| Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis
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Author:
Date:
2021-02-05
[Abstract] Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex to post-transcriptionally silence host target genes. We hereby describe the methodological details of how we recovered cross kingdom sRNA effectors of the oomycete pathogen Hyaloperonospora arabidopsidis during infection of its host plant Arabidopsis thaliana. This Bio-protocol contains two parts: first, a detailed description on the procedure of plant AGO/sRNA co-immunopurification and sRNA recovery for Illumina high throughput sequencing ...
[摘要] [摘要]在过去的十年中，已经注意到，微生物病原体和害虫将小RNA（sRNA）效应子传递到宿主植物中，以操纵植物生理学和免疫力，称为跨界RNA干扰。在此过程中，真菌和卵菌寄生虫sRNA劫持了植物ARGONAUTE（AGO）/ RNA诱导的沉默复合体，以转录后沉默宿主靶基因。我们在此描述方法学的细节，我们如何在宿主植物拟南芥感染期间恢复卵菌病原体拟南芥的跨界sRNA效应子。该生物协议包含两个部分：第一，关于植物AGO / sRNA co- 免疫纯化和sRNA回收，用于Illumina高通量测序分析。其次，我们解释了如何进行生物信息学小号斯尔纳序列分析读取可使用Galaxy服务器。原则上，该协议适用于研究来自多种宿主植物和植物相互作用（微生物）的AGO结合的sRNA。
[背景]小RNA（sRNA）可以充当病原体效应物，劫持植物ARGONAUTE（AGO）/ RNA诱导的沉默复合物（RISC），并使宿主mRNA沉默以进行感染，这种病毒被称为跨界RNA干扰的毒力机制（Weiberg等。，2015； Zeng等，2019）。分析感染期间与植物AGO结合的sRNA的库是一种选择方法，以全面了解可能通过宿主AGO / RISC起作用的植物入侵性病原体sRNA。基于抗体的植物AGO / ...
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| In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors
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Author:
Date:
2020-11-05
[Abstract] Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin (PR-Ub) ligase to inhibit SdeA’s activity. The attachment of multiple glutamate amino acids to the catalytic glutamate residue of SdeA by SidJ inhibits SdeA’s modification of ubiquitin (Ub) and ligation activity. In this protocol, we will discuss a SidJ non-radioactive, in vitro glutamylation assay using its substrate SdeA. This will also include a second reaction to assay the inhibition of SdeA by using both modification of free Ub and ligation ...
[摘要] [摘要]谷氨酰化是翻译后修饰，其中游离谷氨酸氨基酸的氨基与目标蛋白内谷氨酸侧链的羧基缀合。SidJ是一种军团菌激酶样蛋白，最近被发现可对军团菌SdeA磷酸核糖泛素（PR- Ub ）连接酶进行蛋白多谷氨酰化，从而抑制SdeA的活性。SidJ将多个谷氨酸氨基酸附着到SdeA的催化谷氨酸残基上，从而抑制了SdeA对泛素的修饰（Ub ）和结扎活动。在此协议中，我们将讨论使用其底物SdeA的SidJ非放射性，体外谷氨酰化测定。这也将包括一个第二反应以测定抑制SdeA通过使用免费的两个修改泛素和结扎ADP-核糖基化的泛素（ADPR-泛素），以SdeA的基板Rab33b。在鉴定和公布SidJ的活性之前，尚无SdeA抑制试验。我们的小组和其他小组演示了各种方法来抑制SdeA的活性。备选方案包括使用放射性NAD测量Ub的ADP核糖基化，NAD水解以及SdeA对HA- Ub连接的蛋白质印迹分析。该方案将描述使用廉价的标准凝胶和考马斯染色对SdeA的泛素修饰和PR- Ub连接的抑制。
[背景]嗜肺军团菌是感染性细菌，其机会性感染肺泡巨噬细胞。这是通过吸入被污染的水气溶胶而发生的，引起潜在的致命性肺炎，称为军团菌病（McDade et ...
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| Surgical Induction of Endometriosis in Female Mice
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Author:
Date:
2020-09-20
[Abstract] Endometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. It is frequently associated with pain, infertility and a reduced quality of life, and it lacks adequate treatment. Several rodent models of endometriosis have been developed through heterologous and homologous transplantation of endometrial tissue into the abdominal compartment. Here we describe a surgical procedure to generate a syngeneic model of endometriosis in immunocompetent mice with intact uterine and ovarian tissues. In this model, four uterine fragments from a donor mouse at diestrus are sutured to the abdominal wall of a recipient mouse. One month after surgeries, endometrial implants develop into cysts with glandular epithelium and stroma, mimicking ...
[摘要] [摘要 ] 子宫内膜异位症是一种常见的妇科疾病，其特征是子宫腔外存在子宫内膜组织。它经常与疼痛，不育和生活质量下降有关，并且缺乏适当的治疗。子宫内膜异位症的几种啮齿动物模型是通过将子宫内膜组织异源和同源移植到腹腔中而开发的。在这里，我们描述了在具有完整子宫和卵巢组织的免疫功能小鼠中产生子宫内膜异位症的同基因模型的手术程序。在该模型中，将来自供体小鼠在二头肌的四个子宫碎片缝合到受体小鼠的腹壁上。手术一个月后，子宫内膜植入物发展成具有腺上皮和间质的囊肿，模仿了子宫内膜异位症女性中观察到的子宫内膜异位病变。因此，该小鼠模型为研究子宫内膜异位症的病理生理学和潜在治疗方法的有效性提供了有价值的工具。
[背景 ] 子宫内膜异位是由子宫外子宫内膜组织的生长所定义的一种慢性妇科疾病，主要在骨盆和腹部表面生长（Zondervan et al。，2018）。它影响了育龄妇女的10％，并且与疼痛，不孕症和生活质量下降有关（Fourquet 等人，2011;Márki 等人，2017; Zondervan 等人，2018）。子宫内膜异位症的可用药物和手术疗法具有不良作用，并且无法长期缓解症状（Falcone和Flyckt，2018年）。因此，需要新的治疗策略，其发展依赖于动物模型的建立，该模型概括了临床子宫内膜异位症的病理生理和行为特征。
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