这项工作是由奥地利科学基金会（FWF，P20619，P22988，以BZ）的支持。

微波样品制备<ol><li>开始前的样品制备1需要编程的自动微波组织处理电子显微镜（Leica EM AMW的徕卡显微系统，维也纳，奥地利）（表1）微波样品制备TEM以下协议： <img src="/files/ftp_upload/2950/2950table1.jpg" alt="表1" /> 表1。样品制备微波辐射的协议。不同的列显示（从左至右）： <ol><li>样品瓶号（ 样品瓶的nr个。）代表小瓶被加载到该圆盘传送器的处理器中的顺序。 </li><li>步的实际过程。 </li><li>在小瓶试剂。 </li><li>时间的实际步骤。 </li><li>达到的最高温度，在小瓶中的微波照射之前被关闭。 </li><li>微波辐射设置：连续快速的温度增量酶，保持设定的温度;斜率​​温和的温度升高，最后结束时达到的温度;脉冲温度迅速上升，电源关闭，直到温度下降5°C，电源恢复达到温度。 </li><li>最大功率的微波辐射。 </li></ol></li><li>刚准备在样品制备协议（琼脂100环氧树脂见1.7）描述的不同步骤的解决方案，填补他们根据设定的协议（表1）到指定的小瓶，装入小瓶传送带上，然后将圆盘传送带到的微波组织处理器，和最后加载的第一小瓶到单模式室。 </li><li> 烟草感染烟草花叶病毒（TMV）用刀片在造型蜡板，60MM索伦森下降了3％的戊二醛（琼脂科学有限公司，斯坦斯特德，英国）切出小部分叶片（1毫米2）磷酸盐缓冲液（pH 7.2）中，在室温下erature。 </li><li>转移部分用细镊子，立即到指定的篮子的网格宽度约200μm。在彼此顶部堆放的篮子，并将其插入到单声道模式室。必须小心的样品都在不断固定液覆盖在装载和堆叠筐的盖子，使他们不干燥。 </li><li>启动以前编程的微波样品制备协议进行固定，脱水和渗透。 </li><li>虽然样品制备由微波组织处理器自动执行继续与其它的植物材料，如在第3条（负染色）描述的负染色。 </li><li>新鲜制备琼脂100环氧树脂混合作为所述的下列组分：填充24克琼脂100，16克十二碳烯基琥珀酸酐，和10克甲基降冰片烯二酸酐（所有组件看到，斯坦斯特德琼脂科学有限公司，英国）在塑料杯中，热它40°C，拌匀。添加1.2克苄基二甲胺，调匀。到指定的聚合填充琼脂100环氧树脂形成之前的样品制备协议来的端部（例如，在表1中的步骤22）。 </li><li>协议完成后（在表1中的步骤22）后释放的层叠的篮子从单声道模式室到该圆盘传送器的最后小瓶含有渗透的样品。取下转盘微波器件，以便将篮子，并加载它们用细镊子到指定的聚合形式。样品总是覆盖着琼脂100环氧树脂在拆垛和加载，使他们不干燥，必须小心。 </li><li>堆栈彼此的顶部上的聚合形式。必须小心，样品总是覆盖着琼脂100环氧树脂在堆叠和加载，使他们不干燥。 </li><li>删除先前使用的小瓶从传送带的微波TISSUE处理器，加载它堆积的篮子，并插入到的微波组织的处理器的旋转木马。 </li><li>启动预先编程的聚合协议（表1）。 </li><li>虽然聚合时自动进行由微波组织处理器中，用透射电子显微镜检查负染网格[如Philips CM10透射电子显微镜，FEI（前身为飞利浦），埃因霍温，荷兰]，进行图像分析，如在第3和4（描述负染色和图像分析）。 </li><li>协议之后，完成后，除去聚合形式从单声道模式室，拆散聚合形式并删除含有样品的聚合块。它们现在可以用切片机切片。 </li></ol> 2。修整和切片<ol><li>插入到单独的采样保持器的一个或多个块与粘附在上面的样本超薄切片约1cm的持有人。 </li><li>修剪块与用于TEM的标本修剪机（例如徕卡赖克特Ultratrim; Leica公司制），使一个块面最大。 1mm的长度和200μm的宽度，其中包含尽可能多叶材料尽可能实现（阻止面部尺寸，可能需要进行调整，以大小的钻石刀）。 </li><li>组块的超薄切片机（如赖克特徕卡ULTRACUT S;徕卡显微系统）通过使用钻石刀，刀角45°（例如，GröplDiatome超45图尔恩，奥地利）。