1。从烟草植物的病毒接种，病毒颗粒的纯化<ol><li>生产马铃薯X病毒 （PVX）载体质粒携带的上限从T7 RNA的转录：MRFV野生型（WT）和半胱氨酸突变外壳蛋白基因（CP）12，使用Ambion公司的的T7-mMessage mMachine。 </li><li>对于每个T7转录反应，接种两个完全展开的叶片，N.本生烟用10μl反应孵育10天的植物在温室中，在湿度60％，16小时的光（25,000-30,000勒克斯），在25℃，在20℃下8小时黑暗</li><li>收割机吨本生叶病毒感染后10天接种点数（dpi），衡量植物组织（50克），置于冰上。 </li><li>均质在掺混机中在存在下，100毫升的冷冻溶液的0.5M的Na-柠檬酸盐缓冲液，pH值5.5（用2毫升缓冲液每克植物组织）中的植物材料。 </li><li>过滤匀浆通过3层cheeseclOTH和以27,000 xg离心30分钟，通过离心澄清。将上清液转移到一个冷冻量筒，测量体积，然后将其转移到冷冻无菌烧杯。弃沉淀。 </li><li>处理该上清液，加入10％聚乙二醇8000（PEG 8000），在4℃下搅拌15分钟，并孵育45分钟，在4℃沉淀的病毒样颗粒（VLPs）-PEG矩阵。 </li><li>离心样品在4℃下以27,000 xg离心20分钟，除去上清液。 </li><li>加入8毫升的0.05M的Na-柠檬酸盐缓冲液，pH 5.5，含有2％的Triton X-100处理该沉淀。涡流离心管以及释放颗粒。颗粒和缓冲器传输冷藏的无菌烧杯中。冲洗管，立即用2毫升的相同的缓冲液中，并添加至烧杯中。搅拌的悬浮液在4℃，直到颗粒被溶解（通常为1小时）。 </li><li>澄清，通过离心分离，以27,000 xg离心5分钟，弃沉淀，和process通过离心的上清液在140,000 xg离心2小时，在4℃下</li><li>将沉淀重悬于1毫升0.05M的钠 - 柠檬酸盐缓冲液，pH值为5.5，并在0.05M的Na-柠檬酸盐缓冲液，pH值5.5的10-40％的蔗糖梯度的悬浮液放置在顶部。 </li><li> 3小时梯度离心140000 XG在4°C。 </li><li>照亮一盏灯的离​​心管的顶部，并从该管的顶部收集的光散射的上部频带在4厘米，稀释的0.05M的Na-柠檬酸盐缓冲液，pH值5.5，和离心机在140,000 xg离心3小时，在4 °C。 </li><li>将沉淀重悬于0.5ml的无菌水和存储在4℃达6个月。 </li><li>执行Bradford蛋白测定，定量的病毒样颗粒浓度。的产率是在1和3之间的VLPs微克/克植物组织。 </li></ol> 2。荧光素-5 - 马来酰亚胺标记反应的VLPs <ol><li>准备1mM溶液的荧光素-5 - 马来酰亚胺（427.37克/摩尔）在50mM钠phosphatë缓冲液，pH7.0，1mM EDTA中。混合使用的旋涡，，验证完全溶解。光用铝箔保护管。 </li><li>添加到在步骤1中，混合的VLPs产生荧光素-5 - 马来酰亚胺。巯基的摩尔量，使用了25倍摩尔过量的荧光素-5 - 马来酰亚胺。一般来说，使用的浓度为1μg/μl和荧光素-5 - 马来酰亚胺的溶液的60微升30微升的VLPs产生可接受的结果。 </li><li>在4℃下孵育2小时，在室温或过夜反应</li><li>终止该反应通过添加二硫苏糖醇（DTT）至终浓度为50mM。 </li><li>移除未反应的荧光素使用脱盐离心柱（赛默飞世尔科技公司，罗克福德IL），并在室温下以1,500×g离心2分钟离心。 </li><li>对于SDS-PAGE分析，添加到每个反应的10μl的10微升的2×Laemmli样品缓冲液的SDS-PAGE。标记蛋白在4℃下避光保存长达一个月或在单次使用的等分试样在-20℃下，长达3个月。 </li></ol> 3。标记反应和生物素掺入测定<ol><li>使用脱盐离心柱（赛默飞世尔科技公司，罗克福德IL）进行缓冲液交换，从水向磷酸盐缓冲盐水（PBS），pH值为7.0，在步骤1中产生的病毒样颗粒。 </li><li>准备的马来酰亚胺-PEG2-生物素的20mM原液，通过加入190μl的PBS在No-称量马来酰亚胺-PEG2-生物素和混合通过移液向上和向下。 </li><li>马来酰亚胺-PEG2-生物素到的VLPs和组合。使用10倍摩尔过量的试剂的VLPs解决方案≤2毫克/毫升。 </li><li>温育在室温下反应4小时。 </li><li>除去非反应的马来酰亚胺-PEG2-生物素使用脱盐离心柱（赛默飞世尔科技公司，罗克福德IL），并在室温下以1,500×g离心2分钟离心。 </li><li>确定摩尔每摩尔生物素的使用，皮尔斯生物素定量试剂盒（Thermo Scientific的）和FO的VLPsllowing制造商的说明。 </li></ol> 4。交联的半胱氨酸2 - 的VLPs和F肽<ol><li>在下面的步骤中使用的VLPs导致可用的半胱氨酸残基进行交联，在步骤2（半胱氨酸2 - 的VLPs）。准备一个新的交联的原液（28.63毫摩尔）在680微升二甲亚砜中溶解10mg的NHS-PEG4-马来酰亚胺交联剂。 </li><li>溶解（F）的一个17个氨基酸长的的肽（LLPNMOKDKEACAKAPL）在50μl的共轭缓冲液（1×PBS，pH值7.2，1mM EDTA）中的浓度为0.1mM。 </li><li>将4微升交联剂添加到50微升的溶解的肽（蛋白质-NH 2）在1mM的最终浓度（这是10倍摩尔过量的肽溶液的0.1 mM的）。 </li><li>在4℃下孵育2小时的反应</li><li>使用脱盐柱（赛默飞世尔科技公司，罗克福德IL）与缀合缓冲液（1×PBS，pH值7.2，1mM EDTA）中，通过离心平衡在1,500 xg离心纯化的交联的肽在室温下的1分钟。 </li><li>设置结合的VLPs（蛋白质-SH）和交联的肽（蛋白质-NH 2）的摩尔比确定的硫醇的数量和活化胺参与的反应和VLPs和肽之间的分子量的差异的反应混合物。 </li><li>在4℃下孵育反应混合物在室温下搅拌2小时</li><li> Western blot分析，混合产生的共轭产品1:1 Laemmli缓冲液，煮沸10分钟，并进行SDS-PAGE上一个预制10-20％Tris-甘氨酸凝胶（Invitrogen公司），电泳到硝酸纤维素膜（Invitrogen公司）。阻止膜，用1×PBS，pH值为7.2，含有0.05％Tween-20的PBS（PBS-吐温）和5％脱脂奶粉和探头，然后由相应的磷酸酶标记的二次抗体的肽特异性抗体。 </li></ol>

