| Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders
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Author:
Date:
2021-03-05
[Abstract] The high attrition rate in drug development processes calls for additional human-based model systems. However, in the context of brain disorders, sampling live neuronal cells for compound testing is not applicable. The use of human induced pluripotent stem cells (iPSCs) has revolutionized the field of neuronal disease modeling and drug discovery. Thanks to the development of iPSC-based neuronal differentiation protocols, including tridimensional cerebral organoids, it is now possible to molecularly dissect human neuronal development and human brain disease pathogenesis in a dish. These approaches may allow dissecting patient-specific treatment efficacy in a disease-relevant cellular context. For drug discovery approaches, however, a highly reproducible and cost-effective cell model is ...
[摘要] [摘要]药物开发过程中的高流失率要求使用其他基于人的模型系统。但是，在脑部疾病的情况下，不适合对活的神经元细胞进行采样以进行化合物测试。人类诱导的多能干细胞（iPSC ）的使用彻底改变了神经元疾病建模和药物发现领域。由于基于iPSC的神经元分化方案（包括三维脑类器官）的发展，现在可以在一个碟子中分子解剖人神经元发育和人脑疾病的发病机理。这些方法可以允许在与疾病相关的细胞环境中解剖患者特异性的治疗功效。但是，对于药物发现方法，需要高度可复制且具有成本效益的细胞模型。在这里，我们描述了一种一步-步骤，用于从人产生健壮和可膨胀的神经祖细胞（NPC）工艺的iPSC 。用此协议生成的NPC是同质的且高度增殖。这些功能使NPC适合开发用于药物发现的高通量化合物筛选。人iPSC衍生的NPC示出了代谢依赖于线粒体活性，因此可也用于研究神经病症，其中线粒体功能受到影响。该协议涵盖了制备，培养和表征人iPSC来源的NPC所需的所有步骤。
图形摘要：
示意性的协议的所述发电机密封的离子人类源自iPSC的的NPC
[背景技术]近年来，目标为中心的药物发现的缺点已经用于寻址的神经系统疾病的方案变得明显，特别是（保罗等人，2010） ...
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| Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
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Author:
Date:
2018-05-05
[Abstract] This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments.
Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli
[摘要] 该协议提供了分步说明（图1），用于在大肠杆菌中异源表达新西兰弗朗西斯菌弗朗西丝菌Cas12a（以前称为Cpf1）。 它还包括一个高纯度纯化方案，并简要介绍如何进行活性测定。 这些方案也可以用于其他Cas12a同系物的纯化，并且纯化的蛋白质可以用于随后的基因组编辑实验。
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图1.从大肠杆菌 异源表达和纯化<弗朗西斯弗朗西丝菌 Cas12a（FnCas12a）的活动时间表
【背景】原核CRISPR-Cas免疫系统通过使用CRISPR RNA（crRNA）作为外源DNA或RNA的序列特异性靶向的指导来提供针对病毒和质粒的保护（van der Oost等人，2014; Marraffini ，2015）。 1类CRISPR-Cas系统（包含I型，III型和IV型）通常形成多亚基蛋白-cRNA效应复合物，而2类系统（包含II型，V型和VI型）依赖于单个crRNA-引导的效应物核酸酶用于目标干扰（Mohanraju et al。 2016年）。
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| Aspergillus terreus Infection of Fruits and Terrein Quantification by HPLC Analysis
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Author:
Date:
2016-06-20
[Abstract] The opportunistic fungal human and plant pathogen Aspergillus terreus (A. terreus) can be isolated from sea water, soil or decaying organic matter such as rotting leaves and fruits. While growing on fruits A. terreus produces secondary metabolites such as terrein, which may ease its penetration into plant tissues. In addition, biological activities of terrein may support competition against other microorganisms. In summary, terrein is a small polyketide that reduces germination of seedlings, induces lesions on fruit surfaces but also shows moderate antifungal activity. With this manuscript we provide a fruit infection protocol with Aspergillus terreus with subsequent determination of terrein production rates on infected fruits using an HPLC-based ...
[摘要] 机体真菌人和植物病原菌土曲霉（A. terreus）可以从海水，土壤或腐烂的有机物质如腐烂的叶子和果实中分离出来。 当在水果上生长时，A. terreus产生次生代谢物如土曲杆菌，这可以缓解其渗透到植物组织中。 此外，土耳特蛋白的生物活性可能支持与其他微生物的竞争。 总而言之，土豆蛋白是一种减少幼苗发芽的小聚酮，诱发水果表面的病变，但也显示出中等的抗真菌活性。 使用这份手稿，我们提供了一种水果感染方案与土曲霉，随后使用基于HPLC的量化方法测定感染果实的土豆油生产率。
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