the PG, and this mechanism activates preferentially BAM complexes at sites of active PG synthesis. Here we present protocols to purify soluble Bam proteins and full-length BamABCDE, isolate PG and soluble PG fragments, and study BAM-PG interactions with the isolated components. We also describe the protocol to detect interactions between Bam proteins and PG in cells.

革兰氏阴性细菌协调不同细胞包膜成分的生物合成。外膜(OM)的生长需要重要的β-管组装机(BAM),它将 OM 蛋白(OMPs)插入 OM。底层肽聚糖(PG)囊通过新生糖链的插入而生长。我们之前在大肠杆菌中发现了 BAM 和 PG 之间的相互作用,并证明这些相互作用协调了 OM 的生物生成和 PG 的生长。BAM 会对 PG 的成熟状态做出反应,这种机制会优先激活 PG 合成活跃部位的 BAM 复合物。在此,我们介绍了纯化可溶性 Bam 蛋白和全长 BamABCDE、分离 PG 和可溶性 PG 片段以及研究 BAM-PG 与分离成分相互作用的方案。我们还介绍了检测细胞中 Bam 蛋白和 PG 之间相互作用的方案。
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引用次数: 0
In Vitro Tumorigenic Assay: A Tumor Sphere Assay for Cancer Stem Cells. 体外致癌测定:癌症干细胞的肿瘤球试验。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_7
Amani Yehya, Hisham Bahmad, Wassim Abou-Kheir

Cancer stem cells (CSCs) represent a subpopulation of tumor cells that are thought to be responsible for therapy resistance, recurrence, and metastasis through their capacity to self-renew and differentiate into heterogeneous downstream lineages of cancer cells. Understanding the features of CSCs is crucial for managing cancer disease and establishing potential targeted therapeutics. Tumor sphere formation assay is a widely used in vitro method that selects and enriches the CSC subpopulation from the total population of cancer cells, based on their inherent ability to grow and clonally expand in serum-free and nonadherent culture conditions. Here we provide a detailed methodology to generate and propagate spheres from isolated cell suspensions of tumor tissues and cell lines using a semisolid MatrigelTM-based three-dimensional (3D) culture system.

癌症干细胞(CSCs)代表了肿瘤细胞的一个亚群,它们具有自我更新和分化成异质下游癌细胞系的能力,被认为是导致耐药性、复发和转移的罪魁祸首。了解癌细胞间充质干细胞的特征对于控制癌症疾病和建立潜在的靶向疗法至关重要。肿瘤球形成试验是一种广泛使用的体外方法,它能根据癌细胞在无血清和非粘附培养条件下生长和克隆扩增的内在能力,从癌细胞总数中筛选和富集 CSC 亚群。在这里,我们提供了一种详细的方法,利用基于 MatrigelTM 的半固体三维(3D)培养系统,从肿瘤组织和细胞系的分离细胞悬浮液中生成和繁殖球体。
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引用次数: 0
Methods to Study the Role of Mechanical Signals in the Induction of Cancer Stem Cells. 研究机械信号在诱导癌症干细胞中的作用的方法。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_13
Alessandro Gandin, Paolo Contessotto, Tito Panciera

Microenvironmental mechanical signals are fundamental regulators of cell behavior both in physiological and in pathological context, particularly in the induction and maintenance of tumorigenic properties. It is thus of utmost importance to experimentally recreate conditions that mimic the physical attributes of real tissues to study their impact on cell behavior and in particular on the induction of cancer stem cell (CSC) properties. Here we present protocols to investigate the role of mechanical stiffness on reprogramming of primary mammary gland cells into CSCs, including the synthesis of hydrogel substrates of the desired stiffness, the isolation and culture of primary differentiated normal cells derived from the human mammary gland, and the assessment of their CSC attributes after oncogene-mediated transformation.

微环境机械信号是细胞生理和病理行为的基本调节器,尤其是在诱导和维持致瘤特性方面。因此,最重要的是在实验中模拟真实组织的物理属性,研究它们对细胞行为的影响,特别是对诱导癌症干细胞(CSC)特性的影响。在这里,我们介绍了研究机械硬度对原代乳腺细胞重编程为 CSC 的作用的方案,包括合成所需硬度的水凝胶基底、分离和培养来自人类乳腺的原代分化正常细胞,以及评估它们在癌基因介导的转化后的 CSC 属性。
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引用次数: 0
Mechanical Stimulation of Adipose-Derived Stromal/Stem Cells for Functional Tissue Engineering of the Musculoskeletal System via Cyclic Hydrostatic Pressure, Simulated Microgravity, and Cyclic Tensile Strain. 通过循环静水压、模拟微重力和循环拉伸应变对脂肪基质/干细胞进行机械刺激,以实现肌肉骨骼系统的功能性组织工程学。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3762-3_25
Rachel C Nordberg, Josie C Bodle, Elizabeth G Loboa

It is critical that human adipose-derived stromal/stem cell (hASC) tissue engineering therapies possess appropriate mechanical properties in order to restore the function of the load-bearing tissues of the musculoskeletal system. In an effort to elucidate hASC response to mechanical stimulation and develop mechanically robust tissue-engineered constructs, recent research has utilized a variety of mechanical loading paradigms, including cyclic tensile strain, cyclic hydrostatic pressure, and mechanical unloading in simulated microgravity. This chapter will describe the methods for applying these mechanical stimuli to hASC to direct differentiation for functional tissue engineering of the musculoskeletal system.

