| Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures
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Author:
Date:
2021-04-20
[Abstract] Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer’s Disease (AD). We have recently developed a novel optically-responsive tool (the ‘CofActor’ system) that couples cofilin and actin (key players in early stage cytoskeletal abnormalities associated with neurodegenerative disorders) with light-gated optogenetic proteins to provide spatial and temporal resolution of oxidative and energetic stress-dependent biochemical events. In contrast to currently available small-molecule based biosensors for monitoring changes in the redox environment of the cell, CofActor is a ...
[摘要] [摘要]参与神经变性蛋白质可具有耦合光遗传学试剂来创建快速且灵敏的记者到provid Ë洞察介导的神经变性疾病，包括进展的生化过程阿尔茨海默氏病（AD）。我们最近开发了一种新型光学-响应工具（“辅”系统）夫妇COF伊林和行动中使用（与神经退行性疾病相关的早期阶段，细胞骨架异常关键球员）光门控光遗传学 蛋白质提供时空分辨率的氧化和高能应激依赖的生化事件。与目前可用的基于小分子的生物传感器来监测细胞氧化还原环境的变化相比，CofActor是一种光激活的，遗传编码的氧化还原传感器，可以通过精确的空间和时间控制来激活。在这里，我们描述了从新生小鼠制备的解离海马神经元培养物中CofActor系统的表达和激活的协议。将培养物转染用大号ipofectamine上的第五天体外（DIV5），然后暴露于细胞应激诱导刺激，导致的肌动蛋白的形成丝切蛋白可使用活细胞成像技术可以观察到杆。本文所述的方案可用于研究暴露于神经退行性刺激（例如毒性Aβ42低聚物）的活神经元中与压力相关的细胞骨架失调。此外，从AD的转基因小鼠模型和/或与KO相关的小鼠KO小鼠分离的神经元中传感器的表达可以促进我们对与神经变性相关的早期细胞骨架功能障碍的分子基础的理解。
[背景]神经变性疾病的生化标志（神经原纤维，团块和缠结，提高活性氧物质（ROS） ...
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| Organotypic Brain Cultures: A Framework for Studying CNS Infection by Neurotropic Viruses and Screening Antiviral Drugs
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Author:
Date:
2017-11-20
[Abstract] According to the World Health Organization (WHO), at least 50% of emerging viruses endowed with pathogenicity in humans can infect the Central Nervous System (CNS) with induction of encephalitis and other neurologic diseases (Taylor et al., 2001; Olival and Daszak, 2005). While neurological diseases are progressively documented, the underlying cellular and molecular mechanisms involved in virus infection and dissemination within the CNS are still poorly understood (Swanson and McGavern, 2015; Ludlow et al., 2016). For example, measles virus (MeV) can infect neural cells, and cause a persistent brain infections leading to lethal encephalitis from several months to years after primary infection with no available treatment (Reuter and Schneider-Schaulies, 2010; Laksono et ...
[摘要] 根据世界卫生组织（WHO）的统计，至少有50％的新发病毒具有致病性，可感染中枢神经系统（CNS），并诱发脑炎和其他神经系统疾病（Taylor et al。 >，2001; Olival和Daszak，2005）。虽然神经系统疾病逐渐被记录下来，但涉及病毒感染和在CNS内传播的潜在细胞和分子机制仍然知之甚少（Swanson and McGavern，2015; Ludlow等人，2016）。例如，麻疹病毒（MeV）可以感染神经细胞，并在原发感染后几个月至数年导致持续的脑感染，导致致死性脑炎，而没有可用的治疗（Reuter和Schneider-Schaulies，2010; Laksono等人。，2016）。器官型脑文化（OBC）是病毒学领域的一个合适的模型，以更好地理解中枢神经系统感染。实际上，它不仅可以研究中枢神经系统内嗜神经病毒的感染和传播，而且还可以作为创新性抗病毒策略或分子的筛选模型，如我们最近发表的有关融合抑制肽和HSP90伴侣蛋白活性抑制剂的研究， 17-DMAG（Welsch等人，2013; ...
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| Extraction and Quantification of Tryptophan and Kynurenine from Cultured Cells and Media Using a High Performance Liquid Chromatography (HPLC) System Equipped with an Ultra-sensitive Diode Array Detector
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Author:
Date:
2016-04-05
[Abstract] Evidence of the involvement of tryptophan and its metabolite, kynurenine, in various biological processes including cancer is constantly expanding. Analysis of cell extracts and culture media can allow for quick snapshots of the metabolic fluctuations occurring in vitro. Here, we describe a method for metabolite extraction from mammalian cells and analysis of extracted metabolites and cell culture media by HPLC with detection using an ultra-sensitive diode array detector.
[摘要] 色氨酸及其代谢物，犬尿氨酸参与包括癌症在内的各种生物过程的证据不断扩大。 细胞提取物和培养基的分析可以允许在体外发生的代谢波动的快速快照。 在这里，我们描述了一种从哺乳动物细胞代谢物提取的方法，提取代谢物和细胞培养基通过HPLC检测使用超敏二极管阵列检测器分析。
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