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50 ml conical tubes

Company: BD
Catalog#: 352098
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In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals
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Date:
2018-08-20
[Abstract]  We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid ... [摘要]  我们使用体内和体外磷-31核磁共振( 31 P-NMR)光谱来跟踪运输,分区和 外生菌根真菌 Hebeloma cylindrosporum 中的磷酸盐代谢响应来自宿主( Pinus pinaster )或非宿主( Zea mays )的根信号植物。 开发了一种用于体内研究的装置,其允许连续氧化的矿物质溶液在含有菌丝体的NMR管中循环。 在几个连续的处理步骤(在液氮中冷冻;用高氯酸压碎;消除高氯酸;冷冻干燥;在适当的液体培养基中溶解)后,对真菌材料进行体外研究。

【背景】 菌根真菌和植物之间的关联改善了宿主植物的P营养(Smith和Read,2008; Plassard和Dell,2010; Cairney,2011; Smith 等人,,2015)。这种积极效应主要归因于真菌菌丝对磷酸盐(Pi)的吸收,探测了在活跃吸收根周围的耗竭区以外的大量土壤(Smith和Read,2008; Cairney,2011; Smith et al。< em="">,2015)和真菌细胞分泌细胞外磷酸酶(Quiquampoix和Mousain,2005)。吸收的Pi部分地掺入磷酸化的代谢物,磷脂和核酸中,并且部分地浓缩成多磷酸盐(PolyP),其中它们构成液泡中的储存池(Ashford 等人,,1994)。该协议详述了一种装置,该装置允许通过 31 ...

CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus
Author:
Date:
2018-08-20
[Abstract]  Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are ... [摘要]  大型DNA病毒(例如非洲猪瘟病毒(ASFV))的基因编辑传统上依赖于由报道盒组成的供体质粒与周围同源病毒DNA的同源重组。然而,这种导致所需修饰病毒的同源重组是罕见的事件。我们最近报道了使用CRISPR / Cas9编辑ASFV。使用CRISPR / Cas9修饰非洲猪瘟病毒基因组导致了引入遗传变化的快速且相对简单的方法。为了实现这一目标,我们首先用田间分离株ASFV-G感染原代猪巨噬细胞,并用CRISPR / Cas9供体质粒转染质粒,该质粒将表达靶向我们基因的特异性gRNA被删除。通过插入报告盒,我们能够通过有限稀释和噬菌斑纯化从亲本中纯化我们的重组病毒。我们以前曾报道将传统的同源重组方法与CRISPR / Cas9进行比较,结果导致重组增加超过4个对数。

【背景】 非洲猪瘟(ASF)是一种由ASF病毒(ASFV)引起的高度致命的猪传染性病毒性疾病。 ASFV的基因组由大约180-190千碱基对的双链DNA基因组组成。 ASFV引起一系列疾病,从高度致命到亚临床,取决于宿主特征和病毒株(Tulman et al。,2009)。 ASFV没有商业疫苗;实验上,2007年格鲁吉亚爆发的唯一能够抵御目前流行病毒株的疫苗(ASFV-G)是含有一个或多个病毒基因组缺失的减毒活疫苗,例如:( O'Donnell et ...

Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry
Author:
Date:
2018-07-20
[Abstract]  After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways–one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow ... [摘要]  在识别细胞外细菌脂多糖(LPS)后,Toll样受体4(TLR4)-CD14信号传导复合物启动两种不同的信号传导途径 - 一种来自质膜,另一种来自信号传导内体(Kagan 等。,2008)。因此,了解TLR4信号转导的早期阶段需要一种稳健且定量的方法来测量LPS触发的TLR4和CD14受体内吞作用,这是LPS检测中最早发生的事件之一。在这里,我们描述了一种基于流式细胞术的方法,我们最近用它来研究离子通道TRPM7在TLR4内吞作用中的作用(Schappe et al。,2018)。该测定依赖于用LPS刺激细胞并使用流式细胞术在不同时间点测量TLR4(或CD14)的细胞表面水平。尽管我们详细描述了来自鼠骨髓来源的巨噬细胞的TLR4和CD14的方法,但它可以很容易地适应于在各种其他信号传导环境中评估受体内吞作用。

【背景】先天免疫细胞,包括巨噬细胞和树突细胞,使用各种模式识别受体(PRR)来调查其环境中的危险和病原体相关分子模式。来自各种亚细胞区室的PRR的贩运和信号传导实现了更广泛的免疫监视,并且已成为先天免疫的重要设计原则(Brubaker et al。,2015)。细菌内毒素LPS的检测高度依赖于TLR4及其共同受体CD14。 TLR4复合物的内吞作用需要CD14,并且对于LPS诱导的巨噬细胞活化是必需的(Zanoni 等人,2011; Tan ...

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