断面的厚度应调整到约70到90nm和切割速度应该是1mm左右/ s的。拿起一个福尔瓦（琼脂科学有限公司）涂层的铜或镍200平方米网格的多个部分。 </li><li>染色后的网格上的各个部分与柠檬酸铅（琼脂科学的有限公司;1.1克柠檬酸铅溶于双蒸水和8毫升42毫升1N氢氧化钠）部分地填充在培养皿中，用NaOH，以创建一个CO 2 -自由5分钟环境和15英里nutes溶解在蒸馏水中，在室温下，用1％乙酸双氧铀（琼脂科学有限公司）。网格用蒸馏水洗净1分钟之间每个后染色步骤。风干在方格框的网格。 </li><li>检查部分用透射电子显微镜（如Philips CM10透射电子显微镜，FEI，艾恩德霍芬，荷兰）。 </li></ol> 3。负染色<ol><li>约100mg的烟草花叶病毒感染的叶片材料，制备粗汁液60MMSøfrensen磷酸盐缓冲液（pH值7.2）中100μl的显微镜载片上用刀片2分钟均质材料的收获。 </li><li> 20μl的所得到的匀浆转移到第一阱的具有4个或更多的井（或者它也可以转移匀浆一块石蜡）涂特氟隆的显微镜载玻片。 </li><li>顶部与福尔瓦（琼脂科学有限公司）涂层的一面朝向下拉和INCU匀浆放置一个福尔瓦涂覆的网格上大怒5分钟。 </li><li>网格洗净2次，每次2分钟，通过两个滴加入200μl60MM索伦森磷酸盐缓冲液（pH值7.2）的顶部放置网格。 </li><li>孵育网格与新鲜制备的2％磷钨酸（琼脂科学有限公司）60MM索伦森磷酸盐缓冲液（pH为6.5）的溶液中1分钟。 </li><li>删除网格，并让它自然风干即可在方格框。 </li><li>检查网格用透射电子显微镜（如Philips CM10透射电子显微镜; FEI，埃因霍温，荷兰）。以至少10个随机选择的负染病毒粒子的图像（应该是可见的每一个图像上的至少10个或更多的病毒粒子）用透射电子显微镜在初级倍率21000X或更高。必须注意，所有的图像都有相同的放大倍率。 </li></ol> 4。图像分析<ol><li>从采取的显微照片测量的长度和宽度中的至少100个随机选择的单个病毒粒子[EG细胞D（奥林巴斯，生命和材料科学欧洲有限公司，汉堡，德国）的粒子分析工具或OPTIMAS 6.5.1负染色样品使用任何图像分析计算机软件（Media Cyber​​netics的公司，马里兰州贝塞斯达， USA）]。 </li><li>计算平均值和标准偏差，以实现通过负染方法和TEM观察的病毒颗粒的平均长度和宽度。 </li><li>比较长度和超微结构特点（2.5）中获得的文献中已知的，以便清楚地识别病毒疾病引起的病毒病的病毒和超微结构改变大小。 </li></ol> 5。代表性的成果： 经过微波样品制备典型的烟草花叶病毒引起的超微结构变化，如大面积的病毒颗粒排列在平行的形式，用透射电子显微镜可以观察到，在细胞质中受感染的抽烟者一个tabacum的细胞（图1A）。此外，在粗汁液TMV-感染叶烟草花叶病毒颗粒可以观察到的曲折，棒状结构负染后（图1B）。图像分析显示100个病毒颗粒的平均尺寸为280nm的长度和17nm的宽度（图2）为TMV。超微结构的烟草花叶病毒感染的细胞和在这项研究中观察到的病毒粒子的大小被认为是根据TMV-诱导的烟草和烟草花叶病毒颗粒的大小范围的超微结构物业先前报道的文献9-15。 <img src="/files/ftp_upload/2950/2950fig1.jpg" alt="图1" /> 图1。透射电子显微镜的烟草花叶病毒感染的叶片细胞和病毒粒子。 A）图片显示TMV感染的叶肉微波设备样品制备后的烟草叶片细胞的超微结构。注意平行对准的病毒粒子在细胞质中积累的大面积的。C = M =线粒体叶绿体与淀粉（ST），N =核，V =液泡，酒吧= 2微米。 B）图像显示在受感染的叶子的汁液通过负染病毒颗粒进行检测。 <img src="/files/ftp_upload/2950/2950fig2.jpg" alt="图2" /> 图2。病毒粒子的相对大小。相对分布的病叶汁液，他们出现在电子显微镜负染后的TMV粒子的长度和宽度。平均值（平均长度/宽度±标准偏差），计算从100病毒颗粒。

