| Resolving Structural Changes of Photoreceptors in Living Escherichia coli via In-cell Infrared Difference Spectroscopy
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Author:
Date:
2021-02-05
[Abstract] Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions in vivo differ dramatically from those selected for in vitro experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules. Fourier transform infrared (FTIR) difference spectroscopy is a well-suited method to study the light-induced structural responses of photoreceptors including changes in cofactor, side chains and secondary structure. Here, we describe a protocol to study the response of cofactor and protein in living E. coli cells via in-cell infrared difference (ICIRD) spectroscopy using the attenuated total reflection (ATR) ...
[摘要] [摘要]最近开发了几种细胞内光谱技术，以研究蛋白质在其天然环境中的结构和机理。体内条件与为体外实验选择的条件大不相同。因此，细胞环境可例如通过分子拥挤或小分子结合来影响蛋白质机制。傅立叶TRANSF ORM红外（FTIR）光谱差异是非常适合的方法来研究的光诱导结构响应小号光感受器，包括辅因子的变化，侧链和二级结构。在这里，我们描述了一种协议，用于研究活大肠杆菌中辅因子和蛋白质的响应 通过使用衰减全反射（ATR）配置的细胞内红外差（ICIRD）光谱分析细胞。蛋白质在大肠杆菌中过表达，将细胞转移到盐溶液中，并使用荧光光谱法确定每个细胞的拷贝数。将悬浮液离心，并将浓缩的细胞转移到FTIR光谱仪内的ATR池中。该恒温细胞密封并从与LED的顶部照明。在照射之前和之后记录强度光谱，以产生活细胞内部受体的差异光谱。利用ICIRD光谱，可在近乎自然的环境中解决可溶性感光体的结构变化。该方法在环境条件下可在H 2 O中运行，无标记，对蛋白质大小没有任何限制，并且不需要任何纯化步骤。
图形摘要：
使用衰减全反射对活大肠杆菌中的感光细胞进行细胞内红外差异光谱分析。
[背景]光感受器在从细菌，真菌，藻类和植物到动物的各种生物体的光感测和光适应中起着至关重要的作用（Möglich等，2010 ; ...
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| Characterization of Hippocampal Adult-borne Granule Cells in a Transient Cerebral Ischemia Model
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Author:
Date:
2021-01-20
[Abstract] Long-term consequences of stroke significantly impair the quality of life in a growing population of stroke survivors. Hippocampal adult neurogenesis has been hypothesized to play a role in the pathophysiology of cognitive and neuropsychiatric long-term sequelae of stroke. Reliable animal models of stroke are paramount to understanding their biomechanisms and to advancing therapeutic strategies. We present a detailed protocol of a transient cerebral ischemia model which does not cause direct ischemic damage in the hippocampus, allowing investigations into the pathophysiology of long-term neurocognitive deficits of stroke. Furthermore, we describe a protocol for obtaining acute hippocampal slices for the purpose of electrophysiological and morphological characterization of adult-borne ...
[摘要] [摘要]中风的长期后果严重损害了越来越多的中风幸存者的生活质量。假定海马成年神经发生在中风的认知和神经精神病学长期后遗症的病理生理学中起作用。可靠的中风动物模型对于理解其生物机制和推进治疗策略至关重要。我们提出 暂时性脑缺血模型的详细协议，该模型不会在海马体中引起直接缺血损伤，从而可以研究中风的长期神经认知缺陷的病理生理学。此外，我们描述了一种用于获取成人海马颗粒细胞的电生理学和形态学特征的急性海马切片的协议。还讨论了与从小细胞（例如未成熟的成年颗粒细胞）进行电生理记录有关的特殊性。本协议可以通过多模式研究（行为，形态结构，生化）加以补充，以期有希望促进研究和中风的长期后遗症的发展以及发现新的治疗机会。
[背景]中风是在发达国家发病率和死亡率的重要原因引起急性和延迟赤字。虽然针对缺血性中风恢复血流的干预策略在降低急性发病率和死亡率方面已经变得更加有效，但中风的长期后果（如中风后抑郁和中风后认知功能障碍和痴呆症）目前正在逃避临床治疗（Wang等。，2010; Loubinoux等人，2012; Mijajlovic ...
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| Live-cell Imaging and Quantitative Analysis of Meiotic Divisions in Caenorhabditis elegans Males
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Author:
Date:
2020-10-20
[Abstract] Live-imaging of meiotic cell division has been performed in extracted spermatocytes of a number of species using phase-contrast microscopy. For the nematode Caenorhabditis elegans, removal of spermatocytes from gonads has damaging effects, as most of the extracted spermatocytes show a high variability in the timing of meiotic divisions or simply arrest during the experiment. Therefore, we developed a live-cell imaging approach for in situ filming of spermatocyte meiosis in whole immobilized C. elegans males, thus allowing an observation of male germ cells within an unperturbed environment. For this, we make use of strains with fluorescently labeled chromosomes and centrosomes. Here we describe how to immobilize male worms for live-imaging. Further, we describe ...
[摘要] [摘要] 用相差显微镜对一些物种的精母细胞进行了减数分裂的实时成像。对于秀丽隐杆线虫来说，去除生殖腺中的精母细胞具有破坏性作用，因为大多数精母细胞在减数分裂的时间上表现出高度的变异性，或者只是在实验中停止。因此，我们开发了一种活体细胞成像方法，用于原位拍摄固定化线虫雄性精母细胞减数分裂过程，从而可以在不受干扰的环境中观察雄性生殖细胞。为此，我们利用带有荧光标记染色体和中心体的菌株。在这里我们描述如何固定男性蠕虫进行实时成像。此外，我们描述了获取和处理数据的工作流程，以获得有关精母细胞减数分裂I和II中染色体分离动态的定量信息。此外，我们最新开发的方法允许我们在电子显微镜中重新定位胶片上的主轴，而不管蜗杆的初始3D方位如何，并以统计稳健的方式分析活蠕虫的纺锤动力学。我们的实时成像方法也适用于秀丽隐杆线虫雌雄同体，并且可以扩展到其他荧光标记的线虫或其他完全透明的小型模型生物。
[背景] ...
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