Infectious Diseases Research Technologies Core - RML

传染病研究技术核心 - RML

• 批准号: 8946613
• 负责人: Robert Hohman
• 金额: $ 607.31万
• 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/8946613
• 关键词: 3-Dimensional; Address; Alternative Splicing; Antigens; Area; Arts; Bacteria; Bioinformatics; Biological; Biological Assay; Biological Preservation; Biotechnology; Blood capillaries; Cell physiology; Cells; ChIP-seq; Chemicals; Collaborations; Communicable Diseases; Communication; Complex; Computer software; Computers; Confocal Microscopy; Containment; Contrast Media; Copy Number Polymorphism; Coupling; Custom; DNA Microarray Chip; DNA Sequence; Data; Data Analyses; Data Set; Detection; Development; Dimensions; Discipline; Discrimination; Electron Microscope; Electron Microscopy; Electrons; Environment; Equipment; Eukaryotic Cell; Event; Experimental Designs; Explosion; Fixatives; Flow Cytometry; Freeze Substitution; Freezing; Genes; Genetic Markers; Genome; Genomics; Genotype; Health Sciences; Hour; Human; Ice; Image; Imagery; Imaging technology; Immune; Industry; Infectious Diseases Research; Institutes; Internet; Intervention; Intramural Research; Intramural Research Program; Investigation; Label; Laboratories; Lasers; Legal patent; Life; Light; Link; Macromolecular Complexes; Maps; Medical; Methods; Microscope; Microscopy; Molecular Biology; National Institute of Allergy and Infectious Disease; Optics; Organelles; Organic solvent product; Parasites; Phenotype; Play; Preparation; Process; Proteomics; Protocols documentation; Publications; Publishing; Research; Research Personnel; Resolution; Rest; Retrieval; Role; SNP genotyping; Sampling; Scanning; Scientist; Services; Site; Small RNA; Solutions; Sorting - Cell Movement; Specimen; Specimen Handling; Structure; System; Techniques; Technology; Testing; Time; Titania; Titanium; Training; Transmission Electron Microscopy; Transportation; United States National Institutes of Health; Vaccines; Validation; Variant; Virus; Visual; animation; base; biodefense; capillary; cellular imaging; cold temperature; comparative; data management; data mining; design; exome sequencing; flexibility; genome sequencing; image reconstruction; improved; innovation; instrument; instrumentation; interest; light microscopy; microwave electromagnetic radiation; new technology; next generation; next generation sequencing; pathogen; pressure; programs; red fluorescent protein; research study; simulation; technology/technique; transcriptomics; transmission process; web site

In 2014, 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 technologies supported include flow cytometry, confocal and electron microscopy, DNA microarray, DNA sequencing, Next Generation sequencing, bacterial phenotyping and quantitative PCR. Many of these technologies are used in high containment laboratories critical to the Institute's infectious diseases and biodefense research agenda. The RTB also provides advanced training in all aspects of the technologies in the Branch's portfolio. Sequencing: Capillary DNA sequencing uses the AB 3730XL with 96 well high throughput processing. Applications are developed in close collaboration with DIR investigators. All data is uploaded to a server, which tracks and manages all of the sequencing data generated for the Institute. Next Generation sequencing employs the Illumina GA II, the Illumina HiSeq 2000, and the 454 FLX Titanium FLX sequencers towards small RNA discovery, ChIP-Seq, transcriptomics, exome sequencing, de novo and ref-map genome sequencing, and copy number variation studies. Microarrays: The RTB develops project-specific research applications on Affymetrix microarray platform including custom chip design, experimental design, sample processing and chip processing. The RTB also develops statistical analysis, data management, and data mining solutions for DIR research programs; focusing on interpreting data generated by highly parallel detection systems used in genomics. The RTB performs QPCR for high throughput microarray data validation, sample optimization, and Next gen data validation Human/Pathogen Genotyping: Several technologies are used for human and pathogen genotyping depending on the scope of the genotyping project and they range from capillary-based re-sequencing of entire human genes for de novo SNP or In/Del discovery, to high throughput targeted SNP genotyping via allelic discrimination assays using Taqman quantitative PCR, to SNPlex multiplexed assays (3730XL sequencer), to Affymetrix SNP chip-based arrays and custom pathogen SNP (MIP technology) arrays. Next Generation sequencing technologies play a large role in de novo SNP, InDel, copy number, alternative splice variant and new expressed region discovery for both human and pathogen genomes. Bioinformatics data analysis is performed for Next Generation, microarrays and QPCR data analysis. High RAM, task-specific servers loaded with state-of-the-art software are used by skilled bioinformaticians towards providing DIR scientists with finished, publishable data sets where discoveries are clearly highlighted and presented for ease of interpretation. Data sets are stored in multiple formats for quick retrieval and re analysis and or comparative analysis with new or recently published competing datasets. Public data submissions and corrections to submissions are also performed by our bioinformatician. Flow Cytometry Project-specific research applications are developed for flow cytometry analysis and sorting in BSL-2 and BSL-3 environments. Electron Microscopy Project-specific research applications are developed in the 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 two principle areas: 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) introduction of advanced imaging technologies including high resolution transmission and scanning electron microscopes. The Unit has been developing correlative techniques useful 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 recently acquired by the EM Unit is the most advanced instrument for high resolution 3-D biological imaging configured to have optimal flexibility to respond to the needs of investigators and provide them with the highest quality images. It is the platform upon which improvements in ultrastructural imaging will likely be made over the next decade resulting in 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 our ability to relate structure to function, providing information which 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. EM however 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 developed 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 Unit This Unit at RML provides highly specialized and technical services to 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.

