Infectious Diseases Research Technologies Core - RML

传染病研究技术核心 - RML

• 批准号: 9161789
• 负责人: Robert Hohman
• 金额: $ 462.66万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9161789
• 关键词: Address; Alternative Splicing; Antigens; Area; Arts; Bacteria; Bioinformatics; Biological; Biological Assay; Biological Preservation; Biotechnology; Blood capillaries; Cell physiology; Cells; ChIP-seq; Chemicals; Collaborations; Communication; Complex; Computers; Confocal Microscopy; Consumption; Contrast Media; Copy Number Polymorphism; Coupling; Custom; DNA Sequence; Data; Data Analyses; Development; Dimensions; Discipline; Discrimination; Electron Microscope; Electron Microscopy; Electrons; Environment; Equipment; Eukaryotic Cell; Event; Experimental Designs; Explosion; Fixatives; Flow Cytometry; Freeze Substitution; Freezing; Genetic Markers; Genome; Genomics; Genotype; Goals; Health Sciences; Hour; Human; Ice; Image; Imagery; Imaging technology; Immune; Individual; Industry; Infectious Diseases Research; Institutes; Internet; Intervention; Intramural Research; Intramural Research Program; Investigation; Label; Laboratories; Lasers; Legal patent; Length; Life; Light; Link; Macromolecular Complexes; Maps; Medical; Methods; Microscope; Microscopy; Molecular Biology; National Institute of Allergy and Infectious Disease; Online Systems; Optics; Organelles; Organic solvent product; Parasites; Preparation; Process; Proteomics; Protocols documentation; Publications; Reading; Research; Research Personnel; Resolution; Rest; SNP genotyping; Sampling; Scanning; Scientist; Services; Site; Small RNA; Solutions; Sorting - Cell Movement; Specimen; Specimen Handling; Structure; Techniques; Technology; Testing; Time; Training; Transmission Electron Microscopy; Transportation; United States National Institutes of Health; Vaccines; Validation; Variant; Virus; Visual; Work; animation; base; capillary; cellular imaging; cold temperature; data management; data mining; design; exome sequencing; flexibility; genome sequencing; image reconstruction; improved; innovation; instrumentation; interest; light microscopy; microwave electromagnetic radiation; new technology; pathogen; pressure; red fluorescent protein; research study; simulation; technology/technique; transcriptomics; transmission process; web site

In 2015, this project provides state-of-the-art research technologies that are developed, validated, and then applied in support of NIAID research. Technologies developed outside the NIH are likewise tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the intramural program. The Genomics Unit provides applications in NGS sequencing, microarray, human and pathogen genotyping, and bioinformatic data analysis. More than 400,000 samples were processed by the Unit in the last 4 years. The Units Capillary DNA sequencing uses the AB 3730XL with 96-well high throughput processing with reads lengths out to 900bps. NGS employs the Illumina HiSeq 2500, and the MiSeq towards small RNA discovery, ChIP-Seq, transcriptomics, exome sequencing, de novo and ref-map genome sequencing, and copy number variation studies. Applications are developed in close collaboration with DIR investigators. The Unit develops project-specific research applications on the Affymetrix microarray platform including custom chip design, experimental design, sample processing and chip processing. Statistical analysis, data management, and data mining solutions are available as well, including from experimental concept to publication and public data submission. qPCR is performed for microarray and NGS data validation, expression analysis, sample optimization, and data support. Several technologies are available for human and pathogen genotyping, ranging from capillary-based re-sequencing, to high throughput targeted SNP genotyping via allelic discrimination assays. NGS for de novo SNP, In/Del, copy number, alternative splice variant analysis and newly expressed region discovery for both human and pathogen genomes is available. The Unit provides bioinformatics support for all of the offered technologies and applications. Flow Cytometry Project-specific research applications are developed for flow cytometry analysis and sorting in BSL-2 and BSL-3 environments. Electron Microscopy The Electron Microscopy Unit develops project-specific research applications