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这里介绍的方法，使一个非常敏感的和快速的分析本植物生产的VLPs的表面上，以及对其他蛋白复合物的反应性半胱氨酸。马来酰亚胺是硫醇的特定的试剂，与自由的含巯基的分子反应以形成稳定的硫醚键。此方法绘制的马来酰亚胺与巯基反应，不参与与其它氨基酸的相互作用的特异性。保的天然结构的VLPs在整个过程中是非常重要的。在描述的应用程序中，反应是在非变性条件下进行，并在pH为7?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td>薄壁，超清晰的管</td><td>贝克曼</td><td> 344059 </td><td></td></tr><tr><td> mMESSAGE mMACHINE T7套装</td><td>生活Tecnologies </td><td> AM1344M </td><td></td></tr><tr><td>荧光素-5 - 马来酰亚胺</td><td>赛默飞世尔科技生命科技</td><td> 46130 F150 </td><td> 46130是为了替代F150 </td></tr><tr><td>皮尔斯生物素定量试剂盒</td><td> Thermo Scientific的</td><td> 28005 </td><td></td></tr><tr><td> EZ-Link的马来酰亚胺-PEG2-生物素，称格式</td><td> Thermo Scientific的</td><td> 21901 </td><td></td></tr><tr><td> SM（PEG）N交联剂</td><td> Thermo Scientific的</td><td> 22107 </td><td></td></tr><tr><td> 10-20％Tris-甘氨酸凝胶</td><td> Invitrogen公司</td><td> EC61352 </td><td></td></tr><tr><td> Laemmli缓冲液</td><td> Bio-Rad公司</td><td> 1610737 </td><td></td></tr><tr><td>三甘氨酸SDS电泳缓冲液</td><td> Invitrogen公司</td><td> LC2675 </td><td></td></tr><tr><td>三甘氨酸传输缓冲区</td><td> Invitrogen公司</td><td> LC3675 </td><td></td></tr><tr><td>硝酸纤维素膜滤纸三明治</td><td> Invitrogen公司</td><td> LC2001 </td><td></td></tr><tr><td>磷酸酶标记的亲和纯化的兔IgG抗体</td><td> Kirkegaard和佩里实验室</td><td> 0751516 </td><td></td></tr><tr><td> NBT / BCIP显磷酸酶底物</td><td> Kirkegaard和佩里实验室</td><td> 508107 </td><td></td></tr></tbody></table>

analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps

病毒特性和物理化学和物理特性的模仿和开发持有世界上一些最紧迫的挑战提供解决方案的承诺。前所未有的范围和种类，加上有趣的特性的病毒可能给病毒为基础的技术的应用带来无穷的机遇。病毒有标注自组装成离散形状和大小，对称的特异性，多价，并稳定的特性在宽范围的温度和pH条件下的颗粒。毫不奇怪的是，这样一个显着的性能范围，病毒被建议使用的生物材料，疫苗，14，15，电子化学材料，工具和分子电子容器4，5，10，11，16，18，12。 为了利用纳米技术的病毒，他们必须修改其自然的形式注入新的功能。这一具有挑战性的公关ocess可以通过几种机制，包括遗传修饰的病毒基因组和化学外国或期望的分子附着到病毒颗粒的反应性基团8执行。修改病毒的能力主要取决于病毒的理化性质和物理性质。此外，在遗传或理化修改需要被执行，而不会不利地影响该病毒的天然结构和病毒的功能。 玉米rayado菲诺的病毒 （MRFV）外壳蛋白的自组装生产稳定和空的病毒样颗粒是稳定的蛋白质-蛋白质在大肠杆菌中的相互作用，这可用于在病毒为基础的技术的应用程序8。在烟草植物中产生的VLPs检查作为支架的多种肽可以通过共价键显示13。这里，我们描述的步骤1）确定的溶剂可在病毒衣壳的半胱氨酸可用于修饰的BIOCONJUGATE肽的阳离子，和2）的变形的衣壳。通过使用天然的或突变体插入的氨基酸残基和标准耦合技术，有各种各样的材料被植物如， 雀麦花叶病毒 3， 康乃馨斑驳病毒 12， 豇豆褪绿斑驳病毒 6， 烟草花叶病毒的表面上显示病毒 17， 芜菁黄花叶病毒 ，和MRFV 13。

瞬时表达的突变体MRFV的外壳蛋白（CP）基因N.在PVX基于矢量产生的VLPs benthamiana植物是图1中描述。的改性MRFV外壳蛋白基因，通过PCR扩增，然后放置在PVX基于的矢量，pP2C2S 2，（D. Baulcombe，塞恩斯伯里实验室，英格兰诺里奇礼物）的重复的亚基因组的CP启动子的转录控制下。体外 RNA转录产生的RNA转录物，然后用于接种N. benthamiana植物。在受感染的植物（...

Watch this Scientific Journal Video about Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

的方法来分析半胱氨酸残基的巯基的溶剂可及<em&gt;玉米rayado菲诺病毒</em&gt;（MRFV） - 病毒样颗粒（VLPs）随后的肽交联反应的说明。的方法，利用几个化学基团的表面上的，可以是特定的反应，目标的病毒样颗粒的可用性。

半胱氨酸残基的溶剂可及性分析<em&gt;玉米rayado菲诺病毒</em病毒样颗粒<em&gt;烟草</em植物和交联的肽类病毒颗粒

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

analysis-solvent-accessibility-cysteine-residues-on-maize-rayado-fino

Research

JoVE Journal

Immunology and Infection

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

11.0K 次观看.  Agricultural Research Service, United States Department of Agriculture. 的方法来分析半胱氨酸残基的巯基的溶剂可及玉米rayado菲诺病毒（MRFV） - 病毒样颗粒（VLPs）随后的肽交联反应的说明。的方法，利用几个化学基团的表面上的，可以是特定的反应，目标的病毒样颗粒的可用性。

视频: Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

科研

免疫与感染

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

造型埃博拉病毒的生命周期在生物安全二级条件下的病毒样颗粒包含Tetracistronic小基因组

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

粗病毒提取中 DNA 的组合分析及对感染叶 RNA 的分析发现新的病毒基因组

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&amp;#947;R Cross-linking

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

巨噬细胞 ros 产生的流式细胞仪测量对 fcγr 交联的响应

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...