2014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。 支持的技术包括流式细胞仪，共聚焦和电子显微镜，DNA微阵列，DNA测序，下一代测序，细菌表型和定量PCR。这些技术中的许多技术用于对该研究所的传染病和生物形式研究议程至关重要的高遏制实验室。 RTB还提供了分支机构投资组合中各个方面的高级培训。 测序：毛细管DNA测序使用AB 3730xL，具有96个高吞吐量处理。应用程序是与DIR调查人员密切合作开发的。所有数据都将上传到服务器，该服务器跟踪和管理为研究所生成的所有测序数据。下一代测序采用Illumina GA II，Illumina HISEQ 2000和454 FLX钛FLX测序仪用于小RNA发现，ChIP-Seq，Transcriptomics，Exomememics，Exome序列，DE从头开始和REF-MAP基因组测序以及副本数量变异研究。 微阵列：RTB在Affymetrix微阵列平台上开发了特定项目的研究应用程序，包括自定义芯片设计，实验设计，样品处理和芯片处理。 RTB还为DIR研究计划开发了统计分析，数据管理和数据挖掘解决方案；着重解释由基因组学中使用的高度平行检测系统产生的数据。 RTB对高吞吐量微阵列数据验证，样本优化和下一个Gen数据验证执行QPCR 人/病原体基因分型：根据基因分型项目的范围，将几种技术用于人类和病原体基因分型，范围从基于毛细血管的整个人类基因的重新续订为de Novo SNP或/del Discovery的整个人类基因，或使用高吞吐量的靶向SNP基因分型通过使用TAQ7量子量的高吞吐量SNP基因分量，并Sequencer），用于Affymetrix SNP基于SNP的阵列和自定义病原体SNP（MIP Technology）阵列。 下一代测序技术在从头SNP，Indel，拷贝数，替代剪接变体以及人类和病原体基因组的新表达区域发现中起着重要作用。 对下一代，微阵列和QPCR数据分析进行了生物信息学数据分析。熟练的生物信息学家使用了装有最先进软件的高级特定于任务的服务器，用于为DIR科学家提供完工的，可发布的数据集，其中明显强调并提出了发现，以便于解释。数据集以多种格式存储，以快速检索和重新分析和 /或最近发布的竞争数据集进行比较分析。 我们的生物信息学家还执行了公共数据提交和提交的更正。 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 项目特异性研究应用是在样本制备和分析领域开发的，从基本的结构研究到各种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。最新的技术进步集中在两个主要领域：1）引入和优化先进的准备技术和技术，以改善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的保留和可视化，以及2）引入高级成像技术，包括高分辨率传输和扫描电子微型Microscopes。 该单元一直在开发相关技术，可用于检查光学显微镜的瞬态或动态事件，以识别感兴趣的区域，然后可以通过电子显微镜可视化，以确定将结构与功能测定法相关联。 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。 通过高压冻结的冷冻保护在低温下在称为冻结替代的过程中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。 EM单元最近获得的300 kV TEM显微镜是用于高分辨率3-D生物成像的最先进的仪器，该仪器配置为具有最佳的灵活性，可以响应研究人员的需求，并为其提供最高质量的图像。在接下来的十年中，可能会改善超微结构成像的平台，从而可以表征大分子，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到的宿主病原体的能力。 这些技术提高了我们将结构与功能联系起来的能力，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，由于在试样处理过程中固定了样品，因此仅提供一个快照，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。 该单元已经开发了创新的方法，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启用识别和成像单元。 视觉和医学艺术部门 RML的该单元为DIRS科学和支持人员提供高度专业化和技术服务。 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。