in areas of sample preparation and analysis, ranging from basic structural studies to immune-localization of selected antigens for a wide array of specimens. A variety of methods, protocols, and equipment are employed to accommodate different preparative and imaging needs. Recent technological advancements have focused on 1) the introduction and optimization of sophisticated preparative technologies and techniques for improved retention and visualization of labile structures often lost during routine processing and improving structural preservation and 2) the introduction of advanced imaging technologies including high resolution transmission and scanning electron microscopes. The Unit has been developing correlative techniques for examining transient or dynamic events by light microscopy to identify regions of interest, which can then be prepared for visualization by electron microscopy to definitively correlate structures with functional assays. Conventional specimen processing for examination by electron microscopy requires use of chemicals that often extract or alter structures of interest. Cryo-preservation through high pressure freezing followed by chemical exchange at low temperature in a process called freeze substitution, has become the preferred technique allowing retention of fragile structures. It is a lengthy process of replacing vitreous ice in rapidly-frozen hydrated samples with an organic solvent containing fixatives and electron-dense contrasting agents. The EM Unit developed and assessed methods for maintaining cryo conditions in a laboratory microwave processor. Further development resulted in the fabrication of a thermally controllable unit decreasing processing periods from several days to a few hours while achieving excellent structural and antigenic preservation. The Microwave Assisted Freeze Substitution concept resulted in a patent application. The 300 kV TEM microscope for high-resolution 3D biological imaging is configured to have optimal flexibility to respond to the needs of investigators and provide them with the highest quality images. The main goal is to achieve the highest-level resolution possible for the characterization of macromolecular complexes, cellular organelles, viruses, bacteria and other parasites, as well as the ability to observe in three dimensions the host pathogen interactions occurring within eukaryotic cells. These technologies improve investigators ability to relate structure to function, providing information that may identify vaccine targets or other intervention strategies. Current projects include high-resolution imaging and reconstruction of bacteria, viruses, macromolecular complexes and eukaryotic cells. The ability to assign cellular function to structures provides valuable information in the study of host pathogen interactions. Fluorescent labeling antigens of interest or use of green and red fluorescent proteins as genetic markers allows visualization of transient and dynamic events by light microscopy (LM). Although advances in technology have improved resolution by LM, electron microscopy (EM) still provides superior ability for resolving small structures. However, EM provides only a snapshot since specimens are immobilized during specimen processing, limiting information about cellular dynamics. Correlative light and electron microscopy bridges this gap by coupling dynamic or transient information available through LM with the ability to resolve ultrastructural details by EM. Innovative methods have been established by the Unit that enable identification and imaging cells of interest first by confocal laser or epifluorescent microscopy, and then by scanning or transmission electron microscopy. Visual and Medical Arts The Visual and Medical Arts Unit (VMA) at RML provides highly specialized and technical services to the DIRs scientific and support staff. The Unit assists investigators in the use of images, interactive technologies, and animation/simulation to effectively communicate complex science and health topics to a range of audiences in scientific publications and new media platforms. The VMA is integral in providing support for complex web-based applications developed by the RTB. They work closely with scientists on individual projects, as well as produce video and other visualizations for public consumption.