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没有利益冲突的声明。

微波设备已被证实为TEM样品制备提供快速和可靠的超微结构数据在几个小时内3,4,16。本研究中所用的方法取得的细胞器和膜的细微结构保存类似于常规和cryofixed样品5,15与一个巨大的优点是减少在样品固定和嵌入3天或更长的时间从约2小时。这代表目前现有文献中的TEM样品制备最快的协议。本研究中所描述的方法结合微波植物具有负的染色方法允许一个明确的和快速鉴定TMV-诱导的超微结构改建和抗病毒剂本身用于TEM的样品制备。 TMV诱导的超微结构的变化可以进行调查后修剪，切片和染色后，在约4小时内开始后固定在透射电子TRON显微镜。使用完全自动试样制备模式释放研究者在过渡期间进行负染色，以确定病毒剂的大小和宽度。因此，我们可以得出这样的结论：这种方法允许一个明确的和快速诊断的植物病毒病的一半左右，这一天是非常重要的未来在农业和科学实验的植物植物病理学。由于此方法也可用于动物和人类疾病的快速诊断，它有一个大的潜在的未来的应用在医疗和兽医病理学。

<table border="1"><tbody><tr><td> 的试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>戊二醛</td><td>琼脂科学有限公司</td><td> R1312 </td><td></td></tr><tr><td>四氧化锇</td><td>琼脂科学有限公司</td><td> R1022 </td><td></td></tr><tr><td>琼脂100树脂</td><td>琼脂科学有限公司</td><td> R1043 </td><td></td></tr><tr><td>十二烯基琥珀酸酐</td><td>琼脂科学有限公司</td><td> R1051 </td><td></td></tr><tr><td>甲基纳迪克酸酐</td><td>琼脂科学有限公司</td><td> R1081 </td><td></td></tr><tr><td>苄基二甲胺</td><td>琼脂科学有限公司</td><td> R1060 </td><td></td></tr><tr><td>柠檬酸铅</td><td>琼脂科学有限公司</td><td> R1210 </td><td></td></tr><tr><td>醋酸铀</td><td>琼脂科学有限公司</td><td> R1260A </td><td></td></tr><tr><td>磷钨酸</td><td>琼脂科学有限公司</td><td> R1213 </td><td></td></tr><tr><td>福尔瓦</td><td>琼脂科学有限公司</td><td> R1202 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td>徕卡EM AMW </td><td>徕卡显微系统</td><td></td><td></td></tr><tr><td>徕卡（赖克特）Ultratrim“ </td><td>徕卡显微系统</td><td></td><td>较新的型号是</td></tr><tr><td>徕卡（赖克特）ULTRACUT小号</td><td>徕卡显微系统</td><td></td><td>较新的型号是</td></tr><tr><td> Diatome超45 </td><td> Gröpl </td><td></td><td></td></tr><tr><td>飞利浦CM10 TEM的</td><td> FEI（原飞利浦） </td><td> &lt;/ TD&gt; <td>较新的型号是</td></tr><tr><td>孔D </td><td>奥林巴斯公司</td><td></td><td></td></tr><tr><td> OPTIMAS 6.5.1 </td><td>媒体控制论公司</td><td></td><td>较新的版本可用</td></tr></tbody></table>

microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy

由病毒引起的超微结构变化往往是必要的，清楚地识别植物病毒病的调查。与传统的样品制备透射电子显微镜（TEM），这样的调查需要数天时间，1,2，因此不适合用于植物病毒病快速诊断。微波炉固定可以使用相似的超微结构常规样品制备3-5后所观察到的结果用于TEM调查样品制备时间大幅度减少。许多不同的定制的微波器件是目前可用的，这可用于生物样品用于TEM调查5-8成功地固定和嵌入。在这项研究中，我们表现出对烟草花叶病毒（TMV）感染烟草植物，它有可能在大约半天时间，通过使用微波辅助样品制备的T诊断叶片超微结构的改变EM。我们选择了与市售的微波装置进行本研究，因为它执行几乎完全自动样品制备5相反，其他可用的设备，其中许多步骤仍然要手动执行6-8，因此更费时费力。作为执行剩余的TMV病叶及以下检查超微结构和大小的汁液中的病毒颗粒完全自动染色阴性样品制备过程中可进行固定和嵌入。

Watch this Scientific Journal Video about Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy at JoVE.com

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

这项研究描述了使用微波设备样品制备透射电子显微镜和负染色方法相结合的方法，允许快速和清晰的大约半天的植物病毒病的诊断。

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

microwave-assisted-rapid-diagnosis-plant-virus-diseases-transmission

Research

JoVE Journal

Immunology and Infection

13.4K 次观看.  University of Graz. 这项研究描述了使用微波设备样品制备透射电子显微镜和负染色方法相结合的方法，允许快速和清晰的大约半天的植物病毒病的诊断。

视频: Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

在野生鸟类禽流感病毒快速诊断：使用便携式的RRT - PCR和在外地的冻干试剂

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

科研

免疫与感染

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

HIV - 1衣壳大会通过低温电子显微镜的结构和迭代螺旋实时空间重构

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

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

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

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

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

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 ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

的高分辨电子显微<em&gt;幽门螺杆菌</em&gt;恰IV型分泌系统的霹雳产于铁可用性的条件变

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

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 ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

协议侦办主机组织分布，传播模式和效果上浓核病毒的主机健身的棉铃虫

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

喷墨打印机辅助自动跳棋阵列和手动消时法的抗菌协同试验

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...