人体脂肪基质/干细胞(hASC)组织工程疗法必须具备适当的机械特性,才能恢复肌肉骨骼系统承重组织的功能。为了阐明 hASC 对机械刺激的反应并开发出具有机械稳健性的组织工程构建物,最近的研究利用了多种机械加载范例,包括循环拉伸应变、循环静水压和模拟微重力下的机械卸载。本章将介绍对 hASC 施加这些机械刺激的方法,以引导分化,从而实现肌肉骨骼系统的功能性组织工程。
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引用次数: 0
Repurposing Decellularized Lung to Generate Vascularized Fat. 重新利用脱细胞肺来生成血管化脂肪。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3762-3_18
Lindsey K Huff, Zihan Ling, Megan K DeBari, Xi Ren, Rosalyn D Abbott

Conventional therapies to address critically sized defects in subcutaneous adipose tissue remain a reconstructive challenge for surgeons, largely due to the lack of graft pre-vascularization. Adipose tissue relies on a dense microvasculature network to deliver nutrients, oxygen, nonadipose tissue-derived growth factors, cytokines, and hormones, as well as transporting adipose tissue-derived endocrine signals to other organ systems. This chapter addresses these vascularization issues by combining decellularized lung matrices with a step-wise seeding of patient-specific adipose-derived stem cells and endothelial cells to develop large-volume, perfusable, and pre-vascularized adipose grafts.

解决皮下脂肪组织严重缺损的传统疗法仍然是外科医生面临的重建挑战,这主要是由于缺乏移植前血管化。脂肪组织依赖于密集的微血管网络来输送营养、氧气、非脂肪组织衍生的生长因子、细胞因子和激素,并将脂肪组织衍生的内分泌信号输送到其他器官系统。本章通过将脱细胞肺基质与患者特异性脂肪来源干细胞和内皮细胞的分步播种相结合,来开发大容量、可灌注和预血管化的脂肪移植物,从而解决这些血管化问题。
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引用次数: 0
Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial β-Barrel Assembly Machine. 通过特定位点光交联研究细菌β-管状组装机介导的毒素传输
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3734-0_8
Emily M Bouzan, Christine L Hagan

Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative organisms that relies on a specific interaction between a CdiA protein on the surface of one cell and a β-barrel protein on the surface of a neighboring cell. This interaction triggers the transport of a protein toxin into the neighboring cell where it exerts its lethal activity. Several classes of CdiA proteins that bind to different β-barrel receptors have been identified, but the molecular mechanism by which they deliver their toxins across the outer membranes of their target cells is poorly understood. Here we describe the use of site-specific photocrosslinking to characterize the interaction between a CdiA protein and its receptor. We describe the method for an E. coli CdiA that utilizes BamA as its receptor. BamA's central role in assembling β-barrel proteins in the outer membrane makes its role in CDI particularly intriguing; it suggests that these two different protein transport processes might share mechanistic features. Our in vitro photocrosslinking method is useful in elucidating early steps in the CDI mechanism, but it could be adapted to study later steps or to study other CdiA-receptor pairs.

接触依赖性抑制(CDI)是革兰氏阴性菌的一种细菌间竞争机制,它依赖于一个细胞表面的 CdiA 蛋白与邻近细胞表面的 β 管蛋白之间的特异性相互作用。这种相互作用会引发蛋白质毒素转运到邻近细胞,并在那里发挥致命的作用。目前已经发现了几类与不同的β-管受体结合的CdiA蛋白,但它们通过靶细胞外膜传递毒素的分子机制却鲜为人知。在这里,我们描述了利用位点特异性光交联来描述 CdiA 蛋白与其受体之间相互作用的特征。我们描述了利用 BamA 作为受体的大肠杆菌 CdiA 的研究方法。BamA 在组装外膜中的β-管状蛋白方面起着核心作用,因此它在 CDI 中的作用特别引人关注;这表明这两种不同的蛋白质转运过程可能具有共同的机理特征。我们的体外光交联方法有助于阐明 CDI 机制的早期步骤,但也可用于研究后期步骤或研究其他 CdiA 受体对。
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引用次数: 0
Canine Adult Adipose Tissue-Derived Multipotent Stromal Cell Isolation, Characterization, and Differentiation. 犬成体脂肪组织衍生多能基质细胞的分离、表征和分化。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3762-3_8
Takashi Taguchi, Mandi J Lopez

Adult mesenchymal stromal/stem cells (MSCs) are a standard component of de novo tissue generation to treat and study injury, disease, and degeneration. Canine patients constitute a major component of veterinary practice, and dogs share numerous pathologic conditions with humans. The relative abundance of adipose-derived stromal/stem cells (ASCs) in various canine adipose tissue depots is well described. Refined isolation, characterization, and differentiation techniques contribute to the collective knowledge of ASC phenotypes and subpopulations for specific tissue targets. Continued efforts to advance the knowledge of canine ASC behavior in vivo are critical to harnessing the full potential of primary cell isolates. This chapter contains a description of techniques to isolate, characterize, and differentiate canine ASCs.