在2015年，该项目提供了最先进的研究技术，这些技术经过开发，验证，然后应用于支持NIAID研究。同样对NIH之外开发的技术进行了测试，评估，验证，并在适当的情况下纳入壁内计划的技术组合中。 基因组学单元在NGS测序，微阵列，人类和病原体基因分型以及生物信息学数据分析中提供了应用。在过去的四年中，该单元处理了40万多个样品。毛细管DNA测序单元使用的AB 3730XL具有96孔高吞吐量处理，读取长度为900bps。 NGS采用Illumina HISEQ 2500，将Miseq用于小RNA发现，CHIP-SEQ，Transcriptomics，Exome序列，从头开始和参考映射基因组测序以及拷贝数变化研究。应用程序是与DIR调查人员密切合作开发的。 该单元在Affymetrix微阵列平台上开发了项目特定的研究应用程序，包括自定义芯片设计，实验设计，样品处理和芯片处理。还提供统计分析，数据管理和数据挖掘解决方案，包括从实验概念到出版物和公共数据提交。进行QPCR用于微阵列和NGS数据验证，表达分析，样本优化和数据支持。 从基于毛细血管的重新序列到高吞吐量靶向的SNP基因分型，可用于人类和病原体基因分型的几种技术。对于人类和病原体基因组的NGS，/del，/del，/del，副本编号，替代剪接变体分析和新表达的区域发现。该单元为所有提供的技术和应用提供了生物信息学支持。 流式细胞仪 在BSL-2和BSL-3环境中开发了特定于项目的研究应用程序，用于流式细胞仪分析和分类。 电子显微镜 电子显微镜单元在样品制备和分析领域开发了项目特异性研究应用，从基本的结构研究到广泛标本的选定抗原的免疫定位。采用多种方法，协议和设备来满足不同的制备和成像需求。最近的技术进步集中在1）引入和优化复杂的制备技术和技术，以改善保留率和可视化不稳定结构在常规处理和改进结构保存过程中经常丢失的不稳定结构； 2）引入高级成像技术，包括高分辨率传播和扫描电子显微镜。 该单元一直在开发相关技术，用于通过光学显微镜检查瞬态或动态事件，以识别感兴趣的区域，然后可以为通过电子显微镜可视化而准备这些区域，以将结构与功能分析确定相关。 通过电子显微镜检查进行检查的常规样品处理需要使用通常提取或改变感兴趣结构的化学物质。 在称为冻结替代的过程中，通过高压冻结的冷冻保护，然后在低温下进行化学交换，已成为允许保留脆弱结构的首选技术。这是一个漫长的过程，它可以用含有固定剂和电子致密对比剂的有机溶剂替换快速冻结水合样品中的玻璃冰。 EM单元开发并评估了在实验室微波处理器中维持冷冻条件的方法。进一步的发展导致制造可热控制的单元从几天减少到几个小时，同时实现出色的结构和抗原保存。微波辅助冻结替代概念导致了专利申请。 用于高分辨率3D生物成像的300 kV TEM显微镜配置为具有最佳灵活性，可以响应研究人员的需求，并为其提供最高质量的图像。主要目标是实现表征大分子复合物，细胞器细胞器，病毒，细菌和其他寄生虫的最高水平分辨率，以及在三个维度中观察的能力，在三个维度中宿主的病原体相互作用发生在真核细胞内。 这些技术提高了研究人员将结构与功能联系起来的能力，从而提供了可以识别疫苗目标或其他干预策略的信息。 当前的项目包括细菌，病毒，大分子复合物和真核细胞的高分辨率成像和重建。 将细胞功能分配到结构的能力在宿主病原体相互作用的研究中提供了有价值的信息。荧光标记感兴趣或使用绿色和红色荧光蛋白作为遗传标记的抗原允许通过光学显微镜（LM）可视化瞬态和动态事件。 尽管技术的进步改善了LM的分辨率，但电子显微镜（EM）仍然为解决小结构提供了较高的能力。但是，EM仅提供一个快照，因为在试样处理过程中固定了样品，从而限制了有关细胞动力学的信息。相关光和电子显微镜通过通过LM获得的动态或瞬态信息与EM解决超微结构细节的能力来弥合此间隙。 该单元已经建立了创新的方法，该单元首先通过共聚焦激光或表荧光显微镜，然后通过扫描或透射电子显微镜来启用感兴趣的细胞。 视觉和医学艺术 RML的视觉和医学艺术部门（VMA）为DIRS科学和支持人员提供了高度专业化的技术服务。该部门协助研究人员使用图像，交互式技术和动画/模拟，以有效地将复杂的科学和健康主题传达给科学出版物和新媒体平台中的一系列观众。 VMA在为RTB开发的基于Web的复杂应用程序提供支持方面是不可或缺的。他们与科学家紧密合作，从事各个项目，并制作视频和其他可视化供公众消费。