| Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime
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Author:
Date:
2017-01-20
[Abstract] NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzyme-binding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM). These protocols allow differences in metabolism to be detected between cell types and altered physiological and pathological states.
[摘要] NADH和NADPH分别是分解代谢和合成代谢过程的氧化还原辅因子。此外，NADPH在细胞抗氧化防御中起着重要作用。在活细胞和组织中，其光谱相同的自发荧光（称为NAD（P）H）的强度可用于探测线粒体氧化还原状态，而其不同的酶结合特征可用于将其相对贡献与总共分离使用荧光寿命成像显微镜（FLIM）的NAD（P）H强度。这些方案允许在细胞类型和改变的生理和病理状态之间检测代谢的差异。
背景 氧化还原辅因子烟酰胺腺嘌呤二核苷酸（NADH）及其磷酸化对应物NADPH的还原形式本质上是荧光的，两者都吸收波长为340（±30）nm并在460（±50）nm处发射的光（Patterson等人。，2000）。这些光谱特征在氧化成NAD（上标+）或NADP（superson），（2007））时损失。单独的NAD和NADP池的氧化还原平衡决定了对比的代谢过程（Ying，2008），如图1所示。NAD作为电子受体，用于通过三羧酸氧化线粒体中的糖，脂质和氨基酸底物（TCA）循环，并作为内线粒体膜（IMM）上的电子传递链（ETC）的电子供体，促使将质子泵送到膜间隙中，作为合成三磷酸腺苷（ATP）的电源，通过F 1 F 0 O 3 ATP合成酶（Osellame等人，2012）。因此，线粒体中NADH与NAD + 的平衡反映了TCA循环与ETC活性的平衡。 ...
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| DNA in situ Hybridizations for VEGFA Gene Locus (6p12) in Human Tumor Tissue
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Author:
Date:
2015-08-05
[Abstract] Over the last decades numerous regulators of angiogenesis have been identified and characterized. Among the others the vascular endothelial growth factor (VEGFA) appears undoubtedly important in several pathophysiological processes. Moreover, VEGFA represents one of the most attractive targets of anticancer therapy, given its major role in the growth and development of different tumor types. Here we describe a method to detect the copy number variation (CNV) status of the VEGFA gene by fluorescence in situ hybridization (FISH). FISH analysis is a reliable method for investigating VEGFA amplification or increased gene copy number and may represent an alternative method to immunohistochemical analysis for investigating the deregulation of VEGFA expression levels.
[摘要] 在过去的几十年中，已经鉴定和表征了许多血管生成的调节剂。 其中血管内皮生长因子（VEGFA）在几种病理生理过程中无疑是重要的。 此外，VEGFA代表抗癌治疗的最有吸引力的靶标之一，因为其在不同肿瘤类型的生长和发展中的主要作用。 在这里我们描述了通过荧光原位杂交（FISH）检测VEGFA基因的拷贝数变异（CNV）状态的方法。 FISH分析是用于研究VEGFA扩增或增加的基因拷贝数的可靠方法，并且可以代表用于研究VEGFA表达水平的失调的免疫组织化学分析的替代方法。
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| Establishment of a Symbiotic in vitro System between a Green Meadow Orchid and a Rhizoctonia-like Fungus
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Author:
Date:
2015-05-20
[Abstract] Symbiotic orchid seed germination in an in vitro system allows the growth of mycorrhizal protocorms and plantlets for scientific purposes. Orchids in nature need to establish a mycorrhizal symbiosis with fungal partners to germinate and develop into adult plants. Here we present a protocol for symbiotic germination of the terrestrial Mediterranean green meadow orchid Serapias vomeracea. The fungal symbiont Tulasnella calospora (T. calospora) (Basidiomycetes, Cantharellales) was chosen because of its common occurrence (Girlanda et al., 2011), its ability to grow in culture and compatibility in germination assays. T. calospora is one of the most common rhizoctonia-like fungi associated with terrestrial as well as epiphytic orchids.
[摘要] 在体外系统中的共生兰花种子萌发允许用于科学目的的菌根原球茎和小植物的生长。 兰花本质上需要建立与真菌合作者的菌根共生，以发芽和发育成成虫植物。 在这里我们提出了地中海地中海绿色草地兰花 Serapias vomeracea的共生萌发的协议。 选择真菌共生菌（Talsnella calospora）（ T。calospora ）（担子菌门，Cantharellales），因为其常见的发生（Girlanda等人，2011 ），其在培养中生长的能力和在发芽测定中的相容性。 T。 calospora 是与陆生以及附生兰花相关的最常见的丝孢菌样真菌之一。
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