成人间充质基质/干细胞(MSCs)是治疗和研究损伤、疾病和退化的新生组织的标准组成部分。犬类患者是兽医工作的主要组成部分,犬类与人类有许多共同的病理状况。各种犬类脂肪组织储层中脂肪衍生基质/干细胞(ASCs)的相对丰度已得到充分描述。经过改进的分离、表征和分化技术有助于人们对 ASC 表型和特定组织目标亚群有更深入的了解。要充分发挥原代细胞分离物的潜力,就必须继续努力增进对犬 ASC 体内行为的了解。本章介绍了分离、表征和分化犬间叶干细胞的技术。
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引用次数: 0
In Vitro Culture of White Adipose Tissue. 白色脂肪组织的体外培养
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3762-3_19
Jake J Fontenot, Frank H Lau

White adipose tissue (WAT) plays a crucial endocrine organ that regulates blood glucose and lipid levels, satiety, and inflammation. Before the described technique, primary white adipocytes could not be stably cultured in vitro. The lack of a reliable primary culture model impeded research in WAT metabolism and drug development. We have developed a novel technique for WAT primary culture called "sandwiched white adipose tissue" (SWAT). SWAT overcomes the natural buoyancy of adipocytes by sandwiching minced WAT between sheets of adipose-derived stromal cells. The resulting constructs are viable for at least 8 weeks in culture. SWAT maintains the intact extracellular matrix, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

白色脂肪组织(WAT)是调节血糖和血脂水平、饱腹感和炎症的重要内分泌器官。在采用所述技术之前,原代白色脂肪细胞无法在体外稳定培养。缺乏可靠的原代培养模型阻碍了白脂肪代谢和药物开发方面的研究。我们开发了一种新型的白脂肪原代培养技术,称为 "夹层白脂肪组织"(SWAT)。SWAT 通过在脂肪基质细胞薄片之间夹入切碎的白脂肪组织,克服了脂肪细胞的自然浮力。由此产生的构建体在培养过程中至少可存活 8 周。SWAT 保持了完整的细胞外基质、细胞间接触和体内 WAT 条件下的物理压力;此外,SWAT 还保持了强大的转录谱、对外源化学信号的敏感性和整体组织功能。SWAT 是一种简单、可重复且有效的原代脂肪培养方法。它有可能成为一个广泛适用于脂肪细胞生理学、病理生理学、新陈代谢和药物开发研究的平台。
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引用次数: 0
Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting. 通过 Western Blotting 验证 ELISpot 抗体特异性的当前趋势。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3690-9_2
Biji T Kurien, R Hal Scofield

The enzyme-linked immunospot (ELISpot) assay is a highly useful and sensitive method to detect total immunoglobulin and antigen-specific antibody-secreting cells. In addition, this method can measure biological activity and immunological secretions from immune cells. In general, membrane-bound antigen allows binding of antibody secreted by B cells, or a membrane-bound analyte-specific antibody binds to the specific analyte (e.g., cytokines) elicited from cells added to the well containing the bound antibody. The response from added cells is then detected by using an anti-Ig antibody and a colorimetric substrate, while in the case of non-B cells, the elicited antigen is detected with appropriate antibodies and enzyme-conjugated antibodies. Specificity of antibodies binding the protein of interest is necessary to achieve correct results. Western blotting can be used for this with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. Despite its general simplicity, western blotting is a powerful technique for immunodetection of proteins (notably low abundance proteins) as it provides simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Now, we have plethora of immunoblotting methods to validate antibodies for ELISpot.

酶联免疫斑点(ELISpot)检测法是检测总免疫球蛋白和抗原特异性抗体分泌细胞的一种非常有用和灵敏的方法。此外,这种方法还能检测免疫细胞的生物活性和免疫分泌物。一般来说,膜结合抗原可与 B 细胞分泌的抗体结合,或膜结合特异性分析物抗体可与添加到含有结合抗体的孔中的细胞激发的特异性分析物(如细胞因子)结合。然后使用抗 Ig 抗体和比色底物检测加入细胞的反应,而对于非 B 细胞,则使用适当的抗体和酶结合抗体检测激发的抗原。要获得正确的结果,与相关蛋白结合的抗体必须具有特异性。在使用 siRNA 或使用肽抑制剂抑制特异性抗体与目标蛋白结合的情况下,可使用 Western 印迹法检测相关蛋白。尽管Western印迹技术一般比较简单,但它是一种强大的蛋白质(特别是低丰度蛋白质)免疫检测技术,因为它能同时解析样品中的多种免疫原性抗原,以便用特异性抗体进行检测。现在,我们有大量的免疫印迹方法来验证 ELISpot 的抗体。
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引用次数: 0
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Methods in molecular biology
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